Microcapsule encapsulating water-soluble substance

Abstract

The main purpose of the present disclosure is to provide a preparation which contains a polymer and in which a water-soluble substance exhibits excellent stability. The inventors of the present invention found that a preparation in which a water-soluble substance exhibits excellent stability can be provided by a microcapsule encapsulating a water-soluble substance, said microcapsule comprising the water-soluble substance and a polymer that has a chemical structure in which polyalkylene glycol and polyhydroxyalkanoic acid are bonded, wherein the total volume of an aqueous phase in a W / O emulsion prepared from the polymer by the following method is 0.025-0.25 cm3 / g, as measured by differential scanning calorimetry (heating method).<sp / > W / O emulsion preparation method: 50 mg of the polymer is dissolved in 500 μL of a 0.5w / v% sorbitan monooleate / dichloromethane solution, 100 μL of water is added, and then a resultant solution is irradiated with ultrasonic waves to prepare the W / O emulsion.

Description

Microcapsules containing water-soluble substances

[0001] The present disclosure relates to microcapsules encapsulating water-soluble substances, compositions containing the microcapsules, and methods for screening sustained-release polymers.

[0002] In recent years, the importance of polymeric drugs such as peptide drugs, protein drugs, and nucleic acid drugs has increased. However, polymeric drugs are generally less stable than conventional small molecule drugs. For this reason, techniques for stable formulation of polymeric drugs have been investigated.

[0003] For example, Patent Document 1 attempts to improve the stability of antibody molecules by using dry antibody molecule-loaded polymeric microspheres that contain antibody molecules, a polymer, and cyclodextrin, and optionally further contain a buffer and / or a surfactant.

[0004] JP 2022-533038 A, WO 2018 / 062464

[0005] Ishikiriyama, K., Utilizing thermal analysis in polymer material development. Thermal Measurements. 46(4): 2019.10, pp.155-162. Iijima, M., Sugimoto, R., Sakai, S., Topics of DSC for polymer materials. Thermal Measurements. 36(5): 2009.11, pp.247-254.

[0006] However, even if we limit ourselves to the poly(lactide-co-glycoside) copolymer (hereinafter in this disclosure, sometimes referred to as lactic acid / glycolic acid copolymer) used in the examples of Patent Document 1, the polymers represented by this name include polymers with a wide variety of structures depending on the polymerization method, molecular weight, etc. Among the wide variety of polymer structures, there has not been sufficient research to date into what characteristics a polymer should have to be used to obtain a formulation that stably contains a water-soluble substance such as a polymeric drug.

[0007] Therefore, the present inventors investigated what characteristics of a polymer would be required to impart stability to a formulation of a water-soluble substance, and their main objective was to provide a formulation containing a polymer and having excellent stability of a water-soluble substance.

[0008] As a result of the above investigation, the present inventors have found that a water-soluble substance is contained in a W / O emulsion containing a polymer having a chemical structure in which a polyalkylene glycol and a polyhydroxyalkanoic acid are bonded, and the polymer is such that when a W / O emulsion prepared by a predetermined method using the polymer is measured by differential scanning calorimetry (temperature programming), the total volume of the aqueous phase in the W / O emulsion is 0.025 cm ３ / g ~ 0.25 cm ３ / g, it was found that a formulation with excellent stability of water-soluble substances could be provided. Further improvements were made, and the present disclosure was completed.

[0009] The present disclosure includes, for example, the subject matter described in the following items: Item 1. A water-soluble polymer comprising a polymer having a chemical structure in which a polyalkylene glycol and a polyhydroxyalkanoic acid are bonded, and a water-soluble substance, wherein the polymer is such that when a W / O emulsion prepared by the following method is measured by differential scanning calorimetry (temperature programming), the total volume of the aqueous phase in the W / O emulsion is 0.025 cm ３ / g ~ 0.25 cm ３ / g microcapsules: 50 mg of the polymer is dissolved in 500 μL of a 0.5 w / v % sorbitan monooleate / dichloromethane solution, 100 μL of water is added, and the mixture is irradiated with ultrasound to prepare a W / O emulsion. Item 2. The microcapsules according to Item 1, wherein the polyhydroxyalkanoic acid is a homopolymer of a monomer selected from the group consisting of lactic acid, glycolic acid, and 6-hydroxycaproic acid, or a copolymer containing two or more of the monomers. Item 3. The microcapsules according to Item 1 or 2, wherein the polyhydroxyalkanoic acid is a lactic acid-glycolic acid copolymer. Item 4. The microcapsules according to any one of Items 1 to 3, wherein the polyalkylene glycol has a chemical structure in which a linear polyalkylene glycol is bonded to a polyhydric alcohol and has 3 to 10 terminal hydroxy groups per molecule. Item 5. The microcapsules according to any one of Items 1 to 4, wherein the polyalkylene glycol is polyethylene glycol. Item 6. Item 7. The microcapsules according to any one of Items 1 to 5, wherein the polyalkylene glycol is a 4- or 8-branched polyethylene glycol. Item 8. The microcapsules according to any one of Items 1 to 6, wherein the polymer has a chemical structure in which a 4- or 8-branched polyethylene glycol is bonded to a lactic acid-glycolic acid copolymer. Item 9. The microcapsules according to any one of Items 1 to 7, wherein the number-average molecular weight of the polyalkylene glycol is 5,000 to 20,000. Item 10. The microcapsules according to any one of Items 1 to 9, wherein the ratio of the number-average molecular weight of the polyalkylene glycol to the number-average molecular weight of the polyhydroxyalkanoic acid is 1:1 to 1:6.Item 11. The microcapsules according to any one of Items 1 to 10, wherein the polymer has a chemical structure in which 4- or 8-branched polyethylene glycol and lactic acid / glycolic acid copolymer are bonded, the 4- or 8-branched polyethylene glycol has a number average molecular weight of 5,000 to 20,000, the number average molecular weight of the lactic acid / glycolic acid copolymer is 10,000 to 70,000, and the ratio of the number average molecular weight of the polyethylene glycol to the number average molecular weight of the lactic acid / glycolic acid copolymer is 1:1 to 1:6. Item 12. The microcapsules according to any one of Items 1 to 11, wherein the water-soluble substance is at least one selected from the group consisting of proteins, peptides, and nucleic acids. Item 13. A composition comprising the microcapsules according to any one of Items 1 to 12. Item 14. The composition according to Item 13, which is in the form of a sphere, sheet, needle, rod, or coil. Item 15. The composition according to Item 13 or 14, which is a sustained-release pharmaceutical composition. Item 16. A method for screening sustained-release polymers, comprising the following steps (a) to (c): step (a) of dissolving a test polymer in a solution comprising a surfactant and a water-immiscible organic solvent to prepare a test polymer solution; step (b) of adding water to the test polymer solution prepared in step (a) and then irradiating the solution with ultrasound to prepare a W / O emulsion; and step (c) of measuring the W / O emulsion prepared in step (b) by differential scanning calorimetry (temperature-programming method) to find that the total volume of the aqueous phase in the W / O emulsion is 0.025 cm. ３ / g ~ 0.25 cm ３ / g as an index to screen for sustained release polymers.

[0010] According to the technology of the present disclosure, a preparation with excellent stability of water-soluble substances can be provided.

[0011] Figure 2 shows a scanning electron microscope (hereinafter sometimes abbreviated as SEM) image of bevacizumab-loaded microspheres (polymer 1PEG (2 kDa)-PLGA (11.5 kDa)) obtained in Test 2.1.1. Bar = 10 μm. Figure 2 shows an SEM image of bevacizumab-loaded microspheres (polymer 1PEG (10 kDa)-PLGA (40 kDa)) obtained in Test 2.1.1. Bar = 10 μm. Figure 2 shows an SEM image of bevacizumab-loaded microspheres (polymer 2PEG (10 kDa)-PLGA (40 kDa)) obtained in Test 2.1.1. Bar = 10 μm. Figure 2 shows an SEM image of bevacizumab-loaded microspheres (polymer 4PEG (10 kDa)-PLGA (40 kDa)) obtained in Test 2.1.1. Bar = 10 μm. Figure 2 shows an SEM image of bevacizumab-loaded microspheres (polymer 4PEG (10 kDa)-PLGA (69 kDa)) obtained in Test 2.1.1. Bar = 10 μm. Figure 2 shows an SEM image of bevacizumab-loaded microspheres (polymer 4PEG (10 kDa)-PLGA (130 kDa)) obtained in Test 2.1.1. Bar = 10 μm. Figure 2 shows an SEM image of bevacizumab-loaded microspheres (polymer 8PEG (10 kDa)-PLGA (40 kDa)) obtained in Test 2.1.1. Bar = 10 μm. Figure 1 shows an SEM image of cetuximab-loaded microspheres (polymer: PLGA (45 kDa)) obtained in Test 2.1.1. Bar = 10 μm. Figure 2 shows an SEM image of cetuximab-loaded microspheres (polymer: 4PEG (10 kDa)-PLGA (40 kDa)) obtained in Test 2.1.1. Bar = 10 μm. Figure 3 shows an SEM image of ranibizumab-loaded microspheres (polymer: 4PEG (10 kDa)-PLGA (40 kDa)) obtained in Test 3.1.1. Bar = 10 μm. Figure 4 shows an SEM image of faricimab-loaded microspheres (polymer: 4PEG (10 kDa)-PLGA (40 kDa)) obtained in Test 3.2.1. Bar = 10 μm. This shows an SEM image of brolucizumab-loaded microspheres (polymer: 4PEG (10 kDa)-PLGA (40 kDa)) obtained in Test 3.3.1. Bar = 10 μm. Left: shows the two-dimensional structure of calcitonin. Right: shows the three-dimensional structure of calcitonin. This shows the results of Test 5. The vertical axis shows the IgG release rate (cumulative %), and the horizontal axis shows the period (number of days) from the start of the release test.8 shows the results of Test 6. The vertical axis indicates the nucleic acid release rate (cumulative %), and the horizontal axis indicates the period (number of days) from the start of the release test. 8 shows the results of Test 7. The vertical axis indicates the model dye release rate (cumulative %), and the horizontal axis indicates the period (number of days) from the start of the release test. 8 shows SEM images of each microsphere obtained in Test 8.1. 8 shows the results of Test 8.3. The vertical axis indicates the release rate (cumulative %) of siRNA compound 1, and the horizontal axis indicates the period (number of weeks) from the start of the release test. 8 shows the results of Test 8.4. The vertical axis indicates the activity retention rate (%) of siRNA compound 1, and the horizontal axis indicates the period (number of weeks) from the start of the test.

[0012] Each embodiment of the present disclosure will be described in more detail below. The present disclosure preferably includes, but is not limited to, microcapsules encapsulating water-soluble substances, compositions containing the microcapsules, and methods for screening sustained-release polymers. The present disclosure includes all of the disclosures herein that would be recognized by a person skilled in the art.

[0013] The microcapsules included in the present disclosure comprise a polymer having a chemical structure in which polyalkylene glycol and polyhydroxyalkanoic acid are bonded, and a water-soluble substance, and the polymer is such that when a W / O emulsion prepared by the following method is measured by differential scanning calorimetry (temperature-programming method), the total volume of the aqueous phase in the W / O emulsion is 0.025 cm ３ / g to 0.25 cm ３ / g: 50 mg of the polymer is dissolved in 500 μL of a 0.5 w / v % sorbitan monooleate / dichloromethane solution, 100 μL of water is added, and then ultrasonic waves are applied to prepare a W / O emulsion. Hereinafter, the microcapsules included in the present disclosure may be referred to as "microcapsules of the present disclosure."

[0014] A. Polymer of the Present Disclosure As described above, the microcapsules of the present disclosure comprise a polymer having a chemical structure in which a polyalkylene glycol and a polyhydroxyalkanoic acid are bonded, and a water-soluble substance, and the polymer is such that, when a W / O emulsion prepared by the following method is measured by differential scanning calorimetry (temperature-programming method), the total volume of the aqueous phase in the W / O emulsion is 0.025 cm ３ / g to 0.25 cm ３ / g: 50 mg of the polymer is dissolved in 500 μL of a 0.5 w / v % sorbitan monooleate / dichloromethane solution, 100 μL of water is added, and then ultrasonic waves are applied to prepare a W / O emulsion. This polymer may be referred to as the "polymer of the present disclosure." The polymer of the present disclosure is described in detail below.

[0015] A-1. Polyalkylene glycol In the technology of the present disclosure, the polyalkylene glycol constituting the polymer of the present disclosure may be linear or branched. In particular, branched polyalkylene glycols are preferred from the viewpoint of imparting excellent sustained release properties of water-soluble substances to the microcapsules of the present disclosure.

[0016] In addition, as described below, the microcapsules of the present disclosure can be a dry product.When the microcapsules of the present disclosure are made into a particulate dry product such as microspheres, if the polyalkylene glycol constituting the polymer of the present disclosure is a branched polyalkylene glycol, the particulate dry product can have low adhesion (the property of not easily adhering to other particles or container materials) and excellent fluidity.Therefore, from the viewpoint of facilitating the handling of the particulate dry product, the polyalkylene glycol constituting the polymer of the present disclosure is preferably a branched polyalkylene glycol.

[0017] When the polyalkylene glycol is a branched polyalkylene glycol, the branched polyalkylene glycol has a structure in which polyalkylene glycol is ether-bonded to at least some of the hydroxy groups of a polyhydric alcohol. In particular, the branched polyalkylene glycol preferably has a structure in which polyalkylene glycol is ether-bonded to all of the hydroxy groups of the polyhydric alcohol. Furthermore, the branched polyalkylene glycol more preferably has a structure in which linear polyalkylene glycol is ether-bonded to all of the hydroxy groups of the polyhydric alcohol.

