JPH083055A - Production of sustained release preparation - Google Patents

Abstract

PURPOSE:To obtain a sustained release preparation by permeating a water-soluble polypeptide into a matrix decomposable in an organism. CONSTITUTION:A water-soluble polypeptide (e.g. interferon) having 200-50,000 molecular weight is allowed to stand or lightly stirred and permeated into a finely graduated matrix decomposable in an organism in the ratio of about 1:10-20:1 at 4-25 deg.C for several hours to tens hours and then lyophilized to pharmaceutically manufacture the water-soluble polypeptide in the matrix. The matrix decomposable in an organism is obtained by mixing a polymer decomposable in an organism (e.g. lactic acid-glycolic acid copolymer) with a water-soluble aliphatic carboxylic acid metal salt. The water-soluble polypeptide can be permeated into the matrix decomposable in an organism without bringing the water-soluble polypeptide into contact with an organic solvent and the water-soluble peptide can be pharmaceutically manufactured without impairing physiological activity of the water-soluble polypeptide. Permeation ratio can be raised by using the water-soluble aliphatic carboxylic acid metal salt. When this sustained release preparation, for example, is used as an injection, sustained release is shown for several days to one or two weeks.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a sustained-release preparation prepared by allowing a water-soluble polypeptide to penetrate a biodegradable matrix.

2. Description of the Related Art It is known that proteins (also called polypeptides) exhibit various pharmacological actions in the living body,
Some of these have been produced in large quantities using organisms such as Escherichia coli, yeast, animal cells, or hamsters due to the development of genetic engineering and cell engineering techniques, and are being applied as pharmaceuticals. Examples of these protein drugs include interferon (alpha, beta, gamma), interleukin 2, erythropoietin, granulocyte colony stimulating factor (G-CSF) and the like. However, since these proteins generally have a short half-life in vivo, frequent administration is required and the physical burden on patients due to injection cannot be ignored. Various attempts have been made to develop sustained-release preparations to solve this problem. Since it is necessary to carefully administer proteins represented by cytokines while determining their therapeutic effects, the development of injectable sustained-release preparations, particularly microcapsule sustained-release preparations, having a release period of about 1 to 2 weeks has been developed. Is desired. However, it is generally known that proteins are denatured by heat, organic solvent, strong shearing force, etc., and their biological activity is reduced. For example, heating the aqueous protein solution at 60 ° C. for 20 minutes may cause rapid loss of its biological activity. Further, even at a lower temperature of 50 ° C., the biological activity may be reduced by heating for about 1 hour. Similarly, it is known that the biological activity of a protein may be reduced by an organic solvent such as ethanol or dichloromethane.

[0003] WO 93/06872 discloses a formulation in which void-forming particles made of a biodegradable polymer enabling the long-term release of bone morphogenetic proteins, bone morphogenetic proteins, and autologous hemagglutination are combined. The technology is disclosed. In the present technology, a method of controlling the release by adsorbing the bone morphogenetic protein, which is the main drug, to the particles immediately before administration, and further adding autologous blood to form an aggregate, is adopted. The sustained release period is about several weeks. Not commonly used to use autologous blood. Journal of Controlled Releas
e), 23, 157, (1993) (A. Supersax
o et al.) prepared a microcapsule with a large porosity using a biodegradable polymer, and then impregnated the macromolecule into the microcapsule without contacting the organic solvent with the macromolecule. The technology to incorporate is described. Specifically, since the polylactic acid used is hydrophobic, an operation of first wetting the microcapsules with 50% ethanol, which is an organic solvent, then substituting with water, and then substituting with a polymer solution It is carried out. Japanese Patent Laid-Open No. 5-
No. 32559 discloses that the constituents of the pharmaceutical composition and the physiologically active substance are dissolved in an organic solvent, or the constituents of the pharmaceutical composition and the physiologically active substance are uniformly dispersed in an organic medium or an aqueous medium. A method for producing a pharmaceutical composition by dispersing and drying this is disclosed.

[0003]

Although various attempts have been made to produce sustained-release preparations while retaining the biological activity of proteins and the like as described above, the penetration efficiency of the drug into the matrix is high. In terms of suppression of initial burst of drug, constant drug release rate over a long period of time, etc., it is not yet satisfactory.

[0004]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to solve the above-mentioned problems, and found that biodegradable polymers which disappeared in vivo after administration for about 1 to 3 weeks. The concentration of the water-soluble polypeptide in blood can be obtained by preliminarily producing a biodegradable matrix in the form of fine particles, and injecting a preparation obtained by allowing the water-soluble polypeptide to penetrate into the biodegradable matrix in water. About 1-2
The present invention has been completed as a result of further research based on the finding that it can be maintained for a week. That is, the present invention provides (1) a method for producing a sustained-release preparation, which comprises permeating a water-soluble polypeptide into a biodegradable matrix in water, and (2) the biodegradable matrix is biodegradable. The method for producing a sustained-release preparation according to item 1, which is produced by mixing a polymer and a water-soluble metal salt of aliphatic carboxylic acid, (3) the item 2 in which the aliphatic carboxylic acid is an aliphatic monocarboxylic acid The method for producing a sustained-release preparation described in (4), the method for producing a sustained-release preparation as described in (2), wherein the metal salt is a polyvalent metal salt, and (5) a water-soluble polypeptide in a biodegradable matrix in water. The method for producing the sustained-release preparation according to item 1, which is followed by drying, and (6) the method for producing the sustained-release preparation according to item 5, wherein the drying is freeze-drying. The method for producing a sustained-release preparation according to item 1, which is a cytokine, (8) site The Inn is an interferon 7
The method for producing a sustained release preparation according to item 1, (9) The method for producing a sustained release preparation according to item 1, wherein the biodegradable matrix is in the form of fine particles, and (10) the biodegradable matrix is biodegradable. (11) A method for producing a sustained-release preparation, which is produced from a polymer, (11) A method for producing a sustained-release preparation, as described in (10), wherein the biodegradable polymer is an aliphatic polyester.
2) The method for producing a sustained release preparation according to item 11, wherein the aliphatic polyester is a copolymer derived from α-hydroxycarboxylic acid, and (13) The copolymer is a lactic acid-glycolic acid copolymer. Item 12. A method for producing a sustained-release preparation according to item 12,
4) A sustained-release preparation in which a water-soluble polypeptide is impregnated into a biodegradable matrix, (15) The biodegradable matrix is prepared by mixing a biodegradable polymer and a water-soluble aliphatic carboxylic acid metal salt. The sustained-release preparation according to Item 14, which is manufactured by the method, and (16) The sustained-release preparation according to Item 14, which is for injection.

The water-soluble polypeptide in the present invention is preferably a water-soluble polypeptide having a molecular weight of about 200 to about 50,000. The water-soluble polypeptide is more preferably a water-soluble polypeptide having a molecular weight of about 5,000 to about 40,000. The water-soluble polypeptide may have a hormonal action and be endocrine. Examples of such water-soluble polypeptides include cytokines, hematopoietic factors, various growth factors, enzymes and the like. Examples of cytokines include lymphokines and monokines. Examples of lymphokines include interferons (alpha, beta, gamma), interleukins (IL-2 to IL-12), and the like. Examples of monokine include interleukin (IL-1) and tumor necrosis factor. Examples of hematopoietic factors include erythropoietin and granulocyte colony stimulating factor (GC).
SF), macrophage colony stimulating factor (M-CS
F), thrombopoietin, platelet growth stimulating factor, megakaryocyte potentiator and the like. Examples of various growth factors include basic or acidic fibroblast growth factor (FGF) or their families (eg, FGF-9, etc.) [Molecular and Cellular Biolog.
y), 13, 7, 4251 (1993)], nerve cell growth factor (NGF) or a family thereof, insulin-like growth factor (eg, IGF-1, IGF-).
2, etc.), a factor involved in bone growth (BMP), or a family of these. Examples of the enzyme include superoxide dismutase (SOD), tissue plasminogen activator (TPA)
And the like. In addition to the above, for example, growth hormone, insulin, natriuretic peptide, gastrin, prolactin, adrenocorticotropic hormone (ACT)
H), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), motilin, kallikrein, and Reg protein which is a protein involved in pancreatic regeneration [Japanese Patent Publication 1- No. 132388, Febbs Letters (FE
BS Letters), 272, 85 (1990)] and the like are also used as the water-soluble polypeptide of the present invention. The water-soluble polypeptide may be naturally-occurring or produced by gene recombination. The water-soluble polypeptide is
It is not limited to the above water-soluble polypeptides. That is, the water-soluble polypeptide may or may not have a sugar chain, and may have a sugar chain having a different structure. Further, the water-soluble polypeptide is a mutant substance or derivative (agonist, agonist,
Antagonistic) or fragments.

The water-soluble polypeptide is preferably a cytokine or the like. Examples of the cytokine include lymphokines and monokines. Examples of lymphokines include interferon (alpha, beta, gamma) and interleukin (IL-2 to IL-1).
2) and the like. Examples of monokine include interleukin (IL-1) and tumor necrosis factor. The water-soluble polypeptide is more preferably lymphokine. Examples of the lymphokines include interferons (alpha, beta, gamma), interleukins (IL-2 to IL-12) and the like.
The water-soluble polypeptide is particularly preferably interferon (alpha, beta, gamma) and the like.

In the present invention, the biodegradable matrix is preferably in the form of fine particles. The biodegradable matrix may be one that can be passed through an injection needle used for ordinary subcutaneous or intramuscular injection, and the particle size of the biodegradable matrix is, for example, about 0.1 as an average diameter.
˜300 μm, preferably about 1-150 μm, particularly preferably about 2-100 μm.

