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

Abstract

The present invention relates to a microsphere composition comprising at least one type of microsphere comprising: a GLP-1 RA peptide or a pharmaceutically acceptable salt thereof; and a biocompatible polymer, methods for preparing the microspheres and the microsphere composition, and a pharmaceutical composition comprising same for use in the prevention or treatment of diabetes, obesity, hypertension, hyperlipidemia, non-alcoholic fatty liver disease, metabolic dysfunction-associated steatotic liver disease, 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 agents that mimic the action of glucagon-like peptide-1 (GLP-1), a bioactive peptide derived from preproglucagon secreted by L-cells, thereby stimulating insulin secretion from pancreatic islet β-cells to stabilize postprandial blood glucose levels. 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), Wegovy ® (Semaglutide), Mounjaro ® (Tirzepatide), Zepbound ® (Tirzepatide), Trulicity ® (Dulaglutide), Victoza ® (Liraglutide), Saxenda® (Liraglutide), Bydureon ® (Exenatide), 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 rates 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-type peptides in a long-acting manner are being actively researched. As part of this effort, technologies have been developed to manufacture sustained-release formulations by encapsulating peptides in drug delivery systems such as microspheres. However, maintaining long-term efficacy requires a high drug loading capacity within the microspheres, which acts as a technical limitation that can exacerbate the initial burst phenomenon and cause safety issues. Another problem arises when high-viscosity polymers are used to delay release in order to reduce the initial burst; this may result in a failure to reach the therapeutically effective concentration at the time of administration or cause a lag phase, leading to a gap in efficacy.

[0006] Furthermore, when peptide therapeutics such as semaglutide and tyrzepatide are formulated into long-term sustained-release injectable formulations, loss of the active drug may occur due to changes in the local environment caused by the characteristics of the formulation, which leads to a problem of significantly reduced exposure to the active drug and bioavailability in the body.

[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 stably maintaining a therapeutically effective concentration of a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof over a long period while ensuring excellent bioavailability, a pharmaceutical composition containing said microspheres, 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 80% 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 invention, the biocompatible polymer may be composed of a single polymer or a mixture of two or more polymers. For example, the two or more may be a mixture of two, three, or three or more polymers.

[0019] In one embodiment, the polymers included in the plurality of polymer mixtures may have different intrinsic viscosities. In one embodiment, the difference in intrinsic viscosity between polymers having different intrinsic viscosities may be 0.1 dL / g or more, but is not limited thereto.

[0020] In one embodiment, the plurality of polymer mixtures may be a mixture of a low-viscosity polymer and a high-viscosity polymer. In this specification, the low-viscosity polymer refers to a polymer having a relatively low intrinsic viscosity, and the high-viscosity polymer refers to a polymer having a relatively high intrinsic viscosity.

[0021] In one embodiment, when the biocompatible polymer is composed of a mixture of two types of polymers, the weight ratio of each polymer may be 1:0.1 to 1:50. In one embodiment, when the biocompatible polymer is composed of a mixture of three types of polymers, the weight ratio of each polymer may be 1:0.1:0.1 to 1:50:50.

[0022] According to one embodiment of the present invention, the biocompatible polymer may be included in an amount of 20% to 98% by weight based on the total weight of the microspheres.

[0023] 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.

[0024] 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 0.5 dL / g, and the second microsphere may include a biocompatible polymer having an intrinsic viscosity of 0.5 dL / g or more and less than 2.0 dL / g.

[0025] The first microsphere may comprise a biocompatible polymer having an intrinsic viscosity of 0.1 dL / g or more and less than 0.6 dL / g, and the second microsphere may comprise a biocompatible polymer having an intrinsic viscosity of 0.6 dL / g or more and less than 2.0 dL / g.

[0026] The first microsphere comprises 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 comprise 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.

[0027] 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 0.4 dL / g, the second microsphere may include a biocompatible polymer having an intrinsic viscosity of 0.4 dL / g or more and less than 0.6 dL / g, and the third microsphere may include a biocompatible polymer having an intrinsic viscosity of 0.6 dL / g or more and less than 2.0 dL / g.

[0028] The first microsphere may comprise a biocompatible polymer having an intrinsic viscosity of 0.1 dL / g or more and less than 0.4 dL / g, the second microsphere may comprise a biocompatible polymer having an intrinsic viscosity of 0.4 dL / g or more and less than 0.7 dL / g, and the third microsphere may comprise a biocompatible polymer having an intrinsic viscosity of 0.7 dL / g or more and less than 2.0 dL / g.

[0029] The first microsphere may comprise 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 comprise 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 comprise a biocompatible polymer having an intrinsic viscosity of 0.7 dL / g or more and less than 2.0 dL / g.

[0030] The first microsphere may comprise a biocompatible polymer having an intrinsic viscosity of 0.1 dL / g or more and less than 0.6 dL / g, the second microsphere may comprise a biocompatible polymer having an intrinsic viscosity of 0.6 dL / g or more and less than 0.7 dL / g, and the third microsphere may comprise a biocompatible polymer having an intrinsic viscosity of 0.7 dL / g or more and less than 2.0 dL / g.

[0031] 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.

[0032] According to one embodiment of the present invention, the microspheres may further include a bioavailability improver.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] According to one embodiment of the present invention, the organic acid may be one or more selected from the group consisting of stearic acid, tartaric acid, citric acid, succinic acid, and fumaric acid.

[0040] 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.

[0041] According to a second aspect of the present invention,

[0042] The present invention provides a method for manufacturing microspheres comprising the following steps:

[0043] (a) a step of preparing a dispersed phase by dispersing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer in a dispersion solvent;

[0044] (b) a step of introducing the dispersed phase prepared in step (a) above into a continuous phase and stirring it to form microspheres;

[0045] (c) a step of removing the dispersion solvent from the microspheres of step (b) above;

[0046] (d) a step of recovering the microspheres formed in step (c) above; and

[0047] (e) A step of drying the microspheres recovered in step (d) above.

[0048] 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 pamoate salt, acetate salt, benzathine salt, formate salt, and combinations thereof.

[0049] According to one embodiment of the present invention, step (b) may further include the step of introducing a bioavailability enhancer.

[0050] According to one embodiment of the present invention, an extraction step may be further included after step (d) or after step (e).

[0051] 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: purified water, a buffer solution, alcohols, ketones, esters, and inorganic salts; or it may be 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.

[0052] According to the third aspect of the present invention,

[0053] The present invention provides a method for preparing a microsphere composition comprising the following steps:

[0054] (a) a step of preparing a dispersed phase by dispersing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer in a dispersion solvent;

[0055] (b) a step of introducing the dispersed phase prepared in step (a) into a continuous phase and stirring it to form microspheres;

[0056] (c) a step of removing the dispersion solvent from the microspheres formed in step (b) above;

[0057] (d) A step of mixing one or more types of microspheres prepared by steps (a) to (c) above.

[0058] According to one embodiment of the present invention, an extraction step may be further included after step (c) or after step (d).

[0059] According to the fourth aspect of the present invention,

[0060] 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 neurodegenerative disease, comprising a microsphere composition of the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0061] 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 5,000 mg per 1 mL of pharmaceutically acceptable carrier.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] According to one embodiment of the present invention, the pharmaceutical composition of the present invention may further include hyaluronidase.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] According to one embodiment of the present invention, the pharmaceutical composition of the present invention may be a subcutaneous or intramuscular injection formulation.

[0071] The microspheres according to the present invention encapsulate a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof in a biocompatible polymer, thereby ensuring a balance between initial release and sustained release and providing the effect of constant and continuous release over a long period, and can exhibit high bioavailability.

[0072] In addition, the microspheres according to the present invention can control the release period of the entire microsphere by combining a plurality of microspheres with different release periods.

[0073] Furthermore, the pharmaceutical composition according to the present invention can provide excellent effects in the prevention or treatment of obesity or diabetes by including the microspheres.

[0074] Figure 1 shows the semaglutide acetate salt prepared in Example 50. 1 This is a diagram showing the results of H-NMR analysis.

[0075] Figure 2 shows the semaglutide benzathine salt prepared in Example 1. 1 This is a diagram showing the results of H-NMR analysis.

[0076] FIG. 3 is of the semaglutide formate prepared in Example 67. 1 This is a diagram showing the results of H-NMR analysis.

[0077] FIG. 4 is of the tyrzepatide acetate salt prepared in Example 80. 1 This is a diagram showing the results of H-NMR analysis.

[0078] Figure 5 shows the tyrzepatide benzathine salt prepared in Example 69. 1 This is a diagram showing the results of H-NMR analysis.

[0079] FIG. 6 is of the tyrzepatide formate prepared in Example 85. 1 This is a diagram showing the results of H-NMR analysis.

[0080] FIG. 7 shows the tyrzepatide pamoate salt prepared in Example 89. 1 This is a diagram showing the results of H-NMR analysis.

[0081] terminology

[0082] Hereinafter, each term used in this specification is explained.

[0083] Unless otherwise defined, terms and words used in the description and claims of the present invention are used in the sense generally understood by those skilled in the art; however, in the event of a conflict, the definitions in this specification shall prevail. Furthermore, terms used in this specification are for illustrative purposes only and should not be interpreted as intended to be limiting.

[0084] In this specification, "microsphere" means a particle containing or encapsulating various active ingredients, and said active ingredients may be peptides, proteins, low molecular weight compounds, nucleic acids, or pharmaceutically acceptable salts, derivatives, or isomers thereof.

[0085] In this specification, "dispersion" refers to a state in which one or more substances are spread out in the form of fine particles within another continuous medium. In this case, the dispersed substances may be called the dispersed phase, the medium surrounding them may be called the dispersion medium, and the system in which the dispersed phase exists in the dispersion medium may be called the dispersion system. Depending on the particle size of the dispersed phase, a state in which particles are dissolved at the molecular or ionic level with a particle size of about 1 nm or less may be called molecular dispersion or true solution; a state in which particles are dissolved at a level of about 1 nm to 0.5 μm may be called colloidal dispersion; and a state in which particles are dissolved at a level of about 0.5 μm or more may be called coarse dispersion or particulate dispersion.

[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 (IV)" means 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 administration, delivery, or introduction 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 RA by temporarily increasing the permeability of subcutaneous tissue.

[0094]

[0095] Hereinafter, the present invention will be described in more detail to aid in understanding the invention.

[0096] Methods or samples similar or equivalent to those described in the present invention are also included within the scope of the present invention. The contents of all publications cited as references in this specification are incorporated herein by reference in their entirety.

[0097]

[0098] microspheres

[0099] The microspheres of the present invention comprise a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer.

[0100] In one embodiment, pharmaceutically acceptable salts of GLP-1 RA series peptides may be selected from pamoate salt, acetate salt, benzathine salt, formate salt, and combinations thereof, but are not limited to these.

[0101] In one embodiment, the GLP-1 RA series peptide may be one or more selected from the group consisting of GLP-1 receptor agonists, GIP / GLP-1 receptor dual agonists, and GLP-1 / GIP / GCG receptor triple agonists.

[0102] Specifically, GLP-1 RA family peptides may be selected from semaglutide, tirzepatide, retatrutide, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, cotadutide, taspoglutide, pemvidutide, survodutide, petrelintide, ellarintide, and combinations thereof, but are not limited to these.

[0103] 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.

[0104] Specifically, the GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof may be included in an amount of 2% to 80% by weight, 5% to 75% by weight, or 10% to 70% 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 80 wt% or less, 75 wt% or less, 70 wt% or less, 65 wt% or less, 60 wt% or less, 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, It may be included in an amount of 47.0 wt% or less, 46.5 wt% or less, 46.0 wt% or less, 45.5 wt% or less, 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.

[0105] 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 80% 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.

[0106] 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.

[0107] 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.

