WO1999045953A1 - Heparin cofactor ii-containing compositions and method for stabilizing the same - Google Patents
Heparin cofactor ii-containing compositions and method for stabilizing the same Download PDFInfo
- Publication number
- WO1999045953A1 WO1999045953A1 PCT/JP1999/001102 JP9901102W WO9945953A1 WO 1999045953 A1 WO1999045953 A1 WO 1999045953A1 JP 9901102 W JP9901102 W JP 9901102W WO 9945953 A1 WO9945953 A1 WO 9945953A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hcii
- composition
- heparin cofactor
- nonionic surfactant
- adjuster
- Prior art date
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- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 239000003868 thrombin inhibitor Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/55—Protease inhibitors
- A61K38/57—Protease inhibitors from animals; from humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
Definitions
- the present invention relates to a stable composition containing heparin cofactor II (hereinafter, referred to as “HCII”) and a method for stabilizing an HCII-containing composition.
- HCII heparin cofactor II
- HCII like antithrombin III (hereinafter referred to as ⁇ ), is an important anticoagulant protein existing in the body (Matsuo et al .: Biomedical Perspectives, 2, 269-274 (1993)), and its molecular weight is According to SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis, it is a single-chain plasma glycoprotein of 72 kDa (Dougls M. Toll fsen et al .: J. Biol. Chemistry, 257, 2162-2169 (1982) ).
- HCII As a physiological function of HCII, it is known that it inhibits proteases such as thrombin, but it has a different site of action from ⁇ and seems to function as a thrombin inhibitor outside the blood vessel rather than inside the blood vessel . Therefore, since HCII exhibits an anticoagulant effect in specific organs, tissues or cells, the present inventors have found that systemic administration of HCII to veins, arteries and the like may cause sepsis, diseases causing abnormal blood flow, They found that they were effective in preventing and treating diseases such as organ disorders such as liver, lung, kidney, and brain, and filed a patent application (Japanese Patent Application Laid-Open No. 9-176040).
- HCII is also effective for the treatment of a disease caused by abnormal dysfunction of cells in a living body such as macrophages and the like, and is effective in treating the disease.
- the application was filed on October 10, 19197 7, with a filing date of July 7, 1998).
- a pharmaceutical composition containing HCII as an active ingredient is expected to be useful as a pharmaceutical.
- an HCII-containing pharmaceutical composition actually suitable for use as a medicament has not yet been marketed.
- the problems of the stability of the HCII-containing composition include a decrease in the activity of HCII, polymerization of HCII, and insolubility. It has been found that product formation can occur. Decreased activity of HC II is highly correlated with polymerization of HC II, but even when HC II activity is not reduced, production of polymerized HC II may be observed. Polymerized HCII is generally inactive and administration of HCII polymers may cause undesirable side effects such as shock and hypotension. In addition, shaking of the HCII-containing composition or the like may cause insolubles to be generated together with the decrease in the activity of HCII, which poses a problem as a pharmaceutical composition.
- the HCII-containing composition it is desired to suppress the reduction of the activity of HCII, the polymerization of HCII, the generation of insolubles, and the like, and to stabilize the composition.
- Such a problem relating to the stability of the HC II-containing composition has not been studied so far, and the present invention itself is novel.
- the pH of the HCII-containing composition is 6 to 12, preferably 6 to 10, more preferably 6.5 to 10, and particularly preferably 6.5 to 10. ?
- HC II activity reduction and HC II polymerization could be suppressed.
- the present inventors have also found that by adding a nonionic surfactant to the HCII-containing composition, a decrease in the activity of HCII can be suppressed, and generation of insolubles can be suppressed.
- the present inventors have found that by setting the storage temperature of the HCII-containing composition to 37 or lower, it is possible to suppress the decrease in HCII activity and the polymerization of HCII.
- the present invention relates to the following.
- An H C II-containing composition comprising a pH adjuster.
- An HC II-containing composition comprising a nonionic surfactant.
- An H C II-containing composition comprising a pH adjuster and a nonionic surfactant.
- nonionic surfactant is at least one selected from the group consisting of polyalkylene glycol, polyoxyethylene-polyoxypropylene copolymer and polyoxyethylene sorbitan fatty acid ester.
- the composition containing HC II according to any one of the above.
- a method for stabilizing an HCII-containing composition comprising adding a pH adjuster to the HCII-containing composition.
- a method for stabilizing an HCII-containing composition which comprises adding a nonionic surfactant to the HCII-containing composition.
- a method for stabilizing an HCII-containing composition comprising adding a pH adjuster and a nonionic surfactant to the HCII-containing composition.
- nonionic surfactant is at least one selected from the group consisting of polyalkylene glycols, polyoxyethylene-polyoxypropylene copolymers and polyoxyethylene sorbitan fatty acid esters. the method of.
- a method for storing an HCII-containing composition comprising storing the HCII-containing composition according to any one of the above (1) to (14) at a temperature of 37 ° C or lower.
- the present invention also provides an injection kit described in the following (i) to (iii) c
- An injection kit comprising at least the following components (a) and (b):
- a lyophilized composition comprising HCII and a pH adjuster, wherein the pH adjuster brings the pH of the solution to 6 to 10 when the composition is dissolved in an injectable aqueous solvent.
- a lyophilized composition that can be adjusted,
- an injection kit comprising at least the following components (c) and (d):
- An injection kit comprising at least the following components (e) and (ii):
- a lyophilized composition comprising a non-ionic surfactant and a pH adjuster, wherein the pH adjuster comprises a pH of the solution when the composition is dissolved in an injectable aqueous solvent.
- a lyophilized composition capable of adjusting H to 6 to 10,
- the method for stabilizing the HCII-containing composition includes a method for suppressing a decrease in the activity of HCII, a method for suppressing the polymerization of HCII, a method for suppressing the generation of insolubles in the composition, and the like. Is included.
- HCII in the HCII-containing composition of the present invention shows a single band visually at a position of a molecular weight of about 72 kDa by SDS-PAGE, has an isoelectric point of about 5, and has thrombin, chymotrypsin and cathepsin.
- a protein having a G inhibitory action Therefore, the HCII of the present invention is not necessarily limited to a full-length molecule as long as the above properties are satisfied.
- the origin of HCII is not particularly limited, and includes both those derived from natural plasma and those derived from recombinant host cells genetically engineered to produce HCII.
- plasma-derived substances include, for example, whole plasma, plasma fraction having high HCII activity, preferably purified from Eff.I, Eff.11 + III or Fr.IV, particularly waste fraction in ATIII purification. Things.
- the biological origin of the plasma is not particularly limited, and may be derived from humans, but is preferably derived from humans.
- HCII contained in HCII-containing fractions of cultures obtained by culturing host cells transformed with the transferred genes for example, those purified from culture solutions, cell extracts, culture supernatants and the like.
- the host cell is not particularly limited, but preferably includes bacteria such as Escherichia coli, Bacillus subtilis, and lactic acid bacteria, fungi such as yeast, animal and plant cells, and insect cells.
- HCII derived from a recombinant host cell examples include natural HCII (for example, Blinder et al., Biochemistry, 27: 752-759, 1988) or mutant HCII having retained or improved antithrombin activity (for example, Examples thereof include those produced by culturing host cells transformed with a recombinant vector containing DNA encoding the DNA encoding E. coli (see 3-139280).
- the method of purifying HCII is not particularly limited.
- the method of Blinder et al. J. Biol. Chem., Vol. 264, 5128-5133, 1989
- the method described in JP-A-9-286797, international Application No. PC TZ JP 98/04939 filamenting date: October 30, 1998)
- Method for obtaining HCII-containing composition from which depolymerizing factors Pla or host cell-derived protease, etc.
- the method described in the Japanese patent application Japanese Patent Application No. 11-10911, filed on Jan. 19, 1999
- the HCII crude product is brought into contact with an insoluble carrier having a hydrophobic group as a ligand to obtain a salt concentration.
- an HCII-containing composition from which contaminating proteins derived from HCII have been removed by treating with a buffer solution having a different pH. Further, these purification methods can be appropriately combined.
- the HCII-containing composition of the present invention may be any composition as long as it contains HCII, and may be in the form of a lyophilized composition or a liquid composition.
- the HCII-containing composition of the present invention also includes a final preparation of an administrable drug, a composition during a manufacturing process of a pharmaceutical formulation, and a composition during a purification process of HC II.
- HCII-containing composition of the present invention c present invention to provide an HC II containing composition characterized by containing a p H adjusting agent pH6 ⁇ l 2, preferably pH6 ⁇ l 0, favored more Or pH 6.5-1 to 10. Particularly preferably, the pH is adjusted to pH 7 to 10.
- a suitable solvent such as an injectable aqueous solvent
- the pH of the solution when dissolved in a suitable solvent is pH 6 to 12, preferably pH 6 to 10, and more preferably.
- a suitable solvent such as an injectable aqueous solvent
- Adjustment of pH can be performed using a pH adjusting agent.
- the pH adjuster used in the present invention may be any as long as it can adjust the pH of the composition to pH 6 to 12.
- the pH adjuster is capable of adjusting the pH of the composition to preferably pH 6 to 10, more preferably pH 6.5 to 10, and particularly preferably pH 7 to 10.
- a buffer is usually used.
- the buffer include a phosphate buffer (sodium phosphate buffer), a citrate buffer, a Tris-HCl buffer, a getylethanolamine chloride buffer, and the like.
- a phosphate buffer (sodium phosphate buffer) and a tris-HCl buffer are used.
- the concentration of the pH adjusting agent in the composition is usually 0.001 to 100 mM, preferably 0.1 to 10 OmM in the case of a liquid composition.
- a suitable solvent such as an injectable aqueous solvent
- suitable solvents for dissolving the lyophilized composition include sterile, injectable aqueous solvents free of pyrogenic substances, etc. Specifically, distilled water for injection, injection Physiological saline and the like.
- the HC II-containing composition is a lyophilized composition
- HC adjusted to 156 to 12, preferably pH 6 to 10, more preferably pH 6.5 to 10, and particularly preferably pH 7 to 10 with 11 regulators
- the II-containing solution is prepared by lyophilization. When used, it is dissolved in a suitable solvent (for example, distilled water for injection, etc.) and the pH is adjusted to pH 6 to 12, preferably pH 6 to 10, more preferably pH 6.5 to 10, and particularly preferably pH 7 to 10.
- a liquid composition is obtained.
- the HC II-containing composition is a liquid composition
- 15 15 (: 11 is about 0.01 to about 400 U
- It can be prepared as a solution containing Zml, preferably from about 0.1 to about 100 UZml.
- an appropriate solvent for example, distilled water for injection
- the polymerized HCII means a polymer of HCII molecules. More specifically, it is a dimer or higher polymer.
- the present invention also provides an H C II-containing composition comprising a nonionic surfactant.
- a nonionic surfactant By including a nonionic surfactant, it is possible to suppress a decrease in the activity of HCII and to suppress the generation of insolubles.
