Nasal delivery pharmaceutical composition of long-acting GLP-1 / GIP dual agonist

Abstract

Provided are a nasal delivery pharmaceutical composition comprising a long-acting GLP-1 / GIP dual agonist and a kit comprising the pharmaceutical composition.

Description

Long-acting GLP-1 / GIP dual agonist nasal delivery pharmaceutical composition Technical Field

[0001] The present invention belongs to the field of polypeptide drug preparations. Specifically, the present invention provides a nasal delivery pharmaceutical composition and a kit containing the pharmaceutical composition. Background Art

[0002] Glucagon-like peptide 1 (GLP-1) peptide analogs have shown efficacy in treating type 2 diabetes. Semaglutide is a long-acting GLP-1 derivative developed by Novo Nordisk. Semaglutide is currently available in two dosage forms: injection and oral formulation.

[0003] The gastrointestinal system contains enzymes and a pH environment that degrade peptides. Peptide drugs do not easily penetrate the gastrointestinal membrane. Semaglutide's oral bioavailability is low, and it also has significant gastrointestinal adverse reactions and requires a strict dosing regimen. Therefore, peptide protein drugs, such as GLP-1 analogs, are more often administered by injection. However, injections have many drawbacks, such as the short biological half-life of insulin. Once or twice daily insulin injections can cause side effects such as subcutaneous fat atrophy, localized redness and swelling, and subcutaneous nodules, resulting in significant pain and poor compliance for patients requiring long-term medication.

[0004] GIP is an incretin hormone associated with metabolic diseases such as diabetes and obesity. In the GLP-1R / GIPR field, Eli Lilly's telpotide has been approved for marketing by the US Food and Drug Administration (FDA).

[0005] Glucagon (GCG) is a hormone produced in the α cells of the pancreas. Long-term administration can exhibit weight loss effects by increasing energy metabolism. However, the beneficial effects of GCG on energy metabolism have not been widely used due to its inherent hyperglycemic effect.

[0006] Designing a non-injectable formulation that is simple to prepare, mild and stable, and can promote the absorption of peptide drugs has always been a challenge for pharmaceutical developers. There is a need for a pharmaceutical composition with better bioavailability, more convenient administration, and higher compliance. Summary of the Invention

[0007] In one aspect of the present invention, a nasal delivery pharmaceutical composition is provided, comprising a GLP-1R agonist, a first penetration enhancer, and a second penetration enhancer, wherein the first penetration enhancer is a chelating agent, and the second penetration enhancer is selected from fatty acids and bile acids; or

[0008] The composition comprises a GLP-1R agonist and an alkyl saccharide absorption enhancer.

[0009] The nasal delivery pharmaceutical composition of the present invention can gently deliver polypeptide GLP-1R agonists to the nasal cavity without causing irritation to the nasal cilia and nasal mucosa, promote the absorption of the polypeptide compound in the nasal mucosa, and allow the drug to enter the blood directly, thus avoiding the first-pass effect of oral solid dosage forms. The pharmaceutical composition of the present invention not only fully guarantees the efficacy of the drug, but also helps to improve the convenience of administration, enhance compliance, and ensure complete absorption of the drug in the nasal mucosa. In particular, it can reduce the incidence of non-compliance with oral solid medications in the elderly, thereby achieving better therapeutic effects.

[0010] The nasal delivery pharmaceutical composition of the present invention has the characteristics of being non-irritating to the nasal mucosa and promoting the absorption of polypeptide drugs in the nasal cavity, thereby reducing the pain of drug administration for subjects.

[0011] In one aspect of the present invention, a kit is provided, comprising a container and a pharmaceutical composition for nasal delivery of the present invention contained in the container.

[0012] Preferably, the container is a nasal spray device. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG1 shows the blood glucose monitoring results of a rat efficacy experiment in which the composition containing semaglutide was administered nasally.

[0014] FIG2 shows the results of the blood drug concentration measurement in a rabbit pharmacokinetic experiment, in which the composition containing semaglutide was administered nasally.

[0015] FIG3 shows the blood glucose monitoring results of a rat efficacy experiment, in which the composition containing the polypeptide compound P016 of the present invention was administered nasally.

[0016] FIG4 shows the results of the blood drug concentration measurement in a rabbit pharmacokinetic experiment, in which the composition containing the polypeptide compound P016 of the present invention was administered nasally. DETAILED DESCRIPTION

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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 measurement or determination is made (i.e., the limitations of the measurement system). For example, "about" or "substantially comprising" can mean a 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.

[0022] Unless otherwise specified, all materials and reagents used in the present invention are commercially available.

[0023] 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.

[0024] In the context of the present invention, unless otherwise stated or clearly contradictory, the terms "pharmaceutical composition for nasal delivery", "pharmaceutical composition" and "composition" are used interchangeably.

[0025] As used herein, "compounds" and "conjugates" may exist as one or more stereoisomers. Stereoisomers are isomers that have identical constitution but differ in the arrangement of their atoms in space, and include enantiomers, diastereomers, cis-trans isomers (also known as geometric isomers), and atropisomers.

[0026] In the context of the present invention, unless otherwise specified or clearly contradictory, the unit "mg / mL" is based on the total volume of the pharmaceutical composition.

[0027] [Pharmaceutical composition and preparation method thereof]

[0028] The present invention discloses a nasal delivery drug composition, which comprises a GLP-1R agonist, a first penetration enhancer and a second penetration enhancer, wherein the first penetration enhancer is a chelating agent, and the second penetration enhancer is selected from fatty acids and bile acids; or

[0029] The composition comprises a GLP-1R agonist and an alkyl saccharide absorption enhancer.

[0030] In some embodiments, the composition is any one of a nasal spray, nasal drops, and a powder inhaler.

[0031] In some embodiments, the composition is in the form of an aqueous solution.

[0032] In some embodiments, the pH of the composition is from about 5.0 to about 9.0, such as from about 5.8 to about 9.0.

[0033] In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients or carriers.

[0034] In some embodiments, the excipients include: an osmotic pressure regulator, a buffer, a surfactant, an optional pH regulator, an optional viscosity regulator, and optional other excipients or carriers. The other excipients or carriers include preservatives, antioxidants, or combinations thereof.

[0035] In some embodiments, the pharmaceutical composition for nasal delivery is an aqueous solution having a pH of about 5.0-9.0, for example, about 5.8-about 9.0, and comprises: based on the total volume of the aqueous solution,

[0036] about 0.1 to about 50 mg / mL, e.g., about 1 to about 40 mg / mL, of a GLP-1R agonist,

[0037] about 0.1 to about 5 mg / mL of a first penetration enhancer,

[0038] about 0.5 to about 20 mg / mL of a second penetration enhancer,

[0039] about 0.5 to about 100 mg / mL of an osmotic pressure regulator,

[0040] about 0.01 to about 50 mg / mL of a surfactant,

[0041] about 0.5 to about 50 mM of a buffer,

[0042] optionally a pH adjuster, and

[0043] water.

[0044] In some embodiments, the pharmaceutical composition for nasal delivery is an aqueous solution having a pH of about 5.0-9.0, for example, about 5.8-about 9.0, and comprises: based on the total volume of the aqueous solution,

[0045] about 0.1 to about 50 mg / mL, e.g., about 1 to about 40 mg / mL, of a GLP-1R agonist,

[0046] about 0.1 to about 5 mg / mL of a first penetration enhancer,

[0047] about 0.5 to about 20 mg / mL of a second penetration enhancer,

[0048] about 0.5 to about 100 mg / mL of an osmotic pressure regulator,

[0049] about 0.01 to about 50 mg / mL of a surfactant,

[0050] about 0.5 to about 50 mM of a buffer,

[0051] optionally a pH adjuster,

[0052] 0 to about 5 mg / mL viscosity modifier, and

[0053] water.

[0054] In some embodiments, the mass ratio of the first permeation enhancer to the second permeation enhancer is about 1:1.2 to about 1:15, about 1:2 to about 1:10, preferably about 1:3 to about 1:5, for example, about 1:1.2, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14 or about 1:15.

[0055] In some embodiments, the pharmaceutical composition for nasal delivery is an aqueous solution having a pH of about 5.0-9.0, for example, about 5.8-about 9.0, and comprises: based on the total volume of the aqueous solution,

[0056] about 0.1 to about 50 mg / mL, e.g., about 1 to about 40 mg / mL, of a GLP-1R agonist,

[0057] About 0.5 to about 20 mg / mL of an alkyl saccharide absorption enhancer

[0058] about 0.5 to about 100 mg / mL of an osmotic pressure regulator,

[0059] about 0.01 to about 50 mg / mL of a surfactant,

[0060] about 0.5 to about 50 mM of a buffer,

[0061] optionally a pH adjuster, and

[0062] water.

[0063] In some embodiments, the pharmaceutical composition for nasal delivery is an aqueous solution having a pH of about 5.0 to about 9.0, for example about 5.8 to about 9.0, and comprises: based on the total volume of the aqueous solution,

[0064] about 0.1 to about 50 mg / mL, e.g., about 1 to about 40 mg / mL, of a GLP-1R agonist,

[0065] about 0.5 to about 20 mg / mL of an alkyl saccharide absorption enhancer,

[0066] about 0.5 to about 100 mg / mL of an osmotic pressure regulator,

[0067] about 0.01 to about 50 mg / mL of a surfactant,

[0068] about 0.5 to about 50 mM of a buffer,

[0069] optionally a pH adjuster,

[0070] 0 to about 5 mg / mL viscosity modifier, and

[0071] water.

[0072] In some embodiments, the water is purified water, deionized water, or water for injection.

[0073] In some embodiments, the method for preparing the pharmaceutical composition comprises:

[0074] Provides a buffer,

[0075] adding a first permeation enhancer, a second permeation enhancer, an osmotic pressure regulator, a surfactant, and optionally a viscosity regulator to a buffer to obtain a mixture,

[0076] mixing the mixture with water to obtain an aqueous mixture,

[0077] adding a GLP-1R agonist or an aqueous solution of a GLP-1R agonist to the aqueous mixture to obtain a mixture of GLP-1R agonists,

[0078] testing the pH of the mixture containing the GLP-1R agonist, and optionally adding a pH adjuster based on the deviation of the tested pH of the mixture from the target pH to adjust the pH of the mixture to the target pH, and

[0079] Add additional water to bring to volume.

