JP3449738B2 - Water-soluble composition - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a water-soluble composition useful as a sustained-release preparation of a pharmaceutically active substance.

[0002]

2. Description of the Related Art The purpose of a sustained-release preparation of a pharmaceutically active substance, especially a peptidic drug, is to maintain the concentration of peptide in a body fluid continuously within a certain effective concentration range. a) that the pharmacological effect does not depend so much on the effective concentration in body fluid, and that the excessive pharmacological effect is not harmful to the living body; b) that the pharmacological effect appears depending on the effective concentration in body fluid, and In some cases, it is necessary to adjust the dose regularly because the expression and the expression of excessive pharmacological effects are harmful to the living body.

Various types of bases for sustained-release preparations are known, and hyaluronic acid is one of them.
-213 discloses a technique relating to a sustained-release preparation of a physiologically active peptide containing hyaluronic acid and a non-toxic salt thereof. According to the publication, a decrease in calcium concentration in blood after subcutaneous administration of calcitonin or elcatonin continuous preparation having a sodium hyaluronate concentration of 5% to male rats continued for a minimum of 12 hours. There is disclosed an example in which the human growth hormone concentration preparation in the blood after administration to a male rat of a human growth hormone sustained-release preparation with 5% is maintained for at least 12 hours. In all of these, a clear extension of the duration is recognized as compared with the case where sodium hyaluronate of Comparative Example is not present. On the other hand, looking at another example of this publication, no sustained effect was observed even if a tegafur formulation having a sodium hyaluronate concentration of 5% was subcutaneously administered to male rats. By the way, these inventions utilize the diffusion delay of a substance in a solution due to the viscosity of hyaluronic acid at the site of administration, and, in the case of a substance containing a cation group, a hyaluronic acid macromolecule having a carboxyl group Ion exchange can occur at, and this exchange can further slow the diffusion of the material. In JP-A-2-213, it is said that the concentration of hyaluronic acid itself is most preferably 3 to 7%. However, due to its high viscosity, the removal of air bubbles becomes a big problem, and degassing by centrifugation or reduced pressure becomes necessary. At the same time, because of its high viscosity, it is necessary to use a thick injection needle, and the pain to the patient is not negligible. In addition, as in the case of using tegafur, there are cases where the sustained effect cannot be obtained even if the concentration of hyaluronic acid is raised to a high level. On the other hand, JP-A 1-2
Japanese Patent No. 87041 discloses an example using 1% concentration of hyaluronic acid. In the case of this intra-articular injection solution of sodium hyaluronate having a concentration of 1%, it is necessary to use a thick injection needle of about 18 to 20 G, which causes great pain to patients as an injection preparation for subcutaneous administration.

JP-A-62-129226 discloses that the concentration of hyaluronic acid in an injectable product is about 0.05% to 4%.
It is stated that it can be in the range of (weight) and can be made higher depending on the final use of the product, but there is no description about the application to a specific injection. However, as mentioned above, it is easily inferred that the sustained effect of the pharmaceutically active substance cannot be obtained without using a relatively high concentration of hyaluronic acid. That is, the sustained release effect of the pharmaceutically active substance may be obtained depending on the viscosity of the high concentration hyaluronic acid, and by lowering the pH to the acidic side (for example, near pH 2.5), the hyaluronic acid By utilizing the fact that the viscosity is increased, there is a possibility that the sustained release effect of the pharmaceutically active substance can be similarly obtained. However, as the viscosity of hyaluronic acid increases, it becomes more difficult to administer it as an injection, and the pH of the solution of hyaluronic acid becomes physiological.
It is feared that the more it deviates from the region, the more it affects the stability of the pharmaceutically active substance and the more it damages the living tissue site to which it is administered. JP-A-3-4790 describes a method of stabilizing an enzyme by adding a proteinaceous substance and a polysaccharide to an aqueous solution of a proteolytic enzyme produced by Bacillus licheniformis. It is not a peptide secreted by.

[0005]

Although it has been attempted to use hyaluronic acid for the purpose of preparing a sustained-release formulation of a pharmaceutically active substance as described above, its compatibility when administered to a living body has not yet been solved. There are no problems left.

[0006]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to solve the above-mentioned problems and found that water-soluble hyaluronic acid as a pharmaceutically active substance and substantially no pharmacological activity were injected into body fluids. By administering a water-soluble preparation containing a water-soluble protein, it is possible to maintain the pharmacological effect while suppressing the rapid onset of the pharmacological effect in the initial period of administration without impairing the pharmacological activity of the pharmaceutically active substance. As a result of the discovery and further research based on this, the present invention was completed.

The present invention relates to (i) a pharmaceutically active substance or a chemically synthesized pharmaceutically active substance which is a polypeptide secreted by a living body or a derivative thereof, other than erythropoietin,
(B) A water-soluble composition comprising water-soluble hyaluronic acid or a non-toxic salt thereof and (c) a water-soluble protein which has substantially no pharmacological activity and can be injected into a body fluid.

In the present invention, a pharmaceutically active substance which is a polypeptide secreted by the living body or a derivative thereof, or a chemically synthesized pharmaceutically active substance may be simply referred to as "pharmaceutical active substance" in the present specification. In the present invention, the polypeptide as a pharmaceutically active substance secreted by the living body is produced in the living body and secreted into the bloodstream (endocrine) or into surrounding tissue fluid (paracrine or autocrine secretion). ), Or a secretory peptide or protein secreted outside the body (exocrine). Furthermore, in the present invention, the polypeptide as a pharmaceutically active substance may be any one that can be produced by a living body, and when used, it may be one extracted from a living body or one obtained by a genetic engineering technique.

The living body in the present invention includes a living body composed of a single eukaryotic cell, a living body composed of a plurality of eukaryotic cells, a tissue or an organ excised from the living body, and a cell isolated from the living body. . Yeast is mentioned as an example of the living body which consists of this single eukaryotic cell. Examples of living bodies composed of the plurality of eukaryotic cells include vertebrates and invertebrates. Examples of the above-described isolated eukaryotic cells include cancer cells isolated from humans and mice. Examples of the vertebrates include mammals (eg, humans, rodents (eg, rat, mouse), cows, horses, sheep), birds (eg, poultry (eg,
Chicken), fish (eg salmon, tuna), reptile (eg snake)
Is mentioned. Examples of the invertebrates include leeches and spiders. The above-mentioned living body is preferably a living body composed of a plurality of eukaryotic cells, more preferably a human,
Examples include snakes, spiders, and leeches.

The peptide preferably has, for example, an octanol / water partition ratio of about 0.1 or less, and more preferably a partition ratio of about 0.05 or less. When the pharmaceutically active substance is a peptide, the peptide preferably has a molecular weight of about 200 to 200,000, particularly preferably about 300 to 90,000. The peptide may be a naturally produced peptide, a gene recombinant method, or a chemically synthesized peptide.

Examples of the polypeptide include, for example, cytokines, peptide hormones, growth factors, factors acting on the cardiovascular system, cell adhesion factors, factors acting on the central and peripheral nervous systems, fluid electrolytes and blood organic substances. Acting factors, bone and skeleton acting factors, digestive system acting factors, renal and urinary system acting factors, connective tissue and skin acting factors, sensory organ acting factors, immune system acting Factors, factors that affect the respiratory system,
Factors that act on the reproductive system, and enzymes.

The polypeptide includes, among others, cytokines, peptide hormones, growth factors, factors acting on the cardiovascular system, factors acting on the central and peripheral nervous systems,
Factors that affect body fluid electrolytes and organic blood substances, factors that affect bones and skeletons, factors that affect the digestive system, factors that affect the immune system, factors that affect the respiratory system, factors that affect the reproductive system, and Enzymes are preferred.

