WO1994022471A1 - Liquid antithrombin iii preparation and method of stabilizing the same - Google Patents
Liquid antithrombin iii preparation and method of stabilizing the same Download PDFInfo
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- WO1994022471A1 WO1994022471A1 PCT/JP1994/000563 JP9400563W WO9422471A1 WO 1994022471 A1 WO1994022471 A1 WO 1994022471A1 JP 9400563 W JP9400563 W JP 9400563W WO 9422471 A1 WO9422471 A1 WO 9422471A1
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- 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
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- 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
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- 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
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- 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
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- 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
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- 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
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- 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
Definitions
- the present invention relates to a liquid preparation of antithrombin-III which is stable in long-term storage and a method of stabilizing a liquid preparation of antithrombin-III.
- Antithrombin-III (hereinafter referred to as AT-III) is a type of glycoprotein belonging to ⁇ : globulin present in plasma and has a molecular weight of 65.000 to 68.000.
- ⁇ - ⁇ has a protease inhibitory activity, has a strong inhibitory effect on thrombin clotting activity, and also has an inhibitory effect on other blood clotting factors, activated factor X, activated factor IX, etc. I have. In addition, it has been reported that it has an inhibitory effect on plasmin-tribusin. It is known that these inhibitory effects generally progress more rapidly in the presence of heparin.
- AT-III which has such pharmacological actions, is used for correcting hypercoagulability, specifically for treating generalized vascular abnormalities (DIC).
- DIC generalized vascular abnormalities
- AT-III has poor stability in the dissolved state, and may cause side effects in intravenous administration due to polymerization. Therefore, AT-III has been formulated in the form of lyophilization.
- liquid preparations can be easily administered without the need to dissolve in distilled water for injection at the time of use as compared with dry preparations, and they are economical in production because they do not require lyophilization in the production process.
- AT-III is inferior in stability in a solution state, and the practical application of AT-III liquid preparation has been delayed.
- AT- presence of III f heparin merely confirmed and in solution 4 e C 7 days can be stored Attako (JP 55- No. 103463).
- AT-III After extensive research into the stabilization of AT-III, the stability of AT-III in solution was improved by adding an organic acid or its salt, a sugar sulfate or a surfactant as a stabilizer. Was significantly improved, and AT-III was found to be stable even during long-term storage. In addition, the present inventors have found that AT-III is extremely stable in a solution having a pH of 9 to 10 without adding a stabilizer. Further, the present inventors have found that the AT-II liquid preparation thus prepared has no problem in pharmacological effects and safety in clinical terms, and completed the present invention. In particular, AT-III has poor stability in a solution of pH 7 to 8, which is a preferable pH range for an injection, but by adding the above compound as a stabilizer, AT-III in a solution of pH 7 to 8 can be obtained. Is significantly improved.
- a liquid preparation of AT-II I which comprises AT-111 and an organic acid or a salt thereof,
- AT-III liquid preparation characterized by containing AT-III and a surfactant
- a method for preserving and stabilizing AT-III liquid preparations comprising adding an organic acid or a salt thereof as a stabilizer to AT-11 liquid preparations,
- a method for preserving and stabilizing an AT-III liquid preparation comprising adding a sugar sulfate as a stabilizer to the AT-III liquid preparation and adjusting the pH to 7 to 10,
- AT-III a method for preserving and stabilizing a liquid formulation comprising adjusting the pH of a liquid formulation to pH 9 to 10;
- AT-II A method for preserving and stabilizing AT-II liquid formulations, which comprises adding a surfactant to the liquid formulation.
- FIG. 1 shows the results of Experimental Example 3 in which the stability of AT-III in an aqueous solution prepared by dissolving the AT-III dry preparation was compared with the stability of AT-III in the liquid preparation of the present invention. It is a graph.
- the AT-II used in the present invention is not particularly limited as long as it is derived from human and purified to the extent that it can be used as a medicament.
- human whole blood, plasma, serum or coagulation It can be purified from serum squeezed from the extracted blood.
- the blood used is preferably one that is particularly negative for the HBs antigen and anti-HIV antibody and has a GTP of twice or less the normal value.
