Microcapsule encapsulating drug

Abstract

A main purpose of the present disclosure is to provide a formulation containing a polymer and having excellent stability in terms of at least one type of drug selected from the group consisting of cetuximab, bevacizumab, faricimab, ranibizumab, and brolucizumab. The inventors of the present invention found that it is possible to provide a formulation which contains a polymer and has excellent stability in terms of at least one type of drug selected from the group consisting of cetuximab, bevacizumab, faricimab, ranibizumab, and brolucizumab by providing a microcapsule encapsulating said drug, wherein the microcapsule contains a polymer having a chemical structure in which a branched polyalkylene glycol having 3 to 10 terminal hydroxy groups per molecule and a polyhydroxyalkanoic acid are bonded, and in the polymer, the number of units of the polyhydroxyalkanoic acid monomer per single polyhydroxyalkanoic acid chain is 50 to 230.

Description

Microcapsules containing drugs

[0001] The present disclosure relates to microcapsules encapsulating drugs, and compositions containing the microcapsules.

[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, polymers, and cyclodextrin, and optionally further contain a buffer and / or surfactant.

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

[0005] However, even if we limit ourselves to the poly(lactide-co-glycoside) copolymer (hereinafter referred to as lactic acid-glycolic acid copolymer in this disclosure) 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. Furthermore, even if we limit ourselves to peptide drugs or protein drugs that contain amino acids as building blocks among polymer drugs, properties such as stability can vary greatly depending on the amino acid sequence, etc.

[0006] However, the specific structure of a polymer that is compatible with a specific polymer drug, more specifically, at least one drug selected from the group consisting of cetuximab, bevacizumab, faricimab, ranibizumab, and brolucizumab, which are used in the treatment of age-related macular degeneration, has not yet been fully investigated.

[0007] Therefore, the main object of the present inventors was to provide a polymer-containing formulation that provides excellent stability for at least one drug selected from the group consisting of cetuximab, bevacizumab, faricimab, ranibizumab, and brolucizumab.

[0008] As a result of the above-mentioned investigations, the present inventors have discovered that microcapsules encapsulating at least one drug selected from the group consisting of cetuximab, bevacizumab, faricimab, ranibizumab, and brolucizumab, which contain a polymer having a chemical structure in which a branched polyalkylene glycol having 3 to 10 terminal hydroxy groups per molecule is bonded to a polyhydroxyalkanoic acid, and in which the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain in the polymer is 50 to 230, may provide a formulation with excellent stability of the drug. Further improvements were made, leading to the completion of the present disclosure.

[0009] The present disclosure includes, for example, the subject matter described in the following items. Item 1. Microcapsules encapsulating a drug, comprising: a polymer having a chemical structure in which a branched polyalkylene glycol having 3 to 10 terminal hydroxy groups per molecule is bonded to a polyhydroxyalkanoic acid; and the drug, wherein the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain in the polymer is 50 to 230; and the drug is at least one selected from the group consisting of bevacizumab, faricimab, ranibizumab, and brolucizumab. Item 2. Microcapsules according to Item 1, wherein the branched polyalkylene glycol is a branched polyethylene glycol. Item 3. Microcapsules according to Item 1 or 2, wherein the branched polyalkylene glycol is a 4- or 8-branched polyethylene glycol. Item 4. Microcapsules according to any one of Items 1 to 3, wherein the polyhydroxyalkanoic acid is a lactic acid-glycolic acid copolymer. Item 5. Item 6. The microcapsules according to any one of Items 1 to 4, wherein the branched polyalkylene glycol in the polymer has a number average molecular weight of 5,000 to 20,000. Item 7. The microcapsules according to any one of Items 1 to 5, wherein the polyhydroxyalkanoic acid in the polymer has a number average molecular weight of 10,000 to 60,000. Item 8. The microcapsules according to any one of Items 1 to 6, wherein the ratio of the number average molecular weight of the branched polyalkylene glycol to the number average molecular weight of the polyhydroxyalkanoic acid in the polymer is 1:1 to 1:6. Item 9. The microcapsules according to any one of Items 1 to 7, wherein the polyhydroxyalkanoic acid is a lactic acid / glycolic acid copolymer, and wherein 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. Item 10. The microcapsules according to any one of Items 1 to 8, wherein the number average molecular weight of the polymer is 30,000 to 70,000. Item 11. Item 11. A microcapsule according to any one of Items 1 to 9, wherein the drug is ranibizumab. Item 12. A composition comprising the microcapsule according to any one of Items 1 to 10. Item 13. The composition according to Item 11, which is a sustained-release pharmaceutical composition.Item 13. The composition according to Item 11 or 12, which is a microsphere.

[0010] According to the technology of the present disclosure, there can be provided a formulation containing at least one drug selected from the group consisting of cetuximab, bevacizumab, faricimab, ranibizumab, and brolucizumab, which has excellent drug stability. The formulation can also have excellent drug sustained release properties.

[0011]

[0049] Figure 1 shows a scanning electron microscope (hereinafter sometimes referred to as SEM) image of cetuximab-loaded microspheres (the polymer is 4-branched PEG-PLGA) obtained in Test 1.1.1. Bar = 10 μm. Figure 2 shows an SEM image of cetuximab-loaded microspheres (the polymer is PLGA) obtained in Test 1.2.1. Bar = 10 μm. Figure 3 shows an SEM image of bevacizumab-loaded microspheres (the polymer is 4-branched PEG-PLGA) obtained in Test 2.1.1. Bar = 10 μm. Figure 4 shows an SEM image of bevacizumab-loaded microspheres (the polymer is 8-branched PEG-PLGA) obtained in Test 2.2.1. Bar = 10 μm. Figure 5 shows an SEM image of bevacizumab-loaded microspheres (the polymer is monobranched PEG-PLGA) obtained in Test 2.3.1. Bar = 10 μm.

