JP3283288B2 - Bioactive peptide preparation - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a physiologically active peptide preparation and a preparation containing hyaluronic acid or a non-toxic salt thereof and a high molecular substance, which is suitable for clinical application. It also relates to a preparation containing interferon (IFN), which is particularly useful for treating cancer and viral diseases.

[0002]

2. Description of the Related Art In recent years, studies on various physiologically active peptides have been actively conducted, and the mechanism of action has been clarified, and progress has been made in the development of new pharmaceuticals. However, many physiologically active peptides have a short effect duration in vivo, and it is extremely difficult to maintain an effective concentration. Therefore, when bioactive peptides are used clinically, administration forms have been studied so that expensive drugs can be used with as little drug as possible, and the number of administrations can be kept low and the concentration can be maintained at a constant concentration for long-term effects. Was. Pharmaceutical preparations containing hyaluronic acid include those in which hyaluronic acid and bioactive peptides are chemically bonded and those in which hyaluronic acid and a drug are mixed. Examples of patent applications in which hyaluronic acid and a drug are mixed are disclosed in JP-A-61-236732 and JP-A-62-12922.
6, JP-A-1-319427, 2-213, 2-2
There are 31429 and the like, but problems remain due to practical effects and the like.

[0003] For example, in the case of IFN, the duration of the effect in vivo is particularly short, and it is extremely difficult to maintain an effective concentration.
It is necessary to use a large amount of N. Since it is not practical to administer IFN in a large amount and frequently as described above, it is necessary to reduce the number of administrations of the drug and maintain it at a constant concentration for a long time, while making it possible to demonstrate the efficacy of the agent with as little as possible. There is a strong demand for sustained effects. For such a purpose, for example, liposome encapsulation of interferon [JP-A-63-50211]
7], collagen embedding [US Pat. No. 4,855,13
4] and antibody binding [Japanese Patent Application Laid-Open No. 60-502104] to increase the half-life in blood, but it is not sufficient for the length of half-life in blood, antigenicity and safety as a drug. Was not satisfactory.

[0004]

The present invention provides an antiviral effect, an anticancer effect,
It is composed of a bioactive peptide having a wound healing action, an anti-inflammatory action or a bone-related disease healing action, hyaluronic acid or a non-toxic salt of hyaluronic acid, and a polymer substance which can be administered to a living body together with hyaluronic acid or a non-toxic salt thereof. Providing a pharmaceutical formulation.

[0005] The present inventors have found that physiologically active peptide formulations, in particular, the super Okishidoji Sum synthetase (SOD), epidermal growth factor (EGF), fibroblast growth factor (FG
F) Various attempts have been made to improve a physiologically active peptide preparation containing any one of interleukin 1 (IL-1) and interferon (IFN). It has been found that superior properties can be obtained as compared with the case of using the preparation alone.

[0006] In particular, the preparations thus obtained can be expected to have effects such as an excellent healing effect, an excellent drug release effect and an excellent stability.

[0007] Furthermore, the present inventors have proceeded with research on the basis of such findings, super Okishidoji Sum synthetase (SO
D), epidermal growth factor (EGF), fibroblast growth factor (FGF), parathyroid hormone, adrenocortical hormone,
Adrenocorticotropic hormone, adrenocorticotropic hormone-releasing factor, renin, angiotensin, blood clotting factor, human growth hormone, calcitonin, gonadotropin, gonadotropin, insulin, vasopressin, oxytocin, somatostatin, secretin, gastrin, gastrin-secreting peptide, gastric acid Secretion inhibitory peptide, motilin,
Hematopoietic factors, erythropoietin, tissue plasminogen activator (tPA), urokinase, prourokinase, streptokinase, thrombomodulin, protein C, hirudin, colony stimulating factor (G
-CSF, GM-CSF, M-CSF), insulin-like growth factor (IGF), hepatocyte growth factor (HG
F), bone morphogenetic factor (BMF), nerve growth / nutritional factor, natriuretic peptide, stem cell growth factor, platelet growth factor, immunosuppressant, non-specific immunostimulant, thymocyte activating factor (TAF), interleukin Bioactive peptide consisting of one or more of kidney growth factor, tumor necrosis factor (TNF-α and β), and interferon (IFN-α, IFN-β and IFN-γ) (Excluding transforming growth factors and protease inhibitors), it was found that by adding hyaluronic acid to the preparation, superior properties can be obtained as compared with the case of using the bioactive peptide preparation alone. Furthermore, the present inventors proceeded with research based on such findings, and in the formulation of the bioactive peptide and hyaluronic acid, by further coexisting an appropriate amount of one or more polymer substances, It has been found that more excellent characteristics can be obtained.

