JPH05238949A - Composition for injection - Google Patents

Abstract

PURPOSE:To obtain a medicinal composition capable of shortening the duration of physiological action of a peptide in a living body having a short duration of the physiological action in the living body. CONSTITUTION:The objective water-soluble medicinal composition for injection is obtained by blending a water-soluble peptide having >=30ml/hr clearance in a rat based on 1kg body weight with polyethylene glycol which is a waxy solid at ordinary temperature and has 2000-6000 average molecular weight. Thereby, the physiological action of the peptide in a living body is sustained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an injectable pharmaceutical composition useful as a preparation for sustaining the in vivo pharmacological action of a peptide which is rapidly metabolized and excreted.

[0002]

Despite the strong pharmacological effect, peptides are often decomposed and excreted after being administered into body fluids, so that frequent administration such as injection is often necessary. Therefore, the development of sustained-release formulations of these substances reduces the pain of the patient by reducing the number of injections, and enhances the efficacy by continuously maintaining the concentration in the body fluid within a certain effective concentration range, or increases the concentration in the body fluid. It is necessary to reduce the side effects due to rapid fluctuations in Attempts have been made to use polyethylene glycol for the purpose of producing such a sustained-release preparation, but this is not yet sufficient.

For example, British Patent No. 1385914
In the publication, hydrolyzates such as casein, collagen and keratin are acylated (palmitoylated, lauroylated,
A technique of external preparation for skin that can effectively permeate into subcutaneous tissue by dissolving a fat-soluble substance obtained by stearoylation) in an amphipathic solvent such as polyoxyethylene glycol is disclosed. , There is no indication of formulating water-soluble peptides,
Moreover, it has not been shown to be used as an injection at all. PCT International Application Publication No. WO89 / 09610 discloses a technique for a freeze-dried preparation having a low water content, which contains polyethylene glycol or the like for the purpose of reducing association and aggregation of tumor necrosis factors (TNFs). However, the molecular weight and blending ratio of polyethylene glycol are not specifically shown. Moreover, the effect of the blended polyethylene glycol on the in vivo kinetics of tumor necrosis factor (TNF) after administration to a living body is not shown.

PCT International Application Publication No. WO89 / 0515
In JP-A-8, the molecular weight is 900 to 1600, more preferably the molecular weight is 190 to 420, and particularly preferably the molecular weight is 2.
85% to 315 (usually referred to as polyethylene glycol 300) non-electrolyte polyethylene glycol 5% to 50% (v / v), particularly preferably 25% (v / v)
The technology of interferon beta (IFN-β) protein injection containing v) as a stabilizer or solubilizer in a high concentration is disclosed. Since polyethylene glycol which is liquid at room temperature is used in this publication, the vacuum drying or freeze drying method which is usually adopted as a formulation capable of stably storing peptides and proteins cannot be applied. That is, it is described that freeze-drying is impossible with the combination of the concentration and molecular weight of polyethylene glycol used in the application. Moreover, the effect of polyethylene glycol on the in vivo kinetics of interferon beta protein after administration to a living body is not shown. Since the smaller the molecular weight of polyethylene glycol is, the stronger the irritation is, it is considered that it is better to avoid a preparation containing a high concentration of polyethylene glycol having a low molecular weight. JP 61
Japanese Patent Publication No. 97229 discloses a technique for a stable erythropoietin preparation prepared by blending polyethylene glycol or the like. However, although a preparation containing 0.25% or 0.5% of polyethylene glycol 4000 is shown here, a method for preparing an effective preparation such as the molecular weight of polyethylene glycol to be used and the concentration in the final preparation is disclosed. It has not been. JP-A-63-122
No. 628 discloses interleukin 2 and a polyethylene glycol monostearate compound, and PCT International Application Publication No. WO89 / 02750 discloses interferon beta and a polyethylene glycol monostearate compound as stabilizers and solubilizers, respectively. A blending technique is disclosed. That is, polyethylene glycol is used here as a derivative having a nonionic interfacial action.

[0005]

If the duration of the pharmacological action of a water-soluble peptide having a large clearance (that is, the duration of the pharmacological action in vivo is short) can be prolonged, the number of administrations by injection can be increased. By reducing the amount, the pain of the patient is alleviated, and the drug concentration in the body fluid is continuously maintained within a certain effective concentration range, so that the drug efficacy is enhanced.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that by mixing a polyethylene glycol having a wax-like solid with an average molecular weight of 2000 to 6000 at room temperature, the rat Clearance is not less than 30 milliliters / hour per kilogram of body weight per hour, and it does not impair the pharmacological activity of the water-soluble pharmaceutically active peptide, and it is a lyophilizable water-soluble sustainability that can maintain its pharmacological action under low viscosity. It was found that an injection preparation can be obtained, and further intensive research was conducted based on this, resulting in completion of the present invention.
The present invention (1), the clearance in rats is 1
Water-soluble peptide having a volume of 30 ml / hour or more per kilogram and a wax-like solid having an average molecular weight of 20 at room temperature
A water-soluble injectable pharmaceutical composition comprising (2) a polyethylene glycol of 0-6000, and a water-soluble protein having substantially no pharmacological activity, which is injectable into a living body (1). (3), and (3) the pharmaceutical composition of (1) or (2) above, which further comprises an acidic mucopolysaccharide.

In the composition of the present invention, the main drug is
A water-soluble peptide having a clearance of 30 ml / hour or more per kilogram body weight in rats is used. The degree of water solubility referred to above is that the octanol / water partition ratio is 0.1 or less.
The water-soluble polypeptide referred to in the present invention has a clearance in rats of 30 ml / kg of body weight /
There is no particular limitation as long as it is a peptide that has physiological activity over time. For example, antibiotics, blood boosters, infectious disease treatment agents, anti-dementia agents, anti-viral agents, anti-tumor agents, antipyretics, analgesics, anti-inflammatory agents, anti-ulcer agents, anti-allergic agents, anti-depressants, psychotropic agents, Cardiotonic agents, antiarrhythmic agents, vasodilators, antihypertensive agents such as antihypertensive diuretics, antidiabetic agents, anticoagulants, cholesterol lowering agents, osteoporosis therapeutic agents, hormone agents, vaccines, etc., Examples include those specified by the above clearance.

The peptide used in the present invention includes
Peptides having a pharmaceutical activity composed of two or more amino acids and derivatives thereof are used, and have a molecular weight of about 200 to 2
Those of 00000 are preferable. The action mechanism of these peptides also includes antagonists, agonists, or soluble receptors thereof and derivatives thereof. Further, those having a sugar chain include those having a different sugar chain structure. Further, it may be modified with a synthetic polymer such as polyethylene glycol or a natural polymer such as hyaluronic acid, or may be modified with any sugar or non-peptidic compound. The substance added in the modification may be capable of binding to any receptor, antibody or the like. Further, a hybrid peptide in which, for example, a peptide capable of binding to an arbitrary receptor, antibody or the like is added to the amino acid structure necessary for expressing the original pharmacological activity is also included. Examples of the peptide include natural products or interferons (eg, interferon alphas, interferon betas, interferon gammas) produced by gene recombination, hematopoietic factors such as granulocyte colony stimulating factor (G
-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), interleukins (eg IL-
1, IL-2, IL-3, IL-4, IL-5, IL-
6, IL-7, IL-8, IL-9, IL-10, IL
-11) and the like. Examples of the peptide include growth factors derived from natural products or produced by gene recombination, such as nerve growth factor (NGF), neurotrophic factor (NTF), peptides having an action on cerebral nerve cells, and epidermal growth factor ( EGF), insulin-like growth factors (IGF), growth hormone (GH), hepatocyte growth factor (HGF), fibroblast growth factor (FGF)
And so on. Examples of the peptide include parathyroid hormone (PTH) and endothelin.
Further, the peptide has a sugar chain, these factors having different activities of sugar chain structure, or these factors not containing a sugar chain, or these muteins, or their derivatives, analogs, or these Active fragments are mentioned. The peptide is a physiologically active peptide having a platelet proliferating action, and may or may not have a sugar chain.

