JPH11322631A - Sustained release preparation of physiologically active polypeptide and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a sustained release preparation for improving handleability of physiologically active polypeptide powder in a pharmaceutical preparation process, facilitating the mass production of the sustained release preparation, raising inclusion ratio of a physiologically active polypeptide in the sustained release preparation and showing high retention concentration in blood having stability for a long period of time. SOLUTION: This method for producing a sustained release preparation comprises adding a water-miscible organic solvent and/or a volatile salt to an aqueous solution of a physiologically active polypeptide, lyophilizing the aqueous solution to give physiologically active polypeptide powder, dispersing the powder into an organic solvent solution of a degradable in an organism and removing the organic solvent. The physiologically polypeptide has <=10 μm average particle diameter. The physiologically active polypeptide is a growth hormone and the growth hormone is a human growth hormone. The water miscible organic solvent is an alcohol and the alcohol is methanol or ethanol.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a sustained-release preparation containing a physiologically active polypeptide and a method for producing the same.

[0002]

2. Description of the Related Art Physiologically active polypeptides are known to exhibit various pharmacological actions in living organisms, and some of them are Escherichia coli, yeast, animal cells, or goats due to the development of genetic engineering and cell engineering techniques. It is produced in large quantities using living organisms such as hamsters and the like, and is being applied as pharmaceuticals. However, since these bioactive polypeptides generally have a short half-life in vivo, frequent administration is required, and the physical burden on the patient involved in the injection cannot be ignored. For example, growth hormone (hereinafter,
GH) is a typical hormone originally produced and secreted by the anterior pituitary gland, which works to promote body growth, and is involved in sugar / lipid metabolism, protein assimilation, cell proliferation and differentiation. Is a biologically active polypeptide with a wide variety of physiological actions. Currently, it is mass-produced by Escherichia coli using genetic recombination technology and is widely clinically applied worldwide as a pharmaceutical product. It is necessary to administer frequent doses to maintain effective blood levels. Especially in the case of pituitary dwarfism, infants and young patients can be treated for a long period of several months to 10 years or more. The fact is that it is administered subcutaneously.

[0003] JP-A-8-3055 (EP-A-6)
No. 33020) discloses a method for producing a sustained-release preparation in which a water-soluble polypeptide is penetrated in water into a biodegradable matrix comprising a biodegradable polymer and a fatty acid metal salt, and the sustained-release preparation prepared by the production method. Microcapsules are disclosed. Japanese Patent Application Laid-Open No. 8-217691 (WO 96/0
7399) discloses a method for producing a sustained-release preparation comprising a water-soluble peptidic physiologically active substance converted into a water-insoluble or poorly water-soluble polyvalent metal salt with an aqueous zinc chloride solution or the like and a biodegradable polymer. It has been disclosed. WO 94/12
158 describes a method for producing a sustained-release preparation of human GH (hereinafter may be abbreviated as hGH) and a biodegradable polymer in an amount of 0.1 to 30% (W / W) based on the polymer.
It is described that a polymer decomposition accelerator such as zinc hydroxide can be added to the polymer solution. Also disclosed is a method of preparing a sustained-release microcapsule by spraying an organic solvent solution containing hGH and a polymer into liquid nitrogen and maintaining bioactivity as porous particles. Also, W
O 92/17200 and Nature Medicine (Nat
ure Medicine), Volume 2, p. 795 (1996)
A method for producing a sustained-release preparation using a zinc salt of hGH is disclosed. Further, WO 95/29664 discloses that a metal salt such as zinc carbonate is dispersed in a polymer solution in a solid state, and then a physiologically active substance (hormone or the like) is added, and the physiologically active substance and the metal cation are separately biodegraded. A method for producing sustained-release microcapsules dispersed in a conductive polymer is disclosed.

[0004]

As described above, various attempts have been made to produce sustained-release preparations while maintaining the biological activity of the bioactive polypeptide. A formulation that is still clinically satisfactory in that it has a low bioactive polypeptide uptake rate, has an excessive release in the initial stage of administration, does not achieve a long-term stable release, and cannot maintain a sufficient blood concentration for a long period of time. Has not been obtained. At present, the manufacturing method does not match industrialization on the premise of mass production. A bioactive polypeptide powder (solid phase: hereinafter, sometimes abbreviated as S phase) is gradually added to and dispersed in an organic solvent (O phase) in which a polymer is dissolved, using a so-called S / O emulsion. In a method for producing a release preparation, it is possible to relatively stably enclose a bioactive polypeptide, but it is necessary to efficiently handle a large amount of bioactive polypeptide powder as a solid in the preparation process. In addition, since the particle size of the powder in the S / O dispersion greatly affects the quality of the obtained sustained-release preparation, there are many production problems such that control is essential. Therefore, maintaining the stability of the physiologically active polypeptide during the formulation process, creating a powder with good operability, satisfying all of the atomization of the powder, etc.
Moreover, there is a demand for a sustained-release preparation having a high yield and stable quality, and a production method capable of mass-producing the sustained-release preparation.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and added a water-miscible organic solvent and / or volatile salts to a physiologically active polypeptide aqueous solution. It has been found for the first time that freeze-drying can provide a bioactive polypeptide powder having a small particle size with good operability in the formulation step while unexpectedly retaining the physiological activity of the polypeptide by freeze-drying. Furthermore,
The bioactive polypeptide powder thus obtained is
When dispersed in an organic solvent solution of a biodegradable polymer and then the organic solvent is removed to produce a sustained-release preparation, unexpectedly, the encapsulation rate and sustained-release of the bioactive polypeptide in the sustained-release preparation are improved. The present invention was completed based on these.

That is, the present invention provides (1) a bioactive polypeptide powder obtained by adding a water-miscible organic solvent and / or volatile salts to an aqueous solution of the bioactive polypeptide and freeze-drying the powder; A method for producing a sustained-release preparation, comprising dispersing the biodegradable polymer in an organic solvent solution and removing the organic solvent, (2) the bioactive polypeptide powder having an average particle diameter of 10 μm or less. (3) The method according to (1), wherein the physiologically active polypeptide is a growth hormone, and (4) the method according to (3), wherein the growth hormone is human growth hormone. Manufacturing method,
(5) The method according to (1), wherein the water-miscible organic solvent is an alcohol, (6) the method according to (5), wherein the alcohol is methanol or ethanol, and (7) the method, wherein the alcohol is ethanol. (5) The production method described above,
(8) The above (1), wherein the volatile salt is an ammonium salt.
(9) The method according to (8), wherein the ammonium salt is ammonium acetate, ammonium bicarbonate or ammonium carbonate, (10) the method according to (8), wherein the ammonium salt is ammonium acetate. (11)
0.03% concentration in aqueous solution of physiologically active polypeptide
(5) The method according to the above (5), wherein the alcohol is added so as to be (V / V) to 0.5% (V / V). (12) The molar ratio to the bioactive polypeptide is 10 to 80.
(13) the production method according to (1), wherein the biodegradable polymer is a lactic acid-glycolic acid polymer; (14) lactic acid-glycolic acid; (1) wherein the lactic acid / glycolic acid composition ratio (mol%) of the polymer is 100/0 to 40/60.
(15) The method according to (13), wherein the weight average molecular weight of the lactic acid-glycolic acid polymer is 3,000 to 50,000, and (16) the lactic acid-glycolic acid polymer is large. (13) The method according to (13), wherein the polyvalent metal is zinc, (18) the method according to (1), wherein the sustained release preparation is a microcapsule. (19) The method according to (1), wherein the average particle size of the microcapsules is 0.1 μm to 300 μm.
8) The production method according to the above (1), wherein the sustained release preparation is for injection, (21) the sustained release preparation produced by the production method according to the above (1), (22) (23) A process for producing a bioactive polypeptide powder, which comprises adding a water-miscible organic solvent and / or volatile salts to an aqueous solution of the bioactive polypeptide, followed by freeze-drying.
The bioactive polypeptide powder obtained by the production method described in (24), wherein the bioactive polypeptide powder has an average particle size of 1
(23) Use of the bioactive polypeptide powder according to (23) for producing a pharmaceutical preparation, which is 0 μm or less, (25)
6) Use of a water-miscible organic solvent and / or volatile salts to produce a bioactive polypeptide powder having a small particle size. (27) The bioactive polypeptide powder has an average particle size of 1
Use according to the above (26), which is 0 μm or less. (28) A sustained-release preparation obtainable by the production method according to claim 1. (2
9) a sustained-release preparation comprising a bioactive polypeptide and a polyvalent metal salt of a biodegradable polymer, wherein the initial release rate of the bioactive polypeptide is 40% or less; The sustained release preparation according to the above (29), which is a powder having a diameter of 10 μm or less, (31) the sustained release preparation according to the above (29), wherein the bioactive polypeptide is a growth hormone, (32) biodegradability The sustained release preparation according to the above (29), wherein the polymer is a lactic acid-glycolic acid polymer, (3)
3) The aforementioned (29), wherein the sustained-release preparation is a microcapsule.
(34) The sustained release preparation according to (33), wherein the microcapsules have an average particle size of 0.1 to 300 μm, and (35) a one week to one month type sustained release preparation. The sustained release preparation according to the above (29), and (36)
The sustained-release preparation according to the above (29), which comprises 0.1 to 30% (W / W) of a physiologically active polypeptide in the sustained-release preparation.

