Microspheres in which physiologically active substances are uniformly dispersed, and sustained release preparation containing same

Abstract

Provided are microspheres in which a physiologically active substance is homogeneously dispersed, and a sustained release preparation containing the same. The present application provides microspheres having a lactic acid / glycolic acid copolymer (PLGA) or polylactic acid (PLA) as the main component and in which a physiologically active substance is uniformly dispersed, characterized in that the average volume-based particle diameter of the microspheres is 1-150 [mu] m, and a cross-sectional observation sample obtained by cutting the microspheres is prepared, the cross-sectional observation sample is subjected to electron microscope observation at or above a magnification at which the physiologically active substance in the microspheres can be identified, an image of the electron microscope cross-sectional observation is divided into six regions, and the ratio (s / A) * 100 (%) of the region area (A) of each of the divided regions to the sum (s) of the cross-sectional areas of the physiologically active substance contained in the regions is calculated. The coefficient of variation of the ratio calculated in the six regions is 0.35 or less. The microspheres of the present invention can appropriately control the initial release amount of the physiologically active substance and the release rate during the subsequent release period, and can continuously release the physiologically active substance in a living body for a certain period of time.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202080100595.1, application date September 11, 2020, and invention name “Microspheres uniformly dispersed with physiologically active substances and sustained-release preparations containing the same”. Technical Field

[0002] The present invention relates to microspheres uniformly dispersed with physiologically active substances and sustained-release preparations containing the microspheres, and in particular to microspheres uniformly dispersed with physiologically active substances and mainly composed of lactic acid / glycolic acid copolymer (PLGA) or polylactic acid (PLA) and sustained-release preparations containing the microspheres. Background Art

[0003] Microspheres or nanospheres have recently attracted much attention as sustained-release preparations such as medicines containing physiologically active substances. Microspheres generally refer to preparations with a particle size of about 1 μm to 150 μm, and preparations smaller than 1 μm are called nanospheres. For example, they can encapsulate physiologically active substances in biodegradable synthetic polymers or natural polymers, locally release physiologically active substances, or target physiologically active substances to tissues.

[0004] In a sustained-release microsphere preparation that slowly releases a physiologically active substance at a certain rate, for example, it is necessary to appropriately control the preparation of biodegradable polymers, physiologically active substances, additives, solvents, etc. In order for the sustained-release microsphere preparation to effectively exhibit a pharmacological effect in a certain period of time in the body, it is necessary to appropriately control the initial release amount of the physiologically active substance and the release rate during the subsequent release period, so that the physiologically active substance is continuously released in the body for a certain period of time.

[0005] One of the important factors that determine the release rate of the physiologically active substance is the type of biodegradable polymer. In particular, the most widely used lactic acid / glycolic acid copolymer (Polylactide-co-glycolide Acid, PLGA) and polylactic acid (PLA) as a polymer of lactic acid have different decomposition rates in the body according to their physicochemical properties, such as the ratio of lactic acid and glycolic acid as constituent components, molecular weight, water affinity, etc., so they can be used to adjust the release period to a desired period (Patent Document 1).

[0006] Furthermore, on this basis, in order to suppress the abnormal initial release amount (initial burst) of the physiologically active substance and control the release rate during the release period to be constant, the particle size of the microspheres and the dispersion state of the physiologically active substance in the microspheres are involved. The particle size of the microspheres has a yield problem, but it can be adjusted to the target particle size by filtering and other operations. However, the dispersion state of the physiologically active substance in the microspheres is only considered to be uniform and has not been confirmed.

[0007] Microspheres of PLGA or PLA can be produced, for example, by using a liquid drying method, a spray drying method, a spray freeze drying method, a drying method using a supercritical fluid process, a double emulsion method, etc. Among them, the most commonly used production method is a liquid drying method in which, when the physiologically active substance is lipophilic, PLGA or PLA and the physiologically active substance are dissolved or dispersed in an organic solvent, the mixture is mixed with an aqueous solution in which polyvinyl alcohol (PVA) is dissolved to form an emulsion, and the solvent is removed from the emulsion.

[0008] Patent Document 1 discloses a method for producing sustained-release microspheres containing a biodegradable polymer such as PLGA and a peptide drug by spray drying, spray freeze drying, or a drying method using a supercritical fluid process. However, there is no description of the extent to which the particle size of the sustained-release microspheres fluctuates, whether the peptide drug is uniformly dispersed in the sustained-release microspheres, or whether a uniform product can be obtained.

[0009] Patent Document 2 discloses a method for producing PLGA microparticles by a liquid drying method using a mixed solvent consisting of a halogenated hydrocarbon and a non-aqueous miscible organic solvent having a solubility of the drug of 0.3% (W / V) or more. Although the particle diameters (median particle diameters) of the microparticles obtained in Production Examples 1 and 2 are described as 14 and 16 μm, there is no description of the dispersion state of the drug in the microparticles.

[0010] Patent document 3 discloses PLGA nanoparticles containing drugs. These nanoparticles are mainly used for targeting specific tissues, and therefore are nanoparticles of about tens to hundreds of nm that can pass through tiny holes in capillaries. However, in Patent document 3, there is no record of microspheres of 1 μm or more that are much larger than these nanoparticles. In addition, even if the technology of Patent document 3 is used, those skilled in the art cannot produce microspheres with a particle size of more than 1 μm.

[0011] Patent Document 4 discloses a preparation that releases leuprolide acetate, a luteinizing hormone-releasing hormone derivative, for about one to several months by subcutaneous injection. This preparation has a problem that the particle size distribution is very wide, ranging from 1 μm to 400 μm. Therefore, Patent Document 5 proposes a method of using a double emulsion method to produce microspheres in which a physiologically active substance is enclosed in a carrier polymer as a method for solving this problem. However, for the microspheres containing leuprolide acetate obtained in Examples 1 to 5, there is no description of the dispersion state of the drug in the particles.

[0012] Patent document 6 discloses microspheres that relieve chronic pain for at least 28 days (672 hours). The microspheres contain a biodegradable polymer and a local anesthetic (physiologically active substance), and about 75% of the local anesthetic is released until about 72 hours, and about 80-90% of the local anesthetic is released until about 120 hours. This suggests that the distribution of the local anesthetic in the microspheres is uneven in the microspheres and is biased toward the outside. Figure 2 The SEM (scanning electron microscope) image of the cross section of the microspheres described in does not allow confirmation of the dispersion state of the local anesthetic.

[0013] Patent Document 7 discloses a core-shell structured microsphere, wherein the core contains solid aripiprazole and the shell containing a biodegradable polymer covers the surface of the core. Thus, the microsphere of Patent Document 7 is not a microsphere in which the physiologically active substance is uniformly dispersed in the microsphere. Figure 5 In the electron micrograph of the cross section of the microspheres obtained in the described examples, the dispersion state of aripiprazole in the shell could not be confirmed.

[0014] Prior art literature

[0015] Patent Literature

[0016] Patent Document 1: Japanese Patent Application Publication No. 2005-035994

[0017] Patent Document 2: Japanese Patent Application Publication No. 2005-015476

[0018] Patent Document 3: Japanese Patent No. 4856752

[0019] Patent Document 4: Japanese Patent No. 2653255

[0020] Patent Document 5: Japanese Patent Application Publication No. 2014-224114

[0021] Patent Document 6: Japanese Patent Application Publication No. 2016-069378

[0022] Patent Document 7: Japanese Patent Application No. 2010-531303 Summary of the invention

[0023] Problems to be solved by the invention

[0024] For biodegradable polymer microspheres with an average volume reference particle size of more than 1 μm and less than 150 μm, if the distribution of physiologically active substances (sometimes also recorded as drugs) in the microspheres is not controlled, the release period cannot be achieved as designed. For example, if the physiologically active substance is biased toward the surface of the microspheres, a large amount of physiologically active substances are released from the microspheres in the early stage after administration, resulting in the problem of initial burst. On the contrary, if the physiologically active substance is biased toward the center of the microspheres, or if it is in a core-shell state, it cannot be released continuously from the initial stage. Therefore, it is desired that the physiologically active substances of the microparticles are uniformly dispersed. In a dispersed state such as a large mass of physiologically active substances, it is impossible to release continuously from the initial stage. Similarly, if the pores are not controlled, the same problem occurs in the release of physiologically active substances.

