JPH107583A - Production of sustained-release preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a sustained-release preparation capable of suppressing the leakage of the preparation at an early stage of administration and exhibiting a fixed release rate for a long period of time, by dispersing a physiologically active polypeptide into a solution of a metal salt of a biodegradable polymer in an organic solvent and tableting the solution. SOLUTION: A physiologically active polypeptide such as insulin, growth hormone or interferon is dispersed into a solution of a biodegradable polymer previously made into a metal salt such as zinc salt or calcium salt in an organic solvent (preferably a mixed solvent of a halogenated hydrocarbon and an acetonitrile or an alcohol in the volume ratio of (40:1) to (1:1)) and molded. The content of the metal salt is preferably 0.01-10%(W/W).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a sustained-release preparation comprising a metal salt of a biodegradable polymer and a physiologically active polypeptide, and a method for producing the same.

[0002]

2. Description of the Related Art Physiologically active polypeptides or derivatives thereof are known to exhibit various pharmacological actions in living organisms, and some of them are Escherichia coli, yeast and animal It is produced in large quantities using living cells such as cells or hamsters, and is being applied as a pharmaceutical. However, since these bioactive polypeptides generally have a short half-life in vivo, frequent administration is required, and the physical burden on the patient involved in the injection cannot be ignored. To solve this problem, various attempts have been made to develop sustained-release preparations containing a bioactive polypeptide. JP-A-4-4611
JP-A-6-65063 and JP-A-6-65063 disclose a water-soluble physiologically active polypeptide, a biodegradable polymer and a fatty acid salt in an organic solvent to form an o / w emulsion. A method for producing a sustained-release preparation with an increased incorporation rate of a physiologically active peptide is disclosed.

[0003]

As described above, various attempts have been made to produce a sustained-release preparation while maintaining the biological activity of the bioactive polypeptide. Bioactive polypeptide uptake rate,
A clinically satisfactory sustained-release preparation has not yet been obtained in terms of the suppression of leakage at the early stage after administration and the constant release rate of the bioactive polypeptide over a long period of time.

[0004]

Means for Solving the Problems The inventors of the present invention have made intensive studies to solve the above-mentioned problems, and found that a bioactive polydegradable polymer in the form of a metal salt was added to an organic solvent solution of a bioactive polymer. When the peptide is dispersed and molded, a sustained-release preparation that unexpectedly improves the uptake rate of the bioactive polypeptide dramatically, suppresses the initial leakage after administration, and shows a constant release rate over a long period of time It was found that a product having excellent properties was obtained. The present invention has been completed based on these findings. That is, the present invention provides (1) a method for producing a sustained-release preparation, which comprises dispersing a physiologically active polypeptide in an organic solvent solution of a metal salt of a biodegradable polymer and shaping it; The method according to (1), wherein the metal salt is a polyvalent metal salt, (3) the method according to (1), wherein the metal salt is a zinc salt or a calcium salt,
(4) The production method according to the above (1), wherein the organic solvent in the organic solvent liquid is a mixed solution of a halogenated hydrocarbon and acetonitrile or alcohol, and (5) a volume of the halogenated hydrocarbon and acetonitrile or alcohol. The method according to (4), wherein the ratio is about 40: 1 to about 1: 1; (6) the method according to (1), wherein the bioactive polypeptide is a hormone; and (7) the hormone is insulin. (8) the production method according to (6), wherein the hormone is a growth hormone; (9) the production method according to (1), wherein the bioactive polypeptide is a cytokine; The production method according to (9), wherein the cytokine is interferon, (11) the production method according to (1), wherein the biodegradable polymer is an aliphatic polyester, (12) aliphatic Wherein Riesuteru is α- hydroxy carboxylic acid polymer (11) The process according (1
3) The method according to (11), wherein the aliphatic polyester is a lactic acid-glycolic acid copolymer, and (14) the lactic acid / glycolic acid copolymer has a lactic acid / glycolic acid composition ratio of about 100/0. (13) The method according to (13), wherein the weight-average molecular weight is from about 3,000 to about 20,000, and (15) the sustained-release preparation is fine particles.
(16) The fine particles have an average particle size of about 0.1
The production method according to the above (15), wherein
7) the production method according to the above (1), wherein the sustained-release preparation is an injection, (18) a dispersion obtained by dispersing a bioactive polypeptide in an organic solvent liquid of a metal salt of a biodegradable polymer,
(19) The above (18) substantially free of metal salts of fatty acids.
(20) a sustained-release preparation produced by the production method described in (1), (21) a metal salt of a biodegradable polymer having a metal content of about 0.01 to about 10; % (W /
(20) the sustained release preparation according to the above (20), wherein the content of the physiologically active polypeptide is about 0.001 to about 30%;
(W / w) the sustained-release preparation according to the above (20), and (23) a growth hormone dispersed in an organic solvent solution containing a zinc salt of a biodegradable polymer, and molded by dispersing the growth hormone. And the sustained release preparation according to the above (20).

[0005] In the present invention, the biodegradable polymer is a polymer which is hardly soluble or insoluble in water, for example, an aliphatic polyester [eg, α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, 2- Hydroxybutyric acid, valic acid, leucic acid, etc.), hydroxydicarboxylic acids (eg, malic acid etc.), hydroxytricarboxylic acids (eg,
Polymers, copolymers or mixtures thereof synthesized from one or more of such as citric acid, etc.), poly-α-cyanoacrylate (eg, poly-α-cyanoacrylate methyl ester, poly-α- Ethyl cyanoacrylate, butyl poly-α-cyanoacrylate, etc.),
A polyamino acid (eg, poly-γ-benzyl-L-glutamic acid or the like) or a mixture thereof is used. The mode of polymerization of these biodegradable polymer may be any of random, block and graft. The biodegradable polymer is preferably an aliphatic polyester [eg, α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, 2-
Hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg,
A polymer or copolymer synthesized from at least one kind of malic acid, etc., hydroxytricarboxylic acid (eg, citric acid, etc.),
Or a mixture thereof].

A polymer synthesized from one or more α-hydroxycarboxylic acids in the aliphatic polyester described above;
Copolymers are preferred from the viewpoint of reliable biodegradability and biocompatibility. The aliphatic polyester is particularly preferably a copolymer produced from two or more α-hydroxycarboxylic acids. These copolymers may be used as a mixture. The α-hydroxycarboxylic acids,
When the α-hydroxycarboxylic acid is a chiral compound, any of D-form, L-form and D, L-form may be used, but D-form / L-form (mol%) is about 75/25 to about 25
/ 75 is preferred. More preferably, D
-Body / L-body (mol%) is about 60/40 to about 30/70
Α-hydroxycarboxylic acid in the range of As an example of the polymer of the α-hydroxycarboxylic acid, for example, a polymer of lactic acid (hereinafter sometimes referred to as polylactic acid) is used. Examples of the copolymer of the α-hydroxycarboxylic acids include, for example, a copolymer of glycolic acid and another α-hydroxycarboxylic acid, and the α-hydroxycarboxylic acid includes lactic acid and 2-hydroxybutyric acid. preferable. The copolymer of α-hydroxycarboxylic acids is
Preferably, it is a lactic acid-glycolic acid copolymer or a 2-hydroxybutyric acid-glycolic acid copolymer. The copolymer of α-hydroxycarboxylic acids is particularly preferably lactic acid-
It is a glycolic acid copolymer.

The polylactic acid may be any of D-form, L-form and a mixture thereof, but D-form / L-form (mol%) is in the range of about 75/25 to about 20/80. Are preferred. More preferably, the polylactic acid has a D-form / L-form (mol%) in the range of about 60/40 to about 25/75. Particularly preferred is polylactic acid having a D-form / L-form (mol%) in the range of about 55/45 to about 25/75. The polylactic acid has a weight average molecular weight of about 1,500 to about 10,000.
00 is preferred, and more preferably the weight average molecular weight is from about 2,000 to about 8,000. Particularly preferred is a polylactic acid having a weight average molecular weight in the range of about 3,000 to about 6,000. The polylactic acid preferably has a degree of dispersion (weight average molecular weight / number average molecular weight) of about 1.2 to about 4.2.
0, particularly preferably about 1.5 to about 3.5.
Polylactic acid can be produced by a method known per se, for example, JP-A-61-2
No. 8521 (for example, a production method by a dehydration polycondensation reaction in the absence of a catalyst or a dehydration polycondensation reaction in the presence of an inorganic solid acid catalyst). The polylactic acid is preferably produced by non-catalytic dehydration polycondensation.

