JPH0859506A - Preparation containing modified antithrombin - Google Patents

Abstract

PURPOSE: To obtain a modified antithrombin-containing preparation stabilized for a long period. CONSTITUTION: The modified antithrombin-containing preparation contains a modified antithrombin and one or more substances selected from the group consisting of organic acid salts, saccharides, amino acids and sodium chloride. The organic acid salt is preferably sodium succinate or sodium citrate. The saccharide is preferably D-mannitol, lactose, glucose and D-sorbitol. The amino acid is preferably glycine, sodium glutamate and sodium alginate. These substances are used in amounts of 0.1-50 pts.wt., preferably 0.5-15 pts.wt., respectively, per pt.wt. of the modified antithrombin. The modified antithrombin is preferably an antithrombin in which one or more amino acids are different from those of a human antithrombin, especially a human antithrombin III in whose amino sequence the No.132-arginine, the No.133 lysine, the No.391 alanine and the No.392 glycine are converted into glutamic acid, asparaginic acid, phenylalanine and proline, respectively.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a preparation containing modified antithrombin.

[0002]

BACKGROUND OF THE INVENTION and Prior Art The hemostatic mechanism in which thrombin acts in the blood coagulation system to form a thrombus is well known. Thrombin normally exists in the body as inactive prothrombin, and active thrombin does not cause immediate blood coagulation. However, in blood coagulation disorder syndrome (DIC) and the like, prothrombin activity is abnormally increased in the body, blood coagulation occurs in the body, and thrombus occurs. Therefore, an antithrombin action can be reinforced to treat a blood coagulation disorder such as DIC.
With regard to human-derived antithrombin separated and purified from human blood, Japanese Patent Application Laid-Open No. 59-7693 discloses a preparation and a method for producing the same. On the other hand, human-derived antithrombin is extremely difficult to purify because it is extracted from human serum, and the amount of purification is limited. In recent years, with the development of biotechnology, human-derived antithrombin has also been able to be produced by applying genetic recombination technology, but with it, it has also become possible to produce modified antithrombin having a different amino acid composition from human-derived antithrombin. It was

[0003]

[Problems to be Solved by the Invention] Modified antithrombin is different from human-derived antithrombin in activity, physicochemical properties, etc., and therefore formulation thereof is more difficult than human-derived antithrombin. The inventors of the present invention have conducted intensive research into the formulation of modified antithrombin, and as a result, have found that a modified antithrombin formulation stabilized for a long period of time can be produced by the following method, and completed the present invention.

[0004]

The present invention comprises a modified antithrombin and a modified antithrombin containing one or more substances selected from the group consisting of organic acid salts, saccharides, amino acids and sodium chloride. It is a thrombin-containing preparation. The present invention is further a freeze-dried preparation containing modified antithrombin and one or more substances selected from the group consisting of organic acid salts, sugars, amino acids and sodium chloride.

The modified antithrombin according to the present invention is not particularly limited as long as it differs from human antithrombin by at least one amino acid, and is specifically described in, for example, JP-A-5-339292. It can be manufactured by the disclosed method. Particularly preferred modified antithrombin is a modified antithrombin in which 125th lysine in the amino acid sequence of human antithrombin III is converted to glutamic acid, 391st alanine is converted to phenylalanine, and 392nd glycine is converted to proline. Of the amino acid sequence of thrombin III, modified antithrombin in which arginine at position 132 is converted to glutamic acid, lysine at position 133 is aspartic acid, alanine at position 391 is phenylalanine, and glycine at position 392 is proline, or human Antithrombin III
Of the amino acid sequence of No. 132, arginine is glutamic acid, No. 391 alanine is phenylalanine,
A modified antithrombin in which glycine at position 392 is converted to proline is most preferable, and among the amino acid sequences of human antithrombin III, arginine at position 132 is glutamic acid, lysine at position 133 is aspartic acid, and 391st position is 391st. Is a modified antithrombin in which phenylalanine is converted to phenylalanine and glycine at 392 is converted to proline.

Preferred examples of the organic acid salt in the present invention include sodium succinate and sodium citrate. Further, preferred examples of the saccharide in the present invention include D mannitol, lactose, glucose, D sorbitol and the like, and the amino acids include glycine, sodium glutamate and sodium aspartate as preferred examples. You can Further, in the present invention, these substances may be used alone or in combination of two or more.

The amount of the modified antithrombin in the present invention is not particularly limited, but is generally 1 per 1 vial.
~ 500 mg, preferably 2-300 mg. Organic acid salts, sugars, amino acids and / or
Alternatively, the amount of sodium chloride used with respect to the modified antithrombin is generally 0.1 to 50 parts by weight, preferably 0 to 50 parts by weight each of an organic acid salt, a saccharide, an amino acid and / or sodium chloride, relative to 1 part by weight of the modified antithrombin. ．
It is 2 to 20 parts by weight, more preferably 0.5 to 15 parts by weight. When two or more of these substances are used, it is desirable that the total amount be within the above range.

The pH of the solution according to the present invention is 7-10, more preferably 7-9. The substance for adjusting the pH is not particularly limited, but sodium hydroxide solution, hydrochloric acid solution and the like are often used.

The method for producing the solution or freeze-dried preparation according to the present invention can be based on a generally used method. For example, an organic acid salt, a saccharide, an amino acid and / or sodium chloride is added to the modified antithrombin, and distilled water for injection is added to dissolve the antithrombin, the pH is adjusted, the bacteria are filtered, and the mixture is dispensed into a vial bottle. To obtain a freeze-dried preparation, the dispensed vial bottle is frozen, dried under reduced pressure, and sealed. When the freeze-dried preparation is used, it is dissolved in distilled water for injection, physiological saline, etc. before use.

