JPH10231252A - Production of sustained release preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a sustained release preparation, capable of increasing uptake ratio of physiologically active polypeptide and exhibiting high blood concentration over a long period by dispersing a physiologically active polypeptide into an organic solvent solution of an in vivo degradable polymer and zinc oxide and removing an organic solvent. SOLUTION: A physiologically active polypeptide, preferably having about 1,000-50,000 molecular weight (e.g. growth hormone or insulin) is dispersed, preferably in a ratio of about 1-50% into an organic solvent (e.g. dichloromethane) solution containing in vivo degradable polymer (e.g. lactic acid-glycolic acid copolymer) and about 0.001-2wt.% zinc oxide, and then, an organic solvent is removed by a method such as underwater drying method, a phase separation method, spray-drying method to provide the objective sustained release preparation. The daily dose of the sustained release preparation can properly be selected from a range of about 0.0001-10mg/kg weight adult as an active ingredient in the case of one week-type preparation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002]

2. Description of the Related Art Physiologically active polypeptides or derivatives thereof are known to exhibit various pharmacological actions in living organisms, and some of them are Escherichia coli, yeast and animal Produced in large quantities using living organisms such as cells or goats, hamsters,
It is being applied as a pharmaceutical. However, since these bioactive polypeptides generally have a short half-life in vivo, frequent administration is required, and the physical burden on the patient involved in the injection cannot be ignored. For example, growth hormone (hereinafter sometimes abbreviated as GH) is a typical hormone originally produced and secreted by the anterior pituitary gland,
In addition to promoting body growth, sugar and lipid metabolism, protein assimilation,
It is a biologically active polypeptide that has a wide variety of physiological actions, such as being involved in cell growth and differentiation.However, it is currently mass-produced by Escherichia coli using genetic recombination technology and is being widely clinically applied worldwide as a pharmaceutical. . However, GH has a short in vivo half-life and requires frequent administration to maintain an effective blood concentration. Particularly in the case of pituitary dwarfism, it can be used for infants or young patients for several months. The fact is that daily subcutaneous administration is performed over a long period of 10 years or more. In order to solve this problem, various attempts have been made to develop sustained-release preparations containing a physiologically active polypeptide.

[0003] JP-A-8-3055 (EP-A 6330)
20) A method for producing a sustained-release preparation in which a water-soluble polypeptide is penetrated in water into a biodegradable matrix comprising a biodegradable polymer and a fatty acid metal salt, and the sustained-release preparation prepared by the method. Microcapsules (hereinafter sometimes abbreviated as MC) are disclosed. JP-A-8-21
7691 (WO 96/07399) discloses that a water-soluble peptide-based physiologically active substance is made into a water-insoluble or poorly water-soluble polyvalent metal salt using an aqueous solution of zinc chloride or the like, and a sustained release containing this and a biodegradable polymer. A method for the manufacture of a pharmaceutically acceptable formulation is disclosed. WO 94/12158 discloses a method for producing a sustained-release preparation of human GH and a biodegradable polymer, in which a polymer decomposition accelerator such as 0.1-30% (w / w) zinc hydroxide is added to the polymer. It is stated that it can be added to a polymer solution. Further, a method is disclosed in which an organic solvent solution containing human GH and a polymer is sprayed into liquid nitrogen to prepare MC as porous particles while retaining biological activity. WO 92/17200 and Nature Medicine, vol. 2, p. 795 (1996) disclose a method for producing a sustained-release preparation using a zinc salt of human GH. WO 95/29664
After dispersing a metal salt such as zinc carbonate in a solid state in a polymer solution, a bioactive substance (hormone etc.) is added, and the bioactive substance and the metal cation are separately dispersed in a biodegradable polymer. A method for manufacturing an MC is disclosed.

[0004]

As described above, various attempts have been made to produce a sustained-release preparation while maintaining the biological activity of the physiologically active polypeptide. Is still clinically satisfactory in that the uptake rate of bioactive polypeptides is low, there is excessive release at the beginning of administration, stable release over a long period is not achieved, and sufficient blood concentration cannot be maintained for a long period of time. No formulation has been obtained. At present, many production methods do not conform to industrialization on the premise of mass production.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and have found that a lactic acid-glycolic acid copolymer (hereinafter sometimes abbreviated as PLGA) used as an MC base. ) And zinc oxide coexist in an organic solvent, surprisingly, zinc oxide insoluble in the organic solvent by itself dissolves in the organic solvent, and PLGA.
When a zinc oxide form is obtained and the bioactive polypeptide is directly dispersed and molded as a powder in an organic solvent solution of the PLGA / zinc oxide form, the uptake rate of the bioactive polypeptide is improved, and the initial release after administration is suppressed. In addition, it has been found that a sustained-release preparation having excellent sustainability can be obtained. Also,
The production method was found to be a method very suitable for industrialization with a small number of steps, and further studies were made, thereby completing the present invention. That is, the present invention provides (1) a sustained-release preparation characterized in that a physiologically active polypeptide is dispersed in an organic solvent solution containing a biodegradable polymer and zinc oxide, and then the organic solvent is removed. The production method, (2) the production method according to (1), wherein the bioactive polypeptide is a growth hormone, and (3) the production method according to (1), wherein the biodegradable polymer is a lactic acid-glycolic acid copolymer. , (4) Lactic acid-
The production method according to the above (3), wherein the lactic acid / glycolic acid composition ratio (mol%) of the glycolic acid copolymer is from about 85/15 to about 50/50.

(5) The method according to (3), wherein the lactic acid-glycolic acid copolymer has a weight average molecular weight of about 8,000 to about 20,000, (6) biodegradation in an organic solvent solution (7) The method according to the above (1), wherein the content of zinc with respect to the reactive polymer is about 0.001 to about 2% (W / W).
The production method according to (1), wherein the average particle size of the sustained-release preparation is about 0.1 to about 300 μm; (8) the production method according to (1), wherein the sustained-release preparation is for injection; (3) An o / w-type emulsion in which a growth hormone is dispersed in an organic solvent solution containing a lactic acid-glycolic acid copolymer and zinc oxide as an oil phase is dried in water. (10) The production method according to (1), wherein the sustained release preparation is a sustained release microcapsule, (11) an organic solvent containing a lactic acid-glycolic acid copolymer and zinc oxide. Solution, (12) an organic solvent-soluble lactic acid-glycolic acid copolymer / zinc oxide obtained by allowing a lactic acid-glycolic acid copolymer and zinc oxide to coexist in an organic solvent, (1)
3) a dispersion in which a bioactive polypeptide is dispersed in an organic solvent solution containing a lactic acid-glycolic acid copolymer and zinc oxide; (14) the dispersion according to (13), wherein the bioactive polypeptide is a growth hormone And (15) a sustained-release preparation produced by the production method according to (1).

