JP2002255857A - Sustained release preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sustained release preparation and its dispersing medium, capable of suppressing the initial release of a physiologically active substance directly after its administration and releasing a constant amount of the physiologically active substance for a long period of time. SOLUTION: This sustained release preparation and its dispersing medium, capable of suppressing the initial release of a physiologically active substance directly after its administration and releasing a constant amount of the physiologically active substance for a long period of time is obtained by adding a cationic substance or polyols to the outside of a matrix or its dispersing medium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a sustained-release preparation which releases a small amount of a physiologically active substance over a long period of time and has a small initial release immediately after administration, and a dispersion medium thereof.

[0002]

2. Description of the Related Art Biologically active peptides are known to exhibit various pharmacological actions in living organisms, and are being applied to pharmaceuticals. However, since these bioactive peptides 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. For example, growth hormone (hereinafter,
GH) is a typical hormone originally produced and secreted by the anterior pituitary gland, which promotes the growth of the body, and is involved in sugar / lipid metabolism, protein assimilation, cell proliferation and differentiation. It is a physiologically active peptide having a wide variety of physiological actions, and is currently mass-produced by Escherichia coli using gene recombination technology, and is widely clinically applied worldwide as a pharmaceutical. However, GH has a short half-life in vivo, and requires frequent administration to maintain an effective blood concentration.
In particular, in the case of short stature with GH secretion deficiency, it is a fact that daily subcutaneous administration for a long period of several months to 10 years or more is performed to infants or young patients. In order to address such problems inherent in bioactive peptide-based drugs, various studies on drug delivery systems have been conducted. For example, it is a sustained release agent that releases a bioactive peptide over a long period of time. JP-A-8-2176
No. 91 (WO96 / 07399) discloses that a water-soluble peptidic physiologically active substance is converted into a water-insoluble or poorly water-soluble polyvalent metal salt by using an aqueous zinc chloride solution or the like, and a gradual mixture containing this and a biodegradable polymer. A method for making a release formulation is disclosed. JP-A-11-322631 discloses a bioactive peptide powder obtained by adding a water-miscible organic solvent and / or volatile salts to an aqueous solution of the bioactive peptide and freeze-drying the bioactive peptide powder. A method for producing a sustained-release preparation characterized by removing an organic solvent after dispersing the polymer in an organic solvent solution is disclosed.
Japanese Patent Application Laid-Open No. 9-132524 discloses a method for producing a sustained-release microcapsule containing a physiologically active substance and a biodegradable polymer. About 24 ~
A method for producing sustained-release microcapsules having extremely low residual organic solvent by heating and drying for 120 hours and having extremely excellent clinical properties as a drug is disclosed.

[0003]

It is desirable that a sustained-release preparation can release a constant amount of a physiologically active substance over a long period of time while maintaining the activity of the physiologically active substance. Is required.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and obtained a finely powdered physiologically active substance obtained by adjusting the concentration of alkali metal ions to about 10 μg / mL or less. In the case of sustained-release preparations containing biodegradable polymers, and by coexisting cationic substances or polyols with matrices such as microcapsules, the initial release of physiologically active substances immediately after administration is dramatically reduced unexpectedly. The present inventors have found that a constant release amount of a physiologically active substance can be released over a long period of time and that a sustained-release preparation having extremely excellent clinical properties can be produced as a pharmaceutical, and the present invention has been completed based on these.

That is, the present invention provides (1) a sustained-release preparation comprising a combination of a matrix containing a physiologically active substance and a cationic substance and / or a polyol, wherein the initial release of the physiologically active substance is suppressed; A) a sustained-release preparation according to the above (1), which comprises mixing a matrix containing a physiologically active substance with a cationic substance and / or a polyol; And / or the sustained release preparation according to the above (1), wherein the polyol is retained, (4) the sustained release preparation according to the above (1), wherein the cationic substance is a basic substance or a water-soluble polyvalent metal salt, (5) The sustained-release preparation according to the above (4), wherein the basic substance is a basic amino acid, and (6) the above-mentioned (5), wherein the basic amino acid is arginine or lysine.
The sustained release preparation according to the above, (7) the sustained release preparation according to the above (4), wherein the basic substance is a basic additive, and (8) the basic additive is benzalkonium chloride or N-methylglucamine. (7) the sustained release preparation according to the above (7), (9) the sustained release preparation according to the above (4), wherein the basic substance is a basic peptide, a basic polyamine or a basic polysaccharide, and (10) a basic peptide. Is the protamine or a salt thereof, (11) the sustained release formulation according to the above (9), wherein the basic polyamine is spermidine or spermine, and (12) the chitosan is a basic polysaccharide. (9)
The sustained-release preparation according to the above (13), wherein the water-soluble polyvalent metal salt is a water-soluble zinc salt, the sustained-release preparation according to the above (4), and (14) the water-soluble zinc salt is zinc chloride or zinc acetate. (1
The sustained-release preparation according to 3), (15) the sustained-release preparation according to (1), wherein the polyol is polyethylene glycol or propylene glycol, and (16) the above-mentioned (1), wherein the bioactive substance is a bioactive peptide. Sustained release formulation,
(17) The bioactive peptide has a molecular weight of about 200 to about 50
(16) the sustained release preparation according to the above (16),
8) The bioactive peptide has a molecular weight of about 5,000 to about 50
(16) the sustained release preparation according to the above (16),
9) The sustained release preparation according to the above (16), wherein the bioactive peptide is a hormone, a cytokine, a hematopoietic factor, a growth factor, or an enzyme, and (20) the sustained release according to the above (16), wherein the bioactive peptide is a human growth hormone. (21) The above-mentioned (1), wherein the matrix base is a biodegradable polymer.
(22) the biodegradable polymer is α
-The sustained release preparation according to the above (21), which is a homo- or copolymer of a hydroxycarboxylic acid or a mixture thereof, (23) the biodegradable polymer has a composition ratio of lactic acid / glycolic acid of about 100/0 to about 100/0. The sustained-release preparation according to the above (21), which is a copolymer of 40/60 mol%, (24) the above-mentioned (2), wherein the biodegradable polymer is a lactic acid homopolymer.
(25) The sustained release preparation according to the above (21), wherein the biodegradable polymer has a weight average molecular weight of about 3,000 to about 50,000; The sustained release preparation according to the above (1), which is a capsule; (27) the sustained release preparation according to the above (1), which is for injection; (28) a matrix containing a physiologically active substance, a cationic substance and / or a polyol (1) the sustained release preparation according to the above (1), which contains a compound and a dispersion medium; (29) the dispersion medium containing a cationic substance and / or a polyol for producing the sustained release preparation according to the above (28), 30) the dispersion medium according to the above (29), wherein the cationic substance is a basic substance or a water-soluble polyvalent metal salt; (31) the dispersion medium according to the above (30), wherein the basic substance is a basic amino acid;
2) The dispersion medium according to (31), wherein the basic amino acid is arginine or lysine, (33) the dispersion medium according to (30), wherein the basic substance is a basic additive, and (34) the dispersion medium according to (30). (33) The dispersion medium according to the above (33), which is benzalkonium chloride or N-methylglucamine; (35) The dispersion medium according to the above (30), wherein the basic substance is a basic peptide, a basic polyamine or a basic polysaccharide. (36) wherein the basic peptide is protamine or a salt thereof;
The dispersion medium according to 5), (37) the dispersion medium according to (35), wherein the basic polyamine is spermidine or spermine, and (38) the dispersion medium according to (3), wherein the basic polysaccharide is chitosan.
The dispersion medium according to 5), (39) the dispersion medium according to (30), wherein the water-soluble polyvalent metal salt is a water-soluble zinc salt, and (40) the dispersion medium according to (39), wherein the water-soluble zinc salt is zinc chloride or zinc acetate. )
(41) The dispersion medium according to (2), wherein the polyol is polyethylene glycol or propylene glycol.
9) The dispersion medium according to the above (29), which contains an osmotic pressure adjusting agent, (43) the dispersion medium according to the above (42), wherein the osmotic pressure adjusting agent is a saccharide or a salt. )
The dispersion medium according to the above (29), which comprises a thickening agent, (4)
5) The dispersion medium according to the above (44), wherein the thickening agent is a water-soluble polysaccharide, (46) the dispersion medium according to the above (29), which contains a surfactant, and (47) the surfactant is nonionic. The dispersion medium according to the above (46) which is a surfactant, (48) the dispersion medium according to the above (29) which is for injection, and (49) the sustained release preparation containing a matrix containing a physiologically active substance are cationic. A method for suppressing the initial release of a physiologically active substance, which comprises mixing a substance and / or a polyol;
0) Fine particles of a physiologically active substance obtained by adjusting the concentration of alkali metal ions in a physiologically active substance solution to about 10 μg / mL or less, (51) Fine particles of the above (50), wherein the physiologically active substance is a physiologically active peptide (52) The fine particles according to the above (51), wherein the bioactive peptide is a hormone, a cytokine, a hematopoietic factor, a growth factor or an enzyme;
(53) the fine particles according to the above (51), wherein the physiologically active peptide is human growth hormone; (54) the fine particles according to the above (50), wherein the weight average particle diameter is about 0.5 μm to about 2.0 μm; 55) A method for producing fine particles of a physiologically active substance, characterized by using a physiologically active substance solution having an alkali metal ion concentration of about 10 μg / mL or less in the physiologically active substance solution. (56) Ammonium acetate is further added to the solution. (50) The fine particles described in (50) above, (57)
(5) The sustained-release preparation containing fine particles according to (5), wherein the base of the sustained-release preparation is a biodegradable polymer (5).
(60) The sustained-release preparation according to (58), wherein the biodegradable polymer is a homo- or copolymer of α-hydroxycarboxylic acids or a mixture thereof. The biodegradable polymer has a composition ratio of lactic acid / glycolic acid of about 100/0 to about 40/60 mol%.
The sustained release preparation according to the above (58), which is a copolymer of
1) The sustained-release preparation according to the above (58), wherein the biodegradable polymer is a lactic acid homopolymer, and (62) the biodegradable polymer has a weight average molecular weight of about 3,000 to about 50,000.
(58), and (63).
It is intended to provide a sustained release preparation according to the above (57), which is a microcapsule.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The physiologically active substance in the present invention is not particularly limited, but, for example, a peptide compound having a physiological activity (hereinafter, referred to as “bioactive peptide”), other antibiotics, antifungals, anti- Lipemic, antineoplastic, antipyretic, analgesic, anti-inflammatory, antitussive expectorant, sedative,
Muscle relaxants, antiepileptic drugs, antiulcer drugs, antidepressants, antiallergic drugs, inotropic drugs, antiarrhythmic drugs, vasodilators, antihypertensive diuretics, antidiabetic drugs, anticoagulants, hemostatic drugs, antiplatelet drugs ,
Examples include antituberculous drugs, hormonal drugs, narcotic antagonists, bone resorption inhibitors, bone formation promoters, and angiogenesis inhibitors. this house,
Particularly, a peptide compound is preferable. Examples of the bioactive peptide in the present invention include various peptides or proteins that have useful bioactivity for mammals and can be used clinically. The "bioactive peptide"
As the monomer, those having a molecular weight of, for example, about 200 to 500,000 are used, and those having a molecular weight of about 1,000 to 500,000 are generally used. More preferably, the molecular weight is from 5,000 to about 50.
000 peptides are used. A typical example of the activity of a physiologically active peptide is a hormonal action. The physiologically active peptide may be a natural product, a synthetic product, a semi-synthetic product, or a derivative or analog thereof. The mode of action of the physiologically active peptide may be either agonistic or antagonistic. As the physiologically active peptide in the present invention, for example, peptide hormones, cytokines, peptide neurotransmitters, hematopoietic factors, various growth factors, enzymes, peptide antibiotics, analgesic peptides and the like are used. Examples of peptide hormones include insulin, somatostatin, somatostatin derivatives (Sandostatin, U.S. Pat. Nos. 4,087,390, 4,093,574, 4,100,117, 4,253,998). No.), growth hormone (G
H), natriuretic peptide, gastrin, prolactin, adrenocorticotropic hormone (ACTH), ACTH derivatives (such as eviratide), melanocyte stimulating hormone (MSH), thyroid hormone releasing hormone (TRH),
Salts and derivatives thereof (Japanese Patent Laid-Open No. 50-123273)
Thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), thymosin (thymosin), motilin, vasopressin, Vasopressin derivative @ desmopressin [Journal of the Endocrine Society of Japan, Vol. 54, No. 5, No. 676-6]
91 (1978)], oxytocin, calcitonin, parathyroid hormone (PTH), glucagon, secretin, pancreozymine, cholecystokinin, angiotensin, human placental lactogen, glucagon-like peptide (GLP-1) and derivatives thereof. (JP-A-6-8
0584, JP-A-7-2695, EP658568
JP-A-8-245696, JP-A-8-26909
7, WO97 / 15296, WO97 / 31943
No., WO98 / 19698, WO98 / 43658
No., Tokuyohei 10-511365, WO 99/5531
No. 0, Tokio Hei 11-513983, CA227032
No. 0, WO 99/64061, Tokuyohei 11-5149
No. 72, Special Table 2000-500505, WO2000
No./66138, WO2000 / 66142, WO2
000/78333, JP-A-2001-11095,
Tissue Eng. 7 (1) 35-44 (2001), Diabet
ologia 43 (10) 1319-1328 (200
0), WO2000 / 34331, WO2000 / 3
No. 4,332, U.S. Pat. No. 6,268,343, U.S. Publication No. 200101071, U.S. Publication No. 20010069.
No. 43, EP 0733644, WO2000 / 770
No. 39, WO99 / 43707, WO99 / 4334
No. 1, WO99 / 43706, WO99 / 43708
No., WO99 / 43705, WO99 / 29336
No., WO2000 / 37098, EP0969016
No. 5,981,488; U.S. Pat.
958,909, WO93 / 25579, WO98
No./43658, EP0869135, US Pat. No. 5,614,492, US Pat. No. 5,545,618.
No. 5,120,712; U.S. Pat.
118,666, WO95 / 05848, WO91
/ 11457, EP0708179, WO96 / 0
No. 6628, EP 0658568, WO 87/069
No. 41), metastin and its derivatives (WO20)
00/24890) and the like. Preferable peptide hormones include insulin and growth hormone. As the cytokine, for example, lymphokine, monokine and the like are used. Examples of lymphokines include interferons (alpha type, beta type, gamma type, etc.) and interleukins (eg, IL
−2,3,4,5,6,7,8,9,10,11,12
Etc.) are used. Monokines include, for example, interleukin-1 (IL-1), tumor necrosis factor (TN)
F) and the like are used. The cytokine is preferably lymphokine or the like, more preferably interferon or the like, particularly preferably interferon alpha or the like. As the peptide neurotransmitter, for example, substance P, serotonin, GABA and the like are used.