[0080] In some embodiments, the method for preparing the pharmaceutical composition comprises:

[0081] Provides a buffer,

[0082] adding an absorption enhancer, an osmotic pressure regulator, a surfactant, and optionally a viscosity regulator to a buffer to obtain a mixture,

[0083] mixing the mixture with water to obtain an aqueous mixture,

[0084] adding a GLP-1R agonist or an aqueous solution of a GLP-1R agonist to the aqueous mixture to obtain a mixture of GLP-1R agonists,

[0085] testing the pH of the mixture containing the GLP-1R agonist, and optionally adding a pH adjuster based on the deviation of the tested pH of the mixture from the target pH to adjust the pH of the mixture to the target pH, and

[0086] Add additional water to bring to volume.

[0087] 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.

[0088] In some embodiments, the pharmaceutical composition comprises semaglutide, a first permeation enhancer, a second permeation enhancer, a buffer, an osmotic pressure regulator, a surfactant, water, and optionally a pH adjuster.

[0089] In some embodiments, the pharmaceutical composition contains semaglutide, an alkyl saccharide absorption enhancer, a buffer, an osmotic pressure regulator, a surfactant, water, and optionally a pH adjuster.

[0090] In some embodiments, the pharmaceutical composition comprises a polypeptide compound of Formula I, a first permeation enhancer, a second permeation enhancer, a buffer, an osmotic pressure regulator, a surfactant, water, and optionally a pH adjuster.

[0091] In some embodiments, the pharmaceutical composition comprises a polypeptide compound of formula I, an alkyl saccharide absorption enhancer, a buffer, an osmotic pressure regulator, a surfactant, water, and optionally a pH adjuster.

[0092] In some embodiments, the pharmaceutical composition contains:

[0093] Semaglutide or a compound of formula I,

[0094] a first penetration enhancer, wherein the first penetration enhancer is sodium ethylenediaminetetraacetate (EDTA),

[0095] a second penetration enhancer selected from the group consisting of sodium decanoate, sodium N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC), and deoxycholic acid (DCA),

[0096] an optional pH adjuster selected from aqueous sodium hydroxide or hydrochloric acid,

[0097] Surfactant, the surfactant is Tween, preferably Tween 20,

[0098] a buffer selected from trisodium citrate, trisodium citrate dihydrate, citric acid-trisodium citrate, citric acid monohydrate-trisodium citrate dihydrate, and combinations thereof;

[0099] an osmotic pressure regulator selected from the group consisting of mannitol, sodium chloride, propylene glycol, and combinations thereof, and

[0100] water.

[0101] In some embodiments, the pharmaceutical composition is an aqueous solution comprising, based on the total volume of the aqueous solution:

[0102] 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 to about 40 mg / mL, about 4.8 to about 40 mg / mL L, 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 of a GLP-1R agonist as a pharmaceutically active ingredient,

[0103] 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,

[0104] about 1 to about 100 mg / mL of mannitol,

[0105] about 0.01 to about 50 mg / mL of a surfactant,

[0106] optionally aqueous sodium hydroxide or hydrochloric acid, and

[0107] water,

[0108] The pH value of the aqueous solution is about 5.0-9.0, such as about 5.8-about 9.0, preferably about 6.0-8.8, more preferably about 6.5-about 7.4.

[0109] 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.

[0110] [GLP-1R agonists]

[0111] According to the present invention, the pharmaceutical active ingredient is a GLP-1R agonist, which is a GLP-1R (single) agonist, a GLP-1R / GIPR dual agonist, or a GLP-1R / GIPR / GCGR triple agonist.

[0112] In some embodiments, the GLP-1R agonist is selected from semaglutide, liraglutide, and exenatide.

[0113] In some embodiments, the GLP-1R agonist is semaglutide. "Semaglutide" refers to N-ε 26 -[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib 8 Arg 34 ]GLP-1(7-37).

[0114] In some embodiments, the GLP-1R / GIPR dual agonist is tilportide.

[0115] In some embodiments, the GLP-1R / GIPR dual agonist is a compound of Formula I below, or a pharmaceutically acceptable salt thereof, or a physiologically acceptable salt thereof, or a precursor compound thereof:

[0116] Y-Aib-EGT-αMePhe-TSDY-Aib-I-Aib-LDKQAQAEFVK 24 -WLLK 28 -GGPSSGAPPPSK formula I (SEQ ID NO: 12);

[0117] in,

[0118] 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.

[0119] [Polypeptide compound of formula I]

[0120] In some embodiments, the compound of Formula I is: Y-Aib-EGT-αMePhe-TSDY-Aib-I-Aib-LDKQAQAEFVK 24 -WLLK 28 -GGPSSGAPPPSK formula I (SEQ ID NO: 12);

[0121] in,

[0122] One of the K positions at position 24 or 28 is selected by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c-Z is chemically modified by being conjugated 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, each c is independently selected from an integer of 16, 18, or 20, and wherein Z is independently selected from -CH3 or -COOH; and the C-terminal amino acid is amidated to form a C-terminal primary amide.

[0123] 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 side chain chemical modification imparts charge, hydrophilicity, and hydrophobicity to the polypeptide backbone, while retaining the common characteristics of the polypeptide backbone itself. The compound of Formula I of the present invention has dual agonist effects on both the GLP-1 receptor and the GIP receptor, and can be used as a GLP-1 / GIP dual agonist, exhibiting excellent efficacy in lowering blood sugar and / or reducing weight, and exhibiting a long half-life.

[0124] In some embodiments, the "active pharmaceutical ingredient" (API) is a polypeptide compound of Formula I, which is a GLP-1 / GIP dual agonist.

[0125] In some embodiments, the API refers to the polypeptide compounds of the present invention P007, P008, P014, P019, P013, P015, P016, P017, P018, P020, and combinations thereof.

[0126] In some embodiments, the API refers to the polypeptide compound P016 of the present invention.

[0127] In the context of the present invention, unless otherwise specified or clearly contradictory, the terms "polypeptide compound" and "polypeptide" and "pharmaceutical active ingredient" can be used interchangeably, and can be a GIP-1 agonist, a GLP-1R / GIPR dual agonist, or a GLP-1R / GIPR / GCGR triple agonist.

[0128] In some embodiments, in the compound of Formula I,

[0129] 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

[0130] 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.

[0131] 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.

[0132] 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.

[0133] In some embodiments, the compound of Formula I is one or more compounds selected from:

[0134] 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:

[0135] 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:

[0136] 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:

[0137] 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:

[0138] 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:

[0139] 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:

[0140] 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:

[0141] 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:

[0142] 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:

[0143] and

[0144] 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:

[0145] In some embodiments, the compound of Formula I is 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)-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, that is, Y 1 -Aib 2 -E 3 -G 4 -T 5 -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 -P36 -P 37 -P 38 -S 39 -K 40 , molecular formula C 243 H 382 N 52 O 73 , the structure is as follows:

[0146] 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.

[0147] In some embodiments, the concentration of the polypeptide compound of Formula I in the pharmaceutical composition is at least about 0.1 mg / mL, about 1 mg / mL, 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.

[0148] 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.

[0149] [Penetration enhancer]

[0150] In some embodiments, the pharmaceutical composition for nasal delivery comprises a GLP-1R agonist, a first penetration enhancer, and a second penetration enhancer, wherein the first penetration enhancer is a chelating agent and the second penetration enhancer is selected from fatty acids and bile acids.

[0151] The term "permeation enhancer" refers to an excipient that increases the permeability of a drug substance or active pharmaceutical ingredient through cell membranes in vitro or in vivo. The permeation enhancers described herein may act by the following mechanisms: (1) increasing disordering of hydrophobic regions of the cell membrane, leading to increased transcellular transport; (2) extracting membrane proteins to increase transcellular transport; and / or (3) increasing intercellular pore size to increase paracellular transport.

[0152] The nasal delivery pharmaceutical composition of the present invention contains both a first penetration enhancer and a second penetration enhancer. Since water-soluble macromolecules of polypeptide compounds are primarily transported across membranes via intercellular junctions (adherens junctions + tight junctions), the use of either the first penetration enhancer or the second penetration enhancer alone can only partially open intercellular junctions. Surprisingly, the simultaneous use of the first penetration enhancer and the second penetration enhancer produces a synergistic effect, significantly improving the oral bioavailability of the polypeptide and reducing medication costs.

[0153] In some embodiments, the first penetration enhancer is a chelating agent selected from aminotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, cyclohexanediaminetetraacetic acid, ethylenediaminedi-o-phenylacetic acid, hydroxyethylethylenediaminetriacetic acid, and soluble salts thereof.

[0154] In some embodiments, the first penetration enhancer is ethylenediaminetetraacetic acid or a soluble salt thereof, preferably sodium ethylenediaminetetraacetate (EDTA).

[0155] In some embodiments, the second permeation enhancer is selected from fatty acids and bile acids, further selected from N-(8-[2-hydroxybenzoyl]amino)caprylic acid, N-(10-[2-hydroxybenzoyl]amino)decanoic acid, myristic acid, myristoleic acid, palmitoleic acid, stearic acid, oleic acid, hexanoic acid, caprylic acid, capric acid, lauric acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, and soluble salts thereof.

[0156] In some embodiments, the second permeation enhancer is selected from sodium decanoate, sodium N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC), and sodium deoxycholate (DCA).

[0157] In some embodiments, the mass ratio of the first permeation enhancer to the second permeation enhancer is about 1:1.2 to 1:15, about 1:2 to about 1:10, preferably about 1:3 to about 1:5, for example, about 1:1.2, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14 or about 1:15.

[0158] [Absorption enhancer]

[0159] In some embodiments, a pharmaceutical composition for nasal delivery comprises a GLP-1R agonist and an alkyl saccharide absorption enhancer.

[0160] The term "alkyl saccharide absorption enhancer" refers to an alkyl saccharide having an alkyl chain attached to sucrose or maltose via a glycosidic bond, the alkyl chain having a length of 8-20, such as 10-18, 11-16, or 12-14 carbon atoms.

[0161] In some embodiments, the alkyl saccharide absorption enhancer is selected from one or more of C12-C20 alkyl maltoside and C12-C20 alkyl sucrose side, for example, selected from dodecyl maltoside, tridecyl maltoside, tetradecyl maltoside, dodecyl sucrose side, tridecyl sucrose side, and tetradecyl sucrose side.

[0162] The "alkyl saccharide absorption enhancer" may exist as one or more stereoisomers. In some embodiments, the alkyl saccharide absorption enhancer is selected from dodecyl-β-D-maltoside, tridecyl-β-D-maltoside, tetradecyl-D-maltoside, or a combination thereof.

[0163] Alkyl saccharide absorption enhancers are generally recognized as safe (GRAS) substances and are non-irritating enhancers for transmucosal absorption. At concentrations up to 25%, alkyl saccharide absorption enhancers have been shown to be odorless, tasteless, non-toxic, non-mutagenic, and non-sensitizing in the rabbit eye irritation test (Draize test). They also reversibly open the tight junctions of epithelial cells, promoting the absorption of peptides in the nasal cavity.