Specific examples of preferred biodegradable polymers used in the present invention include, for example, α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.), hydroxytricarboxylic acid (Eg, citric acid, etc.) and a polymer having a free carboxyl group or a mixture thereof, a poly-α-cyanoacrylate, a polyamino acid (eg, poly) -γ-benzyl-L-glutamic acid, etc.), and maleic anhydride polymers (eg, styrene-maleic acid polymers, etc.). 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, any of D-, L-, and DL-forms can be used. Among these, biodegradable polymers having a free carboxyl group at the terminal, for example, polymers synthesized from α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, etc.) (eg, lactic acid-glycolic acid copolymer) Etc.) and poly-α-cyanoacrylate. The biodegradable polymer is more preferably a polymer synthesized from α-hydroxycarboxylic acids, and particularly preferably a lactic acid-glycolic acid copolymer.

When a lactic acid-glycolic acid copolymer or a homopolymer is used as the biodegradable polymer, its composition ratio (mol%) is preferably about 100/0 to about 40/60, and about 85/15 to about 40/60. 50/50 is more preferred.
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. The weight average molecular weight of the lactic acid-glycolic acid polymer is preferably from about 3,000 to about 25,000, more preferably from about 5,000 to about 20,000.
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 to 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 was performed using a GPC column KF804L x 2 (manufactured by Showa Denko),
Using RI Monitor L-3300 (manufactured by Hitachi, Ltd.)
This was performed using chloroform as a mobile phase. In addition, the biodegradable polymer having a free carboxyl group at the terminal is a polymer in which the number average molecular weight by terminal group quantification and the number average molecular weight by the above GPC measurement are almost the same,
The number average molecular weight by terminal group quantification 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 groups in the solution to 0.05N alcoholic. The mixture was rapidly titrated with a potassium hydroxide solution at room temperature (20 ° C.) with stirring, and the number average molecular weight by terminal group quantification was calculated by the following formula. Number average molecular weight determined by terminal group determination = 20,000 A / B A: Mass of biodegradable polymer (g) B: 0.05 N alcoholic potassium hydroxide solution added by the end of titration (ml) Number average molecular weight determined by terminal group determination Is an absolute value, whereas the number average molecular weight obtained by GPC measurement depends on various analysis and analysis conditions (for example, type of mobile phase, type of column, reference material, selection of slice width, selection of baseline, etc.). Because of the relative values that fluctuate, it is difficult to make a unique numerical value.However, it is considered that the number average molecular weights obtained by the two measurements are almost the same, for example, in a polymer synthesized from α-hydroxycarboxylic acids, by quantifying the terminal group. Number average molecular weight is G
It means that the number average molecular weight is in the range of about 0.5 to about 2 times the number average molecular weight determined by PC measurement. Preferably, it is in the range of about 0.7 times to about 1.5 times. For example, in a polymer synthesized from one or more α-hydroxycarboxylic acids by a noncatalytic dehydration polycondensation method and having a free carboxyl group at a terminal, the number average molecular weight by GPC measurement and the number average molecular weight by terminal group quantification are different. Almost match. In contrast,
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 about the number average molecular weight determined by GPC. Largely more than twice. 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.

Lactic acid having a terminal free carboxyl group
-Glycolic acid polymer can be produced by a method known per se, for example, a method described in JP-A-61-28521 (for example, a production by a dehydration polycondensation reaction in the absence of a catalyst or a dehydration polycondensation reaction in the presence of an inorganic solid acid catalyst). Method). The decomposition / elimination rate of the lactic acid-glycolic acid polymer greatly varies depending on the composition ratio or the weight average molecular weight, but generally, the lower the glycolic acid fraction, the slower the decomposition / elimination, and therefore the lower the glycolic acid fraction. Alternatively, the release period can be extended by increasing the molecular weight. Conversely, the release period can be shortened by increasing the glycolic acid fraction or decreasing the molecular weight.
In order to obtain a long-term (for example, 1 to 4 months) sustained-release preparation, lactic acid-
Glycolic acid polymers are preferred.

Therefore, in the present invention, the composition of the biodegradable polymer to be used is preferably selected according to the kind of the target bioactive polypeptide, the desired sustained release period, and the like. As a specific example, for example, when GH is used as a physiologically active polypeptide, it is preferable to use a lactic acid-glycolic acid copolymer, and the lactic acid-glycolic acid copolymer has a lactic acid / glycolic acid composition The ratio (mol%) is preferably about 85/15 to about 50/50, and more preferably about 75/25 to about 50/50. Its weight average molecular weight is about 8,000 to about 20,
000 is preferred, and more preferably about 10,000 to
It is about 20,000. The dispersity (weight average molecular weight / number average molecular weight) of the lactic acid-glycolic acid copolymer is preferably about 1.2 to about 4.0, more preferably about 1.5.
~ 3.5. The lactic acid-glycolic acid copolymer to be used can be produced according to a known method such as the method described in the above publication. The copolymer is preferably produced by non-catalytic dehydration polycondensation. Lactic acid, in which the number average molecular weight according to the GPC measurement method and the number average molecular weight according to the terminal group quantification method are almost identical.
It is preferable to use a glycolic acid copolymer (PLGA). Further, the copolymer may be used by mixing two kinds of lactic acid-glycolic acid copolymers having different composition ratios and weight average molecular weights at an arbitrary ratio. As such an example, for example, the composition ratio (lactic acid / glycolic acid) (mol%) is about 75%.
Lactic acid-glycolic acid copolymer having a weight average molecular weight of about 10,000 / 25 and a composition ratio (lactic acid / glycolic acid)
(Mol%) is about 50/50 and the weight average molecular weight is about 12,
A mixture with 000 lactic acid-glycolic acid copolymer is used. The weight ratio when mixing is preferably about 25.
/ 75 to about 75/25.

