JPH04124127A - Microcapsule type sustained releasable preparation and its production - Google Patents

Abstract

PURPOSE:To obtain a microcapsule type sustained releasable preparation gradually releasable in vivo for many hours, having fine and uniform particle diameter, containing a physiologically active substance and a polymer substance compatible with living body. CONSTITUTION:0.01-50wt.% physiologically active substance such as protein, polypeptide or a derivative thereof is contained as an inner substance of microcapsule. 20-99wt.% polymer substance compatible with living body (e.g. polylactic acid glycolic acid copolymer) to be a shell substance of microcapsule is contained. An aqueous solution of the physiologically active substance and a solution of said polymer substance in an organic solvent are adjusted to W/O type emulsion, which is secondarily emulsified with an aqueous solution of a water-soluble polymer substance. In the operation, a salt substance having 0.1M to saturated concentration is made to exist in the outer water layer to prepare a W/O/W type emulsion. The objective substance having <=150mum maximum particle diameter, <=5-100mum average particle diameter and <=50% coefficient of variation is obtained and is directly used as an injection as it is without especially carrying out classifying and adjusting operation of particles.

Description

[Detailed Description of the Invention] [Industrial Field of Application] This invention is intended for administration to warm-blooded mammals such as humans, horses, and cows,
The present invention relates to a microcapsule-type sustained release preparation that gradually releases encapsulated physiologically active substances in the bodies of these warm-blooded mammals over a long period of time, and a method for producing the same.

[Conventional technology]

Physiologically active substances that require long-term administration are microencapsulated, and the bioactive substances encapsulated in the microcapsules are gradually released, thereby improving the sustainability of the medicinal effects of the bioactive substances. Various methods have been proposed for microencapsulating physiologically active substances.

For example, in Japanese Patent Publication No. 1-57.087, W10/
- W10 type emulsion in which the inner aqueous layer containing a water-soluble drug and drug-retaining substance is thickened or solidified to a viscosity of approximately 5,000 cp or more when forming a type emulsion and manufacturing microcapsules by drying in water. A method for manufacturing microcapsules is disclosed in which a drug is efficiently incorporated into the microcapsules.In addition, JP-A No. 62-201.816 discloses a W/O/W type When manufacturing microcapsules by forming an emulsion and drying in water, the viscosity of the W10 emulsion is set to 150 to 10,000.
A method for manufacturing microcapsules is disclosed in which the cp is adjusted to efficiently incorporate drugs into the microcapsules. Furthermore, JP-A-2-124.81
Publication No. 4 discloses that microcapsules with less initial burst can be obtained efficiently by adding a basic drug-retaining substance to the inner aqueous layer containing a water-soluble drug. In addition, Japanese Patent Publication No. 63-36.290 describes a process for dissolving or dispersing an active agent such as a biologically active agent in a shell material in a solvent, and a process for dissolving or dispersing an active agent such as a biologically active agent in a shell material, and dispersing the resulting suspension or solution from the solvent. A step of dispersing in a continuous phase forming medium such as high boiling point water to form a W2C type or ○/W type emulsion compound, and a step of evaporating a part of the solvent from the dispersion obtained in the previous step to microcapsule. This process consists of a step of producing microcapsules, a step of separating the microcapsules, and a step of extracting the remaining solvent from the microcapsules obtained. A method of making capsules is disclosed.

[Problem to be solved by the invention]

However, in any of the above methods, there is a problem that the obtained microcapsules not only have a large particle size but also are irregular and have a wide particle size distribution. Therefore, when microcapsules produced by such a method are used as, for example, a medical injection, 16. Since it is stated in item (4) of the injection that "particles in a suspended injection solution should generally be 150 particles or less," it is necessary to separate and size the particles using a sieve to reduce the particle size to 150 particles or less. This sorting and sizing operation not only significantly reduces the yield of the product, but is itself an extremely troublesome operation, and since it is intended for medical injections, it must be carried out in a sterile and dust-free atmosphere. Must be done below;
This causes an increase in costs, and solving this problem has become a serious issue in industrial production.

Therefore, the present inventors developed a microcapsule-type sustained-release formulation that can obtain microcapsule-type sustained-release preparations of physiologically active substances with fine and uniform particle sizes without the need for such sorting and sizing operations. As a result of extensive research into sexual preparations and their manufacturing methods, we found that in the secondary emulsification step of preparing a W/O/W type emulsion from a W10 type emulsion, a salt substance with a concentration of 0.1M to saturated concentration is added to the outer aqueous layer of the W10 type emulsion. It was discovered that a microcapsule-type sustained-release preparation having a fine and uniform particle size can be obtained by the presence of .

