Method of producing sustained-release microcapsules

Description: TECHNICAL FIELD [Detailed description of the invention]

【0001】

[Industrial application field]

The present invention relates to a method for producing sustained-release microcapsules in which an excessive initial release of a physiologically active substance is suppressed and a constant amount of the physiologically active substance is released for a long period of time immediately after administration.

【0002】

[Conventional technology]

Methods for producing sustained-release microcapsules from W / O emulsions using biodegradable polymers are described in, for example, JP-A-57-118512 and JP-A-57-150609. However, there is no description about a method for producing sustained-release microcapsules of a physiologically active substance by heating the microcapsules to a temperature above the glass transition temperature of the biodegradable polymer so that the particles of the microcapsules do not adhere to each other. ..

【0003】

[Problems to be Solved by the Invention]

It is desirable that sustained-release microcapsules using a biodegradable polymer suppress the initial release of a drug, particularly an excessive amount within one day, and can arbitrarily control the drug release over a long period of time. However, with conventional sustained-release microcapsules, the amount of drug released within a day is excessive and is not yet sufficiently satisfactory.

【0004】

[Means for solving problems]

The present invention 1) Physiology in a method for producing sustained-release microcapsules of physiologically active substances from W / O-type emulsions in which a solution containing a physiologically active substance is used as an internal aqueous phase and a solution containing a biodegradable polymer is used as an oil phase. Physiological activity characterized in that microcapsules obtained by microencapsulating an active substance with a biodegradable polymer are heated to a temperature above the glass transition temperature of the polymer so that the particles of the microcapsules do not adhere to each other. Method for manufacturing sustained-release microcapsules of substances, 2) The production method according to paragraph 1, wherein the physiologically active substance is a peptide having a molecular weight of 200 to 80,000. 3) The production method according to paragraph 1, wherein the physiologically active substance is luteinizing hormone-releasing hormone or a derivative thereof. 4) The production method according to paragraph 1, wherein the biodegradable polymer is an aliphatic polyester. 5) The production method according to Item 4, wherein the aliphatic polyester is a single or copolymer of α-hydroxy acids, or a mixture of these homopolymers and / or copolymers. 6) The production method according to paragraph 4, wherein the weight average molecular weight of the aliphatic polyester is 3,000 to 30,000. 7) The production method according to paragraph 4, wherein the dispersity of the aliphatic polyester is 1.2 to 4.0. 8) The production method according to paragraph 1, wherein the microcapsules are heated at a temperature 5 to 40 ° C higher than the glass transition temperature of the biodegradable polymer. 9) The production method according to paragraph 1, wherein the average particle size of the microcapsules is 1 to 300 μm. 10) The production method according to paragraph 1, wherein the microcapsules obtained by microencapsulation are further added with an anti-aggregation agent and then heated. 11) Physiological activity obtained by microencapsulating a physiologically active substance with a biodegradable polymer and heating the microcapsules to a temperature above the glass transition temperature of the polymer so that the particles of the microcapsules do not adhere to each other. Sustained release microcapsules of material, 12) Sustained-release microcapsules of physiologically active substances according to Item 11, which is obtained by further adding an anti-aggregation agent to the microcapsules obtained by microencapsulation, and 13) The present invention relates to an injection containing sustained-release microcapsules of the physiologically active substance described in paragraph 11.

【0005】

The physiologically active substance used in the present invention is not particularly limited, but is a physiologically active peptide, antitumor agent, antibiotic, antipyretic, analgesic, anti-inflammatory agent, antitussive expectorant, sedative, muscle relaxant, antiepileptic agent, Anti-ulcer, antidepressant, anti-allergic agent, cardiotonic agent, arrhythmia therapeutic agent, vasodilator, antihypertensive diuretic agent, diabetic therapeutic agent, anticoagulant, hemostatic agent, antituberculous agent, hormone agent, drug antagonist, bone Examples include absorption inhibitors and angiogenesis inhibitors.

【0006】

As the physiologically active substance used in the present invention, a peptide having physiological activity is preferable. The peptide is preferably a peptide having a molecular weight of about 200 to 80,000. Specific examples of the peptide include, for example, luteinizing hormone-releasing hormone (LH-RH) or a derivative thereof, and formula (I) (Pyr) Glu-R.₁-Trp-Ser-R₂-R₃-R₄-Arg-Pro-R₅ (I) [In the formula, R₁Is His, Tyr, Trp or p-NH₂-Phe, R₂Is Tyr or Phe, R₃Is a Gly or D-type amino acid residue, R₄Is Leu, Ile or Nle, R₅Is Gly-NH-R₆(R₆Is a lower alkyl group with or without H or hydroxyl group) or NH-R₆(R₆Has the same meaning as above). ], Examples of peptides or salts thereof [US Pat. No. 3,853,837, US Pat. No. 4,008,209, US Pat. No. 3,972,859, UK Pat. No. 1,423,083, Proceedings of the National Academy of Science of the See Proceedings of the National Academy of Sciences of the United States of America, Vol. 78, pp. 6509-6512 (1981)]. In the above equation (I), R₃Examples of D-type amino acid residues represented by are α-D-amino acids having up to 9 carbon atoms (eg, D-Leu, Ile, Nle, Val, Nval, Abu, Phe, Phg, Ser, Thr, Met). , Ala, Trp, α-Aibu) and the like, which may optionally have protecting groups (eg, t-butyl, t-butoxy, t-butoxycarbonyl, etc.). Of course, acid salts of peptide (I) (eg, carbonates, bicarbonates, acetates, propionates, etc.) and metal complex compounds (eg, copper complexes, zinc complexes, etc.) are also used in the same manner as peptide (I). sell.

