Microspheres comprising GLP-1 ra-based peptides, method for preparing same, and pharmaceutical composition comprising same

Abstract

The present invention relates to microspheres comprising GLP-1 RA-based peptides, a method for preparing same, and a pharmaceutical composition comprising same. Provided are microspheres having excellent bioavailability, comprising GLP -1 RA-based peptides, a bioavailability enhancer, and a biocompatible polymer.

Description

Microspheres comprising GLP-1 RA-based peptide series, method for preparing the same, and pharmaceutical compositions comprising the same

[0001] This application claims the benefit of priority to Korean Patent Application No. 10-2024-0012693, filed January 26, 2024, and Korean Patent Application No. 10-2025-0009554, filed January 22, 2025, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to microspheres comprising GLP-1 RA-based peptides, a method for preparing the same, and a pharmaceutical composition comprising the same. More particularly, the present invention relates to microspheres comprising GLP-1 RA-based peptides, a bioavailability improving agent, and a biocompatible polymer, a method for preparing the same, and a pharmaceutical composition comprising the same.

[0003] Obesity is a chronic disease characterized by metabolic dysregulation, particularly abnormal glucose metabolism, and associated with long-term complications. It requires long-term drug treatment. Several anti-obesity therapies are available, including human insulin and other GLP-1 RA-based peptides.

[0004] For example, semaglutide and tirzepatide are among the treatments used to treat obesity. Semaglutide and tirzepatide are approved and marketed as once-weekly subcutaneous injections to improve weight control in patients with obesity and to control blood sugar levels in patients with diabetes.

[0005] The frequent administration of these injections causes significant discomfort to patients, reducing medication compliance. To reduce the frequency of administration for obesity patients, long-acting injectables based on GLP-1 RA-based peptides are being continuously researched.

[0006] In this regard, various formulation technologies have been employed to provide sustained and controlled release of pharmacologically active substances. Among these, injectable formulations, including long-acting microspheres, are injectables in which the pharmacologically active substance is encapsulated within a polymer matrix in the form of microspheres. These formulations are designed to ensure uniform drug release from the microspheres upon subcutaneous or intramuscular injection.

[0007] In order for a release formulation using such long-acting microparticles to actually lead to the effects of extending the dosing cycle and reducing the administered dose, it is necessary to secure bioavailability as well as efficient release control.

[0008] However, when peptide drugs such as semaglutide and tirzepatide are developed as long-acting injections to reduce the number of administrations, the problem of significantly reduced bioavailability occurs when stored in vivo for a long period of time due to the nature of the peptide.

[0009] To solve these problems, research is ongoing to manufacture long-acting injectables with high bioavailability in vivo for long periods of time, and development of injectable formulations containing long-acting microspheres that can increase bioavailability is necessary.

[0010] [Prior Art Literature]

[0011] [Patent Document]

[0012] Republic of Korea Publication Patent No. 10-2021-0147413

[0013] The present invention is intended to solve the above-described problems of the prior art, and provides microspheres containing a GLP-1 RA-based peptide series using a bioavailability improving agent as an additive.

[0014] In addition, the present invention provides microspheres having excellent bioavailability and a method for manufacturing the same.

[0015] In addition, the present invention provides a pharmaceutical composition for preventing or treating diseases such as obesity and diabetes, including the above-described microparticles.

[0016] To solve the above problem,

[0017] The present invention provides microspheres comprising a GLP-1 RA-based peptide; a bioavailability improving agent; and a biocompatible polymer.

[0018] The present invention provides microspheres, wherein the GLP-1 RA-based series peptide is at least one selected from the group consisting of a GLP-1 receptor agonist, a GIP / GLP-1 receptor dual agonist, and a GLP-1 / GIP / GCG receptor triple agonist.

[0019] The present invention provides microspheres in which the bioavailability improving agent is at least one selected from the group consisting of a surfactant, a polyhydric alcohol, an amino acid, and a fatty acid.

[0020] The present invention provides microspheres containing the bioavailability improving agent in an amount of 0.05 wt% to 50 wt% based on the total weight of the microspheres.

[0021] The present invention provides microspheres in which the surfactant is at least one selected from among Poloxamer 188, Poloxamer 407, Polysorbate 20, Polysorbate 60, Polysorbate 80, Polyoxyethylene oleyl ether, Polyoxyethylene cetyl ether, Polyoxyethylene stearyl ether, and Polyoxyethylene lauryl ether.

[0022] The present invention provides microspheres in which the polyhydric alcohol is at least one selected from ethylene glycol, propylene glycol, polyethylene glycol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, and maltitol.

[0023] The present invention provides microspheres in which the amino acid is at least one selected from lysine, arginine, aspartic acid, histidine, alanine, phenylalanine, serine, glutamine, glycine, tryptophan, valine, leucine, isoleucine, cysteine, methionine, and proline.

[0024] The present invention provides microspheres in which the fatty acid is at least one selected from stearic acid, palmitic acid, lauric acid, myristic acid, oleic acid, and arachidonic acid.

[0025] The present invention relates to a GLP-1 RA-based peptide series comprising semaglutide, tirzepatide, retatrutide, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, cotadutide, taspoglutide, pembidutide, survodutide, orfoglipron, mazdutide, mariTide, ecnoglutide, cagrilintide / semaglutide, Provided are microspheres selected from efpeglenatide, dapiglutide, pramlintide and combinations thereof.

[0026] The present invention provides microspheres comprising the GLP-1 RA-based peptide in an amount of 0.1 wt% to 50 wt% based on the total weight of the microspheres.

[0027] The present invention provides microspheres in which the biocompatible polymer is at least one selected from among polylactic acid, polylactide, polylactic-co-glycolic acid, polylactide-co-glycolide (PLGA), polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, copolymer of lactic acid and caprolactone, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid, and copolymer of lactic acid and amino acid.

[0028] The present invention provides microspheres containing the biocompatible polymer in an amount of 50 wt% to 99.9 wt% based on the total weight of the microspheres.

[0029] In addition, the present invention provides a method for producing microspheres, comprising: (a1) a step of dissolving a biocompatible polymer in at least one solvent to produce an oil phase (O); (b1) a step of dissolving or dispersing a GLP-1 RA-based series peptide; and a bioavailability improver in a solvent to produce an inner water phase (W1); (c1) a step of dispersing the inner water phase (W1) in the oil phase (O) to produce a water-in-oil (W1 / O) type emulsion; (d1) a step of dispersing the water-in-oil (W1 / O) type dispersed phase prepared in step (c1) in an external continuous phase (W2) to produce a water-in-oil-in-water (W1 / O / W2) type emulsion solution to solidify microspheres; and (e1) a step of removing the solvent.

