JPH11158200A - Human growth hormone/zinc complex and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new complex useful for a sustained release preparation having a high enclosed ratio of human growth hormone, effective for treating dwarfism, Turner's syndrome, osteoporosis, etc., comprising a human growth hormone/zinc complex composed of a human growth hormone and zinc in a specific molar ratio. SOLUTION: This new human growth hormone/zinc complex contains a human growth hormone and zinc in the molar ratio of about 1:1.6 to 1:2.4, is effective for treating pituitary dwarfism, Turner's syndrome, chronic renal disease of childhood, chondralloplasia, adult growth hormone hyposecretion, osteoporosis, congestive heart disease, etc., useful as a raw material for sustained release preparation having high enclosed ratio of human growth hormone. The complex is obtained by formulating a human growth hormone with zinc in the molar ratio of about 1:1.6 to 1:2.4 to form a complex and grinding the complex. The complex is dispersed into an oil phase containing a biodegradable polymer to give an S/O type dispersion, which is added to a water phase and dried as an S/O/W type emulsion in water to give the objective sustained release preparation of human growth hormone.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a human growth hormone / zinc complex containing human growth hormone and zinc in a molar ratio of about 1: 1.6 to about 1: 2.4.
The present invention relates to a sustained-release preparation containing a zinc complex and a biodegradable polymer.

[0002]

2. Description of the Related Art In recent years, human growth hormone (hereinafter sometimes abbreviated as GH) can be produced in large quantities by genetic engineering techniques. In addition, it is widely used in clinical applications such as Turner's syndrome, pediatric chronic kidney disease, cartilage dystrophy and adult GH secretion deficiency. It is also expected to be applied to remarkable osteoporosis in an aging society and further to congestive heart disease. GH has been conventionally used in repeated administration by intramuscular or subcutaneous injection in consideration of its stability in a living body, and is often administered over a long period of time. There is a problem that the burden is very large. For example, in the case of pituitary dwarfism, the fact is that daily subcutaneous administration is performed for a long period of several months to 10 years or more to infants or young patients. On the other hand, it has been reported that a GH-containing sustained-release preparation exhibiting a sufficient drug efficacy by administration once every several weeks to several months (STPPha).
rma. Sci., 4 (6), pp. 437-441, 199.
4 years; Nature Med., 2 (7), 79-79.
9, 1996; J. Pharm. Exp. Ther.,
281, 1431-1439, 1997; WO 9
4/12158; WO 95/29664; WO 97 /
01331).

[0003]

In a conventional GH-containing sustained-release preparation, a so-called W / W / GH is prepared by converting GH, which is the main drug, into an aqueous solution (aqueous phase) and then adding it to an organic solvent (oil phase) in which a polymer is dissolved. O-emulsions have been formed and sustained-release preparations have been produced using these emulsions. However, GH denaturation during the manufacturing process or storage is remarkable, and sufficient encapsulation rates and sustained-release properties cannot be obtained. On the other hand, GH powder is directly added to and dispersed in an organic solvent (oil phase) in which a polymer is dissolved.
There is also a method of producing a sustained-release preparation using an / O dispersion, but it is necessary to ensure stability by spraying the S / O dispersion into liquid nitrogen, which is not suitable for mass production. It is. In addition, GH is generally used after being crushed with a large particle size.
It was difficult to prepare a uniform S / O type dispersion having a high GH content. Thus, the maintenance of GH stability during the formulation process and the GH
It is very difficult to atomize the compound with a small decrease in activity, and it is extremely difficult to mass-produce a sustained-release preparation having a high yield and a high and uniform GH content without reducing the activity of GH. Met. Therefore, a clinically useful preparation of human growth hormone exhibiting stable sustained release over a long period of time, which overcomes these difficulties, and a method of mass-producing such a sustained release preparation in good yield are desired.

[0004]

Means for Solving the Problems The present inventors have conducted various studies and found that GH and zinc were converted from about 1: 1.6 to about 1: 2.4.
The first production of a GH / zinc composite containing a molar ratio of
Unexpectedly, this complex is substantially water-soluble, can reduce the activity of GH, can advantageously produce fine powder compared to GH itself, and has a smaller particle size.
-When a sustained-release preparation is manufactured using a zinc complex, it is possible to mass-produce a stable-quality sustained-release preparation having an improved GH encapsulation rate and improved sustained-release properties without denaturing the GH during the manufacturing process. And so on. That is, the present invention provides (1) a human growth hormone / zinc complex containing human growth hormone and zinc in a molar ratio of about 1: 1.6 to about 1: 2.4, and (2) a water-soluble complex. (1) the human growth hormone / zinc complex according to (1), (3) the human growth hormone / zinc complex according to (1) having an average particle size of about 10 μm or less, (4) the human growth according to (1) above A sustained release preparation comprising a hormone / zinc complex and a biodegradable polymer, (5) the sustained release preparation according to (4), wherein the biodegradable polymer is an aliphatic polyester, (6) (5) the sustained release preparation according to the above (5), wherein the aliphatic polyester is a lactic acid / glycolic acid polymer; and (7) the composition (mol%) of the lactic acid / glycolic acid polymer is 100/0 to about 40/60. 6) The sustained release preparation according to the above,

(8) The above (5), wherein the weight average molecular weight of the aliphatic polyester is from about 3,000 to about 20,000.
(10) The sustained-release preparation according to (5), wherein the aliphatic polyester is a salt with a polyvalent metal.
The sustained release preparation according to the above (9), wherein the polyvalent metal is zinc,
(11) The sustained release preparation according to (4), which is a microcapsule, (12) the sustained release preparation according to (11), wherein the microcapsule is for injection, and (13) the initial release rate of human growth hormone. The sustained release preparation according to the above (4), which is about 50% or less, and (14) human growth hormone and zinc at about 1:
A method for producing a human growth hormone / zinc complex, comprising mixing at a molar ratio of 1.6 to about 1: 2.4;
5) Micronized, characterized by forming and grinding a human growth hormone / zinc complex containing human growth hormone and zinc in a molar ratio of about 1: 1.6 to about 1: 2.4. A method for producing human growth hormone, (16) a human growth hormone-zinc complex comprising human growth hormone and zinc in a molar ratio of about 1: 1.6 to about 1: 2.4. (17) biodegradation of a human growth hormone / zinc complex containing human growth hormone and zinc in a molar ratio of about 1: 1.6 to about 1: 2.4. / Obtained by dispersing in an oil phase containing a hydrophilic polymer
(18) a method for producing a sustained-release preparation, comprising adding an O-type dispersion to an aqueous phase and subjecting it to drying in water as an S / O / W-type emulsion; (18) human growth hormone / zinc according to (1) above A pharmaceutical preparation characterized by containing the complex; and (19) a preventive and therapeutic agent for pituitary dwarfism characterized by containing the human growth hormone / zinc complex described in (1) above. I do.