【0007】

Amino acids, protecting groups, etc. in the peptides represented by the formula (I) and the peptides shown below are abbreviated by the IUPAC-IUB Commission on Biochemical Nomenclature when indicated by abbreviations. Alternatively, it shall be based on the conventional abbreviation in the field, and if there may be an optical isomer with respect to the amino acid, it shall indicate the L-form unless otherwise specified. As a typical peptide represented by the above formula (I), for example, R₁= His, R₂= Tyr, R₃= D-Leu, R₄= Leu, R₅= NHCH₂-CH₃(The acetate of this peptide is commonly called leuprorelin acetate, and may be abbreviated as TAP-144 below). Examples of the peptide having physiological activity include LH-RH antagonists (see US Pat. Nos. 4,086,219, 4,124,577, 4,253,997, 4,317,815). Further physiologically active peptides include, for example, insulin, somatostatin, somatostatin derivative (see US Pat. Nos. 4,087,390, 4,093,574, 4,100,117, 4,253,998), growth hormone, prolactin, and adrenocorticotropic acid. Hormone (ACTH), melanocyte-stimulating hormone (MSH), thyroid hormone-releasing hormone [(Pyr) Glu-His-ProNH₂ It is expressed by the structural formula of, and may be abbreviated as TRH below.] Its salt and its derivative (see JP-A-50-121273, JP-A-52-116465), thyroid stimulating hormone (TSH), luteinizing hormone. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), gasopresin, bassopresin derivative {see desmopressin [Journal of the Endocrine Society of Japan, Vol. 54, No. 5, pp. 676-691 (1978)]}, oxytocin, calciumtonin, parathyroid hormone , Glucagon, Gastrin, Secretin, Puncleozymin, Cholecystokinin, Angiotensin, Human placenta lactogen, Human chorionic gonadotropin (HCG), Enkefarin, Enkefarin derivative [See US Patent No. 4,277,394, European Patent Application Publication No. 31567 ], Endolphin, Kyotorphin, Interferons (eg, α-type, β-type, γ-type, etc.), Interleukins (eg, I, II, III, etc.), Taftsin, thymopoietin, thymosin, thymostimulin, thoracic gland fluid factor (THF), Blood Cholecystokinin Factor (FTS) and its Derivatives (see US Pat. No. 4,229,438), and Other Cholecystokinin Factors [History of Medicine, Vol. 125, No. 10, pp. 835-843 (1983)] , Tumor necrosis factor (TNF), colony inducing factor (CSF), motilin, dynorphin, bombesin, neurotensin, cerulein, bradyquinin, urokinase, asparaginase, calicrane, substance P, nerve growth factor, cell growth factor, neuronutrient factor, Blood coagulation factors Factors VIII, IX, lysozyme chloride, polymyxin B, cholecystokinin, gramicidin, bacitracin and erythropoetin (EPO), peptides with endoserin antagonism (European Patent Publication Nos. 436189, 457195, s) No. 496452, Japanese Patent Application Laid-Open No. 3-94692, Japanese Patent Application Laid-Open No. 3-130299) and the like.

【0008】

Examples of the above antitumor agents include bleomycin, methotrexate, actinomycin D, mitomycin C, vinblastine sulfate, vincristine sulfate, daunorubicin, adriamycin, neocultinostatin, citocin arabinoside, fluorouracil, tetrahydrofuryl-5-fluorouracil, crestin, and pisibanil. , Lentinan, levamizol, bestatin, adimexone, glycyrrhizin, poly I: C, poly A: U, poly ICLC and the like. Examples of the antibiotics include gentamicin, dibecacin, canendomycin, ribidomycin, tobramycin, amikacin, fradiomycin, cisomycin, tetracycline hydrochloride, oxytetracycline hydrochloride, lolitetracycline, doxycycline hydrochloride, ampicillin, piperacillin, ticarcillin, cephalotin and cefazolin. , Cefotiam, cefthrosin, cefmenoxime, cefmethazole, cefazolin, cefotaxime, cefoperazone, ceftizoxime, moxalactam, thienomycin, sulfazecin, azthleonum and the like.

【0009】

Examples of the antipyretic, analgesic and anti-inflammatory agents include salicylic acid, sulpyrine, flufenamic acid, diclophenac, indomethacin, morphine, pethidine hydrochloride, levorphanol tartrate, and oxymorphone. Ephedrine hydrochloride, methylephedrine hydrochloride, noscapine hydrochloride, codeine phosphate, dihydrocodein phosphate, allocramide hydrochloride, clofedanol hydrochloride, picoperidamine hydrochloride, cloperastin, protoquilol hydrochloride, isoproterenol hydrochloride, salbutamoll sulfate, as antitussive sputum. Examples include terbutalin sulfate. Examples of the sedative include chlorpromazine, prochlorperazine, trifloperazine, atropine sulfate, methylscopolamine bromide and the like. Examples of muscle relaxants include pridineol methanesulfonate, tubocurarine chloride, pancuronium bromide and the like. Examples of antiepileptic agents include phenytoin, ethosuximide, acetazolamide sodium, chlordiazepoxide and the like. Examples of the anti-ulcer agent include metoclopromid, histidine hydrochloride and the like. Examples of antidepressants include imipramine, clomipramine, noxiptiline, phenelzine sulfate and the like. Examples of the antiallergic agent include diphenhydramine hydrochloride, chlorpheniramine maleate, tryperenamine hydrochloride, metodilazine hydrochloride, cremizole hydrochloride, diphenylpyraline hydrochloride, methoxyphenamine hydrochloride and the like.

