JP2001151694A - Method for producing protein powder - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply provide stable protein powder highly holding a higher-order structure without bringing the protein powder into contact with a liquefied gas. SOLUTION: This method for producing the protein powder, characterized by freezing a protein-containing solution at a cooling rate of about -300 to -10 deg.C/min and then drying the cooled solution. Thereby, the stable protein powder highly holding a higher-order structure can simply be obtained without bringing the protein powder into contact with a liquefied gas. The obtained protein powder can be processed into particles by a simple particle-forming treatment, and the obtained protein particles are used to provide the sustained release preparation which exhibits a stable and high blood concentration over a long period.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a protein powder, a sustained-release preparation characterized by using the protein powder, and a sustained-release preparation characterized by using a specific base. The present invention relates to a pharmaceutical preparation and the like.

[0002]

2. Description of the Related Art In recent years, with the development of genetic engineering and cell engineering techniques, E. coli, yeast, animal cells or goats,
2. Description of the Related Art Protein pharmaceuticals are produced in large quantities using living bodies such as hamsters, and are being applied to clinical applications. However, these proteins are not orally absorbed due to their extreme reactivity to acidic conditions and enzymatic digestion. Therefore, it is generally administered subcutaneously or intramuscularly. However, since the half-life in vivo is short, frequent administration is necessary, and the physical burden on the patient accompanying 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, which works to promote the growth of the body, as well as sugar / lipid metabolism, protein assimilation, It is a protein that has a wide variety of physiological actions, such as being involved in cell proliferation and differentiation. Currently, it is 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 pituitary dwarfism, several months to 10
The fact is that daily subcutaneous administration is performed for more than a year.

[0003] In order to address such problems inherent in protein pharmaceuticals, various studies on drug delivery systems have been conducted. For example, a sustained release agent that releases a protein over a long period of time. JP-A-8-217691 (WO 96/07399) discloses that a water-soluble peptidic physiologically active substance is converted into a water-insoluble or hardly water-soluble polyvalent metal salt with an aqueous solution of zinc chloride or the like. A method for producing a sustained-release preparation containing is disclosed. In addition, Japanese Patent Publication No. Hei 8-503950 (WO
No. 94/12158) discloses human GH (hereinafter hG).
H) and a biodegradable polymer as a sustained-release preparation, by spraying an organic solvent solution containing hGH and the polymer into liquid nitrogen to maintain bioactivity as porous particles. Discloses a method for preparing a sustained-release microcapsule. Furthermore, Japanese Patent Publication No. 10-50401
No. 7 (WO 95/29664) discloses that after dispersing zinc carbonate or the like in a solid state in a polymer solution, a physiologically active substance (hormone or the like) is added and the physiologically active substance and the metal cation are separately separated. A method for producing sustained-release microcapsules dispersed in a biodegradable polymer is disclosed. JP-A-10-231252 (WO 98/2798)
No. 0) and Japanese Patent Application Laid-Open No. 10-7538 (WO
97/01331) discloses a method for producing a sustained-release preparation containing a physiologically active polypeptide, but does not disclose conditions for freeze-drying the physiologically active polypeptide. Although various attempts have been made to construct a drug delivery system while maintaining the physiological activity of the protein, the inherent problems of proteins having a higher-order structure include denaturation during the formulation process and changes over time in the formulation. And / or denaturation in vivo after administration and the like, and there may be problems with the stability of the protein.
Specifically, in a sustained-release preparation, the efficiency of protein uptake into the preparation is low, excessive drug release in the initial stage of administration, difficulty in controlling drug release over a long period of time, low blood concentration after administration of the preparation, etc. The problem may remain unresolved.

[0004] However, if the protein can be prepared as a fine powder, a further improvement in stability can be expected due to a decrease in molecular mobility. As a method for producing a protein fine powder, Japanese Patent Application Laid-Open No. 4-500527 (WO 90 /
No. 13,285) discloses a method in which an aqueous protein solution is sprayed into a liquefied gas, frozen, and then dried. Other methods for producing protein fine powder include the Journal of Pharmaceutical Sciences (Journa
l of Pharmaceutical Sciences) Vol. 87, p. 152 (199
The spray drying method is reported in 8), but the denaturation of the protein increases in inverse relation to the particle size of the aqueous protein solution obtained by spraying. It states that it must be added. In addition, WO published after the priority date of the present application
No. 99/48519 describes a method for producing a bioactive polypeptide powder obtained by adding a water-miscible organic solvent and / or volatile salts to a bioactive polypeptide aqueous solution and freeze-drying the resultant. I have. Also, Japanese Patent Application Laid-Open No. 9-248
No. 177 describes a method for producing dried microbial cells which is rapidly frozen by dropping small droplets of a microbial cell culture on a metal plate cooled to a freezing temperature or lower. The cooling rate during lyophilization is generally slower than -10 ° C / min. For example, lyophilization of pharmaceuticals IV, Yoji Ohashi, Preparations and Machinery, page 8, 1988
"The freeze-drying in normal vials rarely freezes rapidly or uses high-concentration solutions of sugars that produce a glassy state unless special equipment is used. That is, the cooling rate is 0.3 to 5.0.
° C / min. " On the other hand, when an aqueous solution is sprayed on a liquefied gas such as liquid nitrogen, the cooling rate is extremely high. For example, in the case of liquid nitrogen, the cooling rate is higher than -300 ° C / min. In the present specification, the cooling rate is expressed with a minus sign, but this is simply minus for the purpose of cooling. Therefore, for example, a cooling rate of -300 ° C / min indicates that the object to be measured is cooled by 300 ° C per minute, and when the object to be measured is cooled by 150 ° C in 30 seconds, the cooling rate is- Shown as 300 ° C./min. Specifically, when the measurement object is cooled from 20 ° C. to −130 ° C. in 30 seconds, the cooling rate is indicated as −300 ° C./min.

[0005]

In a method for producing a protein powder which is sprayed into a liquefied gas, frozen and then dried, similar to the spray-drying method, there is a high possibility of denaturation of the protein. Therefore, large-scale and expensive facilities are required for heat insulation, for coping with expansion and contraction of the material of the device due to a temperature difference, for maintaining sterility, and for discharging liquefied gas. Further, by using a large amount of a surfactant, a protein fine powder having an average particle diameter of several microns can be obtained, but its use is limited because it contains a large amount of a surfactant. Therefore, there is a demand for easily providing a stable protein powder having a high-order structure at a high level without contact with a liquefied gas.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and by adjusting the cooling rate for freezing a protein-containing solution when producing a protein powder, It was found that a protein powder having a high-order structure was maintained at a high level. Further, they have found that a fine powder can be obtained by subjecting the obtained protein powder to an atomization treatment. In addition, the present inventors use the protein fine powder thus obtained in a sustained-release preparation to improve the encapsulation rate of protein in the preparation, excessive drug release in the early stage of administration, and sustained release. I found something. It has also been found that by applying or dropping a protein-containing solution during freezing, the cooling rate is adjusted and a protein powder having a high-order structure is maintained at a high level. Furthermore, it has been found that the above-mentioned freezing can be carried out more cheaply and more easily by using a shelf of a freeze-dryer usually used for freeze-drying of a drug. Based on these findings, the present inventors have completed the invention.

That is, the present invention provides (1) a method for producing a protein powder, which comprises filling a protein-containing solution into a refrigerant, freezing it at a cooling rate of about -300 to -10 ° C / min, and drying it. (2) The method according to the above (1), wherein the protein-containing solution is applied or dropped onto the refrigerant.
(3) When applying or dripping, the diameter of the dripping fluid is about 0.1
(4) The method according to (1), wherein the protein-containing solution is frozen without directly contacting the liquid refrigerant, and (4) the protein-containing solution is frozen. Wherein the volatile salt or a water-miscible organic solvent is added; (6) the method according to (5), wherein the volatile salt is ammonium acetate; (7) The protein powder obtained by the production method according to (1), (8) the protein powder according to (7), wherein the protein has a molecular weight of about 5,000 to 1,000,000 daltons, (9) the protein is a hormone, (1) the protein powder according to the above (7), which is a cytokine, a hematopoietic factor, a growth factor or an enzyme; (10) the protein powder according to the above (7), wherein the protein is a growth hormone or insulin; 1) The protein
About the α-helix content in the protein-containing solution
The protein powder according to the above (7), wherein the protein powder retains an α-helix of 45% or more, and (12) the protein powder obtained by subjecting the protein powder according to the claim 7 to atomization treatment. (13) The method according to the above (12), wherein the fine powder is subjected to atomization treatment so that the average particle diameter of the protein fine powder is about 0.5 to 20 μm. (12) A sustained-release preparation containing the protein fine powder obtained by the production method according to (12), (15) the sustained-release preparation according to (14), wherein the base of the sustained-release preparation is a biological substance or a synthetic polymer. (16) The sustained-release preparation according to the above (15), wherein the biological substance or the synthetic polymer is a biodegradable polymer, and (17) the molar ratio of lactic acid to glycolic acid is about 60:40 to 70:30. Lactic acid-glycolic acid copolymer And (18) a method for producing a sustained-release preparation characterized by using the protein fine powder obtained by the production method according to (12) above, and (19) a protein-containing preparation. Use of the protein powder according to the above (7) for producing a sustained-release preparation,
(20) The production method according to the above (1), wherein the protein-containing solution is sprayed and not frozen, (21) about 10% per 1300 cm ^{2 of} the refrigerant cooled to about -25 ° C or lower before coating or dripping. The production method according to (1), wherein the protein-containing solution is applied or dropped at a rate of about 250 mL / 5 minutes, and (22) the production method according to (1), characterized by drying under reduced pressure. And (23) the method according to the above (1), wherein the freeze-drying is performed using a shelf of a freeze dryer.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The protein in the present invention may be any of natural products, synthetic products, semi-synthetic products, and those produced by genetic recombination, and may also be derivatives, analogs or muteins thereof. . Generally, in order to obtain a large amount of high-purity protein, a gene recombination method is often used. The protein of the present invention preferably has a molecular weight of about 5,000 to about 1,000.
000 daltons, more preferably a molecular weight of about 6,0
00 to about 200,000 dalton proteins. As the protein in the present invention, specifically, hormones, cytokines, hematopoietic factors, growth factors,
Enzymes and the like.

