JP5560719B2 - Method for producing fine particles containing hydrophilic physiologically active substance - Google Patents

Description

  The present invention relates to a method for producing fine particles composed of an amphiphilic polymer and encapsulating a hydrophilic physiologically active substance.

  A microparticle formulation in which a drug is encapsulated in microparticles called nanoparticle, microparticle, nanosphere, microsphere, or microcapsule has been developed and is being studied for use as a drug carrier.

  As the microparticle formulation based on a polymer compound, biodegradable polylactic acid or polylactic acid / polyglycolic acid microparticles, and an amphiphilic polymer in which a hydrophilic polymer and a hydrophobic polymer are covalently bonded were used. Examples include polymer micelles and nanoparticles. In such a microparticle formulation, the compartment in which the drug is encapsulated is a hydrophobic environment, and the efficiency of encapsulating the hydrophilic drug in the microparticle is low. In particular, it is difficult to encapsulate a protein / peptide drug having a hydrophilic property and a large molecular weight in a state in which the particle retains physiological activity.

  In order to solve such a problem, an aqueous solution of a hydrophilic physiologically active substance is solubilized in an organic solvent to which an amphiphilic polymer is added by utilizing the surface active action of the amphiphilic polymer. A method of forming fine particles after forming reverse micelles (see Patent Documents 1 to 3) has been proposed.

  In Patent Document 1, a biodegradable polymer as a hydrophobic segment and an amphiphilic block copolymer having a polyamino acid as a hydrophilic segment are used to form reversed-phase micelles. The inside is a hydrophilic environment and the outer layer is hydrophobic. Certain particulates are described. However, no specific production method is mentioned for such fine particles.

  In Patent Document 2, a solution in which a biodegradable amphiphilic block copolymer is dissolved in an organic solvent is used, a hydrophilic physiologically active substance is contained in the inner aqueous phase, and a surface modifier is bound. Disclosed are fine particles that can be administered into the body, become an aqueous dispersion, and have an average particle size of 1 micron or less. However, there is no mention of a method for making the particle diameter uniform.

  In Patent Document 3, a solid content is produced from a reverse emulsion composed of a biodegradable amphiphilic block copolymer, and the S / O / W or S / O / O type emulsion having the solid content as an S phase is used. A method for producing microparticles is disclosed. However, it is difficult to reduce the average particle size to 200 nm or less by the fine particle production method via emulsion.

  In Patent Document 4, an organic solvent in which an amphiphilic block copolymer is dissolved is used to produce fine particles by drying a W / O / W emulsion containing a hydrophilic physiologically active substance in an inner aqueous phase. Yes. However, since such particles use an organic solvent that cannot be removed even under dry conditions, the particle size is very large from 100 microns to several millimeters, and it is difficult to obtain a uniform particle size.

  Moreover, when administering microparticles | fine-particles in the living body, the particle size becomes very important. Non-Patent Documents 1 and 2 describe differences in interaction and uptake into cells due to differences in the size of various particles, and the intracellular kinetics of microparticles administered in vivo vary greatly depending on their size. Has been reported. In particular, Non-Patent Document 2 reports the effects on macrophage cells that are greatly involved in the inflammatory reaction in the body. From these points, the fine particles to be administered into a living body are required to be a particle group having a particle size as uniform as possible from the viewpoint of reducing the risk of side effects such as inflammation.

Japanese Patent Laid-Open No. 11-269097 International Publication No. 2006/095668 Pamphlet JP 2008-88158 A Japanese translation of PCT publication No. 2004-511631

Joanna Rejman and three others, “Size-Dependent internationalization of the partial via the theways of the Bio-69th, Bio-therapeutic of the Bio-Ninety-seventh, Bio-Ninety-seventh, Bio-Ninety-seventh” James M. Brewer and three others, “Vicele size infusions the trafficking, Processing, and presentation of antigens in lipid vestiges”. Volume 173, p6143-6150

  The present invention provides a method for producing hydrophilic bioactive substance-containing microparticles having a hydrophilic portion therein and having a narrow particle size distribution so that the hydrophilic bioactive substance can be efficiently encapsulated. Is an issue.

  As a result of intensive studies to solve the above problems, the present inventors have completed the present invention. That is, the present invention has the following configuration.

(1) A method for producing fine particles containing a hydrophilic physiologically active substance,
(A) A water-based solvent in which a hydrophilic physiologically active substance is dissolved is mixed with a water-immiscible organic solvent in which an amphiphilic polymer composed of a hydrophobic segment of poly (hydroxy acid) and a hydrophilic segment of polysaccharide or polyethylene glycol is dissolved. Forming a reverse emulsion by
(B) removing the solvent from the reversed-phase emulsion to obtain a hydrophilic bioactive substance-containing solid content;
(C) a step of dispersing a solid content containing a hydrophilic physiologically active substance in acetone, acetonitrile, tetrahydrofuran or a solvent A which is a mixed solvent containing these as a main component , and (d) a hydrophilic physiological activity obtained in the step (c). A method comprising: adding a substance-containing solid dispersion to a solvent B in which the surface modifier is dissolved and the hydrophobic segment of the amphiphilic polymer is insoluble and compatible with the solvent A.

( 2 ) The solvent B in the step (d) is water, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol or a mixed solvent containing these as a main component. The manufacturing method as described in (1 ) .