In the present invention, the zinc oxide used for producing the PLGA / zinc oxide is a poorly water-soluble zinc compound, and is itself insoluble or hardly soluble in an organic solvent such as dichloromethane. When zinc oxide coexists with PLGA in an organic solvent such as dichloromethane, the PLGA / zinc oxide is completely and unexpectedly efficiently formed and dissolved in the organic solvent. These operations are accomplished by simply adding PLGA and zinc oxide to the organic solvent,
Separation operation of LGA / zinc oxide is unnecessary. The bioactive polypeptide-containing MC can be easily prepared by directly adding the bioactive polypeptide to the thus obtained organic solvent solution of PLGA / zinc oxide. Further, the obtained MC keeps the biologically active polypeptide biologically stable and provides a sustained-release preparation having a small initial release and excellent sustainability.

The physiologically active polypeptide used in the present invention preferably has a molecular weight of about 1,000 to about 5
000, more preferably from about 5,000 to about 4 molecular weight
000 bioactive polypeptides are used. As typical activities of the bioactive polypeptide,
Hormonal action. The physiologically active polypeptide may be any of a natural product, a synthetic product, and a semi-synthetic product, and may be a derivative or an analog thereof. The mode of action of the bioactive polypeptide may be either agonistic or antagonistic. As the physiologically active polypeptide of the present invention,
For example, peptide hormones, cytokines, peptide neurotransmitters, hematopoietic factors, various growth factors, enzymes, polypeptide antibiotics, analgesic peptides and the like are used.

Examples of peptide hormones include insulin, somatostatin, somatostatin derivatives (Sandostatin, US Pat. Nos. 4,087,390 and 4,047).
No. 93,574, No. 4,100,117, No. 4,25
No. 3,998), growth hormone (GH), 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 (JP-A-50-121273, JP-A-52-1)
No. 16465), thyroid stimulating hormone (TS
H), luteinizing hormone (LH), follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG), thymosin (thymosin), motilin, vasopressin, a vasopressin derivative ｛desmopressin [Journal of the Japan Endocrine Society,
Vol. 54, No. 5, pp. 676 to 691 (1978)]
Reference II, oxytocin, calcitonin, parathyroid hormone (PTH), glucagon, secretin, pancreozimine, cholecystokinin, angiotensin, human placental lactogen, and the like are used. Peptide hormones are preferably 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.). As the monokine, for example, interleukin-1 (IL-1), tumor necrosis factor (TNF) and the like are used. The cytokine is preferably lymphokine or the like, and more preferably interferon or the like. The cytokine is particularly preferably interferon alpha or the like. As the peptide neurotransmitter, for example, substance P, serotonin, GABA and the like are used.

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. Examples of various growth factors include basic or acidic fibroblast growth factor (FG).
F) or their families (eg, EGF, TGF
-Α, TGF-β, PDGF, 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), and families thereof. Examples of enzymes include superoxide dismutase (SOD), urokinase, tissue plasminogen activator (TPA),
Asparaginase, kallikrein and the like are used. As the polypeptide 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. 4,277,3
No. 94, European Patent Application Publication No. 31567], endorphins, kyotorhuin and the like. Other bioactive polypeptides include thymopoietin, dynorphin, bombesin, caerulein,
Thystimimulin, thymic humoral factor (THF), blood thymic factor (FTS) and its derivatives (US Pat. No. 4,22
9,438), and other thymic factors [Progress of Medicine, Vol. 125, No. 10, 835-843 (1.
983)], peptides having an antagonistic action on neurotensin, bradykinin and endothelin (European Patent Publication Nos. 436189, 457195, 496452, JP-A-3-94692, and JP-A-3-1313).
0299) and the like. Bioactive polypeptides particularly preferably applied to the present invention include growth hormone, insulin and the like.

In the present invention, when the physiologically active polypeptide contains a metal, the metal content is preferably at most 0.1%, more preferably at most 0.01%, particularly preferably at most 0.001%. Thus, a bioactive polypeptide substantially free of metals is optimal. For example, crystalline insulin usually contains small amounts of heavy metals such as zinc, nickel, cobalt, cadmium and the like. 0.4% (w /
w) It is thought that insulin containing zinc exists as a hexamer, exists stably on its own, and weakens the interaction with the metal salt of the biodegradable polymer. If necessary, the metal contained in the physiologically active polypeptide may be removed in advance, and a known method is used for removing the metal. For example, an insulin-hydrochloric acid aqueous solution is dialyzed against water or an ammonium acetate solution and then freeze-dried to obtain an amorphous state of insulin with a minimum amount of metal. The growth hormone may be derived from any species, but is preferably derived from humans. In addition, natural sources extracted from the pituitary gland and the like are also used in the present invention, but preferably, recombinant GH (Japanese Patent Publication No. 6-12996, Japanese Patent Publication No.
-48987). More preferably, it is a recombinant human GH having the same structure as a natural type having no methionine at the N-terminus. The GH may be a metal salt, but a GH substantially not containing a metal is also used.

The organic solvent used in the present invention has a boiling point of 120 ° C.
The following is preferred. Examples of the organic solvent include halogenated hydrocarbons (eg, dichloromethane, chloroform, carbon tetrachloride, etc.), alcohols (eg, ethanol, methanol, 1,4-butanediol, 1,5-pentanediol, etc.), acetic acid Ethyl, acetonitrile and the like can be mentioned. These may be mixed and used at an appropriate ratio. When the organic solvent is used alone, for example, dichloromethane, acetonitrile and the like are preferable. When an organic solvent is used as a mixed solvent, for example, a halogenated hydrocarbon (eg, dichloromethane, chloroform, etc.) and an alcohol (eg, ethanol, methanol, 1,4-butanediol, 1,5-pentanediol, etc.) or acetonitrile Is preferred. In particular, a combination of dichloromethane and acetonitrile is widely used. The mixing ratio (volume ratio) between the halogenated hydrocarbon and the alcohol or acetonitrile is about 40: 1 to about 1:
1, preferably about 20: 1 to about 1: 1. In particular, it is desirable to use a halogenated hydrocarbon (such as dichloromethane) alone.