[0009] The hormone may have an agonistic or antagonistic action mechanism. As the hormone, specifically, insulin, growth hormone (GH), prolactin, thyroid stimulating hormone (TS)
H), luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), thymosin (thymosin), parathyroid hormone (PTH), etc.
Preferably, insulin and growth hormone are used, and more preferably, growth hormone is used. The growth hormone may be derived from any species, but is preferably human growth hormone. Naturally-derived GHs 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). More preferably, it is a recombinant hGH having the same structure as a natural type having no methionine at the N-terminus. Such a recombinant hGH having the same structure as the natural type having no methionine at the N-terminus is disclosed in JP-A-10-72489 (EP-
A-81856) or WO 00/20439.
Can be obtained by the method described in Japanese Patent Application Publication No. The GH may be a metal salt (including a metal complex; for example, the metal includes zinc and the like), but GH substantially containing no metal is also used. As hGH, not only molecular weight of about 22K dalton but also molecular weight of about 20K
K Daltons (see JP-A-7-101877 and JP-A-10-265404) may be used.
Further, a derivative of hGH or a related protein thereof (see WO 99/03887) may be used as hGH.

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. Examples of the monokine include 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.

Examples of the hematopoietic factor include erythropoietin (EPO), colony stimulating factor (G-CSF, G
M-CSF, M-CSF, etc.), thrombopoietin (TP
O), platelet growth stimulating factor, megakaryocyte potentiator and the like are used. Examples of the growth factor include basic or acidic fibroblast growth factor (FG).
F) or their families (eg, EGF, T
GF-α, TGF-β, PDGF, acidic FGF, basic FGF, FGF-9, etc.), hepatocyte growth factor (HGF),
Vascular endothelial growth factor (VEGF), nerve cell growth factor (N
GF) or their families (eg, BDN
F, NT-3, NT-4, CNTF, GDNF, etc.), insulin-like growth factors (eg, IGF-1, IGF-2)
Etc.), factors involved in bone growth (BMP) or their families, etc. are used.

As the enzyme, for example, superoxide dismutase (SOD), urokinase, tissue plasminogen activator (TPA), asparaginase, kallikrein and the like are used. In addition, thymopoietin, blood thymic factor (FTS) and its derivatives (see US Pat. No. 4,229,438), and other thymic factors [Ayumi of Medicine, Vol. No. 10, 835-84
3 (1983)]. As the protein in the present invention, a hormone, more preferably a growth hormone or insulin, and further preferably a human growth hormone is used.

The protein in the present invention may be a protein containing a metal. The metal content of the metal-containing protein is preferably about 0.1% (w / w)
Or less, more preferably about 0.01% (w / w) or less,
More preferably 0.001% (w / w) or less, especially a protein substantially free of metal is optimal. Examples of the protein containing a metal include crystalline insulin. Crystalline insulin usually contains small amounts of heavy metals such as zinc, nickel, cobalt, cadmium and the like. Insulin containing 0.4% (w / w) of zinc exists as a hexamer and is stable on its own, weakening its interaction with metal salts of biodegradable polymers. It is believed that. The protein in the present invention may be a protein from which a metal has been removed in advance. A known method is used as a method for removing the metal of the protein. For example, in the case of insulin, an insulin in an amorphous state having a very low metal content can be obtained by dialyzing an aqueous hydrochloric acid solution of insulin against water or an ammonium acetate salt solution and freeze-drying the resulting solution. When obtaining a protein, if it is necessary to purify and purify the protein from a tissue / body fluid containing the desired protein, a chemically synthesized crude preparation, or a recombinant cell / bacterium, a general protein may be used. (“Protein”, written by Kazuo Satake, Asakura Shoten; “Bioactive peptide”, Pharmacia Review, No. 3, Japan Pharmaceutical Association). Among these separation and purification methods, among others, the combination of several liquid chromatography methods ("High Performance Liquid Chromatography of Proteins and Peptides", edited by Nobuo Ui et al., Chemical Doujinshi) enables high-purity proteins to have their physiological activities. A good yield can be obtained without any loss. In that case, it is preferable to be subjected to desalting operation in the final step of the separation and purification method.

The solvent of the protein-containing solution of the present invention is not particularly limited as long as it dissolves the protein. The solvent preferably has a freezing point of about −130 ° C. or higher. Specific examples of the solvent include water, alcohols (eg, methanol, ethanol, isopropanol, etc.), acetone, a mixed solvent of the alcohols and water, and a mixed solvent of an organic solvent such as acetone and water. And preferably water is used. Alcohols,
An organic solvent such as acetone can also be used as a “water-miscible organic solvent” described later.

The protein-containing solution using the above-mentioned solvent is
It is preferable that volatile salts or a water-miscible organic solvent is added. By further freeze-drying the aqueous protein solution obtained by adding such volatile salts or a water-miscible organic solvent, handling is easy (good operability) and fine (small particle size). Protein powder can be produced. Examples of the volatile salts added to the protein-containing solution 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 added to the protein-containing solution is, for example, about 1 to about 80 moles (specifically, about 10 to about 80 moles), preferably about 10 times, in a molar ratio. Or about 7
0 mole, more preferably about 15 to about 7 moles.
0 mole, more preferably about 15 to about 50 moles.
The molar ratio is about 15 times, and most preferably about 15 times to about 40 times. Examples of the water-miscible organic solvent added to the protein-containing solution include, for example, the alcohols described above,
Acetone or the like is used, and 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 protein-containing solution 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). The water-miscible volatile salts and / or organic solvents added to the protein-containing solution 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 protein-containing solution according to the respective addition amounts described above. Preferably, volatile salts are added to the protein-containing solution, more preferably ammonium salts are added, and even more preferably ammonium acetate is added.

The concentration of the protein-containing solution to be subjected to the freezing operation is not particularly limited, but may be, for example, 0.01% (W /
V) to 30% (W / V), preferably 0.03%
(W / V) to 10% (W / V), particularly preferably 0.05% (W / V) to 3% (W / V).
For example, when the protein is hGH, the concentration of the protein-containing solution is preferably about 0.01 to about 5%.
(W / V), more preferably from about 0.05 to about 2
% (W / V), more preferably from about 0.05 to about 0.5%.
5% (W / V). The protein in the protein-containing solution subjected to the freezing operation is preferably a single protein.

The method for freezing and drying the protein-containing solution in the present invention is not particularly limited, but drying under reduced pressure (eg, vacuum drying) is preferred. For example,
The protein-containing solution that has been frozen in a separate step from the freeze dryer may be dried using a freeze dryer. The device for the freezing is not particularly limited, but from the viewpoint of inexpensively and easily producing the protein powder, a shelf (freeze-drying) of a freeze-drying machine usually used when freeze-drying a pharmaceutical injection is used. Shelf). For the freeze dryer, see Masakazu Kobayashi, "Pharmaceutical Production and Freeze-Drying Technology"
~ 23, 25-35, 38-46). According to that, it is possible to lower the temperature of a freeze-drying shelf to -70 ° C by cooling using ordinary brine. These freeze dryers include, for example, Kyowa Vacuum Engineering Co., Ltd. (eg, RL series, RLC series, RLE series, R2L series, R2L
W series or Triomaster series), manufactured by Japan Vacuum Engineering Co., Ltd. (eg, DF series, DFM series). Further, the preliminary freezers of these freeze dryers are originally designed to be able to handle aseptic and dust-free products for the production of pharmaceutical injections, and are therefore suitable for the production of protein powder. By using a liquefied gas as the primary medium of the lyophilizer and introducing it through the secondary medium, it is also possible to lower the temperature of the lyophilization shelf further than is achieved with normal brine. For example, liquid nitrogen is used as the primary medium, and hydrofluoroether (HF
E: 3M (made by 3M)
It becomes possible to cool to -135 ° C with 100 (made by 3M (3M)) and -117 ° C with HFE-72100 (made by 3M (3M)), respectively. By using such a method, direct contact of the protein-containing solution with the liquefied gas is avoided, and it is not necessary to face the difficult problem of aseptic and dust-free treatment of the liquefied gas. Preferred shelf temperature is about -130 to -20
° C, more preferably about -100 to -30 ° C, more preferably about -80 to -40 ° C. By freezing the protein-containing solution using the freeze-dryer shelf as described above, it is possible to immediately proceed to the vacuum drying step. The freezing is preferably performed on a refrigerant placed on the shelf of the freeze dryer. The refrigerant is not particularly limited, but preferably includes a refrigerant to which a protein-containing solution can be applied or dropped, such as a plate or a tray, and preferably a tray. The plate, tray, etc. are not limited to flat surfaces, and may have irregularities or may be curved. The material of the plate, the tray, and the like may be any material as long as it can withstand the cooling rate of the present invention, but is preferably made of metal (for example, stainless steel). The temperature of the refrigerant (eg, tray) at the start of coating or dropping is preferably about −25 ° C. or less (eg, −25 ° C. to −100 ° C .; specifically, −2 ° C.).
(5 ° C. to −50 ° C.). In addition, you may further apply or drip on the frozen protein-containing solution. The surface temperature of the frozen protein-containing solution at the start of coating or dropping is preferably about −25 ° C. or lower (for example, −25 ° C. to −100 ° C .; specifically −2 ° C.).
(5 ° C. to −50 ° C.). The surface temperature of the refrigerant and the frozen protein-containing solution can be determined, for example, by using a temperature sensor [for example, a thermocouple (TYPE T: manufactured by Okazaki Seisakusho)].
Can be measured. When the protein-containing solution frozen in a place different from the freeze-dryer is dried by a freeze-dryer, for example, the protein-containing solution is frozen and then transferred to a freeze-dryer shelf while maintaining the state. And drying.