- nonionic surfactant examples include polyalkylene glycol [eg, polyethylene glycol (PEG), polypropylene glycol, etc.], polyoxyethylene-polyoxypropylene copolymer [eg, Poloxamer (trade name: Pluronic)], poly Oxyethylene sorbitan fatty acid esters [for example, polysorbate (trade name: Vienna)] and the like are exemplified.
- the average molecular weight of these nonionic surfactants is preferably from 2,000 to 20,000.
- Fatty acids of polyoxyethylene sorbitan fatty acid esters include fatty acids having 12 to 18 carbon atoms such as stearic acid, palmitic acid, myristic acid, lauric acid, and oleic acid.
- nonionic surfactant examples include PEG having an average molecular weight of 4000 to 600 (for example, PEG4000, PEG6000), poloxamer 188 (trade name: Pull mouth nick F 68), polysorbate 80 ( Brand name Tween 80). More preferably, it is PEG (for example, PEG4000, PEG600).
- the concentration of the nonionic surfactant in the composition is usually 0.001 to 2.0 w / v%, preferably 0.01 to: L. 0 w / v% for a liquid composition. More preferably, it is 0.02 to 5 w / v%.
- a suitable solvent for example, distilled water for injection
- the liquid composition is preferably pH 6 to l2, more preferably a pH adjuster as described above. It is adjusted to pH 6-10, particularly preferably pH 6.5-10, most preferably pH 7-10.
- the H C II-containing composition is a lyophilized composition
- it is prepared by lyophilizing an aqueous solution containing H C II and a nonionic surfactant.
- HCII and non-hydrochloric acid are adjusted to pH 6 to 12, more preferably pH 6 to 10, particularly preferably pH 6.5 to 10, most preferably pH 7 to 10 with a pH adjusting agent as described above.
- An aqueous solution containing an ionic surfactant is prepared by freeze-drying.
- a liquid composition is obtained by dissolving in an appropriate solvent (for example, distilled water for injection).
- the HC II-containing composition When the HC II-containing composition is a liquid composition, it can be prepared in the same manner as described above, as a solution containing 11 ⁇ 11 in an amount of about 0.01 to about 400 UZml, preferably about 0.11 to about 100 UZml. In the case of a lyophilized composition, it is prepared so that the HCII concentration in the solution when dissolved in an appropriate solvent (for example, distilled water for injection, etc.) at the time of use is in the same concentration range as the above liquid composition.
- an appropriate solvent for example, distilled water for injection, etc.
- the above-described HC II-containing composition of the present invention can maintain the stability of the HC II-containing composition, particularly when the storage temperature is 37 ° C. or less.
- the storage temperature is preferably -20 to 37 ⁇ , more preferably 0 to 15.
- the HCII-containing composition of the present invention may contain additives used in ordinary pharmaceutical compositions, for example, tonicity agents (eg, sucrose, sorbitol, mannitol, glycerin, glucose, sodium chloride, etc.), preservatives and disinfectants [For example, benzalkonium chloride, benzethonium chloride, paraoxybenzoic acid esters (eg, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate), benzyl alcohol, parachlorometoxylenol, -, Phenethyl alcohol, sorbic acid or its salt, thimerosal, black butanol, etc.), chelating agents (sodium edetate, etc.), thickeners (polyvinyl Loridone, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, sodium polyacryl
- the H C II-containing composition of the present invention can be prepared by a method known per se. If desired, treatments such as heating, sterilization, virus inactivation, sterilization filtration, virus removal, aliquoting, and freeze-drying can be performed.
- the HCII-containing composition of the present invention can also be provided as an injection kit comprising the above lyophilized composition and an injectable aqueous solution.
- the HCII-containing composition of the present invention includes various diseases causing abnormal blood flow such as thrombosis, microcirculation failure, ischemic disease, sepsis, various organ dysfunctions such as liver, lung, kidney, and brain, glomerulonephritis, ⁇ It is useful for the prevention and treatment of various inflammations such as inflammation accompanied by inflammation and edema formation.
- the HCII-containing composition of the present invention is preferably administered parenterally as an injection or infusion.
- the administration method of the HCII-containing composition of the present invention includes a method of intravenous injection or intravenous drip infusion.
- the dose of the HCII-containing composition of the present invention is generally 1 to 1000 OU / kg body weight, preferably 10 to 200 OUZ kg body weight per day for an adult, and this amount is administered once or divided into several times. be able to.
- HCII of the purified product PD- 1 0 column (Pharmacia, Inc.) 5 mM tri scan HCl buffer with (pH 8. 0, 25) after desalting, the absorbance at 280 nm at each buffer (A 28 ) Force was adjusted to be 0, and the stability was examined.
- HCII activity and polymerization HCII ratio was measured by the following method. (1) Measurement of HC II activity
- the sample was diluted with 40 mM Tris buffer (pH 8.56) containing 60 mM EDTA, 0.2% human serum albumin (HSA) and 0.1 mg / mL dermatan sulfate.
- the protein concentration of 0.1 mg mgZmL obtained from the above was defined as 1 U].
- the HCII activity was calculated from the prepared calibration curve.
- the ratio of the polymerized HC II is shown as a ratio (%) of the area of the peak detected on the polymer side from the peak of the full-length HC II in the gel filtration HPLC analysis to the total peak area.
- pH 66 HCII is stable at I 0, since no increase in polymerized HCII and no decrease in HCII activity is observed.
- HCII A 28 in 0.1 M Tris-HCl buffer (at pH 8.0, 25) containing 0.1 wZv% polyethylene glycol 4000. 1.0.
- the HCII solution was horizontally shaken at 125 rpm for 1 hour at room temperature, and subsequently shaken for 1 minute at room temperature with a vortex mixer.
- HCII solution was prepared with a 10 mM sodium phosphate buffer (pH 7.2) containing 0.15 M sodium chloride so that the HCII was 50 U / ml. 10 ml of this solution was dispensed into glass vials for pharmaceuticals and freeze-dried to prepare a freeze-dried preparation.
- the HCII solution was prepared with 1 OmM sodium phosphate buffer (pH 7.2) containing 0.15 M sodium chloride so that HCII was 10 to 50 U / ml.
- a liquid preparation was prepared by dispensing 10 ml of this solution into a glass vial for pharmaceuticals.
- HCII solution with 1 OmM sodium phosphate buffer (pH 7.2) containing 0.15 M sodium chloride and 0.1 w / v% polyethylene glycol 4,000 so that HCII is 5 OU / m1.
- 10 ml of this solution is Dispensed into glass vials and lyophilized to prepare a lyophilized formulation.
- HCII solution 10 mM sodium phosphate buffer containing 0.15 M sodium chloride and 0.1 w / v% polyethylene glycol 4,000 so that HCII is 10-50 U / m1 (PH 7.
- the HCII solution was prepared in 2).
- a liquid preparation was prepared by dispensing 10 ml of this solution into a glass vial for pharmaceuticals.
- An injection kit comprising the lyophilized preparation prepared in Preparation Example 1 and 10 ml of distilled water for injection was prepared.
- An injection is prepared by dissolving the lyophilized formulation in distilled water for injection.
- An injection kit comprising the lyophilized preparation prepared in Preparation Example 3 and 10 ml of distilled water for injection was prepared.
- An injection is prepared by dissolving the lyophilized formulation in distilled water for injection.
- 11 of the ⁇ 111-containing composition is adjusted to ⁇ 16 ⁇ 12, preferably pH 6 ⁇ : 10, more preferably pH 6.5 ⁇ 10, particularly preferably pH 7 ⁇ 10.
- a nonionic surfactant added to the H C II-containing composition, a decrease in the activity of H C II can be suppressed, and the generation of insolubles can be suppressed.
- HCII-containing composition of the present invention by setting the storage temperature of the HCII-containing composition of the present invention to 37X: or less, it is possible to suppress the decrease in HCII activity and the polymerization of HCII.
- ADVANTAGE OF THE INVENTION According to this invention, HCII containing composition excellent in stability which suppressed HCII activity fall, polymerization of HCII, and generation
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Abstract
Stable heparin cofactor II (HCII)-containing compositions. More particularly, HCII-containing compositions containing pH regulating agents as a stabilizer. These HCII-containing compositions are characterized by being adjusted to pH 6 to 12, preferably 6 to 10 and still preferably 6.5 to 10 by the pH regulating agent. HCII-containing compositions containing nonionic surfactant as a stabilizer. In these HCII-containing compositions, a decrease in HCII activity, polymerization of HCII and formation of insoluble matters are inhibited, thus achieving a high stability.
Description
明細書 Specification
へパリンコファクタ一II含有組成物およびその安定化方法 Composition containing heparin cofactor-II and method for stabilizing the same
技術分野 Technical field
本発明は、 へパリンコファクタ一 II (以下、 「HCII」 という） を含有する安 定な組成物および HCII含有組成物の安定化方法に関する。 The present invention relates to a stable composition containing heparin cofactor II (hereinafter, referred to as “HCII”) and a method for stabilizing an HCII-containing composition.
背景技術 Background art
HCIIは、 アンチトロンビン III (以下 ΓΑΤΙΠ 」 という） と同様に、 生体 内に存在する重要な抗凝固活性蛋白であり （松尾ら： Biomedical Perspectives, 2， 269-274 (1993) ) 、 その分子量は、 SDSポリアクリルアミ ドゲル電気泳動 (SDS - PAGE)分析によると、 72k D aの一本鎖血漿糖蛋白である （Dougls M. Toll fsenら： J. Biol. Chemistry, 257, 2162 - 2169 (1982) ) 。 HCII, like antithrombin III (hereinafter referred to as ΓΑΤΙΠ), is an important anticoagulant protein existing in the body (Matsuo et al .: Biomedical Perspectives, 2, 269-274 (1993)), and its molecular weight is According to SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis, it is a single-chain plasma glycoprotein of 72 kDa (Dougls M. Toll fsen et al .: J. Biol. Chemistry, 257, 2162-2169 (1982) ).
HCIIの生理機能としては、 トロンビン等のプロテア一ゼを阻害する事が知ら れているが、 ΑΤΠΙ とは作用部位が異なり血管内よりも血管外におけるトロン ビン阻害物質として機能していると思われる。 そこで、 HCIIが特定の臓器、 組 織または細胞で抗凝固作用を示すことから、 本発明者らは、 HCIIの静脈内、 動 脈内等の全身投与が、 敗血症、 血流異常を生じる疾患、 肝、 肺、 腎、 脳等の臓器 障害等の疾患の予防および治療に有効であることを見出し、 特許出願 （特開平 9 - 1 76040号公報） を行った。 さらに、 本発明者らは、 HCIIをマクロファ —ジ等の生体内細胞の機能異常亢進に起因する疾患に関して局所投与した場合に も該疾患の治療に有効であることを見出し、 特許出願 （特願平 10- 1 9 197 7、 出願日 1998年 7月 7日） を行った。 As a physiological function of HCII, it is known that it inhibits proteases such as thrombin, but it has a different site of action from ΑΤΠΙ and seems to function as a thrombin inhibitor outside the blood vessel rather than inside the blood vessel . Therefore, since HCII exhibits an anticoagulant effect in specific organs, tissues or cells, the present inventors have found that systemic administration of HCII to veins, arteries and the like may cause sepsis, diseases causing abnormal blood flow, They found that they were effective in preventing and treating diseases such as organ disorders such as liver, lung, kidney, and brain, and filed a patent application (Japanese Patent Application Laid-Open No. 9-176040). Furthermore, the present inventors have found that HCII is also effective for the treatment of a disease caused by abnormal dysfunction of cells in a living body such as macrophages and the like, and is effective in treating the disease. The application was filed on October 10, 19197 7, with a filing date of July 7, 1998).
