Method for preparing sustained-release microparticles containing high content of water-soluble peptide drug

Abstract

The present invention relates to a method for preparing sustained-release microspheres containing a drug using a W / O / W emulsion, and has the effect of achieving a stable sustained-release profile with a low initial release rate while loading a high content of various drugs.

Description

Method for manufacturing sustained-release microspheres containing a high content of water-soluble peptide drug

[0001] The present invention relates to a method for manufacturing sustained-release microspheres containing a drug using a W / O / W emulsion.

[0002] Water-soluble drugs such as peptides, proteins, antibodies, and nucleic acids, when developed as oral formulations, face issues with drug instability and low absorption rates, so most are developed as injectable formulations. However, long-term administration of these water-soluble drugs once or twice daily or once a week for the treatment of chronic diseases such as diabetes, obesity, and dementia not only causes pain to patients but also reduces medication compliance. Therefore, there has been a persistent need for the development of sustained-release formulations that can maintain the release of water-soluble drugs for one to six months or more with a single administration.

[0003] Microsphere formulations using biocompatible polymers have attracted considerable attention as sustained-release formulations, leading to active research interest and clinical applications. To achieve long-term, sustained drug efficacy using these microspheres, large doses of drug must be administered, taking into account the duration and dosage of administration. Microspheres must also be administered in corresponding doses. However, if the bioavailability of the encapsulated drug is low or the drug content within the microsphere is low, higher doses of microspheres are necessary to achieve long-term, effective pharmacological effects. However, administering large amounts of microspheres in vivo presents challenges, such as the difficulty of subcutaneous injection, making self-administration difficult for patients. Furthermore, the biodegradable polymers can significantly increase pain and inflammation at the injection site.

[0004] Meanwhile, microspheres loaded with high concentrations of water-soluble drugs using a reduced amount of biodegradable polymers may not have a sufficiently long release period to achieve the desired administration period, or may cause an 'initial burst' in which the drug is released too quickly in the early stages of release. In this case, various side effects may occur due to a rapid increase in the blood concentration of the drug.

[0005] To prevent the above-mentioned problems, conventional technologies manufacture microspheres with a low drug content, typically 20% or less or 10% or less, when manufacturing water-soluble drug microspheres to control the initial release and obtain a sufficient release period.

[0006] Therefore, there is a need for the development of sustained-release microspheres that can encapsulate a high content of water-soluble drug without causing an initial burst and have stable drug release characteristics for a long period of time.

[0007] In order to solve the above problems, the inventor of the present invention developed a method for manufacturing microspheres loaded with a high content of drugs, such as water-soluble peptide drugs, using a W / O / W emulsion. It was verified that microspheres manufactured through this manufacturing method can achieve a low initial release rate and a stable sustained-release profile while loading various drugs at a high content.

[0008] Accordingly, one object of the present invention is to provide a method for manufacturing sustained-release microspheres containing a drug using a W / O / W emulsion.

[0009] Another object of the present invention is to provide sustained-release microspheres containing a drug, a biodegradable polymer, and an early-release inhibitor. The microspheres may be manufactured using the above-described manufacturing method.

[0010] Another object of the present invention is to provide a long-acting injectable composition comprising the above-described sustained-release microparticles.

[0011] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0012] Method for manufacturing sustained-release microspheres containing a drug

[0013] In order to achieve the above purpose, the present invention,

[0014] (a) A step of preparing a W1 / O emulsion by mixing a first phase (W1 phase) solution in which a drug is dissolved in an aqueous solvent and an oil phase (O phase) solution in which a biodegradable polymer is dissolved in an organic solvent;

[0015] (b) a step of preparing a W1 / O / W2 emulsion solution by adding the W1 / O emulsion of step (a) to a second phase (W2 phase) solution containing an early release inhibitor added to an aqueous solution containing a surfactant; and

[0016] (c) A method for producing sustained-release microspheres containing a drug using a W / O / W emulsion is provided, comprising a step of forming microspheres by extracting an organic solvent from the W1 / O / W2 emulsion of step (b) as a second phase (W2 phase) solution.

[0017] In one example, in step (a), the drug may be included in an amount of 4 to 25 w / w% relative to the weight of the first phase (W1 phase) solution.

[0018] In one example, in the step (a), the biodegradable polymer may be included in an amount of 2 to 10 w / w% relative to the weight of the O phase solution.

[0019] In one example, in step (a), the weight ratio of the aqueous solvent and the organic solvent may be 1:1.1 to 1:6.

[0020] In one example, in step (b), the initial release inhibitor may be included in an amount of 1 to 6 w / v% relative to the volume of the second phase (W2 phase) solution.

[0021] In one example, the drug may be a water-soluble peptide having a molecular weight of 2,000 to 10,000 Da or a pharmaceutically acceptable salt thereof.

[0022] In one example, the drug may be present in an amount of 20 to 75 wt% relative to 100 wt% of the sustained-release microspheres.

[0023]

[0024] As another example, the present invention

[0025] (a) a step of preparing a first phase (W1 phase) by dissolving a water-soluble drug or a pharmaceutically acceptable salt thereof in an aqueous solvent, preparing an oil phase (O phase) by dissolving one or more biodegradable polymers in an organic solvent, and preparing a first W1 / O emulsion by mixing the water phase (W1 phase) and the oil phase (O phase) as a dispersed phase (DP);

[0026] (b) a step of preparing a continuous phase (W2 phase, Continuous phase, CP) by adding an early release inhibitor to an aqueous solution containing a surfactant, and adding the dispersed phase of step (a) to prepare a second W1 / O / W2 emulsion solution; and

[0027] (c) a step of forming microspheres by extracting an organic solvent as a continuous phase (W2 phase) from the second W1 / O / W2 emulsion of the above step (b);

[0028] In the first phase (W1) of the above step (a), the water-soluble drug is 4-25 wt%,

[0029] In the O phase of the above step (a), the biodegradable polymer is 2-10 wt%,

[0030] The weight ratio of the aqueous solvent used in the first phase of the above step (a) and the organic solvent used in the oil phase is 1:1.1 to 1:6,

[0031] The concentration of the initial release inhibitor in the continuous phase of the above step (b) is 1-6 w / v%,

[0032] Containing 20-75 wt% of the drug based on the weight of the manufactured microspheres,

[0033] A method for manufacturing microspheres loaded with a high content of a water-soluble drug using a W / O / W emulsion is provided.

[0034]

[0035] The present invention will be described in more detail below.

[0036]

[0037] In one example, the present invention provides a method for preparing sustained-release microspheres capable of significantly suppressing initial release and stably releasing sustained-release drugs despite containing a high drug content.

[0038] In the manufacturing method according to the present invention, step (a) is a step of preparing a first phase (W1 phase) solution (which may also be simply referred to as “first phase”) by dissolving a drug or a pharmaceutically acceptable salt thereof in an aqueous solvent, preparing an oil phase (O phase) solution (which may also be simply referred to as “oil phase”) by dissolving one or more biodegradable polymers in an organic solvent, and preparing a W1 / O emulsion by mixing the water phase (W1 phase) and oil phase (O phase) solutions.

[0039] In this specification, the W1 / O emulsion may be referred to as a “primary emulsion” or a “dispersed phase (DP).”

[0040] In one example, in step (a), the drug may be included in an amount of 4 to 25 w / w% (weight %) relative to the total weight of the first phase (W1 phase) solution. In other words, the concentration of the drug in the first phase may be 4 to 25 w / w% relative to the weight of the first phase.

[0041] More specifically, in the step (a), the concentration of the drug in the first phase can be selected as a lower limit of 4, 5, 6, 7, 7.4, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20.7, 21, 22, 23 or 24 wt% or more, and can be selected as an upper limit of 25, 24, 23, 22, 21, 20.7, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 wt% or less. The concentration of the drug in the first phase can be included in a range formed by a combination of the lower limits and the upper limits. For example, 4-25 wt%, 4-23 wt%, 4-22 wt%, 4-21 wt%, 4-20 wt%, 4-19 wt%, 4-17 wt%, 4-15 wt%, 4-14 wt%, 4-13 wt%, 4-11 wt%, 4-10 wt%, 4-9 wt%, 4-7 wt%, 5-18 wt%, 5-10 wt%, 6-25 wt%, 6-23 wt%, 6-22 wt%, 6-21 wt%, 6-20 wt%, 6-19 wt%, 6-17 wt%, 6-15 wt%, 6-14 wt%, 6-13 wt%, 6-11 wt%, 6-10 wt%, 6-9 wt%, 6-7 wt%, 7-25 wt%, 7-23 wt%, 7-21 wt%, 7-19 wt%, 7-17 wt%, 7-15 wt%, 7-14 wt%, 7-13 wt%, 7-11 wt%, 7-10 wt%, 7.4-25 wt%, 7.4-23 wt%, 7.4-21 wt%, 7.4-19 wt%, 7.4-17 wt%, 7.4-15 wt%, 7.4-14 wt%, 7.4-13 wt%, 7.4-11 wt%, 7.It may be included in an amount of 4-10 wt%, 10-25 wt%, 10-23 wt%, 10-21 wt%, 10-18 wt%, 10-19 wt%, 10-17 wt%, 10-15 wt%, 10-14 wt%, 10-13 wt%, 10-11 wt%, 14-25 wt%, 14-23 wt%, 14-21 wt%, 14-19 wt%, 14-17 wt%, 14-15 wt%, 15-25 wt%, 15-23 wt%, 15-21 wt%, 15-19 wt%, 15-18 wt%, 15-17 wt%, but is not limited thereto. If the concentration range of the drug in the first award exceeds the above-described range, there may be a problem of the drug encapsulation rate being reduced and / or an initial release burst occurring.

[0042] In one example, in step (a), the biodegradable polymer may be included in an amount of 2 to 10 w / w% (weight %) relative to the weight of the oil phase (O phase). In other words, the concentration of the biodegradable polymer in the oil phase may be 2 to 10 wt% relative to the weight of the oil phase.

[0043] More specifically, in the step (a), the concentration of the biodegradable polymer in the oil phase may be selected as a lower limit of 2, 2.5, 2.7, 3, 4, 5, 6, 7, 8, or 9 wt% or more, and may be selected as an upper limit of 10, 9, 8, 7.5, 7, 6, 5, 4, or 3 wt% or less. The concentration of the biodegradable polymer in the oil phase may be included in a range formed by a combination of the lower and upper limits. For example, 2-10 wt%, 2-9 wt%, 2-10 wt%, 2-9 wt%, 2-8 wt%, 2-7 wt%, 2-6 wt%, 2-5 wt%, 2-4 wt%, 2-3 wt%, 2.7-10 wt%, 2.7-9 wt%, 2.7-10 wt%, 2.7-9 wt%, 2.7-8 wt%, 2.7-7.5 wt%, 2.7-7 wt%, 2.7-6 wt%, 2.7-5 wt%, 2.7-4 wt%, 2.7-3 wt%, 2.5-10 wt%, 2.5-9 wt%, 2.5-10 wt%, 2.5-9 wt%, 2.5-8 wt%, 2.5-7.5 wt%, 2.5-7 %, 2.5-6 wt%, 2.5-5 wt%, 2.5-4 wt%, 2.5-3 wt%, 4-10 wt%, 4-9 wt%, 4-10 wt%, 4-9 wt%, 4-8 wt%, 4-7.5 wt%, 4-7 wt%, 4-6 wt%, 4-5 wt%, 5-10 wt%, 5-9 wt%, 5-10 wt%, 5-9 wt%, 5-8 wt%, 5-7.5 wt%, 5-7 wt%, or 5-6 wt%, but is not limited thereto. If the concentration range of the biodegradable polymer in the oil phase is out of the above-mentioned range, there may be a problem of a decrease in the encapsulation rate of the drug and / or a problem of an initial release burst occurring.

[0044] Preferably, in the step (a), the biodegradable polymer may be included in an amount of 2 to 10 w / w% relative to the weight of the oil phase (O phase), and more preferably, it may be included in an amount of 2.5 to 7.5 w / w%. If the concentration of the biodegradable polymer in the oil phase exceeds the upper limit of the above range, the viscosity of the solution may become excessively high, preventing the production of microspheres. If it is less than the above range, the amount of organic solvent used may become excessively large, making it difficult to remove the organic solvent from the produced microspheres and causing environmental pollution.

[0045] In one example, the weight ratio of the aqueous solvent and the organic solvent in step (a) may be 1:1.1 to 1:6 (aqueous solvent:organic solvent). In other words, the organic solvent may be 1.1 to 6 parts by weight relative to 1 part by weight of the aqueous solvent. For example, when distilled water (DW) is used as the aqueous solvent in step (a) and dichloromethane (DCM) is used as the organic solvent, the DW:DCM weight ratio may be 1:1.1 to 1:6.

[0046] More specifically, in the step (a), the weight part of the organic solvent relative to 1 part by weight of the aqueous solvent may be selected as a lower limit of 1.1, 1.5, 1.7, 2, 2.5, 3, 4, 4.96 or 5 parts by weight or more, and may be selected as an upper limit of 6, 5.5, 5.3, 5, 4.96, 4, 3 or 2 parts by weight or less. The weight part of the organic solvent relative to 1 part by weight of the aqueous solvent may be included in a range formed by a combination of the lower and upper limits. For example, 1.1-6 times, 1.1-5.5 times, 1.1-5.3 times, 1.1-5 times, 1.1-6 times, 1.1-5 times, 1.1-4.96 times, 1.1-4 times, 1.1-3 times, 1.1-2 times, 1.5-6 times, 1.5-5.5 times, 1.5-5.3 times, 1.5-5 times, 1.5-6 times, 1.5-5 times, 1.5-4.96 times, 1.5-4 times, 1.5-3 times, 1.5-2 times, 1.7-6 times, 1.7-5.5 times, 1.7-5.3 times, 1.7-5 times, 1.7-6 times, 1.7-5 times, 1.7-4.96 times, It may be 1.7-4 times, 1.7-3 times, 1.7-2 times, 2-6 times, 2-5 times, 2-6 times, 2-5 times, 2-4.96 times, 2-4 times, 2-3 times, 2.5-6 times, 2.5-5 times, 2.5-4.96 times, 2.5-6 times, 2.5-5 times, 2.5-4 times, 2.5-3 times, 3-6 times, 3-5 times, 3-4.96 times, 3-6 times, 3-5 times, or 3-4 times, but is not limited thereto. If the weight part of the organic solvent relative to 1 weight part of the aqueous solvent is out of the above-mentioned range, there may be a problem of a decrease in the encapsulation rate of the drug and / or a problem of an initial release explosion occurring.

[0047] In the above step (a), a homogenizer, a static mixer, an inline mixer, etc. can be used to form a W1 / O emulsion by mixing the primary aqueous solution and the oily solution, and any known method used to form an emulsion can be used without limitation.

[0048] In one example, when forming a W1 / O emulsion by mixing the primary aqueous solution and the oily solution, the emulsion can be formed using a homogenizer under conditions of 15,000 to 35,000 rpm, and preferably 17,500 to 32,500 rpm, 20,000 to 30,000 rpm, 22,500 to 27,500 rpm, 23,500 to 26,500 rpm, 24,000 to 26,000 rpm, 24,500 to 25,500 rpm, 25,000 to 27,000 rpm, 23,000 to 25,000 rpm, 21,000 to 25,500 rpm, or 25,000 to 25,000 Emulsions can be formed under rpm conditions.