[0006] The GH used in the present invention may be a natural type (extract or the like) or a recombinant type (Nature 281).
Volume, p. 544 (1979); and p. 293, p. 408 (1)
981), Proc. Natl. Acad. Sci. U
SA, 80, 397 (1983), Biotech.
No. 5, p. 161 (1981)), but from the viewpoint of safety and quality, recombinant GH is preferred. In the present invention, these muteins, derivatives, analogs and active fragments (J. Bi
ol. Chem., 253, 2679 (1978),
BBRC 92, 511 (1980), End
ocrinol., 109, 1301 (198
1), Protein Eng. 3, p. 49 (198)
9) etc.) may be used. The GH / zinc composite of the present invention may be synthesized by any synthesis method as long as the final molar ratio of GH to zinc is from about 1: 1.6 to about 1: 2.4, For example, the complex may be synthesized according to a synthesis method generally used. Usually, the GH / zinc complex of the present invention can be obtained by contacting GH with, for example, a water-soluble zinc salt. This contact (reaction) is preferably performed in a solvent, particularly an aqueous solvent. The contact time may be about 1 minute to about 1 hour, and the contact temperature may be about 4 to about 37 ° C. (preferably room temperature). Examples of the water-soluble zinc salt used in this reaction include a salt of zinc and an inorganic acid or a salt of zinc and an organic acid.Examples of the inorganic acid include hydrochloric acid, sulfuric acid, and nitric acid. Examples thereof include aliphatic carboxylic acids and aromatic carboxylic acids. Here, examples of the aliphatic carboxylic acid used as the organic acid include an aliphatic monocarboxylic acid, an aliphatic dicarboxylic acid, and an aliphatic tricarboxylic acid. These aliphatic carboxylic acids may be either saturated or unsaturated. The aliphatic carboxylic acid is preferably an aliphatic carboxylic acid having 2 to 9 carbon atoms.

Examples of such aliphatic monocarboxylic acids include saturated aliphatic monocarboxylic acids having 2 to 9 carbon atoms (eg, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargone). Acids, capric acid, etc.) and unsaturated aliphatic monocarboxylic acids having 2 to 9 carbon atoms (eg, acrylic acid, propiolic acid, methacrylic acid, crotonic acid, isocrotonic acid, etc.). Examples of the aliphatic dicarboxylic acid include saturated aliphatic dicarboxylic acids having 2 to 9 carbon atoms (for example, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, etc.) and unsaturated aliphatic dicarboxylic acids having 2 to 9 carbon atoms. Acids (eg, maleic acid, fumaric acid, citraconic acid, mesaconic acid, etc.); Examples of the aliphatic tricarboxylic acid include a saturated aliphatic tricarboxylic acid having 2 to 9 carbon atoms (eg, tricarballylic acid, 1,2,3-butanetricarboxylic acid, and the like). The above-mentioned aliphatic carboxylic acid is
It may have one or two hydroxyl groups. Examples of such a group include glycolic acid, lactic acid, glyceric acid, tartronic acid, malic acid, tartaric acid, citric acid and the like. The aliphatic carboxylic acid is preferably an aliphatic monocarboxylic acid. The aliphatic carboxylic acid is more preferably an aliphatic monocarboxylic acid having 2 to 9 carbon atoms, particularly preferably a saturated aliphatic monocarboxylic acid having 2 or 3 carbon atoms. Particularly preferred specific examples of the aliphatic carboxylic acid include:
For example, acetic acid and the like can be mentioned. Further, as the aromatic carboxylic acid used as the organic acid, for example, benzoic acid,
Salicylic acid and the like are preferable, and benzoic acid is preferable.

More specifically, the GH / zinc complex of the present invention comprises, for example, an aqueous solvent (for example, about GH and a water-soluble zinc salt that does not affect the solubility of GH and the water-soluble zinc salt (for example, about GH and zinc in an aqueous solution which may contain 1 to 10% (w / w) ethanol or acetone, preferably water, etc.) from about 1: 1.6 to about 1: 2.4,
Preferably, it is produced by mixing so as to have a mixing ratio (molar ratio) of about 1: 1.8 to about 1: 2.2. The complex may be a compound (complex salt, double salt, salt, organometallic compound, or the like) formed by the bonding between GH and zinc molecules, or a mixture of compounds having different bonding modes. The composition ratio (molar ratio) of GH to zinc is in the range of about 1: 1.6 to about 1: 2.4, preferably about 1: 1.8 to about 1: 2.2, more preferably about 1: 1.2. 2 is better. In the GH / zinc composite of the present invention, it is preferable that all of GH and zinc contained in a molar ratio of about 1: 1.6 to about 1: 2.4 form a composite. , GH and / or zinc may be included without forming a complex. PH of aqueous solution at the time of mixing
PH is adopted which does not impair the biological activity of GH and does not significantly reduce the solubility of each of GH and zinc salt. The mixing operation is usually performed in distilled water, but may be performed in an aqueous solution adjusted to weak acidity, neutrality or weak alkalinity (pH 6 to 9) as necessary. Also GH
The concentration of the water-soluble zinc salt used in water may be any as long as it is within a single solubility range. The aqueous GH / zinc composite solution obtained in this manner is substantially water-soluble with almost no precipitation. GH of the present invention
The fact that the zinc complex is water-soluble means that, for example, water (p
H6 to 8) The complex may be dissolved in 1 ml or more in an amount of about 2 mg or more. The GH / zinc complex aqueous solution is vacuum-dried or freeze-dried and powdered, and then used for the production of a pharmaceutical preparation, preferably a sustained-release preparation. GH obtained
The zinc complex powder is very low in bulk, easy to handle as compared to the bulky powder of zinc-free free GH, and is extremely advantageous in mass-producing a sustained-release preparation. Specifically, the GH / zinc composite can be a powder having an average particle size of about 10 μm or less, preferably about 4 to about 7 μm. When dispersed in an organic solvent solution in which a biodegradable polymer described later is dissolved, the particle size is small.
It is also very advantageous in improving the encapsulation rate of H and the sustained release. Specifically, in the sustained release preparation, it is possible to achieve a GH encapsulation rate of about 90% or more and an initial release rate of about 50% or less as a GH sustained release property.