- Methods for purifying AT-III from blood and plasma include, for example, JP-A-48-35017 (US Pat. No. 3,842,061), JP-B-59-7693 (US Pat. No. 4,340,589), and JP-A-11-11 No. 275600 (EP 339919) and the method disclosed in EP 551084 are exemplified.
- the AT-III liquid preparation of the present invention (1) is a liquid preparation containing AT-III and an organic acid or a salt thereof, preferably a liquid preparation in which the organic acid is dibasic acid or citric acid. is there. More preferably, it is a liquid preparation containing AT-III, dibasic acid or a salt thereof, and citric acid or a salt thereof.
- the organic acid refers to a compound having at least one, preferably one to three, carboxyl groups (one COOH) in a molecule.
- Monobasic acid, dibasic acid and tribasic acid refer to compounds having one, two or three carboxyl groups, respectively.
- the organic acid used in the present invention may be any of aliphatic or aromatic, saturated or unsaturated, monobasic acid (monocarboxylic acid), dibasic acid (dicarboxylic acid) and tribasic acid (tricarboxylic acid). Preferred are those having 2 to 10 carbon atoms, more preferably 2 to 6.
- Monobasic acids include saturated aliphatic monocarboxylic acids such as acetic acid, propionic acid, butyric acid, and valeric acid, and glycine.
- Examples include monobasic amino acids such as, alanine, norin, leucine, isoleucine, etc.
- dibasic acids examples include saturated aliphatic dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, glutaric acid, and adipic acid Amides of unsaturated aliphatic dicarboxylic acids such as maleic acid, fumaric acid, aromatic dicarboxylic acids such as phthalic acid, dibasic acids such as aspartic acid and glutamic acid Examples include hydroxy acids of dibasic acids such as acid, malic acid, tartaric acid, etc. Examples of tribasic acids include hydroxy acids of tribasic acid such as citric acid, preferably malic acid, tartaric acid, maleic acid , Aspartic acid and cuenic acid.
- organic acids may be in the form of a salt.
- the salt of an organic acid include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt, and organic salts such as ammonium salt. Preferably, it is made with sodium salt.
- the organic acid salts used in the present invention are more preferably sodium malate and sodium citrate.
- the organic acid it is preferable to use a combination of a dibasic acid or a salt thereof and citric acid or a salt thereof. More preferred is a combination of a dibasic acid or a salt thereof and a citrate salt.
- the citrate include sodium citrate, Alkali metal salts such as potassium salts, magnesium salts of citrate, calcium salts and the like can be mentioned. Preferred is sodium citrate.
- ⁇ of the liquid preparation of the present invention (1) is generally pH 6 to 10, preferably pH 7 to 9, and even more preferably pH ⁇ to 8. Formulations having a pH of 7 to 8 are suitable for use as injections because pain upon injection is reduced.
- the preparation of the present invention (1) is an AT-III liquid preparation that is stable at pH 6 to 10, and is especially an AT-III liquid preparation that is stable for long-term storage even at pH 7 to 8. .
- the pH can be adjusted by a usual method, for example, using a hydroxide or an appropriate buffer as an adjusting agent.
- a hydroxide include sodium hydroxide and potassium hydroxide.
- Examples of the mild solution include a phosphate buffer, a bicarbonate buffer, and a Tris buffer.
- the AT-III liquid preparation of the present invention (1) contains AT- ⁇ at a ratio of usually 1 to 1000 units Zm1, preferably 1 to 200 units Zml, more preferably 25 to 100 units / ml. It is.
- 1 unit of AT-III refers to the amount of AT-III corresponding to the amount of AT-III contained in 1 ml of normal human plasma.
- the concentration of the organic acid or salt thereof in the liquid preparation is generally 0.1 to 10 wZv%, preferably 1 to 5 wZv%, more preferably 1 to 3 w / v% in total.
- concentration of the dibasic acid or a salt thereof is preferably 1 to 5 / v%, more preferably 1 to 3 w / v%
- concentration of citric acid or a salt thereof is preferably 0.5 to 5 wZv%, more preferably 1 to 3 wZv%.
- the AT-III liquid preparation of the present invention (2) contains AT-III and a sugar sulfate, and has a pH of? It is characterized that it is ⁇ 10.
