WO1992004891A1

WO1992004891A1 - Microencapsulated sustained-release preparation and production

Info

Publication number: WO1992004891A1
Application number: PCT/JP1991/001224
Authority: WO
Inventors: Masayuki Arakawa; Takashi Uchio; Yasushi Sato
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 1990-09-14
Filing date: 1991-09-13
Publication date: 1992-04-02
Also published as: AU8491291A; JPH04124127A; KR100202073B1

Abstract

A microencapsulated sustained-release preparation comprising a physiologically active substance as the core and a biocompatible polymer which is decomposable in vivo and/or is compatible with the tissues in vivo as the shell, said preparation being produced from a W/O/W emulsion which is prepared by first preparing a W/O emulsion from an aqueous solution of the physiologically active substance and a solution of the biocompatible polymer in an organic solvent, and conducting secondary emulsification between the W/O emulsion and an aqueous solution of a water-soluble polymer in the presence of a salt substance with the concentration of 0.1 M or saturation concentration in the outer water layer. The microcapsule has a maximum particle diameter of 150 νm or less, a mean particle diameter of 5 to 100 νm, and a coefficient of variation of the particle diameter of 50% or less, so that it can be used as an injection as such without the necessity for conducting particular fractionation and particle size adjustment.

Description

Specification

Mic mouth capsule type sustained release preparation and method for producing the same

〔Technical field〕

The present invention relates to a micro-powered capsule which is administered to warm-blooded mammals such as humans, horses, cattle, etc., and gradually releases encapsulated physiologically active substances in the body of these warm-blooded mammals over a long period of time. The present invention relates to a release preparation and a method for producing the same.

[Background 'technology]

For bioactive substances that require long-term administration, they are encapsulated in a micro-mouth capsule, and the bioactive substance encapsulated in the microcapsules is gradually released, thereby improving the efficacy of the bioactive substance. Improvements in sustainability have been made, and various methods for microencapsulating bioactive substances in this way have been proposed.

For example, Japanese Patent Publication No. 1-57,087 discloses that when a WZO .ZW type emulsion is formed and a microcapsule is produced by an underwater drying method, the water-soluble drug and the drug-retaining substance are prepared. A method for producing microcapsules in which a water layer is thickened or solidified to a viscosity of about 5,000 c.F or more to prepare a W / 0 type emulsion, and thereby the drug is efficiently incorporated into the microcapsules. It has been disclosed. Also, Japanese Patent Application Laid-Open No. 62-201,816 discloses that the viscosity of a W / 0 emulsion is reduced by 150% when a WZO / W emulsion is formed and a π-force microphone is produced by a water drying method. A method for producing a micro: ÷ —cell, which is adjusted to 取り込む 10,000 cp to thereby efficiently incorporate a drug into a my: ′ ″ c: capsule, is disclosed. 2-124, 814 The gazette discloses that by adding a basic drug-retaining substance to an inner aqueous layer containing a water-soluble drug, microcapsules with a low initial burst can be obtained efficiently. In addition, JP-B-63-36,290 discloses a process of dissolving or dispersing an active agent such as a biologically active agent in a shell material in a solvent, and disposing the obtained suspension or solution in the solvent. A step of dispersing in a continuous phase forming medium such as water having a boiling point higher than that of the solvent to form a WZO-type or OZW-type emulsion; and a step of evaporating a part of the solvent from the dispersion obtained in the previous step to form a microcapsule. , A step of separating the microcapsules, and a step of extracting the remaining solvent from the obtained microcapsules, thereby providing a high-quality microcapsule having a high active agent content. Disclosed is a method for producing a microforce cell that can be obtained.

[Disclosure of the Invention]

However, any of the above methods has a problem that the obtained micro force cell not only has a large particle size, but also is irregular and has a wide particle size distribution. For this reason, when microcapsules produced by such a method are to be used as, for example, a medical injection, “suspension properties” are described in section (4) of “16. The particles in the injection solution are usually set to 150 or less, and it is necessary to perform a sorting and sizing operation using a sieve to a particle size of 150 or less. Not only significantly lowers the yield of the product, but also is an extremely cumbersome operation in itself, and must be performed in a sterile and dust-free atmosphere because it is used as a medical injection. Cause cost up Therefore, solving this point has been a serious issue in industrial production.