[0049] Figure 1 shows an SEM image of bevacizumab-loaded microspheres (polymer: 2-arm PEG-PLGA) obtained in Test 2.4.1. Bar = 10 μm. Figure 2 shows an SEM image of ranibizumab-loaded microspheres (polymer: 4-arm PEG-PLGA) obtained in Test 3.2.1. Bar = 10 μm. Figure 3 shows an SEM image of faricimab-loaded microspheres (polymer: 4-arm PEG-PLGA) obtained in Test 3.3.1. Bar = 10 μm. Figure 4 shows an SEM image of brolucizumab-loaded microspheres (polymer: 4-arm PEG-PLGA) obtained in Test 3.4.1. Bar = 10 μm. Figure 5 shows an SEM image of aflibercept-loaded microspheres (polymer: 4-arm PEG-PLGA) obtained in Test 3.5.1. Bar = 10 μm.

[0046] Figure 1 shows the results of the sustained drug release evaluation performed in Test 6. The horizontal axis shows the number of days from the start of the drug release test, and the vertical axis shows the ranibizumab activity retention rate (%). An SEM image of ranibizumab-loaded microspheres (polymer: PLGA) obtained in Test 7.1.1 is shown. Bar = 10 μm. An SEM image of ranibizumab-loaded microspheres (polymer: 2PEG (10 kDa)-PLGA (40 kDa)) obtained in Test 7.1.1 is shown. Bar = 10 μm. An SEM image of ranibizumab-loaded microspheres (polymer: 8PEG (10 kDa)-PLGA (40 kDa)) obtained in Test 7.1.1 is shown. Bar = 10 μm.

[0012] The present disclosure preferably includes, but is not limited to, microcapsules containing drugs and compositions containing the microcapsules, and the present disclosure includes everything disclosed in the present specification and recognizable by a person skilled in the art.

[0013] The microcapsules encompassed by the present disclosure contain a drug, and the microcapsules comprise a polymer having a chemical structure in which a branched polyalkylene glycol having 3 to 10 terminal hydroxy groups per molecule is bonded to a polyhydroxyalkanoic acid, wherein the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain in the polymer is 50 to 230, and the drug is at least one selected from the group consisting of cetuximab, bevacizumab, faricimab, ranibizumab, and brolucizumab. Hereinafter, the microcapsules encompassed by 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 branched polyalkylene glycol having 3 to 10 terminal hydroxy groups per molecule is bonded to a polyhydroxyalkanoic acid, and in the polymer, the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain is 50 to 230. 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. Branched Polyalkylene Glycol The branched polyalkylene glycol constituting the polymer of the present disclosure 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.

[0016] The number of hydroxy groups contained in the polyhydric alcohol may be, for example, 3 to 20. The upper or lower limit of the range may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The number of hydroxy groups contained in the polyhydric alcohol is preferably 2 to 15, more preferably 3 to 10, even more preferably 4 to 8, and particularly preferably 4 or 8.

[0017] 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.

[0018] The branched polyalkylene glycol has 3 to 10 branches per molecule. The upper or lower limit of this range may be 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 drug in the microcapsules of the present disclosure and imparting excellent sustained drug release properties to the microcapsules of the present disclosure, the branched polyalkylene glycol preferably has 4 to 8 terminal hydroxy groups per molecule, and particularly preferably has 4 or 8 terminal hydroxy groups per molecule.

[0019] More specifically, examples of the branched polyalkylene glycol constituting the polymer of the present disclosure include branched polyethylene glycol, branched polypropylene glycol, and branched polybutylene glycol. Among these, from the viewpoint of biocompatibility, the branched polyalkylene glycol constituting the polymer of the present disclosure is preferably branched polyethylene glycol. In general, the alkylene group having fewer carbon atoms has higher hydrophilicity.

[0020] The number average molecular weight of the branched 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, 3000 to 50000. From the viewpoint of being able to stably retain a drug in the microcapsules of the present disclosure and being able to impart excellent sustained drug release properties to the microcapsules of the present disclosure, the number average molecular weight of the branched polyalkylene glycol constituting the polymer of the present disclosure in the polymer is preferably 3000 to 30000, more preferably 5000 to 20000, even more preferably 7000 to 15000, and particularly preferably 8000 to 13000.

[0021] A preferred embodiment of the branched 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 5000 to 20000. A more preferred embodiment of the branched 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 of the polyethylene glycol in the polymer of 7000 to 15000.

[0022] Branched 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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. The upper or lower limit of the range may be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 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. In general, 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 degrada- tion rate decreases as the composition ratio of lactic acid increases.

[0027] 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, 8000 to 80000. From the viewpoint of being able to stably retain a drug in the microcapsules of the present disclosure and being able to impart excellent sustained drug release properties 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 10000 to 65000, more preferably 10000 to 60000, even more preferably 15000 to 55000, and particularly preferably 20000 to 50000.

[0028] 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 65,000, 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 15,000 to 55,000, 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.

[0029] 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 branched polyalkylene glycol having 3 to 10 terminal hydroxy groups per molecule is bonded to a polyhydroxyalkanoic acid. More specifically, the polymer has a chemical structure in which the hydroxy group of the branched polyalkylene glycol and the carboxy group of the polyhydroxyalkanoic acid are ester-bonded. Furthermore, in the polymer of the present disclosure, the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain is 35 to 233, and preferably 50 to 230.

[0030] In the present disclosure, the term "the 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 branched polyalkylene glycol.

[0031] As described above, in the polymers of the present disclosure, the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain is 35 to 233, and preferably 50 to 230. The upper or lower limit of the range may be 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, or 230. From the viewpoint of being able to stably retain a drug within the microcapsules of the present disclosure and being able to impart excellent sustained drug release properties 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 is preferably 70 to 220, more preferably 80 to 210, even more preferably 90 to 205, and particularly preferably 100 to 200.

[0032] The ratio of the number average molecular weight of the branched 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. The upper or lower limit of this range may be 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, or 1:10. From the viewpoint of being able to stably retain a drug within the microcapsules of the present disclosure and being able to impart excellent sustained drug release properties to the microcapsules of the present disclosure, the ratio of the number average molecular weight of the branched 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 1:1 to 1:6.5, even more preferably 1:1.5 to 1:6, and particularly preferably 1:1.5 to 1:5.5.

[0033] 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, 20,000 to 100,000. From the viewpoint of being able to stably retain a drug within the microcapsules of the present disclosure and being able to impart excellent sustained drug release properties to the microcapsules of the present disclosure, 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.

[0034] The number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain in the polymer of the present disclosure, the number average molecular weight of the polymer of the present disclosure, the number average molecular weight of the branched 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).

