Preparation composition, preparation method, use and nasal applicator of glucagon-like peptide-1 receptor agonist for nasal mucosal administration

Abstract

The present invention provides a preparation composition of a glucagon-like peptide-1 receptor agonist for administration through the nasal mucosa, and specifically relates to the technical field of biopharmaceutical materials. The preparation composition mainly includes a glucagon-like peptide-1 receptor agonist, an alkyl glycoside and a phospholipid, wherein the ratio of the glucagon-like peptide-1 receptor agonist to the alkyl glycoside to the phospholipid is (0.25-17.5): (0.25-10): (0.1-10) by mass. The active pharmaceutical ingredient in the preparation composition provided by the present invention is delivered through the nasal mucosa, directly enters the blood circulation from the absorption site, can take effect quickly, has a high bioavailability, and improves patient compliance.

Description

Technical Field

[0001] The present invention relates to the technical field B01F of preparing a composition solution for specific purposes by using a step-by-step dissolution method, and is used for preparing a stable high-content polypeptide aqueous solution.

[0002] The present invention relates to the technical field of biomedical materials, and in particular to a preparation composition of a glucagon-like peptide-1 receptor agonist for administration through the nasal mucosa. Background Art

[0003] Glucagon-like peptide-1 receptor agonists (GLP-1RA) stimulate insulin secretion and inhibit glucagon secretion in a glucose concentration-dependent manner by activating glucagon-like peptide-1 (GLP-1) receptors, while increasing glucose uptake in muscle and adipose tissue and inhibiting glucose production in the liver to exert a hypoglycemic effect. They can also inhibit gastric emptying and appetite. GLP-1 receptors are widely distributed in pancreatic islet cells, gastrointestinal tract, lungs, brain, kidneys, hypothalamus, cardiovascular system, liver, adipocytes and skeletal muscle. Glucagon-like peptide-1 receptor agonists can effectively lower blood sugar, partially restore pancreatic β-cell function, reduce body weight, improve blood lipid profile and lower blood pressure.

[0004] The glucagon-like peptide-1 receptor agonists currently on the market and under development include Exenatide, Liraglutide, Dulaglutide, Semaglutide, Tirzepatide, Retatrutide, etc. From short-acting Exenatide to long-acting Semaglutide, from GLP-1 single target to GLP-1, GIP, GCG triple targets, glucagon-like peptide-1 receptor agonists are becoming more and more mature, but all glucagon-like peptide-1 receptor agonists need to be administered by injection in the early stages of development.

[0005] Ozempic ® It is a subcutaneous injection marketed by Novo Nordisk and is administered once a week. The injection pen is relatively complex and requires experienced medical staff to train patients. Long-term and frequent injections of semaglutide can cause inconvenience and discomfort to patients, and are accompanied by the risk of infection and injection site reactions (such as erythema).

[0006] Compared with subcutaneous injections, non-invasive treatments are more easily accepted by patients. ®It is an oral tablet launched by Novo Nordisk, providing an alternative treatment option for patients who are unwilling or unable to inject hypoglycemic drugs themselves. However, due to the poor transmembrane absorption capacity of the gastrointestinal tract for large molecule drugs, and the physiological environment of the gastrointestinal tract will lead to the degradation and metabolism of peptide drugs, the bioavailability of semaglutide is very low (0.4-1%) and the coefficient of variation is high. In order to achieve similar systemic absorption, the weekly dose of oral semaglutide tablets is 21 to 350 mg, which is much higher than the weekly dose of 0.25 to 2.4 mg for injection (about 100 times). In addition, oral tablets must be taken on an empty stomach every morning and strictly follow the amount of drinking water on the instructions. The high-dose permeation enhancer (sodium 8-(2-hydroxybenzamido) octanoate, abbreviated as SNAC) of 300 mg per tablet may cause gastrointestinal mucosal irritation.

[0007] There are no marketed drugs other than injection for multi-targeted glucagon-like peptide-1 receptor agonists such as tirpotide. The development of a non-invasive macromolecular delivery system can greatly improve the pain of injection and enhance patient compliance.

[0008] Nasal drug delivery system refers to a type of preparation that is directly applied to the nasal cavity and exerts local or systemic therapeutic effects after being absorbed through the nasal mucosa. Nasal drug delivery system has the advantages of effectively avoiding the first-pass effect, rapid absorption and onset of action, non-invasive and convenient administration, and high patient compliance. Currently, nasal delivery peptide preparation products such as salmon calcitonin, desmopressin, buserelin, oxytocin, glucagon, etc. have been launched on the market, but there are no glucagon-like peptide-1 receptor agonist nasal spray products on the market.

[0009] WO2023209662A1 discloses a nasal semaglutide composition, specifically disclosing the use of dodecylphosphocholine (DPC), edetate disodium (EDTA-2Na), 2,6-dimethyl-β-cyclodextrin and cellulose for the formulation of nasal spray preparations, with a pH value between 5.0 and 7.0. However, the invention only gives bioavailability data, fails to prove the safety and stability of the prescription, and its drugability is questionable. In the process of drug development and production, the development of the prescription should be considered from the comprehensive perspective of safety, effectiveness and quality control.

[0010] In the patent WO2023110833A1 of Novo Nordisk, the developer of semaglutide, it is disclosed that the degradation rate of semaglutide in a pH 6.0 system is much greater than that in a pH 7.4 system. Too low a pH will make physical aggregation and chemical degradation more likely to occur. Therefore, up to 25% of hydroxypropyl-β-cyclodextrin was added during the development of the prescription, but this still cannot effectively reduce the rate of chemical degradation, and can only increase physical stability. EDTA-2Na, as a metal ion chelator, can not only chelate with calcium ions in the tight junction proteins between cells of the nasal mucosal epithelial cells to open the intercellular gaps and increase the intercellular permeability of the drug, but also inhibit the activity of enzymes in the nasal cavity. However, in actual research, it was found that EDTA-2Na had compatibility problems with raw materials and excipients in the semaglutide prescription. The addition of 0.1% EDTA-2Na resulted in the generation of a large amount of impurities, and the semaglutide content dropped sharply. The prescription disclosed in WO2023209662A1 added 1% EDTA-2Na, which caused serious compatibility problems in the subsequent formulation stability, and the drugability was questionable. In addition, chelating agents such as EDTA-2Na are prone to irritation problems and cause inflammatory reactions. For example, MFZScelza et al. reported that in animal models, EDTA-2Na caused more inflammatory cells to form. At the same time, EDTA-2Na is an anticoagulant, and the nasal mucosa is very weak, which increases the risk of capillary rupture and bleeding under the stimulation of high concentrations of EDTA-2Na. Therefore, the prescription disclosed in WO2023209662A1 has defects in safety and stability.

[0011] In view of this, the present invention is proposed. Summary of the invention

[0012] One of the purposes of the present invention is to provide a preparation composition of a glucagon-like peptide-1 receptor agonist for administration through the nasal mucosa. The use of a specific ratio combination of alkyl glycoside and phospholipid can greatly improve the bioavailability. Compared with the bioavailability of a composition using only alkyl glycoside or only phospholipid at the same concentration, the bioavailability of the technical solution provided by the present invention is more than 3 times higher; at the same time, the present invention provides a stable mixed solubilization system of alkyl glycoside and phospholipid, which solves the problem that alkyl glycoside is easy to precipitate when used alone; in addition, the present invention also solves the problem of poor stability of high-concentration polypeptide drug preparations (the content of semaglutide in the present invention is higher than 0.134% by mass of the commercially available semaglutide injection) existing in the prior art, and has better stability and higher drugability.