In the lactic acid-glycolic acid copolymer,
Its composition ratio (lactic acid / glycolic acid, mol%) is about 10
0/0 to about 40/60 are preferred, and more preferably about 90/10 to about 45/55. The composition ratio is particularly preferably from about 60/40 to about 40/60. The weight average molecular weight of the copolymer of glycolic acid and lactic acid is about 3,
It is preferably from 000 to about 20,000, more preferably from about 4,000 to about 15,000. Further, the degree of dispersion (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 from about 1.5 to about 3.5. The lactic acid-glycolic acid copolymer can be produced by a method known per se, for example,
No. 521 (for example, a production method by a dehydration polycondensation reaction without a catalyst or a dehydration polycondensation reaction in the presence of an inorganic solid acid catalyst). The copolymer is preferably produced by non-catalytic dehydration polycondensation.

In the present invention, two kinds of lactic acid-glycolic acid copolymers having different composition ratios and different weight average molecular weights may be used by mixing at an arbitrary ratio. As such an example, for example, lactic acid having a composition ratio (lactic acid / glycolic acid) (mol%) of about 75/25 and a weight average molecular weight of about 6,000.
A mixture of a glycolic acid copolymer and a lactic acid-glycolic acid copolymer having a composition ratio (lactic acid / glycolic acid) (mol%) of about 50/50 and a weight average molecular weight of about 4,000 is used. The weight ratio upon mixing is preferably from about 25/75 to about 75/25.

In the 2-hydroxybutyric acid-glycolic acid copolymer, the composition ratio of glycolic acid is from about 10 to about 7
Preferably, 5 mol%, the balance being 2-hydroxybutyric acid, more preferably from about 20 to about 75 glycolic acid.
Particularly preferably, the amount of glycolic acid is about 3 mol%.
0 to about 70 mol%. The weight average molecular weight of the 2-hydroxybutyric acid-glycolic acid copolymer is preferably from about 2,000 to about 30,000, more preferably from about 3,000 to about 20,000. The weight average molecular weight is particularly preferably from about 4,000 to about 15,000. The dispersity (weight average molecular weight / number average molecular weight) of the 2-hydroxybutyric acid-glycolic acid copolymer is preferably about 1.2 to about 4.0, and particularly preferably about 1.5 to about 3.0.
5 The 2-hydroxybutyric acid-glycolic acid copolymer can be produced by a known production method, for example, a method described in JP-A-61-28521 (for example, a dehydration polycondensation reaction without a catalyst or a dehydration polymerization under an inorganic solid acid catalyst). Production method by condensation reaction). The copolymer is preferably produced by non-catalytic dehydration polycondensation.

The above-mentioned glycolic acid copolymer (eg, lactic acid-glycolic acid copolymer, 2-hydroxybutyric acid-glycolic acid copolymer, etc.) may be used as a mixture with polylactic acid. When the glycolic acid copolymer and polylactic acid are used as a mixture, the mixing ratio (glycolic acid copolymer / polylactic acid, weight%) is, for example, about 10/90 to about 90/10.
It is. The mixing ratio is preferably from about 20/80 to about 80
/ 20, more preferably about 30/70 to about 70/30.

In the present specification, the weight average molecular weight and the number average molecular weight refer to weight average molecular weights of 120,000 and 52,000, respectively.
0, 22,000, 9,200, 5,050, 2,950,
Gel permeation chromatography (GPC) using nine kinds of polystyrenes of 1,050, 580 and 162 as reference materials
Means the weight average molecular weight and number average molecular weight in terms of polystyrene measured in the above. The measurement was performed using a GPC column KF80.
4L x 2 (manufactured by Showa Denko), RI monitor L-3300
(Manufactured by Hitachi, Ltd.) using chloroform as a mobile phase.

In the present invention, the biodegradable polymer produced by non-catalytic dehydration polycondensation has a free carboxyl group at a terminal. A biodegradable polymer having a free carboxyl group at the end is a polymer in which the number average molecular weight by the end group quantification method and the number average molecular weight by the GPC measurement method using standard polystyrene having a known molecular weight are almost the same. Things.

The number average molecular weight by the terminal group quantification method is calculated as follows. About 1 g to 3 g of the biodegradable polymer is dissolved in acetone (25 ml) and methanol (5 ml).
ml) and dissolved in a mixed solvent with room temperature (about 0 to about 30 ° C.)
Under stirring with phenolphthalein as an indicator, the carboxyl group in this solution was quickly titrated with a 0.05N alcoholic potassium hydroxide solution, and the number average molecular weight was calculated by the following formula using the terminal group determination. Number average molecular weight determined by terminal group determination = 20,000 A / B A: Mass (g) of biodegradable polymer B: 0.05 N alcoholic potassium hydroxide solution (ml) added by the end of titration, for example, 1 For biodegradable polymers produced from non-catalytic dehydration polycondensation method from at least one kind of α-hydroxy acids and having a free carboxyl group at the terminal, the number average molecular weight by GPC measurement method and the number average molecular weight by terminal group determination method And almost match. On the other hand, in the case of a biodegradable polymer produced by a ring-opening polymerization method using a catalyst from a cyclic dimer of an α-hydroxy acid and having essentially no free carboxyl group at the terminal, a terminal group determination method is used. Is significantly higher than the number average molecular weight determined by GPC. Due to this difference,
A biodegradable polymer having a free carboxyl group at a terminal can be clearly distinguished from a biodegradable polymer having no free carboxyl group at a terminal.

While the number average molecular weight determined by the terminal group quantification method is an absolute value, the number average molecular weight determined by the GPC measurement method is determined by various analysis and analysis conditions (for example, the type of mobile phase, the type of column, the reference material, and the slice width). Selection, baseline selection, etc.), it is difficult to make a clear numerical value. However, for example, when the number average molecular weight by GPC measurement method and the number average molecular weight by end group quantification method are almost the same, Means that the number average molecular weight by the terminal group quantification method is in the range of about 0.5 times to about 2 times the number average molecular weight by the GPC measurement method. Preferably, it is within the range of about 0.8 times to about 1.5 times. The term "number-average molecular weight determined by the terminal group quantification method greatly exceeds the number-average molecular weight determined by the GPC measurement method" means that the number-average molecular weight determined by the terminal group quantification method is determined by GPC.
It refers to a case where the number average molecular weight exceeds about twice as measured by the measuring method. In the present invention, a polymer in which the number average molecular weight according to the GPC measurement method and the number average molecular weight according to the terminal group quantification method are almost the same is preferable.

The metal salt used to convert the biodegradable polymer into a metal salt is not particularly limited as long as it does not adversely affect the living body. As metal salts,
For example, unit metals such as alkali metals (eg, sodium, potassium, etc.), or alkaline earth metals (eg, calcium, magnesium, etc.), zinc (II), iron (II, II)
Salts of polyvalent metals such as I (valent), copper (II), tin (II, IV), and aluminum (II, III) and inorganic acids or organic acids are used. The metal is preferably a polyvalent metal, more preferably an alkaline earth metal, zinc. Particularly preferred specific examples of the metal include, for example, calcium, zinc and the like.