Therefore, an object of the present invention is to provide a microcapsule-type sustained release preparation having a fine and uniform particle size that does not require separate sizing operations, and a method for producing the same.

Another object of the present invention is to provide a microcapsule-type sustained release preparation that can achieve a high rate of incorporation of a physiologically active substance into microcapsules, and a method for producing the same.

Furthermore, another object of the present invention is to provide a microcapsule type that exhibits good sustained release properties regardless of the concentration of an aqueous solution of a physiologically active substance as an inclusion material or the concentration of an organic solvent solution of a biocompatible polymeric substance as an outer shell material. It is an object of the present invention to provide a microcapsule-type sustained release preparation that can be manufactured and thereby make it possible to appropriately change the release rate of a physiologically active substance, and a method for manufacturing the same.

[Means to solve the problem]

That is, the present invention has an inner material containing a physiologically active substance and an outer shell material formed of a biocompatible polymeric material having biodegradability and/or in vivo tissue compatibility, and has a maximum particle size of It is a microcapsule type sustained release preparation having a particle size of 1504 or less, an average particle size of 5 to 100, and a coefficient of variation of 50% or less.

Moreover, the present invention prepares a W10 type emulsion with an aqueous solution containing a physiologically active substance and an organic solvent solution containing a biocompatible polymeric substance that is biodegradable and/or tissue compatible in the living body, and then This W10 type emulsion and an aqueous solution of a water-soluble polymer substance are secondary emulsified to prepare a W/O/W type emulsion, and the organic solvent is evaporated from the obtained W/O/W type emulsion. When preparing microcapsule-type sustained-release preparations that contain physiologically active substances by solidification, further washing, and drying, and have a biocompatible polymeric substance as an outer shell material, the secondary emulsification process This is a method for producing a microcapsule type sustained release preparation in which a salt substance is present in the outer aqueous layer at a concentration of 0.1 M to saturation.

In the present invention, the physiologically active substance to be included in the microcapsules only needs to have excellent hydrophilicity and be soluble in water, and is not particularly limited by pharmacological action. proteins, polypeptides, and their derivatives, as well as antibiotics, antitumor agents, antipyretics, analgesics, anti-inflammatory agents, antitussive expectorants, sedatives, muscle relaxants, antiepileptics, antiulcer agents, and antidepressants. , antiallergic agents, cardiotonic agents, antiarrhythmia agents, vasodilators, antihypertensive diuretics, antidiabetic agents, anticoagulants, hemostatic agents, antituberculous agents, hormonal agents, and narcotic antagonists. can. Among these, particularly preferred physiologically active substances are the microcapsule-type sustained-release preparation of the present invention, which has a fine and uniform particle size and can be made into an injectable preparation without the need for separation and sizing operations. Therefore, these proteins, polypeptides, and their derivatives need to be made into injections and require relatively long-term administration.Specifically, they include erythropoietin (EPO), granulocytes, Colony stimulating factor (G-C3F), macrophage colony stimulating factor (M-C3F), granulocyte macrophage colony stimulating factor (GM-C3F), interferon (IFNα, IFNβ, IFNγ), interleukin (TL-1, IL-2) , IL-3, IL-4, IL
-5, IL-6>, tumor necrosis factor (TNF), growth hormone (GH or ST former), insulin, calcitonin (
CT) and their derivatives. These physiologically active substances may be of natural origin extracted and purified from animals such as mammals, fish, and birds, or other plants, or may be produced by means such as genetic recombination, synthesis, or semi-synthesis. It may be manufactured by. These physiologically active substances may be used alone or in combination of two or more, if necessary. The physiologically active substance is usually encapsulated in microcapsules in a range of 0.01 to 50% by weight, although it varies depending on its type and properties.