In particular, it has been found that a complex can be formed by selecting the physiologically active peptide, hyaluronic acid, and the high molecular substance from each other, and the complex obtained in this way exhibits more excellent properties. The composite formed in this way is very excellent in terms of its utility and increased sustainability. A new type of complex preparation comprising hyaluronic acid and a polymer substance obtained in this way has a better healing effect, better drug absorption, better drug release, In addition to excellent drug stability and excellent targeting effect, various advantages in the preparation and processing of pharmaceuticals can be expected. Typical of these properties are the extension of the duration of the effect, the control of the effect expression pattern, the storage of the preparation, the increase in stability, the prevention of degradation of the preparation,
And so on.

The physiologically active peptides used in the present invention include physiologically active substances having peptide chains extracted and purified from animal tissues, such as hormones, enzymes, lymphokines, and growth factors, or obtained by cell culture or gene recombination techniques. Point to. However, transforming growth factors and protease inhibitors are excluded.

[0010] As the physiologically active peptide used in the present invention, super Okishidoji Sum synthetase (SOD), epidermal growth factor (EGF), fibroblast growth factor (FG
F), parathyroid hormone, adrenocortical hormone, adrenocorticotropic hormone, adrenocorticotropic hormone-releasing factor, renin, angiotensin, blood coagulation factor, human growth hormone, calcitonin, gonadotropin, gonadotropin,
Insulin, vasopressin, oxytocin, somatostatin, secretin, gastrin, gastrin secretion peptide, gastric acid secretion inhibitory peptide, motilin, hematopoietic factor,
Erythropoietin, tissue plasminogen activator (tPA), urokinase, prourokinase, streptokinase, thrombomodulin, protein C, hirudin, colony stimulating factor (G-CSF,
GM-CSF, M-CSF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), bone morphogenetic factor (BMF), nerve growth and nutrition factor, natriuretic peptide, stem cell growth factor, platelet growth factor , Immunosuppressants, non-specific immunostimulants, thymocyte activating factor (TA
F), interleukins, kidney growth factor, tumor necrosis factor (TNF-α and β), and interferon (IFN
-Α, IFN-β and IFN-γ), and physiologically active peptides (excluding transforming growth factors and protease inhibitors) comprising one or more selected from the group consisting of: The physiologically active peptide used in the present invention, particularly preferred are super Okishido <br/> di soum synthetase (SOD), tumor necrosis factor (TNF-alpha and beta), interferon (IFN-α, IFN-β and IFN-γ), epidermal growth factor (EGF), fibroblast growth factor, interleukins such as interleukin 1, colony stimulating factors (G-CSF, GM-CSF, M
-CSF) and the like. In the present invention, preferred bioactive peptides include gastric acid secretion inhibitory peptide, motilin, hematopoietic factor, erythropoietin, tissue plasminogen activator (tPA), urokinase, prourokinase, streptokinase, thrombomodulin, protein C, hirudin, insulin -Like growth factor (IGF), hepatocyte growth factor (HGF), bone morphogenetic factor (BMF), nerve growth and nutritional factor, natriuretic peptide, stem cell growth factor, platelet growth factor, immunosuppressant, non-specific immunostimulation Agent, thymocyte activating factor (TA
F), kidney growth factor and the like can be used. In the present invention, as bioactive peptides, parathyroid hormone, adrenocortical hormone, adrenocorticotropic hormone, adrenocorticotropic hormone releasing factor, renin, angiotensin, blood coagulation factor,
Human growth hormone, calcitonin, gonadal hormone, gonadotropin, insulin, vasopressin, oxytocin, somatostatin, secretin, gastrin, gastrin secreting peptide, and the like can also be used. These physiologically active peptides may be derived from natural products, chemically synthesized or partially modified, or obtained by genetic recombination. In particular, those obtained by a gene recombination method are preferred.

