JP3730667B2 - Injectable composition - Google Patents

Description

この結果より、ラットにおけるクリアランスが体重１キログラム当たり５３. ０〜７９.８ミリリットル／時間であるＧ−ＣＳＦに平均分子量２０００のポリエチレングリコールを添加することにより、薬理効果が増強されていることが 判明した。一旦、増加した末梢好中球は通常の生理的メカニズムにより末梢血から消失していくことが知られており、ポリエチレングリコール添加による末梢好中球数増加効果が持続することは明らかである。
【００７２】
【発明の効果】
本発明の注射用組成物は、主薬である水溶性ペプチドの生体内における薬理作用を持続させる。
そのため、注射による投与回数を減らすことが可能になる。このことは、医療行為による患者の苦痛を軽減し、かつ医療従事者の負担をも軽減できるという特徴がある。
【００７３】
【図面の簡単な説明】
【図１】は、実験例１で得られた、実施例１の製剤（△）、比較製剤１（○）または比較製剤２（○を黒く塗りつぶした印）を投与後の血清中グルコース濃度の時間推移を示したものである。
【図２】は、実験例３で得られた、実施例８の製剤（○）、比較製剤４（□）、比較製剤５（○を黒く塗りつぶした印）または比較製剤６（△）を投与後の血清中グルコース濃度の時間推移を示したものである。
【図３】は、実験例４で得られた、実施例９のインスリン溶液をポリエチレングリコール２０００の濃度が２％（Ｗ／Ｖ）の溶液で２倍希釈したもの（○を黒く塗りつぶしたもの）、１０％（Ｗ／Ｖ）の溶液で２倍希釈したしたもの（□）または２０％（Ｗ／Ｖ）の溶液で２倍希釈したもの（△）を投与後の血清中グルコース濃度の時間推移を示したものである。
【図４】は、実験例５で得られた、比較製剤８（○を黒く塗りつぶした印）または実施例１０の製剤（○）を投与後の血清中インターフェロンアルファ濃度の時間推移を示したものである。
【図５】は、実験例６で得られた、比較製剤９（○を黒く塗りつぶした印）、実施例１１の製剤（○）または実施例１２の製剤（□）を投与後の血清中インターフェロンアルファ濃度の時間推移を示したものである。
【図６】は、実験例８で得られた、実施例１４の製剤（○）、比較製剤１１（○を黒く塗りつぶした印）または比較製剤１２（□）を投与後の血清中グルコース濃度の時間推移を示したものである。[0001]
[Industrial application fields]
The present invention relates to an injectable pharmaceutical composition useful as a preparation for maintaining a pharmacological action in vivo of a peptide that is rapidly metabolized and excreted.
[0002]
[Prior art]
Although peptides have strong pharmacological effects, they are frequently decomposed and excreted after being administered into body fluids, and therefore often require frequent administration by injection or the like. Therefore, the development of sustained-release preparations of these substances can reduce patient pain by reducing the number of injections, increase the effectiveness by continuously maintaining the concentration in the body fluid within a certain effective concentration range, or the concentration in the body fluid It is necessary to reduce the side effects caused by sudden fluctuations.
Attempts have been made to use polyethylene glycol for the purpose of producing such a sustained-release preparation, but it is still not sufficient.
[0003]
For example, British Patent No. 1385914 discloses a polyoxyethylene glycol having increased liposolubility by acylating a hydrolyzate such as casein, collagen and keratin (palmitoylation, lauroylation, stearoylation, etc.). Although a technique for topical skin preparation that can effectively penetrate into subcutaneous tissue by dissolving in an amphipathic solvent such as is disclosed, formulation of a water-soluble peptide is not shown at all. It has not been shown to be used as an injection.
PCT International Application Publication No. WO 89/09610 discloses a technique of a freeze-dried preparation having a low water content and containing polyethylene glycol or the like for the purpose of reducing the association and aggregation of tumor necrosis factor (TNF). However, the molecular weight and blending ratio of polyethylene glycol are not specifically shown. Moreover, the influence of the blended polyethylene glycol on the in vivo kinetics of tumor necrosis factor (TNF) after administration to the living body is not shown.
[0004]
In PCT International Application Publication No. WO 89/05158, polyethylene glycol which is a nonelectrolytic substance having a molecular weight of 900 to 1600, more preferably a molecular weight of 190 to 420, particularly preferably a molecular weight of 285 to 315 (usually referred to as polyethylene glycol 300). Of interferon beta (IFN-β) protein injection is disclosed as a stabilizer or solubilizer at a high concentration of 5% to 50% (v / v), particularly preferably 25% (v / v) ing. In this publication, since polyethylene glycol which is liquid at normal temperature is used, a vacuum drying or freeze drying technique which is usually employed as a preparation capable of stably storing peptides and proteins cannot be applied. That is, it is described that freeze-drying is impossible with the combination of polyethylene glycol concentration and molecular weight used in the application. Moreover, the influence of polyethylene glycol on the in vivo kinetics of interferon beta protein after administration to the living body is not shown. Since the molecular weight of polyethylene glycol is smaller, the irritation is stronger, so it is considered that preparations containing a high concentration of polyethylene glycol having a low molecular weight should be avoided.
Japanese Patent Application Laid-Open No. 61-97229 discloses a technique for a stable erythropoietin preparation comprising polyethylene glycol or the like. However, although only a preparation containing 0.25% or 0.5% polyethylene glycol 4000 is shown here, an effective preparation method such as the molecular weight of polyethylene glycol used and the concentration in the final preparation is disclosed. It has not been.
JP-A 63-122628 discloses interleukin 2 and polyethylene glycol monostearate compound, and PCT International Application Publication No. WO 89/02750 discloses interferon beta and polyethylene glycol monostearate compound. A technique for blending as a solubilizer is disclosed. That is, here, polyethylene glycol is used as a derivative having a nonionic interface action.
[0005]
[Problems to be solved by the invention]
If the duration of pharmacological action of a water-soluble peptide with large clearance (ie, short duration of pharmacological action in vivo) can be increased, patient pain can be reduced by reducing the number of administrations by injection. In addition, since the drug concentration in the body fluid is continuously maintained within a certain effective concentration range, the effectiveness of the drug is enhanced.
[0006]
[Means for Solving the Problems]
As a result of diligent research to solve the above problems, the inventors have formulated a polyethylene glycol having an average molecular weight of 2000 to 6000, which is a wax-like solid at room temperature, so that the clearance in rats is 30 ml per kg body weight. It has been found that a lyophilized water-soluble sustained injection preparation capable of maintaining its pharmacological action under low viscosity can be obtained as well as not impairing the pharmacological activity of a water-soluble pharmaceutically active peptide that is longer than / hour, As a result of further intensive studies based on this, the present invention has been completed.
The present invention relates to (1) a water-soluble injection comprising a water-soluble peptide having a clearance of 30 ml / hour or more per kilogram of body weight in a rat and polyethylene glycol having an average molecular weight of 2000 to 6000 which is a wax-like solid at room temperature. (1) a pharmaceutical composition comprising (2) a water-soluble protein having substantially no pharmacological activity that can be injected into a living body; The pharmaceutical composition according to the above (1) or (2), which comprises a saccharide.
[0007]
In the composition of the present invention, a water-soluble peptide having a clearance in rats of 30 ml / hour or more per kilogram body weight is used as the main drug.
The degree of water solubility mentioned above includes an octanol / water partition ratio of 0.1 or less.