The cooling rate of the protein-containing solution in the present invention depends on the average particle diameter of the target protein fine powder,
It is appropriately controlled by the type of the protein-containing solution, the protein concentration, the additive concentration, and the like. The cooling rate of the protein-containing solution in the present invention is generally about -300 to -10 ° C / min, preferably about -250 to -20 ° C / min, more preferably about -210 ° C / min.
Min to -30 ° C / min, more preferably -210 to -40 ° C / min, particularly preferably -210 to -70 ° C / min. The cooling rate in the present invention is calculated based on the temperature of the protein-containing solution before coating or dropping, the temperature at the time of freezing of the protein-containing solution after dropping or coating, and the time until freezing. In addition,
The temperature at the time of freezing of the protein-containing solution after dripping or coating can be measured, for example, using the same temperature sensor as described above. As a method for obtaining the cooling rate, for example, before coating or dripping, about -25 ° C or less (preferably about -10
0 to-25 ° C., more preferably from about -100 to-30 ° C., more preferably from about -80 to-40 ° C.) to the cooled refrigerant 1300 cm ² per
A method in which the protein-containing solution is applied or dropped at a rate of about 10 to 250 mL / 5 minutes, preferably about 15 to 200 mL / 5 minutes, and more preferably about 20 to 175 mL / 5 minutes. The rate at which the protein-containing solution is filled (applied or dropped) can be appropriately selected from the range in which the cooling rate can be obtained. During the filling, the speed of applying or dropping the protein-containing solution may be appropriately changed. The temperature of the refrigerant during application or dropping may be about −2 ° C.

The coating in the present invention means that the protein-containing solution is filled as a continuous fluid without forming droplets from an opening (for example, a nozzle for filling the protein-containing solution). The term “dropping” in the present invention means that a droplet of a protein-containing solution is formed from an opening (for example, a nozzle for filling the protein-containing solution) and is filled as a discontinuous fluid. When applying or dripping, the entire amount of the protein-containing solution to be frozen can be applied or dropped at a stretch, but intermittent application or dripping in several steps lowers the temperature of the frozen part Preferred for. As a result, a desired cooling rate can be maintained. When applying or dropping intermittently, it is preferable to provide a time interval between coating and coating or dropping and dropping until the surface temperature of the refrigerant or the frozen portion becomes -25 ° C or lower. In the present invention, the dropping fluid refers to both a continuous fluid that is filled without forming droplets at the time of application and a discontinuous fluid at the time of dropping. When the protein-containing solution is applied or dropped, the diameter (the maximum length of the horizontal cross section) of the dropping fluid of the protein-containing solution is, for example, about
0.1-40 mm, preferably about 0.2-40 mm, more preferably about
It is 0.3 to 20 mm, more preferably about 0.3 to 10 mm. The shape of the dropping fluid when applying the protein-containing solution is preferably cylindrical, but may be various shapes depending on the shape of the opening such as a polygonal prism (for example, a triangular prism, a quadrangular prism, a pentagonal prism, a hexagonal prism, etc.). Can be. The diameter of the droplet when the protein-containing solution is dropped is preferably about 0.1 to 10 mm, more preferably about 0.7 to 7 mm, and more preferably about 1 to 5 mm.
It is. The diameter of the opening (for example, a filling nozzle) when the protein-containing solution is dropped is preferably about 0.05 to 10 mm,
More preferably, it is about 0.1 to 5 mm. The weight of the droplet when the protein-containing solution is dropped is preferably about 0.0005.
500500 mg, more preferably about 0.2-180 mg, even more preferably about 0.5-65 mg. In the present invention, when the protein-containing solution is applied or dropped, an ice sheet (ice laying) may be prepared in advance on the refrigerant. In the present invention, when the protein-containing solution is frozen, it is also possible to freeze the protein-containing solution in layers. The thickness of the frozen layer at that time is preferably about 0.5 to 1
00 mm, more preferably about 1-80 mm, more preferably about 3
~ 50mm.

In order to make the protein powder obtained by the method of the present invention into finer particles, it is preferable that the protein powder is further subjected to atomization treatment. For the atomization treatment, various powder pulverization treatment methods known in the preparation of pharmaceutical preparations can be used. Examples of the powder pulverizing method include a jet mill method as a dry pulverizing method, and a wet pulverizing method in which a protein fine powder is dispersed in an insoluble solvent, followed by ultrasonic treatment (probe). Type and bath type), stirring type pulverizer (Polytron (Kinematica)),
A method of removing the solvent after treatment with a mini-mixer, Fillmix (manufactured by Tokushu Kika Co., Ltd.), CLEARMIX (M-Tech Co., Ltd.) or the like can be used. Further, the protein powder obtained by the method of the present invention can also be atomized by lightly stirring or lightly shaking in an insoluble solvent for the protein powder (for example, dichloromethane or the like).
When applying the obtained protein powder to a sustained-release preparation, the protein powder is directly added to a base (eg, a biodegradable polymer solution), and then finely divided by ultrasonic waves or a stirring-type pulverizer. It is preferable to carry out a chemical treatment. For example, when a polytron (Kinematica) having a rotation diameter of about 9 mm is used as the stirring-type pulverizer, it is preferably about 500 to 4000.
0 rpm, more preferably about 1000-35000 rpm
m, more preferably at about 5,000 to 30,000 rpm. The stirring time at that time is preferably about 5 seconds to 30 minutes, more preferably about 10 seconds to 20 minutes, and still more preferably about 15 seconds to 10 minutes. For example, when a polytron (Kinematica) having a rotation diameter of about 9 mm is used as the stirring-type pulverizer, it is preferably about 500 to 40000 rpm for about 5 seconds to 30 minutes, and more preferably about 1000 to 35000 rpm for about 10 seconds to
20 minutes, more preferably about 5000 to 30000 r
It is preferable to perform the atomization treatment at pm for about 15 seconds to 10 minutes. The average particle size of the protein fine powder after the micronization treatment varies depending on the drug delivery system to which it is applied, but is generally preferably about 0.5 to 20 μm, more preferably 0.7 to 10 μm, and more preferably about 1 to 5 μm. is there. The average particle diameter of the protein fine powder in the present invention can be measured by a laser diffraction type particle size distribution analyzer (SALD 2000A: manufactured by Shimadzu Corporation). In the measurement, the protein fine powder is dispersed in an insoluble solvent (for example, dichloromethane or the like) of the protein fine powder, and then appropriately diluted with the solvent to a measurable range of the particle size distribution measuring device.

The protein powder and protein fine powder produced according to the present invention have a higher order structure than proteins in a protein-containing solution (eg, a protein-containing aqueous solution) before the production method of the present invention is performed. Are held at a high rate. Secondary structures in protein powder and protein fine powder can be confirmed by FT-IR spectrum analysis. This analysis is based on a review by Carpenter and colleagues (European Genial of Pharmaceuticals and Biophor Matheutics (Europe)
an Journal of Pharmaceutics and Biopharmaceutics)
45 231-238 1998). According to the report, it was reported that the content of α-helix, one of the secondary structures in protein powder and protein fine powder obtained by freeze-drying, was lower than the content of protein in aqueous solution. This indicates that the higher the degree of denaturation, the higher the decrease rate. Therefore, α of protein in a protein-containing solution (eg, a protein-containing aqueous solution) obtained by FT-IR spectrum analysis before the production method of the present invention is performed.
The degree of denaturation of the higher order structure of the protein can be defined from the ratio of the α-helix content in the protein powder or protein fine powder obtained by the production method of the present invention to the helix content. When the degree of denaturation of the protein is defined by the above method, the protein powder and the protein fine powder obtained by the present study preferably retain α-helix of about 45% or more, and more preferably about 50% or more. It is more preferable that

The protein powder and protein fine powder having a higher-order structure (specifically, a secondary structure, more specifically, α-helix) obtained by the present invention are applied to various drug delivery systems. You. The routes of administration include pulmonary, transmucosal (eye, buccal, nasal, uterine, vaginal, rectal), oral,
Percutaneous, intramuscular, subcutaneous, and injections or implants into organs and the like. Protein powders and protein fine powders can be administered as powders, but can be formulated into various dosage forms (eg, tablets, granules, sustained-release preparations, etc.) and used. It is administered in a release preparation. The sustained-release preparation is made up of a tableting method, a spray chilling method, a spray drying method (spray drying method), an emulsifying method, and an underwater drying method using protein powder or protein fine powder and various bases described below. Method (S / O / W method)
Alternatively, it can be formulated by a phase separation method (coacervation method) or the like.