( 3 ) The production method according to (1) or (2) , wherein the method for removing the solvent from the reversed-phase emulsion in the step (b) is a freeze-drying method.

( 4 ) Any of (1) to ( 3 ), wherein the surface modifier in the step (d) is a lipid modified with polyvinyl alcohol, polyethylene glycol-polypropylene glycol copolymer, polyethylene glycol or a derivative thereof. The manufacturing method as described in.

( 5 ) In any one of (1) to ( 4 ), in the step (d), the amount of the solvent B is 3 times or more by volume with respect to the amount of the hydrophilic active substance-containing solid component dispersion. Manufacturing method.

(6) the amphiphilic polymer is a graft type amphiphilic polymer over comprised of hydrophobic segments of the hydrophilic segment and a poly (hydroxy acid) graft chain of the polysaccharide backbone, either (1) to (5) The manufacturing method of crab.

( 7 ) The production method according to any one of (1) to ( 6 ), wherein the polysaccharide in the hydrophilic segment of the amphiphilic polymer is dextran.

( 8 ) The production method according to any one of (1) to ( 5 ), wherein the amphiphilic polymer is a block polymer comprising a hydrophilic segment of polyethylene glycol and a hydrophobic segment of poly (hydroxy acid).

( 9 ) The production method according to any one of (1) to ( 8 ), wherein the poly (hydroxy acid) in the hydrophobic segment of the amphiphilic polymer is poly (lactic acid-glycolic acid).

( 10 ) The production method according to any one of (1) to ( 9 ), wherein the hydrophilic physiologically active substance is a peptide, protein or nucleic acid.

  The hydrophilic bioactive substance-containing microparticles produced according to the present invention are useful as microparticles used as a so-called drug delivery system (Drug Delivery System). In addition, fine particles stably encapsulating physiologically active substances such as proteins and nucleic acids can be obtained, and the narrow particle size distribution can reduce extra side effects due to particle size variation. Then, it is possible to solve the difficulty in inducing an immune reaction and controlling the blood drug concentration derived from particles having a large or small particle size slightly contained in the administered fine particle group, and a patient-friendly drug treatment is possible.

  The present invention relates to a method for producing fine particles composed of an amphiphilic polymer and containing fine hydrophilic bioactive substances (hereinafter, also simply referred to as “fine particles”) encapsulating a hydrophilic bioactive substance. First, the components of the hydrophilic bioactive substance-containing fine particles will be described.

  The amphiphilic polymer constituting the fine particles of the present invention is an amphiphilic polymer comprising a hydrophobic segment of poly (hydroxy acid) and a hydrophilic segment of polysaccharide or polyethylene glycol. Amphiphilic here means having both hydrophilic and hydrophobic properties, and hydrophilic means that when the solubility in water at any site is higher than other segments, The site is said to be hydrophilic. The hydrophilic segment is preferably soluble in water. However, even if it is poorly soluble, the hydrophilic segment only needs to have a higher solubility in water than other sites. Also, the term “hydrophobic” means that a segment is hydrophobic when the solubility in water at any site is lower than at other sites. The hydrophobic segment is preferably insoluble in water, but may be soluble as long as it has a lower solubility in water than other sites.

  In addition, the amphiphilic polymer is preferably a biocompatible polymer since it is preferable that the amphiphilic polymer does not significantly adversely affect fine particles when administered in vivo. Here, the biocompatible polymer refers to a polymer that does not have a significantly harmful effect when administered to a living body, and more specifically, when the polymer is orally administered to a rat, the LD50 is 2,000 mg / mg. Say something more than kg.

  Therefore, poly (hydroxy acid) which is a hydrophobic segment of the amphiphilic polymer includes polyglycolic acid, polylactic acid, poly (2-hydroxybutyric acid), poly (2-hydroxyvaleric acid), poly (2-hydroxy acid). Specific examples include caproic acid), poly (2-hydroxycapric acid), poly (malic acid), or derivatives and copolymers of these polymer compounds. A copolymer of lactic acid or poly (lactic acid-glycolic acid) is preferred. The composition ratio of poly (lactic acid-glycolic acid) (lactic acid / glycolic acid) (mol / mol%) in the case of poly (lactic acid-glycolic acid) is not particularly limited as long as the object of the present invention is achieved. 100/0 to 30/70 are preferable, and 60/40 to 40/60 are particularly preferable.

  Both the polysaccharide and polyethylene glycol, which are hydrophilic segments of the amphiphilic polymer, are biocompatible polymers. Specific examples of the polysaccharide include cellulose, chitin, chitosan, gellan gum, alginic acid, hyaluronic acid, pullulan, and dextran, with dextran being preferred.

  When the hydrophilic segment of the amphiphilic polymer is a polysaccharide, the amphiphilic polymer is preferably a graft-type amphiphilic polymer in which a graft chain of poly (hydroxy acid) is graft-polymerized to a polysaccharide main chain. The molecular weight of the polysaccharide main chain is preferably 1,000 to 100,000, and more preferably 1,000 to 50,000. The molecular weight of the poly (hydroxy acid) is preferably from 500 to 100,000, more preferably from 1,000 to 10,000. The average molecular weight of the poly (hydroxy acid) relative to the average molecular weight of the polysaccharide is preferably 0.01 times to 100 times, preferably 0.02 times to 10 times, and 0.02 times to 1 times. More preferably, it is doubled. The number of poly (hydroxy acid) graft chains bonded to the polysaccharide main chain is preferably 1 to 50. The number of grafts referred to here can be determined from the graft type amphiphilic polymer, the polysaccharide main chain, and the poly (hydroxy acid) graft chain average molecular weight.