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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, and in which the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain in the polymer is 50 to 230. In the polymer, the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain is preferably 70 to 220, more preferably 80 to 210, even more preferably 90 to 205, and particularly preferably 100 to 200; further, in the 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; and In the 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.

[0039] 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 ２ where 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.

[0040] 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. 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).

[0041] 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.

[0042] In the above polymer, the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain is expressed as the number-average value of s + t of each chain. Therefore, in the technology of the present disclosure, s + t is 35 to 233, and preferably 50 to 230. For example, if the number of polyhydroxyalkanoic acid units in the structure of the above polymer is 50, the number-average value of s + t of each polyhydroxyalkanoic acid chain is 50.

[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 ２where 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 ４ and 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.

[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. Properties of the Polymer of the Present Disclosure The polymer of the present disclosure can stably retain a drug within the microcapsules of the present disclosure and can also impart excellent sustained drug release properties to the microcapsules of the present disclosure. Drug stability and sustained drug release properties can be evaluated by conventionally known methods or methods that can be easily derived from conventionally known methods. For example, when the drug is a protein, the total protein amount in a composition containing microcapsules can be quantified by the BCA method, Bradford method, 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. Furthermore, sustained drug release properties can be evaluated, for example, by mixing a composition containing microcapsules with a release test solution and quantifying the amount of drug released into the release test solution over time.

[0049] 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. In the present disclosure, the average particle size refers to the average of the maximum diameters of individual particles. Generally, the average particle size can be measured by laser diffraction scattering, dynamic light scattering, image analysis using a scanning electron microscope (SEM), sieving tests, or the like. In particular, in the present disclosure, the average particle size refers to the volume-based average particle size measured by laser diffraction scattering.

[0050] The average particle size of the microcapsules can be adjusted by a conventionally known method or a method that can be easily derived by a person skilled in the art from a conventionally known method. 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.

[0051] 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 drug, and then mixing the two to prepare a W / O emulsion. The individual emulsified particles contained in the W / O emulsion are preferably included 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 drug. The capsule-like structures in the dried product are also preferably included in the microcapsules of the present disclosure.

[0052] 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 the drug is encapsulated in the space surrounded by the membrane. Here, the "membrane" refers to the part that separates the internal and external phases of each emulsified particle in the W / O emulsion.

[0053] B-2. Drugs The drug encapsulated in the microcapsules of the present disclosure is at least one selected from the group consisting of cetuximab, bevacizumab, faricimab, ranibizumab, and brolucizumab. Cetuximab is an anti-EGFR antibody, bevacizumab is an anti-VEGF antibody, faricimab is a bispecific antibody of anti-VEGF and anti-Ang-2, and brolucizumab is known to function as a single-chain fragment of an anti-VEGF antibody. Ranibizumab is a Fab fragment of an anti-VEGF antibody. These drugs, particularly anti-VEGF antibodies and fragments thereof, are used to treat age-related macular degeneration and the like. Although not particularly limited, it is particularly preferred that the drug encapsulated in the microcapsules of the present disclosure is ranibizumab.

[0054] Among the above-mentioned drugs, there are some that can easily decompose, denature, or change in quality and lose their physiological activity. According to the technology of the present disclosure, even such drugs can maintain their activity in a formulation. Whether a drug 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 drug 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, and the activity retention rate (%) can be calculated using the formula [active protein amount / total protein amount] × 100.

[0055] 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.

[0056] 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 the drug 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] Examples of pH adjusters include citric acid, phosphoric acid, malic acid, pyrophosphoric acid, lactic acid, tartaric acid, glycerophosphoric acid, acetic acid, nitric acid, and chemically possible salts thereof, as well as sodium hydroxide and potassium hydroxide.

[0063] 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.

[0064] 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. Among these dosage forms, microspheres are particularly preferred. In the present disclosure, "microspheres" refers to a spherical composition containing a polymer and a drug and having an average particle size of approximately 1 to 100 μm. Nanoparticles, microparticles, nanospheres, and microcapsules are also preferred dosage forms of the composition of the present disclosure.

[0065] The composition of the present disclosure is preferably a sustained-release pharmaceutical composition. In the composition of the present disclosure, the drug is encapsulated in the microcapsules of the present disclosure, so the drug can be stably maintained. In addition, in the composition of the present disclosure, the drug is encapsulated in the microcapsules of the present disclosure, so the composition of the present disclosure can have sustained drug release properties. Therefore, when the composition of the present disclosure is applied to a subject, the drug 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.

[0066] 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.

[0067] The composition of the present disclosure contains microcapsules encapsulating at least one drug selected from the group consisting of cetuximab, bevacizumab, faricimab, ranibizumab, and brolucizumab. These drugs, particularly anti-VEGF antibodies, are used to treat age-related macular degeneration and the like. Therefore, the composition of the present disclosure is suitable for use in improving and treating age-related macular degeneration. In this disclosure, the terms "improving age-related macular degeneration" and "treating age-related macular degeneration" encompass the suppression of progression of age-related macular degeneration, the halting of progression of age-related macular degeneration, the reduction of lesions, and the elimination of lesions, when administered to a subject diagnosed with age-related macular degeneration.

[0068] 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 directly administered locally, such as into the vitreous body, by injection, or may be administered intravenously, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intrapleurally, or by infusion, or may be administered by perfusion via a catheter, or may be applied to the skin, mucosa, tissue, or the like. It is particularly preferred that the composition of the present disclosure be administered directly into the vitreous body of a subject by injection.

[0069] The administration frequency of the composition of the present disclosure is not particularly limited. For example, it may be administered once or multiple times a day, once or multiple times a week, once or multiple times a month, or once or multiple times a year. The administration period of the composition of the present disclosure is also not particularly limited, as long as the effects of the present disclosure are achieved. Furthermore, the dosage of the composition of the present disclosure is also not particularly limited, as long as the effects of the present disclosure are achieved. The application frequency, application period, and dosage can be adjusted appropriately by those skilled in the art depending on the condition of the subject, the course of treatment, etc.

[0070] The composition of the present disclosure may be optionally combined with other pharmaceutical compositions and / or treatment methods that can be applied to a subject with age-related macular degeneration. When the composition of the present disclosure is combined with other pharmaceutical compositions and / or treatment methods, they may be applied to a subject simultaneously, or may be applied separately at any time.