[0011] IFN is a physiologically active substance produced by lymphocytes and fibroblasts, and is known to have an antiviral effect and an anticancer effect. This IFN includes IFN-α, I
FN-β and IFN-γ are known to be present, and have become available in large quantities due to advances in cell culture technology and genetic recombination.

This IFN has been started to be used as a therapeutic agent for multiple myeloma, renal cancer, glioblastoma, cutaneous malignant melanoma, hepatitis B and the like, and furthermore, non-A non-B hepatitis, AIDS and other It is expected to be used as a therapeutic drug for cancer and rheumatism.

Since excellent preparations can be obtained according to the present invention, application to antiviral and antitumor activities other than these uses, and further to arthritis healing effects can be expected. In the present invention, various IFNs can be used.

Furthermore, since excellent preparations can be obtained according to the present invention, applications in which the range of use is expanded can be expected. In the present invention, excellent results can be expected especially for those obtained by genetic recombination techniques, which have been pointed out as having problems of antigenicity and rapid blood elimination rate.

Hyaluronic acid is a polymer composed of repeating units of N-acetylglucosamine and glucuronic acid,
It is widely found in nature and is found in many mammals. For example, hyaluronic acid extracted from cockscomb is used as a therapeutic agent for osteoarthritis of the knee, and hyaluronic acid produced by certain bacteria such as Streptococcus is also being developed as a pharmaceutical. The hyaluronic acid used in the present invention may be in a free form or a salt thereof, and examples thereof include non-toxic salts such as sodium salt, potassium salt and calcium salt. The source of the hyaluronic acid used in the present invention is not particularly limited.

However, as described above, hyaluronic acid produced by certain bacteria, such as Streptococcus, is preferred because a relatively uniform, higher molecular form is obtained with high purity.

In particular, in the preparation of the present invention, a close relationship was observed between the duration of the effect of the drug and the molecular weight of the hyaluronic acid used, and high-purity hyaluronic acid other than interferon was used. Is preferably about 200,000 to 3,000,000, and in some cases, about 500,000 to 3,000,000. In the case of interferon, preferably 50
About 3 million to 3 million, and in some cases 100
Those having about 10,000 to 3,000,000 are particularly preferable. in this way,
By using a specific molecular weight as the hyaluronic acid used in the preparation of the present invention, it is possible to obtain various pharmaceutical preparations having desirable characteristics.

[0018] Hyaluronic acid or a salt thereof can be used in various amounts with respect to the bioactive peptide. The concentration of hyaluronic acid or its salt depends on its molecular weight, but can be used in the range of 0.1 to 50% by weight per preparation. In the present invention, particularly preferred hyaluronic acid salts include sodium salts, potassium salts, magnesium salts, calcium salts and the like, and those which are pharmaceutically stable are preferred.

According to the present invention, there is obtained a pharmaceutical preparation further comprising a combination of the above-mentioned bioactive peptide and hyaluronic acid or a non-toxic salt thereof, together with a hyaluronic acid or a salt thereof and a polymer which can be administered in vivo. Can be One or two or more, for example, three or four such polymer substances can be blended. Such a formulation can be selected depending on the bioactive peptide.

Examples of the high molecular substance include chitin, chondroitin, such as atelocollagen, gelatin, albumin, fibrin, fibrinogen, polylysine, polyglutamic acid, and other naturally derived or artificially synthesized proteins, or hydrolyzed or modified products thereof. Sulfuric acid, colominic acid or their deacetylated products, dextran,
Examples include polysaccharides such as DEAE-dextran, cyclodextran, inulin, laminaran, and polylactic acid, polyglycolic acid, or copolymers or heteropolymers thereof, and synthetic polymers such as heteropolymers, hydroxypropyl cellulose, carboxyvinyl polymers, and carboxymethyl cellulose.

The polymer substance can be used in various amounts depending on the purpose. For example, depending on its type, it can be blended in an amount of 10 to 200% by weight based on hyaluronic acid. For example, a polymer substance is 0.1 to 1.5 times the amount of hyaluronic acid when it is a protein, and 0.1 to 1.5 times the amount of hyaluronic acid when it is a polysaccharide. In the case of a molecule, excellent results can be obtained by blending it in an amount of 0.1 to 1.5 times the amount of hyaluronic acid. In particular, these high molecular substances interact with hyaluronic acid and play a desirable role in achieving the effect. For example, these macromolecules interact with hyaluronic acid to form a stronger polymer matrix or, particularly in the case of proteins, bind tightly to hyaluronic acid, causing hyaluronic acid to become spinnable. Changes in physical properties, such as viscosity, have the effect of regulating the effect retention time and expression pattern of the drug and improving the stability of the drug. Appropriate polymer substances and production conditions are selected according to the form of use such as granules, tablets or liquid preparations. Generally, the amount of the polymer substance used is 0.1 to 1.5 times that of hyaluronic acid. The complex formation pH is 3 to 10,
Preferably it is 4-8.