  When the hydrophilic segment of the amphiphilic polymer is polyethylene glycol, the amphiphilic polymer is preferably a block polymer of polyethylene glycol and poly (hydroxy acid). In the present invention, the term “block” refers to a part of a polymer molecule, which is composed of at least 5 or more monomer units and is different in chemical structure or configuration from other parts adjacent to the part. A polymer formed by linearly connecting the above blocks is called a block polymer. Each block itself constituting the block polymer may be a random, alternating, or gradient polymer composed of two or more types of monomer units. When the hydrophilic segment of the amphiphilic polymer is polyethylene glycol, the amphiphilic polymer is preferably a block polymer in which polyethylene glycol and poly (hydroxy acid) are connected one by one.

  Specific examples of the polyethylene glycol include linear or branched polyethylene glycol or a derivative thereof, and a linear polyethylene glycol derivative is preferable. A preferable example of the polyethylene glycol derivative is polyethylene glycol monoalkyl ether. Examples of the alkyl group of the polyethylene glycol monoalkyl ether include a linear or branched alkyl group having 1 to 10 carbon atoms, and a linear or branched alkyl group having 1 to 4 carbon atoms is preferable, methyl, ethyl, propyl An isopropyl group is more preferable. Although there is no restriction | limiting in particular in the molecular weight of polyethyleneglycol, It is preferable that it is 2,000-15,000, It is more preferable that it is 2,000-12,000, It is 4,000-12,000. More preferred is 5,000 to 12,000.

  The average molecular weight of the poly (hydroxy acid) when the hydrophilic segment of the amphiphilic polymer is polyethylene glycol is not particularly limited, but is preferably 5,000 to 200,000, and 15,000 to 150,000. More preferably, it is 20,000-100,000. The average molecular weight of the poly (hydroxy acid) relative to the average molecular weight of polyethylene glycol is preferably 1 or more, more preferably 2 or more, still more preferably 4 or more, and particularly preferably 4 times. More than 25 times.

  The average molecular weight in this specification refers to the number average molecular weight unless otherwise specified, and the number average molecular weight is an average molecular weight calculated by a method that does not consider weighting of the molecular size, and is an amphiphilic polymer. The average molecular weights of polysaccharides and polyethylene glycol can be determined as polystyrene or pullulan equivalent molecular weights measured by gel permeation chromatography (GPC). Moreover, the average molecular weight of poly (hydroxy acid) can be calculated | required from the ratio of the peak integrated value of a terminal residue, and the integrated values other than a terminal residue by nuclear magnetic resonance (1H-NMR) measurement.

  About the said amphiphilic polymer, it can manufacture according to the manufacturing method of the amphiphilic polymer described in international publication 2009/095668.

  Examples of the hydrophilic physiologically active substance in the present invention include low molecular weight compounds, proteins, peptides, DNA, RNA, and modified nucleic acids. In addition, even if it is a hydrophobic physiologically active substance, if hydrophilicity was provided with solubilizers, such as cyclodextrin and its analogs, it corresponds to the hydrophilic physiologically active substance said here.

  Specific examples of proteins or peptides that are hydrophilic physiologically active substances include peptide hormones, physiologically active proteins, enzyme proteins, antibodies, etc. Specific examples include parathyroid hormone (PTH), calcitonin, insulin, insulin-like Growth factors, angiotensin, glucagon, GLP-1 receptor agonist peptides represented by GLP-1 and exendin 4, bombesin, motilin, gastrin, growth hormone, prolactin (lutein stimulating hormone), gonadotropin (gonadotropic hormone), thyotropic Hormone, adrenocorticotropic hormone (ACTH), ACTH derivatives (such as shrimp tide), melanocyte stimulating hormone, follicle stimulating hormone (FSH), sermorelin, vasopressin, oxytocin, protillin, luteinizing phor (LH), corticotropin, secretin, somatropin, thyrotropin (thyroid stimulating hormone), somatostatin, gonadotropin releasing hormone (GnRH), G-CSF, erythropoietin (EPO), thrombopoietin (TPO), megacaryocytopotentiator, HGF , EGF, VEGF, interferon α, interferon β, interferon γ, interleukins, FGF (fibroblast growth factor), BMP (bone morphogenetic protein), thymic humoral factor (THF), blood thymic factor (FTS) ), Superoxide dismutase (SOD), urokinase, lysozyme, tissue plasminogen activator, asparaginase, kallikrein, ghrelin, adiponectin, leptin, atrial natriuretic peptide, atrium Natriuretic factor, brain natriuretic peptide (BNP), conantokin G, dynorphin, endorphin, kyotorphin, enkephalin, neurotensin, angiostatin, bradykinin, substance P, kallidin, hemoglobin, protein C, factor VIIa, glucocerebrosidase, Streptokinase, staphylokinase, thymosin (thymosin), pancreosimine, cholecystokinin, human placental lactogen, tumor necrosis factor (TNF), polymyxin B, colistin, gramicidin, bacitracin, thymopoietin, bombesin, cerulein, thymostimulin, selectin , Resistin, hepcidin, neuropeptide Y, neuropeptide S, cholecystokinin-pancreosaimine (CCK- PZ), brain-derived neurotrophic factor (BDNF), vaccines, and the like. These may be natural proteins or peptides, or derivatives obtained by modifying a part of the sequence, such as polyethylene glycol, It may be modified with a sugar chain or the like.

