Microspheres comprising GLP-1 ra-based peptide or pharmaceutically acceptable salt thereof, method for preparing same, and pharmaceutical composition comprising same microspheres

Abstract

The present invention relates to microspheres comprising a GLP-1 RA-based peptide or a pharmaceutically acceptable salt thereof, a biocompatible polymer, and a bioavailability enhancer, a method for preparing same, and a pharmaceutical composition comprising same, the pharmaceutical composition being for use in preventing or treating diabetes, obesity, hypertension, hyperlipidemia, non-alcoholic steatohepatitis, metabolic dysfunction-associated steatohepatitis, cardiovascular diseases, or neurodegenerative diseases.

Description

Microspheres comprising a GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof, a method for preparing the same, and a pharmaceutical composition comprising said microspheres

[0001] The present invention relates to microspheres comprising a GLP-1 receptor agonist (GLP-1 RA) or a pharmaceutically acceptable salt thereof, a method for manufacturing the same, and a pharmaceutical composition comprising said microspheres.

[0002] More specifically, the invention relates to microspheres comprising a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer, a method for manufacturing the same, and a pharmaceutical composition comprising said microspheres.

[0003] GLP-1 receptor agonists (GLP-1 RAs) are agonists that mimic the action of glucagon-like peptide-1 (GLP-1), a bioactive peptide derived from preproglucagon secreted by L-cells, and stabilize postprandial blood glucose levels by stimulating insulin secretion from pancreatic islet β-cells. Based on this mechanism of action, GLP-1 RAs are known to be effective in the treatment of diseases such as type 2 diabetes and obesity, and Ozempic ® (Semaglutide), Mounjaro ® (Tirzepatide), Victoza ® (Liraglutide), Adlyxin ® Preparations such as lixisenatide are currently on the market.

[0004] However, since GLP-1 RAs are peptide-based preparations, they are significantly affected by biological barriers such as degradation by gastrointestinal enzymes, low absorption rate in the intestinal mucosa, and first-pass metabolism in the liver, resulting in very low oral bioavailability. For this reason, most GLP-1 RAs are administered via subcutaneous injection; for example, semaglutide or tyrzepatide are used via once-weekly subcutaneous injection. Such frequent injections can cause significant inconvenience to patients and reduce medication adherence, leading to an increasing demand for formulations that can reduce the frequency of administration for patients, including obese patients.

[0005] To meet these demands, technologies for delivering GLP-1 RA class peptides in a long-acting manner are being actively researched, and as part of this, technologies for manufacturing sustained-release formulations by encapsulating peptides in drug delivery systems such as microspheres have been developed. However, maintaining long-term efficacy requires a high drug loading capacity within the microspheres, which acts as a technical limitation as it can exacerbate the initial burst phenomenon and cause safety issues.

[0006] Furthermore, peptide therapeutics such as semaglutide and tyrzepatide may experience a decline in structural and chemical stability when maintained in the body for a long period, which leads to a problem of significantly reduced bioavailability.

[0007] Therefore, there is a need for technical development of injectable formulations containing long-acting microspheres that can effectively suppress initial excessive release, control the release period to a desired range, and maintain high bioavailability in the body.

[0008] [Prior Art Literature]

[0009] [Patent Literature]

[0010] (Patent Document 1) Republic of Korea Published Patent No. 10-2021-0147413

[0011] The present invention is proposed to solve the above-mentioned problems and aims to provide microspheres capable of preventing the initial excessive release of GLP-1 RA series peptides and ensuring excellent bioavailability, pharmaceutically acceptable salts thereof, pharmaceutical compositions containing the same, and a method for manufacturing said microspheres.

[0012] According to the first aspect of the present invention,

[0013] The present invention provides a microsphere composition comprising at least one microsphere comprising a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer.

[0014] According to one embodiment of the present invention, the pharmaceutically acceptable salt of the GLP-1 RA series peptide may be selected from pamoate salt, acetate salt, benzathine salt, formate salt, and combinations thereof.

[0015] According to one embodiment of the present invention, the GLP-1 RA series peptide may be selected from semaglutide, tirzepatide, retatrutide, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, cotadutide, taspoglutide, pemvidutide, survodutide, petrelintide, ellarintide, and combinations thereof.

[0016] According to one embodiment of the present invention, the GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof may be included in an amount of 2% to 50% by weight based on the total weight of the microspheres.

[0017] According to one embodiment of the present invention, the biocompatible polymer may be one or more selected from the group consisting of poly(lactic acid), polylactide, poly(lactic-co-glycolic acid), polylactide-co-glycolide (PLGA), polyphosphazene, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, copolymer of lactic acid and caprolactone, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid, and copolymer of lactic acid and amino acid.

[0018] According to one embodiment of the present invention, the biocompatible polymer may be included in an amount of 50% to 98% by weight based on the total weight of the microspheres.

[0019] According to one embodiment of the present invention, the intrinsic viscosity of the biocompatible polymer may be 0.1 dL / g to 2.0 dL / g.

[0020] According to one embodiment of the present invention, the microsphere composition may include a first microsphere and a second microsphere. The first microsphere may include a biocompatible polymer having an intrinsic viscosity of 0.1 dL / g or more and less than 0.7 dL / g, and the second microsphere may include a biocompatible polymer having an intrinsic viscosity of 0.7 dL / g or more and less than 2.0 dL / g. The weight ratio of the first microsphere and the second microsphere may be 1:0.1 to 1:10 or 0.1:1 to 10:1.

[0021] According to one embodiment of the present invention, the microsphere composition may include a first microsphere, a second microsphere, and a third microsphere. The first microsphere may include a biocompatible polymer having an intrinsic viscosity of 0.1 dL / g or more and less than 0.5 dL / g, the second microsphere may include a biocompatible polymer having an intrinsic viscosity of 0.5 dL / g or more and less than 0.7 dL / g, and the third microsphere may include a biocompatible polymer having an intrinsic viscosity of 0.7 dL / g or more and less than 2.0 dL / g. The weight ratio of the first microsphere, the second microsphere, and the third microsphere may be 1:0.1:0.1 to 1:10:10.

[0022] According to one embodiment of the present invention, the microspheres may further include a bioavailability improver.

[0023] According to one embodiment of the present invention, the bioavailability improver may be included in an amount of 0.01% to 20% by weight based on the total weight of the microspheres.

[0024] According to one embodiment of the present invention, the bioavailability improving agent may be one or more selected from the group consisting of surfactants, polyalcohols, amino acids, fatty acids, organic acids, and sodium salts of organic acids.

[0025] According to one embodiment of the present invention, the surfactant may be one or more selected from the group consisting of Poloxamer 188, Poloxamer 407, Polysorbate 20, Polysorbate 60, Polysorbate 80, Polyoxyethylene oleyl ether, Polyoxyethylene cetyl ether, Polyoxyethylene stearyl ether, and Polyoxyethylene lauryl ether.

[0026] According to one embodiment of the present invention, the polyalcohol may be one or more selected from the group consisting of ethylene glycol, propylene glycol, polyethylene glycol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, and maltitol.

[0027] According to one embodiment of the present invention, the amino acid may be one or more selected from the group consisting of lysine, arginine, aspartic acid, histidine, alanine, phenylalanine, serine, glutamine, glycine, tryptophan, valine, leucine, isoleucine, cysteine, methionine, and proline.

[0028] According to one embodiment of the present invention, the fatty acid may be one or more selected from the group consisting of stearic acid, palmitic acid, lauric acid, myristic acid, oleic acid, and arachidonic acid.

[0029] According to one embodiment of the present invention, the organic acid may be one or more selected from the group consisting of citric acid, stearic acid, tartaric acid, citric acid, succinic acid, and fumaric acid.

[0030] According to one embodiment of the present invention, the sodium organic acid salt may be one or more selected from the group consisting of sodium acetate, sodium citrate, sodium tartrate, sodium succinate, sodium propionate, sodium bicarbonate, and sodium butyrate.

[0031] According to a second aspect of the present invention,

[0032] The present invention provides a method for manufacturing microspheres comprising the following steps:

[0033] (a) A GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer are dispersed in a dispersion solvent according to the first aspect of the present invention,

[0034] The present invention provides microspheres comprising a GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer.

[0035] According to one embodiment of the present invention, pharmaceutically acceptable salts of GLP-1 RA series peptides may be selected from benzathine salt, acetate salt, formate salt, pamoate salt, and combinations thereof.

[0036] According to one embodiment of the present invention, a GLP-1 RA series peptide may be selected from semaglutide, tirzepatide, retatrutide, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, cotadutide, taspoglutide, pemvidutide, survodutide, petrelintide, ellarintide, and combinations thereof.

[0037] According to one embodiment of the present invention, a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof may be included in an amount of 2% to 50% by weight based on the total weight of the microspheres.

[0038] According to one embodiment of the present invention, the biocompatible polymer may be one or more selected from the group consisting of poly(lactic acid), polylactide, poly(lactic-co-glycolic acid), polylactide-co-glycolide (PLGA), polyphosphazene, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, copolymer of lactic acid and caprolactone, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid, and copolymer of lactic acid and amino acid.

[0039] According to one embodiment of the present invention, the biocompatible polymer may be included in an amount of 50% to 98% by weight based on the total weight of the microspheres.

[0040] According to one embodiment of the present invention, the intrinsic viscosity of the biocompatible polymer may be 0.1 dL / g to 1.9 dL / g.

[0041] According to one embodiment of the present invention, the microspheres may further include a bioavailability improver.

[0042] According to one embodiment of the present invention, the bioavailability improver may be included in an amount of 0.01% to 20% by weight based on the total weight of the microspheres.

[0043] According to one embodiment of the present invention, the bioavailability improver may be one or more selected from the group consisting of surfactants, polyalcohols, amino acids, fatty acids, organic acids, and sodium salts of organic acids.

[0044] According to one embodiment of the present invention, the surfactant may be one or more selected from the group consisting of Poloxamer 188, Poloxamer 407, Polysorbate 20, Polysorbate 60, Polysorbate 80, Polyoxyethylene oleyl ether, Polyoxyethylene cetyl ether, Polyoxyethylene stearyl ether, and Polyoxyethylene lauryl ether.

[0045] According to one embodiment of the present invention, the polyhydric alcohol may be one or more selected from the group consisting of ethylene glycol, propylene glycol, polyethylene glycol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, and maltitol.

[0046] According to one embodiment of the present invention, the amino acid may be one or more selected from the group consisting of lysine, arginine, aspartic acid, histidine, alanine, phenylalanine, serine, glutamine, glycine, tryptophan, valine, leucine, isoleucine, cysteine, methionine, and proline.

[0047] According to one embodiment of the present invention, the fatty acid may be one or more selected from the group consisting of stearic acid, palmitic acid, lauric acid, myristic acid, oleic acid, and arachidonic acid.

[0048] According to one embodiment of the present invention, the organic acid may be one or more selected from the group consisting of citric acid, stearic acid, tartaric acid, citric acid, succinic acid, and fumaric acid.

[0049] According to one embodiment of the present invention, the sodium organic acid salt may be one or more selected from the group consisting of sodium acetate, sodium citrate, sodium tartrate, sodium succinate, sodium propionate, sodium bicarbonate, and sodium butyrate.

[0050] According to a second aspect of the present invention,

[0051] The present invention provides a method for manufacturing microspheres comprising the following steps:

[0052] (a) a step of preparing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof;

[0053] (b) a step of preparing a dispersed phase by dissolving the GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof prepared in step (a) and a biocompatible polymer in a dispersion solvent;

[0054] (c) a step of introducing the dispersed phase prepared in step (b) above into a continuous phase and stirring to form microspheres;

[0055] (d) a step of removing the dispersion solvent from the microspheres of step (c);

[0056] (e) a step of recovering the microspheres formed in step (d) above; and

[0057] (f) A step of drying the microspheres recovered in step (e) above.

[0058] According to one embodiment of the present invention, step (a) includes reacting a GLP-1 RA series peptide with an acid or basic compound to form a salt, and separating and purifying the formed salt, wherein the formed salt may be selected from benzathine salt, formate salt, acetate salt, pamoate salt, and combinations thereof.

[0059] According to one embodiment of the present invention, step (b) may further include the step of introducing a bioavailability enhancer.

[0060] According to one embodiment of the present invention, the method for manufacturing microspheres may further include an extraction step after step (e) or after step (f).

[0061] According to one embodiment of the present invention, the extraction step may be performed using an aqueous solution or a mixture of the following as an extraction solvent, which includes purified water, a buffer solution, alcohols, ketones, esters, and inorganic salts, or may be performed using an extraction solvent which includes at least one selected from the group consisting of ethanol, methanol, acetone, methyl ethyl ketone, and ethyl acetate, and an aqueous solution of disodium hydrogen phosphate.

[0062] According to the third aspect of the present invention,

[0063] The present invention provides a pharmaceutical composition for the prevention or treatment of diabetes, obesity, hypertension, hyperlipidemia, non-alcoholic fatty liver disease (NAFLD), metabolic disorder fatty liver disease (MAFLD), cardiovascular disease, or degenerative neurological disease, wherein the pharmaceutical composition comprises microspheres of the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0064] According to one embodiment of the present invention, the pharmaceutical composition of the present invention may contain microspheres in an amount of 0.01 mg to 2,000 mg per 1 mL of pharmaceutically acceptable carrier.

[0065] According to one embodiment of the present invention, the pharmaceutically acceptable carrier may be one or more selected from the group consisting of a water-soluble liquid, a fluid oil containing a fatty acid ester, a surfactant, and a polyalcohol.

[0066] According to one embodiment of the present invention, a fluid oil containing a fatty acid ester may include one or more selected from the group consisting of coconut oil, palm oil, palm kernel oil, sesame oil, soybean oil, almond oil, rapeseed oil, corn oil, sunflower oil, peanut oil, olive oil, castor oil, safflower oil, cottonseed oil, and ethyl oleate.