The water-soluble polypeptide referred to in the present invention is not particularly limited as long as it has a physiological activity with a clearance in rats of 30 ml / hour or more per kilogram of body weight. For example, antibiotics, blood boosters, infection treatment agents, anti-dementia agents, antiviral agents, antitumor agents, antipyretic agents, analgesics, anti-inflammatory agents, anti-ulcer agents, antiallergic agents, antiepileptics, psychotropic drugs, It is used as an antihypertensive agent, antiarrhythmic agent, vasodilator, antihypertensive diuretic, antidiabetic agent, anticoagulant agent, anticoagulant agent, cholesterol-lowering agent, osteoporosis agent, hormone agent, vaccine, etc. The thing specified by said clearance is mentioned.
[0008]
As the peptide used in the present invention, a peptide having a pharmaceutical activity composed of two or more amino acids and derivatives thereof are used, and those having a molecular weight of about 200 to 200,000 are preferable. The mechanism of action of these peptides also includes antagonists, agonists, or soluble receptors thereof and derivatives thereof. Moreover, what has a sugar chain structure is contained about what has a sugar chain. 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 or antibody. Moreover, in addition to the amino acid structure necessary for expressing the original pharmacological activity, a hybrid peptide in which a peptide capable of binding to any receptor, antibody or the like is added is also included.
Examples of the peptide include interferons derived from natural products or produced by genetic recombination (eg, interferon alpha, interferon beta, and interferon gamma), hematopoietic factors such as granulocyte colony-stimulating factor (G-CSF), granules Sphere-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 peptides include growth factors derived from natural products or produced by genetic recombination, such as nerve growth factor (NGF), neurotropic factor (NTF), peptides having an action on brain neurons, epithelial cell growth factor ( EGF), insulin-like growth factors (IGF), growth hormone (GH), hepatocyte growth factor (HGF), fibroblast growth factor (FGF) and the like. Examples of the peptide include parathyroid hormone (PTH) and endothelin. In addition, as the peptide, those having a sugar chain, those factors having activities with different sugar chain structures, these factors not containing a sugar chain, or these muteins, or their derivatives, analogs, or these Active fragments.
The peptide is a physiologically active peptide having a platelet proliferating action, and may or may not have a sugar chain.
[0009]
Furthermore, the peptides include transforming growth factor (TGF), insulin, lymphotoxin, platelet-derived cell growth factor (PDGF), 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 derivatives, Neurotensin, Oxytocin, Vasopressin, Vasopressin derivative {Desmopressin (Japanese Journal of Endocrine Society, Vol. 54, No. 5, pp. 676-691 (1978))}, hirudin or a derivative thereof, glucagon, gastrin, calcitonin, calcitonin residue. Child-related peptide (CGRP), prolactin, adrenocorticotropic hormone (ACTH), melanocyte stimulating hormone (MSH), thyroid hormone releasing hormone (TRH) salts and derivatives thereof (JP-A-50-121273, JP-A-52- 116465), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), punkreozymine, cholecystokinin, human chorionic gonadotropin (HCG), tuftsin, thymosin, thymostimulin, Examples include motilin, bombesin, cerulein, bradykinin, kallikrein, substance P, tumor necrosis factor (TNF) or their muteins, derivatives, analogs and active fragments.
Examples of the peptide include atrial natriuretic hormone (ANP) and related peptides thereof.
The peptide may be an enzyme derived from a natural product or produced by genetic recombination, may or may not have a sugar chain, may have a different sugar chain structure, and these muteins or derivatives, Or it may be an active fragment, such as, but not limited to, urokinase, superoxide dismutase (SOD), tissue type plasminogen activator (TPA), asparaginase, lysozyme, etc. Other enzymes.
[0010]
Vaccines include peptides, proteins derived from natural products that are antigens, or synthetically or genetically produced non-peptidic antigenic substances, or mixtures thereof, either alone or conjugated to haptens or adjuvants. It is administered as an injection together with the vant, and examples thereof include cedar pollen and swab pollen, but are not limited thereto.
[0011]
It should be noted that peptides with low clearance, which is an indicator of drug metabolism and excretion ability in humans and other warm-blooded animals, such as peptides whose clearance in humans is about 15 milliliters / hour or less per kilogram of body weight, are originally delayed from the body. Sustained enough. In addition, it is necessary to carefully administer a peptide whose side effects are feared or whose pharmacological effect is very strong and excessive administration causes a disadvantage to the patient while observing the patient. For this reason, long-acting preparations are not necessary for peptides that meet these two conditions. For example, the average value of clearance calculated using the serum erythropoietin concentration when recombinant human erythropoietin is administered to humans at the usual dosage of 10 micrograms (1800 international units) or 20 micrograms intravenously in humans. Are reported to be 758 ml / hour or 739 ml / hour, respectively (Yujiaki Uji et al., Clinical Practice and New Drugs, Vol. 26, p. 1, 1989). Since the average weight of each group is 65.8 kilograms, or 69.3 kilograms, the clearance per kilogram of body weight is calculated to be 11.5 milliliters / hour, or 10.7 milliliters / hour, respectively. In this paper, the dose is reduced to 2 and 0.2 micrograms and administered intravenously to humans. The area under serum concentration (AUC) is proportional to the dose, and the pharmacokinetics is It was reported that the linearity was maintained, and it was reported that the clearance of erythropoietin was not dose-dependent within the dose range of 0.2 to 20 micrograms. By the way, the average half-life (from 2 to 8 hours after administration) of erythropoietin after intravenous administration to humans is 3.3 hours in the 10 microgram administration group and 5.2 hours in the 20 microgram administration group. The serum remained several picogram / milliliter to several tens of picogram / milliliter even 48 hours after administration. From these facts, it is said that erythropoietin preparations used in clinical practice may be administered at a frequency of about 1 to 3 times a week. In addition, it is required to measure the hemoglobin concentration or hematocrit value once a week or once every two weeks so as not to increase the blood more than necessary, and to stop the drug when the blood increase is more than necessary. For this reason, even if a sustained-release preparation of erythropoietin is developed, it cannot be controlled when the blood increase occurs more than necessary, which does not benefit the patient. In addition, the clearance of erythropoietin when rats are used as experimental animals has been reported to be 19.4 milliliters / hour per kilogram of body weight (the dose is 500 international units per kilogram of body weight). Is late (Kazuo Okumura et al., Pharmacology and Treatment, Vol. 18, Supplement, pages 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 (Boxesbaum et al., Journal of Pharmacokinetics and Bioforma Shootics, Vol. 10, pages 201-227). (1982) is given as a representative document), and it is clear that the clearance relationship between rat and human body weight per kilogram in erythropoietin is almost the same in other peptides. That is, it can be almost certainly estimated that a peptide having a small clearance in rats has a small clearance in humans. The clearance in the present invention is defined as the rate at which the entire 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 treat drugs (G, Earl, Wilkinson, Pharmacology Review, 39, 1 pp. 1987. Manabu Hanano et al., Pharmacokinetic Applications (Nanyamado), 1989). When the drug concentration in plasma (serum) is used as a reference, it is expressed as CLP, and when the drug concentration in whole blood is used as a reference, it is expressed as CLB. The clearance in the present invention may be referred to as total clearance (CLTOT), but is read as CLP or CLB depending on whether it is in plasma (serum) or whole blood. Usually, these values are expressed as the amount of drug administered intravenously divided by the area under drug concentration (AUC) in plasma (serum) or whole blood. The clearance may be defined by other well-known scientific terms used in the field of pharmacokinetics, but the meanings are the same.
[0012]
In the present invention, polyethylene glycol is a wax-like solid at room temperature and has an average molecular weight of 2000 to 6000.
The normal temperature as used in the field of this invention is a temperature range prescribed | regulated by the Japanese Pharmacopoeia, and specifically says 15 to 25 degreeC.
Wax-like solids include solids that are not liquid but similar in appearance to white wax.