[0164] [pH adjusters and buffers]

[0165] In some embodiments, the pharmaceutical composition comprises a pH adjusting agent.

[0166] In some embodiments, the pharmaceutical composition does not contain a pH adjusting agent.

[0167] In some embodiments, the pharmaceutical composition comprises a buffer.

[0168] In some embodiments, during the preparation of the pharmaceutical composition, after mixing the active ingredient and excipients other than the pH adjuster, 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 the pH adjuster added is very small and can be adjusted according to the target pH value.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] In the context of the present invention, acids, bases and salts may be in the form of themselves or of their hydrates.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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 ...

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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 2.0 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, about 6.0 to about 7.5 mM.

[0185] 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.

[0186] In some embodiments, the molar concentration of tromethamine in the pharmaceutical composition is from about 0.5 to about 50 mM, for example, from about 0.5 mM to about 40.0 mM, from about 0.5 mM to about 30.0 mM, from about 0.5 mM to about 20.0 mM, from about 0.5 mM to about 10.0 mM, from about 1.0 mM to about 40.0 mM, from about 1.0 mM to about 35.0 mM, from about 1.0 mM to about 30.0 mM, from about 1.0 mM to about 25.0 mM, from about 1.0 mM to about 20.0 mM, from about 1.0 mM to about 15.0 mM, from 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, about 6.0 to about 7.5 mM.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] In the context of the present invention, "citric acid" and "citric acid" have the same meaning and can be used interchangeably. "Trisodium citrate" and "trisodium citrate" have the same meaning and can be used interchangeably.

[0191] In some embodiments, the citric acid-citrate buffer is citric acid-trisodium citrate or a hydrate thereof, and the molar concentration of citric acid-trisodium citrate in the pharmaceutical composition is about 0.5-about 50 mM, for example, about 0.5 mM-about 40.0 mM, about 0.5 mM-about 30.0 mM, about 0.5 mM-about 20.0 mM, about 0.5 mM-about 10.0 mM, about 1.0 mM-about 40.0 mM, about 1.0 mM-about 35.0 mM, about 1.0 mM-about 30.0 mM, about 1.0 mM-about 25.0 mM, about 1.0 mM-about 20.0 mM, about 1 .0mM to about 15.0mM, about 1.0mM to about 10.0mM, about 2.0mM to 40.0mM, about 2.0mM to about 35.0mM, about 2.0mM to about 30.0mM, about 2.0mM to about 25.0mM, about 2.0mM to about 20.0mM, about 2.0mM to about 15.0mM, about 2.0mM to about 10.0mM, about 5.0mM to about 20.0mM, about 5.0mM to about 15.0mM, about 5.0mM to about 10.0mM, about 6.0 to about 9.0mM, about 6.0 to about 8.5mM, about 6.0 to about 8.0mM, about 6.0 to about 7.5mM.

[0192] 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.

[0193] In some embodiments, the total molar concentration of citric acid-trisodium 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.

[0194] In some embodiments, in the citric acid-trisodium citrate buffer, the molar ratio of citric acid to trisodium citrate is about 1:1-50, such as about 1:4-50, about 1:5-50, about 1:6-50, about 1:1-20, about 1:5-20, or about 1:5-10. 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.

[0195] 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.

[0196] In some 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.

[0197] In some 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.

[0198] In some 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.

[0199] 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.

[0200] In the context of the present invention, "aqueous HCl solution" and "hydrochloric acid" have the same meaning and can be used interchangeably.

[0201] In some embodiments, the pH adjuster is about 0.1 to about 0.5 M aqueous sodium hydroxide solution or aqueous HCl solution.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] [Osmotic pressure regulator]

[0206] In some embodiments, the pharmaceutical composition, preferably an aqueous solution of the pharmaceutical composition, contains an osmotic pressure regulating agent.

[0207] 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.

[0208] 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.

[0209] 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, borate, and sodium tartrate.

[0210] In some embodiments, the osmotic pressure adjusting agent includes one or more of mannitol, sodium chloride, propylene glycol, and glycerol.

[0211] In some embodiments, the osmotic pressure regulating agent is present in the pharmaceutical composition in an amount of about 1 to about 100 mg / mL based on the total volume of the pharmaceutical composition.

[0212] In some embodiments, the pharmaceutical composition has an osmotic pressure of about 200 to about 500 mOsm / kg, preferably about 250 to 350 mOsm / kg, and preferably about 260 to 320 mOsm / kg.

[0213] In some embodiments, osmotic pressure is measured by an osmometer.

[0214] In some embodiments, the osmotic pressure regulating agent is sodium chloride.

[0215] In some embodiments, the sodium chloride concentration is 1-20 mg / mL, preferably 5-20 mg / mL, more preferably 6-15 mg / mL, and even more preferably 6-10 mg / mL.

[0216] 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 7.5 mg / mL to about 9.5 mg / mL.

[0217] In some embodiments, the osmotic pressure regulating agent is propylene glycol.

[0218] 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.

[0219] In some embodiments, the concentration of propylene glycol in the pharmaceutical composition is from 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 12 mg / mL to about 16 mg / mL, for example, about 14 mg / mL.

[0220] In some embodiments, the osmotic regulator is mannitol.

[0221] In some embodiments, the concentration of mannitol in the pharmaceutical composition is from 10 mg / mL to about 60 mg / mL, e.g., from about 15 mg / mL to about 48 mg / mL, from about 20 mg / mL to about 48 mg / mL, from about 20 mg / mL to about 48 mg / mL, from about 25 mg / mL to about 48 mg / mL, from about 30 mg / mL to about 48 mg / mL, from about 15 mg / mL to about 40 mg / mL, from about 15 mg / mL to about 35 mg / mL, from about 20 mg / mL to about 40 mg / mL, from about 20 mg / mL to about 35 mg / mL, from about 25 mg / mL to about 40 mg / mL, from about 25 mg / mL to about 35 mg / mL, from about 30 mg / mL to about 40 mg / mL, and from 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.

[0222] [Viscosity modifier]

[0223] The pharmaceutical compositions of the present invention may optionally contain a viscosity modifier.

[0224] In some embodiments, the pharmaceutical composition contains a viscosity modifier.

[0225] In some embodiments, the pharmaceutical composition is free of a viscosity modifier.

[0226] In some embodiments, the viscosity modifier is selected from one or more of hypromellose, sodium carboxymethylcellulose, and povidone.

[0227] In some embodiments, the viscosity modifier may be present in an amount of about 0 to about 5 mg / mL based on the total volume of the pharmaceutical composition.

[0228] [preservative]

[0229] The pharmaceutical compositions of the present invention may optionally contain a preservative.

[0230] In some embodiments, the pharmaceutical composition contains a preservative.

[0231] In some embodiments, the pharmaceutical composition is preservative-free.

[0232] 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.

[0233] In some embodiments, phenol, m-cresol, cresol, chlorobutanol, benzyl alcohol, and any combination thereof are included.

[0234] [Antioxidants]

[0235] The pharmaceutical compositions of the present invention may optionally contain an antioxidant.

[0236] In some embodiments, the pharmaceutical composition contains an antioxidant.

[0237] In some embodiments, the pharmaceutical composition is free of antioxidants.

[0238] 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.

[0239] 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.

[0240] In some embodiments, the pharmaceutical composition does not contain vitamin E, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, sodium bisulfite, sodium sulfite, or sodium thiosulfate.

[0241] [Surfactant]

[0242] In some embodiments, the pharmaceutical composition contains a surfactant to inhibit physical aggregation or precipitation of the polypeptide.

[0243] Any surfactant commonly used in medicines 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.

[0244] Surfactants such as nonionic surfactants and / or ionic surfactants, specific examples of which can be lipid excipients (e.g., oleoyl polyoxyethylene glycerides Labrafil, caprylic capric macrogol glycerides Labrasol, propylene glycol dicaprylate Labrafac, Gelucire), polysorbates (also known as Tweens, such as polysorbate 20 (Tween 20), Tween 40, Tween 80), poloxamers (e.g., poloxamer 188), Triton, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glucoside, lauryl-, myristyl-, linoleyl-, or stearyl Acyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, or stearoyl-sarcosine, linoleyl-, myristyl-, or cetyl-betaine, lauramidopropyl-, cocoamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitoylpropyl-, or isostearamidopropyl-betaine (e.g., lauramidopropyl), myristamidopropyl-, palmitoylpropyl-, 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.).

[0245] In some embodiments, the pharmaceutical composition comprises Tween 20.

[0246] In some embodiments, the content of the surfactant can be about 0.01 mg / mL to about 50 mg / mL, such as about 0.5 mg / mL to about 50 mg / mL, about 0.5 mg / mL to about 40.0 mg / mL, about 0.5 mg / mL to about 30.0 mg / mL, about 0.5 mg / mL to about 20.0 mg / mL, about 0.5 mg / mL to about 10.0 mg / mL, about 1.0 mg / mL to about 40.0 mg / mL, about 1.0 mg / mL to about 35.0 mg / mL, about 1.0 mg / mL to about 30.0 mg / mL, about 1.0 mg / mL to about 25.0 mg / mL, about 1.0 mg / mL to about 20.0 mg / mL, about 1.0 mg / mL to about 15.0 mg / mL, about 1.0 mg / mL to about 25.0 mg / mL. 0 mg / mL to about 10.0 mg / mL, about 2.0 mg / mL to 40.0 mg / mL, about 2.0 mg / mL to about 35.0 mg / mL, about 2.0 mg / mL to about 30.0 mg / mL, about 2.0 mg / mL to about 25.0 mg / mL, about 2.0 mg / mL to about 20.0 mg / mL, about 2.0 mg / mL to about 15.0 mg / mL, about 2.0 mg / mL to about 10.0 mg / mL, about 5.0 mg / mL to about 20.0 mg / mL, about 5.0 mg / mL to about 15.0 mg / mL, about 5.0 mg / mL to about 10.0 mg / mL, about 6.0 to about 9.0 mg / mL, about 6.0 to about 8.5 mg / mL, about 6.0 to about 8.0 mg / mL, about 6.0 to about 7.5 mg / mL.

[0247] In some embodiments, the content of the surfactant is about 0.01 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, based on the total volume of the pharmaceutical composition. L, 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 35 mg / mL, about 40 mg / mL, about 45 mg / mL, or about 50 mg / mL.

[0248] [Use of the pharmaceutical composition for treating diseases, pharmaceutical use, and method for treating diseases]

[0249] In some embodiments, the present invention provides use of a pharmaceutical composition in the preparation of a medicament for preventing or treating a disease.

[0250] In some embodiments, the pharmaceutical compositions of the present invention are used to prevent or treat a disease.