[0067] According to one embodiment of the present invention, the surfactant may be one or more selected from Poloxamer 188, Poloxamer 407, Polysorbate 20, Polysorbate 60, Polysorbate 80, Polyoxyethylene oleyl ether, Polyoxyethylene cetyl ether, Polyoxyethylene stearyl ether, and Polyoxyethylene lauryl ether.

[0068] According to one embodiment of the present invention, the polyhydric alcohol may include one or more selected from ethylene glycol, propylene glycol, polyethylene glycol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, and maltitol.

[0069] According to one embodiment of the present invention, the pharmaceutical composition of the present invention may further include hyaluronidase.

[0070] According to one embodiment of the present invention, hyaluronidase may be included in a pharmaceutically acceptable carrier or may be included in a pharmaceutical composition separately from the pharmaceutically acceptable carrier.

[0071] According to one embodiment of the present invention, the pharmaceutical composition of the present invention may further comprise a GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof.

[0072] According to one embodiment of the present invention, the pharmaceutical composition of the present invention may further include one or more excipients selected from buffers, isotonic agents, preservatives, and pH adjusters.

[0073] According to one embodiment of the present invention, the pharmaceutical composition of the present invention may be for subcutaneous or intramuscular injection.

[0074] The microspheres according to the present invention provide the effect of releasing continuously and consistently over a long period without rapid initial release by encapsulating a pharmaceutically acceptable salt of a GLP-1 RA series peptide in a biocompatible polymer, and have high bioavailability.

[0075] In addition, the pharmaceutical composition according to the present invention provides excellent effects in the prevention or treatment of obesity or diabetes by including the microspheres.

[0076] Figure 1 shows the semaglutide benzathine salt prepared in Example 1. 1 This is a diagram showing the H-NMR analysis results.

[0077] FIG. 2 is of the semaglutide acetate salt prepared in Example 50. 1 This is a diagram showing the H-NMR analysis results.

[0078] FIG. 3 is of the semaglutide formate prepared in Example 67. 1 This is a diagram showing the H-NMR analysis results.

[0079] Figure 4 shows the tyrzepatide benzathine salt prepared in Example 69. 1 This is a diagram showing the H-NMR analysis results.

[0080] FIG. 5 is of the tyrzepatide acetate salt prepared in Example 80. 1 This is a diagram showing the H-NMR analysis results.

[0081] FIG. 6 is of the tyrzepatide formate prepared in Example 85. 1 This is a diagram showing the H-NMR analysis results.

[0082] FIG. 7 shows the tyrzepatide pamoate salt prepared in Example 89. 1 This is a diagram showing the H-NMR analysis results.

[0083] The present invention will be described in more detail below.

[0084] terminology

[0085] Each term used in this specification is explained.

[0086] In this specification, "pharmaceuticalally acceptable salt" means any organic or inorganic addition salt of said compound that is available for use at a concentration that exhibits a relatively non-toxic and harmless active effect when administered to a patient, and for which adverse effects caused by said salt do not substantially impede the beneficial efficacy of the active ingredient.

[0087] In this specification, "biocompatible polymer" refers to a polymer with ensured in vivo safety that does not induce high cytotoxicity or inflammatory responses when administered in vivo, and is also referred to simply as "polymer" in this specification.

[0088] In this specification, "intrinsic viscosity" means that the viscosity is measured at a concentration of 0.05% (w / v) to 3% (w / v) in chloroform at 25°C using a Ubbelohde viscometer.

[0089] In this specification, "bioavailability enhancer" refers to a substance that improves bioavailability by being encapsulated within microspheres together with a GLP-1 RA-type peptide or a pharmaceutically acceptable salt thereof, and in particular, means a substance that causes the area under the blood concentration-time curve (AUC) of the GLP-1 RA-type peptide to be higher than that of microspheres not containing the bioavailability enhancer when microspheres containing the GLP-1 RA-type peptide or a pharmaceutically acceptable salt thereof are administered into the body.

[0090] In this specification, "continuous phase" refers to a phase that exists continuously surrounding another phase in an emulsion or dispersion system, and in which the dispersion phase is distributed.

[0091] In this specification, "dispersed phase" refers to a phase that exists in the form of droplets or particles dispersed within a continuous phase in an emulsion or dispersion system.

[0092] In this specification, "carrier" means a compound that facilitates the addition of a compound into a cell or tissue.

[0093] In this specification, "tissue diffusion promoter" refers to a pharmacologically active enzyme component that does not function as a simple physical carrier but rather contributes to the dispersion and absorption of the GLP-1 receptor agonist by temporarily increasing the permeability of subcutaneous tissue.

[0094] All technical terms used herein are used in the sense generally understood by those skilled in the art, unless otherwise defined; however, in the event of a conflict, the definitions in this specification shall prevail. Furthermore, terms used herein are for illustrative purposes only and should not be interpreted as being limiting. Similar or equivalents to the preferred methods or samples described herein are also included within the scope of the invention. The contents of all publications cited as references in this specification are incorporated by reference in their entirety.

[0095] microspheres

[0096] The microspheres of the present invention comprise a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer.

[0097] In one embodiment, the pharmaceutically acceptable salt of the GLP-1 RA family peptide may be selected from benzathine salt, formate salt, acetate salt, pamoate salt, and combinations thereof, but is not limited to these.

[0098] In one embodiment, the GLP-1 RA family peptide may be selected from semaglutide, tirzepatide, retatrutide, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, cotadutide, taspoglutide, pemvidutide, survodutide, petrelintide, ellarintide, and combinations thereof, but is not limited thereto.

[0099] In one embodiment, a GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof may be included in an amount of 2% to 50% by weight based on the total weight of the microspheres.

[0100] Specifically, the GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof may be included in an amount of 2% to 50% by weight, 5% to 45% by weight, or 10% to 40% by weight based on the total weight of the microspheres. More specifically, the GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof is included in an amount of 2.0 wt% or more, 2.5 wt% or more, 3.0 wt% or more, 3.5 wt% or more, 4.0 wt% or more, 4.5 wt% or more, 5.0 wt% or more, 5.5 wt% or more, 6.0 wt% or more, 6.5 wt% or more, 7.0 wt% or more, 7.5 wt% or more, 8.0 wt% or more, 8.5 wt% or more, 9.0 wt% or more, based on the total weight of the microspheres, or 50 wt% or less, 49.5 wt% or less, 49.0 wt% or less, 48.5 wt% or less, 48.0 wt% or less, 47.5 wt% or less, 47.0 wt% or less, 46.5 wt% or less, 46.0 wt% or less, 45.5 wt% or less, It may be included in an amount of 45.0 wt% or less, 44.5 wt% or less, 44.0 wt% or less, 43.5 wt% or less, 43.0 wt% or less, 42.5 wt% or less, 42.0 wt% or less, 41.5 wt% or less, 41.0 wt% or less, 40.5 wt% or less, and 40.0 wt% or less.

[0101] As mentioned above, if a GLP-1 RA-type peptide or a pharmaceutically acceptable salt thereof is included in an amount of less than 2% by weight based on the total weight of the microspheres, there may be a problem where administration becomes difficult because the relative proportion of biocompatible polymers increases, resulting in an excessive dosage in the body, and it is undesirable because a single administration cannot exert a long-term therapeutic effect. In addition, if a GLP-1 RA-type peptide or a pharmaceutically acceptable salt thereof is included in an amount exceeding 50% by weight based on the total weight of the microspheres, there may be a problem where the initial release of the GLP-1 RA-type peptide in the body environment is excessively high, causing the blood concentration of the drug to rise rapidly.

[0102] In one embodiment, the biocompatible polymer may be one or more selected from the group consisting of poly(lactic acid), polylactide, poly(lactic-co-glycolic acid), polylactide-co-glycolide (PLGA), polyphosphazene, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, copolymer of lactic acid and caprolactone, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid, and copolymer of lactic acid and amino acid.

[0103] In one embodiment, the biocompatible polymer contained within the microspheres may be included in an amount of 50% to 98% by weight based on the total weight of the microspheres.

[0104] Specifically, the biocompatible polymer may be included in an amount of 50% to 98% by weight, 55% to 90% by weight, or 60% to 80% by weight based on the total weight of the microspheres. More specifically, the biocompatible polymer may be included in an amount of 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more by weight based on the total weight of the microspheres, or in an amount of 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, or 93% or less by weight, but is not limited to these ranges.

[0105] As mentioned above, if the biocompatible polymer exceeds 98% by weight based on the total weight of the microspheres, the drug release becomes too slow, making it difficult to secure a consistent blood drug profile, and if the biocompatible polymer is included in an amount of less than 50% by weight based on the total weight of the microspheres, the distribution of semaglutide increases relatively, which may result in an initial over-release or a failure to maintain the drug effect for the desired period.

[0106] In one embodiment, the microspheres contain a GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer in a ratio of 1:1-50, 1:1.5-50, 1:1.6-50, 1:1.7-50, 1:1-47, 1:1.5-47, 1:1.6-47, 1:1.7-47, 1:1-45, 1:1.5-45, 1:1.6-45, 1:1.7-45, 1:1-42, 1:1.5-42, 1:1.6-42, 1:1.7-42, 1:1-40, 1:1.5-40, 1:1.6-40, 1:1.7-40, 1:1-38, 1:1.5-38, It may be included in weight ratios of 1:1.6-38, 1:1.7-38, 1:1-35, 1:1.5-35, 1:1.6-35, or 1:1.7-35, but is not limited thereto.

[0107] In the above, if the weight ratio of the biocompatible polymer to the GLP-1 RA series peptide or its pharmaceutically acceptable salt is less than 1, it is undesirable as it may lead to fatal side effects due to the excessive release of the initial drug, and if the weight ratio exceeds 50, the release of the drug becomes too slow, making it difficult to secure a consistent blood drug profile.

[0108] In one embodiment, the intrinsic viscosity of the biocompatible polymer may be 0.10 dL / g to 1.9 dL / g, 0.10 dL / g to 1.3 dL / g, or 0.10 dL / g to 1.2 dL / g.

[0109] As mentioned above, if the intrinsic viscosity of the biocompatible polymer is less than 0.10 dL / g, the molecular weight of the polymer is insufficient, making it difficult to exhibit a sustained-release effect of semaglutide benzathine salt; conversely, if the intrinsic viscosity exceeds 1.9 dL / g, the release of semaglutide benzathine salt may be excessively delayed. Furthermore, when the intrinsic viscosity of the biocompatible polymer is high, a problem arises where an excessive amount of manufacturing solvent must be used during the preparation of the pharmaceutical composition due to the high viscosity of the polymer, which also makes it difficult to produce reproducible microspheres.

[0110] In one embodiment, the microspheres may further include a bioavailability enhancer.

[0111] In one embodiment, the bioavailability improver may be included in an amount of 0.01% to 20% by weight based on the total weight of the microspheres.

[0112] Specifically, the bioavailability improver may be 0.01 wt% or more, 0.015 wt% or more, 0.02 wt% or more, 0.025 wt% or more, 0.03 wt% or more, 0.035 wt% or more, 0.04 wt% or more, 0.045 wt% or more, or 0.05 wt% or more based on the total weight of the microspheres, or 20.0 wt% or less, 19.5 wt% or less, 19.0 wt% or less, 18.5 wt% or less, 18.0 wt% or less, 17.5 wt% or less, 17.0 wt% or less, 16.5 wt% or less, 16.0 wt% or less, or 15.5 wt% or less.

[0113] In one embodiment, the bioavailability improver may be one or more selected from the group consisting of surfactants, polyalcohols, amino acids, fatty acids, organic acids, and sodium salts of organic acids.

[0114] Specifically, the surfactant may be one or more selected from the group consisting of Poloxamer 188, Poloxamer 407, Polysorbate 20, Polysorbate 60, Polysorbate 80, Polyoxyethylene oleyl ether, Polyoxyethylene cetyl ether, Polyoxyethylene stearyl ether, and Polyoxyethylene lauryl ether.

[0115] Additionally, specifically, the polyalcohol may be one or more selected from the group consisting of ethylene glycol, propylene glycol, polyethylene glycol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, and maltitol.

[0116] Additionally, specifically, the amino acid may be one or more selected from the group consisting of lysine, arginine, aspartic acid, histidine, alanine, phenylalanine, serine, glutamine, glycine, tryptophan, valine, leucine, isoleucine, cysteine, methionine, and proline.

[0117] Additionally, specifically, the fatty acid may be one or more selected from the group consisting of stearic acid, palmitic acid, lauric acid, myristic acid, oleic acid, and arachidonic acid.

[0118] Specifically, the organic acid may be one or more selected from the group consisting of citric acid, stearic acid, tartaric acid, citric acid, succinic acid, and fumaric acid.

[0119] Specifically, the sodium organic acid salt may be one or more selected from the group consisting of sodium acetate, sodium citrate, sodium tartrate, sodium succinate, sodium propionate, sodium bicarbonate, and sodium butyrate.

[0120] Method for manufacturing microspheres

[0121] The method for manufacturing microspheres according to the present invention may be manufactured by conventional methods for manufacturing microspheres known in the art, such as O / W type, O / O type or W / O / W type, S / O / W solvent evaporation or solvent extraction methods, methods for manufacturing microspheres by spray drying, methods for manufacturing microspheres by phase separation, etc.

[0122] In one embodiment, the method for manufacturing microspheres of the present invention may include the step of manufacturing an emulsion and the step of aggregating the emulsion by solvent evaporation or solvent extraction.

[0123] Specifically, the solvent evaporation method may include, but is not limited to, methods described in U.S. Patents No. 5,271,945, 5,985,309 and 6,471,996. More specifically, the solvent evaporation method may include the steps of: dissolving a polymer compound in an organic solvent, dispersing or dissolving a drug in the organic solvent, and then emulsifying the mixture in a dispersion medium such as water to prepare an O / W type emulsion; and diffusing the organic solvent contained in the emulsion into the dispersion medium and evaporating it through an air / water interface to form polymer microspheres containing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof.

[0124] Additionally, specifically, the solvent extraction method may include solvent extraction methods conventional in the art used for the preparation of GLP-1 RA series peptides or microspheres containing pharmaceutically acceptable salts thereof. For example, the solvent extraction method may include, but is not limited to, a method of effectively extracting an organic solvent contained in a droplet using a large amount of solubilizing solvent.