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 2000-4500. Moreover, you may mix and use the thing of different molecular weight. The molecular weight as used in the field of this invention means the molecular weight calculated | required by the average molecular weight determination test of the macrogol 400 described in the Japanese Pharmacopoeia.
Examples of the polyethylene glycol include those having an average molecular weight of 2000, an average molecular weight of 3000, and an average molecular weight of 4000.
[0013]
Peptide preparations are easy to handle at room temperature, but degradation is often promoted in aqueous solution compared to low-temperature storage. It is known that there are those that lose their pharmacological activity upon receiving. Therefore, a peptide preparation, particularly an injectable preparation, is preferably vacuum-dried, particularly freeze-dried, and is usually distributed and stored at room temperature. When the aqueous solution of the water-soluble pharmaceutical composition of the present invention is formulated by vacuum drying or freeze-drying, if polyethylene glycol that is liquid at room temperature is used, the liquid remains at room temperature after freeze-drying. There is a high possibility of impairing activity. On the other hand, when polyethylene glycol that is solid at room temperature is used, it remains in a solid state at room temperature even after lyophilization, so that it does not impair the pharmacological activity of the peptide, particularly the peptide, and is excellent in storage stability. When the melting point of solid polyethylene glycol at room temperature was measured using a differential scanning calorimeter (DSC7) manufactured by PerkinElmer Co., Ltd. (temperature rising rate was 10 ° C./min), polyethylene glycol 1000 (Wako first grade, average molecular weight 1000) was 26. Heat transfer peaks were observed at 78 ° C and 36.58 ° C. The peak of heat transfer of polyethylene glycol 1540 (first class Wako, average molecular weight 1500) was 47.32 ° C., but it was confirmed that significant heat transfer occurred from around 30 ° C. In the Japanese Pharmacopoeia, the room temperature is defined as 1 ° C. to 30 ° C., and it is well known in Japan that the temperature rises to 30 ° C. or higher in the midsummer. In the distribution and storage of a pharmaceutical preparation that is not stored in a cold place under such conditions, there is a sufficient chance that the pharmaceutical preparation is exposed to a temperature of 30 ° C. or higher. Therefore, the use of polyethylene glycol 1000 or 1540 is not preferred because it is expected that these polyethylene glycols will melt, for example, in lyophilized formulations. On the other hand, in polyethylene glycol 2000 (Wako first grade, average molecular weight 2000), the peak of heat transfer was 52.39 ° C., and significant heat transfer occurred from around 40 ° C. The peak of heat transfer of polyethylene glycol 400 (Wako primary, average molecular weight 3000) is 58.94 ° C., and the peak of heat transfer of polyethylene glycol 6000 (Wako primary, average molecular weight 7500) is 63.56 ° C. there were. From these results, polyethylene glycol having an average molecular weight of 1540 or less is not suitable for use in a pharmaceutical composition that is formulated by vacuum drying or freeze drying. On the other hand, by using polyethylene glycol having an average molecular weight of 2000 or higher and having a significant heat transfer from around 40 ° C. in the melting point measurement by the differential scanning calorimeter used in the present invention, the room temperature or room temperature can be obtained. It is possible to prevent melting of polyethylene glycol during distribution and storage.
[0014]
Examples of water-soluble proteins having substantially no pharmacological activity that can be injected into body fluids in the present invention include serum albumin, polyclonal or monoclonal immunoglobulins or fragments thereof, collagen or gelatin, preferably serum albumin. is there.
The blending ratio of the peptide cannot be generally determined depending on the activity of the substance and the therapeutically required amount, but a normal dosage is blended in the unit dosage composition.
The weight ratio of peptide to polyethylene glycol is preferably from 0.0001: 1 to 100: 1. More preferably, it is 0.0001: 1-10: 1. Similarly, the mixing ratio of the water-soluble protein cannot be determined unconditionally, but an amount usually added to a pharmaceutical composition for injection can be added, and the weight ratio of the peptide to the water-soluble protein is 0.0000 1: 1 to 100. : 1 is preferred. More preferably, it is 0.0001 to 10: 1. The concentration of polyethylene glycol in the aqueous pharmaceutical composition solution 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, and 0.1% (W / V) or more.
[0015]
Hyaluronic acid having a high negative charge due to a carboxyl group as an acidic mucopolysaccharide that can be further blended in the composition of the present invention, or chondroitin-4-sulfate, dermatan sulfate, chondroitin-6-sulfate as an acidic mucopolysaccharide having a sulfate group , Keratan sulfate, heparin, heparan sulfate and the like. Moreover, the desulfurization oxide of the natural type acidic mucopolysaccharide which has a sulfate group is mentioned.
The acidic mucopolysaccharide may be a long linear complex polysaccharide composed of a repeating structure of disaccharide units of amino sugar and uronic acid. These include a part of uronic acid, which is replaced with galactose, and are polyanions having 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 it may be one in which a sulfate group is bonded to the amino group instead of the acetyl group. Or these derivatives may be sufficient.
[0016]
The acidic mucopolysaccharide may be extracted from a living tissue such as cartilage or may be produced from a microorganism. These derivatives may also be used.
In the case of hyaluronic acid, the molecular weight is 10,000 to 3,000,000, preferably 500,000 to 2,500,000, and more preferably about 1,000,000 to 2,500,000. The molecular weight of the mucopolysaccharide having a sulfate group is 1,000 to 1,000,000, preferably 1,000 to 300,000, and more preferably about 1,000 to 100,000.
Examples of the mucopolysaccharide having a sulfate group include those having a sulfate group number of about 0.01 to 4.0 per disaccharide unit, and about 0.1 to 3.0 per disaccharide unit. Those are preferably used.
A natural acidic mucopolysaccharide having a sulfate group may be desulfated, and as a method thereof, a method of desulfating with an acidic methanol solution (Schubert, Messo's Incarbonate Hydrate Chemistry, 5, 109, 1965). ). 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.
[0017]
Specific examples of the desulfurized oxide of a natural acidic mucopolysaccharide having a sulfate group used in the present invention include chondroitin, desulfated heparin, and heparan. Of these, chondroitin and heparan are preferable.
The acidic mucopolysaccharide may be a salt, such as an alkali metal salt or an alkaline earth metal salt. Examples of the alkali metal salt include sodium salt and potassium salt. A sodium salt is preferable. Examples of the alkaline earth metal salt include magnesium salt and calcium salt, and sodium hyaluronate 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, most preferably about 0.01 to 1: 1.
[0018]
The sustained-release injectable pharmaceutical composition of the present invention contains a water-soluble peptide and polyethylene glycol, a pharmaceutical additive as necessary, or further a water-soluble protein. These may be dissolved or suspended in a saline solution, or may be vacuum-dried or lyophilized depending on the purpose. In addition, aqueous solutions containing peptides and additives, water-soluble proteins, etc., if necessary, or vacuum-dried or freeze-dried powders that have been re-dissolved in sterile water or sterile saline, and polyethylene glycol or What added the additive for pharmaceutical preparations and melt | dissolved by adding sterilized water or sterilized physiological saline may be mixed. Moreover, it is not limited to these, You may employ | adopt another preparation method, and the point should just be a method which does not impair the activity of a peptide.
[0019]
Add water-soluble peptide or serum albumin as a stabilizer to it, and add hyaluronic acid sodium salt as a pharmaceutical additive, then add distilled water for injection or physiological saline for injection and mix gently. The resulting solution or suspension (I) may be mixed with distilled water for injection of polyethylene glycol or physiological saline solution for injection (II) and formulated by vacuum drying or freeze drying. In addition, the product obtained by vacuum drying or freeze-drying (I) may be dispersed in (II) and rapidly formulated into a formulation by vacuum-drying or freeze-drying. In addition, a formulation may be prepared in which molding dehydration (spray drying) is performed by spraying into a heated air stream from two separate fluid nozzles with (I) as the main and (II) as the sub.