[0025] In fact, when investigating pharmacokinetics using small animals such as rats, there are sometimes large fluctuations in release rate and release curve, and the reason is often concluded to be individual differences among rats, etc. However, if the dispersion state of the physiologically active substance in the microspheres is uniform, the frequency of fluctuations can be further improved, and the release of the physiologically active substance from the microspheres can be achieved as designed by controlling the decomposition rate through the type and molecular weight of PLGA.

[0026] Uniformly dispersing physiologically active substances in microspheres is an absolute condition for sustained release of physiologically active substances in vivo for a certain period of time. However, the current situation is that uniform dispersion has not yet been explored. Since the particle size of microspheres is different from that of nanoparticles and is larger, it is generally difficult to homogenize. Therefore, it is necessary to confirm the dispersion state of the physiologically active substances in the microspheres. For this reason, a cross-sectional observation sample obtained by cutting the particles of the microspheres is made, and electron microscopic observation is performed at a magnification that can confirm the physiologically active substances in the microspheres, and it is easy to implement and reliable to confirm that the dispersion state thereof.

[0027] Figure 1 This is an electron micrograph of a microcapsule-type sustained-release preparation, Lepra (registered trademark), for injection 1.88 mg (manufactured by Takeda Pharmaceutical Company, Ltd.), which corresponds to the long-term sustained-release microcapsule of the LH-RH derivative described in Patent Document 4.

[0028] The present preparation includes various particles ranging from large particles to small particles, but a particle of about 6 μm is selected as a representative particle. The SEM (scanning electron microscope) image of the cross section of the particle is as follows: Figure 2 By confirming the edge effect in the image, it can be seen from SEM-EDS (energy dispersive X-ray spectrometer) that the large dispersed bodies indicated by the arrows in the figure are pores. Figure 3 Yes Figure 2The SEM cross-sectional image was divided into 6 regions (regions 1 to 6) on the image, and the commercially available image processing and analysis software iTEM (TEM camera control, image analysis software, manufactured by EMSIS) was used to average the pixels in a range of 3×3, and then the edge portions were emphasized to optimize the contrast. After that, the image was binarized, and noise was removed and particles with low contrast were emphasized during image processing. The image was then averaged again in a range of 3×3 to emphasize the edge portions. Based on Figure 3 , the coefficient of variation of the ratio (s / A) × 100 (%) of the area (A) of each divided region to the sum of the cross-sectional areas (s) of the above-mentioned physiologically active substances contained in the region was calculated, and the result was 1.06. By implementing this, the dispersion state of the physiologically active substances can be confirmed from the particle cross section. This coefficient of variation exceeds 0.35, indicating that the physiologically active substances dispersed in the particles are not uniformly dispersed, so the release rate during the release period cannot be properly controlled in this preparation.

[0029] Therefore, an object of the present invention is to provide microspheres capable of appropriately controlling the initial release amount of a physiologically active substance and the release rate during the subsequent release period, thereby continuously releasing the physiologically active substance for a certain period of time in vivo.

[0030] Means for solving problems

[0031] The inventors of the present invention have conducted in-depth research to solve the above-mentioned problems, and found that the microspheres in which the coefficient of variation of the area ratio of the physiologically active substance in the six regions of the electron microscope cross-section observation image of the microspheres is 0.35 or less, the physiologically active substance is uniformly dispersed in the microspheres without uneven pores, so that the initial release amount of the physiologically active substance and the release rate during the subsequent release period can be appropriately controlled, and the physiologically active substance can be continuously released in the body for a certain period of time, and the present invention has been completed. That is, the present invention is as follows.

[0032] [1] A first aspect of the present invention is a microsphere comprising a lactic acid / glycolic acid copolymer (PLGA) or a polylactic acid (PLA) as a main component in which a physiologically active substance is uniformly dispersed, wherein:

[0033] The average volume particle size of the microspheres is 1 μm to 150 μm or less.

[0034] A cross-sectional observation sample is prepared by cutting the microspheres, and the cross-sectional observation sample is observed by an electron microscope at a magnification greater than that capable of confirming the physiologically active substances in the microspheres. The electron microscope cross-sectional observation image is divided into 6 parts, and the ratio (s / A) × 100 (%) of the area (A) of each divided region to the sum of the cross-sectional areas (s) of the physiologically active substances contained in the region is calculated, and the coefficient of variation of the calculated ratio in the 6 regions is less than 0.35.

[0035] [2] A second aspect of the present invention is the microsphere according to [1], wherein the physiologically active substance is a lipophilic physiologically active substance.

[0036] [3] A third aspect of the present invention is the microsphere according to [1] or [2], wherein the dispersed physiologically active substance has an average volume-based particle size of 5 nm to 500 nm.

[0037] [4] A fourth aspect of the present invention is the microsphere according to any one of [1] to [3], wherein the content of the physiologically active substance in the microsphere is 0.35 to 1.5% by mass.

[0038] [5] A fifth aspect of the present invention is a sustained-release preparation comprising the microspheres according to any one of [1] to [4].

[0039] The microspheres of the present invention can appropriately control the initial release amount of the physiologically active substance and the release rate during the subsequent release period, thereby continuously releasing the physiologically active substance in vivo for a certain period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a SEM (scanning electron microscope) image of Leuproin (registered trademark) injection 1.88 mg (manufactured by Takeda Pharmaceutical Company, Ltd.).

[0041] Figure 2 This is a SEM (scanning electron microscope) image of a cross section of representative particles of Lepra (registered trademark) for injection 1.88 mg (manufactured by Takeda Pharmaceutical Company, Ltd.).

[0042] Figure 3 Yes Figure 2 The cross-sectional image is divided into six regions, and the image is binarized in order to obtain the ratio (s / A)×100(%) of the area (A) of each divided region to the total cross-sectional area (s) of the physiologically active substance contained in the region.

[0043] Figure 4 This is a SEM image of a cross section of the microspheres of Reference Example 1 that do not contain a physiologically active substance.

[0044] Figure 5 Examples of methods for dividing microspheres are shown. (A) is divided into 6 on concentric circles. (B) is divided into 6 in the longitudinal direction. (C) is divided into 6 in the circumferential direction.

[0045] Figure 6-1 is a SEM image of a cross section of the microsphere of Example 1.

[0046] Figure 6-2 Yes Figure 6-1 The cross-sectional image is divided into six regions, and the image is binarized in order to obtain the ratio (s / A)×100(%) of the area (A) of each divided region to the total cross-sectional area (s) of the physiologically active substance contained in the region.

[0047] Figure 7-1 is a SEM image of a cross section of the microsphere of Example 3.

[0048] Figure 7-2 Is magnification Figure 7-1 The cross-sectional image is binarized.

[0049] Figure 8-1 is a SEM image of a cross section of the microsphere of Example 4.

[0050] Figure 8-2 Yes Figure 8-1 The cross-sectional image is divided into six regions, and the image is binarized in order to obtain the ratio (s / A)×100(%) of the area (A) of each divided region to the total cross-sectional area (s) of the physiologically active substance contained in the region.

[0051] Fig. 9 The cross-sectional image of Comparative Example 1 is divided into six regions and binarized to obtain the ratio (s / A)×100(%) of the area (A) of each divided region to the total cross-sectional area (s) of the physiologically active substances contained in the region.

[0052] Fig.10 This is a SEM image of a cross section of the microsphere of Comparative Example 2.

[0053] Fig.11 This is a SEM image of a cross section of the microsphere of Comparative Example 3.