[0108] Specifically, the biocompatible polymer may be included in an amount of 20% to 98% by weight, 25% to 90% by weight, or 30% to 80% by weight based on the total weight of the microspheres. More specifically, the biocompatible polymer may be included in an amount of 20% or more by weight, 25% or more by weight, 30% or more by weight, 40% or more by weight, 50% or more by weight, 55% or more by weight, 60% or more by weight, 65% or more by weight, 70% or more by weight, or 75% or more by weight based on the total weight of the microspheres, or in an amount of 98% or less by weight, 97% or less by weight, 96% or less by weight, 95% or less by weight, 94% or less by weight, or 93% or less by weight, but is not limited to these ranges.

[0109] As described above, if the content of the biocompatible polymer exceeds 98% by weight based on the total weight of the microspheres, the release of the drug may be 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 20% by weight based on the total weight of the microspheres, the distribution of semaglutide may increase relatively, leading to an initial over-release or a problem of not being able to maintain the drug effect for the desired period.

[0110] 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 1:0.25–50, 1:0.5–50, 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, It may be included in a weight ratio of 1:1~38, 1:1.5~38, 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.

[0111] 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.

[0112] In one embodiment, the intrinsic viscosity of the biocompatible polymer may be 0.10 dL / g to 2.0 dL / g, 0.10 dL / g to 1.7 dL / g, or 0.10 dL / g to 1.5 dL / g.

[0113] 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; if the intrinsic viscosity exceeds 2.0 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 due to the high viscosity of the polymer when preparing the pharmaceutical composition, which makes it difficult to produce reproducible microspheres.

[0114] In one embodiment, the microspheres may further include a bioavailability enhancer.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] Specifically, the organic acid may be one or more selected from the group consisting of stearic acid, tartaric acid, citric acid, succinic acid, and fumaric acid.

[0123] 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.

[0124] According to one embodiment, the average particle diameter of the microspheres may be about 1 μm to about 200 μm, for example, about 10 μm to about 100 μm. However, the particle diameter is merely an exemplary range and is not limited thereto, and the present invention can be applied to microspheres of various commonly used particle sizes.

[0125]

[0126] Microparticle composition

[0127] The microsphere composition of the present invention comprises at least one type of microsphere comprising a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer. Specific details regarding the composition and characteristics of the microsphere are applied in the same manner as the description of the microsphere above.

[0128] In one embodiment, the biocompatible polymers included in each of the plurality of microspheres included in the microsphere composition of the present invention may have different intrinsic viscosities. The intrinsic viscosity of a biocompatible polymer is correlated with the molecular weight of the polymer, and generally, polymers with high intrinsic viscosity tend to have a slow degradation rate and a delayed drug release rate; therefore, by combining microspheres containing biocompatible polymers having different intrinsic viscosities, the initial release and sustained release can be controlled to achieve a desired release profile.

[0129] Specifically, each of the above microspheres is 0.1 dL / g or more and less than 0.2 dL / g, 0.2 dL / g or more and less than 0.3 dL / g, 0.3 dL / g or more and less than 0.4 dL / g, 0.4 dL / g or more and less than 0.5 dL / g, 0.5 dL / g or more and less than 0.6 dL / g, 0.6 dL / g or more and less than 0.7 dL / g, 1.0 dL / g or more and less than 1.1 dL / g, 1.1 dL / g or more and less than 1.2 dL / g, 1.2 dL / g or more and less than 1.3 dL / g, 1.3 dL / g or more and less than 1.4 dL / g, 1.4 dL / g or more and less than 1.5 dL / g, and 1.5 It may be dL / g or more and less than 1.6 dL / g, 1.6 dL / g or more and less than 1.7 dL / g, 1.7 dL / g or more and less than 1.8 dL / g, 1.8 dL / g or more and less than 1.9 dL / g, or 1.9 dL / g or more and less than 2.0 dL / g.

[0130] In one embodiment, the microsphere composition of the present invention may include a plurality of microspheres in which the intrinsic viscosity of the biocompatible polymer belongs to a high intrinsic viscosity region and a low intrinsic viscosity region, respectively. In this specification, "high intrinsic viscosity region" refers to a region in which the intrinsic viscosity is relatively high, and "low intrinsic viscosity region" refers to a region in which the intrinsic viscosity is relatively low. The plurality of microspheres may be of two or more types, three or more types, or four or more types.

[0131] In one embodiment, the microsphere composition of the present invention may include two types of microspheres in which the intrinsic viscosity of the biocompatible polymer belongs to a high intrinsic viscosity region and a low intrinsic viscosity region, respectively. By adjusting the mixing ratio of the two types of microspheres, a release profile with a release period of one month or more can be realized. For example, by adjusting the mixing ratio of microspheres containing biocompatible polymers having different intrinsic viscosities, a release profile with a release period of one month or more can be realized.

[0132] In one embodiment, when the microsphere composition of the present invention comprises two types of microspheres, the first microsphere, in which the intrinsic viscosity of the biocompatible polymer belongs to a low intrinsic viscosity region, may have an intrinsic viscosity of 0.1 dL / g or more and less than 0.7 dL / g, and the second microsphere, in which the intrinsic viscosity of the biocompatible polymer belongs to a high intrinsic viscosity region, may have an intrinsic viscosity of 0.7 dL / g or more and less than 2.0 dL / g. The biocompatible polymer contained in the first microsphere above may have an intrinsic viscosity of 0.1 dL / g or more, 0.2 dL / g or more, 0.3 dL / g or more, 0.4 dL / g or more, 0.5 dL / g or more, or 0.6 dL / g or more, and may be less than 0.2 dL / g, less than 0.3 dL / g, less than 0.4 dL / g, less than 0.5 dL / g, less than 0.6 dL / g, or less than 0.7 dL / g. The biocompatible polymer contained in the first microsphere may have an intrinsic viscosity of 0.1 dL / g or more and less than 0.2 dL / g, 0.2 dL / g or more and less than 0.3 dL / g, 0.3 dL / g or more and less than 0.4 dL / g, 0.4 dL / g or more and less than 0.5 dL / g, 0.5 dL / g or more and less than 0.6 dL / g, or 0.6 dL / g or more and less than 0.7 dL / g. The biocompatible polymer contained in the second microsphere above has an intrinsic viscosity of 0.7 dL / g or more, 0.8 dL / g or more, 0.9 dL / g or more, 1.0 dL / g or more, 1.1 dL / g or more, 1.2 dL / g or more, 1.3 dL / g or more, 1.4 dL / g or more, 1.5 dL / g or more, 1.6 dL / g or more, 1.7 dL / g or more, 1.8 dL / g or more, or 1.9 dL / g or more, and is less than 0.8 dL / g, less than 0.9 dL / g, less than 1.0 dL / g, less than 1.1 dL / g, less than 1.2 dL / g, less than 1.3 dL / g, less than 1.4 dL / g, or 1.5 dL / g It may be less than, less than 1.6 dL / g, less than 1.7 dL / g, less than 1.8 dL / g, less than 1.9 dL / g, or less than 2.0 dL / g.The biocompatible polymer contained in the second microsphere above has an intrinsic viscosity of 0.7 dL / g or more and less than 0.8 dL / g, 0.8 dL / g or more and less than 0.9 dL / g, 0.9 dL / g or more and less than 1.0 dL / g, 1.0 dL / g or more and less than 1.1 dL / g, 1.1 dL / g or more and less than 1.2 dL / g, 1.2 dL / g or more and less than 1.3 dL / g, 1.3 dL / g or more and less than 1.4 dL / g, 1.4 dL / g or more and less than 1.5 dL / g, 1.5 dL / g or more and less than 1.6 dL / g, 1.6 dL / g or more and less than 1.7 dL / g, and 1.7 dL / g or more and 1.8 dL / g It may be less than, 1.8 dL / g or more but less than 1.9 dL / g, or 1.9 dL / g or more but less than 2.0 dL / g.

[0133] In one embodiment, when the microsphere composition of the present invention comprises two types of microspheres, the weight ratio of the first microsphere to the second microsphere may be 1:0.1 to 1:10, 0.1:1 to 10:1, 1:0.5 to 1:5, 0.5:1 to 5:1, 1:1 to 1:3, or 1:1 to 3:1. If the proportion of low intrinsic viscosity microspheres is excessively high, the initial release may increase and the release period may be shortened, whereas conversely, if the proportion of high intrinsic viscosity microspheres is excessively high, the initial release may be insufficient and the release may be concentrated in the later stages.

[0134] In one embodiment, the microsphere composition of the present invention may include three types of microspheres in which the intrinsic viscosity of the biocompatible polymer belongs to a high intrinsic viscosity region, a medium intrinsic viscosity region, and a low intrinsic viscosity region, respectively. In this specification, "medium intrinsic viscosity region" refers to an intermediate intrinsic viscosity region. By adjusting the mixing ratio of the three types of microspheres, a release profile with a release period of one month or more can be achieved.

[0135] In one embodiment, when the microsphere composition of the present invention comprises three types of microspheres, the first microsphere, in which the intrinsic viscosity of the biocompatible polymer belongs to a low intrinsic viscosity region, may have an intrinsic viscosity of 0.1 dL / g or more and less than 0.5 dL / g, the second microsphere, in which the intrinsic viscosity of the biocompatible polymer belongs to a medium intrinsic viscosity region, may have an intrinsic viscosity of 0.5 dL / g or more and less than 0.7 dL / g, and the third microsphere, in which the intrinsic viscosity of the biocompatible polymer belongs to a high intrinsic viscosity region, may have an intrinsic viscosity of 0.7 dL / g or more and less than 2.0 dL / g. The biocompatible polymer contained in the first microsphere above may have an intrinsic viscosity of 0.1 dL / g or more, 0.2 dL / g or more, 0.3 dL / g or more, or 0.4 dL / g or more, and may be less than 0.2 dL / g, less than 0.3 dL / g, less than 0.4 dL / g, or less than 0.5 dL / g. The biocompatible polymer contained in the first microsphere above may have an intrinsic viscosity of 0.1 dL / g or more and less than 0.2 dL / g, 0.2 dL / g or more and less than 0.3 dL / g, 0.3 dL / g or more and less than 0.4 dL / g, or 0.4 dL / g or more and less than 0.5 dL / g. The biocompatible polymer contained in the second microsphere above may have an intrinsic viscosity of 0.5 dL / g or more, 0.6 dL / g or more, or 0.7 dL / g or more, and may be less than 0.6 dL / g or less than 0.7 dL / g. The biocompatible polymer contained in the second microsphere above may have an intrinsic viscosity of 0.5 dL / g or more and less than 0.6 dL / g, or 0.6 dL / g or more and less than 0.7 dL / g. The biocompatible polymer contained in the third microsphere above has an intrinsic viscosity of 0.7 dL / g or more, 0.8 dL / g or more, 0.9 dL / g or more, 1.0 dL / g or more, 1.1 dL / g or more, 1.2 dL / g or more, 1.3 dL / g or more, 1.4 dL / g or more, 1.5 dL / g or more, 1.6 dL / g or more, 1.7 dL / g or more, 1.8 dL / g or more, or 1.9 dL / g or more, and is less than 0.9 dL / g, 1.It may be less than 0 dL / g, less than 1.1 dL / g, less than 1.2 dL / g, less than 1.3 dL / g, less than 1.4 dL / g, less than 1.5 dL / g, less than 1.6 dL / g, less than 1.7 dL / g, less than 1.8 dL / g, less than 1.9 dL / g, or less than 2.0 dL / g. The biocompatible polymer contained in the third microsphere above has an intrinsic viscosity of 0.7 dL / g or more and less than 0.8 dL / g, 0.8 dL / g or more and less than 0.9 dL / g, 0.9 dL / g or more and less than 1.0 dL / g, 1.0 dL / g or more and less than 1.1 dL / g, 1.1 dL / g or more and less than 1.2 dL / g, 1.2 dL / g or more and less than 1.3 dL / g, 1.3 dL / g or more and less than 1.4 dL / g, 1.4 dL / g or more and less than 1.5 dL / g, 1.5 dL / g or more and less than 1.6 dL / g, 1.6 dL / g or more and less than 1.7 dL / g, and 1.7 dL / g or more and 1.8 dL / g It may be less than, 1.8 dL / g or more but less than 1.9 dL / g, or 1.9 dL / g or more but less than 2.0 dL / g.