[0049] In the present invention, the type of the drug is not limited, and a person skilled in the art can select and use an appropriate drug according to the purpose.

[0050] In one example, the drug may be a water-soluble drug.

[0051] In one example, the drug is selected from the group consisting of: an anti-dementia drug; an anti-Parkinson's disease drug; an anti-cancer drug; an antipsychotic drug such as an anxiolytic, an antidepressant, a tranquilizer, and a psychotropic drug; a cardiovascular drug such as an anti-hyperlipidemic drug, an anti-hypertensive drug, an anti-hypertensive drug, an antithrombotic drug, a vasodilator, and an anti-arrhythmic drug; an anti-epileptic drug; an anti-epilepsy drug; a gastrointestinal drug such as an anti-ulcer drug; an anti-rheumatic drug; an antispasmodic; an anti-tuberculosis drug; a muscle relaxant; an anti-osteoporosis drug; an anti-erectile drug; a hemostatic drug; a hormonal drug such as a sex hormone; an anti-diabetic drug; an anti-obesity drug; a non-alcoholic steatohepatitis drug; an antibiotic; an antifungal drug; an antiviral drug; an antipyretic, analgesic, and anti-inflammatory drug; an autonomic nervous system regulator; a diuretic; an antidiuretic; an analgesic; an anesthetic; an antihistamine; an antiprotozoal drug; an anti-anemia drug; an antiasthmatic drug; an anticonvulsant; an antidote; an antimigraine drug; an antiemetic; an anti-Parkinson's drug; It may be at least one selected from the group consisting of anticonvulsants; antiplatelet agents; expectorants; bronchodilators; cardiotonic agents; immunomodulators; protein drugs; and genetic drugs.

[0052] In one example, the drug may be at least one selected from the group consisting of antidiabetic agents, antiobesity agents, antidementia agents, nonalcoholic steatohepatitis agents, and anticardiovascular agents.

[0053] In one example, the drug may be a peptide drug having a molecular weight of 2,000 to 10,000 Da. That is, the drug may be a water-soluble peptide having a molecular weight of 2,000 to 10,000 Da. More specifically, the molecular weight of the water-soluble peptide as the drug can be selected as a lower limit of 2, 2.5, 3, 3.5, 4, 4.1, 4.4, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 kDa or more, and an upper limit of 10, 9, 8, 7.9, 7.8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.5, 3 or 2.5 kDa or less.For example, 2,000-10,000 Da, 2,000-9,500 Da, 2,000-9,000 Da, 2,000-8,500 Da, 2,000-8,000 Da, 2,000-7,500 Da, 2,000-7,000 Da, 2,000-6,500 Da, 2,000-6,000 Da, 2,000-5,500 Da, 2,000-5,000 Da, 2,000-4,500 Da, 2,000-4,000 Da, 2,000-3,500 Da, 2,000-3,000 Da, 2,000-2,500 Da, 3,000-10,000 Da, 3,000-9,500 Da, 3,000-9,000 Da, 3,000-8,500 Da, 3,000-8,000 Da, 3,000-7,500 Da, 3,000-7,000 Da, 3,000-6,500 Da, 3,000-6,000 Da, 3,000-5,500 Da, 3,000-5,000 Da, 3,000-4,500 Da, 3,000-4,000 Da, 3,000-3,500 Da, 4,000-10,000 Da, 4,000-9,500 Da, 4,000-9,000 Da, 4,000-8,500 Da, 4,000-8,000 Da, 4,000-7,500 Da, 4,000-7,000 Da, 4,000-6,500 Da, 4,000-6,000 Da, 4,000-5,500 Da, 4,000-5,000 Da, 4,000-4,500 Da, 4,500-10,000 Da, 4,500-9,500 Da, 4,500-9,000 Da, 4,500-8,500 Da, 4,500-8,000 Da, 4,500-7,500 Da, 4,500-7,000 Da, 4,500-6,500 Da, 4,500-6,000 Da, It may be a drug of the water-soluble peptide series having a molecular weight of 4,500-5,500 Da, or 4,500-5,000 Da. If the molecular weight of the drug exceeds the range of water-soluble peptides, there may be a problem of reduced drug loading rate and / or occurrence of an initial burst of release.

[0054]

[0055] In one example, the drug may be a peptide drug in the form of a chemically bonded chain of 15 to 45 amino acids, a linker, and a fatty acid. For example, the drug may be at least one selected from the group consisting of, but not limited to, a GLP-1 receptor agonist, a GLP-2 receptor agonist, an amylin analog, and amicretin.

[0056] In one example, the drug is exenatide, liraglutide, lixisenatide, semaglutide, capriglintide, terzepatide, letaturutide, mazdutide, insulin glargine, insulin degludec, insulin icodec, amycretin, eloralintide (LY-3841136 from Eli Lilly), DACRA QW II (LY-3541105 from Eli Lilly), nisotirostide (LY-3457263 from Eli Lilly), servodutide, ecnoglutide, dapiglutide, ZP8396 from Zealand, Viking therapeutics It may be at least one selected from the group consisting of VK2735, Pemvidutide, Bamadutide, Cotadutide, Utreglutide, PYY1875 of Novo Nordisk, and CT-388 of Roche / Carmot therapeutics, but is not limited thereto. When there are two or more of the above drugs, the two or more drugs may be mixed and used. For example, when manufacturing microspheres using the manufacturing method according to the present invention, semaglutide and capriglintide may be mixed to manufacture microspheres loaded with the two drugs. Here, either one of the two drugs may be loaded into a single microsphere, or the two drugs may be mixed and loaded into a single microsphere.

[0057] Among the above drugs, semaglutide is a GLP-1 receptor agonist. 6.26-{18-[N-(17-carboxy-heptadecanoyl)-L-γ-glutamyl]-10-oxo 3,6,12,15-tetraoxa-9,18-diazaoctadecanoyl}-[8-(2-amino-2-propanoic acid), 34-L-arginine]human glucagon-like peptide 1(7-37), which may also be called N-epsilon26-[2-(2-{2-[2- (2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy} ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37). The structure of semaglutide is as shown in chemical formula 1 below.

[0058] [Chemical Formula 1]

[0059]

[0060] When semaglutide is used as a drug in the manufacturing method of the present invention, the semaglutide can be manufactured as described in Manufacturing Example 4 of International Patent Publication No. WO2006 / 097537, and commercially available semaglutide can also be used.

[0061] Among the above drugs, Cagrilintide is a long-acting amylin analog that targets the calcitonin receptor, a receptor activator that modifies RAMP 1-3. Cagrilintide is a non-selective agonist designed for once-weekly subcutaneous administration at low pH. The structure of Cagrilintide is shown in Chemical Formula 2 below.

[0062] [Chemical Formula 2]

[0063]

[0064] In one example, the free base of the drug, as well as a derivative of the drug or a pharmaceutically acceptable salt of the drug, can be used as the active substance.

[0065] The term "pharmaceutically acceptable salt" as used herein means any organic or inorganic addition salt of the compound, which has an effective action at a concentration that is relatively non-toxic and harmless to the patient, and the side effects attributable to the salt do not diminish the beneficial effects of the active ingredient.

[0066] The term “derivative” as used herein includes pharmaceutically acceptable salts, pharmaceutically acceptable solvates, pharmaceutically acceptable hydrates, pharmaceutically acceptable anhydrides, pharmaceutically acceptable enantiomers, pharmaceutically acceptable esters, pharmaceutically acceptable polymorphs, pharmaceutically acceptable prodrugs, pharmaceutically acceptable complexes, and the like.

[0067] The pharmaceutically acceptable salt may include a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base.

[0068] Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid.

[0069] Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0070] In one example, the pharmaceutically acceptable salt may be, but is not limited to, sodium salt, acetate salt, benzoate salt, hydroxynaphthoate salt, napadiylate salt, or pamoate salt.

[0071] In one example, the drug or pharmaceutically acceptable salt thereof may be in various forms, for example, amorphous or crystalline.

[0072] In one example, the first and / or second phase may further comprise a steroidal anti-inflammatory agent.

[0073] In one example, in step (a) of the manufacturing method according to the present invention, a steroidal anti-inflammatory agent may be further dissolved in the aqueous solvent in addition to the drug.

[0074] In one example, in step (a) of the manufacturing method according to the present invention, a steroidal anti-inflammatory agent may be further dissolved in the organic solvent in addition to the polymer.

[0075] That is, the sustained-release microspheres manufactured by the manufacturing method of the present invention may further include a steroidal anti-inflammatory agent in addition to the aforementioned drug (e.g., water-soluble peptide), polymer, and initial release inhibitor. Accordingly, the sustained-release microspheres manufactured by the manufacturing method of the present invention may be in the form of co-encapsulated microspheres that simultaneously contain the steroidal anti-inflammatory agent along with the drug.

[0076] In one example, the steroidal anti-inflammatory agent may act to alleviate the inflammatory response caused by the biodegradable polymer contained in the sustained-release microspheres manufactured by the manufacturing method of the present invention.

[0077] In one example, the first aqueous phase and / or the oil phase may contain the steroidal anti-inflammatory agent in an amount of 0.1 part by weight or less, 0.05 part by weight or less, 0.01 part by weight or less, 0.005 part by weight or less, or 0.001 part by weight or less, as an upper limit, and 0.0001 part by weight or more, 0.0002 part by weight or more, 0.0003 part by weight or more, 0.0004 part by weight or more, or 0.0005 part by weight or more, as a lower limit, relative to 1 part by weight of the water-soluble peptide. The content of the steroidal anti-inflammatory agent relative to 1 part by weight of the water-soluble peptide may be included in the first aqueous phase and / or the oil phase in a range consisting of a combination of the lower limits and the upper limits. For example, when dexamethasone acetate is used as a steroidal anti-inflammatory agent, the dexamethasone acetate may be included in the first aqueous phase and / or oil phase in an amount of 0.0001-0.0020 parts by weight, 0.0002-0.0018 parts by weight, 0.0003-0.0016 parts by weight, 0.0004-0.0013 parts by weight, or 0.0005-0.0010 parts by weight of the steroidal anti-inflammatory agent relative to 1 part by weight of the water-soluble peptide.

[0078] Specific examples of the above steroidal anti-inflammatory agents include 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximethasone, dexamethasone, dexamethasone acetate, dexamethasone phosphate, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone Acetonide, fluocinonide, flucortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, Prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide,Beclomethasone dipropionate, betamethasone, budesonide, deflazacort, dexamethasone, dexamethasone acetate, dexamethasone phosphate, difluprednate, epinephrine, fludrocortisone, fluocinolone acetonide, fluocortin, fluorometholone, fluticasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, Triamcinolone, etc. can be used alone or in combination of two or more. As an example of the present invention, dexamethasone acetate can be used as the steroidal anti-inflammatory agent.

[0079] The above biodegradable polymer is not limited to a specific type, and a person skilled in the art can select an appropriate biodegradable polymer by considering factors such as the release characteristics of the drug and the manufacturing process according to the purpose.

[0080] In one example, the biodegradable polymer is polylactide (PLA), polyglycolide (PGA), polylactide-co-glycolide (PLGA), polydioxanone, polycaprolactone (PCL), polylactide-co-glycolide-co-caprolactone (PLGC), polylactide-co-hydroxymethyl glycolide (PLGMGA), polyalkylcarbonate, polytrimethylenecarbonate (PTMC), polylactide-co-trimethylenecarbonate (PLTMC), A polymer selected from the group consisting of block copolymers of polyhydroxybutyric acid (PHB), polyhydroxybutyrate-co-hydroxyvalerate (PHBV), polyorthoester, polyanhydride, polyanhydride-co-imide, polypropylene fumarate, pseudo polyaminoacid, polyalkyl cyanoacrylate, polyphosphazene, polyphosphoester, polysaccharide and poly(butylene succinate lactide) (PBSLA); a copolymer or simple mixture of two or more polymers selected from the above group;It may be at least one selected from the group consisting of a copolymer of the above polymer and polyethylene glycol (polyethylenglycol, PEG); and a polymer-sugar complex in which the above selected polymer or copolymer is bound to a sugar.

[0081] For example, the biodegradable polymer may be at least one selected from the group consisting of a polymer selected from the group consisting of polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), and polycaprolactone (PCL); a copolymer or simple mixture of two or more polymers selected from the group; a copolymer of the polymer and polyethylene glycol; and a polymer-sugar complex in which the selected polymer or copolymer is bound to sugar.

[0082] As another example, the biodegradable polymer may be at least one selected from the group consisting of a polymer selected from the group consisting of polylactide (PLA), and poly(lactide-co-glycolide) (PLGA); a copolymer or simple mixture of two or more polymers selected from the group; a copolymer of the polymer and polyethylene glycol; and a polymer-sugar complex in which the selected polymer or copolymer is bound to a sugar.

[0083] As another example, the biodegradable polymer may be poly(lactide-co-glycolide) (PLGA) alone, or two or more types of PLGA with different physical properties may be mixed.

[0084] Here, the above properties may be selected from intrinsic viscosity, molar ratio of repeating units, weight average molecular weight, and terminals, but are not limited thereto.

[0085] In one example, the biodegradable polymer may satisfy one or more of the following:

[0086] (i) The intrinsic viscosity of the biodegradable polymer is 0.16 to 1.7 dL / g;

[0087] (ii) the weight average molecular weight of the biodegradable polymer is a weight average molecular weight of 4,000 to 240,000; and

[0088] (iii) The biodegradable polymer contains a hydroxyl group, a carboxyl group, a methoxy group, a methyl group, an amine group, or an ester group at one or both ends.

[0089] Specifically, in one example, the biodegradable polymer may have an intrinsic viscosity of 0.16 to 1.7 dL / g.

[0090] If the intrinsic viscosity of the biodegradable polymer is lower than the lower limit of the above-described range (i.e., less than 0.16 dL / g), the molecular weight of the polymer may be insufficient to exhibit a sustained-release effect of the drug. In addition, if the intrinsic viscosity exceeds the upper limit of the above-described range (i.e., more than 1.7 dL / g), the release of the drug may be excessively delayed, the high viscosity may require the use of an excessive amount of organic solvent, and the reproducibility of microsphere production may deteriorate.