[0054] Fig.12 This is a SEM image of a cross section of the microsphere of Comparative Example 4. DETAILED DESCRIPTION

[0055] 1. Microspheres

[0056] The microspheres of the present invention are microspheres with lactic acid / glycolic acid copolymer (PLGA) or polylactic acid (PLA) as the main component in which physiologically active substances are uniformly dispersed, characterized in that the average volume particle size of the microspheres is 1 μm to 150 μm or less, a cross-sectional observation sample is prepared by cutting the microspheres, the cross-sectional observation sample is observed by electron microscopy at a magnification greater than that capable of confirming the physiologically active substances in the microspheres, the image observed by the electron microscopic cross-section is divided into 6 parts, and the ratio (s / A) × 100 (%) of the area (A) of each divided region to the sum of the cross-sectional areas (s) of the physiologically active substances contained in the region is calculated, and the coefficient of variation of the calculated ratio in the 6 regions is less than 0.35.

[0057] If the physiologically active substance is segregated in the surface layer or the center, or there are coarse particles, aggregates or large pores, the coefficient of variation of the above ratio becomes larger. If the coefficient of variation of the above ratio is 0.35 or less, the dispersion of the physiologically active substance is uniform. In the microspheres involved in the present invention, the coefficient of variation of the ratio of the area occupied by the physiologically active substance to the area of ​​each region in each of the six regions in which the cross section of the above microsphere is divided is 0.35 or less, preferably 0.25 or less, and more preferably 0.20 or less.

[0058] The microspheres of the present invention can appropriately control the initial release amount of the physiologically active substance and the release rate during the subsequent release period, thereby continuously releasing the physiologically active substance in vivo for a certain period of time.

[0059] <Observation of the cross section of microsphere>

[0060] The following describes a method for confirming the dispersion state of the physiologically active substance in the microspheres.

[0061] It can be implemented by observing the cross section of the microspheres using an electron microscope at a magnification that can clearly confirm the dispersed microparticles of the physiologically active substance. Electron microscopes include transmission electron microscopes (TEM) that use transmitted electrons as information sources and scanning electron microscopes (SEM) that detect secondary electrons (reflected electrons). It is selected according to the sample to be observed, but for nanospheres, etc., transmission electron microscopes can observe fine structures. Specifically, a thin film of gold, platinum, platinum-palladium alloy, etc. is first coated on the microspheres. In the embodiment, osmium is used for coating. Then, the microspheres are frozen using liquid nitrogen. After freezing, a cross section is made using FIB (focused ion beam). That is, using a FIB device, a focused ion beam is irradiated on the sample, and the structure of the desired position inside the sample is cut out, thereby making a cross-sectional observation sample of the microspheres. The preferred particle size of the microparticles of the physiologically active substance dispersed in the matrix of PLGA or PLA is several 10nm to several 100nm, but there are also cases of several μm. The whole cross section of the microsphere is observed with an electron microscope observation magnification that can confirm the dispersed particles of this size. Usually, the observation magnification of the electron microscope is more than 2500 times to hundreds of thousands times. In addition, when using a high magnification that cannot observe the whole microsphere, the observed parts can be connected and observed as a whole.

[0062] When the cross-sectional image is divided into six parts, for example, Figure 5 As shown in A to C of FIG. 1 , the structure may be divided into six parts in a concentric circle shape, in a longitudinal direction or in a transverse direction, or in a circumferential direction passing through the center. It is preferred to adopt a division method that significantly shows the segregation state of the physiologically active substance. For example, in the case of dividing into six parts in a concentric circle shape ( Figure 5 A), the radius must be divided into 6 equal parts from the center point of the maximum diameter of the microsphere cross section to form concentric circles. When dividing in the longitudinal or transverse direction ( Figure 5 B), must be divided into 6 equally spaced parts. Must be divided into 6 parts in any direction parallel or perpendicular to the above maximum diameter. In the case of dividing into 6 parts in the circumferential direction passing through the center ( Figure 5C), it must be divided into 6 parts every 60 degrees with the center point of the above-mentioned maximum diameter as the center. Although it also varies according to the constituent elements of the physiologically active substance, it is possible to use EDS (energy dispersive X-ray spectrometer) to perform elemental analysis on the cross section of the microsphere, and it is also possible to confirm whether it is a pore. In the case of pores, it cannot be accumulated in the area of ​​the physiologically active substance. In the absence of EDS detection elements, ruthenium tetroxide, osmium tetroxide, phosphotungstic acid, uranyl acetate, iodine, etc. can be used to stain the cross-sectional observation sample. In addition, by comparing with the microspheres to which the physiologically active substance is not added, the physiologically active substance can be determined. In the case where it is difficult to obtain contrast in the electron microscope cross-sectional observation image, the staining method is effective. The above is an example, and the sample can also be embedded in a resin, or a microtome can be used in the cross-sectional preparation of the microsphere.

[0063] In addition, with regard to the calculation of cross-sectional area, method is not particularly limited, preferably commercially available image processing analysis software is used. As commercially available image processing analysis software, various software such as Image-ProPlus (manufactured by Media Cybernetics), iTEM (TEM camera control, image analysis software, EMSIS company manufacture) can be used.

[0064] <PLGA and PLA>

[0065] PLGA is a lactic acid / glycolic acid copolymer having a structural unit derived from lactic acid and a structural unit derived from glycolic acid. PLA is a polymer of lactic acid. PLGA may include other biodegradable polymers such as polylactide (PLA) and polyglycolide (PGA). The PLGA described in this specification is a substance described as an example and is not limited to the described substances.

[0066] The molar ratio (L:G) of the structural unit (L) derived from lactic acid and the structural unit (G) derived from glycolic acid in PLGA is not particularly limited and can be appropriately selected according to the purpose, preferably 1:99 to 99:1, more preferably 25:75 to 99:1, further preferably 30:70 to 90:10, and particularly preferably 50:50 to 85:15. In addition, only PLA may be used. In order to achieve a uniform dispersion state of the physiologically active substance, the selection of the molar ratio is important, and the selection of the molecular weight of PLGA or PLA is equally important.

[0067] The PLGA used in the microspheres of the present invention can be produced, for example, by heating and polycondensing lactic acid and glycolic acid under weak reduced pressure using an ion exchange resin as a catalyst. In this case, lactide can also be used instead of lactic acid. PLGA can be a commercially available product. As a commercially available product, for example, it can be purchased from Fujifilm Wako Pure Chemical Industries (strain), Duomu Chemical (strain), EvonicRohm GmbH, Merck, Sigma-Aldrich, etc.

[0068] The content of PLGA or PLA in the microspheres of the present invention is not particularly limited and can be appropriately selected according to the purpose, and is preferably 1 mass % or more, more preferably 30 mass % or more and 95 mass % or less, and particularly preferably 50 mass % or more and 90 mass % or less.

[0069] <Microsphere>

[0070] The microspheres of the present invention contain PLGA or PLA and a physiologically active substance. Furthermore, as required, a dispersant and other components are contained. In the microspheres, a physiologically active substance, a dispersant, other components, etc. are dispersed in the matrix of PLGA or PLA.

[0071] [Physiologically active substances]

[0072] There is no particular limitation on the physiologically active substance contained in the microspheres of the present invention, and it can be appropriately selected according to the purpose, for example, pharmaceutical compounds, functional food compounds, functional cosmetic compounds, etc. can be listed. Microspheres containing pharmaceutical compounds can be used as sustained-release pharmaceutical preparations, for example. The physiologically active substance includes any one of a lipophilic physiologically active substance and a hydrophilic physiologically active substance. As preferred physiologically active substances, lipophilic physiologically active substances can be listed. Lipophilic physiologically active substances refer to substances with a logP value of 3 or more in the water / octanol partition coefficient, and physiologically active substances not contained in lipophilic physiologically active substances are classified as hydrophilic physiologically active substances. The water / octanol partition coefficient can be measured according to the flask oscillation method of JIS Z7260-107 (2000). As long as the physiologically active substance is a substance desired for a sustained-release preparation, there is no particular limitation, and it can be appropriately selected according to the purpose. Physiologically active substances also include any form of salts, hydrates, etc.