[0030] In addition, the present invention provides a method for producing microspheres, comprising: (a2) dissolving a biocompatible polymer and a bioavailability improver in at least one solvent to prepare an oil phase (O); (b2) dissolving or dispersing a GLP-1 RA-based peptide in a solvent to prepare an inner water phase (W1); (c2) dispersing the inner water phase (W1) in the oil phase (O) to prepare a water-in-oil (W1 / O) type emulsion; (d2) dispersing the water-in-oil (W1 / O) type dispersed phase prepared in step (c2) in an external continuous phase (W2) to prepare a water-in-oil-in-water (W1 / O / W2) type emulsion solution to solidify microspheres; and (e2) removing the solvent.

[0031] In addition, the present invention provides a method for producing microspheres, comprising: (a3) ​​a step of dissolving a biocompatible polymer and a bioavailability improver in at least one solvent to prepare an oil phase (O); (b3) a step of dissolving or dispersing a GLP-1 RA-based series peptide and a bioavailability improver in a solvent to prepare an inner water phase (W1); (c3) a step of dispersing the inner water phase (W1) in the oil phase (O) to prepare a water-in-oil (W1 / O) type emulsion; (d3) a step of dispersing the water-in-oil (W1 / O) type dispersed phase prepared in step (c3) in an external continuous phase (W2) to prepare a water-in-oil-in-water (W1 / O / W2) type emulsion solution to solidify microspheres; and (e3) a step of removing the solvent.

[0032] In addition, the present invention provides a pharmaceutical composition for preventing or treating obesity, diabetes, hypertension, hyperlipidemia, non-alcoholic steatohepatitis, metabolic steatohepatitis, cardiovascular disease, or degenerative neurological disease, comprising the above microspheres.

[0033] The present invention provides a pharmaceutical composition for subcutaneous or intramuscular injection.

[0034] The microspheres according to the present invention can increase the bioavailability of GLP-1 RA-based peptides, thereby providing the effect of reducing the frequency of administration and the dosage of administration.

[0035] In addition, microspheres comprising a GLP-1 RA-based peptide manufactured by the method for manufacturing microspheres according to the present invention can increase bioavailability by containing a bioavailability improving agent, and provide the effects of reducing administration frequency and administration dose.

[0036] In addition, the pharmaceutical composition according to the present invention provides an excellent effect in preventing or treating obesity or diabetes by including the microspheres.

[0037] Hereinafter, the present invention will be described in more detail.

[0038] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention. Furthermore, methods or samples similar to or equivalent to those described herein are also included within the scope of the present invention. The contents of all publications cited herein are incorporated herein by reference in their entirety.

[0039]

[0040] Among the treatments used to treat obesity, semaglutide or tirzepatide are administered as a subcutaneous injection once a week, which causes great inconvenience to patients due to the frequent number of administrations and also reduces medication compliance.

[0041] To solve these problems, the present inventors developed a double emulsification method that can include a bioavailability improving agent as an additive in long-acting microspheres containing GLP-1 RA-based peptides, thereby completing microspheres that can achieve high bioavailability and minimize administration dose.

[0042]

[0043] Microspheres according to one embodiment of the present invention may include a GLP-1 RA-based peptide; a bioavailability enhancer; and a biocompatible polymer.

[0044] The above microspheres are characterized in that they contain a bioavailability improving agent as an additive in addition to a GLP-1 RA-based series peptide and a biocompatible polymer.

[0045] In one embodiment of the present invention, the bioavailability improving agent may include at least one selected from the group consisting of a surfactant, a polyhydric alcohol, an amino acid, and a fatty acid.

[0046] In one embodiment of the present invention, the surfactant may be at least one 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. More specifically, the surfactant may be, but is not limited to, Poloxamer 188, Poloxamer 407, Polyoxyethylene oleyl ether, Polyoxyethylene cetyl ether, Polyoxyethylene stearyl ether or Polyoxyethylene lauryl ether.

[0047]

[0048] In one embodiment of the present invention, the polyhydric alcohol may be at least one selected from ethylene glycol, propylene glycol, polyethylene glycol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, and maltitol. More specifically, the polyhydric alcohol may be ethylene glycol, propylene glycol, or polyethylene glycol, but is not limited to these examples.

[0049]

[0050] In one embodiment of the present invention, the amino acid may be one or more selected from lysine, arginine, aspartic acid, histidine, alanine, phenylalanine, serine, glutamine, glycine, tryptophan, valine, leucine, isoleucine, cysteine, methionine, and proline, but is not limited to these examples.

[0051]

[0052] In one embodiment of the present invention, the fatty acid may be at least one selected from stearic acid, palmitic acid, lauric acid, myristic acid, oleic acid, and arachidonic acid, but is not limited to these examples.

[0053]

[0054] In one embodiment of the present invention, the microspheres may contain a bioavailability improving agent in an amount of 0.05 wt% to 50 wt% based on the total weight of the microspheres. More specifically, the content of the bioavailability improver included in the microspheres is 0.05 wt% or more, 0.1 wt% or more, 0.5 wt% or more, 1.0 wt% or more, 1.5 wt% or more, 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, 9.5 wt% or more, 10.0 wt% or more, or 50 wt% or less, 49.5 wt% or less, 49.0 wt% or less, 48.5 wt% or less, 48.0 wt% or less Below, 47.5 wt% or less, 47.0 wt% or less, 46.5 wt% or less, 46.0 wt% or less, 45.5 wt% or less, 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, 40.0 wt% or less.

[0055]

[0056] In one embodiment of the present invention, the GLP-1 RA-based peptide may be at least one selected from the group consisting of a GLP-1 receptor agonist, a GIP / GLP-1 receptor dual agonist, and a GLP-1 / GIP / GCG receptor triple agonist.

[0057] In one embodiment of the present invention, the GLP-1 RA-based peptide is semaglutide, tirzepatide, retatrutide, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, cotadutide, taspoglutide, pembidutide, survodutide, orfoglipron, mazdutide, MariTide, ecnoglutide, dapiglutide, It may be selected from Cagrilintide / semaglutide, Efpeglenatide, Pramlintide, and combinations thereof.

[0058] The above GLP-1 RA based peptide series may be manufactured in various forms, for example, amorphous or crystalline.