[0091] More specifically, the biodegradable polymer has a pH of 0.16 to 1.7 dL / g, 0.16 to 1.5 dL / g, 0.16 to 1.3 dL / g, 0.16 to 1.1 dL / g, 0.16 to 1 dL / g, 0.16 to 0.9 dL / g, 0.16 to 0.7 dL / g, 0.16 to 0.5 dL / g, 0.16 to 0.3 dL / g, 0.16 to 0.1 dL / g, 0.16 to 0.6 dL / g, 0.16 to 0.5 dL / g, 0.16 to 0.48 dL / g, 0.16 to 0.45 dL / g, 0.16 to 0.44 dL / g, 0.2 to 1.7 dL / g, 0.2 to 1.5 dL / g, 0.2 to 1.3 dL / g, 0.2 to 1.1 dL / g, 0.2 to 1 dL / g, 0.2 to 0.9 dL / g, 0.2 to 0.7 dL / g, 0.2 to 0.5 dL / g, 0.2 to 0.3 dL / g, 0.2 to 0.1 dL / g, 0.2 to 0.6 dL / g, 0.2 to 0.5 dL / g, 0.2 to 0.48 dL / g, 0.2 to 0.45 dL / g, 0.2 to 0.44 dL / g, 0.24 to 1.7 dL / g, 0.24 to 1.5 dL / g, 0.24 to 1.3 dL / g, 0.24 to 1.1 dL / g, 0.24 to 1 dL / g, 0.24 to 0.9 dL / g, 0.24 to 0.7 dL / g, 0.24 to 0.5 dL / g, 0.24 to 0.3 dL / g, 0.24 to 0.1 dL / g, 0.24 to 0.6 dL / g, 0.24 to 0.5 dL / g, 0.2 to 0.48 dL / g, 0.24 to 0.45 dL / g, 0.24 to 0.44 dL / g, 0.28 to 1.7 dL / g, 0.28 to 1.5 dL / g, 0.28 to 1.3 dL / g, 0.28 to 1.1 dL / g, 0.28 to 1 dL / g, 0.28 to 0.9 dL / g, 0.28 to 0.7 dL / g, 0.28 to 0.5 dL / g, 0.28 to 0.3 dL / g, 0.28 to 0.1 dL / g, 0.28 to 0.6 dL / g, 0.28 to 0.5 dL / g, 0.2 to 0.48 dL / g, 0.28 to 0.45 dL / g, 0.28 to 0.44 dL / g, 0.3 to 1.7 dL / g, 0.3 to 1.5 dL / g, 0.3 to 1.3 dL / g, 0.3 to 1.1 dL / g, 0.3 to 1 dL / g, 0.3 to 0.9 dL / g, 0.3 to 0.7 dL / g, 0.3 to 0.5 dL / g, 0.3 to 0.3 dL / g, 0.3 to 0.1 dL / g, 0.3 to 0.6 dL / g, It may be a biodegradable polymer having an intrinsic viscosity of, but not limited to, 0.3 to 0.5 dL / g, 0.2 to 0.48 dL / g, 0.3 to 0.45 dL / g, 0.3 to 0.44 dL / g, or 0.32 to 0.44 dL / g. The intrinsic viscosity may be measured at a concentration of 0.1% (w / v) in chloroform at 25°C using an Ubbelohde viscometer.

[0092] The weight average molecular weight of the biodegradable polymer is not particularly limited, but in one example, the lower limit of the weight average molecular weight may be 4,000 or more, 5,000 or more, 10,000 or more, 15,000 or more, 20,000 or more, 21,000 or more, 22,000 or more, 23,000 or more, or 24,000 or more, and the upper limit may be 500,000 or less, 500,000 or less, 450,000 or less, 400,000 or less, 350,000 or less, 300,000 or less, 250,000 or less, 240,000 or less, 200,000 or less, 150,000 or less, 100,000 or less, 50,000 or less, or 40,000 Below, or 38,000, may be a range consisting of a combination of the above upper and lower limits.

[0093] Specifically, the weight average molecular weight of the biodegradable polymer may be a weight average molecular weight of 4,000 to 100,000, a weight average molecular weight of 7,000 to 50,000, a weight average molecular weight of 5,000 to 20,000, a weight average molecular weight of 10,000 to 18,000, or a weight average molecular weight of 18,000 to 28,000, and may correspond to a lower numerical range within the above range, but is not limited thereto.

[0094] In one example, the biodegradable polymer may include a hydroxyl group, a carboxyl group, a methoxy group, a methyl group, an amine group, or an ester group at one or both ends.

[0095] In one example, the biodegradable polymer may contain two or more repeating units. An example of the biodegradable polymer containing two or more repeating units is poly(lactide-co-glycolide).

[0096] In one example, when poly(lactide-co-glycolide) is used as the biodegradable polymer, the molar ratio of lactide to glycolide in the copolymer is 40:60 to 90:10, 40:60 to 85:15, 40:60 to 80:20, 40:60 to 75:25, 40:60 to 70:30, 40:60 to 65:35, 40:60 to 55:45, 40:60 to 50:50, 45:55 to 90:10, 45:55 to 85:15, 45:55 to 80:20, 45:55 to 75:25, 45:55 to 70:30, 45:55 to 65:35, 45:55 It may be 55:45, 45:60 to 50:50, 50:50 to 90:10, 50:50 to 85:15, 50:50 to 80:20, 50:50 to 75:25, 50:50 to 70:30, 50:50 to 65:35, or 50:50 to 55:45. More specifically, when poly(lactide-co-glycolide) is used as the biodegradable polymer, the molar ratio of lactide to glycolide in the copolymer may be, but is not limited to, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15 or 90:10.

[0097] In one example, the biodegradable polymer may be a commercially available biodegradable polymer, a specific example being Resomer from Evonik. ® PURASORB from Corbion, a subsidiary of Corbion ®You can use series, etc., but they are not limited to this. As a specific example, the biodegradable polymers of the Resomer series of Evonik may be RG502H, RG503H, RG504H, RG502, RG503, RG504, RG653H, RG752H, RG752S, RG755S, RG756S, RG858S, R202H, R203H, R205H, R202S, R203S, R205S, etc., and the biodegradable polymers of the PURASORB series of Corbion may be PDL 02A, PDL 02, PDL 04, PDL 05, PDLG 7502A, PDLG 7502, PDLG 7507, PDLG 5002A, PDLG 5002, PDLG 5004A, PDLG 5004, PDLG 5010, PL 10, PL 18, PL 24, PL 32, PL 38, PDL 20, PDL 45, PC 02, PC 04, PC 12, PC 17, PC 24, etc. can be used, but are not limited thereto.

[0098] In one example, in the oil-based manufacturing process of step (a), one type of biodegradable polymer may be dissolved in the organic solvent, but two or more types of biodegradable polymers may also be dissolved. The two or more types of biodegradable polymers may be a copolymer of two or more types of biodegradable polymers, or a simple mixture of two or more types of biodegradable polymers.

[0099] In this specification, a simple mixture of two or more polymers may be referred to as a “polymer blend.” That is, in one example, the biodegradable polymer dissolved in the organic solvent may be a blend of two or more biodegradable polymers.

[0100] In one example, the blending of the two or more biodegradable polymers may be a combination of two or more biodegradable polymers in which at least one selected from the group consisting of the repeating unit constituting each polymer, the molar ratio of the repeating unit (in the case of a polymer including two or more repeating units), the intrinsic viscosity of the polymer, the weight average molecular weight of the polymer, and the terminal group of the polymer is different from each other.

[0101] Specifically, the blending of two or more biodegradable polymers may be a combination of polymers of different classifications (i.e., polymers having different repeating units or polymers having different types / combinations of monomers) among the polymers exemplified above without limitation, or may mean a combination of polymers of the same classification but having different molar ratios of repeating units, intrinsic viscosities, weight average molecular weights, and / or terminal groups.

[0102] More specifically, the blend of two or more biodegradable polymers may be a combination of polymers of different classifications (e.g., PLA and PLGA); a combination of polymers containing two or more repeating units but having the same classification but different molar ratios of the repeating units (e.g., two types of PLGA having lactide:glycolide ratios of 50:50 and 65:35, respectively); a combination of polymers of the same classification but having different intrinsic viscosities (e.g., two types of PLGA having different intrinsic viscosities); a combination of polymers of the same classification but having different weight average molecular weights; a combination of polymers of the same classification but having different terminal groups (e.g., a polymer having an ester, carboxylic acid, or methyl-blocked terminal group), or a combination of polymers having different intrinsic viscosities, a repeating unit molar ratio, a weight average molecular weight, and terminal groups.

[0103] The combination of two or more polymers having different repeating units includes a combination of a biodegradable polymer having only one type of repeating unit and a biodegradable polymer having two or more repeating units. In this case, any one of the repeating units of the biodegradable polymer having two or more repeating units may be identical to the repeating unit of the biodegradable polymer having only one type of repeating unit (e.g., a combination of PLGA and PLA).

[0104] In one example, when a blend of two or more biodegradable polymers having different classifications, intrinsic viscosity, repeat unit molar ratio, weight average molecular weight, and / or terminal groups is used as the biodegradable polymer, the content ratio of these biodegradable polymers may be, but is not limited to, a weight ratio of 0.5:10 to 10:0.5, 0.5:8 to 8:0.5, 1:10 to 10:1, 1:4 to 4:1, 1:3 to 3:1, 1:1.5 to 1.5:1, 1:1.2 to 1.2:1, 1:1.1 to 1.1:1, or 1:2 to 2:1.

[0105] In one example, in the microspheres manufactured by the manufacturing method according to the present invention, the content (weight %) of the biodegradable polymer among 100% by weight of the sustained-release microspheres may be the remainder excluding the weight of the drug and the early-release inhibitor based on 100% by weight of the microspheres.

[0106] The content of the biodegradable polymer in the microspheres manufactured by the manufacturing method according to the present invention may be selected as a lower limit of 24.8, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 wt% or more based on the total weight of the microspheres, and may be selected as an upper limit of 79.8, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 wt% or less. The content of the biodegradable polymer in the microspheres may be included in a range formed by a combination of the lower and upper limits. For example, it may be 24.8-80 wt%, 24.8-75 wt%, 24.8-70 wt%, 24.8-65 wt%, 24.8-60 wt%, 24.8-55 wt%, 24.8-50 wt%, 24.8-45 wt%, 24.8-40 wt%, or 24.8-35 wt%, but is not limited thereto.

[0107] In one example, when a blend of two or more biodegradable polymers (e.g., PLA and PLGA) is dissolved in an organic solvent in step (a), polymer blend microspheres comprising both of the two or more biodegradable polymers can be produced (e.g., microspheres comprising both PLA and PLGA).

[0108] In one example, in step (a), when organic solvents are prepared separately in two tanks and two types of biodegradable polymers are prepared and each type is dissolved in the two tanks, a microsphere blend can be produced, which is a combination of microspheres A composed of one type of the two biodegradable polymers and microspheres B composed of another type of biodegradable polymer (e.g., a mixture of microspheres containing PLA and microspheres containing PLGA).

[0109] In one example, the aqueous solvent of step (a) may be at least one selected from the group consisting of distilled water, PBS (Phosphate buffered saline), TBS (Triss buffered saline), acetate buffer, citrate buffer, glycine-HCl buffer, ammonium bicarbonate buffer, sodium hydroxide aqueous solution, and urea aqueous solution, but is not limited thereto.

[0110] In one example, the organic solvent of step (a) may be at least one selected from the group consisting of dichloromethane, dimethyl carbonate, chloroform, ethyl acetate, methyl ethyl ketone, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, n-methylpyrrolidone, acetic acid, methyl alcohol, ethyl alcohol, propyl alcohol, and benzyl alcohol. Here, when two or more organic solvents are used, a mixed organic solvent of the two or more organic solvents may be used.

[0111] In one example, the mixed organic solvent may be a mixed organic solvent of an organic solvent that is miscible with water and an organic solvent that is immiscible with water. In this case, it is preferable to use the organic solvent that is immiscible with water in an amount of at least 50% (v / v) or more, 60% or more (v / v), 50 to 99.9% (v / v), 50 to 90% (v / v), 50 to 80% (v / v), 50 to 70% (v / v), 60 to 90% (v / v), or 60 to 80% (v / v) based on the total volume of the mixed organic solvent. Preferably, i) dichloromethane or ethyl acetate is used alone, or ii) dichloromethane or ethyl acetate; And at least one of dimethyl sulfoxide, N-methyl pyrrolidone, methyl alcohol, benzyl alcohol and acetic acid can be used in combination. More preferably, i) dichloromethane is used alone, or ii) dichloromethane; and at least one of dimethyl sulfoxide, N-methyl pyrrolidone, methyl alcohol, benzyl alcohol and acetic acid can be used in combination.

[0112] In one example, the W1 / O emulsion of step (a) may further comprise at least one release-controlling agent selected from the group consisting of butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, heptadecylic acid, stearic acid, nonadecylic acid, behenic acid, arachidic acid, isocrotonic acid, oleic acid, elaidic acid, sorbic acid, linoleic acid, arachidonic acid, benzoic acid, hydroxynaphthoic acid, napadicylic acid, naphthalene sulfonic acid, methanesulfonic acid, and pamoic acid. Here, the release-controlling agent may be added to the first aqueous phase and / or the oil phase and used.

[0113] In the manufacturing method according to the present invention, step (b) is a step of preparing a second phase (W2 phase) solution (which may also be simply referred to as “second phase”) by adding an early release inhibitor to an aqueous solution containing a surfactant, and adding the W1 / O emulsion (i.e., dispersed phase) of step (a) to the second phase to prepare a W1 / O / W2 emulsion solution.

[0114] In this specification, the secondary phase may also be referred to as a “continuous phase (CP)”, and the W1 / O / W2 emulsion may be referred to as a “secondary emulsion”.

[0115] The method for homogeneously mixing the dispersed phase and the continuous phase in the above step (b) is not particularly limited, but may be performed using a high-speed stirrer, an inline mixer, a membrane emulsion method, a microfluidic emulsion method, an ultrasonic mixer, or a static mixer. When forming an emulsion using a high-speed stirrer, an inline mixer, an ultrasonic mixer, or a static mixer, it is difficult to obtain a uniform emulsion, so a sieving process, etc. may be additionally performed between steps (c) and (d) described below.

[0116] In the above step (b), the early release inhibitor may be included in an amount of 1 to 6 w / v% relative to the volume of the secondary aqueous solution (continuous phase). In other words, the concentration of the early release inhibitor in the secondary aqueous solution may be 1 to 6 w / v% relative to the volume of the secondary aqueous solution. In the present invention, the early release inhibitor refers to a substance included in the continuous phase to suppress the rapid release of an active ingredient in a microparticle manufacturing process.

[0117] More specifically, in the step (b), the initial release inhibitor is present in an amount of 1-6 w / v%, 1-5.5 w / v%, 1-5 w / v%, 1-4.5 w / v%, 1-4 w / v%, 1-3.5 w / v%, 1-3 w / v%, 1-2.5 w / v%, 1-2 w / v%, 1-1.5 w / v%, 1.5-6 w / v%, 1.5-5.5 w / v%, 1.5-5 w / v%, 1.5-4.5 w / v%, 1.5-4 w / v%, 1.5-3.5 w / v%, 1.5-3 w / v%, 1.5-2.5 w / v%, 1.5-2 w / v%, 2-6 w / v%, 2-5.5 w / v%, 2-5 w / v%, 2-4.5 w / v%, 2-4 w / v%, 2-3.5 w / v%, 2-3 w / v%, or 2-2.5 w / v%, but is not limited thereto.

[0118] If the content of the initial release inhibitor in the continuous phase exceeds 6.0 w / v%, the emulsion droplets may burst during the production of microspheres, and thus the microspheres may not be produced. Conversely, if the content of the initial release inhibitor in the continuous phase is lower than 1 w / v%, an initial burst of drug release may occur during the administration of the microspheres.

[0119] In addition, the microspheres manufactured by the manufacturing method according to the present invention are characterized in that the early-release inhibitor is present in a less than certain amount. In one example, the early-release inhibitor may be contained in the sustained-release microspheres manufactured by the manufacturing method of the present invention in an amount of 5 ppm to 2,000 ppm.

