JPWO2005089928A1 - Preparation kit for coated fine particles - Google Patents

Abstract

The present invention can be mixed with a liquid (liquid A) containing a polar organic solvent in which core fine particles are dispersed and a coating layer component constituting a coating layer for coating the core fine particles is dissolved. Provided is a kit for preparing coated fine particles at the time of use comprising a solvent (liquid B) that does not contain an organic solvent or contains a polar organic solvent in a proportion lower than that of liquid A and a means for mixing liquid A and liquid B. .

Description

  The present invention relates to a kit for preparing coated fine particles at the time of use.

  Conventionally, many techniques have been disclosed regarding methods for producing coated fine particles in pharmaceuticals, foods, agricultural chemicals, veterinary drugs and the like. The coating of the fine particles (coated fine particles) with the coating layer is, for example, in order to suppress the influence of external factors, to selectively accept the influence of external factors, and to cause changes in the fine particles as a trigger, etc. It is done for the purpose of imparting functions. As one of them, a method of coating fine particles with a lipid film in a liquid has been reported (see Patent Document 1). In this method, the fine particles are coated with a lipid film by reducing the proportion of the polar organic solvent in the aqueous solution containing the polar organic solvent in which the fine particles are dispersed and the lipid is dissolved, and the coating is performed in the liquid. For example, coated fine particles having a size suitable for fine particles for intravenous injection and the like are produced with excellent efficiency.

On the other hand, kits for preparation at the time of use are known for microparticle preparations manufactured by a method that does not depend on coating (see, for example, Patent Document 2 and Patent Document 3).
International Publication No. 02/28367 Pamphlet JP 2000-247868 A Japanese Patent No. 2688235

  An object of the present invention is to provide a kit for preparing coated fine particles in which core fine particles are coated with a coating layer.

The present invention relates to the following (1) to (16).
(1) A liquid (liquid A) containing a polar organic solvent in which core fine particles are dispersed and a coating layer component constituting a coating layer for coating the core fine particles is dissolved,
A solvent that can be mixed with liquid A and does not contain a polar organic solvent or contains a polar organic solvent in a proportion lower than liquid A (liquid B),
And a kit for preparation of coated fine particles, including an instrument comprising means for mixing liquid A and liquid B.
(2) An instrument comprising means for mixing the liquid A and the liquid B is composed of a means for containing the liquid A, a means for containing the liquid B, an in-line mixing means, and a means for containing the liquid A. For in-use preparation of coated fine particles according to the above (1), which is a device provided with a liquid feeding means to the inlet of the inline mixing means and a liquid feeding means from the means for containing the liquid B to the inlet of the inline mixing means kit.
(3) A device including means for mixing liquid A and liquid B contains a syringe for storing liquid A, a syringe for storing liquid B, a syringe for storing liquid A, and the liquid B A kit for preparing coated fine particles according to the above (1), which is an instrument provided with an in-line mixing means having two inlets to which syringes can be connected respectively.
(4) The instrument provided with the means for mixing the liquid A and the liquid B is a multi-chamber syringe provided in parallel with chambers for storing the liquid A and the liquid B, respectively, and the discharge port of the syringe is an in-line mixing means The kit for the preparation of coated fine particles according to the above (1), which is a syringe.
(5) The kit for preparation of coated fine particles according to any one of (2) to (4), wherein the inline mixing means is an inline mixing means in which a static mixer is connected or incorporated.
(6) A device including means for mixing the liquid A and the liquid B contains the liquid A from the means for containing the liquid A, the means for containing the liquid B, and the means for containing the liquid B. A kit for preparing coated fine particles according to the above (1), which is an instrument comprising means for adding liquid B to liquid A in the means for the purpose.
(7) An instrument including means for mixing the liquid A and the liquid B is a syringe for storing the liquid A and a syringe for storing the liquid B, and the liquid A is connected to the liquid A by connecting the two syringes. The kit for preparation of coated fine particles according to (1) above, to which can be added.
(8) The instrument provided with the means for mixing the liquid A and the liquid B is a multi-chamber syringe provided with series chambers for storing the liquid A and the liquid B, respectively. The kit for preparation of coated fine particles according to the above (1), which is a syringe that can be added.
(9) When using the coated fine particles according to any one of the above (2) to (8), the instrument comprising means for mixing the liquid A and the liquid B is an instrument comprising a transfusion needle or a co-infusion connector at the outlet. Preparation kit.
(10) The kit for preparation of coated fine particles according to any one of (1) to (9), wherein the coating layer component is one or more substances selected from lipids, surfactants and polymers.
(11) The kit for preparation of coated fine particles according to any one of (1) to (10), wherein the coating layer is a lipid membrane.
(12) The coated fine particle according to any one of (1) to (11), wherein the core fine particle is a fine particle comprising a drug, a lipid aggregate, a liposome, an emulsion fine particle, a polymer, a metal colloid, or a fine particle preparation. Preparation kit for use.
(13) The above-mentioned (1) to (11), wherein the core fine particle is a fine particle comprising a complex comprising a combination of two or more drugs, lipid aggregates, liposomes, emulsion fine particles, polymers, metal colloids or fine particle preparations. A kit for preparation of coated fine particles according to any one of the above.
(14) The kit for preparation of coated fine particles according to any one of (1) to (11), wherein the core fine particles are fine particles containing a complex of a drug and a liposome as a constituent component.
(15) The kit for preparing coated fine particles according to any one of (1) to (14), wherein the polar organic solvent is one or more selected from alcohols, glycols and polyalkylene glycols.
(16) Coated fine particles that can be prepared by the on-use preparation kit for coated fine particles according to any one of (1) to (15).

  According to the present invention, a kit for preparing coated fine particles in which core fine particles are coated with a coating layer is provided.

The schematic diagram (1) of one example of the kit of this invention, and its usage. The schematic diagram (2) of the other example of the kit of this invention, and its usage. The schematic diagram (3) of the other example of the kit of this invention, and its usage. The schematic diagram (4) of the other example of the kit of this invention, and its usage. The schematic diagram (5) of the other example of the kit of this invention, and its usage. The schematic diagram (6) of the other example of the kit of this invention, and its usage. The schematic diagram (7) of the other example of the kit of this invention, and its usage. The schematic diagram (8) of the other example of the kit of this invention, and its usage. The schematic diagram of the conventional coating fine particle manufacturing apparatus. Schematic diagram of an example of the kit of the present invention and its usage in the case where each preparation kit for use in preparing liquid A and core fine particles at the time of use is included

Explanation of symbols

1, 2 Containers 3, 4 Pump 5 Three-way joint 6 Static mixer 7, 8 Infusion bottle 9 Three-way joint 10 Static mixer 11, 12 Syringe 13 Three-way joint 14 Static mixer 15 Multi-chamber syringe 16 Static mixer 17 Container 18 Syringe 19 Needle 20, 21 Infusion bottle 22 Mixed injection tube 23 Syringe with female connector 24 Syringe with male connector 25 Serial multi-chamber syringe 26, 27 Container 28 Pump 29 Container 30, 31, 32, 33, 34 Syringe and transfer Injection needle 35 Syringe 36 Three-way joint, static mixer and flow path liquid A Liquid B containing a polar organic solvent in which core fine particles are dispersed and a coating layer component constituting a coating layer for coating the core fine particles is dissolved Can be mixed with A and does not contain polar organic solvents Is a suspension obtained by mixing a liquid A and a liquid B containing a polar organic solvent in a proportion lower than that of the liquid A

  The kit for preparing coated fine particles of the present invention is a liquid (liquid A) containing a polar organic solvent in which core fine particles are dispersed and a coating layer component constituting a coating layer for coating the core fine particles is dissolved, A kit comprising a liquid (liquid B) that can be mixed with liquid A and does not contain a polar organic solvent or contains a polar organic solvent at a lower ratio than liquid A, and a device comprising means for mixing liquid A and liquid B is there.

