JPH09315997A - Sustained release pharmaceutical preparation and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To suppress initial release of a physiologically active polypeptide without impairing physiological activity and produce a sustained release pharmaceutical preparation exhibiting a definite release rate over a long period by dispersing a rapidly dried product containing a specific compound into an oil phase and forming the dispersant. SOLUTION: This pharmaceutical preparation is produced by dispersing a rapidly dried product (A) containing a physiologically active polypeptide (A1 ) such as hormones (e.g. growth hormones, insulins), cytokines (e.g. interferons or interleukins) and a surfactant (A2 ) such as a polyoxyethylene- polyoxypropylene copolymer into (B) an oil phase of an organic solvent containing an in vivo compatible polymer [a lactic acid-glycolic acid copolymer having 3000-70000 molecular weight and (100/0) to (30/70) composition ratio of lactic acid/glycolic acid] and forming the dispersant into microcapsules, etc., having 1.0 to 200μm average particle diameter.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a sustained-release preparation suitable for sustained release of a physiologically active polypeptide.

[0002]

2. Description of the Related Art It is known that physiologically active polypeptides or their derivatives exhibit various pharmacological actions in the living body. On the other hand, recent advances in genetic engineering or cell engineering techniques have produced large amounts of highly pure polypeptides. It has become possible to do so, and the number of clinically applied drugs is increasing. However, when these physiologically active polypeptides are orally administered, their absorption rate is extremely low because they are easily decomposed by enzymes in the digestive tract. Many also have a short half-life in vivo. For this reason, a method of repeated injection intramuscularly or subcutaneously, or a method of intravenous drip injection is usually used.However, these methods are well tolerated when a limited number of administrations are required. However, in the case of frequent administration for chronic diseases, the physical burden on the patient is very large. For example, in the case of hepatitis C patients, interferon α (IFNα) is continuously administered for 4 weeks or longer, and in the case of pituitary dwarfism, it can be administered to infants or young patients for several months to 10 years or longer. Administered subcutaneously or intramuscularly every day or every other day for a long time. Moreover, in these diseases, it is said that it is necessary to sustain a clinically useful amount of the active ingredient for a long period of time in order to obtain the effects of relieving symptoms, completely curing the symptoms or increasing the height. In order to solve this problem, many attempts have been made to develop sustained-release preparations containing a physiologically active polypeptide [Clinical Reviews in Therapeutic Drug Carrier Systems (Critical R
eviews in Therapeutic Drug Carrier Systems), 12
No., pp. 1-99 (1995)].

WO94 / 19020 discloses a method for stabilizing a polypeptide by dissolving a mixture of the polypeptide and a polyol in an organic solvent. WO94
/ 12158 discloses a sustained-release preparation obtained by adding a mixture of a polymer and growth hormone to an organic solvent and rapidly freezing. Japanese Patent Laid-Open No. 63-2930 discloses a matrix in which a polypeptide is dispersed in polylactide. JP-A-4-46116 and JP-A-6-65063 disclose a method for producing a sustained release preparation by dissolving a biodegradable polymer and a fatty acid salt in an organic solvent to prepare an O / W emulsion. . Japanese Patent Publication No. 6-57658 discloses a sustained release preparation in which a physiologically active polypeptide is uniformly contained in a carrier composed of atelocollagen or a mixture of atelocollagen and gelatin. Pharmaceutical Research, Volume 9, No. 1, 37-
39 (1992), Pharmaceutical Research, Vol. 11, No. 2, 3
Pages 37-340 (1994) and Journal of Controlled Rele
ase), Vol. 33, pp. 437-445 (1995), sustained release obtained from W / O / W emulsion obtained by adding aqueous solution of polypeptide to organic solvent solution of surfactant and polymer. The formulation is described. As described above, most of the conventional techniques employ the W / O / W method in which a surfactant and a biodegradable polymer are dissolved in an organic solvent and an aqueous solution of a physiologically active polypeptide is added to the oil phase. ing. Further, an S / O / W method has also been reported in which a powder of a physiologically active polypeptide is directly added to an oil phase containing a biodegradable polymer and a surfactant. However, in any of the methods, the stability of the physiologically active polypeptide is impaired, which greatly affects the yield,
Alternatively, because the quality of the obtained sustained release preparation is inferior, LHR
Except for some peptides such as H analog, none of them has been clinically put into practical use as a drug. As described above, various attempts to produce sustained-release preparations while retaining the physiological activity of the physiologically active polypeptide have been reported. However, except for some peptides such as LHRH analogues, poly-polypeptides having a particularly high-order structure have been reported. In the case of peptides, clinically satisfactory preparations have not yet been obtained in terms of the rate of bioactive polypeptide uptake into biocompatible polymers, the suppression of initial release, or the constant sustained release over a long period of time. That is, the blood concentration at the initial stage of administration was unexpectedly high, the drug release rate was not constant during the sustained release period, or the physiologically active polypeptide was denatured during the production process, and sufficient yield and effect were obtained. Not not. Further, in the case of a sustained-release implant, it is feared that sufficient compliance cannot be obtained due to pain at the administration site and that antigenicity derived from the different animal collagen used as a base is obtained.

[0004]

It is desired to develop a clinically useful sustained-release preparation having a constant sustained-release property for a long period of time while retaining the physiological activity of a physiologically active polypeptide and a method for producing the same.

[0005]

[Means for Solving the Problems] The inventors of the present invention have made extensive studies to solve the above-mentioned problems. As a result, by mixing a physiologically active polypeptide and a surfactant in advance and rapidly drying the mixture. When the obtained fine particles are dispersed in the oil phase and then molded into a sustained-release preparation, unexpectedly the rate of uptake of the physiologically active polypeptide into the polymer is improved,
It was found that an excellent sustained-release preparation showing a rapid release for the initial release and a constant release rate over a long period of time was obtained, and the present invention was completed based on these findings. That is, the present invention relates to (1) a method for producing a sustained-release preparation, which comprises dispersing a rapidly dried product containing a physiologically active polypeptide and a surfactant in an oil phase and molding the product; The method according to (1) above, wherein the dispersed rapid-dried product has an average particle size of about 0.05 μm to about 50 μm; (3) the weight ratio of the physiologically active polypeptide to the surfactant is about 1: 0.00.
The production method according to (1) above, which is 1 to about 1: 1000;
(4) The production method according to (1) above, wherein the oil phase is an organic solvent phase containing a biocompatible polymer; (5) the concentration of the biocompatible polymer in the organic solvent is about 0.01% (W / W) to about 80% (W / W), the production method according to (4) above;
(6) The ratio of the surfactant to the total amount of the bioactive polypeptide, the surfactant and the biocompatible polymer is about 0.002% (W / W) to about 50% (W / W). 4) The manufacturing method described above;

(7) The production method described in (1) above, wherein the physiologically active polypeptide is water-soluble; (8) The production method described in (1) above, wherein the physiologically active polypeptide is a hormone; (9) Hormones Is a growth hormone, wherein the production method according to (8) above;
(10) The method according to (8) above, wherein the hormones are insulins; (11) The method according to (1) above, wherein the bioactive polypeptide is a cytokine; (12) The cytokines are interferons or interferons. The production method according to (11) above, which is a leukin; (13) The production method according to (4), wherein the biocompatible polymer is a biodegradable polymer; (14) The biodegradable polymer is a fatty acid polyester. (15) the fatty acid polyester is a lactic acid-glycolic acid copolymer; (16) the lactic acid-glycolic acid copolymer has a molecular weight of about 3, This lactic acid /
The composition ratio of glycolic acid is about 100/0 to about 30/7
The production method according to (14) above, which is 0; (17) the production method according to (14), wherein the fatty acid polyester is a hydroxybutyric acid-glycolic acid copolymer; (18) the molecular weight of the hydroxybutyric acid-glycolic acid copolymer. Is about 3,000 to about 70,
000, wherein the hydroxybutyric acid / glycolic acid composition ratio is about 100/0 to about 40/60.