Therefore, the present inventors have been able to obtain a microclob cell-type sustained-release preparation of a bioactive substance having a fine and uniform particle size without requiring such a sorting and sizing operation. As a result of intensive studies on the microcapsule-type sustained-release formulation and its manufacturing method, the outer aqueous layer was 0.1 M concentration or saturated in the secondary emulsification step of preparing a type-emulsion from W-type zero-emulsion. The present inventors have found that a microcapsule-type sustained-release preparation having a fine and uniform particle size can be obtained by the presence of a salt substance at a concentration, and arrived at the present invention. Accordingly, an object of the present invention is to provide a micro-mouth capsule-type sustained-release preparation having a fine and uniform particle size that does not require a sorting and sizing operation, and a method for producing the same.

Another object of the present invention is to provide a microcapsule-type sustained-release preparation capable of achieving a high incorporation rate of a physiologically active substance to be included therein into a microcapsule, and a method for producing the same. .

Furthermore, another object of the present invention is to provide a good micro-mouth regardless of the concentration of an aqueous solution of a physiologically active substance as an inclusion substance or the concentration of an organic solvent solution of a biocompatible polymer substance as an outer shell substance. The present invention provides a microcell-based sustained-release preparation capable of producing a capsule-type sustained-release preparation, whereby the release rate of a physiologically active substance can be appropriately changed, and a method for producing the same. And there.

That is, the present invention has an inclusion having a bioactive substance and an outer shell material formed of a biocompatible polymer substance having biodegradability and / or tissue compatibility in vivo, and has a maximum particle size. 1 5 0 or less It is a microforced sustained-release preparation having an average particle diameter of 5 to 100 and a coefficient of variation of 50% or less.

The present invention also provides a W0-type emulsion comprising an aqueous solution containing a physiologically active substance and an organic solvent solution containing a biocompatible polymer substance having biodegradability and / or histocompatibility in vivo. Then, the W / 0 emulsion and the aqueous solution of a water-soluble polymer substance are subjected to secondary emulsification to prepare a W0 / W emulsion, and the organic solvent is evaporated from the obtained wzo zw emulsion, The secondary emulsification step described above is used to prepare a micro-alpha capsule-type sustained-release preparation containing a bioactive substance as an inclusion substance and a biocompatible polymer substance as an outer shell substance by solidifying, further washing and drying. This is a process for producing a microcapsule-type sustained-release preparation in which a salt substance having a concentration of 0.1% or saturation is present in the outer aqueous layer.

In the present invention, the physiologically active substance to be included in the micro force cell is not particularly limited as long as it has excellent hydrophilicity and is soluble in water, and is not particularly restricted by pharmacological action or the like. , Physiologically active protein polypeptides and their derivatives, as well as antibiotics, antitumor agents, antipyretics, analgesics, anti-inflammatory agents, antitussive expectorants, sedatives, muscle relaxants, antiepileptics, anti- Ulcers, antidepressants, antiallergics, cardiotonic, arrhythmic, vasodilators, antihypertensive diuretics, diabetes, anticoagulants, hemostats, antituberculosis, hormones, narcotics And various agents such as agents. Of these, particularly preferred physiologically active substances are the microforced sustained-release preparations of the present invention, which have a fine and uniform particle size, and are particularly suitable for injection preparations without the necessity of fractionating and sizing. Is possible Therefore, they are proteins, polypeptides and their derivatives that need to be injected and need to be administered over a relatively long period of time. Slovodin (EP type), granulocyte colony stimulating factor (G-CSF), macrophic zico oral knee stimulating factor (M-CSF), granulocyte Mac phagocylonic stimulating factor (GM-CSF), interferon (IFN, IFN, IFN r), interleukin (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6), tumor death factor (TNF), growth hormone (GH or STH), insulin, calcitonin (CT), and the like, and derivatives thereof. These physiologically active substances may be of natural origin extracted and purified from animals such as mammals, fish, birds, and other plants, or may be obtained by means of genetic recombination, synthesis, semi-synthesis, etc. May be manufactured. One of these physiologically active substances may be used alone, or two or more of them may be used in combination, if necessary. The physiologically active substance differs depending on the type and properties of the substance, or is usually contained in a π-capsule in the range of 0.01 to 50% by weight.