Examples of the above-mentioned cytokines include lymphokines, monokines, and hematopoietic factors. Examples of the lymphokines include interferons (eg, interferon-α, -β, -γ), interleukins (eg, interleukin-2 to -11) and the like. Examples of the monokine include interleukin-1, tumor necrosis factor (eg, TNF-α,-
β), malignant leukocyte inhibiting factor (LIF) and the like. Examples of hematopoietic factors include granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and the like. Examples of the hematopoietic factor further include a factor having a thrombopoiesis (proliferation) action, and examples thereof include leukocyte growth factor preparations (Leukoprol, Morinaga Milk Industry), thrombopoietin, platelet growth stimulating factor and megakaryocyte growth (stimulating) factor. Is mentioned.

Examples of factors acting on bone and skeleton include, for example, calcitonin, bone Gla peptide, parathyroid hormone or active fragments thereof (osteostatin, Endocrinology, 129 , 324 (1991)), histone H4-related bone formation. Proliferative peptide (OGP, The E
MBO Journal, 11 , 1867 (1992)), or muteins thereof, or derivatives thereof, or analogs thereof. Examples of the growth factors include nerve growth factors (NGF, NGF-2 /
NT-3), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IG
F), transforming growth factor (TGF), platelet-derived cell growth factor (PDGF) and the like. Examples of the peptide hormone include insulin, growth hormone, luteinizing hormone-releasing hormone (LH-RH), adrenocorticotropic hormone (ACTH), amylin, oxytocin, luteinizing hormone or their derivatives, analogs and homologues. And so on. Examples of the LH-RH analogs include known ones, and among them, for example, those described in US Pat. No. 4,008,209 can be mentioned.

Factors acting on the cardiovascular system include factors controlling blood pressure and arteriosclerosis, such as endothelin, endothelin inhibitor, vasopressin, renin, angiotensin I, angiotensin II, angiotensin III, angiotensin I inhibitor,
Examples include angiotensin II receptor antagonists, atrial natriuretic peptide (ANP), antiarrhythmic peptides and the like. Examples of the factor having an action on the central and peripheral nervous system include, for example, analgesic anesthetic peptides (eg, enkephalin, endorphin, kyotorphin), neurotrophic factor (NTF), calcitonin gene-related peptide (CGRP), pituitary adenylate. Cyclase-activating polypeptide (PACAP), thyroid hormone-releasing hormone (TRH), salts of TRH, and derivatives thereof (Japanese Patent Laid-Open No. 1271273 (US Pat. No. 39).
59247), JP-A-52-116465 (US Patent N
4100152)), neurotensin and the like.

Examples of factors that act on the digestive system include secretin and gastrin. Examples of factors that act on the immune system include factors that control inflammation and malignant neoplasms, factors that attack infectious microorganisms, and the like. Examples of factors that act on body fluid electrolytes and blood organic substances include factors that control blood coagulation, plasma cholesterol concentration and metal ion concentration, and the like. These peptides or factors include in its concept soluble receptors for the polypeptide. Examples of cell adhesion factors include laminin and intercellular adhesion factor 1 (ICAM1).

Examples of the polypeptide include peptides or proteins that are naturally produced or can be produced by chemical synthesis or genetic engineering techniques and serve as antigens, for example, cedar pollen or futabusa pollen. These are administered alone, or in the state of being bound to a hapten, or together with an adjuvant, in the composition of the present invention as an injection. The peptide may further include an enzyme of natural origin or produced by a gene recombination method, and examples of administable enzymes include, but are not limited to, superoxide dismutase (SOD), asparaginase, and kallikrein. It is not something that will be done.
Each of these peptides or factors may include synthetic polymers such as polyethylene glycol, or natural polymers such as chondroitin, polysaccharides, or chemically modified with non-peptidic substances. The non-peptidic substance here may be a ligand for a receptor or an antigen for an antibody.

Further, the above-mentioned polypeptide or factor may include a plurality of peptides linked by a chemical method or a genetic recombination technique. The above-mentioned peptide or factor may be a mutein having a different amino acid sequence or sugar chain, a factor having no sugar chain, or a derivative, analog (analog) or homologue thereof, and It may be an active fragment and they may have either mechanism of action of antagonist or agonist. In the above-mentioned polypeptide, interferons, interleukins, G-CSF, GM-CSF, M-CSF, FGF,
More preferred are TNF, parathyroid hormone, calcitonin, insulin, LH-RH or muteins, analogs, homologues or active fragments thereof. Examples of chemically-synthesized pharmaceutically active substances include those acting on the cardiovascular system, those acting on cell adhesion, those acting on the central and peripheral nervous systems, those acting on body fluid electrolytes and organic substances in blood, bone and skeleton. Acting on the body, those acting on the digestive system, those acting on the renal-urinary system, those acting on connective tissue and skin, those acting on the sensory organs, those acting on the immune system, acting on the respiratory system And those that act on infectious microorganisms and those that act on the reproductive system.

As the above-mentioned chemically-synthesized pharmaceutically active substances, those acting on the cardiovascular system, those acting on cell adhesion, those acting on the central and peripheral nervous systems, those acting on body fluid electrolytes and organic substances in blood, bones. Also preferred are those acting on the skeleton, those acting on the digestive system, those acting on the immune system, those acting on the respiratory system, and those acting on infectious microorganisms. Examples of the chemically-synthesized pharmaceutically active substances that act on the above cardiovascular system include calcium antagonists, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists, potassium channel openers, and the like. An RGD antagonist is mentioned as an example of the chemosynthetic pharmaceutical active substance which acts on the above-mentioned cell adhesion. Examples of the chemically-synthesized pharmaceutically active substances that act on the above-mentioned central and peripheral nerves include psychotropic or antipsychotic agents such as diazepam, antiemetics, and anti-dementia agents such as benzoquinone derivatives.

Examples of the chemically synthesized pharmaceutically active substance acting on the above-mentioned body fluid electrolyte and organic substance in blood include those which control blood coagulation and plasma cholesterol concentration such as pravastatin sodium salt. Examples of the chemosynthetic pharmaceutically active substances that act on the bones and skeletons mentioned above include bisphosphonates, ipriflavone, anti-osteoporosis agents such as vitamin D ₃ , and antirheumatic agents. Examples of the chemically-synthesized pharmaceutically active substances that act on the above-mentioned digestive system include proton pump inhibitors such as lansoprazole and H2 receptor antagonists such as cimetidine. Examples of the chemically-synthesized pharmaceutically active substances that act on the above-mentioned immune system include anti-inflammatory drugs such as indomethacin, anticancer drugs such as adriamycin and cisplatin, antiallergic agents, and non-peptidic antigen substances.

Examples of the chemically synthesized pharmaceutically active substances that act on the above-mentioned respiratory system include anti-asthma drugs. Examples of the chemically synthesized pharmaceutically active substance acting on the above-mentioned infectious microorganism include antiviral agents, antibiotics such as cephalosporins and penicillins. Examples of the chemically-synthesized pharmaceutically active substances that act on the above-mentioned reproductive system include sex steroids such as testosterone. Examples of the above-mentioned chemically synthesized pharmaceutically active substances further include hormones (eg dexamethasone, prednisolone) and non-peptidic antigen substances. Examples of the above-mentioned chemically synthesized pharmaceutically active substance include inorganic substances and organic substances. Further, as the above-mentioned chemically synthesized pharmaceutically active substance, prostaglandin, leukotriene, saccharide, polysaccharide, metal chelate, or nucleic acid such as antisense RNA or DNA may be contained. The above-mentioned chemically synthesized non-peptidic antigen substance is administered as an injection alone or in combination with a hapten, or with an adjuvant in the composition of the present invention.