In the sustained-release preparation of the present invention, the content of the GH / zinc complex is generally about 0.1 to about 40% (W /
W), preferably from about 1 to about 20% (W / W). Examples of the biodegradable polymer include high-molecular-weight polymers (polymers) that are hardly soluble or insoluble in water, such as aliphatic polyesters (eg, α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.), A homopolymer (homopolymer), a copolymer (copolymer), or a mixture thereof) synthesized from at least one of dicarboxylic acids (for example, malic acid and the like), hydroxytricarboxylic acids (for example, citric acid and the like), Poly-α
-Cyanoacrylates, polyamino acids (e.g.,
Poly-γ-benzyl-L-glutamic acid). These may be mixed and used at an appropriate ratio. The type of polymerization may be random, block, or graft. The biodegradable polymer is preferably an aliphatic polyester [eg, α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.), hydroxytricarboxylic acid (eg, A homopolymer, a copolymer, or a mixture thereof). Among the aliphatic polyesters described above, homopolymers and copolymers synthesized from one or more α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.) are preferable from the viewpoint of biodegradability and biocompatibility. The aliphatic polyester is particularly preferably a copolymer synthesized from one or more α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.). Further, these copolymers may be used as a mixture.

The α-hydroxycarboxylic acids are D-
Or D- and L-forms, but D-form / L-form (mol / mol%) is about 75/25 to about 25
/ 75 is preferred. The D-form / L-form (mol / mol%) is more preferably from about 60/40 to about 3
0/70. Examples of the copolymers of the α-hydroxycarboxylic acids include copolymers of glycolic acid and other α-hydroxy acids. Lactic acid and 2-hydroxybutyric acid are preferred as the α-hydroxy acids. α-
The copolymer of hydroxycarboxylic acids is preferably a lactic acid-glycolic acid copolymer, a 2-hydroxybutyric acid-glycolic acid copolymer, and particularly preferably a lactic acid-glycolic acid copolymer.
Glycolic acid copolymer and the like.

In a lactic acid-glycolic acid homopolymer or copolymer (both are collectively referred to simply as lactic acid-glycolic acid polymer or PLGA), the composition ratio (lactic acid / glycolic acid ratio, hereinafter abbreviated as L / G). (Mol / mol%) is about 100/0 to about 40 /
60 is preferred. The composition ratio is more preferably about 90 /
10 to about 45/55. The composition ratio is particularly preferably from about 80/20 to about 45/55. The weight average molecular weight of the lactic acid-glycolic acid polymer is from about 3,000 to about 20,000, preferably from about 3,000 to about 1
6,000, more preferably from about 6,000 to about 14,
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, it is from about 1.5 to about 3.5. The lactic acid-glycolic acid polymer can be synthesized according to a production method known per se, for example, a method described in JP-A-61-28521. The polymer is preferably synthesized by non-catalytic dehydration polycondensation. In the 2-hydroxybutyric acid-glycolic acid copolymer, it is preferred that the glycolic acid is from about 10 to about 75 mol%, with the balance being 2-hydroxybutyric acid. More preferably, the amount of glycolic acid is about 20 to about 75 mol%. Particularly preferred is when glycolic acid is from about 30 to about 70 mol%. The weight average molecular weight of the 2-hydroxybutyric acid-glycolic acid copolymer is from about 2,000 to about 20,000
0 is preferred. The degree of dispersion (weight average molecular weight / number average molecular weight) of the 2-hydroxybutyric acid-glycolic acid copolymer is preferably from about 1.2 to about 4.0. The degree of dispersion is particularly preferably from about 1.5 to about 3.5. The 2-hydroxybutyric acid-glycolic acid copolymer can be synthesized according to a known production method, for example, the methods described in JP-A-61-28521 and JP-A-5-112465. The copolymer is preferably synthesized by non-catalytic dehydration polycondensation. Preferred examples of the homopolymer of the α-hydroxycarboxylic acids include a homopolymer of lactic acid (polylactic acid). The weight average molecular weight of polylactic acid is about 3,000 to about 20,000
00, preferably about 3,000 to about 14,000. Polylactic acid can be produced by a method known per se, for example,
Can be synthesized according to the method described in JP-A-285521.
The homopolymer is preferably synthesized by non-catalytic dehydration polycondensation.

The above-mentioned 2-hydroxybutyric acid-glycolic acid copolymer may be further used by mixing with polylactic acid. The polylactic acid may be any of D-form, L-form and a mixture thereof, but those having D-form / L-form (mol / mol%) in the range of about 75/25 to about 20/80. Is preferred. The D-form / L-form (mol / mol%) is more preferably from about 60/40 to about 25/75. D-
Isomer / L-isomer (mol / mol%) is particularly preferably about 55
/ 45 to about 25/75. The weight average molecular weight of the polylactic acid is about 1,500 to about 20,000, preferably about 1,500 to about 10,000. The polylactic acid preferably has a degree of dispersion of about 1.2 to about 4.0. The degree of dispersion is particularly preferably from about 1.5 to about 3.5.
As a method for producing polylactic acid, a method of ring-opening polymerization of lactide, which is a dimer of lactic acid, and a method of dehydrating and polycondensing lactic acid are known. In order to obtain polylactic acid having a relatively low molecular weight used in the present invention, a method of directly dehydrating and polycondensing lactic acid is preferred. This method is described in, for example, JP-A-61-28521. When using a mixture of 2-hydroxybutyric acid-glycolic acid copolymer and polylactic acid,
The mixing ratio is, for example, about 10/90 to about 90/10
(% By weight). The mixing ratio is preferably about 20/80
Or about 80/20. The mixing ratio is more preferably from about 30/70 to about 70/30.

In the present specification, the weight-average molecular weight means that the weight-average molecular weight is 120,000, 52,000, 22,000.
0, 9,200, 5,050, 2,950, 1,050,5
It refers to the molecular weight in terms of polystyrene measured by gel permeation chromatography (GPC) using nine kinds of polystyrenes of 80 and 162 as reference substances. The number average molecular weight is also calculated by GPC measurement. The degree of dispersion is calculated from the weight average molecular weight and the number average molecular weight. GPC measurement is G
A PC column KF804L × 2 (manufactured by Showa Denko) and an RI monitor L-3300 (manufactured by Hitachi, Ltd.) are used, and chloroform is used as a mobile phase.