[0013] In order to prepare the above-mentioned stable solution containing high concentration of polypeptide drugs, the present invention provides a mixed dissolution method for a special mixed solubilization system of alkyl glycoside and phospholipid, the core of which is step-by-step mixing-dissolution through five steps:

[0014] a. Mix and stir other easily soluble parts (such as buffer salts, antibacterial agents, etc.) and part of the water until fully dissolved to obtain solution a;

[0015] b. Adding the main substances of the mixed solubilization system, alkyl glycoside and phospholipid, into the above solution a and mixing and stirring until fully dissolved to obtain a mixed solubilization system solution b;

[0016] c. Add other insoluble excipients (such as bioadhesives and antioxidants) into the mixed solubilization system solution b and mix and stir until fully dissolved to obtain solution c;

[0017] d. adding the polypeptide drug semaglutide to be dissolved (substance to be dispersed) into solution c and mixing and stirring until fully dissolved to obtain solution d containing a high concentration of polypeptide drug semaglutide;

[0018] e. Add appropriate amounts of pH regulator and osmotic pressure regulator and the remainder of water to solution d, adjust the pH and osmotic pressure to the target range and fix the volume, and finally obtain a stable aqueous solution containing a high concentration of the polypeptide drug semaglutide solubilized by the alkyl glycoside-phospholipid mixed solubilization system.

[0019] The second object of the present invention is to provide a method for preparing a preparation composition of a glucagon-like peptide-1 receptor agonist for administration through the nasal mucosa.

[0020] A third object of the present invention is to provide a nasal applicator for administering drugs to the nasal mucosa.

[0021] A fourth object of the present invention is to provide an application of a preparation composition of a glucagon-like peptide-1 receptor agonist for administration through the nasal mucosa.

[0022] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0023] The first aspect of the present invention provides a preparation composition of a glucagon-like peptide-1 receptor agonist for administration through the nasal mucosa, wherein the glucagon-like peptide-1 receptor agonist is selected from semaglutide, and the main components of the preparation composition include semaglutide, alkyl glycoside and phospholipid, wherein the ratio of semaglutide to alkyl glycoside to phospholipid is (0.25-17.5) by mass: (0.25-10) by mass: (0.1-10), and the mass of semaglutide accounts for 0.25%-17.5% of the total mass of the preparation composition.

[0024] The applicant can clarify through the examples and comparative examples 4-9 that the use of a specific ratio of alkyl glycoside and phospholipid can greatly improve the bioavailability. Compared with the bioavailability of the composition using alkyl glycoside alone (Comparative Examples 4-6) or phospholipid alone (Comparative Examples 7-9) at the same concentration, the bioavailability of the technical solution provided by the present invention is more than 3 times higher. The applicant can clarify through the examples and comparative examples 10-11 that when alkyl glycoside is used alone, the prescription preparation has the risk of precipitation at low temperature; when the amount of phospholipid added is less than 10% of the alkyl glycoside, the solution will also precipitate.

[0025] In summary, when alkyl glycoside and phospholipid are used together and the content ratio is within the range defined in the present invention (the phospholipid content shall not be less than 10% of the alkyl glycoside, that is, the ratio of alkyl glycoside to phospholipid by mass is not greater than 10:1), the solution stability is good and the bioavailability is greater than that of using alkyl glycoside alone or phospholipid alone.

[0026] The ratio of the alkyl glycoside to the phospholipid is preferably in the range of 1:10 to 10:1, more preferably 1:4 to 10:1, by mass.

[0027] In certain embodiments, the ratio of alkyl glycoside to phospholipid is 0.1 (i.e., the ratio of the two is 1:10), 0.25 (i.e., the ratio of the two is 1:4), 1 (i.e., the ratio of the two is 1:1), 1.67 (i.e., the ratio of the two is 3:5), 2 (i.e., the ratio of the two is 2:1), 2.5 (i.e., the ratio of the two is 5:2), 5 (i.e., the ratio of the two is 5:1), and 10 (i.e., the ratio of the two is 10:1) by mass.

[0028] Preferably, the mass of semaglutide accounts for 0.50%-15.00% of the total mass of the formulation composition.

[0029] Preferably, the mass of semaglutide accounts for 2.00%-15.00% of the total mass of the formulation composition.

[0030] Preferably, the mass of semaglutide accounts for 3.50%-15.00% of the total mass of the formulation composition.

[0031] The alkyl glycoside is octyl-β-D-maltoside, dodecyl-β-D-maltoside, dodecyl-β-D-maltoside, tetradecyl-β-D-maltoside or a combination thereof.

[0032] Preferably, the mass of the alkyl glycoside accounts for 0.25%-10.00% of the total mass of the formulation composition.

[0033] The phospholipid is decanylphosphocholine, dodecylphosphocholine, tetradecylphosphocholine, hexadecylphosphocholine, lyso-lauroylphosphatidylcholine, dilauroylphosphatidylglycerol or a combination thereof.

[0034] Preferably, the mass of the phospholipid accounts for 0.10%-10.00% of the total mass of the formulation composition.

[0035] Needless to say, the above-mentioned preparation composition as a pharmaceutical preparation, in addition to the above-mentioned main ingredients, also includes other necessary auxiliary materials suitable for administration through the nasal mucosa:

[0036] Furthermore, the preparation composition comprises a bioadhesive, an antioxidant, an antibacterial agent, an osmotic pressure regulator, a buffer, a pH regulator and water.

[0037] Preferably, the bioadhesive agent is methyl-β-cyclodextrin, 2,6-dimethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin or a derivative thereof or a combination thereof.

[0038] Preferably, the mass of the bioadhesive agent accounts for 0%-20% of the total mass of the formulation composition.

[0039] Preferably, the antioxidant is butylated hydroxyanisole, sodium thiosulfate, phenol or a combination thereof.

[0040] Preferably, the mass of the antioxidant accounts for 0.0005%-0.3% of the total mass of the formulation composition.

[0041] Preferably, the antibacterial agent is benzalkonium chloride, benzyl alcohol, phenylethyl alcohol, parabens or a combination thereof.

[0042] Preferably, the mass of the antibacterial agent accounts for 0%-1.0% of the total mass of the preparation composition.

[0043] Preferably, the osmotic pressure regulator is sodium chloride, potassium chloride, glucose, sucrose, sorbitol, mannitol or a combination thereof.

[0044] Preferably, the buffer is phosphate buffer, citrate buffer, bicarbonate buffer, tartrate buffer, acetate buffer or a combination thereof.

[0045] Preferably, the pH adjuster is phosphoric acid, sodium hydroxide, potassium hydroxide, hydrochloric acid or a combination thereof.

[0046] Further, the pH of the formulation composition is from 6.80 to 8.50, preferably from 6.99 to 8.50.

[0047] Further, the osmotic pressure of the formulation composition is 150 to 450 mOsmol / kg, preferably 164 to 450 mOsmol / kg.

[0048] Compared with the prior art, the present invention has at least the following beneficial effects:

[0049] The active pharmaceutical ingredient in the preparation composition of the glucagon-like peptide-1 receptor agonist for nasal mucosal administration provided by the present invention is delivered through the nasal mucosa, can directly enter the blood circulation from the absorption site, and take effect quickly. Compared with injections, repeated injections are avoided, non-invasive administration is achieved, it is convenient and safe, and patient compliance is improved. Compared with oral tablets, the problem of high gastrointestinal degradation can be avoided, the adverse gastric reactions caused by a high proportion of SNAC are reduced, and it has the advantages of high bioavailability and reduced frequency of administration.