[0018] The number of hydroxy groups contained in the polyhydric alcohol may be, for example, 2 to 20. The number of hydroxy groups contained in the polyhydric alcohol is preferably 2 to 15, more preferably 2 to 10, even more preferably 2 to 8, and particularly preferably 2, 4, or 8.

[0019] Specific examples of the polyhydric alcohol include glycerin, polyglycerin, and pentaerythritol; as well as sugars such as glucose, fructose, xylose, galactose, mannose, erythrose, arabinose, sucrose, maltose, lactose, trehalose, and cellobiose. Examples of the polyglycerin include dimers to hexamers of glycerin. The upper or lower limit of the range may be 2, 3, 4, 5, or 6. Furthermore, the polyhydric alcohol may have a structure in which two or more of the above polyhydric alcohols are polymerized, such as dipentaerythritol.

[0020] The branched polyalkylene glycol has, for example, 2 to 10 branches per molecule. The upper or lower limit of this range may be 2, 3, 4, 5, 6, 7, 8, 9, or 10. The number of branches in the branched polyalkylene glycol can be expressed in other words as the number of terminal hydroxy groups possessed by the branched polyalkylene glycol. That is, for example, a 4-branched polyalkylene glycol has 4 terminal hydroxy groups per molecule. From the viewpoint of stably retaining a water-soluble substance within the microcapsules of the present disclosure and imparting excellent sustained-release properties for the water-soluble substance to the microcapsules of the present disclosure, the branched polyalkylene glycol preferably has 3 to 10 terminal hydroxy groups per molecule, more preferably 4 to 8 terminal hydroxy groups per molecule, and particularly preferably 4 or 8 terminal hydroxy groups per molecule.

[0021] More specifically, examples of the polyalkylene glycol constituting the polymer of the present disclosure include polyethylene glycol, polypropylene glycol, and polybutylene glycol. Among these, from the viewpoint of biocompatibility, the polyalkylene glycol constituting the polymer of the present disclosure is preferably polyethylene glycol. In general, the alkylene group having fewer carbon atoms has higher hydrophilicity.

[0022] The number average molecular weight of the polyalkylene glycol constituting the polymer of the present disclosure in the polymer is not particularly limited as long as the effects of the present disclosure are achieved, and may be, for example, 1000 to 50000. From the viewpoint of being able to stably retain a water-soluble substance in the microcapsules of the present disclosure and being able to impart excellent sustained release properties of a water-soluble substance to the microcapsules of the present disclosure, the number average molecular weight of the polyalkylene glycol constituting the polymer of the present disclosure in the polymer is preferably 1000 to 30000, more preferably 5000 to 20000, even more preferably 7000 to 15000, and particularly preferably 8000 to 13000. In particular, when the polyalkylene glycol constituting the polymer of the present disclosure is a branched polyalkylene glycol, the number average molecular weight of the branched polyalkylene glycol in the polymer is preferably 3,000 to 30,000, more preferably 5,000 to 20,000, even more preferably 7,000 to 15,000, and particularly preferably 8,000 to 13,000.

[0023] A preferred embodiment of the polyalkylene glycol constituting the polymer of the present disclosure is a linear polyethylene glycol having a number average molecular weight in the polymer of 1,000 to 3,000. Another preferred embodiment of the polyalkylene glycol constituting the polymer of the present disclosure is a 4- or 8-branched polyethylene glycol having a chemical structure in which a linear polyethylene glycol is bonded to a polyhydric alcohol and having a number average molecular weight in the polymer of 5,000 to 20,000. A more preferred embodiment of the polyalkylene glycol constituting the polymer of the present disclosure is a 4- or 8-branched polyethylene glycol having a chemical structure in which a linear polyethylene glycol is bonded to a polyhydric alcohol and having a number average molecular weight in the polymer of 7,000 to 15,000.

[0024] Polyalkylene glycols can be produced by known chemical synthesis methods, and are also commercially available. Examples of commercially available products include NOF Corporation's "SUNBRIGHT (registered trademark) PTE" series and "SUNBRIGHT (registered trademark) HGEO" series. The "SUNBRIGHT (registered trademark) PTE" series is a 4-branched polyethylene glycol having a structure in which a linear polyethylene glycol chain is bonded to each of the four hydroxy groups of pentaerythritol. The "SUNBRIGHT (registered trademark) HGEO" series is an 8-branched polyethylene glycol having a structure in which a linear polyethylene glycol chain is bonded to each of the eight hydroxy groups of polyglycerin, which is a tetramer of glycerin.

[0025] A-2. Polyhydroxyalkanoic acid In the technology of the present disclosure, the polyhydroxyalkanoic acid constituting the polymer of the present disclosure may be a single polymer (i.e., a homopolymer) composed of only one type of monomer, or a copolymer (i.e., a copolymer) composed of two or more types of monomers. When the polyhydroxyalkanoic acid is a copolymer, it may be either a random copolymer or a block copolymer.

[0026] Specific examples of monomers constituting the polyhydroxyalkanoic acid include lactic acid, glycolic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and malic acid. Specific examples of raw material monomers for producing polyhydroxyalkanoic acid include ε-caprolactone. The monomers constituting the polyhydroxyalkanoic acid may be either L- or D-isomers, and the polymer of the present disclosure may contain a mixture of D- and L-isomers. From the perspective of imparting excellent mechanical strength to the polymer of the present disclosure, it is preferable that the monomers constituting the polyhydroxyalkanoic acid consist exclusively of D- or L-isomers.

[0027] The polyhydroxyalkanoic acid is preferably a homopolymer of a monomer selected from the group consisting of lactic acid, glycolic acid, and 6-hydroxycaproic acid, or a copolymer containing two or more of the above monomers. From the viewpoint of imparting excellent biodegradability to the polymer of the present disclosure, the polyhydroxyalkanoic acid is more preferably a copolymer of lactic acid and glycolic acid (i.e., a lactic acid-glycolic acid copolymer), and particularly preferably a random copolymer of lactic acid and glycolic acid.

[0028] When the polyhydroxyalkanoic acid is a lactic acid-glycolic acid copolymer, the ratio of the number of moles of lactic acid contained in the lactic acid-glycolic acid copolymer to 1 mole of glycolic acid contained in the lactic acid-glycolic acid copolymer may be, for example, 1 to 10. From the viewpoint of biodegradability, the ratio of the number of moles of lactic acid contained in the lactic acid-glycolic acid copolymer to 1 mole of glycolic acid contained in the lactic acid-glycolic acid copolymer is preferably 2 to 8, more preferably 3 to 6, and particularly preferably 3 to 5. Generally, the biodegradation rate is fastest when the ratio of the number of moles of lactic acid contained in the lactic acid-glycolic acid copolymer to 1 mole of glycolic acid contained in the lactic acid-glycolic acid copolymer is 1, and the degradation rate decreases as the composition ratio of lactic acid increases.

[0029] The number average molecular weight of the polyhydroxyalkanoic acid constituting the polymer of the present disclosure in the polymer is not particularly limited as long as the effects of the present disclosure are achieved, and may be, for example, 8,000 to 80,000. From the viewpoint of being able to stably retain a water-soluble substance within the microcapsules of the present disclosure and being able to impart excellent sustained release properties of a water-soluble substance to the microcapsules of the present disclosure, the number average molecular weight of the polyhydroxyalkanoic acid constituting the polymer of the present disclosure in the polymer is preferably 10,000 to 70,000, more preferably 10,000 to 60,000, and particularly preferably 10,000 to 50,000. In particular, when the polyalkylene glycol constituting the polymer of the present disclosure is a branched polyalkylene glycol, the number average molecular weight of the polyhydroxyalkanoic acid constituting the polymer of the present disclosure in the polymer is preferably 20,000 to 70,000, more preferably 20,000 to 60,000, even more preferably 25,000 to 55,000, and particularly preferably 30,000 to 50,000.

[0030] A preferred embodiment of the polyhydroxyalkanoic acid constituting the polymer of the present disclosure is a lactic acid / glycolic acid copolymer having a number average molecular weight of 10,000 to 70,000, in which the ratio of the number of moles of lactic acid contained in the lactic acid / glycolic acid copolymer to 1 mole of glycolic acid contained in the lactic acid / glycolic acid copolymer is 2 to 8. A particularly preferred embodiment of the polyhydroxyalkanoic acid constituting the polymer of the present disclosure is a lactic acid / glycolic acid copolymer having a number average molecular weight of 10,000 to 60,000, in which the ratio of the number of moles of lactic acid contained in the lactic acid / glycolic acid copolymer to 1 mole of glycolic acid contained in the lactic acid / glycolic acid copolymer is 3 to 6.

[0031] A-3. Structure of the Polymer of the Present Disclosure As described above, the polymer of the present disclosure has a chemical structure in which a polyalkylene glycol and a polyhydroxyalkanoic acid are bonded together. More specifically, the polymer has a chemical structure in which a hydroxy group of the polyalkylene glycol and a carboxy group of the polyhydroxyalkanoic acid are ester-bonded together.

[0032] In the polymer of the present disclosure, the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain may be 15 to 240. In the present disclosure, the term "number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain" refers to the value obtained by dividing the number average of all polyhydroxyalkanoic acid monomer units contained in the polymer by the number of terminal hydroxy groups of the polyalkylene glycol. From the viewpoint of being able to stably retain a water-soluble substance within the microcapsules of the present disclosure and being able to impart excellent sustained-release properties of a water-soluble substance to the microcapsules of the present disclosure, the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain in the polymer of the present disclosure may be 35 to 233, or may be 50 to 230, preferably 70 to 220, more preferably 80 to 210, even more preferably 90 to 205, and particularly preferably 100 to 200.

[0033] The ratio of the number average molecular weight of the polyalkylene glycol in the polymer of the present disclosure to the number average molecular weight of the polyhydroxyalkanoic acid in the polymer is not particularly limited as long as the effects of the present disclosure are achieved, and may be, for example, 1:0.5 to 1:10. From the viewpoint of being able to stably retain a water-soluble substance in the microcapsules of the present disclosure and being able to impart excellent sustained release properties of a water-soluble substance to the microcapsules of the present disclosure, the ratio of the number average molecular weight of the polyalkylene glycol in the polymer of the present disclosure to the number average molecular weight of the polyhydroxyalkanoic acid in the polymer is preferably 1:1 to 1:8, more preferably 1:2 to 1:6, and particularly preferably 1:3 to 1:5.

[0034] The number average molecular weight of the polymer of the present disclosure is not particularly limited as long as the effects of the present disclosure are achieved, and may be, for example, 10,000 to 100,000. From the viewpoint of being able to stably retain a water-soluble substance within the microcapsules of the present disclosure and being able to impart excellent sustained release properties of a water-soluble substance to the microcapsules of the present disclosure, the number average molecular weight of the polymer of the present disclosure is preferably 10,000 to 80,000, more preferably 11,000 to 70,000, and particularly preferably 12,000 to 60,000. In particular, when the polyalkylene glycol constituting the polymer of the present disclosure is a branched polyalkylene glycol, the number average molecular weight of the polymer of the present disclosure is preferably 20,000 to 80,000, more preferably 30,000 to 70,000, even more preferably 35,000 to 65,000, and particularly preferably 40,000 to 60,000.

[0035] The number average molecular weight of the polymer of the present disclosure, the number average molecular weight of the polyalkylene glycol in the polymer, the number average molecular weight of the polyhydroxyalkanoic acid in the polymer, and the molar ratio of each monomer when the polyhydroxyalkanoic acid is a copolymer can be measured by known methods. １ Examples of the molecular weight include a method using HNMR and a method of determining the molecular weight in terms of polystyrene or polymethyl methacrylate (PMMA) measured by gel permeation chromatography (GPC).

[0036] １ A general method for analyzing a polymer using H NMR is described in detail below. １ ​The peak area per proton is calculated from the HNMR chart. The peak area per proton is calculated by dividing the sum of the areas of the peaks derived from the protons of a certain substituent by the number of protons contained in the group. In this case, the group is selected such that the amount present in one molecule of the monomer is known in advance; Next, the number of other monomer units is calculated. The number of monomer units is calculated by dividing the sum of the areas of the peaks derived from the protons of the monomer unit by the number of protons contained in the monomer unit, and then dividing this by the peak area per proton calculated previously; Once the number of monomer units is determined, the molecular weights of the monomers constituting the polymer and the molecular weights of the portions other than the repeating portion are known, and therefore the number average molecular weight of the entire polymer and the number average molecular weight of each repeating portion, etc. can be calculated.

[0037] A typical method for analyzing a polymer using GPC is described in detail below: First, the polymer is dissolved in a solvent such as chloroform and filtered to obtain a sample solution. Using a high-performance liquid chromatograph equipped with a GPC column, the sample solution is injected, separated using a column adjusted to a constant temperature, and detected using a differential refractometer. The number-average molecular weight can be measured using a data analysis workstation or the like, and can be calculated using a calibration curve obtained from the relationship between the molecular weight of standard polystyrene or polymethyl methacrylate and the column elution time.

[0038] The polymer of the present disclosure can also be analyzed according to the above-mentioned general analytical methods, or by a method that a person skilled in the art can easily derive from the above-mentioned general methods.