  The coated fine particle in the present invention is a fine particle formed of at least a core fine particle and a coating layer, and a coating layer component constituting the coating layer is formed by coating the outer side of the core fine particle. It is produced by mixing the liquid A and the liquid B with the instrument to coat the core fine particles with the coating layer component, and is obtained in the form of a suspension (liquid C).

  The core fine particles in the present invention are fine particles containing, for example, drugs, lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle preparations, etc., preferably, at least the drug as a component. Fine particles. Further, the core fine particles in the present invention may comprise a complex comprising a combination of two or more drugs, lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle preparations, etc. , Liposomes, emulsion particles, polymers, metal colloids, fine particle preparations and the like and other compounds (eg, sugars, lipids, inorganic compounds, etc.) may be used as a constituent, preferably Examples thereof include fine particles containing a composite as a constituent component.

  The drug includes a drug in the form of fine particles in the solvent in the liquid A, a drug in the form of fine particles in the solvent in the liquid A by forming a complex with other components constituting the core fine particles, for example, Examples include proteins, peptides, nucleic acids, low molecular compounds, saccharides, high molecular compounds, lipid compounds, metal compounds and the like, which have pharmacological activity, preferably nucleic acids, and more preferably genes, One or more substances selected from DNA, RNA, oligonucleotide, plasmid, and siRNA can be used.

  Examples of the protein or peptide include bradykinin, angiotensin, oxytocin, vasopressin, adrenocorticotropin, calcitonin, insulin, glucagon, cholecystokinin, β-endorphin, melanocyte inhibitor, melanocyte stimulating hormone, gastrin antagonist, neurotensin, somatostatin, brucine , Cyclosporine, enkephalin, transferrin, arginine-glycine-aspartic acid (RGD) peptide, thyroid hormone, growth hormone, gonadotropin, luteinizing hormone, asparaginase, arginase, uricase, carboxypeptidase, glutaminase, superoxide dismutase, tissue plasminose Genactivator, streptokinase, interferon Ikin, interferon, muramyl dipeptide, thymopoietin, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, erythropoietin, thrombopoietin, trypsin inhibitor, lysozyme, epidermal growth factor (EGF), insulin-like growth factor, nerve growth factor, platelet derived Examples include growth factor, transforming growth factor, endothelial cell growth factor, fibroblast (fibroblast) growth factor, glial cell growth factor, thymosin, and specific antibodies (for example, anti-EGF receptor antibody).

  Examples of the nucleic acid include oligonucleotides such as antisense oligonucleotides and sense oligonucleotides, genes, DNA, RNA, plasmids, siRNA and the like, and the nucleic acids are contained in the phosphate part, ester part, etc. in the structure of the nucleic acid. Including derivatives in which the oxygen atom is substituted with another atom such as a sulfur atom. In addition, siRNA is short double stranded RNA.

  Examples of low molecular weight compounds include epsilon-aminocaproic acid, arginine hydrochloride, L-aspartate potassium, tranexamic acid, bleomycin sulfate, vincristine sulfate, cefazolin sodium, cephalothin sodium, citicoline, cytarabine, gentamicin sulfate, vancomycin hydrochloride, kanamycin sulfate, Amikacin sulfate and the like.

  Examples of the saccharide include sodium chondroitin sulfate, heparin sodium, dextran fluorescein and the like.

Examples of the polymer compound include sodium polyethylene sulfonate, divinyl ether-maleic anhydride copolymer (DIVEMA), styrene maleic anhydride copolymer-neocarinostatin conjugate (SMANCS), and the like.
Examples of lipid compounds include vitamin D and vitamin E.
Examples of the metal compound include cisplatin.

  The lipid aggregate or liposome is composed of, for example, a lipid and / or a surfactant, and the lipid may be any of a simple lipid, a complex lipid, or a derived lipid, such as a phospholipid, a glyceroglycolipid, a sphingo. Examples include glycolipids, sphingoids, sterols, and the like, and preferably phospholipids. Examples of the lipid include a surfactant (synonymous with the surfactant described later), a polymer (synonymous with the polymer described later, specifically dextran, etc.), a polyoxyethylene derivative (specifically polyethylene glycol). And the like, and polyethylene glycolated phospholipids are preferable. Examples of the surfactant include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants.

  Examples of phospholipids include phosphatidylcholine (specifically soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, etc.), phosphatidylethanolamine (specifically distearoyl). Phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine, dioleoylphosphatidylethanolamine, etc.), glycerophospholipid (specifically phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine, etc.), sphingophospholipid ( Specifically, Sphingomyelin and Sera Dophosphoethanolamine, ceramide phosphoglycerol, ceramide phosphoglycerophosphate, etc.), glycerophosphonolipid, sphingophosphonolipid, natural lecithin (specifically egg yolk lecithin, soybean lecithin, etc.), hydrogenated phospholipid (specifically Natural or synthetic phospholipids such as hydrogenated phosphatidylcholine).

Examples of the glyceroglycolipid include sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride and the like.
Examples of the glycosphingolipid include galactosyl cerebroside, lactosyl cerebroside, ganglioside and the like.
Examples of sphingoids include sphingan, icosasphingan, sphingosine, and derivatives thereof. As the derivative, for example, —NH ₂ such as sphingan, icosasphingan, sphingosine and the like —NHCO (CH ₂ ) _x CH ₃ (wherein x represents an integer of 0 to 18, among which 6, 12 or 18 is preferable. ) And the like.

  Examples of sterols include cholesterol, dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol, brassicasterol, ergocasterol, fucosterol, and 3β- [N- (N′N′-dimethylaminoethyl) carbamoyl. ] Cholesterol (DC-Chol) and the like.

  Other lipids include, for example, N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride (DOTAP), N- [1- (2,3-dioleo). Oilpropyl)]-N, N-dimethylamine (DODAP), N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride (DOTMA), 2,3-dio Railoxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid (DOSPA), N- [1- (2,3-ditetradecyloxypropyl) ] -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N- [1- (2,3-dioleyloxypropyl)]-N, N-dimethyl-N-hydro Lipids such Shiechiru ammonium bromide (DORIE) may be mentioned.

  Nonionic surfactants include, for example, polyoxyethylene sorbitan monooleate (specifically polysorbate 80 etc.), polyoxyethylene polyoxypropylene glycol (specifically Pluronic F68 etc.), sorbitan fatty acid (specifically Sorbitan monolaurate, sorbitan monooleate, etc.), polyoxyethylene derivatives (specifically, polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, etc.), glycerin fatty acid esters and the like.

  Examples of the anionic surfactant include acyl sarcosine, sodium alkyl sulfate, alkyl benzene sulfonate, and fatty acid sodium having 7 to 22 carbon atoms. Specific examples include sodium dodecyl sulfate, sodium lauryl sulfate, sodium cholate, sodium deoxycholate, sodium taurodeoxycholate and the like.

  Examples of the cationic surfactant include alkylamine salts, acylamine salts, quaternary ammonium salts, amine derivatives and the like. Specifically, benzalkonium chloride, acylaminoethyl diethylamine salt, N-alkylpolyalkylpolyamine salt, fatty acid polyethylene polyamide, cetyltrimethylammonium bromide, dodecyltrimethylammonium bromide, alkylpolyoxyethyleneamine, N-alkylaminopropylamine, And fatty acid triethanolamine ester.

  Examples of amphoteric surfactants include 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid, N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid, and the like. can give.