Examples of the biodegradable polymer used in the present invention include α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.), and hydroxytricarboxylic acids (eg, citric acid). A polymer having a free carboxyl group or a mixture thereof, a poly-α-cyanoacrylate, a polyamino acid (eg, poly-γ-benzyl) -L-
Glutamic acid, etc.), maleic anhydride polymers (for example,
Styrene-maleic acid polymer). These may be either homopolymers or copolymers. The type of polymerization may be random, block, or graft. When the α-hydroxycarboxylic acids, hydroxydicarboxylic acids, and hydroxytricarboxylic acids have an optically active center in the molecule, D-,
Both L- and DL-forms can be used. Among these, biodegradable polymers having a terminal free carboxyl group, for example, polymers synthesized from α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, etc.) (eg, lactic acid polymers, lactic acid-glycols) Acid copolymer) and poly-α-cyanoacrylate. More preferably, the biodegradable polymer is α
-A polymer synthesized from hydroxycarboxylic acids, particularly preferably a lactic acid-glycolic acid polymer.

In this specification, lactic acid-glycolic acid polymer may be simply referred to as lactic acid-glycolic acid copolymer, as well as a homopolymer such as polylactic acid and polyglycolic acid. When a lactic acid-glycolic acid polymer (lactic acid-glycolic acid copolymer or homopolymer) is used as the biodegradable polymer, its composition ratio (mol%) is about 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, preferably from 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 from about 1.2 to about 4.0, more preferably from about 1.5 to about 3.5.

[0009] Regarding the weight average molecular weight and the degree of dispersion in the present specification, the former has a weight average molecular weight of 120,000,
52,000, 22,000, 9,200, 5,05
A value in terms of polystyrene measured by gel permeation chromatography (GPC) using nine kinds of polystyrenes of 0, 2,950, 1,050, 580, and 162 as a reference substance, and the latter is a value calculated from this value. The measurement was performed using a GPC column KF804L x 2 (manufactured by Showa Denko),
Using RI Monitor L-3300 (manufactured by Hitachi, Ltd.)
This is performed using chloroform as the mobile phase.

The term "biodegradable polymer having a free carboxyl group at the end" refers to a polymer whose number average molecular weight determined by the terminal group determination and the number average molecular weight determined by the above-mentioned GPC measurement substantially coincide with each other. The average 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), and the carboxyl group in this solution was adjusted to 0.05 N using phenolphthalein as an indicator.
Titration was carried out promptly with an alcoholic potassium hydroxide solution at room temperature (20 ° C.) with stirring, and the number average molecular weight by terminal group quantification was calculated by the following formula. Number average molecular weight determined by terminal group = 20,000 × A / B A: Mass of biodegradable polymer (g) B: 0.05N alcoholic potassium hydroxide solution added by the end of titration (ml) Number determined by terminal group determination While the average molecular weight is an absolute value, the number average molecular weight obtained by GPC measurement is determined by various analysis and analysis conditions (eg, mobile phase type, column type, reference material, selection of slice width, selection of baseline, etc.). ), It is difficult to make a unique numerical value, but the number average molecular weights obtained by both measurements are almost the same, for example, when a polymer synthesized from α-hydroxycarboxylic acids has a terminal group Number average molecular weight by quantification is G
It means that the number average molecular weight is in the range of about 0.5 to about 2 times, preferably about 0.7 to about 1.5 times the number average molecular weight determined by PC measurement. For example, one or more types of α-
In a polymer synthesized from hydroxycarboxylic acids by a noncatalytic dehydration polycondensation method and having a terminal free carboxyl group, the number average molecular weight measured by GPC and the number average molecular weight determined by terminal group quantification are almost the same. On the other hand, in a polymer synthesized from a cyclic dimer by a ring-opening polymerization method using a catalyst and having essentially no free carboxyl group at the terminal, the number average molecular weight determined by the terminal group is determined by the GPC measurement. Significantly more than about twice the average molecular weight. By this difference, a polymer having a free carboxyl group at a terminal can be clearly distinguished from a polymer having no free carboxyl group at a terminal.

Lactic acid having a terminal free carboxyl group
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
Although it changes greatly depending on the composition ratio or the weight average molecular weight, generally, the lower the glycolic acid fraction, the slower the decomposition and disappearance. Therefore, the release period is lengthened by lowering the glycolic acid fraction or increasing the molecular weight. (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 physiologically active polypeptide, it is preferable to use a lactic acid-glycolic acid polymer, and the lactic acid-glycolic acid polymer has a lactic acid / glycolic acid composition ratio (mol%). ) Is about 85/15 to about 50/50 lactic acid-
Glycolic acid copolymers are preferred, more preferably about 7
5/25 to about 50/50 lactic acid-glycolic acid copolymer. The weight average molecular weight is preferably about 8,000 to about 20,000, more preferably about 10,
000 to about 20,000. The dispersity (weight average molecular weight / number average molecular weight) of the lactic acid-glycolic acid polymer is preferably about 1.2 to about 4.0, more preferably about 1.5 to about 3.5. The lactic acid-glycolic acid polymer to be used can be produced according to a known method such as the method described in the above publication. The polymer is preferably produced by non-catalytic dehydration polycondensation. It is preferable to use a lactic acid-glycolic acid polymer (PLGA) in which the number average molecular weight by the GPC measurement method and the number average molecular weight by the terminal group quantification method are almost the same. The polymer may be used by mixing two kinds of lactic acid-glycolic acid polymers having different composition ratios and / or weight average molecular weights at an arbitrary ratio. As such an example, the composition ratio (lactic acid / glycolic acid) (mol%) is about 7%.
Lactic acid-glycolic acid copolymer having a weight average molecular weight of about 10,000 at 5/25 and a composition ratio (lactic acid / glycolic acid)
(Mol%) is about 50/50 and the weight average molecular weight is about 12,
A mixture with 000 lactic acid-glycolic acid copolymer is used. The weight ratio upon mixing is preferably about 25 /
75 to about 75/25.