As hyaluronic acid in the present specification,
Hyaluronic acid, which is a type of mucopolysaccharide composed of N-acetylglucosamine and glucuronic acid, and a non-toxic salt thereof can be mentioned. Further, a hyaluronic acid derivative or a non-toxic salt thereof may be used. Examples of the non-toxic salt include alkali metal salts such as sodium and potassium, and alkaline earth metal salts such as magnesium and calcium. Especially, the sodium salt is most preferably used. Hyaluronic acid and its non-toxic salts have a molecular weight of 10,000 to 5,000,000 (viscosity method: limiting viscosity and Mark-Houvinc)
(Using a formula), preferably about 100,000 to 3,000,000. .

Examples of the water-soluble protein (which may be hereinafter referred to as "water-soluble protein") which has substantially no pharmacological activity and can be injected into a body fluid in the composition of the present invention include serum albumin, globulin, and the like. Examples include collagen and gelatin. Of these, serum albumin is preferable. It is preferable that the water-soluble protein is not chemically modified. The pharmacological activity as used herein is defined as the substantial pharmacological activity caused by the concentration of the water-soluble protein used in the unit dose.

The water-soluble composition of the present invention is adjusted to have a viscosity of about 500 centipoise (cp) or less when made into an aqueous solution, and preferably adjusted to a range of about 50 to 400 cp. The viscosity is affected by the molecular weight and concentration of hyaluronic acid, the concentration of water-soluble proteins, the concentration of pharmaceutically active substances, and the concentration of salts such as salt (used as a tonicity agent described later), and the water-soluble composition. The pH of the product is also affected to some extent. Therefore, it is only necessary to select the conditions that give the above viscosity in accordance with the desired water-soluble composition.

The viscosity in this specification means the viscosity measured at a temperature of 25 ° C. using a cone LD using an E-type viscometer (manufactured by Tokyo Keiki). The preferred concentrations of hyaluronic acid and water-soluble protein used in the present water-soluble composition are listed below. The concentration of water-soluble hyaluronic acid used is preferably about 0.
01 to 3% (W / V), more preferably about 0.05
~ 2% (W / V). In the composition of the present invention, for example, when serum albumin is used as the water-soluble protein, its use concentration is about 0.001 to 5% (W / V),
More preferably, it is about 0.005 to 2% (W / V). When globulin is used as the water-soluble protein, its concentration is about 0.001-5% (W / V), more preferably about 0.005-2% (W / V).
When collagen is used as the water-soluble protein, the concentration used is about 0.001 to 1% (W / V), more preferably about 0.005 to 0.2% (W / V). When gelatin is used as the water-soluble protein, the concentration used is about 0.001 to 1% (W / V), more preferably about 0.005 to 0.5% (W / V).

The pH of the solution of the water-soluble composition of the present invention does not significantly affect the activity of the pharmaceutically active substance, is within the range acceptable as an injectable solution, changes the viscosity of the solution to a large extent, and precipitates. A pH that does not cause the formation of It is preferably pH4 to pH8. More preferably, pH5
Is about pH8. The compounding ratio of the pharmaceutically active substance may be an effective amount depending on the activity of the substance and the therapeutically necessary amount. Generally, the weight ratio of the pharmaceutically active substance to hyaluronic acid is preferably about 0.0001. : 1 to 10:
1, preferably about 0.0002: 1 to 5: 1, more preferably about 0.0002: 1 to 1: 1 and even more preferably about 0.0002: 1 to 0.1: 1. Similarly, the mixing ratio of the water-soluble protein cannot be unconditionally determined, but the amount usually added to an injectable pharmaceutical composition can be added.
The weight ratio of water soluble protein to hyaluronic acid is about 0.001:
1 to 100: 1 is preferable, and more preferably about 0.0.
1: 1 to 10: 1, most preferably about 0.1 to 10: 1
Is.

The composition of the present invention is preferably such that the pharmaceutically active substance, hyaluronic acid and water-soluble protein are present in a unit dose. For example, dissolve or suspend in sterile water or saline in ampoules or vials so that the three are present. In this case, it may be prepared by mixing a solution of a pharmaceutically active substance, a solution of hyaluronic acid and a solution of a water-soluble protein, or by adding a powder of hyaluronic acid and a powder of a water-soluble protein to the solution of a pharmaceutically active substance. Alternatively, the preparation method by other combinations may be used. Alternatively, it may be prepared by adding sterilized water or sterilized physiological saline to freeze-dried or vacuum-dried powder in which a pharmaceutically active substance, hyaluronic acid and a water-soluble protein are mixed. Additives conventionally used in this unit dosage, for example, pH regulators (eg, glycine, hydrochloric acid, sodium hydroxide), local anesthetics (eg, xylocaine hydrochloride, chlorobutanol), isotonic agents (eg, salt) , Mannitol, sorbitol), an adsorption inhibitor (eg, Tween 80), etc. may be contained.
The unit dose may further include additives such as pharmaceutically acceptable polyethylene glycol 400 and dextran.

In the sustained-release preparation of the present invention, it is desirable that when the main components are mixed, the activity of the pharmaceutically active substance is retained and almost no bubbles are generated in the solution during the mixing operation. The respective main components are put in a container (for example, a bottle, a dough, or the like. The material is selected so as not to impair the activity of the pharmaceutically active substance) simultaneously or in an appropriate order.
The total volume of each major component is preferably three-fourths of the container, more preferably at most three-fifths, even more preferably at most one-half and most preferably at most one-third. . The vessel is agitated under mild conditions or, preferably, tumbled about the major axis of the vessel. The number of rotations of the container is determined in consideration of, for example, the volume of the container, the sum of the volumes of the main components, the concentration of hyaluronic acid or its non-toxic salt, the temperature of the atmosphere of the container, and the like. When the container is rotated about the long axis of the container, the preferable number of rotations is 10 to 1,000 rotations / minute, but the number of rotations is not limited to this. The atmosphere in the container may be sterilized normal air or sterilized clean nitrogen gas. The solution obtained by the above operation may be transferred to a small-capacity vial or ampoule and the like and further subjected to freeze-drying operation.

The water-soluble composition of the present invention is preferably a parenteral preparation. Examples of the parenteral preparations include injection preparations, transmucosal solutions, nasal drops, ear drops. The injection preparations include intravenous administration, subcutaneous administration,
Examples include intra-articular administration, intramuscular administration, intraocular administration, etc. Since the viscosity of the sustained release preparation is adjusted to about 500 cp or less, it is possible to easily aspirate the sustained release preparation from the ampoule or the vial into the syringe using the 25G or 26G injection needle. The bubbles generated during suction can be easily removed by simply leaving them standing for a short time. The composition of the present invention may be dissolved in water, or may be freeze-dried with a crystalline substance such as mannitol. The lyophilized product is made into an aqueous solution by adding sterile water or sterile physiological saline. In the case of a freeze-dried composition, it may be adjusted so that the viscosity and the concentration when made into an aqueous solution are as described above.

The liquid composition of the present invention filled in a soft capsule or the freeze-dried powder filled in a hard capsule or formed into a tablet may be administered to the stomach, large intestine or rectum. For example, poly (lactic acid-glycolic acid)
Liquid or powder composition of the invention dissolved or dispersed in a solution of a biodegradable polymer such as a copolymer, polyhydroxybutyric acid, poly (hydroxybutyric acid-glycolic acid) copolymer, or mixtures thereof. Are, for example, films, microcapsules (microspheres), nanocapsules (nanospheres) according to a method known per se.
Can be molded into Furthermore, the composition of the present invention comprises phospholipid, cholesterol or a derivative thereof, and is encapsulated in a liposome prepared by a method known per se, and then in a physiological saline solution or a hyaluronic acid solution dissolved in a physiological saline solution. May be dispersed in. Furthermore, the composition of the present invention, which is a liquid agent, has an appropriate viscosity and thus exhibits resistance to physical disturbance such as vibration, so that a syringe with a needle is pre-filled with the composition of the present invention. Prefilled Syringe prescribed to the patient, allowing self-administration by the patient.