このように、 H C IIを有効成分とする医薬組成物は医薬品としての有用性が考 えられる。 しかしながら、 実際に医薬としての使用に適した HCII含有医薬組成 物は未だ市販されていない。 Thus, a pharmaceutical composition containing HCII as an active ingredient is expected to be useful as a pharmaceutical. However, an HCII-containing pharmaceutical composition actually suitable for use as a medicament has not yet been marketed.
発明の開示 Disclosure of the invention
本発明者らによる HCII含有組成物の安定性に関する研究によれば、 HCII含 有組成物の安定性の問題として、 HCIIの活性低下、 HCIIのポリマ一化、 不溶
物の生成が起きることがあることが判明した。 H C IIの活性低下は H C IIのポリ マ一化と高い相関性を有するが、 HCIIの活性低下がない場合でもポリマ一化し た H C IIの生成が認められることがある。 ポリマ一化した H C IIは一般に活性が なく、 また HCIIのポリマ一はその投与によりショックゃ血圧降下などの好まし くない副作用を生じるおそれがある。 更に、 HCII含有組成物の振とう等により H C IIの活性低下と共に不溶物が生成することがあり、 医薬組成物として問題と なる。 従って、 HCII含有組成物に関しては、 HCIIの活性低下、 HCIIのポリ マー化、 不溶物の生成等を抑制し、 当該組成物の安定化を図ることが望まれる。 このような H C II含有組成物の安定性に関する課題はこれまで検討されておらず、 本発明は課題自体が新規なものである。 According to the study of the stability of the HCII-containing composition by the present inventors, the problems of the stability of the HCII-containing composition include a decrease in the activity of HCII, polymerization of HCII, and insolubility. It has been found that product formation can occur. Decreased activity of HC II is highly correlated with polymerization of HC II, but even when HC II activity is not reduced, production of polymerized HC II may be observed. Polymerized HCII is generally inactive and administration of HCII polymers may cause undesirable side effects such as shock and hypotension. In addition, shaking of the HCII-containing composition or the like may cause insolubles to be generated together with the decrease in the activity of HCII, which poses a problem as a pharmaceutical composition. Therefore, with respect to the HCII-containing composition, it is desired to suppress the reduction of the activity of HCII, the polymerization of HCII, the generation of insolubles, and the like, and to stabilize the composition. Such a problem relating to the stability of the HC II-containing composition has not been studied so far, and the present invention itself is novel.
本発明の目的は、 安定な HCII含有組成物を提供することである。 すなわち、 本発明の目的は、 HCIIの活性低下が抑制された HCII含有組成物を提供するこ とである。 また本発明の目的は、 HCIIのポリマ一化が抑制された HCII含有組 成物を提供することである。 さらに本発明の目的は、 不溶物の生成が抑制された H C II含有組成物を提供することである。 It is an object of the present invention to provide a stable HCII-containing composition. That is, an object of the present invention is to provide an HCII-containing composition in which a decrease in the activity of HCII is suppressed. Another object of the present invention is to provide an HCII-containing composition in which polymerization of HCII is suppressed. It is a further object of the present invention to provide an H C II-containing composition in which the generation of insolubles is suppressed.
本発明者らは、 上記目的を達成するために鋭意研究を重ねた結果、 HCII含有 組成物の pHを 6〜12、 好ましくは 6〜： 10、 より好ましくは 6. 5-10, 特に好ましくは？〜 10に調整することにより HC IIの活性低下と H C IIのポリ マ一化を抑制できることを見いだした。 また本発明者らは、 HCII含有組成物に 非イオン性界面活性剤を含有させることにより、 HCIIの活性低下を抑制し、 不 溶物の生成を抑制し得ることを見いだした。 さらに本発明者らは、 HCII含有組 成物の保存温度を 37 以下とすることにより、 HCIIの活性低下と HCIIのポ リマー化を抑制し得ることを見いだした。 The present inventors have conducted intensive studies in order to achieve the above object, and as a result, have found that the pH of the HCII-containing composition is 6 to 12, preferably 6 to 10, more preferably 6.5 to 10, and particularly preferably 6.5 to 10. ? By adjusting the value to ~ 10, it was found that HC II activity reduction and HC II polymerization could be suppressed. The present inventors have also found that by adding a nonionic surfactant to the HCII-containing composition, a decrease in the activity of HCII can be suppressed, and generation of insolubles can be suppressed. Furthermore, the present inventors have found that by setting the storage temperature of the HCII-containing composition to 37 or lower, it is possible to suppress the decrease in HCII activity and the polymerization of HCII.
すなわち、 本発明は、 以下に関する。 That is, the present invention relates to the following.
( 1 ) p H調整剤を含有することを特徴とする H C II含有組成物。 (1) An H C II-containing composition comprising a pH adjuster.
(2) p H調整剤を安定化剤として含有する H C II含有組成物。 (2) An H C II-containing composition containing a pH adjuster as a stabilizer.
(3) 非ィォン性界面活性剤を含有することを特徴とする H C II含有組成物。
(4) 非ィォン性界面活性剤を安定化剤として含有する H C II含有組成物。 (3) An HC II-containing composition comprising a nonionic surfactant. (4) An HC II-containing composition containing a nonionic surfactant as a stabilizer.
(5) p H調整剤および非ィォン性界面活性剤を含有することを特徴とする H C II含有組成物。 (5) An H C II-containing composition comprising a pH adjuster and a nonionic surfactant.
(6) p H調整剤および非イオン性界面活性剤を安定化剤として含有する H C II 含有組成物。 (6) An H C II-containing composition containing a pH adjuster and a nonionic surfactant as a stabilizer.
(7) 非イオン性界面活性剤がポリアルキレングリコール、 ポリオキシエチレン —ポリオキシプロピレン共重合体およびポリオキシエチレンソルビタン脂肪酸ェ ステルからなる群より選ばれる少なくとも 1つである上記 （3) 〜 （6) のいず れかに記載の H C II含有組成物。 (7) The above (3) to (6), wherein the nonionic surfactant is at least one selected from the group consisting of polyalkylene glycol, polyoxyethylene-polyoxypropylene copolymer and polyoxyethylene sorbitan fatty acid ester. ) The composition containing HC II according to any one of the above.
(8) 非イオン性界面活性剤がポリエチレングリコール、 ポロクサマ一またはポ リソルべ一トである上記 （3) 〜 （6) のいずれかに記載の HCII含有組成物。 (8) The HCII-containing composition according to any one of the above (3) to (6), wherein the nonionic surfactant is polyethylene glycol, poloxamer or polysorbate.
(9) 非イオン性界面活性剤がポリエチレングリコールである上記 （3) 〜 （6) のいずれかに記載の H C II含有組成物。 (9) The HCII-containing composition according to any one of the above (3) to (6), wherein the nonionic surfactant is polyethylene glycol.
(10) 非イオン性界面活性剤がポリソルベートである上記 （3) 〜 （6) のい ずれかに記載の H CII含有組成物。 (10) The HCII-containing composition according to any one of the above (3) to (6), wherein the nonionic surfactant is polysorbate.
(1 1) pH調整剤により pHが 6〜12に調整された上記 （1) 、 （2) 、 (5) ~ (10) のいずれかに記載の HC II含有組成物。 (11) The HC II-containing composition according to any one of the above (1), (2), (5) to (10), wherein the pH is adjusted to 6 to 12 by a pH adjuster.
(12) pH調整剤により pHが 6〜1 0に調整された上記 （1) 、 （2) 、 (5) ~ (10) のいずれかに記載の HC II含有組成物。 (12) The HC II-containing composition according to any one of the above (1), (2), (5) to (10), wherein the pH is adjusted to 6 to 10 with a pH adjuster.
(13) pH調整剤により pHが 6. 5〜10に調整された上記 （1) 、 （2) 、 (5) ~ (10) のいずれかに記載の HC II含有組成物。 (13) The HC II-containing composition according to any one of the above (1), (2), (5) to (10), wherein the pH is adjusted to 6.5 to 10 with a pH adjuster.
(14) 組成物が液状組成物または凍結乾燥組成物である上記 （1) 〜 （1 3) のいずれかに記載の HCII含有組成物。 (14) The HCII-containing composition according to any one of the above (1) to (13), wherein the composition is a liquid composition or a lyophilized composition.
(15) HCII含有組成物に pH調整剤を含有させることを特徴とする HCII含 有組成物の安定化方法。 (15) A method for stabilizing an HCII-containing composition, comprising adding a pH adjuster to the HCII-containing composition.
(1 6) HCII含有組成物に非イオン性界面活性剤を含有させることを特徴とす る H C II含有組成物の安定化方法。
(1 7) HCII含有組成物に pH調整剤および非イオン性界面活性剤を含有させ ることを特徴とする H C II含有組成物の安定化方法。 (16) A method for stabilizing an HCII-containing composition, which comprises adding a nonionic surfactant to the HCII-containing composition. (17) A method for stabilizing an HCII-containing composition, comprising adding a pH adjuster and a nonionic surfactant to the HCII-containing composition.
(1 8) 非イオン性界面活性剤がポリアルキレングリコール、 ポリオキシェチレ ンーポリオキシプロピレン共重合体およびポリオキシエチレンソルビタン脂肪酸 エステルからなる群より選ばれる少なくとも 1つである上記 （16) または （17) 記載の方法。 (18) The above (16) or (17), wherein the nonionic surfactant is at least one selected from the group consisting of polyalkylene glycols, polyoxyethylene-polyoxypropylene copolymers and polyoxyethylene sorbitan fatty acid esters. the method of.
(19) 非イオン性界面活性剤がポリエチレングリコール、 ポロクサマーまたは ポリソルべ一卜である上記 （16) または （1 7) 記載の方法。 (19) The method according to the above (16) or (17), wherein the nonionic surfactant is polyethylene glycol, poloxamer or polysorbate.
(20) 非イオン性界面活性剤がポリエチレングリコールである上記 （1 6) ま たは （1 7) 記載の方法。 (20) The method according to the above (16) or (17), wherein the nonionic surfactant is polyethylene glycol.
(2 1) 非イオン性界面活性剤がポリソルベー卜である上記 （16) または （17) 記載の方法。 (21) The method according to the above (16) or (17), wherein the nonionic surfactant is polysorbate.