In the present invention, the zinc oxide used for producing the sustained-release preparation is preferably in the form of fine powder. The smaller the particle size, the shorter the reaction time can be expected. The above problems arise. The particle size of zinc oxide is generally about 0.001 μm to about 10 μm, preferably about 0.005 μm to about 1 μm, and more preferably about 0.01 μm to about 0.1 μm. In the organic solvent solution of the biodegradable polymer and zinc oxide, the content of zinc (Zn) with respect to the biodegradable polymer is preferably about 0.001 to about 2% (W / W) by weight ratio, more preferably. Is about 0.01 to about 2% (W / W), particularly preferably about 0.1 to about 2% (W / W). The zinc content in the organic solution of the biodegradable polymer and zinc oxide is
For example, it is quantified by a generally known analytical method such as an atomic absorption method. In the present specification, the sustained-release preparation may be fine particles containing a physiologically active polypeptide and a microcapsule base (biodegradable polymer / zinc oxide). A microcapsule containing one drug core in one particle, a polynuclear microcapsule containing many drug cores in one particle, or a drug dissolved or dispersed in a microcapsule base in molecular form Such microspheres are exemplified.

The sustained-release preparation of the present invention is produced by dispersing a physiologically active polypeptide in an organic solvent solution of a biodegradable polymer and zinc oxide and removing the organic solvent. In the present specification, a product formed from a biodegradable polymer and zinc oxide, which is formed in a clear solution in which a biodegradable polymer and zinc oxide are dissolved in an organic solvent, is referred to as “biodegradable polymer”. Polymer / Zinc Oxide "
Called. The biodegradable polymer / zinc oxide may be a compound or composition formed by intermolecular bonding, such as a normal salt, complex salt, double salt, or organometallic compound.
The biodegradable polymer / zinc oxide has the property of dissolving in an organic solvent and imparting an excellent sustained-release property to a sustained-release preparation as an end product. Further, the one in which the biodegradable polymer is PLGA is referred to as “PLGA / zinc oxide”. As used herein, the term “dispersion” indicates that the biologically active polypeptide is homogeneously dispersed in an organic solvent. Contained in liquid.
In the production method of the present invention, the organic solvent can be removed by, for example, (a) an in-water drying method (O / W method), (b) a phase separation method (coacervation method), (c) a spray drying method, and the like. And the like. Hereinafter, a method for producing a microcapsule as a sustained-release preparation will be described. In the production method of the present invention, a biodegradable polymer and zinc oxide are first made to coexist in an organic solvent to produce a biodegradable polymer / zinc oxide solution in an organic solvent. At this time, the concentration of the biodegradable polymer in the solution varies depending on the molecular weight, the type of organic solvent, and the like, but is, for example, about 0.1 to about 80% (W / W), preferably about 1 to about 70% (W / W). / W), more preferably about 2 to
It is about 60% (W / W). The amount of zinc oxide to be added varies depending on the type of the organic solvent. (W / W), more preferably about 0.1 to about 2.5% (W / W). The order of adding the biodegradable polymer and zinc oxide to the organic solvent is as follows.
Zinc oxide may be added to an organic solvent solution of the biodegradable polymer in the form of a powder or suspended in the organic solvent. An organic solvent solution may be added. Further, an organic solvent may be added after mixing both in powder form.

The conditions for forming a solution of a biodegradable polymer / zinc oxide such as PLGA / zinc oxide from the biodegradable polymer and zinc oxide are determined by the type of the biodegradable polymer to be used and the zinc oxide. Although it is appropriately changed depending on the particle diameter, the type of the organic solvent, their composition, and the like, for example, when PLGA is used as the polymer, it is usually about 0 to about 3
0 ° C., preferably at about 2 to about 25 ° C., for about 1 hour to about 168
By reacting for about 12 hours to about 96 hours, more preferably for about 24 hours to about 72 hours.
PLGA / zinc oxide can be obtained. However, the production of the PLGA / zinc oxide body of the present invention can be visually confirmed by dissolving the zinc oxide in a suspension state in the organic solvent at the time of the addition and forming a clear solution state. Without being limited to the range, the reaction time may be determined using visual observation of the liquid as an index. Although the reaction proceeds by simply allowing PLGA and zinc oxide to coexist in an organic solvent, it is advantageous to carry out the reaction with stirring and shaking by a suitable stirring and shaking means in terms of shortening the reaction time. is there. It is also preferable to carry out the reaction under ultrasonic irradiation. Here, the higher the reaction temperature is, the shorter the reaction time is, but at the same time, there is a disadvantage that the decomposition rate of PLGA is accelerated. In the present invention, the obtained biodegradable polymer / zinc oxide is preferably used in the next step in the form of an organic solvent solution. However, if desired, the organic solvent may be removed and the solid form may be used once.

Next, the physiologically active polypeptide is added to the organic solvent solution of the biodegradable polymer and the zinc oxide obtained as described above, preferably in a powder form, for example, in an amount of about 0% of the biodegradable polymer. 0.1 to about 50% (W / W), preferably about 1 to about 20% (W / W), more preferably about 3 to about 15% (W / W), and dissolve or disperse. An organic solvent dispersion containing a degradable polymer, zinc oxide and a biologically active polypeptide (hereinafter sometimes simply referred to as a biologically active polypeptide dispersion) is produced. When the physiologically active polypeptide does not dissolve in the organic solvent solution of the biodegradable polymer and zinc oxide, and becomes turbid when added as a powder, and has a property that it is difficult to disperse,
The physiologically active polypeptide is preferably dispersed in an organic solvent in advance. In the organic solvent, for example, a bioactive polypeptide stabilizer (eg, serum albumin,
Gelatin, protamine sulfate, etc.). In order to uniformly disperse the bioactive polypeptide in the organic solvent, it is preferable to apply external physical energy. For example, ultrasonic irradiation, a turbine type stirrer,
Homogenizer and the like can be mentioned. At this time, the bioactive polypeptide particle size in the organic solvent is about 0.5.
01 to about 200 μm, preferably about 0.05 to about 100 μm
m, more preferably about 0.1 to about 50 μm. At this time, the concentration of the physiologically active polypeptide in the organic solvent is about 1 to about 50%, preferably about 2 to about 20%. By such treatment, the particle size of the physiologically active polypeptide in the organic solvent can be kept constant, and the bioactive polypeptide can be uniformly dispersed in the organic solvent solution of the biodegradable polymer and zinc oxide. The bioactive polypeptide may be previously dispersed in an organic solvent independently of the biodegradable polymer / zinc oxide. In this case, the organic solvent used may have the same composition as the organic solvent in which the biodegradable polymer and the zinc oxide are dissolved, or may have a different composition. For example, the biodegradable polymer / zinc oxide may be dissolved in dichloromethane, the physiologically active polypeptide may be dispersed in acetonitrile, and both may be mixed. At this time, the ratio (volume ratio) between the bioactive polypeptide and the biodegradable polymer / zinc oxide is, for example, about 1: 1000.
To about 1: 1, preferably about 1: 200 to about 1: 5, particularly preferably about 1: 100 to about 1: 5.