In addition, the biocompatible polymeric material that forms the outer shell material of the microcapsules used in the present invention is biodegradable or tissue compatible with the body and cannot be absorbed into the living body of humans, horses, cows, etc. Any substance may be used as long as it is compatible with biodegradable biodegradable or biocompatible polymer substances, such as polylactic acid and polyglycolic acid. , homopolymers and copolymers of fatty acid esters such as polycitric acid, polymalic acid, and polylactic acid-glycolic acid copolymers, polyacrylic acid esters such as polyα-cyanoacrylic acid ester, and polyβ
- Hydroxybutyric acids such as hydroxybutyric acid, polyalkylene oxalates such as polytrimethylene oxalate, polyorthoesters, polyorthocarbonates,
Polycarbonates such as polyethylene carbonate,
There are polyamino acids such as poly-γ-benzyl monoglutamic acid and poly-L-alanine, and examples of biocompatible polymeric substances that are tissue compatible with the body include polystyrene,
Polyacrylic acid, polymethacrylic acid, copolymer of styrene and acrylic acid or methacrylic acid, copolymer of acrylic acid and methacrylic acid, nylon, tetron, polyamino acid, silicone polymer, polyurethane, polyvinyl acetate, polyvinyl alcohol, Examples include polyacrylamide, ethylene-vinyl acetate copolymer, maleic anhydride copolymer, dextran stearate, ethyl cellulose, acetyl cellulose, nitrocellulose, and the like. These biocompatible polymeric substances are
Only one type thereof may be used, or two or more types may be used in combination if necessary. Among these, biocompatible polymeric substances that are particularly preferred are biodegradable biocompatible polymeric substances, especially polylactic acid, polylactic acid, etc., since the microcapsule-type sustained-release preparation of the present invention is especially suitable as an injection preparation. These are homopolymers and copolymers of fatty acid esters such as glycolic acid, polycitric acid, polymalic acid, and polylactic acid-glycolic acid copolymers. The average molecular weight of these biocompatible polymeric substances is preferably about 1,000 to
200,000, more preferably 2,
The range is 000 to i, oo, , ooo. This biocompatible polymeric substance is usually used in a microcapsule in an amount of 20 to 99% by weight, preferably 40 to 95% by weight, although it varies depending on its type.

Furthermore, in the present invention, water-soluble stabilizers and excipients may be added as necessary to ensure the stability of the physiologically active substance or to increase the amount of the physiologically active substance and make it easier to handle. (excipients) etc. can be encapsulated in microcapsules. Stabilizers used for this purpose include, for example, proteins such as albumin and gelatin, amino acids such as glycine, arginine, histidine, lysine, glutamic acid, and aspartic acid, or their salts, sorbitan fatty acid esters, and polyoxygenates. Surfactants such as ethylene fatty acid ester and glycerin fatty acid ester, chelating agents such as ethylenediaminetetraacetate and citric acid, and sugars or sugar alcohols such as glucose, sucrose, fructose, trehalose, dextrin, dextran, mannitol, and sorbitol. etc. can be mentioned. Examples of excipients include natural water-soluble gums such as gum arabic and xanthan gum, casein, gelatin, collagen, albumin, cellulose,
Examples include natural water-soluble polymer compounds such as starch and agar, and synthetic water-soluble polymer compounds such as carboxymethyl cellulose and polysaccharide. The amount of these stabilizers and excipients to be used is determined depending on the type, purpose, etc., but is usually about 0 to 500% of the biocompatible polymeric substance.

The microcapsule-type sustained release preparation of the present invention is prepared by preparing a W10 type emulsion with an aqueous solution of a physiologically active substance and an organic solvent solution of a biocompatible polymeric substance, and combining this W10 type emulsion with a water-soluble polymeric substance. When secondary emulsifying with an aqueous solution, a salt substance with an O, IM concentration or a saturated concentration is present in the outer aqueous layer.
/O/W type emulsion is prepared, and from the obtained W/O/W type emulsion, microcapsules having a physiologically active substance as an inclusion material and a biocompatible polymeric substance as an outer shell material are prepared. The obtained microcapsules have a maximum particle size of 1504 or less, usually 75 or less, more preferably 30/JIn or less, and an average particle size of 5 to 100, Normally, the range is 5 to 25, more preferably 5 to 15, and the coefficient of variation is 5.
It is 0% or less and can be used as an injection as it is without any special separation and sizing operation. The maximum particle size is affected by emulsification conditions and solution concentration, and may show a value close to 15,074 depending on these conditions. In addition, the maximum particle size is determined by determining the particle size of the largest particle among 10 particles.
The average particle size is calculated by calculating the average value of the particle sizes of 500 particles, and the coefficient of variation is (standard deviation) ÷ (average particle size) x
This is a value obtained by calculating from the formula 100.