[0007] Examples of hematopoietic factors include erythropoietin (EPO) and colony stimulating factors (G-CSF, GM-
CSF, M-CSF, etc.), thrombopoietin (TP
O), platelet growth stimulating factor, megakaryocyte potentiator and the like are used. Various growth factors include, for example, basic or acidic fibroblast growth factor (FGF)
Alternatively, these families (eg, EGF, TGF
-Shiori ATGF-Toyo APDGF, acidic FGF, basic FG
F, FGF-9, etc.), nerve cell growth factor (NGF) or a family thereof (eg, BDNF, NT-
3, NT-4, CNTF, GDNF, etc.), insulin-like growth factors (eg, IGF-1, IGF-2, etc.), factors involved in bone growth (BMP), or a family of these. Examples of enzymes include superoxide dismutase (SOD), urokinase, tissue plasminogen activator (TPA),
Asparaginase, kallikrein and the like are used. As the peptide antibiotic, for example, polymyxin B, colistin, gramicidin, bacitracin and the like are used.
Examples of the analgesic peptide include enkephalin and enkephalin derivatives [US Pat. No. 4,277,394,
European Patent Application Publication No. 31567], endorphin, kyotorphin and the like. Other physiologically active peptides include thymopoietin, dynorphin, bombesin, caerulein, thymostimulin, thymic factor (THF), blood thymic factor (FTS)
And its derivatives (see U.S. Pat. No. 4,229,438), and other thymic factors [Ayumi, 12
5, No. 10, pages 835-843 (1983)],
Peptides having neurotensin, bradykinin and endothelin antagonism (European Patent Publication 43
No. 6189, No. 457195, No. 496452
And JP-A-3-94692 and JP-A-3-130299). The physiologically active peptides particularly preferably applied to the present invention include luteinizing hormone-releasing hormone (LH-RH) and derivatives having the same action or LH-RH antagonists, growth hormones,
Insulin and the like can be mentioned, and among them, growth hormone, especially human growth hormone is preferable.

In the present invention, when the bioactive peptide contains a metal, if necessary, the metal contained in the bioactive peptide may be removed in advance.
As a method for removing the metal, a known method is used.
For example, an aqueous hydrochloric acid solution of insulin is dialyzed against water or an ammonium acetate solution and then lyophilized to obtain insulin in an amorphous state and a minimum amount of metal. The growth hormone may be derived from any species, but is preferably human growth hormone. In addition, natural sources extracted from the pituitary gland and the like are also used in the present invention, but are preferably recombinant GH (see Japanese Patent Publication Nos. 6-12996 and 6-48987), and more preferably. Is a recombinant hGH having the same structure as the natural type having no methionine at the N-terminus. The GH may be a metal salt, but GH substantially containing no metal is also used. As hGH,
Not only those having a molecular weight of about 22K daltons but also those having a molecular weight of about 20K daltons (see JP-A-7-101877 and JP-A-10-265404) may be used. Further, hGH derivatives or their related proteins (WO
99/03887) may be used.

The amount of the physiologically active substance in the sustained-release preparation of the present invention varies depending on the kind of the physiologically active substance and the like.
(W / W), preferably about 0.2 to 30% (W / W).
W), more preferably about 0.5 to 20% (W /
W). The matrix in the present invention is a solid substance containing a physiologically active substance as a base (for example, a biodegradable polymer) and optionally containing additives, and is substantially a sustained-release substance. , For example, in the form of a microcapsule, an embedding rod, or the like. Examples of the biodegradable polymer used in the present invention include α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.), and hydroxytricarboxylic acids (eg, citric acid, etc.). And the like, a polymer having a free carboxyl group or a mixture thereof, a poly-α-cyanoacrylate, a polyamino acid (for example, poly-γ-benzyl-L-
Glutamic acid, etc.), maleic anhydride polymers (for example,
Styrene-maleic acid polymer). These may be either homopolymers or copolymers. The type of polymerization may be random, block, or graft. When the α-hydroxycarboxylic acids, hydroxydicarboxylic acids, and hydroxytricarboxylic acids have an optically active center in the molecule, D-,
Both L- and DL-forms can be used. Among these, biodegradable polymers having a terminal free carboxyl group, for example, polymers synthesized from α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, etc.) (eg, lactic acid polymers, lactic acid-glycols) Acid copolymer) and poly-α-cyanoacrylate. As the biodegradable polymer, more preferably, such as a polymer synthesized from α-hydroxycarboxylic acids,
Particularly preferred are lactic acid-glycolic acid polymers and the like.

In this specification, lactic acid-glycolic acid polymer may be simply referred to as lactic acid-glycolic acid copolymer, as well as a homopolymer such as polylactic acid and polyglycolic acid. When a lactic acid-glycolic acid polymer (lactic acid-glycolic acid copolymer or homopolymer) is used as the biodegradable polymer, its composition ratio (mol%) is about 10%.
0/0 to about 40/60 is preferred, and about 85/15 to about 50/50 is more preferred. The weight average molecular weight of the lactic acid-glycolic acid polymer is from about 3,000 to about 5
Of about 3,000 to about 25,000.
00 is more preferred, from about 5,000 to about 20,000
Is more preferred. The dispersity (weight average molecular weight / number average molecular weight) of the lactic acid-glycolic acid polymer is preferably about 1.2 to about 4.0, and more preferably about 1.5 to about 3.5.