  In the liposome, these lipids and surfactants are used alone or in combination, preferably in combination. As a combination when used in combination, for example, a combination of two or more components selected from hydrogenated soybean phosphatidylcholine, polyethylene glycolated phospholipid and cholesterol, a combination of two or more components selected from distearoyl phosphatidylcholine, polyethylene glycolated phospholipid and cholesterol , A combination of EPC and DOTAP, a combination of EPC, DOTAP and polyethylene glycolated phospholipid, a combination of EPC, DOTAP, cholesterol and polyethylene glycolated phospholipid, and the like.

  Moreover, the liposome may contain a film stabilizer such as sterols such as cholesterol, for example, an antioxidant such as tocopherol, if necessary.

  Examples of lipid aggregates include spherical micelles, spherical reverse micelles, sausage-like micelles, sausage-like reverse micelles, plate-like micelles, plate-like reverse micelles, hexagonal I, hexagonal II, and aggregates composed of two or more lipid molecules. .

  Examples of the emulsion particles include oil emulsions such as fat emulsions, emulsions composed of a nonionic surfactant and soybean oil, lipid emulsions, lipid nanospheres, and water-in-oil-in-water (W / O / W) Emulsion particles and the like.

  Examples of the polymer include natural polymers such as albumin, dextran, chitosan, dextran sulfate, and DNA, such as poly-L-lysine, polyethyleneimine, polyaspartic acid, styrenemaleic acid copolymer, isopropylacrylamide-acrylpyrrolidone copolymer. Examples thereof include synthetic polymers such as polymers, polyethylene glycol-modified dendrimers, polylactic acid, polylactic acid polyglycolic acid, and polyethylene glycolated polylactic acid, and salts thereof.

  Here, the salts in the polymer include, for example, metal salts, ammonium salts, acid addition salts, organic amine addition salts, amino acid addition salts and the like. Examples of the metal salt include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, zinc salt and the like. Examples of ammonium salt include ammonium salt. Examples of acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate and phosphate, and acetate, maleate, fumarate and citrate. Examples of organic amine addition salts include addition salts such as morpholine and piperidine, and examples of amino acid addition salts include addition salts such as glycine, phenylalanine, aspartic acid, glutamic acid, and lysine. can give.

  Examples of the metal colloid include metal colloids containing gold, silver, platinum, copper, rhodium, silica, calcium, aluminum, iron, indium, cadmium, barium, lead and the like.

  Examples of the fine particle preparation include microspheres, microcapsules, nanocrystals, lipid nanoparticles, and polymer micelles.

  Further, when the core fine particle is a core fine particle containing a drug, the core fine particle preferably contains a charged substance having an electrostatic charge opposite to that of the drug, more preferably electrostatically opposite to the drug. And a lipid having the following charge (a cationic lipid or an anionic lipid described later). Here, the electrostatic charge opposite to the drug includes a charge in the drug molecule, a charge that generates an electrostatic attraction with respect to the intramolecular polarization, a surface polarization, and the like.

  The charged substance contained in the core fine particles is classified into a cationic substance exhibiting a cationic property and an anionic substance exhibiting an anionic property, but is an amphoteric substance having both a cationic group and an anionic group. However, since the relative negative degree changes depending on pH, binding with other substances, etc., it can be classified as a cationic substance or an anionic substance depending on the time. These charged substances may be used as a constituent component of the core fine particles or may be used in addition to the constituent components of the core fine particles.

  As the cationic substance, for example, a cationic substance among those exemplified in the definition of the core fine particles [specifically, a cationic lipid, a cationic surfactant (as defined above), a cationic polymer, etc.], Examples include proteins or peptides that can form a complex at a pH lower than the isoelectric point.

  Examples of the cationic lipid include DOTAP, DODAP, DOTMA, DOSPA, DMRIE, DORIE, and DC-Chol.

  Examples of the cationic polymer include poly-L-lysine, polyethyleneimine, polyfect, and chitosan.

  A protein or peptide capable of forming a complex at a pH below the isoelectric point is not particularly limited as long as it is a protein or peptide capable of forming a complex at a pH below the isoelectric point of the substance. Not. Examples include albumin, orosomucoid, globulin, fibrinogen, pepsin, ribonuclease T1, and the like.

  As the anionic substance, for example, anionic substances among those exemplified in the definition of the above-mentioned core fine particles [specifically, anionic lipid, anionic surfactant (as defined above), anionic polymer, etc.], Examples thereof include proteins, peptides, nucleic acids, and the like that can form a complex at a pH equal to or higher than the isoelectric point.

  Examples of the anionic lipid include phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid and the like.

  Examples of the anionic polymer include polyaspartic acid, styrene maleic acid copolymer, isopropylacrylamide-acryl pyrrolidone copolymer, polyethylene glycol modified dendrimer, polylactic acid, polylactic acid polyglycolic acid, polyethylene glycolated polylactic acid, dextran sulfate. Dextran sulfate sodium, chondroitin sulfate, chondroitin sulfate sodium, hyaluronic acid, chondroitin, deltaman sulfate, heparan sulfate, heparin, ketalan sulfate, dextran fluorescein anionic and the like.

  The protein or peptide capable of forming a complex at a pH of the isoelectric point or higher is particularly limited as long as it is a protein or peptide capable of forming a complex at a pH higher than the isoelectric point of the substance. Not. Examples include albumin, orosomucoid, globulin, fibrinogen, histone, protamine, ribonuclease, lysozyme and the like.

  Examples of the nucleic acid as the anionic substance include DNA, RNA, plasmid, siRNA, oligonucleotide and the like, and any nucleic acid having any length and sequence may be used as long as it does not exhibit physiological activity.

  The core fine particles can be obtained as a commercial product, or can be produced according to a known method or a modification thereof. For example, a known method for preparing liposomes can be applied to the production of core fine particles containing liposome as one of the core fine particles. Known methods for preparing liposomes include, for example, Bangham et al., “Liposome preparation method [J. Mol. Biol.”, 1965, Vol. 13, p. 238-252], ethanol injection method ["J. Cell Biol.", 1975, Vol. 66, p. 621-634], French press method ["FEBS Lett.", 1979, Vol. 99, p. 210-214], freeze-thaw method ["Arch. Biochem. Biophys.", 1981, Vol. 212, p. 186-194], reverse phase evaporation [Procedure of the National Academy of Sciences United States of America (Proc. Natl. Acad. Sci. USA)], 1978. 75, p. 4194-4198], pH gradient method (see, for example, Japanese Patent No. 2572554, Japanese Patent No. 2659136, etc.) and the like. As a solution for suspending the liposome during the production of the liposome, for example, water, acid, alkali, various buffers, physiological saline, amino acid infusion, and the like can be used. In the production of liposomes, for example, antioxidants such as citric acid, ascorbic acid, cysteine, and ethylenediaminetetraacetic acid (EDTA), for example, isotonic agents such as glycerin, glucose, and sodium chloride can be added. is there. Liposomes can also be produced by dissolving lipids and the like in an organic solvent such as ethanol and distilling off the solvent, and then adding physiological saline and the like, and stirring to form liposomes.

  In addition, for example, non-ionic surfactant (as defined above), cationic surfactant (as defined above), anionic surfactant (as defined above), polymer, polyoxyethylene derivative, etc. These surface-modified liposomes can also be used as constituents of the core fine particles in the present invention [D. D. Lasic, edited by F. Martin, “Stealth Liposomes ( Stealth Liposomes) "(USA), CRC Press Inc, 1995, p. 93-102]. Examples of the polymer include dextran, pullulan, mannan, amylopectin, and hydroxyethyl starch. Examples of the polyoxyethylene derivatives include polysorbate 80, Pluronic F68, polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy ( Polyethylene glycol) -2000] (PEG-DSPE) and the like.

