RU2181999C2 - Sustained-release preparation, method of its preparing - Google Patents

Abstract

FIELD: medicine, pharmacy. SUBSTANCE: invention discloses the sustained-release preparation containing water-insoluble and difficultly soluble salt formed by polyvalent metal and physiologically active substance of water-soluble peptide type with exception for endothelin antagonist and biologically degradable polymer. Preparation is prepared by dispersion of peptide polyvalent metal salt in oily phase containing biodegradable polymer, emulsion added to an aqueous phase and drying. The sustained-release preparation shows ability to release physiologically active substance of water-soluble peptide type with exception for endothelin antagonist being at retention of its biological activity after its administration in vivo. Except for, physiologically active substance of water-soluble peptide with exception for endothelin antagonist as a component of the sustained-release preparation retains stability for a long time and loss of its biological activity is insignificant. EFFECT: improved method of preparing, valuable properties. 25 cl

Description

 The invention relates to a delayed release preparation containing a water-insoluble or slightly soluble salt of a polyvalent metal and a physiologically active substance such as a water-soluble peptide with the exception of an endothelin antagonist, and a biodegradable polymer.

BACKGROUND OF THE INVENTION
Physiologically active substances, in particular peptides or their derivatives, are known to possess various pharmacological functions in vivo. Some of these substances were produced for pharmaceutical use in large quantities by chemical synthesis methods or more modern methods of genetic and cell engineering using microorganisms such as Escherichia coli and yeast, as well as animal cells, such as hamsters. However, since these peptides have a short biological half-life, they must be administered frequently to achieve a therapeutic effect, which creates certain difficulties for injection into the patient. To solve this problem, various attempts have been made to develop drugs with a delayed release.

 The first attempt to solve this problem was to develop a delayed release formulation containing a water-soluble physiologically active substance, in particular a water-soluble peptide (hereinafter referred to as the "peptide"), and providing regulation of the solubility of the peptide, i.e. regulation of the rate of its release.

 International Patent Application 500286/1991 describes an insoluble zinc protamine-α-interferon complex.

 Japanese Patent Application Laid-Open No. 2930/1988 describes a system comprising a polylactide in which a macromolecular polypeptide is dispersed.

 Japanese Patent Applications Laid-open No. 221855/1993 and 172208/1994 describe a method by which a water-soluble peptide is converted into a water-insoluble peptide salt, followed by its suspension in an organic medium containing a biodegradable polymer to effectively incorporate the water-soluble peptide into small granules. The water-insoluble peptide described in these patent publications is an organic acid salt formed by the main part of a water-soluble peptide molecule, where pamoate, tannic acid, stearic acid or palmitate are used as an illustrative example.

 Although, as mentioned above, various attempts have been made to obtain drugs with a delayed release containing water-soluble physiologically active substances, however, these attempts have not led to satisfactory drugs with a delayed release, and therefore, the need remains to create a drug with a delayed release, which provides highly effective introduction of a water-soluble physiologically active substance, prevents the initial release of water-soluble physiologically active substance provides a constant rate of release of water-soluble physiologically active substance and contributes to maintaining the biological activity of the water soluble physiologically active substance.

Description of the invention
Thanks to intensive research aimed at solving the above problems, the inventors of the present invention found that a drug with a delayed release containing a peptide of the type of a physiologically active substance such as a water-soluble peptide with the exception of the endothelin antagonist, and providing highly efficient incorporation of this water-soluble peptide into a biodegradable polymer and very little drug release immediately after administration of this drug to the patient’s body, can be obtained by the formation of a water-insoluble salt of a polyvalent metal and a physiologically active substance such as a water-soluble peptide (with the exception of the endothelin antagonist) (hereinafter, this salt will be referred to as a “complex”), and this salt is formed as a result of the interaction of a physiologically active substance such as a water-soluble peptide (with the exception of an endothelin antagonist) having an acidic group or a water-soluble salt thereof (hereinafter also referred to as "physiologically active substance" substance "), with a water-soluble salt of a polyvalent metal; and by subsequently dispersing or dissolving the resulting complex in a biodegradable polymer. After further studies based on this discovery, the present invention has been developed.

In accordance with the foregoing, the present invention relates to:
(1) to a drug with a delayed release, which contains:
(a) a water-insoluble or slightly soluble salt of a polyvalent metal and a physiologically active substance such as a water-soluble peptide, with the exception of an endothelin antagonist, and
(b) a biodegradable polymer;
(2) the preparation described in paragraph (1), in which the physiologically active substance is a water-soluble peptide or its derivative;
(3) the preparation according to (2), wherein the peptide is a hormone, cytokine, hematopoietic factor, growth factor, enzyme, soluble or solubilized receptor, antibody, antigen-containing peptide, blood coagulation factor or adhesive molecule;
(4) the preparation according to (2), wherein the peptide is a hormone;
(5) the preparation according to (4), wherein the hormone is growth hormone;
(6) the preparation according to (3), wherein the hormone is insulin;
(7) the preparation according to (2), wherein the peptide is a cytokine;
(8) the preparation according to (7), wherein the cytokine is interferon;
(9) the preparation according to (2), wherein the peptide is a growth factor;
(10) the preparation according to (1), wherein the polyvalent metal salt is a transition metal salt;
(11) the preparation according to (1), wherein the polyvalent metal salt is a zinc salt;
(12) the preparation according to (1), wherein the solubility of the polyvalent metal salt in water is from about 0 to about 0.1% (w / w) at 20 ^° C;
(13) the preparation according to (1), wherein the solubility of the polyvalent metal salt in water is from about 0 to about 0.01% (w / w);
(14) a preparation according to claim (1), containing from about 0.1 to about 50% (w / w) of a polyvalent metal salt;
(15) a preparation according to (1), comprising from about 1 to about 30% (w / w) of a polyvalent metal salt;
(16) the preparation according to (1), wherein the biodegradable polymer is an aliphatic polyester;
(17) the preparation according to (16), wherein the aliphatic polyester is a polymer of lactic acid and glycolic acid;
(18) the preparation according to (17), wherein the ratio of lactic acid to glycolic acid is from 100/0 to about 40/60 (mol%);
(19) the preparation according to (18), wherein said ratio is from about 90/10 to about 45/55 (mol%);
(20) the preparation according to (17), wherein the average molecular weight of the polymer is from about 3,000 to about 20,000;
(21) the preparation according to (17), wherein the average molecular weight of the polymer is from about 3,000 to about 14,000;
(22) the preparation according to (16), wherein the aliphatic polyester is a homopolymer of lactic acid;
(23) the preparation according to (22), wherein the average molecular weight of the homopolymer is from about 3,000 to about 20,000;
(24) the preparation according to (22), wherein the average molecular weight of the homopolymer is from about 3,000 to about 14,000;
(25) the drug according to (1), wherein said drug is a microcapsule;
(26) the preparation according to (25), wherein said microcapsule is for injection;
(27) the preparation according to (1), which is injectable;
(28) the use of a water-insoluble or slightly soluble salt of a polyvalent metal and a physiologically active substance such as a water-soluble peptide (with the exception of the endothelin antagonist); and biodegradable polymer to obtain a drug with a delayed release and
(29) a method for producing a delayed release preparation comprising dispersing a water-insoluble or slightly soluble salt of a polyvalent metal and a physiologically active substance such as a water-soluble peptide (with the exception of an endothelin antagonist) in an oil phase containing a biodegradable polymer to obtain a solid-type emulsion oil, and then this emulsion is added to the aqueous phase to obtain a solid-oil-water emulsion, after which the resulting solid-mass emulsion layer water "drained by evaporation of water.