[0010]

[Effect] According to the present invention, the modified antithrombin, which is a physiologically active substance, is stably maintained in a solution, and when freeze-dried, it is stable even during long-term storage.

Experimental Example 1 2400 mg of modified antithrombin created by biotechnology (In the amino acid sequence of human antithrombin III, arginine at position 132 was glutamic acid, lysine at position 133 was aspartic acid, and position 391 was 391). Alanine to phenylalanine and glycine at position 392 converted to proline (modified antithrombin: hereinafter referred to as modified antithrombin A) are dissolved in 120 mL of distilled water to prepare an aqueous solution, and 60 mL of the solution contains 2Na succinate. 1000 mg was added. The aqueous solution was heated at 55 ° C. for 30 minutes and the activity was compared with that of the non-additive solution. The activity of the sodium citrate-added aqueous solution did not decrease, and the activity of the non-addition aqueous solution decreased. The modified antithrombin activity was measured according to the following documents. Reiko Honma, "Antithrombin titration method", Im
munohaemetology, 1987 (9), pp341-347.

[Experimental Example 2] Modified antithrombin A2
Dissolve 400 mg in 120 mL of distilled water to prepare an aqueous solution, and add 60 mL of the solution to 2 m of succinate 2Na.
4.5 g of g, D mannitol was added. The aqueous solution was heated at 55 ° C. for 30 minutes and the activity was compared with that of the non-additive solution. No activity loss was observed in the aqueous solution containing sodium citrate and D-mannitol, and the activity was decreased in the aqueous solution without addition.

[Experimental Example 3] Modified antithrombin A1
An aqueous solution of mg / mL was prepared, 1 mL of each of the following additives was added in an amount of 50 mg, and the mixture was heated at 55 ° C. for 30 minutes to compare the activity with the additive-free solution. The effect of the stabilizer was judged as × when the residual activity ratio was equal to or less than that of no addition, and as ○ when the improvement tendency was observed.

[0014]

[Table 1]

[Experimental Example 4] Modified antithrombin A1
An aqueous solution of mg / mL was prepared, and 1 mL of each of the following additives (50 mg) was lyophilized to obtain a powder solid. After heating at 45 ° C. for 1 month, the activity was compared with the non-addition solution. The effect of the stabilizer was judged as × when the residual activity ratio was equal to or less than that of no addition, and as ○ when the improvement tendency was observed.

[0016]

[Table 2]

[0017]

Example 1 2400 mg of modified antithrombin A was dissolved in 120 mL of distilled water to prepare an aqueous solution, and 60 mL of this solution to which 1000 mg of 2Na succinate was added was freeze-dried to obtain a powder solid. After heating at 45 ° C. for 1 month, the activity was compared with the non-addition solution. When the residual activity ratio was compared with that without addition, the residual activity ratio of the additive-free lyophilized preparation sample was 50% or less. On the other hand, 100% of the freeze-dried powder solid added with 2Na succinate remained and no activity decrease was observed.

[0018]

Example 2 2400 mg of modified antithrombin A was dissolved in 120 mL of distilled water to prepare an aqueous solution, and 60 mL of the solution to which 1000 mg of 2Na succinate and 4.5 g of D mannitol were added were freeze-dried. It was a powder solid. After heating at 45 ° C. for 1 month, the activity was compared with the non-addition solution. When the residual activity ratio was compared with that without addition, the residual activity ratio of the additive-free lyophilized preparation sample was 50% or less. On the other hand, 100% of the freeze-dried powder solid to which 2Na succinate and D-mannitol were added remained, and no decrease in activity was observed.

[0019]

Example 3 1200 mg of modified antithrombin A was dissolved in 60 mL of distilled water to prepare an aqueous solution, and solution 3 thereof was prepared.
What added 1000 mg of 3Na citrates to 0 mL was freeze-dried and it was set as the powder solid. After heating at 45 ° C. for 1 month, the activity was compared with the non-addition solution. When the residual activity ratio was compared with that without addition, the residual activity ratio of the additive-free lyophilized preparation sample was 50% or less. On the other hand, when 1000 mg of 3Na citrate was added, 100% of the freeze-dried powder solid remained and no activity decrease was observed.

[0020]

[Example 4] Modified antithrombin A (1200 mg) was dissolved in 60 mL of distilled water to prepare an aqueous solution.
What added 500 mg of 2Na citrate and 4.5 g of D mannitol to 0 mL was freeze-dried and it was set as the powder solid. After heating at 45 ° C. for 1 month, the activity was compared with the non-addition solution. When the residual activity ratio was compared with that without addition, the residual activity ratio of the additive-free lyophilized preparation sample was 50% or less. On the other hand, 100% of the powder solid obtained by freeze-drying the one to which 3Na citrate and D mannitol had been added remained, and no decrease in activity was observed.

Continuation of front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical display area A61K 47/02 J 47/12 J 47/16 J 47/26 J A61K 9/14 L

Claims (5)

[Claims] 1. A modified antithrombin and an organic acid salt,
A modified antithrombin-containing preparation comprising one or more substances selected from the group consisting of sugars, amino acids and sodium chloride. 2. The modified antithrombin-containing preparation according to claim 1, wherein the organic acid salts are sodium succinate and sodium citrate. 3. The modified antithrombin-containing preparation according to claim 1, wherein the saccharides are D mannitol, lactose, glucose and D sorbitol. 4. The modified antithrombin-containing preparation according to claim 1, wherein the amino acids are glycine, sodium glutamate and sodium aspartate. 5. A modified antithrombin and an organic acid salt,
A freeze-dried preparation containing one or more substances selected from the group consisting of sugars, amino acids and sodium chloride.