[0039] A preferred embodiment of the polymer of the present disclosure is a polymer having a chemical structure in which 4- or 8-branched polyethylene glycol and a lactic acid / glycolic acid copolymer are bonded. In this polymer, the number-average molecular weight of the polyethylene glycol in the polymer is preferably 3,000 to 30,000, more preferably 5,000 to 20,000, even more preferably 7,000 to 15,000, and particularly preferably 8,000 to 13,000. In addition, in this polymer, the number-average molecular weight of the lactic acid / glycolic acid copolymer in the polymer is preferably 20,000 to 70,000, more preferably 20,000 to 60,000, even more preferably 25,000 to 55,000, and particularly preferably 30,000 to 50,000. Furthermore, in the polymer, the ratio of the number of moles of lactic acid contained in the lactic acid-glycolic acid copolymer to 1 mole of glycolic acid contained in the lactic acid-glycolic acid copolymer is preferably 2 to 8, more preferably 3 to 6, and particularly preferably 3 to 5.

[0040] A preferred embodiment of the polymer of the present disclosure is a polymer represented by the following formula (I): The polymer has a chemical structure in which a 4-branched polyethylene glycol and a lactic acid / glycolic acid copolymer are bonded together. In formula (I), k, m, n, and p are each independently a natural number of 16 to 170, preferably 28 to 110, more preferably 39 to 84, and even more preferably 45 to 73. For example, when the molecular weight of the 4-branched polyethylene glycol constituting the polymer is 30,000, the average of k, m, n, and p is approximately 170. This value is calculated by the formula [(30,000-132) / 44] / 4 (30,000: molecular weight of the 4-branched polyethylene glycol, 132: branched moiety C(CH ２ O) ４ molecular weight of 44:CH ２ CH ２where k is the molecular weight of the 4-branched polyethylene glycol constituting the polymer, m is the molecular weight of the 4-branched polyethylene glycol, and p is the number of branches. Similarly, when the molecular weight of the 4-branched polyethylene glycol constituting the polymer is 3,000, the average of k, m, n, and p is about 16, and when the molecular weight of the 4-branched polyethylene glycol constituting the polymer is 10,000, the average of k, m, n, and p is about 56.

[0041] X １ , X ２ , X ３ , and X ４ are each independently a group represented by the following formula (II): In formula (II), s is a natural number of 19 to 193, preferably 25 to 189, and more preferably 27 to 184. Furthermore, t is a natural number of 3 to 115, preferably 4 to 74, and more preferably 5 to 55. X １ , X ２ , X ３ , and X ４ Each s may be the same or different. １ , X ２ , X ３ , and X ４ Each t may be the same or different. s+t may be 35 to 233, and preferably 50 to 230. Note that a portion enclosed in square brackets ([ ]: brackets) means that the monomer units enclosed in parentheses (() : parentheses) are randomly polymerized (the same applies hereinafter in this specification).

[0042] The above formula (II) is more preferably the following formula (III): In formula (III), v is a natural number from 9 to 97, preferably from 12 to 95, and more preferably from 13 to 92. Furthermore, w is a natural number from 1 to 58, preferably from 2 to 37, and more preferably from 2 to 28.

[0043] A preferred embodiment of the polymer of the present disclosure is a polymer represented by the following formula (IV): When r is 4, the polymer has a chemical structure in which an eight-branched polyethylene glycol and a lactic acid / glycolic acid copolymer are bonded together. In formula (IV), k, m, n, p, and q are each, and independently of one another in each repetition, a natural number from 7 to 84, preferably from 10 to 56, more preferably from 12 to 41, and even more preferably from 13 to 36. In formula (IV), r is a natural number from 1 to 6. The upper or lower limit of the range may be 1, 2, 3, 4, 5, or 6.

[0044] For example, when the above r is 4 and the molecular weight of the 8-branched polyethylene glycol constituting the polymer is 30,000, the average of k, m, n, p, and q is approximately 84. This value is calculated by the formula [(30,000-454) / 44] / 8 (30,000: molecular weight of 8-branched polyethylene glycol, 454: molecular weight calculated by the formula below). The molecular weight of the branched portion represented by 44:CH ２ CH ２ where r is the molecular weight of the 8-branched polyethylene glycol constituting the polymer, and 8 is the number of branches. Similarly, when r is 4 and the molecular weight of the 8-branched polyethylene glycol constituting the polymer is 3,000, the average of k, m, n, p, and q is about 7, and when r is 4 and the molecular weight of the 8-branched polyethylene glycol constituting the polymer is 10,000, the average of k, m, n, p, and q is about 27.

[0045] X １ , X ２ , X ３ , X ４ and X ５ are each, and independently of each other in each repetition, a group represented by the following formula (II): In formula (II), s is a natural number of 10 to 96, preferably 12 to 95, and more preferably 14 to 92. Furthermore, t is a natural number of 2 to 58, preferably 2 to 37, and more preferably 3 to 27. X １ , X ２ , X ３ , X ４and X ５ Each s may be the same or different. １ , X ２ , X ３ , X ４ and X ５ Each t may be the same or different, and s+t may be 18 to 117.

[0046] The above formula (II) is more preferably the following formula (III): In formula (III), v is a natural number of 5 to 48, preferably 6 to 48, and more preferably 7 to 46. Furthermore, w is a natural number of 1 to 29, preferably 1 to 19, and more preferably 1 to 14.

[0047] A-4. Method for Producing the Polymer of the Present Disclosure The bonding of polyalkylene glycol and polyhydroxyalkanoic acid can be carried out by a conventionally known method or a method that can be easily derived from a conventionally known method. Examples include a method of polymerizing hydroxyalkanoic acid starting from the terminal hydroxy group of a polyalkylene glycol chain, and a method of bonding polyalkylene glycol and polyhydroxyalkanoic acid by a condensation reaction. More specifically, the polymer of the present disclosure can be obtained by ring-opening polymerization of a cyclic ester intermediate of hydroxyalkanoic acid, such as lactide, in the presence of polyalkylene glycol using a catalyst such as tin octoate under reduced pressure. Also known in the art are methods for removing moisture and low-molecular-weight compounds from the reaction system by adjusting the conditions for heating and refluxing in an organic solvent used for the polymerization reaction, as well as methods for suppressing depolymerization by deactivating the catalyst after the polymerization reaction is complete. For example, the unreacted cyclic ester intermediate can be sublimated and removed by heat-treating the resulting polymer under reduced pressure. More specifically, the polymer of the present disclosure can be prepared, for example, according to the method described in International Publication No. 2018 / 062464 (Patent Document 2), or by appropriately modifying the method described in Patent Document 2.

[0048] A-5. Characteristics of the Polymer of the Present Disclosure As described above, when a W / O (water-in-oil) emulsion prepared using the polymer by the following method is measured by differential scanning calorimetry (temperature-programming method), the total volume of the water phase in the W / O emulsion is 0.025 cm ３ / g ~ 0.25 cm ３ / g: 50 mg of the polymer is dissolved in 500 μL of a 0.5 w / v % sorbitan monooleate / dichloromethane solution, 100 μL of water is added, and the mixture is irradiated with ultrasound to prepare a W / O emulsion. The measurement of the total volume of the aqueous phase in the W / O emulsion is described in detail below.

[0049] A-5-1. Preparation of W / O Emulsion A 0.5 w / v% sorbitan monooleate / dichloromethane solution can be prepared by adding and dissolving 50 mg of sorbitan monooleate per 10 mL of dichloromethane. Sorbitan monooleate is a nonionic surfactant with an HLB of approximately 4.3. It is sold commercially under the name Span (registered trademark) 80, etc. The structure of sorbitan monooleate is shown below.

[0050] 50 mg of the polymer to be measured is dissolved in 500 μL of the 0.5 w / v% sorbitan monooleate / dichloromethane solution prepared as described above, and then 100 μL of water is added to prepare a W / O emulsion by ultrasonic irradiation. High-purity water, such as distilled water for reverse-phase chromatography (HPLC), is used. The W / O emulsion is then prepared by ultrasonic irradiation. Specifically, for example, the vibration horn of an ultrasonic homogenizer is brought into contact with the solution, and ultrasonic irradiation is performed for approximately 30 seconds. Examples of ultrasonic homogenizers that can be used include the Handy Sonic (UR-20P, manufactured by Tomy Seiko Co., Ltd.) and equivalents.

[0051] A-5-2. Measurement of the total volume of the aqueous phase in a W / O emulsion As described in Non-Patent Document 1, the melting point of ice trapped in pores is lowered due to interfacial tension. Furthermore, not only freezable water but also non-freezable water (water that cannot be frozen) exists in the pores. Since non-freezable water exists along the pore surface, it is believed that the amount of non-freezable water increases as the pore diameter decreases and the specific surface area increases. The microenvironment within a W / O emulsion prepared as described above is thought to be similar to the environment within the pores described in Non-Patent Document 1. Therefore, it is believed that within this W / O emulsion, there exists ice whose melting point has been lowered due to interfacial tension, and non-freezable water existing along the interface with the external oil phase.

[0052] The volume of ice with a reduced melting point present in the W / O emulsion can be determined by differential scanning calorimetry (hereinafter, sometimes referred to as DSC) according to the method described in Non-Patent Document 1. In the present disclosure, DSC is performed using a temperature-programmed method. In the present disclosure, the "total volume of the aqueous phase in the W / O emulsion" refers to the volume of ice with a reduced melting point present in the W / O emulsion measured by DSC (temperature-programmed method). As can be understood from the above description, the "total volume of the aqueous phase in the W / O emulsion" in the present disclosure corresponds to the volume of "freezable pore water" described in Non-Patent Document 1. Furthermore, in Non-Patent Document 2, water with a reduced melting point measured by DSC (temperature-programmed method) similar to that in Non-Patent Document 1 is referred to as "frozen water clusters." The volume of the "frozen water clusters" in Non-Patent Document 2, like the volume of the "freezable pore water" described in Patent Document 1, corresponds to the "total volume of the aqueous phase in the W / O emulsion" in the present disclosure.

[0053] The specific measurement conditions for differential scanning calorimetry (temperature-programmed method) are not particularly limited as long as the desired effect is obtained. For example, measurement can be performed under conditions of a temperature-programming rate of about 0.3°C / min and a measurement temperature range of about -55°C to about 5°C.

[0054] The polymer of the present disclosure has a W / O emulsion in which the total volume of the aqueous phase measured by the above method is 0.025 cm ３ / g ~ 0.25 cm ３ / g. The total volume of the aqueous phase in the W / O emulsion is calculated as the value per 1 g of components other than water contained in the measurement sample. If microcapsules encapsulating a polymer and a water-soluble substance are prepared using a polymer whose total volume of the aqueous phase in the W / O emulsion is within the above range, microcapsules with excellent stability of the water-soluble substance can be provided. The upper or lower limit of the range may be 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. From the viewpoint of being able to stably retain a water-soluble substance in the microcapsules of the present disclosure and being able to impart excellent sustained release properties of a water-soluble substance to the microcapsules of the present disclosure, the polymer of the present disclosure is used when the total volume of the aqueous phase in the W / O emulsion measured by the above method is 0.025 cm ３ / g ~ 0.23 cm ３ / g, and preferably 0.028 cm ３ / g ~ 0.22 cm ３ / g, and more preferably 0.03 cm ３ / g to 0.2 cm ３ In particular, when the polyalkylene glycol constituting the polymer of the present disclosure is a branched polyalkylene glycol, the total volume of the aqueous phase in the W / O emulsion is preferably 0.025 cm 3 / g. ３ / g to 0.1 cm ３ / g, and preferably 0.025 cm ３ / g to 0.05 cm ３ / g, more preferably 0.028 cm ３ / g to 0.05 cm ３ / g, and more preferably 0.03 cm ３ / g to 0.04 cm ３ It is particularly preferred that the SiO2 content is 1 / g.

[0055] The polymer of the present disclosure having the above characteristics can stably retain the water-soluble substance within the microcapsules of the present disclosure, and can also impart excellent sustained release of the water-soluble substance to the microcapsules of the present disclosure. The stability and sustained release of the water-soluble substance can be evaluated by a conventionally known method or a method that can be easily derived from a conventionally known method. For example, when the water-soluble substance is a protein, the total protein amount in a composition containing the microcapsules can be quantified by the BCA method, the Bradford method, the Lowry method, or the like, and the active protein amount in the composition can be quantified by the ELISA method or the like, and the activity retention rate (%) can be calculated using the formula [active protein amount / total protein amount] x 100. In addition, the sustained release of the water-soluble substance can be evaluated, for example, by mixing a composition containing the microcapsules with a release test solution and quantifying the amount of the water-soluble substance released into the release test solution over time.

[0056] B. Microcapsules of the Present Disclosure B-1. Definition In the present disclosure, the term "microcapsules" refers to particles having an average particle size in the nanometer to micrometer range. In this respect, the average particle size of the microcapsules of the present disclosure is not particularly limited and may be, for example, about 10 nm to 1000 μm, about 50 nm to 100 μm, about 100 nm to 50 μm, or about 1 μm to 10 μm. The average particle size of the microcapsules can be adjusted by conventionally known methods or methods that can be easily conceived by those skilled in the art from conventionally known methods. For example, the average particle size of the microcapsules can be adjusted by changing the pore size of the emulsifying membrane used to prepare the W / O emulsion or W / O / W emulsion. Alternatively, the average particle size of the microcapsules can be adjusted by changing the stirring speed of the stirrer used to prepare the W / O emulsion or W / O / W emulsion.

[0057] In the present disclosure, the term "average particle size" refers to the average of the maximum diameters of individual particles. Generally, the average particle size can be measured by a laser diffraction scattering method, a dynamic light scattering method, an image analysis method using a scanning electron microscope (SEM), a sieving test, or the like. In particular, in the present disclosure, the term "average particle size" refers to the volume-based average particle size measured by a laser diffraction scattering method.