[0125] Additionally, specifically, the solvent evaporation method and the solvent extraction method may be applied in parallel, and may include, but are not limited to, methods described in, for example, U.S. Patents No. 4,389,840, 4,530,840, 6,368,632, 6,544,559 and 6,572,894.

[0126] In one embodiment, the method for manufacturing microspheres of the present invention may include the step of preparing an O / W (oil-in-water) or S / O / W (solid-in-oil-in-water) type emulsion comprising a biocompatible polymer, a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a solvent; and the step of aggregating the emulsion into polymer microspheres by an O / W or S / O / W (solid-in-oil-in-water) type solvent evaporation method.

[0127] Specifically, the method for manufacturing the microspheres may include the step of preparing an O / W type emulsion comprising a biocompatible polymer, a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a solvent; and the step of forming microspheres containing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof by aggregating the emulsion into polymer microspheres.

[0128] Additionally, specifically, the O / W type emulsion may comprise a biocompatible polymer, a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a solvent, and conventional methods known in the art may be used to manufacture the O / W type emulsion.

[0129] Additionally, specifically, the step of preparing the O / W type emulsion can be carried out by adding a dispersed phase comprising a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer to a solvent.

[0130] Additionally, specifically, the step of aggregating the O / W type emulsion into polymer microparticles may be carried out by solvent extraction and / or solvent evaporation; an ammonolysis reaction in which ammonia is added to the emulsion; or a hydrolysis reaction in which an acid or base is added.

[0131] Additionally, specifically, when using the amonolithosis reaction or hydrolysis reaction described above, the step of adding an organic solvent that converts a water-insoluble organic solvent into a water-soluble solvent during the reaction process when preparing the emulsion may be further included.

[0132] More specifically, the aggregation by the amonolithic reaction described above may be performed by the method described in Patent No. 918092, but is not limited thereto. Even more specifically, the aggregation by the amonolithic reaction described above may be performed by a reaction in which ammonia is added to an O / W type emulsion containing a water-insoluble organic solvent to induce an amonolithic reaction, thereby converting the water-insoluble organic solvent into a water-soluble solvent and consequently causing the fine particles to aggregate.

[0133] Furthermore, more specifically, the aggregation by the hydrolysis reaction described above may be carried out by the method described in Korean Patent Applications No. 2009-109809 and No. 2010-70407, but is not limited thereto.

[0134] More specifically, the aggregation by the hydrolysis reaction described above can be carried out by adding a basic solution such as NaOH, LiOH, or KOH, or an acidic solution such as HCl or H2SO4, to an O / W type emulsion containing a water-insoluble organic solvent to induce a hydrolysis reaction, which is a type of ester hydrolysis reaction, thereby converting the water-insoluble organic solvent into a water-soluble solvent and consequently causing the fine particles to aggregate.

[0135] In one embodiment, the method for manufacturing microspheres of the present invention may include the step of manufacturing an emulsion by a double emulsion evaporation method and the step of aggregating the emulsion into polymer microparticles.

[0136] Specifically, the step of preparing an emulsion by the above-described double emulsion evaporation method may be performed by forming a W1 / O (water-in-oil) type emulsion comprising an inner phase (W1) in which a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof is dispersed or dissolved in an aqueous solvent, and an oil phase (O) in which a biocompatible polymer is dissolved in a non-aqueous solvent, then dispersing the emulsion in an outer phase (W2) to prepare a W1 / O / W2 (water-in-oil-in-water) type emulsion, and then aggregating the W1 / O / W2 type emulsion into polymer microparticles.

[0137] More specifically, the W1 / O / W2 type emulsion may be prepared to include a biocompatible polymer, a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a dispersion solvent.

[0138] More specifically, conventional methods known in the art may be used to prepare the above W1 / O / W2 type emulsion, for example, by adding a dispersed phase comprising a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer to a dispersion solvent, but is not limited thereto.

[0139] In one embodiment, as a method for manufacturing microspheres of the present invention, a method using solvent evaporation and solvent extraction may include a method comprising the following steps:

[0140] (a) a step of preparing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof;

[0141] (b) a step of preparing a dispersed phase by dissolving the GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof prepared in step (a) and a biocompatible polymer in a dispersion solvent;

[0142] (c) a step of introducing the dispersed phase prepared in step (b) above into a continuous phase and stirring to form microspheres;

[0143] (d) a step of removing the dispersion solvent from the microspheres of step (c);

[0144] (e) a step of recovering the microspheres formed in step (d) above; and

[0145] (f) A step of drying the microspheres recovered in step (e) above.

[0146] In the above manufacturing method, the GLP-1 RA series peptide or its pharmaceutically acceptable salt and biocompatible polymer used have been described above, so a description is omitted in this section.

[0147] In one embodiment, step (a) may include reacting a GLP-1 RA series peptide with an acid or basic compound to form a salt, and separating and purifying the formed salt.

[0148] Specifically, the formed salt may be selected from benzathine salts, formate salts, acetate salts, pamoate salts, and combinations thereof, but is not limited thereto.

[0149] Additionally, specifically, the above-mentioned GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof can be prepared by a conventional technique of obtaining semaglutide benzathine salt by adding benzathine diacetate to semaglutide sodium salt. More specifically, semaglutide sodium salt is dissolved in a solvent, and then benzathine diacetate is dissolved in a solvent in another vial. Subsequently, the semaglutide sodium salt solution is added to the benzathine diacetate solution to react, and then the semaglutide benzathine salt is crystallized to produce the salt.

[0150] More specifically, the semaglutide benzathine salt can be prepared according to the following reaction scheme 1:

[0151] [Reaction Equation 1]

[0152]

[0153] In addition, specifically, it can be prepared by a conventional technique of obtaining tyrzepatide pamoate salt by adding pamoate to tyrzepatide sodium salt. More specifically, it can be prepared by dissolving tyrzepatide sodium salt in a solvent, adding pamoate to react, and then crystallizing tyrzepatide pamoate salt.

[0154] More specifically, the tyrzepatide pamoate salt can be prepared according to the following reaction scheme 2:

[0155] [Reaction Equation 2]

[0156]

[0157] In one embodiment, step (b) can be performed by first dissolving a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof in a solvent, and then dissolving a biocompatible polymer in a dispersion solvent.

[0158] In one embodiment, the dispersion solvent for dissolving the GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and the biocompatible polymer of step (b) may be a solvent that is miscible with water, a solvent that is not miscible with water, or a mixed solvent of a solvent that is miscible with water and a solvent that is not miscible with water.

[0159] In one embodiment, the dispersion solvent may be an organic solvent.

[0160] Specifically, the solvent that is miscible with water may be one or more selected from the group consisting of acetone, acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), formic acid, acetic acid, methanol, ethanol, propanol, and benzyl alcohol.

[0161] Additionally, specifically, the solvent that is not miscible with water may be one or more selected from the group consisting of dichloromethane, chloroform, ethyl acetate, and methyl ethyl ketone.

[0162] In one embodiment, the dispersion solvent may be one or more mixed solvents selected from the group consisting of a solvent that is miscible with water and a solvent that is not miscible with water.

[0163] In one embodiment, an organic solvent that is immiscible with water may be used in an amount of 50% (v / v) or more of the total solvent. When the property of the organic solvent being immiscible with water is utilized in this way, the dispersed phase can be homogeneously mixed in the continuous phase to efficiently form an emulsion.

[0164] In one embodiment, the organic solvent may be mixed with an organic acid solvent.

[0165] In one embodiment, the stirring of step (c) may be performed using a homogenizer such as a high-speed stirrer, an inline mixer, a membrane emulsion method, a microfluidic emulsion method, an ultrasonic mixer, or a static mixer.

[0166] In one embodiment, the continuous phase of step (c) may include a surfactant. The type of surfactant is not particularly limited, and any GLP-1 RA series peptide or a pharmaceutically acceptable salt and polymer solution thereof that can help form a dispersed phase of stable droplets within the continuous phase may be used.

[0167] Specifically, the surfactant may be a hydrophilic polymer selected from the group consisting of methylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, lecithin, gelatin, polyvinyl alcohol, polyoxyethylene-polyoxypropylene block copolymer, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene castor oil derivative, and mixtures thereof, and preferably may be polyvinyl alcohol.

[0168] Additionally, specifically, the surfactant may be added to the continuous phase at a concentration of 0.1 to 3% (w / v). More specifically, the surfactant may be added to the continuous phase at a concentration of 0.3 to 2% (w / v).

[0169] In the above, if the concentration of the surfactant is less than 0.1% (w / v), a dispersed phase or emulsion in the form of droplets may not be sufficiently formed in the continuous phase, and conversely, if it exceeds 3% (w / v), it may be difficult to remove the surfactant after forming microspheres.

[0170] In one embodiment, water may be used as the continuous phase in step (c), and the pH of the aqueous solution may be in the range of 4.0 to 10, but is not limited thereto. Specifically, NaCl may be used to control the initial release in the aqueous solution, acting as an osmotic regulator.

[0171] In one embodiment, if the emulsion of step (c) is maintained at a temperature below the boiling point of the organic solvent for a certain period of time or stirred, the organic solvent can be extracted from the dispersed phase into the continuous phase.

[0172] Specifically, the emulsion may be maintained or stirred for 2 to 48 hours.

[0173] In one embodiment, a portion of the organic solvent extracted in the continuous phase of step (c) may evaporate from the surface, and in this process, the dispersed phase in the form of droplets may solidify to form microspheres.

[0174] In one embodiment, the continuous phase may be heated for a certain period of time to remove the dispersion solvent of step (d). The heating temperature is not particularly limited and can be appropriately set by a person skilled in the art depending on the type of organic solvent used.

[0175] In one embodiment, between step (d) and step (e), the steps may further include a filtration and washing step for removing residual surfactant and a step of recovering microspheres by filtering again. Specifically, the washing step may be performed using water, but is not limited thereto, and may be repeated several times as needed.

[0176] In one embodiment, the method of recovering the microspheres of step (e) may be performed using conventional techniques known in the art. Specifically, the recovery of the microspheres of step (e) may be performed using methods such as filtration or centrifugation, but is not limited thereto.

[0177] In one embodiment, the microspheres obtained after step (e) are dried in step (f) to finally obtain dried microspheres. Specifically, the drying of the microspheres in step (f) can be performed using a conventional drying method.

[0178] In one embodiment, the method for manufacturing microspheres may further include an extraction step after step (e) or after step (f).

[0179] In one embodiment, the extraction step may be performed using an aqueous solution containing purified water, a buffer solution, alcohols, ketones, esters, or inorganic salts, or a mixture thereof, as an extraction solvent.

[0180] Specifically, the extraction solvent may be a solvent composition mixed with at least one of ethanol, methanol, acetone, methyl ethyl ketone, and ethyl acetate, and an aqueous solution containing disodium hydrogen phosphate.

[0181] In one embodiment, as a method for manufacturing microspheres of the present invention, a method using a double-emulsion evaporation method may include the following steps:

[0182] (1) A step of preparing an inner aqueous phase (W1) by dissolving or dispersing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof in a dispersion solvent;

[0183] (2) A step of preparing an oil phase (O) by dissolving a biocompatible polymer in one or more solvents;

[0184] (3) A step of preparing an oil-in-water (W1 / O) type dispersed phase by dispersing an internal water phase (W1) in the above oil phase (O);

[0185] (4) A step of manufacturing microspheres by introducing the above water-in-oil (W1 / O) type dispersed phase into an external continuous phase (W2) and dispersing it to form a water-in-oil (W1 / O / W2) type emulsion; and

[0186] (5) Step of removing the above solvent.

[0187] In the above manufacturing method, since all conditions corresponding to the manufacturing method described above regarding the microspheres and the method of manufacturing the microspheres, such as the type of biocompatible polymer and intrinsic viscosity, can be applied, redundant details are omitted below.

[0188] In one embodiment, the inner aqueous phase (W1) of step (1) may be prepared by dissolving a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof in distilled water.

[0189] In one embodiment, the type of dispersion solvent in step (2) is not particularly limited. Specifically, the dispersion solvent in step (2) may be dimethyl sulfoxide, dichloromethane, or a mixture thereof, and preferably may be dichloromethane.

[0190] In one embodiment, the water-in-oil (W1 / O) type dispersion phase of step (3) can be prepared by dispersing the internal water phase (W1) prepared in step (1) into the oil phase (O) prepared in step (2). Specifically, the weight ratio of the oil phase (O) to the internal water phase (W1) in step (3) may be 1:10 or less.

[0191] In one embodiment, the water-in-oil-in-water (W1 / O / W2) type emulsion of step (4) can be prepared by dispersing the water-in-oil (W1 / O) type dispersion phase prepared in step (3) into the external continuous phase (W2).

[0192] In one embodiment, the outer continuous phase (W2) of step (4) may include a hydrophilic polymer as a surfactant. The type of surfactant is not particularly limited, and any of the following may be used as long as it can assist in forming a dispersed phase in the form of stable droplets within the outer continuous phase, such as a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer.

[0193] In one embodiment, the outer continuous phase (W2) of step (4) may be an aqueous solution of a hydrophilic polymer of 0.1 to 6% (w / v), preferably 0.1 to 4% (w / v).

[0194] In one embodiment, the water-in-oil (W1 / O / W2) type dispersion phase of step (4) can be prepared by adding the water-in-oil (W1 / O) type dispersion phase, which includes a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer prepared in step (3), to an external continuous phase containing the hydrophilic polymer by a drop-by-drop method or by using an in-line mixer, and then vigorously stirring.

[0195] In one embodiment, the step (5) may further include a step of removing the solvent and obtaining the desired microspheres through a conventional filtration and washing step. Specifically, the step (5) may further include a step of washing the obtained microspheres with an organic solvent, such as ethanol, as needed to improve the initial release inhibition effect.