[0020]
In this case, a water-soluble peptide or a combination of serum albumin and hyaluronic acid sodium salt complex (III) is dispersed in polyethylene glycol, or polyethylene glycol is coated on the surface of (III). Is obtained. Disperse (I) or a vacuum-dried or freeze-dried powder of polyethylene glycol heated and melted (temperature slightly higher than the melting point), and drop or stir in a cold solvent immediately after injection. Even if the collected fine particles are collected or rapidly formed into a gas phase by spray cooling (spray chilling) or sprayed in a stream of air heated from a two-fluid nozzle, a formulation to be dehydrated (spray drying) Good. In addition, according to a known method, a formulation was prepared by molding injectable microparticles in which a complex of water-soluble peptide or serum albumin added to solid polyethylene glycol and sodium hyaluronate complex was dispersed. Also good. The obtained fine particles may be further subjected to a dehydration operation by vacuum drying or the like. In addition, 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 aggregation of the fine particles during the production of the fine particles. The microparticles thus obtained are dispersed and immediately administered in a solvent usually composed of a substance acceptable as an injection solvent.
[0021]
The pharmaceutical composition of the present invention may contain a nucleic acid (RNA, DNA), and the nucleic acid may be used as an antisense (Minwitian et al., Journal of Biological Chemistry, Vol. 266, pages 19162-1871 ( (1991) is cited as a representative document). Conventionally used additives may be added to the unit dosage thus obtained. For example, arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid as a pH adjusting agent, benzyl alcohol as a local anesthetic, inorganic salts such as sodium chloride as an isotonic agent, carbohydrates such as mannitol and sorbitol, Tween 80 as an adsorption inhibitor (Such as surfactants), cyclodextrins as solubilizers, serum albumin as stabilizers, glutathione as reducing agents, and the like.
[0022]
The present invention is a pharmaceutical composition in which a peptide and polyethylene glycol that is a wax-like solid at room temperature are mixed, and an additive that is acceptable for constituting a preparation may be contained. Usually, these substances are unit compositions. It is prepared to be present in. Furthermore, a solution formulation obtained by adding a solvent for injection to this preparation, or a vacuum-dried product or a freeze-dried product of this solution, and a formulation obtained by re-dissolving these dried products with a pharmaceutically acceptable solvent for injection. Good. In addition, a pharmaceutical composition to which the peptide or an additive acceptable for constituting the preparation is added, a solution in which the peptide is dissolved with an injectable solvent, a lyophilized product of this solution, and a lyophilized product are obtained as pharmaceutical products. Re-dissolved in an injectable solvent for injection, a wax-like solid polyethylene glycol dissolved in an injectable solvent at room temperature, or a pharmaceutically acceptable additive added thereto or freezing It is a long-lasting preparation in which a dry product dissolved in an injectable solvent is mixed. Further, by adding a water-soluble protein such as serum albumin having substantially no pharmacological effect that can be injected into a living body to the sustained-release preparation, enhancement of the action can be expected.
[0023]
The pharmaceutical composition of the present invention is a water-soluble peptide or a water-soluble peptide that is homogenized by adding a water-soluble protein such as serum albumin and / or an acidic mucopolysaccharide such as sodium hyaluronate, and is solidified at room temperature after being heated and melted. It is also possible to disperse in polyethylene glycol and spray it into a solvent or spray it in the gas phase to form fine particles, which can be dispersed in an injection solvent at the time of administration.
The pharmaceutical composition of the present invention is preferably stored and distributed in a vacuum or lyophilized state, but can be stored and distributed in a solution state. The storage temperature may be room temperature, but is preferably stored in a cold place. The cold place in the present invention refers to 15 ° C. or lower (Japanese Pharmacopoeia), but depending on the preparation, it may be better to avoid freezing.
[0024]
The pH of the pharmaceutical composition of the present invention does not greatly affect the activity of the peptide, and a pH within the physiologically acceptable range is used. Preferably it is about pH2-pH10. More preferably, it is about pH3 to pH9. Particularly preferred is pH 3.5 to pH 8.
The pH is adjusted by adding a solution containing, for example, hydrochloric acid or sodium hydroxide so that the final pH is within the above range in the presence of a water-soluble peptide, polyethylene glycol, and various additives. It is done by doing. In addition, in order to ensure stability in the presence of the peptide, the pH adjustment solution may be used as a separate container, and this operation may be performed immediately before use.
[0025]
One characteristic of the pharmaceutical composition of the present invention is that it is water-soluble and durable, and the viscosity of the solution is very low, and it is easy to handle during injection. The viscosity in the present invention refers to a viscosity measured using a cone LD with an E-type viscometer (manufactured by Tokyo Keiki). The water-soluble injectable composition of the present invention is prepared so that the viscosity when administered to a living body is usually 200 centipoise (cp) or less, preferably 100 cp or less, more preferably 70 cp or less.
As the adjustment method, for example, a water-soluble peptide, polyethylene glycol, or various additives are mixed by the above method, and diluted with an injectable solvent such as sterile physiological saline as necessary.
[0026]
As a method of administration of the pharmaceutical composition of the present invention, administration by injection is performed, and administration by subcutaneous injection is particularly preferable. Depending on use, other injection routes such as intramuscular administration, intravenous administration, intraarticular cavity administration, Intraocular administration and further intra-tissue administration are also performed.
The dosage of the composition of the present invention, the administration target, the administration target disease, and the like are determined based on those of the water-soluble peptide that is the main drug.
Since the present invention does not use an organic solvent, the present invention can be applied to peptides whose three-dimensional structure is important for the expression of physiological activity.
The pharmaceutical composition of the present invention can maintain the pharmacological action of the peptide after administration, but can be administered in a solution state, so that the dosage can be easily adjusted, and a thin injection needle can be used. The pain given to you is small.
Further, in the present invention, by using polyethylene glycol in a limited range of an average molecular weight of 2000 to 6000 that is solid at room temperature, it becomes possible to form a peptide by vacuum drying or freeze drying in the presence of polyethylene glycol, In addition, a sustained-release preparation having a sustained pharmacological action can be obtained. For this reason, it is easy for medical staff to handle, and since it is a water-soluble composition for patients, it is not necessary to use a thick needle, and the number of injections can be reduced, so that the pain caused by the therapeutic action is reduced.
[0027]
【Example】
The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.
[0028]
Example 1. Insulin long-acting preparation
Ten milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands), clearance in rats: 1416 ml / hr / kg) 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 2000 (Wako Pure Chemical Industries) having an average molecular weight of 2000 was dissolved.
[0029]
Example 2 Insulin long-acting preparation
Ten milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 5 milliliters 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) having an average molecular weight of 2000 was dissolved at a concentration of 2% (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 to this and dissolved. 1 ml of this preparation was transferred to a glass vial and subjected to a known freeze-drying operation (freeze-drying apparatus: Riobak GT-3, Reebold).
[0030]
Example 3 Insulin long-acting preparation
Ten milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 5 milliliters 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) having an average molecular weight of 3000 was dissolved at a concentration of 2% (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 to this and dissolved. 1 ml of this preparation was transferred to a glass vial and subjected to a known freeze-drying operation (freeze-drying apparatus: Riobak GT-3, Reebold).
[0031]
Example 4 Insulin long-acting preparation
Ten milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 5 milliliters 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) 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 to this and dissolved. 1 ml of this preparation was transferred to a glass vial and subjected to a known freeze-drying operation (freeze-drying apparatus: Riobak GT-3, Reebold).