The biodegradable matrix is produced, for example, from a biodegradable polymer by a method known per se. Examples of such a method include an in-water drying method, a phase separation method, a spray drying method or a method similar thereto, which will be described below. The biodegradable matrix is preferably produced by mixing a biodegradable polymer and a water-soluble metal salt of an aliphatic carboxylic acid. (A) In-water drying method (W / O / W method) As the inner aqueous phase, for example, water or an aqueous solution containing a water-soluble component is used. Examples of the water-soluble component include inorganic salts (eg, common salt, sodium hydrogen phosphate, disodium hydrogen phosphate, etc.), sugars (eg, mannitol, glucose, inulin, etc.), organic salts (eg, sodium carbonate, magnesium carbonate, acetic acid). Ammonium, etc.), amino acids (eg,
Glycine, arginine, histidine, etc.) and the like. The concentration of the water-soluble component in the aqueous solution is, for example, about 0.1.
-10% (w / v), preferably about 0.5-5% (w /
v). Particularly when the water-soluble component is salt, it is preferable to use, for example, 0.9% (w / v) physiological saline. Further, calcium carbonate or the like may be dispersed in the inner aqueous phase instead of the water-soluble component. Preferably, an aqueous solution containing a water-soluble aliphatic carboxylic acid metal salt is used as the inner aqueous phase. The concentration of the metal salt in the aqueous solution varies depending on the solubility of each metal salt, but is, for example, about 10 to 90%.
(W / v), preferably about 20-80% (w / v). An aqueous solution containing the above water or a water-soluble component, a polymer synthesized from α-hydroxycarboxylic acid,
A biodegradable polymer such as a copolymer is emulsified and dispersed in an organic solvent solution to prepare a W / O type emulsion. The concentration of the biodegradable polymer in the organic solvent solution varies depending on the molecular weight of the biodegradable polymer and the type of organic solvent, but is generally selected from about 0.01 to 90% (w / w). The concentration of the biodegradable polymer in the organic solvent solution is more preferably about 0.1 to 80% (w / w)
Is. The concentration of the biodegradable polymer in the organic solvent solution is particularly preferably about 1 to 70% (w / w). The ratio of water or an aqueous solution containing a water-soluble component to a biodegradable polymer organic solvent solution is 1: 1,000 (v /
v) to 1: 1 (v / v), preferably 1: 100 (v /
v) to 1: 5 (v / v), particularly preferably 1:50 (v
/ V) to 1: 5 (v / v). A known dispersion method is used for the emulsification operation. The method is performed using, for example, a turbine stirrer, a homogenizer, or the like. Then,
The W / O emulsion thus prepared is further added to the aqueous phase (outer aqueous phase) to form a W / O / W emulsion, and then the oil phase solvent is evaporated to form a biodegradable matrix. Prepare. Evaporation of the oil phase solvent is performed by stirring using, for example, a turbine type stirrer. The volume of the water phase at this time is generally about 1 to 10,000 of the volume of the oil phase.
It is selected from 0 times. More preferably, about 2 to 5,000
It is selected from 0 times. Particularly preferably, it is about 5 to 2,000.
Selected from double.

An emulsifier may be added to the outer water phase. The emulsifier may be any one that can form a generally stable O / W emulsion. Specific examples thereof include anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid and the like. One of these may be used, or some of them may be used in combination. The concentration at the time of use is about 0.001 to 20% (w /
It can be appropriately selected from the range of w). More preferably, it is used in the range of about 0.01 to 10% (w / w). It is particularly preferably used in the range of about 0.05-5% (w / w). When calcium carbonate is dispersed in the inner aqueous phase instead of the water-soluble component, calcium carbonate is dissolved by adding dilute hydrochloric acid to the outer aqueous phase. Further, a water-soluble aliphatic carboxylic acid metal salt which is the same as or different from the water-soluble aliphatic carboxylic acid metal salt used in the inner aqueous phase may be added to the outer water phase. At this time, the concentration of the water-soluble aliphatic carboxylic acid metal salt in the outer aqueous phase is about 0.01 to 20% (w / w), more preferably about 0.1 to 10% (w / w). It is preferable to add a water-soluble aliphatic carboxylic acid metal salt to. The elution of the water-soluble aliphatic carboxylic acid metal salt from the biodegradable matrix can be controlled by changing the concentration of the water-soluble aliphatic carboxylic acid metal salt in the external water phase. The biodegradable matrix thus obtained is separated by centrifugation or filtration, and then the emulsifier and the like adhering to the surface of the biodegradable matrix is repeatedly washed several times with distilled water and distilled again. Disperse in water and freeze-dry. The surface of the obtained biodegradable matrix is not smooth, and there are holes of various sizes, and some of the holes extend to the inside of the biodegradable matrix. The proportion of the volume of these holes (porosity) in the biodegradable matrix can be quantified by, for example, the mercury intrusion method or the BET method. The porosity varies depending on the components in the inner water phase, their concentrations, the ratio of the inner water phase solution to the biodegradable polymer organic solvent solution, the ratio of the outer water phase volume to the oil phase volume, the outer water phase temperature, etc. Moreover, the structure of the holes inside the biodegradable matrix is different. The content of the water-soluble aliphatic carboxylic acid metal salt in the biodegradable matrix is preferably about 0.01 to 10% (w / w) as metal.
w), more preferably about 0.05-7% (w / w),
Particularly preferred is about 0.1 to 5% (w / w). The content of the water-soluble aliphatic carboxylic acid metal salt in the biodegradable matrix is quantified as a metal by a method such as an atomic absorption method.

(B) In-water drying method (O / W method) In the present invention, as a method for producing a biodegradable matrix, there is a method which does not use an internal water phase. In the method, first, a solution of a biodegradable polymer in an organic solvent is prepared. At this time, the concentration of the biodegradable polymer in the organic solvent solution varies depending on the molecular weight of the biodegradable polymer, the type of the organic solvent, etc., but is generally about 0.01-9.
It is selected from 0% (w / w). The concentration of the biodegradable polymer in the organic solvent solution is more preferably about 0.1-
80% (w / w). The concentration of the biodegradable polymer in the organic solvent solution is particularly preferably about 1 to 70% (w
/ W). Calcium carbonate may be added and dispersed in the organic solvent solution of the biodegradable polymer. On this occasion,
The amount of calcium carbonate added is such that the weight ratio of calcium carbonate: biodegradable polymer is about 5: 1 to about 1: 100, preferably about 2: 1 to about 1:10. Preferably, the water-soluble aliphatic carboxylic acid metal salt is added and dispersed in the organic solvent solution of the biodegradable polymer. On this occasion,
The amount of the water-soluble aliphatic carboxylic acid metal salt added is such that the weight ratio of the water-soluble aliphatic carboxylic acid metal salt: biodegradable polymer is about 5: 1 to about 1: 100, preferably about 2: 1 to about 1. :
50, more preferably about 1: 1 to about 1:10. Then, the organic solvent solution thus prepared is further added to the aqueous phase, an O / W emulsion is formed using a turbine stirrer, etc., and then the oil phase solvent is evaporated to prepare a biodegradable matrix. To do. At this time, the volume of the aqueous phase is generally selected from about 1 to 10,000 times the volume of the oil phase. More preferably, it is selected from about 2-5,000 times. Particularly preferably, it is selected from about 5-2,000 times.

An emulsifier may be added to the outer water phase. The emulsifier may be any one that can form a generally stable O / W emulsion. Specific examples thereof include anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid and the like. One of these may be used, or some of them may be used in combination. The concentration at the time of use is about 0.001 to 20% (w /
It can be appropriately selected from the range of w). More preferably, it is used in the range of about 0.01 to 10% (w / w). It is particularly preferably used in the range of about 0.05-5% (w / w). When calcium carbonate is added and dispersed in the organic solvent solution of the biodegradable polymer, dilute hydrochloric acid is added to the outer aqueous phase. Further, a water-soluble aliphatic carboxylic acid metal salt which is the same as or different from the water-soluble aliphatic carboxylic acid metal salt added and dispersed in the organic solvent solution of the biodegradable polymer may be added to the outer aqueous phase. At this time, the concentration of the water-soluble aliphatic carboxylic acid metal salt in the external water phase is about 0.01-2.
0% (w / w), more preferably about 0.1-10%
It is preferable to add a water-soluble aliphatic carboxylic acid metal salt so as to be (w / w). The elution of the water-soluble aliphatic carboxylic acid metal salt from the biodegradable matrix can be controlled by changing the concentration of the water-soluble aliphatic carboxylic acid metal salt in the external water phase. Further, as a method for producing a biodegradable matrix, a solution of a biodegradable polymer in an organic solvent is added to an outer aqueous phase containing a water-soluble aliphatic carboxylic acid metal salt, and O /
The method of forming W emulsion is mentioned. The biodegradable matrix thus obtained is separated by centrifugation or filtration, and then the emulsifier and the like adhering to the surface of the biodegradable matrix is repeatedly washed several times with distilled water and distilled again. Disperse in water and freeze-dry.
The content of the water-soluble aliphatic carboxylic acid metal salt in the biodegradable matrix is preferably about 0.01 to 1 as a metal.
0% (w / w), more preferably about 0.05-7%
(W / w), particularly preferably about 0.1-5% (w / w)
Is.

(C) Phase Separation Method (Coacervation Method) When the biodegradable matrix is produced by the phase separation method, the W / O emulsion or the solution of the biodegradable polymer in an organic solvent is used as a core cell. The basation agent is gradually added with stirring to precipitate and solidify the biodegradable polymer. The coacervation agent is added in a volume of about 0.01 to 1,000 times the volume of the w / o emulsion or the solution of the biodegradable polymer in the organic solvent. More preferably, the volume is about 0.05 to 500 times. Particularly preferably, the volume is about 0.1 to 200 times. The coacervation agent may be a polymer-based, mineral oil-based, or vegetable oil-based compound that is miscible with the solvent of the biodegradable polymer and does not dissolve the polymer. Specific examples thereof include silicone oil, sesame oil, soybean oil, corn oil, cottonseed oil, coconut oil, linseed oil, mineral oil, n-hexane, n-heptane and the like. You may use these in mixture of 2 or more types. The biodegradable matrix thus obtained is collected by filtration and then repeatedly washed with heptane or the like to remove the coacervation agent. Further, washing is performed by the same method as the in-water drying method, and then the biodegradable matrix is freeze-dried.
The solvent can be removed by a method known per se. As such a method, for example, a method of evaporating the solvent by stirring with a propeller stirrer or a magnetic stirrer at atmospheric pressure or gradually reducing the pressure, or by evaporating the solvent while adjusting the degree of vacuum using a rotary evaporator or the like Method etc. are mentioned. The content of the water-soluble aliphatic carboxylic acid metal salt in the biodegradable matrix is preferably about 0.01 to 10 as a metal.
% (W / w), more preferably about 0.05-5% (w
/ W), particularly preferably about 0.1-5% (w / w).