As the inorganic acid, for example, hydrogen halide (eg, hydrochloric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, etc.), sulfuric acid, nitric acid, thiocyanic acid and the like are used. As the organic acid, for example, an aliphatic carboxylic acid, an aromatic acid or the like is used. As the aliphatic carboxylic acid, an aliphatic carboxylic acid having 1 to 9 carbon atoms, for example, an aliphatic monocarboxylic acid, an aliphatic dicarboxylic acid, an aliphatic tricarboxylic acid and the like are preferably used. The aliphatic carboxylic acid may be either saturated or unsaturated. Examples of the aliphatic monocarboxylic acid include saturated aliphatic monocarboxylic acids having 1 to 9 carbon atoms (eg, carbonic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid) And C2 to C9 unsaturated aliphatic monocarboxylic acids (eg, acrylic acid, propiolic acid, methacrylic acid, crotonic acid, isocrotonic acid, etc.). Aliphatic dicarboxylic acids include, for example, those having 2 carbon atoms.
To 9 saturated aliphatic dicarboxylic acids (eg, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, etc.) and C2 to C9 unsaturated aliphatic dicarboxylic acids (eg,
Maleic acid, fumaric acid, citraconic acid, mesaconic acid, etc.)
Are used. As the aliphatic tricarboxylic acid, for example, a saturated aliphatic tricarboxylic acid having 2 to 9 carbon atoms (eg, tricarballylic acid, 1,2,3-butanetricarboxylic acid and the like) is used.

The above-mentioned aliphatic carboxylic acid has one hydroxyl group.
Or may have two, such as:
Examples include glycolic acid, lactic acid, glyceric acid, tartronic acid, malic acid, tartaric acid, citric acid and the like. The aliphatic carboxylic acid is preferably an aliphatic monocarboxylic acid. The aliphatic carboxylic acid is more preferably an aliphatic monocarboxylic acid having 2 to 9 carbon atoms, 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. As the aromatic acid, for example, benzoic acid,
Salicylic acid, phenolsulfonic acid and the like are used.
Further, in order to convert the biodegradable polymer into a metal salt, acetylacetonate or metal oxide of a polyvalent metal (as described above) may be used as the metal compound. Preferred are zinc acetylacetonate and zinc oxide.

The metal salt used to convert the biodegradable polymer into a metal salt is preferably a salt of a polyvalent metal and an inorganic acid or an organic acid (hereinafter abbreviated as a polyvalent metal salt). Specific examples of polyvalent metal salts include, for example, salts of zinc and inorganic acids [eg, zinc halides (zinc chloride, zinc bromide,
Zinc iodide, zinc fluoride, etc.), zinc sulfate, zinc nitrate, zinc thiocyanate, etc.), salts of zinc with organic acids [eg, zinc salts of aliphatic carboxylic acids (eg, zinc carbonate, zinc acetate, zinc glycolate) , Zinc lactate, zinc tartrate, etc.), aromatic zinc salts (eg, zinc benzoate, zinc salicylate, zinc phenolsulfonate, etc.) and the like. Salts of calcium and inorganic acids [eg, calcium halides (calcium chloride, calcium bromide, calcium iodide, calcium fluoride, etc.), calcium sulfate, calcium nitrate, calcium thiocyanate, etc.], salts of calcium with organic acids [Example,
Calcium aliphatic carboxylate (eg, calcium carbonate,
Calcium acetate, calcium propionate, calcium oxalate, calcium tartrate, calcium lactate, calcium citrate, calcium gluconate, etc., aromatic calcium salts (eg, calcium benzoate, calcium salicylate, etc.) and the like are used. As the polyvalent metal salt, zinc acetate, calcium acetate and the like are preferably used.

In the present invention, the physiologically active polypeptide preferably has a molecular weight of about 1,000 to about 50,000.
0, more preferably from about 5,000 to about 40,000 molecular weight.
0 bioactive polypeptides are used. A typical example of the activity of a physiologically active polypeptide is a hormonal action. The physiologically active polypeptide may be a natural product, a synthetic product, a semi-synthetic product, or a derivative thereof. The mode of action of the bioactive polypeptide may be either agonistic or antagonistic. As the physiologically active polypeptide of the present invention, for example, peptide hormones, cytokines, haemostatic factors, various growth factors, enzymes and the like are used.

Examples of hormones include insulin, growth hormone, natriuretic peptide, gastrin, prolactin, corticotropin (ACTH), thyroid stimulating hormone (TSH), and luteinizing hormone (L).
H), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), motilin and the like. The hormone is preferably insulin and growth hormone. As the cytokine, for example, lymphokine, monokine and the like are used. As the lymphokine, for example, interferon (alpha, beta, gamma), interleukin (IL-2 to IL-12) and the like are used. Monokines include, for example, interleukin-
1 (IL-1), tumor necrosis factor and the like are used. The cytokine is preferably lymphokine or the like, and more preferably interferon or the like. The cytokine is particularly preferably interferon alpha or the like.

Examples of the hematopoietic factor include erythropoietin, granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), thrombopoietin, platelet growth stimulating factor, megakaryocyte potentiator and the like. Examples of various growth factors include basic or acidic fibroblast growth factor (FG).
F) or their families (eg, FGF-9), nerve cell growth factor (NGF) or their families, insulin-like growth factors (eg, IGF-1, IGF)
GF-2), factors involved in bone growth (BMP), or a family of these factors. As the enzyme, for example, superoxide dismutase (SO
D), tissue plasminogen activator (TPA), kallikrein and the like are used.

In the present invention, when the physiologically active polypeptide contains a metal, the metal content is preferably 0.1% or less, more preferably 0.01% or less, particularly preferably 0.001% or less. Thus, a bioactive polypeptide substantially free of metals is optimal. For example, crystalline insulin usually contains small amounts of heavy metals such as zinc, nickel, cobalt, cadmium and the like. 0.4% (w /
w) It is thought that insulin containing zinc exists as a hexamer, exists stably on its own, and weakens the interaction with the metal salt of the biodegradable polymer. If necessary, the metal contained in the physiologically active polypeptide may be removed in advance, and a known method is used for removing the metal. For example, an insulin-hydrochloric acid aqueous solution is dialyzed against water or an ammonium acetate solution and then freeze-dried to obtain an amorphous state of insulin with a minimum amount of metal. In the present invention, it is desirable that additives other than the metal salt of the biodegradable polymer do not form a metal salt, and it is particularly preferable that the additive does not substantially contain a metal salt of a fatty acid.

In the present invention, the metal salt of the biodegradable polymer is, for example, emulsified and dispersed by dissolving an aqueous solution of the metal salt or a solid metal salt in an organic solvent solution of the biodegradable polymer. To prepare a w / o or o / w type emulsion (emulsion) or an organic solvent solution or suspension of a biodegradable polymer containing a metal salt, and then wash and dry them. Alternatively, they are produced by subjecting them to an underwater drying method, a phase separation method, a spray drying method or a method analogous thereto, followed by washing and drying. In this manufacturing process, it is preferable to remove metal salts that do not form a salt with the biodegradable polymer. The organic solvent in the organic solvent liquid 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 (eg, ethanol,
Methanol, 1,4-butanediol, 1,5-pentanediol, etc.), acetonitrile and the like. These may be mixed and used at an appropriate ratio. When the organic solvent is used alone, for example, dichloromethane, acetonitrile and the like are preferable. When an organic solvent is used as a mixed solvent, for example, a halogenated hydrocarbon (eg, dichloromethane or the like) and acetonitrile or an alcohol (eg,
Methanol, ethanol, etc.).
In particular, a combination of dichloromethane and acetonitrile is widely used. The mixing ratio (volume ratio) between the halogenated hydrocarbon and acetonitrile or alcohol is about 40: 1.
To about 1: 1 and preferably from about 20: 1 to about 1: 1. The metal content in the metal salt of the biodegradable polymer is preferably about 0.01 to about 10% (w / w), more preferably about 0.05 to about 7% (w / w), Particularly preferred is about 0.1 to about 5% (w / w). The metal content in the metal salt of the biodegradable polymer is determined by, for example, an atomic absorption method. Hereinafter, a method for producing a metal salt of a biodegradable polymer, for example, an in-water drying method, a phase separation method, and a spray drying method will be described.