The base used in formulating the sustained-release preparation may be any of those derived from living organisms and those obtained by synthesis (eg, synthetic polymers), but synthetic polymers are often used. Examples of biological bases include gelatin, collagen, fats (lipids, triglycerides, etc.), serum-derived proteins (albumin, globulin, etc.), keratin, chitin, chitosan, pullulan, and celluloses (hydroxymethylcellulose, carboxymethyl). Cellulol, etc.). As the synthetic polymer, any of a biodegradable polymer and a non-biodegradable polymer can be used, but a biodegradable polymer is preferably used.

Examples of the biodegradable polymer used as a base of the sustained-release preparation include α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.), A polymer having a free carboxyl group or a mixture thereof, a poly-α-cyanoacrylate, a polyamino acid (for example, synthesized from one or more kinds of tricarboxylic acids (for example, citric acid and the like) by dehydration-free polycondensation without catalyst, , Poly-γ-
Benzyl-L-glutamic acid, etc.), and maleic anhydride polymers (eg, styrene-maleic acid polymers). 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, any of D-, L-, and DL-forms can be used. As the biodegradable polymer, preferably,
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 polymer, lactic acid-glycolic acid copolymer, etc.), poly-
α-cyanoacrylate and the like, more preferably α-
Polymers synthesized from hydroxycarboxylic acids and the like are more preferable, and lactic acid-glycolic acid polymers are more preferable. In this specification, a lactic acid-glycolic acid polymer may be simply referred to as a 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 100 /%.
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 50,000
00 is preferred, about 3,000 to about 25,000 is more preferred, and about 5,000 to about 20,000 is even 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.

In the present specification, the weight average molecular weight of the lactic acid-glycolic acid polymer is 120,000,
52,000, 22,000, 9,200, 5,05
The values in terms of polystyrene measured by gel permeation chromatography (GPC) using nine types of polystyrenes of 0, 2,950, 1,050, 580 and 162 are shown. The degree of dispersion of the lactic acid-glycolic acid polymer is a value calculated from the value of the weight average molecular weight of the lactic acid-glycolic acid polymer. The weight average molecular weight of the lactic acid-glycolic acid polymer was measured using a GPC column KF804.
L x 2 (Showa Denko), RI monitor L-3300
(Manufactured by Hitachi, Ltd.) using chloroform as the mobile phase. In this specification, the weight average molecular weight of a lactic acid-glycolic acid polymer may be indicated as a polystyrene equivalent weight average molecular weight.

The biodegradable polymer having a free carboxyl group at the terminal is a polymer having a number average molecular weight almost identical to the number average molecular weight determined by the above-mentioned GPC measurement, and a number average molecular weight determined by the above-mentioned GPC measurement. The molecular weight is calculated as follows. About 1 g to about 3 g of the biodegradable polymer is mixed with acetone (25 ml) and methanol.
(5 ml) with phenolphthalein as an indicator to reduce the carboxyl group in this solution to 0.05N.
Titration was carried out quickly with stirring in an alcoholic potassium hydroxide solution at room temperature (20 ° C.), and the number average molecular weight by terminal group quantification was calculated by the following formula. Number average molecular weight by terminal group quantification =
20,000 × A / BA: mass of biodegradable polymer (g) B: 0.05N alcoholic potassium hydroxide solution added by the end of titration (ml) Although the number average molecular weight determined by terminal group determination is an absolute value On the other hand, the number average molecular weight determined by GPC is a relative value that varies depending on various analysis and analysis conditions (for example, the type of mobile phase, the type of column, the reference material, the selection of a slice width, the selection of a baseline, and the like). Although it is difficult to make a unique numerical value, the number average molecular weights obtained by the two measurements are almost the same, for example, in a polymer synthesized from α-hydroxycarboxylic acids, the number average molecular weight determined by the terminal group is determined by the GPC measurement. The number average molecular weight is in the range of about 0.5 times to about 2 times, preferably 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. 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.

Lactic acid having a free carboxyl group at the terminal
The 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, etc.). The rate of decomposition and disappearance of lactic acid-glycolic acid polymer is
It varies greatly depending on the composition ratio or the weight average molecular weight, but generally, the lower the glycolic acid fraction, the slower the decomposition and disappearance. (Eg, about 6 months). Conversely, the release period can be shortened (eg, about 1 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-mentioned 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 according to the kind of the desired physiologically active polypeptide, the desired sustained release period, and the like. As a specific example, for example, when GH is used as a protein, 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%) of about Preferred is an 85/15 to about 50/50 lactic acid-glycolic acid copolymer, more preferably about 75/25 to about 50/50 lactic acid-glycolic acid copolymer.
Its weight average molecular weight is about 8,000 to about 20,000.
00, more preferably from about 10,000 to about 20,000. The degree of dispersion (weight average molecular weight / number average molecular weight) of the lactic acid-glycolic acid polymer is about 1.
Preferably from 2 to about 4.0, more preferably from about 1.5 to about 3.5. The lactic acid-glycolic acid polymer 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. The polymers differ in composition ratio and / or weight average molecular weight.
Various kinds of lactic acid-glycolic acid polymers may be mixed and used in 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 the composition ratio (lactic acid / glycolic acid) (mol%) is about 50.
For example, a mixture with a lactic acid-glycolic acid copolymer having a weight average molecular weight of about 12,000 is used. The weight ratio upon mixing is preferably about 25/75 to about 75 /
25.

The biodegradable polymer used as the base of the sustained-release preparation may be a metal salt of the above-mentioned biodegradable polymer. A polyvalent metal salt of a polymer 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, etc., more preferably, a zinc salt, etc.) 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 trivalent (eg, iron, aluminum, manganese, etc.) and tetravalent (eg, tin, etc.) 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.For example, when the lactic acid-glycolic acid polymer is a polyvalent metal salt, the lactic acid-glycolic acid polymer is also used. 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 zinc salt of the biodegradable polymer can be produced, for example, by the following method. First, a biodegradable polymer and zinc oxide are made to coexist in an organic solvent to produce an organic solvent solution of a biodegradable polymer / zinc oxide. On this occasion,
The concentration of the biodegradable polymer in the solution varies depending on the molecular weight, the type of the organic solvent, and the like.
0% (W / W), preferably about 1 to about 70% (W / W).
W), more preferably about 2 to about 60% (W / W). Further, the amount of zinc oxide to be added is described in JP-A-10-23.
As described in JP-A-1252, although it depends on the type of the organic solvent, for example, about 0.001 to about 2% (W / W), preferably about 0.0, of the biodegradable polymer amount.
1 to about 1.5% (W / W), more preferably about 0.1
Or about 1% (W / W). The order of adding the biodegradable polymer and zinc oxide to the organic solvent may be such that zinc oxide may be added to the organic solvent solution of the biodegradable polymer 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. Further, an organic solvent may be added after mixing both in powder form.

The organic solvent used for dissolving the biodegradable polymer in the production of the sustained-release preparation preferably has a boiling point of 120 ° C. or lower. Examples of the organic solvent include halogenated hydrocarbons (eg, dichloromethane, chloroform, carbon tetrachloride, etc.), alcohols (eg, ethanol, methanol, 1,4-butanediol, 1,5-pentanediol, etc.), acetic acid Ethyl, acetonitrile and the like can be mentioned. These may be mixed and used at an appropriate ratio. When an 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.), 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) of the halogenated hydrocarbon to 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 alone (such as dichloromethane) or a mixed solvent of a halogenated hydrocarbon and acetonitrile in a ratio of 9: 1 to 1: 1. The concentration of the biodegradable polymer in the solution varies depending on the molecular weight, the type of the organic solvent, and the like.
01 to about 80% (W / W), preferably about 0.1 to about 70% (W / W), and more preferably about 1 to about 60% (W / W).

The sustained-release preparation contains a water-miscible organic solvent and / or
Alternatively, the powder (S) obtained by freeze-drying a protein-containing solution (for example, a physiologically active polypeptide solution) to which volatile salts have been added is dissolved in a biological substance or a synthetic polymer (for example, a biodegradable polymer). It is manufactured by removing the solvent from the S / O type dispersion liquid dispersed in the organic solvent liquid (O). Examples of the production method include (a) an underwater drying method (S / O / W method), (b) a phase separation method (coacervation method), and (c) a spray drying method, or a method analogous thereto. No. Hereinafter, a method for producing a microcapsule as a sustained-release preparation will be described.