In the production by the in-water drying method and the coacervation method, an anti-agglomeration agent may be added to prevent the particles from aggregating. Examples of the aggregation preventing agent include mannitol, lactose, glucose, starches (eg, corn starch, etc.), water-soluble polysaccharides such as hyaluronic acid or alkali metal salts thereof, proteins such as glycine, fibrin and collagen, sodium chloride, Examples thereof include inorganic salts such as sodium hydrogen phosphate.

(D) Spray drying method When a biodegradable matrix is produced by a spray drying method, W / O emulsion or biodegradation containing water or an aqueous solution containing a water-soluble component and a biodegradable polymer is used. A solution of a water-soluble polymer in an organic solvent is sprayed into the drying chamber of a spray drier (spray dryer) using a nozzle, and the organic solvent in the atomized droplets is volatilized in an extremely short time, resulting in a fine granular biodegradable capsule. To prepare. Examples of the nozzle include a two-fluid nozzle type, a pressure nozzle type, and a rotating disk type. At this time, if desired, at the same time as W / O emulsion containing water or an aqueous solution containing a water-soluble component and a biodegradable polymer or an organic solvent solution of the biodegradable polymer, to prevent aggregation of the biodegradable matrix. It is also effective to spray the above-mentioned aqueous solution of the aggregation preventing agent from another nozzle. The biodegradable matrix is preferably W / O containing an aqueous solution containing a water-soluble aliphatic carboxylic acid metal salt and a biodegradable polymer.
It is manufactured using an emulsion or an organic solvent solution of a biodegradable polymer containing a water-soluble aliphatic carboxylic acid metal salt. The biodegradable matrix thus obtained is further subjected to removal of water and organic solvent in the biodegradable matrix under heating and reduced pressure, if necessary.
The amount of residual organic solvent in the biodegradable matrix used in the present invention is about 1,000 ppm or less, preferably about 500 ppm or less, more preferably 250 ppm or less, and particularly preferably about 100 ppm or less.

The raw material used for the biodegradable matrix in the present invention is preferably a biodegradable polymer. As the biodegradable polymer, a high molecular weight polymer which is hardly soluble or insoluble in water, for example, aliphatic polyester [eg, α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc., valic acid, leucic acid ), Hydroxydicarboxylic acids (eg, malic acid, etc.),
Polymers, copolymers, or mixtures thereof synthesized from at least one kind of hydroxytricarboxylic acid (eg, citric acid, etc.)], poly-α-cyanoacrylic acid ester, polyamino acid (eg, poly-γ-) Benzyl-L-glutamic acid and the like). Alternatively, a mixture thereof may be used. The type of polymerization may be random, block or graft.

The biodegradable polymer is preferably an aliphatic polyester [eg, α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.), hydroxytricarboxylic acid. (Eg, a polymer synthesized from one or more kinds such as citric acid, etc.), a copolymer, or a mixture thereof]
Is. Among the above-mentioned aliphatic polyesters, polymers and copolymers synthesized from one or more kinds of α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.) have reliable biodegradability and biocompatibility. It is preferable from the viewpoint. The aliphatic polyester is more preferably a copolymer synthesized from one or more kinds of α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.). Particularly preferably α-hydroxycarboxylic acids (eg,
It is a copolymer synthesized from two or more of glycolic acid, lactic acid, hydroxybutyric acid, etc.). Further, the biodegradable polymer in the present invention is produced by a known method, and when molded into a biodegradable matrix that can be administered using a usual injection needle, without using an organic solvent such as ethanol. It is preferable that water can penetrate into the biodegradable matrix and swell.

The above α-hydroxycarboxylic acids are D-
Body, L-body, and D, L-body may be used, but D-
Those having a ratio of body / L-body (mol%) of about 75/25 to 25/75 are preferable. More preferably, D-form / L-
It is a hydroxycarboxylic acid having a body (mol%) in the range of about 60/40 to 30/70. Examples of the α-hydroxycarboxylic acid copolymers include, for example, copolymers of glycolic acid and other α-hydroxy acids.
As the hydroxy acid, lactic acid, 2-hydroxybutyric acid, valic acid and leucic acid are preferable. The α-hydroxycarboxylic acid copolymer is preferably a lactic acid-glycolic acid copolymer or a 2-hydroxybutyric acid-glycolic acid copolymer. The α-hydroxycarboxylic acid copolymer is particularly preferably a lactic acid-glycolic acid copolymer.

In the lactic acid-glycolic acid copolymer, the composition ratio (lactic acid / glycolic acid) (mol%) is about 100.
/ 0 to 40/60 is preferable. More preferably, the composition ratio is about 90/10 to 45/55. Particularly preferably, the composition ratio is about 60/40 to 45/55. The weight average molecular weight of the copolymer of glycolic acid and lactic acid is preferably about 3,000 to 12,000. More preferably, it is about 4,000 to 10,000. In the present invention, the rate of permeation of a water-soluble polypeptide into a biodegradable matrix produced using the copolymer and the disappearance rate of the biodegradable matrix after in vivo administration is a combination of a composition ratio and a weight average molecular weight. Affected by. Assuming that the disappearance period after administration in vivo (subcutaneous administration, etc.) is about 2 weeks and there is no problem in permeation of water into the biodegradable capsule, for example, a composition ratio of about 50/50 and a weight average molecular weight. Is about 4,000 to 9,000, preferably about 5.0
The thing of 00 to 9,000 is mentioned. In the present invention, two types of lactic acid having different composition ratios and weight average molecular weights
You may mix and use a glycolic acid copolymer in arbitrary ratios. As such an example, for example, a lactic acid-glycolic acid copolymer having a composition ratio (lactic acid / glycolic acid) (mol%) of about 75/25 and a weight average molecular weight of about 6,000, and a composition ratio (lactic acid / glycol acid). Acid) (mol%) is about 50/50
And a mixture with a lactic acid-glycolic acid copolymer having a weight average molecular weight of about 4,000. The weight ratio upon mixing is preferably about 25/75 to about 75/25.
The dispersity (weight average molecular weight / number average molecular weight) of the lactic acid-glycolic acid copolymer is preferably about 1.2 to about 4.0. More preferably, it is about 1.5 to about 3.5. The lactic acid-glycolic acid copolymer can be synthesized according to a method known per se, for example, the method described in JP-A-61-28521. The copolymer is preferably synthesized by catalyst-free dehydration polycondensation.

In the 2-hydroxybutyric acid-glycolic acid copolymer, it is preferable that the glycolic acid is about 10 to 75 mol% and the balance is 2-hydroxybutyric acid. More preferably, glycolic acid is about 20 to 75 mol%. Particularly preferably, glycolic acid is about 30-7.
This is the case when it is 0 mol%. The weight average molecular weight of the 2-hydroxybutyric acid-glycolic acid copolymer is about 2,000-
20,000 is preferred. The weight average molecular weight is more preferably about 3,000 to 10,000. The weight average molecular weight is particularly preferably about 4,000 to 8,000. The degree of dispersion (weight average molecular weight / number average molecular weight) of these glycolic acid copolymers is preferably about 1.2 to 4.0. The dispersity is particularly preferably about 1.5 to 3.5.
This glycolic acid copolymer is a production method known per se, for example,
It can be synthesized according to the method described in JP-A-61-28521 (production method by dehydration polycondensation reaction in the absence of a catalyst or dehydration polycondensation reaction in the presence of an inorganic solid acid catalyst). The copolymer is preferably synthesized by catalyst-free dehydration polycondensation.

The glycolic acid copolymer may be mixed with polylactic acid before use. The polylactic acid may be any of D-form, L-form, and a mixture thereof, but the D-form and L-form (mol%) are about 75/25 to 2.
The range of 0/80 is preferable. More preferably,
D-form and L-form (mol%) are about 60/40 to 25/75
The range of polylactic acid. Particularly preferred is D-form, L
-Polylactic acid in the form (mol%) in the range of about 55/45 to 25/75. The polylactic acid has a weight average molecular weight of about 1,
Those of 500 to 10,000 are preferable. More preferred is polylactic acid having a weight average molecular weight in the range of about 2,000 to 8,000. Particularly preferred is polylactic acid having a weight average molecular weight in the range of about 3,000 to 6,000. The polylactic acid preferably has a dispersity of about 1.2 to 4.0. The dispersity of polylactic acid is particularly preferably about 1.5 to 3.5. As a method for producing polylactic acid, a method of ring-opening polymerization of lactide which is a dimer of lactic acid and a method of dehydration polycondensation of lactic acid are known. In order to obtain the relatively low molecular weight polylactic acid used in the present invention, a method of directly dehydrating and polycondensing lactic acid is preferable. The method is described in, for example, JP-A-61-285.
No. 21 publication. When the glycolic acid copolymer and polylactic acid are used as a mixture, the mixing ratio is, for example, in the range of about 10/90 to 90/10 (% by weight).
The mixing ratio is preferably in the range of about 20/80 to 80/20 (% by weight). More preferably, from about 30/70
It is in the range of 70/30 (% by weight).

In the present invention, the biodegradable polymer synthesized by catalyst-free dehydration polycondensation has a free carboxyl group at the terminal. The biodegradable polymer having a free carboxyl group at the terminal is a polymer in which the number average molecular weight determined by GPC and the number average molecular weight determined by the end group determination are substantially the same. About 1 to 3 g of biodegradable polymer was dissolved in a mixed solvent of acetone (25 ml) and methanol (5 ml), and phenolphthalein was used as an indicator to adjust the carboxyl group in this solution to 0.05N alcoholic potassium hydroxide solution. With stirring at room temperature, the mixture was rapidly titrated to calculate the number average molecular weight by quantifying the end group by the following formula. Number average molecular weight by end group quantification = 20,000 A / B A: mass of biodegradable polymer (g) B: 0.05N alcoholic potassium hydroxide solution added by the end of titration (ml) It is expressed as the number average molecular weight by quantification. For example, in a polymer synthesized from one or more kinds of α-hydroxy acids by a non-catalytic dehydration polycondensation method and having a free carboxyl group at the terminal, the number average molecular weight measured by GPC and the number average molecular weight measured by the end group are almost the same. To do. On the other hand, in a polymer synthesized from a cyclic dimer by a ring-opening polymerization method using a catalyst and having essentially no free carboxyl group at the terminal, the number average molecular weight measured by the end group is the number average molecular weight measured by GPC. Much higher than the molecular weight. Due to this difference, the polymer having a free carboxyl group at the terminal can be clearly distinguished from the polymer having no free carboxyl group at the terminal.