(19) The method according to (1) above, wherein the surfactant is nonionic; (20) The hydrophilic-lipophilic ratio (HLB) of the nonionic surfactant is 10 or more. (21) The surfactant is a polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ether,
And one or two selected from polyvinylpyrrolidone
The method according to (1) above, which comprises at least one nonionic surfactant; (22) the nonionic surfactant is a polyoxyethylene-polyoxypropylene copolymer (19).
(23) The production method according to (1) above, wherein the sustained-release preparation is a microcapsule; (24) The above-mentioned (23) wherein the microcapsule has an average particle size of about 1.0 to about 200 μm. (25) The production method according to (1) above, wherein the sustained-release preparation is an injection; (26) A dispersion in which a rapidly dried product containing a physiologically active polypeptide and a surfactant is dispersed in an oil phase. (27) The dispersion according to the above (26), wherein the rapidly dried product dispersed in the oil phase has an average particle size of about 0.05 μm to 50 μm; (28) an aqueous solution containing a physiologically active polypeptide and a surfactant. A raw material for a sustained-release preparation in which a rapidly dried suspension is dispersed in an oil phase containing a biocompatible polymer; and (29) a sustained-release preparation for medicine manufactured by the production method according to (1) above. (30) The method according to (29) above, wherein the physiologically active polypeptide is a growth hormone. (31) Growth hormone deficiency, pituitary dwarfism, Turner syndrome, chronic kidney disease, chondrodystrophy, adult pituitary deficiency, Down syndrome, Silver syndrome, osteogenesis imperfecta, osteoporosis, or The present invention relates to the preparation of the above (30), which is used for the prevention or treatment of juvenile chronic arthropathy.

In the present specification, when an abbreviation is used for an amino acid, a peptide, etc., it is based on an abbreviation by IUPAC-IUB Commission on Biochemical Nomenclature or a conventional abbreviation in the art. To do. If the amino acid may have optical isomers,
Unless otherwise specified, the L form is shown. Examples of the physiologically active polypeptide that is a constituent of the present invention include various peptides or proteins that have useful physiological activities for mammals and can be used clinically. The "bioactive polypeptide" has a molecular weight of, for example, about 2,000 to about 200,000 as a monomer, and preferably about 5,000 to about 5 is used.
Those of 10,000, especially about 5,500 to about 30,000 are commonly used. Preferred physiologically active polypeptides include macromolecules classified into proteins having higher-order structures in the field of biochemistry. The type of the physiologically active polypeptide used in the present invention is not particularly limited as long as the object of the present invention is achieved, and typical examples thereof include growth factors, hormones, cytokines and enzymes. Can be mentioned. More specifically, for example, the following high molecular peptides and proteins can be mentioned.

(1) Examples of growth factors include nerve growth factor (NGF-1, NGF-1 etc.), neurotrophic factor (NTF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), Insulin-like growth factor (IGF
-1, IGF-2, IGF-3, etc.), fibroblast growth factor (aFGF, bFGF), osteogenic growth factor (BM)
P-1, BMP-2, BMP-3, BMP-4, etc.),
Atrial natriuretic factor (ANP), cartilage inducing factor and the like can be mentioned. (2) Examples of cytokines include interferons (IFN-α, -β, -γ, etc.), interleukins (IL-1 to IL-11, etc.), cachectin, oncostatin, colony stimulating factors (G- , M-, GM-
CSF, etc.), thrombopoietin (TPO), erythropoietin (EPO) and the like. (3) Examples of enzymes include tissue plasminogen
Activator (tPA), urokinase (UK), streptokinase, protein C, metalloproteases, superoxide dismutase (SOD), factor VIII and factor IX and the like can be mentioned. (4) Examples of hormones include growth hormone (G
H), growth hormone releasing factor (GRF), insulin,
Glucagon, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TS
H), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), calcitonin and the like.

The physiologically active polypeptide used in the present invention preferably has a certain degree of water solubility, and its solubility in water at 20 ° C. is about 1 mg / 100 ml or more, preferably about 100 mg / 100 ml or more. Some are commonly used. Preferred examples of the “bioactive polypeptide” include hormones (eg, growth hormones (human growth hormones, etc.), insulins (human insulin, etc.), cytokines (eg,
Interferons, interleukins, etc.) and the like. The physiologically active polypeptide used in the present invention may be either naturally derived or obtained by a gene recombination technique [for example, gene recombinant human growth hormone (hereinafter sometimes abbreviated as rhGH)].
These may have sugar chains and may have different sugar chain structures. In addition, these are muteins, derivatives,
Also included are analogs and active fragments. In the following, "bioactive polypeptide", "growth hormones",
References to “insulins”, “interferons”, “interleukins” and the like include muteins, derivatives, analogs and active fragments having these sugar chains, respectively.

The physiologically active polypeptide used in the present invention may be a metal salt. The metal salt is not particularly limited as long as it has no adverse effect on the living body, but a metal salt of a physiologically active polypeptide obtained by contacting a physiologically active polypeptide with a water-soluble polyvalent metal salt is preferable. In the present specification, the term "bioactive polypeptide" including those in the form of metal salts is simply referred to. Examples of the polyvalent metal in the “water-soluble polyvalent metal salt” include alkaline earth metals (for example, calcium,
Magnesium, etc.), zinc (II value), iron (II value, III)
Valence), copper (II valence), tin (II valence, IV valence), aluminum (II valence, III valence) and the like, and zinc, calcium and the like are commonly used. Examples of the "water-soluble polyvalent metal salt" include a salt of a polyvalent metal and an acid, for example, a salt of a polyvalent metal and an inorganic acid or a salt of a polyvalent metal and an organic acid. The "salt of a polyvalent metal and an acid" is preferably a salt having a solubility in water of about 20 mg / ml or more at room temperature (20 ° C.), more preferably a salt of about 100 mg / ml or more, A salt having a solubility of about 200 mg / ml or more is generally used.
Examples of the inorganic acid in the salt of “polyvalent metal and inorganic acid” include hydrochloric acid, sulfuric acid, nitric acid, thiocyanic acid and the like. Examples of the organic acid in the “salt of a polyvalent metal and an organic acid” include aliphatic carboxylic acid and aromatic acid. Examples of the “aliphatic carboxylic acid” include an aliphatic carboxylic acid having 2 to 9 carbon atoms (eg, aliphatic monocarboxylic acid, aliphatic dicarboxylic acid, aliphatic tricarboxylic acid, etc.). These aliphatic carboxylic acids may be saturated or unsaturated. Examples of the "aliphatic monocarboxylic acid" include saturated aliphatic monocarboxylic acids having 2 to 9 carbon atoms (for example, 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.) and the like. Examples of the "aliphatic dicarboxylic acid" include saturated aliphatic dicarboxylic acids having 2 to 9 carbon atoms (eg, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, etc.) and unsaturated compounds having 2 to 9 carbon atoms. Examples thereof include aliphatic dicarboxylic 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 "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,
Examples include citric acid. The "aliphatic carboxylic acid"
Is preferably an aliphatic monocarboxylic acid, more preferably an aliphatic monocarboxylic acid having 2 to 9 carbon atoms, and acetic acid is commonly used. Examples of the “aromatic acid” include benzoic acid and salicylic acid, and benzoic acid is widely used. Examples of the salt of a polyvalent metal and an inorganic acid, that is, an inorganic acid polyvalent metal salt include, for example, halogenated salts (eg, zinc chloride, calcium chloride, etc.), sulfates,
Examples thereof include nitrates and thiocyanates. As the salt of a polyvalent metal and an aliphatic carboxylic acid, that is, an aliphatic carboxylic acid polyvalent metal salt, for example, calcium acetate, zinc acetate,
Examples thereof include calcium propionate, zinc glycolate, calcium lactate, zinc lactate, and zinc tartrate. For example, calcium acetate, zinc acetate, etc. are preferable, and zinc acetate is particularly widely used. Examples of the salt of a polyvalent metal and an aromatic acid, that is, an aromatic acid polyvalent metal salt include benzoates and salicylates, and zinc benzoate is particularly widely used.