【0010】

Examples of the cardiac stimulant include transpyoxocamphor, theophilol, aminophylline, etilefrine hydrochloride and the like. Examples of the arrhythmia therapeutic agent include propranol, alprenolol, bufetrol, oxyprenolol and the like. Examples of the vasodilator include oxyfedrin hydrochloride, diltiazem, tolazoline hydrochloride, hexobendin, and bamethane sulfate. Examples of the antihypertensive diuretic include hexamethonium bromide, pentolinium, mechamilamine hydrochloride, ecarazine hydrochloride, clonidine and the like. Examples of the therapeutic agent for diabetes include sodium glymidin, glypizzaide, fenformin hydrochloride, buformin hydrochloride, metformin and the like. Examples of the anticoagulant include sodium heparin and sodium citrate. Examples of the hemostatic agent include thromboplastin, thrombin, menadione sodium bisulfite, acetomenafton, ε-aminocaproic acid, tranexamic acid, sodium carbazochrome sulfonate, adrenochrome monoaminoguanidine methanesulfonate and the like. Examples of anti-tuberculosis agents include isoniazid, ethambutol, para-aminosalicylic acid and the like. Examples of the hormonal agent include prednisolone, sodium prednisolone, sodium dexamethasone sulfate, betamethasone sodium phosphate, hexestrol phosphate, hexestrol acetate, and methimazole.

【0011】

Examples of the narcotic antagonist include levallorphan tartrate, nalophine hydrochloride, naloxone hydrochloride and the like. Examples of the bone resorption inhibitor include (sulfur-containing alkyl) aminomethylene bisphosphonic acid. Examples of angiogenesis-suppressing agents include angiogenesis-suppressing steroids [see Science Vol. 221, p. 719 (1983)], fumagirin (see European Patent Publication No. 325199), and fumagirole derivatives (European Patent Publication No. 357061). No. 3, No. 359036, No. 386667, No. 415294). Of these, the present invention is more preferably applied to water-soluble drugs because excessive initial release is often observed in the case of preparations of water-soluble drugs. The water solubility of a drug is defined by the oil-water distribution ratio of water and n-octanol, and is preferably applied to a drug having an n-octanol / water solubility ratio of 1 or less, and more preferably 0.1 or less.

【0012】

The oil-water distribution ratio may be measured according to the method described in "Physical Chemistry Experimental Method" by Misaburo Samejima, published by Shokabo, 1958. That is, first, a buffer solution of n-octanol and pH 5.5 (a 1: 1 equal amount mixture) is placed in a test tube. Examples of the buffer solution include Sφerensen buffer solution [Ergeb. Physiol. 12,393 (1912)], Clark-Lubs buffer solution [J. Bact. 2, (1), 109,191 (1917)], and McClubane. (Macllvaine) buffer [J. Biol. Chem. 49,183 (1921)], Michaelis buffer [Die Wasser-stoffionenkonzentration, p.186 (1914)], Kolthoff buffer [Biochem. Z, 179,410 (1926)] and the like. Add an appropriate amount of drug to this, plug it further, immerse it in a constant temperature bath (25 ° C), and often shake vigorously. Then, when the drug dissolves between the layers of both liquids and it seems that equilibrium has been reached, the liquid is allowed to stand or centrifuge, and a certain amount of liquid is taken out separately from each of the upper and lower layers with a pipette, and this is analyzed to analyze each layer. The oil-water distribution ratio is obtained by determining the concentration of the drug in the n-octanol layer and taking the ratio of the concentration of the drug in the n-octanol layer / the concentration of the drug in the aqueous layer. The drug itself may be a pharmacologically acceptable salt (eg, if the drug has a basic group such as an amino group, an inorganic acid such as hydrochloric acid, sulfuric acid, nitrate or an organic acid such as carbonic acid, When a salt with succinic acid or the like or a drug has an acidic group such as a carboxy group, an inorganic base such as an alkali metal such as sodium or potassium or an organic base compound, such as an organic amine such as triethylamine, or a basic amino acid such as arginine. It may be a salt with a kind). The amount of the above-mentioned drug used varies depending on the type of drug, the desired pharmacological effect and the duration of the effect, etc., but is about 0.001% to about 90% (w / w) with respect to the biodegradable polymer of the base. More preferably, it is selected from about 0.01% to about 80% (w / w).

【0013】

The biodegradable polymer used in the present invention is not particularly limited as long as it is sparingly soluble or insoluble in water and is biocompatible and decomposes in vivo. Slightly soluble in water means that the solubility in water is about 3% (w / v) or less.

【0014】

As these biodegradable polymers, those having a weight average molecular weight of about 3,000 to 30,000, preferably about 5,000 to 2,500, and particularly preferably about 5,000 to 20,000 are used. The dispersity of the biodegradable polymer is preferably about 1.2 to 4.0, particularly preferably about 1.5 to 3.5. The weight average molecular weight and the degree of dispersion used in the present specification mean values measured by gel permeation chromatography (GPC).

【0015】

The amount of these biodegradable polymers used is determined by the strength of the pharmacological activity of the bioactive substance, the rate and period of release of the physiologically active substance, and the like, for example, about 0.5 to 10,000 times (weight ratio) with respect to the physiologically active substance. However, it is preferable to use a polymer in an amount of about 1 to 100 times (weight ratio) as the microcapsule base. Examples of preferred biodegradable polymers include, for example, aliphatic polyesters [eg, α-hydroxy acids (eg, glycolic acid, lactic acid, 2-hydroxybutyric acid, 2-hydroxyvaleric acid, 2-hydroxy-3-methylbutyric acid, 2). -Hydroxycaproic acid, 2-hydroxyisocaproic acid, 2-hydroxycaprylic acid, etc.), cyclic dimer of α-hydroxyic acid (eg, glycolide, lactide, etc.), hydroxydicarboxylic acids (eg, malic acid), hydroxy A homopolymer such as tricarboxylic acid (eg, citric acid) (eg, lactic acid polymer, etc.) or two or more copolymers (eg, lactic acid / glycolic acid copolymer, 2-hydroxybutyric acid / glycolic acid copolymer). Etc.), or a mixture of these homopolymers and / or copolymers (eg, a mixture of a lactic acid polymer and a 2-hydroxybutyric acid / glycolic acid copolymer, etc.)], poly-α-cyanoacrylic acid ester, poly Amino acids (eg, poly-γ-benzyl-L-glutamic acid, poly-L-alanine, poly-γ-methyl-L-glutamic acid, etc.), maleic anhydride-based copolymers (eg, styrene / maleic acid copolymer, etc.) ) Etc. can be mentioned. Of these, aliphatic polyesters and poly-α-cyanoacrylic acid esters are preferable. Further, aliphatic polyesters are particularly preferable.