(22) HCII含有組成物に pH調整剤を含有させることにより、 組成物の pH を 6〜12に調整することを特徴とする上記 （15) 、 （1 7) 〜 （21) のい ずれかに記載の方法。 (22) Any of the above (15), (17) to (21), wherein the pH of the composition is adjusted to 6 to 12 by adding a pH adjuster to the HCII-containing composition. The method described in.
(23) HCII含有組成物に pH調整剤を含有させることにより、 組成物の pH を 6〜1 0に調整することを特徴とする上記 （15) 、 （17) 〜 （2 1) のい ずれかに記載の方法。 (23) Any of the above (15), (17) to (21), wherein the pH of the composition is adjusted to 6 to 10 by adding a pH adjuster to the HCII-containing composition. The method described in Crab.
(24) HCII含有組成物に pH調整剤を含有させることにより、 組成物の pH を 6. 5〜10に調整することを特徴とする上記 （15) 、 （1 7) 〜 （2 1) のいずれかに記載の方法。 (24) The method according to (15), (17) to (21), wherein the pH of the composition is adjusted to 6.5 to 10 by adding a pH adjuster to the HCII-containing composition. The method according to any of the above.
(25) 上記 （1) 〜 （14) のいずれかに記載の HCII含有組成物を 37 ¾以 下の温度で保存することを特徴とする H C II含有組成物の保存方法。 (25) A method for storing an HCII-containing composition, comprising storing the HCII-containing composition according to any one of the above (1) to (14) at a temperature of 37 ° C or lower.
本発明はまた、 以下の （i) 〜 （i i i) に記載する注射剤キッ トを提供する c The present invention also provides an injection kit described in the following (i) to (iii) c
(i) 少なくとも下記 （a) および （b) の構成要素からなる注射剤キッ 卜。 (i) An injection kit comprising at least the following components (a) and (b):
(a) HCIIおよび pH調整剤を含有する凍結乾燥組成物であって、 該 pH調 整剤は該組成物を注射可能な水性溶剤に溶解したときに溶液の p Hを 6〜 1 0に
調整し得るものである凍結乾燥組成物、 (a) A lyophilized composition comprising HCII and a pH adjuster, wherein the pH adjuster brings the pH of the solution to 6 to 10 when the composition is dissolved in an injectable aqueous solvent. A lyophilized composition that can be adjusted,
(b) 注射可能な水性溶剤。 (b) Injectable aqueous solvents.
( i i ) 少なくとも下記 （c) および （d) の構成要素からなる注射剤キッ ト。 (ii) an injection kit comprising at least the following components (c) and (d):
( c ) H C IIおよび非ィォン性界面活性剤を含有する凍結乾燥組成物、 (c) a lyophilized composition containing H C II and a nonionic surfactant,
(d) 注射可能な水性溶剤。 (d) Injectable aqueous solvents.
( i i i) 少なくとも下記 （e) および （ί) の構成要素からなる注射剤キッ ト。 (ii) An injection kit comprising at least the following components (e) and (ii):
(e) HCII. 非イオン性界面活性剤および pH調整剤を含有する凍結乾燥組 成物であつて、 該 p H調整剤は該組成物を注射可能な水性溶剤に溶解したときに 溶液の p Hを 6〜 10に調整し得るものである凍結乾燥組成物、 (e) HCII. A lyophilized composition comprising a non-ionic surfactant and a pH adjuster, wherein the pH adjuster comprises a pH of the solution when the composition is dissolved in an injectable aqueous solvent. A lyophilized composition capable of adjusting H to 6 to 10,
( f ) 注射可能な水性溶剤。 (f) Injectable aqueous solvents.
本発明において、 HC II含有組成物の安定化方法には、 HC IIの活性低下を抑 制する方法、 HCIIのポリマー化を抑制する方法、 組成物中の不溶物の生成を抑 制する方法等が含まれる。 In the present invention, the method for stabilizing the HCII-containing composition includes a method for suppressing a decrease in the activity of HCII, a method for suppressing the polymerization of HCII, a method for suppressing the generation of insolubles in the composition, and the like. Is included.
以下、 本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明の H C II含有組成物中の H CIIは、 SDS - PAGEにより分子量約 7 2 kD aの位置に肉眼上単一のバンドを示し、 等電点が約 5であり、 トロンビン、 キモトリブシンおよびカテブシン G阻害作用を有する蛋白質をいう。 したがって、 本発明の HCIIは、 上記の性質を満たす限り必ずしも完全長の分子に限定されな い。 HCII in the HCII-containing composition of the present invention shows a single band visually at a position of a molecular weight of about 72 kDa by SDS-PAGE, has an isoelectric point of about 5, and has thrombin, chymotrypsin and cathepsin. A protein having a G inhibitory action. Therefore, the HCII of the present invention is not necessarily limited to a full-length molecule as long as the above properties are satisfied.
本発明において、 HCIIの由来に特に限定はなく、 天然の血漿由来のものも、 また、 H C IIを産生するように遺伝子操作された組換え宿主細胞由来のものもす ベて包含される。 血漿由来のものとしては、 例えば全血漿、 HCII活性の高い血 漿分画、 好ましくは Eff. I、 Eff.11 + III または Fr. IV、 特に ATIII精製にお ける廃棄画分などから精製されたものが挙げられる。 また、 血漿の生物起源も特 に制限はなくゥシゃヒト由来のものなどが挙げられるが、 好ましくはヒト由来の ものである。 In the present invention, the origin of HCII is not particularly limited, and includes both those derived from natural plasma and those derived from recombinant host cells genetically engineered to produce HCII. Examples of plasma-derived substances include, for example, whole plasma, plasma fraction having high HCII activity, preferably purified from Eff.I, Eff.11 + III or Fr.IV, particularly waste fraction in ATIII purification. Things. The biological origin of the plasma is not particularly limited, and may be derived from humans, but is preferably derived from humans.
組換え宿主細胞由来のものとしては、 上記の天然血漿中に含まれる HCIIをコ
一ドする遺伝子で形質転換された宿主細胞を培養して得られる培養物の H C II含 有画分、 例えば培養液、 細胞抽出液、 培養上清などから精製されたものが挙げら れる。 宿主細胞は特に制限はないが、 大腸菌、 枯草菌、 乳酸菌などの細菌、 酵母 などの真菌類、 動植物細胞、 昆虫細胞などが好ましく例示される。 また、 HCII をコードする遺伝子を組み込んだトランスジエニック動物の体液または乳汁など から精製された HC IIが挙げられる。 As for those derived from recombinant host cells, HCII contained in HCII-containing fractions of cultures obtained by culturing host cells transformed with the transferred genes, for example, those purified from culture solutions, cell extracts, culture supernatants and the like. The host cell is not particularly limited, but preferably includes bacteria such as Escherichia coli, Bacillus subtilis, and lactic acid bacteria, fungi such as yeast, animal and plant cells, and insect cells. HCII purified from body fluids or milk of transgenic animals into which the gene encoding HCII has been incorporated.
組換え宿主細胞由来の HCIIとしては、 天然の HCII (例えば、 Blinder et a 1.， Biochemistry, 27: 752-759, 1988)または抗トロンビン活性が保持もしくは 改善された変異型 HCII (例えば、 特開平 3 - 139280号公報参照） をコ— ドする D N Aを含む組換えべクタ一で形質転換された宿主細胞を培養することに よって生産されるもの等が例示される。 Examples of HCII derived from a recombinant host cell include natural HCII (for example, Blinder et al., Biochemistry, 27: 752-759, 1988) or mutant HCII having retained or improved antithrombin activity (for example, Examples thereof include those produced by culturing host cells transformed with a recombinant vector containing DNA encoding the DNA encoding E. coli (see 3-139280).
HCIIの精製方法は、 特に限定されず、 例えば Blinder ら （J. Biol. Chem.， Vol. 264, 5128-5133, 1989)の方法、 特開平 9 - 28679 7号公報に記載の方 法、 国際出願番号 PC TZ J P 98 / 04939 (出願日 1998年 10月 30日） に記 載の方法 〔低分子化因子 （血漿または宿主細胞由来のプロテアーゼ等） が除去さ れた HCII含有組成物を得る方法〕 、 日本特許出願 （特願平 1 1一 109 1 1、 出願日 1999年 1月 19日） に記載の方法 〔HCII粗製物を疎水性基をリガンドとす る不溶性担体に接触させ、 塩濃度を変えた緩衝液で処理することにより、 HCII 由来の夾雑蛋白が除去された HCII含有組成物を得る方法〕 等が挙げられる。 ま たこれらの精製方法を適宜組み合わせて行うこともできる。 The method of purifying HCII is not particularly limited. For example, the method of Blinder et al. (J. Biol. Chem., Vol. 264, 5128-5133, 1989), the method described in JP-A-9-286797, international Application No. PC TZ JP 98/04939 (filing date: October 30, 1998) [Method for obtaining HCII-containing composition from which depolymerizing factors (plasma or host cell-derived protease, etc.) have been removed The method described in the Japanese patent application (Japanese Patent Application No. 11-10911, filed on Jan. 19, 1999) [The HCII crude product is brought into contact with an insoluble carrier having a hydrophobic group as a ligand to obtain a salt concentration. To obtain an HCII-containing composition from which contaminating proteins derived from HCII have been removed by treating with a buffer solution having a different pH. Further, these purification methods can be appropriately combined.
本発明の HCII含有組成物は、 HCIIが含有されている組成物であればいかな るものであってもよく、 凍結乾燥組成物または液状組成物のいずれの形態であつ てもよい。 また本発明の HCII含有組成物には、 投与可能な医薬の最終製剤、 さ らには医薬製剤の製造工程中の組成物ならびに H C IIの精製工程中の組成物も含 まれる。 The HCII-containing composition of the present invention may be any composition as long as it contains HCII, and may be in the form of a lyophilized composition or a liquid composition. The HCII-containing composition of the present invention also includes a final preparation of an administrable drug, a composition during a manufacturing process of a pharmaceutical formulation, and a composition during a purification process of HC II.