[0196] In one embodiment, microspheres containing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof prepared by the above manufacturing method may be prepared by aggregating an emulsion into microspheres by solvent extraction and / or solvent evaporation, or by aggregation by an amonolithosis reaction with the addition of ammonia or a hydrolysis reaction with the addition of an acid or a base.

[0197] In one embodiment, a water-insoluble organic solvent that is converted into a water-soluble solvent by an amonolithosis reaction or a hydrolysis reaction during emulsion preparation may be further included.

[0198] The above solvent evaporation method, solvent extraction method, aggregation by amonolithosis reaction, and aggregation by hydrolysis reaction can be performed in the same manner as described above.

[0199] Pharmaceutical composition containing microspheres

[0200] The pharmaceutical composition of the present invention is a pharmaceutical composition for the prevention or treatment of diabetes, obesity, hypertension, hyperlipidemia, non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated fatty liver disease (MAFLD), cardiovascular disease, or neurodegenerative disease, and comprises the microspheres of the present invention and / or a pharmaceutically acceptable carrier.

[0201] Specifically, the use of the above pharmaceutical composition may be for the prevention or treatment of diabetes, obesity, hypertension, hyperlipidemia, non-alcoholic fatty liver disease, metabolic fatty liver disease, cardiovascular disease, or degenerative neurological disease. More specifically, the diabetes may be type 2 diabetes.

[0202] Additionally, specifically, the pharmaceutical composition may comprise the microspheres according to the present invention alone, or may comprise the microspheres according to the present invention together with a pharmaceutically acceptable carrier.

[0203] The details regarding the GLP-1 RA series peptide or its pharmaceutically acceptable salt included in the above pharmaceutical composition are the same as those previously described, so a redundant explanation thereof is omitted.

[0204] In one embodiment, the pharmaceutical composition of the present invention may contain microspheres in an amount of 0.01 mg to 2,000 mg per 1 mL of carrier.

[0205] Specifically, the pharmaceutical composition may contain microspheres in an amount of 0.01 mg to 2,000 mg, 0.01 mg to 1,000 mg, 0.01 mg to 900 mg, 0.01 mg to 800 mg, 0.01 mg to 700 mg, 0.01 mg to 600 mg, 0.01 mg to 500 mg, 0.01 mg to 400 mg, 0.01 mg to 300 mg, 0.01 mg to 200 mg, and 0.01 mg to 100 mg per 1 mL of carrier.

[0206] In one embodiment, pharmaceutically acceptable carriers may include all types of solvents, dispersion media, oil-in-water (O / W) or water-in-oil (W / O) emulsions, aqueous compositions, liposomes, microbeads, and microsomes.

[0207] Specifically, the above pharmaceutically acceptable carrier may include one or more selected from the group consisting of water-soluble liquids, fluid oils containing fatty acid esters, surfactants, and polyalcohols.

[0208] More specifically, the fluid oil containing the fatty acid ester may include one or more selected from the group consisting of coconut oil, palm oil, palm kernel oil, sesame oil, soybean oil, almond oil, rapeseed oil, corn oil, sunflower oil, peanut oil, olive oil, castor oil, safflower oil, cottonseed oil, and ethyl oleate.

[0209] More specifically, the surfactant may include one or more selected from the group consisting of Poloxamer 188, Poloxamer 407, Polysorbate 20, Polysorbate 60, Polysorbate 80, Polyoxyethylene oleyl ether, Polyoxyethylene cetyl ether, Polyoxyethylene stearyl ether, and Polyoxyethylene lauryl ether.

[0210] More specifically, the polyalcohol may include one or more selected from the group consisting of ethylene glycol, propylene glycol, polyethylene glycol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, and maltitol.

[0211] In one embodiment, the pharmaceutically acceptable carrier may further include a diluent.

[0212] In one embodiment, the pharmaceutically acceptable carrier may further include a carrier for parenteral administration.

[0213] Specifically, the parenteral administration carrier may include water, suitable oil, saline solution, aqueous glucose, or glycol, and may further include a stabilizer or a preservative.

[0214] More specifically, the stabilizer may be an antioxidant such as sodium bisulfite, sodium sulfite, or ascorbic acid, but is not limited thereto.

[0215] Also, more specifically, the preservative may be benzalkonium chloride, methyl- or propyl-paraben, or chlorobutanol, but is not limited thereto.

[0216] In one embodiment, the pharmaceutical composition of the present invention may further include a tissue diffusion promoter.

[0217] Specifically, the tissue diffusion promoter may be one or more from the group consisting of hyaluronidase, chondroitinase, and heparinase, but preferably is hyaluronidase.

[0218] In one embodiment, the tissue diffusion promoter may be included in a pharmaceutically acceptable carrier or may be included in the pharmaceutical composition of the present invention separately from the pharmaceutically acceptable carrier.

[0219] Specifically, when the tissue diffusion promoter is hyaluronidase, the hyaluronidase may be used by being included in a pharmaceutically acceptable carrier, or may be used substantially alone as a carrier without being included in a pharmaceutically acceptable carrier. In this case, the hyaluronidase is provided in the form of a dissolved aqueous solution for injection and may be mixed with a formulation containing a GLP-1 receptor agonist immediately before administration to form a composition suitable for subcutaneous administration.

[0220] In one embodiment, hyaluronidase may be provided in powder form, but is not limited thereto.

[0221] In one embodiment, the pharmaceutical composition of the present invention may further comprise a GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof.

[0222] Specifically, the GLP-1 RA series peptide or pharmaceutically acceptable salt thereof further included above may be in a liquid formulation or a powder formulation, but is not limited thereto, and may be in various formulations.

[0223] In one embodiment, the pharmaceutical composition of the present invention may further comprise water and a pharmaceutically acceptable excipient. In this case, water may be included in an amount of 60% by weight or more, 70% by weight or more, or 80% by weight or more.

[0224] Specifically, the excipient may include, but is not limited to, buffers, isotonic agents, preservatives, pH adjusters, or thickeners, and excipients known in the art may be used without limitation.

[0225] Additionally, specifically, the buffer may be a phosphate buffer, TRIS, or citrate, but is not limited thereto. More specifically, the phosphate buffer may be sodium dihydrogen phosphate, disodium hydrogen phosphate, or sodium phosphate. Additionally, specifically, the buffer may be included at a concentration of 0.01 to 50 mM, but is not limited thereto.

[0226] Additionally, specifically, the isotonic agent may be propylene glycol, sodium chloride, mannitol, sucrose, sorbitol, trehalose, or lactose, but is not limited thereto, and any isotonic agent known in the art may be used without limitation. Additionally, specifically, the isotonic agent may be included in the pharmaceutical composition at a concentration of 8 mg / mL to 50 mg / mL, preferably 8 mg / mL to 30 mg / mL.

[0227] In addition, specifically, the pH adjuster may be HCl, NaOH, or acetate, but is not limited thereto, and any pH adjuster known in the art may be used without limitation. In addition, specifically, the pH of the pharmaceutical composition of the present invention may be adjusted to a range of 3 to 10, 6 to 9, 6.5 to 8.5, or 7 to 7.8 by the pH adjuster, but is not limited thereto.

[0228] Additionally, specifically, the thickening agent may be a cellulose derivative, but is not limited thereto, and any thickening agent known in the art may be used without limitation.

[0229] In addition, specifically, the preservative may be phenol, but is not limited thereto, and any preservative known in the art may be used without limitation.

[0230] In one embodiment, the pharmaceutically acceptable carrier, excipient, and diluent may be substantially inactive so as not to interact significantly with the microspheres and non-toxic so as not to have an adverse effect on the patient.

[0231] Specifically, the pharmaceutically acceptable carrier, excipient, and diluent may not dissolve the biocompatible polymer forming the microspheres. More specifically, the pharmaceutically acceptable carrier, excipient, and diluent may not dissolve the drug contained within the microspheres. Even more specifically, the pharmaceutically acceptable carrier, excipient, and diluent may improve bioavailability compared to an injectable formulation that does not contain them.

[0232] In one embodiment, the pharmaceutical composition may further include, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, etc.

[0233] For other pharmaceutically acceptable carriers and formulations, refer to the contents of Remington's Pharmaceutical Sciences (19th ed., Mack Publishing Company, Easton, PA, 1995), a known publication in the pharmaceutical field.

[0234] In one embodiment, the pharmaceutical composition may be formulated into various forms of preparations, for example, as a parenteral administration preparation.

[0235] Specifically, the parenteral formulation may be formulated in the form of an injection, cream, lotion, topical ointment, oil, moisturizer, aerosol, patch, or nasal inhaler according to methods known in the art. Such formulation methods are as described in the literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995), which is a prescription generally known in the field of pharmaceutical chemistry.

[0236] In one embodiment, the pharmaceutical composition may be administered to mammals, including humans, in various ways, for example, parenterally.

[0237] Specifically, the above parenteral administration methods may include, but are not limited to, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal administration. Preferably, the above pharmaceutical composition may be administered by subcutaneous injection or intramuscular injection.

[0238] Examples

[0239] In this embodiment, the following abbreviations are defined to represent the following terms.

[0240] IV: Intrinsic Viscosity

[0241] HPLC: High Performance Liquid Chromatography

[0242] LC-MS / MS: Liquid chromatography-tandem mass spectrometry

[0243] NMR: Nuclear Magnetic Resonance Spectroscopy

[0244] ACN: Acetonitrile

[0245] DCM: Dichloromethane

[0246] NaOH: Sodium hydroxide

[0247] PBS: Phosphate buffered saline

[0248] PLGA: Poly(lactic-co-glycolic acid)

[0249] PVA: Polyvinyl alcohol

[0250] TFA: Trifluoroacetic acid

[0251] The present invention will be explained in more detail below through the following examples and experimental examples.

[0252] Examples 1-18: Preparation of microspheres containing semaglutide benzathine salt

[0253] a. Preparation of Semaglutide Benzatin Salt

[0254] Semaglutide (Manufacturer: JYMed) ® ) 200 mg and 1 ml of distilled water were added to a vial and stirred to dissolve. In another vial, benzathine diacetate (Manufacturer: TCI ® 200 mg of ) and 1 ml of distilled water were added and stirred to dissolve. Once the solution was completely dissolved, the semaglutide solution was slowly added dropwise to the benzathine diacetate solution using a syringe to obtain semaglutide benzathine salt. The obtained semaglutide benzathine salt was washed with water and dried at 25°C for at least 20 hours.

[0255] b. Preparation of microspheres containing semaglutide benzathine salt

[0256] Preparation of the dispersed phase

[0257] Weighed semaglutide benzathine salt prepared in 'a. Preparation of semaglutide benzathine salt' and acetic acid were added to a 50ml vial and dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at a speed of 300 rpm for 20 minutes to prepare a dispersed phase.

[0258] The usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the usage amounts of semaglutide benzathine salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 1.

[0259] Continuous phase manufacturing

[0260] 1,000 ml of distilled water, 10 g of PVA, and 5 g of sodium chloride were weighed and added to a reactor. Then, a 1% (w / v) aqueous PVA solution was prepared by stirring at a speed of 1,000 rpm for 60 minutes using an overhead stirrer.

[0261] Preparation and Filtration of Microspheres

[0262] The prepared dispersed phase was placed in a syringe. The continuous phase was injected into a homogenizer, and the dispersed phase contained in the syringe was injected into the homogenizer within 60 seconds. Once the injection of the dispersed phase was complete, the solvent was removed while stirring in the homogenizer, and the mixture was solidified for 24 hours. Subsequently, the mixture was filtered through a 5 μm membrane filter to obtain semaglutide benzathine salt microspheres.

[0263] Washing and Drying of Microspheres

[0264] The microspheres were washed repeatedly with distilled water several times to remove residual PVA. The obtained microspheres were freeze-dried for more than 20 hours.

[0265] No. Acetic acid: DCM solvent Bisemaglutide benzathine salt (g) PGLA (g) PLGA IV (dl / g) Example 11: 1.50.1564 1.40.32 -0.44 Example 21: 1.50.177 1.238 0.32 -0.44 Example 31: 1.50.247 1.40.32 -0.44 Example 41: 1.50.35 1.40.32 -0.44 Example 51: 1.50.1160.660.8 or more Example 61: 1.90.1160.660.32 -0.44 Example 71: 1.90.1160.660.8 or more Example 81: 1.90.207 1.1730.8 Example 91:1.90.2071.1730.8 Example 101: 20.2331.320.45-0.6 Example 111: 20.1281.1550.4-0.6 Example 121: 20.1281.1550.60-0.80 Example 131: 20.1281.1550.4-0.6 Example 141: 2.50.5631.3130.32-0.44 Example 151: 2.50.2130.9650.32-0.44 Example 161: 50.2471.40.32-0.44 Example 171: 90.1160.660.32-0.44 Example 181: 90.1450.660.32-0.44

[0266] Examples 19-49: Preparation of microspheres containing semaglutide benzathine salt and a bioavailability improver

[0267] The steps of preparing semaglutide benzathine salt, preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Examples 1-18, and only the <preparation of dispersed phase> step was carried out as follows.

[0268] <Preparation of Dispersed Phase>

[0269] In a 50ml vial, the semaglutide benzathine salt prepared in 'a. Preparation of semaglutide benzathine salt' of Example 1-18, a bioavailability improver, and acetic acid were weighed and added, and then dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial, and the mixture was stirred at a speed of 300 rpm for 20 minutes to prepare a dispersed phase.

[0270] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 2, and the usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the amounts of semaglutide benzathine salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 3.