Further, the peptides include transforming growth factor (TGF), insulin, lymphotoxin, and platelet-derived cell growth factor (PDG).
F), Angiotensin I, Angiotensin II, Angiotensin III, Angiotensin I inhibitor,
Bradykinin, dynorphin, kyotorphin, endorphin, enkephalin, secretin, vasopressin, growth hormone releasing factor (GRF), luteinizing hormone releasing hormone (LHRH) or its derivative, neurotensin, oxytocin, vasopressin, vasopressin derivative {desmopressin (Japan endocrine Academic Journal, Vol. 54, No. 5, pp. 676-691 (1978))}, hirudin or its derivatives, glucagon, gastrin, calcitonin, calcitonin gene-related peptide (CGRP), prolactin, adrenocorticotropic hormone (ACTH), Melanocyte-stimulating hormone (MSH),
Thyroid hormone-releasing hormone (TRH) and its salts and their derivatives (Japanese Patent Laid-Open Nos. 50-12173 and 52-52)
116465), thyroid stimulating hormone (TS
H), luteinizing hormone (LH), follicle stimulating hormone (FSH), pancreozyme, cholecystokinin,
Human chorionic gonadotropin (HCG), tuftsin, thymosin, thymostimulin, motilin, bombesin,
Examples include cerulein, bradykinin, kallikrein, substance P, tumor necrosis factor (TNF) or muteins, derivatives, analogs and active fragments thereof. Further, examples of the peptide include atrial natriuretic hormone (ANP) or a related peptide thereof. Further, the peptide may be an enzyme derived from a natural product or produced by gene recombination, may or may not have a sugar chain, and may have a different sugar chain structure, and these muteins or derivatives, Alternatively, it may be an active fragment, for example, urokinase, superoxide dismutase (SOD), tissue type plasminogen activator (TPA), asparaginase, lysozyme, etc., but not limited thereto, and other administrable Enzymes are also included.

A vaccine is a peptide derived from a natural product serving as an antigen, or a peptide produced by synthesis or gene recombination,
Non-peptide antigenic substances derived from proteins or natural products,
Alternatively, a mixture of these may be used, and these may be used alone or bound to a hapten, or may be administered as an injection together with an adjuvant, and examples thereof include cedar pollen, butterbur pollen, but are not limited thereto.

Peptides with a small clearance, which is an index of the metabolism and excretion ability of drugs in humans and other warm-blooded animals, such as peptides having a clearance in humans of about 15 ml / hour or less per kilogram of body weight are originally from the body. The discharge is slow and the sustainability is good enough. In addition, it is necessary to carefully administer a peptide that may cause side effects or has a very strong pharmacological effect, and that administration of an excessive amount may be detrimental to the patient while observing the patient. Therefore, a sustained release formulation is not required for peptides that meet these two conditions. For example, the usual human dosage of recombinant human erythropoietin is 10 micrograms (1800
It is reported that the average clearance calculated using the serum erythropoietin concentration after intravenous administration of 20 micrograms (in international units) to humans is 758 ml / hr or 739 ml / hr, respectively (Uji Yasuaki et al., Medical Care and New Drugs, Vol. 26, 1
Page ~, 1989). Average weight of each group is 6
Since it is 5.8 kilograms or 69.3 kilograms, the clearance per kilogram body weight is calculated to be 11.5 milliliters / hour or 10.7 milliliters / hour, respectively. In this paper, the dose is 2
It was administered intravenously to humans at doses down to micrograms and 0.2 micrograms, but the area under serum concentration (AUC) was proportional to the dose, and the pharmacokinetics remained linear. It has been reported that the clearance of erythropoietin is not dose-dependent within the dose range of 0.2 microgram to 20 microgram. Incidentally, the half-life of the erythropoietin after intravenous administration to humans (2 hours to 8 hours after administration)
The average value of (up to time) is 3.3 hours in the 10-microgram administration group and 5.2 hours in the 20-microgram administration group, which is very long, and even 48 hours after administration, several picograms / milliliter to several tens of milliliters in serum. Picogram / ml remained. From these facts, it is considered that the erythropoietin preparation which is clinically used may be administered once to three times a week. Further, it is required to measure the hemoglobin concentration or hematocrit value once a week or once every two weeks so as not to cause excessive blood increase, and to discontinue the drug if excessive blood increase is observed. For this reason, even if a sustained-release preparation of erythropoietin is developed, it cannot be controlled when an excessive blood increase occurs, and it does not benefit the patient. In addition, the clearance of erythropoietin when rats were used as experimental animals was reported to be 19.4 ml / hour per kilogram body weight (the dose was 5 per kilogram body weight).
Metabolism and excretion are slow as in humans (Kazuo Okumura et al., Pharmacology and Treatment, Vol. 18, Supplement, pp. 141-162 (1990)). It has been reported that the clearance of warm-blooded animals such as rats and humans can be expressed as a function of body weight (Boxsenbaum et al.,
Journal of Pharmacokinetics and Bioformauxics, Volume 10, 201
Page to page 227 (1982) is cited as a representative document), and it is clear that the relationship of clearance per kilogram of body weight between rat and human in erythropoietin substantially holds for other peptides. That is, it can be almost certainly estimated that a peptide having a small clearance in rat has a small clearance in human. Clearance in the present invention is defined as the rate at which the whole body processes the drug divided by the circulating blood concentration at that time, and represents the volume of body fluid that the body can process the drug per unit time. The body has a high ability to process drugs (Gee, Earl, Wilkinson, Pharmacology Review, Vol. 39, page 1-, 1987. Manabu Hanano et al., Applied Pharmacokinetics (Nanzandou), 1989). It is described as CLP when the drug concentration in plasma (serum) is used as a reference, and as CLB when the drug concentration in whole blood is used as a reference. The clearance in the present invention is sometimes referred to as total clearance (CLTOT), but can be read as CLP or CLB depending on which of plasma (serum) or whole blood is used as a reference. Usually, these values are expressed as the amount of drug administered by intravenous administration divided by the area under the drug concentration (AUC) in plasma (serum) or whole blood. Clearance may also be defined by other known scientific terms used in the field of pharmacokinetics, but their meanings are the same.

In the present invention, polyethylene glycol is a wax-like solid at room temperature and has an average molecular weight of 20.
The thing of 00-6000 is used. The room temperature referred to in the present invention refers to a temperature range defined by the Japanese Pharmacopoeia, specifically, 15 ° C to 25 ° C. Wax-like solids include solids that resemble white wax in appearance rather than liquids. In the present invention, polyethylene glycol having an average molecular weight of 2000 to 6000 is used. More preferably, the average molecular weight is 2000 to 5000. More preferably, the average molecular weight is 2000 to 450.
It is 0. Further, those having different molecular weights may be mixed and used. The molecular weight referred to in the present invention refers to the molecular weight obtained by the average molecular weight determination test of Macrogol 400 described in the Japanese Pharmacopoeia. Examples of the polyethylene glycol include those having an average molecular weight of 2000, those having an average molecular weight of 3000 and those having an average molecular weight of 4000.