As for the weight average molecular weight and the degree of dispersion in the present specification, the former has a weight average molecular weight of 12,000,
0,52,000,22,000,9,200,5,0
A value in terms of polystyrene measured by gel permeation chromatography (GPC) using nine kinds of polystyrenes of 50, 2,950, 1,050, 580, and 162 as a reference substance, and the latter is a value calculated from this value. The measurement is performed using GPC column KF804L x 2 (manufactured by Showa Denko) and RI monitor L-3300 (manufactured by Hitachi, Ltd.) using chloroform as a mobile phase.

The term "biodegradable polymer having a free carboxyl group at the terminal" refers to a polymer having a number average molecular weight almost identical to the number average molecular weight determined by the above-described GPC measurement. The average molecular weight is calculated as follows. About 1 g to about 3 g of a biodegradable polymer is dissolved in a mixed solvent of acetone (25 mL) and methanol (5 mL), and phenolphthalein is used as an indicator to reduce the carboxyl group in this solution to 0.05 N alcoholic potassium hydroxide. The solution was rapidly titrated with stirring at room temperature (20 ° C.), and the number average molecular weight by terminal group quantification was calculated by the following equation. Number average molecular weight by terminal group quantification = 20,000 × A / B A: Mass of biodegradable polymer (g) B: 0.05N alcoholic potassium hydroxide solution (mL) added up to the end of titration Number by terminal group quantification While the average molecular weight is an absolute value, the number average molecular weight determined by GPC is determined by various analysis and analysis conditions (eg, mobile phase type, column type, reference material, selection of slice width, selection of baseline, etc.). ), It is difficult to obtain a unique numerical value, but it is considered that the number average molecular weights obtained by both measurements are almost the same, for example, in a polymer synthesized from α-hydroxycarboxylic acids. Number average molecular weight by quantification is G
It is in the range of about 0.5 times to about 2 times the number average molecular weight by PC measurement, preferably about 0.7 times to about 1.5 times.
It is within the range of double. For example, one or more types of α
In a polymer synthesized from -hydroxycarboxylic acids by a non-catalytic dehydration polycondensation method and having a free carboxyl group at a terminal, the number average molecular weight measured by GPC and the number average molecular weight determined by terminal group quantification are almost the same. On the other hand, in a polymer synthesized from a cyclic dimer by a ring-opening polymerization method using a catalyst and having essentially no free carboxyl group at the terminal, the number average molecular weight determined by the terminal group is determined by the GPC measurement. Significantly more than about twice the average molecular weight. By this difference, a polymer having a free carboxyl group at a terminal can be clearly distinguished from a polymer having no free carboxyl group at a terminal.

A lactic acid-glycolic acid polymer having a free carboxyl group at a terminal can be prepared by a method known per se, for example, a method described in JP-A-61-28521 (for example, a dehydration polycondensation reaction without inorganic catalyst or an inorganic polymer). Production method by a dehydration polycondensation reaction in the presence of a solid acid catalyst). The decomposition / elimination rate of the lactic acid-glycolic acid polymer greatly varies depending on the composition ratio or the weight average molecular weight.
Due to the slow elimination, the release period can be extended (eg, about 6 months) by lowering the glycolic acid fraction or increasing the molecular weight. Conversely, the release period can be shortened (eg, about one week) by increasing the glycolic acid fraction or decreasing the molecular weight. For example, in order to prepare a one-week to two-month sustained-release preparation, it is preferable to use a lactic acid-glycolic acid polymer having the above composition ratio and weight average molecular weight.

Therefore, it is preferable that the composition of the biodegradable polymer used in the present invention is appropriately selected depending on the kind of the desired physiologically active peptide, a desired sustained release period and the like. As a specific example, for example, when GH is used as a physiologically active peptide, it is preferable to use a lactic acid-glycolic acid polymer, and the lactic acid-glycolic acid polymer has a lactic acid / glycolic acid composition ratio (mol%). Is preferably a lactic acid-glycolic acid copolymer of 85/15 to about 50/50, more preferably about 75/2.
5 to about 50/50 lactic acid-glycolic acid copolymer. The weight average molecular weight is preferably about 8,000 to about 20,000, more preferably about 10,000.
00 to about 20,000. Further, the dispersity (weight average molecular weight / number average molecular weight) of the lactic acid-glycolic acid polymer is preferably about 1.2 to about 4.0, and more preferably about 1.5 to about 3.5. The lactic acid-glycolic acid polymer to be used can be produced according to a known method such as the method described in the above publication. The polymer is preferably produced by non-catalytic dehydration polycondensation. It is preferable to use a lactic acid-glycolic acid polymer (PLGA) in which the number average molecular weight by the GPC measurement method and the number average molecular weight by the terminal group quantification method are almost the same. Further, the polymer may be used by mixing two kinds of lactic acid-glycolic acid polymers having different composition ratios and / or weight average molecular weights at an arbitrary ratio. As such an example, the composition ratio (lactic acid / glycolic acid) (mol%) is about 75/25 and the weight average molecular weight is about 10,000.
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 12,000. Can be The weight ratio upon mixing is preferably from about 25/75 to about 75/25.

The biodegradable polymer used in the present invention may be a metal salt of the above-mentioned biodegradable polymer. For example, various biodegradable polymers described in WO 97/01331 may be used. A valent metal salt or the like is used. Preferably, a polyvalent metal salt of a lactic acid-glycolic acid polymer (further preferably a zinc salt, a calcium salt, a magnesium salt or the like, more preferably a zinc salt or the like) is used. The metal species of the polyvalent metal salt is not particularly limited as long as it is a compound that does not adversely affect the living body.
Polyvalent metals such as iron, zinc, copper, calcium, magnesium, aluminum, tin, and manganese), trivalent (eg, iron, aluminum, manganese, and the like) and tetravalent (eg, tin and the like) can also be used. In the present specification, the biodegradable polymer may be referred to as a biodegradable polymer including the case where the biodegradable polymer is a metal salt. Sometimes referred to as coalescence. These polyvalent metal salts of a biodegradable polymer can be produced by the method described in WO 97/01331 or a method analogous thereto. When the polyvalent metal salt of the biodegradable polymer is a zinc salt, it can be produced by reacting the biodegradable polymer with zinc oxide in an organic solvent. The order of adding the biodegradable polymer and the zinc oxide to the organic solvent may be such that zinc oxide may be added to the solution of the biodegradable polymer in the organic solvent in the form of powder or suspended in the organic solvent. An organic solvent solution of a biodegradable polymer may be added to a suspension of zinc oxide in an organic solvent. Also,
After mixing both in powder form, an organic solvent may be added. The content of the biodegradable polymer contained in the sustained-release preparation of the present invention is usually about 30 to 99.9% (W / W), preferably about 60 to 97% (W / W), and more preferably about 60 to 97% (W / W). 70 to 90% (W / W).

The organic solvent used for dissolving the biodegradable polymer during production of the sustained-release preparation of the present invention has a boiling point of 1%.
The temperature is preferably 20 ° C. or lower. Examples of the organic solvent include halogenated hydrocarbons (eg, dichloromethane, chloroform, etc.), alcohols (eg, ethanol, methanol, etc.), ethyl acetate, acetonitrile, and the like. These may be mixed and used at an appropriate ratio. When an organic solvent is used alone, for example, dichloromethane, ethyl acetate, acetonitrile and the like are preferable. When an organic solvent is used as a mixed solvent, for example, a halogenated hydrocarbon (eg, dichloromethane, chloroform, etc.)
And alcohols (eg, ethanol, methanol, etc.) or acetonitrile are preferred. The mixing ratio (volume ratio) between the halogenated hydrocarbon and the alcohol or acetonitrile is about 100: 1 to about 1: 1 and preferably about 30: 1 to about 2: 1.
It is desirable to use a mixed solvent of The concentration of the biodegradable polymer in the solution varies depending on the molecular weight, the type of the organic solvent, and the like.
/ W), preferably from about 0.1 to about 70% (W /
W), more preferably from about 1 to about 60% (W / W)
It is.

The cationic substance in the present invention means a basic substance or a water-soluble polyvalent metal salt. Examples of the basic substance include a basic amino acid (eg, arginine, lysine, etc.), a basic peptide (eg, protamine such as protamine, protamine sulfate, protamine hydrochloride, protamine phosphate or a salt thereof), a basic polyamine (eg, spermidine) , Spermine, etc.), basic polysaccharides (eg,
Chitosan, etc.) and basic additives (eg, benzalkonium chloride, N-methylglucamine (meglumine), etc.). Examples of the water-soluble polyvalent metal salt include a water-soluble zinc salt (eg, zinc chloride, zinc acetate), a water-soluble calcium salt, a water-soluble magnesium salt, and the like. Examples of the polyols in the present invention include polyethylene glycol and propylene glycol. The dose (addition amount) of the cationic substance and / or the polyol in the present invention depends on the type of the cationic substance and the polyol,
Although it varies depending on the subject animal, administration site, etc., it can be suitably selected from the range of preferably about 0.0001 to about 100 mg / kg body weight per child or adult. Dosage (addition amount) of cationic substances and polyols
Is preferably the amount of the cationic substances and polyols used. For example, when the cationic substance is protamine sulfate, the dosage for children or adults is preferably 3.5 mg or less in the case of subcutaneous administration. When the cationic substance is arginine, the dosage for children or adults is preferably 40 mg or less for subcutaneous administration and 1620 mg or less for intramuscular administration. The content of the cationic substance and / or the polyols contained in the sustained-release preparation of the present invention is about 0.0001 to 80% (W / W), preferably about 0.001 to 40%, based on the whole preparation. (W / W), more preferably about 0.01 to 20% (W / W).