[0271] No. Bioavailability Improver Bioavailability Improver (mg) Example 19 Lysine 3.08 Example 20 Lysine 3.3 Example 21 Lysine 6.6 Example 22 Lysine 3.1 Example 23 Lysine 3.1 Example 24 Lysine 3 Example 25 Lysine 2.6 Example 26 Lysine 1.56 Example 27 Lysine 6.6 Example 28 Lysine 9.9 Example 29 Lysine / Sodium Acetate 3.3 / 1.76 Example 30 Sodium Acetate 1.76 Example 31 Sodium Acetate 1.54 Example 32 Sodium Acetate 0.8 Example 33 Sodium Acetate 2.4 Example 34 Acetic Acid Sodium 0.78 Example 35 Sodium acetate 3.5 Example 36 Sodium acetate 1.8 Example 37 Citric acid 1.54 Example 38 Citric acid 8.3 Example 39 Lysine 2.9 Example 40 Lysine 3.3 Example 41 Lysine 6.2 Example 42 Lysine 9.9 Example 43 Sodium acetate 1.7 Example 44 Sodium acetate 1.7 Example 45 Sodium acetate 1.7 Example 46 Sodium acetate 1.3 Example 47 Sodium acetate 1.7 Example 48 Sodium acetate 1.6 Example 49 Sodium acetate 1.6

[0272] (Note) Example 29 used two types of bioavailability improvers.

[0273] No. Acetic acid: DCM solvent Bisemaglutide benzathine salt (g) PLGA IV (dl / g) PGLA (g) Example 191: 1.5 0.15 6 40.32 - 0.4 41.4 Example 201: 1.5 0.24 70.32 - 0.4 41.4 Example 211: 1.5 0.24 70.32 - 0.4 41.4 Example 221: 2.0 14 70.45 - 0.6 1.32 Example 231: 2.0 23 30.45 - 0.6 1.32 Example 241: 2.5 0.14 60.32 - 0.4 41.313 Example 251: 2.5 0. 230.45-0.61.05 Example 261: 1.30.1160.32-0.440.66 Example 271: 50.2470.32-0.441.4 Example 281: 50.2470.32-0.441.4 Example 291: 1.50.2470.32-0.441.4 Example 301: 1.50.2470.32-0.441.4 Example 311: 1.50.15640.32-0.441.4 Example 321:1.50.1160.8 or more 0.66 Example 331:1.50.3460.16 -0.241.96 Example 341:1.30.1160.32 -0.440.66 Example 351:50.2470.32 -0.441.4 Example 361:50.2470.32 -0.441.4 Example 371:1.50.15640.32 -0.441.4 Example 381:50.2470.32 -0.441.4 Example 391:1.50.2170.16 -0.24 + 0.45 -0.60.615 + 0.615 Example 401: 20.16 50.32 -0.44 + 0.45 -0.6 0.743 + 0.743 Example 411: 20.23 30.32 -0.44 + 0.45 -0.6 0.264 + 1.056 Example 421: 50.24 70.32 -0.44 + 0.45 -0.6 1.12 + 0.28 Example 431: 1.5 0.24 70.16 -0.24 + 0.32 -0.4 40.93 + 0.47 Example 441: 1.5 0.24 70.16 -0.24 + 0.32 -0.4 40.84 + 0.56 Example 451: 1.5 0.24 70.16 -0.24 + 0.32 -0.44 0.7 + 0.7 Example 461: 1.5 0.24 70.16 -0.24 + 0.45 -0.6 0.224 + 0.896 Example 471: 1.5 0.24 70.16 -0.24 + 0.32 -0.44 0.7.23 + 1.17 Example 481: 2.5 0.2 47 0.32 -0.44 + 0.60 -0.8 0.8 98 + 0.449 Example 491: 2.5 0.2 17 0.60 -0.8 1.347.

[0274] (Note) Examples 39 to 48 used two types of PLGA.

[0275] Examples 50-55: Preparation of microspheres containing semaglutide acetate salt

[0276] a. Preparation of Semaglutide Acetate Salt

[0277] Semaglutide (Manufacturer: JYMed) ® 100 mg of ) and 1 mL of acetic acid were placed in a vial and dissolved by stirring. Once the solution was completely dissolved, the vial was left in a fume hood with the lid open for 30 minutes to 1 hour to vaporize some or all of the remaining acetic acid. Subsequently, the vial was freeze-dried and left at 25°C to obtain semaglutide acetate salt. The obtained semaglutide acetate salt was washed with water and dried at 25°C for at least 20 hours.

[0278] b. Preparation of microspheres containing semaglutide acetate salt

[0279] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Examples 1-18, and only the <preparation of the dispersed phase> step was carried out as follows.

[0280] <Preparation of Dispersed Phase>

[0281] Weighed semaglutide acetate salt and acetic acid prepared in 'a. Preparation of semaglutide acetate salt' described above were added to a 50ml vial and dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at a speed of 300 rpm for 20 minutes to prepare a dispersed phase.

[0282] The usage ratio of each solvent in the dispersion solvent used to prepare the dispersed phase, the usage amounts of semaglutide acetate salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 4.

[0283] No. Acetic acid:DCM solvent Bisemaglutide acetate salt (g) PGLA (g) PLGA IV (dl / g) Example 501: 1.20.26 1.176 0.32 -0.44 Example 511: 2.50.146 1.072 0.32 -0.44 Example 521: 1.40.208 1.176 0.32 -0.44 Example 531: 1.70.118 0.67 0.32 -0.44 Example 541: 1.70.116 0.66 0.8 or more Example 551: 1.90.207 1.173 0.8 or more

[0284] Examples 56-66: Preparation of microspheres containing semaglutide acetate salt and bioavailability improver

[0285] The steps of preparing semaglutide acetate salt, preparing the continuous phase, preparing microspheres, filtration, and drying were carried out in the same way as in Examples 50-55, and only the <preparation of dispersed phase> step was carried out as follows.

[0286] <Preparation of Dispersed Phase>

[0287] In a 50ml vial, the semaglutide acetate salt prepared in 'a. Preparation of semaglutide acetate salt' of Examples 50-55, a bioavailability improver, and acetic acid were weighed and added, and then dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial, and the mixture was stirred at a speed of 300 rpm for 20 minutes to prepare a dispersed phase.

[0288] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 5, and the usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the amounts of semaglutide acetate salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 6.

[0289] No. Bioavailability Improver Bioavailability Improver (mg) Example 56 Lysine 3.74 Example 57 Sodium acetate 1.98 Example 58 Citric acid 2.77 Example 59 Citric acid 7.8 Example 60 Citric acid 3.9 Example 61 Citric acid 7.92 Example 62 Citric acid 8.36 Example 63 Citric acid 8.36 Example 64 Citric acid 4.2 Example 65 Citric acid 4.2 Example 66 Citric acid 3.7

[0290] No. Acetic acid:DCM solvent bisema glutide acetate salt (g) PGLA (g) PLGA IV (dl / g) Example 561: 1.5 0.18 70.32 - 0.44 1.68 Example 571: 1.5 0.18 70.32 - 0.44 1.68 Example 581: 1.1 0.2 0 80.32 - 0.44 1.176 Example 591: 1.2 0.2 360.32 - 0.44 1.34 Example 601: Example 611: 1.50.31280.32-0.442.8 Example 621: 1.50.2470.32-0.441.4 Example 631: 1.70.1180.32-0.440.67 Example 641: 1.50.2470.16-0.24 + 0.32-0.440.23 + 1.17 Example 651: 1.50.2470.16-0.24 + 0.32-0.440.14 + 1.26 Example 661: 1.50.2170.16-0.24 + 0.45-0.60.615 + 0.615

[0291] (Note) Examples 64 to 66 used two types of PLGA.

[0292] Example 67: Preparation of microspheres containing semaglutide formate

[0293] a. Preparation of Semaglutide Formate

[0294] Semaglutide (Manufacturer: JYMed) ® 500 mg of ) and 5 ml of distilled water were placed in a vial and stirred to dissolve. Subsequently, formic acid was added dropwise to the semaglutide solution to precipitate semaglutide formate. The semaglutide formate was obtained and washed with water. After washing, it was dried at 25°C for at least 20 hours.

[0295] b. Preparation of microspheres containing semaglutide formate

[0296] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Examples 1-18, and only the <preparation of the dispersed phase> step was carried out as follows.

[0297] <Preparation of Dispersed Phase>

[0298] Weighed semaglutide formate and formic acid prepared in 'a. Preparation of semaglutide formate' described above were added to a 50ml vial and dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at a speed of 300 rpm for 20 minutes to prepare a dispersed phase.

[0299] The usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the usage amounts of semaglutide formate and PLGA, and the intrinsic viscosity of PLGA are as described in Table 7.

[0300] No. Formic acid:DCM solvent Bisemaglutide formate (g) PGLA (g) PLGA IV (dl / g) Example 6 7 1.5 5 0.1 4 6 1.3 1 3 0.3 2 - 0.44

[0301] Example 68: Preparation of Semaglutide Pamoate Salt

[0302] Semaglutide (Manufacturer: JYMed) ® 100 mg of ) and 1 mL of water were placed in a vial and stirred to dissolve. Once the solution was completely dissolved, pamosan was added to the vial and stirred to obtain the precipitate, semaglutide pamoate salt. The obtained semaglutide pamoate salt was washed with water and dried at 25°C for at least 20 hours.

[0303] Examples 69-71: Preparation of microspheres containing tyrzepatide benzathine salt

[0304] a. Preparation of tyrzepatide benzathine salt

[0305] 400 mg of tyrzepatide (Manufacturer: Zhejiang Peptide Biotech Co. LTd) and 1 ml of distilled water were placed in a vial and dissolved by stirring. In another vial, benzathine diacetate (Manufacturer: TCI ® 200 mg of ) and 1 ml of distilled water were added and stirred to dissolve. Once the solution was completely dissolved, the semaglutide solution was slowly added dropwise to the benzathine diacetate solution using a syringe to obtain semaglutide benzathine salt. The obtained semaglutide benzathine salt was washed with water and dried at 25°C for at least 20 hours.

[0306] b. Preparation of microspheres containing tyrzepatide benzathine salt

[0307] The steps of preparing, filtration, and drying the microspheres were carried out in the same way as in Examples 1-18, and only the <preparation of dispersed phase> step was carried out as follows.

[0308] Continuous phase manufacturing

[0309] 1,000 ml of distilled water, 30 g of PVA, and 5 g of sodium chloride were weighed and added to a reactor. Then, a 3% (w / v) aqueous PVA solution was prepared by stirring at a speed of 1,000 rpm for 60 minutes using an overhead stirrer.

[0310] <Preparation of Dispersed Phase>

[0311] In a 50ml vial, the tyrzepatide benzathine salt prepared in 'a. Preparation of tyrzepatide benzathine salt' described above, a bioavailability improver, and acetic acid were weighed and added, and then dissolved by stirring at a speed of 150 rpm. Subsequently, the biocompatible polymers PLGA and DCM were added to the vial, and the mixture was stirred at a speed of 300 rpm for 20 minutes to prepare a dispersed phase.

[0312] The amount of tyrzepatide benzathine salt and PLGA used in the preparation of the dispersed phase, as well as the intrinsic viscosity of PLGA, are as described in Table 8.

[0313] No. Acetic acid: DCM solvent bityrzepatide benzathine salt (g) PGLA (g) PLGA IV (dl / g) Example 691: 1.5 0.28 91.15 50.32 -0.44 Example 701: 1.5 0.38 51.15 50.32 -0.44 Example 711: 20.21 50.5 0.45 -0.6

[0314] Examples 72-79: Preparation of microspheres containing tyrzepatide benzathine salt and a bioavailability improver

[0315] The steps of preparing the tyrzepatide benzathine salt, preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Examples 69-71, and only the <preparation of the dispersed phase> step was carried out as follows.

[0316] <Preparation of Dispersed Phase>

[0317] In a 50ml vial, the tyrzepatide benzathine salt prepared in 'a. Preparation of tyrzepatide benzathine salt' of Examples 69-71, a bioavailability improver, and acetic acid were weighed and added, and then dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial, and the mixture was stirred at a speed of 300 rpm for 20 minutes to prepare a dispersed phase.

[0318] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 9, and the usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the amounts of tyrzepatide benzathine salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 10.

[0319] No. Bioavailability Improver Bioavailability Improver (mg) Example 72 Sodium acetate 2.9 Example 73 Sodium acetate 5.7 Example 74 Sodium acetate 3.0 Example 75 Sodium acetate 1.8 Example 76 Sodium acetate 2.5 Example 77 Sodium acetate 2.7 Example 78 Sodium acetate 2.2 Example 79 Sodium acetate 2.5

[0320] No. Acetic acid: DCM solvent Vitirzepatide benzathine salt (g) PLGA IV (dl / g) PGLA (g) Example 721: 1.5 0.28 90.32 - 0.44 1.155 Example 731: 1.5 0.28 90.32 - 0.44 1.155 Example 741: 1.5 0.38 50.32 - 0.44 1.155 Example 751: 2.26 0.6 - 0.8 0.6 5 Example 761: 2.5 0.37 70.6 - 0.8 0.88 Example 771: 2.5 0.47 40.6 - 0.8 0.88 Example 781: 1.5 0.38 50.32 - 0.44 + 0.45 - 0.6 0.42 + 0.42 Example 791: 2.5 0.3 7 0.3 2 - 0.44 + 0.45 - 0.6 0.44 + 0.44

[0321] (Note) Examples 78 to 79 used two types of PLGA.

[0322] Examples 80-82: Preparation of microspheres containing tyrzepatide acetate salt

[0323] a. Preparation of tyrzepatide acetate salt

[0324] 500 mg of tyrzepatide (Manufacturer: Zhejiang Peptide Biotech Co. LTd) and 2 mL of acetic acid were placed in a vial and dissolved by stirring. Once the solution was completely dissolved, the vial was left open in a fume hood for 30 minutes to 1 hour to vaporize some or all of the remaining acetic acid. Subsequently, the vial was freeze-dried and left at 25°C to obtain tyrzepatide acetate salt. The obtained tyrzepatide acetate salt was washed with water and dried at 25°C for at least 20 hours.

[0325] b. Preparation of microspheres containing tyrzepatide acetate salt

[0326] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Examples 69-71, and only the <preparation of the dispersed phase> step was carried out as follows.

[0327] <Preparation of Dispersed Phase>

[0328] Weighed and added the tyrzepatide acetate salt prepared in 'a. Preparation of tyrzepatide acetate salt' and methanol to a 50ml vial, and then dissolved them by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at a speed of 300 rpm for 20 minutes to prepare a dispersed phase.