[0050] The applicant can clarify through the examples and comparative examples 4-9 that the use of a specific ratio combination of alkyl glycoside and phospholipid can greatly improve the bioavailability. Compared with the bioavailability of the composition using alkyl glycoside alone (Comparative Examples 4-6) or phospholipid alone (Comparative Examples 7-9) at the same concentration, the bioavailability of the technical solution provided by the present invention is more than 3 times higher. The applicant can clarify through the examples and comparative examples 10-11 that when alkyl glycoside is used alone, the prescription preparation has the risk of precipitation at low temperature; when the amount of phospholipid added is less than 10% of the alkyl glycoside, the solution will also precipitate. Comprehensive explanation When alkyl glycoside and phospholipid are used in combination and the content ratio is within the range defined by the present invention (the phospholipid content shall not be less than 10% of the alkyl glycoside, that is, the ratio of alkyl glycoside to phospholipid is not more than 10:1 by mass), the solution has good stability and the bioavailability is greater than that of alkyl glycoside or phospholipid alone.

[0051] In particular, the inventors have found through experimental research that the solubility of alkyl glycoside, as a new type of penetration enhancer, drops sharply at 2-8°C. When the concentration of alkyl glycoside is higher than 1%, it will crystallize and precipitate at low temperatures. This solubility reduction effect becomes more obvious with the increase of carbon chain length. Therefore, under low-temperature storage conditions, adding a large amount of alkyl glycoside to the prescription will pose a risk of low-temperature stability. The inventors have found through experimental research that the use of multi-component mixed solubilization technology has obtained a stable mixed solubilization system of alkyl glycoside and phospholipid, which can avoid the precipitation of alkyl glycoside at low temperatures and improve the stability of the glucagon-like peptide-1 receptor agonist preparation composition.

[0052] Furthermore, the inventors found through experimental research that the semaglutide composition provided by the invention is more effective than the commercially available injection Ozempic ®More stable, the technical solution provided by the present invention reduces the possibility of physical aggregation, thereby reducing the generation of high molecular weight polymers, which may produce immune responses in polypeptide drugs and induce immunogenicity.

[0053] As is common knowledge in the field of biopharmaceuticals, it is well known that the stability of peptides decreases significantly with increasing concentrations. High-concentration peptide formulations are more likely to aggregate and cause content loss. Therefore, the stability of high-concentration peptide preparations is a difficult problem in biopharmaceuticals. The concentration of semaglutide in the present invention is higher than that of the commercially available injection Ozempic ® It is many times higher, but shows higher stability, solving the physical stability problem of high-concentration peptide preparations.

[0054] Furthermore, pharmacokinetic data also show that the technical solution provided by the present invention has great advantages in bioavailability. When a single dose of 1.0 mg / 50 μL is administered, the bioavailability of the semaglutide composition is much higher than that of oral tablets, and reaches the same level as the maximum blood drug concentration of the original injection of 0.25 mg subcutaneous injection.

[0055] The second aspect of the present invention provides a method for preparing a preparation composition of a glucagon-like peptide-1 receptor agonist for administration through the nasal mucosa, comprising the following steps:

[0056] a. Mix the buffer salt, antibacterial agent and part of the water at room temperature at a rate of 200-1200 rpm until fully dissolved to obtain solution a;

[0057] b. Add the alkyl glycoside and phospholipid to solution a, and mix and stir at a rate of 200-1200 rpm until fully dissolved to obtain solution b;

[0058] c. Add the bioadhesive and antioxidant to solution b, and mix and stir at a rate of 200-1200 rpm until fully dissolved to obtain solution c;

[0059] d. adding semaglutide to solution c and mixing and stirring until fully dissolved to obtain solution d;

[0060] e. Add appropriate amounts of pH regulator and osmotic pressure regulator to solution d to adjust the pH and osmotic pressure to the target range;

[0061] f. Add excess water, stir evenly, and perform sterile filtration.

[0062] The third aspect of the present invention provides a nasal applicator for nasal mucosal administration, the nasal applicator comprising a composition preparation reservoir, a composition preparation according to the present invention contained in the reservoir, and a metering pump for discharging the preparation from the reservoir.

[0063] The fourth aspect of the present invention provides an application of a preparation composition of a glucagon-like peptide-1 receptor agonist for administration through the nasal mucosa, including its use in the preparation of a medicament for treating diabetes, obesity, cardiovascular disease, non-alcoholic fatty hepatitis, Alzheimer's disease, chronic kidney disease, knee arthritis, metabolic dysfunction-related fatty hepatitis or osteoarthritis.

[0064] Further, the formulation composition is administered once a week or more frequently.

[0065] Furthermore, the weekly administered preparation composition contains 0.25-30 mg of semaglutide.

[0066] The fifth aspect of the present invention provides a preparation composition of a glucagon-like peptide-1 receptor agonist for administration through the nasal mucosa, wherein the glucagon-like peptide-1 receptor agonist is selected from liraglutide or tilportide in addition to semaglutide. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0068] Figure 1 Circular dichroism spectra of Example 1 and semaglutide API.

[0069] Figure 2 This is the dynamic light scattering spectrum of Example 1. The three colors in the figure represent three parallel tests.

[0070] Figure 3 This is the dynamic light scattering spectrum of Comparative Example 1. The three colors in the figure represent three parallel tests.

[0071] Figure 4 The following are typical chromatograms (the upper figure is the extracted ion chromatogram of semaglutide and semaglutide-d8 blank plasma matrix, and the lower figure is the extracted ion chromatogram of a typical matrix sample).

[0072] Figure 5 is the standard curve and linear regression equation of semaglutide.

[0073] Figure 6 It is the average blood drug concentration diagram of Example 1 and Comparative Example 1.

[0074] Figure 7 It is the average blood drug concentration diagram of Example 1 and Comparative Examples 4-6.

[0075] Figure 8 It is the average blood drug concentration diagram of Example 1 and Comparative Examples 7-9.

[0076] Fig. 9 The following are typical chromatograms (the upper figure is the extracted ion chromatogram of semaglutide and telpotide blank plasma matrix, and the lower figure is the extracted ion chromatogram of 0.2 ng / mL matrix spiked sample).

[0077] Fig.10 is the standard curve and linear regression equation of telportolide.

[0078] Fig.11 This is the average blood drug concentration diagram of Example 37. DETAILED DESCRIPTION

[0079] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0080] In some embodiments, the semaglutide is N-ε 26 -[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1(7-37), which can be prepared by the method described in Example 4 of WO2006097537.

[0081] In some embodiments, the liraglutide is Arg 34 , Lys 26 (N-ε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-37), which can be prepared by the method described in Example 37 of WO1998008871.

[0082] In some embodiments, the telpotide refers to a GIP / GLP1 co-agonist peptide described in US9474780 and described by CAS Registry No. 2023788-19-2, which can be prepared by the method described in Example 1 of US9474780.

[0083] The composition of the present invention is administered nasally and is suitable for being provided in a nasal applicator designed according to the purpose, and the nasal applicator delivers the composition to the nasal mucosa of the user in the form of a spray. Such applicators are known in the art and generally have a composition preparation reservoir, and a preparation contained in the reservoir, and a metering pump for expelling the preparation from the reservoir.

[0084] In certain embodiments, the material of the reservoir can be any material that is compatible with the drug, such as glass, plastic, stainless steel, and other polymer materials.

[0085] In some embodiments, the metering pump can be eDevices (Aptar Pharma), VP7 (AptarPharma), Classic Nasal Device (Aptar Pharma), APF (Aptar Pharma), CPS (AptarPharma), etc.

[0086] In one embodiment, the present invention relates to a method for preventing and / or treating all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, maturity-onset diabetes of the young (MODY), gestational diabetes, and / or lowering glycated hemoglobin (HbA1c), comprising administering to a subject an effective dose of the above-mentioned composition.