  The average particle size of the liposome can be freely selected as desired. Examples of the method for adjusting the average particle diameter include an extrusion method and a method in which large multilamellar liposomes (MLV) are mechanically pulverized (specifically, using a manton gourin, a microfluidizer, etc.) [Müller (R. Edited by H. Muller, Benita, B. Bohm, “Emulsion and Nanosuspensions for Emerging and Nanosuspensions for Drugs” the Formulation of Poorly Soluble Drugs ", Scientific Publishers Stuttgart, Germany, 19 1998, p. 267-294] and the like.

  In addition, a complex comprising two or more selected from, for example, drugs, lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, microparticle preparations, and the like constituting the core microparticles (specifically, for example, cationic lipids) Liposomes containing lipids or complexes of lipid aggregates and nucleic acids, complexes of polymers and nucleic acids containing cationic polymers such as poly-L-lysine, liposomes or lipids containing anionic lipids such as phosphatidic acid Complexes of aggregates and proteins, complexes of polymers containing anionic polymers such as styrenemaleic acid and proteins, liposomes containing cationic lipids, complexes of lipid aggregates and proteins, poly- (For example, a complex of a polymer containing a cationic polymer such as L-lysine and a protein and a protein, etc.) Coalescence, liposomes may be just a production method of mixing the polymer and the like, this time, it is also possible to add further granule sizing step or aseptic processes like if necessary. It is also possible to perform complex formation in various solvents such as acetone and ether. For example, a nucleic acid complex can be formed by dissolving nucleic acid and lipid in an organic solvent such as ethanol and distilling off the solvent, then adding physiological saline and the like, and stirring with shaking. As another method of forming the complex, for example, a cationic substance and polyethylene glycolated phospholipid (specifically, polyethylene glycol-phosphatidylethanolamine (more specifically, 1,2-distearoyl-sn-glycero) are used in water. -3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE), etc.), polyoxyethylene hydrogenated castor oil 60, Cremophor EL, etc.) and the like, Thereafter, for example, a nucleic acid is added, and further, for example, an anionic polymer can be added to form a multiple complex.

  As for the size of the core fine particles, the average particle diameter is preferably several nm to several hundred μm, more preferably 10 nm to 5 μm, still more preferably 50 nm to 300 nm, and more preferably 50 nm to 200 nm. Most preferred.

  Examples of the coating layer component include lipids, surfactants, and polymers listed in the definition of the core fine particles, and preferably selected from the lipids and surfactants listed in the definition of the core fine particles. More preferably, one or more substances selected from lipids and surfactants whose coating layer becomes a lipid membrane and surfactants are more preferable, and phospholipids are more preferable.

  Examples of the lipid used when the coating layer is a lipid membrane include synthetic lipids. Examples of the synthetic lipid include fluorinated phosphatidylcholine, fluorinated surfactant, dialkylammonium bromide and the like, and these may be used alone or in combination with other lipids. When the coating layer is a lipid membrane, it is preferable to use a water-soluble polymer derivative as one of the coating layer components. Examples of water-soluble polymer derivatives include polyethylene glycolated lipids [specifically, polyethylene glycol-phosphatidylethanolamine (more specifically, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ Methoxy (polyethylene glycol) -2000] (PEG-DSPE, etc.), polyoxyethylene hydrogenated castor oil 60, Cremophor EL, etc.], polyethylene glycol sorbitan fatty acid ester (specifically polyoxyethylene monooleate) Sorbitan, etc.), polyethylene glycol fatty acid esters, polyglycerinized lipids (specifically polyglycerin-phosphatidylethanolamine, etc.), polyglycerin fatty acid esters, etc. Chi glycol lipids, and the like.

  The solvent in the liquid A is preferably a solvent in which the core fine particles are not dissolved and the coating layer component is dissolved. In the liquid C in which the liquid A and the liquid B are mixed, the core fine particles are not dissolved and the coating layer component is dissolved. Do not gather or gather. Liquid A is a liquid containing a polar organic solvent, and liquid B is a liquid containing a solvent other than the polar organic solvent, but liquid A is a solvent other than the polar organic solvent and a solvent other than the polar organic solvent in liquid B. The liquid B may contain a polar organic solvent if it is lower than the ratio of the polar organic solvent in the liquid A.

  In the present invention, the dispersion of the core fine particles means a state in which the core fine particles are suspended, emulsified or emulsified, preferably suspended, and most of the core fine particles are dispersed and the remaining portion is dispersed. Although it includes a dissolved state or a partially precipitated state, it is preferable that almost all or all of the core fine particles are dispersed. In addition, dissolution of the coating layer component includes a state in which most of the coating layer component is dissolved and the remaining portion is dispersed, but almost all or all of the coating layer component is dissolved. preferable.

  Examples of the polar organic solvent in the liquid A and the liquid B include alcohols such as methanol, ethanol, n-propanol, 2-propanol, n-butanol, 2-butanol, and tert-butanol, glycerin, ethylene glycol, propylene glycol, and the like. Glycols, polyalkylene glycols such as polyethylene glycol, and the like, and preferably ethanol. Examples of the solvent other than the polar organic solvent in the liquid A and the liquid B include water, liquid carbon dioxide, liquid hydrocarbon, halogenated carbon, halogenated hydrocarbon, and the like, and preferably water. . Moreover, the liquid A and the liquid B may contain ions, buffer components, and the like.

  The combination of the polar organic solvent and the solvent other than the polar organic solvent is preferably a combination that can be mixed with each other. The solubility of the core fine particles, the solubility of the coating layer component, and the like in the solvents in the liquids A to C are determined. It can be selected in consideration. On the other hand, the core fine particles preferably have low solubility in any of the solvents in the liquids A to C, and also have low solubility in any of the polar organic solvent and any solvent other than the polar organic solvent. Preferably, the coating layer component preferably has a low solubility in the solvent in the liquid B and the liquid C, preferably has a high solubility in the solvent in the liquid A, and has a high solubility in the polar organic solvent. It is preferable that the solubility is high, and it is preferable that the solubility in a solvent other than the polar organic solvent is low.

  The ratio of the polar organic solvent in the solvent in the liquid A is not particularly limited as long as the core fine particles are present without being dissolved and the coating layer component covering the core fine particles is satisfied. However, it is preferably 30 vol% or more, more preferably 60 to 90 vol%, although it varies depending on the solvent used, the core fine particles, the type of coating layer components, and the like. Further, the ratio of the polar organic solvent in the solvent in the liquid C is not particularly limited as long as it is a ratio that can be coated with the coating layer component in which the core fine particles are dissolved, but is preferably 50 vol% or less, More preferably, it is 30-50 vol%.

  The combination of the core microparticle and the coating layer component in the present invention is not particularly limited, but the core microparticle is a microparticle having a drug-liposome complex as a constituent component, and the coating layer component is a lipid and / or a surfactant. A combination is preferred. The coated fine particles in which the core fine particle is a fine particle having a liposome or a drug-liposome complex as a constituent component, the coating layer component is a lipid and / or a surfactant, and the coating layer is a lipid film, Coatings that are classified as liposomes in a narrow sense and whose core microparticles are not liposomes or microparticles containing a drug-liposome complex, the coating layer components are lipids and / or surfactants, and the coating layers are lipid membranes Microparticles are classified as broadly defined liposomes. In the present invention, the coated fine particles are more preferably liposomes in a narrow sense.

  The ratio of the core fine particles to the liquid A and the liquid C used in the kit for preparing coated fine particles of the present invention is not particularly limited as long as the core fine particles can be coated with the coating layer component, but 1 μg / mL to 1 g / mL is preferable, and 0.1 to 500 mg / mL is more preferable. Further, the ratio of the coating layer component (for example, lipid) used to the liquid A and the liquid C is not particularly limited as long as the core fine particles can be coated, but preferably 1 μg / mL to 1 g / mL, 0.1 -400 mg / mL is more preferred. The weight ratio of the coating layer component to the core fine particles is preferably 1: 0.1 to 1: 1000, more preferably 1: 1 to 1:10.