The copolymer synthesized by the above-mentioned noncatalytic dehydration polycondensation generally has a free carboxyl group at a terminal. In the present invention, the biodegradable polymer preferably has a free carboxyl group at a terminal. A biodegradable polymer having a free carboxyl group at the terminal is
It is a biodegradable polymer in which the number average molecular weight measured by GPC and the number average molecular weight determined by terminal group quantification are almost the same. The number average molecular weight by terminal group quantification is calculated as follows. About 1 to 3 g of a biodegradable polymer is dissolved in a mixed solvent of acetone (25 ml) and methanol (5 ml), and phenolphthalein is used as an indicator to make the carboxyl group in this solution 0.05N alcoholic potassium hydroxide solution. The mixture is rapidly titrated under stirring at room temperature, and the number average molecular weight by terminal group quantification is calculated by the following formula. Number average molecular weight by terminal group determination = 20,000 A / B A: mass of biodegradable polymer (g) B: 0.05N alcoholic potassium hydroxide solution added by the end of titration (ml) For example, one type In a polymer synthesized from the above α-hydroxy 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 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 GPC.
It greatly exceeds the number average molecular weight measured. 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.

While the number average molecular weight determined by terminal group quantification is an absolute value, the number average molecular weight determined by GPC is determined by various analysis and analysis conditions (eg, selection of mobile phase type, column type, reference substance, slice width, Since it is a relative value that fluctuates depending on the selection of the baseline, etc., it is difficult to make a clear numerical value. For example, it is considered that the number average molecular weight obtained by GPC measurement and the number average molecular weight obtained by terminal group quantification are almost the same. It means that the number average molecular weight by quantification is in the range of about 0.5 to about 2 times the number average molecular weight by GPC measurement. Preferably, the number average molecular weight determined by terminal group determination is about 0.8 to about 1.5 of the number average molecular weight determined by GPC measurement.
It is within the range of double. Further, the expression that the number average molecular weight determined by the terminal group determination greatly exceeds the number average molecular weight determined by the GPC measurement means that the number average molecular weight determined by the terminal group determination exceeds about twice the number average molecular weight determined by the GPC measurement. Further, as the biodegradable polymer of the present invention, a metal salt of the above-described biodegradable polymer is also preferably used. For example,
Polyvalent metal salts of various biodegradable polymers described in WO 97/01331, preferably divalent metal salts, particularly zinc salts of lactic acid-glycolic acid copolymer are preferably used. These biodegradable polymers are described in WO 97/0
It can be produced by the method described in JP-A-1331 and a modified method according to the method. When the polyvalent metal salt of the biodegradable polymer is zinc, 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 made to coexist in an organic solvent to produce a biodegradable polymer / zinc oxide complex in an organic solvent. At this time, the concentration of the biodegradable polymer in the solution varies depending on the molecular weight, the type of 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). The amount of zinc oxide to be added varies depending on the type of the organic solvent.
001 to about 2% (W / W), preferably about 0.01
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.

The conditions for the method of producing a solution of a biodegradable polymer / zinc oxide complex such as PLGA / zinc oxide complex from the biodegradable polymer and zinc oxide are determined by the type of the biodegradable polymer used. The particle size of zinc oxide, the type of organic solvent, and the composition thereof are appropriately changed,
For example, when using PLGA as the polymer, it is usually at about 0 to about 30 ° C., preferably about 2 to about 25 ° C., for about 1 to about 168 hours, preferably about 12 to about 96 hours.
By reacting for a period of time, more preferably for about 24 to about 48 hours, a PLGA / zinc oxide complex can be obtained. Here, the generation of the PLGA / zinc oxide complex is as follows:
At the time of addition, zinc oxide in a suspended state is dissolved in an organic solvent, and can be visually confirmed by being in a clear solution state, and is not limited to the above range. The reaction time may be determined as an index. Although the reaction proceeds by simply allowing PLGA and zinc oxide to coexist in an organic solvent, it is advantageous to carry out the reaction under appropriate stirring and shaking means under stirring and shaking in terms of shortening the reaction time. is there. It is also preferable to carry out the reaction under ultrasonic irradiation. In the present invention, the obtained biodegradable polymer / zinc oxide composite is preferably used in the next step in the form of an organic solvent solution. However, if desired, the organic solvent may be removed and the compound may be once solidified.

The sustained-release preparation of the present invention comprises a GH / zinc complex (preferably powder) containing GH and zinc in a molar ratio of about 1: 1.6 to about 1: 2.4, which is in vivo. A dispersion liquid (preferably, an oil phase) in which a degradable polymer (hereinafter, abbreviated as “biodegradable polymer” including a case where the biodegradable polymer is a metal salt) is dissolved. S /
O-type dispersion) is produced by removing the solvent. Examples of the production method include an in-water drying method, a phase separation method, a spray drying method, a method analogous thereto, and the like. Hereinafter, a case where a microcapsule is produced as a sustained-release preparation will be described. (A) Underwater drying method (S / O / W method): In this method, first, an organic solvent solution of a biodegradable polymer is prepared. The organic solvent used when producing the sustained-release preparation of the present invention has a boiling point of 120 ° C.
The following is preferred. Examples of the organic solvent include halogenated hydrocarbons (eg, dichloromethane, chloroform, carbon tetrachloride), alcohols (eg, ethanol, methanol), and acetonitrile. These may be mixed and used at an appropriate ratio. The organic solvent is preferably dichloromethane, acetonitrile,
Particularly preferably, dichloromethane is used. The concentration of the biodegradable polymer in the organic solvent solution varies depending on the molecular weight of the biodegradable polymer, the type of the organic solvent, and the like, but is generally selected from about 0.01 to about 80% (W / W). It is. More preferably, about 0.1 to about 70%
(W / W), particularly preferably about 1 to about 60% (W / W).
W).

The GH / zinc complex is added and dispersed in the organic solvent solution (oil phase) of the biodegradable polymer thus obtained. At this time, the amount of the GH / zinc composite added is such that the upper limit of the weight ratio of the GH / zinc composite to the biodegradable polymer is up to about 0.4, preferably up to about 0.2. Further, as a dispersion method, it may be added as a GH / zinc composite powder and uniformly dispersed.
The freeze-dried mass of the GH / zinc composite may be directly added, and may be uniformly dispersed by grinding and mixing in the oil phase.
Next, the organic solvent dispersion (S /
O-type dispersion) is further added to an aqueous solvent (aqueous phase), and S / O /
A W-type emulsion is formed. 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, from about 5 to about 2,000
Selected from double. Particularly preferably, about 10 to about 1.0
Selected from 00 times. An emulsifier may be added to the outer aqueous phase. As the emulsifier, generally stable S / O /
Any material can be used as long as it can form a W-type 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 outer aqueous phase is preferably from about 0.001 to about 20% (W / V). More preferably, it is about 0.01 to about 10% (W / V), particularly preferably about 0.05 to about 5% (W / V).