- sugar sulfate examples include heparin, dextran sulfate and the like, and preferably heparin.
- the AT-III liquid preparation of the present invention (2) comprises AT-III 1 to 1000 units Zml, preferably 10 to 100 units Zml and heparin 1 to 1000 units Zml, preferably 10 to 100 units Zml. Contains ⁇ 100 units Zml. Examples of the composition ratio of these are 0.1 to 100 units, preferably 1 to 5 units of hetero and phosphorus per unit of AT-III.
- Examples of the pH of the liquid preparation of the present invention (2) are 10 to 10, preferably 8 to 10.
- Formulations having a pH of 8 to 10 are preferred because they have excellent storage stability in a solution of AT-III.
- the pH can be adjusted by a usual method, for example, using a hydroxide or a buffer as in the preparation of the above invention (1).
- the preparation of the present invention (2) may further contain an organic acid or a salt thereof used in the preparation of the above-mentioned invention (1) as a stabilizer.
- the amount of the organic acid or salt thereof may be referred to the description of the preparation of the invention (1).
- the AT-III liquid preparation of the present invention (3) is characterized in that the pH is 9 to 10.
- the pH is between 9.3 and 9.8.
- the pH can be adjusted by a usual method, for example, using a hydroxide or a buffer as in the preparation of the above invention (1).
- the AT-III liquid preparation of the present invention (3) contains ⁇ - ⁇ in a proportion of usually 1 to 1,000 units Zm1, preferably 1 to 200 units Zm1, more preferably 25 to 100 units Zm1. Things.
- the liquid preparation of the present invention (3) is a preparation that is stable for long-term storage even in the absence of a stabilizing agent.
- a stabilizing agent for example, citrate, citrate, amino acid
- the amount of the organic acid or salt thereof may be referred to the description of the preparation of the invention (1).
- the liquid preparation of the present invention (4) is characterized by containing AT-III and a surfactant.
- a surfactant can prevent the generation of insoluble foreign substances during storage.
- a nonionic surfactant is preferable.
- polyoxyethylene sorbitan fatty acid ester for example, trade name: Tween
- polyoxyethylene-polyoxypropylene copolymer for example, trade name: Bruronic
- polyalkylene glycol For example, polyethylene glycol, polypropylene glycol, etc.
- polyoxyethylene alkyl ether for example, trade name: Triton
- the molecular weight of these surfactants is preferably from 2,000 to 20,000.
- the fatty acid of the polyoxyethylene sorbitan fatty acid ester include fatty acids having 12 to 18 carbon atoms, such as stearic acid, palmitic acid, myristic acid, lauric acid, and oleic acid.
- a polyoxyethylene sorbitan fatty acid ester or a polyoxyethylene-polyoxybutapyrene copolymer is used.
- the AT-III liquid preparation of the present invention (4) contains AT-III in an amount of usually 1 to 1000 units Zm1, preferably 1 to 200 units Zm, more preferably 25 to 100 units Zm1. It is contained in the percentage of.
- the concentration of the surfactant is usually in the range of 0.0 l to 1 w / v%, preferably in the range of 0.01 to 0.1 w // v%, and more preferably in the range of 0.02 to 0.05 w v%.
- the osmotic pressure of the liquid preparation of the present invention is preferably the same as or close to the physiological conditions of humans and animals.
- compositions other than AT-III can be added to the liquid preparation of the present invention as long as the object of the present invention is not violated.
- liquid liquid preparation of the present invention contains additives usually used for liquid preparations, such as isotonic agents (sorbitol, mannitol, glycerin, polyethylene glycol, propylene glycol, glucose, chloride, etc.).
- isotonic agents sorbitol, mannitol, glycerin, polyethylene glycol, propylene glycol, glucose, chloride, etc.
- antiseptic disinfectants benzalkonium chloride, paraoxybenzoic acid esters, benzyl alcohol, parachloromethaxenol, chlorcresol, phenethyl alcohol, sorbic acid or its salts, thimerosal, chlorobutanol
- Chelating agents sodium edetate, condensed sodium phosphate, etc.
- thickeners boinylpyrrolidone, methylcellulose, sodium carboxymethylcellulose, hydroxybutyl cellulose, polyvinyl alcohol, polyacrylic acid May be blended tri ⁇ beam, etc.