Storage of peptide preparations is easy at room temperature, but decomposition is often promoted in the state of aqueous solution as compared with storage at low temperature, and in the case of peptides and the like, physical properties such as vibration in the liquid state are caused. It is known that some of them lose their pharmacological activity by being stimulated. Therefore, it is preferred that the peptide formulation, particularly the injectable formulation, be prepared by vacuum drying, particularly by freeze-drying, and usually distributed and stored at room temperature. When polyethylene glycol that is liquid at room temperature is used when the aqueous solution of the water-soluble pharmaceutical composition of the present invention is formulated by vacuum drying or freeze-drying, the liquid remains at room temperature after freeze-drying. Highly likely to impair activity. On the other hand, when polyethylene glycol, which is solid at room temperature, is used, it remains in a solid state at room temperature even after freeze-drying, so that it does not impair the pharmacological activity of the peptide, particularly the peptide, and thus has excellent storage stability. When the melting point of solid polyethylene glycol was measured at room temperature using a Perkin Elmer differential scanning calorimeter (DSC7) (temperature rising rate was 10 ° C./min), polyethylene glycol 1000 (Wako first class, average molecular weight 1000) was 26.
Peaks of heat transfer were seen at 78 ° C and 36.58 ° C. The heat transfer peak of polyethylene glycol 1540 (Wako first class, average molecular weight 1500) was 47.32 ° C, but it was confirmed that significant heat transfer occurred from around 30 ° C. Room temperature is 1 to 30 degrees Celsius in the Japanese Pharmacopoeia
It is well known that in Japan, the temperature rises to 30 ° C or higher during the summer.
In the distribution and storage of a pharmaceutical preparation which is not stored under cold conditions under such conditions, there is a sufficient opportunity to expose the pharmaceutical preparation to a temperature of 30 ° C. or higher. Therefore, the use of polyethylene glycol 1000 or 1540 is not preferred, as it is expected that these polyethylene glycols will melt, for example in freeze-dried formulations. On the other hand, in polyethylene glycol 2000 (Wako first class, average molecular weight 2000), the peak of heat transfer was 52.39 ° C., and significant heat transfer occurred from around 40 ° C. The heat transfer peak of polyethylene glycol 4000 (Wako first class, average molecular weight 3000) was 58.94 ° C, and the heat transfer peak of polyethylene glycol 6000 (Wako first class, average molecular weight 7500) was 63.56 ° C. From these results,
Polyethylene glycol having an average molecular weight of 1540 or less is unsuitable when used in a pharmaceutical composition prepared by vacuum drying or freeze drying. On the other hand, by using polyethylene glycol having an average molecular weight of 2,000 or more and having an average molecular weight of more than 40 ° C. in the melting point measurement by the differential scanning calorimeter used in the present invention, significant heat transfer occurs at room temperature or room temperature. It is possible to prevent the melting of polyethylene glycol during distribution and storage in

The water-soluble protein having substantially no pharmacological activity which can be injected into the body fluid according to the present invention includes serum albumin, polyclonal or monoclonal immunoglobulins or fragments thereof, collagen or gelatin, and the like. Serum albumin. The proportion of the peptide to be blended cannot be determined unconditionally depending on the activity of the substance and the therapeutically necessary amount, but a usual dosage is blended in the unit dosage composition. The weight ratio of peptide to polyethylene glycol is preferably 0.
It is 00001: 1 to 100: 1. More preferably, it is 0.0001: 1 to 10: 1. Similarly, the mixing ratio of the water-soluble protein cannot be unconditionally determined, but the amount usually added to the injectable pharmaceutical composition can be added, and the weight ratio of the peptide to the water-soluble protein is 0.000001: 1 to 100: 1. 1 is preferred. More preferably, it is 0.0001 to 10: 1. The concentration of polyethylene glycol in the aqueous solution of the pharmaceutical composition of the present invention is preferably 20% (W / V) or less,
0.01% (W / V) or more, more preferably 10%
(W / V) or less, 0.05% (W / V) or more, particularly preferably 5% (W / V) or less, 0.1% (W / V) or more.

Hyaluronic acid having a high negative charge due to a carboxyl group as an acidic mucopolysaccharide that can be further added to the composition of the present invention, or chondroitin-4-sulfate, dermatan sulfate as an acidic mucopolysaccharide having a sulfate group,
Chondroitin-6-sulfate, keratan sulfate, heparin,
Examples include heparan sulfate. Further, a desulfated product of a natural acidic mucopolysaccharide having a sulfate group can also be mentioned. The acidic mucopolysaccharide may be a long linear complex polysaccharide having a repeating structure of an amino sugar and an uronic acid disaccharide unit. These are polyanions that include a part of uronic acid replaced with galactose and have a high negative charge due to a sulfate group or a carboxyl group. The amino group of the amino sugar residue is usually acetylated, but a sulfate group may be bonded to the amino group instead of the acetyl group. Alternatively, these derivatives may be used.

The above-mentioned acidic mucopolysaccharide may be extracted from a living tissue such as cartilage or may be produced from a microorganism. Moreover, these derivatives may be used. In the case of hyaluronic acid, its molecular weight is 10,000 to 3,000,000, preferably 500,000 to 2,500,000, and more preferably 1,000,000.
Some 2.5 million items are listed. The molecular weight of the mucopolysaccharide having a sulfate group is 1,000 to 1,000,000, preferably 1,000 to 300,000, more preferably 1,000 to 10.
There are about ten thousand. The number of sulfate groups of the above-mentioned sulfated-mucopolysaccharide is about 0.
01 to 4.0 can be mentioned, and about 0.1 per disaccharide unit.
The number of 1 to 3.0 is preferably used. The natural acidic mucopolysaccharide having a sulfate group may be desulfated, and as a method thereof, a method of desulfating with an acidic methanol solution (Schwert, Mesozz-In-Carbohydrate Chemistry, vol. 5, p. 109, 1965) ) Is mentioned.
The number of sulfate groups in the desulfated mucopolysaccharide of the present invention is about 0 to 0.1 per disaccharide unit, preferably about 0 to 0.05.

Specific examples of the desulfated natural acid mucopolysaccharide having a sulfate group used in the present invention include chondroitin, desulfated heparin and heparan. Among them, chondroitin and heparan are preferable. The acidic mucopolysaccharides mentioned above may be salts, for example alkali metal salts or alkaline earth metal salts. Examples of the alkali metal salt include sodium salt and potassium salt. The sodium salt is preferred. Examples of the alkaline earth metal salt include magnesium salt and calcium salt, and hyaluronic acid sodium salt is advantageously used in the present invention. The mixing ratio of the acidic mucopolysaccharide is, for example, a weight ratio of mucopolysaccharide to polyethylene glycol of about 0.0001: 1 to 100: 1, preferably about 0.001: 1 to 10: 1, and most preferably about. It is 0.01 to 1: 1.

The sustained-release injectable pharmaceutical composition of the present invention contains a water-soluble peptide and polyethylene glycol, and, if necessary, a pharmaceutical additive or a water-soluble protein. When adjusting the composition, sterile water is placed in an ampoule or a vial. Alternatively, they may be dissolved or suspended in sterile physiological saline to allow them to exist, or may be dried in vacuum or freeze-dried depending on the purpose. In addition, an aqueous solution in which a peptide and, if necessary, additives, water-soluble proteins, etc. are mixed, or a solution obtained by re-dissolving this in vacuum-dried or freeze-dried powder with sterile water or sterile physiological saline, polyethylene glycol or this You may mix what added the sterilization water or the sterilized physiological saline to the thing to which the additive for pharmaceuticals was added, and was made to melt | dissolve. Further, the method is not limited to these, and other preparation methods may be adopted, and any method may be used as long as the activity of the peptide is not impaired.

Further, to the water-soluble peptide or to which serum albumin is added as a stabilizer, for example, hyaluronic acid sodium salt is further added as a formulation additive,
Solution or suspension obtained by adding distilled water for injection or physiological saline for injection and gently mixing (I)
Distilled water for injection of polyethylene glycol or physiological saline solution for injection (II) may be mixed with the above and the formulation may be prepared by vacuum drying or freeze drying. Also, (I) is dried in vacuum or freeze-dried and dispersed in (II),
The formulation may be carried out by rapid vacuum drying or freeze drying. In addition, mainly (I), (II)
Formulation may be carried out by spraying into a heated air flow from separate two-fluid nozzles, each of which is a secondary component, to perform molding dehydration (spray drying).