[0087] In one embodiment, the present invention relates to a method for delaying or preventing the progression of diabetic disease, such as the progression of type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to type 2 diabetes requiring insulin, and / or delaying the progression of non-insulin requiring type 2 diabetes to type 2 diabetes requiring insulin, the method comprising administering to a subject an effective dose of the above-mentioned composition.

[0088] In one embodiment, the present invention relates to a method for preventing and / or treating eating disorders, such as obesity, for example by reducing food intake, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa and / or obesity caused by taking antipsychotics or steroids; reducing gastric motility; and / or delaying gastric emptying, the method comprising administering to a subject an effective dose of the above-mentioned composition.

[0089] In one embodiment, the present invention relates to a method for preventing and / or treating cardiovascular diseases, comprising administering an effective dose of the above composition to a subject.

[0090] In one embodiment, the present invention relates to a method for preventing and / or treating non-alcoholic steatohepatitis, comprising administering an effective dose of the above composition to a subject.

[0091] In one embodiment, the present invention relates to a method for preventing and / or treating Alzheimer's disease, comprising administering an effective dose of the above composition to a subject.

[0092] In one embodiment, the present invention relates to a method for preventing and / or treating chronic kidney disease, comprising administering an effective dose of the above composition to a subject.

[0093] In one embodiment, the present invention relates to a method for preventing and / or treating knee arthritis, comprising administering an effective dose of the above composition to a subject.

[0094] In one embodiment, the present invention relates to a method for preventing and / or treating metabolic dysfunction-associated steatohepatitis, comprising administering an effective dose of the above composition to a subject.

[0095] In one embodiment, the present invention relates to a method for preventing and / or treating osteoarthritis, comprising administering an effective dose of the above composition to a subject.

[0096] In one embodiment, there is provided one of the above compositions for use in the form of a medicament.

[0097] In one embodiment, one of the above compositions for treating diabetes is provided. In one embodiment, one of the above compositions for delaying or preventing the progression of diabetic disease is provided. In one embodiment, one of the above compositions for treating obesity is provided. In one embodiment, one of the above compositions for treating cardiovascular disease is provided. In one embodiment, one of the above compositions for treating nonalcoholic fatty hepatitis is provided. In one embodiment, one of the above compositions for treating Alzheimer's disease is provided. In one embodiment, one of the above compositions for treating knee arthritis is provided. In one embodiment, one of the above compositions for treating chronic kidney disease is provided. In one embodiment, one of the above compositions for treating metabolic dysfunction-related fatty hepatitis is provided. In one embodiment, one of the above compositions for treating osteoarthritis is provided.

[0098] In one embodiment, the composition of the present invention can be administered once a week or more frequently, such as once a day. In some embodiments, the composition of the present invention is administered at any time of the day. In some embodiments, the content of semaglutide contained in the composition administered weekly of the present invention is in the range of 0.25 to 30.0 mg.

[0099] The present invention is further illustrated below by specific examples and comparative examples, however, it should be understood that these examples are only for the purpose of more detailed description, and should not be understood as limiting the present invention in any form. The preferred embodiments described below are only for example, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention. Semaglutide, liraglutide, and telportide used in the present invention are all purchased commercially. The raw materials used in the examples and comparative examples of the present invention, if the specific conditions are not specified, are carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be purchased commercially.

[0100] [Example 1]

[0101]

[0102] All are mass percentages, the same below.

[0103] Note: qs after NaOH, HCl and sodium chloride means adding appropriate amount of pH adjusters NaOH and HCl and osmotic pressure adjuster sodium chloride to the solution so that the pH is adjusted to 7.40 and the osmotic pressure is adjusted to 350 mOsmol / kg, the same below.

[0104] Example 1 provides a semaglutide composition for administration via nasal mucosa, and the preparation process is as follows:

[0105] a. Mix NaH2PO4·H2O, benzalkonium chloride and part of water at room temperature at a rate of 500 rpm and stir until fully dissolved to obtain solution a;

[0106] b. Add dodecyl-β-D-maltoside and dodecylphosphocholine to solution a, and mix and stir at a rate of 300 rpm until fully dissolved to obtain solution b;

[0107] c. Add hydroxypropyl-β-cyclodextrin and butylhydroxyanisole to solution b, and mix and stir at a rate of 300 rpm until fully dissolved to obtain solution c;

[0108] d. Add semaglutide to solution c and mix and stir until fully dissolved to obtain solution d;

[0109] e. Add appropriate amounts of pH regulators NaOH and HCl and osmotic pressure regulator sodium chloride to solution d, adjust the pH of the prescription to 7.40, and adjust the osmotic pressure to 350 mOsmol / kg;

[0110] f. Add excess water, stir evenly, and perform sterile filtration.

[0111] [Example 2-5]

[0112]

[0113] Example 2-5 provides a semaglutide composition for administration through the nasal mucosa, and the preparation process is the same as that of Example 1.

[0114] [Examples 6-10]

[0115]

[0116] Examples 6-10 provide a semaglutide composition for administration through the nasal mucosa, and the preparation process is the same as that of Example 1.

[0117] [Examples 11-15]

[0118]

[0119] Examples 11-15 provide a semaglutide composition for administration through the nasal mucosa, and the preparation process is the same as that of Example 1.

[0120] [Examples 16-19]

[0121]

[0122] Examples 16-19 provide a semaglutide composition for administration through the nasal mucosa, and the preparation process is the same as that of Example 1.

[0123] [Examples 20-23]

[0124]

[0125] Examples 20-23 provide a semaglutide composition for administration through the nasal mucosa, and the preparation process is the same as that of Example 1.

[0126] [Examples 24-26]

[0127]

[0128] Examples 24-26 provide a semaglutide composition for administration through the nasal mucosa, and the preparation process is the same as that of Example 1.

[0129] [Examples 27-30]

[0130]

[0131] Examples 27-30 respectively provide a semaglutide composition for administration through the nasal mucosa, and the preparation process is the same as that of Example 1.

[0132] [Examples 31-35]

[0133]

[0134] Examples 31-35 respectively provide a semaglutide composition for administration through the nasal mucosa, and the preparation process is the same as that of Example 1.

[0135] [Examples 36-37]

[0136]

[0137] Examples 36-37 respectively provide a liraglutide and telpotide composition for transnasal administration, and the preparation process is the same as that of Example 1.

[0138] [Comparative Example 1]

[0139] Comparative Example 1 is the commercially available semaglutide injection Ozempic ® , the specification is 1.34 mg / mL, that is, the mass percentage of semaglutide is about 1.34 / 1000=0.134%, and the batch number is 202402BTA1. The prescription published by FDA is as follows:

[0140]

[0141] [Comparative Example 2]

[0142]

[0143] Comparative Example 2 is the optimal formulation disclosed in the patent specification of WO2023209662A1, numbered PT-N04, and the preparation process is as follows: (1) completely dissolving 200 mg of semaglutide in about 8 mL of PBS 7.0 buffer solution at ambient temperature;

[0144] (2) adding dodecylphosphocholine, 2,6-dimethyl-β-cyclodextrin and EDTA-2Na and dissolving them in a buffer solution under stirring at ambient temperature;

[0145] (3) Add hydroxypropyl methylcellulose and stir until dissolved;

[0146] (4) Finally, add PBS 7.0 buffer solution to 10 mL. The final concentration of semaglutide in the solution is 20 mg / mL.

[0147] That is, the mass percentage of semaglutide is about 200 / 10000=2.0%

[0148] The prescription of comparative example 2 lacks alkyl glycoside and contains 1% EDTA-2Na. From the results of test example 1b, it can be seen that the stability data of Example 4 provided by the present invention is better than that of comparative example 2 (PT-N04 prescription disclosed in WO2023209662A1 patent).