 It should be noted that the abbreviations of amino acids, peptides, etc. available in the present description were used in accordance with the biochemical nomenclature adopted by 1IRAC-1IV, or commonly used in this field; and the possible optical isomers of amino acids are L-isomers, unless otherwise indicated.

The physiologically active substance used in the formation of a water-insoluble or slightly soluble salt of a polyvalent metal is a physiologically active substance having an acid group. An example of such an acid group is a carboxyl group and a sulfo group. The physiologically active substance is preferably a physiologically active substance having a peptide bond or an amino acid and acid group. Such an acid group may be derived from an amino acid. A more preferred physiologically active substance is a water-soluble peptide having an acid group, or a derivative thereof. The solubility of the physiologically active substance in water is 1% (wt./wt.) Or more at 25 ^o C.

 A preferred physiologically active substance has two or more carboxyl groups.

 The molecular weight of the physiologically active substance is about 200-200000, preferably from about 200 to about 50,000, and more preferably from about 500 to about 40,000.

 A characteristic activity of a physiologically active substance is a hormonal effect. The physiologically active substance may be a natural, synthetic, semi-synthetic or genetically engineered product or its derivative. Moreover, by their mechanism of action, these physiologically active substances can be agonists or antagonists.

 Examples of physiologically active substances of the present invention, and in particular a water-soluble peptide or its derivative, are hormones, cytokines, hematopoietic factors, growth factors, enzymes, soluble or solubilized receptor, antibody or fragment thereof, antigen-containing peptide, blood coagulation factor, adhesive molecule, agonists or antagonists capable of binding to receptors of physiologically active substances, and the like.

 Examples of hormones are insulin, growth hormone, natriuretic peptide, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), human chorionic gonacotropin (HCG), etc. The preferred hormone is insulin and growth hormone.

 Examples of cytokines are lymphokines, monokines, and the like. Examples of lymphokines are interferons (alpha, beta and gamma interferon), interleukins (1L-2 - 1L-12), etc. Examples of monokines are interleukin 1 (1L-1), tumor necrosis factor, and the like. A preferred cytokine is lymphokine, and a more preferred cytokine is interferon (alpha, beta, gamma interferon).

 Examples of hematopoietic factors are erythropoietin, granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), thrombopoietin, platelet growth factor, megakaryocyte potentiator, etc.

 Examples of growth factors are basic or acidic fibroblast growth factors (FGF), as well as members of their family (e.g., FGF-9, etc.), nerve tissue growth factors (NGF), as well as members of their family, insulin-like growth factors ( for example, 1GF-1, 1GF-2), proteins involved in osteogenesis (BMP), or members of their family, etc.

 Examples of enzymes are superoxide dismutase (SOD), tissue plasminogen activator (TPA), and the like.

 Examples of soluble receptors are the soluble receptor 1L-6; protein binding to insulin-like growth factor (1GFBP); soluble tumor necrosis factor receptor (TNF); soluble epidermal growth factor receptor (EGF); soluble interleukin-1 receptor, etc.

 Examples of solubilized receptors are known receptors such as the interleukin-1 1L-1 receptor, the interleukin-6 1L-6 receptor, the tumor necrosis factor receptor (TNF) or the Fas ligand, and the like, where these receptors are solubilized by genetic engineering methods.

 Examples of antibodies are human monoclonal antibodies; a human-mouse chimeric monoclonal antibody in which a variable region derived from a murine antibody is connected to a constant region derived from a human antibody, or a fragment thereof, and the like. Such antibodies may be antibodies such as IgM, IgG, IgE, and the like. Examples of antigens recognized by the above antibodies are platelets, viruses, and the like.

 Examples of coagulation factors are factor VIII and the like.

 Examples of adhesive molecules are fibronectin, 1 SAM-1, and the like.

 In addition, endothelin is an example of physiologically active substances; Arg-Gly-Asp-Ser (RGDS); pituitary polypeptide activating adenylate cyclase (PACAP), etc.

 The physiologically active substance is converted into a water-insoluble or slightly soluble salt of a polyvalent metal by the interaction of the specified physiologically active substance with a water-soluble polyvalent metal.

Examples of such polyvalent metals are divalent, trivalent, tetravalent or the like. metals such as alkaline earth metals (e.g., calcium, magnesium, etc.), transition group metals (e.g., iron (II, III), copper (II), zinc (II), etc.), group metals III _c (for example, aluminum (II, III), etc.), Group IV _B metals (for example, tin (II, III), etc.), etc. Preferred polyvalent metals are alkaline earth metals or transition metals, more preferred are calcium or zinc, and zinc is most preferred.

 Water-soluble salts of polyvalent metals are salts formed by polyvalent metals and acids, for example, salts formed by polyvalent metals and inorganic acids, and salts formed by polyvalent metals and organic acids.

A preferred polyvalent metal acid salt is one which has a solubility in water at a normal temperature (20 ^{° C.} ) of not lower than about 20 mg / ml, more preferably not lower than about 100 mg / ml, and even more preferably not lower than approximately 200 mg / ml.

 Inorganic acids that can be used to produce a polyvalent metal salt are hydrochloric acid, sulfuric acid, nitric acid, thiocyanic acid, and the like.

 Organic acids that can be used to produce a polyvalent metal salt are aliphatic carboxylic acids and aromatic acids. Aliphatic carboxylic acids which have from 2 to 9 carbon atoms are preferred. Examples of aliphatic carboxylic acids are aliphatic monocarboxylic acids, aliphatic dicarboxylic acids, aliphatic tricarboxylic acids, and the like. These aliphatic carboxylic acids may be saturated or unsaturated.

 Examples of aliphatic monocarboxylic acids are saturated aliphatic monocarboxylic acids having 2-9 carbon atoms (e.g., acetic acid, propionic acid, butyric acid, valerianic acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, and the like. ); and unsaturated aliphatic monocarboxylic acids having 2-9 carbon atoms (for example, acrylic acid, propiolic acid, methacrylic acid, crotonic acid, isocrotonic acid, and the like).

 Examples of aliphatic dicarboxylic acids are saturated aliphatic dicarboxylic acids having 2-9 carbon atoms (for example, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, etc.); and unsaturated aliphatic dicarboxylic acids having 2-9 carbon atoms (for example, maleic acid, fumaric acid, citraconic acid, mesaconic acid and the like).

 Examples of aliphatic tricarboxylic acids are saturated aliphatic tricarboxylic acids having 2-9 carbon atoms (e.g., tricarballylic acid, 1,2,3-butanetricarboxylic acid, and the like).