- Sugar may be further added to the liquid preparation of the present invention as a co-stabilizer.
- the sugar used in the present invention include monosaccharides, disaccharides, sugar alcohols, amino sugars and the like.
- Monosaccharides include glucose, fructose, galactose, mannose, arabinose, and inositol
- disaccharides include saccharose and rata.
- sugar alcohol include mannitol, sorbitol and xylitol.
- the amino sugar include glucosamine and N-acetyl-D-glucosamine which is an amino sugar derivative.
- saccharose lactose
- sorbitol inositol
- maltose N-acetyl-D-glucosamine
- mannitol Preferred are saccharose, lactose, sorbitol, inositol, maltose, N-acetyl-D-glucosamine, and mannitol.
- the concentration is usually 0.1 to 40 wZv%, preferably 0.5 to 20 wZv%, more preferably 5 to 10 wZv%.
- a surfactant may be further added to the liquid preparations of the present invention (1) to ()) as a co-stabilizer.
- the addition of a surfactant can prevent the generation of insoluble foreign matter during storage.
- a nonionic surfactant is preferable, for example, a polyoxyethylene sorbitan fatty acid ester (for example, trade name: Tween), a boro- oxyethylene-polyoxydipropylene copolymer (for example, trade name: Pull Mouth Nick).
- examples thereof include polyalkylene glycol (eg, polyethylene glycol, polypropylene glycol, etc.), polyoxyethylene alkyl ether (eg, trade name: triton) and the like.
- the molecular weight of these surfactants is preferably from 2,000 to 20,000.
- the fatty acid of the polyoxyethylene sorbitan fatty acid ester include fatty acids having 12 to 18 carbon atoms, such as stearic acid, amino acid, lumitic acid, myristic acid, lauric acid, and oleic acid.
- a surfactant When a surfactant is incorporated, its concentration is usually 0.0 l to lwZv%, preferably 0.01 to 1 wZv%, more preferably 0.02 to 0.05 wZv%.
- the liquid preparation of the present invention may further contain other stabilizers.
- other stabilizers for example, inorganic salts, albumin, abrotinin, ethylenediaminetetraacetic acid (EDTA) or a salt thereof and the like can be mentioned.
- the inorganic salt is not particularly limited, but examples thereof include sodium chloride, potassium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, and the like. Is done.
- the liquid preparation of the present invention includes AT-III dissolved in water together with other components.
- AT-III dissolved in water together with other components.
- examples of the embodiment include injections and infusions.
- examples of the water to be dissolved include distilled water for injection and sterilized purified water.
- the liquid preparation of the present invention can be prepared by a means known per se, depending on the type of the liquid preparation. If desired, treatments such as heat treatment and sterilization filtration can be performed.
- the liquid preparation of the present invention thus prepared can be stored at a temperature of usually 4 to room temperature for a long period of time. Usually, it can be stored for at least 2 years below 10'C and for at least 6 months at room temperature. Preferably, the storage is 1 O'C or less.
- the AT-III suspension preparations of the present invention typically contain at least 80% of the AT-111 activity in the preparation of the formulation, even after storage for at least 6 months at 25 and more preferably at least 3 years at 4. , Preferably 90% or more.
- the liquid preparation of the present invention is useful for the treatment of general intravascular coagulation syndrome (DIC) accompanied by a tendency of thrombus formation due to congenital AT- ⁇ ⁇ ⁇ deficiency and a decrease in AT-III.
- the administration method of the present drug can be in accordance with the conventional formulation of AT-III injection or infusion, and examples include a method of slowly injecting the liquid preparation of the present invention or intravenous drip.
- the preparation of the present invention is administered at a rate of 1,000 to 3,000 units per day (or 20 to 60 units Zkg). It may be increased or decreased.
- this product When this product is used as an emergency treatment in obstetrics, surgical DIC, etc., it is preferable to administer 40 to 60 units / kg once a day.
- the AT- ⁇ liquid formulation of the present invention comprising AT-IU and a stabilizer (organic acid or salt thereof, sugar sulfate or surfactant), the AT- during the heat treatment and during long-term storage can be obtained.