In addition, the biocompatible macromolecule material used as the outer shell material of the microcapsule used in the present invention is biodegradable or biocompatible, and can be used in humans, horses, cows, and other living organisms. Any substance can be used as long as it is decomposed or taken into tissues when it enters, and any substance can be used.Specifically, biodegradable biocompatible high molecular substances include, for example, polylactic acid. Polyglycolic acid, polytaenoic acid, polylingic acid, polylactic acid glycolic acid copolymer, etc. © Homopolymers and copolymers of fatty acid esters, polyacrylic acid esters such as poly- or cyanoacrylic acid esters, hydroxybutyric acids such as poly-hydroxy dramanic acid, and polytriols Polyalkylene oxalates such as methylene oxide, polyorthoesters, polycarbonates such as polyorthocarbonate and polyethylenecarbonate, and poly-τ "-benzyl-L-glutamic acid And polyamino acids such as poly-L-alanine, etc. Examples of biocompatible high-molecular-weight biocompatible materials that are biocompatible include, for example, polystyrene, polyacrylic acid, polymethacrylic acid, styrene, and styrene. Copolymer of acrylic acid and methacrylic acid, copolymer of acrylic acid and methacrylic acid, nylon, tetron, polyamino acid, silicone Copolymer, polyurethane, polybutyl acetate, polyvinyl alcohol, polyacrylamide, ethylene-vinyl acetate copolymer, maleic anhydride copolymer, dextran stearate Examples thereof include ethyl cellulose, acetyl cellulose, nitrocellulose, etc. These biocompatible high-molecular substances may be used alone or in combination of two or more as necessary. Of these, particularly preferred biocompatible polymer substances are biodegradable biodegradable capsules of the present invention, which are particularly suitable for injection. Biocompatible polymer substances, especially fatty acid esters such as polylactic acid, polylactic acid, polycunic acid, polylingic acid, and polylactic glycolic acid copolymer The average molecular weight of these biocompatible polymer substances is preferably about 1,000 to 200,000, and more preferably about 1,000 to 200,000. It is in the range of 2,000 to 100,000. The biocompatible polymer substance is used depending on the type and the like, but is usually used in a microcapsule in an amount of 20 to 99% by weight, preferably 40 to 95% by weight.

Furthermore, in the present invention, a water-soluble stabilizer or excipient may be used, if necessary, for the purpose of ensuring the stability of the above-mentioned physiologically active substance, or increasing the amount of this physiologically active substance to facilitate handling. (Shaping agent) can be encapsulated in microcapsules. Examples of stabilizers used for this purpose include proteins such as albumin and gelatin; amino acids such as glycine, arginine, histidine, lysine, glutamic acid, and aspartic acid; Salts, surfactants such as sorbitan fatty acid ester, polyoxyethylene fatty acid ester, and dalyserine fatty acid ester; chelating agents such as ethylenediaminetetraacetate and citric acid; and darcose and sucrose. And sugars such as fructose, trehalose, dextrin, dextran, mannitol and sorbitol, and sugar alcohols. Examples of excipients include natural water-soluble gums such as arabic gum and xanthan gum, and natural water-soluble gums such as casein, gelatin, collagen, albumin, cellulose, starch, and agar. Examples include a high molecular compound and a synthetic water-soluble high molecular compound such as lipoxymethylcellulose and polysaccharide. The amount of these stabilizers and excipients used is determined depending on the type and purpose thereof, but is usually about 0 to 500% based on the biocompatible polymer. The microcapsule-type sustained-release preparation of the present invention A WZO-type emulsion is prepared from an aqueous solution and an organic solvent solution of a biocompatible polymer, and when the W0-type emulsion is secondarily emulsified with an aqueous solution of a water-soluble polymer, the outer aqueous layer is formed. In the presence of a salt substance at a concentration of 0.1 M or a saturated concentration, a WZO-no W-type emulsion is prepared, and a physiologically active substance is included from the obtained W / OZW-type emulsion. It is obtained by preparing a microcapsule containing a molecular substance as an outer shell substance, and the obtained microcapsule has a maximum particle size of 150 or less, usually 75 or less, and a more preferable form is 30 or less, the average particle size is 5 to 100, usually 5 to 25, and more preferably in the range of 5 to 15 / m, and the coefficient of variation is 50% or less. It can be used as it is without injection. The maximum particle size is affected by the conditions of emulsification and the concentration of the solution, and may be around 150 depending on these conditions. Note that the maximum particle size is obtained by calculating the particle size of the largest particle in one lot, the average particle size is obtained by calculating the average value of 500 particle sizes, and the coefficient of variation is (standard deviation) ÷ (Average particle size) It is a value obtained by calculating from the formula of X100.