The physiologically active polypeptide used in the present invention is added to the oil phase after being contacted with a surfactant in advance. By mixing the surfactant and the physiologically active polypeptide in advance, the physiologically active polypeptide is
The physiological activity is maintained in a clinically useful range, for example, at least 50% or more, and the particles become stable and fine particles, and the dispersibility in the oil phase becomes very good. As a result, a sustained release preparation having a good drug uptake rate, suppressed initial release after administration, and a constant release rate can be obtained. As the "oil phase", for example, an organic solvent phase containing a biocompatible polymer is used.

As the "surfactant", ionic and nonionic surfactants are used, and nonionic surfactants are particularly preferable. Above all, a nonionic surfactant having a hydrophilic / lipophilic ratio (HLB) of 10 or more (preferably 12 to 30) is preferable. For example, polyoxyethylene-polyoxypropylene copolymer [poloxamers (Asahi Denka Kogyo): Pluronic F-68, F-8
7, F-108, F-127, L-44, etc.], polyoxyethylene sorbitan fatty acid ester [polysorbates (Nikko Chemicals): Tween 80, 60, 40, 2
0, etc.), polyoxyethylene hydrogenated castor oils [Nikko Chemicals: HCO-60, -50, -40, etc.], polyoxyethylene alkyl ethers [Nikko Chemicals:
Polyoxyethylene lauryl ether, etc.], sorbitan fatty acid ester [spans (Nikko Chemicals): span 80, 60, 40, 20, sorbitan sesquioleate, etc.], sucrose fatty acid ester [DK ester (Daiichi Kogyo): SS , F50, F10, etc.], polyvinyl alcohols, and polyvinylpyrrolidones. Preferable examples include polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ether, polyvinylpyrrolidone, and the like, particularly polyoxyethylene-such as poloxamers (for example, Pluronic F-68). Polyoxypropylene copolymers and the like are commonly used. In addition, these nonionic surfactants have an appropriate hydrophilic / lipophilic ratio (HLB:
In order to obtain a hydrophile-lipophile balance), two or more kinds may be appropriately mixed and used. When two or more nonionic surfactants are used, the HLB thereof is preferably 10 or more (preferably 12 to 30). The organic solvent used in the “oil phase” may be, for example, one having a boiling point of 120 ° C. or lower and having a property of being immiscible with water, and capable of dissolving a high molecular polymer. Specific examples thereof include halogenated hydrocarbons (eg, dichloromethane, chloroform, carbon tetrachloride, etc.), fatty acid esters (eg, ethyl acetate, butyl acetate, etc.), ethers (eg, ethyl ether, isopropyl ether, etc.) ), Aromatic hydrocarbons (eg, benzene, toluene, xylene, etc.) and the like. You may use these in mixture of 2 or more types in an appropriate ratio.

The mixing operation of the surfactant and the physiologically active polypeptide is usually carried out in an aqueous solution of the physiologically active polypeptide. At this time, the aqueous solution may contain a water-soluble component, for example, a pH adjuster (for example, ammonium carbonate,
Sodium hydrogen carbonate, sodium phosphate, acetic acid, hydrochloric acid,
Sodium hydroxide), stabilizers (eg serum albumin, gelatin etc.), preservatives (eg paraoxybenzoic acids etc.), salts (eg sodium chloride etc.), sugars (eg mannitol, trehalose, dextrose etc.) , Amino acids (eg, glycine, alanine, etc.) and the like. The ratio of the surfactant to the physiologically active polypeptide is not particularly limited as long as it is judged that the physiological activity of the physiologically active polypeptide is clinically useful (for example, 50% or more). For example, the weight ratio of the physiologically active polypeptide and the surfactant mixed in water is, for example, about 1: 0.001 to about 1: 100.
0, preferably about 1: 0.01 to about 1:50, more preferably about 1: 0.05 to about 1:20. In the production of the sustained-release preparation of the present invention, the ratio of the surfactant to the total amount of the biodegradable polymer, physiologically active polypeptide and surfactant is generally about 0.
002% (W / W) to about 50% (W / W), preferably about 0.05% (W / W) to about 20% (W / W)
It is. The "quick-dried product" refers to a preparation obtained by rapidly drying a mixture containing a physiologically active polypeptide and a surfactant, and examples of the rapid-drying method include freeze-drying, spray-drying, and vacuum drying. Examples of the treatment include drying or a combination thereof. Rapid drying means that the physiological activity of the physiologically active polypeptide in the mixture is maintained within a clinically useful range (for example, 50% or more), and the average particle size of the rapidly dried product dispersed in the oil phase is about 0.05 μm. To about 5
The condition is not particularly limited as long as it is 0 μm, preferably about 0.1 μm to about 30 μm. Particularly preferably about 0.1
μm to about 10 μm. In general, it is preferable to carry out the treatment in a temperature range where, for example, the physiologically active polypeptide is not inactivated by heat denaturation.

The "biocompatible polymer" is not particularly limited as long as it is compatible with living tissue and does not show a damaging reaction to the living body after administration into the living body. For example,
Those that are metabolically decomposed in the body and finally excreted out of the body are preferable. Among them, high molecular weight polymers which are hardly soluble or insoluble in water are generally used. For example, fatty acid polyesters [for example, α-hydroxycarboxylic acids (for example,
Glycolic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg, malic acid), hydroxytricarboxylic acids (eg, citric acid), and other polymers, copolymers, or mixtures thereof. ], Poly-α-cyanoacrylic acid esters, polyamino acids (for example, poly-γ-benzyl-L
-Glutamic acid, etc.) and the like. These may be mixed and used at an appropriate ratio. The type of polymerization may be random, block or graft. Biodegradable polymers include fatty acid polyesters [eg, α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid,
(Eg, hydroxybutyric acid), hydroxydicarboxylic acids (eg, malic acid), hydroxytricarboxylic acids (eg, citric acid), and the like, polymers, copolymers, or a mixture thereof are generally used. . Among the above-mentioned fatty acid polyesters, a homopolymer or a copolymer synthesized from one or more α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.) is biodegradable and biocompatible. preferable. In addition, these polymers may be appropriately mixed and used.