[0149] [Comparative Example 3]

[0150]

[0151] Comparative Example 3 is to add 0.1% EDTA-2Na on the basis of the prescription example 4 provided by the present invention, and the preparation process is:

[0152] a. Mix NaH2PO4·H2O, Na2HPO4·H2O, EDTA-2Na and part of water at 500 rpm and stir until fully dissolved to obtain solution a.

[0153] b. Add dodecyl-β-D-maltoside and dodecylphosphocholine to solution a, and mix and stir at a rate of 300 rpm until fully dissolved to obtain solution b;

[0154] c. Add hydroxypropyl-β-cyclodextrin and butylhydroxyanisole to solution b, and mix and stir at a rate of 300 rpm until fully dissolved to obtain solution c;

[0155] d. adding semaglutide to solution c and mixing and stirring until fully dissolved to obtain solution d;

[0156] e. Add appropriate amounts of pH regulators NaOH and HCl and osmotic pressure regulator sodium chloride to solution d, adjust the pH of the prescription to 7.45, and adjust the osmotic pressure to 349 mOsmol / kg;

[0157] f. Add excess water, stir evenly, and perform sterile filtration.

[0158] Comparative Example 3 is to add 0.1% EDTA-2Na on the basis of Prescription Example 4 provided by the present invention. It can be seen from the results of Test Example 1b that the content of semaglutide is significantly reduced and impurities are significantly increased. This is because the addition of EDTA-2Na leads to chemical compatibility problems, so the prescription disclosed in WO2023209662A1 patent has stability risks. In other words, the composition will have better stability if EDTA-2Na is not artificially / deliberately added. Obviously, the EDTA-2Na here also includes similar EDTA acid and EDTA-4Na, which are similar to EDTA-2Na, and have similar chemical compatibility problems that will cause the polypeptide content to decrease, that is, EDTA-2Na analogs include EDTA acid, EDTA-4Na and EDTA-2Na.

[0159] [Comparative Examples 4-9]

[0160]

[0161] Comparative Examples 4-9 respectively provide a semaglutide composition, and the preparation process is the same as that of Example 1.

[0162] Comparative Examples 4-6 lack phospholipids, and use only alkyl glycosides, and the content of alkyl glycosides gradually increases; Comparative Examples 7-9 lack alkyl glycosides, and use only phospholipids, and the content of phospholipids gradually increases. It can be seen from the results of Test Example 4 that the bioavailability of the combination of alkyl glycoside-phospholipid (Examples 1) is more than 3 times higher than the bioavailability of the composition using only alkyl glycoside (Comparative Examples 4-6) or only phospholipids (Comparative Examples 7-9) at the same concentration.

[0163] [Comparative Examples 10-11]

[0164]

[0165] Comparative Examples 10-11 respectively provide a semaglutide composition, and the preparation process is the same as that of Example 1. Comparative Example 10 is an alkyl glycoside content exceeding 10% and a phospholipid content less than 10% of the alkyl glycoside content, and Comparative Example 11 contains only alkyl glycoside and no phospholipid. The composition prepared in Comparative Examples 10-11 was placed in a drug stability test box (5°C ± 3°C) and left for 3 days. Crystals precipitated in the solution, indicating that the alkyl glycoside solution of the prescription amount itself is not stable at low temperatures. When the alkyl glycoside is used alone, the prescription preparation has the risk of precipitation at low temperatures; when the amount of phospholipid added is less than 10% of the alkyl glycoside, the solution will also precipitate, indicating that when the ratio of the alkyl glycoside and phospholipid content exceeds the range specified in the present invention, there is a problem of solution instability.

[0166] In particular, the inventors have found through experimental research that the solubility of alkyl glycoside, as a new type of penetration enhancer, drops sharply at 2-8°C. When the concentration of alkyl glycoside is higher than 1%, it will crystallize and precipitate at low temperatures. This solubility reduction effect becomes more obvious with the increase of carbon chain length. Therefore, under low-temperature storage conditions, adding a large amount of alkyl glycoside to the prescription will pose a risk of low-temperature stability. The inventors have found through experimental research that the use of multi-component mixed solubilization technology has obtained a stable mixed solubilization system of alkyl glycoside and phospholipid, which can avoid the precipitation of alkyl glycoside at low temperatures and improve the stability of the glucagon-like peptide-1 receptor agonist composition.

[0167] [Comparative Examples 12-15]

[0168]

[0169] Comparative Examples 12-15 respectively provide a semaglutide composition, and the preparation process is the same as that of Example 1.

[0170] Comparative Example 12 is a formulation without an antioxidant, and Comparative Examples 13-15 are formulations containing different amounts of vitamin E. It can be seen from Test Example 1i that when no antioxidant is present, or when the antioxidant is selected outside the range specified in the present invention, there is a risk of formulation stability.

[0171] Through experimental exploration, it was determined that the antioxidant vitamin E (VE) may have an impact on the stability of the composition: when vitamin E is not intentionally / artificially added to the composition, the composition solution has better stability.

[0172] [Test Example 1]

[0173] The compositions of Examples 1-37 and Comparative Examples 1-3 and 12-15 were placed in a drug stability test box to examine the decrease in the content of glucagon-like peptide-1 receptor agonist and the growth of impurities and polymers.

[0174] The test method is:

[0175] (1) The content and impurity chromatographic conditions are:

[0176] HPLC: Agilent1260; chromatographic column: C18 4.6×150 mm, 2.6 μm; mobile phase A: NH4H2PO4: ACN = 92:8; mobile phase B: ACN: IPA: water = 60:20:20; flow rate: 0.7 mL / min; column temperature: 30℃; detection wavelength: 210 nm; injection volume: 10 μL; acquisition time: 65 min; sample concentration: 2 mg / mL; diluent: methanol: water: Tween 80 = 60:40:0.1;

[0177] Elution gradient:

[0178]

[0179] (2) Polymer chromatography conditions are:

[0180] HPLC: Agilent1260; chromatographic column: TSK gel G2000SWxl 7.8×300 mm, 5 μm; mobile phase: acetonitrile: water: trifluoroacetic acid = 40:60:0.07; flow rate: 0.7 mL / min; column temperature: 30℃; detection wavelength: 210 nm; injection volume: 20 μL; acquisition time: 30 min; sample concentration: 0.5 mg / mL; diluent: mobile phase.

[0181]

Test Example 1a

[0182] The compositions of Example 1 and Comparative Example 1 were placed in a drug stability test chamber (40°C ± 2°C, 75% ± 5% relative humidity RH) to examine the decrease in semaglutide content and the growth of impurities and polymers at 7, 14 and 28 days. The results are recorded in Table 1.

[0183] Table 1

[0184]

[0185] As can be seen from Table 1, at different sampling test time points, the rate of decrease of semaglutide content in Example 1 is lower than that in Comparative Example 1, and the growth of impurities and polymers is lower than that in Comparative Example 1. At 28 days, the rate of decrease of semaglutide content in Comparative Example 1 is more than 2% higher than that in Example 1, and the growth of polymer in Comparative Example 1 is more than 2 times that in Example 1. As is common knowledge in the field of biopharmaceuticals, it is well known that the stability of proteins decreases significantly with increasing concentration, and high-concentration protein prescriptions are more likely to aggregate in large quantities and cause content loss. The concentration of semaglutide in Example 1 is nearly 15 times that of Comparative Example 1, but it shows higher stability, and the prescription has a significant advantage.

[0186]

Test Example 1b

[0187] The compositions of Example 4 and Comparative Examples 2-3 were placed in a drug stability test chamber (40°C ± 2°C, 75% ± 5% relative humidity RH) to examine the decrease in the content of semaglutide and the increase in related substances after 21 days. The results are recorded in Table 2.