  The liquid A in the preparation kit for use of the coated fine particles of the present invention may be prepared at the time of use. As a method for preparing the liquid A at the time of use, for example, a liquid (liquid D) in which core fine particles are dispersed is prepared, and a liquid (liquid E) in which a coating layer component is dissolved in a solvent containing a polar organic solvent is prepared. And a method of mixing the liquid D and the liquid E. More preferably, the liquid in which the core fine particles are dispersed (liquid D), the liquid in which the coating layer component is dissolved in the solvent containing the polar organic solvent (liquid E), and , A method of preparing using a preparation kit at the time of use comprising an instrument having means for mixing the liquid D and the liquid E, where each solvent in the liquid D and the liquid E is the core fine particles in the liquid A after mixing. Any solvent may be used as long as is dispersed and the coating layer components are dissolved.

  Furthermore, the core fine particles in the liquid D may also be prepared at the time of use, and examples of the preparation method include the core fine particle production method described in the description of the core fine particles, More preferably, a method for producing a composite comprising two or more selected from, for example, drugs, lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle preparations and the like constituting the core fine particles can be mentioned, It is preferable to prepare using a pre-use preparation kit.

  The on-use preparation kit for coated fine particles of the present invention may include the above-mentioned preparation kit for on-use preparation of the liquid A and, if desired, the above-mentioned preparation kit for use of the core fine particles.

  As an instrument provided with the means for mixing the liquid A and the liquid B in the present invention, for example, means for containing the liquid A, means for containing the liquid B, in-line mixing means, and for containing the liquid A A means for feeding liquid from the means to the inlet of the in-line mixing means and a means for feeding the liquid B from the means for containing the liquid B to the inlet of the in-line mixing means, means for containing the liquid A, liquid B And a device including means for adding the liquid B to the liquid A in the means for containing the liquid A from the means for containing the liquid B. The instrument may include a flow path and may be provided with mixing means. Moreover, you may equip the exit of this instrument with the transfusion needle or the connector for co-infusion.

  Examples of the in-line mixing means include a T-shaped tube, a Y-shaped tube, a three-way joint, a three-way cock, an in-line mixer, an in-line mixer assembly, and the like.

  Examples of the means for containing the liquid A and the means for containing the liquid B include a vial, an infusion bag, and a syringe, and each may be provided with a mixing means.

Examples of liquid feeding means include gear pumps, tube pumps, syringe pumps, suction pumps, pressurization pumps, plunger pumps, etc., including syringes that are manually pressed and natural drops using height differences. The means may also function as a flow path.
The flow path may be any flow path such as a metal pipe or a resin tube.

Hereinafter, a kit for preparing coated fine particles of the present invention and a method for producing coated fine particles using the kit will be described with reference to the drawings. However, the preparation kit for coated fine particles of the present invention is not limited to those shown in these drawings.
FIG. 1 shows an instrument provided with means for mixing liquid A and liquid B in the kit of the present invention, means for containing liquid A, means for containing liquid B, in-line mixing means, and liquid A An example of a kit which is a device including a liquid feeding means from the means for containing the liquid to the inlet of the in-line mixing means and a liquid feeding means from the means for containing the liquid B to the inlet of the in-line mixing means; The usage is shown. The kit
I. Liquid A,
II. Liquid B, and
III. It consists of a container (1) containing liquid A, a container (2) containing liquid B, pumps (3) and (4), a three-way joint (5), a static mixer (6) and a flow path.
To prepare coated fine particles using the kit, the liquid A in (1) is sent to (5) by (3), and the liquid B in (2) is sent to (5) by (4). Liquid. In (5) and (6), liquid A and liquid B are mixed to obtain coated fine particles as a suspension (liquid C).

FIG. 2 shows that, in the kit of the present invention, an instrument having a means for mixing the liquid A and the liquid B, a means for containing the liquid A, a means for containing the liquid B, an in-line mixing means, and the liquid Other parts of the kit which is an instrument comprising a means for feeding A from the means for containing A to the inlet of the in-line mixing means and a means for feeding the means from the means for containing B to the inlet of the in-line mixing means Examples and usage are given. The kit
I. Liquid A,
II. Liquid B, and
III. It consists of an infusion bottle (7) containing liquid A, an infusion bottle (8) containing liquid B, a three-way joint (9), a static mixer (10), and a flow path.
In order to prepare coated fine particles using the kit, the liquid A in (7) is sent to (9) by the height difference, and the liquid B in (8) is sent to (9) by the height difference. . In (9) and (10), liquid A and liquid B are mixed to obtain coated fine particles as a suspension (liquid C).

FIG. 3 shows that, in the kit of the present invention, an instrument having means for mixing the liquid A and the liquid B, means for containing the liquid A, means for containing the liquid B, in-line mixing means, the liquid Other parts of the kit which is an instrument comprising a means for feeding A from the means for containing A to the inlet of the in-line mixing means and a means for feeding the means from the means for containing B to the inlet of the in-line mixing means The example and its usage are shown. Specifically, the instrument comprises a syringe for containing the liquid A, a syringe for containing the liquid B, a syringe for containing the liquid A, and the liquid B. An instrument comprising in-line mixing means having two inlets each capable of connecting syringes for storage. The kit
I. Liquid A,
II. Liquid B, and
III. It consists of a syringe (11) containing liquid A, a syringe (12) containing liquid B, a three-way joint (13), a static mixer (14) and a flow path.
In order to prepare coated fine particles using the kit, liquid A in (11) is fed to (13) by pushing the plunger, and liquid B in (12) is pushed by pushing the plunger ( 13). In (13) and (14), liquid A and liquid B are mixed to obtain coated fine particles as a suspension (liquid C).

FIG. 4 shows that, in the kit of the present invention, an instrument having a means for mixing the liquid A and the liquid B, a means for containing the liquid A, a means for containing the liquid B, an in-line mixing means, and the liquid Other parts of the kit which is an instrument comprising a means for feeding A from the means for containing A to the inlet of the in-line mixing means and a means for feeding the means from the means for containing B to the inlet of the in-line mixing means An example and its usage are shown. Specifically, the instrument is a multi-chamber syringe provided with a chamber for storing liquid A and liquid B in parallel, and the discharge port of the syringe is mixed in-line. It is a syringe that is a means. The kit
I. Liquid A,
II. Liquid B, and
III. It consists of a multi-chamber syringe (15) and a static mixer (16).
In order to prepare coated fine particles using the kit, the liquid A in one chamber of (15) and the liquid B in the other one of (15) are respectively sent to (16) by pushing the plunger. Liquid. In (16), liquid A and liquid B are mixed to obtain coated fine particles as a suspension (liquid C).

FIG. 5 shows that, in the kit of the present invention, an instrument including means for mixing liquid A and liquid B contains means for containing liquid A, means for containing liquid B, and liquid B. 1 shows an example of a kit which is a device including means for adding the liquid B to the liquid A in the means for containing the liquid A from the means and the use thereof. The kit
I. Liquid A,
II. Liquid B, and
III. It consists of a container (17) for storing liquid A, a syringe (18) for storing liquid B, and a transfer needle (19).
In order to prepare coated fine particles using the kit, the liquid B in (18) is fed to (17) through (19) by pushing the plunger. The liquid B sent to the liquid A in (17) is added to obtain coated fine particles as a suspension.

FIG. 6 shows that, in the kit of the present invention, an instrument having means for mixing liquid A and liquid B also contains means for containing liquid A, means for containing liquid B, and liquid B. The other example of the kit which is an instrument provided with the means to add the liquid B to the liquid A in the means for accommodating the liquid A from the means for, and its usage are shown. The kit
I. Liquid A,
II. Liquid B, and
III. It consists of an infusion bottle (20) that contains liquid A, an infusion bottle (21) that contains liquid B, and a mixed injection tube (22).
In order to prepare coated fine particles using the kit, the liquid B in (21) is fed to (20) through (22) by the height difference. The liquid B sent to the liquid A in (20) is added to obtain coated fine particles as a suspension.