(A) In-water drying method (o / w method) The physiologically active polypeptide dispersion prepared as described above is further added to an aqueous phase to form an o / w emulsion, The solvent in the phase is volatilized to produce microcapsules. At this time, an emulsifier may be added to the external aqueous phase. Generally, any emulsifier can be used as long as it can form a stable o / w emulsion. Specifically, for example, anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid and the like are used. The emulsifier is preferably polyvinyl alcohol. The emulsifier may be used alone or in combination of two or more. The concentration of the emulsifier in the external aqueous phase is from about 0.001 to about 20% (w / w), preferably about 0.01
About 10% (w / w), more preferably about 0.05%
~ 5% (w / w).

The microcapsules obtained in this manner are separated by centrifugation or filtration, and then the emulsifier and the like adhering to the surface of the microcapsules are removed by washing with distilled water, and the microcapsules are again added to distilled water or the like. Disperse and freeze-dry. Thereafter, if necessary, the mixture is heated to further remove the water and the organic solvent in the microcapsules. It may be heated under reduced pressure. As the heating conditions, the microcapsules are heated and dried at a temperature not lower than the glass transition temperature of the biodegradable polymer used and at such a level that the particles of the microcapsules do not adhere to each other. Preferably, heating and drying are performed in a range from the glass transition temperature of the biodegradable polymer to about 30 ° C. higher than the glass transition temperature. Here, the glass transition temperature refers to an intermediate point obtained when the temperature is raised at a heating rate of 10 to 20 ° C. per minute using a differential scanning calorimeter.

(B) Phase Separation Method (Coacervation Method) When producing MC by this method, a coacervation agent is gradually added to the above-mentioned physiologically active polypeptide dispersion under stirring to precipitate MC. Let it solidify. The added amount of the coacervation agent is about 0.01 times to about 1,000 times the dispersion.
The volume is 0 times, preferably about 0.05 times to about 500 times, more preferably about 0.1 times to about 200 times. As a coacervation agent, a polymer system that is miscible with an organic solvent that dissolves a biodegradable polymer,
Any compound may be used as long as it does not dissolve the biodegradable polymer used in the mineral oil or vegetable oil compound. Specifically, for example, silicone oil, sesame oil, soybean oil, corn oil, cottonseed oil, coconut oil, linseed oil, mineral oil, n-hexane, n-heptane and the like are used. These may be used as a mixture of two or more. After the thus obtained MC is collected by filtration, it is 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 the above (a). In the production of MC by the in-water drying method and the coacervation method, an anti-agglomeration agent may be added at the time of washing the MC in order to prevent aggregation of the particles. Examples of the agglomeration inhibitor include water-soluble polysaccharides such as mannitol, lactose, glucose, starches (eg, corn starch), hyaluronic acid and alkali metal salts thereof; amino acids such as glycine and alanine; fibrin, collagen and the like. Protein; inorganic salts such as sodium chloride and sodium hydrogen phosphate are used as appropriate.

(C) Spray drying method When producing MC by this method, a physiologically active polypeptide dispersion liquid is sprayed into a drying chamber of a spray drier (spray drier) using a nozzle, and the dispersion is performed in an extremely short time. The organic solvent in the atomized droplets is volatilized to produce MC. 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 liquid for the purpose of preventing the aggregation of MC particles. The MC thus obtained is washed in the same manner as in the above (a), and if necessary, further heated (under reduced pressure if necessary) to further remove water and the organic solvent.

In the present invention, when PLGA / zinc oxide is used as a microcapsule base, it is desirable that the uptake rate of a physiologically active polypeptide, for example, GH into MC is about 50% or more. The content of the physiologically active polypeptide contained in the sustained-release preparation of the present invention is, for example, about 0.1 to about 30.
% (W / w), preferably from about 0.2 to about 20% (w / w).
w), more preferably from about 0.5 to about 10% (w / w)
It is. The sustained-release preparation of the present invention can be prepared in various dosage forms, such as parenteral preparations (eg, intramuscular, subcutaneous) Injections or implants for organs, etc., transmucosal preparations for the nasal cavity, rectum, uterus, etc.), oral preparations (eg, capsules (eg, hard capsules, soft capsules, etc.), granules, powders, etc.) Solid preparations, liquid preparations such as suspensions, etc.). The preparations in these dosage forms are produced by known methods generally used for the production of preparations.

The sustained-release preparation of the present invention is particularly preferably an injection. In order to use the microparticles such as microcapsules obtained by the above method as an injection, the microparticles may be used as a dispersant (for example, a surfactant such as Tween 80 or HCO-60, carboxymethylcellulose, sodium alginate, sodium hyaluronate or the like). Polysaccharides, protamine sulfate, polyethylene glycol 400, etc.), preservatives (eg, methyl paraben, propyl paraben, etc.), tonicity agents (eg, sodium chloride, mannitol, sorbitol, glucose, etc.), local anesthetics (xylocaine hydrochloride, chloro Butanol) or an aqueous suspension, or a vegetable oil such as sesame oil or corn oil, or a mixture of this with a phospholipid such as lecithin, or a medium-chain fatty acid triglyceride (eg, Miglyol 812, etc.) to form an oily suspension. Sustained-release injections are used as suspensions. It is particularly preferable that the sustained-release preparation is fine particles. The particle size of the sustained-release preparation, when used as a suspension injection, may be within a range that satisfies the degree of dispersion and needle penetration, for example, about 0.1 to about 300 μm as the average particle diameter, preferably Is about 1 to about 150 μm,
More preferably, it is about 2 to about 100 μm. In order to make the above-mentioned fine particles 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.