(A) Underwater drying method (S / O / W method) According to this method, a water-miscible organic solvent and / or volatile salts are first added to an aqueous protein solution, and then the protein powder is freeze-dried. (S) is created. Next, the biodegradable polymer is dissolved in an organic solvent, and the protein fine powder is added and dispersed in the organic solvent liquid. At this time, the ratio (weight ratio) between the protein and the biodegradable polymer
Is, for example, from about 1: 1000 to about 1: 1, preferably from about 1: 200 to about 1: 5, more preferably about 1: 1.
00 to about 1: 5. In order to uniformly disperse and atomize the protein 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 protein in the organic solvent solution is preferably about 0.5 to 20 μm, more preferably about 0.7 to 10 μm, and more preferably about 1 to 5 μm. This is achieved by using a protein powder obtained by the method. Next, the organic solvent dispersion (S / O type dispersion) thus prepared is further added to the aqueous solvent (W), and the same external physical energy as described above, for example, ultrasonic irradiation, turbine type An S / O / W emulsion is formed using a stirrer or a homogenizer. Thereafter, the oil phase solvent is evaporated to produce microcapsules. The volume of the water phase at this time is generally about 1 to about 1 times the volume of the oil phase.
It is selected from 0000 times. More preferably, it is selected from about 2 times to about 5,000 times. Particularly preferably, it is selected from about 5 times to about 2,000 times. An emulsifier may be added to the 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 between about 0.001% and 20% (w / w)
It is. More preferably, about 0.01% to 10% (w
/ W), particularly preferably from 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. Thereafter, if necessary, the mixture is heated to further remove 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, the glass transition temperature of the biodegradable polymer is 10
The heating and drying are performed at a temperature in a range from a low temperature to about 20 ° C. higher than a glass transition temperature. Here, the glass transition temperature means 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) In this method, a microcapsule is precipitated and solidified by gradually adding a coacervation agent to the S / O-type dispersion of (a) under stirring. The coacervation agent is added in an amount of about 0.01 times to about 1,000 times the volume of the dispersion. More preferably, about 0.05 times to about 500 times.
Times, particularly preferably about 0.1 to about 200 times the volume. 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 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 or the like is 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 in the same manner as in (a) and then freeze-dried. 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. Examples of the coagulation inhibitor include water-soluble polysaccharides such as mannitol, lactose, glucose, starches (eg, corn starch), hyaluronic acid and alkali metal salts thereof, proteins such as glycine, fibrin, collagen, sodium chloride, and phosphate. Inorganic salts such as sodium hydrogen and the like are appropriately used.

(C) Spray drying method In this method, the S / O type dispersion of (a) is sprayed into a drying chamber of a spray drier (spray drier) using a nozzle, and the fine particles are sprayed in a very short time. The organic solvent in the droplets is volatilized 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-aggregation agent from another nozzle simultaneously with the above-mentioned dispersion liquid for the purpose of preventing the aggregation of the microcapsule particles. The microcapsules thus obtained are washed in the same manner as in the above (a) and, if necessary, are heated (if necessary under reduced pressure) to further remove water and the organic solvent.

The sustained-release preparation is preferably in the form of fine particles. This is because the sustained-release preparation is not excessively painful 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 as an average particle size.
To 300 μm, preferably about 1 to 150 μm, particularly preferably about 2 to 100 μm. The content of the protein contained in the sustained-release preparation is, for example, about 0.1 to 40% (W / W), preferably about 0.2 to 20%.
(W / W). The average particle size of the protein is preferably about 0.5 to 20 μm or less, more preferably about 0.5 to 20 μm.
It is 7 to 10 μm, more preferably about 1 to 5 μm. The content of the biological substance or synthetic polymer (preferably biodegradable polymer) contained in the sustained-release preparation is, for example, about 30 to 99.9% (W / W), preferably about 60 to 97% (W / W). W / W), more preferably about 70
To 90% (W / W). The initial release rate of the protein of the sustained release preparation [the release rate up to one day (24 hours) after administration] is preferably about 50% or less, more preferably about 1 to 40%, and more preferably about 3 to 35%. It is. In addition, the initial release rate was determined by measuring the blood concentration of AUC (Area Under the C
oncentration) is applied to the dose-AUC straight line obtained from the AUC up to 24 hours after the subcutaneous administration of the protein solution to obtain the initial release amount, and further calculate the initial release rate.

[0038] Sustained-release preparations are formulated into various dosage forms, for example, as microcapsules or using the microcapsules as a starting material, and 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 sustained-release preparation is particularly preferably for injection. For example, when the sustained-release preparation is a microcapsule, the microcapsule is used as a dispersant (for example, Tween 80, HC
Surfactants such as O-60, carboxymethylcellulose, sodium alginate, polysaccharides such as hyaluronic acid, etc.), preservatives (eg, methyl paraben, propyl paraben, etc.), tonicity agents (eg, sodium chloride, mannitol, sorbitol, Practical sustained-release preparations for injection can be obtained by preparing an aqueous suspension together with glucose and the like.
In addition, vegetable oils such as sesame oil and corn oil, or a mixture thereof with a phospholipid such as lecithin, or dispersed with medium-chain fatty acid triglyceride (eg, miglyol 812) can be used as a sustained-release injection which can be actually used as an oily suspension. Agent.

When the sustained-release preparation is, for example, a microcapsule, the average particle size of the microcapsule may be in a range satisfying the degree of dispersion and needle penetration when used as a suspension injection. For example, an average particle diameter of about 0.1
To about 300 μm. The average particle size preferably ranges from about 1 to about 150 μm, particularly preferably from about 2 to about 100 μm. In order to make the above-mentioned microcapsules into a sterile preparation, a method of sterilizing the whole production process, a method of sterilizing with gamma rays, a method of adding a preservative, and the like are mentioned, but are not particularly limited.

The sustained-release preparation has low toxicity and can be used safely in mammals (eg, human, cow, pig, dog, cat, mouse, rat, rabbit, etc.). The indication of the sustained-release preparation varies depending on the protein used. When the protein is, for example, insulin, diabetes, etc., and when the protein is interferon-α, viral hepatitis (eg, hepatitis C, HBe antigen-positive active hepatitis, etc.), cancer (eg, kidney cancer, multiple occurrences) Anemia in the case of erythropoietin (eg, anemia during renal dialysis)
In case of G-CSF, neutropenia (for example, at the time of anticancer drug treatment), infectious disease, etc., in the case of IL-2, cancer (for example, hemangioendothelioma, etc.), and in the case of FGF, fracture ,wound
(Throat sickness), periodontal disease, gastrointestinal ulcer, etc., thrombocytopenia etc. in the case of FGF-9, senile dementia, neuropathy (neuropathy) in the case of NGF, thrombosis etc. in the case of TPA In the case of tumor necrosis factor, it is effective for treatment or prevention of cancer and the like. In addition, in the GH-containing sustained-release preparation, GH secretion deficient short stature (pituitary dwarfism), Turner syndrome, chronic renal disease, cartilage dysplasia (cartilage Nutritional illness), adult growth hormone deficiency (adult GHD), and wasting 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 for treating or preventing congestive heart failure and the like.
In addition, GH-containing sustained-release preparations are applicable to:
Hematopoiesis during organ transplantation and drug treatment of AIDS patients, improvement of malnutrition, renal anemia, angina, hyperlipidemia, obesity, promotion of treatment for burns, wounds, ulcers, surgical invasion (surgery and trauma) / Early recovery after surgery, prevention of fracture of sepsis and osteoporosis, early recovery of postoperative muscular strength of fracture patients due to osteoporosis, amyotrophic lateral sclerosis (ALS), pressure ulcer 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 as a neuroprotective effect of hGH, it is used to suppress and improve the progress of neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, cerebrovascular disorders, etc.) Can also be expected to be effective. By formulating a sustained-release preparation of GH, superior efficacy to these indications can be obtained as compared with daily GH subcutaneous injection.

The dosage of the sustained-release preparation varies depending on the type and content of the protein, the duration of release, the target disease, the target animal, and the like, but it is sufficient that the effective concentration of the protein is maintained in the body. . The dose of the protein is, for example, when the sustained release preparation is a two-week preparation, preferably about 0.0001 to about 10 mg / k per adult.
It can be appropriately selected from the range of g weight. More preferably, it can be appropriately selected from the range of about 0.05 to about 1 mg / kg body weight. For example, when the sustained release preparation is a one-month preparation, the dose of the protein can be appropriately selected from the range of preferably about 0.0002 to about 20 mg / kg body weight per adult. More preferably, it can be appropriately selected from the range of about 0.1 to about 2 mg / kg body weight. The frequency of administration is once a week, once every two weeks, once a month.
Time, once every two months, etc., can be appropriately selected depending on the type and content of the protein, 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. For example, in the case of a sustained release preparation having a sustained release period of about 2 weeks, the molar ratio of lactic acid to glycolic acid is about 55:45 to 4 as a base material of the sustained release preparation.
It is preferable to use a lactic acid-glycolic acid copolymer having a ratio of 5:55 (for example, about 50:50). The lactic acid-glycolic acid copolymer has a weight average molecular weight of about 1,000.
It is preferably from 0 to 15000 (for example, 13000). When the protein which is the active ingredient of the sustained-release preparation is, for example, insulin, the dose for a diabetic adult is usually about 0.001 to about 1 m as the active ingredient.
g / kg body weight, preferably about 0.01 to about 0.2 mg / kg
It is preferred that the dose be appropriately selected from the range of body weight and administered once a week.