FIG. 7 shows that, in the kit of the present invention, an instrument including means for mixing liquid A and liquid B contains means for containing liquid A, means for containing liquid B, and liquid B. FIG. 4 shows another example of a kit which is a device provided with means for adding the liquid B to the liquid A in the means for containing the liquid A from the above means, and the usage thereof. In the two syringes, the liquid B can be added to the liquid A by connecting them to each other. The kit
I. Liquid A,
II. Liquid B, and
III. It consists of a syringe (23) containing liquid A with a female connector at the outlet and a syringe (24) containing liquid B with a male connector at the outlet.
In order to prepare coated fine particles using the kit, the female connector (23) and the male connector (24) are connected, and the liquid B in (24) is sent to (23) by pushing the plunger. To do. The liquid B sent to the liquid A in (23) is added to obtain coated fine particles as a suspension.

FIG. 8 shows that, in the kit of the present invention, an instrument including means for mixing liquid A and liquid B contains means for containing liquid A, means for containing liquid B, and liquid B. FIG. 4 shows another example of a kit which is a device provided with means for adding the liquid B to the liquid A in the means for containing the liquid A from the above means, and the usage thereof. And a chamber for accommodating the liquid B in series, and a multi-chamber syringe capable of adding the liquid B to the liquid A. The kit
I. Liquid A,
II. Liquid B, and
III. It consists of a serial multi-chamber syringe (25).
In order to prepare coated fine particles using the kit, the plunger of (25) is pushed to move the indoor gasket (a in the figure) to the bypass (b in the figure), and then the plunger is pushed by pushing the plunger. Liquid B in the side chamber is fed to the outlet side chamber through the bypass. The liquid B sent to the liquid A in the chamber on the outlet side is added to obtain coated fine particles as a suspension.

  On the other hand, FIG. 9 shows a coated fine particle manufacturing apparatus in the prior art (see Patent Document 1), which includes a container (26) for storing liquid A, a container (27) for storing liquid B, and a pump (28). The coated fine particle manufacturing apparatus is represented. In order to prepare coated fine particles using the production apparatus, the liquid B in (27) is fed to (26) by (28). The liquid A in (26) and the fed liquid B are mixed to obtain coated fine particles as a suspension.

  Although the syringe used for this invention may be produced specially, a commercially available thing can also be used and it is not specifically limited. Specifically, a one-component syringe (commercially available from Terumo, Nipro, etc.), a two-component syringe (commercially available from Vetter (Germany), Becton Dickinson (USA), etc.), a two-component mixed syringe (TAH) Industries (USA), Plas-Pak Industries (USA), etc.) and the like, and liquid A and / or liquid B may be pre-filled as a prefilled syringe. Moreover, the mixing ratio of the liquid A and the liquid B can be arbitrarily changed by combining syringes having different inner diameters.

Next, a method for preparing coated fine particles in the case where the kit for preparation of coated fine particles of the present invention includes a kit for preparation of each use for preparing liquid A and core fine particles at the time of use. This will be specifically described with reference to an example of a kit for preparing coated fine particles when the coating layer component is a lipid and a complex of a liposome and a coating layer component. However, the on-use preparation kits for preparing the liquid A and the core fine particles at the time of use in the on-use preparation kit of the coated fine particles of the present invention are not limited to these.
-Composition of preparation kit for use (Fig. 10)
Raw material for core fine particles; a. Liposome or raw material of liposome
b. Drug (preferably an aqueous solution of the drug)
Solvent of liquid D; a. water
b. Polar organic solvent Liquid E: Liquid containing a polar organic solvent in which lipid is dissolved (preferably an aqueous solution containing a polar organic solvent that is the same as or different from solvent b of liquid D, more preferably containing the same polar organic solvent. Aqueous solution)
Liquid B; water appliance; a. A container (for example, a vial or the like) for containing the liposome or the raw material of the liposome and preparing the liquid A at the time of use (29)
b. Syringe and liquid transfer needle (30) containing drug (preferably, aqueous solution of drug)
c. Syringe and solvent transfer needle (31) containing solvent a of liquid D
d. Syringe and liquid transfer needle (32) containing solvent b of liquid D
e. Syringe containing liquid E and needle for transfer (33)
f. Syringe and liquid transfer needle (34) for containing liquid A
g. Syringe containing liquid B (35)
h. Three-way joint, static mixer and flow path (36)

・ Preparation method of coated microparticles The solvent a of the liquid D in (31) is put into the liposome in (29) or the raw material of the liposome and shaken, and then the drug in (30) is put in (29) Shake and mix to prepare core microparticles. Similarly, the solvent B of the liquid D in (32) and the liquid F in (33) are put into (29) in this order and mixed by shaking to prepare the liquid A. The liquid A thus obtained is transferred to (34) and connected to (36) as shown in FIG. Similarly, the syringe (35) containing the liquid B is also connected to (36) as shown in FIG. 10, and the plungers of the syringes are respectively pushed to send the liquid A and the liquid B to (36). A and liquid B are mixed to obtain coated fine particles as a suspension (liquid C).

  The coated fine particles of the present invention preferably have an average particle size of 300 nm or less, more preferably 200 nm or less, and specifically, for example, an injectable size.

  The coated microparticles of the present invention are, for example, biological components such as blood components, stabilization of drugs against gastrointestinal fluids, reduction of side effects, increased drug accumulation in target organs such as tumors, and oral and transmucosal drug It can be used as a preparation for the purpose of improving absorption. For the purpose, the coated fine particles of the present invention are preferably coated fine particles containing a drug that can be encapsulated in a biological component for a long time.

  When the coated microparticles of the present invention are used as a preparation, a suspension of the coated microparticles prepared by the above-described method can be used as it is in the form of, for example, an injection. It is also possible to use after removing the solvent by separation or the like.

  In the case of an injection, the suspension of the coated fine particles of the present invention or the coated fine particles from which the solvent has been removed is mixed with, for example, water, acid, alkali, various buffers, physiological saline, amino acid infusion, or the like. It is preferable to prepare an injection. In addition, for example, it is also possible to prepare an injection by adding an antioxidant such as citric acid, ascorbic acid, cysteine, EDTA, and an isotonic agent such as glycerin, glucose, and sodium chloride. Moreover, it can also be cryopreserved by adding a cryopreservation agent such as glycerin.

  In addition, the suspension or the injection is mixed with various solutions for infusion or infusion using, for example, a physiological saline bottle, a double bag kit (Nipro), a drug vial storage type full kit (Nipro). It is also possible. In this case, it is preferable that the instrument provided with the means for mixing the liquid A and the liquid B is an instrument provided with a transfusion needle or a mixed injection connector at the outlet.

  Next, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.