[0073] Physiologically active substances are uniformly dispersed in the microspheres of the present invention. By adopting this structure, the initial release amount of the physiologically active substance and the release rate during the subsequent release period can be appropriately controlled, and the physiologically active substance is continuously released in the body for a certain period of time. The uniform dispersion of the physiologically active substance in the microspheres can be controlled by the content of the physiologically active substance relative to the total amount of the microspheres. As the preferred content of the physiologically active substance, it varies according to the physiologically active substance, and for example, 0.1 to 3% by mass can be listed relative to the total amount of the microspheres, preferably 0.3 to 2% by mass, more preferably 0.35 to 1.5% by mass, and further preferably 0.5 to 1.25% by mass can be listed.

[0074] The average volume-based particle size of the dispersed microparticles of the physiologically active substance is preferably 5 nm to 500 nm, more preferably 10 nm to 400 nm, and even more preferably 20 nm to 200 nm.

[0075] [Dispersant]

[0076] In order to disperse the physiologically active substance, a dispersant can be used. As the dispersant, it can be a low molecular weight dispersant or a high molecular weight dispersant polymer. The low molecular weight dispersant means a compound with a weight average molecular weight of less than 15,000, and the high molecular weight dispersant polymer means a compound containing repeated covalent bonds between more than one monomer and a weight average molecular weight of more than 15,000.

[0077] As the low molecular weight dispersant, as long as it is a dispersant that is permissible in pharmaceutical compounds, functional food compounds, functional cosmetic compounds, etc., there is no particular limitation and it can be appropriately selected according to the purpose. Specifically, lipids, sugars, cyclodextrins, amino acids, organic acids, other components, etc. can be listed. They can be used alone or in combination of two or more.

[0078] As lipids, there are no particular restrictions and they can be appropriately selected according to the purpose, for example, medium-chain or long-chain monoglycerides, diglycerides or triglycerides, phospholipids, vegetable oils (such as soybean oil, avocado oil, squalene oil, sesame oil, olive oil, corn oil, rapeseed oil, safflower oil, sunflower oil, etc.), fish oil, seasoning oil, water-insoluble vitamins, fatty acids and mixtures thereof, derivatives thereof, etc. These can be used alone or in combination of two or more.

[0079] The sugars are not particularly limited and can be appropriately selected according to the purpose, and examples thereof include glucose, mannose, idose, galactose, fucose, ribose, xylose, lactose, sucrose, maltose, trehalose, turanose, raffinose, maltotriose, acarbose, water-soluble cellulose, synthetic cellulose, sugar alcohol, glycerol, sorbitol, lactitol, maltitol, mannitol, xylitol, erythritol, or polyols, or derivatives thereof, etc. These can be used alone or in combination of two or more.

[0080] There is no particular limitation on other components and they may be appropriately selected depending on the intended purpose, but components conventionally used in medicines are preferred.

[0081] <Average volume-based particle size>

[0082] The average volume reference particle size of the microspheres of the present invention is 1 μm or more and 150 μm or less, preferably 10 μm or more and 100 μm or less, more preferably 20 μm or more and 75 μm or less. The average volume reference particle size can be measured using a laser diffraction particle size distribution measuring device. In the present invention, if the average volume reference particle size exceeds 150 μm, the problem of initial outbreak will occur due to the uneven dispersion of the physiologically active substances in the microspheres, and it is easy to condense and settle, and the processing of the subsequent process becomes difficult. If it is less than 1 μm, the problem of initial outbreak will be significantly generated.

[0083] The microspheres of the present invention can appropriately control the initial release amount of the physiologically active substance and the release rate during the subsequent release period, thereby continuously releasing the physiologically active substance in vivo for a certain period of time.

[0084] 2. Sustained-release preparations

[0085] The microspheres of the present invention can be used to prepare sustained-release preparations containing microspheres. The sustained-release preparations of the present invention can appropriately control the initial release amount of the physiologically active substance and the release rate during the subsequent release period, and continuously release the physiologically active substance in the body for a certain period of time, effectively showing a pharmacological effect.

[0086] The sustained-release preparation of the present invention can be used as injection and implant, or as percutaneous agent, and is easily directly administered to lesions such as intramuscular, subcutaneous, blood vessel, organ, joint cavity, tumor, etc. It can also be administered as other various preparation forms. For example, in order to make the sustained-release preparation of the present invention into injection, it is made into aqueous suspension together with dispersant (Tween80, HCO-60, carboxymethyl cellulose, sodium alginate, etc.), preservative (methylparaben, propylparaben, etc.), isotonic agent (sodium chloride, mannitol, sorbitol, glucose, etc.), or dispersed with vegetable oils such as soybean oil, sesame oil, corn oil and made into oily suspension, thereby making sustained-release injection.

[0087] 3. Method for manufacturing microspheres

[0088] <Microsphere production process>

[0089] The method for producing microspheres of the present invention includes at least a particle formation step, and may further include a filtration sterilization step, a good solvent removal step, and other steps as necessary.

[0090] [Particle Formation Process]

[0091] The particle formation process preferably uses a particle processing device described in Japanese Patent Publication No. 2009-132871 or Japanese Patent Publication No. 2011-189348 for particle processing between a plurality of processing surfaces that are relatively arranged to be close or far away and at least one of which rotates relative to the other. The particle formation process is implemented, for example, in the following manner: using the above-mentioned particle processing device, a solution of PLGA or PLA and a physiologically active substance obtained by dissolving or dispersing PLGA or PLA and a physiologically active substance in a good solvent for PLGA or PLA is continuously added with a solution containing a poor solvent for PLGA or PLA to produce emulsified particles, and the good solvent is removed from the produced particles to precipitate the microspheres of the present invention. Here, "dispersion" includes: dispersing the physiologically active substance in a good solvent for PLGA or PLA in a solid state; emulsifying the physiologically active substance in a good solvent for PLGA or PLA; forming a w / o type emulsion containing an aqueous solution of a hydrophilic physiologically active substance and a good solvent for PLGA or PLA, etc.

[0092] As a solution of PLGA or PLA and a physiologically active substance, there is no particular limitation as long as it is a solution in which PLGA or PLA and a physiologically active substance are dissolved or dispersed in a good solvent for PLGA or PLA, and it can be appropriately selected according to the purpose. As a good solvent, there is no particular limitation and it can be appropriately selected according to the purpose, for example, halogenated aliphatic hydrocarbons, aliphatic esters, alcohols, ketones, ethers, acetonitrile, etc. can be listed. As halogenated aliphatic hydrocarbons, for example, dichloromethane, chloroform, carbon tetrachloride, ethyl chloride, 2,2,2-trichloroethane, etc. can be listed. As aliphatic esters, for example, ethyl acetate, propyl acetate, butyl acetate, etc. can be listed. As alcohols, for example, alcohols with low solubility in water such as benzyl alcohol, phenyl alcohol, and n-butanol can be listed. As ketones, for example, ketones with 3 to 6 carbon atoms (such as acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, etc.) can be listed. As ethers, for example, ethers with 2 to 6 carbon atoms (such as dimethyl ether, methyl ethyl ether, diethyl ether, etc.) can be listed. From the viewpoint of the content of physiologically active substances and the purpose of preventing initial outbreak, it is preferred to select a solvent with low solubility in water. As preferred good solvents, halogenated aliphatic hydrocarbons, ketones and mixed solvents thereof can be listed, and more preferably dichloromethane, acetone and mixed solvents thereof can be listed. In addition, they can be used alone or in combination of two or more. By changing the type of solvent and the mixing amount, the particle size can be controlled.