The sustained-release preparation of the present invention refers to a preparation which has been processed (for example, freeze-dried) by adding an excipient (for example, mannitol) as necessary after forming the matrix. The initial release rate of the physiologically active substance in the present invention indicates the ratio of the amount of the physiologically active substance released within one day after administration of the sustained-release preparation to an animal (rat) to the administered dose. The sustained-release preparation of the present invention only needs to coexist with a matrix containing a physiologically active substance and a cationic substance and / or a polyol. It may be contained and prepared (suspended) at the time of use. Further, for example, in one container (for example, a dual-chamber prefilled syringe or the like), the matrix and the cationic substance and / or the polyols and the dispersion medium independently exist without coming into contact with each other;
It is also possible to take such a form that the three are mixed immediately before administration. The cationic substance and / or polyol in the sustained-release preparation of the present invention does not exist inside the matrix, but is held (adhered) to the surface (outside) of the matrix, or contained in the dispersion medium. It may be.

The dispersion medium in the present invention refers to a liquid medium used when a sustained-release preparation is injected as a suspension, and preferably a water-soluble medium. The dispersion medium usually contains an osmotic pressure adjusting agent (isotonizing agent), a thickening agent (suspension agent), a surfactant, a preservative (stabilizing agent), a soothing agent, a local anesthetic, and the like. . Examples of the osmotic pressure adjusting agent (isotonizing agent) include sodium chloride, mannitol, sorbitol, glucose, and the like, and examples of the thickening agent (suspension agent) include sodium carboxymethylcellulose, sodium alginate,
Polysaccharides such as hyaluronic acid and dextran are examples of surfactants such as polysorbate 80 (Tween8).
0), HCO-60, etc., as preservatives (stabilizers), for example, methyl paraben, propyl paraben, etc .; Is used. Further, if necessary, it contains a pH adjuster (for example, hydrochloric acid, acetic acid, sodium hydroxide, or various buffers). In addition to the above aqueous dispersion medium, an oily dispersion medium is also used. Vegetable oil such as sesame oil and corn oil, or a mixture thereof with a phospholipid such as lecithin, or medium-chain fatty acid triglyceride (for example, Miglyol 812) can be used as the oily dispersion medium.

The matrix containing a physiologically active substance in the present invention is obtained by dispersing a powder (S phase) obtained by freeze-drying a physiologically active substance solution in an organic solvent liquid (O phase) in which a biodegradable polymer is dissolved. By removing the solvent from the S / O-type dispersion, or dispersing the aqueous phase (W-phase) containing an aqueous solution of a physiologically active substance in an organic solvent (O-phase) in which a biodegradable polymer is dissolved. It is also produced by removing the solvent from the W / O emulsion or by removing the solvent from a solution in which a physiologically active substance is dissolved in an organic solvent solution (O phase) together with a biodegradable polymer. .
Examples of the production method include (a) an underwater drying method (S / O
/ W method and W / O / W method or O / W method), (b)
Phase separation method (coacervation method) and (c) spray drying method, or a method similar thereto, and the like. Hereinafter, a method for producing microcapsules as a matrix containing a physiologically active substance will be described.

(A-1) Underwater drying method (S / O / W method) According to this method, a water-miscible organic solvent and / or volatile salts are first added to a physiologically active substance aqueous solution, and then freeze-dried. To prepare a physiologically active substance powder (S phase). At this time, in order to obtain a fine powder, the salt concentration in the physiologically active substance solution, for example, an alkali metal (sodium, potassium,
Preferably, the ion concentration is low. For example, when the alkali metal is sodium, its ion concentration is preferably about 10 μg / mL or less. Next, the biodegradable polymer is dissolved in an organic solvent, and the above-mentioned bioactive substance powder is added and dispersed in the organic solvent liquid. At this time, the ratio (weight ratio) of the bioactive substance to the biodegradable polymer is, for example, about 1: 1000 to about 1: 1, preferably about 1: 200 to about 1: 5, and more preferably about 1: 1: 100 to about 1: 5. Further, in order to uniformly disperse the physiologically active substance powder in the organic solvent liquid, it is preferable to apply external physical energy. As the method, for example, ultrasonic irradiation, a turbine type stirrer, a homogenizer and the like are used. At this time, the average particle size of the physiologically active substance in the organic solvent liquid is about 10
μm or less, more preferably about 0.1 μm to about 5 μm.
m, more preferably about 0.5 μm to about 2 μm, which is easily achieved by using the bioactive substance powder obtained according to the present invention. The average particle size of the physiologically active substance in the present invention is determined by dispersing the physiologically active substance in an organic solvent such as dichloromethane using a homogenizer, and then using a laser analysis type particle size distribution analyzer (SAL).
D2000A: Shimadzu). At this time, the physiologically active substance is added to an organic solvent such as dichloromethane at a concentration of, for example, about 20 to 100 mg / mL, and then a homogenizer (for example, Polytron (Kinematica))
The mixture is stirred at about 20,000 rpm for about 30 seconds to about 1 minute to form a dispersion. The dispersion is further appropriately diluted with the organic solvent so as to be within the measurable range of the particle size distribution measuring device, and then subjected to a test. You. Next, the organic solvent dispersion liquid (S / O type dispersion liquid) thus prepared is further added to an aqueous solvent (W phase), and the same external physical energy as described above, for example, ultrasonic irradiation, turbine, An S / O / W emulsion is formed using a mold stirrer or a homogenizer. Thereafter, the oil phase solvent is evaporated to produce microcapsules. In this case, the volume of the aqueous phase is generally selected from about 1 to about 10,000 times the volume of the oil phase. More preferably, it is selected from about 2 times to about 5,000 times. It is particularly preferably selected from about 5 times to about 2,000 times. An emulsifier may be added to the outer aqueous phase. The emulsifier may be any as long as it can form a generally stable S / O / W emulsion. Examples of the emulsifier include anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid, and the like. These may be used in appropriate combination.
The concentration of the emulsifier in the external aqueous phase is preferably about 0.001%
To 20% (W / W). More preferably, it is about 0.01% to 10% (W / W), particularly preferably about 0.05% to 5% (W / W). The microcapsules thus obtained are separated by centrifugation or filtration, and then the emulsifiers and the like adhering to the surface of the microcapsules are removed by washing with distilled water and dispersed again in distilled water or the like. Lyophilize.

Examples of the water-miscible organic solvent added to the aqueous solution of a physiologically active substance in the present method include alcohols (eg, methanol, ethanol, isopropanol, etc., preferably methanol, ethanol, etc.), acetone and the like. These may be mixed and used at an appropriate ratio, but it is preferable to use alcohols, particularly ethanol alone. The amount (concentration) of addition to the aqueous solution of the physiologically active substance is about 0.03 to 0.5% (V / V) in volume ratio, preferably about 0.06%.
To 0.25% (V / V), more preferably about 0.
1 to 0.15% (V / V). By further freeze-drying the aqueous solution of the physiologically active substance obtained by adding such a water-miscible organic solvent, it is easy to handle (has good operability) and is fine (small particle size). Powder can be made. Examples of the volatile salts added to the aqueous solution of a physiologically active substance in the present method include ammonium salts (for example, ammonium acetate, ammonium bicarbonate, ammonium carbonate, ammonium chloride, and the like, preferably ammonium acetate and the like). These may be mixed and used at an appropriate ratio. The amount of the volatile salt to be added to the aqueous solution of the physiologically active substance is about 10-fold to about 80-fold by mole, preferably about 10-fold to about 70-fold, and more preferably about 15-fold to about 70-fold. 70 times, more preferably about 20 times to about 70 times, most preferably about 20 times to about 5 times.
It is 0 times mol. As in the case where a water-miscible organic solvent is added, the aqueous solution of a physiologically active substance obtained by adding a volatile salt is further freeze-dried, so that it is easy to handle (good in operability) and fine ( A fine bioactive substance powder (with a small particle size) can be prepared. In this method, the water-miscible organic solvent and / or the volatile salts added to the aqueous solution of the physiologically active substance may be used alone or in an appropriate combination. When a water-miscible organic solvent and volatile salts are used in combination, they can be added to the aqueous solution of the physiologically active substance according to the respective addition amounts described above.

(A-2) Underwater drying method (W / O / W method) According to this method, first, water or an appropriate buffer is added to a physiologically active substance to prepare a physiologically active substance solution (W phase). I do. Next, the biodegradable polymer is dissolved in an organic solvent, and the above-mentioned physiologically active substance solution is added and dispersed in the organic solvent liquid. The W / O type emulsion obtained in this manner is further added to an aqueous solvent (W phase), and W
Microcapsules are obtained via an / O / W emulsion. (A-3) Underwater drying method (O / W method) According to this method, first, a biodegradable polymer is dissolved in an organic solvent together with a physiologically active substance, and the organic solvent liquid (O phase) is further dissolved in an aqueous solvent (W Phase) to obtain microcapsules via an O / W emulsion in the same manner as in the above S / O / W method.

(B) Phase separation method (coacervation method) In the present method, the S / O type dispersion of (a-1) or the W / O type emulsion of (a-2) or (a- A coacervation agent is gradually added to the oil phase solution of 3) with stirring to precipitate and solidify microcapsules. The coacervation agent is used in an amount of about 0.01 times to about 1,000 times the dispersion.
Zero times the volume is added. More preferably, about 0.1.
The volume is from 05 to about 500 times, particularly preferably from about 0.1 to about 200 times. The coacervation agent may be any as long as it does not dissolve the biodegradable polymer used in the polymer, mineral oil or vegetable oil-based compound that is miscible with the organic solvent that dissolves the biodegradable polymer. Specifically, for example, silicon 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. After the microcapsules thus obtained are collected by filtration, they are repeatedly washed with heptane or the like to remove the coacervation agent. Further, it is washed and freeze-dried in the same manner as in (a) above. In the production of microcapsules by the in-water drying method and the coacervation method, an aggregation inhibitor may be added to prevent aggregation of particles. As the anticoagulant,
For example, mannitol, lactose, glucose, starches (eg, corn starch), water-soluble polysaccharides such as hyaluronic acid or alkali metal salts thereof, proteins such as glycine, fibrin, collagen, and inorganic salts such as sodium chloride and sodium hydrogen phosphate. Is appropriately used.