Sustained-release preparations have low toxicity and are used in mammals (eg, humans, cows, pigs, dogs, cats, mice, rats, rabbits, etc.).
Used safely against The indication of the sustained-release preparation depends on the bioactive polypeptide used. Sustained-release preparations include, for example, diabetes when the bioactive polypeptide is insulin, and viral hepatitis (eg, hepatitis C, HBe antigen-positive active hepatitis, etc.) when it is interferon-α. ), Cancer (eg, renal cancer, multiple myeloma, etc.), anemia in the case of erythropoietin (eg, anemia during renal dialysis, etc.), and neutropenia in the case of G-CSF (eg, an anticancer drug) In the case of IL-2, cancer (eg, hemangioendothelioma, etc.), FGF, fracture, wound (bedsore, etc.), periodontal disease, gastrointestinal ulcer, etc.
TP such as thrombocytopenia in the case of FGF-9, senile dementia and neuropathy in the case of NGF
A is effective for treatment or prevention of thrombosis and the like, and tumor necrosis factor is a cancer or the like in the case of tumor necrosis factor. In addition, GH-containing sustained-release preparations are indicated not only for pituitary dwarfism, but also for Turner syndrome, chronic kidney disease, chondrodystrophy, and adult pituitary dysfunction based on the growth hormone action of GH. it can. It has also been reported that GH has been applied to diseases such as Down syndrome, Silver syndrome, osteogenesis imperfecta, and juvenile chronic arthropathy, and has obtained an effective therapeutic effect.

The dosage of the sustained-release preparation varies depending on the type and content of the physiologically active polypeptide, the duration of release, the target disease, the target animal, etc., but the effective concentration of the physiologically active polypeptide is maintained in the body. The amount may be any amount. For example, when the sustained release preparation is a one-week preparation, the dose of the physiologically active polypeptide can be appropriately selected from the range of preferably about 0.0001 to about 10 mg / kg body weight per adult. More preferably about 0.0005
To about 1 mg / kg body weight. The frequency of administration can be appropriately selected depending on the kind and content of the bioactive polypeptide, dosage form, duration of release, target disease, target animal, etc., such as once a week and once every two weeks. When the physiologically active polypeptide that is the active ingredient of the sustained release preparation is, for example, insulin, the dose to a diabetic adult is usually about 0.001 as the active ingredient.
To about 1 mg / kg body weight, preferably from about 0.01 to about 0.2.
The dose is appropriately selected from the range of mg / kg body weight and is preferably administered once a week.

When the physiologically active polypeptide which is the active ingredient of the sustained-release preparation is GH, the dosage varies depending on the type and content of GH, duration of release, target disease, target animal and the like. The effective concentration of the GH may be an amount that can be retained 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 in the range of about 0.01 to about 5 mg / kg body weight per child or adult. And can be safely administered. 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 can be appropriately selected depending on the GH content, dosage form, duration of release, target disease, target animal, etc., such as once a week, once every two weeks or once a month. The sustained-release preparation is preferably stored at room temperature or in a cold place. More preferably, the sustained-release preparation is stored in a cold place. The normal temperature or cold place here is defined in the Japanese Pharmacopoeia. That is, normal temperature means 15 to 25 ° C, and cold place means 15 ° C or less.

[0032]

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

【Example】

Example 1 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 10,000) 1 g
And 6.6 mg of zinc oxide were added to 1.7 ml of dichloromethane, and the mixture was stirred at 25 ° C. for 3 days (60 rpm) to give clear lactic acid-
An organic solvent solution of a glycolic acid copolymer / zinc oxide was obtained. To this solution was added human growth hormone lyophilized powder 53.0.
The resulting mixture was mixed with a vortex mixer and a small homogenizer and subjected to ultrasonic treatment to obtain an organic solvent solution containing human growth hormone and a lactic acid-glycolic acid copolymer / zinc oxide. This organic solvent solution was previously
The mixture was poured into 400 ml of a 0.1% (w / v) aqueous solution of polyvinyl alcohol (PVA) adjusted to ° C., and an o / w emulsion was obtained using a turbine homomixer. This o / w emulsion was stirred at room temperature to evaporate dichloromethane to prepare microcapsules. The obtained microcapsules are centrifuged (about 1500 r).
pm). Next, after washing twice with 400 ml of distilled water, it was freeze-dried to obtain 521 mg of human growth hormone-containing microcapsules in powder form.

Example 2 Lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 5)
0/50 (mol%), weight average molecular weight 10,000) 1 g
And 13.1 mg of zinc oxide were dissolved in 2.3 ml of dichloromethane to obtain an organic solvent solution of a lactic acid-glycolic acid copolymer / zinc oxide. 53.3 mg of human growth hormone freeze-dried powder was added to this solution, and the mixture was treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 53.
6 mg were obtained. Example 3 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 10,000) 1 g
And 21.9 mg of zinc oxide were dissolved in 2.8 ml of dichloromethane to obtain a lactic acid-glycolic acid copolymer / zinc oxide organic solvent solution. To this solution, 53.8 mg of human growth hormone freeze-dried powder was added, and treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 58.
9 mg were obtained.

Example 4 Lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 5)
0/50 (mol%), weight average molecular weight 12000) 1 g
And 5.1 mg of zinc oxide were dissolved in 1.9 ml of dichloromethane to obtain an organic solvent solution of a lactic acid-glycolic acid copolymer / zinc oxide. To this solution, 52.9 mg of human growth hormone freeze-dried powder was added, and treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 506.
mg was obtained. Example 5 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 12000) 1 g
Was dissolved in 2.5 ml of dichloromethane to obtain a solution of a lactic acid-glycolic acid copolymer / zinc oxide in an organic solvent. To this solution, 53.2 mg of freeze-dried human growth hormone powder was added, and treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 56.
8 mg were obtained.