[0251] 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.

[0252] In some embodiments, the disease comprises diabetes, obesity, a disease associated with diabetes, and / or a disease associated with obesity.

[0253] In some embodiments, the unit dosage or unit dose of the pharmaceutical composition is from about 0.5 mg to about 50 mg, for example, about 1-40 mg, about 10-35 mg, about 20-30 mg, about 1 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. Unless otherwise indicated or clearly contradictory, the unit dosage or unit dose is with respect to the pharmaceutical active ingredient.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] Unless otherwise specified or clearly contradictory, the pharmaceutical composition of the present invention can be administered to humans and animals, including monkeys, mice, dogs, cats, rabbits, pigs, alpacas, horses, sheep, and cattle.

[0261] In some embodiments, the nasal delivery pharmaceutical composition of the present invention is used to treat or prevent type II diabetes.

[0262] In some embodiments, the nasal delivery pharmaceutical composition of the present invention, which is a nasal spray and / or nasal drops, is used to treat or prevent type II diabetes.

[0263] [Container and kit]

[0264] In some embodiments, the present invention provides an article of manufacture or kit comprising a container, and a pharmaceutical composition for nasal delivery of the present invention contained in the container.

[0265] In some embodiments, the container is a nasal spray device.

[0266] In some embodiments, the container is an Aptar nasal spray device.

[0267] [Specific embodiment of the pharmaceutical composition containing polypeptide compound P016]

[0268] Embodiment A1. A pharmaceutical composition for nasal delivery, wherein the pH of the pharmaceutical composition is about 5.0 to about 9.0, for example, about 5.8 to about 9.0, and the pharmaceutical composition comprises, based on the total volume of the pharmaceutical composition:

[0269] About 0.1 to about 50 mg / mL of compound P016: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr10 -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 (SEQ ID NO: 7)

[0270] About 0.1 to about 5 mg / mL of a first penetration enhancer and about 0.5 to about 20 mg / mL of a second penetration enhancer, the first penetration enhancer is a chelating agent, and the second penetration enhancer is a fatty acid and a bile acid.

[0271] Embodiment A2. A pharmaceutical composition for nasal delivery, wherein the pH of the pharmaceutical composition is about 5.8 to about 9.0, and the pharmaceutical composition comprises, based on the total volume of the pharmaceutical composition:

[0272] About 0.1 to about 50 mg / mL of compound P016: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr10 -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 (SEQ ID NO: 7)

[0273] and about 0.5 to about 20 mg / mL of an alkyl saccharide absorption enhancer.

[0274] Embodiment A3. A nasal delivery pharmaceutical composition as described in embodiment A1, wherein the first penetration enhancer is selected from aminotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, cyclohexanediaminetetraacetic acid, ethylenediaminedi-o-phenylacetic acid, hydroxyethylethylenediaminetriacetic acid, and soluble salts thereof.

[0275] Embodiment A4. A nasal delivery pharmaceutical composition as described in embodiment A1 or A3, wherein the second penetration enhancer is selected from N-(8-[2-hydroxybenzoyl]amino)caprylic acid, N-(10-[2-hydroxybenzoyl]amino)decanoic acid, myristic acid, myristoleic acid, palmitoleic acid, stearic acid, oleic acid, hexanoic acid, caprylic acid, capric acid, lauric acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, and soluble salts thereof.

[0276] Embodiment A5. A nasal delivery pharmaceutical composition as described in embodiment A2, wherein the alkyl saccharide absorption promoter is selected from one or more of C12-C20 alkyl maltoside and C12-C20 alkyl sucrose sideroside, for example, selected from dodecyl maltoside, tridecyl maltoside, tetradecyl maltoside, dodecyl sucrose sideroside, tridecyl sucrose sideroside and tetradecyl sucrose sideroside.

[0277] Embodiment A6. A pharmaceutical composition for nasal delivery as described in any of Embodiments A1-A5, wherein the pharmaceutical composition further contains an osmotic pressure regulator, a buffer, a surfactant and water, an optional pH regulator, and optionally other excipients or carriers, wherein the other excipients or carriers include one or more of a viscosity regulator, a preservative, and an antioxidant.

[0278] Embodiment A7. A pharmaceutical composition for nasal delivery as described in embodiment A6, wherein the buffering agent is selected from phosphate, citrate or tromethamine, for example, selected from disodium hydrogen phosphate, tromethamine, trisodium citrate, disodium citrate, trisodium citrate dihydrate, citric acid-trisodium citrate, citric acid monohydrate-trisodium citrate dihydrate, and combinations thereof.

[0279] Embodiment A8. A pharmaceutical composition for nasal delivery according to embodiment A6 or A7, wherein the molar concentration of the buffer is about 0.5 to about 50 mM based on the total volume of the aqueous solution.

[0280] Embodiment A9. A pharmaceutical composition for nasal delivery as described in any of Embodiments A6-A8, wherein the osmotic pressure regulating agent is selected from mannitol, sodium chloride, propylene glycol, and combinations thereof.

[0281] Embodiment A10. A pharmaceutical composition for nasal delivery according to any one of embodiments A6 to A9, wherein the osmotic pressure regulating agent is present in an amount of about 1 to about 100 mg / mL based on the total volume of the aqueous solution.

[0282] Embodiment A11. A pharmaceutical composition for nasal delivery as described in any one of embodiments A6-A10, wherein the surfactant is Tween, such as Tween 20, Tween 40, Tween 80, or a combination thereof.

[0283] Embodiment A12. A pharmaceutical composition for nasal delivery as described in any of Embodiments A6-A11, wherein the surfactant is present in an amount of about 0.01 to about 50 mg / mL based on the total volume of the aqueous solution.

[0284] Embodiment A13. A pharmaceutical composition for nasal delivery as described in any of Embodiments A6-A12, wherein the pH adjusting agent is aqueous sodium hydroxide solution or hydrochloric acid.

[0285] Embodiment A14. A pharmaceutical composition for nasal delivery according to any one of Embodiments A1 and A3-A13, wherein the pharmaceutical composition is an aqueous solution comprising, based on the total volume of the aqueous solution:

[0286] about 0.1 to about 50 mg / mL, for example about 1 to about 40 mg / mL of compound P016,

[0287] about 0.1 to about 5 mg / mL of EDTA,

[0288] about 0.5 to about 20 mg / mL of sodium decanoate, sodium N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC), deoxycholic acid (DCA), or a combination thereof,

[0289] The total molar concentration of citric acid and trisodium citrate is about 0.5 to about 50 mM, and the molar ratio of citric acid to trisodium citrate is about 1:4 to 50, preferably about 1:4 to 20,

[0290] about 1 to about 100 mg / mL of mannitol,

[0291] About 0.01 to about 50 mg / mL of Tween 20,

[0292] optionally aqueous sodium hydroxide or hydrochloric acid, and

[0293] water,

[0294] 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.

[0295] Embodiment A15. A pharmaceutical composition for nasal delivery according to any one of Embodiments A2-A13, wherein the pharmaceutical composition is an aqueous solution comprising, based on the total volume of the aqueous solution:

[0296] about 0.1 to about 50 mg / mL, for example about 1 to about 40 mg / mL of compound P016,

[0297] about 0.5 to about 20 mg / mL of dodecyl maltoside (DDM),

[0298] citric acid and trisodium citrate at a total molar concentration of about 0.5 to about 50 mM,

[0299] about 1 to about 100 mg / mL of mannitol,

[0300] About 0.01 to about 50 mg / mL of Tween 20,

[0301] optionally aqueous sodium hydroxide or hydrochloric acid, and

[0302] water,

[0303] 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.

[0304] Embodiment A16. The nasal delivery pharmaceutical composition as described in any one of Embodiments A1 and A3-A14, wherein the mass ratio of the first penetration enhancer to the second penetration enhancer is about 1:1.2 to 1:15, such as about 1:2 to about 1:10, preferably about 1:3 to about 1:5.

[0305] Embodiment A17. A kit comprising a container, and a nasal delivery pharmaceutical composition as described in any one of embodiments A1-A16 contained in the container,

[0306] Preferably, the container is a nasal spray device.

[0307] Embodiment A18. The kit of embodiment A17, wherein the unit dose of the pharmaceutical composition is from about 0.5 mg to about 50 mg.

[0308] Embodiment A19. A pharmaceutical composition for nasal delivery, wherein the pharmaceutical composition is an aqueous solution having a pH of about 5.8 to about 9.0, and the pharmaceutical composition comprises about 0.1 to about 50 mg / mL of the following polypeptide compound:

[0309] Compound P016: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -αMePhe 6 -Thr 7 -Ser 8 -Asp9 -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 (SEQ ID NO: 7)

[0310] Embodiment A20. The pharmaceutical composition according to embodiment A19, wherein the pharmaceutical composition further comprises an alkyl saccharide absorption enhancer, or

[0311] The pharmaceutical composition also contains a combination of a first penetration enhancer and a second penetration enhancer.

[0312] Embodiment A21. The pharmaceutical composition of embodiment A20, wherein the pharmaceutical composition further comprises an osmotic pressure regulator, a buffer and water, and optionally a pH regulator.

[0313] Embodiment A22. The pharmaceutical composition of embodiment A20 or A21, wherein the buffer is selected from disodium hydrogen phosphate, tromethamine, disodium citrate, trisodium citrate, trisodium citrate dihydrate, citric acid-trisodium citrate, citric acid monohydrate-trisodium citrate dihydrate, and combinations thereof.

[0314] Embodiment A23. The pharmaceutical composition of any one of Embodiments A20-A22, wherein the molar concentration of the buffer is about 0.5 to about 50 mM based on the total volume of the aqueous solution.

[0315] Embodiment A24. The pharmaceutical composition of any one of Embodiments A20-A23, wherein the osmotic pressure regulating agent is selected from mannitol, sodium chloride, propylene glycol, and combinations thereof.

[0316] Embodiment A25. The pharmaceutical composition of any one of Embodiments A20-A24, wherein the osmotic pressure regulator is present in an amount of about 1 to about 100 mg / mL based on the total volume of the aqueous solution.

[0317] Embodiment A26. The pharmaceutical composition of any one of Embodiments A20-A25, wherein the pH adjuster is aqueous sodium hydroxide solution or hydrochloric acid.

[0318] Embodiment A27. A pharmaceutical composition as described in any of Embodiments A20-A26, wherein the pharmaceutical composition further contains a combination of a first penetration enhancer and a second penetration enhancer, the first penetration enhancer being selected from aminotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, cyclohexanediaminetetraacetic acid, ethylenediaminedi-o-phenylacetic acid, hydroxyethylethylenediaminetriacetic acid, and soluble salts thereof.