【0016】

Among the aliphatic polyesters, α-hydroxy acids, homopolymers of cyclic dimers of α-hydroxy acids, or two or more copolymers, or a mixture of these homopolymers and / or copolymers is preferable. .. Further, a single or copolymer of α-hydroxy acids, or a mixture of these homopolymers and / or copolymers is particularly preferable. When the α-hydroxy acids, cyclic dimers of α-hydroxy acids, hydroxydicarboxylic acids, and hydroxytricarboxylic acids have an optically active center in the molecule, any of the D-, L-, and DL-forms can be used. it can. The above-mentioned aliphatic polyester can be produced without any problem by a known production method (see, for example, Japanese Patent Application Laid-Open No. 61-28521). The type of polymerization may be random, block, or graft. The weight average molecular weight of the aliphatic polyester is preferably about 3,000 to 30,000, more preferably about 5,000 to 25,000, and particularly preferably about 5,000 to 20,000. The dispersity of the aliphatic polyester is preferably about 1.2 to 4.0, particularly preferably about 1.5 to 3.5.

【0017】

When a lactic acid / glycolic acid copolymer is used as the aliphatic polyester, the composition ratio is preferably about 100/0 to about 50/50 (weight ratio), and when a 2-hydroxybutyric acid / glycolic acid copolymer is used. , The composition ratio is preferably about 100/0 to about 25/75 (weight ratio). The weight average molecular weight of the lactic acid polymer, the lactic acid / glycolic acid copolymer, and the 2-hydroxybutyric acid / glycolic acid copolymer is preferably about 3,000 to 30,000. Further, about 5,000 to 20,000 are particularly preferable.

【0018】

When, for example, a mixture of a lactic acid polymer (A) and a glycolic acid / 2-hydroxybutyric acid copolymer (B) is used as the aliphatic polyester, the mixing ratio represented by (A) / (B) is about 10 /. It is used in the range of 90 to about 90/10 (weight ratio). It is preferably in the range of about 25/75 to about 75/25 (weight ratio). The weight average molecular weight of the lactic acid polymer is preferably about 3,000 to 30,000. Further, about 5,000 to 20,000 are particularly preferable. The composition of the glycolic acid / 2-hydroxybutyric acid copolymer is preferably about 40 to 70 mol of glycolic acid and the rest is 2-hydroxybutyric acid. The weight average molecular weight of the glycolic acid / 2-hydroxybutyric acid copolymer is preferably about 5,000 to 25,000. Further, about 5,000 to 20,000 are particularly preferable.

【0019】

In the present invention, as a method for producing sustained-release microcapsules of a physiologically active substance from a W / O type emulsion in which a solution containing a physiologically active substance is used as an internal aqueous phase and a solution containing a biodegradable polymer is used as an oil phase. For example, the following methods of microencapsulating a physiologically active substance by a known microencapsulation method, for example, a water drying method, a phase separation method, a spray drying method, or the like, or a method similar thereto can be mentioned. First, a physiologically active substance is dissolved in water in an amount to reach the above concentration, and if necessary, a drug-holding substance such as gelatin, agar, alginic acid, polyvinyl alcohol or a basic amino acid is added to dissolve or suspend the substance. Use as an internal aqueous phase liquid. In these internal aqueous phases, carbonic acid, acetic acid, oxalic acid, citric acid, phosphoric acid, hydrochloric acid, sodium hydroxide, arginine, and lysine are used as pH adjusters to maintain the stability and solubility of physiologically active substances. And their salts and the like may be added. Further, as a stabilizer for physiologically active peptides, a polyol compound such as albumin, gelatin, citric acid, sodium ethylenediaminetetraacetate, dextrin, sodium bisulfite, polyethylene glycol, or the like, or as a preservative, paraoxybenzoic acid generally used. Esters (methylparaben, propylparaben, etc.), benzyl alcohol, chlorobutanol, thimerosal, etc. may be added.

【0020】

The internal aqueous phase liquid thus obtained is added to a solution (oil phase) containing a biodegradable polymer, and then an emulsification operation is performed to prepare a W / O type emulsion (emulsion). The emulsification operation is performed by a known dispersion method, for example, an intermittent shaking method, a method using a mixer such as a propeller type agitator or a turbine type agitator, a colloid mill method, a homogenizer method, an ultrasonic irradiation method, or the like. Be done. As the solution (oil phase) containing the biodegradable polymer, a solution in which the polymer is dissolved in an organic solvent is used. The solvent may be any solvent having a boiling point of about 120 ° C. or less and immiscible with water and dissolving a biodegradable polymer, for example, halogenated hydrocarbons (eg, dichloromethane, chloroform, etc.). Chloroform, dichloromethane, trichloroethane, 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.) etc. These may be used by mixing two or more kinds at an appropriate ratio. The W / O emulsion thus prepared is then subjected to a microencapsulation step.

【0021】

When the W / O type emulsion is made into microcapsules by the water drying method, the W / O emulsion is further added to the third phase aqueous phase to form a W / O / W type three-phase emulsion. , Evaporate the solvent in the oil phase to prepare microcapsules.