Example 6 Lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 6)
5/35 (mol%), weight average molecular weight 12000) 1 g
And 17.0 mg of zinc oxide were dissolved in 3.0 ml of dichloromethane to obtain an organic solvent solution of lactic acid-glycolic acid copolymer / zinc oxide. To this solution, 53.5 mg of freeze-dried human growth hormone powder was added, and treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 56.
1 mg was obtained. Example 7 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 15000) 1 g
And 4.5 mg of zinc oxide were dissolved in 2.0 ml of dichloromethane to obtain a solution of a lactic acid-glycolic acid copolymer / zinc oxide in an organic solvent. To this solution, 52.9 mg of freeze-dried human growth hormone powder was added, and treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 540.
mg was obtained.

Example 8 Lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 5)
0/50 (mol%), weight average molecular weight 15000) 1 g
And 8.9 mg of zinc oxide were dissolved in 2.6 ml of dichloromethane to obtain an organic solvent solution of a lactic acid-glycolic acid copolymer / zinc oxide. 53.1 mg of human growth hormone lyophilized powder was added to this solution, and the mixture was treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 559.
mg was obtained. Example 9 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 15000) 1 g
And 14.9 mg of zinc oxide were dissolved in 3.1 ml of dichloromethane to obtain an organic solvent solution of a lactic acid-glycolic acid copolymer / zinc oxide. To this solution, 53.4 mg of human growth hormone freeze-dried powder was added, and treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 46.
4 mg were obtained.

Example 10 Lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 20,000) 1 g
And 4.0 mg of zinc oxide were dissolved in 2.5 ml of dichloromethane to obtain a solution of a lactic acid-glycolic acid copolymer / zinc oxide in an organic solvent. To this solution was added 52.8 mg of human growth hormone freeze-dried powder, and the mixture was treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 595.
mg was obtained. Example 11 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 20,000) 1 g
And 7.9 mg of zinc oxide were dissolved in 3.6 ml of dichloromethane to obtain an organic solvent solution of a lactic acid-glycolic acid copolymer / zinc oxide. To this solution, 53.1 mg of human growth hormone freeze-dried powder was added, and treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 478.
mg was obtained.

Example 12 Lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 5)
0/50 (mol%), weight average molecular weight 20,000) 1 g
And 13.2 mg of zinc oxide were dissolved in 5.2 ml of dichloromethane to obtain an organic solvent solution of a lactic acid-glycolic acid copolymer / zinc oxide. 53.3 mg of human growth hormone freeze-dried powder was added to this solution, and the mixture was treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 53.
4 mg were obtained. Example 13 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 7
5/25 (mol%), weight average molecular weight 10500) 1 g
And 6.6 mg of zinc oxide were dissolved in 3.0 ml of dichloromethane to obtain an organic solvent solution of a lactic acid-glycolic acid copolymer / zinc oxide. To this solution, 53.0 mg of human growth hormone freeze-dried powder was added, and treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 521.
mg was obtained.

Example 14 Lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 8)
5/15 (mol%), weight average molecular weight 12000) 1 g
And 5.8 mg of zinc oxide were dissolved in 2.0 ml of dichloromethane to obtain a solution of a lactic acid-glycolic acid copolymer / zinc oxide in an organic solvent. To this solution, 53.0 mg of human growth hormone freeze-dried powder was added, and treated in the same manner as in Example 1 to obtain a powdered human growth hormone-containing microcapsule 503.
mg was obtained.

Example 15 Lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 5)
0/50 (mol%), weight average molecular weight 10,000) 1.
89 g and zinc oxide (10 mg) in dichloromethane (3.4 m)
to give a solution of a lactic acid-glycolic acid copolymer / zinc oxide in an organic solvent. 100 mg of freeze-dried human growth hormone powder was added to this solution, and the mixture was treated in the same manner as in Example 1 to obtain 1.41 g of human growth hormone-containing microcapsules in powder form. Example 16 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 12000) 1.
89 g and zinc oxide (10 mg) in dichloromethane (3.5 m)
to give a solution of a lactic acid-glycolic acid copolymer / zinc oxide in an organic solvent. 100 mg of freeze-dried human growth hormone powder was added to this solution, and the mixture was treated in the same manner as in Example 1 to obtain 1.41 g of human growth hormone-containing microcapsules in powder form.

Example 17 Lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 5)
0/50 (mol%), weight average molecular weight 14000) 1.
89 g and zinc oxide (10 mg) in dichloromethane (4.0 m)
to give a solution of a lactic acid-glycolic acid copolymer / zinc oxide in an organic solvent. To this solution was added 100 mg of freeze-dried human growth hormone powder, and the same treatment as in Example 1 was performed to obtain 1.40 g of human growth hormone-containing microcapsules in powder form. Example 18 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 16000) 1.
89 g and zinc oxide (10 mg) in dichloromethane (4.2 m)
to give a solution of a lactic acid-glycolic acid copolymer / zinc oxide in an organic solvent. To this solution was added 100 mg of human growth hormone freeze-dried powder, and the mixture was treated in the same manner as in Example 1 to obtain 1.34 g of human growth hormone-containing microcapsules in powder form.

Comparative Example 1 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 15000) was dissolved in dichloromethane (950 mg / ml) to obtain a lactic acid-glycolic acid copolymer organic solvent solution. This solution 1
The mixture was mixed with 1 ml of a human growth hormone freeze-dried powder in dichloromethane solution (50 mg / ml) and treated in the same manner as in Example 1 to obtain 490 mg of human growth hormone-containing microcapsules in powder form.

Comparative Example 2 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 10,000) 1.
90 g was dissolved in 2.6 ml of dichloromethane to obtain an organic solvent solution of a lactic acid-glycolic acid copolymer. To this solution was added 100 mg of human growth hormone freeze-dried powder, and the same treatment as in Example 1 was performed to obtain 1.28 g of human growth hormone-containing microcapsules in powder form. Comparative Example 3 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 12000) 1.
90 g was dissolved in 2.8 ml of dichloromethane to obtain an organic solvent solution of a lactic acid-glycolic acid copolymer. 100 mg of freeze-dried human growth hormone powder was added to this solution, and the same treatment as in Example 1 was performed to obtain 1.18 g of human growth hormone-containing microcapsules in powder form.