When the protein 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 is maintained in the body. In the treatment of the above-mentioned diseases, for example, a sustained-release preparation
In the case of a weekly preparation, the dose of GH as an active ingredient is preferably about 0.0 per child or adult.
1 to about 5 mg / kg body weight (about 0.03 to about 15 I
(U / kg body weight). More preferably, about 0.05 to about 1 mg / k
g body weight (about 0.15 to about 3 IU / kg body weight). The frequency of administration is once a week,
It can be appropriately selected depending on the GH content, dosage form, duration of release, target disease, target animal, etc., such as 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 is 15 to 25 ° C, and cold place is 15 ° C.
Means: Among cold places, 2 to 8 ° C. is particularly preferred.

On the other hand, there is a need to provide a GH-containing sustained-release preparation having a sustained release period of about 3 to 5 weeks. 5
It has been found that a GH-containing sustained-release preparation having a sustained release period of one week can be obtained by using a specific sustained-release preparation base. The GH-containing sustained-release preparation having a sustained release period of about 3 to 5 weeks can be obtained, for example, according to the above-described production method for producing microcapsules. The GH powder used for the production of microcapsules may be obtained by any method as well as the production method of the present invention. The average average particle size of the GH powder is preferably about 0.5 to 2
0 μm, more preferably 0.7-10 μm, more preferably about
1 to 5 μm. Further, when the degree of denaturation of GH is defined, G
The H powder preferably retains about 45% or more of α-helix, more preferably about 50% or more. As a base of the sustained release preparation, the molar ratio of lactic acid to glycolic acid is about 60:40 to 70:30 (for example, about 6:40).
5:35) is preferably used. The weight average molecular weight of the lactic acid / glycolic acid copolymer is about 10,000 to 18,000 (for example, 1 to 18,000).
4500). When the obtained GH-containing sustained release microcapsules having a sustained release period of about 3 to 5 weeks are used for the treatment of the above-mentioned diseases for injection, the dose of GH is preferably set as an active ingredient, preferably per child or adult. About 0.02 to about 10 mg / kg body weight (about 0.0
0.6 to about 30 IU / kg body weight) for safe administration. More preferably, about 0.1 to about 2 mg / kg body weight (about 0.3 to about 6 IU
/ Kg body weight).

[0043]

The present invention will be described more specifically with reference to Examples, Comparative Examples, Reference Examples and Test Examples, which do not limit the present invention. In calculating the cooling rate,
If it is difficult to confirm freezing, an arbitrary unit time (10
(Seconds or more) was determined, and the calculated maximum value was used as the cooling rate of each example.

Example 1 Freezing of an aqueous solution of bovine serum albumin (BSA) and subsequent addition of a 20-fold molar equivalent of ammonium acetate to an aqueous solution of BSA (final BSA concentration = 2 mg / mL), followed by filtration at 0.22 μm and freezing A dry liquid formulation (Formulation 1) was prepared. After cooling the liquid to 10 ° C. or less, a fixed amount was placed in a tray (about 1300 cm ^{2 in} area) at −45 to −40 ° C. every 5 minutes on a freeze-drying shelf, and the diameter of the fluid was about 0.3 to 0.5 mm. And then freeze-dried (Triomaster A04: Kyowa Vacuum (coagulation amount 10 kg type)) to produce a BSA powder. The tray temperature during coating was between -40 and -30C. 140 mg of this BSA powder (S) was mixed with lactic acid-glycolic acid copolymer (PLGA) (lactic acid / glycolic acid (molar ratio) =
About 50/50, polystyrene equivalent weight average molecular weight = about 1
3000) 1.85 g and zinc oxide 10 mg in dichloromethane
In a liquid (O) obtained by dissolving in 2.7 mL, it was dispersed and atomized by polytron (Kinematica) treatment. This S / O
After adding 2.5 mL of dichloromethane to 100 μL of the dispersion,
The average particle diameter of the SA fine powder was measured using a laser diffraction particle size distribution analyzer (SALD2000A: Shimadzu). The average particle size of the fine BSA powder when Formulation 1 was frozen by the above method is shown in [Table 1]. Apply 10mL / 5min ~ 80 to tray
By controlling to 5 mL / 5 min, the average value of the cooling rate of the prescription 1 was from -108.7 ° C / min (max -156 ° C / min) to -35.1 ° C / min (min -3).
2.4 ° C / min), and the average particle size of the BSA fine powder is 1.2μ.
It was possible to control from m to 6.1 μm.

[Table 1]

Example 2 Freezing of BSA aqueous solution and subsequent vacuum drying A BSA aqueous solution of Formulation 1 was prepared in the same manner as in Example 1, and this solution was brought to room temperature.
BSA is applied to a tray (area of about 1300 cm ² ) cooled to 45 to -40 ° C. with a fluid diameter of about 0.3 to 0.5 mm, and freeze-dried (RL-603BS: Kyowa Vacuum (coagulation amount 60 kg type)). The powder was produced aseptically. The tray temperature during coating was between -40 and -30C. Using this BSA powder, the average particle diameter of the BSA fine powder was measured by the method described in Example 1. Table 2 shows the average particle size of the fine BSA powder when Formulation 1 was frozen by the above method. 30mL / 5min ~
By controlling to 60 mL / 5 minutes, the average value of the cooling rate of the prescription 1 is from -98.9 ° C / min (maximum -101.1 ° C / min) to -80.6 ° C / min (min.
-70.3 ° C / min), and the average particle size of the BSA fine powder is 1.2
It was possible to control from μm to 5.0 μm.

[Table 2]

Example 3 Freezing of hGH aqueous solution and subsequent vacuum drying Genetically modified hGH aqueous solution (final hGH concentration = 2 mg / h)
mL), add 20-fold molar equivalent of ammonium acetate, add 0.22
After filtration through μm, a lyophilized liquid formulation (formulation 2) was prepared.
This solution was cooled to 10 ° C. or less, and a predetermined amount thereof was cooled every 5 minutes to a temperature of −45 to −40 ° C. on a freeze-drying tray (area).
1300 cm ² ), apply a fluid with a diameter of about 0.3 to 0.5 mm, freeze-dry (Triomaster A04: Republican vacuum (coagulation amount 10 kg type)) to freeze-dry powder (hereinafter abbreviated as hGH powder). Manufactured. The tray temperature during coating was between -40 and -30C. Using this hGH powder, hGH was obtained in the same manner as described in Example 1.
The average particle size of the fine powder was measured. [Table 3] shows the average particle size of the fine hGH powder when Formulation 2 was frozen by the above method. By controlling the amount of application to the tray from 10 mL / 5 min to 86 mL / 5 min, the average value of the cooling rate of the prescription 2 becomes -20.
1.0 ° C / min (max -203.7 ° C / min) to -72.5 ° C / min (min -54.6 ° C / min)
Min), and the average particle size of the hGH fine powder is 1.4 μm to 4.7 μm.
It was possible to control down to μm.

[Table 3]

Example 4 Freezing of hGH Aqueous Solution and Subsequent Vacuum Drying An aqueous hGH solution of Formulation 2 was prepared, and the solution was brought to room temperature. In a cooled tray (area about 1300 cm ² ), the fluid diameter is about 0.3 to 0.5 m
mGH and freeze-dried (RL-603BS: Kyowa Vacuum (coagulation amount: 60 kg type)) to produce aseptic hGH powder. The tray temperature during coating was between -40 and -30C. This hGH
Using the powder, the average particle size of the hGH fine powder was measured by the method described in Example 1. Table 4 shows the average particle size of the fine hGH powder when Formulation 2 was frozen by the above method. By controlling the amount of application to the tray from 50 mL / 5 min to 80 mL / 5 min, the average value of the cooling rate of the prescription 1 becomes -84.6 ° C / min.
(Maximum −87.4 ° C./min) to −67.3 ° C./min (minimum −54.9 ° C./min), and it was possible to control the average particle size of the hGH fine powder from 2.7 μm to 5.5 μm.

[Table 4]

Example 5A Freezing and Subsequent Vacuum Drying of BSA Aqueous Solution A BSA aqueous solution of Formulation 1 was prepared, and the solution was brought to room temperature. ° C
A BSA powder was produced by dropping a dropping fluid with a diameter of about 2 to 3 mm onto a cooled tray (area: about 1300 cm ² ) and freeze-drying (Triomaster A04: Republican vacuum (coagulation amount: 10 kg type)).
The temperature of the tray during dropping is 60 mL per tray.
-40 to -32 ° C for / 5 minutes, -32 to -22 ° C for 80 mL / 5 minutes, 1
-34 ~ -9 ℃ for 40mL / 5min, -26 ~ -8 for 160mL / 5min
The temperature was -22 to -4 ° C at 150 ° C for 5 minutes. This BSA
Using the powder, the particle size of the BSA fine powder was measured by the method described in Example 1. In addition, the drop amount per tray is
At 150 mL / 5 min, the silicon tube diameter of the filling nozzle was changed from 2 mm to 4 mm. Table 5 shows the average particle size of the fine BSA powder when Formulation 1 was frozen by the above method. By controlling the amount of dropping onto the tray from 60 mL / 5 min to 160 mL / 5 min, the average value of the cooling rate of the prescription 1 is -92.6 ° C./min.
(Maximum -101.1 ° C./min) to −33.4 ° C./min (minimum −32.6 ° C./min), and it was possible to control the average particle diameter of the BSA fine powder from 1.3 μm to 20.0 μm.