[0059]

[0060] In one embodiment of the present invention, the GLP-1 RA-based peptide may be included in an amount of 0.1 to 50 wt% based on the total weight of the microparticles. More specifically, the content of the GLP-1 RA-based series peptide is 0.1 wt% or more, 0.5 wt% or more, 1.0 wt% or more, 1.5 wt% or more, 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, 9.5 wt% or more, 10.0 wt% or more, or 50 wt% or less, 49.5 wt% or less, 49.0 wt% or less, 48.5 wt% or less, 48.0 wt% or less, 47.5 % by weight or less, 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, 40.0 wt% or less.

[0061] When the content of the GLP-1 RA-based peptide series exceeds 50 wt% based on the total weight of the microspheres, the initial release amount of the GLP-1 RA-based peptide series in the body environment may be excessively high, which may cause a problem in that the blood concentration of the drug may rapidly increase. In addition, when the content of the GLP-1 RA-based peptide series is less than 0.1 wt% based on the total weight of the microspheres, the proportion of biocompatible polymers may be relatively high, which may cause the body to be administered excessively, which may cause a problem in that administration may be difficult.

[0062]

[0063] Microspheres according to one embodiment of the present invention mean microspheres manufactured using a biocompatible polymer in which the GLP-1 RA-based series peptide is encapsulated, and in the present specification, these are simply referred to as GLP-1 RA-based series peptide-containing microspheres, GLP-1 RA-based series peptide microspheres, or microspheres.

[0064]

[0065] In one embodiment of the present invention, a biocompatible polymer means a polymer that is guaranteed to be safe in vivo and does not cause high cytotoxicity or inflammatory response when administered into a living body, and is also referred to simply as a polymer in this specification.

[0066] The biocompatible polymer may be at least one selected from among polylactic acid, polylactide, polylactic-co-glycolic acid, polylactide-co-glycolide (PLGA), polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, copolymer of lactic acid and caprolactone, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid, and copolymer of lactic acid and amino acid.

[0067]

[0068] In one embodiment of the present invention, the biocompatible polymer may have an intrinsic viscosity of 0.1 dL / g to 1.9 dL / g. The intrinsic viscosity is measured at a concentration of 0.05% (w / v) to 3% (w / v) in chloroform at 25°C using an Ubbelohde viscometer.

[0069] If the biocompatible polymer has an intrinsic viscosity of less than 0.1 dL / g, the polymer may decompose too quickly, making it difficult to continuously release the GLP-1 RA-based peptide for a desired period of time. If the polymer has an intrinsic viscosity of more than 1.9 dL / g, the polymer may decompose too slowly, resulting in a small amount of GLP-1 RA-based peptide released, and thus the drug effect may not be observed.

[0070] In one embodiment of the present invention, the biocompatible polymer may be included in an amount of 50 to 99.9 wt% based on the total weight of the microparticles. More specifically, the biocompatible polymer may be included in an amount of 50 wt% or more, 55 wt% or more, 60 wt% or more, 99.9 wt% or less, 99.0 wt% or less, 98.0 wt% or less, 97.0 wt% or less, 96.0 wt% or less, 95.0 wt% or less, 94.0 wt% or less, 93.0 wt% or less, 92.0 wt% or less, 91.0 wt% or less, or 90.0 wt% or less based on the total weight of the microparticles, but is not limited thereto.

[0071] If the biocompatible polymer is included in an amount of less than 50 wt% based on the total weight of the microparticles, the distribution of the GLP-1 RA-based peptide may be relatively increased, which may cause problems such as initial excessive release or inability to maintain the efficacy for a desired period of time. If the biocompatible polymer is included in an amount exceeding 99.9 wt% based on the total weight of the microparticles, the dissolution of the drug may be too slow, making it difficult to administer the drug in the blood at a constant rate.

[0072]

[0073] In one embodiment of the present invention, the microspheres containing the bioavailability improving agent can sustain the release of the encapsulated GLP-1 RA-based peptide series for 20 days or more in an in vivo or in vitro environment.

[0074]

[0075] In one embodiment of the present invention, a method for manufacturing microspheres including a bioavailability improving agent may include the following steps:

[0076] (a1) A step of preparing an oil phase (O) by dissolving a biocompatible polymer in one or more solvents;

[0077] (b1) A step of preparing an inner phase (W1) by dissolving or dispersing a GLP-1 RA-based series peptide; and a bioavailability improving agent; in a solvent;

[0078] (c1) A step of dispersing an internal phase (W1) in the oil phase (O) to prepare a water-in-oil (W1 / O) type emulsion;

[0079] (d1) a step of dispersing the water-in-oil (W1 / O) type dispersed phase prepared in step (c1) in the external continuous phase (W2) to prepare a water-in-oil-in-water (W1 / O / W2) type emulsion solution to solidify microspheres; and

[0080] (e1) Step of removing the above solvent.

[0081]

[0082] In one embodiment of the present invention, a method for manufacturing microspheres including a bioavailability improving agent may include the following steps:

[0083] (a2) a step of preparing an oil phase (O) by dissolving a biocompatible polymer and a bioavailability improving agent in at least one solvent;

[0084] (b2) A step of preparing an inner phase (W1) by dissolving or dispersing a GLP-1 RA-based series peptide in a solvent;

[0085] (c2) A step of dispersing an internal phase (W1) in the oil phase (O) to prepare a water-in-oil (W1 / O) type emulsion;

[0086] (d2) a step of dispersing the water-in-oil (W1 / O) type dispersed phase prepared in step (c2) in the external continuous phase (W2) to prepare a water-in-oil-in-water (W1 / O / W2) type emulsion solution to solidify microspheres; and

[0087] (e2) Step of removing the solvent.

[0088]

[0089] In one embodiment of the present invention, a method for manufacturing microspheres including a bioavailability improving agent may include the following steps:

[0090] (a3) A step of preparing an oil phase (O) by dissolving a biocompatible polymer and a bioavailability improving agent in at least one solvent;

[0091] (b3) A step of preparing an inner phase (W1) by dissolving or dispersing a GLP-1 RA-based series peptide; and a bioavailability improving agent; in a solvent;

[0092] (c3) A step of dispersing an internal phase (W1) in the oil phase (O) to prepare a water-in-oil (W1 / O) type emulsion;

[0093] (d3) a step of dispersing the water-in-oil (W1 / O) type dispersed phase prepared in step (c3) in the external continuous phase (W2) to prepare a water-in-oil-in-water (W1 / O / W2) type emulsion solution to solidify microspheres; and

[0094] (e3) Step of removing the solvent.