While the number average molecular weight determined by quantification of end groups is an absolute value, the number average molecular weight determined by GPC measurement is various analysis and analysis conditions (for example, selection of mobile phase type, column type, reference substance, slice width, Since it is a relative value that fluctuates depending on the selection of the baseline, etc., it is difficult to make a unique numerical value. It means that the number average molecular weight determined by quantification is within a range of about 0.5 to 2 times the number average molecular weight determined by GPC. It is preferably within the range of about 0.8 to 1.5 times. The term "number average molecular weight determined by end group quantitatively significantly higher than the number average molecular weight determined by GPC" means that the number average molecular weight determined by terminal group determination exceeds about twice the number average molecular weight determined by GPC measurement. In the present invention, a polymer in which the number average molecular weight measured by GPC and the number average molecular weight measured by end group determination are substantially the same is preferable. The weight average molecular weight and the number average molecular weight in the present specification mean that the weight average molecular weight is 120,000, 52,000, 22,000, 9,20.
0, 5,050, 2,950, 1,050, 580, 16
2 means the polystyrene-equivalent weight average molecular weight and number average molecular weight measured by gel permeation chromatography (GPC) using 9 kinds of polystyrene as reference substances. For the measurement, GPC column KF804L x2 (manufactured by Showa Denko) and RI monitor L-3300 (manufactured by Hitachi Ltd.) were used, and chloroform was used as a mobile phase. Further, the dispersity is calculated by (weight average molecular weight / number average molecular weight).

The water-soluble aliphatic carboxylic acid metal salt is not particularly limited as long as it is water-soluble and does not adversely affect the living body. The water-soluble aliphatic carboxylic acid metal salt is preferably an aliphatic carboxylic acid metal salt having a solubility in water of about 20 mg / ml or more at room temperature (about 20 ° C.), and more preferably an aliphatic carboxylic acid having a solubility of about 100 mg / ml or more. Acid metal salts, particularly preferably having a solubility of about 20.
It is an aliphatic carboxylic acid metal salt of 0 mg / ml or more. In the water-soluble aliphatic carboxylic acid metal salt, the aliphatic carboxylic acid is preferably an aliphatic carboxylic acid having 2 to 9 carbon atoms. Examples of the aliphatic carboxylic acid include aliphatic monocarboxylic acid, aliphatic dicarboxylic acid, and aliphatic tricarboxylic acid. These aliphatic carboxylic acids may be saturated or unsaturated. Examples of the aliphatic monocarboxylic acid include saturated aliphatic monocarboxylic acid having 2 to 9 carbon atoms (eg, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, etc.) And unsaturated aliphatic monocarboxylic acids having 2 to 9 carbon atoms (eg, acrylic acid, propiolic acid, methacrylic acid, crotonic acid, isocrotonic acid, etc.). Examples of the aliphatic dicarboxylic acid include saturated aliphatic dicarboxylic acid having 2 to 9 carbon atoms (eg, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, etc.) and unsaturated aliphatic dicarboxylic acid having 2 to 9 carbon atoms. Examples thereof include acids (eg, maleic acid, fumaric acid, citraconic acid, mesaconic acid, etc.). Examples of the aliphatic tricarboxylic acid include saturated aliphatic tricarboxylic acid having 2 to 9 carbon atoms (eg, tricarballylic acid, 1,2,3-butanetricarboxylic acid, etc.). The above-mentioned aliphatic carboxylic acid may have 1 or 2 hydroxyl groups, and examples thereof include glycolic acid, lactic acid, glyceric acid, tartronic acid, malic acid, tartaric acid and citric acid. To be The aliphatic carboxylic acid is preferably an aliphatic monocarboxylic acid. The aliphatic carboxylic acid is more preferably a saturated aliphatic monocarboxylic acid having 2 to 9 carbon atoms, and particularly preferably a saturated aliphatic monocarboxylic acid having 2 to 3 carbon atoms. Particularly preferred specific examples of the aliphatic carboxylic acid include acetic acid and the like.

The metal salt of the water-soluble aliphatic carboxylic acid metal salt is, for example, an alkali metal (eg, sodium,
Potassium, etc.), monovalent metal salts such as copper (I valent) salts, alkaline earth metal (eg, calcium, magnesium etc.) salts, zinc (II valent) salts, iron (II valent, III valent) salts, copper ( II value) salt,
Tin (II value, IV value) salt, Aluminum (II value, III value)
Examples thereof include polyvalent metal salts such as salts. The metal salt is preferably a polyvalent metal salt. The metal salt is particularly preferably a calcium salt or a zinc salt. Specific examples of the water-soluble aliphatic carboxylic acid metal salt include, for example, sodium acetate, potassium acetate, calcium acetate, zinc acetate, sodium propionate, calcium propionate, sodium glycolate, zinc glycolate, sodium lactate, calcium lactate, Examples include zinc lactate, sodium tartrate, zinc tartrate and sodium citrate. Specific preferred examples of the water-soluble aliphatic carboxylic acid metal salt include calcium acetate and zinc acetate. A water-soluble aromatic carboxylic acid metal salt is also used in the same manner as the water-soluble aliphatic carboxylic acid metal salt. Specific examples of the water-soluble aromatic carboxylic acid metal salt include sodium benzoate, zinc benzoate, sodium salicylate, zinc salicylate and the like. The organic solvent used in the production of the biodegradable matrix preferably has a boiling point of 120 ° C. or lower. Examples of the organic solvent include halogenated hydrocarbons (eg, dichloromethane, chloroform, carbon tetrachloride, etc.), alcohols (ethanol, methanol), acetonitrile and the like. These may be used as a mixture. The organic solvent is
Dichloromethane and acetonitrile are preferred.

The sustained-release preparation of the present invention can be produced by permeating a water-soluble polypeptide into a biodegradable matrix in water. The sustained-release preparation (eg, microcapsule) can be produced, for example, by the following operation. Hereinafter, the sustained-release preparation produced in this manner may be simply referred to as microcapsules. 1) Prepare an aqueous solution of a water-soluble polypeptide 2) Contact the biodegradable matrix with the aqueous solution of 1) to allow the aqueous solution to penetrate into the biodegradable matrix 3) Biodegradable matrix as necessary To separate biodegradable matrix from water-soluble polypeptide that does not permeate into the body (washing operation) 4) Dry sustained-release preparation (eg, microcapsule) obtained by permeating water-soluble polypeptide into biodegradable matrix In order to increase the solubility of the water-soluble polypeptide or to maintain the biological activity of the water-soluble polypeptide, an aqueous solution of the above-mentioned water-soluble polypeptide, such as inorganic salts (eg, salt , Sodium monohydrogen phosphate, etc., organic salts (eg, ammonia acetate, etc.), amino acids (eg, glycine, arginine, hist) Gin, etc.) may be added. A plurality of the above-mentioned salts may be combined so that the optimum pH of the drug can be obtained. At this time, p
H is generally adjusted to be neutral or weakly acidic, but it may be adjusted to be basic. The concentration of the salt is adjusted so that the aqueous solution of the water-soluble polypeptide has a tonicity of about 1/50 to 5 times that of physiological saline. It is preferably about 1/25 to 3 times. In addition, a surfactant such as Tween 80 may be added. The concentration of the surfactant used is about 0.0001 to 0.2% (w / v), preferably about 0.001 to about 0.1% (w / v).

Serum albumin may be added to the aqueous solution of the water-soluble polypeptide. The addition of serum albumin increases the solubility of water-soluble polypeptides,
The biological activity of the water-soluble polypeptide can be retained. Serum albumin may be added to an aqueous solution of the water-soluble polypeptide or may be premixed with the water-soluble polypeptide. Human serum albumin is preferable as the serum albumin, which may be separated and purified from human blood or may be produced by a genetic engineering method. The mixing ratio (weight ratio) of the water-soluble polypeptide and serum albumin is, for example, about 1: 1,000 to about 100: 1, preferably about 1:10.
0 to about 10: 1. The concentration of the water-soluble polypeptide in the aqueous solution is not particularly limited, but in order to permeate as much of the water-soluble polypeptide as possible into the biodegradable matrix per unit weight, the concentration of the water-soluble polypeptide should be as high as possible below the solubility of the water-soluble polypeptide. Is preferred. The solubility also depends on the salt concentration, temperature, and the presence or absence of additives. It is generally known that the water-soluble polypeptide release pattern of a sustained-release preparation differs depending on the concentration of the water-soluble polypeptide that has penetrated into the biodegradable matrix. From this viewpoint as well, the water-soluble polypeptide concentration is selected. . The water-soluble polypeptide concentration is generally about 100 μg / ml to about 500 mg / m 2.
1, preferably about 1 to 300 mg / ml, more preferably about 5 to 100 mg / ml.

When the biodegradable matrix is produced by mixing a biodegradable polymer and a water-soluble aliphatic carboxylic acid metal salt, when the biodegradable matrix is permeated with an aqueous solution of the water-soluble polypeptide. The pH of the aqueous solution may vary depending on the type of the water-soluble aliphatic carboxylic acid metal salt contained in the biodegradable matrix, the isoelectric point of the water-soluble polypeptide, etc., but cannot be generally stated, but it is preferably about 3 to 10. 9, more preferably about 3-8.
The pH may be adjusted appropriately with an acid such as an inorganic acid (eg, hydrochloric acid), an organic acid (eg, acetic acid), or an alkali such as an alkali metal hydroxide (eg, sodium hydroxide). Here, the amount of the acid or alkali used may be appropriately used depending on the ionization degree of the acid or alkali, the strength, and the target pH. As the water-soluble aliphatic carboxylic acid metal salt, sodium acetate, zinc acetate, calcium acetate or the like is particularly preferable because the biodegradable matrix can be permeated with the aqueous solution of the water-soluble polypeptide at a pH around neutral. The operation of permeating a water-soluble polypeptide into a biodegradable matrix in water is
For example, it is performed by mixing an aqueous solution of a water-soluble polypeptide with a biodegradable matrix. The order of mixing the aqueous solution of the water-soluble polypeptide and the biodegradable matrix can be arbitrarily selected as long as the biological activity of the water-soluble polypeptide is retained. For example, the biodegradable matrix may be immersed in the aqueous solution of the water-soluble polypeptide, or the aqueous solution of the water-soluble polypeptide may be added to the biodegradable matrix.