【0022】

An emulsifier may be added to the aqueous phase of the outer phase, and examples thereof may be any one that generally forms a stable O / W type emulsion. For example, an anionic surfactant (sodium oleate, Sodium stearate, sodium lauryl sulfate, etc.), nonionic surfactant (polyoxyethylene sorbitan fatty acid ester [Tween 80, Tween 60, Atlas Powder], polyoxyethylene castor oil derivative [HCO- 60, HCO-50, Nikko Chemicals], etc.), or polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, lecithin, gelatin, etc., and one of these or a combination of several may be used. .. The concentration at the time of use can be appropriately selected from the range of about 0.01% to 20%, and more preferably the concentration is used in the range of about 0.05% to 10%.

【0023】

A commonly used method is adopted for evaporation of the solvent in the oil phase. The method is carried out at normal pressure or gradually reduced pressure while stirring with a propeller type stirrer or a magnetic stirrer, or is carried out while adjusting the degree of vacuum using a rotary evaporator or the like. The microcapsules thus obtained are separated by centrifugation or filtration, and then the free bioactive peptides, drug-retaining substances, emulsifiers, etc. adhering to the surface of the microcapsules are repeated several times with distilled water. After washing, it is dispersed again in distilled water or the like and freeze-dried. To prevent aggregation of particles during washing, antiaggregating agents [eg, water-soluble sugars such as mannitol, lactose, glucose, starches (eg, corn starch, etc.), amino acids such as glycine, alanine, gelatin, fibrin, collagen Proteins such as, sodium chloride, sodium bromide, inorganic salts such as potassium carbonate, etc.] may be added. The anti-aggregation agent is particularly preferably mannitol. Warm if necessary to more completely desorb water and organic solvents in the microcapsules under reduced pressure.

【0024】

When microcapsules are produced by the phase separation method, a coacervating agent is gradually added to the W / O emulsion under stirring to precipitate and solidify the polymer polymer. The coacervating agent may be a polymer-based, mineral oil-based, or vegetable oil-based compound that is mixed with the solvent of the polymer polymer and does not dissolve the encapsulating polymer. For example, silicon oil. Examples include sesame oil, soybean oil, corn oil, cottonseed oil, coconut oil, flaxseed oil, mineral oil, n-hexane and n-heptane. These may be used by mixing two or more kinds. The microcapsules thus obtained are filtered and separated, and then repeatedly washed with heptane or the like to remove the coacervation agent. Further, the free drug is removed and the solvent is desorbed by the same method as the water drying method.

【0025】

When producing microcapsules by the spray drying method, the above W / O emulsion is sprayed into the drying chamber of a spray dryer device (spray dryer) using a nozzle, and the organic particles in the atomized droplets are contained in an extremely short time. Volatilize the solvent and water to prepare fine-grained microcapsules. Examples of the nozzle include a two-liquid nozzle type, a pressure nozzle type, and a rotating disc type. At this time, if desired, it is also effective to spray the aqueous solution of the above-mentioned anti-aggregation agent from another nozzle for the purpose of preventing the aggregation of the microcapsules at the same time as spraying the W / O emulsion. The microcapsules thus obtained are heated if necessary to more completely remove water and solvent in the microcapsules under reduced pressure.

【0026】

In the method of the present invention, water-soluble sugars such as antiaggregating agents [eg, mannitol, lactose, glucose, starches (eg, corn starch, etc.) are added to the microcapsules obtained by microencapsulating a physiologically active substance with a biodegradable polymer. , Amino acids such as glycine and alanine, proteins such as gelatin, fibrin and collagen, inorganic salts such as sodium chloride, sodium bromide and potassium carbonate, etc.] may be further added. The anti-aggregation agent is particularly preferably mannitol. The particle size of the microcapsules in the method of the present invention may be within a range that satisfies the dispersibility and needle-through property when used as a suspension injection, depending on the degree of sustained release, for example, the average diameter is about about. The range is from 1 to 300 μm, more preferably from about 5 to 150 μm.

【0027】

The microcapsules thus obtained are heated to a temperature equal to or higher than the glass transition temperature of the biodegradable polymer using the microcapsules as a base so that the particles of the microcapsules do not adhere to each other. The glass transition temperature refers to the midpoint glass transition temperature (Tmg) obtained when the temperature is raised at a heating rate of 10 or 20 ° C. per minute using a differential scanning calorimeter (DSC). When the drying step is included in the manufacturing process of the sustained-release microcapsules, the heating period is preferably performed after the drying, but is not particularly limited, and can be performed even after subdivision, for example. When the heating temperature is lower than the glass transition temperature of the biodegradable polymer used as the base, there is no effect of improving the initial release of the excess amount of the physiologically active substance, and when the heating temperature is too high, the fusion, deformation, and physiological activity of the microcapsules The risk of decomposition and deterioration of substances increases. The heating temperature cannot be unequivocally determined, but the physical characteristics of the biodegradable polymer used as the base (eg, molecular weight, stability, etc.), bioactive substances, particle size of microcapsules, heating time, degree of drying of microcapsules, etc. It can be appropriately determined in consideration of the heating method and the like. Preferably, the microcapsules are heated to a temperature equal to or higher than the glass transition temperature of the biodegradable polymer used as a base so that the particles of the microcapsules do not adhere to each other. More preferably, the microcapsules are heated to a temperature of about 5 ° C. or higher, which is higher than the glass transition temperature of the biodegradable polymer used as the base, so that the particles of the microcapsules do not adhere to each other. Particularly preferably, the microcapsules are heated to a temperature of about 10 ° C. or higher than the glass transition temperature of the biodegradable polymer used as the base so that the particles of the microcapsules do not adhere to each other.