(C) Spray drying method In the present method, the S / O type dispersion of (a-1), the W / O type emulsion of (a-2) or the oil phase solution of (a-3) Is sprayed into a drying chamber of a spray dryer (spray dryer) using a nozzle, and the organic solvent in the atomized droplets is volatilized in a very short time to produce microcapsules. Examples of the nozzle include a two-fluid nozzle type, a pressure nozzle type, and a rotating disk type. At this time, if desired,
It is also effective to spray an aqueous solution of the anti-agglomeration agent from another nozzle simultaneously with the above-mentioned dispersion to prevent the microcapsule particles from agglomerating. The microcapsules thus obtained are further washed in the same manner as in (a) above, and if necessary, further heated (under reduced pressure if necessary) to further remove water and the organic solvent. As a production method for producing an implantable rod as a matrix containing a physiologically active substance in the present invention, a mixture of a physiologically active substance and a base is heated to a temperature equal to or higher than the glass transition temperature of the base, and then placed in a mold. Molding method (mold) and extrusion molding method (ex
trude). The shape can be freely selected in addition to the rod shape. Alternatively, a mixture of a physiologically active substance and a base is previously made into a fine powder or microencapsulated by any method, filled in a stainless steel tube or a Teflon tube, and compression molded to produce a rod-shaped implantable preparation. At this time, if necessary, the base may be heated to a temperature higher than the glass transition temperature. For example, a microcapsule obtained by an underwater drying method is filled in a Teflon tube having an inner diameter of 2.0 mm, heated at 60 ° C. for 15 minutes, compressed with a rod having a diameter of 2.0 mm, cooled, and molded to form an outer diameter of 2. A 0 mm rod-shaped formulation is obtained.

The sustained-release preparation of the present invention is preferably in the form of fine particles. This is because the sustained release preparation does not cause undue pain to the patient when administered through a needle usually used for subcutaneous or intramuscular injection. The particle size of the sustained-release preparation is, for example, about 0.1 to 300 μm, preferably about 1 to 15 μm as an average particle diameter.
0 μm, particularly preferably about 2 to 100 μm.
The amount of the physiologically active substance in the sustained-release preparation of the present invention varies depending on the kind of the physiologically active substance and the like. 0.2 to 30% (W / W), more preferably about 0.5 to 20% (W / W). Also,
The content of the biodegradable polymer contained in the sustained-release preparation of the present invention is, for example, about 30 to 99.9% (W / W), preferably about 60 to 97% (W / W), more preferably about 60 to 97% (W / W). 70 to 90% (W / W). Initial release rate of the physiologically active substance of the sustained-release preparation of the present invention [1 after administration
Release rate by day (24 hours)] is preferably about 50% or less of the administered dose, more preferably about 1 to about 30%,
More preferably, it is about 2 to about 20%, most preferably about 2 to about 15%. Note that the initial release rate is calculated based on the AUC (Area Under the Concentration-Time Curve) of the blood concentration up to 24 hours after subcutaneous administration of the sustained-release preparation of the present invention.
By applying e) to the dose-AUC straight line obtained from the AUC up to 24 hours after subcutaneous administration of the physiologically active peptide solution, the initial release amount is obtained, and the initial release rate is calculated.

The sustained-release preparation of the present invention is formulated into various dosage forms, for example, as microcapsules or using the microcapsules as a starting material, and then administered as a parenteral preparation (for example, injection or injection into an intramuscular, subcutaneous, organ, or the like). Implants, nasal cavity,
Transmucosal preparations for rectum, uterus, etc.), oral preparations (for example, capsules (for example, hard capsules, soft capsules, etc.), solid preparations such as granules and powders, liquid preparations for suspensions, etc.), etc. Can be administered as The sustained-release preparation of the present invention is particularly preferably for injection. For example, when the sustained-release preparation is a microcapsule, a practical sustained-release preparation for injection can be obtained by adding the above dispersion medium to form an aqueous suspension. Further, the oily dispersion medium is added to obtain a sustained-release injection which can be actually used as an oily suspension.

When the sustained-release preparation is, for example, a microcapsule, the particle size of the microcapsule may be within a range that satisfies the degree of dispersion and needle penetration when used as a suspension injection. The average particle size ranges from about 0.1 to about 300 μm. The average particle size is preferably about 1 to about 150 μm, particularly preferably about 2 to about 150 μm.
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 mentioned, but are not particularly limited.

The sustained-release preparation of the present invention has low toxicity and can be safely used for mammals (eg, human, cow, pig, dog, cat, mouse, rat, rabbit, etc.). The application of the sustained-release preparation varies depending on the physiologically active substance used. When the bioactive substance is, for example, insulin, diabetes or the like, and when it is interferon alpha, viral hepatitis (for example, hepatitis C, HBe antigen-positive active hepatitis, etc.), cancer (for example, renal cancer, Such as multiple myeloma), anemia in the case of erythropoietin (eg, anemia during renal dialysis), neutropenia (eg, during anticancer drug treatment) in the case of G-CSF, infectious diseases, etc. -2, cancer (for example, hemangioendothelioma), FGF, fracture, wound (bedsore, etc.), periodontal disease, gastrointestinal ulcer, etc.
Treatment or prevention of thrombocytopenia and the like in the case of GF-9, senile dementia and neuropathy (neuropathy) in the case of NGF, thrombosis and the like in the case of TPA, and cancer in the case of tumor necrosis factor It is effective for In addition, GH-containing sustained-release preparations are based on the growth hormone action of GH and, in addition to GH secretion deficient short stature, Turner syndrome, chronic renal failure, cartilage dysgenesis (chondrodystrophy), and adult growth. It is also applicable to treatment of hormone deficiency (adult GHD) and treatment of debilitating diseases such as AIDS. It has also been reported that GH has been applied to diseases such as Down syndrome, Silver syndrome, osteogenesis imperfecta, or juvenile chronic arthropathy, and has obtained an effective therapeutic effect. It is also adaptable to diseases. Furthermore, it is also effective in treating or preventing congestive heart failure and the like. Other subjects to which the GH-containing sustained-release preparation can be applied include hematopoiesis during organ transplantation and drug treatment of AIDS patients, improvement of malnutrition, renal anemia, angina, hyperlipidemia, obesity, burns・ Promote treatment of wounds / ulcers, surgical invasion (surgery / trauma) / early recovery after surgery, prevention of sepsis and osteoporosis fractures, early recovery of postoperative muscle strength in patients with osteoporosis fractures, amyotrophic lateral sclerosis (ALS) , Pressure ulcers and the like. In addition, as an anti-aging drug for the purpose of improving the quality of life (QOL) of frail elderly people, or by hGH's neuroprotective effect, it is used to suppress and improve progression of neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, cerebrovascular disease, etc.) Can also be expected to be effective. By making GH into a sustained release formulation,
Better drug efficacy is obtained for these indications than for daily subcutaneous injections.

The dose of the sustained-release preparation varies depending on the type and content of the physiologically active substance, the duration of release, the target disease, the target animal, and the like. Is good enough. As the dose of the physiologically active substance, for example, when the sustained-release preparation is a two-week preparation,
Preferably from about 0.0001 to about 10 per adult
It can be appropriately selected from the range of mg / kg body weight. More preferably, it can be appropriately selected from the range of about 0.05 to about 1 mg / kg body weight. The frequency of administration is 1 week
Time, once every two weeks, once a month, once every two months, etc., can be appropriately selected depending on the kind and content of the physiologically active substance, dosage form, duration of release, target disease, target animal, etc. . Preferably, a one week to two month sustained release preparation is used, and more preferably, a one week to one month sustained release preparation is used.
When the physiologically active substance as an active ingredient of the sustained-release preparation is, for example, insulin, the dose for an adult with diabetes is usually about 0.001 to about 1 mg as an active ingredient.
/ Kg body weight, preferably about 0.01 to about 0.2 mg
/ Kg body weight range, it is appropriate to administer once a week.

When the physiologically active substance which is the active ingredient of the sustained-release preparation is GH, the dosage varies depending on the type and content of GH, the duration of release, the target disease, the target animal and the like. It is sufficient that the effective concentration of GH is maintained in the body. In the treatment of the above-mentioned diseases, for example, when the sustained-release preparation is a two-week preparation, the dose of GH as an active ingredient is preferably about 0,1 per child or adult.
01 to about 5 mg / kg body weight (about 0.03 to about 1
5IU / kg body weight) and can be safely administered. More preferably, about 0.05 to about 1 mg / kg body weight (about 0.15 to about 3 IU / kg
Weight). The number of administrations is
Once a week, once every two weeks, once a month, etc.
It can be appropriately selected depending on the GH content, dosage form, duration of release, target disease, target animal and the like. Preferably, a one week to two month sustained release preparation is used, and more preferably, a one week to one month sustained release preparation is used. 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 the cold place here is defined in the Japanese Pharmacopoeia. That is, normal temperature is 15 to 25 ° C,
A cool place means below 15 ° C. Among cold places, 2 to 8 ° C. is particularly preferred.

[0032]

The present invention will be described in more detail with reference to Reference Examples, Examples, Comparative Examples and Test Examples, which do not limit the present invention. Reference Example 1 Genetically modified hGH aqueous solution (hGH concentration = 2 mg / m
To L), a 20-fold molar equivalent of ammonium acetate is added,
Using a peristaltic pump, 100 mL was dropped into the inner wall surface of the eggplant-shaped flask cooled in a dry ice-ethanol bath for 30 minutes, rapidly frozen, and then dried under vacuum to obtain hGH powder. Lactic acid-glycolic acid copolymer (lactic acid / glycol = 65/35, viscosity = 0.160 dL /
g) 1.690 g and 10 mg of zinc oxide were dissolved in 2.7 mL of dichloromethane. The above hG is added to this organic solvent liquid.
H powder (300 mg) was added, and the mixture was atomized using a Polytron (Kinematica). 0.1% of this S / O dispersion
It was added to 800 mL of an aqueous polyvinyl alcohol solution, and stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, centrifugation (about 2.0
(00 rpm) to obtain microcapsules. Then, after washing twice with 400 mL of distilled water, D-
0.2 g of mannitol was added and freeze-dried. Furthermore, in order to remove the residual solvent, vacuum drying was performed at 46 ° C. for 3 days, followed by hGH
The resulting microcapsules were obtained.