The biodegradable polymer used in the present invention may be a metal salt of the above-described biodegradable polymer. For example, various biodegradable polymers described in WO 97/01331 may be used. A valent metal salt or the like is used. Preferably, a polyvalent metal salt of a lactic acid-glycolic acid polymer (further preferably, a zinc salt, a calcium salt, a magnesium salt, 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 iron, zinc, copper, calcium, magnesium, aluminum, tin, and manganese), trivalent (eg, iron, aluminum, manganese, and the like), and tetravalent (eg, tin and the like) can also be used. In the present specification, the biodegradable polymer may be referred to as a biodegradable polymer including the case where the biodegradable polymer is a metal salt.For example, when the lactic acid-glycolic acid polymer is a polyvalent metal salt, 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. In the production method, first, a biodegradable polymer and zinc oxide are allowed to coexist in an organic solvent to form a biodegradable polymer.
A solution of zinc oxide in an organic solvent is produced. At this time, the concentration of the biodegradable polymer in the solution varies depending on the molecular weight, the type of the organic solvent, and the like, but is, for example, about 0.1 to about 80%.
(W / W), preferably about 1 to about 70% (W / W).
W), more preferably about 2 to 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 varies depending 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 is added to the biodegradable polymer in an organic solvent solution 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.

In the present invention, the physiologically active polypeptide preferably has a molecular weight of about 1,000 to about 50,000.
0, more preferably a molecular weight of about 5,000 to about 40,0
00 bioactive polypeptides are used. A typical example of the activity of a physiologically active polypeptide is a hormonal action. The physiologically active polypeptide may be any of a natural product, a synthetic product, and a semi-synthetic product, and may be a derivative or an analog thereof. The mode of action of the bioactive polypeptide may be either agonistic or antagonistic. Examples of the physiologically active polypeptide in the present invention include peptide hormones, cytokines, peptide neurotransmitters, hematopoietic factors, various growth factors, enzymes, polypeptide antibiotics, and analgesic peptides. Examples of peptide hormones include insulin, somatostatin, and somatostatin derivatives (Sandostatin, U.S. Pat. Nos. 4,087,390, 4,093,574, 4,100,117, and 4,253,998). No.),
Growth hormone (GH), natriuretic peptide, gastrin, prolactin, adrenocorticotropic hormone (ACT
H), ACTH derivatives (eg, eviratide), melanocyte stimulating hormone (MSH), thyroid hormone releasing hormone (TRH), salts thereof and derivatives thereof (Japanese Patent Application Laid-Open No. 50-1).
No. 21273, JP-A-52-116465), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), thymosin (thymosin), motilin, vasopressin , A vasopressin derivative @ desmopressin [Journal of the Endocrine Society of Japan, Vol. 54, No. 5, No. 6,
76-691 (1978)], oxytocin, calcitonin, parathyroid hormone (PTH), glucagon, secretin, pancreozimine, cholecystokinin, angiotensin, human placental lactogen, and the like. Preferable peptide hormones include insulin and growth hormone. As the cytokine, for example, lymphokine, monokine and the like are used. Examples of lymphokines include interferons (alpha type, beta type, gamma type, etc.) and interleukins (eg, IL-2, 3, 4, 5, 6, 7, 8, 8,
9, 10, 11, 12, etc.). Monokines include, for example, interleukin-1 (IL-1),
Tumor necrosis factor (TNF) and the like are used. The cytokine is preferably lymphokine or the like, more preferably interferon or the like, and particularly preferably interferon alpha or the like. As peptide neurotransmitters,
For example, substance P, serotonin, GABA and the like are used.

Examples of hematopoietic factors include erythropoietin (EPO) and colony stimulating factors (G-CSF, GM-
CSF, M-CSF, etc.), thrombopoietin (TP
O), platelet growth stimulating factor, megakaryocyte potentiator and the like are used. As various growth factors, for example, basic or acidic fibroblast growth factor (FGF)
Alternatively, these families (eg, EGF, TGF
-Α, TGF-β, PDGF, acidic FGF, basic FG
F, FGF-9, etc.), nerve cell growth factor (NGF) or a family thereof (eg, BDNF, NT-
3, NT-4, CNTF, GDNF, etc., insulin-like growth factors (eg, IGF-1, IGF-2, etc.), factors involved in bone growth (BMP), or a family of these. Examples of enzymes include superoxide dismutase (SOD), urokinase, tissue plasminogen activator (TPA),
Asparaginase, kallikrein and the like are used. Examples of polypeptide antibiotics include polymyxin B,
Colistin, gramicidin, bacitracin and the like are used. Examples of analgesic peptides include enkephalin,
Enkephalin derivatives [US Pat. No. 4,277,394
No., European Patent Application Publication No. 31567], endorphin, kyotorphin and the like. Other bioactive polypeptides include thymopoietin, dynorphin, bombesin, caerulein, thymostimulin, thymic humoral factor (THF), blood thymic factor (FTS) and derivatives thereof (US Pat. No. 4,229,4).
No. 38), and other thymic factors [Ayumi,
Vol. 125, No. 10, pp. 835-843 (1983)
Years)], peptides having an antagonistic action on neurotensin, bradykinin and endothelin (European Patent Publication Nos. 436189, 457195 and 4964).
No. 52, JP-A-3-94692 and JP-A-3-130299.
Reference). Examples of the physiologically active polypeptide particularly preferably applied to the present invention include growth hormone, insulin and the like, among which growth hormone, especially human growth hormone, is preferable.

In the present invention, when the physiologically active polypeptide contains a metal, the metal content is 0.1% (w /
w) is preferably not more than 0.01%, more preferably 0.01% (w /
w) or less, particularly preferably 0.001% (w / w) or less and a substantially metal-free bioactive polypeptide is optimal. For example, crystalline insulin is usually zinc,
Contains small amounts of heavy metals such as nickel, cobalt and cadmium. Insulin containing 0.4% (w / w) zinc exists as a hexamer, is stable on its own, and weakens its interaction with metal salts of biodegradable polymers. Conceivable. If necessary, the metal contained in the physiologically active polypeptide may be removed in advance, and a known method is used for removing the metal. For example, an insulin-hydrochloric acid aqueous solution is dialyzed against water or an ammonium acetate solution and then freeze-dried to obtain an amorphous state of insulin with a minimum amount of metal. The growth hormone may be derived from any species, but is preferably human growth hormone. In addition, natural sources extracted from the pituitary gland and the like are also used in the present invention, but are preferably recombinant GH (see Japanese Patent Publication Nos. 6-12996 and 6-48987), and more preferably. Is a recombinant hGH having the same structure as the natural type having no methionine at the N-terminus. The GH may be a metal salt, but GH substantially containing no metal is also used. As hGH,
Not only those having a molecular weight of about 22K daltons but also those having a molecular weight of about 20K daltons (see JP-A-7-101877 and JP-A-10-265404) may be used. In addition, hGH derivatives or their related proteins (WO
99/03887) may be used.

As the biologically active polypeptide, those produced by any method, such as those extracted and purified from living organisms, those chemically synthesized, and those produced by genetic recombination, can be used. However, in order to synthesize a high-purity product in large quantities, it is preferable to use a gene recombination method. In any case, it is necessary to purify and purify the polypeptide from a tissue / body fluid containing a bioactive polypeptide, a crude chemically synthesized preparation, or a recombinant cell / bacterium, respectively. . In this case, a general peptide or protein separation and purification method can be used ("Protein", written by Kazuo Satake, Asakura Shoten; "Bioactive peptide", Pharmacia Review, N.
o.3, Pharmaceutical Society of Japan), especially by combining several liquid chromatography ("High performance liquid chromatography of proteins and peptides", co-edited by Nobuo Ui et al.
Highly pure biologically active polypeptide can be obtained in good yield without impairing its biological activity. Further, if desired, it is preferably subjected to a desalting operation in the final step of the purification method. The concentration of the physiologically active polypeptide in the aqueous solution is not particularly limited, but is, for example, 0.01% (W / V).
To 30% (W / V), preferably 0.03% (W / V).
V) to 10% (W / V), particularly preferably 0.05
% (W / V) to 3% (W / V). When the bioactive polypeptide is hGH, hGH in aqueous solution
The concentration is preferably between about 0.01 and about 5% (W /
V), more preferably from about 0.05 to about 0.5% (W
/ V).