【0028】

Specifically, for example, it is preferable to select a temperature higher than the glass transition temperature of the biodegradable polymer used as a base by about 5 to 40 ° C. More preferably, it is selected from a temperature about 5 to 30 ° C higher than the glass transition temperature of the biodegradable polymer used as the base. Further, it is particularly preferable to select a temperature higher than the glass transition temperature of the biodegradable polymer used as a base by about 10 to 30 ° C. The heating time also varies depending on the heating temperature, the amount of microcapsules to be treated, and the like, but in general, it is preferably within 2 weeks after the temperature of the microcapsules themselves reaches a predetermined temperature. Further, within 24 hours is particularly preferable. The heating method is not particularly limited, but any method may be used as long as the microcapsules are uniformly heated. Preferred specific examples of the heating method include, for example, a method of heating in a constant temperature bath, a fluidized tank, a moving layer or a kiln, a method of heating with microwaves, and the like. Among these, the method of heating in a constant temperature bath is preferable.

【0029】

The microcapsules produced by the method of the present invention have low toxicity and can be used safely. The microcapsules produced by the method of the present invention can be administered to a living body as fine granules as they are, but they can also be molded into various formulations and administered, which is a raw material for producing such formulations. It can also be used as a substance. Examples of the above-mentioned preparations include injections, orally-administered preparations (eg, powders, granules, capsules, tablets), nasal-administered preparations, suppositories (eg, rectal suppositories, vaginal suppositories) and the like. The amount of the physiologically active substance contained in these preparations may vary depending on the type of the physiologically active substance, the dosage form, the target disease, etc., but is usually about 0.001 mg to about 5 g per preparation, preferably about about. From 0.01 mg to about 2 g. These formulations can be produced by known methods commonly used in the formulation process. For example, microcapsules produced by the methods of the invention are dispersants (eg, Tween 80, HCO60 (manufactured by Nikko Chemicals), carboxymethyl cellulose, sodium alginate, etc.), preservatives (eg, methylparaben, propylparaben, benzyl alcohol, chlorobutanol, etc.), isotonic agents (eg, sodium chloride, glycerin, sorbitol, glucose, etc.) ) And the like, or dispersed in olive oil, sesame oil, lacquer oil, cottonseed oil, corn oil and other vegetable oils, propylene glycol and the like to form an oily suspending agent, which can be used as an injection. Further, the above-mentioned sustained release injection of microcapsules is prepared by adding an excipient (for example, mannitol, sorbitol, lactose, glucose, etc.) as a suspension in addition to the above composition, redispersing, and then freezing. A more stable sustained release injection can be obtained by drying or spray-drying to solidify and adding distilled water for injection or a suitable dispersion medium at the time of use.

【0030】

For example, in order to prepare an orally administered preparation, microcapsules produced by the method of the present invention are used, for example, excipients (eg, lactose, sucrose, starch, etc.), disintegrants (eg, starch, calcium carbonate, etc.) according to a method known per se. ), Binders (eg starch, gum arabic, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose, etc.) or lubricants (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.) and compression molding Then, if necessary, it can be made into an orally administered preparation by coating it in a manner known per se for the purpose of masking the taste, enteric or persistent. Examples of the coating agent include hydroxypropylmethyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, polyoxyethylene glycol, Tween 80, Brulonic F68, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxymethylcellulose acetate succinate, and Eudragit (Rome). (Manufactured by West Germany, methacrylic acid / acrylic acid copolymerization) and dyes such as titanium oxide and red iron oxide are used.

【0031】

For example, in order to prepare a nasal administration preparation, the microcapsules produced by the method of the present invention can be made into a solid, semi-solid or liquid nasal administration preparation according to a method known per se. For example, the solid form mentioned above includes the microcapsules as they are, or excipients (eg, glucose, mannitol, starch, microcrystalline cellulose, etc.), thickeners (eg, natural gums, cellulose derivatives, acrylic acid). Polymers, etc.) are added and mixed to obtain a powdery composition. The liquid form is almost the same as that of the injection, and is an oily or aqueous suspension. In the case of semi-solid form, an aqueous or oily gel agent or an ointment form is preferable. In addition, all of these include pH regulators (eg, carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide, etc.), preservatives (eg, paraoxybenzoic acid esters, chlorobutanol, benzalkonium chloride, etc.), etc. May be added.

【0032】

For example, in order to prepare a suppository, the microcapsules produced by the method of the present invention can be made into an oily or aqueous solid, semi-solid or liquid suppository according to a method known per se. The oily base used in the above composition may be any as long as it does not dissolve microcapsules, for example, glycerides of higher fatty acids [eg, cocoa butter, witepsols (dynamite Nobel), etc.], intermediate fatty acids [eg, migliols]. (Dynamite Nobel), etc.], or vegetable oils (eg, sesame oil, soybean oil, cottonseed oil, etc.). Examples of the aqueous base include polyethylene glycols and propylene glycol, and examples of the aqueous gel base include natural gums, cellulose derivatives, vinyl polymers and acrylic acid polymers. The microcapsules produced by the method of the present invention are preferably used as an injection.

【0033】

The dose of the microcapsules produced by the method of the present invention includes the type and content of the main drug, the physiologically active substance, the dosage form, the duration of drug release, the target animal (eg, mouse, rat, horse, cow, human, etc.). (Warm-blooded mammal), although it varies depending on the purpose of administration, it may be an effective amount of the main drug. For example, the dose per adult (body weight 50 kg) can be appropriately selected from the range in which the weight of the microcapsules is about 1 mg to about 10 g, preferably about 10 mg to about 2 g. The volume of the suspension when administered as the above-mentioned injection can be appropriately selected from the range of about 0.1 to 5 ml, preferably about 0.5 to 3 ml.

【0034】

Biodegradable polymers used as the base of microcapsules are known methods, for example, JP-A-50-17525, No. 56-45920, No. 57-118512, No. 57-150609, No. 61-28521, It can be produced according to the method described in Japanese Patent Publication No. 62-54760, European Patent Publication No. 481732, or a method similar thereto.