- a stabilizer organic acid or salt thereof, sugar sulfate or surfactant
- AT-III can be stably maintained in a liquid state for a long period of time even at pH 7 to 8 where it has been difficult to maintain the activity of AT-II in the conventional preparation.
- the AT-III liquid preparation of the present invention having a pH in the range of 9 to 10, the AT-III activity during heat treatment and during long-term storage is reduced and polymerization is prevented even without the presence of a stabilizer. be able to.
- the preparation of the present invention is a liquid preparation that stably retains AT-III even during long-term storage. Therefore, the liquid preparation of the present invention can be provided as it is as a commercial product (such as an injection). Therefore, it can be administered to a patient as an injection or the like without dissolving at the time of use, so that the operation at the time of administration can be simplified and is useful for clinical use. In addition, since the freeze-drying step can be omitted in the manufacturing process, manufacturing efficiency and economy can be improved.
- AT-III liquid formulation 500 units of AT-III, 250 mg of sodium malate and 5 Omg of sodium citrate were dissolved in 5 ml of water for injection, and the pH was adjusted to 7.5 to prepare an AT-III liquid formulation.
- AT-III 500 units sodium tartrate 25 Omg.
- Sodium citrate 50 mg was dissolved in 5 ml of water for injection, and the pH was adjusted to 7.5 to obtain an AT-III liquid preparation.
- AT-IU 500 units sodium aspartate 25 Omg.
- Sodium citrate 5 Omg was dissolved in 5 ml of water for injection, and the pH was adjusted to 7.5 to prepare an AT-III liquid preparation.
- Example 5 500 units of AT-III, 250 mg of sodium malate, and 150 mg of sorbitol were dissolved in 5 ml of water for injection, and the pH was adjusted to 8.0 to obtain an AT-III liquid preparation.
- AT-III liquid preparation 500 units of AT-III, 25 Omg of sodium malate, 250 m of sorbitol and 5 Omg of sodium citrate were dissolved in 5 ml of water for injection, and the pH was adjusted to 7.0 to prepare an AT-III liquid preparation.
- AT- [II (dried formulation, trade name Neuart, manufactured by Green Cross) 500 units and heparin 1000 units were dissolved in an appropriate amount of water for injection to make a total volume of 2 Oml.
- the pH was adjusted to 8 to give an AT-III liquid formulation.
- AT-III 500 units sodium chloride 5 Omg.
- Sodium citrate 52 mg, mannitol 20 Omg and heparin 500 units were dissolved in water for injection 2 Om1 and the pH was adjusted to 8 to prepare AT-III liquid preparation.
- AT-III dry product, trade name Neuart, manufactured by Green Cross
- AT-III dry formulation, trade name Neuart, manufactured by Green Cross 500 units
- saccharose 20 Omg is dissolved in 1 Oml of water for injection
- pH is adjusted to 9.3
- AT-III dry product, trade name Neuart, manufactured by Green Cross 500 units, 5 Omg of ninitol and 5 Omg of sodium citrate were dissolved in 10 ml of water for injection, and the pH was adjusted to 9.8 to prepare an AT-III liquid preparation.
- AT-III liquid preparation 500 units of AT-III, 250 mg of sodium citrate and 500 mg of saccharose were dissolved in 5 ml of water for injection, and the pH was adjusted to 7.5 to prepare an AT-III liquid preparation.
- AT-III 500 units sodium malate 150 mg, citrate 150 mg, saccharose 25 Omg, polyoxyethylene-polyoxypropylene copolymer (trade name Bull Mouth Nick PF 68) 0.5 mg dissolved in 5 ml of water for injection The pH was adjusted to 7.5 to obtain an AT-III liquid preparation.
- AT-III activity was measured using a ⁇ - ⁇ activity assay kit (Test Team AT-III ⁇ 2 kit: manufactured by Daiichi Pure Chemicals), and the AT-III polymerization was measured. The percentage was determined by HPLC (High Performance Liquid Chromatography) analysis.
- Sample diluent 50 // 1 C2.