The mixing ratio of the aqueous solution of the water-soluble polypeptide and the biodegradable matrix is such that the excess amount of the aqueous solution of the water-soluble polypeptide is sufficient so that the aqueous solution of the water-soluble polypeptide permeates the biodegradable matrix. used. That is, all biodegradable matrices are prepared soaked in an aqueous solution of a water-soluble polypeptide. The weight ratio between the aqueous solution of the water-soluble polypeptide and the biodegradable matrix cannot be unconditionally determined because the porosity of the biodegradable matrix is different, but it is preferably about 1:10 to about 20: 1. , And more preferably about 1: 5 to about 10: 1. However, in general, water-soluble polypeptides that do not penetrate the biodegradable matrix are not reused, and in order to reduce the loss of valuable water-soluble polypeptides, the minimum necessary aqueous solution of water-soluble polypeptides is used. Mixing the aqueous solution of the water-soluble polypeptide and the biodegradable matrix is usually performed using a container. As such a container, a container that is less likely to adsorb a water-soluble polypeptide is preferred, and for example, a glass which has been siliconized (siliconized) can be mentioned. Further, an alloy (stainless steel or titanium) whose surface is treated so as not to impair the biological activity of the water-soluble polypeptide is also preferable.

The mixing operation is carried out, for example, by adding the biodegradable matrix to the aqueous solution of the water-soluble polypeptide and allowing it to stand or to carry out a light stirring operation to the extent that the biological activity of the water-soluble polypeptide is not lost. In the operation, vacuum decompression may be performed so that the aqueous solution of the water-soluble polypeptide does not foam excessively. The mixing operation is carried out at a temperature at which the biological activity of the water-soluble polypeptide is not impaired or the biodegradable polymer that is a constituent of the biodegradable matrix is not decomposed, and it is usually performed at room temperature, preferably in a cool place. Done. The temperature in the mixing operation is
Specifically, it is about 1 to 30 ° C, preferably about 4 to 25 ° C. The time of the mixing operation depends on the amount of biodegradable matrix,
The time is several hours to several tens of hours, depending on the composition of the biodegradable polymer, the molecular weight, the temperature and other factors. Specifically, for example, about 4 ° C. for about 10 to 100 hours and about 25 ° C. for about 5 to 50 hours are preferable. This time is arbitrarily selected within the range in which the biological activity of the water-soluble polypeptide is not lost and the range in which the biodegradable polymer is not hydrolyzed more than necessary. When the biodegradable matrix is made by mixing the biodegradable polymer and the water-soluble aliphatic carboxylic acid metal salt, the mixing time is shortened. Specifically, the mixing time is, for example, about 4 ° C. for about 0.5 to 24 hours,
It is about 0.5 to 5 hours at about 25 ° C.

If necessary, a washing operation may be added after the mixing operation. By the washing operation, the water-soluble polypeptide that has not penetrated into the biodegradable matrix is removed.
Although various methods can be considered as a washing method, a method of not destroying the biodegradable matrix, and a water-soluble polypeptide that has penetrated into the biodegradable matrix do not flow out of the biodegradable matrix, and A method that does not lose biological activity is adopted. For example, after the mixing operation is completed, the washing solution is added, and the operation of separating the washing solution from the microcapsules by centrifugation or filtration is repeated. As the washing liquid in this operation, distilled water or an aqueous solution containing a salt (eg, sodium hydrogen phosphate, sodium chloride, etc.) and a sugar (eg, mannitol, etc.) is used. The washing solution is preferably an aqueous solution containing mannitol. The microcapsules thus obtained are then dried. Examples of the drying method include freeze drying and vacuum drying. Freeze-drying is particularly preferable as the drying method. Further, a cleaning liquid containing an anti-agglomeration agent may be used to prevent the particles from aggregating during the drying operation. Examples of the aggregation preventing agent include water-soluble polysaccharides such as mannitol, lactose, glucose, starches (eg, corn starch, etc.), mucopolysaccharides such as hyaluronic acid, proteins such as glycine, fibrin, collagen, sodium chloride, phosphate. Examples thereof include inorganic salts such as sodium hydrogen and phospholipids such as lecithin.

In the drying operation, the drying temperature may be any temperature that does not impair the biological activity of the water-soluble polypeptide and does not destroy the microcapsules. Preferably, it is heated to a temperature above the glass transition temperature of the biodegradable polymer used so that the particles of the microcapsules do not adhere to each other. What is the glass transition temperature? Differential scanning calorimeter (D
SC) is the midpoint glass transition temperature (Tmg) obtained when the temperature is raised at a heating rate of about 10 or 20 ° C. per minute. As a preferable heating temperature, a temperature about 2 to 10 ° C. higher than the glass transition temperature is adopted. Such a temperature is, for example, about 25 to 50 ° C, preferably about 30 to 45 ° C.
Is. The heating time varies depending on the heating temperature, the amount of microcapsules to be treated, etc., but is generally several tens hours, preferably about 24 hours after the temperature of the microcapsules themselves reaches a predetermined temperature. The heating method is not particularly limited, but any method may be used as long as it can uniformly heat the microcapsules. Specific examples include a method of heating in a constant temperature bath and a method of heating with a microwave. The drying operation makes it possible to suppress the initial release of the microcapsules after administration to warm-blooded animals.

In the present invention, since the organic solvent is not used when permeating the water-soluble polypeptide into the biodegradable matrix, and since excessive heating is not performed, the organism of the water-soluble polypeptide is formulated in the process of formulation. Activity is rarely impaired. The organic solvent mentioned here is, for example, a halogenated hydrocarbon, alcohols, acetonitrile, glacial acetic acid, or the like. Further, by using the water-soluble aliphatic carboxylic acid metal salt, the water-soluble polypeptide can be efficiently permeated into the biodegradable matrix. When used as an injection, the sustained-release preparation of the present invention exhibits sustained release at a substantially constant rate for a long period of time, for example, about 1 week to 1 month. The content of the water-soluble polypeptide in the sustained-release preparation of the present invention is, for example, when the sustained-release preparation is microcapsules, the microcapsules are dissolved in a suitable solvent,
Method for separating and quantifying water-soluble polypeptide contained in microcapsules by chromatograph such as HPLC or immunological assay such as enzyme immunoassay, or method for measuring biological activity of separated water-soluble polypeptide It is decided by. The content of the water-soluble polypeptide with respect to the biodegradable polymer in the microcapsules is generally about 0.1 to 30% (w / w), preferably about 1 to 20% (w / w).

The sustained-release preparation of the present invention is formulated into various dosage forms, for example, as microcapsules or by using microcapsules as a raw material, and parenteral preparations (eg, intramuscular, subcutaneous, injectable agents into organs or the like) or Implants, transmucosal agents for nasal cavity, rectum, uterus, etc.), oral agents (eg, capsules (eg, hard capsules, soft capsules, etc.), granules, solid preparations such as powders, suspensions, etc. Solution, etc.) and the like. In the present invention, the sustained release preparation is particularly preferably for injection. For example, when the sustained-release preparation is a microcapsule, the microcapsule can be made into an injection by using the microcapsule as a dispersant (eg, Tween 80, HC
Surfactant such as O-60, carboxymethyl cellulose,
Polysaccharides such as sodium alginate and sodium hyaluronate, protamine sulfate, etc.), preservatives (eg, methylparaben, propylparaben, etc.), isotonic agents (eg, sodium chloride, mannitol, sorbitol, glucose, etc.), local anesthetics ( Xylocaine hydrochloride, chlorobutanol, etc.) or the like as an aqueous suspension agent, or vegetable oil such as sesame oil, corn oil or medium chain fatty acid triglyceride [eg, Miglyol 812 (Frus Actiengeselshaft Co., Ltd.)] or lecithin Dispersed together with a mixture of phospholipids, a sustained-release injection that can be actually used as an oily suspension is prepared.

When the sustained-release preparation is, for example, a microcapsule, the particle size of the microcapsule may be within a range satisfying the degree of dispersion and needle-passability when used as a suspension injection, for example, particles. Diameter is about 0.1 to 300
The range of μm can be mentioned. Preferably about 1 to 150
The particle size is in the range of μm. More preferably, the particle size is in the range of about 2 to 100 μm. Examples of the sterile preparation of the above-mentioned microcapsules include a method of making all the manufacturing steps aseptic, a method of sterilizing with gamma rays, a method of adding a preservative, and the like, but are not particularly limited. The sustained-release preparation of the present invention has low toxicity and is a mammal (eg, human, cow, pig, dog,
It can be safely used for cats, mice, rats, rabbits, etc.). The adaptation of the sustained-release preparation of the present invention depends on the water-soluble polypeptide used. In the sustained-release preparation of the present invention, when the water-soluble polypeptide is interferon alpha, viral hepatitis (eg, hepatitis C, HBe antigen-positive chronic active hepatitis B, etc.), cancer (eg, kidney) Cancer, multiple myeloma, etc.), erythropoietin, anemia (eg, renal dialysis anemia, etc.), G-C
In case of SF, neutropenia (eg, during treatment with anti-cancer drug), infection, etc .; in case of IL-2, cancer (eg, hemangioendothelioma, etc.),
In the case of FGF, gastrointestinal ulcer, etc., in the case of FGF-9, thrombocytopenia, etc., in the case of NGF, senile dementia, neuropathies, etc., and in the case of TPA, thrombosis, etc.
Insulin is effective in treating or preventing diabetes, and tumor necrosis factor is effective in treating or preventing cancer.