[0095]

[0096] In one embodiment of the present invention, the bioavailability improving agent may be manufactured into microspheres by being included in the inner water phase (W1), the oil phase (O), the inner water phase (W1) and the oil phase (O), and there are no particular limitations on the route and method for including the microspheres.

[0097]

[0098] The description of the bioavailability improving agent is the same as described above.

[0099]

[0100] In one embodiment of the present invention, the solvent used in steps (a1), (a2), and (a3) ​​is not particularly limited as long as it can dissolve the biocompatible polymer, and may preferably be a non-aqueous solvent. Non-limiting examples of the non-aqueous solvent include, but are not limited to, dichloromethane, chloroform, acetonitrile, dimethyl sulfoxide, dimethylformamide, and ethyl acetate.

[0101]

[0102] In one embodiment of the present invention, the solvent of the internal water (W1) used in steps (b1), (b2) and (b3) may be any one of distilled water, ethanol, purified water and a combination thereof.

[0103]

[0104] In one embodiment of the present invention,

[0105] In the above steps (d1), (d2) and (d3), a hydrophilic polymer may be included as a surfactant, and the type thereof is not particularly limited, and any hydrophilic polymer that can help the dispersion phase including the GLP-1 RA-based series peptide and the biocompatible polymer to form a stable droplet dispersion phase within the external aqueous phase (W2) may be used. The hydrophilic polymer may preferably be selected from the group consisting of methylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, lecithin, gelatin, polyvinyl alcohol, polyoxyethylene-polyoxypropylene block copolymers, polyoxyethylene sorbitan fatty acid esters and polyoxyethylene castor oil derivatives and mixtures thereof, and most preferably polyvinyl alcohol.

[0106] In the above steps (d1), (d2) and (d3), the external phase (W2) may be a hydrophilic polymer aqueous solution of 0.1 to 6% (w / w), wherein the molecular weight of the hydrophilic polymer may be 7,000 to 40,000, and the degree of hydrolysis may be 80 to 90%.

[0107] In steps (d1), (d2), and (d3), the dispersion phase containing the GLP-1 RA-based peptides and the biocompatible polymer prepared in steps (c1), (c2), and (c3) is added to the external water phase (W2) containing the hydrophilic polymer by drop-by-drop or by using an in-line mixer, and stirred vigorously to prepare an emulsion solution (W1 / O / W2). In this process, the GLP-1 RA-based peptides are encapsulated into biocompatible polymer microparticles.

[0108] Afterwards, the solvent is removed in steps (e1), (e2), and (e3), and the desired microspheres are obtained after conventional filtration and washing. That is, a step of washing the obtained microspheres may be included to enhance the initial release inhibition effect, if necessary.

[0109]

[0110] The method for manufacturing the microspheres of the present invention may be applied equally to all of the above-described microsphere-related information (type of biocompatible polymer, intrinsic viscosity thereof, etc.) that conforms to the manufacturing method. Therefore, any duplicate information will be omitted below.

[0111]

[0112] The present invention can provide a pharmaceutical composition for the prevention or treatment of obesity, diabetes, hypertension, hyperlipidemia, non-alcoholic steatohepatitis, metabolic steatohepatitis, cardiovascular disease, or degenerative neurological disease, comprising the above microspheres. Here, the diabetes may be type 2 diabetes.

[0113] The pharmaceutical composition according to the present invention can be formulated into various forms, for example, as a parenteral administration preparation. The parenteral administration preparation can be formulated into the form of injections, creams, lotions, ointments for topical use, oils, moisturizers, aerosols, patches, and nasal inhalers, for example, by methods known in the art. These formulations are described in the literature (Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995), a generally known prescription manual in all fields of pharmaceutical chemistry.

[0114] The pharmaceutical composition according to the present invention can be administered to mammals, including humans, by any method. For example, it can be administered parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration. More specifically, the pharmaceutical composition according to the present invention can be used as a subcutaneous or intramuscular injection.

[0115] The above parenteral dosage form may contain microspheres alone, or may further contain excipients, diluents, and / or pharmaceutically acceptable carriers in addition to the microspheres. The carriers include all types of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads, and microsomes.

[0116] Pharmaceutically acceptable carriers may further include, for example, carriers for parenteral administration. Carriers for parenteral administration may include water, suitable oils, saline solution, aqueous glucose, and glycols, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, and the like. Other pharmaceutically acceptable carriers and formulations may be referred to those described in the following literature: (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0117] The total effective amount of the pharmaceutical composition according to the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. A preferred total dosage of the pharmaceutical composition of the present invention may be about 0.01 ㎍ to 10,000 mg per 1 kg of patient body weight per day, most preferably 0.1 ㎍ to 500 mg. However, since the dosage of the pharmaceutical composition is determined by taking into consideration various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health status, sex, severity of the disease, diet, and excretion rate, a person having ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention in consideration of these points. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

[0118] In one embodiment of the present invention, the pharmaceutical composition may be administered to the subject at intervals of one month to one year. The pharmaceutical composition of the present invention may be administered once every two weeks, once every three weeks, once every month, once every three months, once every six months, or once every year, but is not limited thereto.

[0119] In one embodiment of the present invention, the pharmaceutical composition can be used for the prevention or treatment of obesity, diabetes, hypertension, hyperlipidemia, non-alcoholic steatohepatitis, metabolic steatohepatitis, cardiovascular disease, or degenerative neurological disease.

[0120] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the present invention is not limited thereto.

[0121]

[0122] Comparative Example 1: Preparation of Semaglutide Microspheres

[0123] (1) Preparation of continuous phase (900 mL)

[0124] 4.5 g of polyvinyl alcohol (manufacturer: sigma-aldrich, hereinafter the same) was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor, and then an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0125] (2) Discontinuous phase manufacturing

[0126] Semaglutide (0.32 g) was weighed into a 50 mL vial, dissolved in distilled water (2.4 mL) and stirred at 150 rpm to prepare solution A. Then, PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) (2.88 g) was weighed and dissolved in dichloromethane (9.6 mL) by stirring at 300 rpm to prepare solution B.

[0127] (3) Primary homogenization

[0128] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0129] (4) Manufacturing of microspheres

[0130] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0131] Comparative Example 2: Preparation of Semaglutide Microspheres

[0132] (1) Preparation of continuous phase (900 mL)

[0133] 900 ml of distilled water, 4.5 g of polyvinyl alcohol, and 4.5 g of sodium chloride were weighed and added to the reactor. Then, an overhead stirrer (Overhead (IKA)) was used to stir at 1,000 rpm to prepare a 0.5% (w / v) polyvinyl alcohol aqueous solution.