In this case, the complex (III) of hyaluronic acid sodium salt and water-soluble peptide or serum albumin added thereto is dispersed in polyethylene glycol, or polyethylene glycol is formed on the surface of (III). What is coated is obtained.
In addition, (I) or a powder obtained by vacuum-drying or freeze-drying it in polyethylene glycol heated and melted (a temperature slightly higher than the melting point) is dispersed in a cold solvent while stirring or dropping, or immediately obtained. Formulation may be carried out by recovering the fine particles, or by rapidly performing spray cooling in the gas phase (spray chilling) or spraying in a heated air stream from a two-fluid nozzle into a mold for dehydration (spray drying). .. Further, according to a known method, a formulation for molding injectable fine particles in which a complex of water-soluble peptide or serum albumin added to solid polyethylene glycol and hyaluronic acid sodium salt is dispersed is formed. Good. Further, the obtained fine particles may be dehydrated by further performing vacuum drying or the like. Further, during the production of the fine particles, an inorganic salt such as polyvinyl alcohol, mannitol, or phosphate may be attached to the surface of the fine particles for the purpose of preventing agglomeration of the fine particles. The fine particles thus obtained are usually dispersed immediately before administration in a solvent composed of a substance that is acceptable as an injection solvent, and then immediately administered.

The pharmaceutical composition of the present invention contains nucleic acid (RNA, D
NA), and the nucleic acid may be one used as an antisense (Minhwiang et al., Journal of Biological Chemistry, Vol. 266, 1).
Pages 9162 to 1871 (1991) are listed as a representative document). Conventionally used additives may be added to the unit dose thus obtained. For example, arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid as a pH regulator, benzyl alcohol as a local anesthetic, inorganic salts such as sodium chloride as a tonicity agent, carbohydrates such as mannitol and sorbitol, Tween 80 ( Examples thereof include, but are not limited to, cyclodextrin as a solubilizer, serum albumin as a stabilizer, and glutathione as a reducing agent.

The present invention is a pharmaceutical composition in which a peptide is mixed with a polyethylene glycol which is a wax-like solid at room temperature and may contain additives which are acceptable for constituting a preparation. Prepared to be present in unit composition. Furthermore, a solution formulation prepared by adding an injectable solvent to this preparation, a vacuum dried product or a lyophilized product of this solution, and a formulation prepared by re-dissolving these dried products in a pharmaceutically acceptable injectable solvent may also be used. Good. In addition, a pharmaceutical composition containing the peptide or an additive acceptable for constituting a preparation to the peptide, a solution obtained by dissolving the peptide in a solvent for injection, a lyophilized product of the solution, or a lyophilized product Re-dissolved in a commercially acceptable solvent for injection, dissolved in wax-like solid polyethylene glycol at room temperature with a solvent for injection, added with pharmaceutically acceptable additives or frozen. It is a long-acting preparation that is a mixture of a dried product and a redissolved solvent for injection. Further, the action can be expected to be enhanced by incorporating a water-soluble protein such as serum albumin, which has substantially no pharmacological effect, which can be injected into the body, into the sustained-release preparation.

The pharmaceutical composition of the present invention is prepared by adding water-soluble peptides or water-soluble peptides to water-soluble proteins such as serum albumin and / or acidic mucopolysaccharides such as sodium hyaluronate and homogenizing them, followed by heating and melting. It can also be used by dispersing it in solid polyethylene glycol at room temperature and injecting it in a solvent or spraying it in the gas phase to form fine particles, and dispersing it in a solvent for injection at the time of administration. The pharmaceutical composition of the present invention is preferably stored and distributed in a vacuum or freeze-dried state, but can be stored and distributed in a solution state. The storage temperature may be room temperature, but it is preferably stored in a cold place. The cold place in the present invention refers to 15 ° C or lower (Japanese Pharmacopoeia), but it may be better to avoid freezing depending on the formulation.

The pH of the pharmaceutical composition of the present invention does not significantly affect the activity of the peptide and is within the physiologically acceptable range.
pH is used. It is preferably about pH 2 to pH 10. More preferably, it is about pH 3 to pH 9. Particularly preferred is pH 3.5 to pH 8. The pH is adjusted by adding a solution containing hydrochloric acid or sodium hydroxide so that the final pH falls within the above range in the presence of water-soluble peptides, polyethylene glycol, various additives and the like. It is done by. Further, in order to ensure stability in the presence of the peptide, the pH adjusting liquid may be placed in a separate container and this operation may be performed immediately before use.

One feature of the pharmaceutical composition of the present invention is that it is water-soluble and persistent, and the viscosity of its solution is very low, and it is easy to handle during injection. The viscosity in the present invention refers to a viscosity measured by using a cone LD with an E-type viscometer (manufactured by Tokyo Keiki). The water-soluble injectable composition of the present invention is prepared to have a viscosity of 200 centipoise (cp) or less, preferably 100 cp or less, more preferably 70 cp or less when administered to a living body.
As the adjusting method, for example, a water-soluble peptide, polyethylene glycol, or various additives are mixed by the above method, and if necessary, diluted with an injectable solvent such as sterile physiological saline.

The administration method of the pharmaceutical composition of the present invention includes:
Administration by injection is performed, but subcutaneous administration is particularly preferable, and other injection routes such as intramuscular administration, intravenous administration, intraarticular administration, intraocular administration, and intratissue administration are also performed depending on the application. .. The dose, administration subject, administration subject disease, etc. of the composition of the present invention are determined based on those of the water-soluble peptide which is the main drug. Since the present invention does not use an organic solvent, it can be applied to peptides and the like whose three-dimensional structure is important for expression of physiological activity. Although the pharmaceutical composition of the present invention can sustain the pharmacological action of the peptide after administration, it can be administered in a solution state, so that the dose can be easily adjusted, and a thin injection needle can be used, so that it can be used by patients. The pain it gives is small. Further, in the present invention, by using polyethylene glycol in a limited range of an average molecular weight of 2000 to 6000, which is solid at room temperature, it becomes possible to formulate a peptide by vacuum drying or freeze drying in the presence of polyethylene glycol, Moreover, a sustained-release preparation having a sustained pharmacological action can be obtained. Therefore, it is easy for medical staff to handle, and it is not necessary for a patient to use a thick needle because it is a water-soluble composition, and the number of injections is reduced, so that the pain caused by a therapeutic action is reduced.

[0027]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1. Insulin long-acting formulation Porcine insulin (26.8 units / milligram: Diosynth (Netherlands), clearance in rat:
1416 ml / hr / kg) was dissolved in 5 ml of 0.1 N hydrochloric acid. 0.7 ml of this solution was added with polyethylene glycol 2 having an average molecular weight of 2000.
000 (Wako Pure Chemical Industries, Ltd.) was dissolved in 4.2 mg of physiological saline for injection and 0.7 ml was added and mixed.

Example 2. Insulin long-acting formulation 10 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 5 ml of 0.1 N hydrochloric acid. 0.7 ml of physiological saline for injection in which polyethylene glycol 2000 (Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 2000 was dissolved at a concentration of 2% (W / V) was added to 0.7 ml of this solution and mixed. In addition, 17 mg of citric acid hydrate,
17 mg sodium citrate, 28 mannitol
Milligram was added and dissolved. 1 ml of this preparation was transferred to a glass vial, and a known freeze-drying operation was performed (freeze-drying device: Riovac GT-3, Rebold).

Example 3. Insulin long-acting formulation 10 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 5 ml of 0.1 N hydrochloric acid. 0.7 ml of physiological saline for injection in which polyethylene glycol 4000 (Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 3000 was dissolved at a concentration of 2% (W / V) was added to 0.7 ml of this solution and mixed. In addition, 17 mg of citric acid hydrate,
17 mg sodium citrate, 28 mannitol
Milligram was added and dissolved. 1 ml of this preparation was transferred to a glass vial, and a known freeze-drying operation was performed (freeze-drying device: Riovac GT-3, Rebold).