[0188] Table 2

[0189]

[0190] From the above data, it can be seen that the stability data of Example 4 provided by the present invention is better than that of Comparative Example 2 (PT-N04 prescription disclosed in WO2023209662A1 patent). In addition, when 0.1% EDTA-2Na is added to the prescription of Example 4 (Comparative Example 3), the content of semaglutide decreases significantly and the impurities increase significantly. This is because the addition of EDTA-2Na causes chemical compatibility problems.

[0191]

Test Example 1c

[0192] The compositions of Examples 2-5 and the composition of Comparative Example 1 were placed in a drug stability test chamber (40°C±2°C, 75%±5% relative humidity RH) to examine the decrease in semaglutide content and the increase in impurities after 21 days. The results are recorded in Table 3.

[0193] Table 3

[0194]

[0195] Examples 2-5 investigated the stability of compositions containing different types of alkyl glycosides under accelerated conditions at 40° C. When the conditions were investigated for 21 days, the evaluation results of the decrease in semaglutide content and the increase in impurities were better than those of Comparative Example 1.

[0196]

Test Example 1d

[0197] The compositions of Examples 6-10 and the composition of Comparative Example 1 were placed in a drug stability test chamber (40°C ± 2°C, 75% ± 5% relative humidity RH) to examine the decrease in semaglutide content and the increase in impurities after 21 days. The results are recorded in Table 4.

[0198] Table 4

[0199]

[0200] Examples 6-10 investigated the stability of compositions containing different types of phospholipids under accelerated conditions at 40°C. When the test was conducted for 21 days, the evaluation results of the decrease in semaglutide content and the increase in impurities were better than those of Comparative Example 1.

[0201]

Test Example 1e

[0202] The compositions of Examples 11-15 and the composition of Comparative Example 1 were placed in a drug stability test chamber (40°C±2°C, 75%±5% relative humidity RH) to examine the decrease in semaglutide content and the increase in impurities after 21 days. The results are recorded in Table 5.

[0203] Table 5

[0204]

[0205] Examples 11-15 investigated the stability of compositions containing different types of bioadhesives under accelerated conditions at 40°C. When the test was conducted for 21 days, the evaluation results of the decrease in semaglutide content and the increase in impurities were better than those of Comparative Example 1.

[0206]

Test Example 1f

[0207] The compositions of Examples 16-23 and the composition of Comparative Example 1 were placed in a drug stability test chamber (60°C±2°C, 75%±5% relative humidity RH) to examine the decrease in semaglutide content and the growth of impurities and polymers after 7 days. The results are recorded in Table 6.

[0208] Table 6

[0209]

[0210] Examples 16-23 investigate the stability of compositions containing alkyl glycosides and phospholipids in different content ratios under accelerated conditions of 60°C. After 7 days of investigation, the evaluation results of the decrease in semaglutide content, impurities and polymer growth were better than those of Comparative Example 1.

[0211]

Test Example 1g

[0212] The compositions of Examples 24-26 were placed in a drug stability test chamber (60°C±2°C, 75%±5% relative humidity RH) to examine the decrease in semaglutide content and the growth of impurities and polymers over a 7-day period. The results are recorded in Table 7.

[0213] Table 7

[0214]

[0215] Examples 24-26 investigated the stability of compositions containing alkyl glycosides and phospholipids in different content ratios under accelerated conditions of 60°C. After 7 days of investigation, the evaluation results of decreased semaglutide content and increased impurities and polymers were better than those of Comparative Example 1.

[0216]

Test example 1h

[0217] The compositions of Examples 27-30 were placed in a drug stability test chamber (40°C±2°C, 75%±5% relative humidity RH) to examine the decrease in semaglutide content and the growth of impurities and polymers over a 7-day period. The results are recorded in Table 8.

[0218] Table 8

[0219]

[0220] Examples 27-30 investigate the stability of compositions containing different amounts of cyclodextrin under accelerated conditions at 40°C. After 7 days of investigation, the evaluation results of the decrease in semaglutide content, impurities and polymer growth were better than those of Comparative Example 1.

[0221]

Test Example 1i

[0222] The compositions of Examples 31-35 and Comparative Examples 12-15 were placed in a drug stability test chamber (40°C ± 2°C, 75% ± 5% relative humidity RH) to examine the decrease in semaglutide content and the growth of impurities and polymers after 7 days. The results are recorded in Table 9.

[0223] Table 9

[0224]

[0225] As can be seen from Table 9, Comparative Example 12 is a prescription without antioxidants. After 7 days at 40°C, the semaglutide content decreased by 1.75%, and the polymer increased by 0.30%. Comparative Examples 13-15 are prescriptions containing vitamin E (VE) in different proportions. After 7 days at 40°C, the semaglutide content decreased by up to 4.17%, and the polymer increased by up to 0.30%. It can be seen that when the antioxidant is selected beyond the range defined in the present invention, there will be a risk of formulation stability.

[0226] In addition, Examples 31-35 are prescriptions containing different proportions of antioxidants butylated hydroxyanisole and sodium thiosulfate. After 7 days of observation at 40°C, the maximum decrease in semaglutide content was 1.36%, and the maximum increase in polymer was 0.11%. The decrease in semaglutide content was much lower than that in Comparative Examples 12-15, and the polymer growth was also much lower than that in Comparative Examples 12-15. The polymer growth value of Comparative Example 12 without antioxidants was 3 times that of Examples 31-35 containing butylated hydroxyanisole and sodium thiosulfate antioxidants. It is worth noting that the semaglutide concentrations in Examples 31-35 were higher than or equal to Comparative Examples 12-15. The high-concentration protein prescription itself has a higher tendency to aggregate, but the antioxidants butylated hydroxyanisole and sodium thiosulfate inhibit this trend, while the antioxidant vitamin E does not have this inhibitory ability. This may be related to its excessive fat solubility, which binds to the side chains of the polypeptide and causes physical aggregation.

[0227]

Test Example 1j

[0228] Examples 36-37 were placed in a drug stability test chamber (40°C ± 2°C, 75% ± 5% relative humidity RH) to examine the decrease in the content of liraglutide and telportide, and the growth of impurities and polymers after 28 days. The results are recorded in Table 10.

[0229] Table 10

[0230]

[0231] It can be seen from Table 10 that when the technical solution of the present invention is applied to tepote and liraglutide, the stability of the preparation can still be guaranteed.

[0232] [Test Example 2]

[0233] The stability samples of Example 1 (40°C ± 2°C, 28 days) and the samples of semaglutide alone were subjected to circular dichroism (CD) analysis. The test results are shown in Tables 11 and Figure 1 shown.

[0234] Test method:

[0235] The experimental instrument was Chirascan Plus V100 circular dichroism spectrometer (Applied Photophysics, UK); the cuvette was (Hellma1mm); PBS was used as the diluent, and the sample concentration was 0.5 mg / mL.

[0236] Instrument parameter setting: The temperature was set to room temperature, the detector bandwidth was 1.0 nm, the scanning step was 1.0 nm, the scanning range was 190-260 nm, and the sample was measured three times. The specific implementation was as follows: the cuvette was soaked in 2M HNO3 overnight, rinsed with deionized water and dried, the background was collected first, then the blank buffer was collected, and then the test sample was added to the cuvette to perform far-ultraviolet scanning at 190-260 nm according to the above parameters and collect data.

[0237] Secondary prediction of the test sample: The scanning spectrum of circular dichroism in the far ultraviolet region reflects the arrangement information of protein peptide bonds, and the calculated value is the proportion of protein secondary structure, that is, the proportion of helix, folding (Antiparallel+Parallel), turn (Betaturn) and random coil. We use CDNN software to fit the secondary structure of the test sample, and calculate the proportion of helix, folding (Antiparallel+Parallel), turn (Betaturn) and random coil in different wavelength ranges in the Milli-Degress mode.