The dose of the sustained-release preparation depends on the kind and content of the water-soluble polypeptide, the duration of release of the water-soluble polypeptide,
The amount varies depending on the target disease, target animal, etc., but may be any amount as long as the pharmacological effect of the water-soluble polypeptide contained therein is exerted. The dose per administration of the water-soluble polypeptide is, for example, when the sustained-release preparation is a one-week type preparation,
Although it depends on the kind of the water-soluble polypeptide, it can be appropriately selected from the range of about 0.0001 to 10 mg / kg body weight per adult. More preferably, it can be appropriately selected from the range of about 0.0005 to 1 mg / kg body weight. The dose of the sustained-release preparation can be appropriately selected from the range of about 0.0005 to 50 mg / kg body weight per adult, preferably once. More preferably about 0.002
It can be appropriately selected from the range of 5 to 10 mg / kg body weight.
The frequency of administration can be appropriately selected once a week, once every two weeks, etc. depending on the type and content of the water-soluble polypeptide, dosage form, duration of release of the water-soluble polypeptide, target disease, target animal, etc. . The preparation of the present invention may be stored at room temperature or in a cold place, but preferably in a cold place. The room temperature or the cold place mentioned here is defined in the Japanese Pharmacopoeia. That is, the normal temperature means 15 to 25 ° C, and the cold place means 15 ° C or lower.

[0036]

EXAMPLES The present invention will be described more specifically below with reference to Reference Examples, Examples and Comparative Examples, but these do not limit the present invention. Hereinafter,% means weight / volume percent unless otherwise specified. Example 1 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), weight average molecular weight 5,400 measured by GPC, number average molecular weight 2,90 measured by GPC
0, number average molecular weight of 2,200 by quantitative determination of end groups, 2.0 g of Wako Pure Chemical Industries, Ltd.) was added to and dissolved in 5.3 g (4 ml) of dichloromethane. As an inner aqueous phase, 1 ml of physiological saline for injection was added and stirred with a homogenizer (Polytron) for about 30 seconds. 0.1 adjusted to 18 ℃ in advance
% (W / w) polyvinyl alcohol (EG-40, manufactured by Nippon Gosei Kagaku) aqueous solution (500 ml), and a W / O / W emulsion was prepared at 4,000 rpm using a turbine homomixer. This W / O / W emulsion was stirred at room temperature for 5 hours to volatilize dichloromethane to solidify the oil phase, and then use a centrifuge (05PR-22, Hitachi) at 2,000 rp.
collected by m. This was again dispersed in distilled water and further centrifuged. The collected lactic acid-glycolic acid copolymer matrix was redispersed by adding a small amount of distilled water, and then the dispersion was freeze-dried to obtain a powder. Interferon alfa 1.08 × in a polyethylene test tube
10 ⁹ IU (containing about 25 mg of human serum albumin) was weighed and dissolved in 200 μl of distilled water. The above-mentioned microcapsules (200 mg) were added thereto, and the containers were tightly closed and then allowed to stand in a refrigerator at 4-8 ° C. for about 4 days. After this operation, 5 ml of distilled water is added and gently stirred for about 1 minute. After centrifugation at about 2,000 rpm for 5 minutes, the supernatant was discarded. A washing operation was performed by repeating this operation three times. To the obtained microcapsules, 44 mg of D-mannitol was added, 2 ml of distilled water was added, and the mixture was slowly stirred. Then, the resulting dispersion was dried under vacuum (temperature: 40 ° C./6 hours).

Example 2 In the same manner as in Example 1, a lactic acid-glycolic acid copolymer matrix was obtained as a powder. Interferon alfa 1.08 × 10 ⁹ IU (containing about 25 mg of human serum albumin) was weighed into a polyethylene test tube, and 200 μl
Dissolved in distilled water. And the microcapsule 2
After adding 00 mg and sealing the container, the mixture was allowed to stand in a refrigerator at 4 to 8 ° C for about 30 hours. After this operation, 5 ml of distilled water is added and gently stirred for about 1 minute. Centrifugation was performed for 5 minutes at about 1,000 rpm, and the supernatant was discarded. A washing operation was performed by repeating this operation three times. 0.1% sodium hyaluronate (molecular weight 1.8 million) was added to the obtained microcapsules.
1 ml of the aqueous solution was added, and the mixture was slowly stirred. afterwards,
The resulting dispersion was freeze dried (16 hours).

Example 3 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), weight average molecular weight 5,900 by GPC measurement, number average molecular weight 2,600 by GPC measurement,
Wako Pure Chemical Industries, Ltd.) 4.0 g, dichloromethane 5.3 g (4
ml) and dissolved. 4 g of calcium carbonate in this solution
After adding, was stirred for about 30 seconds with a vortex mixer to obtain an S / O emulsion. 0.1% (w / w) polyvinyl alcohol (EG
-40, manufactured by Nippon Synthetic Chemical Industry Co., Ltd.) was poured into 800 ml of an aqueous solution, and a turbine homomixer was used to perform S / O / at 6,000 rpm.
It was a W emulsion. This S / O / W emulsion was stirred at room temperature for 5 hours to vaporize dichloromethane to solidify the oil phase. Then 10 ml of 1N hydrochloric acid was added to remove excess calcium carbonate. Centrifugation was performed at about 2,000 rpm (05PR-22, Hitachi, Ltd.) and the supernatant was discarded. This was again dispersed in distilled water and further centrifuged. A small amount of distilled water was added to the collected biodegradable matrix for redispersion, and this dispersion was freeze-dried to obtain a powder (about 2.0 g). Interferon alfa freeze-dried powder 4 mg (about 8 × 10 ⁸ IU in a glass test tube)
(International unit)) was weighed and dissolved with 2 ml of 10 mM hydrochloric acid solution. The biodegradable matrix (50 mg) was added thereto, and the mixture was rotatively mixed on a low profile roller (manufactured by Life Science) at 4 ° C. for about 1 day. After the operation, 4 ml of distilled water was added and gently stirred for about 1 minute, and then about 1,
The mixture was centrifuged at 000 rpm for 5 minutes and the supernatant was discarded (the washing operation was performed by repeating this operation twice). The resulting dispersion was freeze-dried to obtain microcapsules (about 48 mg). In order to examine the interferon alpha content in the obtained microcapsules, the microcapsules were extracted with a 25% Block Ace (Snow Brand Milk Products Co., Ltd.) (blocking agent for immunological experiment) solution containing 10% acetonitrile, followed by enzyme immunology. Measurement method (E
Interferon alpha was quantified by IA). As a result, 1 mg of microcapsules contained 5.7 million IU of interferon alpha.

Example 4 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), weight average molecular weight measured by GPC 5,100, number average molecular weight measured by GPC 2,570,
Wako Pure Chemical Industries, Ltd.) 4.0 g, dichloromethane 5.3 g (4
ml) and dissolved. 0.4 g of zinc acetate (dihydrate) was added to this solution, and the mixture was shaken for 2 hours and then stirred with a homogenizer (Polytron) for about 30 seconds to give S /
O emulsion. This was poured into 800 ml of a 0.1% (w / w) polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry) aqueous solution which had been adjusted to 18 ° C. in advance, and a turbine homomixer was used at 6,000 rpm. This was an S / O / W emulsion. This S / O / W emulsion is used at room temperature for 5
The mixture was stirred for a period of time to evaporate dichloromethane, and the oil phase was solidified. Centrifuge at approximately 2,000 rpm (05PR-2
(2, Hitachi) The supernatant was discarded. This was again dispersed in distilled water and further centrifuged. After adding a small amount of distilled water to the collected biodegradable matrix and redispersing it,
This dispersion was freeze-dried to obtain a powder (about 2.0 g). Weigh 6 mg of interferon alpha lyophilized powder (about 1.2 × 10 ⁹ IU (international unit)) into a glass test tube,
It was dissolved in 3 ml of a 0.5 mM hydrochloric acid solution. The above biodegradable matrix (300 mg) was added thereto, and the mixture was rotatively mixed on a low profile roller (manufactured by Life Science) at 4 ° C. for about 1 day. After the operation, 5% mannitol aqueous solution 1
Add 0 ml and stir gently for about 1 minute, then about 2,000
Centrifugation was performed at 0 rpm for 5 minutes, and the supernatant was discarded (the washing operation was performed by repeating this operation 3 times). D-mannitol (30 mg) and distilled water (0.5 ml) were added to the obtained microcapsules, and the mixture was slowly stirred and the resulting suspension was freeze-dried to give microcapsules (about 310 mg). In order to examine the interferon alpha content in the obtained microcapsules, the microcapsules were extracted with a 25% Block Ace (Snow Brand Milk Products Co., Ltd.) (blocking agent for immunological experiment) solution containing 10% acetonitrile, followed by enzyme immunology. Assay (sandwich method using interferon antibody, hereinafter referred to as EI
Interferon alpha was quantified by A). As a result, 1 mg of microcapsules contained 2.3 million IU of interferon alpha.

Example 5 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), weight average molecular weight 5,900 by GPC measurement, number average molecular weight 2,600 by GPC measurement,
Wako Pure Chemical Industries, Ltd.) 4.0 g, dichloromethane 5.3 g (4
ml) and dissolved. To this solution was added 1 ml of an aqueous solution containing 800 mg of zinc acetate (dihydrate), and the mixture was stirred with a homogenizer (Polytron) for about 30 seconds to give a W / O emulsion. This was adjusted to 18 ° C in advance.
It was poured into 800 ml of a 1% (w / w) polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry) aqueous solution, and a W / O / W emulsion was prepared at 6,000 rpm using a turbine homomixer. The W / O / W emulsion was stirred at room temperature for 5 hours to vaporize dichloromethane to solidify the oil phase. Centrifugation was performed at about 2,000 rpm (05PR-22, Hitachi, Ltd.) and the supernatant was discarded. This was again dispersed in distilled water and further centrifuged. A small amount of distilled water was added to the collected biodegradable matrix for redispersion, and this dispersion was freeze-dried to obtain a powder (about 2.0 g). 6 mg of interferon alfa lyophilized powder in a glass test tube
(Approximately 1.2 × 10 ⁹ IU) Weigh and add 3 ml of 0.5 mM hydrochloric acid solution
It was dissolved in. In addition to the above-mentioned biodegradable matrix 3
00 mg was added, and the mixture was rotatively mixed on a low profile roller (manufactured by Life Science) at 10 ° C. for about 1 day. After this operation, 10 ml of a 5% mannitol aqueous solution was added, and the mixture was gently stirred for about 1 hour, and then centrifuged at about 1,000 rpm for 5 minutes, and the supernatant was discarded (the washing operation was repeated twice. Was done). The resulting microcapsules were freeze dried. To examine the interferon alpha content in the microcapsules, the microcapsules were extracted with 25% Block Ace (Snow Brand Milk Products Co., Ltd.) solution containing 10% acetonitrile, and then EI
Interferon alpha was quantified by A. As a result, 1 mg of microcapsules contained 780,000 IU of interferon alpha.