[0319] Embodiment A28. A pharmaceutical composition according to any one of embodiments A20 to A27, wherein the pharmaceutical composition further comprises a combination of a first penetration enhancer and a second penetration enhancer, the second penetration enhancer being selected from N-(8-[2-hydroxybenzoyl]amino)caprylic acid, N-(10-[2-hydroxybenzoyl]amino)decanoic acid, myristic acid, myristoleic acid, palmitoleic acid, stearic acid, oleic acid, hexanoic acid, caprylic acid, capric acid, lauric acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, and soluble salts thereof.

[0320] Embodiment A29. A pharmaceutical composition as described in any one of Embodiments A20 to A28, wherein the pharmaceutical composition further contains an alkyl saccharide absorption enhancer, wherein the alkyl saccharide absorption enhancer is selected from one or more of C12-C20 alkyl maltoside and C12-C20 alkyl sucrose side, for example, selected from dodecyl maltoside, tridecyl maltoside, tetradecyl maltoside, dodecyl sucrose side, tridecyl sucrose side and tetradecyl sucrose side.

[0321] Embodiment A30. The pharmaceutical composition of any one of Embodiments A19-A29, wherein the pharmaceutical composition is an aqueous solution comprising:

[0322] Based on the total volume of the aqueous solution, about 0.1 to about 50 mg / mL, for example, about 1 to about 40 mg / mL of compound P016,

[0323] 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,

[0324] about 1 to about 100 mg / mL of mannitol based on the total volume of the aqueous solution,

[0325] optionally aqueous sodium hydroxide or hydrochloric acid, and

[0326] water,

[0327] 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.

[0328] Embodiment A31. Use of a pharmaceutical composition according to any one of Embodiments A1 to A30 for the preparation of a medicament for treating a disease, such as diabetes, obesity, a disease associated with diabetes, and / or a disease associated with obesity.

[0329] Embodiment A32. The use according to claim A31, wherein the unit dose of the pharmaceutical composition is from about 0.5 mg to about 50 mg.

[0330] [Specific embodiment of the pharmaceutical composition containing semaglutide]

[0331] Embodiment B1. A pharmaceutical composition for nasal delivery, wherein the pH of the pharmaceutical composition is about 5.0-9.0, for example, about 5.8 to about 9.0, and the pharmaceutical composition comprises, based on the total volume of the pharmaceutical composition:

[0332] about 0.1 to about 50 mg / mL of semaglutide; and

[0333] About 0.1 to about 5 mg / mL of a first penetration enhancer and about 0.5 to about 20 mg / mL of a second penetration enhancer, the first penetration enhancer is a chelating agent, and the second penetration enhancer is a fatty acid and a bile acid.

[0334] Embodiment B2. A pharmaceutical composition for nasal delivery, wherein the pH of the pharmaceutical composition is about 5.0-9.0, for example, about 5.8 to about 9.0, and the pharmaceutical composition comprises, based on the total volume of the pharmaceutical composition:

[0335] about 0.1 to about 50 mg / mL of semaglutide; and

[0336] About 0.5 to about 20 mg / mL of an alkyl saccharide absorption enhancer.

[0337] Embodiment B3. A nasal delivery pharmaceutical composition as described in embodiment B1, wherein the first penetration enhancer is selected from aminotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, cyclohexanediaminetetraacetic acid, ethylenediaminedi-o-phenylacetic acid, hydroxyethylethylenediaminetriacetic acid, and soluble salts thereof.

[0338] Embodiment B4. A nasal delivery drug composition as described in embodiment B1 or B3, wherein the second penetration enhancer is selected from N-(8-[2-hydroxybenzoyl]amino)caprylic acid, N-(10-[2-hydroxybenzoyl]amino)decanoic acid, myristic acid, myristoleic acid, palmitoleic acid, stearic acid, oleic acid, hexanoic acid, caprylic acid, capric acid, lauric acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, and soluble salts thereof.

[0339] Embodiment B5. A nasal delivery drug composition as described in embodiment B2, wherein the alkyl saccharide absorption enhancer is selected from one or more of C12-C20 alkyl maltoside and C12-C20 alkyl sucrose side, for example, selected from dodecyl maltoside, tridecyl maltoside, tetradecyl maltoside, dodecyl sucrose side, tridecyl sucrose side and tetradecyl sucrose side.

[0340] Embodiment B6. A pharmaceutical composition for nasal delivery as described in any of Embodiments B1-B5, wherein the pharmaceutical composition further contains an osmotic pressure regulator, a buffer, a surfactant and water, an optional pH regulator, and optionally other excipients or carriers, wherein the other excipients or carriers include one or more of a viscosity regulator, a preservative, and an antioxidant.

[0341] Embodiment B7. A pharmaceutical composition for nasal delivery as described in embodiment B6, wherein the buffering agent is selected from phosphate, citrate or tromethamine, for example, selected from disodium hydrogen phosphate, tromethamine, trisodium citrate, disodium citrate, trisodium citrate dihydrate, citric acid-trisodium citrate, citric acid monohydrate-trisodium citrate dihydrate, and combinations thereof.

[0342] Embodiment B8. A pharmaceutical composition for nasal delivery as described in embodiment B6 or B7, wherein the molar concentration of the buffer is about 0.5 to about 50 mM based on the total volume of the aqueous solution.

[0343] Embodiment B9. A pharmaceutical composition for nasal delivery as described in any of embodiments B6-B8, wherein the osmotic pressure regulating agent is selected from mannitol, sodium chloride, propylene glycol, and combinations thereof.

[0344] Embodiment B10. A pharmaceutical composition for nasal delivery as described in any one of Embodiments B6 to B9, 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.

[0345] Embodiment B11. A pharmaceutical composition for nasal delivery as described in any one of embodiments B6 to B10, wherein the surfactant is Tween, such as Tween 20, Tween 40, Tween 80, or a combination thereof.

[0346] Embodiment B12. A pharmaceutical composition for nasal delivery as described in any of Embodiments B6-B11, wherein the surfactant is present in an amount of about 0.01 to about 50 mg / mL based on the total volume of the aqueous solution.

[0347] Embodiment B13. A pharmaceutical composition for nasal delivery as described in any of Embodiments B6-B12, wherein the pH adjusting agent is aqueous sodium hydroxide solution or hydrochloric acid.

[0348] Embodiment B14. A pharmaceutical composition for nasal delivery according to any one of Embodiments B1 and B3-B13, wherein the pharmaceutical composition is an aqueous solution comprising, based on the total volume of the aqueous solution:

[0349] about 0.1 to about 50 mg / mL, e.g., about 1 to about 40 mg / mL, of semaglutide,

[0350] about 0.1 to about 5 mg / mL of EDTA,

[0351] about 0.5 to about 20 mg / mL of sodium decanoate, sodium N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC), deoxycholic acid (DCA), or a combination thereof,

[0352] a total molar concentration of citric acid and trisodium citrate of 0.5 to about 50 mM, a molar ratio of citric acid to trisodium citrate of about 1:4 to 50, preferably about 1:4 to 20,

[0353] about 1 to about 100 mg / mL of mannitol,

[0354] About 0.01 to about 50 mg / mL of Tween 20,

[0355] optionally aqueous sodium hydroxide or hydrochloric acid, and

[0356] water,

[0357] 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.

[0358] Embodiment B15. A pharmaceutical composition for nasal delivery according to any one of Embodiments B2 to B13, wherein the pharmaceutical composition is an aqueous solution comprising, based on the total volume of the aqueous solution:

[0359] about 0.1 to about 50 mg / mL, e.g., about 1 to about 40 mg / mL, of semaglutide,

[0360] about 0.5 to about 20 mg / mL of dodecyl maltoside (DDM),

[0361] a total molar concentration of citric acid and trisodium citrate of 0.5 to about 50 mM, a molar ratio of citric acid to trisodium citrate of about 1:4 to 50, preferably about 1:4 to 20,

[0362] about 1 to about 100 mg / mL of mannitol,

[0363] About 0.01 to about 50 mg / mL of Tween 20,

[0364] optionally aqueous sodium hydroxide or hydrochloric acid, and

[0365] water,

[0366] The pH value of the aqueous solution is about 5.8 to about 9.0, about 5.0 to about 9.0, for example, preferably about 6.0 to about 8.8, more preferably about 6.5 to about 7.4.

[0367] Embodiment B16. The nasal delivery pharmaceutical composition of any one of Embodiments B1 and B3-B14, wherein the mass ratio of the first permeation enhancer to the second permeation enhancer is about 1:1.2 to 1:15, for example, about 1:2 to about 1:10, preferably about 1:3 to about 1:5.

[0368] Embodiment B17. A kit comprising a container, and a nasal delivery pharmaceutical composition as described in any one of embodiments B1-B16 contained in the container,

[0369] Preferably, the container is a nasal spray device.

[0370] Embodiment B18. The kit of embodiment B17, wherein the unit dose of the pharmaceutical composition is from about 0.5 mg to about 50 mg.

[0371] Embodiment B19. Use of the pharmaceutical composition of any one of Embodiments B1 to B16 for the preparation of a medicament for treating a disease, such as diabetes, obesity, a disease associated with diabetes, and / or a disease associated with obesity.

[0372] Embodiment B20. The use according to claim B19, wherein the unit dose of the pharmaceutical composition is from about 0.5 mg to about 50 mg.

[0373] The following examples are intended to illustrate the present invention in detail but are not intended to limit the scope of the present invention.

[0374] Example

[0375] [Preparation of polypeptide compounds]

[0376] [Compound Preparation Example 1—Preparation of Polypeptide Compound P015]

[0377] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0378] 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).

[0379] 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.

[0380] The preparation method adopts the Fmoc solid phase peptide synthesis strategy, and the method includes:

[0381] (1) Synthesis of peptide resin intermediate 1 (compound P001, SEQ ID NO: 1)

[0382] 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 later use.

[0383] 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.

[0384] 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.

[0385] 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.

[0386] get:

[0387] 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

[0388] After the above condensation was completed, the Mtt protection was removed with 50% HFIP / DCM solution for 30 minutes, followed by washing and filtration to obtain peptide resin intermediate 1 (compound P001, SEQ ID NO: 1):

[0389] 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.

[0390] (2) Modification chain modification step

[0391] 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.

[0392] 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:

[0393] 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 (SEQ ID NO:6).

[0394] 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.

[0395] [Compound Preparation Example 2—Preparation of Polypeptide Compound P019]

[0396] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0397] 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).

[0398] 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.

[0399] The preparation method adopts the Fmoc solid phase peptide synthesis strategy, and the method includes:

[0400] (1) Synthesis of peptide resin intermediate 2

[0401] 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.

[0402] 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.

[0403] 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.

[0404] 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.