[0120] When a continuous phase containing an early release inhibitor is used in the manufacture of microspheres, the microspheres manufactured therefrom may have a drug release rate of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less from the time of administration to the first day of administration when administered in vivo or in vitro.

[0121] In one example, the initial release inhibitor may be added at the time of preparing the second aqueous solution (continuous phase) of step (b).

[0122] As described below. In one example of the present invention, in order to efficiently remove the organic solvent in step (c), a process of additionally supplying a fresh secondary aqueous solution (continuous phase) and discharging the secondary aqueous solution (continuous phase) mixed with the organic solvent may be performed. Here, the fresh secondary aqueous solution (continuous phase) may be the secondary aqueous solution prepared in step (b), but is not limited thereto. Here, the early release inhibitor may be added at the time of preparing the continuous phase in step (b), or may be added to the fresh continuous phase additionally supplied in step (c), and may be included in both the continuous phases of step (b) and step (c). Here, when the early release inhibitor is included in both the continuous phase of step (b) and the continuous phase of step (c), the concentration of the early release inhibitor in the continuous phase of step (b) may be 1-6 w / v, and the concentration of the early release inhibitor in the continuous phase of step (c) may be 1-6 w / v. At this time, the concentration of the early release inhibitor in each continuous phase may be the same or different.

[0123] In the present invention, the method of introducing the initial release inhibitor into the continuous phase is not particularly limited, and any method known in the art can be used without limitation.

[0124] The above early release inhibitor is a substance that, when dissolved in a continuous phase, can maintain the pH of the continuous phase at 4 or more, 5 or more, 6 or more, 7 or more, more specifically, 4 or more, 4.5 or more, 5 or more, 6 or more, 7 or more, 7.2 or more, 7.4 or more, 8.0 or more, 8.5 or more, 9.0 or more, 4.0 to 9.0, or 4.5 to 8.5, and any substance that can form a monovalent or divalent anion in the continuous phase can be used. In one example, the early release inhibitor may be at least one selected from the group consisting of phosphate salts, hydroxide salts, phosphide salts, phosphite salts, carbonate salts, bicarbonate salts, chromate salts, dichromate salts, oxides, oxalate salts, silicate salts, sulfate salts, sulfide salts, sulfite salts, tartrate salts, tetraborate salts, thiosulfate salts, arsenate salts, arsenite salts, citrate salts, ferricyanide salts, and nitride salts of alkali metals, alkaline earth metals, or ammonium.

[0125] In one example, the early release inhibitor may be at least one selected from the group consisting of, but not limited to, phosphate salts of two or more alkali metals, phosphate salts of one or more alkali metals, bicarbonate salts of one or more alkali metals, and carbonate salts of one or more alkali metals.

[0126] Here, the phosphate salts of the two or more alkali metals may be disodium phosphate (Na2HPO4) or dipotassium phosphate (K2HPO4), the phosphate salt of the one or more alkali metals may be monosodium phosphate (NaH2PO4), the bicarbonate salt of the one or more alkali metals may be sodium bicarbonate (NaHCO3), and the carbonate salt of the one or more alkali metals may be sodium carbonate (Na2CO3). These metal salts may be used alone as an early-release inhibitor, or two or more types may be mixed and used as an early-release inhibitor.

[0127] In one example, the aqueous solution of step (b) may be distilled water. When distilled water (water) is used as the aqueous solution, water containing at least one regulator selected from the group consisting of methyl alcohol, ethyl alcohol, propyl alcohol, and ethyl acetate may be used to control the extraction rate of the organic solvent from the dispersed phase in the emulsion state.

[0128] The surfactant of the above step (b) is not limited to a specific type, and can be used without limitation as long as it can help the dispersed phase form a stable droplet emulsion within the continuous phase.

[0129] In one example, the surfactant of step (b) may be at least one selected from the group consisting of polyvinyl alcohol, methylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, lecithin, gelatin, polyoxyethylene sorbitan fatty acid ester, and polyoxyethylene castor oil derivatives.

[0130] In one example, in the step (b), the surfactant may be included in an amount of 0.01 w / v% to 20 w / v% relative to the total volume of the secondary aqueous solution (continuous phase). In other words, the concentration (content) of the surfactant in the secondary aqueous solution may be 0.01 w / v% to 20 w / v% relative to the volume of the secondary aqueous solution.

[0131] When the content of the surfactant is less than 0.01 w / v%, a dispersed phase emulsion in the form of droplets may not be formed in the continuous phase, and when the content of the surfactant exceeds 20 w / v%, after microspheres are formed in the continuous phase due to an excessive amount of surfactant, it may be difficult to remove the surfactant.

[0132] More specifically, in the step (b), the surfactant is present in an amount of 0.01-20 w / v%, 0.01-20 w / v%, 0.01-18 w / v%, 0.01-16 w / v%, 0.01-14 w / v%, 0.01-12 w / v%, 0.01-10 w / v%, 0.01-8 w / v%, 0.01-6 w / v%, 0.01-4 w / v%, 0.01-2 w / v%, 0.01-1 w / v%, 0.01-0.8 w / v%, 0.01-0.6 w / v%, 0.01-0.4 w / v%, 0.01-0.2 w / v%, relative to the total volume of the secondary aqueous solution (continuous phase). 0.01-0.15 w / v%, 0.03-20 w / v%, 0.03-20 w / v%, 0.03-18 w / v%, 0.03-16 w / v%, 0.03-14 w / v%, 0.03-12 w / v%, 0.03-10 w / v%, 0.03-8 w / v%, 0.03-6 w / v%, 0.03-4 w / v%, 0.03-2 w / v%, 0.03-1 w / v%, 0.03-0.8 w / v%, 0.03-0.6 w / v%, 0.03-0.4 w / v%, 0.03-0.2 w / v%, 0.03-0.15 w / v%, 0.05-20 w / v%, 0.05-20 w / v%, 0.05-18 w / v%, 0.05-16 w / v%, 0.05-15 w / v%, 0.05-14 w / v%, 0.05-12 w / v%, 0.05-10 w / v%, 0.05-8 w / v%, 0.05-6 w / v%, 0.05-4 w / v%, 0.05-2 w / v%, 0.05-1 w / v%, 0.05-0.8 w / v%, 0.05-0.6 w / v%, 0.05-0.4 w / v%, 0.05-0.2 w / v%, 0.05-0.15 w / v%, 0.07-20 w / v%, 0.07-20 w / v%, 0.07-18 w / v%, 0.07-16 w / v%, 0.07-14 w / v%, 0.07-12 w / v%, 0.07-10 w / v%, 0.07-8 w / v%, 0.07-6 w / v%, 0.07-4 w / v%, 0.07-2 w / v%, 0.07-1 w / v%, 0.07-0.8 w / v%, 0.07-0.6 w / v%, 0.07-0.4 w / v%, 0.07-0.2 w / v%, 0.07-0.15 w / v%, 0.1-20 w / v%, 0.1-20 w / v%, 0.1-18 w / v%, 0.1-16 w / v%, 0.1-14 w / v%, 0.1-12 w / v%, 0.1-10 w / v%, 0.1-8 w / v%, 0.1-6 w / v%, 0.1-5 w / v%, 0.1-4 w / v%, 0.1-2 w / v%, 0.1-1 w / v%, 0.1-0.8 w / v%, 0.1-0.6 w / v%, 0.1-0.4 w / v%, 0.1-0.2 w / v%, or 0.1-0.15 It may be included in w / v%, but is not limited thereto.

[0133] In one example, the pH range of the continuous phase may be selected as a lower limit of 4.0 or more, 4.5 or more, 5.0 or more, 6.0 or more, 7.0 or more, 8.5 or more, or 9.0 or more, and may be selected as an upper limit of 12.0 or less, 11.0 or less, or 10.0 or less, or may be a range formed by a combination of the upper and lower limits. That is, the continuous phase may be acidic or basic. For example, the pH range of the continuous phase may be, but is not limited to, 4.0 to 9.0, 4.5 to 8.5, 7.0 to 9.0, or 7.0 to 8.5.

[0134] The temperature of the above continuous phase is not particularly limited, but in one example, the second aqueous solution (continuous phase) may be heated to a level close to the boiling point of the organic solvent used in preparing the W1 / O emulsion (dispersed phase) in step (a). Using a continuous phase heated in this way may be advantageous for selective evaporation of the organic solvent mixed into the second aqueous solution (continuous phase) after the organic solvent of the W1 / O emulsion (dispersed phase) is extracted from the emulsion suspended in the second aqueous solution (continuous phase). In another example, when the drug used is a drug sensitive to high temperatures, the second aqueous solution (continuous phase) may be heated to a level that does not affect the activity or structure of the drug.

[0135] In the manufacturing method according to the present invention, step (c) is a step of forming microspheres by extracting an organic solvent as a secondary aqueous solution (continuous phase) from the W1 / O / W2 emulsion manufactured in step (b).

[0136] In the above step (c), as the organic solvent in the W1 / O / W2 emulsion is extracted toward the continuous phase, the W1 / O / W2 emulsion is gradually hardened to form microspheres. To this end, when the W1 / O / W2 emulsion is maintained or stirred for a certain period of time, for example, 2 to 48 hours, at a temperature below the boiling point of the organic solvent used in the preparation of the oil phase in step (a), the organic solvent can be extracted from the W1 / O / W2 emulsion as a continuous phase. A portion of the organic solvent extracted into the continuous phase can be evaporated from the surface of the continuous phase. As the organic solvent is extracted and evaporated from the drug in the form of droplets (e.g., a water-soluble drug and a pharmaceutically acceptable salt thereof) and the polymer solution, the dispersed phase in the form of droplets can be solidified to form microspheres.

[0137] That is, in one example, the microspheres according to the present invention can be manufactured by solvent extraction and evaporation.

[0138] In the above step (c), the continuous phase may be heated for a certain period of time to efficiently remove the organic solvent. The heating temperature is not limited and can be appropriately adjusted by a person skilled in the art depending on the organic solvent used. For example, when dichloromethane is used as the organic solvent, the heating may be applied so as to maintain the temperature at 30°C or higher, 40°C or higher, 45°C or higher, 30 to 50°C, 40 to 50°C, or 45°C.

[0139] In one example, in order to efficiently remove the organic solvent in the above step (c), a process of supplying a fresh secondary aqueous solution (continuous phase) and discharging the secondary aqueous solution (continuous phase) mixed with the organic solvent may be added.

[0140] In one example, the supply of the fresh continuous phase and the discharge of the continuous phase mixed with the organic solvent can be performed continuously or discontinuously.

[0141] In one example, the supply of the fresh continuous phase and the discharge of the continuous phase mixed with the organic solvent may be performed from the start of extracting the organic solvent from the W1 / O / W2 emulsion into the second aqueous solution (continuous phase) or after a certain period of time (e.g., 5 minutes, 5 minutes to 10 minutes, 10 minutes to 20 minutes, 10 minutes to 30 minutes, 10 minutes to 40 minutes, 10 minutes to 50 minutes, or 10 minutes to 60 minutes).

[0142] In one example, the fresh secondary phase solution (continuous phase) may be the secondary phase solution prepared in step (b).

[0143] Furthermore, the organic solvent discharged in the above step (c) can be separated from the mixed continuous phase, and the separated organic solvent and continuous phase can be recycled, respectively. Adding a process for recycling the organic solvent and the continuous phase has the advantage of being able to manufacture microspheres through an environment-friendly process. The method for separating the organic solvent from the mixed continuous phase is not particularly limited, and any known technology can be used. For example, organic solvent evaporation-condensation recovery method, OSRO (Organic solvent reverse osmosis), OSN (organic solvent nanofiltration), SRNF (solvent-resistant nanofiltration), etc. can be used.

[0144] In one example, the manufacturing method according to the present invention may further include the following steps (d) to (f) after the step (c).

[0145] (d) a step of removing surfactants remaining on the surface of the microparticles through washing;

[0146] (e) a step of recovering the microspheres; and

[0147] (f) A step of drying the obtained microspheres.

[0148] The means of step (d) above is not limited to a specific type, and any known technology may be applied. For example, washing may be performed using water, and the washing may be repeated several times.

[0149] The means of step (e) above are not limited to a specific type, and known techniques can be applied. For example, methods such as filtration and centrifugation can be performed to recover microspheres. For example, microspheres can be recovered using a hydrocyclone utilizing centrifugation.

[0150] The means of the above step (f) is not limited to a specific type, and known techniques can be applied. For example, the microspheres can be dried using methods such as freeze-drying, vacuum freeze-drying, low-temperature drying, room-temperature drying, hot-air drying, ventilation drying, pressurized-reduced-pressure drying, fluidized bed drying, vacuum drying, infrared drying, and / or suction drying.

[0151] In one example, the manufacturing method according to the present invention may additionally include a sieving process between steps (d) and (e). Through this sieving process, uniform microspheres can be obtained. There are no specific limitations on the means for the sieving process, and the process can be performed using known techniques. For example, microspheres of uniform size can be obtained by filtering out small and large particles using sieves of different sizes.

[0152] In one example, the remaining surfactant may be removed through filtration and washing between steps (c) and (d), and the microspheres may be recovered by filtration again. The washing step to remove the remaining surfactant may typically be performed using water, and the washing step may be repeated several times.

[0153] In one example, the manufacturing method according to the present invention may further include, after step (f), the step (g) of filling the dried microspheres into a container.

[0154] For example, step (g) can be used to obtain a final product in the form of a suspension of dried microspheres and filled into a suitable container. For example, a final product in the form of a prefilled syringe can be obtained by filling a microsphere suspension into a disposable syringe.

[0155] As another example, the step (g) may also obtain a final product in the form of a powder filled into a vial with dried microspheres.

[0156] The sustained-release microspheres manufactured using the manufacturing method according to the present invention can contain a high content of the drug. That is, sustained-release microspheres containing a high content of drug (i.e., loaded with a high content of drug) can be manufactured using the manufacturing method according to the present invention.

[0157] In the sustained-release microspheres manufactured by the manufacturing method according to the present invention, the drug may be included in an amount of 20 wt% or more, 30 wt% or more, 35 wt% or more, 40 wt% or more, 45 wt% or more, 50 wt% or more, 55 wt% or more, 60 wt% or more, 65 wt% or more, or 70 wt% or more, based on 100 wt% of the sustained-release microspheres. In other words, the microspheres manufactured by the manufacturing method of the present invention may include the drug in an amount of 20 wt% or more, 30 wt% or more, 35 wt% or more, 40 wt% or more, 45 wt% or more, 50 wt% or more, 55 wt% or more, 60 wt% or more, 65 wt% or more, or 70 wt% or more, based on the total weight of the entire microspheres. The content of the above drug refers to the total weight of drugs loaded into the microspheres with respect to the total weight of the sustained-release microspheres manufactured by the manufacturing method of the present invention.

[0158] Even if the upper limit of the drug content in the sustained-release microspheres is not specified, a person skilled in the art will be able to clearly practice the present invention for the purpose of producing sustained-release microspheres containing a drug, but the upper limit may be, for example, 75 wt% or less, 70 wt% or less, 65 wt% or less, or 60 wt% or less.