The composition of the present invention is stored at a normal temperature or a normal refrigerating temperature, preferably 0 ° C. to room temperature (eg about 2 ° C.).
5 ° C), more preferably + 2 ° C to + 8 ° C. The dose, administration target, administration target disease and the like of the composition of the present invention depend on those of the pharmaceutically active substance. In the composition of the present invention, the components added to the pharmaceutically active substance have low toxicity. The water-soluble composition of the present invention has excellent durability and can sufficiently exhibit this effect even with a low concentration of hyaluronic acid. Therefore, it becomes possible to administer with a thin injection needle, and a patient's pain is reduced. This composition has a low viscosity and therefore the formation of bubbles can be significantly suppressed. Therefore, it is easy to use in clinical situations.

[0032]

The present invention will be described in more detail with reference to the following examples and experimental examples, but the present invention is not limited thereto. Example 1. Continuous preparation of basic fibroblast growth factor mutein Recombinant human basic FGF mutein CS23 (hereinafter sometimes abbreviated as rhbFGF mutein CS23) (EP Publication 281,822) Solution (0.96)
0.36 ml of milligram protein / ml, physiological saline for injection 1.26 ml, sodium hyaluronate (average molecular weight 1.47 million daltons:
Genzyme Co., Ltd.) (8.1 mg) was added, and 6 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin was further added. Viscosity 36
6 cp.

Example 2. Nerve cell growth factor (NGF)
(Biomedical Technology, Inc., USA) 4 ml of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 ml of physiological saline containing 10 μg, and physiological saline 1.4 for injection was added. Add milliliters (Fuso Yakuhin) to bring the total volume to 2.
I made it 1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 285c
p.

Example 3. Epidermal growth factor (EFG)
(Chemicon International Inc., USA) 4 ml of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 ml of physiological saline containing 10 μg, and further 1.4 ml of physiological saline for injection. (Fuso Yakuhin) is added and the total amount is 2.
I made it 1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 305c
p.

Example 4. Insulin-like growth factor (IG
F) 4 ml of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 ml of physiological saline containing 10 μg (Chemicon International) and 1.4 ml of physiological saline for injection. (Fuso Yakuhin) was added to make the total volume 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 298 cp.

Example 5. 4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 milliliters of physiological saline containing 10,000 international units of interferon α (Lee Biomolecular Research Lab, USA), and physiological saline for injection. 1.4 ml of water (Fuso Yakuhin) was added to bring the total volume to 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 312 cp.

Example 6. Interferon β (Pazel, Germany) Saline solution containing 10,000 international units.
4 ml of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 7 ml, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 320 cp.

Example 7. Saline solution containing 10,000 units of interferon γ (Genzyme, USA) 0.7
4 ml of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to ml, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. Also,
Hyaluronic acid sodium salt (average molecular weight 14
70,000 Daltons: Genzyme Inc.) 10.5 milligrams were added. Viscosity 311 cp.

Example 8. Interleukin 2 (IL-
2) (A mixture of a product having Met at the N-terminus and a product not having Met at the N-terminal, which is produced by the method described in JP-A-61-78799 and purified by the method described in JP-A-60-115528). Saline containing microgram 0.7
4 ml of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to ml, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. Also,
Hyaluronic acid sodium salt (average molecular weight 14
70,000 Daltons: Genzyme Inc.) 10.5 milligrams were added. Viscosity 320 cp.

Example 9. Transforming growth factor (TGF-
β) (manufactured by Wako Pure Chemical Industries, Ltd.) 2 ml of human serum albumin was added to 0.7 ml of physiological saline containing 1 microgram.
4 microliters of 0% albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) was added, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. In addition, sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) 1 was added to this solution.
0.5 milligram was added. Viscosity 353 cp.

Example 10. Parathyroid hormone (PTH)
(Bachem Fine Chemicals, Switzerland) 4 ml of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 ml of physiological saline containing 10 μg, and physiological saline for injection 1.4. Add milliliters (Fuso Yakuhin) to bring the total volume to 2.
I made it 1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 251c
p.

Example 11. 4 micron of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin in 0.7 ml of physiological saline containing 5000 units of granulocyte-macrophage colony stimulating factor (GM-CSF) (ICN Biomedicals, USA). Then, 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml.
In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 301 cp.

Example 12 Macrophage colony stimulating factor (M-CSF) (ICN Biomedicals) 5
4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 milliliters of physiological saline containing 000 units, and 1.4 ml of physiological saline for injection was added to make the total amount. I made it 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 2
99 cp.

Example 13. Acidic fibroblast growth factor (a
FGF) (Toyobo) 4 ml of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 ml of physiological saline, and 1.4 ml of physiological saline for injection (Fuso) Chemicals) was added to bring the total volume to 2.1 ml.
In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 274 cp.

Example 14 Tumor necrosis factor (TNF-α)
(Wako Pure Chemical Industries, Ltd.) 0.7 ml of physiological saline for injection (Fuso Yakuhin) was added to 1 microgram, and 4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added and further injected. The physiological saline 1.4 ml (Fuso Yakuhin) was added to bring the total volume to 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 286 cp.

Example 15 Serratia superoxide dismutase (SOD) (JP-A-57-29285, JP-A-58-16685) Albumin Nichiyaku containing 20% of human serum albumin in 0.7 ml of physiological saline containing 400 units (Nippon Pharmaceutical)
4 microliters of the above was added, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 291 cp.

Example 16. Albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin in 0.7 ml of physiological saline containing 10 micrograms of vasopressin (Cambridge Research Biochemical, UK)
4 microliters of the above was added, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 220 cp.

Example 17 20% of human serum albumin was added to 0.7 ml of physiological saline containing 10 micrograms of somatostatin (Bachem Fine Chemicals).
4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) contained therein was added, and further 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 249 cp.

Example 18. Oxytocin (Bachem Fine Chemicals Co., Ltd.) 0.7 ml of physiological saline containing 4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 1.4 ml of physiological saline for injection. (Fuso Yakuhin) was added to make the total volume 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 253 cp.

Example 19 4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 milliliters of physiological saline containing 10 micrograms of luteinizing hormone-releasing hormone (Bachem Fine Chemicals), and physiological saline for injection was further added. 1.4 ml (Fuso Yakuhin) was added to bring the total volume to 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 241 cp.

Example 20. 4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 milliliters of physiological saline containing 10 micrograms of growth hormone-releasing factor (Bachem Fine Chemicals), and physiological saline for injection 1 0.4 ml (Fuso Yakuhin) was added to bring the total volume to 2.1 ml. In addition, sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) 1 was added to this solution.
0.5 milligram was added. Viscosity 265 cp.

Example 21. 4 ml of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 ml of physiological saline containing 10 μg of growth hormone (UCB Bioproducts, Belgium), and physiological saline for injection was added. 1.
4 ml (Fuso Yakuhin) was added to bring the total volume to 2.1 ml. In addition, sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution.
10.5 milligrams was added. Viscosity 293 cp.

Example 22. 20% human serum albumin was added to 0.7 ml of physiological saline containing 10 micrograms of calcitocin (UCB Bioproducts).
4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) contained therein was added, and further 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 238 cp.

Example 23. 4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 milliliters of physiological saline containing 10 micrograms of calcitonin gene-related peptide (UCB Bioproducts) and further physiological saline for injection. Add 1.4 ml of water (Fuso Yakuhin) to bring the total volume to 2.
I made it 1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 229c
p.