  Specific examples of modified nucleic acids that are hydrophilic physiologically active substances include those complexed with cationic surfactants, cationic lipids, cationic polymers, and analogs thereof.

The present invention relates to a method for producing hydrophilic bioactive substance-containing fine particles by the following steps (a) to (d) using the amphiphilic polymer and the hydrophilic bioactive substance as main raw materials.
(A) A water-based solvent in which a hydrophilic physiologically active substance is dissolved is mixed with a water-immiscible organic solvent in which an amphiphilic polymer composed of a hydrophobic segment of poly (hydroxy acid) and a hydrophilic segment of polysaccharide or polyethylene glycol is dissolved. (B) removing the solvent from the reverse phase emulsion to obtain a solid content containing a hydrophilic bioactive substance (c) removing the solid content containing the hydrophilic bioactive substance from the hydrophobic of the amphiphilic polymer The hydrophilic bioactive substance-containing solid content dispersion obtained in the steps (d) and (c) of dispersing in the solvent A in which the hydrophilic segment is soluble and the hydrophilic segment is insoluble is dissolved in the surface modifier. Adding to the solvent B in which the hydrophobic segment of the amphiphilic polymer is insoluble and compatible with solvent A.

  Hereinafter, this invention is demonstrated for every process.

  As the aqueous solvent in the step (a), water or an aqueous solution containing a water-soluble component is used. Examples of the water-soluble component include inorganic salts, saccharides, organic salts, amino acids and the like.

  The water-immiscible organic solvent in the step (a) has a solubility in water of 30 g (organic solvent) / 100 mL (water) or less, preferably 10 g (organic solvent) / 100 mL (water) or less, more preferably 1 g ( Organic solvent) / 100 mL (water) or less, more preferably 0.1 g (organic solvent) / 100 mL (water) or less. Other characteristics of the water-immiscible organic solvent are that the hydrophobic segment of the amphiphilic polymer is soluble and the hydrophilic segment is preferably insoluble or insoluble, and can be volatilized and removed by lyophilization. preferable. Examples of such water-immiscible organic solvents include ethyl acetate, isopropyl acetate, butyl acetate, dimethyl carbonate, diethyl carbonate, methylene chloride, and chloroform.

  In the step (a), the mixing ratio of the aqueous solvent in which the hydrophilic physiologically active substance is dissolved and the water-immiscible organic solvent in which the amphiphilic polymer is dissolved is preferably 1: 1 to 1: 1000, more preferably 1: 3 to 1: 100. The concentration of the hydrophilic physiologically active substance in the aqueous solvent may vary depending on the type of the hydrophilic physiologically active substance, but is preferably 0.01 mg / mL to 100 mg / mL, more preferably 0.1 mg / mL to 10 mg / mL. mL, more preferably 0.5 mg / mL to 5 mg / mL. The concentration of the amphiphilic polymer in the water-immiscible organic solvent varies depending on the type of the organic solvent and the amphiphilic polymer, but is preferably 0.01 to 90% (w / w), more preferably 0.8. It is 1 to 50% (w / w), more preferably 1 to 20% (w / w).

  The reversed-phase emulsion in step (a) is formed by adding an aqueous solvent in which a hydrophilic physiologically active substance is dissolved to a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, and if necessary, For example, a stirring device such as a magnetic stirrer, a turbine-type stirrer, a homogenizer, or a membrane emulsifying device equipped with a porous membrane may be used. In the step of forming the reverse emulsion, the reverse emulsion may be formed with a water-immiscible organic solvent in which two or more amphiphilic polymers are dissolved, depending on the pharmaceutical purpose. In the step of forming the reverse emulsion, a co-agent may be added for the purpose of assisting the formation of the reverse emulsion and forming a uniform and fine reverse emulsion. Examples of the coagent include compounds selected from alkyl alcohols having 3 to 6 carbon atoms, alkylamines having 3 to 6 carbon atoms, and alkylcarboxylic acids having 3 to 6 carbon atoms, and the structure of the alkyl chain of these coagents May have a straight chain structure or a branched structure, and may be saturated alkyl or unsaturated alkyl, but tert-butanol, iso-propanol, and pentanol are preferred.

  The average particle size of the reversed-phase emulsion obtained by the step (a) is not particularly limited, but is preferably 5 μm or less, and preferably 500 nm or less for producing pharmaceutical fine particles that are the object of the present invention. More preferably, it is 200 nm or less. The lower limit of the average particle size of the reversed emulsion is preferably 10 nm, and more preferably 25 nm. The average particle size of the reverse emulsion varies depending on the weight ratio of the aqueous solvent, the water-immiscible organic solvent, the amphiphilic polymer, and the co-agent. Therefore, in order to obtain a preferable average particle size, the ratio of the four components is appropriately set. Just decide.