[0058] As described below, a preferred embodiment of the method for producing microcapsules of the present disclosure involves preparing an oil phase solution containing the polymer of the present disclosure and an aqueous phase solution containing a water-soluble substance, and then mixing the two to prepare a W / O emulsion. The individual emulsified particles contained in the W / O emulsion are preferably encapsulated in the microcapsules of the present disclosure. Furthermore, when the W / O emulsion is dried, the volatile components in the internal and external phases of the emulsified particles volatilize, resulting in a dried product containing capsule-like structures encapsulating the water-soluble substance. The capsule-like structures in the dried product are also preferably encapsulated in the microcapsules of the present disclosure.

[0059] Although not particularly limited, in a preferred embodiment of the microcapsules of the present disclosure, the polymer of the present disclosure is contained in a membrane, and a water-soluble substance is encapsulated in the space surrounded by the membrane. Here, the "membrane" refers to the part that separates the inner and outer phases of each emulsified particle in the W / O emulsion.

[0060] B-2. Water-Soluble Substances The type of water-soluble substance encapsulated in the microcapsules of the present disclosure is not particularly limited. Specific examples of water-soluble substances include low molecular weight compounds, proteins, peptides, and nucleic acids. In the technology of the present disclosure, proteins, peptides, and nucleic acids may be antibodies and / or vaccines. Note that, in this disclosure, the term "antibody" is intended to encompass antibodies and antigen-binding fragments of antibodies unless otherwise specified. Furthermore, proteins and peptides may be modified with polyethylene glycol and / or sugar chains. Nucleic acids may be DNA or RNA, and may be modified with a protecting group or may form a complex with a surfactant, lipid, polymer, or the like.

[0061] Specific examples of water-soluble substances that may be encapsulated in the microcapsules of the present disclosure include parathyroid hormone (PTH), calcitonin, insulin, insulin-like growth factor, angiotensin, glucagon, GLP-1 and GLP-1 receptor agonist peptides represented by exendin 4, bombesin, motilin, gastrin, growth hormone, prolactin (luteinizing hormone), gonadotropin (gonadotropin), thyrotropin, adrenocorticotropic hormone (ACTH), ACTH derivatives (e.g., ebiratide), melanocyte-stimulating hormone, follicle stimulating hormone, and the like. FSH, sermorelin, vasopressin, oxytocin, protirelin, luteinizing hormone (LH), corticotropin, secretin, somatropin, thyrotropin (thyroid-stimulating hormone), somatostatin, gonadotropin-releasing hormone (GnRH), G-CSF, erythropoietin (EPO), thrombopoietin (TPO), megakaryocyte potentiator, HGF, EGF, VEGF, interferon α, interferon β, interferon γ, interleukins, FGF (fibroblast growth factors), BMP (bone morphogenetic proteins), thymic humoral factor (THF), blood thymic factor (FTS), superoxide dismutase (SOD), urokinase, lysozyme, tissue plasminogen activator, asparaginase, kallikrein, ghrelin, adiponectin, leptin, atrial natriuretic peptide, atrial natriuretic factor, brain natriuretic peptide (BNP), conantoxin G, dynorphin, endorphin, kyotorphin, enkephalin, neurotensin, angiostatin, bradykinin, substance P, kallidin, Hemoglobin, protein C, factor VIIa, glucocerebrosidase, streptokinase, staphylokinase, thymosin (thymosin), pancreozymin, cholecystokinin, human placental lactogen, tumor necrosis factor (TNF), polymyxin B, colistin, gramicidin, bacitracin, thymopoietin, bombesin, caerulein, thymostimulin, selectin, resistin, hepcidin, neuropeptide Y, neuropeptide S, cholecystokinin-pancreozymin (CCK-PZ), brain-derived neurotrophic factor (BDNF);Examples include anti-VEGF antibodies such as bevacizumab, faricimab, and brolucizumab, Fab fragments of anti-VEGF antibodies such as ranibizumab, anti-EGFR antibodies such as cetuximab, and vaccines.

[0062] Among the water-soluble substances described above, there are some that easily decompose, denature, or change in quality, resulting in the loss of physiological activity. According to the technology of the present disclosure, even such water-soluble substances can maintain their activity in a formulation. Whether a water-soluble substance maintains its activity can be evaluated by a conventionally known method or a method that can be easily derived from a conventionally known method. For example, when the water-soluble substance is a protein, the total protein amount in the formulation can be quantified by the BCA method, the Bradford method, the Lowry method, or the like, and the active protein amount in the formulation can be quantified by the ELISA method, or the like, to calculate the activity retention rate (%) using the formula [active protein amount / total protein amount] x 100.

[0063] B-3. ​​Method for Producing Microcapsules of the Present Disclosure The microcapsules of the present disclosure can be produced by a conventionally known method or a method that can be easily derived from a conventionally known method. Specific examples include emulsion solvent evaporation, phase separation, phase transition, and polymer spherical crystallization (including spherical granulation (SA) method and emulsion solvent diffusion (ESD) method). The solvent used can be appropriately selected depending on the method.

[0064] For example, when producing the microcapsules of the present disclosure using the emulsion solvent evaporation method, an oil phase solution containing the polymer of the present disclosure and an aqueous phase solution containing a water-soluble substance may be prepared, and then the two may be mixed to prepare a W / O emulsion. Mixing may be performed using a magnetic stirrer, a propeller-type agitator, a turbine-type agitator, a dispersing mixer, a homomixer, an ultrasonic irradiator, a homogenizer, or a membrane emulsifying device equipped with a porous membrane. Alternatively, the W / O emulsion may be dried to volatilize the volatile components in the internal and external phases, resulting in a dried product.

[0065] The oil phase solution preferably contains a water-immiscible organic solvent. In the present disclosure, "water-immiscible organic solvent" refers to an organic solvent having a water solubility of 30 g (water-immiscible organic solvent) / 100 mL (water) or less. The water solubility of the water-immiscible organic solvent used in the production of the microcapsules of the present disclosure is more preferably 0.1 g (water-immiscible organic solvent) / 100 mL (water) or less. Furthermore, although not particularly limited, the water-immiscible organic solvent used in the production of the microcapsules of the present disclosure is preferably volatile, more specifically, one that can be removed by volatilization such as freeze-drying or spray-drying. Specific examples of such water-immiscible organic solvents include ethyl acetate, isopropyl acetate, butyl acetate, dimethyl carbonate, diethyl carbonate, dichloromethane, and trichloromethane. Among these, dichloromethane is particularly preferred.

[0066] The oil phase solution and the aqueous phase solution may further contain other components, such as pharmaceutically acceptable bases, carriers, excipients, polymers other than the polymers of the present disclosure, diluents, surfactants, preservatives, pH adjusters, adjuvants, chelating agents, etc., either alone or in combination of two or more thereof.

[0067] Examples of polymers other than the polymers of the present disclosure include polyalkylene glycols, polyhydroxyalkanoic acids, polyvinylpyrrolidone, polyvinyl alcohol, polyethyleneimine, polyacrylic acid, polymethacrylic acid, poly-1,3-dioxolane, 2-methacryloyloxyethyl phosphorylcholine polymer, and poly-1,3,6-trioxane, as well as polysaccharides, etc. Polymers other than the polymers of the present disclosure can be used alone or in combination of two or more.

[0068] The surfactant can be exemplified by nonionic surfactants, specifically, polyoxyalkylene alkyl ethers, polyoxyalkylene glycols, polyoxyalkylene fatty acid esters, polyoxyalkylene sorbitan fatty acid esters, polyoxyalkylene sorbit fatty acid esters, polyoxyalkylene glycerin fatty acid esters, polyoxyalkylene fatty acid amides, polyoxyalkylene glycol fatty acid esters, polyoxyalkylene castor oil derivatives, polyoxyalkylene hydrogenated castor oil derivatives other than polyoxyethylene hydrogenated castor oil, polyglycerin fatty acid esters, monoglycerin fatty acid esters, sorbitan fatty acid esters, sorbit fatty acid esters, alkylene glycol fatty acid esters, alkyl polyglycosides, sugar fatty acid esters (sucrose fatty acid esters, maltose fatty acid esters, lactose fatty acid esters, etc.), fatty acid alkanolamides, diethyl sebacate, etc. A preferred nonionic surfactant in the technology of the present disclosure can be sorbitan monooleate, which is a type of sorbitan fatty acid esters. Other preferred examples include alkyl sulfates such as sodium lauryl sulfate, ammonium lauryl sulfate, and sodium stearyl sulfate, as well as lecithin and its derivatives. The surfactants can be used alone or in combination of two or more.

[0069] Examples of preservatives include parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, sodium benzoate, phenoxyethanol, alkyldiaminoethylglycine hydrochloride, etc. The preservatives may be used alone or in combination of two or more kinds.

[0070] Examples of pH adjusters include citric acid, phosphoric acid, malic acid, pyrophosphoric acid, lactic acid, tartaric acid, glycerophosphoric acid, acetic acid, nitric acid, amino acids and their chemically acceptable salts, sodium hydroxide, potassium hydroxide, etc. Adding the basic amino acid L-arginine to the aqueous phase solution is particularly preferred because it ionically interacts with the polymer, improving the encapsulation rate of the water-soluble substance contained in the aqueous phase solution. That is, the basic amino acid L-arginine not only adjusts the pH of the formulation, but also improves the encapsulation rate of the water-soluble substance in the microcapsules. pH adjusters can be used alone or in combination of two or more.

[0071] C. Compositions of the Present Disclosure The present disclosure also encompasses compositions containing the microcapsules of the present disclosure. Such compositions may be referred to as "compositions of the present disclosure." The matters described above regarding the microcapsules of the present disclosure are incorporated by reference into the compositions of the present disclosure. The compositions of the present disclosure may be the microcapsules of the present disclosure themselves, or may be microcapsules of the present disclosure mixed with other components. There are no limitations on the components that can be mixed as long as the effects of the present disclosure are achieved, and preferred examples include the components described in the section "B-3. Method for producing microcapsules of the present disclosure." Furthermore, a W / O emulsion containing the microcapsules of the present disclosure may be dispersed in a separate aqueous phase to form a W / O / W emulsion.

[0072] The dosage form of the composition of the present disclosure is not particularly limited and may be, for example, sphere, sheet, needle, rod, or coil form. Of these dosage forms, sphere or sheet form is preferred, and microsphere or film is particularly preferred. In this disclosure, "microsphere" refers to a spherical composition containing a polymer and a water-soluble substance and having an average particle size of approximately 1 to 100 μm. Nanoparticles, microparticles, nanospheres, and microcapsules are also preferred examples of dosage forms of the composition of the present disclosure.

[0073] The composition of the present disclosure is preferably a sustained-release pharmaceutical composition. In the composition of the present disclosure, the water-soluble substance is encapsulated in the microcapsules of the present disclosure, so the water-soluble substance can be stably maintained. In addition, in the composition of the present disclosure, the water-soluble substance is encapsulated in the microcapsules of the present disclosure, so the composition of the present disclosure can have sustained release properties for the water-soluble substance. Therefore, when the composition of the present disclosure is applied to a subject, the water-soluble substance is gradually released from the composition of the present disclosure in the body of the subject, and it is expected that a sustained effect will be achieved.

[0074] The method of applying the composition of the present disclosure to a subject is not particularly limited as long as the effects of the present disclosure are achieved. For example, the composition of the present disclosure may be administered by direct injection into a local area such as the vitreous body, by injection or infusion into a vein, an artery, an intraperitoneal cavity, an intramuscular cavity, a subcutaneous cavity, an intrapleural cavity, or the like, by perfusion via a catheter, or by application to the skin, mucosa, tissue, or the like.

[0075] The compositions of the present disclosure can be applied to, for example, humans and non-human mammals (e.g., rats, mice, rabbits, cows, pigs, dogs, cats, sheep, monkeys, etc.), with humans being preferred.

[0076] D. Screening Method for Sustained-Release Polymers The present disclosure also encompasses a screening method for sustained-release polymers. The screening method comprises the following steps (a) to (c): step (a) of dissolving a test polymer in a solution comprising a surfactant and a water-immiscible organic solvent to prepare the test polymer solution; step (b) of adding water to the test polymer solution prepared in step (a) and then irradiating the solution with ultrasound to prepare a W / O emulsion; and step (c) of determining whether the total volume of the aqueous phase in the W / O emulsion is 0.025 cm when the W / O emulsion prepared in step (b) is measured by differential scanning calorimetry (temperature-programming method). ３ / g to 0.25 cm ３ / g as an index. In the present disclosure, this screening method may be referred to as the "screening method of the present disclosure." The matters described in the sections "A-1. Polyalkylene glycol" to "A-4. Method for producing a polymer of the present disclosure" and the matters related to water-immiscible organic solvents described in the section "B-3. Method for producing microcapsules of the present disclosure" are incorporated by reference as appropriate into the screening method of the present disclosure.

[0077] The polymer to which the screening method of the present disclosure is applied is not particularly limited. For example, it may be a polymer containing polyhydroxyalkane, and preferably a polymer having a structure in which polyhydroxyalkane and polyalkylene glycol are bonded.

[0078] In step (a), a test polymer solution is prepared by dissolving the test polymer in a solution consisting of a surfactant and a water-immiscible organic solvent. The type of surfactant is not particularly limited, as long as a W / O emulsion can be prepared in step (b), described below. Specific surfactants include, for example, the nonionic surfactants described in the section "B-3. Method for producing microcapsules according to the present disclosure." Among these, nonionic surfactants with an HLB of approximately 2 to 7 are preferred. The upper or lower limit of the HLB range may be 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7. The HLB of the surfactant used in the screening method of the present disclosure is preferably approximately 3 to 6, and more preferably approximately 3.5 to 5.5. A particularly preferred example of a specific surfactant is sorbitan monooleate (HLB 4.3), sold under the trade name Span (registered trademark) 80. In the screening method of the present disclosure, surfactants can be used alone or in combination of two or more.