Reference Example 2 5 g of mannitol, 0.5 g of sodium carboxymethylcellulose and 0.1 g of polysorbate 80 were dissolved in about 90 mL of distilled water for injection, and the solution was adjusted to pH 5 with acetic acid.
After adjusting to ７7, the volume was made up to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 1 5 g of mannitol, 2 g of L-arginine hydrochloride, 0.5 g of sodium carboxymethylcellulose and 0.1 g of polysorbate 80 were dissolved in about 90 mL of distilled water for injection, and adjusted to pH 5 to 7 with acetic acid. The volume was made up to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 2 5 g of mannitol, 2 g of L-arginine hydrochloride and 0.1 g of polysorbate 80 were dissolved in about 60 mL of distilled water for injection, and 30 mL of polyethylene glycol 400 was added. did.
The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 3 5 g of mannitol, 0.1 g of benzalkonium chloride and 800.1 g of polysorbate were dissolved in about 60 mL of distilled water for injection, and polyethylene glycol 400 was dissolved in 30 m of water.
After adding L, the volume was made up to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 4 5 g of mannitol, 10 mg of protamine sulfate and 0.1 g of polysorbate 80 were dissolved in about 60 mL of distilled water for injection, 30 mL of polyethylene glycol 400 was added, and the mixture was made up to 100 mL with distilled water for injection.
The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 5 5 g of mannitol, 42 mg of zinc chloride and 0.1 g of polysorbate 80 were dissolved in about 60 mL of distilled water for injection, 30 mL of polyethylene glycol 400 was added, and the mixture was made up to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 6 5 g of mannitol, 1.7 g of lysine hydrochloride and 0.1 g of polysorbate 80 were dissolved in about 60 mL of distilled water for injection, and 30 mL of polyethylene glycol 400 was added. did. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 7 5 g of mannitol, water-soluble chitosan (manufactured by PRONOVA, ultrapure grade, hydrochloride, CL113) 1
0 mg and polysorbate 80 0.1 g for about 60 m
L of distilled water for injection.
After adding 30 mL of 00, the volume was made up to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 8 5 g of mannitol and 0.1 g of polysorbate 80
Was dissolved in about 60 mL of distilled water for injection, and 30 mL of polyethylene glycol 400 was added.
The volume was increased to 00 mL. The obtained solution was adjusted to a pore size of 0.4.
After filtration through a 5 μm filter, a dispersion medium for injection of microcapsules was obtained.

Example 9 A recombinant hGH aqueous solution (hGH concentration = 2 mg / m
To L), a 20-fold molar equivalent of ammonium acetate is added,
Using a peristaltic pump, 100 mL was dropped into the inner wall surface of the eggplant-shaped flask cooled in a dry ice-ethanol bath for 30 minutes, rapidly frozen, and then dried under vacuum to obtain hGH powder. Lactic acid-glycolic acid copolymer (lactic acid / glycol = 65/35, viscosity = 0.160 dL /
g) 1.690 g and 10 mg of zinc oxide were dissolved in 2.7 mL of dichloromethane. The above hG is added to this organic solvent liquid.
H powder (300 mg) was added, and the mixture was atomized using a Polytron (Kinematica). 0.1% of this S / O dispersion
It was added to 800 mL of an aqueous polyvinyl alcohol solution, and stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, centrifugation (about 2.0
(00 rpm) to obtain microcapsules. Then, after washing twice with 400 mL of distilled water, D-
0.2 g of mannitol and 0.4 mg of protamine sulfate
And lyophilized. Furthermore, to remove the residual solvent, 4
The resultant was vacuum-dried at 6 ° C. for 3 days to obtain hGH-containing microcapsules.

Example 10 A recombinant hGH aqueous solution (hGH concentration = 2 mg / m
To L), a 20-fold molar equivalent of ammonium acetate is added,
Using a peristaltic pump, 100 mL was dropped into the inner wall surface of the eggplant-shaped flask cooled in a dry ice-ethanol bath for 30 minutes, rapidly frozen, and then dried under vacuum to obtain hGH powder. Lactic acid-glycolic acid copolymer (lactic acid / glycol = 65/35, viscosity = 0.160 dL /
g) 1.690 g and 10 mg of zinc oxide were dissolved in 2.7 mL of dichloromethane. The above hG is added to this organic solvent liquid.
H powder (300 mg) was added, and the mixture was atomized using a Polytron (Kinematica). 0.1% of this S / O dispersion
It was added to 800 mL of an aqueous polyvinyl alcohol solution, and stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, centrifugation (about 1,5
(00 rpm) to obtain microcapsules. Then, after washing twice with 400 mL of distilled water, D-
Mannitol 0.2 g and L-arginine hydrochloride 0.2
5 g was added and lyophilized. Furthermore, in order to remove the residual solvent, vacuum drying was performed at 46 ° C. for 3 days to obtain hGH-containing microcapsules.

Example 11 Genetically-modified hGH aqueous solution (hGH concentration = 2 mg / m
To L), a 20-fold molar equivalent of ammonium acetate is added,
Using a peristaltic pump, 100 mL was dropped into the inner wall surface of the eggplant-shaped flask cooled in a dry ice-ethanol bath for 30 minutes, rapidly frozen, and then dried under vacuum to obtain hGH powder. Lactic acid-glycolic acid copolymer (lactic acid / glycol = 65/35, viscosity = 0.160 dL /
g) 1.690 g and 10 mg of zinc oxide were dissolved in 2.7 mL of dichloromethane. The above hG is added to this organic solvent liquid.
H powder (300 mg) was added, and the mixture was atomized using a Polytron (Kinematica). 0.1% of this S / O dispersion
It was added to 800 mL of an aqueous polyvinyl alcohol solution, and stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, centrifugation (about 1,5
(00 rpm) to obtain microcapsules. Then, after washing twice with 400 mL of distilled water, D-
0.2 g of mannitol and 0.1 g of N-methylglucamine.
25 g was added and freeze-dried. Furthermore, in order to remove the residual solvent, vacuum drying was performed at 46 ° C. for 3 days to obtain hGH-containing microcapsules.

Example 12 The microcapsules containing protamine sulfate obtained in Example 9 were filled in a Teflon (registered trademark) tube having an inner diameter of 2.0 mm and heated at 60 ° C. for 15 minutes. After heating, it was compressed with a stick and cooled, and then molded. A rod-shaped preparation having an outer diameter of 2.0 mm and a length of about 1 cm was obtained.

Example 13 5 g of mannitol, 0.2 g of L-arginine hydrochloride, 0.5 g of sodium carboxymethylcellulose and 0.1 g of polysorbate 80 were dissolved in about 90 mL of distilled water for injection, and adjusted to pH 5 to 7 with acetic acid. After that, the volume was increased to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 14 5 g of mannitol, 1.2 g of L-arginine hydrochloride, 0.5 g of sodium carboxymethylcellulose and 0.1 g of polysorbate 80 were dissolved in about 90 mL of distilled water for injection and adjusted to pH 5 to 7 with acetic acid. After that, the volume was increased to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 15 5 g of mannitol, 2.4 g of L-arginine hydrochloride, 0.5 g of sodium carboxymethylcellulose and 0.1 g of polysorbate 80 were dissolved in about 90 mL of distilled water for injection, and adjusted to pH 5 to 7 with acetic acid. After that, the volume was made up to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 16 1.5 g of mannitol, 2.4 g of L-arginine hydrochloride,
0.5 g of sodium carboxymethylcellulose and 0.1 g of polysorbate 80 were dissolved in about 90 mL of distilled water for injection, and the volume was made up to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 17 5 g of mannitol, 2.1 g of lysine hydrochloride, 0.5 g of sodium carboxymethylcellulose and 0.1 g of polysorbate 80 were dissolved in about 90 mL of distilled water for injection, and made up to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 18 5 g of mannitol, 1 g of N-methylglucamine, 0.5 g of methylcellulose and 0.1 of polysorbate 80 0.1
g was dissolved in about 90 mL of distilled water for injection, adjusted to pH 6 with hydrochloric acid, and then diluted to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 19 5 g of mannitol, 1 g of zinc acetate, 0.5 g of methylcellulose and 0.1 g of polysorbate 80 were added to about 90%.
Dissolve in mL of distilled water for injection, 100m with distilled water for injection
L was raised to L. The obtained solution was prepared with a pore size of 0.45 μm.
After filtration through a filter described in (1), a dispersion medium for injection of microcapsules was obtained.

Example 20 5 g of mannitol, water-soluble chitosan (manufactured by PRONOVA, ultrapure grade, hydrochloride, CL113)
0.1 g, methyl cellulose 0.5 g, and polysorbate 80 0.1 g were dissolved in about 90 mL of distilled water for injection, and made up to 100 mL with distilled water for injection. The obtained solution was filtered through a filter having a pore size of 0.45 μm to obtain a dispersion medium for injection of microcapsules.

Example 21 Recombinant hG containing 9.6 μg / mL sodium
A 20-fold molar equivalent of ammonium acetate was added to an aqueous H solution (hGH concentration = 2 mg / mL), and 100 mL for 30 minutes was added to the inner wall surface of an eggplant-shaped flask cooled in a dry ice-ethanol bath using a peristaltic pump. After dripping and rapid freezing, the product was vacuum-dried to obtain hGH powder.
Lactic acid-glycolic acid copolymer (lactic acid / glycol = 65
/ 35, viscosity = 0.160 dL / g) 1.690 g and zinc oxide 10 mg were dissolved in dichloromethane 2.7 mL. 300 mg of the above hGH powder was added to this organic solvent solution, and the mixture was pulverized using Polytron (Kinematica). The S / O dispersion was added to 800 mL of a 0.1% aqueous polyvinyl alcohol solution, and the mixture was stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, microcapsules were collected by centrifugation (about 2,000 rpm). Then distilled water 40
After washing twice with 0 mL, D-mannitol 0.2 g
And lyophilized. Furthermore, to remove the residual solvent, 4
The resultant was vacuum-dried at 6 ° C. for 3 days to obtain hGH-containing microcapsules.