[0093] A good solvent means a solvent in which the solubility of PLGA or PLA is large, and a poor solvent means a solvent in which the solubility of PLGA or PLA is small or insoluble. As for good solvents and poor solvents, solvents are selected so that the physiologically active substances in the microspheres do not segregate or do not produce coarse particles or agglomerates. In addition, good solvents and poor solvents can be specified, for example, by the mass of PLGA or PLA that can be dissolved in 100g of solvent at 25°C. In the present invention, a good solvent is preferably a solvent that dissolves more than 0.1g of PLGA or PLA, more preferably more than 0.2g, and further preferably more than 0.5g. A poor solvent is preferably a solvent that dissolves only less than 0.05g of PLGA or PLA, more preferably less than 0.02g, and further preferably less than 0.01g. As a poor solvent, there is no particular limitation and it can be appropriately selected according to the purpose, preferably water.

[0094] The content of PLGA or PLA in the solution of PLGA or PLA and physiologically active substances can be changed according to a good solvent, according to the particle size of the target microsphere, and in addition to the way that the physiologically active substances are uniformly dispersed in the microsphere. The content of PLGA or PLA can be listed as 1 to 30% by mass, preferably 3 to 20% by mass, and more preferably 5 to 15% by mass. The content of the physiologically active substance in the PLGA or PLA solution can be appropriately changed according to the purpose, pharmacological effect, etc., and in addition to the way that the physiologically active substances in the microspheres are uniformly dispersed.

[0095] In order to further ensure the stability of the manufactured microspheres, a stabilizer can be added to the poor solvent. As a stabilizer, there is no particular limitation and it can be appropriately selected according to the purpose. For example, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), carboxymethyl cellulose (CMC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), lecithin, polysorbate 80, etc., preferably polyvinyl alcohol (PVA). In addition, the concentration of the stabilizer added is preferably 0.01 to 20% by mass, more preferably less than 5% by mass. As a preferred poor solvent, for example, PVA aqueous solution, etc. can be mentioned.

[0096] It is desirable to use a device that makes agitators of various shapes such as rod-shaped, plate-shaped, propeller-shaped rotate in a groove, a device with a screen that rotates relatively relative to the agitator, etc., to apply shear force to the fluid, so as to realize the preparation devices such as the rotary dispersion machine of homogeneous mixing to prepare the solution of PLGA or PLA and physiologically active substances and the solution comprising a poor solvent. As a preferred example of a rotary dispersion machine, the agitator disclosed in Japanese Patent No. 5147091 can be applied. In order to disperse the physiologically active substances in the microsphere uniformly, it is necessary to fully mix PLGA or PLA solution with a poor solvent. In order to fully mix, it is necessary to target the homogenization at least at the molecular level, and the reason why mixing insufficiently becomes an uneven dispersion state.

[0097] Rotary dispersion machine can also be carried out in a continuous manner with an intermittent manner. When being carried out in a continuous manner, the supply and discharge of the fluid for the stirred tank can be carried out continuously, or the continuous mixer can be used without using the stirred tank, and the stirring energy can be properly controlled using a known stirrer or stirring means. It should be noted that about stirring energy, it is described in detail in the Japanese Patent Laid-Open No. 04-114725 Gazette of the applicant. The method for stirring in the present invention is not particularly limited, and the stirrer, dissolver, emulsifier, dispersion machine, homogenizer, etc. of various shearing, friction, high pressure jet, ultrasonic, etc. can be used to implement. As an example, continuous emulsifiers such as Ultra-Turrax (manufactured by IKA), Polytron (manufactured by KINEMATICA), TK Homomixer (manufactured by PRIMIX), Ebara Milder (manufactured by EBARA MISSUES CO., LTD.), TK Homomic Line Flow (manufactured by PRIMIX), Colloid Mill (manufactured by Kobelco Environmental Solutions), THRASHER (manufactured by Nippon COKE Industries), Trigonal Wet Micro-Pulverizer (manufactured by Mitsui Miike Chemical Industry Co., Ltd.), Cavitron (manufactured by EUROTEC), and FineFlow Mill (manufactured by Pacific Machinery Co., Ltd.), and batch or continuous dual-purpose emulsifiers such as CLEARMIX (manufactured by M-Technique) and CLEARMIX DISSOLVER (manufactured by M-Technique) can be mentioned. In addition, for the stirring process, it is desirable to use a mixer having a high-speed rotating stirring blade, a screen on the outside of the stirring blade, and a fluid discharged as a jet from the opening of the screen. It is particularly desirable to use the above-mentioned CLEARMIX (manufactured by M-Technique) and CLEARMIX DISSOLVER (manufactured by M-Technique).

[0098] In the above-mentioned particle processing device, the particle size and particle size distribution of PLGA or PLA microparticles can be controlled by adjusting the contact surface pressure when the rotating processing surface stops. The experimental results of the inventors show that the contact surface pressure is preferably 20g / cm 2 ~250g / cm 2 The contact surface pressure is less than 20g / cm 2 In the case of , the film is unstable and the particle size distribution becomes broad. It is clear that if the contact surface pressure is higher than 250g / cm 2 , it becomes difficult to adjust the target particle size. More preferably, 50 g / cm 2 ~200g / cm 2 , and 80 g / cm 2 ~150g / cm 2 .

[0099] It is preferred to prevent the agglomeration of each microsphere formed by the solution of PLGA or PLA and the solution containing a poor solvent from being connected. As a method to prevent its agglomeration, it is preferred to put a solution containing a poor solvent in advance in a solution discharge liquid recovery tank and stir slowly. By stirring, the agglomeration of microspheres can be further suppressed. With regard to stirring, a rotary dispersing machine is preferred, and it is desired to be CLEARMIX DISSOLVER (manufactured by M-Technique). As long as the whole can be made to flow slowly, there is no particular limitation. If the stirring is strong, the emulsified particles of PLGA or PLA are destroyed, the distribution width widens, and the dispersed state of the physiologically active substance is likely to collapse.

[0100] When the physiologically active substance is a lipophilic physiologically active substance, the particle formation process can be suitably implemented according to the above description, so that microspheres can be manufactured. When the physiologically active substance is a hydrophilic physiologically active substance, the hydrophilic physiologically active substance can be dispersed in a good solvent of PLGA or PLA by using, for example, a dispersant, and the particle formation process is similarly implemented to manufacture microspheres.

[0101] In addition, when the physiologically active substance is a hydrophilic physiologically active substance, a w / o type emulsion prepared by dissolving the hydrophilic physiologically active substance together with a stabilizer as required in an aqueous solvent such as water and mixing it with a solution obtained by dissolving PLGA or PLA in a good solvent for PLGA or PLA can be used as a solution of PLGA or PLA and the physiologically active substance, and the above-mentioned particle processing device is used to implement the above-mentioned particle formation process. In the preparation of the w / o type emulsion, an intermittent oscillation method, a method using a mixer using a propeller type stirrer or a turbine type stirrer, a colloid mill method, a homogenizer method, and an ultrasonic irradiation method can be used. Using the above-mentioned particle processing device, a solution of PLGA or PLA and the physiologically active substance as the w / o type emulsion and a solution containing a poor solvent for PLGA or PLA are continuously added to produce emulsified particles as a w / o / w emulsion, and the good solvent is removed from the produced particles, thereby precipitating the microspheres of the present invention. The microspheres can be used directly, but excipients (mannitol, sorbitol, lactose, glucose, etc.) can also be further added, and then dispersed and freeze-dried or spray-dried to solidify. When the solidified microspheres are used, distilled water for injection or an appropriate dispersion medium can be added to obtain a more stable sustained-release injection.

[0102] [Filter sterilization process]

[0103] As required, before the particle formation step, the prepared PLGA or PLA and physiologically active substance solution and the solution containing a poor solvent are preferably sterile filtered. The solution containing a poor solvent can be sterilized by filtration using a hydrophilic filter, and the solution of PLGA or PLA and physiologically active substance can be sterilized by filtration using a hydrophobic filter. The pore size of the filter used for filtration is preferably 0.1 μm to 0.45 μm, more preferably 0.2 μm.