[0329] The usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the usage amounts of tyrzepatide acetate salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 11.

[0330] No. Acetic Acid: DCM Solvent Bityrzepatide Acetate (g) PGLA (g) PLGA IV (dl / g) Example 801: 1.5 0.28 9 1.15 5 0.32 - 0.44 Example 811: 1.5 0.38 5 1.15 5 0.32 - 0.44 No. Methanol: DCM Solvent Bityrzepatide Acetate (g) PGLA (g) PLGA IV (dl / g) Example 821: 1.5 0.11 0.33 + 0.67 0.16 - 0.24 + 0.32 - 0.44

[0331] (Note) Example 82 used two types of PLGA.

[0332] Examples 83-86: Preparation of microspheres comprising tyrzepatide acetate salt and a bioavailability improver

[0333] The steps of preparing the tyrzepatide acetate salt, preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Examples 80-82, and only the <preparation of the dispersed phase> step was carried out as follows.

[0334] <Preparation of Dispersed Phase>

[0335] In a 50ml vial, the tyrzepatide acetate salt prepared in 'a. Preparation of tyrzepatide acetate salt' of Examples 80-82, a bioavailability improver, and methanol were weighed and added, and then dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial, and the mixture was stirred at a speed of 300 rpm for 20 minutes to prepare a dispersed phase.

[0336] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 12, and the usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the amounts of semaglutide acetate salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 13.

[0337] No. Bioavailability Improver Bioavailability Improver (mg) Example 83 Citrate 3.7 Example 84 Citrate 2.6

[0338] No. Methanol: DCM solvent Vitalzepatide Acetate (g) PLGA IV (dl / g) PGLA (g) Example 831: 1.5 0.28 90.32 - 0.44 1.155 Example 841: 1.5 0.3 0.32 - 0.44 0.7

[0339] Example 85: Preparation of microspheres containing tyrzepatide formate

[0340] a. Preparation of tyrzepatide formate

[0341] 500 mg of tircepate (Manufacturer: Zhejiang Peptide Biotech Co. LTd) and 5 ml of distilled water were placed in a vial and dissolved by stirring. Formic acid was dropped into the tircepate solution. The precipitated tircepate formate was obtained and washed with water. After washing, it was dried at 25°C for at least 20 hours.

[0342] b. Preparation of microspheres containing tyrzepatide formate

[0343] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Examples 69-71, and only the <preparation of the dispersed phase> step was carried out as follows.

[0344] <Preparation of Dispersed Phase>

[0345] The tyrzepatide formate and acetic acid prepared in 'a. Preparation of tyrzepatide formate' in a 50ml vial were weighed and added, and then dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at a speed of 300 rpm for 20 minutes to prepare a dispersed phase.

[0346] The usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the usage amounts of tyrzepatide formate and PLGA, and the intrinsic viscosity of PLGA are as described in Table 14.

[0347] No. Acetic acid:DCM solvent bisemaglutide formate (g) PGLA (g) PLGA IV (dl / g) Example 851: 1.5 0.1 46 1.3 13 0.3 2 - 0.44

[0348] Examples 86-88: Preparation of microspheres containing tyrzepatide formate and a bioavailability improver

[0349] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Example 85, and only the <preparation of the dispersed phase> step was carried out as follows.

[0350] <Preparation of Dispersed Phase>

[0351] In a 50ml vial, the tyrzepatide formate prepared in 'a. Preparation of tyrzepatide formate' of Example 85, a bioavailability improver, and acetic acid were weighed and added, and then dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at a speed of 300 rpm to prepare a dispersed phase.

[0352] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 15, and the usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the amounts of tyrzepatide formate and PLGA, and the intrinsic viscosity of PLGA are as described in Table 16.

[0353] No. Bioavailability Improver Bioavailability Improver (mg) Example 86 Citrate 3.7 Example 87 Citrate 3.9 Example 88 Citrate 2.2

[0354] No. Acetic acid:DCM solvent Bityrzepatide formate (g) PGLA (g) PLGA IV (dl / g) Example 881: 1.5 0.28 9 1.15 5 0.32 - 0.44 Example 871: 1.5 0.38 5 1.15 5 0.32 - 0.44 Example 881: 20.26 0.60 5 0.60 - 0.80

[0355] Example 89: Preparation of Tircepate Pamoate Salt

[0356] 400 mg of tyrzepatide (Manufacturer: Zhejiang Peptide Biotech Co. LTd) and 1 mL of DMSO were placed in a vial and stirred to dissolve them. Once the solution was completely dissolved, pamosan was added to the vial and stirred to obtain the precipitate, tyrzepatide pamoate salt. The obtained tyrzepatide pamoate salt was washed with water and dried at 25°C for at least 20 hours.

[0357] Comparative Example 1: Preparation of microspheres containing semaglutide sodium salt

[0358] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Examples 1-18, and only the <preparation of the dispersed phase> step was carried out as follows.

[0359] <Preparation of Dispersed Phase>

[0360] The aforementioned semaglutide sodium salt in a 50ml vial (Manufacturer: JYMed ®Weighed and added ) and formic acid, then stirred at 150 rpm for 20 minutes to dissolve. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at 300 rpm to prepare a dispersed phase.

[0361] The usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the usage amounts of semaglutide sodium salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 17.

[0362] No. Formic acid:DCM solvent bisemaglutide sodium salt (g) PGLA (g) PLGA IV (dl / g) Comparative Example 11: 1.2 0.09 20.8 25 0.32 - 0.44

[0363] Comparative Example 2-6: Preparation of microspheres containing semaglutide sodium salt

[0364] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Examples 1-18, and only the <preparation of the dispersed phase> step was carried out as follows.

[0365] <Preparation of Dispersed Phase>

[0366] Semaglutide sodium salt in a 50ml vial (Manufacturer: JYMed ® Weighed and added ) and acetic acid, then stirred at 150 rpm for 20 minutes to dissolve. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at 300 rpm to prepare a dispersed phase.

[0367] The usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the usage amounts of semaglutide sodium salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 18.

[0368] No. Acetic acid:DCM solvent Bisemaglutide sodium salt (g) PLGA IV (dl / g) PGLA (g) Comparative Example 21: 1.55 0.156 40.32 - 0.44 1.4 Comparative Example 31: 1.9 0.41 40.8 or more 2.346 Comparative Example 41: 20.14 70.45 - 0.61.32 Comparative Example 51: 20.11 60.32 - 0.44 0.66 Comparative Example 61: 20.25 60.4 - 0.62.31

[0369] Comparative Examples 7-12: Preparation of microspheres containing semaglutide sodium salt and a bioavailability improver

[0370] The steps of preparing semaglutide sodium salt, preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Comparative Examples 2-6, and only the <preparation of dispersed phase> step was carried out as follows.

[0371] <Preparation of Dispersed Phase>

[0372] Sodium semaglutide, a bioavailability improver, and acetic acid were weighed and added to a 50 ml vial, and then dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at a speed of 300 rpm to prepare a dispersed phase.

[0373] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 19, and the usage ratios of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the amounts of semaglutide sodium salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 20.

[0374] No. Bioavailability Improver Bioavailability Improver (mg) Comparative Example 7 Lysine 1.54 Comparative Example 8 Sodium acetate 1.54 Comparative Example 9 Citric acid 3.96 Comparative Example 10 Citric acid 4.2 Comparative Example 11 Citric acid 1.5 Comparative Example 12 Citric acid 4.2

[0375] No. Acetic acid: DCM solvent sodium bisema glutide salt (g) PLGA IV (dl / g) PGLA (g) Comparative Example 71: 1.5 0.15 6 40.32 - 0.4 41.4 Comparative Example 81: 1.5 0.15 6 40.32 - 0.4 41.4 Comparative Example 91: 1.5 0.15 6 40.32 - 0.4 41.4 Comparative Example 101: 1.5 0.24 70.32 - 0.4 41.4 Comparative Example 111: 20.14 70.45 - 0.6 1.32 Comparative Example 121: 20.14 70.45 - 0.6 1.49

[0376] Comparative Example 13: Preparation of microspheres containing tyrzepatide sodium salt

[0377] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Examples 69-71, and only the <preparation of the dispersed phase> step was carried out as follows.

[0378] <Preparation of Dispersed Phase>

[0379] Sodium tyrzepatide salt and acetic acid were weighed and added to a 50 ml vial, and then dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at a speed of 300 rpm to prepare a dispersed phase.

[0380] The usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the usage amounts of tyrzepatide sodium salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 21.

[0381] No. Acetic acid:DCM solvent sodium vityrzepatide (g) PGLA (g) PLGA IV (dl / g) Comparative Example 131: 1.5 0.28 9 1.15 5 0.32 - 0.44

[0382] Comparative Example 14: Preparation of microspheres containing tyrzepatide sodium salt

[0383] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were carried out in the same way as in Examples 69-71, and only the <preparation of the dispersed phase> step was carried out as follows.

[0384] <Preparation of Dispersed Phase>

[0385] Sodium tyrzepatide salt and methanol were weighed and added to a 50 ml vial, and then dissolved by stirring at a speed of 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at a speed of 300 rpm to prepare a dispersed phase.

[0386] The usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the usage amounts of tyrzepatide sodium salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 22.

[0387] No. Methanol:DCM Solvent Sodium Bityrzepatide (g) PGLA (g) PLGA IV (dl / g) Comparative Example 141: 1.54 0.11 0.33 + 0.67 0.16 -0.24 + 0.32 -0.44

[0388] (Note) Comparative Example 14 used two types of PLGA.

[0389] Experimental Example 1: Salt Crystal Analysis of Semaglutide and Tircepate

[0390] To measure the nuclear magnetic resonance (NMR) spectra of the semaglutide benzathine salt, semaglutide acetate salt, semaglutide formate salt, tyrzepatide benzathine salt, tyrzepatide acetate salt, tyrzepatide formate salt, and tyrzepatide pamoate salt prepared in the present invention, the following tests were performed.

[0391] 1 HNMR data is from Bruker's Ascend TM Measurements were taken at 500 MHz using a system. The chemical shift (δ) was expressed in ppm (parts per million) for the residual proton signal in the deuterated solvent, and the reference peak was set to 7.27 ppm for CDCl3. Signals in the spectrum were indicated according to commonly used abbreviations, where s represents singlet, d represents doublet, t represents triplet, q represents quadruplet, m represents multiplet, and br represents a broad peak.

[0392] of the above semaglutide benzathine salt 1 H-NMR analysis results of semaglutide acetate salt are shown in Fig. 1. 1 H-NMR analysis results of semaglutide formate are shown in Fig. 2. 1 The results of the H-NMR analysis are shown in Figure 3, respectively. In addition, tyrzepatide benzathine salt, tyrzepatide acetate salt, tyrzepatide formate salt, and tyrzepatide pamoate salt 1 The results of the H-NMR analysis are shown in Figures 4 to 7, respectively.

[0393] From Figures 1 to 7 above, it can be seen that each salt-form compound prepared according to the present invention is a crystal of the corresponding compound.

[0394] Experimental Example 2: In vitro release test and evaluation of initial release rate of microspheres containing semaglutide salt

[0395] 25 mg of microspheres from Examples 1 to 68 and Comparative Examples 1 to 12 were taken and placed in a 25 ml volumetric flask. After sonication with an ACN solution containing 0.1% TFA, the 25 ml volumetric flask was filled to the mark with a distilled water solution containing 0.1% TFA. This solution was filtered through a 0.45 µm syringe filter and detected using an ultraviolet-visible spectrophotometer with an HPLC (equipment name: Agilent 1260). Column packing was performed with L1, an internal diameter of 4.6 mm x 150 mm, and a thickness of 3.5 µm. Through the above experimental results, the loading amount of microspheres containing semaglutide salt was confirmed.

[0396] In addition, 15 mg of microspheres prepared in Examples 1 to 68 and Comparative Examples 1 to 12 were placed in a vial, 3 mL of pH 7.4 PBS solution was added, and the mixture was stirred at 100 rpm while maintaining the temperature at 37°C for a certain period. To measure the release amount after a certain period, the entire supernatant was taken and filtered through a 0.45 μm RC filter to obtain the sample solution. The sample solution was detected using an ultraviolet-visible spectrophotometer with an HPLC (equipment name: Agilent 1260). The column packing was performed with L1, an internal diameter of 4.6 mm x 150 mm, and a thickness of 3.5 μm.

[0397] The above experimental results are shown in Table 23.

[0398] Microsphere Loading Amount (WJ%) Initial Release Rate (%) Example 1 10.7 11.3 Example 2 11.5 12.1 Example 3 14.8 11.0 Example 5 12.5 3.8 Example 6 12.9 14.0 Example 7 15.4 5.3 Example 8 15.2 5.9 Example 9 17.2 1.1 Example 10 14.5 4.2 Example 11 10.7 5.9 Example 12 8.9 2.5 Example 13 10.8 1.2 Example 15 21.8 14.0 Example 16 15.8 7.0 Example 17 15.6 12.5 Example 18 14.8 9.7 Example 19 9.8 3.7 Example Example 2015.214.0 Example 2114.7 11.4 Example 229.4 3.6 Example 2314.8 22.0 Example 248.0 0.0 Example 2518.3 18.5 Example 268.4 33.8 Example 2715.4 22.1 Example 2814.18.3 Example 2914.76.9 Example 3015.3 11.9 Example 319.77.9 Example 3212.5 3.1 Example 349.3 27.0 Example 3514.46.9 Example 3616.1 16.6 Example 3710.28.1 Example 38 16.1 20.9 Example 39 13.7 6.2 Example 40 9.9 13.0 Example 41 14.1 6.2 Example 42 14.7 5.6 Example 46 15.5 15.6 Example 48 12.0 19.9 Example 49 16.2 12.7 Example 50 15.5 8.9 Example 518.6 13.8 Example 52 13.4 12.7 Example 53 13.7 3.6 Example 54 13.4 3.9 Example 55 10.2 6.6 Example 56 10.5 6.5 Example 57 10.3 5.7 Example 58 12.6 5.2 Example 59 14.2 3.3 Example 60 12.9 8.0 Example 61 11.6 11.8 Example 62 17.5 26.1 Example 63 16.5 20.6 Example 67 4.7 1.2 Comparative Example 19.3 1.0 Comparative Example 28.9 3.4 Comparative Example 3 14.4 65.4 Comparative Example 49.7 14.4 Comparative Example 5 13.9 6.6 Comparative Example 66.8 18.8 Comparative Example 7 10.7 14.5 Comparative Example 8 10.3 5.2 Comparative Example 9 11.2 9.4 Comparative Example 10 17.2 14.7 Comparative Example 11 10.5 14.6 Comparative Example 12 11.5 22.9

[0399] From the results of Table 23, it can be confirmed that the microspheres of Examples 1-3, 5-13, 15-42, 46, 48-63, and 67 of the present invention have a lower initial release amount compared to Comparative Examples 1 to 12. That is, it can be seen that the microspheres of Examples 1-3, 5-13, 15-42, 46, 48-63, and 67 of the present invention have excellent sustained release ability.