[Table 5]

Example 5B Freezing of BSA Aqueous Solution and Subsequent Vacuum Drying A BSA aqueous solution of Formula 1 was prepared, and the solution was brought to room temperature.
Drop the fluid with a diameter of about 2 to 3 mm on a tray (area of about 1300 cm ² ) cooled to 0 ° C and freeze-dry (RL-402BS: Kyowa Vacuum)
(Coagulation amount 40 kg type)) to produce aseptically BSA powder. The temperature of the tray during the dropping was 500 mL of Formula 1.
The drop amount per tray when filling the BSA aqueous solution is -40 to -31 ° C for 60 mL / 5 minutes, and -38 to -3 for 80 mL / 5 minutes.
The temperature was -28 ° C to -12 ° C at 0 ° C and 120mL / 5min. This BS
A powder (S) 300 mg was lactic acid-glycolic acid copolymer (PLGA) (lactic acid / glycolic acid (molar ratio) = about 65/3)
5, weight average molecular weight in terms of polystyrene = about 14,500)
In a liquid (O) obtained by dissolving 1.69 g and 10 mg of zinc oxide in 2.7 mL of dichloromethane, the mixture was dispersed and atomized by polytron (Kinematica) treatment. 100 μL of this S / O dispersion
2.5 mL of dichloromethane was added to the mixture, and the average particle size of the BSA fine powder was measured using a laser diffraction type particle size distribution analyzer (SALD2
000A: Shimadzu). Table 6 shows the average particle size of the fine BSA powder when Formulation 1 was frozen by the above method. By controlling the amount of dripping onto the tray from 60 mL / 5 min to 120 mL / 5 min, the average value of the cooling rate of the prescription 1 becomes -9.
3.7 ° C / min (max -104.2 ° C / min) to -59.8 ° C / min (min -57.4 ° C / min)
Min), and the average particle diameter of the BSA fine powder is 1.2 μm to 2.4 μm.
It was possible to control down to μm.

[Table 6]

Example 6A Freezing of hGH Aqueous Solution and Subsequent Vacuum Drying An aqueous hGH solution of Formulation 2 was prepared, the solution was brought to room temperature, and a constant amount of the resulting solution was dropped in small amounts continuously from -45 to -40 on a freeze-drying shelf. ° C
Was dropped into a tray (area of about 1300 cm ² ) having a diameter of about 2 to 3 mm, and was freeze-dried (Triomaster A04: Republic of Vacuum (coagulation amount: 10 kg type)) to produce hGH powder.
The temperature of the tray during dropping is 140 m per drop.
The temperature was -38 to -18 ° C for L / 5 minutes, and -28 to -2 ° C for 160 mL / 5 minutes. Using this hGH powder, the average particle diameter of the fine hGH powder was measured by the method described in Example 1. Table 7 shows the average particle size of the hGH fine powder when Formulation 2 was frozen by the above method. By controlling the amount of dropping onto the tray from 140 mL / 5 min to 160 mL / 5 min, the average value of the cooling rate of Formulation 2 is -74.1 ° C / min (up to -79.2 ° C / min) to -42.6 ° C / min.
(Minimum −39.8 ° C./min), and it was possible to control the average particle size of the hGH fine powder from 1.9 μm to 5.9 μm.

[Table 7]

Example 6B Freezing of hGH aqueous solution and subsequent vacuum drying A 20-fold molar equivalent of ammonium acetate was added to the hGH aqueous solution (final hGH concentration = 5 mg / mL), and the solution was subjected to 0.22 μm filtration. A hGH aqueous solution of Formulation 3) and Formulation 2 was prepared, and this solution was brought to room temperature. A tray (area of about 1300 cm ² ) was cooled to −50 to −40 ° C. on a freeze-drying shelf by dropping a predetermined amount continuously in small amounts. The hGH powder was aseptically produced by dropping a dropping fluid having a diameter of about 2 to 3 mm and freeze-drying (RL-402BS: a reconstituted vacuum (coagulation amount: 40 kg type)). The tray temperature during dropping is when the drop volume per tray is 60 mL / 5 minutes.
(Filling liquid volume 1L) at -35 to -25 ° C, 80mL / 5min (filling liquid volume 500m
L) is -31 to -24 ° C, 94mL / 5min (filling liquid volume 250mL) is about -30
° C. Using this hGH powder, the particle size of the hGH fine powder was measured by the method described in Example 5B. Prescription 2
Table 8 shows the average particle size of the hGH fine powder obtained when the powders of Examples 3 and 3 were frozen by the above method. When the average value of the cooling rate of Formula 2 is -87.4 ° C / min (maximum -95.3 ° C / min) to -83.5 ° C / min (minimum -76.6 ° C / min), the average particle size of the hGH fine powder is 1.3 It was possible to control from μm to 1.8 μm. In the hGH aqueous solution of Formula 3, the average value of the cooling rate was -93.4 ° C /
In the case of minute, it was possible to control the average particle diameter of the hGH fine powder to 1.8 μm.

[Table 8]

Example 6C Freezing of hGH Aqueous Solution and Subsequent Vacuum Drying An aqueous hGH solution of Formulation 2 was prepared, and the solution was brought to room temperature.
Drop the fluid with a diameter of about 2 to 3 mm on a tray (area of about 1300 cm ² ) cooled to 0 ° C, fill 1 L, freeze-dry (RL-40
2BS: hGH by Kyowa vacuum (coagulation amount 40kg type))
The powder was produced aseptically. 60mL / 5 drops per tray
Using the hGH powder obtained in minutes and 80 mL / 5 min, the average particle diameter was measured by adjusting the hGH fine powder by the method described in Example 5B. 120mL / 5min and 1 drop per tray
Using the hGH powder obtained at 40 mL / 5 min, the fine hGH powder was prepared by the method described in Example 1, and the average particle diameter was measured. Table 9 shows the average particle size of the fine hGH powder when Formulation 2 was frozen by the above method. hGH aqueous solution 1
When L is filled, the average particle diameter of the hGH fine powder is 1.6
It was possible to control from μm to 4.0 μm.

[Table 9]

Reference Example 1 Freeze-vacuum drying of BSA aqueous solution A BSA aqueous solution of Formulation 1 was prepared, and this solution was applied to a layer thickness of 1 mm
It was added to the tray so as to be mm and 5 mm, and cooled to about -5 to 0 ° C. The cooled BSA aqueous solution is freeze-dried (Triomaster A0
4: Freeze-dried powder (hereinafter, referred to as `` freeze-dried powder '')
BSA powder). Using the obtained BSA powder, the average particle diameter of the BSA fine powder was measured by the method described in Example 1. The average particle size of the BSA fine powder when the BSA aqueous solution of Formulation 1 was frozen by the above method is shown in [Table 10]. The average value of the cooling rate in the freeze-drying method was about −2.1 to −1.6 ° C./min.
The average particle size of the fine powder was 28.9 to 35.0 μm.

[Table 10]

Reference Example 2 Freeze-vacuum drying of BSA aqueous solution A BSA aqueous solution of Formulation 1 was prepared, and this solution was added to a tray so as to have a layer thickness of 1 mm. And the temperature of the BSA aqueous solution was controlled to 0 ° C. Next, the temperature of the freeze-drying shelf was set to -4 ° C / h.
, And the BSA aqueous solution was frozen and freeze-dried to produce a BSA powder. Using this BSA powder,
The average particle diameter of the BSA fine powder was measured by the method described in Example 1. Table 11 shows the average particle size of the BSA fine powder when the BSA aqueous solution of Formulation 1 was frozen by the above method. The cooling rate of the BSA aqueous solution in the above method is about −4 ° C./h, which is the same as the cooling rate of the freeze-drying shelf. At this time, the average particle size of the BSA fine powder was 32.5 μm.

[Table 11]

As a result of comparison between Examples 1 to 6C and Reference Examples 1 and 2, it was confirmed that the slower the cooling rate, the larger the average particle size of the protein fine powder.

Example 7 Freezing of hGH Aqueous Solution and Subsequent Vacuum Drying An hGH aqueous solution of Formulation 2 was prepared, and the solution was brought to room temperature. (Area about 1300 cm ² ) and freeze-dried (Triomaster A
04: hGH powder was produced by applying a recuperation vacuum (coagulation amount: 10 kg type). Using this hGH powder, the average particle size of the hGH fine powder was measured by the method described in Example 1. [Table 12] shows the average particle size of the hGH fine powder when Formulation 2 was frozen by the above method. By controlling the spray amount of the prescription 2 on the tray from 50 mL / 5 min to 100 mL / 5 min, the average value of the cooling rate of the prescription 2 is -65.3 ° C / min (maximum -73.9 ° C / min)
-37.3 ° C./min (minimum −34.3 ° C./min), and it was possible to control the average particle size of the hGH fine powder from 1.5 μm to 9.5 μm.

[Table 12]

Example 8 Production of hGH-containing microcapsules A lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50)
/ 50, average molecular weight in terms of polystyrene = 13,000, viscosity = 0.145 dl / g) 1.85 g of zinc oxide and 10 mg of zinc oxide were dissolved in 2.7 ml of dichloromethane. In this organic solvent liquid, hGH powder 1 obtained in Example 3 at an application amount (application amount (mL) per 5 minutes / tray) of 10 and 60 was obtained.
40 mg was added, and the mixture was pulverized using Polytron (Kinematica). This 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 1,800 rpm). Next, the microcapsules thus obtained were washed twice with 400 ml of distilled water, and 0.2 g of D-mannitol was added thereto, followed by freeze-drying.
Further, in order to remove the residual solvent, vacuum drying was performed at 46 ° C. for 3 days to obtain two types of hGH-containing microcapsules.