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

First, a biocompatible polymeric substance is dissolved in an organic solvent to prepare an organic solvent solution. The organic solvent used for this purpose may be one that has a relatively low boiling point, is immiscible with water, and dissolves biocompatible polymeric substances, such as dichloromethane, chloroethane, chloroform, trichloroethane, and tetrachloride. Examples include carbon, ethyl acetate, n-hexane, toluene, etc., and these can be used alone or in combination of two or more. The concentration of the biocompatible polymeric substance in this organic solvent solution can be changed as appropriate depending on the type of biocompatible polymeric substance used, and is usually 0.5 to 50% by weight, preferably 1 to 50% by weight. It is about 40% by weight.

In addition, in addition to the organic solvent solution of the biocompatible polymeric substance described above, the physiologically active substance and water-soluble stabilizers and excipients added as necessary can be dissolved in a predetermined water solution. Prepare. When preparing an aqueous solution of this physiologically active substance, a pH adjuster is added if necessary in order to maintain its stability and solubility. Examples of the pH adjuster used for this purpose include acids such as phosphoric acid, citric acid, acetic acid, and carbonic acid, their sodium salts and potassium salts, and various buffers such as phosphate buffers.

The amount of these pH adjusters to be used is not particularly limited, but is usually at a concentration of several tens of mM.

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 W10 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°C, preferably -50 to 20°C.

The W10 type emulsion prepared in this way was then added to the outer aqueous layer at a concentration of 0.1%. Mixed with an aqueous solution of a water-soluble polymer substance prepared in advance in the presence of a salt substance at a concentration ranging from M to saturation;
Secondary emulsification is performed to form a Wlo/W type product.

Examples of water-soluble polymer substances used in this secondary emulsification step include polyvinyl alcohol (PVA), polyvinylpyrrolidone, sodium carboxymethyl cellulose,
Gelatin, chitin and its derivatives, xanthone and its derivatives, hyaluronic acid and its sodium salts, dextran, pectin, chondroitin sulfate and its sodium salts, etc. can be mentioned, and these can be used alone, as well as 2 It is also possible to use more than one species as a mixture. The concentration of this water-soluble polymeric 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 aqueous layer during the above secondary emulsification should be used to stabilize the emulsion formed at the interface between the previously prepared W10 emulsion and the outer aqueous layer. There is no particular limitation, as long as it can improve the biocompatible polymeric substance and suppress the swelling of the biocompatible polymeric substance. For example, sodium acetate, sodium citrate,
Various organic acid salts such as potassium citrate, sodium chloride, potassium chloride, calcium chloride, sodium sulfate,
Examples include various inorganic acid salts such as potassium sulfate, ammonium salts such as ammonium sulfate, and quaternary amine salts such as choline chloride and tetramethylammonium chloride. These salt substances are usually present in the outer aqueous layer at a concentration ranging from 0.1M to saturation concentration, preferably 0.5M.
It is preferable that O,1 be present in the range from the concentration to the saturation concentration.
If the concentration is lower than M, there will be a problem of bursting, and
When the concentration is higher than the saturation concentration, the problem of salt embedding occurs. As for the method of making this salt substance exist in the outer aqueous layer, it is sufficient that it is present in the outer aqueous layer as a result when a W/O/W type emulsion is prepared in the secondary emulsification step, and It may be added to an aqueous solution of a water-soluble polymer substance,
Further, it may be added separately when mixing the W10 type emulsion and the aqueous solution of the water-soluble polymer substance.

The W/O/W emulsion prepared in this way is then usually heated at room temperature or at 0 to 50°C, preferably at 0 to 25°C.
C to evaporate the organic solvent, thereby solidifying the biocompatible polymer material of the outer shell material and producing microcapsules.

Furthermore, the produced microcapsules are separated by means such as centrifugation or filtration, washed and dried if necessary. Thereafter, if necessary, the microcapsules are redispersed in an aqueous solution containing an appropriate excipient such as mannitol, dried by freeze-drying or other means to remove residual organic solvent, and are made into microcapsules with excellent redispersibility during use. to produce sustained release formulations.

The microcapsules obtained as described above have a maximum particle size of 1501#R or less and an average particle size of 5 to 5.
100, and the coefficient of variation is 50% or less, so it can be used as an injection as it is without any special separation and sizing operation.