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. 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 about 10 to about 20 ° C. per minute using a differential scanning calorimeter.

(B) Phase separation method (coacervation method) In this method, a coacervation agent is gradually added to the S / O type dispersion under stirring to precipitate microcapsules.
Let it solidify. The coacervation agent is added in an amount of about 0.01 to about 1,000 times the volume of the dispersion.
More preferably, the volume is about 0.05 to about 500 times the volume. Particularly preferred is a volume of about 0.1 to about 200 times. 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 and the like are used. These may be used as a mixture of two or more. After the microcapsules thus obtained are collected by filtration, they are repeatedly washed with heptane or the like to remove the coacervation agent. Further, it is washed and freeze-dried in the same manner as in the above (a). 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 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 is sprayed into a drying chamber of a spray drier (spray drier) using a nozzle, and the atomized droplets are dispersed in a very short time. The organic solvent is volatilized to produce microcapsules. As the nozzle,
For example, there are a two-fluid nozzle type, a pressure nozzle type, a rotating disk type and the like. At this time, if desired, simultaneously with the above dispersion,
It is also effective to spray an aqueous solution of the anticoagulant from another nozzle for the purpose of preventing the microcapsule particles from coagulating. The microcapsules thus obtained are:
Washing is performed in the same manner as in (a), and if necessary, heating (under reduced pressure if necessary) further removes water and the organic solvent.

The sustained-release preparation used in 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 about 300 μm, preferably about 1 to about 150 μm, particularly preferably about 2 to about 1 as an average particle diameter.
00 μm. In the present specification, a microcapsule may be a fine particle containing a drug (GH / zinc complex) and a microcapsule base (biodegradable polymer) (also referred to as a microsphere). For example, microcapsules containing one drug core in one particle and polynuclear microcapsules containing many drug cores in one particle can be mentioned.

The sustained-release preparation of the present invention is formulated into various dosage forms, for example, as a microcapsule or using the microcapsule as a raw material, and is used as a parenteral preparation (for example, an injection for intramuscular, subcutaneous, injection into organs or the like). 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.). In the present invention, the sustained-release preparation is particularly preferably for injection. For example, when the sustained-release preparation is a microcapsule, the microcapsule may be used as a dispersant (for example, Tween 80 (Bio-Rad), H
Surfactants such as CO-60 (Nikko Chemicals), carboxymethyl cellulose, polysaccharides such as sodium alginate, hyaluronic acid, etc., preservatives (eg, methyl paraben, propyl paraben, etc.), isotonic agents (eg, sodium chloride, Mannitol, sorbitol, glucose, etc.)
A practical sustained-release preparation for injection can be obtained by preparing an aqueous suspension together with the above. Also, vegetable oils such as sesame oil and corn oil or mixtures thereof with phospholipids such as lecithin,
Alternatively, it is dispersed together with a medium-chain fatty acid triglyceride (for example, Miglyol 812 (Huls. AG)) to give a sustained-release injection which can be actually used as an oily suspension.

When the sustained-release preparation is a microcapsule, for example, the particle size of the microcapsule may be within a range satisfying 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 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 of the present invention has low toxicity and can be used in mammals (eg, humans, cows, pigs, dogs, cats, mice, rats,
Rabbits) can be used safely. The sustained-release preparation of the present invention includes growth hormone secretion deficiency, Turner's syndrome, pituitary dwarfism, chronic kidney disease, cartilage dystrophy, adult pituitary deficiency, Down's syndrome, Silver's syndrome, osteogenesis imperfecta Is effective in treating or preventing osteoporosis, osteoporosis, juvenile chronic arthropathy, and congestive heart failure. The dose of the sustained-release preparation varies depending on the duration of release, the target disease, the target animal, and the like, but may be any amount that maintains the effective concentration of GH in the body. For example, when a two-week-type preparation of a human growth hormone sustained-release preparation is administered to a patient with pituitary dwarfism, about 0.01 to about 5 mg is usually used as an active ingredient.
/ Kg body weight, preferably from about 0.03 to about 1 mg / k
The dose is preferably selected from the range of g body weight and administered once every two weeks. Although the GH / zinc complex of the present invention has low toxicity and may be in neat form, it is generally used as a carrier for pharmaceutical preparations, for example, excipients (eg, calcium carbonate, kaolin, sodium hydrogencarbonate, lactose, soluble starch). , Corn starch, crystalline cellulose, talc, granulated sugar, porous substances, etc.), binders (eg, dextrin, gums, pregelatinized starch, gelatin, hydroxypropylcellulose,
Hydroxypropyl methylcellulose, pullulan, etc.),
Thickeners (eg, natural gums, cellulose derivatives, etc.),
Disintegrants (eg, carboxymethylcellulose calcium, croscarmellose sodium, crospovidone,
Low-substituted hydroxypropylcellulose, partially pregelatinized starch, etc.), solvents (eg, water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, sesame oil, corn oil, etc.), dispersants (eg, Tween)
80, HCO60, carboxymethylcellulose, sodium alginate, etc.), suspending agents (eg, sodium lauryl sulfate, benzalkonium chloride, etc.), solubilizing agents (eg, polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, Ethanol, trisaminomethane, triethanolamine, sodium carbonate, sodium citrate, etc.), soothing agent (eg, benzyl alcohol, etc.), buffering agent (eg, phosphate, acetate, carbonate, citrate, etc.) Lubricants (eg, magnesium stearate, calcium stearate, talc, starch, sodium benzoate, etc.), coloring agents (eg, tar dye, caramel, iron sesquioxide, titanium oxide, riboflavins, etc.), flavoring agents (eg, , Sweets,
Perfumes), stabilizers (eg, sodium sulfite, ascorbic acid, etc.) and preservatives (eg, parabens, sorbic acid, etc.) are used in appropriate amounts and administered as pharmaceutical preparations prepared according to conventional methods. You can also. The pharmaceutical preparation of the present invention, which may contain the carrier for a pharmaceutical preparation, appropriately controls an effective amount of the GH / zinc complex of the present invention to prevent or treat the disease similarly to the sustained-release preparation. contains. The content of the GH / zinc complex of the present invention in a pharmaceutical preparation is usually about 0.1 to about 100% by weight of the whole pharmaceutical preparation. The sustained-release preparation is stored at room temperature or in a cold place, preferably in a cold place. The normal temperature or cold place here is defined in the Japanese Pharmacopoeia. That is, room temperature means 15 ° C to 25 ° C, and cold place means 15 ° C or less.