[0104] As the above-mentioned filtration sterilization filter, there is no particular restriction, and it can be suitably selected according to the purpose. For example, in order to sterilize and filter the solution comprising a poor solvent, hydrophilic filters such as polyvinylidene fluoride (PVDF), polyether sulfone can be listed. In order to filter and sterilize the solution of PLGA or PLA and physiologically active substances, hydrophobic filters such as polytetrafluoroethylene (PTFE) can be listed. It is not limited to the material described here, and it is necessary to select according to the adsorption of medicine, the solvent type.

[0105] [Good solvent removal step]

[0106] In the good solvent removal process, the good solvent is removed from the emulsified particles containing PLGA or PLA and the physiologically active substance. As far as the good solvent removal process is concerned, there is no particular limitation as long as the good solvent can be removed from the liquid containing the emulsified particles in a state where the physiologically active substance in the microspheres is uniformly dispersed, and it can be appropriately selected according to the purpose. For example, a method of removing the good solvent from the liquid by evaporating the good solvent by heating the liquid while stirring, flowing a gas such as nitrogen to the liquid surface of the liquid, and decompressing the liquid can be cited, and a method of flowing a gas such as nitrogen to the liquid surface of the liquid is preferably cited. In order to maintain a uniformly dispersed state of the physiologically active substance in the microspheres, it is mostly preferred to remove the good solvent quickly, but sometimes it is appropriate to remove it slowly. As the time for removing the good solvent, for example, 30 minutes to 12 hours can be cited, preferably 1 to 10 hours, and more preferably 1 to 5 hours can be cited.

[0107] In addition, the temperature when removing the good solvent varies depending on the type of good solvent. It is necessary to observe the cross section of the microsphere from a high temperature near the boiling point of the good solvent to a low temperature and to implement it at an appropriate temperature. When removing the good solvent, when the concentration of PLGA or PLA is low, the volume of the particles changes greatly, and it is often seen that the physiologically active substances that have been dispersed with great difficulty aggregate, so it is necessary to pay attention.

[0108] [Other processes]

[0109] As other steps, for example, solvent composition preparation, classification step, particle washing step, etc. can be cited. Usually, coarse powder removal and fine powder removal are performed in the classification step, but the particles produced in the present invention basically do not need to be subjected to the classification step. However, for the sake of caution, the classification step may also be included.

[0110] The above-mentioned manufacturing method can manufacture microspheres having a particle size of 1 μm to 150 μm or less and in which the physiologically active substances in the microspheres are in a uniformly dispersed state. That is, the following microspheres can be manufactured: a cross-sectional observation sample is prepared by cutting the particles of the microspheres, and an electron microscope observation is performed at a magnification that can confirm the physiologically active substances in the microspheres, and the cross-sectional image is divided into 6 parts, and the ratio of the area (A) of each divided area to the sum of the cross-sectional areas (s) of the above-mentioned physiologically active substances contained in the area is (s / A)×100(%), and the coefficient of variation of the area ratio occupied by the physiologically active substances in the divided 6 areas is less than 0.35.

[0111] Example

[0112] Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples.

[0113] (Reference Example 1)

[0114] Microspheres (PLGA microparticles) containing no physiologically active substance were produced in Reference Example 1. The dispersion state of the physiologically active substance in the microspheres of Examples and Comparative Examples was confirmed below by observing the cross-sections of the microspheres of Examples and Comparative Examples using the microspheres of Reference Example 1 as an index by SEM images.

[0115] <Preparation of PLGA solution and PVA aqueous solution>

[0116] Dichloromethane (manufactured by Kanto Chemical) is added in a manner that lactic acid / glycolic acid copolymer (Resomer RG504, manufactured by Evonik) becomes 13% by mass, and a high-speed rotary disperser CLEARMIX DISSOLVER (manufactured by M-Technique) is used to dissolve it to obtain a PLGA solution. Then, filter it with a 0.2μm ventilation filter (φ62, manufactured by Merck). Add ion exchange water in a manner that polyvinyl alcohol (PVA, EG-40P, manufactured by Nippon Synthetic Chemical Industry) becomes 1.5% by mass, and a high-speed rotary disperser CLEARMIX (manufactured by M-Technique) is used to dissolve it to obtain a PVA aqueous solution. Then, filter it with a hydrophilic PVDF membrane filter (φ47, manufactured by Merck). The PVA aqueous solution is pre-placed in the tank for recovering the PLGA emulsified particles and stirred to the extent that the liquid surface moves.

[0117] <Preparation of microspheres (PLGA microparticles)>

[0118] As particle formation operation, use the particle treatment device of recording in Japanese Unexamined Patent Publication No. 2011-189348 communique that PLGA solution and the PVA aqueous solution of preparation are mixed.At this, the particle treatment device of recording in Japanese Unexamined Patent Publication No. 2011-189348 communique is the device of recording in Figure 25 of this communique, and wherein the 2nd introduction port d20 is the concentric annular shape of the opening of the central authorities with face 2 around the processing as annular disk, and disk diameter is 75mm. Specifically, the prepared PVA aqueous solution is introduced into the processing surfaces 1 and 2 from the first introduction part d1 at 0.02MPaG, 65mL / min, and 30°C, while the processing part 10 is rotated at 2000rpm, and the prepared PLGA solution is introduced into the processing surfaces 1 and 2 from the second introduction part d2 at 0.65MPaG, 20mL / min, and 30°C, and the PVA aqueous solution and the PLGA solution are mixed in a forced film to produce PLGA emulsified particles containing dichloromethane between the processing surfaces 1 and 2. The fluid containing the PLGA emulsified particles between the processing surfaces 1 and 2 (hereinafter referred to as the PLGA emulsified particle dispersion) is discharged from the processing surfaces 1 and 2 of the particle processing device. The discharged PLGA emulsified particle dispersion is recovered in a recovery tank.

[0119] Next, as a desolvation step, the effluent was stirred at a circumferential speed of 4.7 m / s using a CLEARMIX DISSOLVER (manufactured by M-Technique) while argon gas was flowed to spray toward the liquid surface, and dichloromethane was removed over 3.5 hours to obtain a suspension containing PLGA microparticles (PLGA microparticle suspension). The average volume-based particle size of the obtained PLGA microparticles was 34.0 μm. After freezing representative particles with liquid nitrogen, FIB cross sections were prepared and SEM images were observed ( Figure 4 ).

[0120] like Figure 4 As shown, it was confirmed that there were no particle-like masses and pores in the above FIB cross section. In addition, it was found that the cross section of the microsphere of Reference Example 1 can be used as an indicator in the observation of the cross section of the microsphere of the Example and the Comparative Example.

[0121] (Example 1)

[0122] <Preparation of PLGA and physiologically active substance solutions and PVA aqueous solutions>

[0123] In a manner where lactic acid / glycolic acid copolymer (Resomer RG752H, manufactured by Evonik) is 10% by mass and curcumin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Wako Special Grade) as a physiologically active substance is 0.5% by mass, 64.5% by mass of dichloromethane (manufactured by Kanto Chemical) and 25% by mass of acetone (manufactured by Kanto Chemical) are added, and a high-speed rotary disperser CLEARMIX DISSOLVER (manufactured by M-Technique) is used to dissolve it to obtain a solution of PLGA and physiologically active substances. Afterwards, it is filtered with a 0.2μm ventilation filter (φ62, manufactured by Merck). The PVA aqueous solution is prepared in the same manner as in Reference Example 1. The PVA aqueous solution is pre-placed in a tank for recovering the emulsified particles of PLGA and physiologically active substances, and stirred to the extent that the liquid surface moves.