Using the following kit, liquid A and liquid B, liquid A was fed at 5.2 mL / h and liquid B was mixed at 11.1 mL / h so that the ethanol concentration in liquid C after mixing was approximately 20 vol%. did. A reservoir in which 18.8 mL of distilled water was previously placed was placed at the outlet of the liquid C in the assembled kit so that the resulting suspension of coated fine particles was dropped. After feeding for 23 minutes, the reservoir was covered and inverted and mixed, and the resulting mixture was ultracentrifuged (1 hour, 110,000 × g, 25 ° C.). Phosphate buffered saline (PBS) was added to the precipitate after ultracentrifugation so that the total lipid concentration after dilution was 30 mg / mL and resuspended to obtain a preparation.
kit;
The kit shown in FIG. 3 was used. A three-way joint for HPLC piping (manufactured by Shimadzu Corporation) is used for (13), and a 2 cm static mixer (quartz wool for gas chromatograph (manufactured by Shimadzu Corporation) on a fluororesin tube having a length of 20 mm and an inner diameter of 1.5 mm is used for (14). ) Was used, and a 20 mL syringe (manufactured by Terumo) loaded in a syringe pump (manufactured by Nikkiso) was used for (11) and (12).
Liquid A;
Dextran fluorescein anionic (FD) (Molecular Probes; the same applies hereinafter) 30 mg, DOTAP (Avanti Polar Lipids; the same applies below) 180 mg and PEG-DSPE 72 mg 9 mL of distilled water was added to the mixture, and the mixture was shaken and stirred with a vortex mixer. The obtained suspension was passed 10 times through a 0.4 μm polycarbonate membrane filter and 10 times through a 0.1 μm polycarbonate membrane filter at room temperature to obtain a suspension of core fine particles. 750 μL of the obtained suspension of core fine particles and 1 mL of ethanol were mixed, and 250 μL of a solution obtained by adding 2 mL of ethanol to 480 mg of EPC and 100 mg of PEG-DSPE and stirring was mixed.
Liquid B;
Distilled water

  Using the same kit, liquid A and liquid B as in Example 1, the ethanol concentration in liquid C after mixing liquid A at 5.2 mL / h and liquid B at 5.6 mL / h is approximately 30 vol%. Each was sent. At the outlet of the liquid C of the assembled kit, a reservoir containing 20.9 mL of distilled water was placed in advance, and the resulting suspension of coated fine particles was dropped. After feeding for 23 minutes, in the same manner as in Example 1, mixing, ultracentrifugation, and resuspension were performed to obtain a preparation.

  Using the same kit, liquid A and liquid B as in Example 1, the ethanol concentration in liquid C after mixing liquid A at 5.2 mL / h and liquid B at 2.9 mL / h is approximately 40 vol%. Each was sent. A reservoir in which 21.9 mL of distilled water was previously placed was placed at the outlet of the liquid C of the assembled kit so that the resulting suspension of coated fine particles was dropped. After feeding for 23 minutes, in the same manner as in Example 1, mixing, ultracentrifugation, and resuspension were performed to obtain a preparation.

  Using the same kit, liquid A and liquid B as in Example 1, the ethanol concentration in liquid C after mixing liquid A at 5.2 mL / h and liquid B at 0.7 mL / h was approximately 55 vol%. Each was sent. A reservoir containing 22.7 mL of distilled water was placed in advance at the outlet of the liquid C of the assembled kit so that the resulting suspension of coated fine particles was dropped. After feeding for 23 minutes, in the same manner as in Example 1, mixing, ultracentrifugation, and resuspension were performed to obtain a preparation.

  Using the same kit, liquid A and liquid B as in Example 1, the ethanol concentration in liquid C after mixing liquid A at 26.0 mL / h and liquid B at 28.0 mL / h is approximately 30 vol%. Each was sent. At the outlet of the liquid C of the assembled kit, a reservoir containing 20.9 mL of distilled water was placed in advance, and the resulting suspension of coated fine particles was dropped. After feeding for 4.6 minutes, in the same manner as in Example 1, mixing, ultracentrifugation and resuspension were performed to obtain a preparation.

  Using the same kit, liquid A and liquid B as in Example 1, the ethanol concentration in liquid C after mixing liquid A at 1.0 mL / h and liquid B at 1.1 mL / h is approximately 30 vol%. Each was sent. At the outlet of the liquid C of the assembled kit, a reservoir containing 20.9 mL of distilled water was placed in advance, and the resulting suspension of coated fine particles was dropped. After feeding for 115 minutes, the mixture was mixed, ultracentrifuged and resuspended in the same manner as in Example 1 to obtain a preparation.

  Using the same kit as Example 1 except the static mixer and the same liquid A and liquid B as in Example 1, liquid A was mixed at 5.2 mL / h and liquid B was mixed at 5.6 mL / h. Each solution was fed so that the ethanol concentration in C was about 30 vol%. At the outlet of the liquid C of the assembled kit, a reservoir containing 20.9 mL of distilled water was placed in advance, and the resulting suspension of coated fine particles was dropped. After feeding for 23 minutes, in the same manner as in Example 1, mixing, ultracentrifugation, and resuspension were performed to obtain a preparation.

Using the following kit, liquid A and liquid B, each was sent at 5.2 mL / h. At the outlet of the liquid C of the assembled kit, a reservoir previously filled with 21.0 mL of distilled water was placed so that the resulting suspension of coated fine particles was dropped. After feeding for 23 minutes, in the same manner as in Example 1, mixing, ultracentrifugation, and resuspension were performed to obtain a preparation.
kit;
The kit shown in FIG. 4 was used. (15) Using a two-component mixing syringe (TAHplus 9 mL Micro dispenser (TAH Plus 9 ml microdispenser): manufactured by TAH Corporation, USA) loaded in a syringe pump (Nikkiso Co., Ltd.) and a static mixer (16) Spiral mixer 190-212: manufactured by USA H.C.
Liquid A;
9 mL of distilled water was added to 30 mg of FD, 180 mg of DOTAP and 72 mg of PEG-DSPE, and the mixture was shaken and stirred with a vortex mixer. The obtained suspension was passed 10 times through a 0.4 μm polycarbonate membrane filter and 10 times through a 0.1 μm polycarbonate membrane filter at room temperature to obtain a suspension of core fine particles. 1000 μL of the obtained suspension of core fine particles and 1.333 mL of ethanol were mixed, and 333 μL of a solution obtained by adding 2 mL of ethanol to 480 mg of EPC and 100 mg of PEG-DSPE was mixed.
Liquid B;
Distilled water

<Comparative Example 1>
Using the following coated fine particle production apparatus, liquid A and liquid B, liquid A is put into (26), liquid B is put into the following syringe pump, and liquid B is added to liquid A at 1 mL / min for 23 minutes (23 mL). It was. The obtained suspension of coated fine particles was subjected to ultracentrifugation (1 hour, 110,000 × g, 25 ° C.). The precipitate after ultracentrifugation was diluted with PBS and adjusted so that the total lipid concentration after dilution was 30 mg / mL to obtain a preparation.
Coated fine particle production equipment;
The coated fine particle manufacturing apparatus shown in FIG. 9 was used. (27) and (28) are 20 mL syringes (made by Terumo) loaded in syringe pumps (Telfusion syringe pump STC-525, made by Terumo), and a glass container with a volume of about 30 mL is used for (26). Put a rotor and stirred with a stirrer.
Liquid A;
9 mL of distilled water was added to 30 mg of FD, 180 mg of DOTAP and 72 mg of PEG-DSPE, and the mixture was shaken and stirred with a vortex mixer. The resulting suspension was passed 10 times through a 0.4 μm polycarbonate membrane filter and 10 times through a 0.1 μm polycarbonate membrane filter at room temperature. 750 μL of the obtained suspension was put in a glass container having a volume of about 30 mL, and 1 mL of ethanol was mixed while stirring with a stirrer to obtain a suspension of core fine particles. To the obtained suspension of core fine particles, 250 μL of a solution obtained by adding 5 mL of ethanol to EPC 1200 mg and PEG-DSPE 250 mg and stirring was added and mixed.
Liquid B;
Distilled water

<Test Example 1>
For each of the preparations obtained in Examples 1 to 8 and Comparative Example 1, the average particle size of the coated fine particles was measured with a dynamic light scattering (DLS) measuring device (A model ELS-800, Otsuka Electronics). The results are shown in Table 1.