Example 6 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), weight average molecular weight 5,900 by GPC measurement, number average molecular weight 2,600 by GPC measurement,
Wako Pure Chemical Industries, Ltd.) 4.0 g of dichloromethane 5.3 g (4 m
l) and dissolved. Add calcium acetate (1
Hydrate) (0.4 g) was added, and the mixture was stirred with a homogenizer (Polytron) for about 30 seconds to give an S / O emulsion.
0.1% (w / w) polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry) aqueous solution adjusted to 18 ° C in advance 8
Pour into 00 ml and use a turbine homomixer,
An S / O / W emulsion was prepared at 6,000 rpm. This S
The / O / W emulsion was stirred at room temperature for 5 hours to strip off dichloromethane and solidify the oil phase. About 2,000 rpm
Centrifuge operation was performed at (05PR-22, Hitachi, Ltd.) and the supernatant was discarded. This was again dispersed in distilled water and further centrifuged. A small amount of distilled water was added to the collected biodegradable matrix for redispersion, and this dispersion was freeze-dried to obtain a powder (about 2.0 g). Add 4 mg (8 × 10 ⁸ I) of interferon alpha lyophilized powder to a glass test tube.
U) Weighed and dissolved in 2 ml of 1 mM hydrochloric acid solution. The biodegradable matrix (50 mg) was added thereto, and the mixture was rotatively mixed on a low profile roller (manufactured by Life Science) at 4 ° C. for about 1 day. After the operation, 4 ml of distilled water was added and gently stirred for about 1 minute, and then 5 rpm at about 1,000 rpm.
Centrifugation was performed for 1 minute, and the supernatant was discarded (the washing operation was performed by repeating this operation twice). Then, the resulting dispersion was freeze-dried to obtain microcapsules (about 48 mg). In order to examine the interferon alpha content in the obtained microcapsules, the microcapsules were extracted with 25% Block Ace (Snow Brand Milk Products Co., Ltd.) solution containing 10% acetonitrile, and then EI
Interferon alpha was quantified by A. As a result, 1 mg of microcapsules contained 9.4 million IU of interferon alpha.

Example 7 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), weight average molecular weight measured by GPC 5,100, number average molecular weight measured by GPC 2,570,
Wako Pure Chemical Industries, Ltd.) 4.0 g, dichloromethane 5.3 g (4
ml) and dissolved. 0.4 g of zinc acetate (dihydrate) was added to this solution, and the mixture was shaken for 2 hours and then stirred with a homogenizer (Polytron) for about 30 seconds to obtain an S / O emulsion. This was poured into 800 ml of a 0.1% (w / w) polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry) aqueous solution which had been adjusted to 18 ° C. in advance, and a turbine homomixer was used at 6,000 rpm. This was an S / O / W emulsion. This S / O / W emulsion was stirred at room temperature for 5 hours to vaporize dichloromethane to solidify the oil phase. Centrifuge at approximately 2,000 rpm (05PR-2
(2, Hitachi) The supernatant was discarded. This was again dispersed in distilled water and further centrifuged. After adding a small amount of distilled water to the collected biodegradable matrix and redispersing it,
This dispersion was freeze-dried to obtain a powder (about 2.0 g). 50 mg of biodegradable matrix was weighed in a glass test tube and the pH was adjusted with hydrochloric acid or sodium hydroxide to prepare a 2 mg / ml interferon alpha solution (about 4.0 x
Interferon-alpha was permeated into the biodegradable matrix by adding 10 ⁸ IU) (4 steps of pH about 2, about 4, about 5, and about 8) and rotatively mixing at 4 ° C. for 24 hours. . After centrifugation at about 1,000 rpm to remove the supernatant, the mixture was washed twice with 4 ml of distilled water, added with 0.5 ml of distilled water, and freeze-dried.

Example 8 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), weight average molecular weight 5,900 by GPC measurement, number average molecular weight 2,600 by GPC measurement,
Wako Pure Chemical Industries, Ltd.) 4.0 g, dichloromethane 5.3 g (4
ml) and dissolved. Add calcium acetate (1
(Hydrate) (0.4 g) was added, and the mixture was stirred with a homogenizer (Polytron) for about 30 seconds to obtain an S / O emulsion. 0.1% (w / w) adjusted to 18 ℃ in advance
It was poured into 800 ml of an aqueous solution of polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry Co., Ltd.) and a S / O / W emulsion was prepared at 6,000 rpm using a turbine homomixer. This S / O / W emulsion was stirred at room temperature for 5 hours to vaporize dichloromethane to solidify the oil phase. About 2,000
Centrifuge operation at 0 rpm (05PR-22, Hitachi)
The supernatant was discarded. This was again dispersed in distilled water and further centrifuged. A small amount of distilled water was added to the collected biodegradable matrix for redispersion, and this dispersion was freeze-dried to obtain a powder (about 4.0 g). Then, in the same manner as in Example 7, interferon alfa was permeated into the biodegradable matrix at various pHs and freeze-dried.

Example 9 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), weight average molecular weight measured by GPC 5,100, number average molecular weight measured by GPC 2,570,
Wako Pure Chemical Industries, Ltd.) 4.0 g, dichloromethane 5.3 g (4
ml) and dissolved. Sodium acetate (3
Hydrate) 0.2 g was added, and the mixture was shaken for 2 hours and then stirred with a homogenizer (Polytron) for about 30 seconds,
It was an S / O emulsion. 0.1% (w / w) polyvinyl alcohol (EG-4
0, manufactured by Nippon Synthetic Chemical Co., Ltd.) Injected into 800 ml of an aqueous solution, and using a turbine homomixer, S / O / W at 6,000 rpm
It was an emulsion. This S / O / W emulsion was stirred at room temperature for 5 hours to vaporize dichloromethane to solidify the oil phase. Centrifugation was performed at about 2,000 rpm (05PR-22, Hitachi, Ltd.) and the supernatant was discarded. This was again dispersed in distilled water and further centrifuged. A small amount of distilled water was added to the collected biodegradable matrix for redispersion, and this dispersion was freeze-dried to obtain a powder (about 2.0 g). Then, in the same manner as in Example 7, interferon alfa was permeated into the biodegradable matrix at various pHs and freeze-dried.

Example 10 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), weight average molecular weight measured by GPC 5,100, number average molecular weight measured by GPC 2,570,
Wako Pure Chemical Industries, Ltd.) 4.0 g, dichloromethane 5.3 g (4
ml) and dissolved. 0.4 g of zinc acetate (dihydrate) was added to this solution, and the mixture was shaken for 2 hours and then stirred with a homogenizer (Polytron) for about 30 seconds to give S /
O emulsion. This was poured into 800 ml of a 0.1% (w / w) polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry) aqueous solution which had been adjusted to 18 ° C. in advance, and a turbine homomixer was used at 6,000 rpm. This was an S / O / W emulsion. This S / O / W emulsion is used at room temperature for 5
The mixture was stirred for a period of time to evaporate dichloromethane, and the oil phase was solidified. Centrifuge at approximately 2,000 rpm (05PR-2
(2, Hitachi) The supernatant was discarded. This was again dispersed in distilled water and further centrifuged. After adding a small amount of distilled water to the collected biodegradable matrix and redispersing it,
This dispersion was freeze-dried to obtain a powder (about 2.0 g). 2 ml of an aqueous solution containing interleukin 2 (20 μg) was weighed in a glass test tube, and then 300 mg of the above-mentioned biodegradable matrix was added to this, and the mixture was heated to 4 ° C. on a low profile roller (Life Science). And mixed by rotation for about 5 hours. The interleukin 2 is produced by the method described in JP-A-61-78799 and purified by the purification method described in JP-A-60-115528, in which methionine is bound to the N-terminal. And a mixture of those not bound with. After this operation, 10 ml of a 5% mannitol aqueous solution was added, and the mixture was gently stirred for about 1 minute, and then centrifuged at about 2,000 rpm for 5 minutes, and the supernatant was discarded (the washing operation was repeated 3 times. Was done). D- in the obtained microcapsules
Add 30 mg mannitol, dissolve in 0.5 ml distilled water,
Stir slowly. Then the resulting suspension was freeze-dried.