[0124] <Preparation of microspheres>

[0125] As a particle formation process, the prepared PLGA and physiologically active substance solution and the PVA aqueous solution were mixed using a particle processing device described in Japanese Patent Publication No. 2011-189348 as in Reference Example 1. Specifically, the prepared PVA aqueous solution was introduced into the processing surface 1 and 2 from the first introduction part d1 at 0.0 MPaG, 50 mL / min, and 25° C., while the processing part 10 was rotated at 5000 rpm, the prepared PLGA and physiologically active substance solution was introduced into the processing surface 1 and 2 from the second introduction part d2 at 0.3 MPaG, 16 mL / min, and 25° C., and the PVA aqueous solution was mixed with the PLGA and physiologically active substance solution in a forced film, and emulsified particles of PLGA and physiologically active substance containing dichloromethane were manufactured between the processing surfaces 1 and 2. The fluid containing emulsified particles of PLGA and a physiologically active substance (hereinafter referred to as an emulsified particle dispersion of PLGA and a physiologically active substance) between the processing surfaces 1 and 2 is discharged from between the processing surfaces 1 and 2 of the particle forming treatment device. The emulsified particle dispersion of PLGA and a physiologically active substance used to capture the discharged emulsified particle dispersion of PLGA and a physiologically active substance is recovered in a recovery tank maintained at a pressure of 0.03 MPaG.

[0126] Next, as a desolvation step, the above-mentioned discharge liquid was stirred at a circumferential speed of 4.7 m / s using CLEARMIX DISSOLVER (manufactured by M-Technique) while argon gas was flowed to spray toward the liquid surface, and dichloromethane and acetone were removed over 3.5 hours to obtain a suspension containing microspheres (microsphere suspension). The average volume-based particle size of the obtained microspheres was 7.5 μm. After freezing representative particles with liquid nitrogen, FIB cross-sections were prepared and SEM images were observed ( Figure 6-1). The particle cross-sections of the obtained SEM images were averaged with a pixel range of 3×3 using iTEM (TEM camera control, image analysis software, manufactured by EMSIS), and the edge portions were emphasized to optimize the contrast. Then, binarization was performed, and noise was removed and particles with low contrast were emphasized in the image processing. The images were averaged again with a pixel range of 3×3 and the edge portions were emphasized. Figure 6-2 2 shows an image in which the particle cross section is divided into six regions (regions 1 to 6) at intervals of 60 degrees with the center point of the maximum diameter as the center.

[0127] Figure 6-2 The coefficient of variation of the ratio (s / A)×100(%) of the area (A) of each region obtained by segmentation in the FIB cross section of the curcumin particle and the total cross-sectional area (s) of the physiologically active substance contained in the region was 0.161.

[0128] (Example 2)

[0129] A suspension containing microspheres was prepared in the same manner as in Example 1, except that polylactic acid (Resomer R202H) was used instead of lactic acid / glycolic acid copolymer (Resomer RG504, manufactured by Evonik). The average volume-based particle size of the obtained microspheres was 7.3 μm, and representative particles were frozen with liquid nitrogen, and FIB cross sections were prepared to observe SEM images.

[0130] The observed SEM image was analyzed by the same method as in Example 1. The coefficient of variation of the ratio (s / A) × 100 (%) of the area (A) of each divided region to the sum of the cross-sectional areas (s) of the above-mentioned physiologically active substances contained in the region was 0.214.

[0131] (Example 3)

[0132] Dichloromethane (manufactured by Kanto Chemical) was added in a manner that lactic acid / glycolic acid copolymer (Resomer RG504, manufactured by Evonik) was 13% by mass and progesterone (manufactured by Sigma-Aldrich) as a physiologically active substance was 1.0% by mass, and a high-speed rotary disperser CLEARMIX DISSOLVER (manufactured by M-Technique) was used to dissolve it to obtain a solution of PLGA and the physiologically active substance. After that, it was filtered with a 0.2 μm ventilation filter (φ62, manufactured by Merck). The PVA aqueous solution was prepared in the same manner as in Reference Example 1. The PVA aqueous solution was pre-placed in a tank for recovering the emulsified particles of PLGA and the physiologically active substance, and stirred to the extent that the liquid surface moved.

[0133] <Preparation of microspheres>

[0134] As a particle formation process, the prepared PLGA and physiologically active substance solution and the PVA aqueous solution were mixed using a particle processing device described in Japanese Patent Publication No. 2011-189348 as in Reference Example 1. Specifically, the prepared PVA aqueous solution was introduced into the processing surface 1 and 2 from the first introduction part d1 at 0.0 MPaG, 50 mL / min, and 30° C., while the processing part 10 was rotated at 1700 rpm, the prepared PLGA and physiologically active substance solution was introduced into the processing surface 1 and 2 from the second introduction part d2 at 0.35 MPaG, 16 mL / min, and 30° C., and the PVA aqueous solution was mixed with the PLGA and physiologically active substance solution in a forced film, and emulsified particles of PLGA and physiologically active substance containing dichloromethane were manufactured between the processing surfaces 1 and 2. The fluid containing emulsified particles of PLGA and a physiologically active substance (hereinafter referred to as an emulsified particle dispersion of PLGA and a physiologically active substance) between the processing surfaces 1 and 2 is discharged from between the processing surfaces 1 and 2 of the particle forming treatment device. The emulsified particle dispersion of PLGA and a physiologically active substance used to capture the discharged emulsified particle dispersion of PLGA and a physiologically active substance is recovered in a recovery tank maintained at a pressure of 0.02 MPaG.

[0135] The desolventizing step was carried out in the same manner as in Examples 1 and 2, and the average volume-based particle size of the obtained microspheres was 34.8 μm. After freezing representative particles with liquid nitrogen, FIB cross sections were prepared and SEM images were observed ( Figure 7-1 ).

[0136] The observed SEM images were analyzed by the same method as in Examples 1 and 2. The SEM cross section after enlargement and binarization is shown in FIG. Figure 7-2 The coefficient of variation of the ratio (s / A) × 100 (%) of the area (A) of each region obtained by dividing the region into 6 regions at 60-degree intervals with the center point of the maximum diameter as the center and the sum of the cross-sectional areas (s) of the above-mentioned physiologically active substances contained in the region was 0.056.

[0137] (Example 4)

[0138] Dichloromethane (manufactured by Kanto Chemical) was added in a manner that lactic acid / glycolic acid copolymer (Resomer RG504, manufactured by Evonik) was 13% by mass and probucol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., for biochemical use) as a physiologically active substance was 1.0% by mass, and a high-speed rotary disperser CLEARMIX DISSOLVER (manufactured by M-Technique) was used to dissolve it to obtain a solution of PLGA and the physiologically active substance. Afterwards, it was filtered with a 0.2 μm ventilation filter (φ62, manufactured by Merck). The PVA aqueous solution was prepared in the same manner as in Reference Example 1. The PVA aqueous solution was pre-placed in a tank for recovering the emulsified particles of PLGA and the physiologically active substance, and stirred to the extent that the liquid surface moved.

[0139] The desolventizing step was carried out in the same manner as in Examples 1 to 3, and the average volume-based particle size of the obtained microspheres was 32.5 μm. After freezing representative particles with liquid nitrogen, FIB cross sections were prepared and SEM images were observed ( Figure 8-1 ).

[0140] The observed SEM images were analyzed in the same manner as in Examples 1 to 3. The SEM cross section after binarization is shown in FIG. Figure 8-2 The coefficient of variation of the ratio (s / A) × 100 (%) of the area (A) of each region obtained by dividing the region into 6 regions at 60 degree intervals with the center point of the maximum diameter as the center and the sum of the cross-sectional areas (s) of the above-mentioned physiologically active substances contained in the region was 0.235.