(A) In-water drying method (w / o / w method) In this method, first, a biodegradable polymer is dissolved in an organic solvent, and an organic solvent liquid (hereinafter sometimes referred to as an oil phase). ) To manufacture. At this time, the concentration of the biodegradable polymer in the organic solvent liquid varies depending on the molecular weight of the biodegradable polymer and the type of the organic solvent, but is, for example, about 0.01 to about 90% (w / W), more preferably about 0.
1 to about 80% (w / w), particularly preferably about 1 to about 70
% (W / w). As the inner aqueous phase, an aqueous solution in which a metal salt is dissolved in water is used. The concentration of the metal salt varies depending on the solubility of each metal salt in water, for example, about 10 to about 9
0% (w / v), preferably about 20 to about 80% (w / v)
v). The aqueous solution of the metal salt is emulsified and dispersed in an organic solvent solution of a biodegradable polymer to produce a w / o emulsion. The ratio (volume ratio) between the aqueous solution of the metal salt and the organic solvent solution of the biodegradable polymer is about 1:
It is from 1,000 to about 1: 1, preferably from about 1: 100 to about 1: 2, particularly preferably from about 1:50 to about 1: 3.
The emulsification method is performed by a known emulsification operation. The emulsification operation is performed using, for example, a turbine type stirrer, a homogenizer, or the like. Then, the w / o thus prepared is
Type emulsion is further added to the aqueous phase (outer aqueous phase), and w
After forming the / o / w emulsion, the oil phase solvent is evaporated to produce a metal salt of a biodegradable polymer.
The external water phase volume at this time is, for example, about 1 to about 1 of the oil phase volume.
It is selected from 0000 times. More preferably, about 2 to
It is selected from about 5,000 times, particularly preferably about 5 to about 2,000 times. The method of removing the solvent can be performed according to a known method. As such a method, for example, a method of evaporating the solvent at normal pressure or gradually reducing the pressure while stirring with a propeller type stirrer or a magnetic stirrer, or evaporating the solvent while adjusting the degree of vacuum using a rotary evaporator or the like And the like.

An emulsifier may be added to the outer aqueous phase. The emulsifier may be any as long as it can form a stable w / o / w emulsion. Specifically, for example, anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid and the like are used. As the emulsifier, polyvinyl alcohol is preferably used. The emulsifier may be used alone or in combination of two or more. The concentration at the time of use can be appropriately selected from the range of about 0.001 to about 20% (w / w) with respect to the external aqueous phase. More preferably, about 0.01 to about 10% (w
/ W), particularly preferably from about 0.05% to about 5% (w /
w) is used.

A metal salt which is the same as or different from the metal salt used in the internal aqueous phase may be added to the external aqueous phase. At this time, the concentration of the metal salt in the external water phase is about 0.01 to about 20% (w /
w), particularly preferably about 0.1 to about 10% (w / w) of the fatty acid metal salt. By changing the concentration of the metal salt in the external aqueous phase, the metal salt used in the internal aqueous phase can be prevented from being eluted from the biodegradable polymer into the external aqueous phase. The metal salt of the biodegradable polymer obtained in this way is separated by centrifugation or filtration, and then an emulsifier or the like is attached to the surface of the metal salt of the biodegradable polymer. Are repeatedly washed several times with distilled water, dispersed again in distilled water or the like, and freeze-dried.

(B) Underwater drying method (o / w method) In this method, first, an organic solvent solution of a biodegradable polymer is produced in the same manner as in the above (A). Next, a metal salt is added, dispersed or dissolved in an organic solvent liquid of the biodegradable polymer. At this time, the amount of the metal salt added is such that the weight ratio of the metal salt to the biodegradable polymer is about 5: 1 to about 1: 100, preferably about 2: 1 to about 1: 5.
0, more preferably about 1: 1 to about 1:10. The organic solvent liquid thus prepared is further added to the aqueous phase, and o / w is added using a turbine-type stirrer or the like.
After forming the emulsion, the oil phase solvent is evaporated in the same manner as in (A) to produce a metal salt of a biodegradable polymer. The volume of the aqueous phase at this time is, for example, about 1 to about 10,000 times the volume of the oil phase, and more preferably about 2 to about 10,000 times.
Double to about 5,000 times. Particularly preferably, it is selected from about 5 times to about 2,000 times. An emulsifier may be added to the aqueous phase as in (A). The same or different metal salt as the metal salt added, dispersed or dissolved in the oil phase may be added to the aqueous phase in the same manner as in the above (A). The metal salt of the biodegradable polymer obtained in this manner is separated, washed and freeze-dried in the same manner as in the above (A).

(C) Phase Separation Method (Coacervation Method) When a metal salt of a biodegradable polymer is produced by this method, the w / o emulsion described in the above (A) or ( A coacervation agent is gradually added to the organic solvent liquid of the biodegradable polymer containing the metal salt described in B) while stirring, to precipitate and solidify the metal salt of the biodegradable polymer. The coacervation agent comprises w / o
A volume of about 0.01 times to about 1,000 times the volume of the organic solvent solution of the emulsion or the biodegradable polymer is added. More preferably, the volume is about 0.05 to about 500 times. Particularly preferably, the volume is about 0.1 times to about 200 times. The coacervation agent may be a polymer-based, mineral oil-based or vegetable oil-based compound that is miscible with an organic solvent that dissolves the biodegradable polymer, and does not dissolve the 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 metal salt of the biodegradable polymer obtained in this manner is separated by filtration and then repeatedly washed with heptane or the like to remove the coacervation agent. Further, washing is carried out in the same manner as in the above (A), followed by freeze-drying.

In the production of a metal salt of a biodegradable polymer by the underwater drying method and the coacervation method, an anti-aggregation agent may be added to prevent aggregation of particles. Examples of the agglomeration inhibitor include mannitol, lactose, glucose, starches (eg, corn starch and the like),
Water-soluble polysaccharides such as hyaluronic acid or alkali metal salts thereof, proteins such as glycine, fibrin and collagen, and inorganic salts such as sodium chloride and sodium hydrogen phosphate are used in appropriate amounts.

(D) Spray drying method In the case where a metal salt of a biodegradable polymer is produced by the present method, the aqueous solution of the metal salt and the organic solvent liquid of the biodegradable polymer are mixed with each other. An organic solvent solution or suspension of a biodegradable polymer containing a / o emulsion or a metal salt is sprayed into a drying chamber of a spray dryer (spray dryer) using a nozzle, and the spray is performed in an extremely short time. The organic solvent in the atomized droplets is volatilized to prepare a finely divided metal salt of a biodegradable polymer. 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 the organic solvent solution or suspension of the biodegradable polymer containing the w / o emulsion or the metal salt, the aggregation of the metal salt of the biodegradable polymer is prevented. For the purpose, it is also effective to spray the aqueous solution of the anti-agglomeration agent from another nozzle. The metal salt of the biodegradable polymer obtained in this manner is washed in the same manner as in the above (A), and if necessary, the water and the organic solvent are further removed under heating and reduced pressure. .

The sustained-release preparation of the present invention is produced by dispersing a physiologically active polypeptide in an organic solvent solution of a metal salt of a biodegradable polymer and shaping it. Examples of the production method of the present invention include the above-mentioned (A) underwater drying method (w /
o / w method), (B) an underwater drying method (o / w method), (C) a phase separation method (coacervation method), and (D) a method according to a spray drying method. The organic solvent in the organic solvent liquid 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 (eg, ethanol, methanol,
1,4-butanediol, 1,5-pentanediol, etc.), acetonitrile and the like. These may be mixed and used at an appropriate ratio. When the organic solvent is used alone, for example, dichloromethane, acetonitrile and the like are preferable. When an organic solvent is used as the mixed solvent, for example, a combination of a halogenated hydrocarbon (eg, dichloromethane or the like) and acetonitrile or an alcohol (eg, methanol, ethanol, or the like) is preferable. In particular, a combination of dichloromethane and acetonitrile is widely used.
The mixing ratio (volume ratio) of the halogenated hydrocarbon to acetonitrile or alcohol is from about 40: 1 to about 1: 1 and preferably from about 20: 1 to about 1: 1. less than,
As a sustained release preparation, for example, a production method for producing microcapsules will be described. (A) Underwater drying method (w / o / w method) According to this method, first, an organic solvent solution of a metal salt of a biodegradable polymer is produced by a method according to the above (A). At this time, the concentration of the metal salt of the biodegradable polymer in the organic solvent liquid varies depending on the type, molecular weight, and type of the organic solvent of the metal salt of the biodegradable polymer. 0.01 to about 80% (w / w), more preferably about 0.1 to about 70% (w / w), and particularly preferably about 1 to about 60% (w / w). An aqueous solution of a physiologically active polypeptide is used as the internal aqueous phase. The concentration of the physiologically active polypeptide in the aqueous solution is, for example, about 0.1% (w / v) to about 5%.
00% (w / v). Preferably about 1% (w / v)
To about 400% (w / v), particularly preferably about 10% (w / v).
/ V) to about 300% (w / v). At this time, a pH adjuster (eg, acetic acid, hydrochloric acid, sodium hydroxide, etc.), a stabilizer (eg, serum albumin, gelatin, protamine sulfate, etc.), a preservative (eg, paraoxybenzoic acid, etc.) in the aqueous solution, etc. May be added. The aqueous solution thus obtained is emulsified and dispersed in an organic solvent liquid of a metal salt of a biodegradable polymer to produce a w / o emulsion.