[0032]
Embodiment 5 FIG. Insulin long-acting preparation
Ten milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 5 milliliters 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) 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 to this and dissolved. 1 ml of this preparation was transferred to a glass vial and subjected to a known freeze-drying operation (freeze-drying apparatus: Riobak GT-3, Reebold).
[0033]
Example 6 Insulin long-acting preparation
Ten milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 5 milliliters 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) having an average molecular weight of 2000 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 to this and dissolved. 1 ml of this preparation was transferred to a glass vial and subjected to a known freeze-drying operation (freeze-drying apparatus: Riobak GT-3, Reebold).
[0034]
Example 7 Insulin long-acting preparation
Ten milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 5 milliliters 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) 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 to this and dissolved. 1 ml of this preparation was transferred to a glass vial and subjected to a known freeze-drying operation (freeze-drying apparatus: Riobak GT-3, Reebold).
[0035]
Example 8 FIG. Insulin long-acting preparation
Ten milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 5 milliliters 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 2000 (Wako Pure Chemical Industries) having an average molecular weight of 2000 was dissolved.
[0036]
Example 9 Insulin long-acting preparation
12 milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 1 milliliter of 0.1N hydrochloric acid, and further diluted with 5 milliliters of physiological saline for injection. 2% (W / V), 10% (W / V) of polyethylene glycol 2000 (Wako Pure Chemical Industries, Ltd.) having an average molecular weight of 2000 of 60 milligrams, 300 milligrams or 600 milligrams in 3 milliliters of physiological saline for injection. Alternatively, a 20% (W / V) polyethylene glycol solution was prepared. Three kinds of insulin injection solutions having different polyethylene glycol concentrations were prepared by adding 1 ml of the above-mentioned insulin aqueous solution to 1 ml of each solution.
[0037]
Example 10 Interferon alfa continuous formulation
1 milliliter of distilled water for injection was added to 2 vials each of interferon alpha preparation containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin (Takeda, clearance in rats: 123 ml / hr / kg). Take 1.2 ml of this, and add polyethylene glycol 4000 (first grade of Wako Pure Chemicals, average molecular weight 3000) to physiological saline for injection (Fusan Yakuhin) to a concentration of 0.45% (W / V). 0.6 milliliter of the dissolved solution was added and mixed.
[0038]
Example 11 Interferon alfa continuous formulation
1 milliliter of distilled water for injection was added to 2 vials of an interferon alpha preparation (Takeda) containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin, and combined. Of this, take 1.2 ml of a solution of polyethylene glycol 2000 (Wako Pure Chemical grade 1, average molecular weight 2000) dissolved in physiological saline for injection (Fuso Yakuhin) to a concentration of 20% (W / V). 0.3 ml and 0.3 ml saline for injection were added and mixed.
[0039]
Example 12 Interferon alfa continuous formulation
1 milliliter of distilled water for injection was added to 2 vials of an interferon alpha preparation (Takeda) containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin, and combined. Take 1.2 ml of this, and use a solution of polyethylene glycol 4000 (Wako Pure Chemical Industries, grade 1, average molecular weight 3000) dissolved in physiological saline for injection (Fuso Yakuhin) to a concentration of 20% (W / V). 0.3 ml and 0.3 ml saline for injection were added and mixed.
[0040]
Example 13 Insulin long-acting preparation
400 milligrams of polyethylene glycol 2000 or 4000 (each Wako first grade) and 500 milligrams D-mannitol (Wako special grade) were dissolved in 10 milliliters of physiological saline for injection (Fuso Yakuhin) and 20% (W / V). 0.1 milliliters of albumin NICHIYAKU (Nippon Pharmaceutical Co., Ltd.) containing human serum albumin was added and mixed. This solution was mixed with 10 milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) dissolved in 2 milliliters of 0.1N hydrochloric acid. Precipitation was not seen in the obtained two types of solutions, and clear solutions were obtained. The two mixtures were lyophilized by a method known per se using a lyophilizer Riobach GT-3 (Reebold Heraeus, Germany).
[0041]
Example 14 Insulin long-acting preparation
4.5 milligrams of pork insulin (26.8 units / milligram: Diosins (Netherlands)) is dissolved in 0.5 milliliters of 0.1N hydrochloric acid, and then 2.5 milliliters of physiological saline for injection is added. Diluted. 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 porcine insulin solution.
[0042]
Example 15. G-CSF sustained-release preparation
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. G-CSF was added to 23 microliters of Filgrastim (Amgen, USA), a formulation containing G-CSF, by adding 1.727 ml of physiological saline for injection and 0.35 ml of this polyethylene glycol solution. An injection solution was prepared. The clearance of G-CSF in rats has been reported to be 53.1-79.8 ml / hour per kilogram of body weight (Hanaji Tanaka et al., The Journal of Pharmacology and Experimental Therapy). Ticks, vol. 255, pages 724-729, 1990).
[0043]
Example 16 SOD lasting preparation
Polyethylene in 0.7 ml of physiological saline containing 400 units (equivalent to 200 micrograms) of Serratia superoxide dismutase (SOD) (Japanese Patent Laid-Open Nos. 57-29285 and 58-1685). A physiological saline solution (10% (w / v)) of glycol (polyethylene glycol 4000 having an average molecular weight of 3000: Wako Pure Chemical Industries, Ltd.) (0.7 ml) was added and mixed thoroughly.
[0044]
Example 17. Hirudin long-lasting formulation
Saline for injection was added to a solution containing 100 ATU (equivalent to about 8 micrograms) hirudin (Peninsula Laboratories, USA) to make a total volume of 1 milliliter. To the glass vial containing the 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.
[0045]
Example 18 IL-2 sustained-release preparation
1 milliliter of Interleukin 2 (IL-2; containing 10 micrograms) is produced by the production method (described in JP-A-61-78799) and purified by the purification method of JP-A-60-115528. did. 0.5 ml of physiological saline was added to a mixture of N-terminal with and without methionine). 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 a glass fine vial containing 1 ml of this solution and mixed thoroughly. .
[0046]
Example 19. Leuprorelin acetate long-lasting formulation
750 micrograms of LH-RH analog leuprorelin acetate (US Pat. No. 4,008,209) was dissolved in 1.5 milliliters of 1/30 molar phosphate buffer (pH 6). To a glass vial containing 1 ml of this solution, 1 ml of 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.
[0047]
Example 20. PTH persistent preparation
450 micrograms of parathyroid hormone (PTH) (Bacchem, 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.
[0048]
Example 21. TRH long-lasting formulation
1. 464 milligrams of thyrotropin-releasing hormone (TRH) tartrate and 50 milligrams of D-sorbitol containing 1 milliliter of hiltonin injection (Takeda) in a glass vial (capacity approx. 5 milliliters) 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.
[0049]
Example 22. FGF mutein sustained-release preparation
TGP-580 (recombinant human basic FGF mutein CS23: EP Publication 281 and 822) solution (0.96 mg protein / ml) in 0.36 ml of saline for injection 1.14 ml, polyethylene glycol (average 1.5 ml of physiological saline solution (10% (w / v)) of polyethylene glycol 4000 (molecular weight 3000: Wako Pure Chemical Industries) and 6 microliters of albumin Nichiyaku (Nippon Pharmaceutical) containing 20% human serum albumin And mixed thoroughly.
[0050]
Example 23. NGF persistent preparation
4 microliters of albumin NICHIYAKU (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin in 0.7 ml of physiological saline containing 10 micrograms of nerve cell growth factor (NGF) (Biomedical Technology, USA) 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.