Example 4. Insulin long-acting formulation 10 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 5 ml of 0.1 N hydrochloric acid. To 0.7 ml of this solution, 0.7 ml of physiological saline for injection in which polyethylene glycol 2000 (Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 2000 was dissolved at a concentration of 10% (W / V) was added and mixed. Further, 17 mg of citric acid hydrate, 17 mg of sodium citrate, and 28 mg of mannitol were added and dissolved therein. 1 ml of this preparation was transferred to a glass vial, and a known freeze-drying operation was performed (freeze-drying device: Riovac GT-3,
Rebold).

Example 5. Insulin long-acting formulation 10 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 5 ml of 0.1 N hydrochloric acid. To 0.7 ml of this solution, 0.7 ml of physiological saline for injection in which polyethylene glycol 4000 (Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 3000 was dissolved at a concentration of 10% (W / V) was added and mixed. Further, 17 mg of citric acid hydrate, 17 mg of sodium citrate, and 28 mg of mannitol were added and dissolved therein. 1 ml of this preparation was transferred to a glass vial, and a known freeze-drying operation was performed (freeze-drying device: Riovac GT-3,
Rebold).

Example 6. Insulin long-acting formulation 10 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 5 ml of 0.1 N hydrochloric acid. 0.7 ml of physiological saline for injection in which polyethylene glycol 2000 (Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 2000 was dissolved at a concentration of 20% (W / V) was added to 0.7 ml of this solution and mixed. Further, 17 mg of citric acid hydrate, 17 mg of sodium citrate, and 28 mg of mannitol were added and dissolved therein. 1 ml of this preparation was transferred to a glass vial, and a known freeze-drying operation was performed (freeze-drying device: Riovac GT-3,
Rebold).

Example 7. Insulin long-acting formulation 10 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 5 ml of 0.1 N hydrochloric acid. To 0.7 ml of this solution, 0.7 ml of physiological saline for injection in which polyethylene glycol 4000 (Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 3000 was dissolved at a concentration of 20% (W / V) was added and mixed. Further, 17 mg of citric acid hydrate, 17 mg of sodium citrate, and 28 mg of mannitol were added and dissolved therein. 1 ml of this preparation was transferred to a glass vial, and a known freeze-drying operation was performed (freeze-drying device: Riovac GT-3,
Rebold).

Example 8. Insulin long-acting formulation 10 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 5 ml of 0.1 N hydrochloric acid. To 0.7 ml of this solution, 0.7 ml of physiological saline for injection in which 4.2 mg of polyethylene glycol 2000 (Wako Pure Chemical Industries) having an average molecular weight of 2000 was dissolved was added and mixed.

Example 9. Insulin long-acting preparation 12 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 1 ml of 0.1N hydrochloric acid, and further diluted with 5 ml of saline for injection. did. Polyethylene glycol 2000 (Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 2000 of 60 milligrams, 300 milligrams, or 600 milligrams is dissolved in 3 milliliters of physiological saline for injection, and 2% (W / V) and 10% (W / V) , Or 2
A 0% (W / V) polyethylene glycol solution was prepared. 1 ml of the above-mentioned insulin aqueous solution was added to 1 ml of each solution to prepare three types of insulin injection solutions having different polyethylene glycol concentrations.

Example 10. Interferon alpha long-acting preparation Interferon alpha preparation containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin (Takeda, rat clearance:
123 ml / hr / kg) 1 vial of distilled water for injection was added to each of 2 vials and combined. 1.2 ml of this was taken, and 0.45% (W / W of polyethylene glycol 4000 (Wako Pure Chemicals first grade, average molecular weight 3000) was added.
V) physiological saline for injection (Fuso Yakuhin)
0.6 ml of the solution dissolved in was added and mixed.

Example 11. Interferon alpha long-acting preparation 1 ml of distilled water for injection was added to 2 vials of interferon alpha preparation (Takeda) containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin, and combined. 1.2 of these
Take a milliliter of polyethylene glycol 2000
(Wako Pure Chemicals first grade, average molecular weight 2000) 20% (W /
V) physiological saline for injection (Fuso Yakuhin)
0.3 ml of the solution dissolved in and 0.3 ml of physiological saline for injection were added and mixed.

Example 12. Interferon alpha long-acting preparation 1 ml of distilled water for injection was added to 2 vials of interferon alpha preparation (Takeda) containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin, and combined. 1.2 of these
Take milliliters and polyethylene glycol 4000
(Wako Pure Chemicals first-class, average molecular weight 3000) 20% (W /
V) physiological saline for injection (Fuso Yakuhin)
0.3 ml of the solution dissolved in and 0.3 ml of physiological saline for injection were added and mixed.

Example 13. Insulin long-acting preparation 400 mg of polyethylene glycol 2000 or 4000 (each Wako first grade) and 500 mg of D-mannitol (Wako special grade) were dissolved in 10 ml of physiological saline for injection (Fuso Yakuhin), and 20% thereof was added.
Albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing (W / V) human serum albumin was added in an amount of 0.1 milliliter and mixed. This solution was mixed with 10 mg of pork insulin (26.8 units / milligram: Diosynth (Netherlands)) dissolved in 2 ml of 0.1 N hydrochloric acid. No precipitation was observed in the obtained two kinds of solutions, and clear solutions were obtained. The two types of the mixture were freeze-dried by a method known per se using a freeze-drying apparatus Lyovac GT-3 (Rebold Heraeus, Germany).

Example 14 Insulin long-acting preparation 4.5 mg of porcine insulin (26.8 units / milligram: Diocins (Netherlands)) was dissolved in 0.5 ml of 0.1 N hydrochloric acid, and 2.5 ml of physiological saline for injection was further dissolved. Water was added to dilute. 600 mg of polyethylene glycol 2000 having an average molecular weight of 2000 (Wako Pure Chemical Industries, Ltd.) was dissolved in 3 ml of physiological saline for injection to prepare a 20% (W / V) polyethylene glycol solution. An insulin injection solution was prepared by adding 0.5 ml of a polyethylene glycol solution and 0.5 ml of physiological saline for injection to 1 ml of a pig insulin solution.

Example 15. G-CSF sustained-release preparation 600 mg of polyethylene glycol 2000 (Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 2000 was dissolved in 3 ml of physiological saline for injection to prepare a 20% (W / V) polyethylene glycol solution. G-CSF was prepared by adding 1.727 ml of physiological saline for injection and 0.35 ml of this polyethylene glycol solution to 23 μl of Filgrastim (Amgen, USA), which is a formulation containing G-CSF. An injection solution was prepared. The clearance of G-CSF in rats is 53.1 to 79.8 per kilogram body weight.
Milliliters / hour reported (Tanaka Hideji et al., The Journal of Pharmacology and Experimental Therapeutics, 25th
Volume 5, 724-729, 1990).

Example 16 SOD Sustainable Preparation 400 units (equivalent to 200 micrograms) of Serratia-derived superoxide dismutase (SO
D) (JP-A-57-29285 and JP-A-58-1)
6685) and 0.7 ml of physiological saline containing polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) (10% (w / v)) in physiological saline was added to 0.7 ml of physiological saline. Completely mixed.

Example 17 Hirudin Sustainable Formulation A saline solution for injection was added to a solution containing 100 ATU (corresponding to about 8 micrograms) of hirudin (Peninsula Laboratory, USA) to make a total volume of 1 ml. A glass vial containing this solution was placed in a polyethylene glycol (polyethylene glycol with an average molecular weight of 3000).
00: Wako Pure Chemical Industries, Ltd. physiological saline solution (10% (w / v))
Add 1 ml and mix thoroughly.

Example 18. IL-2 long-acting formulation 1 ml of interleukin 2 (IL-2; 1
Containing 0 microgram) (JP-A-61-78799)
(Described in Japanese Patent Laid-Open Publication No. 60-11).
5528). 0.5 ml of physiological saline was added to the mixture (with and without methionine at the N-terminus). A glass saline vial containing 1 ml of this solution was added with a physiological saline solution of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) (10% (w / w)).
v)) 1 ml was added and mixed thoroughly.