 The aforementioned aliphatic carboxylic acids may have 1 or 2 hydroxyl groups. Such aliphatic carboxylic acids are glycolic acid, lactic acid, glyceric acid, tartronic acid, malic acid, tartaric acid, citric acid, and the like.

 Preferred aliphatic carboxylic acid is aliphatic monocarboxylic acid, more preferred is aliphatic monocarboxylic acid having 2 to 9 carbon atoms, and even more preferred is saturated aliphatic monocarboxylic acid having 2 or 3 carbon atoms. An example of particularly preferred aliphatic carboxylic acids is acetic acid and the like.

 Examples of aromatic acids are benzoic acid, salicylic acid and the like, with benzoic acid being most preferred.

 Examples of salts formed by polyvalent metals and inorganic acids, i.e., salts of a polyvalent metal and inorganic acid, are halides (e.g., zinc chloride, calcium chloride), sulfates, nitrates, thiocyanates, and the like.

 Examples of salts formed by polyvalent metals and aliphatic carboxylic acids, i.e. salts of aliphatic carboxylic acid and polyvalent metal are calcium acetate, zinc acetate, calcium propionate, zinc glycolate, calcium lactate, zinc lactate, zinc tartrate, and the like. Preferred salts of aliphatic carboxylic acid and polyvalent metal are calcium acetate and zinc acetate. Most preferred is zinc acetate and the like.

 Examples of salts formed by polyvalent metals and aromatic acids, i.e., salts of aromatic acid and polyvalent metal, are benzoates, salicylates, and the like. In this case, zinc benzoate is most preferred.

 A water-insoluble salt of a polyvalent metal and a physiologically active substance is obtained by mixing in a solvent a water-soluble physiologically active substance and a water-soluble salt of a polyvalent metal. The mixing procedure is preferably carried out in water.

 The concentration ratio (molar ratio) of the physiologically active substance and the water-soluble salt of the polyvalent metal in water is, for example, 1: 1-1: 1000, preferably 1: 1-1: 100, more preferably 1: 1-1: 50, and even more preferably 1: 1-1: 10. The exact concentrations of both components in water are not critical, provided that the solubility of the resulting complex does not go beyond the corresponding values.

 The pH of the aqueous solution obtained by the above mixing should be such that it does not adversely affect the biological activity of the physiologically active substance and that it does not contribute to an excessive decrease in the solubilities of the physiologically active substance and the water-soluble salt of the polyvalent metal. Although usually the mixing procedure is carried out in distilled water, however, if necessary, such mixing can be carried out in water with a slightly acidic, neutral or slightly alkaline pH.

Used in the present description, the term "insoluble or sparingly soluble in water" refers not only to irreversibly, but also to reversibly insoluble or sparingly soluble in water, and means that the solubility of this substance in water is very low. Typically, the solubility in water of such a substance is from about 0 to about 0.1% (wt./wt.), And preferably from about 0 to about 0.01% (wt./wt.) At normal temperature (20 ^o C) .

 The insoluble or sparingly soluble salt of the polyvalent metal and the water-soluble physiologically active substance thus obtained is subjected, if necessary, to vacuum drying or freeze-drying before use.

 In a drug with a delayed release of the present invention, the content of the water-insoluble or slightly soluble salt of the polyvalent metal and physiologically active substance is usually from about 0.1 to about 50% (wt./wt.), And preferably from about 1 to about 30% (wt. ./mass.).

 Examples of biodegradable polymers are poorly soluble or water insoluble high molecular weight polymers such as aliphatic polyesters (e.g. homopolymers, copolymers or mixtures thereof, synthesized from one or more α-hydroxycarboxylic acids, such as glycolic acid, lactic acid, hydroxybutyric acid, etc.). p.; hydroxydicarboxylic acids such as malic acid and the like; hydroxytricarboxylic acids such as citric acid and the like and other acids), poly-α-cyanoacrylic esters isloty; polyamino acids such as poly-γ-benzyl-L-glutamic acid, and the like. These polymers can be used in a mixture in an appropriate ratio. Moreover, these polymers can be obtained by statistical polymerization, block polymerization or graft polymerization.

 A preferred biodegradable polymer is an aliphatic polyester (e.g., a homopolymer, copolymer, or a mixture thereof, synthesized from one or more α-hydroxycarboxylic acids, such as glycolic acid, lactic acid, hydroxybutyric acid, and the like; hydroxydicarboxylic acids, such as malic acid, etc .; hydroxytricarboxylic acids such as citric acid, etc., and other acids).

 Of the above aliphatic polyesters, homopolymers or copolymers synthesized from one or more α-hydroxycarboxylic acids (e.g., glycolic acid, lactic acid, hydroxybutyric acid and the like) are preferred in terms of their reliable biodegradability and biological compatibility. A more preferred aliphatic polyester is a copolymer synthesized from one or more α-hydroxycarboxylic acids (e.g., glycolic acid, lactic acid, hydroxybutyric acid, and the like). These copolymers can also be used in a mixture.

 The biodegradable polymer of the present invention can be obtained by a known method.

 Although the aforementioned α-hydroxycarboxylic acids may be present in the D-, L- or D, L-configuration, it is preferred that the ratio of the D- / L-configurations (mol%) is in the range of about 75/25 to about 25/75 . Moreover, a more preferred ratio of D- / L- configurations (mol.%) Is from about 60/40 to about 30/70.

 Examples of the copolymers of the aforementioned α-hydroxycarboxylic acids are glycolic acid and other α-hydroxyacids, with the preferred copolymer being a copolymer of lactic acid or 2-hydroxybutyric acid.

 A preferred copolymer is a lactic acid-glycolic acid copolymer or a 2-hydroxybutyric acid-glycolic acid copolymer.

 A more preferred α-hydroxycarboxylic acid copolymer is a lactic acid-glycolic acid copolymer.

 In the case of using a copolymer of lactic acid and glycolic acid, the preferred ratio of the content of lactic acid / glycolic acid (mol.%) Is from about 100/0 to about 40/60. A more preferred ratio is from about 90/10 to about 45/55, and a most preferred ratio is from about 80/20 to about 45/55. The weight average molecular weight of the copolymer of lactic acid and glycolic acid is from about 3,000 to about 20,000, preferably from about 3,000 to about 14,000, and more preferably from about 3,000 to about 12,000.

 In addition, the degree of dispersion of the copolymer of lactic and glycolic acid (weight average molecular weight / number average molecular weight) is preferably from about 1.2 to about 4.0, and more preferably from about 1.5 to about 3.5.

 A copolymer of lactic and glycolic acid can be synthesized in a known manner, for example, such as the method described in Japanese Patent Application Laid-Open No. 28521/1986. Moreover, the preferred method for the synthesis of this polymer is dehydration polycondensation without the use of catalysts.