The biocompatible polymer used in the present invention is produced by a method known per se. The α-hydroxycarboxylic acid may be any of D-form, L-form and D, L-form, and the D-form / L-form (mol / mol%) is about 75/25 to about 25/75. Those in the range are preferred. This D-form / L-form (mol / mol%) is especially about 6
The range of 0/40 to about 30/70 is generally used. Polymers of the α-hydroxycarboxylic acids (including homopolymers and copolymers; hereinafter simply referred to as copolymers)
Examples thereof include copolymers of glycolic acid and other α-hydroxycarboxylic acids, and the “α-hydroxycarboxylic acids” are preferably lactic acid, 2-hydroxybutyric acid and the like. The copolymer of α-hydroxycarboxylic acids is, for example, lactic acid-glycolic acid copolymer,
A 2-hydroxybutyric acid-glycolic acid copolymer or the like is preferable, and a lactic acid-glycolic acid copolymer is particularly widely used.
The composition ratio (lactic acid / glycolic acid) (mol / mol%) of the "lactic acid-glycolic acid copolymer" is not particularly limited as long as the object of the present invention is achieved, but it is from about 100/0 to about 3.
The one of 0/70 is used. A preferable example of the composition ratio is about 90/10 to about 40/60, and particularly about 80/20 to about 45/55 is generally used.
The "lactic acid-glycolic acid copolymer" has a weight average molecular weight of, for example, about 3,000 to about 70,000, and preferably about 3,000 to about 20,000. Especially, about 5,000 to 15,000 are widely used. The "lactic acid-glycolic acid copolymer" preferably has a dispersity (weight average molecular weight / number average molecular weight) of about 1.2 to about 4.0, particularly about 1.5.
The ones of about 3.5 to about 3.5 are generally used. The "lactic acid-glycolic acid copolymer" can be synthesized according to a method known per se, for example, the method described in JP-A No. 61-28521. The copolymer is preferably synthesized by catalyst-free dehydration polycondensation.

The "2-hydroxybutyric acid-glycolic acid copolymer" is not particularly limited as long as the object of the present invention is achieved. For example, glycolic acid is about 10 to about 75 mol% and the rest is 2-hydroxy. Butyric acid is preferred. More preferably, the amount of glycolic acid is about 20 to about 75 mol%. In particular, those containing about 30 to about 70 mol% of glycolic acid are widely used. The “2-
The "hydroxybutyric acid-glycolic acid copolymer" preferably has a weight average molecular weight of, for example, about 2,000 to about 20,000. The “2-hydroxybutyric acid-glycolic acid copolymer” preferably has a dispersity (weight average molecular weight / number average molecular weight) of, for example, about 1.2 to 4.0, and particularly preferably about 1.5 to 3.0. Five are commonly used. The "2-hydroxybutyric acid-glycolic acid copolymer" can be synthesized according to a method known per se, for example, the method described in JP-A No. 61-28521. The copolymer is preferably synthesized by catalyst-free dehydration polycondensation. The "α-
The “hydroxycarboxylic acid” is not particularly limited as long as the object of the present invention is achieved, but a preferable example of the polymer is a lactic acid polymer. The "lactic acid polymer, that is, polylactic acid" preferably has a weight average molecular weight of, for example, about 3,000 to about 20,000, and particularly about 5,000 to about 15,000 are generally used. The "lactic acid polymer" can be synthesized according to a method known per se, for example, the method described in JP-A No. 61-28521. The polymer is preferably synthesized by catalyst-free dehydration polycondensation.

The "2-hydroxybutyric acid-glycolic acid copolymer" may be mixed with polylactic acid for use. The “polylactic acid” may be any of D-form, L-form and a mixture thereof. For example, D-form / L-form (mol / mol%) of about 75/25 to about 20/80 A range is used. The D-form / L-form (mol / mol%) is preferably about 60/40 to about 25/75, and particularly about 55/45 to about 25/75 is generally used. The "polylactic acid" has a weight average molecular weight of, for example, about 1,500 to about 20,000, preferably about 1,500 to about 10,000. The polylactic acid has a dispersity of about 1.2.
Those of about 1.5 to about 3.5 are generally used. As a method for producing the “polylactic acid”, a method of ring-opening polymerization of lactide which is a dimer of lactic acid, a method of dehydration polycondensation of lactic acid and the like are known. In order to obtain the relatively low molecular weight polylactic acid used in the present invention, for example, the method of directly dehydrating and polycondensing lactic acid as described in JP-A-61-28521 is preferable. When the 2-hydroxybutyric acid-glycolic acid copolymer and polylactic acid are mixed and used, a mixture ratio thereof is, for example, about 10/90 to about 90/10 (% by weight). The mixing ratio is about 20/80 to about 8
The case of 0/20 is preferable, and the case of about 30/70 to about 70/30 is generally used.

In the present specification, the weight average molecular weight means the weight average molecular weight of 120,000, 52,000, 22,000.
0, 9,200, 5,050, 2,950, 1,05
Gel permeation chromatography (GP) using 9 kinds of polystyrenes of 0, 580 and 162 as reference substances.
The molecular weight in terms of polystyrene measured in C). GP
The number average molecular weight is also calculated by C measurement. The dispersity is calculated from the weight average molecular weight and the number average molecular weight. GPC measurement is GPC column KF804Lx2 (Showa Denko), R
I monitor L-3300 (manufactured by Hitachi Ltd.) was used, and chloroform was used as a mobile phase. The biocompatible polymer used in the present invention may be a metal salt.
The metal salt used to convert the “biocompatible polymer” into a metal salt is not particularly limited as long as it does not adversely affect the living body. As the "metal salt", for example, a salt of a monovalent metal or a polyvalent metal with an inorganic acid or an organic acid is used. Examples of the “monovalent metal” include alkali metals (eg, sodium, potassium, etc.) and the like. Examples of the “polyvalent metal” include alkaline earth metals (eg, calcium, magnesium, etc.),
Examples thereof include zinc (II value), iron (II value, III value), copper (II value), tin (II value, IV value), aluminum (II value, III value) salts and the like. Among the "metals", polyvalent metals are preferable. More preferably alkaline earth metal, zinc,
Especially, calcium and zinc are generally used. The "inorganic acid"
Examples thereof include hydrochloric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, sulfuric acid, nitric acid, thiocyanic acid and the like. Examples of the “organic acid” include aliphatic carboxylic acid,
An aromatic acid etc. are mentioned. Examples of the "aliphatic carboxylic acid" include aliphatic carboxylic acids having 1 to 9 carbon atoms (eg, aliphatic monocarboxylic acid, aliphatic dicarboxylic acid, aliphatic tricarboxylic acid, etc.). The "aliphatic carboxylic acid" may be saturated or unsaturated. Examples of the "aliphatic monocarboxylic acid" include saturated aliphatic monocarboxylic acids having 1 to 9 carbon atoms (for example, carbonic acid, 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.) and the like. Examples of the "aliphatic dicarboxylic acid" include saturated aliphatic dicarboxylic acids having 2 to 9 carbon atoms (eg, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, etc.) and unsaturated compounds having 2 to 9 carbon atoms. Examples thereof include aliphatic dicarboxylic acids (eg, maleic acid, fumaric acid, citraconic acid, mesaconic acid, etc.). Examples of the "aliphatic tricarboxylic acid" include saturated aliphatic tricarboxylic acids having 2 to 9 carbon atoms (eg, tricarballylic acid, 1,2,3-butanetricarboxylic acid, etc.) and the like. The "aliphatic carboxylic acid" may have one or two hydroxyl groups, and examples thereof include glycolic acid, lactic acid, glyceric acid, tartronic acid, malic acid, tartaric acid, citric acid and the like. Can be mentioned. The "aliphatic carboxylic acid" is preferably an aliphatic monocarboxylic acid, more preferably an aliphatic monocarboxylic acid having 2 to 9 carbon atoms, and acetic acid is commonly used. Examples of the “aromatic acid” include benzoic acid,
Examples include salicylic acid and phenolsulfonic acid.
The metal salt used for converting the biocompatible polymer into the metal salt is preferably a salt of a polyvalent metal and an inorganic acid or an organic acid (hereinafter, abbreviated as polyvalent metal salt). Examples of the “polyvalent metal salt” include a salt of zinc and an inorganic acid [eg, zinc halide (eg, zinc chloride, zinc bromide, zinc iodide, zinc fluoride, etc.), zinc sulfate, zinc nitrate, Zinc thiocyanate, etc., a salt of zinc and an organic acid [eg, aliphatic carboxylic acid zinc salt (eg, zinc carbonate, zinc acetate, zinc glycolate, zinc lactate, zinc tartrate, etc.), aromatic acid zinc salt (eg, , Zinc benzoate, zinc salicylate, zinc phenolsulfonate, etc.), a salt of calcium and an inorganic acid [for example, calcium halide (for example, calcium chloride, calcium bromide, calcium iodide, calcium fluoride, etc.), Calcium sulfate, calcium nitrate, calcium thiocyanate, etc.], a salt of calcium and an organic acid [eg, aliphatic carboxylic acid calcium salt (eg, Calcium carbonate, calcium acetate, calcium propionate, calcium oxalate, calcium tartrate,
Calcium lactate, calcium citrate, calcium gluconate, etc.), aromatic acid calcium salts (eg, calcium benzoate, calcium salicylate, etc.) and the like are used. As the "polyvalent metal salt", zinc acetate, calcium acetate, etc. are generally used. In the present specification, the term "biocompatible polymer" may be simply referred to as a metal salt.