In the present invention, examples of the water-miscible organic solvent added to the aqueous solution of the physiologically active polypeptide include alcohols (eg, methanol, ethanol, isopropanol, etc., preferably methanol, ethanol, etc.), acetone and the like. . These may be used in a mixture at an appropriate ratio, but it is preferable to use alcohols, particularly ethanol alone. The amount (concentration) of addition to the aqueous solution of the physiologically active polypeptide is about 0.03 to 0.5% (V / V) in volume ratio,
Preferably, it is about 0.06 to 0.25% (V / V), more preferably about 0.1 to 0.15% (V / V).
By further freeze-drying the aqueous solution of the physiologically active polypeptide obtained by adding such a water-miscible organic solvent, it is easy to handle (has good operability) and is fine (small particle size). Polypeptide powder can be made. In the present invention, examples of the volatile salts added to the aqueous physiologically active polypeptide 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 to be added to the aqueous physiologically active polypeptide solution is about 10-fold to about 80-fold by mole, preferably about 10-fold to about 7-fold.
0 mole, more preferably about 15 to about 7 moles.
0-fold mole, more preferably about 20-fold to about 70-fold.
The molar ratio is about 20-fold, most preferably about 20-fold to about 50-fold. As in the case of adding a water-miscible organic solvent, the aqueous solution of a physiologically active polypeptide obtained by adding a volatile salt is further freeze-dried, so that it is easy to handle (has good operability) and is fine. A fine bioactive polypeptide powder (small particle size) can be prepared. In the present invention, the water-miscible organic solvent and / or volatile salts added to the aqueous physiologically active polypeptide solution may be used alone or in an appropriate combination. When a water-miscible organic solvent and a volatile salt are used in combination, the water-miscible organic solvent obtained by the above-mentioned method can be added to the aqueous physiologically active polypeptide solution according to the respective addition amounts described above. An aqueous solution of a physiologically active polypeptide containing volatile salts and / or is subjected to the freeze-drying operation of the present invention, and is used not only in pharmaceuticals and foods but also in all fields related to water. (Development of Pharmaceuticals, Vol. 11, edited by Yoshinobu Nakai, Hirokawa Shoten). For example, an aqueous solution of a physiologically active polypeptide is filled in a vial or an ampule, and then the aqueous solution is frozen and then subjected to a drying treatment. In this case, after freezing outside the machine, it may be subjected to a dryer, but it is usually preferable to freeze the inside of the machine under sufficient temperature control. Further, the drying operation can be carried out stepwise. Recent freeze dryers are capable of controlling these temperatures or vacuum, and optimal conditions are selected for the target bioactive polypeptide. The concentration of the physiologically active polypeptide in the aqueous solution to be subjected to the lyophilization operation is not particularly limited.
1% (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). When the bioactive polypeptide is hGH, the hGH concentration in the aqueous solution is preferably about 0.01 to about 5% (W / V), more preferably about 0.05 to about 0.5% (W / V). V).

The organic solvent used for dissolving the biodegradable polymer in the present invention 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 the organic solvent is used alone, for example, dichloromethane, acetonitrile and the like are preferable. When an organic solvent is used as the mixed solvent, for example, a halogenated hydrocarbon (eg, dichloromethane, chloroform, etc.) and an alcohol (eg, ethanol, methanol, 1,4-butanediol, 1.5)
-Pentanediol, etc.) or acetonitrile. In particular, a combination of dichloromethane and acetonitrile is widely used. The mixing ratio (volume ratio) between the halogenated hydrocarbon and the alcohol or acetonitrile is about 40: 1 to about 1: 1 and preferably about 20: 1 to about 1: 1. Particularly, a halogenated hydrocarbon (such as dichloromethane) alone or a 9: 1 to 1: 1 mixture of a halogenated hydrocarbon and acetonitrile is used.
It is desirable to use one mixed solvent. The concentration of the biodegradable polymer in the solution varies depending on the molecular weight, the type of the organic solvent, and the like. For example, about 0.01 to about 80%
(W / W), preferably from about 0.1 to about 70% (W / W).
W), more preferably from about 1 to about 60% (W / W).

The sustained-release preparation of the present invention comprises a biodegradable powder obtained by freeze-drying a physiologically active polypeptide solution to which a water-miscible organic solvent and / or volatile salts have been added. It is manufactured by removing a solvent from an S / O type dispersion liquid which is dispersed in an organic solvent liquid (O phase) in which a conductive polymer is dissolved. 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 solution of a physiologically active polypeptide, and then the physiologically active polypeptide is freeze-dried. A peptide powder (S phase) is prepared. Next, the biodegradable polymer is dissolved in an organic solvent,
The above-mentioned physiologically active polypeptide powder is added and dispersed in the organic solvent liquid. At this time, the ratio (weight ratio) of the bioactive polypeptide to the biodegradable polymer is, for example, about 1: 1000 to about 1: 1, preferably about 1: 200.
To about 1: 5, more preferably from about 1: 100 to about 1: 5. Further, in order to uniformly disperse the physiologically active polypeptide powder in the organic solvent liquid, it is preferable to apply external physical energy. For example,
Ultrasonic irradiation, a turbine type stirrer, a homogenizer and the like are used. At this time, the average particle size of the physiologically active polypeptide in the organic solvent solution is preferably about 10 μm or less, more preferably about 0.1 μm to about 10 μm, more preferably about 0.5 μm to 5 μm. It is easily achieved by using the bioactive polypeptide powder obtained by the production method of the present invention. The average particle size of the physiologically active polypeptide in the present invention is a value obtained by dispersing the physiologically active polypeptide in an organic solvent such as dichloromethane using a homogenizer and then using a laser analysis type particle size distribution analyzer (SALD2000A: Shimadzu). Is shown. At this time, the physiologically active polypeptide is added to an organic solvent such as dichloromethane at a concentration of, for example, about 20 to 100 mg / ml, and then, using a homogenizer (for example, Polytron (Kinematica)) at about 20,000 rpm for about 30 seconds. The mixture is stirred for 1 minute to form a dispersion liquid, and further appropriately diluted with the organic solvent so as to be within the measurable range of the particle size distribution measuring device, and then subjected to a test. Next, the organic solvent dispersion thus prepared (S / O type dispersion)
Is further added to an aqueous solvent (W phase) to form an S / O / W emulsion by the same external physical energy as described above, for example, by ultrasonic irradiation, a turbine stirrer, or a homogenizer. Thereafter, the oil phase solvent is evaporated to produce microcapsules. In this case, the volume of the aqueous phase is generally selected from about 1 to about 10,000 times the volume of the oil phase. More preferably, it is selected from about 2 times to about 5,000 times. Particularly preferably, about 5 times to about 2,000
It is selected from 0 times. An emulsifier may be added to the outer aqueous phase. As the emulsifier, generally stable S / O / W
Any material that can form an emulsion may be used.
Examples of the emulsifier include anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives,
Examples include 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 outer aqueous phase is preferably between about 0.001% and 20% (w / w). More preferably, it is about 0.01% to 10% (w / w), particularly preferably about 0.05% to 5% (w / w).

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

(B) Phase separation method (coacervation method) In this method, a microcapsule is precipitated and solidified by gradually adding a coacervation agent to the S / O type dispersion of (a) with 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. Any coacervation agent may be used 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 and lyophilized in the same manner as in (a). In the production of microcapsules by underwater drying method and coacervation method,
An aggregation inhibitor may be added to prevent aggregation of the 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 phosphoric acid. 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. In this case, if desired, it is also effective to spray an aqueous solution of the anti-agglomeration agent from another nozzle simultaneously with the above-mentioned dispersion liquid for the purpose of preventing the micro-capsule particles from aggregating. The microcapsules thus obtained are washed in the same manner as in (a) above, and if necessary, further heated (under reduced pressure if necessary) to further remove water and the organic solvent.