[0238] Table 11

[0239]

[0240] According to the results of circular dichroism spectroscopy analysis, the secondary structure of the stability samples of the formulation in the invention has no significant difference from that of the semaglutide raw material, which proves that the formulation excipients and preparation process will not significantly change the secondary structure of semaglutide under accelerated conditions.

[0241] [Test Example 3]

[0242] The particle size analysis of Example 1 and Comparative Example 1 was performed by dynamic light scattering (DLS). The test results are shown in Table 12 and Figure 2 , Figure 3 shown.

[0243] Test method:

[0244] The samples were tested using a Malvern Zetasizer Pro nanoparticle size analyzer equipped with a quartz microsample cell. The test selected a back angle of 173° and a temperature of 20°C. The test was repeated three times to obtain the average value.

[0245] Table 12

[0246]

[0247] From the DLS data, it can be seen that Example 1 has a smaller particle size than Comparative Example 1, which may be related to the characteristic of the technology of the present invention that reduces oligomers and retains more monomers, thereby being able to better penetrate the nasal mucosa; while the particle size of Comparative Example 1 is larger, which may be related to the formation of more oligomers in Comparative Example 1, thereby prolonging the release time of the drug; from the peak area and polydispersity coefficient values, it can be seen that this experiment confirms that the technical solution provided by the present invention is more stable than Comparative Example 1, and has fewer large particle aggregates.

[0248] [Test Example 4]

[0249] Pharmacokinetic study of the compositions of Example 1 and Comparative Examples 1, 4-9.

[0250] Research Methodology:

[0251] (1) Nasal spray administration: 42 beagle dogs weighing 9-12 kg were prepared, with 6 dogs in each group (3 females and 3 males). When administering the drug, the dogs were fixed with their nostrils facing downward. The nasal applicator nozzle was inserted into the dog's left nostril to a depth of about 1.5 cm. The drug solution was sprayed once, with a total of 50 μL of liquid sprayed.

[0252] (2) Subcutaneous injection: Prepare 6 beagle dogs (3 females / 3 males) weighing 9-12 kg. Fix the dogs during administration and give a single subcutaneous injection (0.25 mg / dog).

[0253] (3) Blood collection: 1 mL of whole blood was collected from the forelimb vein of all animals before administration and 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 36 h, 48 h, 56 h, and 72 h after administration. The blood was anticoagulated with EDTA-K2 and centrifuged at 5000 rpm for 10 min to separate the plasma.

[0254] (4) Blood drug concentration determination method

[0255] Sample preparation:

[0256] Take 100 µL of plasma and transfer it to a centrifuge tube (Eppendorf LoBind® Tube), add 5 µL of 2000 ng / mL semaglutide-d8 isotope internal standard (TLC, 89.0%) solution, mix for 30 seconds; then add 700 µL of methanol and mix for 3 minutes; centrifuge at 13000 r / min for 15 minutes, collect the supernatant and detect it on the analyzer.

[0257] Experimental equipment:

[0258] A Waters I-Class UPLC and SCIEX 6500+ MS / MS system were used.

[0259] Chromatographic conditions:

[0260] Chromatographic column: Claris C18-120 (2.1×50 mm, 2.6 μm); mobile phase: A: water (containing 0.1% formic acid); B: acetonitrile (containing 0.1% formic acid); flow rate: 0.4 mL / min; column temperature: 60°C; injection volume: 10 μL; acquisition time: gradient running time;

[0261] Elution gradient:

[0262]

[0263] Mass spectrometry conditions:

[0264] The data acquisition method was MRM (multiple reaction monitoring), the ion source was ESI source positive mode, the curtain gas CUR was 35 psi, the IS voltage was 5500 V, the source temperature TEM was 450 °C, the nebulizer gas Gas1 was 60 psi, the auxiliary gas Gas2 was 60 psi, and the collision gas CAD was High.

[0265] Multiple reaction monitoring ion channels:

[0266]

[0267] Data processing:

[0268] SCIEX OS software, version 3.3, was used. The MQ4 algorithm was used for integration, with a weight of 1 / x 2 .

[0269] Method verification items:

[0270] Sensitivity and linearity

[0271] In this experiment, the beagle plasma standard working solutions were prepared to 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL and 2000 ng / mL. After protein precipitation, the blank was clean and free of interference. ( Figure 4 ). Linear range and regression equation of semaglutide compound ( Figure 5 ), semaglutide showed good linearity in the concentration range of 5~2000ng / mL.

[0272] Reproducibility

[0273] To investigate the reproducibility and accuracy of the method, the processed samples with the limit of quantification (LOQ) concentration of 5 ng / mL were tested for repeatability. Six samples were collected continuously, and the precision (RSD) was 1.8%, and the accuracy was between 94.7% and 107.5%.

[0274] Recovery rate

[0275] Nine portions of beagle dog blank plasma were used to prepare three spiked solutions in parallel, with concentrations of low concentration quality control (within 3 times of the lower limit of quantification, LQC), medium concentration quality control (near the geometric mean of the quantitative range of the calibration curve, MQC), and high concentration quality control (above 75% of the upper limit of quantification, HQC) as quality control samples, and the purified solution was tested on the machine. The accuracy of the spiked samples was between 91.71-101.96%, and the RSD% of the parallel samples was less than 5%. The accuracy and reproducibility met the test requirements, and the test results were good.

[0276] (5) Calculation of pharmacokinetic data

[0277] WinwonLin software, version 8.3, was used to calculate the pharmacokinetic parameters of a single dose. The results are shown in Tables 13-20 and Figure 6-9 shown.

[0278] Table 13

[0279]

[0280] Table 14

[0281]

[0282] Table 15

[0283]

[0284] Table 16

[0285]

[0286] Table 17

[0287]

[0288] Table 18

[0289]

[0290] Table 19

[0291]

[0292] Table 20

[0293]

[0294] From the above results, it can be seen that the bioavailability is proportional to the concentration of alkyl glycoside or phospholipid; compared with the bioavailability of the composition using alkyl glycoside alone (Comparative Examples 4-6) or phospholipid alone (Comparative Examples 7-9) at the same concentration, the bioavailability of the combined alkyl glycoside-phospholipid (Example 1) is more than 3 times higher, and when the concentration of phospholipid in the preparation is less than 0.5%, the blood drug concentration is already less than the quantitative lower limit.

[0295] [Test Example 5]

[0296] Pharmacokinetic study of Example 37.

[0297] Research Methodology:

[0298] (1) Nasal spray administration: Prepare 6 beagle dogs (3 females / 3 males) weighing 9-12 kg. Fix the dogs with their nostrils facing downwards. Insert the nasal applicator nozzle into the left nostril of the dog to a depth of about 1.5 cm. Press once to spray the drug solution, and spray 50 μL of liquid.

[0299] (2) Blood collection: 1 mL of whole blood was collected from the forelimb vein of all animals before administration and 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 36 h, 48 h, 56 h, and 72 h after administration. The blood was anticoagulated with EDTA-K2 and centrifuged at 5000 rpm for 10 min to separate the plasma.

[0300] (3) Blood drug concentration determination method

[0301] Sample preparation:

[0302] 200 µL beagle plasma was mixed with 10 µL 1000 ng / ml semaglutide internal standard (Shenzhen Jianyuan, 98.7%); 400 µL methanol was added to precipitate the protein in the plasma and vortexed for 2 min. Centrifuge at 15000 rpm / min for 10 min. 500 µL of the supernatant was taken, diluted with 400 µL water, vortexed and purified. The purified solution was loaded into an activated 96-well microplate (Peptide-3-MW), rinsed with water, and finally eluted twice with elution solution (5% formic acid ethanol: water = 4:1), vortexed and tested on the machine.