[0051]
Example 24. EGF continuous preparation
4 microliters of albumin NICHIYAKU (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin is added to 0.7 ml of physiological saline containing 10 micrograms of epidermal growth factor (EGF) (Chemicon International, USA), and further injected. Physiological saline 1.4ml (Fuso Yakuhin) was added to a total volume of 2.1ml. 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 mixed thoroughly.
[0052]
Example 25. Interferon beta long-acting preparation
4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin is added to 0.7 ml of physiological saline containing 10 micrograms of interferon beta (Pezel, Germany), and physiological saline for injection 1. 4 ml (fuso chemicals) 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.
[0053]
Example 26. Interferon gamma sustained-release preparation
Add 4 microliters of albumin niyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin to 0.7 milliliter of physiological saline containing 10 micrograms of interferon gamma (Genzyme, USA). 4 ml (fuso chemicals) 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 completely mixed.
[0054]
Example 27. TGF-β persistent preparation
4 microliters of albumin niyak (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin in 0.7 ml of physiological saline containing 1 microgram of transforming growth factor (TGF-β) (manufactured by Wako Pure Chemical Industries, Ltd.) Further, 1.4 ml of physiological saline for injection (Fusan Yakuhin) was added to make the total volume 2.1 ml. To this solution, 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 and thoroughly mixed.
[0055]
Example 28. Parathyroid hormone long-acting preparation
4 microliters of albumin niyak (Nippon Pharmaceutical Co., Ltd.) containing 20% of human serum albumin is added to 0.7 ml of physiological saline containing 10 micrograms of parathyroid hormone (PTH) (Bacchem, Switzerland), and further physiological for injection The total amount was made 2.1 ml by adding 1.4 ml of saline (Fusan Yakuhin). 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 mixed thoroughly.
[0056]
Example 29. TNF-α continuous preparation
Tumor necrosis factor (TNF-α) (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.1 ml of a phosphate buffer solution of pH 7.4 containing 0.1 mg of human serum albumin and 0.1 mL of physiological saline for injection. Add 6 milliliters (Fuso Yakuhin), add 4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin, and then add 1.4 ml of physiological saline for injection (Fusan Yakuhin) for a total volume of 2.1. Made milliliters. 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.
[0057]
Example 30. FIG. Growth hormone sustained-release preparation
4 microliters of albumin niyak (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin was added to 0.7 ml of physiological saline containing 10 micrograms of growth hormone (UCB BioProducts, Belgium), and physiological saline for injection. 1.4 ml of water (fuso chemicals) was added to a total volume of 2.1 ml. To this solution, 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 and thoroughly mixed.
[0058]
Example 31. Calcitonin long-acting formulation
Calcitonin (UCB BioProducts, Belgium) Add 4 microliters of albumin nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin to 0.7 milliliter of physiological saline containing 10 micrograms of physiological saline for injection. 1.4 ml (fuso chemicals) was added to make the total volume 2.1 ml. To this solution, 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 and thoroughly mixed.
[0059]
Example 32. TPA continuous preparation
Tissue plasminogen activator (TPA) (Bioscott, USA) 4 microliters of albumin Nichiyaku (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin in physiological saline 0.7 milliliters containing 10 micrograms, 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.
[0060]
Example 33. IL-3 sustained-release preparation
4 microliters of albumin NICHIYAKU (Nippon Pharmaceutical Co., Ltd.) containing 20% human serum albumin is added to 0.7 ml of physiological saline containing 1000 units of Interleukin 3 (IL-3; R & D Systems, USA), and further injected. Physiological saline 1.4ml (Fuso Yakuhin) was added to a total volume of 2.1ml. 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 mixed thoroughly.
[0061]
Example 34. Interferon alfa continuous formulation
One milliliter of distilled water for injection was added to 10 vials of an interferon alpha preparation (Takeda, clearance in rats: 123 ml / hr / kg) containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin.
To this was added 10 ml of a solution of sodium hyaluronate having a molecular weight of 1,800,000 (Genzyme, USA) dissolved in physiological saline for injection so as to be 1.2% (w / v), and gently mixed. This solution was freeze-dried according to a conventional method, and the resulting powder was added to 10 g of polyethylene glycol 400 (average molecular weight 3000: Wako first grade) heated and melted at 55 ° C. and quickly dispersed. Fine particles were produced by dropping the solution into 200 ml of distilled water cooled to 5 ° C. while stirring at high speed with a homogenizer (Polytron: Kinematica). Subsequently, the fine particles were collected by sieving quickly and lyophilized.
[0062]
Example 35. Interferon alfa continuous formulation
One milliliter of distilled water for injection was added to 10 vials of an interferon alpha preparation (Takeda, clearance in rats: 123 ml / hr / kg) containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin.
To this was added 10 ml of a solution of sodium hyaluronate having a molecular weight of 1,800,000 (Genzyme, USA) dissolved in physiological saline for injection so as to be 1.2% (w / v), and gently mixed. This solution was freeze-dried according to a conventional method, and the resulting powder was added to 10 g of polyethylene glycol 400 (average molecular weight 3000: Wako first grade) heated and melted at 55 ° C. and quickly dispersed. Subsequently, fine particles were produced from this solution using a spray dryer (GS-31: Yamato Science), and further freeze-dried.
[0063]
Experimental example 1
An injection was prepared at the following blending ratio and used in the experiment.
Comparative preparation 1: 10 milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 5 milliliters of 0.1 N hydrochloric acid. To 0.7 ml of this solution, 0.7 ml of physiological saline for injection was added.
Comparative preparation 2: physiological saline for injection 2 ml
The injection of the preparation of Example 1 or 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, the physiological saline injection solution of Comparative preparation 2 was administered in the same manner. Blood was collected approximately 0.4 milliliters before administration and at regular intervals, and serum was separated and collected. Glucose C test Wako (Wako Pure Chemical Industries) quantified the glucose concentration in serum.
FIG. 1 shows the time course of serum glucose concentration. In the physiological saline injection administration group of Comparative preparation 2, the serum glucose concentration hardly changed. In the insulin solution administration group of comparative preparation 1, the 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 polyethylene glycol 2000 of the preparation of Example 1 was added, the serum glucose concentration decreased from 30 minutes after administration, and was almost the same low at 6 hours after administration. In addition, the clearance per kg body weight of insulin in rats is reported to be 1416 ml / hour (Halbang et al., Metabolism, Vol. 28, pages 1097 to 1104 (1979)).
[0064]
Experimental example 2
Comparative preparation 3: 10 milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 5 milliliters 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) 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 to this and dissolved. One milliliter of this preparation was transferred to a glass vial and subjected to a known lyophilization operation (lyophilization apparatus: Riobak GT-3, Reebold).
In comparison to the case where the insulin sustained-release preparation containing polyethylene glycol having an average molecular weight of 2000 or 3000 obtained in Example 2 to Example 7 was obtained, a uniform freeze-dried product in the form of sponge cake was obtained. In the insulin preparation blended with polyethylene glycol 400, the freeze-dried product was wet due to polyethylene glycol 400 which was liquid 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 lyophilization is widely used as a method for preparing unstable peptides.
[0065]
Experimental example 3
Comparative preparation 4: 10 milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 5 milliliters 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.
Comparative preparation 5: 10 milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 5 milliliters 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: 2 ml of physiological saline for injection
The injection solution of the preparation of Example 8, Comparative preparation 4 or Comparative preparation 5 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 6 was similarly administered. Blood was collected approximately 0.4 ml before administration and at regular intervals, and serum was separated and collected. The glucose concentration in serum was quantified with Glucose C Test Wako (Wako Pure Chemical Industries).