The microcapsule-type sustained-release preparation of the present invention is produced by the following method.

First, a biocompatible polymer substance is dissolved in an organic solvent to prepare an organic solvent solution. The organic solvent used for this purpose may be any solvent which has a relatively low boiling point, is immiscible with water, and dissolves a biocompatible polymer.For example, dichloromethane, chloromethane, chlorophore J-REM, trichlorethane, carbon tetrachloride, ethyl acetate And n-hexane and tolnin. These can be used alone, or two or more of them can be used in combination. The concentration of the biocompatible polymer substance in the organic solvent solution can be appropriately changed depending on the type of the biocompatible polymer substance to be used, but is usually 0.5 to 50% by weight, preferably It is about 1 to 40% by weight.

Separately from the above-mentioned organic solvent solution of the biocompatible polymer substance, the above-mentioned physiologically active substance and a water-soluble stabilizing agent or excipient added as necessary are dissolved in predetermined water to prepare an aqueous solution thereof. Prepare. When preparing the aqueous solution of the physiologically active substance, a pH adjuster is added as necessary for the purpose of maintaining its stability and solubility. PH regulators used for this purpose include, for example, acids such as sulfuric acid, citric acid, acetic acid, and carbonic acid, and sodium and calcium salts thereof, and various buffers such as a phosphate buffer. Liquids can be mentioned. The amount of use of these pH adjusters is not particularly limited, but is usually about several tens of mM concentration.

Next, the aqueous solution of the physiologically active substance prepared in this manner is added to the organic solvent solution of the biocompatible polymer substance under high-speed stirring to prepare a WZO type emulsion. The stirring speed at this time is usually 1,000 to 30,000 rpm, preferably 5,000 to 28,000 rPm, and the emulsification temperature is about -100 to 40, preferably about -50 to 20. Is good.

The WZO-type emulsion thus prepared is then mixed with a prepared aqueous solution of a water-soluble polymer substance in the presence of a salt substance having a concentration of 0.1 M to a saturation concentration in the outer aqueous layer, It is secondarily emulsified into a W / 0 emulsion. Examples of the water-soluble polymer used in the secondary emulsification step include polyvinyl alcohol (PVA), polyvinylpyrrolidone carboxymethyl senorelose sodium, gelatin, chitin and its derivatives, xanthonone and Derivatives, hyaluronic acid and its sodium salts, dextran, pectin, chondroitin sulfate and its sodium salts, and the like can be mentioned. In addition to being used alone, two or more can be used as a mixture. The concentration of the water-soluble polymer substance in the aqueous solution is usually in the range of 0.05 to 5.0% by weight, preferably in the range of 0.1 to 2.0% by weight.