Comparative Example 4 Lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 5)
0/50 (mol%), weight average molecular weight 14000) 1.
90 g was dissolved in 3.0 ml of dichloromethane to obtain an organic solvent solution of a lactic acid-glycolic acid copolymer. 100 mg of freeze-dried human growth hormone powder was added to this solution, and the mixture was treated in the same manner as in Example 1 to obtain 0.89 g of human growth hormone-containing microcapsules in powder form. Comparative Example 5 Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5
0/50 (mol%), weight average molecular weight 16000) 1.
90 g was dissolved in 3.2 ml of dichloromethane to obtain an organic solvent solution of a lactic acid-glycolic acid copolymer. To this solution was added 100 mg of freeze-dried human growth hormone powder, followed by treatment in the same manner as in Example 1 to obtain 1.26 g of powdered human growth hormone-containing microcapsules.

[Experimental Example] Experimental Example 1 Human growth hormone and PLGA.
Dispersion medium (composition of the dispersion medium: mannitol (5%), carboxymethylcellulose (0.5%), Tween 20 (0.1%))
Dissolved in distilled water and adjusted to pH 6.8 with acetic acid)
Similarly, 251 mg of the microcapsules containing the human growth hormone and PLGA / zinc oxide obtained in Example 8 were added to 2.25 ml of the dispersion medium, and the human growth hormone and PLGA / zinc oxide obtained in Example 9 were added to 2.25 ml of the dispersion medium. 327 mg of body-containing microcapsules in 1.75 ml of dispersion medium,
Further, human growth hormone and PL obtained in Comparative Example 1
229 mg of GA-containing microcapsules was added to a dispersion medium 1.75.
Dispersed in ml. 0.5 ml of the obtained dispersion (human GH)
Was administered subcutaneously to the back of the rat under ether anesthesia. Blood was collected over time from the tail vein to collect serum. The concentration of human GH in the obtained serum was measured by radioimmunoassay (Ab beads HGH, manufactured by Eiken Chemical Co., Ltd.). The results obtained are shown in [Table 1].

[Table 1] The human GH concentration in the group administered with human growth hormone and PLGA / zinc oxide-containing microcapsules obtained in Examples 7, 8, and 9 was lower than that in the group administered with human growth hormone and PLGA-containing microcapsules obtained in Comparative Example 1. And significantly higher values, and showed long-term persistence.
According to the production method of the present invention, a sustained-release preparation exhibiting excellent release properties can be produced.

Experimental Example 2 The microcapsules containing human growth hormone and PLGA / zinc oxide obtained in Examples 15, 16, 17 and 18 were 550 mg, 556 mg and 576 mg, respectively.
And 573 mg in 3.38 m of the dispersion medium described in Experimental Example 1.
1; 548 mg, 548 mg, 567 mg and 560 m of the microcapsules containing human growth hormone and PLGA obtained in Comparative Examples 2, 3, 4 and 5, respectively.
g was dispersed in 3.38 ml of the same dispersion medium. 0.75 ml of the obtained dispersion (containing 6 mg of human GH) was subcutaneously administered to the back of the rat under ether anesthesia. Blood was collected over time from the tail vein, and serum was collected. Human GH in the obtained serum
The concentration was measured by the radioimmunoassay described in Experimental Example 1. The obtained results are shown in [Table 2] to [Table 5].

[Table 2]

[0047]

[Table 3]

[Table 4]

[0048]

[Table 5] The human growth hormone obtained in Examples 15, 16, 17 and 18 and the human GH concentration in the microcapsule administration group containing PLGA / zinc oxide were the same as those of Comparative Examples 2, 3, 4, and 5, respectively. And PL
The value was significantly higher than the human GH concentration in the group containing the GA-containing microcapsules.

[0049]

According to the present invention, it is possible to provide a sustained-release preparation which increases the uptake rate of a physiologically active peptide such as growth hormone and exhibits a constant high blood concentration over a long period of time.

Claims (15)

[Claims] 1. A method for producing a sustained-release preparation, comprising: dispersing a physiologically active polypeptide in an organic solvent solution containing a biodegradable polymer and zinc oxide; and removing the organic solvent. 2. The method according to claim 1, wherein the physiologically active polypeptide is a growth hormone. 3. The method according to claim 1, wherein the biodegradable polymer is a lactic acid-glycolic acid copolymer. 4. The lactic acid / glycolic acid copolymer has a lactic acid / glycolic acid composition ratio (mol%) of about 85/15 to about 50/50.
The method according to claim 3, wherein the ratio is 50. 5. The method according to claim 3, wherein the lactic acid-glycolic acid copolymer has a weight average molecular weight of about 8,000 to about 20,000. 6. The method according to claim 1, wherein the content of zinc based on the biodegradable polymer in the organic solvent solution is about 0.001 to about 2% (W / W). 7. The sustained-release preparation has an average particle size of about 0.1 to about 3
2. The method according to claim 1, wherein the thickness is 00 μm. 8. The method according to claim 1, wherein the sustained-release preparation is for injection. 9. An o / w-type emulsion in which a growth hormone is dispersed in an organic solvent solution containing a lactic acid-glycolic acid copolymer and zinc oxide and the oil phase is an oil phase is dried in water. The production method according to claim 1, wherein 10. The method according to claim 1, wherein the sustained-release preparation is a sustained-release microcapsule. 11. An organic solvent solution containing a lactic acid-glycolic acid copolymer and zinc oxide. 12. An organic solvent-soluble lactic acid-glycolic acid copolymer / zinc oxide obtained by allowing a lactic acid-glycolic acid copolymer and zinc oxide to coexist in an organic solvent. 13. A dispersion in which a physiologically active polypeptide is dispersed in an organic solvent solution containing a lactic acid-glycolic acid copolymer and zinc oxide. 14. The dispersion according to claim 13, wherein the physiologically active polypeptide is a growth hormone. (15) A sustained-release preparation produced by the production method according to (1).