[0071] 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.

[0072] 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.

[0073] 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. "Number of PLGA units" refers to the number of PLGA units per PEG chain of the polymer.

[0074] In the following examples, various PEG-PLGA polymers were prepared according to the method described in International Publication No. 2018 / 062464 (Patent Document 2).

[0075] 1. Test 1: Polymer-Dependent Stability (During Manufacturing): Comparison of 4-Branch PEG-PLGA and PLGA. First, the inventors evaluated the stability of drugs encapsulated in microspheres during manufacturing using a polymer having a chemical structure in which 4-branched polyethylene glycol and lactic acid / glycolic acid copolymer are bonded (hereinafter, sometimes referred to as "4-branched PEG-PLGA") or lactic acid / glycolic acid copolymer (hereinafter, sometimes referred to as "PLGA"). In this test, the 4-branched PEG-PLGA used had a total molecular weight of 50 kDa, a PEG portion molecular weight of 10 kDa, a PLGA portion molecular weight of 40 kDa, and an LA:GA ratio of 8:2. The number of PLGA units per PEG chain of this polymer was approximately 160. This 4-branched PEG-PLGA is sometimes referred to as 4PEG(10 kDa)-PLGA(40 kDa). Furthermore, as PLGA, "Resomer RG504, 739944, LA:GA = 1:1" (Sigma-Aldrich) with a molecular weight of 45 kDa was used. This PLGA may be referred to as PLGA (45 kDa). Cetuximab was used as the drug to be encapsulated in the microspheres. Details of this test are described below.

[0076] 1.1 Evaluation using 4-branched PEG-PLGA (Example 1) 1.1.1 Preparation of microspheres 0.05 g of Span (registered trademark) 80 and 0.5 g of 4PEG (10 kDa)-PLGA (40 kDa) were dissolved in 20 mL of dichloromethane (oil phase solution). 1 mL of 0.5% cetuximab solution (Erbitux (registered trademark) injection 100 mg, Merck Ltd.) was added to the oil phase 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.

[0077] 1.1.2 Observation by scanning electron microscope The cetuximab-encapsulated microspheres obtained in 1.1.1 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 1.

[0078] 1.1.3 Measurement of total cetuximab amount by MicroBCA Assay 10 mg of the cetuximab-encapsulated microspheres obtained in 1.1.1 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.

[0079] The total amount of cetuximab 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 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 amount of cetuximab calculated from the absorbance is shown in Table 1.

[0080] 1.1.4 Measurement of active cetuximab amount by ELISA 5 mg of the cetuximab-encapsulated microspheres obtained in 1.1.1 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.

[0081] 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.

[0082] After washing, 100 μL of sample solution or calibration curve solution was added to each well and the plate was 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 (Goat Anti-Human IgG (H+L) 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 ABTS Microwell Peroxidase Substrate (SeraCare Life Sciences, Inc.) was added and the plate was 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 1. Table 1 also shows the activity retention rate (%) calculated by the formula [active cetuximab amount / total cetuximab amount]×100.

[0083] 1.2 Evaluation using PLGA (Comparative Example 1) 1.2.1 Microsphere Preparation 0.05 g of Span 80 and 0.5 g of PLGA (45 kDa) were dissolved in 20 mL of dichloromethane (oil phase solution). 1 mL of 0.5% cetuximab solution was added to the oil phase solution, and the mixture was emulsified at 9,500 rpm for 1 minute using a homogenizer. After emulsification, the mixture was spray-dried using a spray dryer (drying conditions were the same as in 1.1) to obtain white particulate cetuximab-encapsulated microspheres.

[0084] 1.2.2 Observation by scanning electron microscope The cetuximab-encapsulated microspheres obtained in 1.2.1 were observed using the same procedure as in 1.1.2. The observed images are shown in Figure 2.

[0085] 1.2.3 Measurement of total cetuximab amount by MicroBCA Assay The total cetuximab amount of the cetuximab-encapsulated microspheres obtained in 1.2.1 was measured using the same procedure as in 1.1.3. The measured total cetuximab amount is shown in Table 1.

[0086] 1.2.4 Measurement of active cetuximab amount by ELISA The amount of active cetuximab in the cetuximab-encapsulated microspheres obtained in 1.2.1 was measured using the same procedure as in 1.1.4. The measured amount of active cetuximab is shown in Table 1. Table 1 also shows the activity retention rate (%) calculated using the formula [active cetuximab amount / total cetuximab amount] × 100.

[0087]

[0088] 1.3 Summary As shown in Table 1, in Comparative Example 1, in which PLGA was used as the polymer in preparing the microspheres, no cetuximab that maintained its activity was detected in the microspheres, and the activity retention rate was 0%. On the other hand, in Example 1, in which 4-branched PEG-PLGA was used as the polymer in preparing the microspheres, a high activity retention rate of 84.6% was obtained.

[0089] This study suggested that when preparing drug-loaded microspheres, using PEG-PLGA as the polymer may result in higher drug stability than using PLGA alone.

[0090] 2. Study 2: Stability (at the time of manufacture) depending on the branch number of PEG-PLGA: Comparison of branch numbers. Based on the results of Study 1, the inventors investigated whether the branch number of PEG-PLGA affects the stability of the drug encapsulated in the microspheres. In this study, bevacizumab-encapsulated microspheres were prepared using mono-, di-, tetra-, or octa-branched PEG-PLGA as the polymer, and the stability of the encapsulated bevacizumab in each prepared microsphere was evaluated. The mono-branched PEG-PLGA used had a molecular weight of 50 kDa for the entire polymer, a molecular weight of 10 kDa for the PEG portion, a molecular weight of 40 kDa for the PLGA portion, and an LA:GA ratio of 8:2. The number of PLGA units per PEG chain of this polymer was approximately 640. This mono-branched PEG-PLGA may be referred to as 1PEG (10 kDa)-PLGA (40 kDa). The bi-branched PEG-PLGA used had a molecular weight of 50 kDa for the entire polymer, a molecular weight of 10 kDa for the PEG portion, and a molecular weight of 40 kDa for the PLGA portion, with an LA:GA ratio of 8:2. The number of PLGA units per PEG chain in this polymer was approximately 320. This bi-branched PEG-PLGA may be referred to as 2PEG (10 kDa)-PLGA (40 kDa). The 4-branched PEG-PLGA used was 4PEG (10 kDa)-PLGA (40 kDa) described in Test 1. The 8-branched PEG-PLGA used had a molecular weight of 50 kDa for the entire polymer, a molecular weight of 10 kDa for the PEG portion, and a molecular weight of 40 kDa for the PLGA portion, with an LA:GA ratio of 8:2. The number of PLGA units per PEG chain of this polymer was approximately 80. This 8-branched PEG-PLGA is sometimes referred to as 8PEG (10 kDa)-PLGA (40 kDa). Details of this test are described below.