Example 24. Brain serum natriuretic peptide (Peptide Institute, Japan) 2 ml of human serum albumin was added to 0.7 ml of physiological saline containing 10 micrograms.
4 microliters of 0% albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) was added, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. In addition, sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) 1 was added to this solution.
0.5 milligram was added. Viscosity 241 cp.

Example 25. 4 ml of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 ml of physiological saline containing 10 μg of atrial natriuretic peptide (Cambridge Research Biochemicals), and physiological saline for injection was added. 1.4 ml of water (Fuso Yakuhin) was added to bring the total volume to 2.1 ml. In addition, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 2
43 cp.

Example 26. 4 ml of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 ml of physiological saline containing 10 μg of osteocalcin analog (American Peptide, USA), and physiological saline for injection 1.
4 ml (Fuso Yakuhin) was added to bring the total volume to 2.1 ml. In addition, sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution.
10.5 milligrams was added. Viscosity 265 cp.

Example 27. To 0.7 ml of physiological saline containing 10 micrograms of hepatocyte growth factor (Peptide Institute), 4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added, and physiological saline for injection was added. 4 ml (Fuso Yakuhin) was added to bring the total volume to 2.1 ml. In addition, sodium hyaluronate (average molecular weight 147
10,000 Dalton: Genzyme Co., Ltd.) 10.5 mg was added. Viscosity 271 cp.

Example 28. Urokinase (Nippon Chemical Research Co., Ltd.) 4 ml of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 ml of physiological saline containing 1.4 ml of physiological saline for injection. (Fuso Yakuhin) was added to make the total volume 2.1 ml.
Further, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 311 cp.

Example 29. To 0.7 ml of physiological saline containing 10 micrograms of tissue plasminogen activator (TPA) (Bioscott, UK), 4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added. 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to bring the total volume to 2.1 ml. Further, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 33
5 cp.

Example 30. Interleukin 3 (IL
-3) (R & D Systems, USA) Albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin in 0.7 ml of physiological saline containing 1 microgram.
4 microliters, and then 1.4 ml of physiological saline for injection (Fuso Yakuhin) is added to make the total volume 2.1 ml. In addition, sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution.
10.5 milligrams was added. Viscosity 259 cp.

Example 31. Interleukin 4 (IL
-4) (R & D Systems, Inc.) To 0.7 ml of physiological saline containing 1 microgram, 4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin was added, and physiological saline for injection 1.4 was added. Add milliliters (Fuso Yakuhin) to make the total volume 2.1 ml. Also, add hyaluronic acid sodium salt to this solution.
(Average molecular weight 1.47 million daltons: Genzyme) 10.
5 milligrams were added. Viscosity 265 cp.

Example 32. Interleukin 5 (IL
-5) (Amgen, USA) 4 ml of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 ml of physiological saline containing 1 microgram, and physiological saline for injection was further added 1. Add 4 ml (Fuso Yakuhin) to make the total volume 2.1 ml. Also, add hyaluronic acid sodium salt to this solution.
(Average molecular weight 1.47 million daltons: Genzyme) 10.
5 milligrams were added. Viscosity 282 cp.

Example 33. Interleukin 6 (IL
-6) (Amgen) 20% human serum albumin in 0.7 ml of physiological saline containing 1 microgram.
4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) contained therein was added, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. Further, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 249 cp.

Example 34. Interleukin 7 (IL
-7) (Genzyme) 20 ml of human serum albumin was added to 0.7 ml of physiological saline containing 1 microgram.
% Of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) is added, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) is added to make the total volume 2.1 ml. Further, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 255 cp.

Example 35. Interleukin 8 (IL
-8) Human serum albumin 20 was added to 0.7 ml of physiological saline containing 1 microgram (Genzyme).
% Of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) is added, and then 1.4 ml of physiological saline for injection (Fuso Yakuhin) is added to make the total volume 2.1 ml. Further, 10.5 mg of sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme) was added to this solution. Viscosity 263 cp.

Example 36. Long-acting insulin preparation Porcine insulin (26.8 units / milligram: DIOSY
(NTH, The Netherlands) 3 mg is dissolved in 0.1 ml of 0.1 N hydrochloric acid to prepare a physiological saline solution for injection 1.65.
Add milliliter and add hyaluronic acid sodium salt (average molecular weight 1.47 million daltons: Genzyme) 1
5.75 milligrams were added. 4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to the obtained solution. Viscosity 384c
p.

Example 37. Granulocyte colony stimulating factor (G
-CSF) A continuous preparation of human granulocyte colony stimulating factor (Nupogen: Amgen) containing 300 micrograms of recombinant human G-CSF (Amgen), 50 milligrams of mannitol and 0.004% of Tween 80 in 1 milliliter of continuous preparation. 4.154 ml of physiological saline for injection was added to 0.0466 ml of Hyaluronic acid sodium salt, and 21 mg of hyaluronic acid sodium salt (average molecular weight 1.47 million daltons: Genzyme) was added. 8 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to the obtained solution. Viscosity 264c
p.

Example 38. Long-acting insulin preparation Porcine insulin (26.8 units / milligram: DIOSYN
TH) (3 mg) is dissolved in 0.1 N hydrochloric acid (1.5 ml), and physiological saline for injection (1.65 ml) is added thereto. Hyaluronic acid sodium salt (average molecular weight 1.47 million daltons: Genzyme) 15. 75 milligrams were added. Human immunoglobulin G (Cappel) was added to the resulting solution in an amount of 0.5 mg. viscosity
328 cp.

Example 3 In 300 milliliters of recombinant human G-CSF, 300 micrograms, mannitol, 50 milligrams, and Tween 80, 0.00
Albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 0.037 ml of a human granulocyte colony stimulating factor preparation (Nupogen: Amgen) containing 4% of physiological saline for injection (1.377 ml) and containing 20% of human serum albumin Was added at 8 microliters. 0.7 ml of a solution prepared by dissolving sodium hyaluronate (average molecular weight 1.47 million daltons: Genzyme Co., Ltd.) in physiological saline for injection (Fuso Yakuhin) to a concentration of 1.5% (W / V). Was added. A glass vial containing these solutions (capacity: about 5 ml) was hermetically sealed, and was rotationally mixed about a long axis in a 200 ml-capacity eggplant-shaped flask attached to a three-one motor (made by Haydon) for about 1 hour ( 20 to 100 revolutions / minute) to prepare a dosing solution of a sustained-release preparation having almost no air bubbles.

Example 4 0.1 microliter of a human granulocyte colony stimulating factor preparation (Nypogen: Amgen) containing 300 micrograms of recombinant human G-CSF, 50 milligrams of mannitol and 0.004% of Tween 80 in 0.1 milliliter. 1.377 ml of physiological saline for injection was added to 023 ml, and 4 μl of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added. To this solution, 0.7 ml of a solution of sodium hyaluronate (average molecular weight of about 500,000 daltons) dissolved in physiological saline for injection (Fuso Yakuhin) to a concentration of 1.5% (W / V) was added. It was A glass vial containing these solutions (capacity: about 5 ml) was hermetically sealed, and was rotationally mixed about a long axis in a 200 ml-capacity eggplant-shaped flask attached to a three-one motor (made by Haydon) for about 1 hour ( Two
(0 to 100 revolutions / minute) to prepare an administration solution of a sustained-release preparation having almost no air bubbles.