The ratio (volume ratio) of the aqueous solution of the physiologically active polypeptide to the organic solvent solution of the metal salt of the biodegradable polymer is about 1: 1,000 to about 1: 1, preferably about 1: 1:
100 to about 1: 5, particularly preferably about 1:50 to about 1:
5 Then, the w / o emulsion thus prepared is further added to the external aqueous phase to form a w / o / w emulsion, and then the solvent in the oil phase is evaporated to prepare microcapsules. . An emulsifier may be added to the external aqueous phase. The emulsifier may be any as long as it can form a stable w / o / w emulsion.
Specifically, for example, anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid and the like are used. As the emulsifier, polyvinyl alcohol is preferably used. The emulsifier is one or two
A combination of more than one species may be used. The concentration at the time of use can be appropriately selected from the range of about 0.001 to about 20% (w / w) with respect to the external aqueous phase. More preferably about 0.01
About 10% (w / w), particularly preferably about 0.05%
It is used in a range of about 5% (w / w). Further, the same or different metal salt as the metal salt used in the inner aqueous phase may be added to the outer aqueous phase. At this time, the concentration of the metal salt in the external aqueous phase is about 0.01 to about 20% (w / w), particularly about 0.1 to about 10%.
It is preferable to add a fatty acid metal salt so as to obtain (w / w). By changing the concentration of the metal salt in the external aqueous phase, the metal salt used in the internal aqueous phase can be prevented from being eluted from the biodegradable polymer into the external aqueous phase.

The microcapsules thus obtained are separated by centrifugation or filtration, and the emulsifier or the like adhering to the surface of the microcapsules is repeatedly washed several times with distilled water, and then again distilled water or the like. And freeze-dried. Thereafter, if necessary, the water and the organic solvent in the microcapsules are further removed by heating under reduced pressure. As the heating conditions, the microcapsules are heated and dried at a temperature not lower than the glass transition temperature of the biodegradable polymer and not causing the particles of the microcapsules to adhere to each other. Preferably, heating and drying are performed in a range from the glass transition temperature of the biodegradable polymer to about 30 ° C. higher than the glass transition temperature. Here, the glass transition temperature, using a differential scanning calorimeter,
The midpoint glass transition temperature obtained when the temperature is raised at a heating rate of 10 to 20 ° C. per minute.

(B) Underwater drying method (o / w method) In this method, first, an organic solvent solution of a metal salt of a biodegradable polymer is produced by a method according to the above (A).
At this time, the concentration of the metal salt of the biodegradable polymer in the organic solvent liquid is the same as that described in (a) above. Subsequently, a bioactive polypeptide is added to the obtained organic solvent solution of the metal salt of the biodegradable polymer, and the resulting solution is dissolved or dispersed to form an organic solution of the metal salt of the biodegradable polymer and the bioactive polypeptide. Produce a solvent solution or suspension. At this time, if the bioactive polypeptide does not dissolve in the organic solvent solution of the metal salt of the biodegradable polymer, that is, if it becomes turbid, it is better to disperse the bioactive polypeptide in the organic solvent in advance. preferable. For example, a stabilizer (eg, serum albumin, gelatin, protamine sulfate, etc.) may be added to the organic solvent. In order to uniformly disperse the bioactive polypeptide in the organic solvent, it is necessary to apply external physical energy. What is that method,
For example, ultrasonic irradiation, a turbine-type stirrer, a homogenizer and the like can be mentioned. At this time, the particle size of the physiologically active polypeptide in the organic solvent is from about 0.01 to about 10
It is desired that the thickness be 0 μm, preferably about 0.5 to about 50 μm, and more preferably about 0.1 to about 10 μm. At this time, the concentration of the physiologically active polypeptide in the organic solvent is about 1 to about 50%, preferably about 2 to about 20%. By performing such a treatment, the particle size of the physiologically active polypeptide in the organic solvent can be made uniform, and the metal salt of the biodegradable polymer can be uniformly dispersed in the organic solvent liquid. Becomes Further, the physiologically active polypeptide may be dispersed in an organic solvent independently of the metal salt of the biodegradable polymer. In this case, the organic solvent to be used may have the same composition as the organic solvent in which the metal salt of the biodegradable polymer has been dissolved, or may have a different composition. For example, a metal salt of a biodegradable polymer may be dissolved in dichloromethane, a physiologically active polypeptide may be dispersed in acetonitrile, and both may be mixed. On this occasion,
The ratio (weight ratio) of the bioactive polypeptide to the biodegradable polymer polymer metal salt is, for example, about 1: 1000 to about 1: 1, preferably about 1: 200 to about 1: 5, and particularly preferably. Is from about 1: 100 to about 1: 5. Then, an organic solvent solution of the metal salt of the biodegradable polymer and the physiologically active polypeptide thus prepared is further added to the aqueous phase to form an o / w emulsion, The solvent in the oil phase is evaporated to produce microcapsules. The microcapsules thus obtained are separated, washed and freeze-dried in the same manner as in the above (a). Thereafter, if necessary, the water and the organic solvent in the microcapsules are further removed by heating under reduced pressure in the same manner as in the above (a).

(C) Phase Separation Method In the case of producing microcapsules according to the present method, the w / o emulsion of (a) or the metal salt of the biodegradable polymer of (b) is treated with physiological salt. A coacervation agent is gradually added to the organic solvent solution containing the active polypeptide under stirring in the same manner as in the above (C) to precipitate and solidify the microcapsules. The microcapsules thus obtained are separated and washed in the same manner as in the above (C) to remove the coacervation agent and free bioactive polypeptide. Thereafter, if necessary, the water and the organic solvent in the microcapsules are further removed by heating under reduced pressure in the same manner as in the above (a). In the production by the underwater drying method and the phase separation method, in order to prevent agglomeration of particles, the (C) is used.
A coagulation inhibitor may be added in the same manner as described above.

(D) Spray-drying method In the case of producing microcapsules by this method, the w / o emulsion of (a) or the metal salt of the biodegradable polymer of (b) is treated with a physiological salt. An organic solvent solution containing the active polypeptide is sprayed in the same manner as in (D) to produce microcapsules. If necessary, the microcapsules thus obtained are heated under reduced pressure in the same manner as in the above (a) to further remove water and the organic solvent in the microcapsules.

In the present invention, the rate of incorporation of the bioactive polypeptide into the metal salt of the biodegradable polymer is preferably about 50% or more. The content of the physiologically active polypeptide contained in the sustained-release preparation of the present invention is, for example, about 0.001 to about 30% (w / w), preferably about 0.
02 to about 20% (w / w), more preferably about 0.1
To about 10% (w / w), particularly preferably about 0.5 to about 5% (w / w). The sustained-release preparation of the present invention may be, for example, the microcapsules obtained as described above, or various dosage forms using the microcapsules as a raw material,
For example, parenteral preparations (eg, injection or implant for intramuscular, subcutaneous, organ, etc., transmucosal preparations for nasal cavity, rectum, uterus, etc.), oral preparations (eg, capsules (eg, hard capsules,
Soft capsules), solid preparations such as granules and powders, and liquid preparations such as suspensions). The sustained-release preparation of the present invention is particularly preferably an injection. In order to use the microcapsules obtained by the above method as an injection, the microcapsules are dispersed in a dispersant (eg, Tween 80, HC
Surfactants such as O-60, polysaccharides such as carboxymethylcellulose, sodium alginate, sodium hyaluronate, protamine sulfate, polyethylene glycol 4
00), preservatives (eg, methyl paraben, propyl paraben, etc.), isotonic agents (eg, sodium chloride, mannitol, sorbitol, glucose, etc.), aqueous suspensions with local anesthetics (xylocaine hydrochloride, chlorobutanol, etc.) Or a mixture of vegetable oils such as sesame oil and corn oil or phospholipids such as lecithin,
Alternatively, it is dispersed with medium-chain fatty acid triglycerides (eg, Miglyol 812, etc.) to give a sustained-release injection as an oily suspension.