The sustained-release preparation of the present invention is prepared by dispersing a solid or semi-solid component obtained by mixing a physiologically active polypeptide and a surfactant in an oil phase containing a biocompatible polymer. Be converted. When the physiologically active polypeptide and the surfactant are mixed, a rapidly dried product obtained by rapidly drying a mixture containing them (for example, an aqueous solution or a suspension is preferable) is preferable. Since the rapidly dried product of the present invention is preferably dispersed in the oil phase and then uniformly dispersed in the oil phase as finer particles, at this time, for example, it is subjected to ultrasonic irradiation or atomization by a homogenizer. It is preferable. Examples of the method for producing the “sustained release preparation” include an in-water drying method, a phase separation method, a spray drying method, and the like, and the like. A method for producing, for example, microcapsules as a sustained-release preparation will be described below.

(A) In-water drying method (S / O / W method) In this method, first, a physiologically active polypeptide and a surfactant are mixed and then rapidly dried (eg, freeze-dried,
Vacuum drying, etc.) to obtain a rapidly dried product. Meanwhile, an organic solvent solution containing a biocompatible polymer is prepared. The organic solvent used for producing the sustained-release preparation of the present invention preferably has a boiling point of 120 ° C. or lower. Examples of the “organic solvent” include halogenated hydrocarbons (eg,
Dichloromethane, chloroform, carbon tetrachloride, etc.), fatty acid esters (eg, ethyl acetate, butyl acetate, etc.) and the like can be mentioned. These may be mixed and used at an appropriate ratio. The "organic solvent" is preferably dichloromethane, ethyl acetate or the like, and dichloromethane is generally used. The concentration of the biocompatible polymer in the organic solvent solution is usually about 0.01% (W / W) to about 80% (W / W) although it varies depending on the molecular weight of the biocompatible polymer and the type of the organic solvent. ) Selected from the range. Preferably about 0.1% (W / W) to about 70% (W /
W) is selected, especially about 1% (W / W) to about 6
0% (W / W) is generally used.

A rapidly dried product of the above-mentioned physiologically active polypeptide and a surfactant is added to and dispersed in the thus obtained organic solvent solution containing a biocompatible polymer. The S / O suspension can be improved in dispersibility by an emulsification operation using a homogenizer such as Polytron (Kinematica) or ultrasonic irradiation. Next, this S / O emulsion is further treated with a surfactant (eg, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose, etc.) if necessary.
Into a water phase containing the above, an S / O / W emulsion is prepared using a turbine stirrer, and then the oil phase solvent is evaporated to produce microcapsules. The volume of the aqueous phase at this time is, for example, about 1 time to about 10,000 times the volume of the oil phase.
It is selected from 0 times. Preferably, about 2 times to about 5.0
It is selected from 00 times, especially from about 5 times to about 2,000 times. Further, at this time, a pH regulator (eg acetic acid, hydrochloric acid, sodium hydroxide etc.), a stabilizer (eg serum albumin, gelatin etc.), a preservative (eg paraoxybenzoic acids etc.) or As the osmotic pressure adjusting agent, for example, salts (for example, sodium chloride),
Sugars (such as mannitol) may be added. The microcapsules thus obtained are collected by centrifugation or filtration, and then the emulsifiers and the like adhering to the surface of the microcapsules are repeatedly washed and removed with distilled water several times, and dispersed again in distilled water, etc. If necessary, additives such as mannitol are added and freeze-dried. Then, if necessary, the mixture is heated under reduced pressure to remove water and organic solvent in the microcapsules. The heating conditions include, for example, heating the microcapsules at a temperature rising rate of 10 to 20 ° C. per minute to a temperature about 5 ° C. higher than the midpoint glass transition temperature of the biocompatible polymer determined by a differential scanning calorimeter. However, after the microcapsules themselves reach a predetermined temperature, the microcapsules are kept at the same temperature for 1 week or less, preferably for 2 to 3 days, and more preferably for about 1 to 24 hours.

(B) Phase Separation Method (Coacervation Method) When microcapsules are produced by this method, S / containing the above-mentioned (a) physiologically active polypeptide, surfactant and biocompatible polymer is used. The coacervation agent is gradually added to the O suspension with stirring to precipitate and solidify the microcapsules. The “coacervation agent” is added in an amount of about 0.01 to about 2,000 times the volume of the emulsion. More preferably about 0.05 times to about 50 times.
It is preferable that the volume is 0 times, especially about 0.1 to about 200 times. Further, the "coacervation agent" is
There is no particular limitation as long as it is a polymer that is miscible with the organic solvent used as the solvent for the biocompatible polymer, a mineral oil, a vegetable oil, or the like, and that does not dissolve the “biocompatible polymer”. Specifically, for example, silicone oil, sesame oil, soybean oil, corn oil, cottonseed oil, coconut oil, linseed oil, mineral oil, n-hexane, n-heptane and the like are used. These may be used as a mixture of two or more. The microcapsules thus obtained are collected by centrifugation or filtration, and then repeatedly washed with heptane or the like,
Remove the coacervation agent. Further, washing is carried out in the same manner as in (a), followed by freeze-drying. In the production of microcapsules by the "in-water drying method" and the "phase separation method", an anti-agglomeration agent may be added to prevent the particles from aggregating. Examples of the "aggregation inhibitor" include water-soluble polysaccharides such as mannitol, lactose, glucose, starches (such as corn starch), hyaluronic acid or alkali metal salts thereof; proteins such as fibrinogen and collagen; sodium chloride and phosphorus. An appropriate amount of inorganic salts such as sodium hydrogenate is used.

(C) Spray drying method In the case of producing microcapsules by this method, an S / O suspension containing the physiologically active polypeptide of (a), a surfactant and a biocompatible polymer is The fine particles are prepared by spraying into the drying chamber of a spray dryer (spray dryer) using a nozzle and volatilizing the organic solvent in the atomized droplets in a very short time. Examples of the "nozzle" include a two-fluid nozzle type, a pressure nozzle type, and a rotating disk type. If desired, it is also possible to spray the aggregation inhibitor described in (b) above from another nozzle for the purpose of preventing aggregation. If necessary, the microcapsules thus obtained are heated under reduced pressure in the same manner as in (a) above to remove water and organic solvent from the microcapsules.