【0035】

[Example]

Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples. In the following description, Tmg indicates the above-mentioned intermediate point glass transition temperature. Reference example 1 A 1000 ml 4-neck flask equipped with a nitrogen introduction tube and a cooling tube is charged with 495.4 g of a 90% (w / w) D, L-lactic acid aqueous solution for 5 hours from 90 ° C, 400 mmHg to 150 ° C, 30 mmHg under a nitrogen stream. Distillate water was removed by heating under reduced pressure. Further, the mixture was heated at 5 to 7 mmHg and 150 to 175 ° C. under reduced pressure for 65 hours and then cooled to obtain an amber-colored lactic acid polymer. The obtained polymer was dissolved in 1000 ml of dichloromethane and injected into warm water at 60 ° C. with stirring. The separated rice cake-shaped polymer polymers were collected and vacuum dried at 30 ° C. The peak molecular weight of the obtained lactic acid polymer measured by GPC was 16,000, and the Tmg measured by DSC was 40 ° C.

【0036】

Reference example 2 90% (w / w) D, L-lactic acid aqueous solution 247.7 g and glycolic acid 190.2 g were charged in a 1000 ml 4-neck flask equipped with a nitrogen introduction tube and a cooling tube, and 90 ° C under a nitrogen stream, 500 mmHg to 150 ° C. , 30 mmHg was heated under reduced pressure for 5 hours to remove distillate. Further, the mixture was heated under reduced pressure for 28 hours at 5 to 7 mmHg and 150 to 180 ° C. and then cooled to obtain an amber-colored lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 50/50 (mol%)). The obtained polymer was dissolved in 1000 ml of dichloromethane and injected into warm water at 60 ° C. with stirring. The separated rice cake-shaped polymer polymers were collected and vacuum dried at 30 ° C. The peak value of the molecular weight of the obtained lactic acid / glycolic acid copolymer measured by GPC was 12,000, and the Tmg measured by DSC was 36 ° C.

【0037】

Reference example 3 145.8 g of D, L-2-hydroxybutyric acid and 177.7 g of glycolic acid were placed in a 1000 ml 4-necked flask equipped with a nitrogen inlet tube and a cooling tube, and the temperature was changed from 100 ° C, 500 mmHg to 150 ° C, 30 mmHg under a nitrogen stream for 3.5 hours. Distilled water was removed by heating under reduced pressure. Furthermore, it is heated under reduced pressure at 5 to 7 mmHg and 150 to 180 ° C for 27 hours and then cooled to obtain an amber 2-hydroxybutyric acid / glycolic acid copolymer (2-hydroxybutyric acid / glycolic acid = 37.5 / 62.5 (mol%). )) Was obtained. The obtained polymer was dissolved in 1000 ml of dichloromethane and injected into warm water at 60 ° C. with stirring. The separated rice cake-shaped polymer polymers were collected and vacuum dried at 25 ° C. The peak value of the molecular weight of the obtained 2-hydroxybutyric acid / glycolic acid copolymer measured by GPC was 14,000, and the Tmg measured by DSC was 26 ° C.

【0038】

Reference example 4 90% (w / w) D, L-lactic acid aqueous solution 300g and 90% (w / w) L-lactic acid aqueous solution 100g were charged in a 1000ml 4-neck flask equipped with a nitrogen introduction tube and a cooling tube, and 100 ° under a nitrogen stream. Distillate was removed by heating under reduced pressure from C, 500 mmHg to 150 ° C, 30 mmHg for 4 hours. Further, the mixture was heated under reduced pressure for 24 hours at 5 to 7 mmHg and 150 to 180 ° C. and then cooled to obtain an amber-colored lactic acid polymer. The obtained polymer was dissolved in 1000 ml of dichloromethane and injected into warm water at 60 ° C. with stirring. The separated rice cake-shaped polymer polymers were collected and vacuum dried at 30 ° C. The peak value of the molecular weight of the obtained lactic acid copolymer measured by GPC was 7,000, and the Tmg measured by DSC was 33 ° C.

【0039】

Reference example 5 400 mg of leuproreline acetate (TAP-144) was dissolved in 0.5 ml of distilled water, 4.0 g of the lactic acid polymer obtained in Reference Example 1 was added to a solution dissolved in 7.5 ml of dichloromethane, and mixed with a small homogenizer for 60 seconds. A W / O type emulsion was obtained. After cooling this emulsion to 17 ° C, it is injected into 1000 ml of a 0.1% (w / v) polyvinyl alcohol aqueous solution that has been adjusted to 19 ° C in advance, and W / O / W using a turbine-type homomixer. It was made into a mold emulsion. This W / O / W type emulsion was stirred at room temperature to volatilize dichloromethane, and the internal W / O type emulsion was solidified and then collected using a centrifuge. After dispersing this in distilled water again, it was further centrifuged to wash free drugs and the like. The collected microcapsules were obtained as a powder by adding 0.3 g of D-mannitol and lyophilizing.

【0040】

Reference example 6 The microcapsules obtained as powder in Reference Example 5 were heated in a constant temperature bath at 90 ° C, which is 50 ° C higher than the Tmg of the base lactic acid polymer, for 2 hours, and then in the same manner as in Example 1 described later. An in vitro elution test was attempted, but the microcapsules were fused and coalesced and could not be dispersed in a phosphate buffer solution at pH 7.0.

【0041】

Reference example 7 400 mg of leuprolerin acetate (TAP-144) was dissolved in 0.4 ml of distilled water, and 4.0 g of the lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 50/50 (mol%)) obtained in Reference Example 2 was added. The solution was added to a solution dissolved in 5.0 ml of dichloromethane and mixed with a small homogenizer for 60 seconds to obtain a W / O type emulsion. Using this emulsion, microcapsules were prepared in the same manner as in Reference Example 5.