本発明は p H調整剤を含有することを特徴とする H C II含有組成物を提供する c 本発明の HCII含有組成物は pH6〜l 2、 好ましくは pH6〜l 0、 より好ま
しくは pH6. 5-1 0.特に好ましくは pH7〜l 0に調整されてなることを 特徴とする。 HC II含有組成物が凍結乾燥組成物の場合は、 適当な溶剤 （注射可 能な水性溶剤等） に溶解したときに溶液の pHが pH 6〜12、 好ましくは pH 6〜1 0、 より好ましくは pH6. 5〜1 0、 特に好ましくは pH7〜l 0とな るように調整する。 かかる P H範囲に調整することにより H C IIの活性低下を抑 制し、 HCIIのポリマ一化を抑制することができる。 HCII-containing composition of the present invention c present invention to provide an HC II containing composition characterized by containing a p H adjusting agent pH6~l 2, preferably pH6~l 0, favored more Or pH 6.5-1 to 10. Particularly preferably, the pH is adjusted to pH 7 to 10. When the HC II-containing composition is a lyophilized composition, the pH of the solution when dissolved in a suitable solvent (such as an injectable aqueous solvent) is pH 6 to 12, preferably pH 6 to 10, and more preferably. Is adjusted to pH 6.5 to 10, particularly preferably pH 7 to 10. By adjusting the pH to such a range, it is possible to suppress a decrease in the activity of HCII and suppress the polymerization of HCII.
p Hの調整は p H調整剤を用いて行うことができる。 本発明で用いる p H調整 剤は組成物の pHを pH6〜12に調整し得るものであればよい。 該 p H調整剤 は組成物の pHを好ましくは pH6〜l 0、 より好ましくは pH6. 5〜1 0、 特に好ましくは p H 7〜 10に調整し得るものである。 pH調整剤としては、 通 常、 緩衝剤が用いられる。 緩衝剤としては、 リン酸塩緩衝剤 （リン酸ナトリウム 緩衝剤） 、 クェン酸塩緩衝剤、 トリス塩酸緩衝剤、 ジェチルエタノールアミン塩 酸緩衝剤等が例示される。 好ましくは、 リン酸塩緩衝剤 （リン酸ナトリゥム緩衝 剤） 、 卜リス塩酸緩衝剤が用いられる。 Adjustment of pH can be performed using a pH adjusting agent. The pH adjuster used in the present invention may be any as long as it can adjust the pH of the composition to pH 6 to 12. The pH adjuster is capable of adjusting the pH of the composition to preferably pH 6 to 10, more preferably pH 6.5 to 10, and particularly preferably pH 7 to 10. As the pH adjuster, a buffer is usually used. Examples of the buffer include a phosphate buffer (sodium phosphate buffer), a citrate buffer, a Tris-HCl buffer, a getylethanolamine chloride buffer, and the like. Preferably, a phosphate buffer (sodium phosphate buffer) and a tris-HCl buffer are used.
pH調整剤の組成物中の濃度は、 液状組成物の場合、 通常 0. 001〜1 00 0mM、 好ましくは 0. 1〜10 OmMである。 凍結乾燥組成物の場合は、 用時 適当な溶剤 （注射可能な水性溶剤等） に溶解したときに溶液中の p H調整剤の濃 度が上記濃度範囲となるように調製する。 The concentration of the pH adjusting agent in the composition is usually 0.001 to 100 mM, preferably 0.1 to 10 OmM in the case of a liquid composition. In the case of a lyophilized composition, it is prepared so that the concentration of the pH adjusting agent in the solution when used in a suitable solvent (such as an injectable aqueous solvent) is within the above-mentioned concentration range.
本発明において、 凍結乾燥組成物を溶解させる適当な溶剤としては、 無菌であ り、 発熱性物質が存在しない注射可能な水性溶剤等が例示され、 具体的には、 注 射用蒸留水、 注射用生理食塩水等が挙げられる。 In the present invention, examples of suitable solvents for dissolving the lyophilized composition include sterile, injectable aqueous solvents free of pyrogenic substances, etc. Specifically, distilled water for injection, injection Physiological saline and the like.
HC II含有組成物が凍結乾燥組成物の場合、 11調整剤で 156〜12、 好ま しくは pH6〜： 10、 より好ましくは pH6. 5〜1 0、 特に好ましくは pH 7 〜10に調整した HC II含有溶液を凍結乾燥して調製する。 用時適当な溶剤 （例 えば、 注射用蒸留水等） に溶解して pH6〜l 2、 好ましくは pH6〜l 0、 よ り好ましくは pH6. 5〜10、 特に好ましくは p H 7〜 10の液状組成物を得 る。 HC II含有組成物が液状組成物の場合は、 15(：11を約0. 0 1〜約 400 U
Zml、 好ましくは約 0. 1〜約 100 UZml含有する溶液として調製できる。 凍 結乾燥組成物の場合は、 用時適当な溶剤 （例えば、 注射用蒸留水等） に溶解した とき溶液中の H C II濃度が上記液状組成物と同様の濃度範囲となるように調製す る。 When the HC II-containing composition is a lyophilized composition, HC adjusted to 156 to 12, preferably pH 6 to 10, more preferably pH 6.5 to 10, and particularly preferably pH 7 to 10 with 11 regulators The II-containing solution is prepared by lyophilization. When used, it is dissolved in a suitable solvent (for example, distilled water for injection, etc.) and the pH is adjusted to pH 6 to 12, preferably pH 6 to 10, more preferably pH 6.5 to 10, and particularly preferably pH 7 to 10. A liquid composition is obtained. When the HC II-containing composition is a liquid composition, 15 (: 11 is about 0.01 to about 400 U It can be prepared as a solution containing Zml, preferably from about 0.1 to about 100 UZml. In the case of freeze-dried composition, adjust so that when dissolved in an appropriate solvent (for example, distilled water for injection), the HC II concentration in the solution will be in the same concentration range as the above liquid composition. .
なお本発明において、 ポリマ一化 HCIIとは、 HCII分子の重合体を意味する。 より具体的には、 二量体またはそれ以上の重合体である。 In the present invention, the polymerized HCII means a polymer of HCII molecules. More specifically, it is a dimer or higher polymer.
本発明はまた、 非ィォン性界面活性剤を含有することを特徴とする H C II含有 組成物を提供する。 非イオン性界面活性剤を含有させることにより、 HCIIの活 性低下を抑制し、 不溶物の生成を抑制することができる。 The present invention also provides an H C II-containing composition comprising a nonionic surfactant. By including a nonionic surfactant, it is possible to suppress a decrease in the activity of HCII and to suppress the generation of insolubles.
非イオン性界面活性剤としては、 例えばポリアルキレングリコール 〔例えば、 ポリエチレングリコール （PEG) 、 ポリプロピレングリコール等〕 、 ポリオキ シエチレン—ポリオキシプロピレン共重合体 〔例えば、 ポロクサマ一 （商品名プ ルロニック） 〕 、 ポリオキシエチレンソルビタン脂肪酸エステル 〔例えば、 ポリ ソルべ一卜 （商品名卜ウィーン） 〕 等が例示される。 これらの非イオン性界面活 性剤の平均分子量は 2， 000〜20, 000が好ましい。 ポリオキシエチレン ソルビタン脂肪酸エステルの脂肪酸としては、 ステアリン酸、 パルミチン酸、 ミ リスチン酸、 ラウリン酸、 ォレイン酸などの炭素数 12〜1 8の脂肪酸が挙げら れる。 非イオン性界面活性剤の具体的な例としては、 平均分子量 4000〜60 00の P E G (例えば、 PEG4000、 PEG6000) 、 ポロクサマ一 18 8 (商品名プル口ニック F 68) 、 ポリソルべ一ト 80 (商品名トウイーン 80) 等が挙げられる。 より好ましくは PEG (例えば、 PEG4000、 PEG 60 00) である。 Examples of the nonionic surfactant include polyalkylene glycol [eg, polyethylene glycol (PEG), polypropylene glycol, etc.], polyoxyethylene-polyoxypropylene copolymer [eg, Poloxamer (trade name: Pluronic)], poly Oxyethylene sorbitan fatty acid esters [for example, polysorbate (trade name: Vienna)] and the like are exemplified. The average molecular weight of these nonionic surfactants is preferably from 2,000 to 20,000. Fatty acids of polyoxyethylene sorbitan fatty acid esters include fatty acids having 12 to 18 carbon atoms such as stearic acid, palmitic acid, myristic acid, lauric acid, and oleic acid. Specific examples of the nonionic surfactant include PEG having an average molecular weight of 4000 to 600 (for example, PEG4000, PEG6000), poloxamer 188 (trade name: Pull mouth nick F 68), polysorbate 80 ( Brand name Tween 80). More preferably, it is PEG (for example, PEG4000, PEG600).
非イオン性界面活性剤の組成物中の濃度は、 液状組成物の場合、 通常 0. 00 1〜2. 0 w/v%、 好ましくは 0. 0 1〜： L. 0 w/v%、 より好ましくは 0 . 02〜 5 w/v%である。 凍結乾燥組成物の場合は、 用時適当な溶剤 （例 えば、 注射用蒸留水等） に (容解したときに溶液中の非イオン性界面活性剤の濃度 が上記濃度範囲となるように調製する。
非イオン性界面活性剤を含有する本発明の H C II含有組成物が液状組成物の場 合、 当該液状組成物は、 好ましくは前記のような pH調整剤で pH6〜l 2、 よ り好ましくは pH6〜10、 特に好ましくは pH 6. 5〜10、 最も好ましくは pH 7〜 10に調整されている。 The concentration of the nonionic surfactant in the composition is usually 0.001 to 2.0 w / v%, preferably 0.01 to: L. 0 w / v% for a liquid composition. More preferably, it is 0.02 to 5 w / v%. In the case of lyophilized composition, prepare in a suitable solvent (for example, distilled water for injection) at the time of use ( so that the concentration of nonionic surfactant in the solution when dissolved is within the above concentration range). I do. When the HC II-containing composition of the present invention containing a nonionic surfactant is a liquid composition, the liquid composition is preferably pH 6 to l2, more preferably a pH adjuster as described above. It is adjusted to pH 6-10, particularly preferably pH 6.5-10, most preferably pH 7-10.
H C II含有組成物が凍結乾燥組成物の場合、 H C IIおよび非イオン性界面活性 剤を含有する水溶液を凍結乾燥して調製する。 好ましくは、 前記のような pH調 整剤で pH6〜l 2、 より好ましくは pH6〜l 0、 特に好ましくは pH 6. 5 〜1 0、 最も好ましくは pH7〜l 0に調整された HCIIおよび非イオン性界面 活性剤を含有する水溶液を凍結乾燥して調製する。 用時適当な溶剤 （例えば、 注 射用蒸留水等） に溶解して液状組成物を得る。 HC II含有組成物が液状組成物の 場合は、 前記と同様に、 11〇11を約0. 01〜約 400 UZml、 好ましくは約 0 • 1〜約 100 UZml含有する溶液として調製できる。 凍結乾燥組成物の場合は、 用時適当な溶剤 （例えば、 注射用蒸留水等） に溶解したとき溶液中の HC II濃度 が上記液状組成物と同様の濃度範囲となるように調製する。 When the H C II-containing composition is a lyophilized composition, it is prepared by lyophilizing an aqueous solution containing H C II and a nonionic surfactant. Preferably, HCII and non-hydrochloric acid are adjusted to pH 6 to 12, more preferably pH 6 to 10, particularly preferably pH 6.5 to 10, most preferably pH 7 to 10 with a pH adjusting agent as described above. An aqueous solution containing an ionic surfactant is prepared by freeze-drying. When used, a liquid composition is obtained by dissolving in an appropriate solvent (for example, distilled water for injection). When the HC II-containing composition is a liquid composition, it can be prepared in the same manner as described above, as a solution containing 11〇11 in an amount of about 0.01 to about 400 UZml, preferably about 0.11 to about 100 UZml. In the case of a lyophilized composition, it is prepared so that the HCII concentration in the solution when dissolved in an appropriate solvent (for example, distilled water for injection, etc.) at the time of use is in the same concentration range as the above liquid composition.