FIG. 2 shows the time course of serum glucose concentration. In the group administered with the physiological saline injection 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 administration group containing polyethylene glycol 400 of comparative preparation 4, the serum glucose concentration decreased from 30 minutes after administration, but started to increase from 240 minutes after administration. On the other hand, in the insulin solution administration group to which polyethylene glycol 2000 of the preparation of Example 8 was added, the serum glucose concentration decreased from 30 minutes after administration, and was almost the same low at 6 hours after administration. From this fact, it was found that the polyethylene glycol added has a higher molecular weight and a longer lasting effect can be obtained.
[0066]
Experimental Example 4
Comparative preparation 7: 12 milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) was dissolved in 1 milliliter of 0.1N hydrochloric acid and further diluted with 5 milliliters of physiological saline for injection. . An insulin injection solution was prepared by adding 1 ml of physiological saline for injection to 1 ml of this insulin aqueous solution.
The formulation of Example 9 and Comparative Formulation 7 were injected subcutaneously in the back of 8-week-old male SD rats at a dose of 200 microgram / rat. Blood was collected approximately 0.4 ml before administration and at regular intervals, and serum was separated and collected. The glucose concentration in serum was quantified by glucose C test Wako (Wako Pure Chemical Industries).
FIG. 3 shows the time course of serum glucose concentration. In the insulin solution administration group not containing polyethylene glycol 2000 of comparative preparation 7, the serum glucose concentration once decreased, but started to increase from 180 minutes after administration, and returned to a normal value at 360 minutes. On the other hand, in the insulin solution administration group containing polyethylene glycol 2000, the serum glucose concentration once decreased, but the recovery of blood glucose level tended to be delayed as the polyethylene glycol concentration increased, and 2% (W / V) polyethylene. In the insulin injection solution administration group diluted 2-fold with an aqueous glycol solution, the blood glucose level began to recover from 240 minutes after administration. In the insulin injection solution administration group diluted 2-fold with a polyethylene glycol 2000 aqueous solution having a concentration of 10% (W / V), the blood glucose level further decreased, and the recovery of the blood glucose level from 240 minutes after administration was weak. However, even if the concentration of polyethylene glycol 2000 used was increased to 20% (W / V), no significant increase in drug efficacy was observed. From this fact, it has been found that the sustained effect of insulin can be obtained by the coexistence of about 5% (W / V) of polyethylene glycol at the most.
[0067]
Experimental Example 5
Comparative preparation 8: 1 ml of distilled water for injection was added to 2 vials each of interferon alpha preparation (Takeda) containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin. Of this, 1.2 milliliters was taken, and 0.6 ml of physiological saline for injection (Fusan Yakuhin) was added and mixed.
The preparation of Example 10 or the injection of Comparative Preparation 8 was administered subcutaneously to the back of 8-week-old male SD rats at a dose of 0.3 ml / rat. After administration, blood was collected at about 0.4 milliliters at regular intervals to separate and collect serum. Serum interferon alpha concentration was quantified by ELISA. The results are shown in FIG. In the group to which interferon alpha was administered alone, the blood concentration rapidly decreased from 1 hour after the administration. On the other hand, in the group administered with polyethylene glycol having an average molecular weight of 3000, the blood concentration was lower in the first and second hours after administration than in the group administered with interferon alfa alone, but at 4 and 6 hours after administration. In the eyes, the blood concentration was higher than in the group treated with interferon alpha alone. By mixing and administering interferon alpha and polyethylene glycol with an average molecular weight of 3000, 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 per kg body weight in rats of interferon alpha was 123 ml / hour.
[0068]
Experimental Example 6
Comparative preparation 9: 1 milliliter of distilled water for injection was added to 2 vials each of interferon alpha preparation (Takeda) containing 3 million international units of interferon alpha and 5 milligrams of human serum albumin. Of this, 1.2 milliliters was taken, and 0.6 ml of physiological saline for injection (Fusan Yakuhin) was added and mixed.
The preparation of Example 11, the preparation of Example 12, or the injection of Comparative Preparation 9 was administered subcutaneously to the back of 8-week-old male SD rats at a dose of 0.3 ml / rat. After administration, blood was collected at about 0.4 milliliters at regular intervals, and serum was separated and collected. Serum interferon alpha concentration was quantified by ELISA. FIG. 5 shows the results. In the group administered with interferon alpha alone, the blood concentration decreased rapidly from 1 hour after administration. On the other hand, in the group administered with polyethylene glycol having an average molecular weight of 2000 or 3000, the blood concentration was lower or almost the same as that in the group administered with interferon alpha alone at 1 hour and 2 hours after administration. At 4 hours and 6 hours, the blood concentration was higher than that in the group treated with interferon alpha alone. This means that interferon alpha can be mixed with polyethylene glycol having an average molecular weight of 2000 or 3000 to maintain a high blood concentration while lowering the blood concentration peak and suppressing undesirable side effects. Indicates that it is possible.
[0069]
Experimental Example 7
Comparative preparation 10: 400 milligrams of polyethylene glycol 6000 (Wako first grade average molecular weight 7500) and 500 milligrams of D-mannitol (Wako special grade) were dissolved in 10 ml of physiological saline for injection (Fusan Yakuhin), and 20% ( 0.1 ml of albumin NICHIYAKU (Nippon Pharmaceutical Co., Ltd.) containing human serum albumin (W / V) was added and mixed. This solution was mixed with 10 milligrams of porcine insulin (26.8 units / milligram: Diosyns (Netherlands)) dissolved in 2 milliliters of 0.1 N hydrochloric acid. No precipitate was observed in the obtained solution, and a clear solution was obtained. The mixture was lyophilized by a method known per se using a lyophilizer Riobach GT-3 (Reebold Heraeus, Germany). The two lyophilized products obtained in Example 13 or Comparative Formulation 10 were each dissolved by adding 10 ml of a mixture of distilled water for injection (Fuso Yakuhin) and 2 ml of 0.1 N hydrochloric acid to dissolve polyethylene glycol. Milky white turbidity was observed only in the group using 6000. Accordingly, polyethylene glycol having an average molecular weight of 6000 or more cannot be used in the present invention as a pharmaceutical composition for injection that can be lyophilized because it denatures the peptide.
[0070]
Experimental Example 8
Comparative formulation 11: 4.5 milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) dissolved in 0.5 milliliters of 0.1N hydrochloric acid, and 2.5 milliliters of physiological saline for injection Diluted with water. 600 milligrams of polyethylene glycol 1540 having an average molecular weight of 1500 (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 porcine insulin solution.
Comparative formulation 12: 4.5 milligrams of porcine insulin (26.8 units / milligram: Diosins (Netherlands)) dissolved in 0.5 milliliters of 0.1N hydrochloric acid, and 2.5 milliliters of physiological saline for injection Diluted with water. An insulin injection solution was prepared by adding 1 ml of physiological saline for injection to 1 ml of this insulin solution.
The injection of the preparation of Example 14 or Comparative preparation 11 was administered subcutaneously to the back of male SD rats of 11 to 12 weeks of age at a dose of 150 microgram / rat. As a control, an injection solution of Comparative preparation 12 was administered. Blood was collected approximately 0.4 ml before administration and at regular intervals, and serum was separated and collected. The glucose concentration in serum was quantified with 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 Formulation 12, the serum glucose concentration decreased until 2 hours after administration, but then increased, and returned to the value before administration almost 5 hours after administration. In the insulin solution administration group containing polyethylene glycol 1540 of comparative preparation 11, the serum glucose concentration showed almost the same change in blood glucose level with time as in the comparison preparation 12 administration group, and no difference was observed. On the other hand, in the insulin solution administration group to which polyethylene glycol 2000 of the preparation of Example 14 was added, the decrease in serum glucose concentration continued until 3 hours after administration, and the comparative preparation 11 or comparison formulation 12 administration group also at 5 hours after administration. The blood glucose level was about half as low. From this, it was found that polyethylene glycol 1540 having an average molecular weight of polyethylene glycol of 1,500 had no sustained effect, and polyethylene glycol 2000 having an average molecular weight of 2000 had a sufficient sustained effect.