[0303] Experimental equipment:

[0304] A Waters I-Class UPLC and SCIEX 6500+ MS / MS system were used.

[0305] Chromatographic conditions:

[0306] Chromatographic column: Phenomenex Aeris Peptide XB-C18 (2.1×100 mm, 2.6 μm); mobile phase: A: water (containing 0.1% formic acid); B: acetonitrile (containing 0.1% formic acid); flow rate: 0.3 mL / min; column temperature: 30°C; detection wavelength: 210 nm; injection volume: 10 μL; acquisition time: gradient running time;

[0307] Elution gradient:

[0308]

[0309] Mass spectrometry conditions:

[0310] The data acquisition method was MRM (multiple reaction monitoring), the ion source was ESI source positive mode, the curtain gas CUR was 35 psi, the IS voltage was 5500 V, the source temperature TEM was 450 °C, the nebulizer gas Gas1 was 60 psi, the auxiliary gas Gas2 was 60 psi, and the collision gas CAD was High.

[0311] Multiple reaction monitoring ion channels:

[0312]

[0313] Data processing:

[0314] SCIEX OS software, version 3.3, was used. The MQ4 algorithm was used for integration, with a weight of 1 / x 2 .

[0315] Method verification items:

[0316] Sensitivity and linearity

[0317] In this experiment, the standard working solutions of beagle plasma were prepared to 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL, and analyzed by the machine after protein precipitation and 96-well plate; the blank was clean and free of interference ( Fig. 9 ). Linear range and regression equation of telportide compound ( Fig.10 ), tepoxetine has good linearity in the concentration range of 0.2~100ng / mL. Three injections were made at each mark point, and the RSD% was less than 10%.

[0318] Reproducibility

[0319] To investigate the reproducibility and accuracy of the method, the processed samples with LOQ concentration of 0.2 ng / mL were tested for repeatability. Six samples were collected continuously, with a precision (RSD) of 3.2% and an accuracy between 92.3% and 110.2%.

[0320] Recovery rate

[0321] Nine beagle blank plasmas were taken and three spiked solutions were prepared in parallel, with concentrations at LQC, MQC and HQC as quality control samples, and the purified solution was tested on the machine. The accuracy of the spiked samples was 96.87-108.32%, and the RSD% of the parallel samples was less than 5%. The accuracy and reproducibility met the test requirements, and the test results were good.

[0322] (5) Calculation of pharmacokinetic data

[0323] WinwonLin software, version 8.3, was used to calculate the pharmacokinetic parameters of a single dose. The results are shown in Table 21 and Fig.11 shown.

[0324] Table 21

[0325]

[0326] When this technical solution is applied to telportide, the expected bioavailability can also be achieved.

[0327] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation composition of a glucagon-like peptide-1 receptor agonist for administration through the nasal mucosa, characterized in that: in, The glucagon-like peptide-1 receptor agonist is selected from semaglutide, and the main components of the preparation composition include semaglutide, alkyl glycoside and phospholipid, wherein the ratio of semaglutide: alkyl glycoside: phospholipid is (0.25-17.5) by mass: (0.25-10) by mass: (0.1-10), the mass of semaglutide accounts for 0.25%-17.5% of the total mass of the preparation composition, the alkyl glycoside is octyl-β-D-maltoside, decanyl-β-D-maltoside, dodecyl-β-D-maltoside, tetradecyl-β-D-maltoside or a combination thereof, and the ratio of the alkyl glycoside to phospholipid is not more than 10:1 by mass.

2. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to claim 1, characterized in that: The mass of the alkyl glycoside accounts for 0.25%-10.00% of the total mass of the preparation composition.

3. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to claim 1, characterized in that: The phospholipid is decanylphosphocholine, dodecylphosphocholine, tetradecylphosphocholine, hexadecylphosphocholine, lyso-lauroylphosphatidylcholine, dilauroylphosphatidylglycerol or a combination thereof.

4. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to claim 3, characterized in that: The mass of the phospholipid accounts for 0.10%-10.00% of the total mass of the preparation composition.

5. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to any one of claims 1 to 4, characterized in that: The composition further comprises a bioadhesive, an antioxidant, a bacteriostatic agent, an osmotic pressure regulator, a buffer, a pH regulator and water.

6. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to claim 5, characterized in that: The bioadhesive is methyl-β-cyclodextrin, 2,6-dimethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin or a combination thereof.

7. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to claim 6, characterized in that: The mass of the bioadhesive agent accounts for 0%-20% of the total mass of the preparation composition.

8. The preparation composition of glucagon-like peptide-1 receptor agonist for transnasal administration according to claim 5, characterized in that: The antioxidant is butylated hydroxyanisole, sodium thiosulfate, phenol or a combination thereof.

9. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to claim 8, characterized in that: The weight percentage of the antioxidant to the total weight of the formulation composition is 0.0005%-0.3%.

10. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to claim 5, characterized in that: The antibacterial agent is benzalkonium chloride, benzyl alcohol, phenylethyl alcohol, parabens or a combination thereof.

11. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to claim 10, characterized in that: The mass of the antibacterial agent accounts for 0%-1.0% of the total mass of the preparation composition.

12. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to claim 5, characterized in that: The osmotic pressure regulator is sodium chloride, potassium chloride, glucose, sucrose, sorbitol, mannitol or a combination thereof.

13. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to claim 5, characterized in that: The buffer is a phosphate buffer, a citrate buffer, a bicarbonate buffer, a tartrate buffer, an acetate buffer or a combination thereof.

14. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to claim 5, characterized in that: The pH adjuster is phosphoric acid, sodium hydroxide, potassium hydroxide, hydrochloric acid or a combination thereof.

15. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to any one of claims 1 to 4, characterized in that: The pH of the formulation composition is 6.80 to 8.

50.

16. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to any one of claims 1 to 4, characterized in that: The osmotic pressure of the formulation composition is 150 to 450 mOsmol / kg.

17. A method for preparing a preparation composition of a glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to any one of claims 5 to 16, the method comprising the following steps: a. Mix the buffer solution, antibacterial agent and part of the water at room temperature at a rate of 200-1200 rpm until fully dissolved to obtain solution a; b. Add the alkyl glycoside and phospholipid to solution a, mix and stir at a rate of 200-1200 rpm until fully dissolved to obtain solution b; c. Add the bioadhesive and antioxidant to solution b, and mix and stir at a rate of 200-1200 rpm until fully dissolved to obtain solution c; d. Add semaglutide to solution c and mix and stir until fully dissolved to obtain solution d; e. Add appropriate amounts of pH regulator and osmotic pressure regulator to solution d to adjust the pH and osmotic pressure to the target range; f. Add excess water, stir evenly, and perform sterile filtration.

18. A nasal applicator for administration to the nasal mucosa, the applicator comprising a composition preparation reservoir, a composition preparation contained in the reservoir, and a metering pump for expelling the preparation from the reservoir, wherein the composition preparation is the preparation composition described in any one of claims 1 to 16.

19. Use of the preparation composition of glucagon-like peptide-1 receptor agonist for nasal administration according to any one of claims 1 to 16 in the preparation of drugs for treating diabetes and obesity, wherein the preparation composition is administered to the nasal mucosa of a subject in a therapeutically effective amount.

20. The use of claim 19, wherein the formulation composition is administered once a week or more frequently.

21. The use according to claim 20, wherein the content of semaglutide in the weekly administered formulation composition is 0.25-30 mg.

22. The preparation composition of glucagon-like peptide-1 receptor agonist for nasal mucosal administration according to any one of claims 1-4 and 6-14, characterized in that: Semaglutide was replaced by either liraglutide or telpotide alone.

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