[0022] Formulations for interferons are those that give better results and are preferred. As such good results, as described above, a high therapeutic effect is achieved, a wide range of pharmaceutical forms can be obtained, drug stability can be obtained, in vivo persistence can be obtained, Painlessness is achieved, formulation processability is improved, and bioavailability is improved.

In the present invention, hyaluronic acid can be used in various amounts depending on the purpose with respect to the physiologically active peptide, and the high-molecular substance depends on the type. Various amounts can be blended and used accordingly. For example, when interferon is used, the amount of interferon can be included in the formulation in an amount of 10 ⁵ to 10 ¹² units, more preferably 10 ⁶ to 10 ¹⁰ units. On the other hand, hyaluronic acid or a non-toxic salt thereof can be used in various amounts depending on the dosage form per preparation, for example, 0.1 to 50% by weight, more preferably 0.1 to 50% by weight.
An amount of 5 to 10% by weight can be used. Hyaluronic acid or a non-toxic salt thereof is 0.1 to 10% by weight in the case of a liquid dosage form, 0.5 to 50% by weight in the case of a solid, and 0.5 to 10% by weight in the case of a semi-liquid. Can be exemplified. In addition, the polymer substance, various things can be used in various amounts, for example, for hyaluronic acid,
0.1 to 1.5% by weight, more preferably 0.5%
Preferred results are obtained with additions of up to 1.2% by weight.

The formulations of the present invention can be in solid or liquid form, as well as in gelled semi-liquid form,
Solid or liquid pharmaceutical carriers or excipients, stabilizers and the like can be added to make the preparation into an appropriate formulation. Examples of such additives include a water-soluble aliphatic carboxylic acid having 2 to 8 carbon atoms, a suitable surfactant, an electrolyte, a preservative, and an antioxidant. As the other additives, a pH adjuster, a base, a gel stabilizer, a wetting agent, and the like can be appropriately selected.

The carboxylic acid includes, for example, monocarboxylic acid, dicarboxylic acid, tricarboxylic acid and the like. Examples of the monocarboxylic acid include lower fatty acids and monocarboxylic acids having 1 to 7 hydroxyl groups. Examples of the di- or tricarboxylic acid include 2 to 3 arbitrarily in an alkane chain.
And carboxyl groups, which may also optionally contain 1 to 3 hydroxyl groups. Further, these carboxylic acids may have an amino group. Specific examples of these carboxylic acids include acetic acid, propionic acid,
Lactic acid, gluconic acid, glucuronic acid, malonic acid, succinic acid, citric acid, ascorbic acid, tartaric acid, ring acid, glutaric acid, aspartic acid, glutamic acid, etc., which are partially salted or esterified Can also be made.

Suitable surfactants include those acceptable as pharmaceutical additives, for example, sorbitan fatty acid esters such as sorbitan monopalmitate, sorbitan tristearate, sorbitan sesquioleate; polyoxyethylene sorbitan monoester Stearate,
Polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene monooleate; glycerin such as glycerin monostearate, glycerin / coconut oil fatty acid ester, glycerin monooleate, acetylated glyceryl monostearate, and acetoacetylated glyceryl monostearate Fatty acid esters; propylene glycol fatty acid esters such as propylene glycol monolaurate; polyoxyethylene glycerin fatty acid esters such as polyoxyethylene glycerin monostearate; polyoxy such as polyoxyethylene monostearate and polyoxyethylene oleate Ethylene fatty acid esters; polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether Polyoxyethylene alkyl ethers such as Le; polyoxyethylene alkyl phenyl ethers such as polyoxyethylene nonylphenyl ether; polyoxyethylene hydrogenated castor oil, polyoxyethylene derivatives such as polyoxyethylene lanolin;
Polyoxyethylene alkylamines and amides such as polyoxyethylene stearylamine and polyoxyethylene oleate; alkyl sulfate salts and salts such as sodium lauryl sulfate, triethanolamine lauryl sulfate and triethanolamine polyoxyethylene lauryl ether sulfate; Oxyethylene alkyl ether sulfates; pyridoxine fatty acid esters; ascorbic acid fatty acid esters such as ascorbyl dipalmitate; sucrose fatty acid esters such as sucrose laurate; carboxylate salts such as iridium laurate; Alkyl phosphates such as sodium polyoxyethylene cetyl ether phosphate and polyoxyethylene alkyl ether phosphates; Sulfuric ester salts such as shea oil fatty acid monoglyceride sodium sulfate; soya lecithin, cephalin, phospholipids such as lipoic inositol;
Examples thereof include saponins such as senegasaponin, and cholic acids and salts thereof such as sodium ursodeoxycholate and sodium glycocholate, and one or more of these are used.