上述の本発明の HC II含有組成物は、 特にその保存温度を 37 ¾以下とするこ とにより当該 H C II含有組成物の安定性を維持することができる。 保存温度は好 ましくは— 20で〜 3 7^、 より好ましくは 0 〜 15でである。 かかる温度条 件下で保存することにより、 HC IIの活性低下を抑制し、 HCIIのポリマ一化を 抑制することができる。 The above-described HC II-containing composition of the present invention can maintain the stability of the HC II-containing composition, particularly when the storage temperature is 37 ° C. or less. The storage temperature is preferably -20 to 37 ^, more preferably 0 to 15. By storing under such temperature conditions, a decrease in the activity of HCII can be suppressed, and the polymerization of HCII can be suppressed.
本発明の HCII含有組成物には、 通常の医薬組成物に用いられる添加剤、 例え ば等張化剤 （例えば、 ショ糖、 ソルビトール、 マンニトール、 グリセリン、 グル コース、 塩化ナトリウム等） 、 防腐殺菌剤 〔例えば、 塩化ベンザルコニゥム、 塩 化べンゼトニゥム、 パラォキシ安息香酸エステル類 （パラォキシ安息香酸メチル、 パラォキシ安息香酸ェチル、 パラォキシ安息香酸プロピル、 パラォキシ安息香酸 ブチル等） 、 ベンジルアルコール、 パラクロロメタキシレノール、 クロ口クレゾ —ル、 フエネチルアルコール、 ソルビン酸またはその塩、 チメロサール、 クロ口 ブタノ一ル等〕 、 キレート剤 （ェデト酸ナトリウム等） 、 粘稠剤 （ポリビニルビ
ロリ ドン、 メチルセルロース、 カルボキシメチルセルロースナトリウム、 ヒ ドロ キシプロピルセルロース、 ヒ ドロキシプロピルメチルセルロース、 ポリビニルァ ルコール、 ポリアクリル酸ナトリウム等） 、 その他通常の蛋白製剤の安定化に用 いられる化合物等を通常使用される添加量で配合することができる。 The HCII-containing composition of the present invention may contain additives used in ordinary pharmaceutical compositions, for example, tonicity agents (eg, sucrose, sorbitol, mannitol, glycerin, glucose, sodium chloride, etc.), preservatives and disinfectants [For example, benzalkonium chloride, benzethonium chloride, paraoxybenzoic acid esters (eg, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate), benzyl alcohol, parachlorometoxylenol, -, Phenethyl alcohol, sorbic acid or its salt, thimerosal, black butanol, etc.), chelating agents (sodium edetate, etc.), thickeners (polyvinyl Loridone, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, sodium polyacrylate), and other compounds commonly used for stabilizing ordinary protein preparations It can be blended in an added amount.
本発明の H C II含有組成物は、 自体既知の手段を用いて調製することができる。 また所望により加熱、 滅菌、 ウィルス不活化、 除菌濾過、 ウィルス除去、 分注小 分け、 凍結乾燥処理などの処理を施すことができる。 The H C II-containing composition of the present invention can be prepared by a method known per se. If desired, treatments such as heating, sterilization, virus inactivation, sterilization filtration, virus removal, aliquoting, and freeze-drying can be performed.
本発明の H CII含有組成物は、 上記の凍結乾燥組成物および注射可能な水性溶 剤からなる注射剤キッ トとして提供することもできる。 The HCII-containing composition of the present invention can also be provided as an injection kit comprising the above lyophilized composition and an injectable aqueous solution.
本発明の HCII含有組成物は、 血栓症、 微小循環不全、 虚血性疾患のような血 流異常を生じる各種疾患、 敗血症、 肝、 肺、 腎、 脳などの各種臓器機能障害、 糸 球体腎炎、 腌炎、 浮腫形成を伴う炎症などの各種炎症などの疾患の予防および治 療に有用である。 The HCII-containing composition of the present invention includes various diseases causing abnormal blood flow such as thrombosis, microcirculation failure, ischemic disease, sepsis, various organ dysfunctions such as liver, lung, kidney, and brain, glomerulonephritis,に It is useful for the prevention and treatment of various inflammations such as inflammation accompanied by inflammation and edema formation.
本発明の HC II含有組成物は、 注射剤または点滴剤として非経口的に投与する ことが好ましい。 本発明の HCII含有組成物の投与方法としては、 静注または点 滴静注する方法が挙げられる。 The HCII-containing composition of the present invention is preferably administered parenterally as an injection or infusion. The administration method of the HCII-containing composition of the present invention includes a method of intravenous injection or intravenous drip infusion.
本発明の HCII含有組成物の投与量は、 通常、 成人 1日あたり HCII1〜10 00 OU/kg体重、 好ましくは 10〜200 OUZkg体重であり、 この量を 1回 または数回に分けて投与することができる。 The dose of the HCII-containing composition of the present invention is generally 1 to 1000 OU / kg body weight, preferably 10 to 200 OUZ kg body weight per day for an adult, and this amount is administered once or divided into several times. be able to.
実施例 Example
本発明を以下の実施例により詳述する。 以下の実験例および製剤例に用いられ た HCIIは、 特開平 9一 286 797号公報に記載の方法により調製した。 The present invention is described in detail by the following examples. HCII used in the following experimental examples and formulation examples was prepared by the method described in JP-A-9-1286797.
以下の実 ¾ ^例において、 本発明の H C II含有組成物の安定性を調べた。 In the following Examples, the stability of the H C II-containing composition of the present invention was examined.
HCIIの精製品を、 PD— 1 0カラム （フアルマシア社） を用いて 5mMトリ ス塩酸緩衝液 （pH 8. 0、 25 ） で脱塩した後、 各緩衝液で 280 nmでの 吸光度 （A28。 ） 力 . 0になるように調製し、 安定性の検討に供した。 HCII 活性およびポリマ一化 H C IIの割合は下記の方法で測定した。
(1) HC II活性の測定 HCII of the purified product, PD- 1 0 column (Pharmacia, Inc.) 5 mM tri scan HCl buffer with (pH 8. 0, 25) after desalting, the absorbance at 280 nm at each buffer (A 28 ) Force was adjusted to be 0, and the stability was examined. HCII activity and polymerization HCII ratio was measured by the following method. (1) Measurement of HC II activity
検体を 60mM EDTA、 0. 2 % ヒト血清アルブミン （H S A) および 0. 1 mg/mL デルマタン硫酸を含む 40 mM トリス緩衝液 （pH 8. 5 6) で希釈し、 この検体希釈液 50 Lに 0. 05% ゥシ血清アルブミ ンおよ び 0. 05% P E G 6000を含む 1. 0 UZm L 卜ロンビン溶液 1 00 / Lを添加し、 37 にて 5分間静置した後、 0. 9 5mg/mL H— D—フヱ 二ルァラ二ルー L―ピペコリルー L—アルギニル— p—二トロアニリ ドニ塩酸塩 溶液 100 /zLを添加して 37 、 5分間インキュベートした。 2% クェン酸 lmLを加えて反応を停止させた後、 405 nmでの吸光度 （A 405 ) を測定し、 従来法により調製された部分精製品を標準品として [吸光係数： E1 =5. 93The sample was diluted with 40 mM Tris buffer (pH 8.56) containing 60 mM EDTA, 0.2% human serum albumin (HSA) and 0.1 mg / mL dermatan sulfate. Add 1.0 UZmL thrombin solution (100 / L) containing 0.05% PB serum albumin and 0.05% PEG 6000, leave at 37 for 5 minutes, and then add 0.95 mg / The solution was added with 100 mL / mL of a solution of mL H-D-phenyl-2-L-pipecolol-L-arginyl-p-nitroanilide-hydrochloride, and incubated at 37, 5 minutes. After stopping the reaction by adding 1 mL of 2% citrate, the absorbance at 405 nm (A 405 ) was measured, and the partially purified product prepared by the conventional method was used as a standard product [extinction coefficient: E 1 = 5. 93
(Arch. Biochem. Biophys. , 259: 331-340, 1997)と A28。 から求めたタンパク 濃度 0. lmgZmLを 1 Uとした] 作成した検量線より H CII活性を算出した。 (.. Arch Biochem Biophys, 259 :. 331-340, 1997) and A 28. The protein concentration of 0.1 mg mgZmL obtained from the above was defined as 1 U]. The HCII activity was calculated from the prepared calibration curve.
(2) ゲル濾過高速液体クロマトグラフィー （以下、 ゲル濾過 HP LCという） 分析 (2) Gel filtration high performance liquid chromatography (hereinafter referred to as gel filtration HP LC) analysis
以下の条件でゲル濾過 H P L C分析を行った。 Gel filtration HPLC analysis was performed under the following conditions.
カラム： G3， 000 SWXL (07. 8 mm x 30 c m、 東ソ一社製） 展開液： 0. 1M酢酸ナトリウム、 0. 3M塩化ナトリウム、 0. lwZv%7 ジ化ナトリウム含有溶液 （pH 6. 6) Column: G3,000 SWXL (07.8 mm x 30 cm, manufactured by Tosoh I) Co., Ltd. Developing solution: 0.1 M sodium acetate, 0.3 M sodium chloride, 0.1 lwZv% 7 A solution containing sodium dioxide (pH 6. 6)
検体量： 25 / 1 Sample volume: 25/1
流速： 1. OmL/分 Flow rate: 1. OmL / min
分析時間： 12分 Analysis time: 12 minutes
ポリマ一化 H C IIの割合は、 ゲル濾過 H PLC分析で完全長 H C IIのピ一クよ りも高分子側に検出されるピークの面積の全ピーク面積に対する比率 （％) で示 す。 The ratio of the polymerized HC II is shown as a ratio (%) of the area of the peak detected on the polymer side from the peak of the full-length HC II in the gel filtration HPLC analysis to the total peak area.
実験例 1 Experimental example 1
11011を0. 1Mトリス塩酸緩衝液 （PH8. 0、 25X0 で A28。 = 1. 0 になるように調製し、 この検体を 37 、 50°Cおよび 60 で 120分間加温
した。 0、 20、 40、 60、 120分後にサンプリングし、 HCIIの残存活性 の測定とゲル濾過 H P L C分析を行った。 0. 1M Tris-HCl buffer 11011 (PH8. 0, A 28 . = 1. adjusted to be zero in 25X0, the specimen 37, 50 ° C and 60 at 120 minutes warming did. After 0, 20, 40, 60, and 120 minutes, sampling was performed, and the residual activity of HCII was measured and gel filtration HPLC analysis was performed.