  Examples of the method for removing the solvent from the reversed phase emulsion in the step (b) include heating, vacuum drying, dialysis, lyophilization, centrifugation, filtration, reprecipitation, and combinations thereof. This is preferable because there is little influence on the structural change due to coalescence of particles in the reverse emulsion, and modification of the hydrophilic physiologically active substance due to high temperature can be avoided.

  The step of freeze-drying is preferably a step that undergoes vacuum drying at room temperature after preliminary freezing, but may be a step of preliminary freezing, primary vacuum drying at low temperature, or secondary vacuum drying at room temperature. For example, it is frozen by cooling below the melting point of the water-based solvent and water-immiscible organic solvent constituting the reverse emulsion, and then dried under reduced pressure to obtain a lyophilized solid containing a hydrophilic physiologically active substance. It is done. The preliminary freezing temperature may be appropriately determined experimentally from the solvent composition, but is preferably −20 ° C. or lower. Further, the degree of reduced pressure in the drying process may be appropriately determined experimentally from the solvent composition, but is preferably 3,000 Pa or less, more preferably 500 Pa or less for shortening the drying time. As the freeze-drying apparatus, it is preferable to use a laboratory freeze-dryer equipped with a cold trap and connectable to a vacuum pump, or a shelf-type vacuum freeze-dryer used for manufacturing pharmaceutical products. After preliminary freezing with liquid nitrogen, a refrigerant, or the like, drying under reduced pressure may be performed with a decompression device such as a vacuum pump under cooling or room temperature.

  In the step (c), the solvent A in which the hydrophilic bioactive substance-containing solid content is dispersed is an amphiphilic polymer and a hydrophilic bioactive substance reflecting the structure of the reverse phase emulsion constituting the hydrophilic bioactive substance-containing solid content. For the purpose of maintaining a fine particle structure containing, the hydrophobic segment of the amphiphilic polymer is soluble and the hydrophilic segment is insoluble. Examples of the solvent A include acetone, acetonitrile, 1,4-dioxane, tetrahydrofuran, and a mixed solvent containing these as a main component, and acetonitrile is preferable. In addition, the main component here means that the content of the poly (hydroxy acid) that is a hydrophobic segment of the amphiphilic polymer is more than the content, preferably 50% by volume or more. Preferably it is 70 volume% or more, More preferably, it is 80 volume% or more. In order to disperse the hydrophilic bioactive substance-containing solid content, the solvent A may be simply added to the hydrophilic bioactive substance-containing solid content, but stirring, ultrasonic treatment, or the like may be performed as necessary. .

  In the step (d), it is important that the solvent B is a group of solvents in which the hydrophobic segment of the amphiphilic polymer constituting the hydrophilic bioactive substance-containing solid content is insoluble and compatible with the solvent A described above. Yes, for example, an aqueous solvent, an alcohol compound solvent, or a mixed solvent containing these as a main component. The main component here means that the poly (hydroxy acid), which is a hydrophobic segment of the amphiphilic polymer, is not less than a content that is substantially insoluble, preferably not less than 50% by volume, more preferably Is 70% by volume or more, more preferably 80% by volume or more.

  Examples of the aqueous solvent include water, hydrochloric acid, and a sodium hydroxide aqueous solution, and water is preferred. The aqueous solvent may contain a water-soluble component, and examples of the water-soluble component include inorganic salts, saccharides, organic salts, amino acids and the like.

  Examples of the alcohol compound solvent include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol, and the like, preferably methanol and ethanol.

  The surface modifier in the step (d) is used for the purpose of improving the colloidal stability of the fine particles by physical adsorption or chemical adsorption on the hydrophilic bioactive substance solid content surface. Here, improving the colloidal stability means preventing or delaying aggregation of fine particles in a solvent. Suitable surface modifiers for the purposes of the present invention include polyvinyl alcohol, polyethylene glycol-polypropylene glycol copolymers, lipids modified with polyethylene glycol or lipids modified with polyethylene glycol derivatives.

  When the surface modifier is polyvinyl alcohol, the molecular weight of polyvinyl alcohol is preferably 5,000 to 100,000, more preferably 10,000 to 50,000.

  When the surface modifier is a polyethylene glycol-polypropylene glycol copolymer, Pluronic (registered trademark of BASF) commercially available from BASF or an equivalent product is preferred. The molecular weight of the polyethylene glycol-polypropylene glycol copolymer is preferably 5,000 to 100,000, and preferably 10,000 to 50,000.

  When the surface modifier is a lipid modified with polyethylene glycol, the molecular weight of polyethylene glycol is preferably 500 to 15,000, more preferably 500 to 10,000, and 1,000 to 10 Is more preferable.

When the surface modifier is a lipid modified with a polyethylene glycol derivative, preferred examples of the polyethylene glycol derivative include those having a functional group introduced at one end of polyethylene glycol. The said functional group, is not particularly _{limited, -OH, -COH, -COOH, -NH} 2, -SH, -NO 2, -SO 3 H, -OSO 3 H, -CH 2 OSO ₃ H, a maleimide group, and an N-hydroxysuccinimide (NHS) group. For the purpose of modifying targeting antibodies and functional peptides on the microparticles produced according to the present invention, polyethylene glycol derivatives having COOH, —NH ₂ , maleimide groups, and NHS groups are preferably used. The molecular weight of the polyethylene glycol derivative is preferably in the same range as the molecular weight of the polyethylene glycol.