[0091] 2.1 Evaluation using 4-branched PEG-PLGA (Example 2) 2.1.1 Preparation of microspheres 0.025 g of Span80 and 0.25 g of 4PEG (10 kDa)-PLGA (40 kDa) were dissolved in 10 mL of dichloromethane (oil phase solution). 0.1 mL of 2.5% bevacizumab solution (Bevacizumab BS Intravenous Drip Infusion 100 mg "Pfizer" (Pfizer Inc.) was used as is) was added to the oil phase 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.

[0092] 2.1.2 Observation by scanning electron microscope The bevacizumab-encapsulated microspheres obtained in 2.1.1 were observed using a scanning electron microscope (JSM-IT300, JEOL Ltd.). The samples used for observation were previously coated with palladium-gold. The observed images are shown in Figure 3.

[0093] 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.

[0094] 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 2.

[0095] 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.

[0096] 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.

[0097] 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 2. Table 2 also shows the activity maintenance rate (%) calculated by the formula [active bevacizumab amount / total bevacizumab amount]×100.

[0098] 2.2 Evaluation using 8-branched PEG-PLGA (Example 3) 2.2.1 Microsphere Preparation 0.025 g of Span80 and 0.25 g of 8PEG (10 kDa)-PLGA (40 kDa) were dissolved in 10 mL of dichloromethane (oil phase solution). 0.1 mL of a 2.5% bevacizumab solution was added to the oil phase solution, and the mixture was emulsified at 20,500 rpm using a homogenizer for 1 minute. After emulsification, the mixture was spray-dried using a spray dryer (drying conditions were the same as in 2.1.1), yielding white particulate bevacizumab-encapsulated microspheres.

[0099] 2.2.2 Observation by scanning electron microscope The bevacizumab-encapsulated microspheres obtained in 2.2.1 were observed using the same procedure as in 2.1.2. The observed images are shown in Figure 4.

[0100] 2.2.3 Measurement of total bevacizumab amount by MicroBCA Assay The total bevacizumab amount of the bevacizumab-encapsulated microspheres obtained in 2.2.1 was measured using the same procedure as in 2.1.3. The measured total bevacizumab amount is shown in Table 2.

[0101] 2.2.4 Measurement of active bevacizumab amount by ELISA The amount of active bevacizumab in the bevacizumab-encapsulated microspheres obtained in 2.2.1 was measured using the same procedure as in 2.1.4. The measured active bevacizumab amounts are shown in Table 2. Table 2 also shows the activity retention rate (%) calculated using the formula [active bevacizumab amount / total bevacizumab amount] × 100.

[0102] 2.3 Evaluation using monobranched PEG-PLGA (Comparative Example 2) 2.3.1 Microsphere preparation 0.025 g of Span 80 and 0.25 g of 1PEG (10 kDa)-PLGA (40 kDa) were dissolved in 10 mL of dichloromethane (oil phase solution). 0.1 mL of 2.5% bevacizumab solution was added to the oil phase solution, and the mixture was emulsified at 20,500 rpm for 1 minute using a homogenizer. After emulsification, the mixture was spray-dried using a spray dryer (drying conditions were the same as in 2.1.1), yielding white particulate bevacizumab-encapsulated microspheres.

[0103] 2.3.2 Observation by scanning electron microscope The bevacizumab-encapsulated microspheres obtained in 2.3.1 were observed in the same manner as in 2.1.2. The observed images are shown in Figure 5.

[0104] 2.3.3 Measurement of total bevacizumab amount by MicroBCA Assay The total bevacizumab amount of the bevacizumab-encapsulated microspheres obtained in 2.3.1 was measured using the same procedure as in 2.1.3. The measured total bevacizumab amount is shown in Table 2.

[0105] 2.3.4 Measurement of active bevacizumab amount by ELISA The amount of active bevacizumab in the bevacizumab-encapsulated microspheres obtained in 2.3.1 was measured using the same procedure as in 2.1.4. The measured active bevacizumab amounts are shown in Table 2. Table 2 also shows the activity retention rate (%) calculated using the formula [active bevacizumab amount / total bevacizumab amount] × 100.

[0106] 2.4 Evaluation using 2-branched PEG-PLGA (Comparative Example 3) 2.4.1 Microsphere Preparation 0.025 g of Span 80 and 0.25 g of 2PEG (10 kDa)-PLGA (40 kDa) were dissolved in 10 mL of dichloromethane (oil phase solution). 0.1 mL of a 2.5% bevacizumab solution was added to the oil phase solution, and the mixture was emulsified at 20,500 rpm for 1 minute using a homogenizer. After emulsification, the mixture was spray-dried using a spray dryer (drying conditions were the same as in 2.1.1), yielding white particulate bevacizumab-encapsulated microspheres.

[0107] 2.4.2 Observation by scanning electron microscope The bevacizumab-encapsulated microspheres obtained in 2.4.1 were observed in the same manner as in 2.1.2. The observed images are shown in Figure 6.

[0108] 2.4.3 Measurement of total bevacizumab amount by MicroBCA Assay The total bevacizumab amount of the bevacizumab-encapsulated microspheres obtained in 2.4.1 was measured using the same procedure as in 2.1.3. The measured total bevacizumab amount is shown in Table 2.

[0109] 2.4.4 Measurement of active bevacizumab amount by ELISA The amount of active bevacizumab in the bevacizumab-encapsulated microspheres obtained in 2.4.1 was measured using the same procedure as in 2.1.3. The measured active bevacizumab amount is shown in Table 2. Table 2 also shows the activity retention rate (%) calculated using the formula [active bevacizumab amount / total bevacizumab amount] × 100.