[0136] In one embodiment, when the microsphere composition of the present invention comprises three types of microspheres, 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, 1:0.1:0.1 to 1:5:5, 1:0.1:0.1 to 1:3:3, 1:0.1:0.1 to 1:2:2, or 1:0.1:0.1 to 1:1:1. If the proportion of low intrinsic viscosity microspheres is excessively high, the initial release may increase and the release period may be shortened, whereas conversely, if the proportion of high intrinsic viscosity microspheres is excessively high, the initial release may be insufficient and the release may be concentrated in the later stages.

[0137]

[0138] Method for manufacturing microspheres

[0139] 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 method, a method for manufacturing microspheres by spray drying, a method for manufacturing microspheres by phase separation, etc.

[0140] 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.

[0141] Specifically, the solvent evaporation method may include, but is not limited to, methods described in U.S. Patents No. 5,271,945, No. 5,985,309 and No. 6,471,996. More specifically, the solvent evaporation method may include the steps of: dispersing a polymer compound in an organic solvent, dispersing or dispersing a drug in the organic solvent, and then emulsifying the mixture in a dispersion medium such as water to produce 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.

[0142] 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.

[0143] 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.

[0144] In one embodiment, the method for manufacturing microspheres of the present invention may include the steps of: preparing an oil-in-water (O / W) or solid-in-oil-in-water (S / O / W) emulsion comprising a biocompatible polymer, a GLP-1 RA-based peptide or a pharmaceutically acceptable salt thereof, and a solvent; and aggregating the emulsion into polymer microspheres by an O / W or solid-in-oil-in-water (S / O / W) solvent evaporation method.

[0145] 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 the GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof by aggregating the emulsion into polymer microspheres.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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 in an aqueous solvent, and an oil phase (O) in which a biocompatible polymer is dispersed 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.

[0155] 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.

[0156] 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.

[0157] 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:

[0158] (a) a step of preparing a GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof;

[0159] (b) a step of preparing a dispersed phase by dispersing the GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof prepared in step (a) and a biocompatible polymer in a dispersion solvent;

[0160] (c) a step of introducing the dispersed phase prepared in step (b) above into a continuous phase and stirring to form microspheres;

[0161] (d) a step of removing the dispersion solvent from the microspheres of step (c);

[0162] (e) a step of recovering the microspheres formed in step (d) above; and

[0163] (f) A step of drying the microspheres recovered in step (e) above.

[0164] In the above manufacturing method, the GLP-1 RA series peptide or its pharmaceutically acceptable salt and biocompatible polymer used are the same as those described above, so an explanation is omitted in this section.

[0165] 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.

[0166] Specifically, the formed salt may be selected from pamoate salt, acetate salt, benzathine salt, formate salt, and combinations thereof, but is not limited thereto.

[0167] 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 dispersed in a solvent in another vial. Subsequently, the semaglutide sodium salt solution is added to a benzathine diacetate solution to react, and then the semaglutide benzathine salt is crystallized to prepare the salt.

[0168] More specifically, the semaglutide benzathine salt can be prepared according to the following reaction scheme 1:

[0169] [Reaction Equation 1]

[0170]

[0171] 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.

[0172] More specifically, the tyrzepatide pamoate salt can be prepared according to the following reaction scheme 2:

[0173] [Reaction Equation 2]

[0174]

[0175] In one embodiment, step (b) can be performed by first dissolving a GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof in a solvent, and then dispersing a biocompatible polymer in a dispersion solvent.

[0176] In one embodiment, the dispersion solvent for dispersing 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.

[0177] In one embodiment, the dispersion solvent may be an organic solvent.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] In one embodiment, the organic solvent may be mixed with an organic acid solvent.

[0183] According to one embodiment of the present invention, step (b) may further include the step of adding and dispersing a bioavailability improver in a dispersion solvent.

[0184] 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. Specifically, it may be one or more selected from the group consisting of lysine, sodium acetate, and citric acid.

[0185] In one embodiment, the bioavailability improver may be included in an amount of 0.01% to 20% by weight, 0.01% to 15% by weight, or 0.01% to 10% by weight based on the total weight of the microsphere composition.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] Additionally, specifically, the surfactant may be added at a concentration of 0.1 to 3% (w / v) with respect to the continuous phase. More specifically, the surfactant may be added at a concentration of 0.3 to 2% (w / v) with respect to the continuous phase.

[0190] 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.

[0191] 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 acting as an osmotic regulator may be used to control the initial release in the aqueous solution.

[0192] 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.

[0193] Specifically, the emulsion may be maintained or stirred for 2 to 48 hours.

[0194] 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.

[0195] 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.

[0196] In one embodiment, between step (d) and step (e), the steps may further include a filtration and washing step to remove residual surfactant and a step to recover 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.

[0197] 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.

[0198] 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.

[0199] In one embodiment, the method for manufacturing microspheres may further include an extraction step after step (e) or after step (f).

[0200] In one embodiment, the extraction step may be performed using purified water, a buffer solution, an aqueous solution containing alcohols, ketones, esters, or inorganic salts, or a mixed solvent thereof, as the extraction solvent.

[0201] 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.

[0202] 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:

[0203] (1) A step of preparing an inner phase (W1) by dispersing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof in a dispersion solvent;

[0204] (2) A step of preparing an oil phase (O) by dispersing a biocompatible polymer in one or more solvents;

[0205] (3) A step of preparing an oil-in-water (W1 / O) type dispersed phase by dispersing an internal aqueous phase (W1) in the above oil phase (O);

[0206] (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

[0207] (5) Step of removing the above solvent.

[0208] In the above manufacturing method, since all conditions corresponding to the manufacturing method among the descriptions regarding the aforementioned microspheres and the method of manufacturing microspheres, such as the type of biocompatible polymer and intrinsic viscosity, can be applied, redundant details are omitted below.

[0209] In one embodiment, the inner aqueous phase (W1) of step (1) may be prepared by dispersing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof in distilled water.

[0210] 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.

[0211] In one embodiment, the water-in-oil (W1 / O) type dispersion phase of step (3) can be prepared by dispersing the internal aqueous 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 aqueous phase (W1) in step (3) may be 1:10 or less.

[0212] 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).

[0213] 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 a stable droplet within the outer continuous phase, such as a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer.

[0214] In one embodiment, the outer continuous phase (W2) of step (4) may be an aqueous solution of a hydrophilic polymer with a content of 0.1 to 6% (w / v), preferably 0.1 to 4% (w / v).

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220]

[0221] Method for preparing a microsphere composition

[0222] The method for preparing the microsphere composition of the present invention may include a method comprising the following steps:

[0223] (a) a step of preparing a dispersed phase by dispersing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer in a dispersion solvent;

[0224] (b) a step of introducing the dispersed phase prepared in step (a) into a continuous phase and stirring it to form microspheres;

[0225] (c) a step of removing the dispersion solvent from the microspheres formed in step (b) above;

[0226] (d) A step of mixing one or more types of microspheres each prepared by steps (a) to (c) above.

[0227] In the above manufacturing method, the microspheres used, the GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof, the biocompatible polymer, the dispersion solvent, the continuous phase, the stirring, and the removal of the dispersion solvent are the same as those described above, so an explanation is omitted in this part.

[0228] According to one embodiment of the present invention, step (a) may further include the step of adding and dispersing a bioavailability improver in a dispersion solvent. Since the type and amount of the bioavailability improver added in step (a) are the same as those described above, a description is omitted here.

[0229] In one embodiment, between steps (c) and (d), a filtration and washing step (step (e)) for removing residual dispersion solvent and a step (step (f)) for filtering again to recover microspheres may be further included. Specifically, the washing step may be performed using water, but is not limited thereto, and may be repeated several times as needed.

[0230] In one embodiment, in step (f), the method of recovering the microspheres may be performed using conventional techniques known in the art. Specifically, the recovery of the microspheres may be performed using methods such as filtration or centrifugation, but is not limited thereto.

[0231] In one embodiment, after step (f), the method may further include a step (step (g)) of drying the microspheres obtained in step (f) to recover the dried microspheres. Specifically, the drying of the microspheres in step (g) may be performed using a conventional drying method.

[0232] In one embodiment, the method for manufacturing microspheres may further include an extraction step after step (f) or after step (g).

[0233]

[0234] Pharmaceutical composition containing microspheres

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] In one embodiment, the pharmaceutical composition of the present invention may contain microspheres in an amount of 0.01 mg to 5,000 mg per 1 mL of carrier.

[0240] Specifically, the pharmaceutical composition may contain microspheres in an amount of 0.01 mg to 5,000 mg, 0.01 mg to 4,900 mg, 0.01 mg to 4,800 mg, 0.01 mg to 4,600 mg, 0.01 mg to 4,500 mg, 0.01 mg to 4,400 mg, 0.01 mg to 4,300 mg, 0.01 mg to 4,000 mg, 0.01 mg to 3,000 mg, 0.01 mg to 2,000 mg, and 0.01 mg to 1,000 mg per 1 mL of carrier.

[0241] In one embodiment, the pharmaceutically acceptable carrier 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] In one embodiment, the pharmaceutically acceptable carrier may further include a diluent.

[0247] In one embodiment, the pharmaceutically acceptable carrier may further include a carrier for parenteral administration.

[0248] 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.

[0249] More specifically, the stabilizer may be an antioxidant such as sodium bisulfite, sodium sulfite, or ascorbic acid, but is not limited thereto.

[0250] Also, more specifically, the preservative may be benzalkonium chloride, methyl- or propyl-paraben, or chlorobutanol, but is not limited thereto.

[0251] In one embodiment, the pharmaceutical composition of the present invention may further include a tissue diffusion promoter.

[0252] Specifically, the tissue diffusion promoter may be one or more from the group consisting of hyaluronidase, chondroitinase, and heparinase, but preferably is hyaluronidase.

[0253] 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.

[0254] Specifically, when the tissue diffusion promoter is hyaluronidase, the hyaluronidase may be used by being contained in a pharmaceutically acceptable carrier or provided separately from the pharmaceutically acceptable carrier. In this case, the hyaluronidase is provided in the form of a dispersed aqueous solution for injection and may be mixed with a formulation containing a GLP-1 RA immediately before administration to form a composition suitable for subcutaneous administration.

[0255] In one embodiment, hyaluronidase may be provided in powder form, but is not limited thereto.

[0256] In one embodiment, the pharmaceutical composition may further comprise a GLP-1 RA family peptide or a pharmaceutically acceptable salt thereof.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] Specifically, the pharmaceutically acceptable carrier, excipient, and diluent may not disperse the biocompatible polymer forming the microspheres. More specifically, the pharmaceutically acceptable carrier, excipient, and diluent may not disperse 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.

[0267] 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.

[0268] For other pharmaceutically acceptable carriers and formulations, refer to the contents described in Remington's PHARMACEUTICAL Sciences (19th ed., Mack Publishing Company, Easton, PA, 1995), a known publication in the pharmaceutical field.

[0269] In one embodiment, the pharmaceutical composition may be formulated into various forms of preparations, for example, as a parenteral administration preparation.

[0270] Specifically, the parenteral formulation may be formulated in the form of an injection, cream, lotion, topical ointment, oil, moisturizer, aerosol, patch, or nasal inhalant 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.

[0271] In one embodiment, the pharmaceutical composition may be administered to mammals, including humans, in various ways, for example, parenterally.