Furthermore, the microcapsule-type sustained release preparation according to the present invention includes:
Depending on the therapeutic purpose and the pharmacological action of the physiologically active substance contained in the microcapsules, it can be formulated into any dosage form such as injections, powders, granules, tablets, suppositories, etc. It is particularly suitable as an injection because it has a fine and uniform diameter and has needle passability and redispersibility as it is without the need for fractionating and sizing operations.

[For production]

The microcapsule type sustained release preparation of the present invention has a fine and uniform particle size. Furthermore, according to the method of the present invention, W10
In the secondary emulsification process in which the type emulsion is emulsified into a W/O/W type emulsion, 0.0% is added to the outer water layer. The presence of a salt substance at IM concentration or saturation concentration improves the properties of the interface between the W10 type emulsion and the aqueous solution of the water-soluble polymer substance, thereby forming a microcapsule-type pellet with fine and uniform particle size. It is believed that a release formulation is obtained.

〔Example〕

Hereinafter, the present invention will be specifically explained based on Examples and Experimental Examples.

Example 1 Polylactic acid/glycolic acid copolymer (lactic acid/glycolic acid ratio = 75/25, weight average molecular weight 10,000) 2
0■ was dissolved in dichloromethane 5- to prepare an organic solvent solution of a biocompatible polymeric substance. Genetically recombinant human erythropoietin (hereinafter abbreviated as rEPo) 1 and 2 high-grade gelatin type B ((manufactured by Rini Sopi, alkali-treated gelatin with an average molecular weight of 7.000) 40
- dissolved in Japanese Pharmacopoeia water for injection, pore size 0, 22. in
The mixture was sterile-filtered using a membrane filter (manufactured by Nippon Millipore Limite Sodo: Milefx Gv) to obtain an aqueous solution of the physiologically active substance. The above aqueous solution was added dropwise to the above organic solvent solution under stirring (28,000 rpm) using a homogenizer (manufactured by Kinematica: Bof Tanron), and further stirred at the same rotation speed for 30 min.
A W10 type emulsion was obtained by stirring and mixing for seconds.

Next, the W10 type emulsion was added to IM-0.5X-polyvinyl alcohol aqueous solution containing sodium chloride, which had been separately ice-cooled in advance, and stirred and mixed at a speed of 10,000 rpm for 30 seconds. A W/O/W type emulsion was produced.

Furthermore, the dispersion containing this W/O/W type emulsion was subsequently gently stirred at room temperature to volatilize the remaining methylene chloride and solidify the polylactic acid-glycolic acid copolymer, and then 3. Microcapsules produced by centrifugation at 00 rpm for 10 minutes were collected.

By repeating the procedure of dispersing the thus obtained microcapsules in water for injection and further centrifugation five times, polyvinyl alcohol, sodium chloride, free rEPo, and polylactic acid/glycolic acid in the outer aqueous layer were dissolved. The polymer was washed.

The finally collected microcapsules were redispersed in a 5x-mannitol aqueous solution 5- and then subjected to freeze-drying. Note that all of the above steps were performed in a sterile and dust-free manner.

The rEPO-containing microcapsules thus obtained had a maximum particle size of 157 m, an average particle size of 8.0, and a coefficient of variation of 40%. Moreover, the uptake rate of rEPO was about 95%.

Experimental Example 10 μ of rEPo-containing microcapsules obtained in Example 1 above were mixed with 0.05 tons of 10 − T w e n 2
The cells were dispersed in a 50 mM phosphate buffer (pH 7,4) containing 0 and rotated inverted at 25 rpm in a 37° C. constant temperature bath using a rotary culture machine (Taiyo Kagaku Kogyo (m:RT-50)).

After a predetermined time, the dispersion was centrifuged (3,000 rpm, 1
microcapsules were collected.

The rEPo content in the microcapsules was determined by dissolving the collected microcapsules with acetonitrile 1-.
The total amount was adjusted to 10- with M-+J acid buffer, and quantified by reversed-phase high-performance liquid chromatography (HPLC).

FIG. 1 shows the changes in rEPO content in the microcapsules determined over time. As is clear from this figure 1,
It was observed that rEPO in the microcapsules was released at a nearly constant rate for 4 weeks.

Example 2 Polylactic acid/glycolic acid copolymer (lactic acid/glycolic acid ratio -75/25, weight average molecular weight 10,000) 20
(2) was dissolved in dichloromethane 5- to obtain an organic solvent solution of a biocompatible polymeric substance. rEPo 1■ 1-50m
Dissolved in M phosphate buffer (pH 6,6), pore size 0,227
The mixture was sterile-filtered using a membrane filter (M) to obtain an aqueous solution of the physiologically active substance. Stir with a homogenizer (28,
The above aqueous solution was added dropwise into the above organic solvent solution under the same rotation speed (000 rpm), and the mixture was stirred and mixed for 30 seconds at the same rotation speed to obtain a W10 type emulsion.