[0159] More specifically, in the sustained-release microspheres manufactured by the manufacturing method according to the present invention, the drug may be included in an amount of 20-75 wt%, 25-75 wt%, 30-75 wt%, 35-75 wt%, 40-75 wt%, 45-75 wt%, or 50-75 wt% based on 100 wt% of the sustained-release microspheres, but is not limited thereto. For example, the manufacturing method of the present invention can be used to manufacture low-content microspheres having a drug content of less than 20 wt%, and in this case, the encapsulation rate and / or initial release inhibition effect of the drug can be improved.

[0160] In one example, the encapsulation rate of the drug in the microspheres manufactured by the manufacturing method according to the present invention may be 80% or more, 82% or more, 84% or more, 85% or more, 86% or more, 88% or more, 90% or more, 92% or more, 94% or more, 96% or more, or 98% or more.

[0161] In one example, the in vitro drug release rate of the microspheres manufactured by the manufacturing method according to the present invention within 24 hours may be 10% or less. In one example, the in vitro drug release rate within 24 hours can be analyzed by detecting the drug present in the supernatant of the test solution 24 hours after the start of culturing the microspheres in a release test solution under conditions similar to a living body. The type of the release test solution is not limited to a specific type, and a person skilled in the art can select an appropriate release test solution depending on the type of drug used based on common sense in the art.

[0162] In one example, the cumulative drug release rate of microspheres manufactured using the manufacturing method of the present invention may be 10% or less for up to 24 hours after in vivo administration. The cumulative drug release rate after in vivo administration can be analyzed by measuring the concentration of the drug in the blood of a subject a certain period of time after in vivo administration.

[0163] In one example, the in vitro drug release rate within 24 hours of the microspheres manufactured by the manufacturing method according to the present invention; or the cumulative drug release rate up to 24 hours after in vivo administration may be 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Even if the lower limit of the drug release rate is not specified, a person skilled in the art will clearly be able to practice the present invention for the purpose of manufacturing sustained-release microspheres containing a drug, but the lower limit may be, for example, 0% or more, 0.1% or more, 0.5% or more, 1% or more, 1.5% or more, 2% or more, or 2.5% or more.

[0164] In one example, the in vitro drug release rate within 6 hours of the microspheres manufactured by the manufacturing method according to the present invention; or the cumulative drug release rate up to 6 hours after in vivo administration may be 2% or less, 1.6% or less, 1.54% or less, 1.2% or less, 1% or less, 0.8% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less. In this case, the lower limit of the drug release rate may be, for example, 0% or more, 0.01% or more, or 0.05% or more, but is not limited thereto.

[0165] In one example, microspheres manufactured by the manufacturing method according to the present invention can release the drug for 4 to 24 weeks after in vivo administration. More specifically, when the microspheres are administered in vivo, the microspheres are administered at 4 to 24 weeks, 5 to 24 weeks, 6 to 24 weeks, 7 to 24 weeks, 8 to 24 weeks, 9 to 24 weeks, 10 to 24 weeks, 11 to 24 weeks, 12 to 24 weeks, 13 to 24 weeks, 14 to 24 weeks, 15 to 24 weeks, 16 to 24 weeks, 17 to 24 weeks, 18 to 24 weeks, 19 to 24 weeks, 20 to 24 weeks, 21 to 24 weeks, 22 to 24 weeks, 23 to 24 weeks, 4 to 5 weeks, 4 to 6 weeks, 4 to 7 weeks, 4 weeks from the time of administration. The drug can be released for 8 weeks, 4 weeks to 9 weeks, 4 weeks to 10 weeks, 4 weeks to 11 weeks, 4 weeks to 12 weeks, 4 weeks to 13 weeks, 4 weeks to 14 weeks, 4 weeks to 15 weeks, 4 weeks to 16 weeks, 4 weeks to 17 weeks, 4 weeks to 18 weeks, 4 weeks to 19 weeks, or 4 weeks to 20 weeks. In the embodiment of the present invention, a target formulation for 4-6 weeks was prepared as an example, but the release period can be extended by appropriately adjusting the composition of the polymer.

[0166] In one example, when the above-described conditions are not satisfied during the manufacturing method according to the present invention, that is, when one or more of (i) the content (w / w%) of the drug in the first phase of step (a); (ii) the weight ratio of the aqueous solvent:organic solvent of step (a); and (iii) the content (w / v%) of the early release inhibitor in the continuous phase of step (b) are not satisfied, the microspheres manufactured from the manufacturing method may have a reduced drug encapsulation rate (e.g., less than 80%), or a cumulative drug release rate within 24 hours after in vivo administration or a drug release rate within 24 hours in vitro may exceed 10%.

[0167] In one example, when the condition of the content (w / v%) of the initial release inhibitor in the continuous phase of step (b) described above is not satisfied during the manufacturing method according to the present invention, the microspheres may be decomposed during the manufacturing process and normal microspheres may not be obtained.

[0168] In one example, when the content (w / w%) of the biodegradable polymer in the oil phase of step (a) (iv) described above is not satisfied during the manufacturing method according to the present invention, the microspheres manufactured from the manufacturing method may have a reduced drug encapsulation rate (e.g., less than 80%), or the cumulative drug release rate within 24 hours after in vivo administration or the drug release rate within 24 hours in vitro may exceed 10%.

[0169] In one example, the microspheres manufactured by the manufacturing method according to the present invention have a size of 5 μm to 100 μm, 5 μm to 90 μm, 5 μm to 80 μm, 10 μm to 90 μm, 10 μm to 80 μm, 15 μm to 100 μm, 15 μm to 90 μm, 15 μm to 80 μm, 70 μm to 100 μm, 70 μm to 90 μm, 70 μm to 80 μm, 60 μm to 100 μm, 60 μm to 80 μm, 60 μm to 70 μm, 20 μm to 90 μm, 20 μm to 70 μm, 20 μm to 60 μm, 30 μm to 80 μm, 30 μm to 60 μm, It is preferable to have a uniform particle distribution, i.e., an average particle size, of 40 μm to 70 μm, 40 μm to 50 μm, 30 μm to 40 μm, 20 μm to 30 μm, 5 μm to 30 μm, 5 μm to 20 μm, 10 μm to 20 μm, or 5 μm to 10 μm. The term “average particle size” used in the present invention refers to a particle size corresponding to 50% of the volume % in a particle size distribution curve, and means median diameter, and is expressed as D50 or D(v, 0.5).

[0170] If the average particle size of the above microspheres is less than 5 μm, the drug release from the microspheres will be too rapid, which is undesirable. If the average particle size exceeds 100 μm, the injection needle may become too thick during human administration, which may cause pain during injection or the drug may leak out of the injection site after injection, which may be undesirable.

[0171] In one example, it is preferable that the microspheres manufactured by the manufacturing method according to the present invention have a uniform particle distribution. Microspheres having a uniform particle distribution have a smaller deviation during injection and can be administered in a more accurate amount compared to microspheres having an uneven particle distribution. It is preferable that the span value of the microspheres manufactured by the manufacturing method of the present invention is 1.5 or less. More preferably, the span value is 1.2 or less. More specifically, the span value of the microspheres manufactured by the manufacturing method of the present invention may be 1.5 or less, 1.2 or less, 0.1 to 1.5, 0.3 to 1.5, 0.5 to 1.5, 0.1 to 1.0, 0.4 to 1.0, 0.6 to 1.0, 0.2 to 0.8, or 0.4 to 0.8. The term “span value” used in the present invention is an index indicating the uniformity of the particle size of microspheres, and means a value obtained by the formula Span value = (Dv0.9-Dv0.1) / Dv0.5. Here, Dv0.1 means a particle size corresponding to 10% of the volume% in the particle size distribution curve of microspheres, Dv0.5 means a particle size corresponding to 50% of the volume% in the particle size distribution curve of microspheres, and Dv0.9 means a particle size corresponding to 90% of the volume% in the particle size distribution curve of microspheres.

[0172] In one example, the total weight of the microspheres manufactured by the manufacturing method of the present invention is 20 to 1000 mg, 20 to 800 mg, 20 to 600 mg, 20 to 400 mg, 20 to 200 mg, 20 to 100 mg, 30 to 1000 mg, 30 to 800 mg, 30 to 600 mg, 30 to 400 mg, 30 to 200 mg, 30 to 100 mg, 40 to 1000 mg, 40 to 800 mg, 40 to 600 mg, 40 to 400 mg, 40 to 200 mg, 40 to 100 mg, 50 to 1000 mg, 50 to 800 mg, It may be 50 mg to 600 mg, 50 mg to 400 mg, 50 mg to 200 mg, or 50 mg to 100 mg.

[0173] In a composition containing the above microspheres (e.g., a long-lasting injection composition described below), when the total weight of the microspheres in the composition is within the above range, the composition has the advantage of minimizing an inflammatory reaction at the site of administration when the composition is administered, and also enabling self-administration by the patient.

[0174] In one example, sustained-release microspheres manufactured by the manufacturing method of the present invention have an area under the blood concentration-time curve (AUC) of the drug for up to 24 hours after in vivo administration. 0-24hr ) is the area under the total blood concentration-time curve (AUC) total ) may be 10% or less, 5% or less, 0.1 to 20%, 1 to 10%, or 1 to 5%.

[0175]

[0176] Sustained-release microspheres containing a drug and long-acting injectable composition containing the same

[0177] The present invention also provides a long-acting injectable composition comprising one or more sustained-release microspheres containing a drug, a biodegradable polymer, and an early-release inhibitor.

[0178] In one example, the drug may be a water-soluble peptide having a molecular weight of 2,000 to 10,000 Da or a pharmaceutically acceptable salt thereof.

[0179] In one example, the drug may be included in an amount of 20 to 75 wt% based on 100 wt% of the sustained-release microspheres.

[0180] In one example, the early release inhibitor may be included in the sustained release microspheres at a concentration of 5 ppm to 2,000 ppm.

[0181] In one example, the sustained-release microspheres may be one type of sustained-release microsphere containing the same biodegradable polymer or two or more types of sustained-release microspheres containing different biodegradable polymers.

[0182] In one example, the sustained-release microspheres may have a cumulative drug release rate of 10% or less for up to 24 hours after in vivo administration.

[0183] In one example, the sustained-release microspheres can release the drug for 4 to 24 weeks after in vivo administration.

[0184] In one example, the above western-type microspheres are

[0185] (a) A step of preparing a W1 / O emulsion by mixing a first phase (W1 phase) solution in which a drug is dissolved in an aqueous solvent and an oil phase (O phase) solution in which a biodegradable polymer is dissolved in an organic solvent;

[0186] (b) a step of preparing a W1 / O / W2 emulsion solution by adding the W1 / O emulsion of step (a) to a second phase (W2 phase) solution containing an early release inhibitor added to an aqueous solution containing a surfactant; and

[0187] (c) It may be manufactured by a manufacturing method including a step of forming microspheres by extracting an organic solvent from the W1 / O / W2 emulsion of the above step (b) as a second phase (W2 phase) solution.

[0188] In one example, in step (a) of the above manufacturing method, the drug may be included in an amount of 4 to 25 w / w% relative to the weight of the first phase (W1 phase) solution.

[0189] In one example, in step (a) of the manufacturing method, the biodegradable polymer may be included in an amount of 2 to 10 w / w% relative to the weight of the O phase solution.

[0190] In one example, the weight ratio of the aqueous solvent and the organic solvent may be 1:1.1 to 1:6.

[0191] In one example, in step (b) of the manufacturing method, the initial release inhibitor may be included in an amount of 1 to 6 w / v% relative to the volume of the second phase (W2 phase) solution.

[0192]

[0193] As another example, the present invention

[0194] (i) sustained-release microspheres comprising a water-soluble peptide or a pharmaceutically acceptable salt thereof, a biodegradable polymer, and an early-release inhibitor as a pharmaceutical agent (or active substance); or

[0195] (ii) A long-acting injection composition comprising a sustained-release microsphere blend, which is a combination of two or three types of sustained-release microspheres of (i) having different compositions from each other.

[0196] In one example, the water-soluble peptide may have a molecular weight of 2,000 to 10,000 Da.

[0197] In one example, the early release inhibitor may be disodium phosphate or monosodium phosphate.

[0198] In one example, the early release inhibitor may be included in the sustained release microspheres at 5 ppm to 2000 ppm.

[0199] In one example, the sustained-release microparticles of (i) or the sustained-release microparticle blend of (ii) may contain 20 to 75 wt% of the active substance based on the total weight.

[0200] In one example, the encapsulation rate of the active substance in the above-described western-type microparticles may be 80% or more.

[0201] In one example, the initial release amount of the active substance on the first day after in vivo administration of the above-described western-type microspheres may be 10% or less.

[0202] In one example, the active substance may be released for 4-24 weeks after in vivo administration of the above-described western-type microspheres.

[0203] In one example, the above (i) western-type microspheres may further comprise a steroidal anti-inflammatory agent.

[0204] In one example, the sustained-release microsphere blend of (ii) may further include a steroidal anti-inflammatory agent in at least one of the 2-3 sustained-release microspheres.

[0205] In one example, the long-acting injectable composition may further comprise sustained-release microspheres containing a steroidal anti-inflammatory agent.

[0206] In one example, the two or three kinds of sustained-release microspheres of (i) having different compositions may mean two or more kinds of sustained-release microspheres having different compositions, at least one of which is selected from the group consisting of a biodegradable polymer and a drug.

[0207]

[0208] The present invention will be described in more detail below.

[0209] The aforementioned matters can be applied to sustained-release microspheres included in the long-acting injection composition.

[0210] In one example, the sustained-release microspheres included in the long-acting injectable composition may be manufactured by the manufacturing method of the present invention described above. In this case, when the long-acting injectable composition includes two or more types of sustained-release microspheres, each sustained-release microsphere may be manufactured through a single manufacturing process or may be manufactured through separate, independent manufacturing processes.

[0211] In one example, the long-acting injectable composition according to the present invention may comprise only one type of sustained-release microsphere as the microsphere. In other words, in one example, the long-acting injectable composition according to the present invention may comprise only microspheres containing the same type of drug, biodegradable polymer, and early-release inhibitor. That is, in the present specification, the type or composition of the microspheres being the same means that each microsphere contains the same type of drug, biodegradable polymer, and early-release inhibitor.

[0212] In another example, the long-acting injectable composition according to the present invention may comprise two or more types (e.g., two to five types, two to four types, or two to three types) of sustained-release microspheres as microspheres. In other words, in one example, the long-acting injectable composition according to the present invention may comprise a mixture (blend) of two or more types of sustained-release microspheres, wherein at least one of the drug, the biodegradable polymer, and the initial release inhibitor contained in the microspheres is different from each other.

[0213] Preferably, the same type of sustained-release microspheres may mean that the biodegradable polymers contained in the microspheres are the same, and two or more types of sustained-release microspheres may mean two or more types of sustained-release microspheres in which the biodegradable polymers contained in the microspheres are different from each other.

[0214] Here, “two or more types of sustained-release microspheres containing different biodegradable polymers” means:

[0215] (i) If at least one biodegradable polymer contained in a microparticle is different from the biodegradable polymer contained in another microparticle;

[0216] (ii) when the number of types of biodegradable polymers contained in each microsphere is different (e.g., a combination of microspheres containing only one type of biodegradable polymer and microspheres containing two or more types of biodegradable polymers), and

[0217] (iii) Microspheres each containing two or more types of biodegradable polymers, the types of biodegradable polymers are the same, but the relative ratios (content ratios) of the two types of biodegradable polymers within the microspheres are different (e.g., microspheres containing PLGA:PLA = 1:3 and microspheres containing PGLA:PLA = 3:1).