[0405] get:

[0406] 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

[0407] 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 peptide resin intermediate 2:

[0408] 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.

[0409] (2) Modification chain modification step

[0410] 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.

[0411] 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:

[0412] 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.

[0413] 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.

[0414] [Compound Preparation Example 3—Preparation of Polypeptide Compound P007]

[0415] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0416] 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).

[0417] 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.

[0418] 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.

[0419] [Compound Preparation Example 4—Preparation of Polypeptide Compound P013]

[0420] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0421] 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).

[0422] 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.

[0423] 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.

[0424] [Compound Preparation Example 5—Preparation of Polypeptide Compound P014]

[0425] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0426] 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).

[0427] 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.

[0428] 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.

[0429] [Compound Preparation Example 6—Preparation of Polypeptide Compound P016]

[0430] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0431] 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).

[0432] 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.

[0433] 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.

[0434] [Compound Preparation Example 7—Preparation of Polypeptide Compound P008]

[0435] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0436] 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: 3).

[0437] 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.

[0438] 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.

[0439] [Compound Preparation Example 8—Preparation of Polypeptide Compound P017]

[0440] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0441] 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).

[0442] 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.

[0443] 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.

[0444] [Compound Preparation Example 9—Preparation of Polypeptide Compound P018]

[0445] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0446] 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).

[0447] 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.

[0448] 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.

[0449] [Compound Preparation Example 10—Preparation of Polypeptide Compound P020]

[0450] Y-X1-EGT-X2-TSDY-X3-I-X4-LDKQAQAEFVKWLLKGGPSSG-APPPSK;

[0451] 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).

[0452] 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.

[0453] 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.

[0454] 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:

[0455] 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

[0456] 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:

[0457] 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 [Agonistic activity of peptide compounds on hGLP-1R and hGIPR]

[0458] For human GLP-1 and GIP receptors, the in vitro functional activity of the compounds of the invention at these receptors was determined in HEK-293 clonal cell lines expressing these receptors.

[0459] 1) Agonist activity on human GLP-1 receptor (cAMP reporter gene method)

[0460] The cAMP reporter gene assay was used to determine the agonist activity of the compounds of the present invention on human GLP-1R, with P001, Tirzepatide and endogenous ligand GLP-1 as references.

[0461] HEK293 / CRE / GLP-1R cells were seeded at 50,000 cells / well (80 μL / well) in a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator. 20 μL / well of assay medium (DMEM containing 0.1% casein) containing a compound (a compound of the present invention, P001, Tirzepatide, or GLP-1) was added to the 96-well plate and incubated in a 37°C, CO2 incubator for another 6 hours. After equilibration to room temperature, the supernatant was removed, and 50 μL / well of Bright-Glo reagent was added. The cells were shaken and lysed at room temperature for 10 minutes. Luminescence was read using an Envision microplate reader to measure luciferase activity.

[0462] The response value of 100 nM GLP-1 was set as 100% response value, and nonlinear regression was performed using GraphPad based on the response percentage and the added compound concentration to obtain the EC50 value of each compound.

[0463] 2) Agonist activity on human GIP receptor (LANCE Ultra cAMP assay)

[0464] The LANCE Ultra cAMP Kit was used to determine the agonist activity of the compounds of the present invention on human GIPR, with P001, Tirzepatide and endogenous ligand GIP as references.

[0465] The in vitro agonist activity of the compounds of the present invention against GIPR was determined in HEK293 cells stably expressing human GIPR (HEK293 / GIPR cells). HEK293 / GIPR cells were prepared with HBSS buffer (0.1% Casein, 500 μM IBMX, 5 mM HEPES) and seeded at 1000 cells / well (5 μL / well) in 384-well cell culture plates. 5 μL of HBSS buffer containing 2× compound was added to the 384-well cell culture plates. The plates were sealed and incubated in a 37°C, 5% CO2 incubator for approximately 30 minutes. After incubation, 5 μL of cAMP-Eu working solution and 5 μL of cAMP-Ulight working solution were added, followed by shaking to mix. The plates were incubated at 25°C for 1 hour. The signals at 665 nm and 615 nm were read on an Envision microplate reader. The 665 nm / 615 nm ratio was calculated and converted to cAMP concentration using a cAMP standard curve. The response value of 1 μM GIP was set as 100% response value, and nonlinear regression was performed using GraphPad based on the response percentage and the concentration of the added compound to obtain the EC value of each compound. 50 value.

[0466] Table 1. Agonist activity at human GLP-1 and GIP receptors, EC 50 value

[0467] NA: Not detected

[0468] EC 50 Value: Endogenous ligand EC 50 EC values ​​of the tested compounds 50 Value ratio

[0469] The agonist activity of all compounds at human GLP-1 and GIP receptors is expressed as EC50 values, with larger EC50 values ​​indicating stronger activity. All compounds of the present invention exhibited stronger agonist activity at human GLP-1R than tilpoitide, and six of the compounds, P007, P008, P013, P016, P018, and P019, also exhibited slightly stronger agonist activity at human GIP receptors than tilpoitide.

[0470] [Pharmacodynamics of polypeptide compounds]

[0471] [Compound Pharmacodynamics Example 1] Pharmacodynamics of Polypeptide Compounds in db / db Mice

[0472] 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.

[0473] 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 vehicle (PBS containing 0.1% Tween 20, pH 7.2-7.4). After a single subcutaneous administration, random blood glucose levels were recorded at designated time points (0-120 h, with random blood glucose recording discontinued when the compound's random blood glucose level was no different from that of the vehicle group), as well as daily body weight and food intake. Statistical analysis of the data was performed using GraphPad Prism 8. Statistical differences between groups were analyzed using T-TEST; differences were considered significant when P < 0.05.

[0474] 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).

[0475] Table 1-1 Duration of hypoglycemic effect of compounds

[0476] *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.

[0477] 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).

[0478] 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).

[0479] 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.

[0480] 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.

[0481] 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.

[0482] [Compound Pharmacodynamics Example 2] Weight Loss Effect of Peptide Compound P016 in the DIO Mouse Obesity Model

[0483] 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 to compare the effects 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.

[0484] 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.

[0485] 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. The polypeptide compound P016 of the present invention significantly reduced fasting blood glucose and insulin levels on D30 at doses of 1, 3, and 30 nmol / kg, improving insulin resistance. The compound of the present invention also significantly reduced liver weight and serum ALT levels, improving liver function.

[0486] Table 1-2 Cumulative food intake, body weight, fat and liver weight of DIO mice 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.

[0487] Table 1-3 Fasting blood glucose, insulin and insulin resistance index of DIO mice Note: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 compared with the Vehicle group; #P ≤ 0.05 compared with the Tirzepatide group, a positive control at the same dose. Results are expressed as Mean ± SEM of 10 mice.

[0488] Table 1-4 Serum TC and ALT in DIO mice Note: *P≤0.05, ***P≤0.001, ****P≤0.0001 compared with the Vehicle group. Results are expressed as Mean ± SEM of 10 mice.

[0489] 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.

[0490] [Composition Preparation Example]

[0491] At room temperature, 1.9 mg of trisodium citrate dihydrate (C6H5Na3O7·2H2O, molecular weight 294) and 0.22 mg of citric acid monohydrate (C6H8O7·H2O, molecular weight 210) were weighed and added to an appropriate amount of purified water, stirred and dissolved to prepare a citric acid-trisodium citrate buffer. 46 mg of mannitol and Tween 20 were added to the buffer and stirred to dissolve, with the final concentration of Tween 20 in the composition being 0.1% (w / w). 2.08 mg of polypeptide compound P016 prepared in Compound Preparation Example 6 above was then added and stirred to dissolve. The pH of the resulting mixture was measured using a pH meter, and the pH was adjusted to 6.8-7.4 by adding 0.5 M NaOH or 0.1 M HCl aqueous solution. The volume was then made up to 1 mL with purified water to obtain Composition A1.

[0492] Compositions A2 to A6 were prepared in the same manner as composition A1, except that the raw materials and their amounts were adjusted according to the formulation shown in Table 2-1 below.

[0493] Unless otherwise stated or clearly contradictory, in the Examples of the present invention, the final concentration of Tween 20 in the composition is 0.1 w / w%, corresponding to about 1 mg / mL, based on the total weight or volume of the composition.

[0494] Table 2-1 Formulations of pharmaceutical compositions A1 to A6

[0495] [Pharmacokinetics of Composition Example] Transmembrane Transport of Permeation Enhancer Combination and Semaglutide (Cellular Transport Experiment)

[0496] [Preparation of Control Compositions B1 to B5, Compositions C1 and C2]

[0497] Control composition B1: Semaglutide was weighed, passed through a 40-mesh sieve, and dissolved in HBSS to a concentration of 0.5 mg / mL.

[0498] HBSS is Hank's balanced salt solution, a phosphate buffered saline solution whose main components are NaCl, KCl, KH2PO4, Na2HPO4, NaHCO3 and glucose, purchased from Maclean.

[0499] Control composition B2: Semaglutide and sodium N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC) were weighed, passed through a 40-mesh sieve, and dissolved in HBSS to prepare an aqueous solution of 0.5 mg / mL semaglutide and 3 mg / mL SNAC.

[0500] Control composition B3: Semaglutide and disodium ethylenediaminetetraacetic acid (EDTA) were weighed, passed through a 40-mesh sieve, and dissolved in HBSS to prepare an aqueous solution of 0.5 mg / mL semaglutide and 1 mg / mL disodium ethylenediaminetetraacetic acid (EDTA).

[0501] Control composition B4: Semaglutide and sodium decanoate were weighed, passed through a 40-mesh sieve, and dissolved in HBSS to prepare an aqueous solution of 0.5 mg / mL semaglutide and 4 mg / mL sodium decanoate.

[0502] Control composition B5: Semaglutide and sodium deoxycholate (DCA) were weighed, passed through a 40-mesh sieve, and dissolved in HBSS to prepare an aqueous solution of 0.5 mg / mL semaglutide and 5 mg / mL sodium deoxycholate (DCA).

[0503] Composition C1 (penetration enhancer combination): Take (% w / w) 1 part of semaglutide, mix it with 2 parts of disodium ethylenediaminetetraacetic acid (EDTA) and 8 parts of sodium decanoate, pass it through a 40-mesh sieve, and dissolve it in purified water to prepare an aqueous solution of 0.5 mg / mL semaglutide, 1 mg / mL disodium ethylenediaminetetraacetic acid (EDTA), and 4 mg / mL sodium decanoate.