Suitable electrolytes include sodium chloride,
Potassium chloride, sodium carbonate, magnesium sulfate and the like can be mentioned. Preservatives include methyl paraben, sorbic acid, benzyl alcohol, chlorbutanol and the like.

Suitable antioxidants include sodium bisulfite, butylhydroxyanisole, ascorbic acid, sodium sulfite, tocopherol and the like.

Suitable pH adjusters include suitable acids, bases and buffers, and include, for example, water-soluble aliphatic carboxylic acids, inorganic acids, and organic or inorganic bases. Examples include acid, phosphoric acid, sulfuric acid, sodium hydroxide, potassium hydroxide, phosphate buffer, citrate buffer and the like.

Among the bases, aqueous gel bases include, for example, natural gums such as tragacanth gum, acacia gum, karaya gum, guaiac gum, xanthan gum, vinyl polymers such as polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl methyl ether and carboxypolymethylene. Examples include starch, pectin, sodium alginate and the like.

Other bases include fats and oils mainly containing triglycerides such as cocoa butter, palm oil, palm kernel oil, coconut oil, fractionated coconut oil, lard, witepsol ^(R) ; lanolin, reduced lanolin Waxes such as petrolatum, hydrocarbons such as petrolatum, squalene, and squalane; medium- and long-chain fatty acids such as capric acid, lauric acid, stearic acid, and oleic acid; lauryl alcohol, cetanol,
Higher alcohols such as stearyl alcohol and oleyl alcohol; fatty acid esters such as butyl stearate and dilauryl malonate; glycerin-substituted carboxylic acids such as glycerin medium-chain carboxylic acid esters such as triolein and tristearin or glycerin acetoacetate esters Glycerin esters such as esters; polyethylene glycols such as macrogol and setmacrogol; and the like, and one or more of these are used.

The preparation of the present invention can be produced by applying a usual method. For example, in the case of a liquid preparation, a solution containing or suspending a biologically active peptide in a solution containing a suitable pH adjuster or the like is added to a solution containing hyaluronic acid or a nontoxic salt thereof, followed by stirring. in this case,
By adding the above-mentioned additives as needed, it can be optimized. The preparation thus obtained can be further made into a better preparation by further adding a polymer substance, and in particular, a complex can be formed to make a very excellent preparation.

The solution thus obtained may further optionally contain a pH adjuster, a preservative, and the like, and may be further freeze-dried, concentrated under reduced pressure, or gelled. It can be made into a solid or powder, or into a gel by adding an agent. According to the present invention, re-dissolving or dispersing treatment can be carried out as needed, and further, a desired preparation can be obtained by appropriately heating or cooling.

The preparation of the present invention can be changed depending on the dosage form, the type of the main drug, the target animal (eg, a warm-blooded animal such as mouse, rat, cow, horse, human, etc.) and the purpose of administration. The amount may be an effective amount, for example, about 1 mg to 500
It can be appropriately selected from the range of mg / kg body weight. The number of doses can be changed in the same manner as above, but once a week.
It can be appropriately selected within a range of about 4 to 1 to 3 times a day.

The preparations obtained according to the invention exhibit excellent properties, but especially preparations with IFN-hyaluronic acid are desirably used in the treatment of cancer and viral diseases which require long-term treatment.

The preparation obtained according to the present invention comprises granules,
It can be administered nasally or orally as a tablet or liquid, can be applied to the skin or the like, and can be administered intravenously, intramuscularly or subcutaneously as an injection. An injection may be prepared and used as a powder at the time of use.