[0218] May include, but is not limited to, the following:

[0219] Here, the fact that the biodegradable polymers of (i) are different may mean that at least one selected from the group consisting of the repeating unit constituting each polymer, the molar ratio of the repeating unit, the intrinsic viscosity of the polymer, the weight average molecular weight of the polymer, and the terminal group of the polymer is different from each other.

[0220] The definitions, types, and specific descriptions of the above drugs, biodegradable polymers, and early-release inhibitors are the same as those described in [Method for Manufacturing Sustained-Release Microspheres Containing Drugs], so duplicate descriptions are omitted.

[0221] In one example, the early release inhibitor may be included in the sustained release microspheres at a concentration of 5 ppm to 2,000 ppm.

[0222] Specifically, the initial release inhibitor is present in an amount of 10 ppm or more, 20 ppm or more, 30 ppm or more, 40 ppm or more, 50 ppm or more, 100 ppm or more, 150 ppm or more, 200 ppm or more, 250 ppm or more, 200 ppm or more, 300 ppm or more, 350 ppm or more, 400 ppm or more, 450 ppm or more, 500 ppm or more, 550 ppm or more, 600 ppm or more, 650 ppm or more, 700 ppm or more, 750 ppm or more, 800 ppm or more, 850 ppm or more, 900 ppm or more, 950 ppm or more, 1000 ppm or more, 1100 ppm or more, 1200 ppm or more, 1300 ppm or more, based on the total weight of the microparticles. The upper limit may include 1400 ppm or more, or 1500 ppm or more, and the lower limit may include 2000 ppm or less, 1900 ppm or less, 1800 ppm or less, 1700 ppm or less, 1600 ppm or less, 1500 ppm or less, 1400 ppm or less, 1300 ppm or less, 1200 ppm or less, 1100 ppm or less, 1000 ppm or less, 900 ppm or less, 800 ppm or less, 700 ppm or less, 600 ppm or less, or 500 ppm or less, and the range may be comprised of a combination of the above upper and lower limits. For example, the early release inhibitor may be included in the sustained release microspheres at a concentration of 10 ppm to 2000 ppm, preferably 20 ppm to 1500 ppm, more preferably 20 ppm to 1000 ppm, and most preferably 30 ppm to 500 ppm, but is not limited thereto.

[0223] In one example, when disodium phosphate or monosodium phosphate is used as the early-release inhibitor, the content of the early-release inhibitor in the microspheres can be measured by measuring the sodium (Na) and phosphorus (P) residues in the microspheres.

[0224] In one example, the long-acting injectable composition may further comprise at least one release-controlling agent selected from the group consisting of butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, heptadecylic acid, stearic acid, nonadecylic acid, behenic acid, arachidic acid, isocrotonic acid, oleic acid, elaidic acid, sorbic acid, linoleic acid, arachidonic acid, benzoic acid, hydroxynaphthoic acid, napadicylic acid, naphthalene sulfonic acid, methanesulfonic acid, and pamoic acid. The release-controlling agent may be included in the sustained-release microspheres, but is not limited thereto.

[0225] In one example, the drug encapsulation rate of the microparticles may be 80% or more, 82% or more, 84% or more, 85% or more, 86% or more, 88% or more, 90% or more, 92% or more, 94% or more, 96% or more, or 98% or more.

[0226] The drug content and encapsulation rate within the microspheres, the cumulative drug release rate within a certain period of time after in vivo administration of the sustained-release microspheres, and the release period after in vivo administration of the sustained-release microspheres are the same as those described in the above-mentioned [Method for producing sustained-release microspheres containing drugs], so duplicate description is omitted.

[0227] In one example, the in vitro drug release rate of the microspheres within 24 hours may be 10% or less. In one example, the in vitro drug release rate within 24 hours can be analyzed by detecting the drug present in the supernatant of the test solution 24 hours after the start of culturing the microspheres in the release test solution. The type of the release test solution is not limited to a specific type, and a person skilled in the art can select an appropriate release test solution based on the type of drug used based on common knowledge in the art.

[0228] Specifically, the in vitro drug release rate of the microspheres within 24 hours may be 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Even if the lower limit of the drug release rate is not specified, a person skilled in the art will clearly be able to practice the present invention for the purpose of preparing sustained-release microspheres containing a drug, but the lower limit may be, for example, 0% or more, 0.1% or more, 0.5% or more, 1% or more, 1.5% or more, 2% or more, or 2.5% or more.

[0229] In one example, the sustained-release microspheres according to the present invention may further include a steroidal anti-inflammatory agent, which may be in the form of co-encapsulated microspheres containing the water-soluble peptide and the steroidal anti-inflammatory agent as active substances in one microsphere.

[0230] In one example, when the long-acting injectable composition of the present invention comprises two or more sustained-release microspheres (i.e., a microsphere blend), at least one of the two or more sustained-release microspheres may further comprise a steroidal anti-inflammatory agent. That is, in the sustained-release microsphere blend, at least one of the microspheres included in the blend may be in the form of a co-encapsulated microsphere containing both a water-soluble peptide as an active substance (active substance) and a steroidal anti-inflammatory agent.

[0231] In one example, the long-acting injectable composition according to the present invention may further comprise sustained-release microspheres containing a steroidal anti-inflammatory agent. Specifically, the sustained-release injectable composition may comprise a microsphere blend comprising sustained-release microspheres containing a water-soluble peptide as a pharmaceutically active substance and sustained-release microspheres containing a steroidal anti-inflammatory agent. That is, in one example, the long-acting injectable composition of the present invention may comprise a microsphere blend of (1) sustained-release microspheres containing a water-soluble peptide or a pharmaceutically acceptable salt thereof as a pharmaceutically active substance; and (2) sustained-release microspheres containing a steroidal anti-inflammatory agent instead of a pharmaceutically active substance.

[0232] The above steroidal anti-inflammatory agent can play a role in alleviating the inflammatory response caused by the biodegradable polymer included in the sustained-release microspheres. The upper limit may be 0.1 part by weight or less, 0.05 part by weight or less, 0.01 part by weight or less, 0.005 part by weight or less, or 0.001 part by weight or less of the steroidal anti-inflammatory agent relative to 1 part by weight of the water-soluble peptide, which is the active substance of the present invention, and the lower limit may be 0.0001 part by weight or more, 0.0002 part by weight or more, 0.0003 part by weight or more, 0.0004 part by weight or more, or 0.0005 part by weight or more. The content of the steroidal anti-inflammatory agent relative to 1 part by weight of the water-soluble peptide may be included in a range consisting of a combination of the above lower limits and upper limits. For example, when using dexamethasone acetate as a steroidal anti-inflammatory agent, 0.0001-0.0020 parts by weight, 0.0002-0.0018 parts by weight, 0.0003-0.0016 parts by weight, 0.0004-0.0013 parts by weight, or 0.0005-0.0010 parts by weight of the steroidal anti-inflammatory agent can be used per 1 part by weight of the water-soluble peptide.

[0233] Specific examples of the above steroidal anti-inflammatory agents include 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximethasone, dexamethasone, dexamethasone acetate, dexamethasone phosphate, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone Acetonide, fluocinonide, flucortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, Prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide,Beclomethasone dipropionate, betamethasone, budesonide, deflazacort, dexamethasone, dexamethasone acetate, dexamethasone phosphate, difluprednate, epinephrine, fludrocortisone, fluocinolone acetonide, fluocortin, fluorometholone, fluticasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, Triamcinolone, etc. can be used alone or in combination of two or more. As an example of the present invention, the steroidal anti-inflammatory agent can be dexamethasone acetate.

[0234] Additionally, the present invention provides a pharmaceutical composition for improving, preventing, and / or treating a disease, comprising a long-acting injectable agent according to the present invention.

[0235] The present invention also provides a use for improving, preventing, and / or treating a disease, comprising a long-acting injectable composition according to the present invention.

[0236] In addition, the present invention provides a method for improving, preventing, and / or treating a disease, comprising administering a long-acting injectable composition according to the present invention to a subject in need thereof.

[0237] The present invention also provides a use of the long-acting injectable formulation for improving, preventing, and / or treating a disease.

[0238] The present invention also provides a use of the long-acting injectable formulation for the manufacture of a medicament for improving, preventing, and / or treating a disease.

[0239] The above disease is not limited to a specific type, but may preferably be a target disease of a drug (e.g., a water-soluble drug or a pharmaceutically acceptable salt thereof) loaded into the microspheres of the present invention, i.e., a disease improved, prevented, and / or treated by the effective substance.

[0240] For example, when the drug is semaglutide or capriglintide, the disease may be, but is not limited to, diabetes, specifically type 2 diabetes, preservation of beta-cell function, hypertension, hyperlipidemia, obesity, non-alcoholic steatohepatitis, and degenerative neurological diseases (e.g., Alzheimer's disease, Parkinson's disease, and dementia).

[0241] The composition (injection composition or pharmaceutical composition, etc.) containing microspheres according to the present invention can be formulated into various forms of preparations, and for example, can be a formulation of a known parenteral administration preparation. Accordingly, the composition according to the present invention can further include a thickener, a stabilizer, an isotonic agent, an early-release inhibitor, a surfactant, an excipient, and / or a carrier in addition to the drug. The usable isotonic agent can be a water-soluble excipient or a sugar such as mannitol, sucrose, sorbitol, trehalose, lactose, sodium chloride, etc., and the thickener can be, for example, sodium carmellose, sodium carboxymethyl cellulose, povidone, etc. In addition, the buffer can be, for example, sodium monohydrogen phosphate, anhydrous citric acid, sodium hydroxide, sodium chloride, etc.

[0242] The composition according to the present invention can be administered in a therapeutically effective amount of the drug, i.e., an amount effective to treat the target disease of the drug. The therapeutically effective amount can be assessed by a physician depending on the type of drug used.

[0243] In one example, the composition according to the present invention may be administered once a month to once a quarter. In some embodiments, the monthly dosage of the composition according to the present invention is, based on the drug, 1 mg to 100 mg, 1 mg to 80 mg, 1 mg to 60 mg, 1 mg to 30 mg, 1 mg to 20 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 6 mg, 2 mg to 100 mg, 2 mg to 80 mg, 2 mg to 60 mg, 2 mg to 30 mg, 2 mg to 20 mg, 2 mg to 10 mg, 2 mg to 8 mg, 2 mg to 6 mg, 4 mg to 100 mg, 4 mg to 80 mg, 4 mg to 60 mg, 4 mg to 30 mg, 4 mg to 20 mg, 8 mg to 100 mg, 8 mg to 80 mg, 8 mg to 60 mg, 8 mg to 30 mg, 10 mg to 100 mg, 10 mg to 80 mg, 10 mg to 60 mg, 10 mg to 30 mg, 20 mg to 100 mg, 20 mg to 80 mg, 20 mg to 60 mg, or 20 mg to 30 mg.

[0244] In one example, the dosage of the composition according to the present invention is 1 mg to 500 mg, 1 mg to 450 mg, 1 mg to 400 mg, 1 mg to 350 mg, 1 mg to 300 mg, 1 mg to 250 mg, 1 mg to 200 mg, 1 mg to 150 mg, 1 mg to 100 mg, 1 mg to 80 mg, 1 mg to 60 mg, 1 mg to 30 mg, 1 mg to 20 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 6 mg, 2 mg to 500 mg, 2 mg to 450 mg, 2 mg to 400 mg, 2 mg to 350 mg, 2 mg to 300 mg, 2 mg to 250 mg, 2 mg to 200 mg, 2 mg 150 mg, 2 mg to 100 mg, 2 mg to 80 mg, 2 mg to 60 mg, 2 mg to 30 mg, 2 mg to 20 mg, 2 mg to 10 mg, 2 mg to 8 mg, 2 mg to 6 mg, 4 mg to 500 mg, 4 mg to 450 mg, 4 mg to 400 mg, 4 mg to 350 mg, 4 mg to 300 mg, 4 mg to 250 mg, 4 mg to 200 mg, 4 mg to 150 mg, 4 mg to 100 mg, 4 mg to 80 mg, 4 mg to 60 mg, 4 mg to 30 mg, 4 mg to 20 mg, 8 mg to 500 mg, 8 mg to 450 mg, 8 mg to 400 mg, 8 mg to 350 mg, 8 mg to 300 mg, 8 mg to 250 mg, 8 mg to 200 mg, 8 mg to 150 mg, 8 mg to 100 mg, 8 mg to 80 mg, 8 mg to 60 mg, 8 mg to 30 mg, 10 mg to 500 mg, 10 mg to 450 mg, 10 mg to 400 mg, 10 mg to 350 mg,10 mg to 300 mg, 10 mg to 250 mg, 10 mg to 200 mg, 10 mg to 150 mg, 10 mg to 100 mg, 10 mg to 80 mg, 10 mg to 60 mg, 10 mg to 30 mg, 20 mg to 500 mg, 20 mg to 450 mg, 20 mg to 400 mg, 20 mg to 350 mg, 20 mg to 300 mg, 20 mg to 250 mg, 20 mg to 200 mg, 20 mg to 150 mg, 20 mg to 100 mg, 20 mg to 80 mg, 20 mg to 60 mg, or 20 mg to 30 mg.

[0245] In one example, when the active ingredient of the composition according to the present invention is semaglutide, the subcutaneous or intramuscular injection dosage of the composition may be selected as 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg or 9.5 mg or more as the lower limit, and 100 mg, 90 mg, 80 mg, 70 mg, 60 mg, 50 mg, 40 mg, 30 mg, 25 mg, 20 mg, 17.5 mg, 15 mg, 12.5 mg or 10 The upper limit may be selected as mg or less. When the active substance of the composition according to the present invention is semaglutide, the subcutaneous or intramuscular injection dosage of the composition may be included in a range consisting of a combination of the lower and upper limits per month based on the drug. For example, 0.25 mg to 50 mg, 0.5 mg to 50 mg, 0.75 to 50 mg, 1 mg to 50 mg, 1.25 mg to 50 mg, 1.5 mg to 50 mg, 1.75 mg to 50 mg, 2 mg to 50 mg, 2.5 mg to 50 mg, 3 mg to 50 mg, 3.5 mg to 50 mg, 4 mg to 50 mg, 4.5 mg to 50 mg, 5 mg to 50 mg, 5.5 mg to 50 mg, 6 mg to 50 mg, 6.5 mg to 50 mg, 7 mg to 50 mg, 7.5 mg to 50 mg, 8 mg to 50 mg, 8.5 mg to 50 mg, 9 mg to 50 mg, 9.5 mg to 50 mg or It can be 10 mg to 50 mg.

[0246] In one example, a composition comprising microspheres according to the present invention may be administered parenterally, for example, via subcutaneous injection or intramuscular injection.

[0247] In one example, a composition according to the present invention may be composed of a pharmaceutical part containing microparticles and a solvent part used to suspend the microparticles, and may be in the form of a dual-chamber syringe having the pharmaceutical part in one chamber and the solvent part in the other chamber, or a prefilled syringe in which the pharmaceutical part is suspended in the solvent part. When composed in the form of a prefilled syringe in which the pharmaceutical part is suspended in the solvent part, the solvent part used may be an injectable oil including medium-chain oil, mineral oil, etc.