[0079] Furthermore, the type of water-immiscible organic solvent used in the screening method of the present disclosure is not particularly limited. Among water-immiscible organic solvents, dichloromethane is particularly preferred.

[0080] The specific contents of the surfactant and the water-immiscible organic solvent in the solution are not particularly limited, as long as a W / O emulsion can be prepared in the next step (b). That is, it is reasonably understood that the technology of the present disclosure does not include embodiments in which the surfactant content in the solution is so extremely low that a W / O emulsion cannot be prepared, or embodiments in which the surfactant content is so extremely high that a W / O emulsion cannot be prepared. As long as a W / O emulsion is prepared in the next step (b), the specific contents of the surfactant and the water-immiscible organic solvent in the solution do not substantially affect the measurement result of the total volume of the aqueous phase in the W / O emulsion.

[0081] The solution consisting of a surfactant and a water-immiscible organic solvent may further contain other components as long as the desired effect is obtained. It is particularly preferable that the solution consisting of a surfactant and a water-immiscible organic solvent consists only of a surfactant and a water-immiscible organic solvent. In this disclosure, the term "consisting only of" means that the solution does not substantially contain other components. In other words, the term "consisting only of" in this disclosure does not exclude embodiments in which unavoidable impurities are contained.

[0082] The amount of surfactant necessary for preparing a W / O emulsion can be easily determined based on common general knowledge in the art and / or by a reasonable amount of trial and error by a person skilled in the art based on said common general knowledge. For example, the concentration of the surfactant in the water-immiscible organic solvent may be 0.01 to 10 w / v%, preferably 0.1 to 5 w / v%, more preferably 0.2 to 3 w / v%, and even more preferably 0.2 to 3 w / v%.

[0083] The amount of test polymer dissolved in the solution comprising the surfactant and water-immiscible organic solvent is not particularly limited, as long as a W / O emulsion can be prepared. For example, the polymer may be dissolved in a ratio of 2 to 100 parts by mass of the solution comprising the surfactant and water-immiscible organic solvent to 1 part by mass of the test polymer. The range is preferably 5 to 50 parts by mass, more preferably 7 to 30 parts by mass, and particularly preferably 10 to 20 parts by mass.

[0084] In step (b), water is added to the test polymer solution prepared in step (a) and then ultrasonically irradiated to prepare a W / O emulsion. The water added is preferably high-purity water, such as distilled water for reverse-phase chromatography (HPLC). The amount of water added is not particularly limited as long as a W / O emulsion can be prepared. For example, 0.5 to 5 parts by mass of water may be used per 1 part by mass of the test polymer. The range is preferably 1 to 4 parts by mass, and more preferably 1.5 to 3 parts by mass. As long as a W / O emulsion is prepared, the specific contents of the polymer and water in the W / O emulsion do not substantially affect the measurement results of the total volume of the aqueous phase in the W / O emulsion.

[0085] The conditions for ultrasonic irradiation are not particularly limited as long as a W / O emulsion can be prepared. For example, irradiation may be performed for about 5 seconds to 5 minutes, preferably for about 10 seconds to 3 minutes, and more preferably for about 15 seconds to 1 minute.

[0086] A specific example of the ultrasonic irradiation method is to bring the vibration horn of an ultrasonic homogenizer into contact with the solution and irradiate the solution with ultrasonic waves for about 30 seconds. Examples of ultrasonic homogenizers that can be used include Handy Sonic (UR-20P, manufactured by Tomy Seiko Co., Ltd.) and equivalents.

[0087] In step (c), the total volume of the aqueous phase in the W / O emulsion prepared as described above is measured by differential scanning calorimetry (temperature programming method), and the total volume of the aqueous phase in the W / O emulsion is determined to be 0.025 cm ３ / g ~ 0.25 cm ３ The total volume of the aqueous phase in a W / O emulsion is determined by the method described in "A-5-2. Measurement of the total volume of the aqueous phase in a W / O emulsion."

[0088] In the present disclosure, the term "sustained release" refers to the property of a water-soluble substance being continuously released from a formulation for at least one week. The term "sustained-release polymer" refers to a polymer that can impart sustained release of a water-soluble substance to a formulation prepared using the polymer. The period during which the water-soluble substance is continuously released from the formulation is preferably two weeks or more, more preferably three weeks or more, even more preferably four weeks or more, and particularly preferably six weeks or more. The upper limit of the period is not particularly limited, and may be, for example, 6, 7, 8, 9, 10, 11, or 12 weeks or less, or 4, 5, 6, or 7 months or less. The sustained release of a water-soluble substance can be evaluated, for example, by mixing the formulation with a release test solution and quantifying the amount of water-soluble substance released into the release test solution over time.

[0089] In this specification, the term "comprising" includes "essentially consisting of" and "consisting of" in addition to "containing." Furthermore, the present disclosure encompasses any and all combinations of the constituent elements described in this specification.

[0090] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0091] Hereinafter, embodiments of the present disclosure will be described in more detail using examples, but the embodiments of the present disclosure are not limited to the following examples. Unless otherwise specified, "molecular weight" refers to number average molecular weight. Furthermore, "PEG" refers to polyethylene glycol, and "PLGA" refers to lactic acid-glycolic acid copolymer. Furthermore, "LA:GA" refers to the molar ratio of lactic acid monomer units to glycolic acid monomer units in the lactic acid-glycolic acid copolymer.

[0092] 1. Test 1: Measurement of the total volume of the aqueous phase in a W / O emulsion First, the inventors prepared W / O emulsions using various polymers and measured the total volume of the aqueous phase in the W / O emulsion by differential scanning calorimetry (hereinafter sometimes referred to as DSC). Details are described below.

[0093] 1.1 Evaluated Polymers The polymers evaluated in this study are shown in the table below. PLGA (45 kDa) was prepared using "Resomer RG504, 739944, LA:GA = 1:1" (Sigma-Aldrich). 1PEG (2 kDa)-PLGA (11.5 kDa) was prepared using "Polyethylene glycol methyl ether-block-poly(lactide-co-glycolide), 764760, LA:GA = 1:1" (Sigma-Aldrich). The other polymers were prepared at an LA:GA = 8:2 ratio according to the method described in WO 2018 / 062464 (Patent Document 2).

[0094]

[0095] 1.2 Preparation of W / O emulsions For each polymer described in 1.1, a W / O emulsion was prepared by the following procedure: 50 mg of polymer was weighed into a 2 cc screw vial, and 500 μL of a 0.5 w / v % sorbitan monooleate / dichloromethane solution (referred to as "oil phase solution" in the table below) was added to dissolve the polymer; after visually confirming dissolution, 100 μL of distilled water for reversed-phase HPLC was added, and the mixture was ultrasonically irradiated for about 30 seconds to prepare a W / O emulsion.

[0096] The composition of the W / O emulsion, converted into weight, is shown in the table below.

[0097]

[0098] 1.3 Measurement of the total volume of the aqueous phase in a W / O emulsion. Nano-sized pores and structures contain ice whose melting point has been lowered due to interfacial tension, and unfrozen water. Each W / O emulsion prepared in 1.2 was subjected to DSC measurement, and the volume of ice whose melting point had been lowered in the W / O emulsion (i.e., the total volume of the aqueous phase in the W / O emulsion) was calculated. Details of the measurement method and the results are shown below.

[0099] 1.3.1 DSC measurement conditions DSC device: DSC Q100 manufactured by TA Instruments Data processing: Analysis program "TRC-THADAP-DSC" manufactured by Toray Research Center Measurement temperature range: Approximately -55°C to 5°C Heating rate: 0.3°C / min Sample amount: Approximately 5 mg Sample container: Sealed aluminum sample container Temperature and calorific value calibration: Pure water (melting point 0.0°C, heat of fusion 79.7 cal / g)

[0100] 1.3.2 Results The calculated total volume of the aqueous phase in the W / O emulsion is shown in the table below.

[0101]

[0102] In addition, DSC measurement was also carried out on a W / O emulsion prepared in the same manner as in 1.2, except that the amount of polymer added was 12.5 mg using the 4PEG (10 kDa)-PLGA (40 kDa) polymer shown in Table 1. The total volume of the aqueous phase in the W / O emulsion calculated for this W / O emulsion was 0.031 cm. ３ This suggests that when W / O emulsions are prepared using the same polymer, the total volume of the aqueous phase in the W / O emulsion calculated by DSC is almost the same even if the polymer content in the W / O emulsion is different.

[0103] 2. Test 2 Preparation of antibody-encapsulated microspheres and evaluation of stability during manufacturing 1 Next, the inventors prepared antibody-encapsulated microspheres using each polymer for which the total volume of the aqueous phase inside the W / O emulsion was measured in Test 1, and evaluated the stability of the antibody in the microspheres during manufacturing. Bevacizumab (Test 2.1) and cetuximab (Test 2.2) were used as antibodies to be encapsulated in the microspheres. Details are described below.

[0104] 2.1. Bevacizumab-encapsulated microspheres 2.1.1 Preparation of microspheres 0.025 g of Span 80 and 0.25 g of any of the polymers shown in Table 1 were added to 10 mL of dichloromethane and dissolved (polymer solution). 0.1 mL of a 2.5% bevacizumab solution (Bevacizumab BS Intravenous Drip Infusion 100 mg "Pfizer" (Pfizer Inc.) was used as is) was added to the polymer solution, and the mixture was emulsified using a homogenizer at 20,500 rpm for 1 minute. After emulsification, the mixture was spray-dried using a spray dryer (two-fluid nozzle φ1.4, N ２ Flow rate: 30 mm, temperature: 10-15°C) to obtain white particulate microspheres containing bevacizumab.

[0105] Furthermore, compared with microspheres using 1PEG (2 kDa)-PLGA (11.5 kDa) polymer, microspheres using 4PEG (10 kDa)-PLGA (40 kDa) polymer showed less adhesion to each other and to the container material, and were more fluid and easier to handle. This suggests that microspheres using 4PEG (10 kDa)-PLGA (40 kDa) polymer are more useful from the perspective of reducing yield loss due to container transfer during the manufacturing process and improving the accuracy of filling product containers.

[0106] 2.1.2 Observation by scanning electron microscope Each bevacizumab-encapsulated microsphere obtained in 2.1.1 was observed using a scanning electron microscope (JSM-IT300, JEOL Ltd.). The samples used for observation were previously coated with palladium-gold. The observed images are shown in Figures 1 to 8.

[0107] 2.1.3 Measurement of total bevacizumab amount by MicroBCA Assay 5 mg of bevacizumab-encapsulated microspheres obtained in 2.1.1 was dissolved in 1 mL of dimethyl sulfoxide / PBS (7:3 mixture). PBS was added to 100 μL of this solution to make a total volume of 10 mL, which was used as the sample solution. In addition, bevacizumab BS intravenous infusion 100 mg "Pfizer" was diluted with PBS to prepare a calibration curve solution of 0.1 to 20 μg / mL.

[0108] The total amount of bevacizumab was measured using a MicroBCA Assay. 100 μL of sample solution or calibration curve solution was added to each well of a 96-well plate, followed by 100 μL of Working Reagent prepared according to the protocol included with the MicroBCA Assay Kit (Thermo Fisher Scientific). The plate was agitated for 30 seconds and then incubated at 37°C for 2 hours. After visually confirming color development, the absorbance at a wavelength of 562 nm was measured using a microplate reader. The total amount of bevacizumab calculated from the absorbance is shown in Table 4.

[0109] 2.1.4 Measurement of active bevacizumab amount by ELISA 5 mg of the bevacizumab-encapsulated microspheres obtained in 2.1.1 was dissolved in 1 mL of dimethyl sulfoxide / PBS (7:3 mixture). This solution was diluted 1000 times with PBS to prepare a sample solution. In addition, bevacizumab BS intravenous infusion 100 mg "Pfizer" was diluted with PBS to prepare a calibration curve solution of 2 to 200 ng / mL.

[0110] The amount of active bevacizumab was measured using ELISA. First, 100 ng / mL human VEGF-A was plated on a 96-well immunoplate. １６５ 100 μL of the solution was added, and the plate was shaken on a shaker for 1 minute, followed by standing at 37°C for 1 hour. After standing, the liquid in the wells was discarded, and a washing solution was added to each well to wash the wells. After washing, 200 μL of 1% BSA solution was added to each well, and the plate was shaken at room temperature for 1 hour. After shaking, the liquid in the wells was discarded, and a washing solution was added to each well to wash the wells.

[0111] After washing, 100 μL of sample solution or calibration curve solution was added to each well and shaken at room temperature for 2 hours. After shaking, the liquid in the wells was discarded, and washing solution was added to each well to wash the wells. After washing, 100 μL of secondary antibody solution was added and shaken at room temperature for 1 hour. After shaking, the secondary antibody solution was discarded, washing solution was added to each well, and the wells were washed. After washing, 100 μL of TMB ELISA Substrate (High Sensitivity) (Abcam.plc) was added and shaken at room temperature for 15 minutes. After visually confirming color development, 100 μL of 1N HCl test solution (Fujifilm Wako Pure Chemical Corporation) was added, and the absorbance at a wavelength of 450 nm was measured using a microplate reader. The amount of active bevacizumab calculated from the absorbance is shown in Table 4. Table 4 also shows the activity maintenance rate (%) calculated by the formula [active bevacizumab amount / total bevacizumab amount]×100.