[0272] 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.

[0273] Examples

[0274] In this embodiment, the following abbreviations are defined to represent the following terms.

[0275] IV: Intrinsic Viscosity

[0276] HPLC: High Performance Liquid Chromatography

[0277] LC-MS / MS: Liquid Chromatography-Tandem Mass Spectrometry

[0278] NMR: Nuclear Magnetic Resonance Spectroscopy

[0279] ACN: Acetonitrile

[0280] DCM: Dichloromethane

[0281] NaOH: Sodium hydroxide

[0282] PBS: Phosphate buffered saline

[0283] PLGA: Poly(lactic-co-glycolic acid)

[0284] PVA: Polyvinyl alcohol

[0285] TFA: Trifluoroacetic Acid

[0286]

[0287] In addition, in this embodiment, regarding the intrinsic viscosity of the PLGA, "first intrinsic viscosity" means an intrinsic viscosity of 0.1 dL / g or more and less than 0.2 dL / g, "second intrinsic viscosity" means an intrinsic viscosity of 0.2 dL / g or more and less than 0.3 dL / g, and "third intrinsic viscosity" means an intrinsic viscosity of 0.3 dL / g or more and less than 0.4 dL / g. In the same manner below, "4th intrinsic viscosity" is 0.4 dL / g or more and less than 0.5 dL / g, "5th intrinsic viscosity" is 0.5 dL / g or more and less than 0.6 dL / g, "6th intrinsic viscosity" is 0.6 dL / g or more and less than 0.7 dL / g, "7th intrinsic viscosity" is 0.7 dL / g or more and less than 0.8 dL / g, "8th intrinsic viscosity" is 0.8 dL / g or more and less than 0.9 dL / g, "9th intrinsic viscosity" is 0.9 dL / g or more and less than 1.0 dL / g, "10th intrinsic viscosity" is 1.0 dL / g or more and less than 1.1 dL / g, "11th intrinsic viscosity" is 1.1 dL / g or more and less than 1.2 dL / g, "12 "Intrinsic viscosity" refers to an intrinsic viscosity of 1.2 dL / g or more and less than 1.3 dL / g, "13th intrinsic viscosity" refers to an intrinsic viscosity of 1.3 dL / g or more and less than 1.4 dL / g, "14th intrinsic viscosity" refers to an intrinsic viscosity of 1.4 dL / g or more and less than 1.5 dL / g, "15th intrinsic viscosity" refers to an intrinsic viscosity of 1.5 dL / g or more and less than 1.6 dL / g, "16th intrinsic viscosity" refers to an intrinsic viscosity of 1.6 dL / g or more and less than 1.7 dL / g, "17th intrinsic viscosity" refers to an intrinsic viscosity of 1.7 dL / g or more and less than 1.8 dL / g, "18th intrinsic viscosity" refers to an intrinsic viscosity of 1.8 dL / g or more and less than 1.9 dL / g, and "19th intrinsic viscosity" refers to an intrinsic viscosity of 1.9 dL / g or more and less than 2.0 dL / g.

[0288]

[0289] The present invention will be explained in more detail below through the following examples and experimental examples.

[0290]

[0291] Example 1-18: Preparation of microspheres containing semaglutide benzathine salt (1)

[0292] a. Preparation of Semaglutide Benzatin Salt

[0293] Semaglutide (Manufacturer: JYMed) ® ) 200 mg and 1 mL of distilled water were added to a vial and dispersed by stirring. In another vial, benzathine diacetate (Manufacturer: TCI ® 200 mg of ) and 1 mL of distilled water were added and stirred to disperse. 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.

[0294] b. Preparation of microspheres containing semaglutide benzathine salt

[0295] Preparation of the dispersed phase

[0296] Weighed semaglutide benzathine salt prepared in 'a. Preparation of semaglutide benzathine salt' and acetic acid were added to a 50 mL vial and dispersed by stirring at 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at 300 rpm for 20 minutes to prepare a dispersed phase.

[0297] The usage ratio of each solvent in the dispersion solvent used to prepare the dispersed phase, the usage amounts of semaglutide benzathine salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 1.

[0298] Continuous phase manufacturing

[0299] 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.

[0300] Preparation and Filtration of Microspheres

[0301] The prepared dispersed phase was taken into 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.

[0302] Washing and Drying of Microspheres

[0303] 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.

[0304] No. Acetic acid: DCM solvent Bisemaglutide benzathine salt (g) PLGA (g) PLGA IV Example 11: 1.50.156 41.4 Third intrinsic viscosity Example 21: 1.50.17 71.238 Third intrinsic viscosity Example 31: 1.50.24 71.4 Third intrinsic viscosity Example 41: 1.50.35 1.4 Third intrinsic viscosity Example 51: 1.50.1160.66 Eighth intrinsic viscosity Example 61: 1.90.1160.66 Third intrinsic viscosity Example 71: 1.90.1160.66 Eighth intrinsic viscosity Example 81: 1.90.20 71.173 Eighth intrinsic viscosity Example 91: 1.90.20 71.173 Eighth Intrinsic Viscosity Example 101: 20.23 31.32 Fifth Intrinsic Viscosity Example 111: 20.128 1.155 Fifth Intrinsic Viscosity Example 121: 20.128 1.15 50.60 - 0.80 Example 131: 20.128 1.155 Fifth Intrinsic Viscosity Example 141: 2.5 0.56 31.313 Third Intrinsic Viscosity Example 151: 2.5 0.21 30.965 Third Intrinsic Viscosity Example 161: 50.24 71.4 Third Intrinsic Viscosity Example 171: 90.11 60.66 Third Intrinsic Viscosity Example 181: 90.14 50.66 Third Intrinsic Viscosity

[0305]

[0306] Examples 94-100: Preparation of microspheres containing semaglutide benzathine salt (2)

[0307] The steps of preparing semaglutide benzathine salt, preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to those in Examples 1-18, and only the <preparation of dispersed phase> step was performed as follows.

[0308] <Preparation of Dispersed Phase>

[0309] In a 50 mL vial, the semaglutide benzathine salt prepared in 'a. Preparation of semaglutide benzathine salt' of Example 1-18 and acetic acid were weighed and added, and then dispersed 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.

[0310] The usage ratio of each solvent in the dispersion solvent used to prepare the dispersed phase, the usage amounts of semaglutide benzathine salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 2.

[0311] No. Acetic acid: DCM solvent Bisemaglutide benzathine salt (g) PLGA IV PLGA (g) Example 941: 1.5 0.884 3rd intrinsic viscosity + 6th intrinsic viscosity 4.6 + 0.4 Example 951: 1.9 0.149 6th intrinsic viscosity 1.34 Example 961: 1.9 0.65 8th intrinsic viscosity 5.865 Example 971: 1.9 0.331 2nd intrinsic viscosity + 6th intrinsic viscosity 0.2 + 1.56 Example 981: 1.9 0.52 2nd intrinsic viscosity + 8th intrinsic viscosity 0.426 + 4.266 Example 991: 1.9 0.207 6th intrinsic viscosity 1.173 Example 1001: 1.9 0.207 5th intrinsic viscosity 1.173

[0312] (Note) Examples 94, 97, and 98 used two types of PLGA.

[0313]

[0314] Example 101: Preparation of Semaglutide Benzathine Salt Microspheres Including an Extraction Step

[0315] The steps of preparing semaglutide benzathine salt, preparing the continuous phase, preparing microspheres, filtration, and drying were performed identically to Examples 1-18, and <preparation of dispersed phase>, <extraction>Only the steps were performed as follows.

[0316] Preparation of the dispersed phase

[0317] 0.207 g of semaglutide benzathine salt prepared in 'a. Preparation of semaglutide benzathine salt' of Example 1-18 and acetic acid were weighed and added to a 50 mL vial, and then dispersed by stirring at 150 rpm. Subsequently, 1.173 g of PLGA (intrinsic viscosity 6), a biocompatible polymer, and DCM were added to the vial, and the mixture was stirred at 300 rpm for 20 minutes to prepare a dispersed phase. The ratio of each solvent in the dispersion solvent used to prepare the dispersed phase was acetic acid:DCM = 1:1.9.

[0318] Continuous phase manufacturing

[0319] 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.

[0320] Preparation and Filtration of Microspheres

[0321] The prepared dispersed phase was taken into 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.

[0322] Washing and Drying of Microspheres

[0323] 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.

[0324] <Extraction>

[0325] 500 mL of distilled water and 0.11 g of Na2HPO4 were weighed and added to a reactor. Subsequently, a 0.022% (w / v) Na2HPO4 solution was prepared by stirring at a speed of 500 rpm using an overhead stirrer. The microspheres dried in the <Washing and Drying of Microspheres> step were placed in the reactor and stirred for 60 minutes, after which the mixture was filtered through a 5 μm membrane filter to obtain semaglutide benzathine salt microspheres. The microspheres were washed repeatedly with distilled water several times, and the obtained microspheres were freeze-dried for more than 20 hours.

[0326]

[0327] Example 102: Preparation of semaglutide acetate microspheres including an extraction step

[0328] The steps of preparing semaglutide acetate, preparing the continuous phase, preparing microspheres, filtration, and drying were performed identically to Examples 50-55, and <preparation of dispersed phase>, <extraction>Only the steps were performed as follows.

[0329] Preparation of the dispersed phase

[0330] 0.207 g of semaglutide acetate prepared in 'a. Preparation of semaglutide acetate' of Examples 50-55 and acetic acid were weighed and added to a 50 mL vial, and then dispersed by stirring at 150 rpm. Subsequently, 1.173 g of PLGA (intrinsic viscosity 6), a biocompatible polymer, and DCM were added to the vial, and the mixture was stirred at 300 rpm for 20 minutes to prepare a dispersed phase. The ratio of each solvent in the dispersion solvent used to prepare the dispersed phase was acetic acid:DCM = 1:1.9.

[0331] Continuous phase manufacturing

[0332] 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.

[0333] Preparation and Filtration of Microspheres

[0334] The prepared dispersed phase was taken into 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, semaglutide acetate microspheres were obtained by filtering through a 5 μm membrane filter.

[0335] Washing and Drying of Microspheres

[0336] 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.

[0337] <Extraction>

[0338] 500 mL of distilled water and 0.11 g of Na2HPO4 were weighed and added to a reactor. Subsequently, a 0.022% (w / v) Na2HPO4 solution was prepared by stirring at a speed of 500 rpm using an overhead stirrer. The microspheres dried in the <Washing and Drying of Microspheres> step were placed in the reactor and stirred for 60 minutes, after which semaglutide acetate microspheres were obtained by filtering through a 5 μm membrane filter. The microspheres were washed repeatedly with distilled water several times, and the obtained microspheres were freeze-dried for more than 20 hours.

[0339]

[0340] Examples 19-49: Preparation of microspheres containing semaglutide benzathine salt and a bioavailability improver

[0341] The steps of preparing semaglutide benzathine salt, preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to those in Examples 1-18, and only the <preparation of dispersed phase> step was performed as follows.

[0342] <Preparation of Dispersed Phase>

[0343] In a 50 mL 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 dispersed by stirring at 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial, and the mixture was stirred at 300 rpm for 20 minutes to prepare a dispersed phase.

[0344] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 3, 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 4.

[0345] 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 Sodium Acetate 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

[0346] (Note) Example 29 used two types of bioavailability improvers.