The sustained-release preparation of the present invention is, for example, the microcapsules obtained above as they are, or various dosage forms such as parenteral preparations (eg, intramuscular, subcutaneous, organ, Injectable or implantable agent for bone margins, joints, etc., transmucosal agent for nasal cavity, rectum, uterus, etc.), oral agent (eg, capsules (eg, hard capsules, soft capsules, etc.), granules Agents, solid preparations such as powders, liquid preparations such as suspensions, etc.] and the like. The sustained-release preparation of the present invention is particularly preferably an injection. In order to use the microcapsules obtained by the method as an injection, the microcapsules are dispersed (for example, Tween 80, a surfactant such as HCO-60, carboxymethyl cellulose, sodium alginate, a polysaccharide such as sodium hyaluronate, Protamine sulfate, polyethylene glycol 400 etc.), preservatives (eg methylparaben, propylparaben etc.),
An isotonic agent (eg, sodium chloride, mannitol, sorbitol, glucose, etc.), a local anesthetic (eg, xylocaine hydrochloride, chlorobutanol, etc.) in an aqueous suspension, a vegetable oil such as sesame oil, corn oil or the like Mixed with phospholipid such as lecithin,
Alternatively, a sustained-release injection is prepared by dispersing an intermediate suspension fatty acid triglyceride (for example, Miglyol 812) into an oily suspension.

When the sustained-release preparation is, for example, a microcapsule, it is particularly preferable that it is a fine particle. When used as a suspension injection, the particle size of the microcapsules may be in a range that satisfies the degree of dispersion and needle penetration, and examples thereof include an average particle size in the range of about 0.1 to 300 μm. . The “fine particles” preferably have an average particle size of about 1 to 200 μm, and more preferably about 2 to 10 μm.
Those having a particle size in the range of 0 μm are generally used. In order to make the above-mentioned microcapsules into sterile preparations, there are, but not limited to, a method of sterilizing all manufacturing steps, a method of sterilizing with gamma rays, a method of adding a preservative, and the like. INDUSTRIAL APPLICABILITY The sustained-release preparation of the present invention has low toxicity and prevents the indication of mammals (for example, humans, cows, pigs, dogs, cats, mice, rats, rabbits, etc.) according to each physiologically active polypeptide and And / or can be safely used for treatment. The indication of the sustained-release preparation depends on the physiologically active polypeptide used. Sustained-release preparations include, for example, diabetes when the "physiologically active polypeptide" is insulin, growth hormone deficiency, Turner syndrome, pituitary dwarfism, and chronic growth hormone. Renal disease,
Chondrodystrophy, adult pituitary deficiency, Down's syndrome, Silver's syndrome, osteogenesis imperfecta, osteoporosis, juvenile chronic arthrosis, etc., in the case of interferon-α, viral hepatitis (eg, hepatitis C) , HBe antigen-positive active hepatitis, etc.), cancer (eg, renal cancer, multiple myeloma, etc.), erythropoietin or thrombopoietin, anemia (eg, anemia during renal dialysis), etc.
-In the case of CSF, neutropenia (eg, during anticancer drug treatment) and infection, and in the case of IL-2, cancer (eg,
Hemangioendothelioma, kidney cancer, etc.), in the case of FGF, gastrointestinal ulcer, etc., and in the case of FGF-9, thrombocytopenia, etc.
In the case of NGF, it is effective for treating or preventing senile dementia and neurological diseases (neuropathy), in the case of tPA, thrombosis, etc., and in the case of tumor necrosis factor, cancer, etc.

The dose of the sustained-release preparation varies depending on the type and content of the physiologically active polypeptide, the duration of release, the target disease, the target animal, etc., but the effective concentration of the "physiologically active polypeptide" in the body is Any amount can be retained. The dose of the “bioactive polypeptide” can be appropriately selected, for example, from the range of about 0.0001 to 10 mg / kg body weight per adult when the sustained-release preparation is a one-week type preparation. . More preferably, it can be appropriately selected from the range of about 0.0005 to 1 mg / kg body weight. The frequency of administration may be once a week, once every two weeks, or once a month. The type and content of the "bioactive polypeptide", dosage form, duration of release, target disease, target animal, etc. Can be selected as appropriate. When the physiologically active polypeptide which is the active ingredient of the sustained-release preparation is, for example, human growth hormone, and the two-week preparation is administered to a patient with pituitary dwarfism, the active ingredient is usually about 0.01. To about 5 mg / kg body weight, preferably about 0.0
It is preferable to appropriately select from the range of 3 to about 1 mg / kg body weight and to administer once every two weeks. When the physiologically active polypeptide is insulin, the dose for diabetic patients is usually about 0.001 to about 1 mg / kg as an active ingredient.
The body weight, preferably selected from the range of about 0.01 to about 0.2 mg / kg, is preferably administered once a week. The sustained-release preparation is stored at room temperature or in a cold place, preferably in a cold place. The room temperature or the cold place mentioned here is defined in the Japanese Pharmacopoeia. That is, room temperature means 15 to 25 ° C. and cold place means 15 ° C. or lower.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to Examples and Test Examples, but these do not limit the present invention.

【Example】

Example 1 60 mg of human growth hormone (rhGH) and 20 mg of Pluronic F-68 were dissolved in 6 ml of distilled water and freeze-dried. The obtained powder was mixed with lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 75/25; polystyrene-equivalent average molecular weight 8400) 1920 mg of methylene chloride 2.
Disperse in 5 ml solution and atomize with Polytron, then
An S / O / W type emulsion was prepared by using a homogenizer in 800 ml of a 0.1% PVA aqueous solution cooled to 0 ° C. After that, the mixture was slowly stirred with an ordinary propeller stirrer for 3 hours, waited for the microcapsules to solidify along with the evaporation of methylene chloride, and then collected with a centrifuge and simultaneously washed with purified water. The collected microcapsules were obtained as a powder by freeze-drying overnight.

Comparative Example 1 60 mg of rhGH was dissolved in 6 ml of distilled water and freeze-dried. Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 75/25; polystyrene-converted average molecular weight 8400) 1940 mg of methylene chloride in 2.5 ml of the obtained powder.
After dispersing in a solution and atomizing with a polytron, an S / O / W type emulsion was prepared by using a homogenizer in 800 ml of a 0.1% PVA aqueous solution cooled to 15 ° C. Microcapsule powder was obtained in the same manner as in Example 1 below. [Table 1] shows the characteristics of the microcapsules obtained by the methods of Example 1 and Comparative Example 1.

[Table 1] Manufacturing method Surfactant rhGH uptake rate Daily release (In vitro dissolution test) Example 1 Pluronic F-68 105% 38% Comparative Example 1 Additive-free 63% 68% From the results of [Table 1], the surfactant (Pluronic F-6 was used.
The addition of 8) improves the uptake rate of rhGH into the microcapsules and improves the initial release (1) in the in vitro dissolution test.
It has become possible to control the daily release amount).

Comparative Example 2 60 mg of rhGH was dissolved in 6 ml of distilled water and freeze-dried.
The obtained powder was previously mixed with 20 mg of Pluronic F-68 and a lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 7
5/25, polystyrene equivalent average molecular weight 8400) 19
20 mg and 20 mg were dispersed in a solution dissolved in 2.5 ml of methylene chloride, and atomized with Polytron. Then, an S / O / W type emulsion was prepared by using a homogenizer in 800 ml of a 0.1% PVA aqueous solution cooled to 15 ° C. Example 1 below
Microcapsule powder was obtained by the same operation as the above method. [Table 2] shows the characteristics of the microcapsules obtained by the methods of Example 1 and Comparative Example 2.

[Table 2] Production method Surfactant rhGH uptake rate Daily release amount (In vitro dissolution test) Example 1 Pluronic F-68 freeze-drying 105% 38% Comparative example 2 Pluronic F-68 oil phase dissolution 69% 62% [Table 2] results From surfactant (Pluronic F-6
The microcapsules prepared by the method of Example 1 using the freeze-dried powder obtained by previously adding 8) to rhGH are the methods of Comparative Example 2 using the oil phase (dichloromethane) in which the same amount of the surfactant is dissolved. As compared with the microcapsules prepared by, the rate of incorporation of rhGH into the microcapsules was improved and it became possible to suppress the initial release (daily release amount) in the in vitro dissolution test.