In particular, in the case of a preparation for interferon, it is preferable to administer a daily dose of 100 to 1,000 mg to an adult, but the dose may be adjusted as appropriate according to age and symptoms. The daily dose of the preparation of the interferon of the present invention can be administered once a day, but a sufficient effect can be expected even if administered once every three or seven days.

Next, the method for producing the physiologically active peptide preparation containing IFN of the present invention will be further described. That is, IFN is added to a solution containing hyaluronic acid or a nontoxic salt and stirred to obtain a hyaluronic acid-IFN mixture. If necessary, a polymer substance may be added and used as a liquid preparation. Further, this product may be used in the form of a gel semi-liquid by concentrating or increasing the concentration of additives, or by selecting a combination, or may be used as a solid powder by freeze-drying or the like. It can be freely selected according to the application.

[0039]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. In the following examples, the interferon titer was measured using cell lines (FL cells) derived from VS virus and human amniotic membrane and converted to international units (IU) by the cytopathic effect method.

Example 1 1 × 10 ⁸ units of human interferon-α (recombinant) was treated with sodium hyaluronate (average molecular weight 2.1 million)
The mixture was added to 5 ml of a 0.5 wt% physiological saline solution and mixed. Deacetylated chitin (chitosan) (average molecular weight 1
000) 1 ml of a 5% aqueous solution was added and mixed to adjust the pH to 6.0.
After maintaining for a time, an IFN-hyaluronic acid complex for injection was obtained. 0.5 ml of the interferon-α-hyaluronic acid complex obtained by the above method was used in rats (Wister strain,
Male, weight 200g, 7 weeks old, Charles River Japan
Was injected intramuscularly into the thigh and the blood titer was measured. The results are shown in FIG. In FIG. 1,-●-indicates an interferon-α-hyaluronic acid complex, and-○-indicates
The measurement results of interferon-α alone as a control are shown. The interferon-α / hyaluronic acid complex preparation has excellent properties.

Example 2 (gel) Sodium hyaluronate (average molecular weight 1,000,000) 1.5
Human interferon in 10 ml of wt% saline solution
After mixing 1 × 10 ⁸ units of β (natural type), 0.5 g of atelocollagen (manufactured by Sigma) was added and dissolved after adjusting the pH. This gel preparation can be used as an oral administration agent.
This formulation has excellent properties.

Example 3 (gel) Sodium hyaluronate (average molecular weight 2.5 million) 1.5
Human EGF (recombinant) in 100 ml of wt% saline solution
After mixing 50 μg, 5 g of atelocollagen was added,
After adjustment, it was dissolved. The gel preparation thus obtained is used as a therapeutic agent for burns.

Example 4 (Powder) 1 mg of human Cu-Zn-SOD (recombinant) was added under aseptic conditions to 1 wt% of sodium hyaluronate (average molecular weight 1.5 million)
After dissolving in 1000 ml of physiological saline solution, 10 g of hydroxypropylcellulose was added, dissolved and freeze-dried. The dried product is powdered and used as a sterile preparation for injection of rheumatoid arthritis, which is prepared at the time of use.

Example 5 (Ointment) Take 840 g of Macrogol ointment of the Japanese Pharmacopoeia on an ointment plate,
50 g of cetanol is kneaded with an ointment plate, and then IL-1β
(Recombinant) (Zenzyme) 1 mg, sodium hyaluronate (average molecular weight 1.5 million) 10 g, polylysine 10
g and an appropriate amount of a preservative were added, and finally physiological saline was added to make 1000 g, which was kneaded well to prepare. This one is
It is used as a treatment for wounds after surgery and bedsores in the elderly.

Example 6 In place of the chitosan of Example 1, gelatin, albumin,
Fibrin, fibrinogen, polylysine, polyglutamic acid, chondroitin sulfate, colominic acid, dextran, cyclodextran, DEAE-dextran,
Inulin, laminaran, polylactic acid (molecular weight: 500
0) and a solution prepared in the same manner using a physiological saline solution containing 1% of one selected from polyglycolic acid (molecular weight: 10,000). The preparation thus obtained has an excellent action.