 In the case of using a copolymer of 2-hydroxybutyric acid and glycolic acid, it is preferable that the concentration of glycolic acid is from about 10 to about 75 mol%, and the rest, respectively, is 2-hydroxybutyric acid. More preferably, the glycolic acid content is from about 20 to about 75 mol%, and even more preferably from about 30 to about 70 mol%. The average molecular weight of the copolymer of 2-hydroxybutyric acid-glycolic acid is preferably from about 2000 to about 20,000. The dispersion degree of the copolymer of 2-hydroxybutyric acid-glycolic acid (weight average molecular weight / number average molecular weight) is preferably from about 1.2 to 4.0 and more preferably from about 1.5 to 3.5. A copolymer of 2-hydroxybutyric acid-glycolic acid can be synthesized in a known manner, for example, by the method described in Japanese Patent Application Laid-Open No. 28521/1986. Moreover, the preferred method for the synthesis of this copolymer is dehydration polycondensation in the absence of catalysts.

 A preferred homopolymer of the aforementioned α-hydroxycarboxylic acid is a lactic acid homopolymer. The weight average molecular weight of the lactic acid homopolymer is from about 3,000 to about 20,000, and preferably from about 3,000 to about 14,000. The lactic acid homopolymer can be synthesized by a known method, for example, the method described in Japanese Patent Application Laid-Open No. 28521/1986. In this case, the preferred method for the synthesis of this homopolymer is dehydration polycondensation in the absence of a catalyst.

 The above copolymer of 2-hydroxybutyric acid and glycolic acid can be used in a mixture with polylactic acid. Although polylactic acid may have a D- or L-configuration or a mixture thereof, a preferred ratio of B- / L-configurations (mol%) is in the range of about 75/25 to about 20/80. A more preferred ratio of the D- / L-configurations (mol.%) Is in the range from about 60/40 to about 25/75, and even more preferred is from about 55/45 to about 25/75. The weight average molecular weight of polylactic acid is preferably from about 1500 to about 20,000, and more preferably from about 1500 to 10000. In addition, the degree of dispersion of polylactic acid is preferably from about 1.2 to 4.0, and more preferably from about 1.5 up to 3.5.

 To obtain polylactic acid, two known methods can be used, namely, the polymerization of lactide, a lactic acid dimer, with ring opening and the method of polycondensation of lactic acid by dehydration. According to the present invention, it is preferable to use the direct dehydration polycondensation of lactic acid to prepare polylactic acid with a relatively low molecular weight. This method is described, for example, in Japanese Patent Application Laid-Open No. 28521/1986.

 In the case when the specified copolymer of 2-hydroxybutyric acid-glycolic acid and polylactic acid are used in the mixture, the ratio of their concentrations is from about 10/90 to about 90/10 (wt.%). Moreover, the preferred concentration ratio is from about 20/80 to 80/20, and a more preferred ratio is from about 30/70 to 70/30.

 In the present description, the mass-average molecular weight is defined as the molecular weight obtained using gel permeation chromatography (GPC), where 9 polystyrenes with corresponding molecular weights of 120,000, 52,000, 22,000, 9,200, 5050, 2950, 1050, 580 and 162 were used as standards. Based on the data obtained by GPC, the number average molecular weight was also calculated. The degree of dispersion was calculated using data obtained for the weight average molecular weight and number average molecular weight. The measurements were carried out using a GF column KF804L • 2 (manufactured by Showa Denko) of an R1 monitor and L-3300 (manufactured by Hitachi, Ltd.) and using chloroform as the mobile phase.

 Typically, the above copolymer synthesized by dehydration polycondensation in the absence of a catalyst has a terminal carboxyl group.

 In the present invention, the biodegradable polymer preferably has a terminal carboxyl group.

 A biodegradable polymer having a terminal carboxyl group is a polymer in which the number average molecular weight determined by gel permeation chromatography and the number average molecular weight determined from the end groups are nearly the same.

 The number average molecular weight was determined by the number of end groups as follows.

About 1-3 g of biodegradable polymer was dissolved in a solvent, which is a mixture of acetone (25 ml) and methanol (5 ml), and the resulting solution was quickly titrated with 0.5 N. an alcoholic solution of potassium hydroxide, stirring simultaneously at room temperature with phenolphthalein, used as an indicator for determining the content of terminal carboxyl groups; while the number average molecular weight was calculated based on the quantification of the end groups according to the following equation:
The number average molecular weight, determined by the number of end groups, = 20,000 A / B, where
A is the weight mass (g) of the biodegradable polymer;
In the amount (ml) of 0.05 N. potassium hydroxide alcohol solution added until the titration end point is reached.

 So, for example, if the polymer has a terminal carboxyl group and if it is synthesized from one or more α-hydroxy acids by hydration polycondensation in the absence of a catalyst, then the number average molecular weight determined by gel permeation chromatography and number average molecular weight determined by the amount end groups almost coincide. On the other hand, if the polymer basically does not have a terminal carboxyl group and if it is synthesized from a cyclic dimer by ring-opening polymerization using a catalyst, then its number average molecular weight, determined by the number of end groups, significantly exceeds the number average molecular weight determined using gel permeation chromatography (GPC). Such a difference in the number average molecular weight allows you to accurately determine whether the polymer has or does not have a terminal carboxyl group.

 The number average molecular weight, determined by the number of end groups, is an absolute value, while the number average molecular weight, determined by GPC, is a relative value, which can vary depending on different analytical conditions (for example, on the particular mobile phase used, such as a column, reference substance, selected slice width, selected zero line, etc.), and therefore it is difficult to imagine such a value in absolute numerical terms. However, the indication that the number average molecular weight determined by gel permeation chromatography and the number average molecular weight determined by the number of end groups almost coincide means that the number average molecular weight determined by the number of end groups is approximately 0.5 -2 times, and preferably about 0.8-1.5 times, differs from the number average molecular weight determined by gel permeation chromatography. In addition, the indication that the number average molecular weight determined by the number of end groups is significantly higher than the number average molecular weight determined by gel permeation chromatography means that the number average molecular weight determined by the number of end groups exceeds the number average molecular weight, determined by gel permeation chromatography, about 2 or more times.

 In accordance with the present invention, a delayed release preparation is prepared by dispersing in a biodegradable polymer a water-insoluble or poorly soluble salt of a polyvalent metal and a physiologically active substance obtained by mixing this physiologically active substance with a water-soluble salt of a polyvalent metal. Methods of obtaining a drug with a delayed release are the method of drying in water, the method of separation of phases, the method of spray drying and modifications of these methods.

 Methods of obtaining a drug with a delayed release, for example, microcapsules are described below.

(i) Method of drying in water (method to obtain an emulsion of the type oil / water)
In this method, a solution of a biodegradable polymer in an organic solvent is first obtained. In accordance with the present invention, the organic solvent used to produce the drug with a delayed release preferably has a boiling point not exceeding 120 ^° C. Such organic solvents are halogenated hydrocarbons (e.g. dichloromethane, chloroform, carbon tetrachloride, etc.), alcohols ( e.g. ethanol, methanol), acetonitrile and the like. These solvents can be used in mixtures in appropriate proportions. Dichloromethane and acetonitrile are the preferred organic solvent, and dichloromethane is most preferred. The concentration of the biodegradable polymer in the organic solvent is usually from about 0.01 to about 80% (w / w), preferably from about 0.1 to about 70% (w / w), and more preferably from about 1 up to about 60% (w / w) depending on the molecular weight of the biodegradable polymer, the type of organic solvent, and the like.