- Human thrombin solution (0.9% sodium chloride, 0.05% ⁇ serum albumin (BSA), 0.05% polyethylene glycol (PEG) 6000, 1 U ml thrombin) 100 // 1
- BSA serum albumin
- PEG polyethylene glycol
- the synthetic substrate solution S-2238: HD-Fenralara L-Pipecoli L-Arginyl-p-Nitroanilide 'dihydrochloride
- the reaction was stopped by adding 1 ml of citrate solution, and the absorbance at a wavelength of 405 nm was measured with a spectrophotometer.
- Normal human plasma (1U AT-III Zml) was measured at the same time as the measurement sample, and the AT-III content of the sample was measured from the calibration curve.
- AT-III activity shows a residual rate of 99.9% or more.
- AT-III solution (AT-III titer: 80.4 UZm 1, containing 3w / v% or 5w / v% of various stabilizers (sodium aspartate, sodium glutamate, sodium tartrate, DL-sodium malate) pH 7.5) was prepared, and the stability of AT-III during heating for 1 to 10 hours was measured over time, and the results were compared. Table 2 shows the results.
- AT-III In an aqueous solution of AT-III dry preparation, AT-III is inactivated to 18% by heating at 55 eC for 1 hour, whereas in a solution containing a stabilizer, AT-III activity is about 70%. ⁇ 100% remained. In particular, when 1 wZv% sodium citrate and 3 wZv% DL-sodium malate were added, the ratio was 55, and the AT-III activity remained over 80% even after heating for 10 hours. (Aqueous solution of (dried formulation)), the stabilizing effect was increased about 60 times or more.
- AT-III titer 1 13.4 U nom1
- AT-III titer 1 13.4 U nom1
- the AT-III activity (titer) of the sample before the heat treatment was set to 13.4 UZml of 100%, and the residual ratio of AT-III activity in each sample was determined.
- AT-III solution AT-III titer: 85, 9 U / m 1, H7.5
- various stabilizers sodium aspartate, sodium glutamate, sodium tartrate, DL-sodium malate, glycine or sodium acetate
- the stabilizing effect of various stabilizers and 0.5 wZv% sodium citrate on AT-III was investigated by measuring the degree of polymer formation using HPLC analysis. Table 5 shows the results.
- Non-heating ⁇ When a combination of dibasic acid and citric acid was used as a stabilizer, polymerization of AT-111 was suppressed, and a high stabilizing effect was observed.
- AT-III lOUZml 3.5 wZv% sodium citrate was added to the AT-III solution (AT-III lOUZml), and the pH was adjusted to 7.5 to prepare an AT-ill liquid preparation. After storing this formulation for 1 week at 40 or for 1 month at 25, the AT-III activity was measured to determine the residual activity. Table 7 shows the results.
- the stabilizer described in Table 9 was added to the AT-III solution (AT-III 100 U / m 1), and the pH was adjusted to 7.5 to prepare an AT-III liquid preparation. After storing these preparations at 4 or 25 for 1 month, AT-III activity was measured to determine the residual activity. Table 9 shows the results.
- the relationship between the pH of the AT-III liquid preparation of the present invention and the stability of AT-III in the preparation is n '.
- AT-III liquid preparations (AT-II titer: 25 units Zm 1) adjusted to pH 6, 7, 8, 9, and 10 were prepared, respectively, and heat-treated at 50 for 30 minutes each. After that, the AT-III activity remaining ratio of each AT-III liquid preparation was examined, and The stability of AT-III was compared.
- AT-III was very unstable in the pH-6 AT-III liquid formulation.
- the residual activity of AT-II was 52%, 75%, 88 and 92%, and the higher the pH in the range of 9-10, the higher AT_II It was confirmed that I was extremely stable.
- the AT-III liquid preparation (AT-III titer: 25 units Zm 1) adjusted to each pH was heated at 55 ° C for 30 minutes and then each AT-III liquid preparation Of the AT-III activity was examined.
- AT-III was very unstable at pH6.
- the pH increased to 7, 8, 9 and 10
- the residual AT-III activity increased to 24%, 45%, 69% and 84%. It was confirmed that III was stable.
- This precipitate was dissolved in about 20 ⁇ of cold saline and injected into a column of heparin sepharose prepared in advance with saline to adsorb AT-III to the column.
- the column was washed with a 0.4 M sodium chloride solution to remove impure proteins, and a 2.0 M sodium chloride solution was passed through the column to collect the eluted portion.