  When the surface modifier is a lipid modified with polyethylene glycol or a polyethylene glycol derivative, the lipid may be a compound having a long-chain fatty acid or a hydrocarbon chain portion and showing no significant toxicity. Examples include phospholipids and oleic acid. Moreover, steroids, such as cholesterol, may be sufficient.

  In the step (d), the mixing ratio of the hydrophilic physiologically active substance-containing solid dispersion and the solvent B is preferably such that the amount of the solvent B is 3 times or more by volume with respect to the amount of the dispersion. It is more preferably at least twice, more preferably at least 5 times and not more than 20 times. Further, when the hydrophilic bioactive substance-containing solid content dispersion is introduced into the solvent B containing the surface modifier, the solvent B containing the surface modifier is preferably in a state of being stirred. For stirring, a stirrer such as a magnetic stirrer or a turbine stirrer can be used, but is not limited thereto. The stirring speed is preferably 100 to 10,000 rpm, more preferably 150 to 5,000 rpm, and even more preferably 200 to 2,000 rpm. In order to introduce the hydrophilic bioactive substance-containing solid content dispersion, a pump or the like may be used or may not be used. Moreover, either addition in liquid or addition outside liquid may be sufficient. The speed at which the hydrophilic bioactive substance-containing solid content dispersion is introduced into the solvent B containing the surface modifier is not particularly limited, but is preferably 0.01 to 1,000 ml / min. 05-500 ml / min is more preferable, and 0.05-100 ml / min is still more preferable.

  The fine particles produced by the present invention include an amphiphilic polymer composed of a hydrophobic segment of poly (hydroxy acid) and a hydrophilic segment of polysaccharide or polyethylene glycol, and a hydrophilic portion composed of a hydrophilic segment of the amphiphilic polymer inside. A hydrophilic bioactive substance-containing fine particle comprising a hydrophobic outer layer composed of a hydrophobic segment of an amphiphilic polymer and having a surface modifier bonded to the hydrophobic outer layer. The shape of the obtained fine particles is not particularly limited, and may be spherical or substantially spherical, but may be elliptical or irregular.

  The fine particles produced according to the present invention are characterized by a narrow particle size distribution. The particle size distribution as used herein refers to what size (average particle size) particles are included in the target particle group in what proportion (relative particle amount with 100% as a whole). It means an indicator of whether or not. Examples of the measurement method of the average particle diameter include a microscopy method, a mass method, a light scattering method, a light blocking method, an electric resistance method, an acoustic method, and a dynamic light scattering method, but preferably by a microscopy method and a dynamic light scattering method. Measured. Examples of the microscope used for the microscopy include a scanning electron microscope and a transmission electron microscope. Examples of the particle measuring apparatus using the dynamic light scattering method include ELS-Z1 / Z2 manufactured by Otsuka Electronics Co., Ltd. The autocorrelation function is obtained from the data obtained by the measurement by the photon correlation method, and can be calculated by, for example, analyzing by the CONTIN method and the histogram method. When the shape of the fine particles is other than spherical, the average value of the major axis diameter can be regarded as the average particle size of the fine particles. The narrow particle size distribution means that there is less variation in the particle size of individual particles contained in the target particle group, and the smaller the particle size distribution, the more uniform the size is considered as a single particle group. It is. The particle size distribution can be evaluated by a polydispersity index (PDI). The polydispersity index is a normalized z-average dispersion of the distribution of particle diffusion coefficients that can be determined by the dynamic light scattering method. The smaller this value, the narrower the particle size distribution and the more uniform the particle size. Means that.

  Examples are shown below, but the present invention is not limited to these examples.

(Reference Example 1) Synthesis of graft polymer of dextran and poly (lactic acid-glycolic acid) copolymer Dextran (Nacalai Tesque Co., Ltd., Nacalai standard special grade product, number average molecular weight 13,000, 5.0 g) formamide (100 ml) ) And heated to 80 ° C. To this solution, 1,1,1,3,3,3-hexamethyldisilazane (100 ml) was added dropwise over 20 minutes. After completion of dropping, the mixture was stirred at 80 ° C. for 2 hours. After completion of the reaction, the reaction solution was returned to room temperature, and the two layers were separated using a separatory funnel. The upper layer was concentrated under reduced pressure, methanol (300 ml) was added, and the resulting solid was filtered and dried to obtain TMS-dextran (11.4 g) as a white solid. TMS-dextran (500 mg) and tert-butoxypotassium (35 mg) were dried under reduced pressure for 1 hour, tetrahydrofuran (10 ml) was added, and the mixture was stirred for 1.5 hours at room temperature. To this solution, a solution of (DL) -lactide (0.78 g) and glycolide (0.63 g) in tetrahydrofuran (15 ml) was added dropwise and stirred for 5 minutes. After completion of the reaction, the solvent was concentrated under reduced pressure, and purified by reprecipitation with a chloroform-methanol system and a chloroform-diethyl ether system to obtain a graft polymer of TMS-dextran and poly (lactic acid-glycolic acid) copolymer (hereinafter TMS). -Dextran-PLGA) was obtained as a white solid. To a solution of TMS-dextran-PLGA in chloroform (9.0 ml) was added trifluoroacetic acid (1.0 ml), and the mixture was stirred at room temperature for 30 minutes. After evaporating the solvent under reduced pressure, the residue was dissolved in chloroform (7 ml) and added dropwise to diethyl ether (200 ml) cooled to 0 ° C. to precipitate the product. The precipitate was filtered off and dried under reduced pressure to obtain a graft polymer (0.65 g) of dextran and poly (lactic acid-glycolic acid) copolymer as a white solid.