Example 22 Recombinant hG containing 7.3 μg / mL sodium
Using an H aqueous solution (hGH concentration = 2 mg / mL), hGH-containing microcapsules were obtained in the same manner as in Example 21.

Comparative Example 1 Recombinant h containing 16.3 μg / mL of sodium
Using an aqueous GH solution (hGH concentration = 2 mg / mL), hGH-containing microcapsules were obtained in the same manner as in Example 21.

Test Example 1 The hGH-containing microcapsules obtained in Reference Example 1 were used in Example 1.
The following tests were carried out using the dispersion obtained by adding the dispersion medium prepared in the above. (1) In vivo release in rats SD rats (male, 6 weeks old) were subjected to immunosuppressive treatment with tacrolimus. 5 mg of Prograf Injection (Fujisawa Pharmaceutical Co., Ltd.) was diluted with physiological saline and administered in microcapsules.
0.4 mg / 0.2 mL / rat before day, 0.2 mg immediately after microcapsule administration, 0.2 mg after 4, 7 and 11 days after administration
/0.2 mL / rat, administration 14, 18, 21, 25,
By subcutaneous administration at a dose of 0.3 mg / 0.2 mL / rat 28 and 32 days later, antibody production against hGH can be suppressed, and hG in rat serum for 5 weeks after administration can be suppressed.
It became possible to make H concentration. The microcapsules obtained in Reference Example 1 were adjusted to 16 mg hGH / mL.
It was dispersed in the dispersion medium produced in Example 1. 0.75 mL of the obtained dispersion was subcutaneously administered to the back of the rat under ether anesthesia. The dose was 12 mg as hGH. After administration of the microcapsules, blood was collected over time from the tail vein, and the serum was collected. The serum hGH concentration was measured by an immunoradiometric assay (Ab beads HGH, Eiken Chemical). (2) Initial release rate The hGH solution was added to immunosuppressed SD rats at 5, 10 and 20 hours.
The solution was subcutaneously administered at a dose of mg / kg, blood was collected over time, and the serum hGH concentration was measured. AUC was calculated by the trapezoidal method. AUC up to 24 hours after microcapsule administration
To hGH equivalent to subcutaneous administration of hGH solution
The dose was calculated and the initial release rate was calculated by dividing by the microcapsule dose of 12 mg. The initial release rate of the dispersion administration group obtained by adding the dispersion medium of Example 1 was 6
%Met. As is clear from these results, in the microcapsule administration group using the L-arginine hydrochloride-containing dispersion medium, the initial release was small, and thereafter the blood h increased over one month.
GH concentration was achieved.

Test Example 2 Using the respective dispersions obtained by adding the respective dispersion media prepared in Example 2, Example 3, Example 4 or Example 5 to the hGH-containing microcapsules obtained in Reference Example 1, Was carried out. (1) In Vivo Release Property in Rats The same operation as in Test Example 1 (1) was used to evaluate the change in serum hGH concentration in immunosuppressed SD rats. (2) Initial Release Rate The initial release rate in immunosuppressed SD rats was evaluated by the same operation as in Test Example 1 (2). The initial release rate in each of the dispersion administration groups obtained by adding each dispersion medium of Example 2, Example 3, Example 4, or Example 5 was 5%, respectively.
6%, 3% and 7%. As is clear from the results, L-arginine hydrochloride, benzalkonium chloride,
In the microcapsule administration group using a dispersion medium containing protamine sulfate or zinc chloride, the initial release is small,
High blood hGH levels were achieved over the following month.
The effect of suppressing the initial release by the dispersion medium containing these basic substances or the water-soluble polyvalent metal salts was clarified.

Test Example 3 Reference Example 2 was applied to the hGH-containing microcapsules obtained in Example 9.
The following tests were carried out using the dispersion obtained by adding the dispersion medium prepared in the above. (1) In Vivo Release Property in Rats The same operation as in Test Example 1 (1) was used to evaluate the change in serum hGH concentration in immunosuppressed SD rats. (2) Initial Release Rate The initial release rate in immunosuppressed SD rats was evaluated by the same operation as in Test Example 1 (2). HG obtained in Example 9
The initial release rate in the dispersion administration group obtained by adding the dispersion medium of Reference Example 2 to the H-containing microcapsules was 8%. As is evident from the results, in the sustained-release microcapsule administration group in which the basic substance protamine sulfate is retained on the surface of the matrix, the initial release was small, and thereafter a high blood hGH concentration was achieved over one month. .

Test Example 4 The following test was carried out using each dispersion obtained by adding the dispersion medium prepared in Example 8 to the hGH-containing microcapsules obtained in Reference Example 1 and Example 10. (1) In Vivo Release Property in Rats The same operation as in Test Example 1 (1) was used to evaluate the change in serum hGH concentration in immunosuppressed SD rats. (2) Initial Release Rate The initial release rate in immunosuppressed SD rats was evaluated by the same operation as in Test Example 1 (2). The initial release rate in each of the dispersion administration groups obtained by adding the dispersion medium of Example 8 to the hGH-containing microcapsules obtained in Reference Example 1 and Example 10 was 7% and 5%, respectively. As is clear from these results, in the group to which the sustained release microcapsule preparation was suspended in the dispersion medium containing the polyethylene glycol 400, which is a polyol, the initial release was small, and
High blood hGH levels over months have been achieved.

Test Example 5 The hGH-containing microcapsules obtained in Reference Example 1 were used in Example 1
3, the following tests were carried out using each dispersion obtained by adding each dispersion medium produced in Example 14 or Example 15. (1) In Vivo Release Property in Rats The same operation as in Test Example 1 (1) was used to evaluate the change in serum hGH concentration in immunosuppressed SD rats. (2) Initial Release Rate The initial release rate in immunosuppressed SD rats was evaluated by the same operation as in Test Example 1 (2). The initial release rate in each of the dispersion administration groups obtained by adding each dispersion medium of Example 13, Example 14, or Example 15 was 11% and 7%, respectively.
And 6%. As is evident from the results, in the microcapsule administration group using the dispersion medium containing L-arginine hydrochloride, the initial release was reduced with an increase in the amount added, and thereafter, the high blood hGH concentration over one month was reduced. Achieved. The effect of suppressing the initial release depending on the amount of L-arginine hydrochloride in the dispersion medium was clarified.

Test Example 6 The hGH-containing microcapsules obtained in Reference Example 1 were used in Example 1
The following test was carried out using each dispersion obtained by adding the dispersion medium prepared in 6. (1) In Vivo Release Property in Rats The same operation as in Test Example 1 (1) was used to evaluate the change in serum hGH concentration in immunosuppressed SD rats. (2) Initial Release Rate The initial release rate in immunosuppressed SD rats was evaluated by the same operation as in Test Example 1 (2). The initial release rate in each of the dispersion administration groups obtained by adding the dispersion medium of Example 16 was 4
%Met. As is clear from the results, in the microcapsule administration group using the dispersion medium containing L-arginine hydrochloride, even if mannitol was reduced to make the dispersion medium isotonic, the initial release was small, High blood hGH levels over time were achieved. It was revealed that the effect of suppressing the initial release depends on the amount of L-arginine hydrochloride, and there is no effect even if the amount of mannitol is reduced.

Test Example 7 The hGH-containing microcapsules obtained in Reference Example 1 were used in Example 1
7, The following tests were carried out using each dispersion obtained by adding each dispersion medium produced in Example 18, Example 19 or Example 20. (1) In Vivo Release Property in Rats The same operation as in Test Example 1 (1) was used to evaluate the change in serum hGH concentration in immunosuppressed SD rats. (2) Initial Release Rate The initial release rate in immunosuppressed SD rats was evaluated by the same operation as in Test Example 1 (2). The initial release rate in each of the dispersion administration groups obtained by adding each dispersion medium of Example 17, Example 18, Example 19 or Example 20 was 8%, 11%, 10% and 7%, respectively. . As is clear from these results, in the microcapsule administration group using the dispersion medium containing lysine hydrochloride, N-methylglucamine, zinc acetate or chitosan, the initial release was small, and thereafter the high blood hGH concentration over one month was obtained. Was achieved. The effect of suppressing the initial release by the dispersing medium containing these basic substances, water-soluble polyvalent metal salts or basic polysaccharides was clarified.

Test Example 8 The following test was performed on the hGH-containing microcapsules obtained in Example 21 or Comparative Example 1. (1) hGH weight average particle size in dichloromethane The average particle size was determined by treating with a homogenizer (Polytron (Kinematica)) at about 20,000 rpm for about 30 seconds.
After hGH was dispersed in dichloromethane, it was appropriately diluted with dichloromethane so as to be within a measurable range of a laser analysis type particle size distribution analyzer (SALD2000A: Shimadzu) and measured. (2) hGH encapsulation rate in microcapsules After adding 1.75 mL of acetonitrile to 10 mg of microcapsules, the mixture was subjected to ultrasonic treatment. 3.25 mL of 10 mM phosphate buffer (pH 8.0) was added to the obtained acetonitrile solution, and the mixture was subjected to ultrasonic treatment again to extract hGH as the main drug. Next, the hGH extract was subjected to size exclusion high-performance liquid chromatography under the following conditions to measure the hGH content and calculate the encapsulation rate. Column: TSKgelG 3000SW _XL (Toso
h) Eluent: 50 mM NH ₄ HCO ₃ Flow rate: 0.6 mL / min Detection wavelength: 214 nm (3) Initial release rate The initial release rate in immunosuppressed SD rats was evaluated by the same operation as in Test Example 1 (2). . The hGH weight average particle diameter in dichloromethane of Example 21 or Comparative Example 1 was
1.2 μm and 2.4 μm, respectively, the hGH encapsulation rate in microcapsules was 85% and 73%, respectively, and the initial release rate in immunosuppressed rats was 18% and 40%.
%Met. As is clear from these results, by setting the sodium concentration in the human growth hormone solution to about 10 μg / mL or less, it is possible to obtain human growth hormone fine particles having a weight average particle diameter of about 0.5 μm to about 2.0 μm. Thus, microcapsules having a high hGH encapsulation rate and a small initial release were obtained. In other words, it was clarified that by reducing the salt concentration in the physiologically active substance solution, the drug was miniaturized, and a sustained-release preparation having a high encapsulation rate and suppressed initial release was obtained.