[0347] No. Acetic acid: DCM solvent Bisemaglutide benzathine salt (g) PLGA IV PLGA (g) Example 191: 1.5 0.15 64 Third intrinsic viscosity 1.4 Example 201: 1.5 0.247 Third intrinsic viscosity 1.4 Example 211: 1.5 0.247 Third intrinsic viscosity 1.4 Example 221: 20.147 Fifth intrinsic viscosity 1.32 Example 231: 20.233 Fifth intrinsic viscosity 1.32 Example 241: 2.5 0.146 Third intrinsic viscosity 1.313 Example 251: 2.5 0.23 Fifth intrinsic viscosity 1.05 Example 261: 1.3 0.116 Third intrinsic viscosity 0.66 Example 271: 50.247 Third Intrinsic Viscosity 1.4 Example 281: 50.247 Third Intrinsic Viscosity 1.4 Example 291: 1.50.247 Third Intrinsic Viscosity 1.4 Example 301: 1.50.247 Third Intrinsic Viscosity 1.4 Example 311: 1.50.1564 Third Intrinsic Viscosity 1.4 Example 321: 1.50.116 Eighth Intrinsic Viscosity 0.66 Example 331: 1.50.346 Second Intrinsic Viscosity 1.96 Example 341: 1.30.116 Third Intrinsic Viscosity 0.66 Example 351: 50.247 Third Intrinsic Viscosity 1.4 Example 361: 50.247 Third Intrinsic Viscosity 1.4 Example 371: 1.50.1564 Third Intrinsic viscosity 1.4 Example 381: 50.247 Third intrinsic viscosity 1.4 Example 391: 1.50.217 Second intrinsic viscosity + Fifth intrinsic viscosity 0.615 + 0.615 Example 401: 20.165 Third intrinsic viscosity + Fifth intrinsic viscosity 0.743 + 0.743 Example 411: 20.233 Third intrinsic viscosity + Fifth intrinsic viscosity 0.264 + 1.056 Example 421: 50.247 Third intrinsic viscosity + Fifth intrinsic viscosity 1.12 + 0.28 Example 431: 1.50.247 Second intrinsic viscosity + Third intrinsic viscosity 0.93 + 0.47 Example 441: 1.50.247 Second intrinsic viscosity + Third intrinsic viscosity 0.84 + 0.56 Example 451: 1.50.247 Second intrinsic viscosity + Third intrinsic viscosity 0.7 + 0.7 Example 461: 1.50.247 Second intrinsic viscosity + Fifth intrinsic viscosity 0.224 + 0.896 Example 471: 1.50.247 Second intrinsic viscosity + Third intrinsic viscosity 0.23 + 1.17 Example 481: 2.50.247 Third intrinsic viscosity + 0.60 - 0.80 0.898 + 0.449 Example 491: 2.50.21 70.60 - 0.80 1.347

[0348] (Note) Examples 39 to 48 used two types of PLGA.

[0349]

[0350] Examples 50-55: Preparation of microspheres containing semaglutide acetate salt

[0351] a. Preparation of Semaglutide Acetate Salt

[0352] Semaglutide (Manufacturer: JYMed) ® 100 mg of ) and 1 mL of acetic acid were placed in a vial and dispersed 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.

[0353] b. Preparation of microspheres containing semaglutide acetate salt

[0354] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to Examples 1-18, and only the <preparation of the dispersed phase> step was performed as follows.

[0355] <Preparation of Dispersed Phase>

[0356] Weighed semaglutide acetate salt and acetic acid prepared in 'a. Preparation of semaglutide acetate salt' described above were added to a 50 mL vial and dispersed 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.

[0357] 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.

[0358] No. Acetic acid:DCM solvent bisemaglutide acetate salt (g)PLGA (g)PLGA IV Example 501: 1.20.26 1.176 Third intrinsic viscosity Example 511: 2.50.146 1.072 Third intrinsic viscosity Example 521: 1.40.208 1.176 Third intrinsic viscosity Example 531: 1.70.118 0.67 Third intrinsic viscosity Example 541: 1.70.116 0.66 Eighth intrinsic viscosity Example 551: 1.90.207 1.173 Eighth intrinsic viscosity

[0359]

[0360] Examples 56-66: Preparation of microspheres containing semaglutide acetate salt and bioavailability improver

[0361] The steps of preparing semaglutide acetate salt, preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to those in Examples 50-55, and only the <preparation of dispersed phase> step was performed as follows.

[0362] <Preparation of Dispersed Phase>

[0363] In a 50 mL 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 dispersed by stirring at 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial, and the mixture was stirred at 300 rpm for 20 minutes to prepare a dispersed phase.

[0364] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 6, 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 7.

[0365] 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

[0366] No. Acetic acid:DCM solvent bisemaglutide acetate salt (g) PLGA IV PLGA (g) Example 561: 1.50.187 Third intrinsic viscosity 1.68 Example 571: 1.50.187 Third intrinsic viscosity 1.68 Example 581: 1.10.208 Third intrinsic viscosity 1.176 Example 591: 1.20.236 Third intrinsic viscosity 1.34 Example 601: 1.50.3128 Third intrinsic viscosity 2.8 Example 611: 1.50.494 Third intrinsic viscosity 2.8 Example 621: 1.50.247 Third intrinsic viscosity 1.4 Example 631: 1.70.118 Third intrinsic viscosity 0.67 Example 641: 1.50.247 Second intrinsic viscosity + Third Intrinsic viscosity 0.23 + 1.17 Example 651: 1.50.247 Second intrinsic viscosity + Third intrinsic viscosity 0.14 + 1.26 Example 661: 1.50.217 Second intrinsic viscosity + Fifth intrinsic viscosity 0.615 + 0.615

[0367] (Note) Examples 64 to 66 used two types of PLGA.

[0368]

[0369] Example 67: Preparation of microspheres containing semaglutide formate

[0370] a. Preparation of Semaglutide Formate

[0371] Semaglutide (Manufacturer: JYMed) ® 500 mg of ) and 5 mL of distilled water were placed in a vial and dispersed by stirring. 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.

[0372] b. Preparation of microspheres containing semaglutide formate

[0373] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to Examples 1-18, and only the <preparation of the dispersed phase> step was performed as follows.

[0374] <Preparation of Dispersed Phase>

[0375] Semaglutide formate and formic acid prepared in 'a. Preparation of semaglutide formate' described above were weighed and added to a 50 mL vial, and then dispersed 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.

[0376] The usage ratio of each solvent in the dispersion solvent used to prepare the dispersed phase, the usage amounts of semaglutide formate and PLGA, and the intrinsic viscosity of PLGA are as described in Table 8.

[0377] No. Formic acid:DCM solvent bisemaglutide formate (g)PLGA (g)PLGA IV Example 671: 1.55 0.146 1.313 3rd intrinsic viscosity

[0378]

[0379] Example 68: Preparation of Semaglutide Pamoate Salt

[0380] Semaglutide (Manufacturer: JYMed) ® 100 mg of ) and 1 mL of water were placed in a vial and dispersed by stirring. 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.

[0381]

[0382] Examples 69-71: Preparation of microspheres containing tyrzepatide benzathine salt

[0383] a. Preparation of tyrzepatide benzathine salt

[0384] 400 mg of tyrzepatide (Manufacturer: Zhejiang Peptide Biotech Co., Ltd) and 1 mL of distilled water were placed in a vial and dispersed by stirring. In another vial, benzathine diacetate (Manufacturer: TCI ® 200 mg of ) and 1 mL of distilled water were added and dispersed by stirring. Once the solution was completely dissolved, the tyrzepatide solution was slowly added dropwise to the benzathine diacetate solution using a syringe to obtain tyrzepatide benzathine salt. The obtained tyrzepatide benzathine salt was washed with water and dried at 25°C for at least 20 hours.

[0385] b. Preparation of microspheres containing tyrzepatide benzathine salt

[0386] The steps of preparing, filtration, and drying of microspheres were performed in the same way as in Examples 1-18, and the steps of <continuous phase preparation> and <dispersed phase preparation> were performed as follows.

[0387] Continuous phase manufacturing

[0388] 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.

[0389] <Preparation of Dispersed Phase>

[0390] Weighed and added the tyrzepatide benzathine salt and acetic acid prepared in 'a. Preparation of tyrzepatide benzathine salt' described above to a 50 mL vial, and then dispersed 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.

[0391] 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 9.

[0392] No. Acetic acid:DCM solvent bityrzepatide benzathine salt (g)PLGA (g)PLGA IV Example 691: 1.5 0.28 9 1.155 3rd intrinsic viscosity Example 701: 1.5 0.38 5 1.155 3rd intrinsic viscosity Example 711: 20.21 5 0.5 5th intrinsic viscosity

[0393]

[0394] Examples 72-79: Preparation of microspheres containing tyrzepatide benzathine salt and a bioavailability improver

[0395] The steps of preparing the tyrzepatide benzathine salt, preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to those in Examples 69-71, and only the <preparation of the dispersed phase> step was performed as follows.

[0396] <Preparation of Dispersed Phase>

[0397] In a 50 mL 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 dispersed by stirring at 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial, and the mixture was stirred at 300 rpm for 20 minutes to prepare a dispersed phase.

[0398] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 10, 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 11.

[0399] 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

[0400] No. Acetic acid: DCM solvent Vitirzepatide benzathine salt (g) PLGA IV PLGA (g) Example 721: 1.5 0.289 Third intrinsic viscosity 1.155 Example 731: 1.5 0.289 Third intrinsic viscosity 1.155 Example 741: 1.5 0.385 Third intrinsic viscosity 1.155 Example 751: 20.26 Sixth intrinsic viscosity 0.605 Example 761: 2.5 0.377 Sixth intrinsic viscosity 0.88 Example 771: 2.5 0.474 Sixth intrinsic viscosity 0.88 Example 781: 1.5 0.385 Third intrinsic viscosity + Fifth intrinsic viscosity 0.42 + 0.42 Example 791: 2.5 0.377 Third intrinsic viscosity + Fifth intrinsic viscosity 0.44 + 0.44

[0401] (Note) Examples 78 to 79 used two types of PLGA.

[0402]

[0403] Examples 80-82: Preparation of microspheres containing tyrzepatide acetate salt

[0404] a. Preparation of tyrzepatide acetate salt

[0405] 500 mg of tyrzepatide (Manufacturer: Zhejiang Peptide Biotech Co., Ltd) and 2 mL of acetic acid were placed in a vial and dispersed 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.

[0406] b. Preparation of microspheres containing tyrzepatide acetate salt

[0407] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to Examples 69-71, and only the <preparation of the dispersed phase> step was performed as follows.

[0408] <Preparation of Dispersed Phase>

[0409] Weighed and added the tyrzepatide acetate salt prepared in 'a. Preparation of tyrzepatide acetate salt' and methanol to a 50 mL vial, and then dispersed 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.

[0410] The usage ratio of each solvent in the dispersion solvent used to prepare the dispersed phase, the usage amounts of tyrzepatide acetate salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 12.

[0411] No. Acetic acid: DCM solvent bityrzepatide acetate (g) PLGA (g) PLGA IV Example 801: 1.5 0.28 9 1.155 Third intrinsic viscosity Example 811: 1.5 0.38 5 1.155 Third intrinsic viscosity No. Methanol: DCM solvent bityrzepatide acetate (g) PLGA (g) PLGA IV (dL / g) Example 821: 1.5 0.11 0.33 + 0.67 Second intrinsic viscosity + Third intrinsic viscosity

[0412] (Note) Example 82 used two types of PLGA.

[0413]

[0414] Examples 83-84: Preparation of microspheres comprising tyrzepatide acetate salt and bioavailability improver

[0415] The steps of preparing the tyrzepatide acetate salt, preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to those in Examples 80-82, and only the <preparation of the dispersed phase> step was performed as follows.

[0416] <Preparation of Dispersed Phase>

[0417] In a 50 mL 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 dispersed by stirring at 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial, and the mixture was stirred at 300 rpm for 20 minutes to prepare a dispersed phase.

[0418] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 13, and the usage ratio of each solvent in the dispersion solvent used in the preparation of the dispersed phase, the amounts of tyrzepatide acetate salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 14.