 Then, a water-insoluble or slightly soluble salt of a polyvalent metal and a physiologically active substance is added to or dissolved in a solution of a biodegradable polymer in an organic solvent thus obtained and previously subjected, if necessary, to freeze-drying or vacuum drying. In this procedure, the specified complex is added in such an amount that the mass ratio "complex: biodegradable polymer" is up to about 1: 2, and preferably about 1: 3.

 A solution thus prepared in an organic solvent is added to the aqueous phase and, using a mechanical stirrer such as a turbine mixer or the like, an oil / water emulsion is obtained, followed by evaporation of the solvent in the oil phase, whereby microcapsules are obtained. The volume of the aqueous phase is usually chosen so that it exceeds the volume of the oil phase by about 1-10000 times, preferably about 2-5000 times, and more preferably about 5-2000 times.

 An emulsifier may be added to the continuous aqueous phase. Such an emulsifier can be, in principle, any emulsifier capable of forming a stable oil / water emulsion. Examples of such emulsifiers include anionic surfactants, nonionic surfactants, castor oil polyoxyethylene derivatives, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, lecithin, gelatin, hyaluronic acid, and the like. Appropriate combinations of these emulsifiers may also be used. The concentration of emulsifier in the continuous aqueous phase is preferably from about 0.001 to about 20% (w / w), more preferably from about 0.01 to about 10% (w / w), and more preferably from about 0.05 up to about 5% (w / w).

 In the method described above, by forming an oil / water emulsion, microcapsules can be prepared by a method in which the complex is dispersed in a solution of a biodegradable polymer in an organic solvent, i.e. by a method by forming a solid / oil / water emulsion.

(ii) Method of drying in water (method to obtain an emulsion of the type water / oil / water)
In this method, a solution of a biodegradable polymer in an organic solvent is first obtained. The concentration of the biodegradable polymer in the organic solvent is usually from about 0.01 to about 80 wt. %, preferably from about 0.1 to about 70 wt.%, and more preferably from about 1 to about 60 wt.%, depending on the molecular weight of the biodegradable polymer, such as an organic solvent and the like. The aqueous dispersion of the complex is used as the internal aqueous phase. The concentration of the complex in the aqueous dispersion is, for example, from about 10 to about 90 wt.%. Using known dispersion methods using a turbine type mechanical stirrer, homogenizer, etc. the above-described aqueous dispersion of the complex is emulsified and dispersed in a solution of a biodegradable polymer in an organic solvent, as a result of which a water / oil emulsion is formed.

 This procedure is carried out so that the mass ratio of the internal aqueous phase and the biodegradable polymer is up to about 1: 2, and preferably about 1: 3. The ratio of the dispersed aqueous phase and the organic solution of the biodegradable polymer is 1: 1000-1: 1 (vol./vol.), Preferably 1: 100-1: 5 (vol./vol.), And more preferably 1:50 -1: 5 (v / v).

 The water / oil emulsion thus obtained is added to another aqueous phase to form a water / oil / water emulsion, after which the solvent is evaporated in the oil phase and microcapsules are obtained. This procedure is carried out under the conditions described above in paragraph (i).

 In accordance with the present invention, a delayed release preparation is preferably used in the form of fine particles. Therefore, this drug with a delayed release during normal subcutaneous or intramuscular injection does not cause any pain to the patient. The average particle diameter of the drug with a delayed release is, for example, from about 0.1 to about 300 microns, preferably from about 1 to about 150 microns, and more preferably from about 2 to about 100 microns.

 In the present description, delayed release formulations made in the form of small particles are also called microcapsules.

 As used herein, the term “microcapsule” may also mean “microsphere”.

 In accordance with the present invention, delayed release preparations may be administered, for example, in the form of microcapsules as such, or they may be administered in various dosage forms not intended for oral administration (e.g., preparations for intramuscular, subcutaneous or visceral injection or other physiologically acceptable injections, as well as preparations for administration through the nose, rectal administration or administration through the mucous membrane of the uterus, etc.), in the form of preparations for oral administration (for example, in e capsules such as hard capsules, soft capsules and the like. p., in the form of solid preparations such as granules and powders, and the like, in the form of liquid preparations such as suspensions etc.).

 In accordance with the present invention, preparations for injection are preferred.

 If the drug with a delayed release is a microcapsule, it can be obtained in the form of an aqueous suspension by suspending the microcapsules in water together with a dispersing agent (for example, a surfactant such as Tween 80 and HCO-60, polysaccharides such as carboxymethyl cellulose, sodium alginate, sodium hyaluronate, etc.), a preservative (such as methyl paraben, propyl paraben, etc.), an isotonic agent (such as sodium chloride, mannitol, sorbitol, glucose, etc.), etc., moreover, the thus obtained aqueous suspension may not be osredstvenno used as a delayed release formulation for injection. Alternatively, the delayed release preparation of the present invention is prepared as an oily suspension by dispersing microcapsules in a vegetable oil, such as sesame or corn oil, in the absence or presence of a phospholipid, such as lecithin, or a medium chain fatty acid triglyceride (e.g., M1G -LVOL 812), and the resulting drug with a delayed release can be directly used for injection.

 If the drug with a delayed release is a microcapsule and if it is intended to be used as an injectable suspension, then the average particle size is chosen, for example, in the range from about 0.1 to about 300 microns, provided that this size meets the requirements presented to the degree of dispersion, and is acceptable for the passage of particles through the channel of the needle of the syringe. A preferred particle size is in the range of from about 1 to about 150 microns, and more preferably from about 2 to about 100 microns.

 The above microcapsules can be obtained in the form of a sterile preparation by any method, without any restrictions, in which the whole process of obtaining such a preparation is sterile, for example, a method in which gamma radiation is used as a sterilizing factor, and also in a method in which antiseptic.

 Due to its low toxicity, the delayed release preparation of the present invention can be used with a high degree of reliability for administration to mammals (e.g., humans, cows, pigs, dogs, cats, mice, rats, rabbits, etc.).

 Indications for use of the drug with a delayed release of the present invention vary depending on the specific physiologically active substance used. So, for example, if the physiologically active substance is insulin, then the drug with a delayed release of the present invention is effective for the treatment or prevention of diabetes mellitus, etc. if the physiologically active substance is interferon-alpha, then the preparation of the present invention can be used to treat kidney cancer, hepatitis C, and the like; if the physiologically active substance is erythropoietin, then the preparation of the present invention can be used to treat anemia, etc. if the physiologically active substance is growth hormone, then the preparation of the present invention can be used to treat developmental insufficiency; and if the physiologically active substance is a granulocyte colony stimulating factor, the drug with a delayed release of the present invention can be used to treat neutropenia after anti-cancer chemotherapy and the like. In the case where the physiologically active substance is erythropoietin, the drug with a delayed release of the present invention is also effective for stimulating hematopoiesis during autohemotransfusion.