The sustained-release preparation of the present invention is preferably in the form of fine particles. This is because the sustained release preparation does not cause undue pain to the patient when administered through a needle usually used for subcutaneous or intramuscular injection. The particle size of the sustained-release preparation is, for example, about 0.1 to 300 μm, preferably about 1 to 150 μm as an average particle diameter.
μm, particularly preferably about 2 to 100 μm. The content of the physiologically active polypeptide contained in the sustained-release preparation of the present invention is, for example, about 0.1 to 30% (W / W), preferably about 0.2 to 20% (W / W), and more preferably. It is about 0.5 to 10% (W / W). The average particle size of the physiologically active polypeptide is preferably about 10 μm or less, more preferably about 0.1 μm to 10 μm, and more preferably about 0.5 μm to 5 μm. The content of the biodegradable polymer contained in the sustained-release preparation of the present invention is, for example, about 30 to 99.9% (W / W), preferably about 60 to 97% (W / W), and more preferably. Is about 70
To 90% (W / W). Initial release rate of the bioactive polypeptide of the sustained-release preparation of the present invention [1 day after administration (24
Time) is preferably about 40% or less, more preferably about 1 to 40%, more preferably about 3%.
Or 35%. Note that the initial release rate is determined by the AUC of the blood concentration up to 24 hours after subcutaneous administration of the sustained-release preparation of the present invention.
By applying (Area Under the Concentration) to the dose-AUC straight line obtained from the AUC up to 24 hours after subcutaneous administration of the physiologically active polypeptide solution, the initial release amount is obtained, and the initial release rate is further calculated.

The sustained-release preparation of the present invention may be formulated into various dosage forms, for example, as microcapsules or using the microcapsules as a raw material, and may be used in parenteral preparations (for example, injections into intramuscular, subcutaneous, organs, etc. Implants, transmucosal preparations for the nasal cavity, rectum, uterus, etc., oral preparations (eg, capsules (eg, hard capsules, soft capsules, etc.), solid preparations such as granules and powders, suspensions, etc.) , Etc.). The sustained-release preparation of the present invention is particularly preferably for injection. For example, when the sustained-release preparation is a microcapsule, the microcapsule is used as a dispersant (for example, a surfactant such as Tween 80 or HCO-60, a polysaccharide such as carboxymethylcellulose, sodium alginate, or hyaluronic acid), and a preservative. (E.g., methylparaben, propylparaben, etc.), tonicity agent (e.g., sodium chloride, mannitol, sorbitol, dextrose, etc.) to give an aqueous suspension, whereby a practical sustained-release preparation for injection can be obtained. Vegetable oils such as sesame oil and corn oil, or mixtures thereof with phospholipids such as lecithin, or medium-chain fatty acid triglycerides (eg, miglyol 8
12) to give a sustained-release injection which can be actually used as an oily suspension.

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

The sustained-release preparation of the present invention has low toxicity and can be safely used for mammals (for example, human, cow, pig, dog, cat, mouse, rat, rabbit, etc.). The indication of the sustained-release preparation varies depending on the bioactive polypeptide used. When the bioactive polypeptide is, for example, insulin, interferon-α such as diabetes
If viral hepatitis (eg, hepatitis C,
HBe antigen-positive active hepatitis, etc.), cancer (eg, renal cancer, multiple myeloma, etc.), anemia in the case of erythropoietin (eg, anemia in renal dialysis, etc.), and favorable in the case of G-CSF Cytopenia (eg, when treating cancer drugs), infectious diseases, etc.
In the case of IL-2, cancer (eg, hemangioendothelioma, etc.)
In the case of GF, fracture, wound (bedsore etc.), periodontal disease, gastrointestinal ulcer, etc., in the case of FGF-9, thrombocytopenia, NGF
In the case of senile dementia, neuropathy (neuropathy), etc.
TPA is effective for treatment or prevention of thrombosis and the like, and tumor necrosis factor is effective for treatment or prevention of cancer and the like. In addition, the GH-containing sustained-release preparation can be applied to Turner syndrome, chronic kidney disease, chondrodystrophy, and adult pituitary insufficiency, in addition to pituitary dwarfism, based on the growth hormone action of GH. . G
H has also been reported to be effective for diseases such as Down syndrome, Silver syndrome, osteogenesis imperfecta, or juvenile chronic arthropathy, and it has been reported that an effective therapeutic effect has been obtained. Is also applicable. Furthermore, it is also effective in treating or preventing congestive heart failure and the like.

The dose of the sustained-release preparation varies depending on the type and content of the physiologically active polypeptide, the duration of release, the target disease, the target animal, etc., but the effective concentration of the physiologically active polypeptide is maintained in the body. The amount may be any amount. For example, when the sustained release preparation is a two-week preparation, the dose of the physiologically active polypeptide can be appropriately selected from the range of preferably about 0.0001 to about 10 mg / kg body weight per adult. More preferably, about 0.05 to about 1 m
It can be appropriately selected from the range of g / kg body weight. The number of administrations is once a week, once every two weeks, once a month, once every two months, etc., such as the type and content of the bioactive polypeptide, dosage form, duration of release, target disease, It can be appropriately selected depending on the target animal and the like. Preferably a 1 week to 1 month sustained release formulation, more preferably a 1 to 3 week sustained release formulation, more preferably a 10 to 20 day sustained release formulation,
Particularly preferred is a two-week sustained-release preparation.
A biologically active polypeptide which is an active ingredient of a sustained-release preparation,
For example, in the case of insulin, the dosage for an adult with diabetes is usually about 0.001 to about 1 mg / kg body weight, preferably about 0.01 to about 0.2 mg as an active ingredient.
/ Kg body weight is appropriately selected and administered once a week.

When the physiologically active polypeptide which is the active ingredient of the sustained-release preparation is GH, the dosage varies depending on the type and content of GH, duration of release, target disease, target animal, etc. The effective concentration of the GH may be an amount that can be retained in the body. In the treatment of the above-mentioned diseases, for example, when the sustained-release preparation is a two-week preparation, the dose of GH as an active ingredient is preferably about 0.01 to about 5 mg / kg body weight per child or adult. 0.03 to about 15 IU / kg body weight) and can be safely administered. More preferably, it can be appropriately selected from the range of about 0.05 to about 1 mg / kg body weight (about 0.15 to about 3 IU / kg body weight). The number of administrations is 1
G once a week, once every two weeks or once a month
It can be appropriately selected depending on the H content, dosage form, duration of release, target disease, target animal, and the like. Preferably, a 1 week to 1 month sustained release formulation, more preferably a 1 to 3 week sustained release formulation, more preferably a 10 to 20 day sustained release formulation, particularly preferably a 2 week sustained release formulation No. The sustained-release preparation is preferably stored at room temperature or in a cold place. More preferably, the sustained-release preparation is stored in a cold place. The normal temperature or cold place here is defined in the Japanese Pharmacopoeia. That is, normal temperature means 15 to 25 ° C, and cold place means 15 ° C or less. Among cold places, 2 to 8 ° C. is particularly preferred.

[0030]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to Reference Examples, Examples, Comparative Examples, and Test Examples, but these do not limit the present invention. In the specification of the present invention, when an abbreviation such as an amino acid is used, IUP
AC-IUB Commission on Biochemical Nomenclatur
This is based on the abbreviation by e or a common abbreviation in the field, examples of which are shown below. When amino acids have optical isomerism, L-form is shown unless otherwise specified. SDS: sodium dodecyl sulfate Gly: glycine Ala: alanine Val: valine Leu: leucine Ile: isoleucine Ser: serine Thr: threonine Cys: cysteine Met: methionine Glu: glutamic acid Gln: paraglutinic acid G: glutamic acid Gln: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asx: Asp + AsnGlx: Glu + Gln

[0031]

EXAMPLES Reference Example 1 Construction of Human Growth Hormone (hGH) Expression Vector Using T7 Promoter The structural gene of hGH is plasmid pHGH107 (described in Japanese Patent Publication No. 6-12996, accession number ATCC31).
538 and ATCC 40011) from EcoRI and Eco
An about 0.75 kb fragment was isolated by digestion with RV. On the other hand, pE
T-3C [Rosenberg et al., Gene, 56
125 (1987)], and a fragment of about 4.6 kb having a T7 promoter and an ampicillin resistance gene was isolated. T4D both fragments
After blunt-ending with NA polymerase (DNA Blunting kit, manufactured by Takara Shuzo Co., Ltd.) and ligating with T4 DNA ligase, the resulting DNA was introduced into Escherichia coli JM109, and transformants were selected using ampicillin resistance as an index. From the colonies that appeared, 12 were picked up, plasmids were prepared therefrom, and the cleavage pattern with the restriction enzyme PstI was examined.
It was found that the hGH gene was inserted in the correct orientation in the plasmids from the individual colonies. The plasmid obtained from one of these strains was named pTGA201.