In addition, the salt substance to be present in the outer water layer during the secondary emulsification is an emulsion formed at the interface between the previously prepared WZO emulsion and the outer water layer. Any material can be used as long as it can improve the stability and suppress the swelling of the biocompatible polymer substance, and is not particularly limited. For example, sodium acetate. And various organic acid salts such as sodium citrate, and various inorganic acid salts such as sodium chloride, potassium chloride, calcium chloride, sodium sulfate, sulfuric acid potassium, and the like. Examples thereof include ammonium salts such as ammonium sulfate, and quaternary amine salts such as link mouth ride and tetramethylammonium chloride. These salt substances are usually present in the outer water layer in the range of 0.1 M concentration to the saturation concentration, preferably in the range of 0.5 M concentration to the saturation concentration, and more preferably in the range of 0.1 M concentration to the saturation concentration. If the concentration is low, the problem of bursting occurs, and if the concentration is higher than the saturation concentration, the problem of embedding of salt occurs. And the method of making this salt substance exist in the outer water layer For the wzo / w-type emulsion to be prepared in the secondary emulsification step, it only needs to be present in the outer water layer as a result, and it may be added to an aqueous solution of a water-soluble polymer in advance. Also, it may be added separately when the W / 0 emulsion is mixed with an aqueous solution of a water-soluble polymer substance.

The W / 0 / W type emulsion thus prepared is then stirred at room temperature or at 0 to 50 ° C, preferably at 0 to 25, to evaporate the organic solvent. As a result, the biocompatible polymer material of the outer shell material is solidified to generate micro force cells.

Further, the produced microcapsules are separated by means such as centrifugation or filtration, washed and dried if necessary. Thereafter, if necessary, the dispersion is re-dispersed in an aqueous solution containing an appropriate excipient such as mannitol, and dried by freeze-drying or the like to remove the residual organic solvent. Produces a micronized capsule-type sustained-release preparation with excellent dispersibility.

The microcapsules obtained as described above have a maximum particle size of 150 or less and an average particle size of 5 to 100, and a coefficient of variation of 50% or less. In particular, it can be used as an injection as it is, without performing a sorting and sizing operation.

In addition, the microcapsule-type sustained-release preparation of the present invention can be used for injections, powders, granules, tablets, tablets, etc., depending on the pharmacological action of a physiologically active substance contained as an inclusion in the microcapsules for therapeutic purposes. Although it can be formulated into any dosage form such as suppositories, it has a fine and uniform particle size and has needle penetration and redispersibility as it is without the need for sorting and sizing. It is suitable as an injection. The microcapsule-type sustained-release preparation of the present invention has a fine and uniform particle size. Further, according to the method of the present invention, in the secondary emulsification step of emulsifying the WZ0-type emulsion into a W / 0 / W-type emulsion, a salt substance having a concentration of 0.1% or more is present in the outer water layer. As a result, the properties of the interface between the WZO-type emulsion and the aqueous solution of a water-soluble polymer are improved, and as a result, a microcellular sustained-release preparation having a fine and uniform particle size is obtained. It is considered to be obtained.

[Brief description of drawings]

FIG. 1 is a graph showing the time-dependent change in the rEPO content of the rEPO-owned microcapsule obtained in Example 1. [Best mode for carrying out the invention]

Hereinafter, the present invention will be specifically described based on examples and experimental examples.

Example 1

1 g of a polylactic acid-glycolic acid copolymer (noglycolic acid lactate-75 / 25s weight-average molecular weight 10,000) was dissolved in dichloromethane 5 to obtain a biocompatible polymer organic solvent solution. Genetically modified human erythropoietin (hereinafter abbreviated as rEPO) 1 rag and two high-grade gelatin type B (manufactured by Nitsubishi, Al-treated gelatin with an average molecular weight of about 7,000) 40ni of 1 ffig It was dissolved in water for injection and sterile-filtered through a membrane filter with a pore size of 0.22 (manufactured by Nippon Millipore Limited: Mylex GV) to obtain an aqueous solution of a physiologically active substance. The above aqueous solution is dropped into the above organic solvent solution under stirring (28,000 rpm) with a homogenizer (manufactured by Kinematics: Polytron), and further stirred and mixed at the same inversion number for 30 seconds. Combined to obtain a WZ O-type emulsion

Next, the above-mentioned W / 0 type emulsion was added to a 0.δ% aqueous polyvinyl alcohol solution 200 containing 1 M sodium chloride, which had been previously ice-cooled, and added at a speed of 10.000 rpm. The mixture was stirred and mixed for 2 seconds to form a W, 0 / W emulsion.