Reference Example 2 Synthesis of block polymer of polyethylene glycol and poly (lactic acid-glycolic acid) copolymer Polyethylene glycol monomethyl ether (manufactured by NOF Corporation, SUNBRIGHT MEH-20H, number average molecular weight 5,128, Mw / Mn = 1.02) (0.3 g), (DL) -lactide (2.16 g) and glycolide (1.74 g) were mixed and heated to 140 ° C. After stirring for 20 minutes, tin octylate (II) (0.05% by weight based on polyethylene glycol monomethyl ether) was added, and the mixture was stirred at 180 ° C. for 3 hours. The reaction solution is returned to room temperature, dissolved in chloroform (concentration to about 100 mg / ml), purified by reprecipitation with diethyl ether cooled to 0 ° C., and the resulting solid is filtered and dried under reduced pressure. A block polymer of polyethylene glycol and poly (lactic acid-glycolic acid) copolymer was obtained as a white solid.

Example 1
Dextran-g-poly (lactic acid-glycolic acid) of Reference Example 1 (average molecular weight of dextran 17,500, average molecular weight of poly (lactic acid-glycolic acid) graft chain 4,290, average number of graft chains 20-26) 5 mg Was dissolved in 100 μL of dimethyl carbonate to prepare a polymer solution. After adding 20 μL of tert-butanol to the polymer solution, 50 μL of a 1 mg / mL FITC-labeled bovine serum albumin (FITC-BSA) solution was dropped, and the mixture was stirred by vortex to produce a reverse phase emulsion. The inverse emulsion was pre-frozen with liquid nitrogen and then freeze-dried at a trap cooling temperature of −45 ° C. and a vacuum of 30 Pa for 12 hours using a freeze dryer (EYELA, FREEZE DRYER FD-1000). The obtained solid content was dispersed in 1 mL of acetonitrile to prepare a solid content dispersion. The solid dispersion was mixed with various surface modifiers (polyvinyl alcohol (PVA) molecular weight 13,000 to 23,000, saponification rate 87 to 89% (manufactured by Aldrich), polyethylene glycol-polypropylene glycol copolymer "Pluronic ( BASF registered trademark F68 "((PEO-PPO-PEO block polymer), manufactured by BASF, molecular weight 8,400), polyethylene glycol modified phospholipid" SUNBRIGHT DSPE-020CN "(1,2-distearoyl-sn-glycero- Each surface modifier binds by adding dropwise with stirring to 5 mL of an aqueous solution containing 3-phosphatidylethanolamine-N-monomethoxy- [poly (ethylene glycol)], manufactured by Nippon Oil & Fats Co., Ltd., polyethylene glycol molecular weight 2,000). FITC-BSA-encapsulated fine particles were obtained, and the average particle size and polydispersity of the obtained fine particles were determined. Measurement was performed using a dynamic light scattering apparatus (ELS-Z manufactured by Otsuka Electronics Co., Ltd.).

(Comparative Example 1)
Dextran-g-poly (lactic acid-glycolic acid) of Reference Example 1 (average molecular weight of dextran 17,500, average molecular weight of poly (lactic acid-glycolic acid) graft chain 4,290, average number of graft chains 20 to 26) 10 mg Was dissolved in 2 mL of ethyl acetate to prepare a polymer solution. To the polymer solution, 100 μL of a 1 mg / mL FITC-BSA solution was dropped, and stirred at 1,600 rpm for 1 hour using a magnetic stirrer to produce a reverse phase emulsion. Next, instead of removing the solvent from the reverse phase emulsion to obtain a solid content, the reverse phase emulsion is added to 20 mL of dioxane, concentrated to 2 mL using a rotary evaporator, and the concentrated solution is subjected to various surface modifiers. (PVA, “Pluronic (registered trademark of BASF) F68”, “SUNBRIGHT DSPE-020CN”) is added dropwise to 10 mL of an aqueous solution with stirring to obtain FITC-BSA-encapsulated fine particles to which the respective surface modifiers are bonded. It was. The average particle diameter and polydispersity of the obtained fine particles were measured using a dynamic light scattering apparatus (ELS-Z manufactured by Otsuka Electronics Co., Ltd.).

<Result>
The measurement results of the fine particles produced by the method of Example 1 and Comparative Example 1 are shown in Tables 1 and 2. Even when any surface modifier was used, particles having a smaller polydispersity value and a narrower particle size distribution could be produced as compared with Comparative Example 1.