- aqueous solution was added sodium citrate to a concentration of 0.6 M, adjusted to pH 7.8, and then subjected to a heat treatment at 60 for 10 hours, followed by sodium chloride (final concentration 3 M) and quinone.
- Sodium acid final concentration 20 mM Adjusted to 7.5.
- an aqueous solution containing AT-III was brought into contact with a butyl-type polyvinyl carrier (Putiltoyopar 650, manufactured by Toyo Soda Co., Ltd.) equilibrated with 2 OmM sodium citrate buffer (pH 7.5) containing 3 M sodium chloride. The mixture was developed with the above buffer solution, and the unadsorbed fraction was recovered. Subsequently, a 0.5% sodium citrate buffer solution (pH 7.5) was dialyzed overnight to obtain purified AT-III.
- a butyl-type polyvinyl carrier Putiltoyopar 650, manufactured by Toyo Soda Co., Ltd.
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP94910601A EP0653212A4 (en) | 1993-04-05 | 1994-04-05 | ANTITHROMBIN III COMPOSITION IN LIQUID FORM AND METHOD FOR STABILIZING IT. |
KR1019940704443A KR100330540B1 (ko) | 1993-04-05 | 1994-04-05 | 안티트롬빈-iii액상제제및그안정화방법 |
US08/351,268 US5589516A (en) | 1993-04-05 | 1994-04-05 | Liquid preparation of antithrombin-III and stabilizing method therefor |
JP52193194A JP3711561B2 (ja) | 1993-04-05 | 1994-04-05 | アンチトロンビン−▲iii▼液状製剤およびその安定化方法 |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP10011793 | 1993-04-05 | ||
JP5/100117 | 1993-04-05 | ||
JP5/328205 | 1993-12-24 | ||
JP32820593 | 1993-12-24 | ||
JP6/5073 | 1994-01-21 | ||
JP507394 | 1994-01-21 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1994022471A1 true WO1994022471A1 (en) | 1994-10-13 |
Family
ID=27276580
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP1994/000563 WO1994022471A1 (en) | 1993-04-05 | 1994-04-05 | Liquid antithrombin iii preparation and method of stabilizing the same |
Country Status (5)
Country | Link |
---|---|
US (1) | US5589516A (ja) |
JP (1) | JP3711561B2 (ja) |
KR (1) | KR100330540B1 (ja) |
CA (1) | CA2137258A1 (ja) |
WO (1) | WO1994022471A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999045953A1 (fr) * | 1998-03-09 | 1999-09-16 | Welfide Corporation | Compositions contenant un cofacteur ii de l'heparine et techniques de stabilisation |
JP2011001273A (ja) * | 2009-06-16 | 2011-01-06 | Eci Inc | eMIPを有効成分とする水溶性製剤 |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6491965B1 (en) | 1995-11-30 | 2002-12-10 | Hamilton Civic Hospitals Research Development, Inc. | Medical device comprising glycosaminoglycan-antithrombin III/heparin cofactor II conjugates |
US6562781B1 (en) * | 1995-11-30 | 2003-05-13 | Hamilton Civic Hospitals Research Development Inc. | Glycosaminoglycan-antithrombin III/heparin cofactor II conjugates |
US7045585B2 (en) * | 1995-11-30 | 2006-05-16 | Hamilton Civic Hospital Research Development Inc. | Methods of coating a device using anti-thrombin heparin |
DE69911629D1 (de) | 1998-10-08 | 2003-10-30 | Childrens Hospital Boston | Zusammensetzungen und deren verwendung zur hemmung von angiogenese |
US20110082083A1 (en) * | 2009-04-10 | 2011-04-07 | Gtc Biotherapeutics, Inc. | Formulations of liquid stable antithrombin |
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JP2011001273A (ja) * | 2009-06-16 | 2011-01-06 | Eci Inc | eMIPを有効成分とする水溶性製剤 |
Also Published As
Publication number | Publication date |
---|---|
CA2137258A1 (en) | 1994-10-13 |
US5589516A (en) | 1996-12-31 |
KR100330540B1 (ko) | 2002-09-04 |
JP3711561B2 (ja) | 2005-11-02 |
KR950701819A (ko) | 1995-05-17 |
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