Reference Example 2 Expression of Met-hGH in Escherichia coli Escherichia coli JM109 was lysogenized with lambda phage (Studier, Supra) recombined with the T7 phage RNA polymerase gene. Thereafter, the hGH expression vector pTGA201 obtained in Reference Example 1 was introduced into Escherichia coli JM109 (DE3), and Escherichia coli JM109 (D
E3) / pTGA201 was obtained. This transformed cell is
LB medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) containing 50 μg / ml ampicillin 1
In a 2 liter flask containing 30 liters, 30 ° C., 16
The cells were cultured with shaking for a time. The obtained culture solution was added to 20 liters of L containing 0.02% Newpole LB-625 (antifoaming agent; manufactured by Sanyo Chemical Industries) and 50 μg / ml of ampicillin.
Transfer to a 50-liter fermenter containing B medium,
Incubation with aeration and stirring was performed at 7 ° C for 6 hours. This culture was used in 360 liters of the main fermentation medium (1.68% sodium monohydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.0246% sulfuric acid). Magnesium, 0.02% Newpole LB-625,
0.0005% thiamine hydrochloride, 1.5% glucose, 1.5
% Casamino acid) was transferred to a 500-liter fermenter, and aeration and agitation culture was started at 37 ° C. When the turbidity of the culture reaches about 500 klet units, 5.95 mg
/ Liter of isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the culture was further continued. After 4 hours, the culture was centrifuged to obtain about 4.5 kg of wet cells. It was stored frozen at 80 ° C. The above transformed E. coli J
M109 (DE3) / pTGA201 has accession number FE
Deposited as RM BP-5632 with the National Institute of Advanced Industrial Science and Technology (NIBH) of the Ministry of International Trade and Industry of Japan, and under the accession number IFO 16001 as a fermentation research institute (I)
FO).

Reference Example 3 Activation of Met-hGH 50 mM Tris / HCl was added to 2 kg of the cells obtained in Reference Example 2.
After adding 6 liters of 8M guanidine hydrochloride solution (pH 8.0) to lyse the cells, centrifugation (10000 rpm, 120 rpm) was performed.
Min). 50 mM was added to 6 liters of the obtained supernatant.
Tris / HCl, 0.28 mM GSGSG, 1.4 mM GGS
After adjusting the pH to 8.0 by adding 18 liters of 0.7 M arginine (pH 8.0), activation was performed at 4 ° C. for 5 days.

Reference Example 4 Purification of Met-hGH The regenerated solution which had been activated in Reference Example 3 was purified from 20 mM Tris / HCl, 2.5 M urea (pH 8.0) using a Pellicon cassette system (PTGC membrane, Millipore). After addition, desalting and concentration were performed until the electric conductivity became 10 mS or less, and the obtained concentrate was centrifuged (10000 rpm, 60 rpm).
For 5 minutes) to obtain 5 L of the supernatant of the concentrated solution. The solution was then added to 20 mM Tris / HCl, 2.5 M urea (pH 8.0).
-Toyopearl 650M column (2
After adsorbing on 0 cmφ × 84 cm (Tosoh Corporation) and washing thoroughly, 0 to 25% B (B = 20 mM Tris / HCl,
Elution was performed with a concentration gradient of 2.5 M urea, 1 M sodium chloride, pH 8.0) for 100 minutes at a flow rate of 300 ml / min.
An eluate of 10 liters was obtained as a Met-hGH fraction.
Further, this eluate is used in a Pellicon cassette system (PTG).
(C membrane, Millipore). Further, this solution was subjected to a DEAE-5PW column (2) by a high performance liquid chromatography (Gilson HPLC system, Gilson).
(1 cm × 30 cm, Tosoh Corporation), and A = 5
0 mM Tris / HCl + 2.5 M urea (pH 8.0), B =
Elution was performed with a 70-85% B pH gradient with 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) for 70 minutes at a flow rate of 320 ml / min. 6 liters of fraction were obtained. To this eluate was added 300 ml of a 2M Tris / HCl (pH 7.8) solution.
l The mixture was adjusted to pH 7.2, and then concentrated and desalted using a Pellicon cassette system (PTGC membrane, Millipore).
9,979 milligrams of Met-hGH were obtained.

Reference Example 5 Removal of N-terminal Met 2.5 M was added to 1650 ml of the Met-hGH solution obtained in Reference Example 4.
Glyoxylic acid, 35 mM copper sulfate, 6 M pyridine solution 4
After adding 13 ml and stirring well, the mixture was reacted at 25 ° C. for 60 minutes. Then, the solution was passed through a Sephadex G-25 column (11.3 cmφ × 125 cm, Pharmacia) equilibrated with 20 mM Tris / HCl, 2.5 M urea (pH 8.0) at a flow rate of 3 liter / h to equilibrate. The eluted Met-hGH diketone fraction developed and eluted with the same buffer as described above was directly mixed with 4 M acetic acid, 4 M sodium acetate, 80 mM Mo-phenylenediamine, 3 M urea solution 4 with good stirring.
In liters. After the elution was completed, 8 liters of the reaction solution was allowed to stand at 4 ° C. for 3 days. After standing, the mixture was concentrated to 4 liters using a Pellicon cassette system (PTGC membrane, Millipore), and 20 mM Tris / HCl, 2.5 M urea (pH 8.0.
0) equilibrated in Sephadex G-25 column (1)
(1.3 cmφ × 140 cm, Pharmacia) at a flow rate of 3 L / h, and 4.7 L of the hGH fraction was collected. Furthermore, high performance liquid chromatography (Gilson HPL
C System, Gilson)
After passing through a -5PW column (21 cm × 30 cm, Tosoh Corporation) and adsorbing, A = 50 mM Tris / HCl + 2.5 M urea (pH 8.0), B = 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2 2.5M urea (pH 4.0)
70-85% B pH gradient with 70 min.
Elution was performed at a flow rate of 320 ml / min to obtain 10 liters of an hGH fraction. This hGH fraction contains 2M Tris / HCl (pH
7.8) After adjusting the pH to 7.2 by adding 500 ml of the solution, the solution was concentrated with Minitan II (PTGC membrane, Millipore), and 500 ml of this concentrated solution was equilibrated with distilled water to obtain Sephacryl S-10.
The solution was passed through a 0 column (113.cmφ × 50 cm, Pharmacia) at a flow rate of 2 liter / h, developed, and developed into a hGH fraction 165.
1 ml was obtained. Further, this solution was filtered through Millipak 60 (Millipore) to obtain 1487 ml of hGH solution (3309 mg of hGH).

Reference Example 6 Confirmation of Properties of hGH (a) Analysis using SDS polyacrylamide gel electrophoresis Sample buffer containing 100 mM DTT in hGH obtained in Reference Example 5 [Laemmli, Nature, 227 , 680 (197)
0)], stirred well, heated at 95 ° C. for 2 minutes, and electrophoresed on Multigel 10/20 (Daiichi Pure Chemicals). Run the gel after electrophoresis with Coomassie Brilliant
As a result of staining with blue (Coomassie brilliant blue),
Since a single band was observed at about 22 kd, the purified h
It was confirmed that GH was almost single.