Example 2 100 mg rhGH and Pluronic F-6810
After dissolving 0 mg in 20 ml of distilled water, it was freeze-dried. A lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50/50; polystyrene-converted average molecular weight: 10) was obtained.
100) Dispersed in 1800 mg of ethyl acetate in 2.5 ml of solution, atomized by Polytron, and then made into S / O / W type emulsion in 800 ml of 0.5% PVA aqueous solution cooled to 15 ° C. using a homogenizer. After that, the mixture was slowly stirred with an ordinary propeller stirrer for 3 hours, waited for volatilization of ethyl acetate and solidification of the microcapsules, and then collected with a centrifuge and simultaneously washed with purified water. The collected microcapsules were obtained as a powder by freeze-drying overnight.

Example 3 50 mg of rhGH, 50 mg of Pluronic F-68 and 10 mg of HCO-60 were dissolved in 10 ml of distilled water and freeze-dried. The resulting powder was hydroxybutyric acid-glycolic acid copolymer (hydroxybutyric acid / glycolic acid =
75/25; polystyrene-converted average molecular weight 12,000
0) dispersed in 1890 mg of methylene chloride in 5 ml,
1000% 0.2% PVA aqueous solution containing 5% mannitol cooled to 15 ° C. after atomization with a Polytron
An S / O / W type emulsion was prepared by using a homogenizer in ml. After that, the mixture was slowly stirred with an ordinary propeller stirrer for 3 hours, waited for the microcapsules to solidify as the methylene chloride volatilized, was collected by a centrifuge, and was simultaneously washed with purified water. The collected microcapsules were obtained as a powder by freeze-drying overnight.

Example 4 Interferon-α 10 mg and Pluronic F
30 mg of -68 was dissolved in 10 ml of distilled water and freeze-dried. The powder obtained by freeze-drying was hydroxybutyric acid-glycolic acid copolymer (hydroxybutyric acid / glycolic acid = 75
/ 25; polystyrene equivalent average molecular weight 12,000) 1
0.2% PV that was dispersed in 960 mg of methylene chloride in 5 ml, atomized with Polytron, and cooled to 15 ° C.
S using a homogenizer in 1000 ml of A solution
/ O / W type emulsion. After that, the mixture was slowly stirred with an ordinary propeller stirrer for 3 hours, waited for the microcapsules to solidify as the methylene chloride volatilized, was collected by a centrifuge, and was simultaneously washed with purified water. The collected microcapsules were obtained as a powder by freeze-drying overnight.

Example 5 To 4.7 ml of an aqueous solution containing 60 mg of rhGH and 2 mg of pluronic F-68 was added 0.5 ml of an aqueous solution containing 100 mg of salmon-derived free protamine, and the mixture was slowly stirred at 25 ° C for 20 minutes to form an insoluble complex. Then, it was freeze-dried. The obtained powder was dispersed in a solution of 1838 mg of polylactic acid polymer (100% lactic acid, polystyrene-converted average molecular weight 9000) in which the carboxyl terminal was completely ethyl esterified in 5 ml of methylene chloride, and first, using a polytron at 15,000 rpm. Atomization was carried out for 1 minute and further for 2 minutes by ultrasonic irradiation. Then 0.1% P containing 5% mannitol cooled to 15 ° C
S / using a homogenizer in 800 ml of VA aqueous solution
It was an O / W emulsion. After that, the mixture was slowly stirred with an ordinary propeller stirrer for 3 hours, waited for the microcapsules to solidify along with the evaporation of methylene chloride, and then collected with a centrifuge and simultaneously washed with purified water. The collected microcapsules were obtained as a powder by freeze-drying overnight.

Example 6 75 mg rhGH and polyoxyethylene hydrogenated castor oil HC
After dissolving 15 mg of O-60 in 15 ml of 5 mM ammonium hydrogen carbonate buffer (pH 7.8), an aqueous zinc acetate solution (5 mg
/ Ml) is gradually added (rhGH: Zn = 1: 7 molar ratio).
An insoluble complex was formed. After centrifugation, the precipitate was redispersed in a small amount of distilled water and freeze-dried. The obtained powder was lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50/5
0, polystyrene equivalent average molecular weight 15,000) 1425
Disperse in 2 ml of methylene chloride solution (2 ml), first sonicate for 5 minutes, then use polytron for 15,000 rpm.
And treated for 1 minute to atomize. Then cooled to 15 ° C 1
An S / O / W type emulsion was prepared by using a homogenizer in 800 ml of a 0.1% PVA aqueous solution containing 0% mannitol. Thereafter, microcapsules were produced by the same method as in Example 5.

Example 7 Method A: rhGH 75 mg and Pluronic F-68 15 mg
And were dissolved in 6 ml of distilled water and freeze-dried. The obtained powder was lactic acid-glycolic acid copolymer (lactic acid / glycolic acid =
50/50, polystyrene equivalent average molecular weight 15,000)
Disperse in 1410 mg of methylene chloride in 3 ml and mix with a vortex mixer for about 30 seconds. After 0.1 ml of this S / O dispersion was diluted with methylene chloride, it was subjected to a laser diffraction particle size distribution analyzer to measure the particle size of rhGH. Above S /
The O dispersion was made into an S / O / W type emulsion by using a homogenizer in 800 ml of a 0.1% PVA aqueous solution containing 10% mannitol cooled to 15 ° C. Thereafter, microcapsules were produced by the same method as in Example 5. Method B: rhGH 75 mg and Pluronic F-68 15 mg
And were dissolved in 6 ml of distilled water and freeze-dried. The obtained powder was lactic acid-glycolic acid copolymer (lactic acid / glycolic acid =
50/50, polystyrene equivalent average molecular weight 15,000)
Disperse in 1410 mg of methylene chloride in 3 ml of solution, first sonicate for 5 minutes, then use polytron for 15.0
It was treated at 00 rpm for 1 minute and sufficiently atomized. This S /
After 0.1 ml of O dispersion was diluted with methylene chloride, it was subjected to a laser diffraction type particle size distribution analyzer to measure the particle size of rhGH.
The above S / O dispersion was treated in the same manner as in Example 7-A to prepare microcapsules.

Comparative Example 3 75 mg of rhGH was dissolved in 6 ml of distilled water and freeze-dried.
The obtained powder was dispersed in a solution of 1410 mg of lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50/50, polystyrene-converted average molecular weight 15,000) in 3 ml of methylene chloride, and mixed with a vortex mixer for about 30 seconds. After 0.1 ml of this S / O dispersion was diluted with methylene chloride, it was subjected to a laser diffraction particle size distribution analyzer to measure the particle size of rhGH. The above S / O dispersion was treated in the same manner as in Example 7A to produce microcapsules. [Table 3] shows the characteristics of the microcapsules obtained by the methods A and B of Example 7 and the method of Comparative Example 3.

[Table 3] Production method rhGH average particle size in S / O dispersion rhGH uptake rate Method A of Example 7 12.1 μm 97% Method B of Example 7 6.2 μm 103% Comparative Example 3 40.3 μm 71% From the results of [Table 3], according to the present invention, the microcapsules prepared by using the S / O dispersion liquid having a rhGH average particle size in the liquid of 20 μm or less do not rapidly dry rhGH together with the surfactant, and RhGH rather than microcapsules prepared using S / O dispersion liquid with diameter of 30 μm or more
The uptake rate has improved.