 Depending on the type and content of the physiologically active substance, the duration of the release of the physiologically active substance, the particular disease, the particular animal being exposed to the drugs with a delayed release, and other factors, such a dose of the drug with a delayed release can be established which ensures the effective action of the physiologically active substance. If the manufactured product is designed to release the active substance within 1 week, then the dose of the physiologically active substance is chosen in the range from about 0.0001 to about 10 mg per 1 kg of weight for each adult individual. A more preferred dose is in the range of about 0.0005 to about 1 mg / kg body weight.

 The dose for administration of the drug with a delayed release is preferably selected in the range of from about 0.0005 to 50 mg / kg body weight for each adult individual. A more preferred dose is in the range of about 0.0025 to about 10 mg / kg body weight. Depending on the type, content and dosage form of the physiologically active substance, on the duration of the release of the physiologically active substance, on the particular disease, the particular animal being treated with the drug with a delayed release, and other factors, the appropriate dosage regimen may be chosen, for example, once a week, once every two weeks or once every four weeks.

The preparation of the present invention can be stored at normal temperature or in a cold place, however, the preferred storage condition is storage in a cold place. Specified in the present description, storage at normal temperature and storage in a cold place is as defined by the Pharmacopoeia of Japan, namely storage at normal temperature means storage at 15-25 ^o C, and storage in a cold place means storage at a temperature below 15 ^o C .

Preferred Embodiments of the Present Invention
The present invention is described in more detail in the following examples, which, however, should not be construed as a limitation of the scope of the invention.

Reference Example 1
A solution containing 0.5 g of pig insulin (27.3 units / mg, DIOSYNTH, Netherlands) in 22 ml of an aqueous solution of 100 mm sodium hydroxide, and a solution containing 1 gram of zinc acetate (dihydrate) in 10 ml of distilled water was mixed and then left for one hour at room temperature. After centrifugation at approximately 3000 rpm (05PR-22, Hitachi, Ltd.), the supernatant was discarded. The resulting residue was again dispersed in distilled water, and then centrifuged. After this, the supernatant was discarded and a small amount of distilled water was added to the residue, and then the mixture was dried by freezing, resulting in about 1 g of crude pig salt of pig insulin in the form of a dry powder.

To determine the insulin content in the powder thus obtained, this powder was extracted with a solution of 50 mM EDTA containing 30% acetonitrile for three hours with shaking, and then quantitative assessment was performed using high-performance liquid chromatography (HPLC). As a result of this procedure, it was found that in 100 mg of dry powder contains 47.6 mg of pig insulin. Reference Example 2
To a mixture containing 168 ml of a 40% aqueous solution of potassium hydroxide and 1000 ml of ethyl ether, 104 g of nitrosoethylurea were gradually added, and then this mixture was stirred under ice-cooling. The resulting yellow ether layer was separated and dried by adding granular potassium hydroxide. Potassium hydroxide was then removed, resulting in approximately 900 ml of a diazoethane solution.

 130 g of a copolymer of lactic acid and glycolic acid (lactic acid / glycolic acid = 50/50 (mol.%); Weight average molecular weight was approximately 5800) were dissolved in 1900 ml of methylene chloride, and this solution was stirred and cooled. The above diazoethane solution was gradually added dropwise to the resulting mixture under ice cooling, and then the mixture was stirred at room temperature for 2 hours. After keeping the mixture overnight, the solvent was distilled off under reduced pressure. The residue was dried in vacuo at room temperature, whereby 131 g of ethyl ester of a copolymer of lactic acid and glycolic acid were obtained.

Reference Example 3
A solution of 1 mg of human growth hormone (Biotechnology General, USA) in 0.9 ml of distilled water and a solution containing 9.98 μg, 29.43 μg, 49.88 μg, 69.84 μg, 79.81 μg or 99, 77 μg of zinc acetate (dehydrate) in 0.1 ml of distilled water, stirred thoroughly. The molar ratio of zinc to growth hormone was 1, 3, 5, 7, 8, and 10. When the molar ratio was 5, approximately 60% of human growth hormone precipitated. And in the case when the molar ratio was 7 or more, almost 100% of human growth hormone was precipitated.

Reference Example 4
One gram of leuprolide acetate (TAP-144) and 157.5 mg of gelatin were dissolved in 1 ml of distilled water at a temperature of 70-80 ^° C. To the aqueous solution, when heated at a temperature slightly above the gelatinization temperature of the aqueous solution, 21 g of a milk copolymer solution was added. acid and glycolic acid, which was obtained by dissolving 7.85 g of a copolymer of lactic acid and glycolic acid [lactic acid / glycolic acid: 75/25 (mol.%); viscosity: 0.142-0.169 centipoise] in 13.15 g of dichloromethane. The resulting mixture was emulsified using a compression homogenizer for several minutes or more to obtain an oil / water emulsion. The resulting water / oil emulsion was cooled to a temperature of 10-20 ^o C. Then the emulsion was poured into 5000 ml of 0.1% (wt./about.) An aqueous solution of polyvinyl alcohol, the temperature of which was brought to 10-20 ^o C, after which the mixture emulsified using a turbine homomixer to obtain a water / oil / water emulsion. This water / oil / water emulsion was stirred at room temperature (15-30 ^{° C.} ) to evaporate dichloromethane, and then the internal water / oil emulsion was solidified, after which the microcapsules were collected by centrifugation. These microcapsules were again dispersed in distilled water, and then centrifuged to remove excess drug and polyvinyl alcohol. The isolated microcapsules were suspended in a small amount of distilled water. 1.5 g of D-mannitol was added to the suspension and then dissolved. The resulting suspension was dried by freezing under reduced pressure, whereby powdered microcapsules were obtained.

After lyophilization, the obtained microcapsules in the form of a powder were dried at 50 ^° С under reduced pressure, namely, at a temperature of 3 ^° С higher than the gelatinization temperature of the main matrix component, a copolymer of lactic and glycolic acid for 24, 48, 96, or 120 hours, as a result, powdered microcapsules with a prolonged action were obtained.

Example 1
To 200 ml of an aqueous solution of interferon -α (containing 40 billion, ME) was added 1 ml of an aqueous solution of zinc acetate (dihydrate) (200 mg / ml) and 1 ml of 1 N. sodium hydroxide. After stirring, the mixture was kept overnight at 4 ^° C. After centrifugation at 3000 rpm, the insoluble complex was isolated and dried by freezing, resulting in approximately 200 mg of crude zinc interferon-α salt.