[0419] No. Bioavailability Improver Bioavailability Improver (mg) Example 83 Citric Acid 3.7 Example 84 Citric Acid 2.6

[0420] No. Methanol: DCM solvent Vitalzepatide acetate (g) PLGA IV PLGA (g) Example 831: 1.5 0.289 Third intrinsic viscosity 1.155 Example 841: 1.5 0.3 Third intrinsic viscosity 0.7

[0421]

[0422] Example 85: Preparation of microspheres containing tyrzepatide formate

[0423] a. Preparation of tyrzepatide formate

[0424] 500 mg of tircepate (manufacturer: Zhejiang Peptide Biotech Co., Ltd) and 5 mL of distilled water were placed in a vial and dispersed 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.

[0425] b. Preparation of microspheres containing tyrzepatide formate

[0426] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to Examples 69-71, and only the <preparation of the dispersed phase> step was performed as follows.

[0427] <Preparation of Dispersed Phase>

[0428] Weighed tyrzepatide formate and acetic acid prepared in 'a. Preparation of tyrzepatide formate' were added to a 50 mL vial and dispersed by stirring at 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at 300 rpm for 20 minutes to prepare a dispersed phase.

[0429] The usage ratio of each solvent in the dispersion solvent used to prepare the dispersed phase, the usage amounts of tyrzepatide formate and PLGA, and the intrinsic viscosity of PLGA are as described in Table 15.

[0430] No. Acetic acid:DCM solvent bityrzepatide formate (g)PLGA (g)PLGA IV Example 851: 1.5 0.1 46 1.3 13 Intrinsic viscosity of the third

[0431]

[0432] Examples 86-88: Preparation of microspheres containing tyrzepatide formate and a bioavailability improver

[0433] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to those in Example 85, and only the <preparation of the dispersed phase> step was performed as follows.

[0434] <Preparation of Dispersed Phase>

[0435] In a 50 mL 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 dispersed by stirring at 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at 300 rpm to prepare a dispersed phase.

[0436] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 16, 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 17.

[0437] No. Bioavailability Improver Bioavailability Improver (mg) Example 86 Citric Acid 3.7 Example 87 Citric Acid 3.9 Example 88 Citric Acid 2.2

[0438] No. Acetic acid:DCM solvent bityrzepatide formate (g)PLGA (g)PLGA IV Example 861: 1.5 0.28 9 1.155 3rd intrinsic viscosity Example 871: 1.5 0.38 5 1.155 3rd intrinsic viscosity Example 881: 20.26 0.605 6th intrinsic viscosity

[0439]

[0440] Example 89: Preparation of Tircepate Pamoate Salt

[0441] 400 mg of tyrzepatide (Manufacturer: Zhejiang Peptide Biotech Co., Ltd) and 1 mL of DMSO were placed in a vial and dispersed by stirring. 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.

[0442]

[0443] Comparative Example 1: Preparation of microspheres containing semaglutide sodium salt

[0444] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to Examples 1-18, and only the <preparation of the dispersed phase> step was performed as follows.

[0445] <Preparation of Dispersed Phase>

[0446] In a 50 mL vial, the aforementioned semaglutide sodium salt (Manufacturer: JYMed ® After weighing and adding ) and formic acid, the mixture was dispersed by stirring at 150 rpm for 20 minutes. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at 300 rpm to prepare a dispersed phase.

[0447] The usage ratio of each solvent in the dispersion solvent used to prepare the dispersed phase, the usage amounts of semaglutide sodium salt and PLGA, and the intrinsic viscosity of PLGA are as described in Table 18.

[0448] No. Formic acid:DCM solvent sodium bisemaglutide (g)PLGA (g)PLGA IV Comparative Example 11: 1.2 0.09 20.8 25 3rd intrinsic viscosity

[0449]

[0450] Comparative Example 2-6: Preparation of microspheres containing semaglutide sodium salt

[0451] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to Examples 1-18, and only the <preparation of the dispersed phase> step was performed as follows.

[0452] <Preparation of Dispersed Phase>

[0453] Semaglutide sodium salt in a 50 mL vial (Manufacturer: JYMed ® After weighing and adding ) and acetic acid, the mixture was dispersed by stirring at 150 rpm for 20 minutes. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at 300 rpm to prepare a dispersed phase.

[0454] 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 19.

[0455] No. Acetic acid:DCM solvent sodium bisema glutide (g) PLGA IV PLGA (g) Comparative Example 21: 1.55 0.1564 3rd intrinsic viscosity 1.4 Comparative Example 31: 1.9 0.414 8th intrinsic viscosity 2.346 Comparative Example 41: 20.147 5th intrinsic viscosity 1.32 Comparative Example 51: 20.116 3rd intrinsic viscosity 0.66 Comparative Example 61: 20.256 5th intrinsic viscosity 2.31

[0456]

[0457] Comparative Examples 7-12: Preparation of microspheres containing semaglutide sodium salt and a bioavailability improver

[0458] The steps of preparing semaglutide sodium salt, preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to Comparative Examples 2-6, and only the <preparation of dispersed phase> step was performed as follows.

[0459] <Preparation of Dispersed Phase>

[0460] Sodium semaglutide, a bioavailability improver, and acetic acid were weighed and added to a 50 mL vial, and then dispersed by stirring at 150 rpm. Subsequently, biocompatible polymers PLGA and DCM were added to the vial and stirred at 300 rpm to prepare a dispersed phase.

[0461] The types and amounts of bioavailability improvers used in the preparation of the dispersed phase are as described in Table 20, and the usage ratio 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 21.

[0462] 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

[0463] No. Acetic acid: DCM solvent sodium bisemaglutide (g) PLGA IV PLGA (g) Comparative Example 71: 1.5 0.1564 3rd intrinsic viscosity 1.4 Comparative Example 81: 1.5 0.1564 3rd intrinsic viscosity 1.4 Comparative Example 91: 1.5 0.1564 3rd intrinsic viscosity 1.4 Comparative Example 101: 1.5 0.247 3rd intrinsic viscosity 1.4 Comparative Example 111: 20.147 5th intrinsic viscosity 1.32 Comparative Example 121: 20.147 5th intrinsic viscosity 1.49

[0464]

[0465] Comparative Example 13: Preparation of microspheres containing tyrzepatide sodium salt

[0466] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to Examples 69-71, and only the <preparation of the dispersed phase> step was performed as follows.

[0467] <Preparation of Dispersed Phase>

[0468] Sodium tyrzepatide salt and acetic acid were weighed and added to a 50 mL vial, and then dispersed 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.

[0469] 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.

[0470] No. Acetic acid:DCM solvent sodium vityrzepatide (g)PLGA (g)PLGA IV Comparative Example 131: 1.5 0.28 9 1.15 5 3 Intrinsic viscosity

[0471]

[0472] Comparative Example 14: Preparation of microspheres containing tyrzepatide sodium salt

[0473] The steps of preparing the continuous phase, preparing the microspheres, filtration, and drying were performed identically to Examples 69-71, and only the <preparation of the dispersed phase> step was performed as follows.

[0474] <Preparation of Dispersed Phase>

[0475] Tircepate sodium salt and methanol were weighed and added to a 50 mL vial, and then dispersed 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.

[0476] 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 23.

[0477] No. Methanol:DCM solvent sodium vityrzepatide (g)PLGA (g)PLGA IV Comparative Example 141: 1.54 0.11 0.33 + 0.67 2nd intrinsic viscosity + 3rd intrinsic viscosity

[0478] (Note) Comparative Example 14 used two types of PLGA.

[0479]

[0480] Experimental Example 1: Salt Crystal Analysis of Semaglutide and Tircepate

[0481] In order to measure the nuclear magnetic resonance (NMR) spectra of the semaglutide acetate salt, semaglutide benzathine salt, semaglutide formate salt, tyrzepatide acetate salt, tyrzepatide benzathine salt, tyrzepatide formate salt, and tyrzepatide pamoate salt prepared in the present invention, the following tests were conducted.

[0482] 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.

[0483] of the above semaglutide acetate salt 1 H-NMR analysis results of semaglutide benzathine 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, the tyrzepatide acetate, tyrzepatide benzathine, tyrzepatide formate, and tyrzepatide pamoate salts 1 The results of the H-NMR analysis are shown in Figures 4 to 7, respectively.

[0484] From Figures 1 to 7 above, the structure of each salt-type compound prepared according to the present invention was confirmed.

[0485]

[0486] Experimental Example 2: In vitro release test and evaluation of initial release rate of microspheres containing semaglutide salt

[0487] 25 mg of microspheres from Examples 1 to 67 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 a UV-Vis detector 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.

[0488] In addition, 15 mg of microspheres prepared in Examples 1 to 67 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 and maintained at 37°C for a certain period of time. To measure the release amount after a certain period of time, the entire supernatant was taken and filtered through a 0.45 μm RC filter, and the filtered liquid was used as the test solution. The test solution was detected using an HPLC (equipment name: Agilent 1260) with an ultraviolet-visible absorbance detector. Column packing was performed with L1, an internal diameter of 4.6 mm x 150 mm, and a thickness of 3.5 μm.

[0489] The above experimental results are shown in Table 24.

[0490] 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 1 110.7 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 2 0 15.2 14.0 Example 2 114.7 11.4 Example 2 29.43.6 Example 23 14.8 22.0 Example 24 8.0 0.0 Example 25 18.3 18.5 Example 26 8.43 3.8 Example 27 15.42 2.1 Example 28 14.18.3 Example 29 14.76.9 Example 30 15.3 11.9 Example 319.77.9 Example 32 12.53.1 Example 349.32 7.0 Example 35 14.46.9 Example 36 16.1 16.6 Example 37 10.28.1 Example 38 16.1 20.9 Example 39 13.76.2 Example 40 9.9 13.0 Example 41 14.16.2 Example 42 14.75 .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 Example 9 416.1 3.9 Example 95 10.13.1 Example 96 10.82.5 Example 97 16.00.7 Example 98 10.60.5 Example 99 15.71.2 Example 100 16.14.8 Example 101 16.01.2 Example 102 15.81.0 Comparative Example 19.31.0 Comparative Example 28.93.4 Comparative Example 3 14.465.4 Comparative Example 49.714.4 Comparative Example 5 13.96.6 Comparative Example 66.818.8 Comparative Example 7 10.714.5 Comparative Example 8 10.35.2 Comparative Example 9 11.29.4 Comparative Example 10 17.214.7 Comparative Example 11 10.514.6 Comparative Example 12 11.52.9

[0491] From the results of Table 23, it can be confirmed that the microspheres of Examples 1-3, 5-13, 15-42, 46, 48-63, 67, and 94-102 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, 67, and 94-102 of the present invention have excellent sustained release ability.

[0492]

[0493] Experimental Example 3: In vivo pharmacokinetics test of microspheres containing semaglutide salt

[0494] 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.

[0495] 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 thereto and mixed to prepare an injectable formulation.

[0496] 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.

[0497] 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.

[0498] 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.

[0499] 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.

[0500] 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 mixed to prepare an injectable formulation.

[0501] Microspheres containing the semaglutide sodium salt and bioavailability improver of Comparative Examples 7-10 were weighed so 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.

[0502] The above injectables were administered subcutaneously to Beagle dogs, 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.

[0503] The results of the above experiment are shown in Table 25.

[0504] 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 8 261,063.3 Example 10 215,330.1 Example 11 248,037.5 Example 15 311,647.2 Example 16 207,205.1 Example 19 166,479.4 Example 20 197,314.7 Example 21 293,972.2 Example 22 153,234.0 Example 23 149,193.7 Example 25 289,074.4 Example 27 262,549.0 Example 28 265,013.8 Example 30282,733.0 Example 31173,488.4 Example 35241,894.0 Example 36294,630.6 Example 37188,989.5 Example 40194,577.4 Example 41174,176.8 Example 42165,875.0 Example 50188,739 .7 Example 5 1165,403.3 Example 5 5418,884.8 Example 6 1207,054.6 Example 6 2288,669.5 Example 6 3341,863.8 Comparative Example 7 152,606.3 Comparative Example 8 16,264.0 Comparative Example 9 136,581.0 Comparative Example 1055,470.1

[0505] 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 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.