When the sustained-release preparation is, for example, a microcapsule, it is particularly preferred to be fine particles. The particle size of the microcapsules, when used as a suspension injection, may be within a range that satisfies the degree of dispersion and needle penetration. For example, the average particle size may range from about 0.1 to about 300 μm. Can be Particle size is preferably from about 1 to about 150μ
m, more preferably the particle size ranges from about 2 to about 100 μm. In order to make the above-mentioned microcapsules into a sterile preparation, a method of sterilizing the whole production process, a method of sterilizing with gamma rays, a method of adding a preservative, and the like are included.
There is no particular limitation.

The sustained-release preparation has low toxicity and can be safely used for mammals (eg, human, cow, pig, dog, cat, mouse, rat, rabbit, etc.). The indication of the sustained-release preparation depends on the bioactive polypeptide used. Sustained-release preparation, when the bioactive polypeptide is, for example, insulin, such as diabetes, when growth hormone is growth hormone secretion deficiency and Turner syndrome, such as interferon-alpha, Anemia in the case of erythropoietin (eg, anemia during renal dialysis) such as viral hepatitis (eg, hepatitis C, HBe antigen-positive active hepatitis, etc.), cancer (eg, kidney cancer, multiple myeloma, etc.)
In the case of G-CSF, neutropenia (eg, during anticancer drug treatment), in the case of infectious disease, etc., in the case of IL-2, cancer (eg,
Such as gastrointestinal ulcer in the case of FGF, thrombocytopenia in the case of FGF-9, senile dementia, neuropathy in the case of NGF, etc.
TPA is effective for treating or preventing thrombosis and the like, and tumor necrosis factor is effective for treating and preventing cancer and the like.

The dosage of the sustained-release preparation varies depending on the type and content of the bioactive polypeptide, the duration of release, the target disease, the target animal, etc., but the effective concentration of the bioactive polypeptide is maintained in the body. The amount may be any amount. For example, when the sustained release preparation is a one-week preparation, the dose of the physiologically active polypeptide can be appropriately selected from the range of preferably about 0.0001 to about 10 mg / kg body weight per adult. More preferably about 0.0005
To about 1 mg / kg body weight. The frequency of administration can be appropriately selected depending on the kind and content of the bioactive polypeptide, dosage form, duration of release, target disease, target animal, etc., such as once a week and once every two weeks. When the physiologically active polypeptide that is the active ingredient of the sustained release preparation is, for example, insulin, the dose to a diabetic adult is usually about 0.001 as the active ingredient.
To about 1 mg / kg body weight, preferably from about 0.01 to about 0.1 mg / kg body weight.
The dose is appropriately selected from the range of 2 mg / kg body weight and is preferably administered once a week. If it is a growth hormone,
The dose for a patient with pituitary dwarfism is appropriately selected from the range of usually about 0.004 mg to about 4 mg / kg body weight, preferably about 0.04 mg to about 0.8 mg / kg body weight as an active ingredient. Preferably, it is administered once a week.
Alternatively, it is preferably selected as appropriate from the range of about 0.008 mg to about 8 mg / kg body weight, preferably about 0.08 mg to about 1.6 mg / kg body weight, and administered once every two weeks.
The sustained-release preparation is preferably stored at room temperature or in a cold place. More preferably, the sustained-release preparation is stored in a cold place. The normal temperature or cold place here is defined in the Japanese Pharmacopoeia. That is, room temperature is 15
~ 25 ° C, cold place means 15 ° C or less.

[0042]

The present invention will be described in more detail with reference to the following Reference Examples and Examples, which do not limit the present invention. Reference Example 1 Lactic acid / glycolic acid copolymer [lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 6000] was dissolved in 4 ml of dichloromethane. 438 m to this solution
1 ml of an aqueous g / ml zinc acetate solution was added and mixed with a small homogenizer to obtain a w / o emulsion. This emulsion was adjusted to 18 ° C. in advance of 0.1.
% (W / v) aqueous solution of polyvinyl alcohol (PVA) 8
100 ml and w
An / o / w emulsion was obtained. Thereafter, dichloromethane was volatilized while stirring the w / o / w emulsion at room temperature to obtain a zinc salt of a lactic acid-glycolic acid copolymer. The zinc salt of the obtained lactic acid-glycolic acid copolymer was separated by centrifugation (about 1000 rpm), and the supernatant was discarded.
Then, the mixture was washed twice with 600 ml of distilled water and freeze-dried to obtain a powdery zinc salt of a lactic acid-glycolic acid copolymer.
When the zinc content of the zinc salt of the lactic acid-glycolic acid copolymer was measured by an atomic absorption method, it was 1.36% (w / w).
Met.

Reference Example 2 Lactic acid / glycolic acid copolymer [lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 10,000] 4 g
Was dissolved in 4 ml of dichloromethane. 292
1.5 ml of an aqueous mg / ml zinc acetate solution was added and mixed with a small homogenizer to obtain a w / o emulsion. This emulsion was treated in the same manner as in Reference Example 1 to obtain a powdery zinc salt of a lactic acid-glycolic acid copolymer. This lactic acid
When the zinc content of the zinc salt of the glycolic acid copolymer was measured by an atomic absorption method, it was 1.1% (w / w).

Reference Example 3 Lactic acid / glycolic acid copolymer [lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 15000] 4 g
Was dissolved in 4 ml of dichloromethane. 292
1.5 ml of an aqueous mg / ml zinc acetate solution was added and mixed with a small homogenizer to obtain a w / o emulsion. This emulsion was treated in the same manner as in Reference Example 1 to obtain a powdery zinc salt of a lactic acid-glycolic acid copolymer. This lactic acid
The zinc content of the zinc salt of the glycolic acid copolymer was measured by an atomic absorption method and found to be 0.99%.

Reference Example 4 Recombinant human insulin (manufactured by Wako Pure Chemical Industries, zinc content: 0.3
(35%) was dissolved in 200 ml of 0.01N hydrochloric acid solution. This solution was dialyzed three times in 10 L of a 0.01N hydrochloric acid solution using a semipermeable membrane having a molecular weight fraction of 6000 (Spectrapor ^™ 7 MWCO 1000, manufactured by Spectrum Medical Industries, USA). Further, 1 L with 30 L of 0.2 M ammonium acetate aqueous solution.
1 time with 30 L of 0.02 M aqueous ammonium acetate solution
After dialysis once with 30 L of distilled water one time, the solution was freeze-dried. The zinc content in the obtained freeze-dried insulin powder was 0.0001% (w / w) or less.

Reference Example 5 Lactic acid / glycolic acid copolymer [lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 6000] 8 g was dissolved in dichloromethane 8 ml. 292m to this solution
1.5 ml of a g / ml aqueous zinc acetate solution was added and mixed with a small homogenizer to obtain a w / o emulsion. 0.1% (w / v) aqueous solution of polyvinyl alcohol 1
The mixture was poured into 800 ml, and a w / o / w emulsion was obtained using a turbine-type homomixer. This emulsion was treated in the same manner as in Reference Example 1 to obtain a powdery zinc salt of a lactic acid-glycolic acid copolymer. The zinc content of the zinc salt of this lactic acid-glycolic acid copolymer was measured by an atomic absorption method and found to be 1.15%.

Reference Example 6 Lactic acid / glycolic acid copolymer [lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 6000] 8 g was dissolved in dichloromethane 8 ml. 292m to this solution
1.5 ml of a g / ml aqueous zinc acetate solution was added and mixed with a small homogenizer to obtain a w / o emulsion. This emulsion was treated in the same manner as in Reference Example 4 to obtain a powdery zinc salt of a lactic acid-glycolic acid copolymer. The zinc content of the zinc salt of the lactic acid-glycolic acid copolymer was measured by an atomic absorption method and found to be 1.24%.