(B) Analysis of amino acid composition The amino acid composition was determined using an amino acid analyzer (L-8500A, Hitachi). As a result, the amino acid composition of the obtained hGH was consistent with the amino acid composition deduced from the nucleotide sequence of cDNA (Table 1).

TABLE 1 hGH base sequence per mole of hGH amino acid residue number predicted from ------------------------- Asx 20.2 20 Thr ¹⁾ 10.0 10 Ser ¹⁾ 16.7 18 Glx 27------------------------- 0.027 Pro 8.18 Gly 8.22 Ala 7.67 Cys ND ²⁾ 4 Val 7.07 Met 3.03 Ile 7.7 8 Leu 27.926 Tyr 8.1 8 Phe 12.7 13 His 3.23 Lys 8.9 9 Arg 10.9 11 Trp 0.8 1 ------------------------------------ --- Acid hydrolysis (6N HCl-4% thioglycolic acid, 1
(Average value of hydrolysis at 10 ° C. for 24 and 48 hours) 1) Extrapolated value at 0 hour 2) Not detected Analysis was performed using about 20 μg.

(C) N-terminal amino acid sequence analysis The N-terminal amino acid sequence was determined using a gas phase protein sequencer (Applied Biosystems, Model 477A). As a result, the N-terminal amino acid sequence of the obtained hGH is the N-terminal amino acid sequence of the hGH deduced from the nucleotide sequence of the cDNA.
It was consistent with the terminal amino acid sequence (Table 2).

TABLE 2 Base sequence of hGH detected Residue No. PTH ¹⁾ -amino acid From ------------------------- Predicted (pmol) Amino acid ------------------------- 1 Phe (949) Phe 2 Pro (404) Pro 3 Thr (422) Thr 4 Ile (744) Ile 5 Pro (283) Pro 6 Leu (514) Leu 7 Ser (136) Ser 8 Arg (36) Arg 9 Leu (377) Leu 10 Phe (408) Phe 11 A Asp 12 Asn (230) Asn 13 Ala (435) Ala 14 Met (334) Met 15 Leu (398) Leu 16 Arg (67) Arg 17 Ala (488) la 18 His (30) His 19 Arg (42) Arg 20 Leu (406) Leu -------------------------------------------- 1) Analysis was performed using 1 nmol of phenylthiohydantoin.

(D) C-terminal amino acid analysis The C-terminal amino acid was determined using an amino acid analyzer (L-8500A, Hitachi). The C-terminal amino acid of the obtained hGH coincided with the C-terminal amino acid deduced from the nucleotide sequence of the cDNA (Table 3).

[Table 3] ------------------- C-terminal amino acid recovery rate (%) ----------------- The analysis was performed using 18 nmol of Phe 52 -------- vapor phase hydrazine decomposition method (100 ° C., 6 hours). (E) Measurement of hGH activity Nb2 cells of the purified hGH obtained in Reference Example 5 [Journal of Clinical Endocrinology and
Metabolism, vol. 51, p. 1058 (1980)] had the same growth promoting effect as the standard product.

Example 1 (1) Production of hGH powder Ethanol [final concentration (V / V%)] was added to a recombinant hGH aqueous solution (final hGH concentration = 2 mg / ml) obtained according to Reference Example 5. = 0.03%, 0.06%, 0.1%, 0.15
%, 0.25%, 0.5%)] and freeze-dried to obtain six kinds of hGH powders. (2) Production of hGH-containing microcapsules Lactic acid-glycolic acid copolymer (lactic acid / glycol = 50)
/ 50, polystyrene equivalent average molecular weight = 12,000,
1.85 g of (viscosity = 0.145 dl / g) and 10 mg of zinc oxide were dissolved in 3.0 ml of dichloromethane. 140 mg of the above hGH powder (1) was added to the organic solvent solution, and the mixture was pulverized using Polytron (Kinematica). This S / O dispersion was added to 800 ml of a 0.1% aqueous solution of polyvinyl alcohol, 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 1,500 rpm). Then, after washing twice with 400 ml of distilled water, 0.2 g of D-mannitol was added and freeze-dried.
Furthermore, in order to remove the residual solvent, vacuum drying was performed at 46 ° C. for 3 days to obtain six kinds of hGH-containing microcapsules.

Example 2 (1) Production of hGH powder A recombinant hGH aqueous solution (final hGH concentration = 2 mg / ml) obtained according to Reference Example 5 was added with 10, 20, 25, 40 ammonium acetate. , 50, 70 and 100-fold molar equivalents were added and freeze-dried to obtain seven kinds of hGH powders. (2) Production of hGH-containing microcapsules The same operation as in Example 1 (2) was carried out, and
Seven kinds of hGH-containing microcapsules were obtained using GH powder.

Comparative Example 1 (1) Production of hGH Powder To a genetically modified hGH aqueous solution (final hGH concentration = 2 mg / ml) obtained according to Reference Example 5, ethanol and / or ammonium acetate were not added. And by freeze-drying, an hGH powder was obtained. (2) Production of hGH-containing microcapsules The same operation as in Example 1 (2) was carried out, and
HGH-containing microcapsules were obtained using GH powder.

Test Example 1 hG obtained in Example 1 (1) and Comparative Example 1 (1)
Regarding the H powder, the ease of handling was determined from the viewpoint of powder volume (bulk height) and chargeability to static electricity. The results are shown in [Table 4]. As is clear from the results of [Table 4],
As compared to the hGH powder without ethanol, the handling of the hGH powder with added ethanol was remarkably improved.

Test Example 2 S / O dispersion 5 of hGH powder / PLGA organic solvent obtained in the intermediate step of Example 1 (2) and Comparative example 1 (2)
After adding 2 ml of dichloromethane to 0 μl, hG
The average particle size of the H powder was measured using a laser diffraction type particle size distribution analyzer (SALD2000A: Shimadzu). The results are shown in [Table 4]. As is clear from the results of Table 4, the hGH powder without ethanol had an average particle diameter of 10%.
μm or more, but hGH added with ethanol
Each of the powders was a fine powder having an average particle diameter of 10 μm or less.

[Table 4]

Test Example 3 The following test was carried out using the hGH-containing microcapsules produced in Example 1 (2) and Comparative Example 1 (2). (1) hGH content To 5 mg of microcapsules, 0.3 ml of acetonitrile was added, followed by sonication. To the resulting acetonitrile solution was added 0.7 ml of 10 mM phosphate buffer (pH 8.
0) was added to extract hGH as the main drug. Next, the hGH extract was subjected to size exclusion high performance liquid chromatography under the following conditions to measure the hGH content and calculate the encapsulation rate. The results are shown in [Table 5]. Column: TSKgelG 3000SW _XL (Tosoh) Extract: 0.2 M phosphate buffer (pH 6.8) -0.2 M
NaCl flow rate: 0.6 ml / min As is clear from the results shown in Table 5, the hGH powder containing ethanol was used in comparison with the hGH-containing microcapsules produced using hGH powder without ethanol. A high hGH encapsulation rate was obtained in the manufactured hGH-containing microcapsules.

[Table 5]

(2) In Vivo Release Microcapsules were administered subcutaneously (6 mg / rat as hGH amount) to immunosuppressed SD rats (male, 6 weeks old), blood was collected over time, and the hGH concentration in the serum was measured. Measured by radioimmunoassay (Ab beads HGH: Eiken Chemical)
The hGH sustained release was evaluated. For the immunosuppressed SD rats, Prograf (Fujisawa Pharmaceutical) was subcutaneously administered 0.4 mg / rat 3 days before administration of the microcapsules, and 0.2 mg / rat each 4 days, 7 days, 11 days and 14 days after administration. Created. The results are shown in FIG. As is clear from the results of FIG. 1, in the hGH-containing microcapsules obtained using the hGH powder to which ethanol was added, the initial release [the amount of release up to 1 day (24 hours) after administration] was small, Thereafter, the sustained concentration [the amount released over one day (24 hours) to two weeks after administration] was high. That is, by using hGH powder to which ethanol was added, suppression of initial release and high blood hGH concentration over two weeks were achieved.