Test Example 9 The following test was carried out on the hGH-containing microcapsules obtained in Example 22. (1) hGH weight average particle diameter in dichloromethane The hGH weight average particle diameter in dichloromethane was evaluated by the same operation as in Test Example 8 (1). (2) hGH encapsulation rate in microcapsules The hGH encapsulation rate in microcapsules was evaluated by the same operation as in Test Example 8 (2). (3) Initial release rate The initial release rate in immunosuppressed SD rats was evaluated by the same operation as in Test Example 1 (2). The hGH weight average particle diameter in dichloromethane of Example 22 was 1.2 μm, the hGH encapsulation rate in microcapsules was 83%, and the initial release rate in immunosuppressed rats was 14%. As is clear from these results, by setting the sodium concentration in the human growth hormone solution to about 10 μg / mL or less, it is possible to obtain human growth hormone fine particles having a weight average particle diameter of about 0.5 μm to about 2.0 μm. Thus, microcapsules having a high hGH encapsulation rate and a small initial release were obtained. In other words, it was clarified that by reducing the salt concentration in the physiologically active substance solution, the drug was miniaturized, and a sustained-release preparation having a high encapsulation rate and suppressed initial release was obtained.

[0066]

According to the present invention, the initial release of a physiologically active substance immediately after administration is drastically suppressed by adding a basic substance or a water-soluble polyvalent metal salt to the outside of a matrix or a dispersion medium thereof, A sustained-release preparation that releases a constant amount of a physiologically active substance over a long period immediately after administration and has extremely low clinically excellent properties as a medicinal product, with very little deterioration of the physiologically active substance and very little residual organic solvent.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61K 38/00 A61K 47/04 38/22 47/12 38/27 47/34 47/04 47/36 47 / 12 47/42 47/34 A61P 3/10 47/36 5/06 47/42 A61K 37/02 A61P 3/10 37/24 5/06 37/36 F term (reference) 4C076 AA11 AA16 AA61 CC21 CC30 DD07 DD23 DD41 DD51 EE17 EE23 EE30 EE37 EE41 FF31 4C084 AA01 DB01 DB52 MA17 MA21 MA38 ZC021 ZC031 ZC331

Claims (63)

[Claims] 1. A sustained-release preparation comprising a combination of a matrix containing a physiologically active substance, a cationic substance and / or a polyol, wherein the initial release of a physiologically active substance is suppressed. 2. The sustained-release preparation according to claim 1, wherein the matrix containing a physiologically active substance is mixed with a cationic substance and / or a polyol. 3. The sustained-release preparation according to claim 1, wherein a cationic substance and / or a polyol is held on the surface of the matrix containing the physiologically active substance. 4. The sustained-release preparation according to claim 1, wherein the cationic substance is a basic substance or a water-soluble polyvalent metal salt. 5. The sustained-release preparation according to claim 4, wherein the basic substance is a basic amino acid. 6. The sustained-release preparation according to claim 5, wherein the basic amino acid is arginine or lysine. 7. The sustained-release preparation according to claim 4, wherein the basic substance is a basic additive. 8. The sustained-release preparation according to claim 7, wherein the basic additive is benzalkonium chloride or N-methylglucamine. 9. The sustained-release preparation according to claim 4, wherein the basic substance is a basic peptide, a basic polyamine or a basic polysaccharide. 10. The sustained-release preparation according to claim 9, wherein the basic peptide is protamine or a salt thereof. 11. The sustained-release preparation according to claim 9, wherein the basic polyamine is spermidine or spermine. 12. The sustained-release preparation according to claim 9, wherein the basic polysaccharide is chitosan. 13. The sustained-release preparation according to claim 4, wherein the water-soluble polyvalent metal salt is a water-soluble zinc salt. 14. The sustained-release preparation according to claim 13, wherein the water-soluble zinc salt is zinc chloride or zinc acetate. 15. The sustained-release preparation according to claim 1, wherein the polyol is polyethylene glycol or propylene glycol. 16. The sustained-release preparation according to claim 1, wherein the physiologically active substance is a physiologically active peptide. 17. The bioactive peptide having a molecular weight of about 200 to
17. The sustained release formulation of claim 16, which is about 500,000. 18. The bioactive peptide has a molecular weight of about 5,000.
17. The sustained release formulation according to claim 16, wherein the amount is from 0 to about 500,000. 19. The physiologically active peptide is a hormone, a cytokine, a hematopoietic factor, a growth factor or an enzyme.
6. The sustained-release preparation according to 6. 20. The sustained-release preparation according to claim 16, wherein the physiologically active peptide is human growth hormone. 21. The sustained-release preparation according to claim 1, wherein the matrix base is a biodegradable polymer. 22. The sustained-release preparation according to claim 21, wherein the biodegradable polymer is a homo- or copolymer of α-hydroxycarboxylic acids or a mixture thereof. 23. The sustained-release preparation according to claim 21, wherein the biodegradable polymer is a copolymer having a composition ratio of lactic acid / glycolic acid of about 100/0 to about 40/60 mol%. 24. The sustained-release preparation according to claim 21, wherein the biodegradable polymer is a lactic acid homopolymer. 25. The sustained-release preparation according to claim 21, wherein the biodegradable polymer has a weight average molecular weight of about 3,000 to about 50,000. 26. The sustained-release preparation according to claim 1, wherein the matrix is a microcapsule. 27. The sustained-release preparation according to claim 1, which is for injection. 28. The sustained-release preparation according to claim 1, comprising a matrix containing a physiologically active substance, a cationic substance and / or a polyol and a dispersion medium. 29. A dispersion medium containing the cationic substance and / or polyol for producing the sustained-release preparation according to claim 28. 30. The dispersion medium according to claim 29, wherein the cationic substance is a basic substance or a water-soluble polyvalent metal salt. 31. The dispersion medium according to claim 30, wherein the basic substance is a basic amino acid. 32. The dispersion medium according to claim 31, wherein the basic amino acid is arginine or lysine. 33. The dispersion medium according to claim 30, wherein the basic substance is a basic additive. 34. The dispersion medium according to claim 33, wherein the basic additive is benzalkonium chloride or N-methylglucamine. 35. The dispersion medium according to claim 30, wherein the basic substance is a basic peptide, a basic polyamine or a basic polysaccharide. 36. The dispersion medium according to claim 35, wherein the basic peptide is protamine or a salt thereof. 37. The dispersion medium according to claim 35, wherein the basic polyamine is spermidine or spermine. 38. The dispersion medium according to claim 35, wherein the basic polysaccharide is chitosan. 39. The dispersion medium according to claim 30, wherein the water-soluble polyvalent metal salt is a water-soluble zinc salt. 40. The dispersion medium according to claim 39, wherein the water-soluble zinc salt is zinc chloride or zinc acetate. 41. The dispersion medium according to claim 29, wherein the polyol is polyethylene glycol or propylene glycol. 42. The dispersion medium according to claim 29, comprising an osmotic pressure adjusting agent. 43. The dispersion medium according to claim 42, wherein the osmotic pressure adjusting agent is a saccharide or a salt. 44. The dispersion medium according to claim 29, comprising a thickening agent. 45. The dispersion medium according to claim 44, wherein the thickening agent is a water-soluble polysaccharide. 46. The dispersion medium according to claim 29, comprising a surfactant. 47. The dispersion medium according to claim 46, wherein the surfactant is a nonionic surfactant. 48. The dispersion medium according to claim 29, which is for injection. 49. A method for suppressing the initial release of a physiologically active substance, comprising mixing a cationic substance and / or a polyol with a sustained-release preparation containing a matrix containing the physiologically active substance. 50. Fine particles of a physiologically active substance obtained by adjusting the concentration of an alkali metal ion in a physiologically active substance solution to about 10 μg / mL or less. 51. The microparticle according to claim 50, wherein the physiologically active substance is a physiologically active peptide. 52. The physiologically active peptide is a hormone, cytokine, hematopoietic factor, growth factor or enzyme.
1. Fine particles according to 1. 53. The fine particle according to claim 51, wherein the physiologically active peptide is human growth hormone. 54. The fine particles of claim 50, wherein the weight average particle size is from about 0.5µm to about 2.0µm. 55. A method for producing fine particles of a physiologically active substance, comprising using a physiologically active substance solution having an alkali metal ion concentration of about 10 μg / mL or less in the physiologically active substance solution. 56. The fine particles according to claim 50, wherein the solution further contains ammonium acetate. 57. A sustained-release preparation containing the fine particles according to claim 50. 58. The sustained release preparation according to claim 57, wherein the base of the sustained release preparation is a biodegradable polymer. 59. The sustained-release preparation according to claim 58, wherein the biodegradable polymer is a homo- or copolymer of α-hydroxycarboxylic acids, or a mixture thereof. 60. The sustained-release preparation according to claim 58, wherein the biodegradable polymer is a copolymer having a composition ratio of lactic acid / glycolic acid of about 100/0 to about 40/60 mol%. 61. The sustained-release preparation according to claim 58, wherein the biodegradable polymer is a lactic acid homopolymer. 62. The sustained release preparation according to claim 58, wherein the biodegradable polymer has a weight average molecular weight of about 3,000 to about 50,000. 63. The sustained-release preparation according to claim 57, which is a microcapsule.