Furthermore, the dispersion containing the W / 0 / W type emulsion is gently stirred at room temperature to volatilize the remaining methylene chloride, and the polylactic acid-glycolic acid is removed. After solidification of the polymer, microcapsules generated by centrifugation at 3,000 rpm for 10 minutes were collected.

The microforce obtained in this manner was dispersed again in water for injection, and the operation of centrifugation was repeated 5 times, so that polybutyl alcohol, sodium chloride and free water were released into the outer water layer. The obtained rEP0, polylactic acid-glycolic acid copolymer was washed.

The microcapsules finally collected were redispersed in 5% aqueous mannitol solution 5 and then freeze-dried. The above steps were all performed aseptically and dustlessly.

The maximum particle size of the rEPO0 Municipal Mikuguchi capsule obtained in this way was 15, the average particle size was 8.0, and the coefficient of variation was 40%. Also, the uptake rate of rEPO was about 95%. Experimental example

The r EP 0 microcapsule obtained in Example 1 above was dispersed in a 50 mM monophosphate buffer (pH 7.4) containing 0.05% of Tween 20 in a rotary culture machine. (Taiyo scientific industrial賴: KT - 50) using, in O thermostatic bath Te 37 and invert rotated at ₂₅ r _P ς - After a predetermined time, the dispersion was subjected to centrifugation (3,000 rpm, 10 minutes) to collect microcapsules.

The amount of r EPO in the microcapsules was adjusted by dissolving the collected microcapsules with 1 rag of acetonitrile and then adjusting the total volume to lOfflg with 50 mM phosphate buffer. It was quantified by liquid chromatography (HPLC).

The changes in the rEPO content in the microcapsules determined over time are shown in FIG. As is clear from FIG. 1, it was observed that r Ε Ρ0 in the microphone was released at a substantially constant rate for 4 weeks.

Example 2

1 g of poly (lactic acid-glycolic acid) copolymer (lactic acid-glycolic acid ratio-75 / 25; weight average molecular weight 10,000) was dissolved in dichloromethane 5 to obtain a biocompatible polymer solution in an organic solvent. r EPO 1 mg was dissolved in 1 fflfi of 50 mM phosphate buffer (pH 6, 6).

The solution was aseptically filtered through a 0.22 membrane filter to obtain an aqueous solution of a physiologically active substance. The aqueous solution was dropped into the organic solvent solution under stirring (28,000 rpm) with a homogenizer, and further stirred and mixed at the same rotation speed for 30 seconds to obtain a WZO-type emulsion.

Next, set up 1 M sodium chloride, which was previously ice-cooled separately.

The above-mentioned WZO-type emulsion was added to 0.5% -polyvinyl alcohol aqueous solution 200, and the mixture was stirred and mixed at a speed of 10,000 rpm for 30 seconds to form a W / 0 / W-type emulsion. Thereafter, in the same manner as in Example 1 above, rEPO0 microcapsules were obtained.

The r EPO house-owned microcapsules obtained in this way are Had a maximum particle size of 40, an average particle size of 22%, and a coefficient of variation of 45%. The uptake rate of rEP0 was about 80%.

Example 3

1 g of polylactic acid dalicholic acid copolymer (lactic acid / daricholic acid ratio = 7525, weight average molecular weight 20,000) was dissolved in dichloromethan (5Λ? £) to prepare an organic solvent solution. In addition, recombinant human granulocyte colony stimulating factor (hereinafter referred to as rG-CSF) la and two-grade high-grade gelatin type B40 mg were dissolved in 1 i of water for injection in the Japanese Pharmacopoeia, and a 0.22 pore size membrane was used. The solution was aseptically filtered with a filter to obtain an aqueous solution. The aqueous solution was dropped into the organic solvent solution under stirring (28,000 rpm) with a homogenizer, and the mixture was further stirred and mixed at the same rotation speed for 30 seconds to obtain a WZO-type emulsion. Next, the above-mentioned W0 type emulsion was added to a 0.5% aqueous solution of 0.5% polybutylpyrrolidone containing 1M sodium sulfate, which had been previously ice-cooled separately, and added at a speed of 10,000 rpm for 30 minutes. The mixture was stirred and mixed for 2 seconds to produce a W / 0 ZW type emulsion. Thereafter, in the same manner as in Example 1 above, rG—CSF microcapsules were obtained.