【0042】

Reference example 8 500 mg of thyrotropin release (TRH) was dissolved in 0.2 ml of distilled water, and 4.5 g of the lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 50/50 (mol%)) obtained in Reference Example 2 was 4.7 ml of dichloromethane. In addition to the solution dissolved in, the mixture was mixed with a small homogenizer for 60 seconds to obtain a W / O emulsion. Using this emulsion, microcapsules were prepared in the same manner as in Reference Example 5.

【0043】

Reference example 9 400 mg of leuproreline acetate (TAP-144) was dissolved in 0.4 ml of distilled water, and the 2-hydroxybutyric acid / glycolic acid copolymer obtained in Reference Example 3 (2-hydroxybutyric acid / glycolic acid = 37.5 / 62.5 (mol)). %)) and added to a solution of equal mixture 4.0g of the resultant lactic acid polymer was dissolved in dichloromethane 5.0ml in reference example 4, were mixed for 60 seconds with a small homogenizer, W / O error was obtained Marujon. Using this emulsion, microcapsules were prepared in the same manner as in Reference Example 5.

【0044】

Example 1 The microcapsules obtained as powder in Reference Example 5 were heated in a constant temperature bath at 45 ° C, which is 5 ° C higher than the Tmg of the base lactic acid polymer, for 2 weeks. The in vitro dissolution test of the obtained microcapsules was carried out in a phosphate buffer solution at 37 ° C. and pH 7.0 at 120 cycles / min. The drug release rate after 1 day obtained is shown in [Table 1]. Example 2 The microcapsules obtained as powder in Reference Example 5 were heat-treated in a constant temperature bath at 60 ° C, which is 20 ° C higher than the Tmg of the base lactic acid polymer, for 4 hours, and then in vitro as in Example 1. An dissolution test was performed. The drug release rate after 1 day obtained is shown in [Table 1].

[table 1]

<img file="JPP3277342B2_D0001.tif" />【0045】

Example 3 The microcapsules obtained as powder in Reference Example 7 were heated in a constant temperature bath at 56 ° C, which is 20 ° C higher than the Tmg of the lactic acid / glycolic acid copolymer as the base, for 1 hour, and then in Example 1 and Similarly, an in vitro dissolution test was conducted. The drug release rate after 1 day is shown in [Table 2]. Example 4 The microcapsules obtained as powder in Reference Example 7 were heated in a constant temperature bath at 56 ° C, which is 20 ° C higher than the Tmg of the lactic acid / glycolic acid copolymer as the base, for 5 hours, and then in Example 1 and Similarly, an in vitro dissolution test was conducted. The drug release rate after 1 day obtained is shown in [Table 2].

[Table 2]

<img file="JPP3277342B2_D0002.tif" />【0046】

Example 5 The microcapsules obtained as powder in Reference Example 8 were heated in a constant temperature bath at 56 ° C, which is 20 ° C higher than the Tmg of the lactic acid / glycolic acid copolymer as the base, for 4 hours, and then in Example 1 and Similarly, an in vitro dissolution test was conducted. The drug release rate after 1 day obtained is shown in [Table 3]. In addition, the drug release rate without heat treatment was shown as a control group.

[Table 3]

<img file="JPP3277342B2_D0003.tif" />【0047】

Example 6 The microcapsules obtained as powder in Reference Example 9 were placed in a constant temperature bath in a Tmg (31) mixture of a base 2-hydroxybutyric acid / glycolic acid copolymer and a lactic acid polymer in a 1: 1 (W / W) mixture. After heating at 55 ° C, which is 24 ° C higher than ° C) for 8 hours, an in vitro dissolution test was performed in the same manner as in Example 1. The drug release rate after 1 day obtained is shown in [Table 4]. Example 7 The microcapsules obtained as powder in Reference Example 9 were placed in a constant temperature bath in a Tmg (31) mixture of a base 2-hydroxybutyric acid / glycolic acid copolymer and a lactic acid polymer in a 1: 1 (W / W) mixture. After heating at 50 ° C, which is 19 ° C higher than ° C) for 1 week, an in vitro dissolution test was performed in the same manner as in Example 1. The drug release rate after 1 day obtained is shown in [Table 4].

[Table 4]

<img file="JPP3277342B2_D0004.tif" />【0048】

Example 8 The microcapsules obtained in Example 1 were subcutaneously administered to rats (n = 5), and the drug release rate calculated by quantifying the residual drug after 1 day is shown in [Table 5].

[Table 5]

<img file="JPP3277342B2_D0005.tif" />【0049】

Example 9 60 mg of interferon α (IFN-α) and 200 mg of human serum albumin were dissolved in 0.5 ml of distilled water, and a lactic acid / glycolic acid copolymer (Wako Pure Chemical Acid, lactic acid / glycolic acid = 50/50 (mol), weight average molecular weight). 6,400, 1.74 g of Tmg 30 ° C) measured by DSC) was added to a solution dissolved in 2.0 ml of dichloromethane and mixed with a small homogenizer for 20 seconds to obtain a W / O type emulsion. Using this emulsion, microcapsules were prepared in the same manner as in Reference Example 5 except that D-mannitol was not added. After adding 87 mg of D-mannitol to 75 mg of the obtained microcapsules, heat treatment was performed at 50 ° C, which is 20 ° C higher than the Tmg of the base lactic acid / glycolic acid copolymer, for 16 hours. The microcapsules thus obtained were subcutaneously administered to rats (n = 4), and the results of measuring the blood IFN-α concentration 1 hour later are shown in [Table 6]. In addition, the blood IFN-α concentration without heat treatment was shown as a control group. The dose of microcapsules was 10 mg in each case.

[Table 6]

<img file="JPP3277342B2_D0006.tif" />【0050】

[Effect of the invention]

According to the method of the present invention, the excessive initial release amount of the physiologically active substance after one day is suppressed, that is, the initial release rate is small, and the excellent sustained release property of releasing a constant amount of the physiologically active substance for a long period of time immediately after administration is achieved. The microcapsules shown can be produced.