[0506]

[0507] Experimental Example 4: Release test and initial release rate evaluation of microspheres containing tyrzepatide salt

[0508] 25 mg of microspheres from Examples 69-88 and Comparative Examples 13 and 14 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 HPLC (equipment name: Agilent 1260) with an ultraviolet-visible absorbance detector. 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.

[0509] 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 and maintained at 37°C for a certain period of time. To measure the release amount after a certain period of time, the entire supernatant was taken and filtered through a 0.45 μm RC filter, and the filtered liquid was used as the test solution. The test solution was detected using an HPLC (equipment name: Agilent 1260) with an ultraviolet-visible absorbance detector. Column packing was performed with L1, an internal diameter of 4.6 mm x 150 mm, and a thickness of 3.5 μm.

[0510] The results of the above experiment are shown in Table 26 below.

[0511] Microsphere Loading Amount (WJ%) 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

[0512] 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 compared to Comparative Examples 13 and 14 and tend to have a lower initial release amount. That is, it can be seen that the microspheres of Examples 69 to 88 of the present invention have excellent sustained-release ability.

[0513]

[0514] Experimental Example 5: In vivo pharmacokinetics test of microspheres containing tyrzepatide salt

[0515] 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.

[0516] 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.

[0517] Microspheres containing the tyrzepatide benzathine salt and bioavailability improver of Examples 72 to 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.

[0518] Microspheres containing the tircepate acetate salt and bioavailability improver of Example 83 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.

[0519] Microspheres containing tircepate formate of Example 85 were weighed so that the dosage of tircepate was 1 mg / kg, the solvent was added thereto, and then mixed to prepare an injectable formulation.

[0520] Microspheres containing tircepate formate and a bioavailability improver of Examples 86 and 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.

[0521] Microspheres containing the sodium salt of tircepate 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.

[0522] The above injectable formulations were administered subcutaneously to Beagle dogs, and approximately 2 mL of blood was collected at a designated time. The blood concentration of tircepate was measured using LC-MS / MS. Based on the measured blood concentration, the area under the blood concentration-time curve (AUC last ) calculated.

[0523] The results of the above experiment are shown in Table 27.

[0524] 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

[0525] 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.

[0526]

[0527] 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

[0528] 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.

[0529] 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.

[0530] The liquid formulations using the above-mentioned water-soluble liquid as a carrier and the liquid formulations using a polyalcohol as a carrier were administered subcutaneously to Beagle dogs 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.

[0531] The results of the above experiment are shown in Table 28 below.

[0532] 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

[0533] 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.

[0534]

[0535] 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

[0536] 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.

[0537] b. Liquid formulation injectable 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 dose 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, a liquid formulation injectable using a water-soluble liquid as a carrier was prepared.

[0538] 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 Beagle dogs, and approximately 2 mL of blood was collected at a designated time. The blood concentration of semaglutide was measured using LC-MS / MS.

[0539] The results of the above experiment are shown in Table 29 below.

[0540] 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

[0541] 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 pharmaceutical composition in a liquid formulation using a fluid oil carrier.

[0542]

[0543] 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

[0544] 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 dose 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.

[0545] b. Injectable in liquid formulation using a water-soluble liquid containing a tissue diffusion promoter as a carrier: A water-soluble liquid containing a tissue diffusion promoter was prepared by dispersing hyaluronidase in 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 water-soluble liquid containing the tissue diffusion promoter was added thereto. By mixing these, an injectable in liquid formulation using a water-soluble liquid containing a tissue diffusion promoter as a carrier was prepared.

[0546] 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.

[0547] 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.

[0548] 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.

[0549] The results of the above experiment are shown in Tables 30 and 31 below.

[0550] Formulation AUC last0-21D (ng·hr / mL) Example 46 + water-soluble liquid carrier (a) 41,645.8 Example 46 + fluid oil carrier (c) 40,992.4

[0551] Formulation AUC last0-14D (ng·hr / mL) Example 46 + water-soluble liquid carrier containing tissue diffusion promoter (b) 24,420.8 Example 46 + fluid oil carrier containing tissue diffusion promoter (d) 38,074.6

[0552] 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.

[0553]

[0554] Experimental Example 9: In vivo pharmacokinetic test of microspheres containing a combined formulation of a GLP-1 RA class peptide salt

[0555] 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.

[0556] b. Injectable formulation of the combination of Example 3: The total dosage of semaglutide not contained in the microspheres was weighed to be 0.05 mg / kg, and the dosage of the microspheres containing the semaglutide benzathine salt of Example 3 was weighed to be 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 to them. These were mixed to prepare an injectable formulation of the combination

[0557] c. Injectable formulation containing a single microsphere of Example 61: Microspheres containing the semaglutide acetate salt and bioavailability improver of Example 61 were weighed so that the total dose of semaglutide 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.

[0558] d. Injectable formulation of the combination formulation of Example 61: The total dose of semaglutide not contained in the microspheres was weighed to be 0.05 mg / kg, and the dose of the microspheres of Example 61 was weighed to be 0.45 mg / kg. To this, the same solvent as in 'a. Injectable formulation containing a single microsphere of Example 3' was added. These were mixed to prepare an injectable formulation of the combination formulation.

[0559] The above injectables were administered subcutaneously to Beagle dogs, 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.

[0560] The results of the above experiment are shown in Table 32 below.

[0561] 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

[0562] 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.

[0563]

[0564] Experimental Example 10: Release test for a composition containing a mixture of multiple GLP-1 RA-based peptide salts and microspheres

[0565] A formulation was prepared by blending microspheres having different intrinsic viscosities as follows.

[0566] Example 90 was prepared by mixing the microspheres of Example 13 and the microspheres of Example 9 in a weight ratio of 1:1.

[0567] Example 91 was prepared by mixing the microspheres of Example 13 and the microspheres of Example 9 in a weight ratio of 1:2.

[0568] Example 92 was prepared by mixing the microspheres of Example 13 and the microspheres of Example 9 in a weight ratio of 1:4.

[0569] Example 93 was prepared by mixing the microspheres of Example 16, Example 13, and Example 9 in a weight ratio of 1:1:1.

[0570] Example 103 was prepared by mixing the microspheres of Example 94, the microspheres of Example 95, and the microspheres of Example 96 in a weight ratio of 1:2:3.

[0571] Example 104 was prepared by mixing the microspheres of Example 46, the microspheres of Example 95, and the microspheres of Example 96 in a weight ratio of 1:2:3.

[0572] Example 105 was prepared by mixing the microspheres of Example 46, the microspheres of Example 97, and the microspheres of Example 98 in a weight ratio of 1:2:3.

[0573] Example 106 was prepared by mixing the microspheres of Example 101 and the microspheres of Example 98 in a weight ratio of 1:4.

[0574] Example 107 was prepared by mixing the microspheres of Example 97 and the microspheres of Example 98 in a weight ratio of 1:4.

[0575] Example 108 was prepared by mixing the microspheres of Example 100, the microspheres of Example 9, and the microspheres of Example 96 in a weight ratio of 1:2:3.

[0576] Example 109 was prepared by mixing the microspheres of Example 100, the microspheres of Example 9, and the microspheres of Example 102 in a weight ratio of 1:2:3.

[0577] Meanwhile, a single microsphere formulation was also prepared for comparison.

[0578] Comparative Example 15 was prepared using only the microspheres of Example 13.

[0579] Comparative Example 16 was prepared using only the microspheres of Example 9.

[0580] Comparative Example 17 was prepared using only the microspheres of Example 16.

[0581] Comparative Example 18 was prepared using only the microspheres of Example 94.

[0582] Comparative Example 19 was prepared using only the microspheres of Example 95.

[0583] Comparative Example 20 was prepared using only the microspheres of Example 96.

[0584] Comparative Example 21 was prepared using only the microspheres of Example 97.

[0585] Comparative Example 22 was prepared using only the microspheres of Example 98...

[0586] Comparative Example 23 was prepared using only the microspheres of Example 100.

[0587] Comparative Example 24 was prepared using only the microspheres of Example 101.

[0588] Comparative Example 25 was prepared using only the microspheres of Example 102.

[0589] Comparative Example 26 was prepared using only the microspheres of Example 46.

[0590] After adding 15 mg of the mixed microspheres of Examples 90 to 109 to a vial, 3 mL of PBS solution with a pH of 7.4 was added, and the mixture was maintained at 37°C for a certain period while stirring at 100 rpm. To measure the release amount after a certain period, the entire supernatant was taken and filtered through a 0.45 μm RC filter to be used as the test solution. The test solution was detected using an ultraviolet-visible spectrophotometer with HPLC (Agilent 1260) and analyzed using an L1 packed column (internal diameter 4.6 mm × length 150 mm, particle size 3.5 μm).

[0591] The cumulative release over time was calculated from the measured drug concentration, and the release rate was calculated by converting this into a percentage (%) relative to the initial total drug content contained in the microspheres. The results are shown in Table 33.

[0592] Day 0 Release Rate (%) Day 7 Release Rate (%) Day 14 Release Rate (%) Day 28 Release Rate (%) Day 42 Release Rate (%) Day 56 Release Rate (%) Day 70 Release Rate (%) Example 90 1.6 2.0 28.1 6 1.9 8 9.3 9 4.8 9 6.6 Example 9 11.5 2.0 18.2 5 5.0 8 7.2 9 3.5 9 5.5 Example 9 21.5 1.8 12.1 46.6 8 4.3 9 0.7 9 2.7 Example 9 32.9 27.1 44.3 6 6.0 8 9.0 9 3.8 9 4.1 Example 10 33.4 20.2 32.9 Example 10 42.6 20.7 Example 10 51.3 24.5 Example 10 62.1 4.8 27.9 Example 10 70.5 13.4 Example 10 81.21. 7 Comparative Example 15 1.6 2.4 6 9.5 95.5 --- Comparative Example 16 1.4 1.6 2.1 45.1 8 4.2 9 1.8 9 4.6 Comparative Example 17 9.6 9 0.3 10 0.0 ---- Comparative Example 18 3.9 8 1.2 9 7.0 Comparative Example 19 3.1 5.4 46.7 Comparative Example 20 2.5 2.8 4.9 36.7 8 4.8 Comparative Example 210.7 34.4 Comparative Example 220.5 1.1 Comparative Example 23 4.8 8.7 8 6.4 Comparative Example 24 1.2 5.3 8 7.2 Comparative Example 25 1.0 1.0 1.5 Comparative Example 26 3.0 7 4.6 10 5.1

[0593] Looking at the results in Table 32, Comparative Examples 15, 17, 19, 23, and 26, which are single microsphere formulations, exhibited a relatively rapid release pattern, with more than 70% released within 14 days or most of the drug released within 7 days, whereas Comparative Example 16 showed a tendency for relatively delayed release to persist until after 56 days. Examples 90 to 93, which mixed microspheres with different intrinsic viscosities, maintained a low release rate until day 7, while gradually increasing release after day 14 and continuing release until day 70. From this, it was confirmed that formulations mixed with microspheres of different intrinsic viscosities exhibit characteristics of maintaining long-term release while suppressing initial release, and that the release rate and duration of release can be controlled depending on the mixing ratio.

[0594]

[0595] Experimental Example 11: In vivo pharmacokinetic test of a composition containing a mixture of multiple GLP-1 RA-type peptide salts and microspheres

[0596] Microspheres of Examples 90 to 93 and Comparative Examples 15 to 17 were prepared using the same method as in Experimental Example 10.