Test Example 1 Two kinds of microcapsules obtained in the above-mentioned Example 1 and Comparative Example 1 were applied to SD rats (male, 6 weeks old) at 15 mg / kg.
Was subcutaneously administered, and the serum concentration was measured by a radioimmunoassay (Ab beads HGH: Eiken Kagaku). The result obtained by this is shown in FIG. The addition of Pluronic F-68 suppresses the increase in serum concentration of rhGH in the early post-dose period, which is often observed in S / O / W type microcapsules, and more stably prolongs the drug release. It has become possible. As a result, 18 days of AUC (Area Under the C
The ratio of AUC for the first day to
Compared to 94% when no surfactant is added, Pluronic F-
When 68 was added, it was suppressed to 56%.

Test Example 2 2 obtained by the method of Example 1 and the method of Comparative Example 2
The seed microcapsules were subcutaneously administered to SD rats (male, 6 weeks old) at 15 mg / kg, and the serum concentration thereof was measured by the radioimmunoassay described in Test Example 1. The results obtained by this are shown in [Fig. 2]. Pluronic F-68
The microcapsules prepared by the method of Example 1 using the freeze-dried powder obtained by previously adding
Compared with the microcapsules prepared by the method of Comparative Example 2 using the oil phase (dichloromethane) in which the same amount of the surfactant was dissolved, the increase in the serum concentration of rhGH in the initial period after administration was smaller, and more stable sustainability was exhibited. . As a result, administration 1
The serum concentration after the lapse of time was 574 ng / ml for the microcapsules of Comparative Example 2 and 188 ng / ml for the microcapsules of Example 1. The ratio of AUC for the first day to AUC for 18 days, which is an index of initial release in vivo, was 67% in the method of Comparative Example 2 in which Pluronic F-68 oil phase was added, whereas that of Pluronic F-68. /
The method of Example 1 of rhGH freeze-drying was suppressed to 56%.

Test Example 3 Three types of microcapsules obtained by the methods A and B of Example 7 and the method of Comparative Example 3 were subcutaneously administered to SD rats (male, 6 weeks old) at 15 mg / kg. Serum concentration was measured by the radioimmunoassay described in Test Example 1. The result obtained by this is shown in FIG. The addition of Pluronic F-68 suppresses the increase in serum concentration of rhGH in the early post-dose period, which is often observed in S / O / W type microcapsules, and more stably prolongs the drug release. It has become possible. As a result, the first 1 to 14 day AUC that is an indicator of in vivo initial release.
The ratio of AUC for a day was 79% when the surfactant was not added, 41% when Pluronic F-68 was added and vortex mixed, and 49 when Pluronic F-68 was added and ultrasonic-polytron treated. It was suppressed to%.

[0041]

EFFECTS OF THE INVENTION According to the present invention, a physiologically active polypeptide and a surfactant are mixed in advance, and then the rapidly dried product is uniformly dispersed in an oil phase and then molded into a sustained release preparation. It is possible to obtain an excellent sustained-release preparation in which the release is suppressed and which shows a constant release rate over a long period of time.

[Brief description of drawings]

FIG. 1 is a test example 1 in which two types of microcapsules obtained in Example 1 and Comparative Example 1 were subcutaneously administered to SD rats.
The results show that the rhGH serum concentration changes with time.

FIG. 2 shows the results of Test Example 2 and shows the time-dependent changes in rhGH serum concentration when two types of microcapsules obtained in Example 1 and Comparative Example 2 were subcutaneously administered to SD rats.

FIG. 3 shows the results of Test Example 3, Method A of Example 7,
3 shows the time course of rhGH serum concentration when three types of microcapsules obtained by Method B and Comparative Example 3 were subcutaneously administered to SD rats.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical indication location A61K 9/52 A61K 31/765 31/765 31/785 31/785 47/14 H 38/22 AEF 47/26 H 38/28 ACV 47/32 H 38/27 ABG 47/34 D 38/21 ABJ H 47/14 37/02 ADT 47/26 37/24 AEF 47/32 37/26 ACV 47/34 37/36 ABG 37/66 ABJH

Claims (31)

[Claims] 1. A method for producing a sustained-release preparation, which comprises dispersing a rapidly dried product containing a physiologically active polypeptide and a surfactant in an oil phase and molding the product. 2. The method according to claim 1, wherein the rapidly dried product dispersed in the oil phase has an average particle size of about 0.05 μm to about 50 μm. 3. The method according to claim 1, wherein the weight ratio of the physiologically active polypeptide and the surfactant is about 1: 0.001 to about 1: 1000. 4. The method according to claim 1, wherein the oil phase is an organic solvent phase containing a biocompatible polymer. 5. The method according to claim 4, wherein the concentration of the biocompatible polymer in the organic solvent is about 0.01% (W / W) to about 80% (W / W). 6. The ratio of the surfactant used to the total amount of the bioactive polypeptide, the surfactant and the biocompatible polymer is about 0.002% (W / W) to about 50% (W / W).
The method according to claim 4, which is W). 7. The method according to claim 1, wherein the physiologically active polypeptide is water-soluble. 8. The method according to claim 1, wherein the physiologically active polypeptide is a hormone. 9. The method according to claim 8, wherein the hormones are growth hormones. 10. The method according to claim 8, wherein the hormones are insulins. 11. The method according to claim 1, wherein the physiologically active polypeptide is a cytokine. 12. The method according to claim 11, wherein the cytokines are interferons or interleukins. 13. The method according to claim 4, wherein the biocompatible polymer is a biodegradable polymer. 14. The method according to claim 13, wherein the biodegradable polymer is a fatty acid polyester. 15. The method according to claim 14, wherein the fatty acid polyester is a lactic acid-glycolic acid copolymer. 16. A lactic acid-glycolic acid copolymer having a molecular weight of about 3,000 to 70,000 and a lactic acid / glycolic acid composition ratio of about 100/0 to about 30/70. Manufacturing method. 17. A fatty acid polyester is hydroxybutyric acid.
The production method according to claim 14, which is a glycolic acid copolymer. 18. A hydroxybutyric acid-glycolic acid copolymer having a molecular weight of about 3,000 to about 70,000 and a hydroxybutyric acid / glycolic acid composition ratio of about 100/0 to about 40/60. 17. The method according to item 17. 19. The surface-active agent is nonionic.
Production method as described. 20. The method according to claim 19, wherein the nonionic surfactant has a hydrophilic / lipophilicity ratio (HLB) of 10 or more. 21. One or more non-ionic surfactants selected from polyoxyethylene-polyoxypropylene copolymers, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ethers, and polyvinylpyrrolidone. The method according to claim 1, which comprises a surfactant. 22. The nonionic surfactant is a polyoxyethylene-polyoxypropylene copolymer.
Production method as described. 23. The method according to claim 1, wherein the sustained-release preparation is a microcapsule. 24. The average particle size of microcapsules is about 1.
24. The method according to claim 23, which is 0 to about 200 .mu.m. 25. The method according to claim 1, wherein the sustained-release preparation is an injection. 26. A dispersion liquid in which a rapidly dried product containing a physiologically active polypeptide and a surfactant is dispersed in an oil phase. 27. The dispersion according to claim 26, wherein the rapidly dried product dispersed in the oil phase has an average particle size of about 0.05 μm to 50 μm. 28. A sustained-release preparation raw material in which a rapidly dried product of an aqueous solution or suspension containing a physiologically active polypeptide and a surfactant is dispersed in an oil phase containing a biocompatible polymer. 29. A sustained-release pharmaceutical preparation, which is produced by the production method according to claim 1. 30. The preparation according to claim 29, wherein the physiologically active polypeptide is growth hormones. 31. Growth hormone deficiency, pituitary dwarfism, Turner syndrome, chronic kidney disease, chondrodystrophy, adult pituitary deficiency, Down syndrome, Silver syndrome, osteogenesis imperfecta, osteoporosis, Alternatively, the preparation according to claim 30, which is used for prevention or treatment of juvenile chronic arthropathy.