[0112]

[0113] 2.2. Cetuximab-Encapsulated Microspheres 2.2.1 Preparation of Microspheres 0.05 g of Span 80 and 0.5 g of PLGA (45 kDa) or 4PEG (10 kDa)-PLGA (40 kDa) listed in Table 1 were dissolved in 20 mL of dichloromethane (polymer solution). 1 mL of 0.5% cetuximab solution (Erbitux (registered trademark) injection 100 mg, Merck) was added to the polymer solution, and the mixture was emulsified using a homogenizer at 9,500 rpm for 1 minute. After emulsification, the mixture was spray-dried (two-fluid nozzle φ1.4, N) using a spray dryer (Mini Spray Dryer B-290, Nippon Buchi Co., Ltd.). ２ Flow rate: 30 mm, temperature: 10-15°C) to obtain white particulate microspheres containing cetuximab.

[0114] 2.2.2 Observation by scanning electron microscope Each cetuximab-encapsulated microsphere obtained in 2.2.1 was observed using a scanning electron microscope (JSM-IT300, JEOL Ltd.). The samples used for observation were previously coated with palladium-gold. The observed images are shown in Figures 8 and 9.

[0115] 2.2.3 Measurement of total cetuximab amount by MicroBCA Assay 10 mg of the cetuximab-encapsulated microspheres obtained in 2.2.1 was dissolved in 1 mL of dimethyl sulfoxide. PBS was added to 100 μL of this solution to make a total volume of 10 mL, which was used as the sample solution. Additionally, 100 mg of Erbitux® injection was diluted with PBS to prepare a calibration curve solution of 0.1 to 10 μg / mL.

[0116] The total cetuximab amount was measured using the MicroBCA Assay. 100 μL of sample solution or calibration curve solution was added to each well of a 96-well plate, followed by 100 μL of Working Reagent prepared according to the protocol included with the MicroBCA Assay Kit (Thermo Fisher Scientific). The plate was agitated for 30 seconds and then incubated at 60°C for 1 hour. After visually confirming color development, the absorbance at a wavelength of 562 nm was measured using a microplate reader (Synergy HTX Multimode Plate Reader, BioTek Instruments, Inc.). The total cetuximab amount calculated from the absorbance is shown in Table 5.

[0117] 2.2.4 Measurement of active cetuximab by ELISA 5 mg of the cetuximab-encapsulated microspheres obtained in 2.2.1 was dissolved in 1 mL of dimethyl sulfoxide. PBS was added to 100 μL of this solution to make a total volume of 10 mL, which was used as the sample solution. Also, 100 mg of Erbitux® injection was diluted with PBS to prepare a calibration curve solution of 2 to 200 ng / mL.

[0118] ELISA was used to measure the amount of active cetuximab. First, 100 μL of 100 ng / mL human EGFR solution was added to a 96-well immunoplate (MaxiSorp Nunc-Immuno Plate, Thermo Fisher Scientific), stirred on a shaker for 1 minute, and then allowed to stand at 37°C for 1 hour. After standing, the liquid in the wells was discarded, and a washing solution (PBS containing 0.05% Tween (registered trademark) 20) was added to each well to wash the wells. After washing, 200 μL of 1% BSA solution was added to each well, and the plate was shaken at room temperature for 1 hour. After shaking, the liquid in the wells was discarded, and a washing solution was added to each well to wash the wells.

[0119] After washing, 100 μL of sample solution or calibration curve solution was added to each well and shaken at room temperature for 2 hours. After shaking, the liquid in the wells was discarded, washing solution was added to each well, and the wells were washed. After washing, 100 μL of secondary antibody solution (Goat Anti-Human IgG (H+L) solution) was added and the wells were shaken at room temperature for 1 hour. After shaking, the secondary antibody solution was discarded, washing solution was added to each well, and the wells were washed. After washing, 100 μL of ABTS Microwell Peroxidase Substrate (SeraCare Life Sciences, Inc.) was added and the wells were shaken at room temperature for 15 minutes. After visually confirming the color development, 100 μL of ABTS Peroxidase Stop Solution (SeraCare Life Sciences, Inc.) was added, and the absorbance at a wavelength of 405 nm was measured using a microplate reader. The amount of active cetuximab calculated from the absorbance is shown in Table 5. Table 5 also shows the activity retention rate (%) calculated by the formula [active cetuximab amount / total cetuximab amount] × 100.

[0120]

[0121] 2.3 Summary As shown in Tables 4 and 5, when preparing microspheres, the total volume of the water phase in the W / O emulsion was 0.025 cm ３ / g to 0.25 cm ３When the encapsulated water-soluble substance was used, a high activity retention rate of 80% or more was obtained in both cases where bevacizumab and cetuximab were used as the water-soluble substance to be encapsulated.

[0122] This study suggested that when preparing microspheres containing water-soluble substances, high stability of the water-soluble substances may be achieved by using a polymer in which the total volume of the aqueous phase in the W / O emulsion is within a certain range.

[0123] 3. Test 3 Preparation of antibody-encapsulated microspheres and evaluation of stability during production 2 The present inventors investigated whether similarly excellent stability of water-soluble substances could be obtained when different water-soluble substances were encapsulated in the polymers that were found to have excellent stability of water-soluble substances in Test 2.

[0124] In Test 2, bevacizumab or cetuximab was used as the water-soluble substance to be encapsulated in the microspheres. On the other hand, in this test, ranibizumab, faricimab, or brolucizumab was used as the water-soluble substance to be encapsulated in the microspheres. Also, 4PEG (10 kDa)-PLGA (40 kDa) was used as the polymer. As described in Test 1, the total volume of the aqueous phase in the W / O emulsion of this polymer was 0.033 cm. ３ The details are described below.

[0125] 3.1 Evaluation with Ranibizumab 3.1.1 Microsphere Preparation 0.025 g of Span80 and 0.25 g of 4PEG (10 kDa)-PLGA (40 kDa) were dissolved in 10 mL of dichloromethane (polymer solution). 0.1 mL of a 0.5% ranibizumab solution (a two-fold diluted solution of Lucentis (registered trademark) intravitreal injection solution 10 mg / mL (Novartis Pharma K.K.)) was added to the polymer solution, and the mixture was emulsified using a homogenizer at 20,500 rpm for 1 minute. After emulsification, the mixture was spray-dried using a spray dryer (two-fluid nozzle φ1.4, N ２ Flow rate: 30 mm, temperature: 10-15°C) to obtain white particulate microspheres containing ranibizumab.

[0126] 3.1.2 Observation by scanning electron microscope The ranibizumab-encapsulated microspheres obtained in 3.1.1 were observed using a scanning electron microscope (JSM-IT300, JEOL Ltd.). The samples used for observation were previously coated with palladium-gold. The observed image is shown in Figure 10.

[0127] 3.1.3 Measurement of total ranibizumab amount by MicroBCA Assay 5 mg of ranibizumab-encapsulated microspheres obtained in 3.1.1 was dissolved in 1 mL of dimethyl sulfoxide / PBS (7:3 mixture). PBS was added to 100 μL of this solution to make a total volume of 10 mL, which was used as the sample solution. Furthermore, 10 mg / mL of Lucentis® intravitreal injection solution was diluted with PBS to prepare a calibration curve solution of 0.1 to 20 μg / mL.

[0128] MicroBCA Assay was used to measure the total amount of ranibizumab. 100 μL of sample solution or calibration curve solution was added to each well of a 96-well plate, followed by 100 μL of Working Reagent prepared according to the protocol attached to the MicroBCA Assay Kit (Thermo Fisher Scientific). The plate was stirred for 30 seconds and then incubated at 37 ° C for 2 hours. After visually confirming the color development, the absorbance at a wavelength of 562 nm was measured using a microplate reader. The total amount of ranibizumab calculated from the absorbance is shown in Table 6.

[0129] 3.1.4 Measurement of active ranibizumab by ELISA 5 mg of ranibizumab-encapsulated microspheres obtained in 3.1.1 was dissolved in 1 mL of dimethyl sulfoxide / PBS (7:3 mixture). This solution was diluted 1000-fold with PBS to prepare a sample solution. Additionally, 10 mg / mL of Lucentis® intravitreal injection solution was diluted with PBS to prepare a calibration curve solution of 2 to 200 ng / mL.

[0130] The amount of active ranibizumab was measured using ELISA. First, 100 ng / mL human VEGF-A was plated on a 96-well immunoplate. １６５100 μL of the solution was added, and the plate was shaken on a shaker for 1 minute, followed by standing at 37°C for 1 hour. After standing, the liquid in the wells was discarded, and a washing solution was added to each well to wash the wells. After washing, 200 μL of 1% BSA solution was added to each well, and the plate was shaken at room temperature for 1 hour. After shaking, the liquid in the wells was discarded, and a washing solution was added to each well to wash the wells.

[0131] After washing, 100 μL of sample solution or calibration curve solution was added to each well and shaken at room temperature for 2 hours. After shaking, the liquid in the well was discarded, and washing solution was added to each well to wash the well. After washing, 100 μL of secondary antibody solution was added and shaken at room temperature for 1 hour. After shaking, the secondary antibody solution was discarded, washing solution was added to each well, and the well was washed. After washing, 100 μL of TMB ELISA Substrate (High Sensitivity) was added and shaken at room temperature for 15 minutes. After visually confirming color development, 100 μL of 1N HCl test solution was added, and the absorbance at a wavelength of 450 nm was measured using a microplate reader. The amount of active ranibizumab calculated from the absorbance is shown in Table 6. Table 6 also shows the activity retention rate (%) calculated using the formula [amount of active ranibizumab / total amount of ranibizumab] × 100.

[0132] 3.2 Evaluation with Faricimab 3.2.1 Microsphere Preparation 0.025 g of Span80 and 0.25 g of 4PEG (10 kDa)-PLGA (40 kDa) were dissolved in 10 mL of dichloromethane (polymer solution). 0.1 mL of a 2.4% faricimab solution (a 5-fold diluted solution of BabySmo® intravitreal injection solution 120 mg / mL (Chugai Pharmaceutical Co., Ltd.)) was added to the polymer solution, and the mixture was emulsified using a homogenizer at 20,500 rpm for 1 minute. After emulsification, the mixture was spray-dried using a spray dryer (drying conditions were the same as in 3.1.1), yielding white, particulate faricimab-encapsulated microspheres.

[0133] 3.2.2 Observation by scanning electron microscope The faricimab-encapsulated microspheres obtained in 3.2.1 were observed using the same procedure as in 3.1.2. The observed images are shown in Figure 11.

[0134] 3.2.3 Measurement of Total Faricimab Amount by MicroBCA Assay Five mg of faricimab-encapsulated microspheres obtained in 3.2.1 was dissolved in 1 mL of dimethyl sulfoxide / PBS (7:3 mixture). PBS was added to 100 μL of this solution to make a total volume of 10 mL, which was used as the sample solution. Additionally, 120 mg / mL of BabySmo® Intravitreal Injection Solution was diluted with PBS to prepare a 0.1-20 μg / mL calibration curve solution. Measurements were performed using the MicroBCA Assay. 100 μL of the sample solution or calibration curve solution was added to each well of a 96-well plate, followed by 100 μL of Working Reagent prepared according to the protocol provided with the MicroBCA Assay Kit (Thermo Fisher Scientific). The plate was agitated for 30 seconds and then incubated at 37°C for 2 hours. After visually confirming color development, the absorbance at a wavelength of 562 nm was measured using a microplate reader. The total amount of faricimab calculated from the absorbance is shown in Table 6.

[0135] 3.2.4 Measurement of active faricimab by ELISA 5 mg of the faricimab-encapsulated microspheres obtained in 3.2.1 was dissolved in 1 mL of dimethyl sulfoxide / PBS (7:3 mixture). This solution was diluted 1000-fold with PBS to prepare a sample solution. Additionally, 120 mg / mL of BabySmo® intravitreal injection solution was diluted with PBS to prepare a calibration curve solution ranging from 2 to 200 ng / mL.

[0136] ELISA was used to measure the amount of active faricimab. First, 100 ng / mL human VEGF-A was plated on a 96-well immunoplate. １６５ 100 μL of the solution was added, and the plate was shaken on a shaker for 1 minute, followed by standing at 37°C for 1 hour. After standing, the liquid in the wells was discarded, and a washing solution was added to each well to wash the wells. After washing, 200 μL of 1% BSA solution was added to each well, and the plate was shaken at room temperature for 1 hour. After shaking, the liquid in the wells was discarded, and a washing solution was added to each well to wash the wells.

[0137] After washing, 100 μL of sample solution or calibration curve solution was added to each well and shaken at room temperature for 2 hours. After shaking, the liquid in the wells was discarded, and washing solution was added to each well to wash the wells. After washing, 100 μL of secondary antibody solution was added and shaken at room temperature for 1 hour. After shaking, the secondary antibody solution was discarded, washing solution was added to each well, and the wells were washed. After washing, 100 μL of TMB ELISA Substrate (High Sensitivity) was added and shaken at room temperature for 15 minutes. After visually confirming color development, 100 μL of 1N HCl test solution was added, and the absorbance at a wavelength of 450 nm was measured using a microplate reader. The amount of active faricimab calculated from the absorbance is shown in Table 6. Table 6 also shows the activity retention rate (%) calculated using the formula [amount of active faricimab / total amount of faricimab] × 100.