[0400] Experimental Example 3: In vivo pharmacokinetics test of microspheres containing semaglutide salt

[0401] A solvent to be used for preparing an injectable formulation was prepared using 1 mg of polysorbate 80, 5 mg of carboxymethylcellulose, 15 mg of mannitol, 14 mg of propylene glycol, and 1 ml of water.

[0402] Microspheres containing the semaglutide benzathine salt of Examples 1, 3, 6, 10, 11, 15, and 16 were weighed so that the dosage of semaglutide was 0.5 mg / kg, and the solvent was added to them and mixed to prepare an injectable formulation.

[0403] Microspheres containing the semaglutide benzathine salt of Examples 7 and 8 were weighed so that the dosage of semaglutide was 1 mg / kg, the solvent was added thereto, and then mixed to prepare an injectable formulation.

[0404] Microspheres containing semaglutide benzathine salt and a bioavailability improver of Examples 19-23, 25, 27, 28, 30, 31, 35-37 and 40-42 were weighed so that the dosage of semaglutide was 0.5 mg / kg, and after adding the solvent to them, they were mixed to prepare an injectable formulation.

[0405] Microspheres containing the semaglutide acetate salt of Examples 50 and 51 were weighed so that the dosage of semaglutide was 0.5 mg / kg, the solvent was added thereto, and then mixed to prepare an injectable formulation.

[0406] Microspheres containing the semaglutide acetate salt of Example 55 were weighed so that the dosage of semaglutide was 1 mg / kg, the solvent was added thereto, and then mixed to prepare an injectable formulation.

[0407] Microspheres containing the semaglutide acetate salt and bioavailability improver of Examples 61-63 were weighed so that the dosage of semaglutide was 0.5 mg / kg, the solvent was added thereto, and the mixture was prepared in an injectable formulation.

[0408] The semaglutide sodium salt and bioavailability improver of Comparative Examples 7-10 were weighed into microspheres such that the dosage of semaglutide was 0.5 mg / kg, the solvent was added thereto, and the mixture was prepared to form an injectable formulation.

[0409] The above injectables were administered subcutaneously to beagles, and approximately 2 mL of blood was collected at a designated time. The blood concentration of semaglutide was measured using LC-MS / MS. Based on the measured blood concentration, the area under the blood concentration-time curve (AUC last ) calculated.

[0410] The results of the above experiment are shown in Table 24.

[0411] No.AUC last(ng·hr / mL) Example 1 237,865.08 Example 3 198,384.8 Example 6 181,760.3 Example 7 115,613.8 Example 8261,063.3 Example 10215,330.1 Example 11248,037.5 Example 15311,647.2 Example 16207,205.1 Example 19166,479.4 Example 20197,314.7 Example 21293,972.2 Example 22153,234.0 Example 23149,193.7 Example 25289,074.4 Example 27262,549.0 Example 28265,013.8 Example 30282,733.0 Example 31173,488.4 Example 352 41,894.0 Example 36 294,630.6 Example 37 188,989.5 Example 40 194,577.4 Example 41 174,176.8 Example 42 165,875.0 Example 50 188,739.7 Example 51 165,403.3 Example 55 418,884.8 Example 61 207,054.6 Example 62 288,669.5 Example 63 341,863.8 Comparative Example 7 152,606.3 Comparative Example 8 16,264.0 Comparative Example 9 136,581.0 Comparative Example 10 55,470.1

[0412] From the results in Table 24, Examples 1, 3, 6-8, 10, 11, 15, 16, 19-23, 25, 27, 28, 30, 31, 35-37, 40-42, 50, 51, 55, and 61-63 of the present invention show the area under the concentration-time curve (AUC) after a certain time compared to Comparative Examples 7-10. last It is confirmed that ) has increased. Generally, bioavailability is proportional to the area under the blood concentration-time graph curve. From these experimental results, it can be seen that the microspheres of Examples 1, 3, 6-8, 10, 11, 15, 16, 19-23, 25, 27, 28, 30, 31, 35-37, 40-42, 50, 51, 55 and 61-63 of the present invention have higher bioavailability compared to the microspheres of Comparative Examples 7-10 containing semaglutide sodium salt.

[0413] Experimental Example 4: Release test and initial release rate evaluation of microspheres containing tyrzepatide salt

[0414] 25 mg of microspheres from Examples 69-88 and Comparative Examples 1-12 were taken and placed in a 25 ml volumetric flask. After sonication with an ACN solution containing 0.1% TFA, the 25 ml volumetric flask was filled to the mark with a distilled water solution containing 0.1% TFA. This solution was filtered through a 0.45 µm syringe filter and detected using a UV-visible spectrophotometer with an HPLC (equipment name: Agilent 1260). Column packing was performed with L1, an internal diameter of 4.6 mm x 150 mm, and a thickness of 3.5 µm. Through the above experimental results, the loading amount of microspheres containing tyrzepatide salt was confirmed.

[0415] In addition, 15 mg of microspheres prepared in Examples 69 to 88 and Comparative Examples 13 and 14 were placed in a vial, 3 mL of pH 7.4 PBS solution was added, and the mixture was stirred at 100 rpm while maintaining the temperature at 37°C for a certain period. To measure the release amount after a certain period, the entire supernatant was taken and filtered through a 0.45 μm RC filter to obtain the sample solution. The sample solution was detected using an ultraviolet-visible spectrophotometer with an HPLC (equipment name: Agilent 1260). The column packing was performed with L1, an internal diameter of 4.6 mm x 150 mm, and a thickness of 3.5 μm.

[0416] The results of the above experiment are shown in Table 25 below.

[0417] Microsphere Loading Amount (Wet%) Initial Release Rate (%) Example 69 17.8 4.8 Example 70 22.5 9.5 Example 71 27.7 32.5 Example 72 18.3 5.9 Example 73 18.2 5.8 Example 74 21.8 17.2 Example 75 24.3 34.4 Example 76 25.6 23.3 Example 77 30.2 38.9 Example 78 22.4 19.2 Example 79 2 7.9 48.9 Example 80 16.9 11.8 Example 81 22.9 13.6 Example 82 7.50 Example 83 19 8.7 Example 84 26.7 36.4 Example 85 26.3 11.3 Example 86 30.9 45.4 Example 87 20.1 9.5 Example 88 21.7 7.7 Comparative Example 13 19.0 21.9 Comparative Example 14 9.3 0.9

[0418] From the results of Table 25, it can be confirmed that the microspheres of Examples 69 to 88 of the present invention contain a higher content and have a lower initial release amount compared to Comparative Examples 13 and 14. That is, it can be seen that the microspheres of Examples 69 to 88 of the present invention have excellent sustained-release ability.

[0419] Experimental Example 5: In vivo pharmacokinetics test of microspheres containing tyrzepatide salt

[0420] A solvent to be used for preparing an injectable formulation was prepared using 1 mg of polysorbate 80, 5 mg of carboxymethylcellulose, 15 mg of mannitol, 14 mg of propylene glycol, and 1 ml of water.

[0421] Microspheres containing the tyrzepatide benzathine salt of Examples 69-71 were weighed so that the dosage of tyrzepatide was 1 mg / kg, the solvent was added thereto, and then mixed to prepare an injectable formulation.

[0422] Microspheres containing the tyrzepatide benzathine salt and bioavailability improver of Examples 72-79 were weighed so that the dosage of tyrzepatide was 1 mg / kg, the solvent was added thereto, and the mixture was mixed to prepare an injectable formulation.

[0423] Microspheres containing the tircepate acetate salt and bioavailability improver of Examples 83, 85, and 86 were weighed so that the dosage of tircepate was 1 mg / kg, the solvent was added thereto, and the mixture was prepared to form an injectable formulation.

[0424] Microspheres containing the tircepate formate and bioavailability improver of Example 88 were weighed so that the dosage of tircepate was 1 mg / kg, the solvent was added thereto, and the mixture was mixed to prepare an injectable formulation.

[0425] Microspheres containing the sodium salt of tircepate and a bioavailability improver of Comparative Example 13 were weighed so that the dosage of tircepate was 1 mg / kg, the solvent was added thereto, and the mixture was mixed to prepare an injectable formulation.

[0426] The above injectable formulations were administered subcutaneously to beagles, and approximately 2 mL of blood was collected at designated times. The blood concentration of semaglutide was measured using LC-MS / MS. Based on the measured blood concentrations, the area under the blood concentration-time curve (AUC last ) calculated.

[0427] The results of the above experiment are shown in Table 26.

[0428] No.AUC last (ng·hr / mL) Example 69995,129.5 Example 701,226,783.5 Example 711,719,774.6 Example 72914,848.4 Example 73664,829.1 Example 741,258,082.2 Example 751,389,900.0 Example 761,474.8 89.7 Example 771,900,711.5 Example 781,519,050.0 Example 791,586,379.9 Example 831,110,598.5 Example 851,144,530.1 Example 861,085,970.7 Example 889,74,079.3 Comparative Example 137,815,34.8

[0429] From the results in Table 26, Examples 69-79, 83, 85, 86, and 88 show the area under the concentration-time curve (AUC) relative to Comparative Example 13. last It is confirmed that ) has increased. Generally, bioavailability is proportional to the area under the blood concentration-time curve. From these experimental results, it can be seen that the microspheres of Examples 69-79, 83, 85, 86, and 88 of the present invention have higher bioavailability compared to the microspheres of Comparative Example 13 containing tyrzepatide sodium salt.

[0430] Experimental Example 6: In vivo pharmacokinetic study of microspheres containing GLP-1 RA class peptide salts in a liquid formulation containing a carrier of a water-soluble liquid or polyalcohol

[0431] a. Injectable in a liquid formulation using a water-soluble liquid as a carrier: Microspheres containing the semaglutide benzathine salt and bioavailability improver of Example 37 were weighed so that the dosage of semaglutide was 0.5 mg / kg. To this, a water-soluble liquid prepared using 1 mg of polysorbate 80, 5 mg of carboxymethylcellulose, 15 mg of mannitol, 14 mg of propylene glycol, and 1 ml of water was added. By mixing these, an injectable in a liquid formulation using a water-soluble liquid as a carrier was prepared.

[0432] b. Liquid formulation injectable using a polyalcohol as a carrier: Microspheres containing the semaglutide benzathine salt of Example 37 and a bioavailability improver were weighed so that the dosage of semaglutide was 0.5 mg / kg. A polyalcohol was added thereto. By mixing these, a liquid formulation injectable using a polyalcohol as a carrier was prepared.

[0433] The liquid formulations using the above-mentioned water-soluble liquid as a carrier and the liquid formulations using a polyalcohol as a carrier were subcutaneously injected into beagles using an injector, and approximately 2 mL of blood was collected at a designated time. The blood concentration of semaglutide was measured using LC-MS / MS. Based on the measured blood concentration, the area under the blood concentration-time curve (AUC last ) calculated.

[0434] The results of the above experiment are shown in Table 27 below.

[0435] Liquid formulation AUC last (ng·hr / mL) Microspheres of Example 37 + water-soluble liquid carrier 188,989.5 Microspheres of Example 37 + polyalcohol carrier 184,689.5

[0436] Looking at the results in Table 27, when administered as an injectable with a polyalcohol liquid formulation as a carrier, the area under the concentration-time curve (AUC) compared to when administered as an injectable with a water-soluble liquid formulation as a carrier last ) appears to be similar or higher. Generally, bioavailability is proportional to the area under the blood concentration-time curve. That is, injectables with water-soluble liquids as carriers and injectables with polyalcohols as carriers have similar bioavailability. From these experimental results, it can be seen that microspheres containing GLP-1 RA-type peptide salts can be prepared in a liquid formulation using a carrier selected from the group consisting of water-soluble liquids or polyalcohols.

[0437] Experimental Example 7: In vivo pharmacokinetic study of microspheres containing a GLP-1 RA class peptide salt in a liquid formulation containing a fluid oil carrier

[0438] a. Liquid formulation injectable using a fluid oil as a carrier: Microspheres containing the semaglutide benzathine salt and bioavailability improver of Example 21 were weighed so that the dosage of semaglutide was 0.5 mg / kg. MCT oil was added to this. By mixing these, a liquid formulation injectable using a fluid oil as a carrier was prepared.

[0439] b. Injectable in liquid formulation using a water-soluble liquid as a carrier: Microspheres containing the semaglutide benzathine salt and bioavailability improver of Example 21 were weighed so that the dosage of semaglutide was 0.5 mg / kg. To this, a water-soluble liquid prepared using 1 mg of polysorbate 80, 5 mg of carboxymethylcellulose, 15 mg of mannitol, 14 mg of propylene glycol, and 1 ml of water was added. By mixing these, an injectable in liquid formulation using a water-soluble liquid as a carrier was prepared.

[0440] The liquid formulation injectable using the above-mentioned fluid oil as a carrier and the liquid formulation injectable using the above-mentioned water-soluble liquid as a carrier were administered subcutaneously to beagles, and approximately 2 mL of blood was collected at a designated time. The blood concentration of semaglutide was measured using LC-MS / MS.