Example 19 Leuprorelin acetate sustained release formulation 750 micrograms of LH-RH analog leuprorelin acetate (US Pat. No. 4,008,209) was added to 1.5 ml of 1/30 molar phosphate buffer (pH).
It was dissolved in 6). To a glass vial containing 1 ml of this solution, 1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added and thoroughly mixed.

Example 20. Persistent PTH preparation 450 micrograms of parathyroid hormone (PTH)
(Bachem, Switzerland) was dissolved in 1.5 ml of saline for injection. To a glass vial containing 1 ml of this solution, 1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added and thoroughly mixed.

Example 21. TRH long-acting formulation 1.464 milligrams of thyrotropin-releasing hormone (TRH) tartrate and 50 milligrams of D-sorbitol containing 1 ml of hiltonin injection (Takeda Yakuhin) in a glass vial. 5 ml) and add polyethylene glycol (polyethylene glycol 4000 with average molecular weight 3000: Wako Pure Chemical Industries) in physiological saline solution (10%
(w / v)) 1 ml was added and mixed thoroughly.

Example 22. FGF mutein sustained-release preparation 0.36 of TGP-580 (recombinant human basic FGF mutein CS23: EP publication 281,822) solution (0.96 mg protein / ml).
1.14 ml of physiological saline for injection, 1.5 ml of physiological saline solution of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) (10% (w / v)), and human serum albumin. 6 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% was added and thoroughly mixed.

Example 23. NGF persistent preparation 4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin in 0.7 milliliters of physiological saline containing 10 micrograms of nerve cell growth factor (NGF) (Biomedical Technology Inc., USA). Then, 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. Further, 2.1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added to this solution and thoroughly mixed.

Example 24. Sustainable EGF preparation 4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 milliliters of physiological saline containing 10 micrograms of epidermal growth factor (EGF) (Chemicon International, USA). Then, 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. Further, 2.1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added to this solution and thoroughly mixed.

Example 25. Interferon beta long-acting preparation 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 interferon beta (Pazel, Germany), and further injected. 1.4 ml of physiological saline (Fuso Yakuhin) was added to bring the total volume to 2.1 ml. Further, 2.1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added to this solution and thoroughly mixed.

Example 26. Interferon gamma long-acting preparation Interferon gamma (Genzyme, USA) 10
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 microgram, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make a total amount. To 2.1 ml. Further, 2.1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added to this solution and thoroughly mixed.

Example 27. TGF-β continuous preparation Transforming growth factor (TGF-β) (Wako Pure Chemical Industries, Ltd.)
4 ml of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin was added to 0.7 ml of physiological saline containing 1 microgram, and 1.4 ml of physiological saline for injection (Fuso Yakuhin)
Was added to bring the total volume to 2.1 ml. Further, 2.1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added to this solution and thoroughly mixed.

Example 28. Parathyroid hormone persistent preparation Parathyroid hormone (PTH) (Bachem, Switzerland) 1
4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin was added to 0.7 milliliters of physiological saline containing 0 microgram, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was further added. The total volume was 2.1 ml. Further, 2.1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added to this solution and thoroughly mixed.

Example 29. TNF-α persistent preparation Tumor necrosis factor (TNF-α) (manufactured by Wako Pure Chemical Industries, Ltd.) 4
Albumin Nichiyaku containing 20% of human serum albumin by adding 0.6 ml of physiological saline for injection to 0.1 ml of phosphate buffer of pH 7.4 containing 00 unit and 0.1 mg of human serum albumin (Nippon Pharmaceutical Co., Ltd.) was added in an amount of 4 microliters, and further 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. Further, 2.1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added to this solution and thoroughly mixed.

Example 30. Sustainable growth hormone growth hormone (UCB BioProducts, Belgium)
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, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was further added. The total volume was 2.1 ml. Further, 2.1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added to this solution and thoroughly mixed.

Example 31. Calcitonin sustained-release formulation Calcitonin (UCB BioProducts, Belgium)
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, and 1.4 ml of physiological saline for injection (Fuso Yakuhin) was further added. The total volume was 2.1 ml. Further, 2.1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added to this solution and thoroughly mixed.

Example 32. TPA long-acting preparation 20 ml of human serum albumin was added to 0.7 ml of physiological saline containing 10 micrograms of tissue plasminogen activator (TPA) (Bioscot, USA).
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. Further, 2.1 ml of a physiological saline solution (10% (w / v)) of polyethylene glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) was added to this solution and thoroughly mixed.

Example 33. IL-3 Sustainable Preparation Interleukin 3 (IL-3; R & D Systems, USA) 4 microliters of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin in 0.7 milliliters of physiological saline containing 1000 units. Then, 1.4 ml of physiological saline for injection (Fuso Yakuhin) was added to make the total volume 2.1 ml. Also,
Polyethylene glycol (average molecular weight 300
0 ml of polyethylene glycol 4000: Wako Pure Chemical Industries, Ltd. (2.1%) in physiological saline solution (10% (w / v)) was added and mixed thoroughly.

Example 34. Interferon alpha long-acting preparation Interferon alpha preparation containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin (Takeda, clearance in rat:
1 ml of distilled water for injection was added to each of 10 vials (123 ml / hr / kg). To this, 10 ml of a solution of sodium salt of hyaluronic acid having a molecular weight of 1.8 million (Genzyme, USA) dissolved in physiological saline for injection at 1.2% (w / v) was added and gently mixed. This solution was freeze-dried according to a conventional method, and the obtained powder was heated to 55 ° C.
It was added to 10 g of polyethylene glycol 400 (average molecular weight 3000: Wako first-class) which was heated and melted and rapidly dispersed. The fine particles were prepared by dropping the solution into 200 ml of distilled water cooled to 5 ° C. with high speed stirring using a homogenizer (Polytron: Kinematica). Subsequently, the particles were recovered by rapidly sieving and freeze-dried.

Example 35. Interferon alpha long-acting preparation Interferon alpha preparation containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin (Takeda, clearance in rat:
1 ml of distilled water for injection was added to each of 10 vials (123 ml / hr / kg). To this, 10 ml of a solution of sodium salt of hyaluronic acid having a molecular weight of 1.8 million (Genzyme, USA) dissolved in physiological saline for injection at 1.2% (w / v) was added and gently mixed. This solution was freeze-dried according to a conventional method, and the obtained powder was heated to 55 ° C.
It was added to 10 g of polyethylene glycol 400 (average molecular weight 3000: Wako first-class) which was heated and melted and rapidly dispersed. Then spray dryer (GS-3
Microparticles were prepared from this solution using 1: Yamato Scientific Co., Ltd., and freeze-dried.

Experimental Example 1 An injection was prepared in the following mixing ratio and used in the experiment. Comparative preparation 1: 10 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 5 ml of 0.1 N hydrochloric acid. To 0.7 ml of this solution was added 0.7 ml of physiological saline for injection. Comparative preparation 2: physiological saline for injection 2 ml The preparation of Example 1 or the injection of Comparative preparation 1 was administered subcutaneously to the back of 8-week-old male SD rats at a dose of 200 microgram / rat. As a control, a physiological saline injection solution of Comparative Preparation 2 was similarly administered. Before administration and at regular intervals, about 0.4 ml of blood was collected and serum was separated and collected. The glucose concentration in serum was quantified by glucose C test Wako (Wako Pure Chemical Industries, Ltd.). [FIG. 1] shows the time course of serum glucose concentration. In the physiological saline injection solution administration group of Comparative Preparation 2, the serum glucose concentration hardly changed. In the insulin solution administration group of Comparative Preparation 1, serum glucose concentration decreased from 30 minutes after administration, but started to increase from 120 minutes after administration. On the other hand, in the insulin solution administration group to which the polyethylene glycol 2000 of the preparation of Example 1 was added, the serum glucose concentration decreased from the 30th minute after the administration, and the value was almost the same at 6 hours after the administration. The clearance of insulin per kilogram of body weight in rats is 1416 ml /
Time is reported (Halban et al., Metabolism, Vol. 28, pp. 1097-1104 (1979).
Year)).