[0134] (2) Discontinuous phase manufacturing

[0135] 0.2 g of semaglutide and 2.4 ml of distilled water were weighed and added to a 50 ml vial, and stirred at 150 rpm to prepare Solution A. Then, 1.8 g of PLGA (inherent viscosity of 0.1 dL / g to 1.9 dL / g) and 7.2 ml of dichloromethane were added to another 50 ml vial, and stirred at 300 rpm to prepare Solution B.

[0136] (3) Primary homogenization

[0137] The B solution obtained in the above first discontinuous phase preparation was taken with a syringe, added to the A solution, and stirred at 20,000 rpm for 1 minute using a homogenizer (IKA).

[0138] (4) Manufacturing of microspheres

[0139] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0140] Comparative Example 3: Preparation of semaglutide microspheres

[0141] (1) Preparation of continuous phase (900 mL)

[0142] 900 ml of distilled water, 4.5 g of polyvinyl alcohol, and 4.5 g of sodium chloride were weighed and added to the reactor. Then, an overhead stirrer (Overhead (IKA)) was used to stir at 1,000 rpm to prepare a 0.5% (w / v) polyvinyl alcohol aqueous solution.

[0143] (2) Discontinuous phase manufacturing

[0144] Semaglutide (0.08 g) and distilled water (0.6 ml) were weighed and added to a 50 ml vial, and stirred at 150 rpm to prepare Solution A. Then, 0.72 g of PLGA (inherent viscosity of 0.1 dL / g to 1.9 dL / g) and 2.4 ml of dichloromethane were added to another 50 ml vial, and stirred at 300 rpm to prepare Solution B.

[0145] (3) Primary homogenization

[0146] The B solution obtained in the above first discontinuous phase preparation was taken with a syringe, added to the A solution, and stirred at 20,000 rpm for 1 minute using a homogenizer (IKA).

[0147] (4) Manufacturing of microspheres

[0148] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0149]

[0150] Example 1: Preparation of microspheres containing semaglutide and poloxamer

[0151] (1) Preparation of continuous phase (900 mL)

[0152] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0153] (2) Discontinuous phase manufacturing

[0154] Semaglutide (0.32 g) was weighed into a 50 mL vial, suspended in distilled water (2.4 mL), and poloxamer188 (0.01 g) was added thereto and stirred at 150 rpm to dissolve, thereby preparing solution A. Subsequently, 2.88 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed and dissolved in dichloromethane (9.6 mL) by stirring at 300 rpm, thereby preparing solution B.

[0155] (3) Primary homogenization

[0156] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0157] (4) Manufacturing of microspheres

[0158] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0159] Example 2: Preparation of microspheres containing semaglutide and poloxamer

[0160] (1) Preparation of continuous phase (900 mL)

[0161] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0162] (2) Discontinuous phase manufacturing

[0163] Semaglutide (0.32 g) was weighed into a 50 mL vial and suspended in distilled water (2.4 mL), then poloxamer188 (0.1 g) was added and stirred at 150 rpm to dissolve, thereby preparing solution A. Subsequently, 2.88 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed and dissolved in dichloromethane (9.6 mL) by stirring at 300 rpm, thereby preparing solution B.

[0164] (3) Primary homogenization

[0165] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0166] (4) Manufacturing of microspheres

[0167] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0168] Example 3: Preparation of microspheres containing semaglutide and poloxamer

[0169] (1) Preparation of continuous phase (900 mL)

[0170] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0171] (2) Discontinuous phase manufacturing

[0172] Semaglutide (0.32 g) was weighed into a 20 mL vial, suspended in distilled water (2.4 mL), stirred at 150 rpm to dissolve, and solution A was prepared. Then, 2.88 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed, suspended in dichloromethane (9.6 mL), and poloxamer 188 (0.1 g) was added, stirred at 300 rpm to dissolve, and solution B was prepared.

[0173] (3) Primary homogenization

[0174] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0175] (4) Manufacturing of microspheres

[0176] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0177] Example 4: Preparation of microspheres containing semaglutide and propylene glycol

[0178] (1) Preparation of continuous phase (900 mL)

[0179] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0180] (2) Discontinuous phase manufacturing

[0181] Semaglutide (0.32 g) was weighed into a 50 mL vial, suspended in distilled water (2.4 mL), and propylene glycol (0.03 mL) was added. The suspension was stirred at 150 rpm to dissolve the suspension, thereby preparing solution A. Subsequently, 2.88 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed and dissolved in dichloromethane (9.6 mL) by stirring at 300 rpm, thereby preparing solution B.

[0182] (3) Primary homogenization

[0183] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0184] (4) Manufacturing of microspheres

[0185] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0186] Example 5: Preparation of microspheres containing semaglutide and propylene glycol

[0187] (1) Preparation of continuous phase (900 mL)

[0188] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0189] (2) Discontinuous phase manufacturing

[0190] Semaglutide (0.32 g) was weighed into a 50 mL vial, suspended in distilled water (2.4 mL), stirred at 150 rpm to dissolve, and solution A was prepared. Then, 2.88 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed, suspended in dichloromethane (9.6 mL), stirred at 300 rpm to dissolve, and solution B was prepared.

[0191] (3) Primary homogenization

[0192] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0193] (4) Manufacturing of microspheres

[0194] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0195] Example 6: Preparation of microspheres containing semaglutide and propylene glycol

[0196] (1) Preparation of continuous phase (900 mL)

[0197] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0198] (2) Discontinuous phase manufacturing

[0199] Semaglutide (0.32 g) was weighed into a 20 mL vial, suspended in distilled water (2.4 mL), and propylene glycol (0.09 mL) was added. The suspension was stirred at 150 rpm to dissolve the suspension, thereby preparing solution A. Subsequently, 2.88 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed and dissolved in dichloromethane (9.6 mL) by stirring at 300 rpm, thereby preparing solution B.

[0200] (3) Primary homogenization

[0201] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0202] (4) Manufacturing of microspheres

[0203] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 30 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0204] Example 7: Preparation of microspheres containing semaglutide and polyoxyethylene oleyl ether

[0205] (1) Preparation of continuous phase (900 mL)

[0206] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0207] (2) Discontinuous phase manufacturing

[0208] Semaglutide (0.32 g) was weighed in a 20 mL vial, suspended in distilled water (2.4 mL), and stirred at 150 rpm to dissolve, thereby preparing solution A. Then, 2.88 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed, and 9.6 mL of dichloromethane was added, followed by polyoxyethylene oleyl ether (Breeze). ® 98(brij ®98)) 0.11 mL was added and stirred at 300 rpm to dissolve, and then solution B was prepared.