結果を表 1〜 3に示す。 温度： 3 7 The results are shown in Tables 1-3. Temperature: 3 7
3 7 では、 120分後においても HCII活性の低下および HP L C分析にお けるポリマー化 HCIIの生成は観察されなかった。 し力、し、 50ででは 20分後
より経時的な HCII活性の低下がみられ、 1 2 0分後の残存活性は 6 4%であつ た。 HP L C分析においても 2 0分後よりポリマ一化 HCIIが検出され、 1 2 0 分後まで経時的に増加した。 6 0ででは HCII活性の低下およびポリマー化が顕 著であり、 2 0分後には 6 %の残存活性しかなく、 HP L C分析においても 9 5 %がポリマ一化 H C IIであつた。 In 37, no decrease in HCII activity and no formation of polymerized HCII in HP LC analysis was observed even after 120 minutes. Power, then at 50 and after 20 minutes The HCII activity decreased over time, and the residual activity after 120 minutes was 64%. Also in the HP LC analysis, polymerized HCII was detected after 20 minutes, and increased with time until after 120 minutes. At 60, the decrease in HCII activity and polymerization were remarkable, and only 20% of the residual activity was observed after 20 minutes, and 95% of the polymerized HCII was also obtained by HP LC analysis.
実験例 2 Experimental example 2
HCIIを、 0. 1M酢酸ナ卜リゥム緩衝液 （pH 5、 6) 、 0. 1Mリン酸ナ ト リ ウム緩衝液 （pH 6. 5、 7. 0、 7. 5) 、 0. 1 Mトリス塩酸緩衝液 （ p H 6. 9、 7. 4、 7. 8、 8. 1) 、 0. 1 Mジェチルエタノールアミン塩 酸緩衝液 （P H 8. 5、 9. 0 ) で A28Q = 1. 0になるように調製し、 これら の検体を 5 0でで 2時間加温した。 2時間加温した後、 HCIIの残存活性の測定 とゲル濾過 HP L C分析を行った。 HCII was treated with 0.1 M sodium acetate buffer (pH 5, 6), 0.1 M sodium phosphate buffer (pH 6.5, 7.0, 7.5), 0.1 M Tris In hydrochloric acid buffer (pH 6.9, 7.4, 7.8, 8.1) and 0.1 M getyl ethanolamine salt buffer (PH 8.5, 9.0), A 28 Q = It was prepared to be 1.0, and these specimens were heated at 50 for 2 hours. After heating for 2 hours, the remaining activity of HCII was measured and gel filtration HPLC analysis was performed.
結果を表 4に示す。 Table 4 shows the results.
表 4 Table 4
実験例 3 Experiment 3
HCIIを、 0. 1Mリン酸ナ ト リウム緩衝液 （pH6、 7、 8) 、 0. 1Mジ ェチルエタノールァミン塩酸緩衝液 （pH 9、 10) で八28。 = 1. 0になるよ うに調製し、 これらの検体を 37°Cで 2時間加温した。 2時間加温後、 HCIIの 残存活性の測定とゲル濾過 H P L C分析を行った。 The HCII, 0. 1M phosphate Sanna preparative potassium buffer (pH6, 7, 8), 0. 1M di E chill ethanol § Min HCl buffer (pH 9, 10) was eight 28. = 1.0, and these specimens were heated at 37 ° C for 2 hours. After heating for 2 hours, the remaining activity of HCII was measured and gel filtration HPLC analysis was performed.
結果を表 5に示す。 Table 5 shows the results.
表 5 Table 5
コン トロール ：非加温 Control: unheated
ポリマー化 HCIIの増加および HCII活性の低下が認められないことから、 3 7で、 pH6〜： I 0で HCIIは安定である。 At 37, pH 66: HCII is stable at I 0, since no increase in polymerized HCII and no decrease in HCII activity is observed.
実験例 4 Experiment 4
HCIIを、 0. 1 wZv%ポリエチレングリコール 4000を含有する 0. 1 Mトリス塩酸緩衝液 （pH8. 0、 25で） で A28。 = 1. 0になるように調製 した。 この HCII溶液を室温にて、 125 r pmで 1時間水平振とうし、 続いて 室温にてボルテックスミキサ一で 1分間振とうした。 HCII A 28 in 0.1 M Tris-HCl buffer (at pH 8.0, 25) containing 0.1 wZv% polyethylene glycol 4000. = 1.0. The HCII solution was horizontally shaken at 125 rpm for 1 hour at room temperature, and subsequently shaken for 1 minute at room temperature with a vortex mixer.
HCIIの残存活性を測定し、 不溶物の有無を目視で評価した。 結果を表 6に示 す。 +は不溶物が観察されたことを示し、 一は不溶物が観察されなかったことを
示す。 The remaining activity of HCII was measured, and the presence or absence of insolubles was visually evaluated. Table 6 shows the results. + Indicates that no insoluble matter was observed, and one indicates that no insoluble matter was observed. Show.
表 6 Table 6
HCIIが 50 U/m 1になるように、 0. 15 M塩化ナトリウムを含む 10 m Mリン酸ナトリウム緩衝液 （pH7. 2) で HCII溶液を調製した。 この溶液の 1 0m 1を医薬品用のガラスバイアルに分注し、 凍結乾燥して凍結乾燥製剤を調 製した。 An HCII solution was prepared with a 10 mM sodium phosphate buffer (pH 7.2) containing 0.15 M sodium chloride so that the HCII was 50 U / ml. 10 ml of this solution was dispensed into glass vials for pharmaceuticals and freeze-dried to prepare a freeze-dried preparation.
製剤例 2 Formulation Example 2
HCIIが 10〜50 U/m 1になるように、 0. 1 5 M塩化ナトリウムを含む 1 OmMリン酸ナトリウム緩衝液 （pH 7. 2 ) で H CII溶液を調製した。 この 溶液の 1 0mlを医薬品用のガラスバイアルに分注して液状製剤を調製した。 製剤例 3 The HCII solution was prepared with 1 OmM sodium phosphate buffer (pH 7.2) containing 0.15 M sodium chloride so that HCII was 10 to 50 U / ml. A liquid preparation was prepared by dispensing 10 ml of this solution into a glass vial for pharmaceuticals. Formulation Example 3
HCIIが 5 OU/m 1になるように、 0. 15 M塩化ナトリウムおよび 0. 1 w/v%ポリエチレングリコール 4， 000を含む 1 OmMリン酸ナトリゥム緩 衝液 （p H 7. 2) で HCII溶液を調製した。 この溶液の 10m lを医薬品用の
ガラスバイアルに分注し、 凍結乾燥して凍結乾燥製剤を調製した。 HCII solution with 1 OmM sodium phosphate buffer (pH 7.2) containing 0.15 M sodium chloride and 0.1 w / v% polyethylene glycol 4,000 so that HCII is 5 OU / m1. Was prepared. 10 ml of this solution is Dispensed into glass vials and lyophilized to prepare a lyophilized formulation.
製剤例 4 Formulation Example 4
HCIIが 1 0〜50 U/m 1になるように、 0. 15 M塩化ナトリウムおよび 0. 1 w/v%ポリエチレングリコール 4, 000を含む 1 0 mMリン酸ナトリ ゥム緩衝液 （PH 7. 2) で HCII溶液を調製した。 この溶液の 10m lを医薬 品用のガラスバイアルに分注して液状製剤を調製した。 10 mM sodium phosphate buffer containing 0.15 M sodium chloride and 0.1 w / v% polyethylene glycol 4,000 so that HCII is 10-50 U / m1 (PH 7. The HCII solution was prepared in 2). A liquid preparation was prepared by dispensing 10 ml of this solution into a glass vial for pharmaceuticals.
製剤例 5 Formulation Example 5
製剤例 1で調製した凍結乾燥製剤、 および注射用蒸留水 10mlから構成され る注射剤キッ トを調製した。 凍結乾燥製剤を注射用蒸留水に溶解することにより 注射剤が調製される。 An injection kit comprising the lyophilized preparation prepared in Preparation Example 1 and 10 ml of distilled water for injection was prepared. An injection is prepared by dissolving the lyophilized formulation in distilled water for injection.
製剤例 6 Formulation Example 6
製剤例 3で調製した凍結乾燥製剤、 および注射用蒸留水 10mlから構成され る注射剤キッ トを調製した。 凍結乾燥製剤を注射用蒸留水に溶解することにより 注射剤が調製される。 本発明によれば、 ^1 11含有組成物の 11を ^16〜12、 好ましくは pH 6 〜： 10、 より好ましくは pH6. 5〜10、 特に好ましくは p H 7〜 1 0に調整 することにより HCIIの活性低下と HCIIのポリマ一化を抑制できる。 また、 H C II含有組成物に非ィォン性界面活性剤を添加することにより、 H C IIの活性低 下を抑制し、 不溶物の生成を抑制することができる。 さらに、 本発明の HCII含 有組成物の保存温度を 3 7 X：以下とすることにより、 HCIIの活性低下と HCII のポリマー化を抑制できる。 本発明によれば、 HCIIの活性低下、 HCIIのポリ マ一化、 および不溶物の生成が抑制された、 安定性に優れた HCII含有組成物が 提供される。 本出願は日本で出願された平成 10年特許願第 56445号を基礎としており- その内容は本明細書に全て包含されるものである。
An injection kit comprising the lyophilized preparation prepared in Preparation Example 3 and 10 ml of distilled water for injection was prepared. An injection is prepared by dissolving the lyophilized formulation in distilled water for injection. According to the present invention, 11 of the ^ 111-containing composition is adjusted to ^ 16 ~ 12, preferably pH 6 ~: 10, more preferably pH 6.5 ~ 10, particularly preferably pH 7 ~ 10. Thus, the decrease in HCII activity and the polymerization of HCII can be suppressed. Further, by adding a nonionic surfactant to the H C II-containing composition, a decrease in the activity of H C II can be suppressed, and the generation of insolubles can be suppressed. Furthermore, by setting the storage temperature of the HCII-containing composition of the present invention to 37X: or less, it is possible to suppress the decrease in HCII activity and the polymerization of HCII. ADVANTAGE OF THE INVENTION According to this invention, HCII containing composition excellent in stability which suppressed HCII activity fall, polymerization of HCII, and generation | occurrence | production of an insoluble matter is provided. This application is based on Japanese Patent Application No. 56445 filed in Japan-the contents of which are incorporated in full herein.
Claims
請求の範囲 The scope of the claims
I . pH調整剤を含有することを特徴とするへパリンコファクタ一 II含有組成 物。 I. A composition containing heparin cofactor-II, which comprises a pH adjuster.
2. p H調整剤を安定化剤として含有するへパリ ンコファクタ一II含有組成物 c 2. Heparin cofactor-II-containing composition c containing a pH adjuster as a stabilizer
3. 非ィォン性界面活性剤を含有することを特徴とするへパリンコフアクター II含有組成物。 3. A composition containing heparin cofactor II, comprising a nonionic surfactant.
4. 非ィォン性界面活性剤を安定化剤として含有するへパリンコフアクター II 含有組成物。 4. A composition containing heparin cofactor II containing a nonionic surfactant as a stabilizer.