(Example 2)
5 mg of polyethylene glycol-b-poly (lactic acid-glycolic acid) of Reference Example 2 (average molecular weight of polyethylene glycol 5,000, average molecular weight of poly (lactic acid-glycolic acid) graft chain 55,000) was dissolved in 100 μL of dimethyl carbonate. A polymer solution was prepared. After adding 20 μL of tert-butanol to the polymer solution, 50 μL of a 1 mg / mL FITC-labeled bovine serum albumin (FITC-BSA) solution was dropped, and the mixture was stirred by vortex to produce a reverse phase emulsion. The inverse emulsion was pre-frozen with liquid nitrogen and then freeze-dried at a trap cooling temperature of −45 ° C. and a vacuum of 30 Pa for 12 hours using a freeze dryer (EYELA, FREEZE DRYER FD-1000). The obtained solid content was dispersed in 1 mL of acetonitrile to prepare a solid content dispersion. The solid dispersion was dropped into 5 mL of an aqueous solution containing various surface modifiers (PVA, “Pluronic (registered trademark of BASF) F68”, “SUNBRIGHT DSPE-020CN”) of Example 1 with stirring, respectively. FITC-BSA-encapsulated fine particles to which the surface modifier was bonded were obtained. The average particle diameter and polydispersity of the obtained fine particles were measured using a dynamic light scattering apparatus (ELS-Z manufactured by Otsuka Electronics Co., Ltd.).

(Comparative Example 2)
10 mg of polyethylene glycol-b-poly (lactic acid-glycolic acid) of Reference Example 2 (average molecular weight of polyethylene glycol 5,000, average molecular weight of poly (lactic acid-glycolic acid) graft chain 55,000) was dissolved in 2 mL of ethyl acetate. A polymer solution was prepared. To the polymer solution, 100 μL of a 1 mg / mL FITC-BSA solution was dropped, and stirred at 1,600 rpm for 1 hour using a magnetic stirrer to produce a reverse phase emulsion. Next, instead of removing the solvent from the reverse-phase emulsion to obtain a solid content, the reverse-phase emulsion is added to 20 mL of dioxane, concentrated to 2 mL that is baked using a rotary evaporator, and the concentrated solution is subjected to various surface modifications. FITC-BSA-encapsulated fine particles to which the respective surface modifiers were bound were obtained by adding dropwise to 10 mL of an aqueous solution containing the agent (PVA, Pluronic F68, SUNBRIGHT DSPE-020CN) with stirring. The average particle diameter and polydispersity of the obtained fine particles were measured using a dynamic light scattering apparatus (ELS-Z manufactured by Otsuka Electronics Co., Ltd.).

<Result>
The measurement results of the fine particles produced by the method of Example 2 and Comparative Example 2 are shown in Tables 3 and 4. Even when any surface modifier was used, particles having a smaller polydispersity and a narrower particle size distribution could be produced as compared with Comparative Example 2.

  The hydrophilic physiologically active substance produced by the present invention can be used as a pharmaceutical composition.

Claims (10)

A method for producing fine particles containing a hydrophilic physiologically active substance,
(A) A water-based solvent in which a hydrophilic physiologically active substance is dissolved is mixed with a water-immiscible organic solvent in which an amphiphilic polymer composed of a hydrophobic segment of poly (hydroxy acid) and a hydrophilic segment of polysaccharide or polyethylene glycol is dissolved. Forming a reverse emulsion by
(B) removing the solvent from the reversed-phase emulsion to obtain a hydrophilic bioactive substance-containing solid content;
(C) a step of dispersing a solid content containing a hydrophilic physiologically active substance in acetone, acetonitrile, tetrahydrofuran or a solvent A which is a mixed solvent containing these as a main component , and (d) a hydrophilic physiological activity obtained in the step (c). A method comprising: adding a substance-containing solid dispersion to a solvent B in which the surface modifier is dissolved and the hydrophobic segment of the amphiphilic polymer is insoluble and compatible with the solvent A. The solvent B in the step (d) is water, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol or a mixed solvent containing these as a main component. 2. The production method according to 1 . The method according to claim 1 or 2 , wherein the method of removing the solvent from the reverse emulsion in the step (b) is a freeze-drying method. The production method according to any one of claims 1 to 3 , wherein the surface modifier in the step (d) is a lipid modified with polyvinyl alcohol, polyethylene glycol-polypropylene glycol copolymer, polyethylene glycol or a derivative thereof. . The production method according to any one of claims 1 to 4 , wherein in the step (d), the amount of the solvent B is 3 times or more as a volume ratio with respect to the amount of the hydrophilic active substance-containing solid dispersion. The amphiphilic polymer is a graft type amphiphilic polymer over comprised of hydrophobic segments of the polysaccharide backbone hydrophilic segment and a poly (hydroxy acid) graft chain, prepared as described in any one of claims 1 to 5 Method. Wherein a polysaccharide is dextran in the hydrophilic segment of the amphiphilic polymer, the production method according to any one of claims 1-6. The amphiphilic polymer is a block polymer composed of a hydrophobic segment of the polyethylene glycol hydrophilic segment and a poly (hydroxy acid), The process according to any one of claims 1-5. Wherein the poly in the hydrophobic segment of the amphiphilic polymer (hydroxy acid) is poly - a (lactic acid-glycolic acid), The process according to any one of claims 1-8. Wherein the hydrophilic bioactive substance is a peptide, protein or nucleic acid, The process according to any one of claims 1-9.