Reference Example 7 Lactic acid / glycolic acid copolymer [lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 15000] 8 g
Was dissolved in 8 ml of dichloromethane. 292
A 1.2 mg / ml aqueous zinc acetate solution was added and mixed with a small homogenizer to obtain a w / o emulsion. This emulsion was treated in the same manner as in Reference Example 4 to obtain a powdery zinc salt of a lactic acid-glycolic acid copolymer. The zinc content of this zinc salt of the lactic acid-glycolic acid copolymer was measured by an atomic absorption method and found to be 0.96%.

Reference Example 8 Lactic acid / glycolic acid copolymer [lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 8000] 8 g was dissolved in dichloromethane 8 ml. 292m to this solution
1.5 ml of a g / ml aqueous zinc acetate solution was added and mixed with a small homogenizer to obtain a w / o emulsion. 0.1% (w / v) aqueous solution of polyvinyl alcohol 1
The mixture was poured into 800 ml, and a w / o / w emulsion was obtained using a turbine-type homomixer. After this, w / o /
While stirring the w-type emulsion at room temperature, dichloromethane was evaporated to obtain a zinc salt of a lactic acid-glycolic acid copolymer. The obtained zinc salt of the lactic acid-glycolic acid copolymer was separated by centrifugation (about 1000 rpm), and the supernatant was discarded. Then, it was washed twice with 1200 ml of distilled water. What was obtained by repeating the above operation once again,
After mixing the components prepared above, the mixture was freeze-dried to obtain 11.8 g of a powdery zinc salt of a lactic acid-glycolic acid copolymer. When the zinc content of the zinc salt of the lactic acid-glycolic acid copolymer was measured by an atomic absorption method, it was 1.19% (w /
w).

Example 1 900 mg of the zinc salt of a lactic acid-glycolic acid copolymer obtained in Reference Example 1 was dissolved in 1 ml of dichloromethane. 100 mg of the freeze-dried Zn-free insulin powder obtained in Reference Example 4 was added to this solution, mixed with a vortex mixer, mixed with a small homogenizer, and contained insulin and a zinc salt of a lactic acid-glycolic acid copolymer. An organic solvent solution was obtained. This organic solvent solution was previously adjusted to 18 ° C. with 0.1% (w / v) polyvinyl alcohol (P
VA) The solution was poured into 800 ml of an aqueous solution to obtain an o / w emulsion using a turbine type homomixer. This o / w
While stirring the emulsion at room temperature, dichloromethane was evaporated to obtain microcapsules. The obtained microcapsules were collected by centrifugation (about 1000 rpm), and the supernatant was discarded. Then, after washing twice with 600 ml of distilled water, it was freeze-dried to obtain 520 mg of powdered insulin-containing microcapsules.

Example 2 900 mg of a zinc salt of a lactic acid-glycolic acid copolymer obtained in Reference Example 2 was dissolved in 1 ml of dichloromethane. To this solution, 100 mg of the lyophilized powder of Zn-free insulin obtained in Reference Example 4 was added and treated in the same manner as in Example 1 to obtain 450 mg of powdered insulin-containing microcapsules.

Example 3 900 mg of a zinc salt of a lactic acid-glycolic acid copolymer obtained in Reference Example 3 was dissolved in 1.5 ml of dichloromethane.
To this solution was added 100 mg of the lyophilized powder of insulin-free insulin obtained in Reference Example 4 and treated in the same manner as in Example 1 to obtain 503 mg of powdered insulin-containing microcapsules.
I got

Example 4 950 mg of the zinc salt of the lactic acid-glycolic acid copolymer obtained in Reference Example 1 was dissolved in 1.5 ml of dichloromethane.
This solution contains human growth hormone (Genotropin ™).
Eight (16 IU / ampoule, manufactured by Sumitomo Pharmaceutical Co., Ltd.)
28IU) of powder and the same treatment as in Example 1 to obtain 500 mg of growth hormone-containing microcapsules.

Example 5 950 mg of the zinc salt of a lactic acid-glycolic acid copolymer obtained in Reference Example 5 was dissolved in 1.5 ml of dichloromethane.
50 mg of human growth hormone freeze-dried powder was added to this solution, and the mixture was treated in the same manner as in Example 1 to obtain 517 mg of powdered growth hormone-containing microcapsules.

Example 6 950 mg of the zinc salt of a lactic acid-glycolic acid copolymer obtained in Reference Example 3 was dissolved in 3 ml of dichloromethane. 50 mg of human growth hormone freeze-dried powder was added to this solution, and the mixture was treated in the same manner as in Example 1 to obtain 415 mg of powdered growth hormone-containing microcapsules.

Example 7 475 mg of a zinc salt of a lactic acid-glycolic acid copolymer obtained in Reference Example 6 and a lactic acid-glycolic acid copolymer [lactic acid / glycolic acid = 50/50 (mol%), weight average molecular weight 6
000] was dissolved in 1.5 ml of a dichloromethane-ethanol mixed solvent [dichloromethane / ethanol = 2/1 (volume ratio)]. 50 mg of human growth hormone freeze-dried powder was added to this solution, and the mixture was treated in the same manner as in Example 1 to obtain a powdery growth hormone-containing microcapsule 2.
49 mg were obtained.

Example 8 475 mg of a zinc salt of a lactic acid-glycolic acid copolymer obtained in Reference Example 7 and a lactic acid-glycolic acid copolymer [lactic acid / glycolic acid = 50/50 (mol%), weight average molecular weight 1
5000] was dissolved in 3 ml of a dichloromethane-ethanol mixed solvent [dichloromethane / ethanol = 2/1 (volume ratio)]. 50 mg of human growth hormone freeze-dried powder was added to this solution, and 447 mg of powdered growth hormone-containing microcapsules were prepared in the same manner as in Example 1.
I got

Example 9 2.12 g of a zinc salt of a lactic acid-glycolic acid copolymer obtained in Reference Example 8 was dissolved in 3.45 ml of dichloromethane. Next, 160 mg of the zinc-free insulin lyophilized powder obtained in Reference Example 4 was added to 2 ml of dichloromethane,
Ultrasonic irradiation was performed for 5 minutes to prepare an insulin suspension. To this suspension, a dichloromethane solution of the lactic acid-glycolic acid copolymer obtained above (the amount containing 1.84 g as a zinc salt of the lactic acid-glycolic acid copolymer) was added, and mixed with a vortex mixer. Thereafter, the mixture was mixed with a small homogenizer to obtain an organic solvent solution containing insulin and a zinc salt of a lactic acid-glycolic acid copolymer. This organic solvent solution was poured into 2000 ml of a 0.1% (w / v) aqueous polyvinyl alcohol solution containing 1.4% zinc acetate dihydrate, which had been previously adjusted to 18 ° C., and o was added using a turbine-type homogenizer. / W type emulsion. This o
While stirring the / w type emulsion at room temperature, dichloromethane was volatilized to obtain microcapsules. The obtained microcapsules were collected by centrifugation (about 1000 rpm), and the supernatant was discarded. Then, after washing twice with 1200 ml of distilled water, 250 mg of mannitol was added and freeze-dried to obtain 1.53 g of powdery insulin-containing microcapsules.