5. p H調整剤および非ィォン性界面活性剤を含有することを特徴とするへパ 5. Heparin characterized by containing a pH adjuster and a nonionic surfactant.
7 7
リ ンコファクタ一II含有組成物。 A composition containing Rincofactor-II.
6. p H調整剤および非ィォン性界面活性剤を安定化剤として含有するへパリ ンコファクタ一 II含有組成物。 6. A heparin cofactor-II-containing composition containing a pH adjuster and a nonionic surfactant as a stabilizer.
7. 非イオン性界面活性剤がポリアルキレングリコール、 ポリオキシエチレン —ポリオキシプロピレン共重合体およびポリオキシエチレンソルビタン脂肪酸ェ ステルからなる群より選ばれる少なくとも 1つである請求項 3〜 6のいずれか 1 項に記載のへパリ ンコファクタ一 II含有組成物。 7. The non-ionic surfactant is at least one selected from the group consisting of polyalkylene glycol, polyoxyethylene-polyoxypropylene copolymer and polyoxyethylene sorbitan fatty acid ester. 2. The composition containing heparin cofactor-II according to item 1.
8. 非イオン性界面活性剤がポリエチレングリコール、 ポロクサマ一またはポ リソルベートである請求項 3〜 6のいずれか 1項に記載のへパリンコファクタ一 II含有組成物。 8. The composition containing heparin cofactor II according to any one of claims 3 to 6, wherein the nonionic surfactant is polyethylene glycol, poloxamer or polysorbate.
9. 非ィォン性界面活性剤がポリエチレングリコールである請求項 3〜 6のい ずれか 1項に記載のへパリ ンコファクタ一 II含有組成物。 9. The heparin cofactor-II-containing composition according to any one of claims 3 to 6, wherein the nonionic surfactant is polyethylene glycol.
10. 非イオン性界面活性剤がポリソルべ一トである請求項 3〜6のいずれか 1項に記載のへパリ ンコファクタ一 II含有組成物。 10. The heparin cofactor-II-containing composition according to any one of claims 3 to 6, wherein the nonionic surfactant is a polysorbate.
I I. pH調整剤により pHが 6~12に調整された請求項 1、 2、 5〜： 1 0 のいずれか 1項に記載のへパリ ンコファクタ一 II含有組成物。 I I. The heparin cofactor-II-containing composition according to any one of claims 1, 2, 5 and 10, wherein the pH is adjusted to 6 to 12 with a pH adjuster.
12. pH調整剤により pHが 6〜10に調整された請求項 1、 2、 5〜1 0 のいずれか 1項に記載のへパリ ンコファクタ一II含有組成物。
12. The heparin cofactor-II-containing composition according to any one of claims 1, 2, and 5 to 10, wherein the pH is adjusted to 6 to 10 with a pH adjuster.
1 3. pH調整剤にょりpHが6. 5〜1 0に調整された請求項 1、 2、 5〜 1 0のいずれか 1項に記載のへパリンコファクタ一II含有組成物。 13. The heparin cofactor-II-containing composition according to any one of claims 1, 2, 5 to 10, wherein the pH is adjusted to 6.5 to 10 with a pH adjuster.
14. 組成物が液状組成物または凍結乾燥組成物である請求項 1〜1 3のいず れか 1項に記載のへパリンコファクタ一II含有組成物。 14. The heparin cofactor-II-containing composition according to any one of claims 1 to 13, wherein the composition is a liquid composition or a lyophilized composition.
1 5. へパリンコファクタ一II含有組成物に pH調整剤を含有させることを特 徵とするへパリンコファクタ一 II含有組成物の安定化方法。 1 5. A method for stabilizing a composition containing heparin cofactor-II, which comprises adding a pH adjuster to the composition containing heparin cofactor-II.
16. へパリンコファクタ一 II含有組成物に非イオン性界面活性剤を含有させ ることを特徴とするへパリンコファクタ一 II含有組成物の安定化方法。 16. A method for stabilizing a composition containing heparin cofactor II, which comprises adding a nonionic surfactant to the composition containing heparin cofactor II.
1 7. へパリンコファクタ一 II含有組成物に pH調整剤および非イオン性界面 活性剤を含有させることを特徴とするへパリンコファクター II含有組成物の安定 化方法。 1 7. A method for stabilizing a composition containing heparin cofactor II, comprising adding a pH adjuster and a nonionic surfactant to the composition containing heparin cofactor II.
1 8. 非イオン性界面活性剤がポリアルキレングリコール、 ポリオキシェチレ ン一ポリオキシプロピレン共重合体およびポリオキシエチレンソルビタン脂肪酸 エステルからなる群より選ばれる少なくとも 1つである請求項 1 6または 1 7記 載の方法。 18. The method according to claim 16, wherein the nonionic surfactant is at least one selected from the group consisting of polyalkylene glycol, polyoxyethylene-polyoxypropylene copolymer, and polyoxyethylene sorbitan fatty acid ester. the method of.
19. 非イオン性界面活性剤がポリエチレングリコール、 ポロクサマ一または ポリソルベートである請求項 1 6または 1 7記載の方法。 19. The method according to claim 16 or 17, wherein the nonionic surfactant is polyethylene glycol, poloxamer or polysorbate.
20. 非ィォン性界面活性剤がポリエチレングリコールである請求項 16また は 1 7記載の方法。 20. The method according to claim 16 or 17, wherein the nonionic surfactant is polyethylene glycol.
2 1. 非イオン性界面活性剤がポリソルべ一トである請求項 1 6または 1 7記 載の方法。 2 1. The method according to claim 16 or 17, wherein the nonionic surfactant is a polysorbate.
22. へパリンコファクタ一 II含有組成物に pH調整剤を含有させることによ り、 組成物の pHを 6〜12に調整することを特徴とする請求項 15、 1 7〜21 のいずれか 1項に記載の方法。 22. The composition according to any one of claims 15, 17 to 21, wherein the pH of the composition is adjusted to 6 to 12 by adding a pH adjuster to the composition containing heparin cofactor II. The method of paragraph 1.
23. へパリンコファクタ一II含有組成物に pH調整剤を含有させることによ り、 組成物の pHを 6〜10に調整することを特徴とする請求項 15、 1 7〜21 のいずれか 1項に記載の方法。
23. The composition according to any one of claims 15, 17 to 21, wherein the pH of the composition is adjusted to 6 to 10 by adding a pH adjuster to the composition containing heparin cofactor-II. The method of paragraph 1.
24. へパリンコファクタ一 II含有組成物に pH調整剤を含有させることによ り、 組成物の pHを 6. 5〜10に調整することを特徴とする請求項 1 5、 1 7 〜 21のいずれか 1項に記載の方法。 24. The composition containing heparin cofactor II containing a pH adjuster to adjust the pH of the composition to 6.5 to 10, wherein the composition has a pH of 6.5 to 10. The method according to any one of the preceding claims.
25. 請求項 1〜14のいずれか 1項に記載のへパリンコファクタ一 II含有組 成物を 3 7 °C以下の温度で保存することを特徴とするへパリ ンコフアクター II含 有組成物の保存方法。
25. A composition containing heparin cofactor II, wherein the composition containing heparin cofactor-II according to any one of claims 1 to 14 is stored at a temperature of 37 ° C or less. How to save.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU27463/99A AU2746399A (en) | 1998-03-09 | 1999-03-05 | Heparin cofactor ii-containing compositions and method for stabilizing the same |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10/56445 | 1998-03-09 | ||
| JP5644598 | 1998-03-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1999045953A1 true WO1999045953A1 (en) | 1999-09-16 |
Family
ID=13027300
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP1999/001102 WO1999045953A1 (en) | 1998-03-09 | 1999-03-05 | Heparin cofactor ii-containing compositions and method for stabilizing the same |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU2746399A (en) |
| WO (1) | WO1999045953A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003342193A (en) * | 2002-05-30 | 2003-12-03 | Otsuka Pharmaceut Co Ltd | Preparation for injection |
| WO2004075913A1 (en) * | 2003-02-28 | 2004-09-10 | Chugai Seiyaku Kabushiki Kaisha | Stabilized preparation containing protein |
| JP2005511520A (en) * | 2001-10-05 | 2005-04-28 | ビスカム・アーゲー | Stable lyophilized crude drug formulation of recombinant carbohydrate-binding polypeptide |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03139280A (en) * | 1989-10-20 | 1991-06-13 | Washington Univ | Modified heparin cofactor 2 |
| WO1994022471A1 (en) * | 1993-04-05 | 1994-10-13 | The Green Cross Corporation | Liquid antithrombin iii preparation and method of stabilizing the same |
| JPH09176040A (en) * | 1995-12-27 | 1997-07-08 | Green Cross Corp:The | Pharmaceutical use of heparin cofactor II |
| JPH09286797A (en) * | 1996-04-18 | 1997-11-04 | Green Cross Corp:The | Heparin cofactor II and purification method thereof |
-
1999
- 1999-03-05 WO PCT/JP1999/001102 patent/WO1999045953A1/en active Application Filing
- 1999-03-05 AU AU27463/99A patent/AU2746399A/en not_active Abandoned
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03139280A (en) * | 1989-10-20 | 1991-06-13 | Washington Univ | Modified heparin cofactor 2 |
| WO1994022471A1 (en) * | 1993-04-05 | 1994-10-13 | The Green Cross Corporation | Liquid antithrombin iii preparation and method of stabilizing the same |
| JPH09176040A (en) * | 1995-12-27 | 1997-07-08 | Green Cross Corp:The | Pharmaceutical use of heparin cofactor II |
| JPH09286797A (en) * | 1996-04-18 | 1997-11-04 | Green Cross Corp:The | Heparin cofactor II and purification method thereof |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005511520A (en) * | 2001-10-05 | 2005-04-28 | ビスカム・アーゲー | Stable lyophilized crude drug formulation of recombinant carbohydrate-binding polypeptide |
| JP2003342193A (en) * | 2002-05-30 | 2003-12-03 | Otsuka Pharmaceut Co Ltd | Preparation for injection |
| JP4610154B2 (en) * | 2002-05-30 | 2011-01-12 | 大塚製薬株式会社 | Injectable preparation |
| WO2004075913A1 (en) * | 2003-02-28 | 2004-09-10 | Chugai Seiyaku Kabushiki Kaisha | Stabilized preparation containing protein |
| JPWO2004075913A1 (en) * | 2003-02-28 | 2006-06-01 | 中外製薬株式会社 | Protein-containing stabilized preparation |
| AU2004216298B2 (en) * | 2003-02-28 | 2009-04-23 | Chugai Seiyaku Kabushiki Kaisha | Stabilized protein-containing formulations |
| US8765124B2 (en) | 2003-02-28 | 2014-07-01 | Chugai Seiyaku Kabushiki Kaisha | Stabilized preparation containing protein |
| US9968677B2 (en) | 2003-02-28 | 2018-05-15 | Chugai Seiyaku Kabushiki Kaisha | Stabilized protein-containing formulations |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2746399A (en) | 1999-09-27 |
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