Example 9 Production of hGH-Containing Microcapsules In the same manner as in Example 8, the spray amount in Example 7 was
The two types of hGH-containing microcapsules obtained at (application amount (mL) per 5 minutes / tray) of 50 and 80 were obtained.

Example 10 Production of hGH-containing microcapsules 1.69 g of lactic acid-glycolic acid copolymer (lactic acid / glycolic acid (molar ratio) = about 65/35, weight average molecular weight in terms of polystyrene = about 14,500) and 10 mg of zinc oxide
Was dissolved in 2.7 ml of dichloromethane. To this organic solvent liquid, 300 mg of hGH powder obtained by freezing and drying Formula 2 in Example 6B at a dripping amount of 94 ml / 5 minutes (filling amount: 250 ml) was added, and fine particles were formed using Polytron (Kinematica). It has become. This 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, centrifugation (about 1,800 rpm)
By doing so, microcapsules were collected. Next, the microcapsules thus obtained were washed twice with 400 ml of distilled water, and 0.2 g of D-mannitol was added thereto, followed by freeze-drying. Further, in order to remove the residual solvent, vacuum drying was performed at 46 ° C. for 3 days to obtain hGH-containing microcapsules.

Example 11 Production of hGH-containing microcapsules 1.69 g of lactic acid-glycolic acid copolymer (lactic acid / glycolic acid (molar ratio) = about 65/35, weight average molecular weight in terms of polystyrene = about 14,500) and 10 mg of zinc oxide
Was dissolved in 2.7 ml of dichloromethane. To this organic solvent solution, 300 mg of hGH powder obtained by freezing and drying Formula 3 in Example 6B at a dripping amount of 80 ml / 5 min (filling amount: 100 ml) and adding the resulting fine powder was added using a polytron (Kinematica). Has become This 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, centrifugation (about 1,800 rpm)
By doing so, microcapsules were collected. Next, the microcapsules thus obtained were washed twice with 400 ml of distilled water, and 0.2 g of D-mannitol was added thereto, followed by freeze-drying. Further, in order to remove the residual solvent, vacuum drying was performed at 46 ° C. for 3 days to obtain hGH-containing microcapsules.

Example 12 Production of hGH-containing microcapsules 1.69 g of lactic acid-glycolic acid copolymer (lactic acid / glycolic acid (molar ratio) = about 65/35, weight average molecular weight in terms of polystyrene = about 14500) and 10 mg of zinc oxide
Was dissolved in 2.565 ml of dichloromethane. HGH obtained by freezing and drying after drying Formula 2 in Example 6B in this organic solvent liquid at a drop amount of 94 ml / 5 min (filling amount 250 ml).
After adding 300 mg of powder, 0.135 ml of ethanol
Was added and the mixture was atomized using a Polytron (Kinematica). This 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 1,800 rpm). Next, the separated microcapsules were washed twice with 400 ml of distilled water,
-0.2 g of mannitol was added and lyophilized. Furthermore, in order to remove the residual solvent, vacuum drying was carried out at 46 ° C. for 3 days, followed by hGH
The resulting microcapsules were obtained.

Test Example 1 In Vivo Release Immunosuppressed SD rats (male, 6 weeks old) were subcutaneously administered with the microcapsules obtained in Examples 8 and 9 (hGH amount: 6 mg / rat), and blood was collected over time. HG in the serum
The H concentration was measured by radioimmunoassay (Ab beads HGH: Eiken Chemical Co., Ltd.) to evaluate the sustained release of hGH. For the immunosuppressed SD rats, 0.4 mg / rat of Prograf (Fujisawa Pharmaceutical) was administered 3 days before the administration of the microcapsules, and 0.2 mg each at 4, 4, 7, 11 and 14 days after the administration.
/ Rat was made subcutaneously. The results are shown in FIG.

As is clear from the results shown in FIG. 1, the blood hGH concentration after administration of the microcapsules prepared using the hGH powder obtained by application was determined using the powder obtained by spraying. It was higher than that after administration of the microcapsules prepared. These results indicate that the bioavailability of the microcapsule preparation made using the hGH powder obtained by application is higher.

Test Example 2 Analysis of Higher-Order Structure of Protein Fine Powder by FT-IR In Example 4, hGH powder obtained at a coating amount (coating amount (mL) per 5 minutes / tray) of 80 was obtained. In Example 7,
Higher order structural analysis by FT-IR was performed on two kinds of hGH powders obtained when the spray amount (coating amount per 5 minutes (mL) / tray) was 80 (Journal of Pharmaceutical Sciences) (Journal of Pharmaceutical
Science) 87, 1412-1420, 1998). The results are shown in [Table 13] as mean ± SD of n = 3. [Table 1
As shown in [3], hGH of Example 4 obtained by coating was obtained.
The α-helix content of the powder was much higher than the α-helix content of the hGH powder of Example 7 obtained by spraying. Since the α-helix content of hGH in heavy water is 59%, Example 7 obtained by spraying was used.
The hGH powder retained 39% α-helix with respect to heavy water. In addition, the hGH powder of Example 4 obtained by coating had an α-helix of 53% with respect to heavy water.

[Table 13]

[0065]

According to the present invention, a stable protein powder having a high-order structure maintained at a high level can be easily produced without contact with a liquefied gas. Therefore, compared to the method of producing protein fine powder which is sprayed into a liquefied gas and dried after freezing, heat insulation, expansion and contraction of equipment materials due to temperature difference,
There is no need to use large and expensive equipment for handling aseptic maintenance and exhausting liquefied gas. Furthermore, the protein powder obtained by the method of the present invention can be made into a fine powder by a simple atomization treatment, and using this protein fine powder, shows a stable and high blood concentration over a long period of time. Sustained-release preparations can be provided. In addition, when manufacturing a growth hormone-containing preparation, a lactic acid-glycolic acid copolymer having a molar ratio of lactic acid to glycolic acid of about 60:40 to 70:30 is used as a base, so that it can be used for about one month. Sustained-release preparations that release growth hormone can be manufactured.

[0066]

[Brief description of the drawings]

FIG. 1 shows the results of application in an immunosuppressed SD rat.
●-)] and spray [spray volume: 50 mL / 5 minutes (-△
-), 80 mL / 5 minutes (-○-)] is a graph showing changes in serum hGH concentration after administration of microcapsules prepared using hGH powder obtained by freezing.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61K 38/28 A61P 5/50 38/43 A61K 37/36 47/30 37/02 47/34 37/24 A61P 5/02 37/26 5/50 37/48 (72) Inventor Shigeyuki Takada 3-20-504 Kasumicho, Nishinomiya-shi, Hyogo F-term (reference) 4C076 AA61 BB01 CC30 EE24A EE48A FF31 GG06 GG21 4C084 AA01 DA01 DA36 DB22 DB34 DC01 MA05 MA38 MA52 NA12 ZB031 ZC031

Claims (19)

[Claims] (1) A method comprising filling a protein-containing solution into a refrigerant and adding
A method for producing a protein powder, comprising freezing at a cooling rate of 00 to -10 ° C / min and drying. 2. The method according to claim 1, wherein the protein-containing solution is applied or dropped onto the cooling medium. 3. The method according to claim 2, wherein the diameter of the dripping fluid when applying or dripping is about 0.1 to 40 mm. 4. The method according to claim 1, wherein the protein-containing solution is frozen without directly contacting the liquid refrigerant. 5. The method according to claim 1, wherein a volatile salt or a water-miscible organic solvent is added to the protein-containing solution. 6. The method according to claim 5, wherein the volatile salt is ammonium acetate. 7. A protein powder obtained by the production method according to claim 1. 8. The method according to claim 8, wherein the protein has a molecular weight of about 5,000 to 1,000.
8. The protein powder according to claim 7, which has a molecular weight of 0,000 daltons. 9. The method according to claim 9, wherein the protein is a hormone, a cytokine,
The protein powder according to claim 7, which is a hematopoietic factor, a growth factor or an enzyme. 10. The protein powder according to claim 7, wherein the protein is growth hormone or insulin. 11. The protein powder according to claim 7, wherein the protein has an α-helix of about 45% or more based on the α-helix content in the protein-containing solution. 12. A method for producing a protein fine powder, comprising subjecting the protein powder according to claim 7 to atomization treatment. 13. The average particle size of the protein fine powder is about 0.5.
13. The method according to claim 12, wherein the particle size is reduced to 5 to 20 [mu] m. 14. A sustained-release preparation containing the protein fine powder obtained by the production method according to claim 12. 15. The sustained release preparation according to claim 14, wherein the base of the sustained release preparation is a biological substance or a synthetic polymer. 16. The sustained-release preparation according to claim 15, wherein the biological substance or the synthetic polymer is a biodegradable polymer. 17. The molar ratio of lactic acid to glycolic acid is about 60:
A sustained-release preparation containing a lactic acid-glycolic acid copolymer of 40 to 70:30 and growth hormone. 18. A method for producing a sustained-release preparation, comprising using the protein fine powder obtained by the production method according to claim 12. (19) Use of the protein powder according to (7) for producing a sustained-release preparation containing protein.