Example 10 1.06 g of a zinc salt of a lactic acid-glycolic acid copolymer obtained in Reference Example 8 was dissolved in 2.3 ml of a dichloromethane / acetonitrile mixed solution (volume ratio: 10/1). next,
Freeze-dried zinc-free insulin powder 8 obtained in Reference Example 4
0 mg was added to 1 ml of a mixed solution of dichloromethane / acetonitrile (volume ratio: 10/1), and ultrasonic irradiation was performed for 5 minutes to prepare an insulin suspension. To this suspension, a dichloromethane solution of the lactic acid-glycolic acid copolymer obtained above (containing 0.92 g as a zinc salt of a lactic acid-glycolic acid urine polymer) was added, and mixed with a vortex mixer. Thereafter, the mixture was mixed with a small homogenizer to obtain an organic solvent solution containing insulin and a zinc salt of a lactic acid-glycolic acid copolymer. This organic solvent solution was previously adjusted to 18 ° C. and contained 0.7% zinc acetate dihydrate containing 0.1%
(W / v) The solution was poured into 1000 ml of an aqueous polyvinyl alcohol solution, and an o / w emulsion was prepared using a turbine homogenizer. While stirring the o / w emulsion at room temperature, dichloromethane was volatilized to obtain microcapsules. The obtained microcapsules were collected by centrifugation (about 1000 rpm), and the supernatant was discarded.
Then, after washing twice with 600 ml of distilled water, 100 mg of mannitol was added and freeze-dried to obtain 0.734 g of powdered insulin-containing microcapsules.

Experimental Example 1 Insulin-containing microcapsules 1 obtained in Example 1
47 mg of a dispersion medium [mannitol 5% (w / v), carboxymethylcellulose 0.5% (w / v), Tween 20 0.1% (w / v), pH with acetic acid
Adjusted to 6.8]. 0.5 ml of the resulting dispersion (containing 100 U of insulin) was subcutaneously administered to the back of streptozotocin-sensitized hyperglycemic rats under ether anesthesia. Blood was collected over time from the tail vein to collect serum. The concentration of insulin in the obtained serum was quantified by an enzyme immunoassay using a two-antibody sandwich method.
As a control, an insulin solution and a commercially available insulin sustained release agent Novolin ^™ U (manufactured by Novo Nordisk, Denmark) (each containing 100 U of insulin) were administered. As a result, as shown in Table 1, the serum concentration of insulin in the group administered with the insulin / zinc-containing microcapsules was significantly longer than that of the insulin solution and novolin ^™ U, and the sustained release produced by the production method of the present invention. Excellent sustained release effect of the formulation was confirmed.

[0061]

[Table 1]

Experimental Example 2 261 mg of the growth hormone-containing microcapsules obtained in Example 6 were dispersed in 1.75 ml of a dispersion medium. 0.5 ml of the obtained dispersion (containing 3 mg of growth hormone) was subcutaneously administered to the back of the rat under ether anesthesia. The blood was collected over time from the tail vein, and the serum was collected. The growth hormone concentration in the obtained serum was quantified by a radioimmunoassay (Ab beads HGH, manufactured by Eiken Chemical Co., Ltd.). As a control, a growth hormone solution (containing 3 mg of growth hormone) was administered. As a result, as shown in Table 2, the growth hormone / serum concentration in the growth hormone / zinc-containing microcapsule administration group was significantly longer than that in the growth hormone solution administration group, and the concentration of the growth hormone / zinc-containing microcapsules was lower than that of the growth hormone solution administration group. An excellent sustained-release sustained effect of the release preparation was confirmed.

Experimental Example 3 The growth hormone-containing microcapsules 211 mg obtained in Example 7 were dispersed in 1.75 ml of a dispersion medium. 0.5 ml of the obtained dispersion (containing 3 mg of growth hormone) was subcutaneously administered to the back of the rat under ether anesthesia. Blood was collected over time from the tail vein, and serum was collected. The growth hormone concentration in the obtained serum was quantified by a radioimmunoassay (Ab beads HGH, manufactured by Eiken Chemical Co., Ltd.). As a result, as shown in [Table 2], the growth hormone serum concentration of the growth hormone / zinc-containing microcapsule administration group significantly suppressed the initial leakage after administration as compared with the growth hormone solution administration group, and was significantly sustained. An excellent sustained release effect of the sustained release preparation produced by the production method of the present invention was confirmed.

[0064]

[Table 2]

Experimental Example 4 Insulin-containing microcapsules 2 obtained in Example 9
36.6 mg was dispersed in 1.75 ml of a dispersion medium. 0.5 ml (containing 100 U of insulin) of the obtained dispersion was subcutaneously administered to the back of the rat under ether anesthesia. Blood was collected over time from the tail vein, and serum was collected. The insulin concentration in the obtained serum was quantified by enzyme-immunoassay. As a result, as shown in [Table 3], insulin
The insulin serum concentration of the zinc-containing microcapsule administration group significantly suppresses the initial leakage after administration and is significantly longer than that of the insulin solution administration group, and the sustained-release preparation produced by the production method of the present invention is excellent. A sustained sustained release effect was also confirmed.

Experimental Example 5 216.4 mg of the insulin-containing microcapsules obtained in Example 10 were dispersed in 1.75 ml of a dispersion medium. 0.5 ml of the obtained dispersion (containing 100 U of insulin) was subcutaneously administered to the back of the rat under ether anesthesia. Blood was collected over time from the tail vein, and serum was collected. The insulin concentration in the obtained serum was quantified by enzyme-immunoassay. As a result, as shown in [Table 3], as compared with the insulin / zinc-containing microcapsule administration group, the initial leakage after administration was significantly suppressed and significantly sustained, and the sustained-release property produced by the production method of the present invention. Excellent sustained release effect of the formulation was confirmed.

[0067]

[Table 3]

[0068]

According to the present invention, it is possible to provide a sustained-release preparation which increases the uptake rate of a physiologically active polypeptide, suppresses early leakage after administration, and shows a constant release rate over a long period of time.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display A61K 38/27 A61K 9/14 K 38/21 L 47/34 37/24 C08G 63/06 NLP 37 / 26 C08L 67/04 LPG 37/36 37/66 (72) Inventor Masafumi Gozen 2-4-14-1103 Hibarigaoka, Takarazuka City, Hyogo Prefecture

Claims (23)

[Claims] 1. A method for producing a sustained-release preparation, comprising dispersing a physiologically active polypeptide in an organic solvent solution of a metal salt of a biodegradable polymer and shaping the same. 2. The method according to claim 1, wherein the metal salt is a polyvalent metal salt. 3. The method according to claim 1, wherein the metal salt is a zinc salt or a calcium salt. 4. The method according to claim 1, wherein the organic solvent in the organic solvent liquid is a mixed solution of a halogenated hydrocarbon and acetonitrile or an alcohol. 5. The process according to claim 4, wherein the volume ratio of the halogenated hydrocarbon to acetonitrile or alcohol is from about 40: 1 to about 1: 1. 6. The method according to claim 1, wherein the physiologically active polypeptide is a hormone. 7. The method according to claim 6, wherein the hormone is insulin. 8. The method according to claim 6, wherein the hormone is a growth hormone. 9. The method according to claim 1, wherein the physiologically active polypeptide is a cytokine. 10. The method according to claim 9, wherein the cytokine is interferon. 11. The method according to claim 1, wherein the biodegradable polymer is an aliphatic polyester. 12. The method according to claim 11, wherein the aliphatic polyester is an α-hydroxycarboxylic acid polymer. 13. The method according to claim 11, wherein the aliphatic polyester is a lactic acid-glycolic acid copolymer. 14. The lactic acid / glycolic acid copolymer has a lactic acid / glycolic acid composition ratio (mol%) of about 100/0 to about 40 /.
14. The method of claim 13, wherein the weight average molecular weight is about 3000 to about 20,000. 15. The method according to claim 1, wherein the sustained-release preparation is fine particles. 16. The fine particles have an average particle size of about 0.1 to about 30.
The method according to claim 15, wherein the thickness is 0 µm. 17. The method according to claim 1, wherein the sustained-release preparation is an injection. 18. A dispersion in which a physiologically active polypeptide is dispersed in an organic solvent liquid of a metal salt of a biodegradable polymer. 19. The dispersion according to claim 18, which is substantially free of metal salts of fatty acids. (20) A sustained-release preparation produced by the production method according to (1). 21. The sustained-release preparation according to claim 20, wherein the metal content of the metal salt of the biodegradable polymer is about 0.01 to about 10% (w / w). 22. The bioactive polypeptide content of about 0.0
21. The sustained release formulation according to claim 20, wherein the amount is from 01 to about 30% (w / w). 23. The sustained-release preparation according to claim 20, which is obtained by dispersing growth hormone in an organic solvent solution containing a zinc salt of a biodegradable polymer and molding the same.