[0026]

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 amino acid is used, IUPA
C-IUB Commission on Bioche
This is based on the abbreviations of medical Nomenclature or commonly used abbreviations in the art, 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 G: glutamic acid G: glutamic acid Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asx: Asp + AsnGlx: Glu + Gln

[0027]

EXAMPLES Reference Example 1 Construction of Human Growth Hormone (GH) Expression Vector Using T7 Promoter The GH structural gene was obtained from plasmid pHGH107 (described in Japanese Patent Publication No. 6-12996, accession numbers ATCC 31538 and ATCC 40011) from EcoRI and EcoRV.
And a fragment of about 0.75 kb was isolated. On the other hand, pE
T-3C [Rosenberg et al., Gene, 56, 125 (1987)]
After digestion with deI and BamHI, an approximately 4.6 kb fragment containing the T7 promoter and the ampicillin resistance gene was isolated. Both fragments were converted to T4 DNA polymerase (DNA
After blunting with Blunting kit (Takara Shuzo Co., Ltd.) and ligation with T4 DNA ligase, E. coli JM109 was introduced, and transformants were selected using ampicillin resistance as an index. From the colonies that appeared, 12 were picked up, a plasmid was prepared therefrom, and the restriction enzyme Pst
Examination of the cleavage pattern by I revealed that the GH gene was inserted in the correct direction in plasmids from six colonies. The plasmid obtained from one of these strains was named pTGA201.

Reference Example 2 Expression of Met-GH in Escherichia coli Escherichia coli JM109 was lysogenized with lambda phage (Studier, Supra) recombined with the T7 phage RNA polymerase gene. Thereafter, the GH expression vector pTGA201 obtained in Reference Example 1 was
M109 (DE3) and E. coli JM109 (DE
3) / pTGA201 was obtained. The transformed cells are
LB medium containing 0 μg / ml ampicillin (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) 1
In a 2 liter flask containing 30 liters, 30 ° C., 16
The cells were cultured with shaking for a time. The obtained culture was transferred to a 50-liter fermenter containing 20 liters of LB medium containing 0.02% Newpole LB-625 (antifoaming agent; manufactured by Sanyo Chemical Industries) and 50 μg / ml ampicillin. hand,
The cells were cultured with aeration and stirring at 37 ° C. for 6 hours. This culture solution is used for 360
One liter 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% magnesium sulfate, 0.02% % New Paul LB-62
5, 0.0005% thiamine hydrochloride, 1.5% glucose,
The mixture was transferred to a 500-liter fermenter charged with 1.5% casamino acid), and aeration and stirring 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-mentioned transformed Escherichia coli JM109 (DE3) / pTGA201 was deposited with the National Institute of Advanced Industrial Science and Technology (NIBH) under the accession number FERM BP-5632,
It has been deposited with the Fermentation Research Institute (IFO) under the accession number IFO 16001.

REFERENCE EXAMPLE 3 Activation of Met-GH 50 kg of Tris / HC was added to 2 kg of the cells obtained in Reference Example 2.
l, 8 L of guanidine hydrochloride solution (pH 8.0) was added to dissolve the cells, followed by centrifugation (10000 rpm).
m, 120 minutes). To 6 liters of the obtained supernatant, 50 mM Tris / HCl, 0.28 mM GSSG, 1.
After adjusting the pH to 8.0 by adding 18 liters of 4 mM GSH and 0.7 M arginine (pH 8.0), activation was performed at 4 ° C. for 5 days.

Reference Example 4 Purification of Met-GH The regenerated solution which had been activated in Reference Example 3 was treated with a Pellicon cassette system (PTGC membrane, Millipore) in 20 mM Tris / HCl, 2.5 M urea (pH 8.0). After addition, desalting and concentration were performed until the electric conductivity became 10 mS or less, and the obtained concentrated liquid was centrifuged (10000 rpm, 6 rpm).
0 minutes) to obtain 5 liters of the supernatant of the concentrated solution. This solution was then added to 20 mM Tris / HCl, 2.5 M urea (pH
8.0) and adsorbed on a DEAE-Toyopearl 650M column (20 cmφ × 84 cm, Tosoh Corporation) equilibrated in
After extensive washing, 0-25% B (B = 20 mM Tris / HCl, 2.5 M urea, 1 M sodium chloride, p
Elution was performed at a flow rate of 300 ml / min for 100 minutes with a concentration gradient of H8.0) to obtain 10 L of an eluate as a Met-GH fraction. Further, the eluate was concentrated and desalted using a Pellicon cassette system (PTGC membrane, Millipore). 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] +2.5 M urea (p
H4.0) with a pH gradient of 70-85% B,
Elution was performed at a flow rate of 320 ml / min for 70 minutes.
6 liters of the GH fraction were obtained. 300 ml of a 2M Tris / HCl (pH 7.8) solution was added to the eluate to obtain a pH 7.2.
And then the Pellicon cassette system (PTGC
The solution was concentrated and desalted using a membrane (Millipore) to obtain 9,979 mg of Met-GH.