[0209] (3) Primary homogenization

[0210] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0211] (4) Manufacturing of microspheres

[0212] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0213] Example 8: Preparation of microspheres containing semaglutide and lysine

[0214] (1) Preparation of continuous phase (900 mL)

[0215] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0216] (2) Discontinuous phase manufacturing

[0217] Semaglutide (0.2 g) was weighed into a 50 mL vial, suspended in distilled water (2.4 mL), lysine (12 mg) was added, and stirred at 150 rpm to dissolve, thereby preparing solution A. Subsequently, PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) (1.8 g) was weighed, dissolved in dichloromethane (7.2 mL) by stirring at 300 rpm, thereby preparing solution B.

[0218] (3) Primary homogenization

[0219] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0220] (4) Manufacturing of microspheres

[0221] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0222] Example 9: Preparation of microspheres containing semaglutide and lysine

[0223] (1) Preparation of continuous phase (900 mL)

[0224] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0225] (2) Discontinuous phase manufacturing

[0226] Semaglutide (0.2 g) was weighed into a 50 mL vial, suspended in distilled water (2.4 mL), and lysine (24 mg) was added and stirred at 150 rpm to dissolve, thereby preparing Solution A. Subsequently, 1.8 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed and dissolved in 7.2 mL of dichloromethane (stirred at 300 rpm), thereby preparing Solution B.

[0227] (3) Primary homogenization

[0228] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0229] (4) Manufacturing of microspheres

[0230] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0231] Example 10: Preparation of microspheres containing semaglutide and lysine

[0232] (1) Preparation of continuous phase (900 mL)

[0233] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0234] (2) Discontinuous phase manufacturing

[0235] Semaglutide (0.2 g) was weighed into a 50 mL vial, suspended in distilled water (2.4 mL), lysine (36 mg) was added, and stirred at 150 rpm to dissolve, thereby preparing solution A. Subsequently, PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) (1.8 g) was weighed, dissolved in dichloromethane (7.2 mL) by stirring at 300 rpm, thereby preparing solution B.

[0236] (3) Primary homogenization

[0237] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0238] (4) Manufacturing of microspheres

[0239] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0240] Example 11: Preparation of microspheres containing semaglutide and arginine

[0241] (1) Preparation of continuous phase (900 mL)

[0242] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0243] (2) Discontinuous phase manufacturing

[0244] Semaglutide (0.16 g) was weighed into a 50 mL vial, suspended in distilled water (1.2 mL), arginine (80 mg) was added, and stirred at 150 rpm to dissolve, thereby preparing solution A. Subsequently, PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) (1.44 g) was weighed, dissolved in dichloromethane (4.8 mL) and stirred at 300 rpm, thereby preparing solution B.

[0245] (3) Primary homogenization

[0246] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 7,600 rpm for 1 minute using a homogenizer.

[0247] (4) Manufacturing of microspheres

[0248] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0249] Example 12: Preparation of microspheres containing semaglutide and stearic acid

[0250] (1) Preparation of continuous phase (900 mL)

[0251] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0252] (2) Discontinuous phase manufacturing

[0253] Semaglutide (0.1 g) was weighed into a 50 mL vial, suspended in distilled water (1.2 mL), and stirred at 150 rpm to dissolve to prepare solution A. Then, 0.9 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) and 6 mg of stearic acid were weighed and dissolved in 3.6 mL of dichloromethane (300 rpm) to prepare solution B.

[0254] (3) Primary homogenization

[0255] The B solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and added to the A solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0256] (4) Manufacturing of microspheres

[0257] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0258]

[0259] Experimental Example 1: Evaluation of loading rate and initial release rate of microspheres

[0260] To evaluate the loading rate of microspheres, the microspheres manufactured in Comparative Examples 1 to 3 and Examples 1 to 12 were taken and placed in a 20 mL volumetric flask, completely dissolved in acetonitrile containing 0.1% TFA (Trifluoracetic acid), and then filtered through a 0.45 ㎛ RC filter until the volume was adjusted to the mark with distilled water containing 0.1% TFA (Trifluoracetic acid). This solution was detected using an ultraviolet-visible spectrophotometer detector using HPLC.

[0261] To evaluate the initial release rate of microspheres, the microspheres manufactured in Comparative Examples 1 to 3 and Examples 1 to 12 were placed in vials, pH 7.4 PBS solution was added, and the mixture was stirred at 100 rpm and maintained at 37°C. To measure the release amount over a certain period of time, the supernatant was collected after centrifugation and filtered through a 0.45 μm RC filter. This solution was placed in a vial and detected using a UV-visible spectrophotometer using HPLC. The results of the experiment are shown in Table 1 below.

[0262] Division Loading Rate (%) Initial Release Rate (%) Comparative Example 110.5 0.6 Comparative Example 210.2 0.8 Comparative Example 310.4 2.1 Example 110.2 2.7 Example 29.6 41.7 Example 39.9 02.0 Example 410.6 2.8 Example 510.6 0.9 Example 610.3 0.7 Example 710.3 3.3 Example 89.8 1.0 Example 99.5 3.4 Example 109.3 7.2 Example 1210.0 0.9

[0263]

[0264] Experimental Example 2: In vivo pharmacokinetics test using beagles

[0265] After subcutaneously injecting the semaglutide microparticle formulations of Comparative Example 3 and Examples 6 to 10 into beagles, blood samples were collected at designated times, and the blood semaglutide concentration was measured using LC-MS / MS. The area under the curve (AUC) was calculated from the blood concentration-time graph of semaglutide. The results of the experiment are shown in Table 2 below.

[0266] Classification AUClast (ng*hr / mL) Increase rate of area under the curve compared to Comparative Example 3 (%) Comparative Example 3 31,759.52 - Example 6 115,862.80 264.74 Example 7 71,801.00 126.08 Example 8 135,892.80 327.74 Example 9 192,499.80 506.15 Example 10 251,885.40 692.99

[0267]

[0268] As confirmed in Table 2, the microspheres including the bioavailability improving agent (Examples 6 to 10) were confirmed to have an area under the concentration curve increased by at least 126% compared to the microspheres without the addition (Comparative Example 3).