Example 41 3 ml of recombinant human G-CSF was prepared in 1 ml.
00 micrograms, mannitol 50 milligrams,
0.023 of a human granulocyte colony stimulating factor preparation (Neupogen: Amgen) containing 0.004% of Tween 80
To the milliliter, 1.039 ml of physiological saline for injection was added, and 4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added. To this solution, 1.039 ml of a 1% sodium hyaluronate salt solution (average molecular weight: about 800,000 daltons to 900,000: Alz Note, Seikagaku Corporation) was added. A glass vial containing these solutions (capacity: about 5 ml) was hermetically sealed, and was rotationally mixed about a long axis in a 200 ml-capacity eggplant-shaped flask attached to a three-one motor (made by Haydon) for about 1 hour ( 20 to 100 revolutions / minute) to prepare a dosing solution of a sustained-release preparation having almost no air bubbles.

Example 4 In 0.1 ml of a human granulocyte colony stimulating factor preparation (Neupogen: Amgen) containing 300 micrograms of recombinant human G-CSF, 50 milligrams of mannitol and 0.004% of Tween 80. 1.377 ml of physiological saline for injection was added to 023 ml, and 4 μl of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added. Hyaluronic acid sodium salt (average molecular weight of about 1.8 million daltons:
A solution of Genzyme Co., Ltd. dissolved in physiological saline for injection (Fuso Yakuhin) to a concentration of 1.2% (W / V).
7 ml was added. A glass vial containing these solutions (capacity of about 5 ml) was hermetically sealed and mounted on a three-one motor (made by Haydon) with a capacity of 200.
By rotating and mixing (20 to 100 revolutions / minute) about the major axis for about 1 hour in a milliliter eggplant-shaped flask, a dosing solution of a sustained-release preparation containing almost no air bubbles was prepared.

Example 4 A human granulocyte colony-stimulating factor preparation (Nypogen: Amgen) containing 300 micrograms of recombinant human G-CSF, 50 milligrams of mannitol, and 0.004% of Tween 80 in 3.1 milliliters was used. 1.377 ml of physiological saline for injection was added to 023 ml, and 4 μl of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added. Hyaluronic acid sodium salt (average molecular weight of about 2.3 million daltons:
Genzyme Co., Ltd.) solution of 0.9% (W / V) in physiological saline for injection (Fuso Yakuhin).
7 ml was added. A glass vial containing these solutions (capacity of about 5 ml) was hermetically sealed and mounted on a three-one motor (made by Haydon) with a capacity of 200.
By rotating and mixing (20 to 100 revolutions / minute) about the major axis for about 1 hour in a milliliter eggplant-shaped flask, a dosing solution of a sustained-release preparation containing almost no air bubbles was prepared.

Example 44. Human parathyroid hormone (PT
Albumin Nichiyak (Japan) containing 300 micrograms of an active fragment (Bachem Co., Ltd.) consisting of amino acids from the amino group end to the 34th amino acid terminal of H) in 1 ml of physiological saline for injection and further containing 20% of human serum albumin. (Pharmaceutical) was added in an amount of 4 microliters. Hyaluronic acid sodium salt (average molecular weight of about 18
200,000 Daltons: Genzyme Inc.) 1.2% (W / V)
Then, 1.5 ml of a solution dissolved in physiological saline for injection (Fuso Yakuhin Co., Ltd.) was added so that the concentration became 1. A glass vial containing these solutions (capacity: about 10 ml) was sealed, and was rotated and mixed about a long axis in a 200 ml-capacity eggplant-shaped flask attached to a three-one motor (made by Haydon) for about 1 hour ( 20 to 100 revolutions / minute) to prepare a dosing solution of a sustained-release preparation having almost no air bubbles.

Example 45. Human parathyroid hormone (PT
300 μg of H) (Bachem Co.) was dissolved in 1 ml of physiological saline for injection, and 4 μl of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was further added. Hyaluronic acid sodium salt (average molecular weight of about 1.8 million daltons:
Genzyme) dissolved in physiological saline for injection (Fuso Yakuhin) to a concentration of 1.2% (W / V) 1.
5 ml was added. A glass vial containing these solutions (capacity: about 10 ml) was sealed, and a capacity of 20 was attached to a three-one motor (made by Haydon).
By rotating and mixing (20 to 100 revolutions / minute) about the long axis in a 0 ml eggplant-shaped flask for about 1 hour, an administration solution of a continuous preparation containing almost no air bubbles was prepared.

Example 46. After adding physiological saline for injection to a solution containing 100 ATU of Hirudin (Peninsula Laboratories, Inc. USA) to bring the total volume to 1.4 ml, 4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin. Added. Hyaluronic acid sodium salt (average molecular weight of about 1.8 million daltons: Genzyme) was added to this solution.
Add 0.7 ml of a solution dissolved in physiological saline for injection (Fuso Yakuhin) to a concentration of 2% (W / V). A glass vial containing these solutions (capacity: about 5 ml) was sealed, and the mixture was rotated and mixed about a long axis in a 200 ml-capacity eggplant-shaped flask attached to a three-one motor (made by Haydon Co.) for about 1 hour. (2
(0 to 100 revolutions / minute) to prepare an administration solution of a sustained-release preparation having almost no air bubbles.

Example 47. Thyroid-stimulating hormone-releasing hormone (TRH, Backchem Fine Chemicals, USA)
Was dissolved in 1.4 ml of physiological saline, and 4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added.
Hyaluronic acid sodium salt (average molecular weight about 1
800,000 Daltons: Genzyme Inc. 1.2% (W /
V) physiological saline for injection (Fuso Yakuhin)
0.7 ml of the solution dissolved in is added. A glass vial containing these solutions (capacity: about 5 ml) was hermetically sealed, and was rotationally mixed about a long axis in a 200 ml-capacity eggplant-shaped flask attached to a three-one motor (made by Haydon) for about 1 hour ( 20 to 100 revolutions / minute) to prepare a dosing solution of a sustained-release preparation having almost no air bubbles.

Example 48. Cefotiam hydrochloride
Dihydrochloride: Takeda Yakuhin) 10 mg of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was dissolved in 1.4 ml of physiological saline.
Microliter was added. A solution of hyaluronic acid sodium salt (average molecular weight of about 1.8 million daltons: Genzyme) dissolved in physiological saline for injection (Fuso Yakuhin) to a concentration of 1.2% (W / V), 0.7 Add milliliters. A glass vial containing these solutions (capacity: about 5 ml) was hermetically sealed, and was rotationally mixed about a long axis in a 200 ml-capacity eggplant-shaped flask attached to a three-one motor (made by Haydon) for about 1 hour ( 20 to 100 revolutions / minute) to prepare a dosing solution of a sustained-release preparation having almost no air bubbles.

Example 49. Interferon alfa 2
1 ml of distilled water was added to and dissolved in 2 vials of Campferon A300 (Takeda Yakuhin) containing 3 million international units of a and 5 mg of human serum albumin. Hyaluronic acid sodium salt (average molecular weight of about 1.47 million daltons: Genzyme) was dissolved in 1.4 ml of this solution in physiological saline for injection (Fuso Yakuhin) so as to have a concentration of 1.5% (W / V). 0.7 ml of the solution was added. A glass vial containing these solutions (capacity: about 5 ml) was hermetically sealed, and was rotationally mixed about a long axis in a 200 ml-capacity eggplant-shaped flask attached to a three-one motor (made by Haydon) for about 1 hour ( 20 to 100 revolutions / minute) to prepare a dosing solution of a sustained-release preparation having almost no air bubbles.