To a solution containing 1.5 g of a copolymer of lactic acid and glycolic acid (the ratio of lactic acid / glycolic acid is 50/50; molecular weight is 5800; from Wako Pure Chemical Industries) and 1.5 g of ethyl ester of a copolymer of lactic acid and glycolic acid (obtained in Reference Example 2) in 4 ml of dichloromethane, 200 mg of the above crude zinc salt of interferon-α was added and the resulting mixture was stirred for about 30 seconds in a homogenizer (Polytron) to obtain a solid / oil emulsion. This emulsion was poured into 700 ml of a 0.1% (w / w) aqueous solution of polyvinyl alcohol (EG-40 obtained by Nippon Synthetic Chemical Industry), previously brought to a temperature of 18 ^{° C.} , and then the mixture was stirred in a turbine homomixer at 6000 rpm, resulting in an emulsion of the type solid / oil / water. The resulting emulsion was stirred at room temperature for 3 hours to remove volatile dichloromethane and solidify the oil phase. After centrifugation at approximately 2000 rpm (05PR-22, Hitachi, Ltd), the supernatant was discarded. The resulting residue was again dispersed in distilled water and centrifuged. After that, the collected microcapsules were again dispersed in a small amount of distilled water in the presence of 50 mg of D-mannitol, and this dispersion was dried by freezing, whereby powdered microcapsules were obtained.

Example 2
3.6 g of a copolymer of lactic acid and glycolic acid (lactic acid / glycolic acid = 75/25 (mol%), weight average molecular weight was 4413, number average molecular weight was 13585; Wako Pure Chemical Industries) was dissolved in 6.6 g (5 ml) dichloromethane. 420 mg of crude zinc salt of pig insulin (containing 200 mg of pig insulin) obtained in Reference Example 1 was dispersed in 6.6 mg (5 ml) of dichloromethane. Both substances were mixed and then mixed for approximately 10 seconds in a homogenizer (Polytron), resulting in a solid / oil emulsion. This emulsion was poured into 800 ml of 0.1 wt. % aqueous solution of polyvinyl alcohol (EG-40, obtained from Nippon Synthetic Chemical Industry), previously brought to a temperature of 18 ^o With, and this mixture was stirred in a turbine homomixer at 6000 rpm, resulting in a solid / oil / emulsion water. The resulting emulsion was stirred for 3 hours at room temperature to remove dichloromethane and solidify the oil phase. After centrifugation at approximately 2000 rpm (05RP-22, Hitachi, Ltd.), the supernatant was discarded. The residue was again dispersed in distilled water and centrifuged. The collected microcapsules were redispersed in a small amount of distilled water in the presence of 50 mg of D-mannitol, and this dispersion was freeze-dried, resulting in powdered microcapsules (about 3 g of substance was isolated).

 To determine the insulin content in the microcapsules thus obtained, the powder was extracted with a solution of 50 mM EDTA containing 30% acetonitrile with shaking for 3 hours, and then quantitative assessment was performed using high-performance liquid chromatography (HPLC). As a result of this procedure, it was found that 100 mg of microcapsules contained 6.2 mg of insulin.

Example 3
One gram of zinc chloride was gradually added to 8 ml of an injection solution containing erythropoietin (12000, ME) (Espo ^™ Injection 3000, supplied by Sankyo); this mixture was kept at room temperature for one hour. After centrifuging the mixture at 3000 rpm, the precipitate was again dispersed in distilled water, and then centrifuged to obtain a precipitate. A small amount of distilled water was added to the obtained precipitate, and this mixture was dried by freezing, whereby a 60 mg mixture of crude zinc salt of erythropoietin and crude zinc salt of albumin was obtained in the form of a powdery substance.

 To a solution of 0.5 g of a copolymer of lactic acid and glycolic acid (lactic acid / glycolic acid = 50/50, molecular weight = 12000; supplied by Wako Pure Chemical Industries) in 1.5 ml of dichloromethane was added 60 mg of the above crude zinc erythropoietin salt mixture and crude albumin zinc salt, after which the mixture was stirred for about 30 seconds in a homogenizer (Polytron) to obtain a solid / oil emulsion. This emulsion was treated in a similar manner to that described in Example 1, whereby 152 mg of powdered microcapsules were obtained.

Example 4
Human growth hormone (Genotropin ^™ 16ME; supplied by Sumitomo Pharmaceuticals) was dissolved in 1 ml of distilled water. To this solution was added 100 μl of an aqueous solution of zinc chloride (10 mg / ml); this mixture was kept for one hour at room temperature. After centrifuging the mixture, the precipitate was again dispersed in distilled water, and then centrifuged, resulting in a precipitate. A small amount of distilled water was added to the resulting precipitate, and then freeze-dried, whereby 5.6 mg of the crude zinc salt of human growth hormone was obtained in powder form.

 To a solution containing 0.5 g of a copolymer of lactic acid and glycolic acid (lactic acid / glycolic acid = 75/25, molecular weight = 9800; supplied by Wako Pure Chemical Industries) and 1.5 ml of dichloromethane, 5.6 mg of the above was added. crude zinc salt of human growth hormone, and this mixture was stirred for approximately 30 seconds in a homogenizer (Polytron), resulting in a solid / oil emulsion. The resulting emulsion was processed by the method described in example 1, with the result that received 121 mg of powdered microcapsules.

Example 5
10 ml of an injection solution containing granulocyte colony stimulating factor (G-CSF) (3 × 10 ⁸ ME) Filgrastin Neupogen, trademark of Amgen, USA) was neutralized with a dilute aqueous solution of sodium hydroxide, and then 1 ml of an aqueous solution of zinc chloride was added (10 mg / ml); the resulting mixture was left for one hour at room temperature. Then the mixture was centrifuged; the precipitate formed was again dispersed in distilled water, and then centrifuged to form a precipitate. A small amount of distilled water was added to the resulting precipitate, and this mixture was freeze-dried to give 4 mg of the crude zinc salt of a granulocyte colony stimulating factor in powder form.

 To a solution containing 0.5 g of a copolymer of lactic acid and glycolic acid (lactic acid / glycolic acid = 50/50, molecular weight = 8000; supplied by Wako Pure Chemical Industries) and 1.5 ml of dichloromethane, 4 mg of the above crude zinc was added salts of granulocyte colony stimulating factor and the resulting mixture was stirred for approximately 30 seconds in a homogenizer (Polytron), resulting in a solid / oil emulsion. The resulting emulsion was processed by the method described in example 1, with the result that received 110 mg of powdered microcapsules.

Example 6
5.21 mg (26 units / mg) of human insulin (human recombinant insulin, supplied by Wako Pure Chemical Industries) was dissolved in 0.63 ml of an aqueous solution (57 mmol) of hydrochloric acid, and 0.35 ml of 0 was added to the resulting mixture. 05 n. an aqueous solution of sodium hydroxide, resulting in a solution of human insulin having an almost neutral pH. To a solution of human insulin was added 0.2 ml of an aqueous solution of zinc acetate (20 mg / ml); this mixture was kept overnight at a temperature of 4 ^o C. Then the mixture was centrifuged at approximately 3000 rpm; the resulting precipitate was again dispersed in distilled water and centrifuged to form the desired precipitate. A small amount of distilled water was added to this precipitate, and the mixture was freeze-dried, whereby 11 mg of crude zinc salt of human insulin was obtained in powder form.

 To a solution containing 0.5 g of a copolymer of lactic acid and glycolic acid (lactic acid / glycolic acid = 50/50, molecular weight = 6000; supplied by Wako Pure Chemical Industries) and 1.5 ml of dichloromethane, 11 mg of the above crude zinc was added salts of human insulin, and then the mixture was stirred for approximately 30 seconds in a homogenizer (Polytron), resulting in a solid / oil emulsion. The resulting emulsion was processed by the method described in example 1, whereby 105 mg of powdered microcapsules were obtained.