The obtained rG—CSF-containing microcapsule capsule had a maximum particle size of 50, an average particle size of 20, and a coefficient of variation of 40%. The uptake rate of rG—CSF was about 90%.

Example 4

1 g of polylactic glycolic acid copolymer (noglycolic acid lactate ratio = 75/25; weight average molecular weight 5,000) was dissolved in dichloromethan 5 ^ to obtain an organic solvent solution. r EP 0 1 nig and Nippi High Grade Gelatin Type B 40 mg dissolved in 1 Japanese Pharmacopoeia Water for Injection Then, the solution was aseptically filtered through a membrane filter having a pore size of 0.22 to obtain an aqueous solution. The aqueous solution was dropped into the above organic solvent solution under stirring (28,000 rpm) with a homogenizer, and further stirred and mixed at the same inversion number for 30 seconds to obtain a W0 emulsion.

Next, the above-mentioned WZO-type emulsion was added to 200 ml of 0.5% -rubiximetyl cell π-sodium sodium water solution containing 1 M ammonium sulfate, which had been separately ice-cooled, and the speed was increased to 10,000 rpm. For 30 seconds to form a WZOZW emulsion. Thereafter, r EPO microcapsules were obtained in the same manner as in Example 1 above. The obtained r カプセル Ρ 0 micro-capsule has the largest particle size.

The average particle size was 55 and the coefficient of variation was 48%. The uptake rate of r r Ε 0 was about 95%.

卖 Example 5

1 g of poly (lactic acid) polymer (weight average molecular weight 5,000) was dissolved in dichloromethane 5 to obtain an organic solvent solution. 0 to 3-11 mg and Nippi High-Grade Gelatin Type B 40 mg were dissolved in 1 ^ water for injection in the Japanese Pharmacopoeia (1 ^), and sterile-filtered with a membrane filter with a pore size of 0.22 / OT to obtain an aqueous solution. The aqueous solution was dropped into the above organic solvent solution with stirring (28,000 rPm) using a homogenizer, and the mixture was stirred and mixed at the same speed for 30 seconds to form a W / 0 emulsion. Obtained.

Next, a 1M-chlorinated reamer, which has been separately ice-cooled, is set up. 0. δ% —200 gelatin aqueous solution of purified gelatin in the Japanese Pharmacopoeia. The above-mentioned W / 0 emulsion is added, and ΙΟ, ΟΟΟ pm The mixture was stirred and mixed at the speed described above for 30 seconds to form a W / 'OZW type emulsion. Hereinafter, in the same manner as in Example 1 above, As a result, microcapsules containing rG—CSF were obtained.

The obtained rG-CSF microcapsule had a maximum particle size of 60, an average particle size of 25, and a coefficient of variation of 44%. The uptake rate of rG—CSF was about 90%.

Example 6

250 rag of poly (lactic acid dalycholic acid) copolymer (ratio of daglycolic acid-75 to 25, weight average molecular weight 10,000) was dissolved in dichloromethane (5 s? £) to prepare an organic solvent solution. r Dissolve 1 mg of EP 0 and 40 mg of Nippi High-Grade Gelatin Type B in 1 ffig of 10 mM phosphate buffer (PH 6.5), filter aseptically through a 0.22 pore size membrane filter, The active substance solution was used. The physiologically active substance solution was dropped into the organic solvent solution under stirring (28,000 rpm) with a homogenizer, and the mixture was further stirred and mixed at the same rotation speed for 30 seconds to obtain a W / 0 emulsion.

Next, the above-mentioned WZO-type emulsion was added to a 0.1M calcium chloride solution containing 0.5M calcium chloride aqueous solution 200 which had been separately cooled in advance and stirred at 7,500 rpm for 30 seconds. By mixing, a W / O ZW type emulsion was formed. Thereafter, in the same manner as in Example 1 above, rEPO Shayu Microcrobusel was obtained.

The obtained r E P 0 有 own microcapsule has the maximum particle size.