Example 50. Genetically modified basic human F
Albumin Nichiyaku containing 2.4 ml of physiological saline for injection and 20% of human serum albumin in 0.4 ml of GF mutein CS23 (EP Publication 281,822) (0.96 mg protein / ml) solution 15 μl of pharmaceutical)
Furthermore, hyaluronic acid sodium salt (average molecular weight about 147
1.4 ml of a solution prepared by dissolving 10,000 Daltons in physiological saline for injection (Fuso Yakuhin) to a concentration of 1.5% (W / V) was added. A glass vial containing these solutions (capacity: about 12 ml) was sealed and had a capacity of 200 attached to a three-one motor (made by Haydon).
By rotating and mixing (20 to 100 revolutions / minute) about the major axis for about 1 hour in a milliliter eggplant-shaped flask, a dosing solution of a sustained-release preparation containing almost no air bubbles was prepared.

Example 5 1. 300 micrograms of parathyroid hormone (PTH) (Bachem) was dissolved in 2 ml of saline for injection. In a glass vial (capacity: about 5 ml) containing this solution, 4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin and hyaluronic acid sodium salt (average molecular weight: about 1.47 million daltons) 1. 5% (W /
V) physiological saline for injection (Fuso Yakuhin)
1 ml of the solution dissolved in was added. The glass vials containing these solutions were sealed and rotatively mixed about 20 minutes (20 to 100 rpm) about the major axis in a 200 ml capacity eggplant-shaped flask attached to a three-one motor (Haydon). By doing so, an administration solution of a sustained-release preparation containing almost no air bubbles was prepared.

Experimental Example 1 An injection was prepared in the following mixing ratio and used in the experiment. Comparative preparation 1: Recombinant human basic FGF mutein CS23 (EP Publication 281,822) solution (0.9
1.255 ml of saline for injection was added to 0.35 ml of 6 mg protein / ml). Comparative preparation 2: Recombinant human basic FGF mutein CS23 (EP Publication 281,822) solution (0.9
0.36 ml of 6 mg protein / ml, physiological saline for injection 1.26 ml, sodium hyaluronate (average molecular weight 1.47 million daltons:
(Genzyme Co.) 8.1 mg was added. 8-week-old male SD rat subcutaneously subcutaneously on the back of 160 micrograms /
The injection solution of Example 1 was administered in a dosage of kilograms. As a control, the injection solutions of Comparative Preparation 1 and Comparative Preparation 2 were similarly administered. Before administration and at regular intervals, about 0.4 ml of blood was collected to separate serum. RhbF in serum
The GF mutein CS23 concentration was quantified by the ELISA method.
The result is shown in FIG. FIG. 1 shows rhbFGF mutein C of Comparative Preparation 3 (○), Comparative Preparation (□) and Example 1 (●).
It is a graph which shows transition of the blood concentration after administering the S23 injection solution. Five rats per group are used in each figure, and the points in each figure are the average values and the bars are the standard errors (S.
E. ) Is shown. In the group administered with the rhbFGF mutein CS23 solution of Comparative preparation 1, rhbFGF mutein CS23 rapidly disappeared from the blood, compared to the hyaluronic acid added group of Comparative preparation 2 due to the effect of hyaluronic acid. It was found that the blood concentration of mutein CS23 was high and maintained. However, in the group administered with the injection solution of rhbFGF mutein CS23 of Example 1 to which hyaluronic acid and serum albumin were added, as compared with the group of Comparative Formulation 2 to which only hyaluronic acid was added, at 1 hour and 4 hours after administration. It was found that an even higher blood concentration was maintained.

Experimental Example 2 An injection was prepared in the following mixing ratio and used in the experiment. Comparative preparation 3: Pig insulin (26.8 units / milligram: DIOSYNTH Co., Ltd.) (3 mg) was dissolved in 0.1 N hydrochloric acid (1.5 ml) to prepare a physiological saline solution for injection.
65 ml was added, and to this was added 15.75 mg of hyaluronic acid sodium salt (average molecular weight 1470,000 Dalton: Genzyme). Comparative formulation 4: Saline for injection 2 ml Male SD strain rat aged 8 weeks was subcutaneously administered to Example 36 and Comparative formulation 3 insulin injection solution at a dose of 200 microgram / rat under the skin. As a control, the physiological saline solution for injection of Comparative Preparation 4 was administered so as to have the same administration liquid volume as in Example 36 and Comparative Preparation 3. Before administration and at regular intervals, about 0.5 ml of blood was collected, and the blood glucose level in serum was measured with a glucose C test kit (Wako Pure Chemical Industries, Ltd.).
The results are shown in [Fig. 2]. In Fig. 2, (○)
Indicates the group administered with the physiological saline of the comparative preparation 4, (●) indicates the group administered with the solution containing the hyaluronic acid of pig insulin of the comparative preparation 3, and (Δ) indicates the hyaluronic acid of the pig insulin of Example 36. 2 shows the time course of blood glucose level in serum of a group to which a solution containing and serum albumin was administered. The rats in [Fig. 2] used 5 animals per group, and each point shows the average value. In the physiological saline-administered group of Comparative Preparation 4, there was almost no change in blood glucose level. In Comparative Preparation 3 and Example 36, it was suppressed to the same degree up to 4 hours after administration,
After 6 hours from the administration, the blood glucose level of Example 36 continued to be suppressed for a longer time in Example 36, and at the 9 hours after the administration, the blood glucose level of the comparative preparation 3 was almost restored to the normal level. hand,
It was suppressed to about 2/3 of the physiological saline-administered group of Comparative Preparation 4. This suggests that the addition of serum albumin to this system results in a longer-lasting pharmacological effect than the same amount of insulin and hyaluronic acid, rather than the combination of insulin and hyaluronic acid, which are said to bring about a prolonged effect of the peptide. Shows.

Experimental Example 3 An injection was prepared in the following mixing ratio and used in the experiment. Comparative formulation 5: Recombinant human G in 1 ml
-300 micrograms CSF, 50 mannitol
Human granulocyte colony stimulating factor preparation containing milligram and 0.004% of Tween 80 (Nypogen: Amgen)
To 0.0233 ml of saline for injection 1.3
77 ml was added. A solution of hyaluronic acid sodium salt (average molecular weight of about 1.47 million daltons: Genzyme) dissolved in physiological saline for injection (Fuso Yakuhin) to a concentration of 1.5% (W / V) was added to this solution 0.7. Added milliliters. A glass vial containing these solutions (capacity: about 5 ml) was hermetically sealed, and was rotationally mixed about a long axis in a 200 ml-capacity eggplant-shaped flask attached to a three-one motor (made by Haydon) for about 1 hour ( 20 to 100 revolutions / minute) to prepare a dosing solution of a sustained-release preparation having almost no air bubbles. Comparative formulation 6: Recombinant human G in 1 ml
-300 micrograms CSF, 50 mannitol
2.077 ml of physiological saline for injection was added to 0.0233 ml of a human granulocyte colony stimulating factor preparation (Nypogen: Amgen) containing 0.004% of milligram and Tween 80. Comparative formulation 7: 1.4 ml of saline for injection,
0.7 ml of a solution prepared by dissolving hyaluronic acid sodium salt (average molecular weight of about 1.47 million daltons: Genzyme Co., Ltd.) in physiological saline for injection (Fuso Yakuhin) to a concentration of 1.5% (W / V) was added. It was A glass vial containing these solutions (capacity: about 5 ml) was hermetically sealed, and was rotationally mixed about a long axis in a 200 ml-capacity eggplant-shaped flask attached to a three-one motor (made by Haydon) for about 1 hour ( 20 to 100 revolutions / minute) to prepare a dosing solution of a sustained-release preparation having almost no air bubbles. The injection solutions of Example 39, Comparative Preparation 5, and Comparative Preparation 6 containing human G-CSF were subcutaneously administered to the back of 8-week-old male SD rats in an amount of 0.3 ml each. As a control, 0.3 ml of a physiological saline solution of Comparative Preparation 7 for injection of sodium hyaluronate was administered. About 0.1 ml of blood was collected before administration and at a predetermined time (using EDTA2Na as an anticoagulant). Peripheral leukocytes, erythrocytes, and platelets are counted by micro cell counter CC-
It measured using 180A type (Toa Medical Electronics). The number of peripheral neutrophils, lymphocytes, monocytes, and eosinophils was calculated by observing a Giemsa-stained smear to classify 200 white blood cells and multiplying the number of white blood cells by the frequency of appearance of each cell. . The results are shown in [Fig. 3]. The number of neutrophils in the peripheral blood of the comparative preparation 7 administered as a control in the hyaluronic acid aqueous solution administration group (∘) hardly changed. In the human G-CSF solution-administered group (●) of Comparative Preparation 6, the number of neutrophils in peripheral blood increased about twice at 16 hours after administration, and hyaluronic acid was added thereto. In the comparative preparation 5 administration group (Δ), the number of neutrophils in peripheral blood was further increased.
In the group (□) administered with the preparation of Example 39 in which hyaluronic acid and human serum albumin were added to human G-CSF, the number of neutrophils in peripheral blood was further increased as compared with the comparative preparation 5 administration group. At 24 hours after administration, the numbers of neutrophils in the peripheral blood of the comparative preparation 5 and the comparative preparation 6 administration groups decreased to almost the same level as those in the control group.
In the group to which the preparation of 9 was administered, the value was almost twice as high as that in the control group. Thus, it is shown that the addition of human serum albumin can further enhance the duration of the pharmacological effect of hyaluronic acid. Each point in the figure is an average value of 5 rats.