Comparative Example
To a solution containing 0.9 g of a copolymer of lactic acid and glycolic acid [lactic acid / glycolic acid = 50/50 (mol.%), The mass-average molecular weight was 6000; the product was supplied by Wako Pure Chemical Industries] and 1.5 ml of dichloromethane, 100 mg of human insulin was added, mainly without zinc [zinc content was 0.0001 wt. %], and this mixture was stirred for approximately 10 seconds in a homogenizer (Polytron) to obtain a solid / oil emulsion. The resulting emulsion was processed by the method described in example 1, whereby powdered microcapsules (470 mg) were obtained.

 To determine the insulin content in the microcapsules thus obtained, the powder was extracted with a solution of 50 mM EDTA containing acetonitrile for three hours with shaking, and then quantitative assessment was carried out using high-performance liquid chromatography (HPLC). As a result of this procedure, it was found that 100.7 microcapsules contained 8.7 mg of insulin.

Experimental Example 1
323 mg of the powdered microcapsules obtained by the method described in example 2 were dispersed in 1 ml of a dispersant for injection (containing 5 mg of carboxymethyl cellulose, 1 mg of polysorbate 80 and 50 mg of mannitol dissolved in one milliliter of distilled water). The resulting dispersion was subcutaneously injected into the back of 6-week-old male rats (insulin was administered in an amount of about 20 mg per rat). After the administration of insulin from the tail of the rat, blood was drawn at regular intervals and analyzed for serum concentration of porcine insulin using an enzyme-linked immunosorbent assay (ELISA) (supplied by Sankyo Junyku). Active porcine insulin was detected in serum for 1 week or more after insulin administration.

Experimental Example 2
70 mg of the powdered microcapsules obtained by the method described in example 4 were dispersed in 0.5 ml of a dispersant for injection (containing 5 g of carboxymethyl cellulose, 2 g of polysorbate 80 and 50 g of mannitol dissolved in 1 liter of distilled water). The resulting dispersion was subcutaneously injected into the backs of 6-week-old SD male rats (growth hormone was administered in an amount of about 3 mg per rat). After the administration of growth hormone, blood was taken from the tail of the rat at fixed constant time intervals and analyzed for serum concentration of growth hormone using radioimmunoassay. The detection of active growth hormone in serum was carried out for one or more weeks after the introduction of growth hormone.

Comparative Experimental Example
154.7 mg of powdered microcapsules obtained by the method described in the comparative example were dispersed in 1.75 ml of a dispersant for injection (containing 5 mg of carboxymethyl cellulose, 1 mg of polysorbate 80 and 50 mg of mannitol dissolved in one milliliter of distilled water). The resulting dispersion was subcutaneously injected into the backs of 6-week-old male SD rats (insulin was administered in an amount of about 44 mg per rat). After the administration of insulin, blood was taken from the tail of the rat at regular time intervals and analyzed for serum insulin concentration by enzyme-linked immunosorbent assay (ELISA). Active serum insulin was detected only one day after administration.

Industrial application
In accordance with the present invention, the drug with a delayed release allows for the highly effective inclusion of a physiologically active substance in suppressing the initial release of a physiologically active substance. This delayed release preparation of the present invention has the ability to release a physiologically active substance while maintaining its biological activity after in vivo administration. In addition, the physiologically active substance contained in the drug with a delayed release, retains its stability for a long period of time, while the loss of its biological activity is very small.

Claims (25)

1. The drug with a delayed release, characterized in that it contains 0.1-50 wt. % insoluble or slightly soluble in water salts of a polyvalent metal of a physiologically active substance such as a water-soluble peptide, with the exception of an endothelin antagonist, the solubility of which in water is from 0 to 0.1 mol. % at 20 ^o C and biodegradable polymer.  2. The drug according to claim 1, characterized in that the physiologically active substance is a water-soluble peptide or its derivative.  3. The drug according to claim 2, characterized in that the peptide is a hormone, cytokine, hematopoietic factor, growth factor, enzyme, soluble or solubilized receptor, antibody, antigen-containing peptide, blood coagulation factor, or an adhesive molecule.  4. The drug according to p. 2, characterized in that the peptide is a hormone.  5. The drug according to claim 4, characterized in that said hormone is growth hormone.  6. The drug according to p. 4, characterized in that said hormone is insulin.  7. The drug according to p. 2, characterized in that the peptide is a cytokine.  8. The drug according to claim 7, characterized in that said cytokine is interferon.  9. The drug according to p. 2, characterized in that the peptide is a growth factor.  10. The drug according to any one of paragraphs. 1-9, characterized in that said polyvalent metal salt is a transition metal salt.  11. The drug according to any one of paragraphs. 1-9, characterized in that said polyvalent metal salt is a zinc salt.  12. The drug according to any one of paragraphs. 1-11, characterized in that the solubility of the polyvalent metal salt in water is from 0 to 0.01% (wt./wt.).  13. The drug according to any one of paragraphs. 1-12, characterized in that it contains from 1 to 30% (wt./wt.) Salt of the polyvalent metal.  14. The drug according to any one of paragraphs. 1-13, characterized in that the specified biodegradable polymer is an aliphatic polyester.  15. The drug according to p. 14, characterized in that said aliphatic polyester is a polymer of lactic acid and glycolic acid.  16. The drug according to p. 15, characterized in that the ratio of lactic acid and glycolic acid is from 100/0 to 40/60 mol. %  17. The drug according to p. 15, characterized in that the ratio is from 90/10 to 45/55 mol. %  18. The drug according to p. 15, characterized in that the mass-average molecular weight of the specified polymer is from 3000 to about 20,000.  19. The drug according to p. 15, characterized in that the mass-average molecular weight of the specified polymer is from 3000 to about 14000.  20. The drug according to p. 14, characterized in that said aliphatic polyester is a homopolymer of lactic acid.  21. The drug according to p. 20, characterized in that the mass-average molecular weight of the specified polymer is from 3000 to about 20,000.  22. The drug according to p. 20, characterized in that the weight average molecular weight of the specified homopolymer is from 3000 to about 14000.  23. The drug according to paragraphs. 1-22, characterized in that it is a microcapsule.  24. The drug according to any one of paragraphs. 1-23, characterized in that it is injectable.  25. The method of producing a drug with a delayed release according to claim 1, comprising dispersing a water-insoluble or slightly soluble salt of a polyvalent metal of a physiologically active substance such as a water-soluble peptide, with the exception of an endothelin antagonist, in an oil phase containing a biodegradable polymer, resulting in an emulsion of the type solid-in-oil, and then a solid-in-oil emulsion is added to the aqueous phase to obtain a solid-in-oil-in-water emulsion, whereupon obtained The emulsion is solid-in-oil-in-water dried.  Pp 1, 2, 4-9, 11-17, 23-25 have priority on 09/09/1994.  Pp 3, 10, 18-22 take priority on September 6, 1995.