[0110]

[0111] 2.5 Summary As shown in Table 2, Example 2, which used 4-branched PEG-PLGA as the polymer in preparing the microspheres, and Example 3, which used 8-branched PEG-PLGA, achieved a higher activity retention rate than Comparative Example 2, which used single-branched PEG-PLGA, and Comparative Example 3, which used two-branched PEG-PLGA.

[0112] This study suggests that the number of branches in PEG-PLGA may affect the stability of drugs encapsulated in microspheres. In particular, it was suggested that high drug stability may be achieved when 4- or 8-branched PEG-PLGA is used.

[0113] 3. Test 3: Comparison of Stability of Different Drugs (Polymer Used During Production: 4-Branched PEG-PLGA) Next, the inventors investigated whether the type of drug encapsulated in PEG-PLGA microspheres would affect the stability of the drug within the microspheres. In this test, microspheres were prepared using the same polymer as in Tests 1 and 2, 4PEG (10 kDa)-PLGA (40 kDa). The drugs encapsulated in the microspheres were bevacizumab, ranibizumab, faricimab, brolucizumab, or aflibercept. Details of this test are described below.

[0114] 3.1 Evaluation with Bevacizumab Bevacizumab-loaded microspheres were prepared and evaluated as described in 2.1.

[0115] 3.2 Evaluation with Ranibizumab (Example 4) 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 (oil phase 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 oil phase 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.

[0116] 3.2.2 Observation by scanning electron microscope The ranibizumab-encapsulated microspheres obtained in 3.2.1 were observed using a scanning electron microscope (JSM-IT300, JEOL Ltd.). The samples used for observation were previously coated with palladium-gold. The observed images are shown in Figure 7.

[0117] 3.2.3 Measurement of total ranibizumab amount by MicroBCA Assay 5 mg of ranibizumab-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, 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.

[0118] 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 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 3.

[0119] 3.2.4 Measurement of active ranibizumab by ELISA 5 mg of ranibizumab-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, 10 mg / mL of Lucentis® intravitreal injection solution was diluted with PBS to prepare a calibration curve solution ranging from 2 to 200 ng / mL.

[0120] The amount of active ranibizumab 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.

[0121] 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 the 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 3. Table 3 also shows the activity maintenance rate (%) calculated by the formula [active ranibizumab amount / total ranibizumab amount]×100.

[0122] 3.3 Evaluation with Faricimab (Example 5) 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 (oil phase solution). 0.1 mL of a 2.4% faricimab solution (a 5-fold dilution of BabySmo® intravitreal injection solution 120 mg / mL (Chugai Pharmaceutical Co., Ltd.)) was added to the oil phase 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 3.2.1), yielding white, particulate faricimab-encapsulated microspheres.

[0123] 3.3.2 Observation by scanning electron microscope The faricimab-encapsulated microspheres obtained in 3.3.1 were observed using the same procedure as in 3.2.2. The observed images are shown in Figure 8.

[0124] 3.3.3 Measurement of Total Faricimab Amount by MicroBCA Assay Five mg of faricimab-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. 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 3.

[0125] 3.3.4 Measurement of active faricimab by ELISA 5 mg of the faricimab-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. 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.

[0126] 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.

[0127] 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 3. Table 3 also shows the activity retention rate (%) calculated using the formula [amount of active faricimab / total amount of faricimab] × 100.

[0128] 3.4 Evaluation with Brolucizumab (Example 6) 3.4.1 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 (oil phase 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 oil phase 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 3.2.1), yielding white particulate microspheres encapsulating brolucizumab.

[0129] 3.4.2 Observation by scanning electron microscope The brolucizumab-encapsulated microspheres obtained in 3.4.1 were observed using the same procedure as in 3.2.2. The observed images are shown in Figure 9.

[0130] 3.4.3 Measurement of total brolucizumab amount by MicroBCA Assay 5 mg of brolucizumab-encapsulated microspheres obtained in 3.4.1 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.

[0131] 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 3.

[0132] 3.4.4 Measurement of active brolucizumab by ELISA 5 mg of brolucizumab-encapsulated microspheres obtained in 3.4.1 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.

[0133] 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.

[0134] 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 brolucizumab calculated from the absorbance is shown in Table 3. Table 3 also shows the activity retention rate (%) calculated by the formula [active brolucizumab amount / total brolucizumab amount]×100.

[0135] 3.5 Evaluation with Aflibercept (Comparative Example 4) 3.5.1 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 (oil phase solution). 0.1 mL of 2.5% aflibercept solution (Zaltrap (registered trademark) intravenous drip infusion 100 mg (Sanofi Corporation) used as is) was added to the oil phase solution, and the mixture was emulsified at 20,500 rpm for 1 minute using a homogenizer. After emulsification, the mixture was spray-dried using a spray dryer (drying conditions were the same as in 3.2.1) to obtain white particulate aflibercept-encapsulated microspheres.

[0136] 3.5.2 Observation by scanning electron microscope The aflibercept-encapsulated microspheres obtained in 3.5.1 were observed in the same manner as in 3.2.2. The observed images are shown in Figure 10.

[0137] 3.5.3 Measurement of total aflibercept amount by MicroBCA Assay 5 mg of aflibercept-encapsulated microspheres obtained in 3.5.1 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, 100 mg of Zaltrap® intravenous infusion was diluted with PBS to prepare a calibration curve solution of 0.1 to 20 μg / mL.

[0138] MicroBCA Assay was used to measure the total amount of aflibercept. 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 attached protocol of 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 aflibercept calculated from the absorbance is shown in Table 3.

[0139] 3.5.4 Measurement of active aflibercept by ELISA 5 mg of aflibercept-encapsulated microspheres obtained in 3.5.1 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. Also, 100 mg of Zaltrap® intravenous infusion was diluted with PBS to prepare a calibration curve solution of 2 to 200 ng / mL.

[0140] The amount of active aflibercept 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.

[0141] 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 aflibercept calculated from the absorbance is shown in Table 3. Table 3 also shows the activity retention rate (%) calculated using the formula [active aflibercept amount / total aflibercept amount] × 100.