Example 11 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), GPC measurement weight average molecular weight 5,800, GPC measurement number average molecular weight 2,805,
5.0 g of Wako Pure Chemical Industries) and 782 mg of zinc benzoate were added to 6.625 g (5 ml) of dichloromethane, and the mixture was mixed at room temperature for 3 hours.
It was shaken for a time to obtain an S / O emulsion. This one in advance
It was poured into 1,000 ml of a 0.1% (w / w) polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry) aqueous solution which had been adjusted to 8 ° C., and 6,000 r using a turbine homomixer.
S / O / W emulsion at pm. This S / O / W emulsion was stirred at room temperature for 5 hours to vaporize dichloromethane to solidify the oil phase. Centrifugation was performed at about 2,000 rpm (05PR-22, Hitachi, Ltd.) and the supernatant was discarded.
This was again dispersed in distilled water and further centrifuged.
A small amount of distilled water was added to the collected biodegradable matrix for redispersion, and this dispersion was freeze-dried to obtain a powder (about 2.0 g). 2 mg (1.7 × 10 ⁸ IU) of interferon-alpha freeze-dried powder was weighed in a glass test tube and dissolved in 3 ml of 0.5 mM hydrochloric acid solution. The biodegradable matrix (302 mg) was added thereto, and the mixture was rotatively mixed on a low profile roller (manufactured by Life Science Co., Ltd.) at 15 ° C. for about 5 hours. After this operation, 10 ml of a 5% mannitol aqueous solution was added, and the mixture was gently stirred for about 1 minute, and then centrifuged at about 2,000 rpm for 5 minutes, and the supernatant was discarded (the washing operation was repeated 3 times. Was done). To the obtained microcapsules, 30 mg of D-mannitol was added, dissolved with 0.5 ml of distilled water, and slowly stirred. Then freeze-dry the resulting suspension,
Microcapsules (about 310 mg) were obtained. In order to examine the interferon alpha content in the obtained microcapsules, the microcapsules were extracted with a 25% Block Ace (Snow Brand Milk Products Co., Ltd.) solution containing 10% acetonitrile, and then interferon alpha was quantified by EIA. As a result, 1 mg of microcapsules contained 3.61 million IU of interferon alpha. Example 12 A matrix of lactic acid-glycolic acid copolymer was obtained as a powder in the same manner as in Example 11 except that zinc benzoate was zinc salicylate 995 mg. Then, microcapsules were obtained in the same manner as in Example 11 except that 304 mg of matrix was used. Each mg of microcapsules contained 1.93 million IU of interferon alpha.

Comparative Example 1 Interferon alfa freeze-dried powder 2 mg 0.5%
It was dissolved in 2 ml of a phosphate buffered saline solution containing bovine albumin (concentration of 175 billion IU / ml as measured by EIA). Comparative Example 2 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), weight average molecular weight 5,900 by GPC measurement, number average molecular weight 2,600 by GPC measurement,
Wako Pure Chemical Industries, Ltd.) 4.0 g, dichloromethane 5.3 g (4
ml) and dissolved. After adding 0.5 ml of an aqueous zinc chloride solution adjusted to a concentration of 2 g / ml to this solution, the mixture was stirred with a homogenizer (Polytron) for about 30 seconds to obtain a W / O emulsion. This was poured into 800 ml of a 0.1% (w / w) polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry) aqueous solution which had been adjusted to 18 ° C. in advance, and a turbine homomixer was used at 6,000 rpm. It was a W / O / W emulsion. The W / O / W emulsion was stirred at room temperature for 5 hours to vaporize dichloromethane to solidify the oil phase. Centrifuge at approximately 2,000 rpm (05PR-2
(2, Hitachi) The supernatant was discarded. This was again dispersed in distilled water and further centrifuged. After adding a small amount of distilled water to the collected biodegradable matrix and redispersing it,
This dispersion was freeze-dried to obtain a powder (about 2.0 g). Then, in the same manner as in Example 7, the biodegradable matrix was permeated with interferon alfa at various pHs,
Lyophilization was performed.

Comparative Example 3 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50
/ 50 (mol%), weight average molecular weight 5,900 by GPC measurement, number average molecular weight 2,600 by GPC measurement,
Wako Pure Chemical Industries, Ltd.) 4.0 g, dichloromethane 5.3 g (4
ml) and dissolved. After adding 0.2 g of zinc carbonate to this solution, it was stirred for about 30 seconds with a vortex mixer to obtain an S / O emulsion. 0.1% (w / w) polyvinyl alcohol (EG
-40, manufactured by Nippon Synthetic Chemical Industry Co., Ltd.) was poured into 800 ml of an aqueous solution, and a turbine homomixer was used to perform S / O / at 6,000 rpm.
It was a W emulsion. This S / O / W emulsion was stirred at room temperature for 5 hours to vaporize dichloromethane to solidify the oil phase. Centrifugation was performed at about 2,000 rpm (05PR-22, Hitachi, Ltd.) and the supernatant was discarded. After dispersing again in distilled water, further centrifugation was performed. A small amount of distilled water was added to the collected biodegradable matrix for redispersion, and this dispersion was freeze-dried to obtain a powder (about 2.0 g). Then, in the same manner as in Example 7, interferon alfa was permeated into the biodegradable matrix at various pHs and freeze-dried.

Experimental Example 1 About 40 mg of the microcapsules obtained in Example 1 was added to 0.5 m.
l dispersion medium (2.5 mg carboxymethylcellulose,
It was dispersed in 0.5 mg of polysorbate 80 and 25 mg of mannitol dissolved in distilled water) and administered subcutaneously to the back of 8-week-old male SD rats with a 22 G needle (dosage as microcapsules: 133 mg / kg). Blood was collected from the rat tail at regular intervals after administration, and the serum interferon alpha concentration was measured by enzyme immunoassay (EIA). Experimental Example 2 Using about 30 mg of the microcapsules obtained in Example 2,
It was administered to rats in the same manner as in Experimental Example 1, and the serum interferon alpha concentration was assayed by enzyme immunoassay (E.
When measured by IA), a substantially constant blood concentration lasted for one week.

Experimental Example 3 About 22 mg of the microcapsules obtained in Example 4 and about 64 mg of the microcapsules obtained in Example 5 were each added to 0.5 ml of a dispersion medium (5 mg of carboxymethylcellulose,
2 mg of polysorbate 80 (surfactant) and 25 mg of mannitol dissolved in 1 liter of distilled water) were dispersed and administered to the back of 8-week-old male SD rats subcutaneously with an 18 G needle (about one rat). 50 million IU of interferon alfa has been administered). Blood was collected from the rat tail at regular intervals after administration, and the concentration of interferon alpha in the serum was measured by EIA. As a control, the interferon-alpha aqueous solution obtained in Comparative Example 1 was subcutaneously administered to rats (interferon-alpha dose per rat: about 50 million IU).
In the microcapsule administration group of Comparative Example 1, serum interferon on the 3rd day after administration was decreased to the detection limit, but in the microcapsule administration groups of Example 4 and Example 5, the initial high blood concentration continued. An almost constant blood concentration lasted for one week.

Experimental Example 4 The relationship between the pH of the interferon alfa solution and various zinc salts on the permeation efficiency of interferon alfa into the biodegradable matrix (interferon content in microcapsules) was investigated. The specific method is shown below. The microcapsules obtained in Example 7 (zinc acetate), Comparative Example 2 (zinc chloride) and Comparative Example 3 (zinc carbonate) were extracted with a 25% block ace solution containing 10% acetonitrile and then interferon by EIA. Alpha was quantified. As a result, as shown in FIG.
When zinc acetate was used, the penetration efficiency of interferon alfa was good under the pH conditions where interferon alfa was physically relatively stable. Experimental Example 5 The relationship between the pH of the interferon alfa solution and the calcium acetate salt on the permeation efficiency of interferon alfa into the biodegradable matrix (interferon content in microcapsules) was investigated. Thereafter, the same procedure as in Experimental Example 3 was performed. As a result, as shown in FIG. 2, the permeation efficiency of interferon alfa was good under the pH condition where interferon alfa was physically relatively stable.

[0052]

EFFECTS OF THE INVENTION According to the production method of the present invention, a water-soluble polypeptide can be permeated into a biodegradable matrix without contact with an organic solvent, and a pharmaceutical preparation can be prepared without impairing the biological activity of the water-soluble polypeptide. Is possible. Further, by using the water-soluble aliphatic carboxylic acid metal salt, the water-soluble polypeptide can be efficiently permeated into the biodegradable matrix. When the sustained-release preparation of the present invention is used as an injection, for example, it exhibits excellent sustained-release properties over a period of several days to 1 month (for example, about 1 to 2 weeks).

[Brief description of drawings]

1 shows the relationship between the pH of an interferon solution and various zinc salts on the interferon content in microcapsules. In the figure, ● indicates zinc acetate (Example 7), ○ indicates zinc chloride (Comparative Example 2), and Δ indicates zinc carbonate (Comparative Example 3).

FIG. 2 shows the relationship between the pH of an interferon solution and calcium acetate (Example 8) on the interferon content in microcapsules.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location A61K 9/52 J 38/21 47/12 C 47/34 C A61K 37/66 H (72) Invention Ichirō Yamamoto 1-7-10-116 Ayame Ikenami, Nara City, Nara Prefecture

Claims (16)

[Claims] 1. A method for producing a sustained-release preparation, which comprises permeating a water-soluble polypeptide into a biodegradable matrix in water. 2. The method for producing a sustained release preparation according to claim 1, wherein the biodegradable matrix is produced by mixing a biodegradable polymer and a water-soluble metal salt of an aliphatic carboxylic acid. 3. The method for producing a sustained-release preparation according to claim 2, wherein the aliphatic carboxylic acid is an aliphatic monocarboxylic acid. 4. The method for producing a sustained release preparation according to claim 2, wherein the metal salt is a polyvalent metal salt. 5. The method for producing a sustained-release preparation according to claim 1, wherein the water-soluble polypeptide is permeated into a biodegradable matrix in water and then dried. 6. The method for producing a sustained-release preparation according to claim 5, wherein the drying is freeze-drying. 7. The method for producing a sustained-release preparation according to claim 1, wherein the water-soluble polypeptide is a cytokine. 8. The method for producing a sustained-release preparation according to claim 7, wherein the cytokine is interferon. 9. The method for producing a sustained-release preparation according to claim 1, wherein the biodegradable matrix is in the form of fine particles. 10. The method for producing a sustained release preparation according to claim 1, wherein the biodegradable matrix is produced from a biodegradable polymer. 11. The method for producing a sustained release preparation according to claim 10, wherein the biodegradable polymer is an aliphatic polyester. 12. The method for producing a sustained release preparation according to claim 11, wherein the aliphatic polyester is a copolymer derived from α-hydroxycarboxylic acid. 13. The method for producing a sustained-release preparation according to claim 12, wherein the copolymer is a lactic acid-glycolic acid copolymer. 14. A sustained-release preparation obtained by permeating a water-soluble polypeptide into a biodegradable matrix. 15. The sustained release preparation according to claim 14, wherein the biodegradable matrix is produced by mixing a biodegradable polymer and a water-soluble metal salt of an aliphatic carboxylic acid. 16. The sustained-release preparation according to claim 14, which is for injection.