<Test Example 2>
For each of the preparations obtained in Examples 1 to 8 and Comparative Example 1, the inclusion rate of FD in the coated fine particles was determined as follows.
30 μL of each preparation was taken, 2970 μL of purified water was added to each, stirred and ultracentrifuged to obtain a supernatant. Each of the above preparations and each of the supernatants were diluted 1000-fold, 50 μL of 10 w / v% Triton X-100 (Triton X-100) and 400 μL of PBS were added to each 50 μL of each diluted solution, and stirred with a vortex mixer. 100 μL of the solution was taken in a 96-well microplate, and the fluorescence intensity at an excitation wavelength of 485 nm and a fluorescence wavelength of 530 nm was measured using a fluorescence plate reader (manufactured by Wallac Co., Ltd., Arvo Sx-4). On the other hand, the fluorescence intensity of each FD aqueous solution which becomes 1, 0.5 and 0.25 μg / mL was measured to obtain a calibration curve. From the calibration curve, the FD concentration in each preparation and the FD concentration in each supernatant after ultracentrifugation were determined, and the encapsulation rate of FD in the coated fine particles was calculated by the following formula (1). The results are shown in Table 2.

<Test Example 3>
The recovery rates with respect to the charged amounts of FD and EPC in the respective preparations obtained in Examples 1 to 8 and Comparative Example 1 were determined as follows.
Each preparation was diluted 1000-fold, 50 μL of 10 w / v% Triton X-100 and 400 μL of PBS were added to 50 μL of each diluted product, and the mixture was stirred with a vortex mixer. 100 μL of the solution was taken in a 96-well microplate, and the fluorescence intensity at an excitation wavelength of 485 nm and a fluorescence wavelength of 530 nm was measured using a fluorescence plate reader (manufactured by Wallac Co., Ltd., Arvo Sx-4). On the other hand, the fluorescence intensity of each FD aqueous solution which becomes 1, 0.5 and 0.25 μg / mL was measured to obtain a calibration curve. The FD concentration in each preparation was obtained from the calibration curve. On the other hand, the EPC concentration in each preparation was determined by measuring using phospholipid C-Test Wako (manufactured by Wako Pure Chemical Industries). The FD recovery rate and EPC recovery rate in each preparation were calculated by the following formula (2). The results are shown in Table 3.

<Test Example 4>
Each formulation obtained in Examples 1 to 8 and Comparative Example 1 was examined for stability in fetal bovine serum (FBS).
2970 μL of FBS was added to 30 μL of each preparation and mixed. Immediately after mixing and after standing at 37 ° C. for 3 hours, 500 μL each was taken and subjected to gel filtration chromatography (Gel Permeation Chromatography, GPC), and fractions (100 drops, 10) were collected. Each fraction was shaken with a vortex mixer to give a sample, and 50 μL of 10 w / v% Triton X-100 and 400 μL of PBS were added to each 50 μL, and the mixture was stirred with a vortex mixer. 100 μL of the solution was taken in a 96-well microplate, and the fluorescence intensity at an excitation wavelength of 485 nm and a fluorescence wavelength of 530 nm was measured using a fluorescence plate reader (manufactured by Wallac Co., Ltd., Arvo Sx-4).
The FD recovery rates after 0 hour and 3 hours were calculated by the following formula (3). The results are shown in Table 4.

  As can be seen from Tables 1 and 2, the coated fine particles in each of the preparations obtained in Examples 1 to 8 and Comparative Example 1 have the same average particle diameter and the same inclusion rate, and the coated fine particles having the same quality in appearance. was gotten. As can be seen from Table 3, the recovery rates of FD and EPC were the same, and coated fine particles were obtained with the same efficiency. On the other hand, as can be seen from Table 4, each of the preparations obtained in Examples 1 to 8 had a higher recovery rate of FD in PBS than the preparation obtained in Comparative Example 1, and stability in PBS. Is good. That is, although the coated microparticles can be easily prepared by the kit of the present invention, the coated microparticles having the same size and the drug encapsulation rate as the conventional method are contained in the drug more firmly with the same efficiency. It was also possible to manufacture in the state.

  According to the present invention, a kit for preparing coated fine particles in which core fine particles are coated with a coating layer is provided.

Claims (16)

A liquid (liquid A) containing a polar organic solvent in which core fine particles are dispersed and a coating layer component constituting a coating layer for coating the core fine particles is dissolved;
A solvent that can be mixed with liquid A and does not contain a polar organic solvent or contains a polar organic solvent in a proportion lower than liquid A (liquid B),
And a kit for preparation of coated fine particles, including an instrument comprising means for mixing liquid A and liquid B. An instrument comprising means for mixing liquid A and liquid B comprises means for containing liquid A, means for containing liquid B, in-line mixing means, means for containing the liquid A to in-line mixing means 2. The kit for preparing coated fine particles at the time of use according to claim 1, wherein the kit comprises a means for feeding liquid to the inlet of the liquid and a means for feeding liquid from the means for containing the liquid B to the inlet of the in-line mixing means. An instrument including means for mixing liquid A and liquid B is a syringe for storing liquid A, a syringe for storing liquid B, a syringe for storing liquid A, and a liquid B for storing the liquid B The kit for preparation of coated fine particles according to claim 1, wherein the kit comprises an in-line mixing means having two inlets each capable of connecting a syringe. The instrument provided with the means for mixing the liquid A and the liquid B is a multi-chamber syringe provided in parallel with chambers for storing the liquid A and the liquid B, respectively, and the discharge port of the syringe is an in-line mixing means. The kit for preparation of coated fine particles according to claim 1, wherein the kit is a syringe. The kit for preparation of coated fine particles according to any one of claims 2 to 4, wherein the in-line mixing means is an in-line mixing means in which a static mixer is connected or incorporated. Means for containing the liquid A from the means for containing the liquid A, the means for containing the liquid B, and the means for containing the liquid B. The kit for preparation of coated fine particles according to claim 1, wherein the kit comprises means for adding the liquid B to the liquid A therein. An instrument including means for mixing the liquid A and the liquid B is a syringe for storing the liquid A and a syringe for storing the liquid B, and the liquid B is added to the liquid A by connecting the two syringes. The kit for the preparation of coated fine particles according to claim 1, which can be used. The instrument provided with the means for mixing the liquid A and the liquid B is a multi-chamber syringe provided with series chambers for storing the liquid A and the liquid B, respectively, and the syringe adds the liquid B to the liquid A. The kit for preparation of coated fine particles according to claim 1, which is a syringe capable of being used. The kit for preparing coated fine particles according to any one of claims 2 to 8, wherein the instrument comprising means for mixing the liquid A and the liquid B is an instrument comprising a transfusion needle or a co-infusion connector at the outlet. The kit for preparation of coated fine particles according to any one of claims 1 to 9, wherein the coating layer component is one or more substances selected from lipids, surfactants and polymers. The kit for preparation of coated fine particles according to any one of claims 1 to 10, wherein the coating layer is a lipid membrane. The kit for preparing coated fine particles according to any one of claims 1 to 11, wherein the core fine particles are fine particles comprising a drug, a lipid aggregate, a liposome, an emulsion fine particle, a polymer, a metal colloid or a fine particle preparation. The coating according to any one of claims 1 to 11, wherein the core fine particle is a fine particle comprising a complex comprising a combination of two or more drugs, lipid aggregates, liposomes, emulsion fine particles, polymers, metal colloids or fine particle preparations. A kit for preparation of fine particles. The kit for preparing coated fine particles according to any one of claims 1 to 11, wherein the core fine particles are fine particles containing a complex of a drug and a liposome as a constituent component. The kit for preparation of coated fine particles according to any one of claims 1 to 14, wherein the polar organic solvent is one or more selected from alcohols, glycols and polyalkylene glycols. Coated microparticles that can be prepared by the on-use preparation kit for coated microparticles according to any one of claims 1 to 15.

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