Next, a 0.5 topolyvinyl alcohol aqueous solution 20011I containing LM-sodium chloride, which had been cooled separately in advance, was prepared.
Add the above W10 type emulsion into 1,000 rpm.
The mixture was stirred and mixed for 30 seconds at a speed of 30 to form a W/O/W type emulsion. Hereinafter, in the same manner as in Example 1 above, rEP
O-containing microcapsules were obtained.

The rEPo-containing microcapsules obtained in this way have a maximum particle size of 40 mm and an average particle size of 22A#n.
The coefficient of variation was 45%. Moreover, the uptake rate of rEPo was about 80%.

Example 3 Polylactic acid/glycolic acid copolymer (lactic acid/glycolic acid ratio = 75/25, weight average molecular weight 20,000) 2
0■ was dissolved in dichloromethane 5- to prepare an organic solvent solution.

In addition, recombinant human granulocyte colony stimulating factor (hereinafter referred to as rG-C3F) 1■ and Nitupi High Grade Gelatin Type 840■ were dissolved in 11 nl of Japanese Pharmacopoeia Water for Injection, and a pore size of 0.22. The mixture was sterile-filtered using a cm membrane filter to obtain an aqueous solution. The aqueous solution was added dropwise to the organic solvent solution while being stirred by a homogenizer (28,000 rpm), and further stirred and mixed for 30 seconds at the same rotation speed to obtain a W10 emulsion.

Next, the above W10 type emulsion was added to 200 d of a 0.5 topolyvinylpyrrolidone aqueous solution containing IM-sodium sulfate that had been ice-cooled separately in advance, and stirred and mixed at a speed of 10,000 rpm for 30 seconds. /W type emulsion was produced. Hereinafter, in the same manner as in Example 1 above, rG-C3F
Containing microcapsules were obtained.

The obtained rG-C3F-containing microcapsules had a maximum particle size in the 50s, an average particle size in the 20s, and a coefficient of variation of 40%. Moreover, the uptake rate of rG-C3F was about 90%.

Example 4 Polylactic acid/glycolic acid copolymer (lactic acid/glycolic acid ratio = 75/25, weight average molecular weight 5,000) 20
(2) was dissolved in dichloromethane 5- to form an organic solvent solution.

rEPo 1■ and Nippi High Grade Gelatin Type B40■ were dissolved in 1- Japanese Pharmacopoeia Water for Injection, and the pore size (
The mixture was sterile filtered using a No. 1,2274 membrane filter to obtain an aqueous solution. While stirring with a homogenizer (28,0
The above aqueous solution was added dropwise to the above organic solvent solution at a rotational speed of 0 rpm), and further stirred and mixed for 30 seconds at the same rotation speed to obtain a W10 type emulsion.

Next, the above W10 type emulsion was added to IM = 0.5% aqueous sodium carboxymethyl cellulose solution containing ammonium sulfate, which had been ice-cooled separately in advance.
Stir and mix for 30 seconds at a speed of 000 rpm, W/O/W
A type emulsion was produced. Thereafter, rEPo-containing microcapsules were obtained in the same manner as in Example 1 above.

The obtained rEPo-containing microcapsules had a maximum particle size of 55 particles, an average particle size of 23 particles, and a coefficient of variation of 48%. Moreover, the uptake rate of rEPo was about 95%.

Example 5 Polylactic acid polymer (weight average molecular weight 5,000) 20■
was dissolved in dichloromethane 5- to obtain an organic solvent solution.

rG-C3F 1■ and Nippi High Grade Gelatin Type 840■ were dissolved in 1- Japanese Pharmacopoeia Water for Injection, and the pore size was 0.22. It was sterile filtered using a C41 membrane filter to obtain an aqueous solution. While stirring with a homogenizer (2
The above aqueous solution was added dropwise to the above organic solvent solution at a rotational speed of 8.00 Orpm), and further stirred and mixed at the same rotation speed for 30 seconds to obtain a W10 type emulsion.