[0504] Composition C2 (penetration enhancer combination): Take 1 part (% w / w) of semaglutide, mix it with 2 parts of disodium ethylenediaminetetraacetic acid (EDTA) and 10 parts of sodium deoxycholate, pass it through a 40-mesh sieve, and dissolve it in purified water to prepare an aqueous solution of 0.5 mg / mL semaglutide, 1 mg / mL disodium ethylenediaminetetraacetic acid (EDTA), and 5 mg / mL sodium deoxycholate (DCA).

[0505] [Transmembrane transport experiment]

[0506] 1. Experimental materials: MDCK (NBL-2) cells were purchased from Beina Biotechnology, passage 8, catalog number BNCC337866.

[0507] 2. Cell culture: MDCK cells were cultured at 6 x 10 4 cells / cm 2Cells were seeded onto polycarbonate membranes in 24-well Corning Transwell plates and allowed to form confluent cell monolayers within 3-4 days.

[0508] 3. Experimental plan: The control compositions B1 to B5 prepared above and the compositions C1 and C2 containing the permeation enhancer combination were dissolved in HBSS and prepared to the corresponding concentrations. In duplicate, the permeability of semaglutide from A to B was determined by adding 0.5 mL of 20 μg / mL fluorescein yellow and semaglutide solution to the top side and 1.5 mL of transport buffer to the B side. The 24-well Corning Transwell plate was incubated at 37±1°C without shaking. A 200 μL sample was taken from the B side at 120 minutes. The concentration of semaglutide was quantified by LC / MS analysis.

[0509] 4. Experimental determination:

[0510] The apparent permeability coefficient Papp (cm / s) was calculated using the following equation:

[0511] Papp=(dCr / dt)x Vr / (A×C0),

[0512] where dCr / dt is the cumulative concentration of semaglutide in side B as a function of time (μM / s); Vr is the volume of solution in side B of the receiving chamber (0.5 mL for side A and 1.5 mL for side B for 12 wells); and A is the surface area of ​​the transport (1.12 cm for a monolayer in a 12-well plate). 2 ); C0 is the initial concentration of semaglutide in the donor compartment (μM). The experimental results are recorded in the following Table 2-2.

[0513] Table 2-2 Transmembrane transport of semaglutide

[0514] As can be seen from Table 2 above, the composition C1 of the present invention contains a combination of the first penetration enhancer (EDTA) and the second penetration enhancer (sodium decanoate), and the Papp is 1.81*10 -6 cm / s, the Papp of the control composition B3 containing only the first penetration enhancer (EDTA) was 0.04*10 -6 cm / s, and the Papp of the control composition B4 containing only the second penetration enhancer (sodium decanoate) was 0.06*10 -6 The Papp of the composition C1 of the present invention is much greater than the sum of the Papps of the control compositions B3 and B4, which unexpectedly produces a synergistic effect of Papp.

[0515] Composition C2 of the present invention contains a combination of a first penetration enhancer (EDTA) and a second penetration enhancer (DCA), and has a Papp of 4.48*10-6 cm / s, the Papp of the control composition B3 containing only the first penetration enhancer (EDTA) was 0.04*10 -6 cm / s, the Papp of the control composition B5 containing only the second penetration enhancer (DCA) was 3.85*10 -6 The Papp of the composition C2 of the present invention is significantly greater than the sum of the Papps of the control compositions B3 and B5, which unexpectedly produces a synergistic effect of Papp.

[0516] [Pharmacological Examples of Compositions]

[0517] [Composition Pharmacodynamics Example 1] Nasal Administration of a Composition Containing Semaglutide (GLP-1R Agonist) in Rats

[0518] 1. Experimental animals and reagents: 15 ZDF male rats, 15-16 weeks old, weighing 350-400 g, fed with Keao Xieli high-fat diet K5008. Supplier: Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0519] Experimental plan: Control composition D1, control composition D2 and composition E1 were prepared in the same manner as the preparation method of the aforementioned composition A1, except that the raw materials and their amounts were adjusted according to the formula shown in Table 2-3 below. Rats were divided into three groups, the first group was a blank nasal drop group, each dosed with control composition D1, the second group was a subcutaneous administration group of 0.05 mpk (mpk represents mg / kg), each dosed with control composition D2, and the third group was a nasal drop administration group of 0.5 mpk, administered with composition E1, as shown in Table 2-3 below. Rats were fasted overnight. On the day of the experiment, isoflurane anesthesia was performed, and the subcutaneous administration group was subcutaneously injected according to the body weight of the day; for the blank nasal drop group and the nasal drop administration group, each composition was divided into two equal parts according to the administration volume converted to 0.125 mL / kg. After anesthesia, the first part was slowly dripped into one nostril of the rat. After the dripping was completed, the second part was slowly dripped into the other nostril of the rat, and anesthesia was maintained for 5 minutes until the drug solution was completely absorbed. The start of drug administration for all groups was recorded as 0 h, and the blood glucose of the test animals was measured at the following time points: before administration (0 h) and 0.5 h, 1 h, 2 h, 6 h, 12 h, and 24 h after administration; the measurement was performed using a handheld blood glucose meter, with two measurements each time. When the deviation was greater than 10% and greater than 0.5 mmol / L, a third measurement was performed and the average value was taken. The test results are recorded in the following Tables 2-3 and Figure 1.

[0520] Table 2-3 Hypoglycemic efficacy of nasal administration of semaglutide in rats

[0521] Figure 1 shows the blood glucose monitoring results of the rat efficacy experiment. As can be seen from Figure 1 and Tables 2-3 above, when the intranasal administration dose is equal to or greater than 10 times the dose of the subcutaneous administration group, the hypoglycemic efficacy iAUC (mmol / L*h) 0-24h of the intranasal administration group is consistent with that of the subcutaneous administration group, indicating that the agonist composition E1 administered via the nasal cavity is well absorbed and produces a good hypoglycemic efficacy. Composition E1 comprises a combination of semaglutide, a first permeation enhancer EDTA, and a second permeation enhancer DCA.

[0522] [Composition Pharmacokinetics Example 1] Pharmacokinetic Study of Nasal Administration of a Composition Containing Semaglutide (GLP-1R Agonist) in Rabbits

[0523] 1. Experimental Animals: Eight male New Zealand white rabbits, 2-3 months old, purchased from Pizhou Dongfang Breeding Co., Ltd. The experimental animals were housed in the animal room of Suzhou Xihua New Drug Development Co., Ltd. (Animal Use License No.: SYXK(Su)2021-0018). Animals were fed normally for at least 2 days after purchase and enrolled in this study only if they met the veterinary criteria for physical examination. Animals were housed in separate cages and fasted for 16 hours before dosing.

[0524] 2. Experimental Protocol: Control composition F1 and composition G1 were prepared using the same method as described for composition A1, with the differences being that the raw materials and their amounts were adjusted according to the formulations shown in Tables 2-4 below. Rabbits were divided into two groups: the first group received subcutaneous administration of 0.5 mpk of control composition F1 per portion, and the second group received intranasal administration of 1 mpk of composition G1 per portion. On the day of the experiment, the animals were pre-anesthetized. Each rabbit received two equal portions of the dosing solution. Before dosing, the anesthetic hose was removed, and one portion was pipetted and slowly dripped into one nostril. After the dripping was complete, the second portion was slowly dripped into the other nostril. Observation was performed for 5 minutes. At 0.5 h, 1 h, 2 h, 4 h, 6 h, 10 h, and 24 h after administration, 0.3 mL of whole blood was collected via the ear vein, placed in a test tube containing anticoagulant, and stored on wet ice. Plasma was then collected by centrifugation within 1 h (3,000 g, 2-8°C, 10 min) and drug concentrations were determined using LC / MS. The test results are recorded in Tables 2-4 and Figure 2 below.

[0525] Table 2-4 Blood concentration of semaglutide in rabbits after nasal administration

[0526] Figure 2 shows the results of a pharmacokinetic study in rabbits with blood drug concentrations. In Tables 2-4 above, the dosage, concentration, and volume are for the active ingredient, semaglutide. As shown in Figure 2 and Tables 2-4 above, the bioavailability of the intranasal administration group was approximately 44% relative to the subcutaneous administration group, demonstrating that intranasally administered GLP-1R agonist composition G1 exhibits good drug absorption and bioavailability. Composition G1 comprises a combination of semaglutide, a first permeation enhancer, EDTA, and a second permeation enhancer, DCA.

[0527] [Composition Pharmacodynamics Example 2] Nasal Administration of a Composition Containing Polypeptide Compound P016 (GlP-1R / GIPR Dual Agonist) in Rats

[0528] 1. Experimental animals: 16 ZDF male rats, 15-16 weeks old, weighing 350-400 g, fed with Keao Xieli high-fat diet K5008. Supplier: Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0529] 2. Experimental plan: The composition A1 and composition A2 prepared above were used. The control composition H1 and control composition H2 were prepared in the same manner as the preparation method of the aforementioned composition A1, except that the raw materials and their amounts were adjusted according to the formula shown in Table 2-5 below. The rats were divided into four groups, and the dosing formula and dosage of each group were shown in Table 2-5 below. The rats were fasted overnight and anesthetized with isoflurane on the day of the experiment. The subcutaneous administration group was subcutaneously injected according to the body weight of the day; for the blank nasal drop group and the nasal drop administration group, each composition was divided into two equal parts according to the administration volume converted to 0.125 mL / kg. After anesthesia, the first part was slowly dripped into one nostril of the rat. After the dripping was completed, the second part was slowly dripped into the other nostril of the rat, and anesthesia was maintained for 5 minutes until the drug solution was completely absorbed. The start of drug administration for all groups was recorded as 0 h, and the blood glucose of the test animals was measured at the following time points: before administration (0 h) and 0.5 h, 1 h, 2 h, 6 h, 12 h, and 24 h after administration; the measurement was performed using a handheld blood glucose meter, with two measurements each time. When the deviation was greater than 10% and greater than 0.5 mmol / L, a third measurement was performed and the average value was taken. The test results are recorded in the following Tables 2-5 and Figure 3.

[0530] Table 2-5 Hypoglycemic efficacy of peptide compound P016 in rats after nasal administration

[0531] Figure 3 shows the blood glucose monitoring results of the rat efficacy experiment. In Tables 2-5 above, the dosage, concentration, and volume are all for the active pharmaceutical ingredient P016. As can be seen from Figure 3 and Tables 2-5 above, the blank nasal drop group showed no hypoglycemic effect, while both nasal drop groups 1 and 2 showed hypoglycemic effects. When the dose of nasal drop group 2 (Composition A2 containing the osmotic agent combination) was equal to or greater than 10 times the dose of the subcutaneous group, the hypoglycemic efficacy iAUC (mmol / L*h) of nasal drop group 2 over 0-24h was consistent with that of the subcutaneous group, indicating that the GLP-1R / GIPR dual agonist composition A2 of the present invention containing the osmotic agent combination was well absorbed and produced a good hypoglycemic effect after nasal administration. Composition A2 of the present invention comprises a combination of the polypeptide compound P016 of the present invention, a first permeation enhancer EDTA, and a second permeation enhancer DCA. Composition A1 of the present invention without the osmotic agent combination produced a good hypoglycemic effect, but the absorption effect was not as good as that of Composition A2 of the present invention containing the osmotic agent combination.