Experimental Example 4 An injection was prepared in the following mixing ratio and used in the experiment. Comparative formulation 8: 1 ml of distilled water was added and dissolved in 2 vials of Campferon A300 (Takeda Pharmaceutical Co., Ltd.) containing 3 million international units of interferon alpha 2a and 5 mg of human serum albumin. Saline solution for injection (Fuso Yakuhin) to 1.4 ml of this solution
0.7 ml was added. 0.3 ml of each injection of Example 49 or Comparative Preparation 8 containing interferon alpha 2a was subcutaneously administered to the back of 8-week-old male SD rats. Before administration and at a predetermined time, about 0.4 ml of blood was collected and serum was separated, and the interferon alpha 2a concentration was determined by the ELISA method. The results are shown in [Fig. 4]. In the case of the aqueous preparation containing the interferon alfa 2a of the comparative preparation 8 administered as a control (○), the interferon alfa 2 from the administration site was rapidly
Absorption of a occurred, and the serum concentration of interferon alfa 2a decreased with time. On the other hand, in the solution administration group (●) containing hyaluronic acid, human serum albumin and interferon alfa 2a of the preparation of Example 49, the concentration of interferon alfa 2a 1 hour after administration was higher than that of the control group, and The sustained effect of adding hyaluronic acid and human serum albumin is remarkable. Each point in the figure is an average value of 5 rats.

Experimental Example 5 An injection was prepared in the following mixing ratio and used in the experiment. Comparative preparation 9: genetically modified basic human FGF mutein CS23 (EP Publication 281,822) (0.96)
To 0.4 ml of the (milligram protein / milliliter) solution was added 3.6 ml of saline for injection. After fasting for 24 hours, 8-week-old SD male rats slightly anesthetized by intraperitoneal administration of pentobarbital sodium salt (Somnopentyl, Pitman Moore, USA) were treated with the preparation of Example 50 or the preparation of Comparative Preparation 9. It was orally administered into the stomach. Thirty minutes after administration, the abdomen was incised and the stomach contents were collected. The stomach contents were immediately subjected to a centrifugation operation (Microfuge B, Beckman, USA), and 50 microliters of the supernatant was added with 100 microliters of a protease inhibitor (aprotinin, Sigma, USA). The sample was stored at -40 ° C until the CS23 concentration was measured by the immunoassay.

[Table 1] CS23 concentration (pg / ml) ──────────────────── Comparative formulation 9 formulation administration group 1768 Formulation administration group of Example 50 8448 ────────────────────

Experimental Example 6 An injection was prepared in the following mixing ratio and used in the experiment. Comparative formulation 10: 300 micrograms of parathyroid hormone (Bachem) was added with 2 ml of saline for injection. The preparation of Example 51 (●) and the preparation of Comparative Preparation 10 (◯) were each subcutaneously administered to the 8-week-old SD male rats subcutaneously on the back of the rat in a volume of 0.2 ml.
About 0.4 ml of blood was collected before administration and at regular intervals after administration, and after separating serum, the concentration of PTH was measured by radioimmunoassay. The results are shown in [Fig. 5]. Serum PTH concentrations at 2 hours and 3 hours after administration were significantly higher in the formulation administration group of Example 51 than in the formulation administration group of comparative formulation 10. These results indicate that the combination of hyaluronic acid, human serum albumin, and PTH is effective in sustaining serum PTH levels.

[0089]

EFFECT OF THE INVENTION By adding hyaluronic acid and a water-soluble protein to a pharmaceutically active substance, a further long-lasting effect can be obtained, and a long-term preparation using low concentration of hyaluronic acid has become possible. As a result, it becomes possible to use a thin injection needle, and it is possible to reduce the pain caused to the patient. Moreover, since the viscosity is low, the inclusion of air bubbles is reduced, and clinical use is simplified. Furthermore, a high lasting effect is obtained as compared with the case of using hyaluronic acid alone.

[Brief description of drawings]

FIG. 1 shows the time course of the concentration of mutein drug in serum obtained in Experimental Example 1.

FIG. 2 shows the time course of serum glucose concentration obtained in Experimental Example 2.

FIG. 3 shows the time course of the number of neutrophils in peripheral blood obtained in Experimental Example 3.

FIG. 4 shows the time course of drug concentration in serum obtained in Experimental Example 4.

FIG. 5 shows the time course of serum PTH concentration obtained in Experimental Example 5.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI A61K 47/42 A61K 37/26 (56) References JP-A-2-213 (JP, A) JP-A-1-287041 (JP, A) JP-A-3-4790 (JP, A) JP-A-63-146827 (JP, A) JP-A-61-236732 (JP, A) JP-A-60-59000 (JP, A) JP-A-60-155136 (JP, A) JP 60-222424 (JP, A) JP 61-197527 (JP, A) JP 5-97694 (JP, A) JP 4-282322 (JP, A) Europe Patent application publication 123291 (EP, A 1) International publication 90/005522 (WO, A1) International publication 91/004058 (WO, A1) (58) Fields investigated (Int.Cl. ⁷ , DB name) A61K 38/00 A61K 9/08 A61K 38/22 A61K 38/28 A61K 47/36 A61K 47/42

Claims (6)

(57) [Claims] 1. (a) Granulocyte colony stimulating factor (G-CS)
F), fibroblast growth factor (FGF), parathyroid form
Activity selected from insulin (PTH) and insulin
(B) Water-soluble hyaluronic acid or its non-toxic salt is added together with the substance in an amount of about 0.05 to 0.5% (W / V) and (c)
A water-soluble composition comprising human serum albumin in an amount of about 0.001 to 5% (W / V) and having a viscosity of about 500 cp or less. 2. Human serum albumin , wherein the weight ratio of pharmaceutically active substance to hyaluronic acid is about 0.0001: 1 to 100: 1.
The weight ratio of emission versus hyaluronic acid of about 0.001: 1 to 10
The water-soluble composition according to claim 1, which is 0: 1. 3. The water-soluble composition according to claim 1, which is a liquid medicine. 4. The water-soluble composition according to claim 1, which is a parenteral preparation. 5. The water-soluble composition according to claim 4, which is an injection preparation. 6. A viscosity of about 50-400 cp.
The water-soluble composition as described.