[0138] 3.3 Evaluation with Brolucizumab 3.3.1 Microsphere Preparation 0.025 g of Span80 and 0.25 g of 4PEG (10 kDa)-PLGA (40 kDa) were dissolved in 10 mL of dichloromethane (polymer solution). 0.1 mL of a 2.4% brolucizumab solution (a 5-fold diluted solution of Beovu® intravitreal injection kit 120 mg / mL (Novartis Pharma K.K.)) was added to the polymer solution, and the mixture was emulsified using a homogenizer at 20,500 rpm for 1 minute. After emulsification, the mixture was spray-dried using a spray dryer (drying conditions were the same as in 3.1.1), yielding white particulate microspheres encapsulating brolucizumab.

[0139] 3.3.2 Observation by scanning electron microscope The brolucizumab-encapsulated microspheres obtained in 3.3.1 were observed using the same procedure as in 3.1.2. The observed images are shown in Figure 12.

[0140] 3.3.3 Measurement of total brolucizumab amount by MicroBCA Assay 5 mg of brolucizumab-encapsulated microspheres obtained in 3.3.1 was dissolved in 1 mL of dimethyl sulfoxide / PBS (7:3 mixture). PBS was added to 100 μL of this solution to make a total volume of 10 mL, which was used as the sample solution. In addition, 120 mg / mL of Beovu® intravitreal injection kit was diluted with PBS to prepare a calibration curve solution of 0.1 to 20 μg / mL.

[0141] The total amount of brolucizumab was measured using a MicroBCA Assay. 100 μL of sample solution or calibration curve solution was added to each well of a 96-well plate, followed by 100 μL of Working Reagent prepared according to the protocol included with the MicroBCA Assay Kit (Thermo Fisher Scientific). The plate was agitated for 30 seconds and then incubated at 37°C for 2 hours. After visually confirming color development, the absorbance at a wavelength of 562 nm was measured using a microplate reader. The total amount of brolucizumab calculated from the absorbance is shown in Table 6.

[0142] 3.3.4 Measurement of active brolucizumab by ELISA 5 mg of brolucizumab-encapsulated microspheres obtained in 3.3.1 was dissolved in 1 mL of dimethyl sulfoxide / PBS (7:3 mixture). This solution was diluted 1000-fold with PBS to prepare a sample solution. In addition, 120 mg / mL of Beovu® intravitreal injection kit was diluted with PBS to prepare a calibration curve solution of 2 to 200 ng / mL.

[0143] The amount of active brolucizumab was measured using ELISA. First, 100 ng / mL human VEGF-A was plated on a 96-well immunoplate. １６５ 100 μL of the solution was added, and the plate was shaken on a shaker for 1 minute, followed by standing at 37°C for 1 hour. After standing, the liquid in the wells was discarded, and a washing solution was added to each well to wash the wells. After washing, 200 μL of 1% BSA solution was added to each well, and the plate was shaken at room temperature for 1 hour. After shaking, the liquid in the wells was discarded, and a washing solution was added to each well to wash the wells.

[0144] After washing, 100 μL of sample solution or calibration curve solution was added to each well and shaken at room temperature for 2 hours. After shaking, the liquid in the wells was discarded, and washing solution was added to each well to wash the wells. After washing, 100 μL of secondary antibody solution was added and the plate was shaken at room temperature for 1 hour. After shaking, the secondary antibody solution was discarded, washing solution was added to each well, and the wells were washed. After washing, 100 μL of TMB ELISA Substrate (High Sensitivity) was added and the plate was shaken at room temperature for 15 minutes. After visually confirming color development, 100 μL of 1N HCl test solution was added, and the absorbance at a wavelength of 450 nm was measured using a microplate reader. The amount of active brolucizumab calculated from the absorbance is shown in Table 6. Table 6 also shows the activity retention rate (%) calculated using the formula [active brolucizumab amount / total brolucizumab amount] × 100.

[0145]

[0146] 3.4 Summary As shown in Table 6, when ranibizumab, faricimab, or brolucizumab was used as the water-soluble substance to be encapsulated in the microspheres, a high activity retention rate of the water-soluble substance was obtained, similar to when bevacizumab or cetuximab was used.

[0147] This study suggested that when preparing microspheres containing water-soluble substances, if a polymer is used in which the total volume of the aqueous phase in the W / O emulsion is within a certain range, high stability of the water-soluble substance can be obtained regardless of the type of water-soluble substance to be encapsulated.

[0148] 4. Test 4: Preparation of Peptide-Encapsulating Film and Evaluation of Stability During Manufacturing Next, the inventors investigated the encapsulation of a water-soluble substance in a film. The polymers used were 4PEG (10 kDa)-PLGA (40 kDa), 4PEG (10 kDa)-PLGA (69 kDa), or 4PEG (10 kDa)-PLGA (130 kDa), the total volume of which was measured for the aqueous phase of the W / O emulsion in Test 1. Salmon calcitonin (calcitonin derived from salmon, hereinafter sometimes simply referred to as "calcitonin"), a type of peptide, was used. Calcitonin is a peptide consisting of 32 amino acid residues (SEQ ID NO: 2). The two-dimensional and three-dimensional structures of calcitonin are shown in Figure 13.

[0149] After preparing the calcitonin-containing film, the inventors extracted the calcitonin from the film and measured the residual rate of calcitonin by HPLC, as described in detail below.

[0150] 4.1 Preparation of Peptide-Encapsulating Films 0.05 g of Span 80 was dissolved in 10 mL of dichloromethane. 0.05 g of 4PEG(10 kDa)-PLGA(40 kDa), 4PEG(10 kDa)-PLGA(69 kDa), or 4PEG(10 kDa)-PLGA(130 kDa) was added to 0.5 mL of this solution and dissolved (polymer solution). 100 μL of PBS(-) was added to 5 mg of salmon calcitonin and dissolved (aqueous phase solution). After adding the aqueous phase solution to the polymer solution, the container was stirred and ultrasonicated for 1 minute using an ultrasonic generator (UR-20P, Tomy Seiko Co., Ltd.) to emulsify the mixture. 0.1 mL of the emulsified mixture was dropped onto a glass plate using a micropipette and dried to obtain a film.

[0151] 4.2 Extraction of peptides 100 μL of dichloromethane was added to one piece of film obtained in 4.1, and 250 μL of aqueous phase (1N HCl:PBS(-) = 1:3) was added, followed by stirring with a vortex mixer, centrifugation, and the supernatant was collected. 250 μL of the aqueous phase was added to the tube from which the supernatant was collected, followed by stirring in the same manner, centrifugation, and the supernatant was collected. The above procedure was repeated four times, and the supernatant was collected (i.e., a total of 1000 μL of aqueous phase was added).

[0152] 4.3 Measurement of residual peptide rate The residual rate of calcitonin in the aqueous phase recovered in 4.2 was measured using HPLC. The measurement conditions are shown in Table 7. The measured residual rate of calcitonin is shown in Table 8.

[0153]

[0154]

[0155] 4.4 Summary As shown in Table 8, the total volume of the water phase in the W / O emulsion as the polymer during film preparation was 0.033 cm ３When 4PEG (10 kDa)-PLGA (40 kDa) was used at 1000 kDa / g, a high calcitonin retention rate of 92.5% was obtained.

[0156] This study suggested that high stability of water-soluble substances can be achieved even when water-soluble substances are encapsulated in the film. It also suggested that high stability of water-soluble substances can be achieved even when peptides with molecular weights smaller than antibodies are encapsulated as water-soluble substances.

[0157] 5. Test 5: Evaluation of Sustained Release of Antibody-Encapsulated Microspheres 5.1 Microspheres Using 4-Arm PEG-PLGA 0.5 g of 4PEG (10 kDa)-PLGA (40 kDa) (the total volume of the internal aqueous phase of the W / O emulsion measured in Test 1) and 0.05 g of a nonionic surfactant ("Span 80") were added to 20 mL of dichloromethane and dissolved. 1 mL of a 0.5% PBS solution of human serum-derived IgG was added to the resulting polymer solution. The resulting mixture was emulsified by stirring at 9,500 rpm for 1 minute using a homogenizer. The emulsified mixture was spray-dried using a spray dryer ("Mini Spray Dryer B-290" manufactured by Nippon Buchi Co., Ltd.), yielding white particles with a yield of 74.1%.

[0158] 5.2 In vitro water-soluble substance release test The IgG-encapsulated microspheres obtained in 5.1 were added to 3 mL of a 1% gellan gum solution and dispersed. The entire amount of the obtained dispersion was injected into a release test solution of 0.02% NaN using a 27G syringe. ３ The resulting mixture was placed in an incubator set at 37°C to release the IgG from the microspheres.

[0159] 1 mL of test solution was collected at each sampling time point. Test solutions containing IgG-encapsulated microspheres were collected 1, 2, 3, 4, 6, 8, and 12 weeks after the start of the test.

[0160] 5.3 Measurement of IgG Release The amount of IgG released in the test solution was measured by the MicroBCA method. Specifically, human serum-derived IgG was diluted with PBS to prepare a calibration curve solution of 0.1 to 20 μg / mL.

[0161] A MicroBCA Assay was used to measure the amount of antibody contained in the sample solution. Specifically, 100 μL of each sample solution and calibration curve solution were added to a 96-well plate, followed by 100 μL of Working Reagent prepared from the MicroBCA Assay Kit (Thermo Fisher Scientific). The plate was stirred for 30 seconds and then incubated at 60°C for 1 hour. After confirming color development, the absorbance at a wavelength of 562 nm was measured using a microplate reader ("Synergy HTX Multimode Plate Reader" manufactured by BioTek Instruments). The absorbance measurements of the sample solution were substituted into the calibration curve to calculate the concentration of IgG contained in the sample solution, and the cumulative IgG release rate at each time point was determined. The cumulative IgG release rate is shown in Figure 14.

[0162] As shown in FIG. 14, the microspheres according to the present disclosure were shown to be capable of sustained release of IgG for approximately three months.

[0163] 6. Test 6 Nucleic Acid-Encapsulating Film 6.1 Preparation of Nucleic Acid-Encapsulating Film Next, the present inventors attempted to encapsulate nucleic acids in a film. Yeast RNA was used as the nucleic acid. As the polymer, the W / O emulsion in Test 1 was used, in which the total volume of the aqueous phase was 0.033 cm ３ 4PEG (10 kDa)-PLGA (40 kDa) was used.

[0164] Specifically, 0.05 g of Span 80 was first dissolved in 10 mL of dichloromethane, and 0.05 g of 4PEG (10 kDa)-PLGA (40 kDa) was added to 0.5 mL of the solution and dissolved (polymer solution).

[0165] A yeast RNA solution with a concentration of 80 mg / mL was prepared as follows: 20 mg of yeast RNA (Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed out, 200 μL of PBS(-) was added, and the mixture was stirred. Approximately 30 μL of 1N NaOH aqueous solution was added, and after stirring, the yeast RNA was confirmed to be dissolved. PBS(-) was then added to bring the total volume to 250 μL. The pH of the prepared yeast RNA solution was approximately 5.

[0166] 25 μL of the yeast RNA solution was added to the polymer solution using a micropipette. The mixture was emulsified by irradiating the container with ultrasound for 1 minute while stirring using an ultrasonic generator (US-50, Nippon Seiki Seisakusho). 0.1 mL of the emulsified mixture was dropped onto a glass plate using a micropipette and dried to obtain a film.

[0167] 6.2 In vitro water-soluble substance release test One nucleic acid-encapsulating film obtained in 6.1 was placed in a standard bottle, and 1 mL of PBS (release test solution) was added. The standard bottle was placed in an incubator set at 37°C to release the yeast RNA from the film.

[0168] The solution was stirred at each sampling time point, and 1 mL of test solution was collected. After the test solution was collected, 1 mL of PBS was added, and the film was placed in an incubator set at 37°C to release the yeast RNA remaining on the film.

[0169] 6.3 Measurement of the amount of nucleic acid released The amount of nucleic acid contained in the test solution was measured using HPLC. The measurement conditions are shown in Table 9. The cumulative release rate of nucleic acid at each time point was determined. The cumulative release rate of nucleic acid from the film is shown in Figure 15.

[0170]

[0171] 7. Test 7 Preparation of W / O / W Microspheres Encapsulating a Model Dye and Evaluation of Sustained Release 7.1 W / O / W Microspheres Encapsulating a Model Dye In Tests 1 to 6, the inventors prepared a W / O emulsion followed by a drying process to prepare each composition. In this test, the inventors attempted to prepare W / O / W microspheres encapsulating a model dye by preparing a W / O / W emulsion followed by a drying process.

[0172] Generally, W / O / W emulsions are prepared by first preparing a W / O emulsion and then dispersing the W / O emulsion in an aqueous phase. In this test, the inventors used a polymer in preparing the W / O emulsion. In Test 1, the total volume of the aqueous phase in the W / O emulsion was 0.033 cm. ３4PEG (10 kDa)-PLGA (40 kDa) with a molecular weight of 1000 kDa / g was used. Polyvinyl alcohol (hereinafter sometimes referred to as PVA) was dissolved in the aqueous phase in which the W / O emulsion was dispersed. Fluorescein isothiocyanate (FITC)-dextran (molecular weight approximately 4000), which has a molecular weight similar to that of a common peptide, was used as a model dye.

[0173] Specifically, 50 mg of 4PEG (10 kDa)-PLGA (40 kDa) was dissolved in 0.5 mL of dichloromethane (polymer solution). A 100 mg / mL FITC solution was prepared as follows: 50 mg of L-arginine (Ajinomoto Co.) was dissolved in 1 mL of PBS to prepare an L-arginine solution; 5 mg of FITC-dextran (FD4, Sigma-Aldrich) was dissolved in 50 μL of the L-arginine solution. 50 μL of the FITC solution was added to the polymer solution, and the mixture was emulsified by stirring for 30 seconds while irradiating with ultrasound (preparation of a W / O emulsion).