Experimental Example 2 Comparative preparation 3: 5 mg of 10 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands))
It was dissolved with milliliter of 0.1 N hydrochloric acid. 0.7 ml of this solution and 0.7 ml of physiological saline for injection in which polyethylene glycol 400 (Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 400 was dissolved at a concentration of 10% (W / V) were added and mixed. Further, 17 mg of citric acid hydrate, 17 mg of sodium citrate, and 28 mg of mannitol were added and dissolved therein. 1 of this formulation
A milliliter was transferred to a glass vial, and a known freeze-drying operation was performed (freeze-drying device: Riovac GT-
3, Rebold). Compared to the insulin-sustaining preparations obtained by mixing the polyethylene glycol having an average molecular weight of 2000 or 3000 obtained in Examples 2 to 7 with sponge cake-like uniform freeze-dried products, comparative preparation 3 was obtained. In the insulin preparation containing the polyethylene glycol 400, the lyophilized product was wet due to the liquid polyethylene glycol 400 at room temperature. This indicates that polyethylene glycol having an average molecular weight of 2000 or 3000 is superior to polyethylene glycol 400 as a compounding agent when freeze-drying, which is widely used as a method for formulating unstable peptides, is performed.

Experimental Example 3 Comparative preparation 4: 10 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 5 ml of 0.1 N hydrochloric acid. To 0.7 ml of this solution was added 0.7 ml of physiological saline for injection in which 4.2 mg of polyethylene glycol 400 (Wako Pure Chemical Industries) having an average molecular weight of 400 was dissolved,
Mixed. Comparative formulation 5: 10 mg of swine insulin (26.8 units / milligram: Diosynth (Netherlands))
It was dissolved with milliliter of 0.1 N hydrochloric acid. To 0.7 ml of this solution was added 0.7 ml of physiological saline for injection. Comparative preparation 6: Saline for injection 2 ml 8 weeks old male SD rat subcutaneously subcutaneously on the back of the preparation of Example 8 at a dose of 200 microgram / rat, Comparative preparation 4
Alternatively, the injection solution of Comparative Preparation 5 was administered. As a control, the physiological saline injection solution of Comparative Preparation 6 was similarly administered. Before administration and at regular intervals, about 0.4 ml of blood was collected and serum was separated and collected. Glucose concentration in serum was quantified by Glucose C Test Wako (Wako Pure Chemical Industries). [FIG. 2] shows the time course of serum glucose concentration. In the physiological saline injection solution administration group of Comparative Preparation 6, the serum glucose concentration hardly changed. In the insulin solution administration group of Comparative Preparation 5, the serum glucose concentration decreased from 30 minutes after administration, but started to increase from 120 minutes after administration. In the insulin solution-administered group containing polyethylene glycol 400 of Comparative preparation 4, the serum glucose concentration was 30 after administration.
Although it decreased from the minute, the increase started from 240 minutes after the administration. On the other hand, in the insulin solution administration group to which the polyethylene glycol 2000 of the preparation of Example 8 was added, the serum glucose concentration decreased from 30 minutes after the administration, and 6 minutes after the administration.
It was almost the same low value at the time. From this, it was found that the polyethylene glycol added had a larger molecular weight and a longer lasting effect was obtained.

Experimental Example 4 Comparative preparation 7: 12 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 1 ml of 0.1 N hydrochloric acid, and further 5 ml of physiological saline for injection. Water was added to dilute. 1 ml of physiological saline for injection was added to 1 ml of this insulin aqueous solution to prepare an insulin injection solution. 8
The injections of the preparation of Example 9 and the comparative preparation 7 were administered subcutaneously to the back of week-old male SD rats at a dose of 200 microgram / rat. Before administration and at regular intervals, about 0.4 ml of blood was collected and serum was separated and collected. The glucose concentration in serum was quantified by glucose C test Wako (Wako Pure Chemical Industries, Ltd.). [FIG. 3] shows the time course of serum glucose concentration. In the insulin solution administration group containing no polyethylene glycol 2000 of Comparative Preparation 7, the serum glucose concentration once decreased, but started increasing at 180 minutes after administration and returned to almost normal value at 360 minutes. On the other hand, in the insulin solution-administered group containing polyethylene glycol 2000, the serum glucose concentration is once lowered, but the higher the concentration of polyethylene glycol is, the more likely the recovery of blood glucose level is delayed, which is 2% (W / V).
In the insulin injection solution-administered group, which was diluted 2-fold with the polyethylene glycol aqueous solution, the blood glucose level began to recover from 240 minutes after administration. The blood glucose level was further decreased in the insulin injection solution-administered group that was diluted 2-fold with a 10% (W / V) concentration of polyethylene glycol 2000 aqueous solution.
Recovery of blood glucose level from the 40th minute was also weak. However, the concentration of polyethylene glycol 2000 used was 20%
Even if it was raised to (W / V), no significant enhancement of drug efficacy was observed. From this, it was revealed that the sustained effect of insulin can be obtained by coexistence of polyethylene glycol of at most about 5% (W / V).

Experimental Example 5 Comparative preparation 8: 1 ml of distilled water for injection was added to 2 vials of an interferon alpha preparation (Takeda Pharmaceutical Co., Ltd.) containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin and combined. 1.2 ml of this was taken, and 0.6 ml of physiological saline for injection (Fuso Yakuhin) was added and mixed. 8
The injection solution of the preparation of Example 10 or Comparative preparation 8 was administered subcutaneously to the back of week-old male SD rats at a dose of 0.3 ml / rat. Approximately 0.4 at regular intervals after administration
Blood was collected in milliliters and serum was separated and collected. Interferon alpha concentration in serum was quantified by ELISA. The results are shown in FIG. In the group administered with interferon alfa alone, the blood concentration rapidly decreased from 1 hour after the administration. On the other hand, average molecular weight 300
In the group administered with polyethylene glycol of 0, the blood concentration was lower than that in the interferon alfa alone administration group at 1 hour and 2 hours after administration, but at 4 and 6 hours after administration, interferon alfa The blood level was higher than that in the single-dose group. This means that by administering interferon alfa and polyethylene glycol having an average molecular weight of 3000 as a mixture, it is possible to maintain a high blood concentration while lowering the blood concentration peak and suppressing the occurrence of undesirable side effects. It shows that. The clearance of interferon alfa in the rat per kilogram body weight was 123 ml / hour.

Experimental Example 6 Comparative preparation 9: 1 ml of distilled water for injection was added to 2 vials of an interferon alfa preparation (Takeda Pharmaceutical Co., Ltd.) containing 3 million international units of interferon alpha and 5 mg of human serum albumin, and combined. 1.2 ml of this was taken, and 0.6 ml of physiological saline for injection (Fuso Yakuhin) was added and mixed. 8
Injections of the formulation of Example 11, the formulation of Example 12, or the comparative formulation 9 were administered subcutaneously to the dorsal skin of week-old male SD rats at a dose of 0.3 ml / rat. After administration, about 0.4 milliliters of blood was collected at regular intervals and the serum was separated and collected. The interferon alpha concentration in serum was quantified by the ELISA method. The results are shown in FIG. In the group administered with interferon alfa alone, the blood concentration rapidly decreased from 1 hour after the administration. On the other hand, in the group administered by mixing with polyethylene glycol having an average molecular weight of 2000 or 3000, the blood concentration was lower than or almost the same as that of the interferon alfa alone administration group at 1 hour and 2 hours after administration. At the 4th and 6th hours, higher blood levels were maintained compared to the interferon alfa alone administration group. This means that by administering a mixture of interferon alpha and polyethylene glycol having an average molecular weight of 2000 or 3000, the peak blood concentration is lowered to suppress the occurrence of undesirable side effects while maintaining a high blood concentration. Is possible.