[0248] In one example, the microspheres included in the composition according to the present invention have a high drug content relative to the content of the microspheres, while suppressing initial excessive release of the drug that may cause fatal side effects and exhibiting sufficient efficacy for a desired period of time, and are therefore useful for preventing, improving, and / or treating the target disease of the loaded drug.

[0249] In one example, the injectable composition may be for intra-articular administration, subcutaneous administration, intradermal administration, intramuscular administration, intratumoral administration, intraocular administration, intravitreal administration, or intratympanic administration, and may be a sustained-release injectable formulation for local administration, but is not limited thereto.

[0250] The term "one or more" used herein means "number" corresponding to one or more. In the present invention, when a certain configuration comprises one or more types, it may preferably be one type, two or more types, three or more types, one to three types, or one to two types, but is not limited thereto. The term "one or more types" may be used interchangeably with the term "one or more" in the present invention.

[0251] The present invention relates to a method for manufacturing sustained-release microspheres loaded with a high content of drugs using a W / O / W emulsion, wherein the microspheres manufactured by the manufacturing method have the effect of achieving a low initial drug release rate and a stable sustained-release profile while loading various drugs with a high content.

[0252] Figure 1 is a graph showing the cumulative release rate of semaglutide in vivo over time from a western-type microparticle according to an example of the present invention.

[0253] Figure 2 is a graph showing the cumulative release rate of in vivo kagurilintide over time of a western-type microparticle according to an example of the present invention.

[0254] Hereinafter, the present application will be described in more detail with reference to the following manufacturing examples and experimental examples. However, these manufacturing examples and experimental examples are merely intended to illustrate the present application and are not intended to limit the scope of the present application.

[0255]

[0256] [Manufacturing example]

[0257] <Manufacturing Example 1> Manufacturing of biodegradable polymer microparticles containing semaglutide and an early-release inhibitor

[0258] Semaglutide freebase (Chengdu Shengnuo Biopharm Co., Ltd, China) or semaglutide sodium salt (manufacturer: Zhejiang Peptides Biotech Co., Ltd, China) as a drug was dissolved in deionized water (DW) to prepare a first phase (W1) solution. The oily phase (O) solution was prepared by dissolving biodegradable polymers Resomer RG503H and RG653H (manufacturer: Evonik, Germany) in a 1:1 weight ratio or by dissolving RG503H alone in dichloromethane (DCM) as an organic solvent. The first phase was dispersed in the oily phase using a homogenizer (manufacturer: IKA Works Inc., Germany, model name: T18 digtal ULTRA TURRAX) at 25,000 rpm to prepare a W1 / O emulsion (dispersed phase, DP).

[0259] The continuous phase (CP; also referred to as the second phase (W2) solution) was used by adding Na2HPO4 (Disodium phosphate) or NaH2PO4 (Monosodium phosphate) as an early release inhibitor to 400-3,000 mL of a 0.1 (w / v)% polyvinyl alcohol (viscosity: 4.8 to 5.8 mPa.s; PVA concentration in the final continuous phase was 0.1 (w / v)%) aqueous solution to a final concentration of 0.5-18.0 (w / v)% relative to the volume of the continuous phase.

[0260] After connecting the continuous phase to an emulsifying device equipped with a 40 ㎛ diameter porous membrane, the prepared dispersed phase was injected into the continuous phase through the porous membrane. At this time, the volume ratio of the dispersed phase and the continuous phase was injected at a level of 1:100 to 1:200 (dispersed phase:continuous phase).

[0261] Through the above process, a W1 / O / W2 emulsion in which biodegradable polymer microdroplets containing semaglutide were dispersed was prepared, and the W1 / O / W2 emulsion solution was placed in a preparation container and stirred at a speed of 200 to 300 rpm.

[0262] The temperature of the preparation vessel was maintained at 25°C, and after the dispersion phase injection was completed, the temperature was maintained at 40°C for 3 hours to remove the organic solvent. After cooling to 25°C, the solution was filtered, and the residual polyvinyl alcohol was removed with triple-distilled water, followed by freeze-drying.

[0263] Comparative examples were manufactured using the same process as above, but by varying the content of biodegradable polymer in the oil phase, the weight ratio of aqueous solvent:organic solvent (i.e., DW:DCM in this manufacturing example), or the concentration (w / v%) of the early-release inhibitor in the continuous phase. In the case of Comparative Example 4, which was manufactured using a higher concentration of the early-release inhibitor than that of the examples, microspheres were temporarily formed and then decomposed during the organic solvent extraction and evaporation process, and thus, final microspheres were not obtained.

[0264] The amount and concentration of the drug (API), polymer (Poly), and early release inhibitor (Na2HPO4 or NaH2PO4) used in this manufacturing example, as well as the amount of each solution used, are shown in Table 1 below.

[0265] Classification T / LW1 phaseO phaseW2 phaseDW:DCMAPIDWAPI in W1PolyDCMPoly in OPVANa2HPO4in W2NaH2PO4in W2(%)Type(g)(g)(wt%)Type(g)(g)(wt%)(w / v%)(w / v%)(w / v%)Weight ratioExample 1-150F0.2502.25010A0.2504.75050.11.0-1:2.11Example 1-250N0.2501.53614A0.2504.75050.11.0-1:3.09Example 1-350N0.2501.53614A0.2504.75050.12.0-1:3.09Example 1-455N0.2751.68914B0.2254.27550.12.0-1:2.53 Example 1-560N0.3001.84314B0.2007.2072.70.12.0-1:3.91 Example 1-650N0.2501.53614B0.2504.75050.1-1.91:3.09 Example 1-750N0.2501.53614B0.2504.75050.1-41:3.09 Example 1-850N0.2501.53614B0.2504.75050.1-61:3.09 Example 1-950N0.2501.53614B0.2504.75050.1-1.51:3.09 Example 1-1050N0.2501.53614B0.2504.75050.1-1.91:3.09 Example 1-1150N0.2501.53614B0.2504.75050.1-2.51:3.09 Example 1-1250N0.2501.53614B0.2504.75050.1-1.91:3.09 Comparative Example 150N0.2501.53614A0.2504.75050.10.5-3.09 Comparative Example 250N0.2504.7505A0.2504.75050.11.0-1.00Comparative example 360N0.3001.84314B0.20018.4921.070.12.0-10.03Comparative example 450N0.2501.53614B0.2504.75050.1-183.09

[0266] * T / L: Target Loading

[0267] * API Type F: Semaglutide Freebase

[0268] * API Type N: Semaglutide sodium salt

[0269] * Polymer type A: RG503H:RG653H = 1:1

[0270] * Polymer type B: RG503H

[0271]

[0272] <Manufacturing Example 2> Manufacturing of biodegradable polymer microparticles containing kagrilintide acetate salt and an early-release inhibitor

[0273] The first phase (W1) solution was prepared by dissolving the drug, Kagrilintide acetate salt (manufacturer: Chengdu Shengnuo Biopharm Co., Ltd., China), in deionized water (DW). The oily phase (O) solution was prepared by dissolving biodegradable polymers, Resomer RG503H and RG653H (manufacturer: Evonik, Germany), in a 1:1 weight ratio or by dissolving RG503H alone in dichloromethane (DCM), an organic solvent. The W1 / O emulsion (dispersed phase, DP) was prepared by dispersing the first phase in the oily phase using a homogenizer (manufacturer: IKA Works Inc., Germany, model name: T18 digtal ULTRA TURRAX) at 25,000 rpm.

[0274] As a continuous phase (CP; also referred to as a second phase (W2) solution), 400-2,000 mL of a 0.1 (w / v)% polyvinyl alcohol (viscosity: 4.8-5.8 mPa.s) aqueous solution was used, and Na2HPO4 (Disodium phosphate) or NaH2PO4 (Monosodium phosphate) was added as an early release inhibitor to a final concentration of 2.0 (w / v)% relative to the volume of the continuous phase.

[0275] After connecting the continuous phase to an emulsifying device equipped with a 40 ㎛ diameter porous membrane, the prepared dispersed phase was injected into the continuous phase through the porous membrane. At this time, the volume ratio of the dispersed phase and the continuous phase was injected at a level of 1:100 to 1:200 (dispersed phase:continuous phase).

[0276] Through the above process, a W1 / O / W2 emulsion in which biodegradable polymer microdroplets containing kagrilintide were dispersed was prepared, and the W1 / O / W2 emulsion solution was placed in a preparation container and stirred at a speed of 200 to 300 rpm.

[0277] The temperature of the preparation vessel was maintained at 25°C, and after the dispersion phase was injected, the temperature was maintained at 40°C for 3 hours to remove the organic solvent. After cooling to 25°C, the solution was filtered to remove the remaining polyvinyl alcohol with triple-distilled water, and freeze-dried.

[0278] Comparative examples were manufactured using the same process as above, but the concentration of the drug in the primary aqueous solution (w / w%) or the weight ratio of aqueous solvent:organic solvent (i.e., DW:DCM in this manufacturing example) was changed.

[0279] The amount and concentration of the drug (API), polymer (Poly), and early release inhibitor (Na2HPO4 or NaH2PO4) used in this manufacturing example, as well as the amount of each solution used, are shown in Table 2 below.

[0280] Classification T / LW1 phaseO phaseW2 phaseDW:DCMAPIDWAPI in W1PolyDCMPoly in OPVANa2HPO4in W2NaH2PO4in W2(%)(g)(g)(wt%)Type(g)(g)(wt%)(w / v%)(w / v%)(w / v%)Weight ratioExample 2-1400.2001.80010.0A0.3005.7005.00.12.0-1:3.17Example 2-2500.2502.25010.0A0.2504.7505.00.12.0-1:2.11Example 2-3500.2501.54014.0B0.2504.7505.00.12.0-1:3.08Example 2-4500.2500.95820.7B0.2504.7505.00.12.0-1:4.96 Example 2-5500.2501.54014.0B0.2503.0807.50.12.0-1:2.00 Example 2-6500.2503.1307.4B0.2509.7502.50.12.0-1:3.12 Example 2-7500.2501.00020.0B0.2503.0807.50.12.0-1:3.08 Example 2-8550.2751.68914.0B0.2254.2805.00.12.0-1:2.53 Example 2-9600.3001.84314.0B0.2003.8005.00.12.0-1:2.06 Example 2-10650.3251.30020.0B0.1753.3305.00.12.0-1:2.56 Example 2-11700.3501.40020.0B0.1502.8505.00.12.0-1:2.04 Example 2-12750.3751.12525.0B0.1252.3805.00.12.0-1:2.12Example 2-13500.2501.53614.0B0.2504.7505.00.1-2.01:3.09Comparative Example 5500.2501.54014.0B0.2509.7502.50.12.0-6.33Comparative Example 6800.4000.93330.0B0.1001.9005.00.12.0-2.04

[0281] * T / L: Target Loading

[0282] * API type: Kagrilintide acetate salt

[0283] * Polymer type A: RG503H:RG653H = 1:1

[0284] * Polymer type B: RG503H

[0285]

[0286] <Manufacturing Example 3> Manufacturing of biodegradable polymer microspheres containing leuprolide as an example of a low-molecular-weight peptide drug (Comparative Example 7)

[0287] To manufacture biodegradable polymer microspheres containing leuprolide (Comparative Example 7), the same process as in Manufacturing Example 1 described above was performed, and the same manufacturing conditions as in Examples 1-4 were used, but the type of drug was replaced with leuprolide acetate (Zhejiang Peptides Biotech Co., Ltd, China).

[0288]

[0289] <Experimental Example 1> Measurement of drug content in microspheres

[0290] (1) Measurement of semaglutide content in microspheres

[0291] In order to measure the semaglutide content of the microspheres manufactured in Manufacturing Example 1, the microspheres were weighed to be 2 mg as a drug. This was dissolved in 1 mL of dimethyl sulfoxide (DMSO) and diluted to 200 μg / mL, which was used as a test solution. 20 μL of the test solution was injected into HPLC and measured at a detection wavelength of 280 nm. In this experimental example, ZORBAX 300SB-C18, 5 μm, 4.6 × 150 mm was used as a column, and 0.1% (w / w) trifluoroacetic acid aqueous solution was used as a mobile phase A, and acetonitrile (gradient: 40-60 (v / v)%) containing 0.1% (w / w) trifluoroacetic acid was used as a mobile phase B.

[0292] The drug content and encapsulation rate measurements are shown in Table 3 below.

[0293]

[0294] (2) Measurement of the content of kagrilintide in microparticles

[0295] In order to measure the content of kagrilintide in the microspheres manufactured in Manufacturing Example 2, 2 mg of the drug was weighed out of the microspheres, completely dissolved in 4.0 mL of acetonitrile, and extracted by adding 6.0 mL of a 0.1% (w / w) trifluoroacetic acid aqueous solution. The solution filtered using a 0.45 μm filter was used as a test solution. 20 μL of the test solution was injected into HPLC and measured at a detection wavelength of 214 nm. The column used in this experimental example was ZORBAX 300SB-C18, 5 μm, 4.6 × 150 mm, and the mobile phase was a mixture of acetonitrile containing 0.1% (w / w) trifluoroacetic acid and 0.1% (w / w) trifluoroacetic acid aqueous solution in a ratio of 40:60 (v / v).

[0296] The drug content (total weight of drug encapsulated in the entire microspheres relative to the total weight of the entire microspheres) and the encapsulation ratio measurements are shown in Table 3 below. The encapsulation ratio was calculated using the following equation.

[0297] Encapsulation rate = (API measured content / API target loading content) X 100

[0298] Target Loading (T / L) content = [API usage / (API usage + polymer usage)] X 100

[0299] Classification Drug content (wt%) Encapsulation rate (%) Example 1-144.0588.1 Example 1-248.9597.9 Example 1-348.196.2 Example 1-454.3498.8 Example 1-557.0695.1 Example 1-646.993.8 Example 1-751.45102.9 Example 1-850.65101.3 Example 1-949.599 Example 1-1048.8597.7 Example 1-1148.7597.5 Example 1-1247.294.4 Example 2-139.8699.66 Example 2-248.6597.3 Example 2-349.2998.57 Example 2-446.893.6 Example 2-545.5991.17 Example 2-640.6381.25 Example 2-742.785.39 Example 2-847.3286.03 Example 2-952.9988.32 Example 2-1056.8287.41 Example 2-1166.5695.09 Example 2-1270.9894.64 Example 2-1344.0788.14 Comparative Example 145.3590.7 Comparative Example 247.795.4 Comparative Example 345.5475.9 Comparative Example 4--Comparative Example 527.6355.25Comparison Example 678.5398.16Comparison Example 727.1649.38

[0300] In the above results, Examples 1-1 to 1-12 and 2-1 to 2-13 had a drug content of about 40% by weight or more relative to the total weight of the microspheres, and the encapsulation rate was measured to be 80% or more, indicating that a high content of drug was loaded into the microspheres.

[0301] On the other hand, when the weight ratio of the organic solvent to the aqueous solvent during the production of microspheres exceeded 6 times or the polymer content in the oil phase was less than 2 w / w%, the drug encapsulation rate significantly decreased (Comparative Examples 3 and 5). In addition, as described above, when the concentration of the initial release inhibitor in the continuous phase exceeded 6 w / v%, normal microspheres were not produced, making it impossible to measure the drug content and encapsulation rate (Comparative Example 4). Furthermore, when leuprolide, which has a significantly lower molecular weight than semaglutide and kagrilintide, was used, the encapsulation rate significantly decreased (Comparative Example 7).