Reference Example 5 Removal of N-terminal Met 2.5 was added to 1650 ml of the Met-GH solution obtained in Reference Example 4.
413 ml of M glyoxylic acid, 35 mM copper sulfate, and 6 M pyridine solution were added, and the mixture was stirred well and reacted at 25 ° C. for 60 minutes. Then Sephadex G-25 equilibrated with 20 mM Tris / HCl, 2.5 M urea (pH 8.0)
The solution was passed through a column (11.3 cmφ × 125 cm, Pharmacia) at a flow rate of 3 L / h, developed using the same buffer solution as for the equilibration, and the eluted Met-GH diketone fraction was thoroughly stirred. 4M acetic acid, 4M sodium acetate, 80 mMo-phenylenediamine, 3M
Added in 4 liters of urea solution. After the elution was completed, 8 liters of the reaction solution was allowed to stand at 4 ° C. for 3 days. After settling, 4 times with Pellicon cassette system (PTGC membrane, Millipore)
Concentrated to 20 liters, 20 mM Tris / HCl, 2.5 M
Sephadex G-2 equilibrated with urea (pH 8.0)
The solution was passed through 5 columns (11.3 cmφ × 140 cm, Pharmacia) at a flow rate of 3 L / h, and the GH fraction was 4.7.
Collected liters. Further, this solution was applied to a DEAE-5PW column (21 cm × 30 cm) by high performance liquid chromatography (Gilson HPLC system, Gilson).
A, 50 mM Tris / HCl + 2.5 M urea (pH 8.0), B = 50 m
MMES [2- (N-morpholino) ethanesulfonic acid]
70-85% with +2.5 M urea (pH 4.0)
Elution was performed at a flow rate of 320 ml / min for 70 minutes with a pH gradient of B to obtain 10 liters of a GH fraction. 500 ml of a 2M Tris / HCl (pH 7.8) solution is added to the GH fraction.
In addition, after adjusting to pH 7.2, Minitan II (PTGC membrane,
(Millipore Co.) and a Sephacryl S-100 column (113.
cmφ × 50cm, Pharmacia) 2 liters / h
The solution was passed through and developed at a flow rate of, and a GH fraction of 1651 ml was obtained. Further, this solution was filtered through Millipak 60 (Millipore) to obtain 1487 ml (3309 mg of GH) of a GH solution.

Reference Example 6 Confirmation of GH properties (a) Analysis using SDS polyacrylamide gel electrophoresis Sample buffer containing 100 mM DTT in GH obtained in Reference Example 5 [Laemmli, Nature, 2
27 , 680 (1970)], stirred well, heated at 95 ° C. for 2 minutes, and electrophoresed on Multigel 10/20 (Daiichi Pure Chemicals). The gel after the electrophoresis is subjected to Coomassie Brilliant Blue (Coomassie).
brilliant blue), about 2
A single band was observed at 2 kd, confirming that the purified 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 GH was consistent with the amino acid composition deduced from the nucleotide sequence of the cDNA (Table 1).

Table 1 Base sequence of GH per 1 mole of GH Amino acid residues Number predicted from the number of residues 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 GH matched the N-terminal amino acid sequence of GH deduced from the nucleotide sequence of cDNA (Table 2).

Table 2 Base Sequence of GH Detected Residue No. __________________________________________________ PTH ¹⁾ -amino acid 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 ) Phe 11 Asp (77) Asp 12 Asn (230) Asn 13 Ala (435) Ala 14 Met (334) Met 15 Leu (398) Leu 16 Arg (67) Arg 17 Ala (488) Ala 18 His 19 Arg (42) Arg 20 Leu (406) Leu -------- -------------------- 1) was analyzed using a phenylthiohydantoin 1 nmol.

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

[Table 3] Gas phase hydrazine decomposition method (100 ° C, 6 hours) 18 nmo
The analysis was performed using l. (E) Measurement of GH activity The growth promoting effect of the purified GH obtained in Reference Example 5 on Nb2 cells [Journal of Clinical Endocrinology and Metabolism, vol. 51, p. 1058 (1980)] is standard. Products (Chemicon International, Temecula, California, US)
It was equivalent to A).

Example 1 (1) Production of GH / Zinc Complex In a 120 ml aqueous recombinant GH solution (2 mg / ml) obtained according to Reference Example 5, the molar ratio of zinc to 1 mol of GH was 1.8. Then, 0.5 ml of a zinc acetate aqueous solution having two concentrations so as to obtain a GH / zinc complex (about 230 mg) was obtained. (2) Production of GH-containing microcapsules Lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 5)
0/50, polystyrene equivalent average molecular weight = 12,000,
(Viscosity = 0.145 dl / g) 1.89 g was dissolved in dichloromethane 4 ml, and zinc oxide 10 mg was added.
The mixture was stirred (60 rpm) at 5 ° C. and completely dissolved. 100 mg of the GH / zinc composite obtained in the above (1) was added to an organic solvent solution of this polymer, and the mixture was pulverized with a polytron (Kinematica). Next, the obtained 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 drying in water at room temperature for 2 hours, GH-containing microcapsules (1.07 g (1: 1.8), 0.9) were washed with distilled water and freeze-dried.
1 g (1: 2.0)) was obtained.

COMPARATIVE EXAMPLE 1 (1) Production of GH Powder In a 120 ml aqueous recombinant GH solution (2 mg / ml) prepared according to Reference Example 5, the molar ratio of zinc to 1 mol of GH was 0.3. 0.5 ml of various concentrations of zinc acetate aqueous solution were added to each of the solutions so that the concentrations became 0, 4.0, 5.0, and 6.0, and lyophilized to obtain GH powder (about 230 mg). (2) Production of GH-containing microcapsules The same operation as in Example 1- (2) was carried out to produce G
GH-containing microcapsules (0.95 g)
(1: 0), 1.23 g (1: 3), 1.13 g (1:
4) 1.28 g (1: 5), 1.27 g (1: 6)) were obtained.

Test Example 1 The following experiment was performed using the microcapsules produced in Example 1- (2) and Comparative Example 1- (2). (1) GH content 300 μl of acetonitrile was added to 4 mg of the microcapsules produced in Example 1- (2) and Comparative example 1- (2), and the lactic acid-glycolic acid copolymer as a base was dissolved. 700 μl of an aqueous solution containing .02% bovine serum albumin-0.05% trifluoroacetic acid was added and stirred to extract GH. The supernatant obtained by centrifugation was subjected to high performance liquid chromatography to determine the GH content in the microcapsules. The GH encapsulation rate is a GH theoretically calculated from the GH content to which the GH content in the obtained microcapsules is added.
It was determined by dividing by the content. [Table 4] shows the results. From the results shown in Table 4, the molar ratio was 1.8 relative to 1 mole of GH.
Alternatively, when the composite of the present invention containing 2.0 zinc was used, a GH encapsulation rate of 90% or more was obtained. From this, it is clear that the sustained-release preparation containing the GH / zinc complex of the present invention has an excellent GH encapsulation rate. (2) In Vivo Release The microcapsules prepared in Example 1- (2) and Comparative Example 1- (2) were subcutaneously administered to 6 mg GH / rat to immunosuppressed SD rats (male, 6 weeks old), followed by aging. And the serum GH concentration was measured with a radioimmunoassay kit (Ab beads HGH: Eiken Chemical). Immunosuppression SD
Rats were treated with Prograf (Fujisawa Pharmaceutical) at 0.4 mg / rat 3 days before microcapsule administration and at the time of administration.
After 7, 11 and 14 days, 0.2 mg / rat was subcutaneously administered, respectively. Based on the AUC and clearance obtained from the change in serum GH concentration, the amount of GH released by 1 day after microcapsule administration and the amount of GH released by 1-18 days thereafter were calculated. The initial release rate was determined by dividing the amount of GH released up to one day after administration of the microcapsule by the amount of GH administered. [Table 4] shows the results.
From the results shown in Table 4, the microcapsules containing the complex in which the molar ratio of zinc to 1 mol of GH was 1.8 or 2.0 was the initial release amount (the release amount up to 1 day (24 hours) after administration). Was small (initial release rate: about 30% or less), and the subsequent release amount (one day after administration (the release amount up to 18 days after 24 hours) was large.) Production in Comparative Example 1- (2) All of the obtained microcapsules had a large initial release amount (initial release rate: about 50% or more), and hence the subsequent release amount was small, and a sufficient sustained release property was not obtained. It is clear that a sustained release formulation containing a zinc complex has excellent sustained release properties.