Test Example 4 hG obtained in Example 2 (1) and Comparative Example 1 (1)
Regarding the H powder, the ease of handling was determined from the viewpoint of powder volume (bulk height) and chargeability to static electricity. The results are shown in [Table 6]. As is clear from the results of [Table 6],
As compared to the hGH powder without ammonium acetate, the handling of the hGH powder with ammonium acetate added was remarkably improved.

Test Example 5 The S / O dispersion of hGH powder / PLGA organic solvent obtained in the intermediate step of Example 2 (2) and Comparative Example 1 (2) was subjected to laser diffraction by the same operation as in Test Example 2. To a particle size distribution analyzer (SALD2000A: Shimadzu)
The average particle size of GH was measured. The results are shown in [Table 6].

[Table 6]

Test Example 6 The following experiment was carried out using the hGH-containing microcapsules produced in Example 2 (2) and Comparative Example 1 (2). (1) hGH Content The hGH encapsulation rate of various microcapsules was measured by the same operation as in Test Example 1 (1). The results are shown in [Table 7]. As is evident from the results in Table 7, compared to the hGH-containing microcapsules produced using the hGH powder without ammonium acetate, the h-capsules containing ammonium acetate were added.
A high hGH encapsulation rate was obtained in the hGH-containing microcapsules manufactured using GH powder. (2) Initial release rate The initial release rate of the microcapsules was calculated by the following method. The results are shown in [Table 7]. HGH aqueous solution (concentration 5, 10, 2) was given to immunosuppressed SD rats (male, 6 weeks old).
0 mg / Kg) was administered subcutaneously and blood was collected over time up to 24 hours. The hGH concentration in the serum was measured by radioimmunoassay (Ab beads HGH: Eiken Chemical Co., Ltd.), and the AUC at each concentration was determined. AUC (Area Under t
he Concentration) A straight line was obtained. In addition, microcapsules (hGH amount: 6 mg) were subcutaneously administered to immunosuppressed SD rats (male, 6 weeks old), blood was collected over time up to 24 hours, and the hGH concentration in the serum was measured by radioimmunoassay (Ab beads HGH: AUC in each microcapsule was determined. AUC obtained
Was applied to the dose-AUC line to obtain the initial release amount, and the initial release rate was calculated.

[Table 7] (2) In vivo release properties The same operation as in Test Example 1 (2) was used to evaluate the sustained release properties of the microcapsules in hGH-suppressed SD rats in hGH. The results are shown in FIG. As is clear from the results of FIG. 2, in the hGH-containing microcapsules obtained by using the hGH powder to which ammonium acetate was added, the initial release [release amount up to 1 day (24 hours) after administration] was small. ,
Thereafter, the sustained concentration [the amount released over one day (24 hours) to two weeks after administration] was high. That is, by using the hGH powder to which ammonium acetate was added, suppression of initial release and high blood hGH concentration over two weeks were achieved.

[0050]

According to the present invention, the ease of handling of the bioactive polypeptide powder in the preparation process is improved, and industrial mass production of sustained-release preparations becomes possible. In addition, it is possible to provide a sustained-release preparation which has a high encapsulation rate of the physiologically active polypeptide in the sustained-release preparation and has a low initial release rate and a stable high blood maintenance concentration over a long period of time.

[0051]

[Brief description of the drawings]

FIG. 1 is a graph showing changes in serum hGH concentration after administration of hGH-containing microcapsules obtained using ethanol-added hGH powder in immunosuppressed SD rats.

FIG. 2 is a graph showing the time course of hGH concentration in serum after administration of hGH-containing microcapsules obtained using hGH powder to which ammonium acetate has been added in immunosuppressed SD rats.

Claims (36)

[Claims] 1. A bioactive polypeptide powder obtained by adding a water-miscible organic solvent and / or a volatile salt to an aqueous solution of a bioactive polypeptide and freeze-drying the resulting solution to obtain an organic biodegradable polymer. A method for producing a sustained-release preparation, comprising removing an organic solvent after dispersing in a solvent solution. 2. The bioactive polypeptide powder has an average particle size of 1
2. The method according to claim 1, wherein the thickness is 0 μm or less. 3. The method according to claim 1, wherein the physiologically active polypeptide is a growth hormone. 4. The method according to claim 3, wherein the growth hormone is human growth hormone. 5. The method according to claim 1, wherein the water-miscible organic solvent is an alcohol. 6. The method according to claim 5, wherein the alcohol is methanol or ethanol. 7. The method according to claim 5, wherein the alcohol is ethanol. 8. The method according to claim 1, wherein the volatile salt is an ammonium salt. 9. The method according to claim 8, wherein the ammonium salt is ammonium acetate, ammonium bicarbonate or ammonium carbonate. 10. The method according to claim 8, wherein the ammonium salt is ammonium acetate. 11. The method according to claim 5, wherein the alcohol is added so that the concentration with respect to the aqueous solution of the physiologically active polypeptide is from 0.03% (V / V) to 0.5% (V / V). 12. The method according to claim 8, wherein an ammonium salt having a molar ratio to the physiologically active polypeptide of 10 to 80 times is added. 13. The method according to claim 1, wherein the biodegradable polymer is a lactic acid-glycolic acid polymer. 14. A lactic acid / glycolic acid polymer having a lactic acid / glycolic acid composition ratio (mol%) of 100/0 to 40/6.
14. The method according to claim 13, wherein 0 is used. 15. The method according to claim 13, wherein the lactic acid-glycolic acid polymer has a weight average molecular weight of 3,000 to 50,000. 16. The method according to claim 13, wherein the lactic acid-glycolic acid polymer is a polyvalent metal salt. 17. The method according to claim 16, wherein the polyvalent metal is zinc. 18. The method according to claim 1, wherein the sustained-release preparation is a microcapsule. 19. The microcapsules having an average particle size of 0.1.
The method according to claim 18, wherein the thickness is from 300 to 300 µm. 20. The method according to claim 1, wherein the sustained-release preparation is for injection. 21. A sustained-release preparation produced by the production method according to claim 1. 22. A process for producing a bioactive polypeptide powder, comprising adding a water-miscible organic solvent and / or volatile salts to an aqueous solution of the bioactive polypeptide and freeze-drying it. 23. A bioactive polypeptide powder obtained by the production method according to claim 22. 24. The bioactive polypeptide powder according to claim 23, wherein the bioactive polypeptide powder has an average particle size of 10 μm or less. 25. A method for producing a pharmaceutical preparation according to claim 23.
Use of the physiologically active polypeptide powder described above. 26. Use of a water-miscible organic solvent and / or a volatile salt for producing a bioactive polypeptide powder having a small particle size. 27. The use according to claim 26, wherein the bioactive polypeptide powder has an average particle size of 10 μm or less. [28] A sustained-release preparation obtainable by the production method according to [1]. 29. The method according to claim 29, wherein the initial release rate of the bioactive polypeptide is 4
A sustained-release preparation containing 0% or less of a bioactive polypeptide and a polyvalent metal salt of a biodegradable polymer. (30) the physiologically active polypeptide has an average particle size of 1;
30. The sustained-release preparation according to claim 29, which is a powder having a particle size of 0 µm or less. 31. The sustained-release preparation according to claim 29, wherein the physiologically active polypeptide is a growth hormone. 32. The sustained-release preparation according to claim 29, wherein the biodegradable polymer is a lactic acid-glycolic acid polymer. 33. The sustained release preparation according to claim 29, wherein the sustained release preparation is a microcapsule. 34. The microcapsules having an average particle size of 0.1.
34. The sustained-release preparation according to claim 33, which has a thickness of from 300 to 300 µm. 35. The sustained release preparation according to claim 29, which is a one week to one month type sustained release preparation. 36. The sustained release preparation according to claim 29, wherein the sustained release preparation contains 0.1 to 30% (W / W) of a physiologically active polypeptide.