Next, the above W10 type emulsion was added to a 0.5x aqueous solution of potassium chloride-containing gelatin purified by the Japanese Pharmacopoeia method, which had been ice-cooled separately in advance, and mixed by stirring at a speed of 0.00 rpm for 30 seconds. , W/O/W type compound was produced. Hereinafter, rG-C3F was prepared in the same manner as in Example 1 above.
Containing microcapsules were obtained.

The obtained rG-C3F-containing microcapsules had a maximum particle size of 60 mm, an average particle size of 25 mm, and a coefficient of variation of 44%. Moreover, the uptake rate of rG-CSF was about 90%.

Example 6 Polylactic acid/glycolic acid copolymer (lactic acid/glycolic acid ratio = 75/25. Weight average molecular weight 10,000) 2
0■ was dissolved in dichloromethane 5- to prepare an organic solvent solution of a biocompatible polymeric substance. rEPo 1■ and Nippi High Grade Gelatin Type 840■ were dissolved in 1- Japanese Pharmacopoeia Water for Injection and sterile filtered through a membrane filter with a pore size of 0.22IJII to obtain an aqueous solution of a physiologically active substance. Stir by homogenizer (28,000 rpm)
The aqueous solution was added dropwise to the organic solvent solution below, and the mixture was further stirred and mixed at the same rotation speed for 30 seconds to form a WZO emulsion.

Next, 200 0.5% polyvinyl alcohol aqueous solution containing sodium chloride of various concentrations, which had been cooled separately in advance, was added.
Add each of the above V10 type emulsions to 10.0
The mixture was stirred and mixed at a speed of 0 rpm for 30 seconds to produce a W/O/W type emulsion.

Furthermore, the dispersion containing these W/O/W emulsions was subsequently gently stirred at room temperature to volatilize the remaining methylene chloride and solidify the polylactic acid glycolic acid copolymer. Microcapsules generated by centrifugation at 3.00 rpm for 10 minutes were collected.

By repeating the procedure of dispersing the thus obtained microcapsules in water for injection and further centrifugation five times, polyvinyl alcohol, sodium chloride, free rEPo, and polylactic acid/glycolic acid in the outer aqueous layer were dissolved. The polymer was washed.

The finally collected microcapsules were redispersed in 5X-mannitol aqueous solution 5- and then subjected to freeze-drying.

The maximum particle diameter, average particle diameter, and coefficient of variation of each rEPo-containing microcapsule prepared by changing only the salt concentration of the outer water layer in the secondary emulsification step were determined, and the shape thereof was observed. The results are shown in Table 1.

Table 1 [Effects of the Invention] According to the present invention, the presence of a salt substance at a predetermined concentration in the outer water layer when producing a W/O/W type emulsion eliminates the need for viscosity adjustment and fractional sizing operations. It is possible to produce microcapsules with fine and uniform particle size without causing any damage, which is industrially advantageous. In addition, the microcapsule-type sustained-release preparation comprising the physiologically active substance and biocompatible polymeric substance of the present invention produced by this method has excellent sustained-release properties, and can be administered for a long period of time by administration once. The medicinal efficacy of the physiologically active substance can be expressed over a period of time.

[Brief explanation of drawings]

FIG. 1 is a graph showing the change over time in the rEPo content in the rEPO-containing microcapsules obtained in Example 1. Patent applicant Chugai Pharmaceutical Co., Ltd.

Claims (2)

[Claims] (1) It has an inner material containing a physiologically active substance and an outer shell material made of a biocompatible polymer material that is biodegradable and/or tissue compatible in the living body, and has a maximum particle size of 150 μm
1. A microcapsule-type sustained release preparation, which has an average particle size of 5 to 100 μm or less, and a coefficient of variation of 50% or less. (2) A W/O emulsion is prepared with an aqueous solution containing a physiologically active substance and an organic solvent solution containing a biocompatible polymeric substance that is biodegradable and/or tissue compatible with the body, and then this emulsion is prepared. A W/O/W emulsion is prepared by secondary emulsification of the W/O emulsion and an aqueous solution of a water-soluble polymer substance, and the organic solvent is evaporated from the obtained W/O/W emulsion. , solidify, further wash and dry to contain physiologically active substances,
In addition, when preparing a microcapsule-type sustained-release preparation using a biocompatible polymeric substance as an outer shell material, it is recommended that a salt substance be present in the outer aqueous layer at a concentration of 0.1M to saturated concentration in the secondary emulsification step. A method for producing a characteristic microcapsule-type sustained-release preparation.