[0071]
Experimental Example 9
Comparative preparation 13: G-CSF injection solution was prepared by adding 2.077 ml of physiological saline for injection to 23 microliters of Filgrastim (Amgen, USA) which is a preparation containing G-CSF. .
Comparative preparation 14: Polyethylene glycol 2000 (Wako Pure Chemical Industries) having an average molecular weight of 2000 of 600 mg was dissolved in 3 ml of physiological saline for injection to prepare a 20% (W / V) polyethylene glycol solution. An injection solution was prepared by adding 1.7 ml of physiological saline for injection and 0.35 ml of this polyethylene glycol solution.
Each of the injections of the preparation of Example 15, the comparative preparation 13, and the comparative preparation 14 containing human G-CSF was subcutaneously administered to the back of 8-week-old male SD rats at 0.3 ml. Seven hours after administration, blood was collected approximately 0.4 ml (using EDTA 2Na as an anticoagulant), and the peripheral white blood cell count, red blood cell count, and platelet count were measured using a microcell counter CC-180A (Toa Medical Electronics). It was measured. Peripheral neutrophil count, lymphocyte count, monocyte count, and eosinophil count are classified into 200 leukocytes by microscopic examination of Giemsa-stained blood smear, and the appearance frequency of each cell is used as the leukocyte count. Calculated by multiplication. The results are shown below (average of 4 rats in each group):

From these results, it was found that the pharmacological effect was enhanced by adding polyethylene glycol having an average molecular weight of 2000 to G-CSF having a clearance of 53.0 to 79.8 ml / hour per kg body weight in rats. did. Once increased peripheral neutrophils are known to disappear from peripheral blood by a normal physiological mechanism, it is clear that the effect of increasing the number of peripheral neutrophils by adding polyethylene glycol is sustained.
[0072]
【The invention's effect】
The injectable composition of the present invention maintains the in vivo pharmacological action of the water-soluble peptide as the main agent.
Therefore, it is possible to reduce the number of administrations by injection. This is characterized in that the patient's pain due to medical practice can be reduced and the burden on the medical staff can be reduced.
[0073]
[Brief description of the 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 a black circle) obtained in Experimental Example 1. It shows the time transition.
FIG. 2 shows administration of the preparation of Example 8 (◯), comparative preparation 4 (□), comparative preparation 5 (marked with a black circle) or comparative preparation 6 (Δ) obtained in Experimental Example 3. The time course of the later glucose concentration in serum is shown.
FIG. 3 is a two-fold dilution of the insulin solution of Example 9 obtained in Experimental Example 4 with a polyethylene glycol 2000 concentration of 2% (W / V) (circled in black). Time course of serum glucose concentration after administration of 10% (W / V) diluted 2-fold (□) or 20% (W / V) 2-fold diluted (△) Is shown.
FIG. 4 shows the time course of serum interferon alpha concentration obtained after administration of comparative preparation 8 (marked with black circles) or preparation of Example 10 (◯) obtained in Experimental Example 5; It is.
FIG. 5 shows serum interferon obtained after administration of comparative preparation 9 (marked with a black circle), preparation of Example 11 (◯) or preparation of Example 12 (□) obtained in Experimental Example 6. It shows the time transition of alpha concentration.
FIG. 6 shows the glucose concentration in serum after administration of the preparation of Example 14 (◯), comparative preparation 11 (marked with a black circle) or comparative preparation 12 (□) obtained in Experimental Example 8. It shows the time transition.

Claims (3)

The following water-soluble peptides:
Interferon alphas, interferon betas, interferon gammas, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, macrophage colony stimulating factor, IL-1, IL-2, IL-3, IL-4, IL-5 , IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, nerve growth factor, neurotrophic factor, peptides that act on brain neurons, epidermal growth factor, insulin-like growth Factors, growth hormone, hepatocyte growth factor, fibroblast growth factor, parathyroid hormone, endothelin, peptide having sugar chain, peptide factor having different sugar chain structure, peptide factor not containing sugar chain , And their muteins, and their derivatives, analogs, and their activities Ragumento, bioactive peptides with good platelet growth function with or without a sugar chain;
Transforming growth factor, insulin, lymphotoxin, platelet-derived cell growth factor, angiotensin I, angiotensin II , angiotensin III , angiotensin I inhibitor, bradykinin, dynorphin, kyotorphin, endorphin, enkephalin, secretin, growth hormone releasing factor, luteinizing hormone Release hormone and its derivatives, neurotensin, oxytocin, vasopressin, vasopressin derivative desmopressin, hirudin and its derivatives, glucagon, gastrin, calcitonin, calcitonin gene related peptide, prolactin, corticotropin, melanocyte stimulating hormone, thyroid hormone releasing hormone and its Salt and its derivatives, thyroid stimulating hormone, luteinization Hormone, follicle stimulating hormone, pancreosimine, cholecystokinin, human chorionic gonadotropin, tuftsin, thymosin, thymostimulin, motilin, bombesin, cerulein, bradykinin, kallikrein, substance P, tumor necrosis factor and their muteins, derivatives, Analogs, active fragments, atrial natriuretic hormone and related peptides thereof,
From urokinase, superoxide dismutase, tissue-type plasminogen activator, asparaginase, lysozyme, enzyme with or without sugar chain, enzyme with different sugar chain structure, these muteins, derivatives, and active fragments A water-soluble pharmaceutical composition for injection, comprising a water-soluble peptide selected from the group consisting of polyethylene glycol having an average molecular weight of 2000 to 6000, which is a wax-like solid at room temperature. The following water-soluble peptides:
Interferon alphas, interferon betas, interferon gammas, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, macrophage colony stimulating factor, IL-1, IL-2, IL-3, IL-4, IL-5 , IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, nerve growth factor, neurotrophic factor, peptides that act on brain neurons, epidermal growth factor, insulin-like growth Factors, growth hormone, hepatocyte growth factor, fibroblast growth factor, parathyroid hormone, endothelin, bioactive peptide with platelet proliferative activity;
Transforming growth factor, insulin, lymphotoxin, platelet-derived cell growth factor, angiotensin I, angiotensin II , Angiotensin III , Angiotensin I inhibitor, bradykinin, dynorphin, kyotorphin, endorphin, enkephalin, secretin, growth hormone releasing factor, luteinizing hormone releasing hormone and derivatives thereof, neurotensin, oxytocin, vasopressin, vasopressin derivatives desmopressin, hirudin and derivatives thereof, glucagon , Gastrin, calcitonin, calcitonin gene-related peptide, prolactin, corticotropin, melanocyte-stimulating hormone, thyroid hormone-releasing hormone and its salts and derivatives, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, punk reozymine, kore Cystokinin, human chorionic gonadotropin, tuftsin, thymosin, thymostrimlin, motilin, cylinder Emissions, caerulein, bradykinin, kallikrein, substance P, tumor necrosis factor, atrial natriuretic hormone,
Urokinase, superoxide dismutase, tissue type plasminogen activator, asparaginase, lysozyme,
A water-soluble pharmaceutical composition for injection, comprising a water-soluble peptide selected from the group consisting of polyethylene glycol having a wax-like solid and an average molecular weight of 2000 to 6000 at room temperature. Furthermore, the pharmaceutical composition of Claim 1 or 2 formed by mix | blending the water-soluble protein which does not have pharmacological activity substantially injectable in the living body.

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