[0269] Since bioavailability is proportional to the area under the blood concentration-time curve (AUClast), these results indicate that the microspheres of the present invention containing the bioavailability enhancing agent provide significantly improved bioavailability.

[0270]

[0271] Comparative Example 4: Preparation of tirzepatide microspheres

[0272] (1) Preparation of continuous phase (900 mL)

[0273] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0274] (2) Discontinuous phase manufacturing

[0275] 0.386 g of tirzepatide was weighed into a 20 mL vial, dissolved in 1.5 mL of distilled water by stirring at 150 rpm, and solution C was prepared. Thereafter, 0.9 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed and dissolved in 3.0 mL of dichloromethane by stirring at 300 rpm, to prepare solution D.

[0276] (3) Primary homogenization

[0277] The D solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and placed in the C solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0278] (4) Manufacturing of microspheres

[0279] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0280]

[0281] Example 13: Preparation of microspheres containing tirzepatide and poloxamer

[0282] (1) Preparation of continuous phase (900 mL)

[0283] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0284] (2) Discontinuous phase manufacturing

[0285] 0.386 g of tirzepatide was weighed into a 20 mL vial, dissolved in 1.5 mL of distilled water by stirring at 150 rpm, and solution C was prepared. Then, 0.9 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed, dissolved in 3.0 mL of dichloromethane by stirring at 300 rpm, and 0.1 g of poloxamer188 was added to prepare solution D.

[0286] (3) Primary homogenization

[0287] The D solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and placed in the C solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0288] (4) Manufacturing of microspheres

[0289] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0290] Example 14: Preparation of microspheres containing tirzepatide and propylene glycol

[0291] (1) Preparation of continuous phase (900 mL)

[0292] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0293] (2) Discontinuous phase manufacturing

[0294] 0.386 g of tirzepatide was weighed into a 20 mL vial, 1.5 mL of distilled water was added, and the mixture was stirred at 150 rpm to prepare an E solution. Then, 0.9 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed and dissolved in 3.0 mL of dichloromethane by stirring at 300 rpm, and 0.11 mL of propylene glycol was added, stirred, and an F solution was prepared.

[0295] (3) Primary homogenization

[0296] The F solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and placed in the E solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0297] (4) Manufacturing of microspheres

[0298] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0299] Example 15: Preparation of microspheres containing tirzepatide and propylene glycol

[0300] (1) Preparation of continuous phase (900 mL)

[0301] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0302] (2) Discontinuous phase manufacturing

[0303] 0.386 g of tirzepatide was weighed into a 20 mL vial, 1.5 mL of distilled water was added, and the vial was stirred at 150 rpm to dissolve. 0.11 mL of propylene glycol was added and stirred to prepare solution E. Subsequently, 0.9 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed and stirred at 300 rpm in 3.0 mL of dichloromethane to prepare solution F.

[0304] (3) Primary homogenization

[0305] The F solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and placed in the E solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0306] (4) Manufacturing of microspheres

[0307] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0308] Example 16: Preparation of microspheres containing tirzepatide, lysine, and propylene glycol

[0309] (1) Preparation of continuous phase (900 mL)

[0310] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0311] (2) Discontinuous phase manufacturing

[0312] 0.321 g of tirzepatide was weighed into a 20 mL vial, dissolved in 1.2 mL of distilled water, 2.4 mg of lysine, and 0.031 mL of propylene glycol by stirring at 150 rpm, thereby preparing solution C. Subsequently, 0.75 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) was weighed and dissolved in 3.0 mL of dichloromethane by stirring at 300 rpm, thereby preparing solution D.

[0313] (3) Primary homogenization

[0314] The D solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and placed in the C solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0315] (4) Manufacturing of microspheres

[0316] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0317] Example 17: Preparation of microspheres containing tirzepatide, lysine, and propylene glycol

[0318] (1) Preparation of continuous phase (900 mL)

[0319] 4.5 g of polyvinyl alcohol was weighed into 900 mL of distilled water (DW) and placed in a reactor. 4.5 g of sodium chloride (NaCl) was weighed and placed in the reactor. Then, an overhead stirrer was used to stir at a speed of 1,000 rpm to prepare a 0.5% polyvinyl alcohol solution.

[0320] (2) Discontinuous phase manufacturing

[0321] 0.321 g of tirzepatide was weighed into a 20 mL vial, and 1 mL of distilled water, 7 mg of lysine, and 0.031 mL of propylene glycol were added and stirred at 150 rpm to dissolve, thereby preparing solution C. Subsequently, 0.375 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) and 0.375 g of PLGA (intrinsic viscosity of 0.1 dL / g to 1.9 dL / g) were weighed and dissolved in 3.0 mL of dichloromethane by stirring at 300 rpm, thereby preparing solution D.

[0322] (3) Primary homogenization

[0323] The D solution obtained in the above (2) discontinuous phase manufacturing process was taken with a syringe and placed in the C solution, and then ground at 20,000 rpm for 1 minute using a homogenizer.

[0324] (4) Manufacturing of microspheres

[0325] After the above (3) first homogenization process was completed, the solution was immediately taken into a syringe, the homogenizer was connected to the reactor, and the solution was injected for 60 seconds. After solidification for 20 hours, microspheres were obtained through a 5 μm filter. The obtained microspheres were freeze-dried in a freeze-dryer for more than 20 hours.

[0326]

[0327] Experimental Example 3: Evaluation of the Initial Release Rate of Microspheres

[0328] To evaluate the loading rate of microspheres, the microspheres prepared in Comparative Example 4 and Examples 13 to 17 were placed in a 20 mL volumetric flask, completely dissolved in acetonitrile containing 0.1% TFA (Trifluoracetic acid), and then filtered through a 0.45 ㎛ RC filter after adjusting the line with distilled water containing 0.1% TFA (Trifluoracetic acid). This solution was detected using an ultraviolet-visible spectrophotometer detector using HPLC.

[0329] To evaluate the initial release rate of microspheres, the microspheres prepared in Comparative Example 4 and Examples 13 to 17 were placed in vials, a pH 7.4 PBS solution was added, and the mixture was stirred at 100 rpm and maintained at 37°C. To measure the release amount over a certain period of time, the supernatant was collected after centrifugation and filtered through a 0.45 μm RC filter. This solution was placed in a vial and detected using a UV-visible spectrophotometer using HPLC. The results of the experiment are shown in Table 3 below.