[0142]

[0143] 3.6 Summary As shown in Table 3, when bevacizumab (Example 2), ranibizumab (Example 4), faricimab (Example 6), or brolucizumab (Example 6) was used as the drug to be encapsulated in the microspheres, a higher activity retention rate was obtained than when aflibercept was used (Comparative Example 4).

[0144] This study suggested that the stability of drugs in PEG-PLGA microspheres may differ depending on the type of drug encapsulated in the microspheres. In particular, it was suggested that high drug stability may be achieved when bevacizumab, ranibizumab, faricimab, or brolucizumab was encapsulated in the microspheres.

[0145] 4. Test 4: Stability as a function of PLGA unit number (drug used: bevacizumab, Example 7 and Comparative Example 5) Next, the inventors investigated whether the number of PLGA units in PEG-PLGA affects the stability of the drug encapsulated in the microspheres. In this test, bevacizumab-encapsulated microspheres were prepared using four-branched PEG-PLGA with various numbers of PLGA units as shown in Table 4 in 4.1, and the stability of the encapsulated bevacizumab in each prepared microsphere was evaluated. The LA:GA ratio of the PEG-PLGA used in this test was 8:2 in all cases. Details of this test are described below.

[0146] 4.1 Details of the 4-arm PEG-PLGA polymer used in this study

[0147] 4.1.1 Microsphere Preparation 0.025 g of Span80 and 0.25 g of 4-branched PEG-PLGA were dissolved in 10 mL of dichloromethane (oil phase solution). The number of PLGA units in the 4-branched PEG-PLGA used was 100, 110, 160, 200, 250, 300, 350, 500, or 1000, as shown in Table 4. 0.1 mL of 2.5% bevacizumab solution was added to each oil phase solution, and the mixture was emulsified at 20,500 rpm for 1 minute using a homogenizer. After emulsification, the mixture was spray-dried using a spray dryer (two-fluid nozzle φ1.4, N ２Flow 30 mm, temperature 10-15°C) to obtain white particulate microspheres containing bevacizumab.

[0148] 4.1.2 Measurement of total bevacizumab amount by MicroBCA Assay 5 mg of each bevacizumab-encapsulated microsphere obtained in 4.1.1 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.

[0149] 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 stirred 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 in each microsphere calculated from the absorbance is shown in Table 5.

[0150] 4.1.3 Measurement of active bevacizumab amount by ELISA 5 mg of each bevacizumab-encapsulated microsphere obtained in 4.1.1 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.

[0151] The amount of active bevacizumab 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.

[0152] 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 bevacizumab in each microsphere calculated from the absorbance is shown in Table 5. Table 5 also shows the activity maintenance rate (%) calculated by the formula [active bevacizumab amount / total bevacizumab amount]×100.

[0153]

[0154] 4.2 Summary As shown in Table 5, in Examples 7(i) to (v), in which a 4-branched PEG-PLGA with 100 to 250 PLGA units was used as the polymer in preparing the microspheres, a higher bevacizumab activity retention rate was obtained than in Comparative Examples 5(i) to (iv), in which a 4-branched PEG-PLGA with 300 to 1000 PLGA units was used. In particular, in Examples 7(i) to (iv), in which a 4-branched PEG-PLGA with 100 to 200 PLGA units was used, a high bevacizumab activity retention rate of approximately 90% was obtained.

[0155] This study suggests that the number of PLGA units in PEG-PLGA may affect the stability of drugs encapsulated in microspheres. In particular, it was suggested that high drug stability may be achieved when PEG-PLGA with 100 to 200 PLGA units is used.

[0156] 5. Experiment 5: Stability as a function of the number of 4-branched PEG-PLGA units (drug used: ranibizumab, Example 8 and Comparative Example 6) Based on the results of Experiment 4, the inventors investigated whether the number of PLGA units in PEG-PLGA affects drug stability when drugs other than bevacizumab are encapsulated, as in the case of encapsulating bevacizumab. In this experiment, ranibizumab-encapsulated microspheres were prepared using 4-branched PEG-PLGA having various numbers of PLGA units as shown in Table 6 in 5.1, and the stability of the encapsulated ranibizumab in each prepared microsphere was evaluated. The LA:GA ratio of the PEG-PLGA used in this experiment was 8:2 in all cases. Details of this experiment are described below.

[0157] 5.1 Details of the 4-arm PEG-PLGA polymer used

[0158] 5.1.1 Microsphere Preparation 0.025 g of Span80 and 0.25 g of 4-branched PEG-PLGA were dissolved in 10 mL of dichloromethane (oil phase solution). The number of PLGA units in the 4-branched PEG-PLGA used was 160, 200, 250, 300, or 350, as shown in Table 6. 0.1 mL of 0.5% ranibizumab solution was added to each oil phase solution, and the mixture was emulsified at 20,500 rpm for 1 minute using a homogenizer. After emulsification, the mixture was spray-dried using a spray dryer to obtain white, particulate ranibizumab-encapsulated microspheres.

[0159] 5.1.2 Measurement of total ranibizumab amount by MicroBCA Assay 5 mg of each ranibizumab-encapsulated microsphere obtained in 5.1.1 was dissolved in 1 mL of a 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.

[0160] 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 in each microsphere calculated from the absorbance is shown in Table 7.

[0161] 5.1.3 Measurement of active ranibizumab amount by ELISA 5 mg of each ranibizumab-encapsulated microsphere obtained in 5.1.1 was dissolved in 1 mL of a dimethyl sulfoxide / PBS (7:3) mixture. This solution was diluted 1000 times 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.

[0162] ELISA was used to measure the activity of ranibizumab. 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.

[0163] 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 in each microsphere calculated from the absorbance is shown in Table 7. Table 7 also shows the activity maintenance rate (%) calculated by the formula [active ranibizumab amount / total ranibizumab amount]×100.

[0164]

[0165] 5.2 Summary As shown in Table 7, in Examples 8(i) and (ii), in which a 4-branched PEG-PLGA with 160 or 200 PLGA units was used as the polymer in preparing the microspheres, a higher ranibizumab activity retention rate was obtained than in Comparative Examples 6(i) to (iii), in which a 4-branched PEG-PLGA with 250 to 350 PLGA units was used.