[Table 4]

Test Example 2 In 2 ml of the recombinant GH aqueous solution (2 mg / ml) obtained according to Reference Example 5, the molar ratio of zinc to 1 mol of GH was 0, 1.4, 1.6, 1.8. , 2.0, 2.2, 2.
50 μl of aqueous zinc acetate solutions of various concentrations were added so as to obtain 4, 3.0, 4.0, 5.0, and 6.0, and lyophilized.
The obtained freeze-dried powder was dispersed in 2 ml of dichloromethane, pulverized and atomized with a vortex mixer, and the particle size distribution was measured using a laser diffraction type particle size distribution analyzer (SALD2000A; Shimadzu). The results are shown in FIG. 1 (in the figure, the particle size (average particle size μm) is indicated by ●).
As shown in the results of FIG. 1, the GH / zinc composite containing zinc at a molar ratio of 1.6 to 2.4 with respect to 1 mol of GH exhibited an average particle diameter of 5 μm or less. From this, it is clear that the GH / zinc composite of the present invention is atomized.

Test Example 3 In 1 ml of the aqueous recombinant GH solution (2 mg / ml) obtained according to Reference Example 5, the molar ratio of zinc to 1 mol of GH was 0, 1.0, 2.0, 3.0. , 4.0, 5.0, 6.
25 μl of aqueous zinc acetate solutions of various concentrations were added so as to obtain 0, 7.0, and 8.0, and centrifuged at 12,000 rpm for 5 minutes. The obtained supernatant was filtered (0.45 μm).
m) After filtration, G was determined by high performance liquid chromatography.
The H concentration was quantified, and the insoluble complex formation rate (ratio (%) of GH contained in the insoluble complex to the total amount of GH added)
Was calculated. The results are shown in FIG. 2 (in the figure, the insoluble complex formation rate is indicated by ○). From the results shown in FIG. 2, when the molar ratio of zinc to 1 mol of GH is 2.0 or less, the coexisting insoluble component is 30% or less, and GH.
It is clear that the zinc complex is substantially water-soluble. From this, it is clear that the GH / zinc composite of the present invention is substantially water-soluble.

[0041]

Industrial Applicability According to the present invention, a human growth hormone sustained-release preparation exhibiting a stable and sustained release with an increased rate of encapsulation of human growth hormone and a suppressed initial release can be obtained.

[Brief description of the drawings]

FIG. 1 shows a change in particle size distribution with a change in the composition ratio of GH and zinc in a GH / zinc composite.

FIG. 2 shows a change in the water solubility of the GH / zinc composite with a change in the composition ratio of GH and zinc in the composite.

Claims (19)

[Claims] 1. The method of claim 1, wherein human growth hormone and zinc are present in an amount of about 1: 1.6.
A human growth hormone / zinc complex containing a molar ratio of from about 1: 2.4. 2. The human growth hormone / zinc complex according to claim 1, which is water-soluble. 3. The human growth hormone / zinc complex according to claim 1, which has an average particle size of about 10 μm or less. 4. A sustained-release preparation comprising the human growth hormone / zinc complex according to claim 1 and a biodegradable polymer. 5. The sustained-release preparation according to claim 4, wherein the biodegradable polymer is an aliphatic polyester. 6. The sustained-release preparation according to claim 5, wherein the aliphatic polyester is a lactic acid / glycolic acid polymer. 7. The composition ratio (mol%) of the lactic acid / glycolic acid polymer is from 100/0 to about 40/60.
The sustained-release preparation according to the above. 8. The sustained-release preparation according to claim 5, wherein the aliphatic polyester has a weight average molecular weight of about 3,000 to about 20,000. 9. The sustained-release preparation according to claim 5, wherein the aliphatic polyester is a salt with a polyvalent metal. 10. The sustained-release preparation according to claim 9, wherein the polyvalent metal is zinc. 11. The sustained-release preparation according to claim 4, which is a microcapsule. 12. The sustained-release preparation according to claim 11, wherein the microcapsules are for injection. 13. An initial release rate of human growth hormone of about 50.
% Or less. 14. The method according to claim 1, wherein human growth hormone and zinc salt are mixed at about 1:
A method for producing a human growth hormone / zinc complex, comprising mixing at a molar ratio of 1.6 to about 1: 2.4. 15. The method of claim 1 wherein human growth hormone and zinc are present in an amount of about 1: 1.
A method for producing micronized human growth hormone, comprising forming and grinding a human growth hormone / zinc complex containing a molar ratio of 6 to about 1: 2.4. 16. The method of claim 1 wherein human growth hormone and zinc are present in an amount of about 1: 1.
Use of a human growth hormone / zinc complex containing a molar ratio of 6 to about 1: 2.4 for the manufacture of a sustained release preparation containing human growth hormone. 17. The method of claim 1 wherein human growth hormone and zinc are present in an amount of about 1: 1.
An S / O dispersion obtained by dispersing a human growth hormone / zinc complex containing a molar ratio of 6 to about 1: 2.4 in an oil phase containing a biodegradable polymer is added to an aqueous phase. And S /
A method for producing a sustained-release preparation, which is dried in water as an O / W emulsion. 18. A pharmaceutical preparation comprising the human growth hormone / zinc complex according to claim 1. (19) A prophylactic / therapeutic agent for pituitary dwarfism, comprising the human growth hormone / zinc complex according to (1).