Experimental Example 7 Comparative preparation 10: 400 mg of polyethylene glycol 6000 (Wako first-class average molecular weight 7500) and 50
Dissolve 0 mg D-mannitol (Wako special grade) in 10 ml physiological saline for injection (Fuso Yakuhin),
To this, 0.1 ml of albumin Nichiyak (Nippon Pharmaceutical Co., Ltd.) containing 20% (W / V) human serum albumin was added and mixed. This solution and pig insulin (26.8
Unit / milligram: 10 mg of Diosynth (Netherlands) dissolved in 2 ml of 0.1 N hydrochloric acid was mixed. No precipitation was observed in the obtained solution, and a clear solution was obtained. The mixture was freeze-dried by a method known per se using a lyophilizer Lyovac GT-3 (Rebold Heraeus, Germany). Each of the two lyophilized products obtained in Example 13 or Comparative Preparation 10 was dissolved by adding 10 ml of a mixture of distilled water for injection (Fuso Yakuhin) and 2 ml of 0.1 N hydrochloric acid. 60
Milky white turbidity was observed only in the group using 00. From this fact, polyethylene glycol having an average molecular weight of 6000 or more causes denaturation of the peptide and cannot be used in the present invention as a lyophilizable injectable pharmaceutical composition.

Experimental Example 8 Comparative preparation 11: 4.5 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 0.5 ml of 0.1 N hydrochloric acid,
Furthermore, 2.5 ml of physiological saline for injection was added to dilute. 600 mg of polyethylene glycol 1540 (Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 1500 was dissolved in 3 ml of physiological saline for injection to prepare a 20% (W / V) polyethylene glycol solution. 0.5 ml polyethylene glycol solution to 1 ml pig insulin solution
An insulin injection solution was prepared by adding milliliters and physiological saline 0.5 ml for injection. Comparative preparation 12: 4.5 mg of porcine insulin (26.8 units / milligram: Diosynth (Netherlands)) was dissolved in 0.5 ml of 0.1 N hydrochloric acid,
Furthermore, 2.5 ml of physiological saline for injection was added to dilute. An insulin injection solution was prepared by adding 1 ml of physiological saline for injection to 1 ml of this insulin solution. Example 1 was subcutaneously administered to the back of 11 to 12-week-old male SD rats at a dose of 150 microgram / rat.
Injections of formulation 4 or comparative formulation 11 were administered. As a control, the injection solution of Comparative Preparation 12 was administered. Before administration and at regular intervals, about 0.4 ml of blood was collected and serum was separated and collected. Glucose concentration in serum was quantified by Glucose C Test Wako (Wako Pure Chemical Industries). [FIG. 6] shows the time course of serum glucose concentration. In the insulin solution administration group of Comparative Preparation 12, the serum glucose concentration was 2 after administration.
Although it decreased to the time point, it increased after that, and was almost restored to the value before the administration at 5 hours after the administration. In the insulin solution-administered group containing polyethylene glycol 1540 of Comparative Preparation 11, the serum glucose concentration showed almost the same time course of blood glucose level as that of Comparative Preparation 12-administered group, and no difference was observed. On the other hand, in the insulin solution administration group to which the polyethylene glycol 2000 of the preparation of Example 14 was added, 3 days after administration
The decrease in serum glucose concentration continued until the time, and even at 5 hours after administration, it was about half the blood glucose level of the comparative preparation 11 or the comparative preparation 12 administration group. Therefore, polyethylene glycol 1540 having an average molecular weight of 1500 of polyethylene glycol to be added does not have a sustaining effect, and polyethylene glycol 20 having an average molecular weight of 2000 cannot be obtained.
It has been found that 00 has a sufficient sustaining effect.

Experimental Example 9 Comparative preparation 13: A physiological saline solution for injection (2.077) was added to 23 microliters of Filgrastim (Amgen, USA) which is a preparation containing G-CSF.
G-CSF injection was prepared by adding milliliter. Comparative formulation 14: 600 mg average molecular weight 2000
Polyethylene glycol 2000 (Wako Pure Chemical Industries, Ltd.) was dissolved in 3 ml of physiological saline for injection to give 20% (W /
A polyethylene glycol solution of V) was prepared. An injection solution was prepared by adding 1.75 ml of physiological saline for injection and 0.35 ml of this polyethylene glycol solution. 8-week-old male SD rats were subcutaneously injected with human G-
The injection solutions of the preparation of Example 15 containing CSF, Comparative Preparation 13 and Comparative Preparation 14 were each administered in an amount of 0.3 ml. Approximately 0.4 ml of blood was collected at 7 hours after administration (using EDTA 2Na as an anticoagulant), and peripheral white blood cell count, red blood cell count, and platelet count were measured using a microcell counter CC-180A type (Toa Medical Electronics). did.
Peripheral neutrophil count, lymphocyte count, monocyte count, and eosinophil count were classified into 200 leukocytes by microscopic examination of Giemsa-stained blood smear, and the frequency of appearance of each cell was taken as the white blood cell count. Calculated by multiplying. The results are shown below (average value of 4 rats in each group): Number of peripheral neutrophils per cubic millimeter of blood (× 100) Preparation of Example 15 107.0 Comparative preparation 13. 84.0 Comparative formulation 14. 52.5 These results indicate that the pharmacological effect is enhanced by adding polyethylene glycol having an average molecular weight of 2000 to G-CSF, which has a clearance of 53.0 to 79.8 ml / hour per kilogram of body weight in rats. It has been found. It is known that the once increased peripheral neutrophils disappear from peripheral blood by a normal physiological mechanism, and it is clear that the effect of increasing the number of peripheral neutrophils by the addition of polyethylene glycol is sustained.

[0072]

INDUSTRIAL APPLICABILITY The injectable composition of the present invention sustains the in vivo pharmacological action of the water-soluble peptide which is the main drug.
Therefore, the number of injections can be reduced. This reduces the suffering of the patient from the medical procedure,
Moreover, there is a feature that the burden on the medical staff can be reduced.

[0073]

[Brief description of drawings]

FIG. 1 shows the glucose concentration in serum after administration of the preparation of Example 1 (Δ), Comparative preparation 1 (◯) or Comparative preparation 2 (marked with black in black) obtained in Experimental Example 1. It shows the time transition.

FIG. 2 shows the administration of the preparation of Example 8 (◯), the comparative preparation 4 (□), the comparative preparation 5 (marked with ○ in black) or the comparative preparation 6 (△) obtained in Experimental Example 3. It shows the time course of the glucose concentration in serum afterwards.

FIG. 3 shows that the insulin solution of Example 9 obtained in Experimental Example 4 had a polyethylene glycol 2000 concentration of 2%.
What was diluted two-fold with the (W / V) solution (black is filled in) and what was diluted two-fold with the 10% (W / V) solution (□) or 20% (W / V) 2 shows the time course of serum glucose concentration after administration of a 2-fold diluted solution (Δ).

FIG. 4 shows the time course of serum interferon alfa concentration after administration of Comparative Preparation 8 (marked with a black circle) or Preparation of Example 10 (○) obtained in Experimental Example 5. Is.

FIG. 5 is serum interferon after administration of the comparative preparation 9 (marked with a black circle), the preparation of Example 11 (○) or the preparation of Example 12 (□) obtained in Experimental Example 6. It shows the time transition of the alpha concentration.

FIG. 6 shows the serum glucose concentration after administration of the preparation of Example 14 (◯), the comparative preparation 11 (marked with black in black) or the comparative preparation 12 (□) obtained in Experimental Example 8. It shows the time transition.

Claims (3)

[Claims] 1. A water-soluble peptide having a clearance of 30 ml / hour or more per kilogram body weight in rats and an average molecular weight of wax-like solid at room temperature of 2000.
A water-soluble pharmaceutical composition for injection, which comprises 6000 to 6000 polyethylene glycol. 2. The pharmaceutical composition according to claim 1, further comprising a water-soluble protein which has substantially no pharmacological activity and is injectable into a living body. 3. The pharmaceutical composition according to claim 1 or 2, further comprising an acidic mucopolysaccharide.