[0166] This study suggested that the number of PLGA units in PEG-PLGA may affect the stability of the drug encapsulated in the microspheres, not only when bevacizumab is encapsulated but also when ranibizumab is encapsulated. Study 4 and this study suggested that high drug stability may be achieved, especially when PEG-PLGA with a PLGA unit number of 100 to 200 is used.

[0167] 6. Test 6: Evaluation of sustained release (activity value) in microspheres using 4-branched PEG-PLGA (Example 9) In this test, the inventors evaluated the sustained drug release of microspheres using 4-branched PEG-PLGA. The evaluation of sustained drug release was performed using the ranibizumab-encapsulated microspheres obtained in 3.2. Details are described below.

[0168] 6.1 In vitro ranibizumab release test 20 mg of the ranibizumab-encapsulated microspheres obtained in 3.2 was weighed out and added to 1 mL of 10% gellan gum solution and dispersed. The entire amount of this dispersion was injected into a release test solution (0.02% NaN ３ After the injection, the sample was placed in a thermostatic chamber set at 37°C, and the drug release test was initiated.

[0169] After the start of the drug release test, sampling was carried out at 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 60 days, 90 days, 120 days, 150 days, and 180 days. Specifically, 1 mL of the release test liquid was collected at each sampling time point (the collected release test liquid may be referred to as the sampling liquid).

[0170] The total amount of ranibizumab released into the sampling solution was measured by the MicroBCA method described in 6.2. The amount of active ranibizumab released into the sampling solution was also measured by the ELISA method described in 6.3. From the measured total amount of ranibizumab and the amount of active ranibizumab, the activity retention rate (%) was calculated using the formula [amount of active ranibizumab / total amount of ranibizumab] x 100. The activity retention rate (%) at each time point is shown in Figure 11.

[0171] 6.2 Measurement of total ranibizumab amount by MicroBCA Assay The sampled solution obtained at each time point in 6.1 was used as the sample solution. In addition, 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.

[0172] 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 color development, the absorbance at a wavelength of 562 nm was measured using a microplate reader. The total amount of ranibizumab in each sample solution was calculated from the absorbance.

[0173] 6.3 Measurement of active ranibizumab by ELISA The sample solutions obtained at each time point in 6.1 were used as sample solutions. In addition, 10 mg / mL of Lucentis® intravitreal injection solution was diluted with PBS to prepare calibration curve solutions ranging from 2 to 200 ng / mL.

[0174] The amount of active ranibizumab 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.

[0175] 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 the 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 in each sampling solution was calculated from the absorbance.

[0176] 6.4 Summary As shown in Figure 11, the ranibizumab-encapsulated microspheres obtained in 3.2 showed a high ranibizumab activity retention rate of 80% or more at 6 weeks (42 days) after the start of the drug release test. In other words, the microspheres continued to release the active drug for a long period of time.

[0177] 7. Test 7: Stability as a Function of Branch Number of PEG-PLGA (Drug Used: Ranibizumab) 7.1.1 Microsphere Preparation 0.025 g of Span 80 and 0.25 g of PLGA (50 kDa), 2PEG (10 kDa)-PLGA (40 kDa), or 8PEG (10 kDa)-PLGA (40 kDa) were dissolved in 10 mL of dichloromethane (oil phase solution). 0.1 mL of a 0.5% ranibizumab solution (a 2-fold diluted solution of Lucentis® intravitreal injection solution 10 mg / mL (Novartis Pharma K.K.)) was added to the oil phase 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 1.1) to obtain white, particulate ranibizumab-encapsulated microspheres.

[0178] 7.1.2 Observation by Scanning Electron Microscope Each of the obtained ranibizumab-encapsulated microspheres 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 12 to 14.

[0179] 7.1.3 Measurement of total ranibizumab amount by MicroBCA Assay 5 mg of each obtained ranibizumab-encapsulated microsphere was dissolved in 1 mL of dimethyl sulfoxide / PBS (7:3 mixture). PBS was added to 200 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.

[0180] MicroBCA Assay was used to measure the total ranibizumab amount. 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 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 ranibizumab amount calculated from the absorbance is shown in Table 8.

[0181] 7.1.4 Measurement of active ranibizumab by ELISA 5 mg of each of the obtained ranibizumab-encapsulated microspheres was dissolved in 1 mL of dimethyl sulfoxide / PBS. This solution was diluted 250-fold with PBS to prepare a sample solution. Furthermore, 10 mg / mL of Lucentis® intravitreal injection solution was diluted with PBS to prepare a calibration curve solution ranging from 1 to 1000 ng / mL.

[0182] The amount of active cetuximab was measured by ELISA. First, 1000 ng / mL human VEGF-A was added to a 96-well immunoplate (MaxiSorp Nunc-Immuno Plate, Thermo Fisher Scientific). １６５ 100 μL of the solution was added, and the plate was shaken on a shaker for 1 minute, followed by standing overnight at 4°C. 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 a 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.

[0183] 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 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 the 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 8. Table 8 also shows the activity maintenance rate (%) calculated by the formula [active ranibizumab amount / total ranibizumab amount]×100.

[0184] 8. Summary of this Example The above test suggests that the technology of the present disclosure may provide a formulation containing at least one drug selected from the group consisting of cetuximab, bevacizumab, faricimab, ranibizumab, and brolucizumab, which has excellent drug stability. It also suggests that the formulation may have excellent sustained drug release properties.

Claims

1. Microcapsules comprising a polymer having a chemical structure in which a branched polyalkylene glycol having 3 to 10 terminal hydroxy groups per molecule is bonded to a polyhydroxyalkanoic acid, and a drug, wherein the number of polyhydroxyalkanoic acid monomer units per polyhydroxyalkanoic acid chain in the polymer is 50 to 230, and the drug is at least one selected from the group consisting of cetuximab, bevacizumab, faricimab, ranibizumab, and brolucizumab.

2. The microcapsules of claim 1, wherein the branched polyalkylene glycol is a 4- or 8-branched polyethylene glycol.

3. Microcapsules according to claim 1 or 2, wherein the ratio of the number average molecular weight of the branched polyalkylene glycol to the number average molecular weight of the polyhydroxyalkanoic acid in the polymer is 1:1 to 1:

6.

4. A composition containing the microcapsules according to claim 1 or 2.

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