[0302]

[0303] <Experimental Example 2> Measurement of the initial (day 1) drug release rate of microspheres in vitro

[0304] In order to confirm the initial drug release of the microspheres manufactured in the above Manufacturing Examples 1 and 2, the following experiment was performed. Microspheres were weighed to be 2 mg of drug, added to 1 mL of a release test solution, and stored in an incubator at 37°C. After 24 hours, centrifugation was performed, and 0.5 mL of the supernatant was taken, which was diluted 10-fold and used as a test solution. Depending on the type of drug, the test solution was analyzed for drug release rate using HPLC under the same analysis conditions as Experimental Example 1-(1) or Experimental Example 1-(2). The release rate was calculated by quantifying the content of the drug contained in the test solution and measuring the ratio of the drug content to the total amount of drug (2 mg) before release.

[0305] The release test solution for semaglutide was a phosphate buffer solution (pH 7.4) containing 0.5% sodium dodecyl sulfate and 0.02% sodium azide, and the release test solution for kagrillintide was a phosphate buffer solution (pH 7.4) containing 0.1% polysorbate 20.

[0306] The in vitro drug release rate measurements (day 1) are shown in Table 4 below.

[0307] In vitro initial release rate (%, 1-day) Example 1-17.6 Example 1-25.3 Example 1-33.4 Example 1-42.7 Example 1-58.6 Example 1-64.61 Example 1-75 Example 1-84.7 Example 1-95.03 Example 1-104.26 Example 1-115.09 Example 1-126.33 Example 2-12.67 Example 2-22.92 Example 2-32.6 Example 2-41.23 Example 2-57.13 Example 2-62.42 Example 2-72.87 Example 2-81.84 Example 2-94.19 Example 2-105.24 Example 2-114.23 Example 2-128.21 Example 2-130.26 Comparative Example 119.3 Comparative Example 250.9 Comparative Example 363 Comparative Example 4-Comparative Example 53.16 Comparative Example 646.9 Comparative Example 717.01

[0308] As shown in Table 4 above, Examples 1-1 to 1-12 and 2-1 to 2-13 showed no problem of excessive initial drug release, with the first-day drug release rate being 10% or less.

[0309] On the other hand, in the case of comparative examples manufactured by changing the concentration of the drug in the primary aqueous solution, the weight ratio of aqueous solvent:organic solvent, or the concentration of the initial release inhibitor in the continuous phase during the manufacturing process, the first-day release rate was significantly increased.

[0310] Specifically, when the concentration of the drug in the primary aqueous solution exceeded 25 wt% during the manufacture of microspheres, the first-day drug release rate was very high at 46.90% (Comparative Example 6), and when the weight ratio of the organic solvent to the aqueous solvent during the manufacture of microspheres was less than 1.1 (Comparative Example 2) or exceeded 6 (Comparative Example 3), the first-day drug release rate was found to exceed 50%. In particular, in the case of Comparative Example 3, which was manufactured by lowering the concentration of the biodegradable polymer in the oily solution compared to the examples or other comparative examples, the initial drug release rate was found to be very high at 63%.

[0311] In addition, even when the concentration of the initial release inhibitor in the continuous phase during the production of microparticles was less than 1 w / v%, the drug release rate was as high as 19.3% (Comparative Example 1), and when the concentration of the initial release inhibitor in the continuous phase exceeded 6 w / v%, it was observed that normal production of microparticles was impossible as described above (Comparative Example 4).

[0312] The above results suggest that in the process of manufacturing microspheres, the drug concentration in the primary aqueous solution, the polymer concentration in the oily phase solution, the weight ratio of the aqueous solvent and the organic solvent, and the concentration of the initial release inhibitor in the continuous phase must satisfy certain conditions to reduce the risk of initial burst of the drug and achieve desirable sustained release.

[0313]

[0314] <Experimental Example 3> Measurement of in vivo drug release rate of microspheres

[0315] In order to evaluate the drug release profile of microspheres according to an embodiment of the present invention, the blood drug concentration was measured after administration to rats.

[0316] In the case of microspheres according to Manufacturing Example 1, the microspheres were measured so that the semaglutide administration dose would be 3.6 mg / head (API raw material solution), dispersed in 0.5 mL of suspension, and then subcutaneous injected into SD rats (Sprague-Dawley rats). In the case of microspheres according to Manufacturing Example 2, the microspheres were measured so that the capriglintide administration dose would be 3.6 mg / head (API raw material solution), dispersed in 0.5 mL of suspension, and then subcutaneously injected into SD rats.

[0317] After injection, 0.5 mL of blood was collected at pre-scheduled time intervals from rats, and the drug concentration in the blood was measured using HPLC. The in vivo cumulative release rate was calculated by measuring the AUC of the blood samples collected at each time interval and calculating the cumulative release rate from the measured values.

[0318] In-vivo cumulative release rate (%) Time (day) Classification 00.040.2514710Example 1-120.00%0.10%1.54%8.82%29.90%44.46%61.84%Example 2-40.0%0.0%0.3%4.8%30.2%46.5%57.1%Example 2-90.0%0.0%0.5%6.6%35.0%48.3%55.2%Example 2-130.0%0.0%0.1%2.6%29.7%52.5%61.2% Time (day) Classification 141721283542 Example 1-1281.07%90.15%96.26%100.00% Example 2-469.4%76.4%83.4%94.2%100.0%- Example 2-965.9%71.7%77.3%90.9%99.6%100.0% Example 2-1371.6%78.3%86.7%98.0%100.0%-

[0319] As shown in Table 5, the microspheres of the present invention demonstrated a cumulative blood concentration of less than 10% on the first day after in vivo drug administration, demonstrating that they do not cause an initial burst of drug release in vivo. Furthermore, an excellent drug release profile was observed, with sustained drug release for as little as 28 days and as long as 42 days (Figs. 1 and 2). These results demonstrate that the microspheres of the present invention can stably release drugs over a long period of time without excessive initial drug release, even when loaded with a high dose of drug.

Claims

1. A long-acting injection composition comprising one or more sustained-release microspheres containing a drug, a biodegradable polymer, and an early-release inhibitor, The above drug is a water-soluble peptide having a molecular weight of 2,000 to 10,000 Da or a pharmaceutically acceptable salt thereof, and is included in an amount of 20 to 75 wt% based on 100 wt% of sustained-release microspheres. The above early release inhibitor is included in the sustained release microspheres at 5 ppm to 2,000 ppm, The above sustained-release microspheres are one type of sustained-release microspheres containing the same biodegradable polymer and drug, or two or more types of sustained-release microspheres containing at least one different type selected from the group consisting of biodegradable polymers and drugs. The above-mentioned sustained-release microspheres have a cumulative drug release rate of 10% or less for up to 24 hours after in vivo administration, A long-acting injection composition wherein the above-mentioned sustained-release microspheres release the drug for 4 to 24 weeks after in vivo administration.

2. In the first paragraph, the western-type microspheres (a) A step of preparing a W1 / O emulsion by mixing a first phase (W1 phase) solution in which a drug is dissolved in an aqueous solvent and an oil phase (O phase) solution in which a biodegradable polymer is dissolved in an organic solvent; (b) a step of preparing a W1 / O / W2 emulsion solution by adding the W1 / O emulsion of step (a) to a second phase (W2 phase) solution containing an early release inhibitor added to an aqueous solution containing a surfactant; and (c) A long-acting injection composition manufactured by a manufacturing method including a step of forming microspheres by extracting an organic solvent from the W1 / O / W2 emulsion of step (b) as a second phase (W2 phase) solution.

3. In the second paragraph, in step (a) of the manufacturing method, the drug is included in an amount of 4 to 25 w / w% relative to the weight of the first phase (W1 phase) solution, In the above step (a), the biodegradable polymer is included in an amount of 2 to 10 w / w% relative to the weight of the oil phase (O phase) solution, The weight ratio of the aqueous solvent and the organic solvent is 1:1.1 to 1:6, A long-acting injection composition, wherein in the above step (b), the initial release inhibitor is included in an amount of 1 to 6 w / v% relative to the volume of the second phase (W2 phase) solution.

4. A long-acting injection composition according to claim 1, wherein the one type of sustained-release microspheres further comprises a steroidal anti-inflammatory agent.

5. A long-acting injection composition according to claim 1, wherein at least one of the two or more types of sustained-release microspheres further comprises a steroidal anti-inflammatory agent. 6.(a) A step of preparing a W1 / O emulsion by mixing a first phase (W1 phase) solution in which a drug is dissolved in an aqueous solvent and an oil phase (O phase) solution in which a biodegradable polymer is dissolved in an organic solvent; (b) a step of preparing a W1 / O / W2 emulsion solution by adding the W1 / O emulsion of step (a) to a second phase (W2 phase) solution containing an early release inhibitor added to an aqueous solution containing a surfactant; and (c) A method for producing sustained-release microspheres containing a drug using a W / O / W emulsion, comprising a step of forming microspheres by extracting an organic solvent as a second phase (W2 phase) solution from the W1 / O / W2 emulsion of step (b), In the above step (a), the drug is included in an amount of 4 to 25 w / w% relative to the weight of the first phase (W1 phase) solution, In the above step (a), the biodegradable polymer is included in an amount of 2 to 10 w / w% relative to the weight of the O phase solution, In the above step (a), the weight ratio of the aqueous solvent and the organic solvent is 1:1.1 to 1:6, In the above step (b), the initial release inhibitor is included in an amount of 1 to 6 w / v% relative to the volume of the second phase (W2 phase) solution, A method for producing microspheres, wherein the drug is a water-soluble peptide or a pharmaceutically acceptable salt thereof having a molecular weight of 2,000 to 10,000 Da, and is included in an amount of 20 to 75 wt% based on 100 wt% of sustained-release microspheres.

7. In the 6th paragraph, the biodegradable polymer is polylactide (PLA), polyglycolide (PGA), polylactide-co-glycolide (PLGA), polydioxanone, polycaprolactone (PCL), polylactide-co-glycolide-co-caprolactone (PLGC), polylactide-co-hydroxymethyl glycolide (PLGMGA), polyalkylcarbonate, polytrimethylenecarbonate (PTMC), polylactide-co-trimethylenecarbonate (PLTMC), A polymer selected from the group consisting of polyhydroxybutyric acid (PHB), polyhydroxybutyrate-co-hydroxyvalerate (PHBV), polyorthoester, polyanhydride, polyanhydride-co-imide, polypropylene fumarate, pseudo polyaminoacid, polyalkyl cyanoacrylate, polyphosphazene, polyphosphoester, polysaccharide, and poly(butylene succinate lactide) (PBSLA); a copolymer or simple mixture of two or more thereof; a copolymer of the above polymer and polyethylenglycol (PEG);And a method for producing microspheres, wherein the microspheres are at least one selected from the group consisting of polymer-sugar complexes in which the polymer or copolymer is combined with sugar.

8. A method for producing microspheres in the 6th paragraph, wherein the biodegradable polymer is at least two types of biodegradable polymers, at least one of which is different from each other in the group consisting of repeating units constituting each polymer, the molar ratio of repeating units, the intrinsic viscosity of the polymer, the weight average molecular weight of the polymer, and the terminal groups of the polymer.

9. In the 6th paragraph, the biodegradable polymer satisfies at least one selected from the group consisting of the following: (i) The intrinsic viscosity of the biodegradable polymer is 0.16 to 1.7 dL / g; (ii) The weight average molecular weight of the biodegradable polymer is a weight average molecular weight of 4,000 to 240,000; (iii) the biodegradable polymer comprises a hydroxyl group, a carboxyl group, a methoxy group, a methyl group, an amine group, or an ester group at one or both ends; and (iv) The biodegradable polymer contains two or more repeating units.

10. In paragraph 6, the drug is exenatide, liraglutide, lixisenatide, semaglutide, capriglintide, terzepatide, letaturutide, mazdutide, insulin glargine, insulin degludec, insulin icodec, amycretin, monlunabant, orforglipron, eloralintide, DACRA QW II, nisotirostide, survodutide, ecnoglutide, dapiglutide, pembidutide, bamadutide, A method for manufacturing microspheres, comprising at least one selected from the group consisting of cotadutide, utreglutide, and pharmaceutically acceptable salts thereof.

11. A method for producing microspheres in claim 6, wherein the first phase and / or oil phase further comprises a steroidal anti-inflammatory agent.

12. A method for producing microspheres in claim 6, wherein the pharmaceutically acceptable salt is sodium salt, acetate salt, benzoate salt, hydroxynaphthoate salt, napadiylate or pamoate.

13. A method for producing microspheres in the 6th paragraph, wherein the aqueous solvent of step (a) is at least one selected from the group consisting of distilled water, PBS (Phosphate buffered saline), TBS (Triss buffered saline), acetate buffer, citrate buffer, glycine-HCl buffer, ammonium bicarbonate buffer, sodium hydroxide aqueous solution, and urea aqueous solution.

14. A method for producing microspheres, wherein the organic solvent of step (a) is at least one selected from the group consisting of dichloromethane, dimethyl carbonate, chloroform, ethyl acetate, methyl ethyl ketone, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, n-methylpyrrolidone, acetic acid, methyl alcohol, ethyl alcohol, propyl alcohol, and benzyl alcohol.

15. A method for producing microspheres, wherein in the step (a), the W1 / O emulsion further comprises at least one release-controlling agent selected from the group consisting of butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, heptadecylic acid, stearic acid, nonadecylic acid, behenic acid, arachidic acid, isocrotonic acid, oleic acid, elaidic acid, sorbic acid, linoleic acid, arachidonic acid, benzoic acid, hydroxynaphthoic acid, napadicylic acid, naphthalene sulfonic acid, methanesulfonic acid, and pamoic acid.

16. A method for producing microspheres in claim 6, wherein the early release inhibitor is at least one selected from the group consisting of phosphate salts, hydroxide salts, phosphide salts, phosphite salts, carbonate salts, bicarbonate salts, chromate salts, dichromate salts, oxides, oxalate salts, silicate salts, sulfate salts, sulfide salts, sulfite salts, tartrate salts, tetraborate salts, thiosulfate salts, arsenate salts, arsenite salts, citrate salts, ferricyanide salts, and nitride salts of alkali metals, alkaline earth metals, or ammonium.

17. A method for producing microspheres in the sixth paragraph, wherein the surfactant of step (b) is at least one selected from the group consisting of polyvinyl alcohol, methylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, lecithin, gelatin, polyoxyethylene sorbitan fatty acid ester, and polyoxyethylene castor oil derivatives.

18. A method for manufacturing microspheres, wherein the drug encapsulation rate of the microspheres manufactured by the manufacturing method in paragraph 6 is 80% or more.

19. A method for manufacturing microspheres, wherein the drug release rate within 24 hours in vitro of the microspheres manufactured by the manufacturing method in paragraph 6 is 10% or less.

20. A method for manufacturing microspheres, wherein the cumulative drug release rate for up to 24 hours after in vivo administration of the microspheres manufactured by the manufacturing method in paragraph 6 is 10% or less.

21. A method for producing microspheres, wherein the microspheres produced by the method in paragraph 6 release the drug for 4 to 24 weeks after in vivo administration.

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