There were 25 lords with an average particle size of 13 lords, and the coefficient of variation was 47%. Also, the uptake rate of rEPO was about 90%.

Example 7

Polylactic acid glycolic acid copolymer (lactic acid / glycolic acid ratio = 75/25, weight average molecular weight 10,000) 1 g to dichloromethan 5 After dissolution, a solution of a biocompatible polymer substance in an organic solvent was obtained. 1 ng of rPO and 40 m of Nibbi Greater Gelatin Type B were dissolved in 1 i of water for injection in the Japanese Pharmacopoeia, and sterile-filtered through a membrane filter with a pore size of 0.22 to obtain an aqueous solution of a physiologically active substance. The above aqueous solution was dropped into the above organic solvent solution under stirring (28,000 rpm) with a homogenizer, and the mixture was further stirred and mixed at the same rotation speed for 30 seconds to obtain a WZ0 type emulsion.

Next, each of the WZ0 emulsions was added to 200% of a 0.5% aqueous solution of sodium borohydride containing sodium chloride of various concentrations, which had been previously ice-cooled separately, and the emulsion was added at a speed of 10,000 rpm. After stirring and mixing for 2 seconds, a WZ0ZW type emulsion was formed.

Further, the dispersion containing these WZOZW-type emulsions was gently stirred at room temperature to volatilize the remaining methylene chloride and solidify the polylactic acid-glycolic acid copolymer. Thereafter, microcapsules generated by centrifugation at 3,000 rpm for 10 minutes were collected.

The microcapsules thus obtained were dispersed again in water for injection, and the operation of centrifugation was repeated 5 times to remove the boryl alcohol, sodium chloride and the sodium chloride in the outer water layer. r EPO, polylactic acid glycolic acid copolymer was washed.

The microcapsules finally collected were redispersed in 5% aqueous mannitol solution 5 and then freeze-dried.

The maximum particle size, average particle size, and variation coefficient of each rEP Hishei micro-power cell prepared by changing only the salt concentration of the outer water layer in the secondary emulsification process were determined, and the shape was observed. . The results are shown in Table 1. ¾- »PfiP 37 to f ¾ Zl fl¾

¾ 2¾ S. nr tiz. 3¾- 1¾ 权ナ

M diameter im diameter several%

Ms J Sho H¾

0 80.5 30.6 55.0

Ω 1 丄 1 丄 1 * 7 f AO q

0,5 17.5 8.9.43.0 Substantially spherical

1.0 15.0 7.7 40.3 Perfect spherical

[Industrial applicability]

According to the present invention, the presence of a predetermined concentration of a salt substance in the outer water layer when a wzozw type emulsion is produced allows the particle size to be fine and fine without the need for viscosity adjustment and sorting and sizing. Uniform micro force can be produced, which is industrially advantageous. In addition, the micro-force Busel-type sustained-release preparation comprising the physiologically active substance of the present invention and the biocompatible polymer produced by this method has excellent sustained-release properties, and can be administered by a single administration. The drug effect of the physiologically active substance can be exhibited over a long period of time.

Claims

The scope of the claims

1. Includes a biologically active substance and an outer shell formed of a biocompatible polymer material having biodegradability and / or tissue compatibility, and has a maximum particle size of 150 or less. A microcapsule-type sustained-release preparation characterized by having an average particle size of 5 to 100 and a coefficient of variation of 50% or less.

2. A W0-type emulsion is prepared from an aqueous solution containing a physiologically active substance and an organic solvent solution containing a biocompatible polymer substance having biodegradability and / or tissue compatibility in vivo. The W / ZW emulsion is secondarily emulsified with an aqueous solution of a water-soluble polymer substance to prepare a W / 0 / W emulsion, and the organic solvent is evaporated and solidified from the obtained W / OZW emulsion. And then washed and dried to prepare a microcapsule-type sustained-release preparation containing a physiologically active substance as an inclusion substance and a biocompatible polymer substance as an outer shell substance. A method for producing a micron π-force busel-type sustained-release preparation, characterized in that a salt substance having a concentration of 0.1 M or a saturated concentration is present in the outer aqueous layer.