[0532] [Composition Pharmacokinetics Example 2] Nasal Administration of a Composition Containing Polypeptide Compound P016 (GlP-1R / GIPR Dual Agonist) in Rabbits

[0533] 1. Experimental Animals: 12 female New Zealand white rabbits, 2-3 months old, purchased from Pizhou Dongfang Breeding Co., Ltd. The experimental animals were housed in the animal room of Suzhou Xihua New Drug Development Co., Ltd. (Animal Use License No.: SYXK(Su)2021-0018). Animals were fed normally for at least 2 days after purchase and enrolled in this study only if they met the veterinary criteria for physical examination. Animals were housed in separate cages and fasted for 16 hours before dosing.

[0534] 2. Experimental Plan: Compositions A3 and A4 prepared above were used. A control composition, I1, was prepared using the same method as for composition A1, except that the raw materials and their amounts were adjusted according to the formulations shown in Tables 2-6 below. Rabbits were divided into three groups, and each group was administered the ingredients and dosages shown in Tables 2-6 below.

[0535] On the day of the experiment, animals were pre-anesthetized. Each animal received two aliquots of the dosing solution. Before dosing, the anesthetic hose was removed, and one aliquot was pipetted and slowly dripped into one nostril. After the aliquot was complete, the second aliquot was slowly dripped into the other nostril. The animals were observed for 5 minutes. At 0.25, 0.5, 1, 2, 4, 6, 10, 24, 48, 72, and 96 hours after dosing, 0.3 mL of whole blood was collected via the ear vein. The blood was placed in a tube containing anticoagulant and stored on wet ice. Within 1 hour, the blood was centrifuged (3,000 g, 2-8°C, 10 minutes). Plasma was collected and drug concentrations were determined using LC / MS. The results are reported in Tables 2-6 and Figure 4.

[0536] Table 2-6 Blood concentration of polypeptide compound P016 in rabbits after nasal administration

[0537] Figure 4 shows the results of a pharmacokinetic study in rabbits with blood drug concentrations. As can be seen from Figure 4 and Tables 2-6 above, the bioavailability of intranasal administration group 3 was approximately 3.7% relative to the subcutaneous administration group. However, the rabbits exhibited potential sensitivity during the experiment, such as scratching their noses. The bioavailability of intranasal administration group 4 was approximately 2.0% relative to the subcutaneous administration group, and no adverse reactions were observed in the animals. This indicates that intranasal administration of the GLP-1R / GIPR dual agonist compositions A3 and A4 of the present invention exhibits good drug absorption and bioavailability. Composition A3 of the present invention comprises a combination of the polypeptide compound P016 of the present invention, a first permeation enhancer EDTA, and a second permeation enhancer DCA; composition A4 of the present invention comprises the polypeptide compound P016 of the present invention and an absorption enhancer DDM. Compared to composition A3 containing a permeation enhancer combination, composition A4 containing an absorption enhancer exhibits comparable bioavailability and is non-irritating.

[0538] The present invention discloses a nasal administration formulation of a GLP-1R related polypeptide. Those skilled in the art can refer to the contents of the present invention, combine the principles of pharmaceutics, and appropriately improve the process parameters or prescription ratios to achieve this. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention. The application of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0539] Amino acid sequence:

Claims

1. A nasal delivery pharmaceutical composition, the pH of the pharmaceutical composition being about 5.0 - 9.0, and based on the total volume of the pharmaceutical composition, the pharmaceutical composition comprises: about 0.1 - about 50 mg / mL of a pharmaceutically active ingredient; and about 0.1 - about 5 mg / mL of a first penetration enhancer and about 0.5 - about 20 mg / mL of a second penetration enhancer, the first penetration enhancer being a chelating agent, and the second penetration enhancer being selected from fatty acids and bile acids, wherein the pharmaceutically active ingredient is a polypeptide compound of 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 of formula I (SEQ ID NO:12); wherein, At the position of any one of the 16-bit, 24-bit, 28-bit, and 40-bit K in Formula I, by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is conjugated to the ε-amino group of the K side chain for chemical modification, 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, 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. A nasal delivery pharmaceutical composition, the pH of the pharmaceutical composition being about 5.0 - 9.0, and based on the total volume of the pharmaceutical composition, the pharmaceutical composition comprises: about 0.1 - about 50 mg / mL of a pharmaceutically active ingredient; and about 0.5 - about 20 mg / mL of an alkyl saccharide absorption enhancer, wherein the pharmaceutically active ingredient is a polypeptide compound of 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 of formula I (SEQ ID NO:12); wherein, At any one of the positions of K among the 16-bit, 24-bit, 28-bit, and 40-bit Ks of Formula I, by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is conjugated to the ε-amino group of the K side chain for chemical modification, 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, 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.

3. The nasal delivery pharmaceutical composition according to claim 1 or 2, 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 of formula I (SEQ ID NO:12), wherein The 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 (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 (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 (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 (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 (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 (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 (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 (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 (SEQ ID NO: 10); or K at position 28 was 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 was amidated to a C-terminal primary amide (SEQ ID NO:11).

4. The nasal delivery pharmaceutical composition according to claim 1, wherein the first penetration enhancer is selected from nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, cyclohexanediaminetetraacetic acid, ethylenediaminedi(o - phenylacetic acid), hydroxyethylethylenediaminetriacetic acid, and their soluble salts.

5. The nasal delivery pharmaceutical composition according to claim 1, 3 or 4, wherein the second penetration enhancer is selected from N-(8-[2 - hydroxybenzoyl]amino)octanoic acid, N-(10-[2 - hydroxybenzoyl]amino)decanoic acid, myristic acid, myristoleic acid, palmitoleic acid, stearic acid, oleic acid, caproic acid, caprylic acid, capric acid, lauric acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, tauro - ursodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, and their soluble salts.

6. The nasal delivery pharmaceutical composition according to claim 2 or 3, wherein the alkyl saccharide absorption enhancer is selected from one or more of C12 - C20 alkyl maltosides and C12 - C20 alkyl sucrosides, for example selected from dodecyl maltoside, tridecyl maltoside, tetradecyl maltoside, dodecyl sucroside, tridecyl sucroside, and tetradecyl sucroside.

7. The nasal delivery pharmaceutical composition according to any one of claims 1 - 6, wherein the pharmaceutical composition further contains an osmotic pressure regulator, a buffer, a surfactant and water, an optionally present pH regulator, and an optionally present other excipient or carrier, the other excipient or carrier including one or more of a viscosity regulator, a preservative and an antioxidant.

8. The nasal delivery pharmaceutical composition according to claim 7, 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), tris(hydroxymethyl)aminomethane (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, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium phosphate, disodium hydrogen phosphate - potassium dihydrogen phosphate, and combinations thereof.

9. The nasal delivery pharmaceutical composition according to claim 7 or 8, wherein based on the total volume of the aqueous solution, the molar concentration of the buffer is about 0.5 - about 50 mM.

10. The nasal delivery pharmaceutical composition according to any one of claims 7 - 9, 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.

11. The nasal delivery pharmaceutical composition according to any one of claims 7 - 10, wherein based on the total volume of the aqueous solution, the content of the osmotic pressure regulator is about 1 - about 100 mg / mL.

12. The nasal delivery pharmaceutical composition according to any one of claims 7 - 11, wherein the surfactant is Tween, such as Tween 20, Tween 40, Tween 80, or combinations thereof.

13. The nasal delivery pharmaceutical composition according to any one of claims 7 - 12, wherein based on the total volume of the aqueous solution, the content of the surfactant is about 0.01 - about 50 mg / mL.

14. The nasal delivery pharmaceutical composition according to any one of claims 7 - 13, wherein the pH regulator is an aqueous sodium hydroxide solution or hydrochloric acid.

15. The nasal delivery pharmaceutical composition according to claim 1 and any one of claims 3 - 14, wherein the pharmaceutical composition is an aqueous solution, and based on the total volume of the aqueous solution, it comprises: about 0.1 - about 50 mg / mL, such as about 1 - about 40 mg / mL of compound P016, P014, or P020, about 0.1 - about 5 mg / mL of EDTA, about 0.5 - about 20 mg / mL of sodium caprate, sodium N-(8-(2 - hydroxybenzoyl)amino)octanoate (SNAC), deoxycholic acid (DCA), or combinations thereof, citric acid and trisodium citrate with a total molar concentration of about 0.5 - about 50 mM, and the molar ratio of citric acid to trisodium citrate is about 1:4 - 50, preferably about 1:4 - 20, about 1 - about 100 mg / mL of mannitol, about 0.01 - about 50 mg / mL of Tween 20, optionally present aqueous sodium hydroxide solution or hydrochloric acid, and water The pH value of the aqueous solution is about 5.0 - about 9.0, such as about 5.8 - about 9.0, preferably about 6.0 - about 8.8, more preferably about 6.5 - about 7.

4.

16. The nasal delivery pharmaceutical composition according to any one of claims 2 - 14, wherein the pharmaceutical composition is an aqueous solution, and based on the total volume of the aqueous solution, it comprises: about 0.1 - about 50 mg / mL, such as about 1 - about 40 mg / mL of compound P016, P014 or P020, about 0.5 - about 20 mg / mL of dodecyl maltoside (DDM), citric acid and trisodium citrate with a total molar concentration of about 0.5 - about 50 mM, and the molar ratio of citric acid to trisodium citrate is about 1:4 - 50, preferably about 1:4 - 20, about 1 - about 100 mg / mL of mannitol, about 0.01 - about 50 mg / mL of Tween 20, optionally present aqueous sodium hydroxide solution or hydrochloric acid, and water, wherein the pH value of the aqueous solution is about 5.0 - about 9.0, about 5.8 - about 9.0, preferably about 6.0 - about 8.8, more preferably about 6.5 - about 7.

4.

17. A kit, which comprises a container, and the nasal delivery pharmaceutical composition according to any one of claims 1 - 16 contained in the container, preferably, the container is a nasal spray device.

18. Use of the pharmaceutical composition according to any one of claims 1 - 16 for the preparation of a medicament for treating a disease, such as diabetes, obesity, diseases related to diabetes, and / or diseases related to obesity.

19. The use according to claim 18, wherein the unit dose of the pharmaceutical composition is about 0.5 mg to about 50 mg.

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