[0174] A NaCl-containing PVA solution was prepared by stirring 1 g of PVA, 1.5 g of NaCl, and 97.5 g of water. While stirring 5 mL of the NaCl-containing PVA solution with a stirrer, the entire amount of the prepared W / O emulsion was added. After addition, the emulsion was emulsified through an SPG membrane by three strokes using a 5 mL syringe with an emulsifying connector (SPG, surface-untreated, 50 μm). While stirring the emulsion with a stirrer, 10 mL of distilled water was added, and the emulsion was then submerged and dried for 3 hours (preparation of a W / O / W emulsion).

[0175] The prepared W / O / W emulsion was transferred to a centrifuge tube and centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. Approximately 5 mL of distilled water was added to the precipitate remaining in the centrifuge tube, and the mixture was stirred. Then, the mixture was centrifuged in the same manner, and the supernatant was collected. This procedure was repeated three times, and the centrifugation was performed. The weight of the model dye contained in the supernatant was 4.0% of the weight of the added model dye, and the encapsulation rate was 94%.

[0176] The precipitate (particles) obtained after the final centrifugation was collected and resuspended in 0.5 mL of 0.1 wt % mannitol aqueous solution, and the suspension was freeze-dried to obtain model dye-encapsulated W / O / W microspheres in a yield of 81.0%.

[0177] 5.8 mg of the model dye-encapsulated W / O / W microspheres obtained in 7.1 was weighed, and 1 mL of PBS (release test solution) was added and stirred. The resulting suspension was placed in a thermostatic chamber set at 37°C to release the model dye from the microspheres.

[0178] At each sampling time, the solution was stirred and centrifuged to collect 1 mL of test solution. After collecting the test solution, 1 mL of PBS was added and stirred, and the solution was placed in a thermostatic chamber set at 37°C to release the model dye remaining in the microspheres.

[0179] 7.3 Measurement of the amount of released model dye The amount of released model dye in the test solution was measured using a fluorescence detector by a known method. The cumulative release rate of the model dye at each time point was calculated. The cumulative release rate of the model dye is shown in Figure 16.

[0180] 8. Test 8 Preparation of nucleic acid-encapsulated W / O / W type microspheres, evaluation of sustained release and stabilization 8.1 Preparation of nucleic acid-encapsulated W / O / W type microspheres

[0181] The preparation of the W / O / W emulsion was carried out in the same manner as in Test 7. In this test, the inventors used the same polymer as in Test 1 when preparing the W / O emulsion, in which the total volume of the water phase in the W / O emulsion was 0.033 cm ３ 4PEG (10 kDa)-PLGA (40 kDa) with a molecular weight of 1000 mg / g was used. siRNA compound 1 having the following structure was used as the nucleic acid.

[0182] <siRNA compound 1> siRNA consisting of a nucleotide sequence represented by 5'-AGCAGAGUACACACAGCAUAUACC-P-GGUAUAUGCUGUGUGUACUCUGCUUC-P-G-3' (SEQ ID NO: 1) (in this sequence, P is represented by the following formula: (The linker shown is a proline derivative linker represented by the formula:

[0183] Specifically, 500 mg of 4PEG (10 kDa)-PLGA (40 kDa) was dissolved in 5 mL of dichloromethane (polymer solution). A 100 mg / mL siRNA Compound 1 solution was prepared by the following method: 1 g of L-arginine (Ajinomoto Co., Inc.) was dissolved in 20 mL of PBS to prepare an L-arginine solution; 100 mg of siRNA Compound 1 was dissolved in 1 mL of the L-arginine solution. 500 μL of the siRNA Compound 1 solution was added to the polymer solution, and the mixture was emulsified by stirring for 30 seconds while irradiating with ultrasound (preparation of a W / O emulsion).

[0184] A NaCl-containing PVA solution was prepared by stirring 1 g of PVA, 1.5 g of NaCl, and 97.5 g of water. While stirring 50 mL of the NaCl-containing PVA solution with a stirrer, the entire amount of the prepared W / O emulsion was added. After addition, the emulsion was reciprocated three times with a syringe using an emulsifying membrane (SPG, surface untreated, pore size 50 μm). While stirring the emulsion with a stirrer, 100 mL of distilled water was added, and the emulsion was then submerged and dried for 3 hours (preparation of a W / O / W emulsion with an average particle size of 8 μm).

[0185] A NaCl-containing PVA solution was prepared by stirring 1 g of PVA, 1.5 g of NaCl, and 97.5 g of water. While stirring 50 mL of the NaCl-containing PVA solution with a stirrer, the entire amount of the prepared W / O emulsion was added. After addition, ultrasonic irradiation was performed for 30 seconds, and the emulsion was reciprocated three times with a syringe using an emulsifying membrane (SPG, surface untreated, pore size 5 μm). While stirring the emulsion with a stirrer, 100 mL of distilled water was added, and the emulsion was then submerged and dried for 3 hours (preparation of a W / O / W emulsion with an average particle size of 3 μm).

[0186] For comparison, when the polymer was changed from 4PEG (10 kDa)-PLGA (40 kDa), the total volume of the aqueous phase in the W / O emulsion in Test 1 was 0.020 cm ３ The emulsion was prepared in the same manner as in the preparation of the W / O / W emulsion with an average particle size of 3 μm, except that the original solubility was changed to PLGA (45 kDa).

[0187] The prepared W / O / W emulsion was centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. Approximately 50 mL of distilled water was added to the remaining precipitate, and after stirring, the mixture was centrifuged in the same manner, and the supernatant was collected. This procedure was repeated for a total of three centrifugations. The obtained W / O / W emulsion was resuspended in 5 mL of 0.1% mannitol aqueous solution and lyophilized to prepare microspheres. SEM images of each prepared microsphere are shown in Figure 17.

[0188] 8.2 Evaluation of drug encapsulation rate and activity retention rate of siRNA compound 1-encapsulated microspheres 5.8 mg of each microsphere prepared in Test 8.1 was weighed out and dissolved in 1 mL of DMSO in an Eppendorf tube, then diluted 100-fold and subjected to high-performance liquid chromatography (HPLC) under the following conditions. The drug encapsulation rate was calculated using the formula [amount of drug in microspheres / amount of drug charged] x 100. The activity retention rate was calculated using the formula [peak area of ​​unchanged substance / total area of ​​peaks of related substances] x 100.

[0189] <HPLC measurement conditions> Column: ACQUITY UPLC Oligonucleotide BEH C18 (size: 4.6 mm I.D. x 5 cm) Mobile phase A: 50 mmol / L TEAA, 2 mmol / L EDTA (pH 7.5) Mobile phase B: Solution A / acetonitrile (30 / 70) Flow rate: 0.8 mL / min Column temperature: 40°C Injection volume: 50 μL Detection wavelength: 260 nm Analysis time: 15 minutes Gradient conditions:

[0190] The results are shown in the table below.

[0191] In Test 1, the total volume of the water phase in the W / O emulsion was 0.020 cm ３ When microspheres were prepared using PLGA (45 kDa) with a molecular weight of 0.033 cm / g, the activity retention rate decreased to 96.3% and the drug encapsulation rate was 30.9%. On the other hand, in Test 1, when the total volume of the aqueous phase in the W / O emulsion was 0.033 cm ３When microspheres were prepared using 4PEG (10 kDa)-PLGA (40 kDa), which had a molecular weight of 1000 mg / g, the activity retention rate was 100%, and the drug encapsulation rates were 67.6% and 87.7%. Furthermore, the particle size of the microspheres could be controlled by preparing W / O / W emulsions using emulsifying membranes with different pore sizes.

[0192] 8.3 Evaluation of the drug sustained-release ability of siRNA compound 1-encapsulated microspheres 5.8 mg of each microsphere prepared in Test 8.1 was weighed, 1 mL of PBS was added, and the mixture was stirred and allowed to stand at 37°C. After a predetermined time, the sample was removed and centrifuged. The entire supernatant containing the released drug (approximately 1 mL) was sampled, and the release rate and activity retention rate of siRNA compound 1 were calculated. 1 mL of PBS was added to the precipitated microspheres, stirred, and allowed to stand at 37°C. This procedure was repeated. The release rate was calculated using the formula [cumulative amount of released drug / amount of drug in microspheres] x 100. The effective sustained-release rate was calculated using the formula [amount released at the final time point - amount released in the initial burst (1 hour)) / amount of drug in microspheres] x 100. The effective sustained-release rate of the unchanged drug was calculated using the formula [sum of (amount released at each time point x activity retention rate) / amount of drug in microspheres] x 100.

[0193] The results are shown in the table below and in FIG.

[0194] According to this study, in Test 1, the total volume of the water phase in the W / O emulsion was 0.033 cm ３ It was confirmed that when siRNA compound 1 was encapsulated in microspheres using 4PEG (10 kDa)-PLGA (40 kDa) as the polymer, which had a molecular weight of 1 / g, there was little initial rapid release and the drug could be released while maintaining its activity over a long period of time.

[0195] 8.4 Evaluation of Stability of siRNA Compound 1-Encapsulated Microspheres Against RNA Degrading Enzymes 5.8 mg of each microsphere (particle size 3 μm, 8 μm) using 4PEG (10 kDa)-PLGA (40 kDa) as the polymer prepared in Test 8.1 was weighed out, 0.5 mL of buffer was added and stirred, and 0.5 mL of 0.5 μg / mL RNase solution was added thereto and stirred and allowed to stand at 37 ° C. After a predetermined time, the microspheres were removed, stirred, centrifuged, and the precipitated microspheres were washed, and the activity retention rate of the drug encapsulated in the microspheres was calculated. In addition, 0.5 mL of 0.5 μg / mL RNase solution was added to 0.5 mL of 0.5 mg / mL siRNA Compound 1 solution diluted with buffer, stirred, and allowed to stand at 37 ° C. The activity retention rate was calculated after a predetermined time.

[0196] The results are shown in Figure 19. In aqueous solution without being encapsulated in microspheres, siRNA compound 1 was rapidly degraded by RNases. On the other hand, in Test 1, when the total volume of the aqueous phase in the W / O emulsion was 0.033 cm ３ When the drug was encapsulated in microspheres using 4PEG (10 kDa)-PLGA (40 kDa) as the polymer, the activity of the drug within the microspheres was maintained at 80% or more for a long period of 3 months.

[0197] 9. Summary of this Example Tests 1 to 4 and 8 suggest that if microcapsules encapsulating a water-soluble substance are prepared using a polymer whose total volume of the aqueous phase in the W / O emulsion, as measured by the method described herein, falls within a certain range, high stability of the water-soluble substance may be achieved regardless of the type of water-soluble substance to be encapsulated. Furthermore, it was shown that compositions containing such microcapsules can exhibit high stability of the water-soluble substance regardless of the dosage form, such as microspheres or films.

[0198] Furthermore, Tests 5 to 7 suggested that the compositions containing the microcapsules not only stabilize the water-soluble substance but also sustained-release the water-soluble substance. It was shown that the sustained-release property can be achieved regardless of whether the encapsulated water-soluble substance is a protein, nucleic acid, or peptide.

[0199] As described above, this example suggests that the technology of the present disclosure can provide a formulation with excellent stability of water-soluble substances, and that the formulation can also have excellent sustained release properties for water-soluble substances.

Claims

1. A water-soluble substance and a polymer having a chemical structure in which polyalkylene glycol and polyhydroxyalkanoic acid are bonded together, wherein the polymer is such that when a W / O emulsion prepared by the following method is measured by differential scanning calorimetry (temperature-programming method), the total volume of the aqueous phase in the W / O emulsion is 0.025 cm ３ / g to 0.25 cm ３ (Method) 50 mg of the polymer is dissolved in 500 μL of a 0.5 w / v % sorbitan monooleate / dichloromethane solution, 100 μL of water is added, and the mixture is irradiated with ultrasound to prepare a W / O emulsion.

2. The microcapsules according to claim 1, wherein the polyhydroxyalkanoic acid is a homopolymer of a monomer selected from the group consisting of lactic acid, glycolic acid, and 6-hydroxycaproic acid, or a copolymer containing two or more of the monomers.

3. Microcapsules according to claim 1 or 2, wherein the polyalkylene glycol has a chemical structure in which a linear polyalkylene glycol is bonded to a polyhydric alcohol and has 3 to 10 terminal hydroxy groups per molecule.

4. Microcapsules according to claim 1 or 2, wherein the polyalkylene glycol is a 4- or 8-branched polyethylene glycol.

5. Microcapsules according to claim 1 or 2, wherein the ratio of the number average molecular weight of said polyalkylene glycol to the number average molecular weight of said polyhydroxyalkanoic acid is 1:1 to 1:

6.

6. The microcapsules according to claim 1 or 2, wherein the water-soluble substance is at least one selected from the group consisting of proteins, peptides and nucleic acids.

7. A composition comprising the microcapsules of claim 1 or 2.

8. A method for screening sustained-release polymers, comprising the following steps (a) to (c): Step (a): preparing a test polymer solution by dissolving a test polymer in a solution comprising a surfactant and a water-immiscible organic solvent; Step (b): adding water to the test polymer solution prepared in step (a) and then irradiating the solution with ultrasonic waves to prepare a W / O emulsion; Step (c): determining whether the total volume of the water phase in the W / O emulsion is 0.025 cm when the W / O emulsion prepared in step (b) is measured by differential scanning calorimetry (temperature-programming method). ３ / g to 0.25 cm ３ / g as an index, and a step of screening sustained release polymers.

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