[0330] Division Loading Rate (%) Initial Release Rate (%) Comparative Example 430.7 1.3 Example 1329.7 0.6 Example 1426.8 2.8 Example 1530.11.4 Example 1631.04.1 Example 1728.3 1.5

[0331]

[0332] Experimental Example 4: In-vivo pharmacokinetics test using beagles

[0333] After subcutaneously injecting the tirzepatide microparticle formulations of Comparative Example 4 and Examples 15 to 17 into beagles, blood was collected at designated times and the blood tirzepatide concentration was measured using LC-MS / MS. The area under the curve (AUC) was calculated from the blood concentration-time graph of tirzepatide.

[0334] AUClast (ng*hr / mL) Increase rate of area under the curve compared to Comparative Example 4 (%) Comparative Example 4468,991.60 - Example 15679,774.60 44.94 Example 16782,749.20 66.90 Example 171,003,198.50 113.91

[0335]

[0336] As confirmed in Table 4, the microspheres including the bioavailability improving agent (Examples 15 to 17) were confirmed to have an area under the concentration curve increased by at least 45% compared to the microspheres without the addition (Comparative Example 4).

[0337] Since bioavailability is proportional to the area under the blood concentration-time curve (AUClast), these results indicate that the microspheres of the present invention containing the bioavailability enhancing agent provide significantly improved bioavailability.

Claims

1. GLP-1 RA-based series peptides; bioavailability enhancers; and Microspheres comprising a biocompatible polymer.

2. In paragraph 1, The above GLP-1 RA-based series peptide is at least one selected from the group consisting of a GLP-1 receptor agonist, a GIP / GLP-1 receptor dual agonist, and a GLP-1 / GIP / GCG receptor triple agonist.

3. In paragraph 1, Microspheres, wherein the bioavailability improving agent is at least one selected from the group consisting of surfactants, polyhydric alcohols, amino acids, and fatty acids.

4. In paragraph 1, Microspheres, wherein the bioavailability improving agent is included in an amount of 0.05 wt% to 50 wt% based on the total weight of the microspheres.

5. In paragraph 3, Microspheres, wherein the surfactant is at least one selected from among Poloxamer 188, Poloxamer 407, Polysorbate 20, Polysorbate 60, Polysorbate 80, Polyoxyethylene oleyl ether, Polyoxyethylene cetyl ether, Polyoxyethylene stearyl ether, and Polyoxyethylene lauryl ether.

6. In paragraph 3, Microspheres, wherein the above polyhydric alcohol is at least one selected from ethylene glycol, propylene glycol, polyethylene glycol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, and maltitol.

7. In paragraph 3, Microspheres, wherein the amino acid is at least one selected from lysine, arginine, aspartic acid, histidine, alanine, phenylalanine, serine, glutamine, glycine, tryptophan, valine, leucine, isoleucine, cysteine, methionine, and proline.

8. In paragraph 3, Microspheres, wherein the fatty acid is at least one selected from stearic acid, palmitic acid, lauric acid, myristic acid, oleic acid, and arachidonic acid.

9. In paragraph 1, The above GLP-1 RA-based series peptides include semaglutide, tirzepatide, retatrutide, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, cotadutide, taspoglutide, pembidutide, survodutide, orfoglipron, mazdutide, MariTide, ecnoglutide, cagrilintide / semaglutide, Microspheres selected from efpeglenatide, dapiglutide, pramlintide, and combinations thereof.

10. In paragraph 1, Microspheres, wherein the GLP-1 RA-based series peptide is contained in an amount of 0.1 wt% to 50 wt% based on the total weight of the microspheres.

11. In paragraph 1, The above biocompatible polymer is at least one selected from among polylactic acid, polylactide, polylactic-co-glycolic acid, polylactide-co-glycolide (PLGA), polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, copolymer of lactic acid and caprolactone, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid, and copolymer of lactic acid and amino acid.

12. In paragraph 1, Microspheres, wherein the biocompatible polymer is contained in an amount of 50 wt% to 99.9 wt% based on the total weight of the microspheres. 13.(a1) A step of preparing an oil phase (O) by dissolving a biocompatible polymer in one or more solvents; (b1) A step of preparing an inner phase (W1) by dissolving or dispersing a GLP-1 RA-based series peptide; and a bioavailability improving agent; in a solvent; (c1) A step of dispersing an internal phase (W1) in the oil phase (O) to prepare a water-in-oil (W1 / O) type emulsion; (d1) a step of dispersing the water-in-oil (W1 / O) type dispersed phase prepared in step (c1) in the external continuous phase (W2) to prepare a water-in-oil-in-water (W1 / O / W2) type emulsion solution to solidify microspheres; and (e1) A method for producing microparticles, comprising the step of removing the solvent. 14.(a2) A step of preparing an oil phase (O) by dissolving a biocompatible polymer and a bioavailability improving agent in at least one solvent; (b2) A step of preparing an inner phase (W1) by dissolving or dispersing a GLP-1 RA-based series peptide in a solvent; (c2) A step of dispersing an internal phase (W1) in the oil phase (O) to prepare a water-in-oil (W1 / O) type emulsion; (d2) a step of dispersing the water-in-oil (W1 / O) type dispersed phase prepared in step (c2) in the external continuous phase (W2) to prepare a water-in-oil-in-water (W1 / O / W2) type emulsion solution to solidify microspheres; and (e2) A method for producing microparticles, comprising a step of removing the solvent. 15.(a3) A step of preparing an oil phase (O) by dissolving a biocompatible polymer and a bioavailability improving agent in at least one solvent; (b3) A step of preparing an inner phase (W1) by dissolving or dispersing a GLP-1 RA-based series peptide; and a bioavailability improving agent; in a solvent; (c3) A step of dispersing an internal phase (W1) in the oil phase (O) to prepare a water-in-oil (W1 / O) type emulsion; (d3) a step of dispersing the water-in-oil (W1 / O) type dispersed phase prepared in step (c3) in the external continuous phase (W2) to prepare a water-in-oil-in-water (W1 / O / W2) type emulsion solution to solidify microspheres; and (e3) A method for producing microparticles, comprising a step of removing the solvent.

16. Comprising a microsphere according to any one of paragraphs 1 to 12, A pharmaceutical composition for the prevention or treatment of obesity, diabetes, hypertension, hyperlipidemia, non-alcoholic steatohepatitis, metabolic steatohepatitis, cardiovascular disease, or degenerative neurological disease.

17. In paragraph 16, The above pharmaceutical composition is for subcutaneous or intramuscular injection, pharmaceutical composition.