[0597] 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. Microspheres containing the semaglutide benzathine salt of Examples 90 to 93 were weighed so that the dosage of semaglutide was 2 mg / kg, and an injectable formulation was prepared by adding the solvent and mixing.

[0598] Microspheres containing the semaglutide benzathine salt of Comparative Example 17 were weighed so that the dosage of semaglutide was 2 mg / kg, and the solvent was added in the same manner to prepare an injectable formulation.

[0599] After subcutaneously administering the above injectable formulation to a beagle dog, blood was collected at regular intervals to measure the blood semaglutide concentration, and the blood drug concentration values ​​over time are shown in Table 34 below.

[0600] Concentration (ng / mL) 0.5 days 1 day 7 days 14 days 21 days 28 days 35 days Example 9 0 7 2.9 2 10 3.7 4 5 3.8 6 2 7 9.6 0 2 0 8 2 8 6.6 1 2 0.8 8 Example 9 1 10 5.4 7 1 3 8.0 1 3 9.2 4 2 6 5.7 8 1 7 3.7 0 1 10.1 7 2 2 8.1 0 Example 9 2 6 2.4 4 78 2.74 29.69 164.22 67.30 54.44 146.96 Example 9 31 61.42 213.14 650.148 66.67 315.44 125.77 166.00 Comparative Example 1 72 37.58 309.69 633.73 538.19 134.67 10.19 0.00 Concentration (ng / mL) 42 days 56 days 63 days 70 days 77 days 84 days Example 90 255.38 233.79 151.44 51.73 18.30 4.30 Example 91 698.144 46.94 162.48 53.48 30.88 15.16 Example 92 481.45 488.83 229.00 79.76 31.60 18.78 Example 93 307.60 234.88 122.77 43.61 17.44 8.28 Comparative Example 17------

[0601] Looking at the results in Table 34, Comparative Example 17, a single microsphere formulation, showed a relatively uniform pattern of decline, reaching a peak concentration of 633.73 ng / mL on day 7, followed by a rapid decrease and the disappearance of blood concentration after day 35. For Examples 90 to 92, which are mixed microsphere formulations, the concentrations on day 7 ranged from 29.69 to 53.86 ng / mL, which were significantly lower than those of Comparative Example 17, confirming that the initial increase in concentration was suppressed. Example 91 showed 265.78 ng / mL on day 14, then rose again to 698.14 ng / mL on day 42 to form a second Cmax, and maintained a high concentration until day 56; Example 92 also maintained a concentration of 480 ng / mL or higher during the period from day 42 to day 56. This indicates multiphase pharmacokinetic behavior, which is not observed in single microsphere formulations.

[0602]

[0603] Experimental Example 12: In vivo pharmacokinetic test of a composition containing a mixture of multiple GLP-1 RA-based peptide salts and microspheres

[0604] Microspheres of Examples 103 to 109 and Comparative Examples 18 to 26 were prepared using the same method as in Experimental Example 10.

[0605] A solvent to be used for preparing an injectable formulation was prepared using 1 mg of polysorbate 80, 15 mg of carboxymethylcellulose, 15 mg of mannitol, 14 mg of propylene glycol, and 1 mL of water. Microspheres containing semaglutide benzathine salt and / or semaglutide acetate of Examples 103 to 109 were weighed so that the dosage of semaglutide was 2 mg / kg, and an injectable formulation was prepared by adding the solvent and mixing.

[0606] Microspheres containing the semaglutide benzathine salt of Comparative Example 26 were weighed so that the dosage of semaglutide was 0.5 mg / kg, and the solvent was added in the same manner to prepare an injectable formulation.

[0607] After administering the above injectable formulation subcutaneously to a beagle dog, blood was collected at regular intervals to measure the blood semaglutide concentration.

[0608] According to the experimental results, the single microsphere formulation (Comparative Example 26) showed a relatively uniform pattern of decline in blood concentration after reaching the peak concentration (Cmax) following administration. The mixed microsphere formulation containing multiple types of microspheres (Examples 103 to 109) maintained an initial concentration at the effective blood concentration level or a significantly lower concentration compared to the Comparative Example. The mixed microsphere formulation showed a pattern of suppressed increase in initial concentration compared to Comparative Example 26. The mixed microsphere formulation maintained a blood concentration for a certain period after the first Cmax, and then formed a second Cmax that rose again due to additional drug release.

[0609] As such, it was confirmed that mixing biocompatible polymers with different intrinsic viscosities in appropriate proportions tends to mitigate the initial excessive rise in concentration and increase the contribution of the drug in the later stages, suggesting that effective blood concentrations can be stably maintained over the target period. Furthermore, considering that GLP-1 RA peptides have a longer half-life in humans than in Beagle dogs, it is expected that blood concentrations can be maintained for a longer period when applied to humans.

[0610]

[0611] Although the embodiments have been described above by means of limited examples, those skilled in the art can apply various technical modifications and variations based on the above. For example, suitable results may be achieved even if the described techniques are performed in a different order than the described method and / or are replaced or substituted by other components or equivalents. Therefore, other implementations, other embodiments, other aspects, and equivalents to the claims are also included within the scope of the claims set forth below.< / extraction> < / extraction>

Claims

1. A microsphere composition comprising at least one microsphere comprising a GLP-1 RA (GLP-1 receptor agonist) series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer.

2. In Paragraph 1, A microsphere composition wherein the above pharmaceutically acceptable salt is selected from pamoate salt, acetate salt, benzathine salt, formate salt, and combinations thereof.

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

4. In Paragraph 1, A microsphere composition comprising the above-mentioned GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof in an amount of 2% to 80% by weight based on the total weight of the microsphere composition.

5. In Paragraph 1, A microsphere composition wherein 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, A microsphere composition in which the above-mentioned biocompatible polymer is included in an amount of 20% to 98% by weight based on the total weight of the microsphere composition.

7. In Paragraph 1, A microsphere composition comprising one type of microsphere, wherein the intrinsic viscosity of the biocompatible polymer contained in the microsphere is 0.1 dL / g or more and less than 2.0 dL / g.

8. In Paragraph 1, The above microsphere composition comprises a first microsphere and a second microsphere, and The first microsphere comprises a biocompatible polymer having an intrinsic viscosity of 0.1 dL / g or more and less than 0.5 dL / g, and the second microsphere comprises a biocompatible polymer having an intrinsic viscosity of 0.5 dL / g or more and less than 2.0 dL / g; The first microsphere comprises a biocompatible polymer having an intrinsic viscosity of 0.1 dL / g or more and less than 0.6 dL / g, and the second microsphere comprises a biocompatible polymer having an intrinsic viscosity of 0.6 dL / g or more and less than 2.0 dL / g; A microsphere composition wherein the first microsphere comprises 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 comprises a biocompatible polymer having an intrinsic viscosity of 0.7 dL / g or more and less than 2.0 dL / g.

9. In Paragraph 8, A microsphere composition in which the weight ratio of the first microsphere and the second microsphere is 1:0.1 to 1:10 or 0.1:1 to 10:

1.

10. In Paragraph 1, The above microsphere composition comprises a first microsphere, a second microsphere, and a third microsphere, and The first microsphere comprises a biocompatible polymer having an intrinsic viscosity of 0.1 dL / g or more and less than 0.4 dL / g, the second microsphere comprises a biocompatible polymer having an intrinsic viscosity of 0.4 dL / g or more and less than 0.6 dL / g, and the third microsphere comprises a biocompatible polymer having an intrinsic viscosity of 0.6 dL / g or more and less than 2.0 dL / g; The first microsphere comprises a biocompatible polymer having an intrinsic viscosity of 0.1 dL / g or more and less than 0.4 dL / g, the second microsphere comprises a biocompatible polymer having an intrinsic viscosity of 0.4 dL / g or more and less than 0.7 dL / g, and the third microsphere comprises a biocompatible polymer having an intrinsic viscosity of 0.7 dL / g or more and less than 2.0 dL / g; A microsphere composition wherein the first microsphere comprises a biocompatible polymer having an intrinsic viscosity of 0.1 dL / g or more and less than 0.5 dL / g, the second microsphere comprises 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 comprises a biocompatible polymer having an intrinsic viscosity of 0.7 dL / g or more and less than 2.0 dL / g.

11. In Paragraph 10, A microsphere composition in which the weight ratio of the first microsphere, the second microsphere, and the third microsphere is 1:0.1:0.1 to 1:10:

10.

12. In Paragraph 1, The above microsphere composition further comprises a bioavailability improver.

13. In Paragraph 12, A microsphere composition in which the above-mentioned bioavailability improver is included in an amount of 0.01% to 20% by weight based on the total weight of the microsphere composition.

14. In Paragraph 12, A microsphere composition wherein the above-mentioned bioavailability improver is one or more selected from the group consisting of surfactants, polyhydric alcohols, amino acids, fatty acids, organic acids, and sodium salts of organic acids.

15. In Paragraph 14, A microsphere composition wherein the above 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.

16. In Paragraph 14, A microsphere composition wherein the 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.

17. In Paragraph 14, A microsphere composition wherein 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.

18. In Paragraph 14, A microsphere composition wherein the 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.

19. In Paragraph 14, A microsphere composition wherein the above organic acid is one or more selected from the group consisting of citric acid, stearic acid, tartaric acid, succinic acid, and fumaric acid.

20. In Paragraph 14, A microsphere composition wherein 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.

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

22. In Paragraph 21, 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. A method for manufacturing microspheres, wherein the salt formed above is selected from benzathine salt, formate salt, acetate salt, pamoate salt, and combinations thereof.

23. In Paragraph 21, A method for manufacturing microspheres, wherein step (a) further comprises the step of introducing a bioavailability enhancer.

24. In Paragraph 21, A method for manufacturing microspheres, further comprising an extraction step after step (d) or after step (e).

25. In Paragraph 24, The above extraction step (i) performed using an aqueous solution containing purified water, a buffer solution, alcohols, ketones, esters, and inorganic salts, or a mixture thereof, as an extraction solvent, or (ii) A method for producing microspheres, wherein the method is 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 (Na2HPO4).

26. A method for preparing a microsphere composition of claim 1, comprising the following steps: (a) a step of preparing a dispersed phase by dispersing a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof and a biocompatible polymer in a dispersion solvent; (b) a step of introducing the dispersed phase prepared in step (a) into a continuous phase and stirring it to form microspheres; (c) a step of removing the dispersion solvent from the microspheres formed in step (b) above; (d) A step of mixing one or more types of microspheres prepared by steps (a) to (c) above. A method for preparing a microsphere composition comprising 27. In Paragraph 26, A method for preparing a microsphere composition, further comprising an extraction step after step (c) or after step (d).

28. 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, A pharmaceutical composition comprising the microsphere composition of claim 1 and a pharmaceutically acceptable carrier.

29. In Paragraph 28, The above pharmaceutical composition comprises the microsphere composition of claim 1 in an amount of 0.01 mg to 5,000 mg per 1 mL of the pharmaceutically acceptable carrier.

30. In Paragraph 28, 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.

31. In Paragraph 30, 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.

32. In Paragraph 30, A pharmaceutical composition comprising one or more surfactants 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.

33. In Paragraph 30, A pharmaceutical composition comprising one or more polyalcohols selected from the group consisting of ethylene glycol, propylene glycol, polyethylene glycol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, and maltitol.

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

35. In Paragraph 34, 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.

36. In Paragraph 28, The above pharmaceutical composition further comprises a GLP-1 RA series peptide or a pharmaceutically acceptable salt thereof.

37. In Paragraph 28, The above pharmaceutical composition further comprises one or more excipients selected from buffers, isotonic agents, preservatives, and pH adjusters.

38. In Paragraph 28, The above pharmaceutical composition is a pharmaceutical composition for subcutaneous or intramuscular injection.

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