[0141] (Comparative Example 1)

[0142] As in Example 4, as a particle formation process, an emulsified particle dispersion of PLGA and a physiologically active substance was prepared. Then, as a desolvation process, a CLEARMIX DISSOLVER (manufactured by M-Technique) was used to stir the recovered discharge at a circumferential speed of 4.7 m / sec, while removing dichloromethane in the atmosphere for 42 hours to obtain a suspension (microsphere suspension) containing microspheres. The average volume reference particle size of the obtained microspheres was 31.8 μm, and after freezing representative particles with liquid nitrogen, a FIB cross section was made and an SEM image was observed.

[0143] The observed SEM images were analyzed in the same manner as in Examples 1 to 3. The SEM cross section after binarization is shown in FIG. Fig. 9 The coefficient of variation of the ratio (s / A) × 100 (%) of the area (A) of each region obtained by dividing the radius into 6 equal parts from the center point of the maximum diameter in a concentric circle and the sum of the cross-sectional areas (s) of the above-mentioned physiologically active substances contained in the region is 0.468.

[0144] (Comparative Example 2)

[0145] The same formulation as in Example 4 was used, but the dissolution of PLGA and the drug was changed to stirring for 10 minutes using a propeller stirrer (manufactured by Three-One Motor), and the particle formation step and the desolvation step were performed under the same conditions as in Example 4 to prepare a suspension containing microspheres. The average volume-based particle size of the obtained microspheres was 29.8 μm. After freezing representative particles with liquid nitrogen, FIB cross sections were prepared and SEM images were observed ( Fig.10 ).

[0146] The observed SEM images were analyzed using the same method as in Examples 1 to 4, and the coefficient of variation (s / A) × 100 (%) of the ratio of the area (A) of each region obtained by dividing the region into 6 regions every 60 degrees with the center point of the maximum diameter as the center to the sum of the cross-sectional areas (s) of the above-mentioned physiologically active substances contained in the region was 0.357.

[0147] (Comparative Example 3)

[0148] In a manner where lactic acid / glycolic acid copolymer (Resomer RG504, manufactured by Evonik) is 5.0% by mass and curcumin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Wako Special Grade) as a physiologically active substance is 0.25% by mass, 69.75% by mass of dichloromethane (manufactured by Kanto Chemical) and 25% by mass of acetone (manufactured by Kanto Chemical) are added, and a high-speed rotary disperser CLEARMIX DISSOLVER (manufactured by M-Technique) is used to dissolve it to obtain a solution of PLGA and physiologically active substances. Afterwards, it is filtered with a 0.2μm ventilation filter (φ62, manufactured by Merck). The PVA aqueous solution is prepared in the same manner as in Reference Example 1. The PVA aqueous solution is pre-placed in a tank for recovering the emulsified particles of PLGA and physiologically active substances, and stirred to the extent that the liquid surface moves.

[0149] The particle formation step and the solvent removal step were carried out under the same conditions as in Example 1 to prepare a suspension containing microspheres. The average volume-based particle size of the obtained microspheres was 6.8 μm. After freezing representative particles with liquid nitrogen, FIB cross sections were prepared and SEM images were observed ( Fig.11 ).

[0150] The observed SEM images were analyzed using the same method as in Examples 1 to 4, and the coefficient of variation (s / A) × 100 (%) of the ratio of the area (A) of each region obtained by dividing the region into 6 regions every 60 degrees with the center point of the maximum diameter as the center to the sum of the cross-sectional areas (s) of the above-mentioned physiologically active substances contained in the region was 0.386.

[0151] (Comparative Example 4)

[0152] 69.5% by mass of dichloromethane (manufactured by Kanto Chemical) and 25% by mass of acetone (manufactured by Kanto Chemical) were added to a lactic acid / glycolic acid copolymer (Resomer RG504, manufactured by Evonik) at 5.0% by mass and a progesterone (manufactured by Sigma-Aldrich) as a physiologically active substance at 0.3% by mass, and the mixture was dissolved using a high-speed rotary disperser CLEARMIX DISSOLVER (manufactured by M-Technique) to obtain a solution of PLGA and the physiologically active substance. Afterwards, the mixture was filtered using a 0.2 μm ventilation filter (φ62, manufactured by Merck). The PVA aqueous solution was prepared in the same manner as in Reference Example 1. The PVA aqueous solution was placed in advance in a tank for recovering the emulsified particles of PLGA and the physiologically active substance, and stirred to the extent that the liquid surface moved.

[0153] The particle formation step and the solvent removal step were carried out under the same conditions as in Example 3 to obtain a suspension containing microspheres. The average volume-based particle size of the obtained microspheres was 21.6 μm. After freezing representative particles with liquid nitrogen, FIB cross sections were prepared and SEM images were observed ( Fig.12 ).

[0154] The observed SEM images were analyzed using the same method as in Examples 1 to 4, and the coefficient of variation (s / A) × 100 (%) of the ratio of the area (A) of each region obtained by dividing the region into 6 regions every 60 degrees with the center point of the maximum diameter as the center to the sum of the cross-sectional areas (s) of the above-mentioned physiologically active substances contained in the region was 1.094.

[0155] Table 1 shows part of the conditions of Examples 1 to 4, Comparative Examples 1 to 4, and Reference Example 1 (PLGA only).

[0156] [Table 1]

[0157]

[0158] Table 2 shows the area ratio % in each region, the standard deviation, the average value, and the coefficient of variation (CV value) of microspheres of Examples 1 to 4, Comparative Examples 1 to 4, and representative particle sizes of Lepra.

[0159] [Table 2]

[0160]

[0161] As shown in Tables 1 and 2, in Examples 1 and 2, regardless of which of PLGA and PLA is used, the coefficient of variation of the area ratio of the physiologically active substance in each region of the particle is less than 0.35, and the distribution of the physiologically active substance in the particle is uniform. In Comparative Example 3, although the particle purification process and the desolvation process are the same conditions as in Example 1, if the PLGA concentration is reduced, the uniformity in the particle is reduced, and the coefficient of variation of the area ratio of the physiologically active substance in each region increases to 0.386.

[0162] In Example 4 and Comparative Example 1, the particle size of the microparticles of the physiologically active substance changed due to the difference in drying conditions and drying time. When the drying time was long, the uniformity of the physiologically active substance distributed in the particles decreased, and the coefficient of variation of the area ratio occupied by the physiologically active substance in each region increased to 0.468. In Comparative Examples 3 and 4, in which the concentrations of PLGA and the drug were reduced compared to the examples, the shrinkage rate during drying increased, the physiologically active substance in the particles deviated, and the coefficient of variation of the area ratio occupied by the physiologically active substance in each region exceeded 0.35.

[0163] Industrial Applicability

[0164] According to the present invention, it is possible to provide microspheres capable of appropriately controlling the initial release amount of a physiologically active substance and the release rate during the subsequent release period, thereby continuously releasing the physiologically active substance for a certain period of time in a living body.

Claims

1. Microspheres, which are microspheres with lactic acid / glycolic acid copolymer (PLGA) or polylactic acid (PLA) as the main component and in which physiologically active substances are uniformly dispersed, characterized in that: The average volume particle size of the microspheres is greater than 1 μm and less than 150 μm. A cross-sectional observation sample is prepared by cutting the microspheres, and the cross-sectional observation sample is observed by an electron microscope at a magnification greater than that capable of confirming the physiologically active substances in the microspheres. The electron microscope cross-sectional observation image is divided into 6 parts, and the ratio (s / A) × 100 (%) of the area (A) of each divided region to the sum of the cross-sectional areas (s) of the physiologically active substances contained in the region is calculated, and the coefficient of variation of the calculated ratio in the 6 regions is less than 0.

35.

2. The microsphere according to claim 1, wherein The physiologically active substance is a lipophilic physiologically active substance.

3. The microsphere according to claim 1 or 2, wherein: The dispersed physiologically active substance has an average volume-based particle size of 5 nm to 500 nm.

4. The microsphere according to any one of claims 1 to 3, wherein The content of the physiologically active substance in the microspheres is 0.35 to 1.5% by mass.

5. A sustained-release preparation comprising the microspheres according to any one of claims 1 to 4.

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