[0441] The results of the above experiment are shown in Table 28 below.

[0442] Liquid formulation blood concentration (ng / mL) after 12 hours Microspheres of Example 21 + fluid oil carrier 235.57 Microspheres of Example 21 + water-soluble liquid carrier 435.45

[0443] Looking at the results in Table 28, blood semaglutide concentrations were lower after 12 hours when administered as an injectable using a fluid oil carrier compared to when administered as an injectable using a water-soluble liquid formulation carrier. From these experimental results, it can be seen that the initial blood concentration can be controlled by using a fluid oil carrier when administering microspheres containing GLP-1 RA-type peptide salts. Furthermore, it was confirmed that microspheres containing GLP-1 RA-type peptide salts can be provided as a suspension-type pharmaceutical composition in a liquid formulation using a fluid oil carrier.

[0444] Experimental Example 8: In vivo pharmacokinetic study of microspheres containing a GLP-1 RA-class peptide salt in a liquid formulation containing a tissue diffusion promoter in a carrier

[0445] a. Injectable in liquid formulation using a water-soluble liquid as a carrier: A water-soluble liquid was prepared using 1 mg of polysorbate 80, 5 mg of carboxymethylcellulose, 15 mg of mannitol, 14 mg of propylene glycol, and 1 ml of water. Microspheres containing the semaglutide benzathine salt and bioavailability improver of Example 46 were weighed so that the dosage of semaglutide was 4 mg, and the above water-soluble liquid was added to them. By mixing these, an injectable in liquid formulation using a water-soluble liquid as a carrier was prepared.

[0446] b. Injectable in liquid formulation using a water-soluble liquid containing a tissue diffusion promoter as a carrier: Hyaluronidase was dissolved in 1 mL of water to prepare a water-soluble liquid containing a tissue diffusion promoter. Microspheres containing the semaglutide benzathine salt and bioavailability improver of Example 46 were weighed so that the dosage of semaglutide was 4 mg, and the water-soluble liquid containing the tissue diffusion promoter was added to them. By mixing these, an injectable in liquid formulation using a water-soluble liquid containing a tissue diffusion promoter as a carrier was prepared.

[0447] c. Liquid formulation injectable using a fluid oil as a carrier: Microspheres containing the semaglutide benzathine salt and bioavailability improver of Example 46 were weighed so that the dosage of semaglutide was 4 mg. MCT oil was added to this. By mixing these, a liquid formulation injectable using a fluid oil as a carrier was prepared.

[0448] d. Liquid formulation injectable using a fluid oil containing a tissue diffusion promoter as a carrier: Hyaluronidase was suspended in 1 mL of MCT oil to prepare a fluid oil containing a tissue diffusion promoter. Microspheres containing the semaglutide benzathine salt and bioavailability improver of Example 46 were weighed so that the dosage of semaglutide was 4 mg. The MCT oil containing the tissue diffusion promoter was added thereto. By mixing these, a liquid formulation injectable using a fluid oil containing a tissue diffusion promoter as a carrier was prepared.

[0449] The above injectables were administered subcutaneously to minipigs, and blood was collected at designated times. The blood concentration of semaglutide was measured using LC-MS / MS.

[0450] The results of the above experiment are shown in Tables 29 and 30 below.

[0451] Formulation AUC last0-21D(ng·hr / mL) Example 6 + water-soluble liquid carrier (a) 41,645.8 Example 6 + fluid oil carrier (c) 40,992.4

[0452] Formulation AUC last0-14D (ng·hr / mL) Example 6 + water-soluble liquid carrier containing tissue diffusion promoter (b) 24,420.8 Example 6 + fluid oil carrier containing tissue diffusion promoter (d) 38,074.6

[0453] Looking at the results in Tables 29 and 30, when administered as an injectable using a water-soluble liquid formulation containing a tissue diffusion promoter or a fluid oil formulation containing a tissue diffusion promoter as a carrier, the area under the concentration-time curve (AUC) compared to when administered as an injectable using a water-soluble liquid formulation or a fluid liquid formulation as a carrier last ) appears similar or high.

[0454] Experimental Example 9: In vivo pharmacokinetic test of microspheres containing a combined formulation of a GLP-1 RA class peptide salt

[0455] a. Injectable formulation containing a single microsphere of Example 3: A solvent to be used for the injectable was prepared using 1 mg of polysorbate 80, 5 mg of carboxymethylcellulose, 15 mg of mannitol, 14 mg of propylene glycol, and 1 ml of water. Microspheres containing the semaglutide benzathine salt of Example 3 were weighed so that the total dose of semaglutide was 0.5 mg / kg, and the above solvent was added to them. By mixing these, an injectable formulation containing a single microsphere was prepared.

[0456] b. Injectable formulation of the complex formulation of Example 3: Semaglutide was weighed to a total dosage of 0.05 mg / kg, and the dosage of microspheres containing the semaglutide benzathine salt of Example 3 was weighed to a dosage of 0.45 mg / kg, and then the same solvent as that used in 'a. Injectable formulation containing a single microsphere of Example 3' was added. All of these were mixed to prepare an injectable formulation of the complex formulation.

[0457] c. Injectable formulation containing a single microsphere of Example 61: Microspheres containing the semaglutite acetate salt and bioavailability improver of Example 61 were weighed so that the total dose of semaglutite was 0.5 mg / kg. To this, the same solvent as in 'a. Injectable formulation containing a single microsphere of Example 3' was added. By mixing these, an injectable formulation containing a single microsphere was prepared.

[0458] d. Injectable formulation of the complex formulation of Example 61: Semaglutide was weighed to a total dosage of 0.05 mg / kg, and the microsphere dosage of Example 61 was weighed to a total dosage of 0.45 mg / kg. To this, the same solvent as in 'a. Injectable formulation containing a single microsphere of Example 3' was added. All of these were mixed to prepare an injectable formulation of the complex formulation.

[0459] The above injectables were administered subcutaneously to beagles, and approximately 2 mL of blood was collected at a designated time. The blood concentration of semaglutide was measured using LC-MS / MS. Based on the measured blood concentration, the area under the blood concentration-time curve (AUC last ) calculated.

[0460] The results of the above experiment are shown in Table 31 below.

[0461] Formulation AUC last (ng·hr / mL) Example 3 Formulation containing a single microsphere (a) 198,384.8 Example 3 Compound Formulation (b) 273,871.2 Example 61 Formulation containing a single microsphere (c) 207,054.6 Example 61 Compound Formulation (d) 275,675.5

[0462] Looking at the results in Table 31, when the microspheres of Examples 3 and 61 were administered as a combined formulation, compared to when they were administered as a formulation containing single microspheres, the area under the concentration-time curve (AUC) was the same even under the same total dosage conditions. last) is found to be high. Generally, bioavailability is proportional to the area under the blood concentration-time curve. That is, when microspheres containing GLP-1 RA-type peptides are administered as a complex formulation, they exhibit improved bioavailability compared to when administered as a formulation containing a single microsphere. From these experimental results, it was confirmed that microspheres containing GLP-1 RA-type peptide salts can be provided as a pharmaceutical composition in a complex formulation.

Claims

1. Microspheres comprising a GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer.

2. In Paragraph 1, Microspheres in which the pharmaceutically acceptable salt of the above GLP-1 RA series peptide is selected from benzathine salt, acetate salt, formate salt, pamoate salt, and combinations thereof.

3. In Paragraph 1, The above GLP-1 RA series peptide is a microsphere selected from semaglutide, tirzepatide, retatrutide, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, cotadutide, taspoglutide, pemvidutide, survodutide, petrelintide, ellarintide, and combinations thereof.

4. In Paragraph 1, Microspheres in which the above-mentioned GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof is included in an amount of 2% to 50% by weight based on the total weight of the microspheres.

5. In Paragraph 1, The above-mentioned biocompatible polymer is one or more selected from the group consisting of poly(lactic acid), polylactide, poly(lactic-co-glycolic acid), polylactide-co-glycolide (PLGA), polyphosphazene, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, copolymer of lactic acid and caprolactone, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid, and copolymer of lactic acid and amino acid.

6. In Paragraph 1, Microspheres in which the above biocompatible polymer is included in an amount of 50% to 98% by weight based on the total weight of the microspheres.

7. In Paragraph 1, Microspheres having an intrinsic viscosity of 0.1 dL / g to 1.9 dL / g of the above biocompatible polymer.

8. In Paragraph 1, The above microspheres are microspheres that further comprise a bioavailability improver.

9. In Paragraph 8, The above-mentioned bioavailability improver is included in an amount of 0.01% to 20% by weight based on the total weight of the microspheres.

10. In Paragraph 8, The above-mentioned bioavailability improving agent is a microsphere selected from the group consisting of surfactants, polyhydric alcohols, amino acids, fatty acids, organic acids, and sodium salts of organic acids.

11. In Paragraph 10, The above-mentioned surfactant is one or more selected from the group consisting of Poloxamer 188, Poloxamer 407, Polysorbate 20, Polysorbate 60, Polysorbate 80, Polyoxyethylene oleyl ether, Polyoxyethylene cetyl ether, Polyoxyethylene stearyl ether, and Polyoxyethylene lauryl ether, and is a microsphere.

12. In Paragraph 10, The above polyalcohol is one or more selected from the group consisting of ethylene glycol, propylene glycol, polyethylene glycol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, and maltitol, and is a microsphere.

13. In Paragraph 10, Microspheres in which the above amino acid is one or more selected from the group consisting of lysine, arginine, aspartic acid, histidine, alanine, phenylalanine, serine, glutamine, glycine, tryptophan, valine, leucine, isoleucine, cysteine, methionine, and proline.

14. In Paragraph 10, Microspheres in which the above fatty acid is one or more selected from the group consisting of stearic acid, palmitic acid, lauric acid, myristic acid, oleic acid, and arachidonic acid.

15. In Paragraph 10, Microspheres in which the above organic acid is one or more selected from the group consisting of citric acid, stearic acid, tartaric acid, citric acid, succinic acid, and fumaric acid.

16. In Paragraph 10, Microspheres in which the sodium organic acid salt is one or more selected from the group consisting of sodium acetate, sodium citrate, sodium tartrate, sodium succinate, sodium propionate, sodium bicarbonate, and sodium butyrate.

17. A method for manufacturing microspheres comprising the following steps: (a) a step of preparing a GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof; (b) a step of preparing a dispersed phase by dissolving the GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof prepared in step (a) and a biocompatible polymer in a dispersion solvent; (c) a step of introducing the dispersed phase prepared in step (b) above into a continuous phase and stirring to form microspheres; (d) a step of removing the dispersion solvent from the microspheres of step (c); (e) a step of recovering the microspheres formed in step (d) above; and (f) A step of drying the microspheres recovered in step (e) above.

18. In Paragraph 17, The above step (a) includes reacting a GLP-1 RA series peptide with an acid or basic compound to form a salt, and separating and purifying the formed salt, wherein A method for manufacturing microspheres, wherein the formed salt is selected from benzathine salt, formate salt, acetate salt, pamoate salt, and combinations thereof.

19. In Paragraph 17, A method for manufacturing microspheres, wherein step (b) further includes the step of introducing a bioavailability enhancer.

20. In Paragraph 17, The method for manufacturing the above microspheres further comprises an extraction step after step (e) or after step (f).

21. In Paragraph 20, The above extraction step It is performed using an aqueous solution containing purified water, buffer solution, alcohols, ketones, esters, and inorganic salts, or a mixture thereof, as an extraction solvent, or A method for producing microspheres, performed using an extraction solvent comprising at least one selected from the group consisting of ethanol, methanol, acetone, methyl ethyl ketone, and ethyl acetate, and an aqueous solution of disodium hydrogen phosphate.

22. A pharmaceutical composition for the prevention or treatment of diabetes, obesity, hypertension, hyperlipidemia, non-alcoholic fatty liver disease (NAFLD), metabolic disorder fatty liver disease (MAFLD), cardiovascular disease or neurodegenerative disease, wherein The above pharmaceutical composition comprises the microspheres of claim 1 and a pharmaceutically acceptable carrier.

23. In Paragraph 22, The above pharmaceutical composition comprises the microspheres of claim 1 in an amount of 0.01 mg to 2,000 mg per 1 mL of the pharmaceutically acceptable carrier.

24. In Paragraph 22, A pharmaceutical composition comprising one or more selected from the group consisting of a water-soluble liquid, a fluid oil containing a fatty acid ester, a surfactant, and a polyalcohol.

25. In Paragraph 24, A pharmaceutical composition comprising one or more selected from the group consisting of coconut oil, palm oil, palm kernel oil, sesame oil, soybean oil, almond oil, rapeseed oil, corn oil, sunflower oil, peanut oil, olive oil, castor oil, safflower oil, cottonseed oil, and ethyl oleate, wherein the fluid oil containing the above fatty acid ester comprises the above fatty acid ester.

26. In Paragraph 24, A pharmaceutical composition comprising one or more surfactants selected from Poloxamer 188, Poloxamer 407, Polysorbate 20, Polysorbate 60, Polysorbate 80, Polyoxyethylene oleyl ether, Polyoxyethylene cetyl ether, Polyoxyethylene stearyl ether, and Polyoxyethylene lauryl ether.

27. In Paragraph 24, A pharmaceutical composition comprising one or more polyalcohols selected from ethylene glycol, propylene glycol, polyethylene glycol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, and maltitol.

28. In Paragraph 22, The above pharmaceutical composition further comprises hyaluronidase.

29. In Paragraph 28, A pharmaceutical composition in which the hyaluronidase is included in the pharmaceutically acceptable carrier or is included in the pharmaceutical composition separately from the pharmaceutically acceptable carrier.

30. In Paragraph 22, The above pharmaceutical composition further comprises a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof.

31. In Paragraph 22, The above pharmaceutical composition further comprises one or more excipients selected from buffers, isotonic agents, preservatives, and pH adjusters.

32. In Paragraph 22, The above pharmaceutical composition is a pharmaceutical composition for subcutaneous or intramuscular injection.

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