Example 7 Bovine erythrocyte-derived Cu-Zn-SOD (Wako Pure Chemical Industries) 500 U
With sodium hyaluronate (average molecular weight 1.8 million)
After dissolving in 5 ml of 6 wt% physiological saline solution, 2 mg of gelatin (Nitta gelatin) was added and dissolved. The stability of lyophilized SOD in the resulting formulation was determined before and after
It was examined by measuring the enzyme activity of D. The results are shown in Table 1.

[0047]

(Equation 1)

[0048]

[Table 1]

The mixed preparation of SOD, hyaluronic acid and gelatin had a better activity retention effect than the other preparations.

Example 8 Macrophage colony stimulating factor (recombinant) 1 mg
Was added to 10 mg of human serum albumin, dissolved in 10 ml of a 0.6% by weight aqueous solution of sodium hyaluronate (average molecular weight: 1.6 million) and freeze-dried. This formulation is used as a ready-to-use type anticancer drug for injection.

Example 9 1 × 10 ⁶ units of TNF-β (recombinant) of tumor necrosis factor was sodium hyaluronate (average molecular weight 1.8 million)
The mixture was added to 3 ml of a 0 wt% phosphate buffer (pH 7.2) solution and mixed. Deacetylated chitin (chitosan)
(Average molecular weight 150,000) 2% hydrochloric acid solution (0.05M) 6ml
Was added and mixed to obtain a TNF-β-hyaluronic acid complex for injection. 1.0 ml of the TNF-β / hyaluronic acid complex obtained by the above method was injected subcutaneously into the back of a rat (Wister strain, male, body weight 200 g, 7 weeks old, Charles River Japan Co., Ltd.), and TNF in the blood was administered. -Β amount was measured by enzyme immunoassay. The results are shown in FIG. In FIG.
●-is the TNF-β / hyaluronic acid complex,-○-
Indicates the measurement result of the mixed preparation of TNF-β and hyaluronic acid as the control, and-□-indicates the measurement result of TNF-β alone as the control. The TNF-β / hyaluronic acid complex preparation has excellent properties.

[0052]

The preparation of the present invention can form a complex with hyaluronic acid by adding a high molecular substance in addition to hyaluronic acid, and is extremely excellent as a therapeutic drug. Furthermore, it is preferable also in the processability as a preparation.

[Brief description of the drawings]

1 shows the effect of delaying clearance in rat serum upon intramuscular injection of the interferon-α-sodium hyaluronate complex shown in Example 1.

FIG. 2 shows the effect of delaying clearance in rat serum upon subcutaneous administration of the TNF-β · sodium hyaluronate complex shown in Example 2.

Continuation of front page (56) References JP-A-1-156912 (JP, A) JP-A-63-146827 (JP, A) JP-A-60-155136 (JP, A) JP-A-60-59000 (JP) JP-A-61-236721 (JP, A) JP-A-59-59625 (JP, A) WO 90/5522 (WO, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/45 A61K 38/00 A61K 38/21 A61K 47/36 A61K 47/42 CA (STN) EMBASE (STN) MEDLINE (STN)

Claims (3)

(57) [Claims] 1. A scan over peroxide dismutase (SO
D), tumor necrosis factor (TNF-α and β), and interferon (IFN-α, IFN-β and IFN-γ)
A bioactive polypeptide preparation comprising a substance selected from the group consisting of: (a) when the bioactive peptide is superoxide dismutase, a non-toxic salt of superoxide dismutase and hyaluronic acid or hyaluronic acid; and hyaluronic acid (B) When the bioactive peptide is selected from the group consisting of tumor necrosis factor and interferon, and is a polymer substance which can be administered to a living body together with an acid or a non-toxic salt thereof, and which is made of gelatin. A non-toxic salt selected from the group consisting of tumor necrosis factor and interferon, hyaluronic acid or a non-toxic salt of hyaluronic acid, and a macromolecular substance which can be administered to a living body together with hyaluronic acid or a non-toxic salt thereof, and deacetylation Specially composed of chitin (chitosan) or atelocollagen A physiologically active polypeptide preparation to be characterized. 2. The preparation according to claim 1, wherein the preparation is in the form of a solid, liquid or gel semi-liquid. 3. The preparation according to claim 1, wherein the preparation is used as an antiviral agent, an anticancer agent, an agent for treating wounds, an antiinflammatory agent or an agent for treating bone-related diseases, depending on the physiologically active peptide contained. Preparations.