WO1994015631A1

WO1994015631A1 - Dry plasminogen preparation

Info

Publication number: WO1994015631A1
Application number: PCT/JP1994/000019
Authority: WO
Inventors: Takashi Kobayashi; Shinobu Motizuki
Original assignee: The Green Cross Corporation
Priority date: 1993-01-11
Filing date: 1994-01-10
Publication date: 1994-07-21
Also published as: JPH06211691A

Abstract

A dry plasminogen preparation more efficacious in treating, for example, various thromboses than before. The preparation comprises either plasminogen and a nonionic surfactant or plasminogen, sucrose, an amino acid and albumin. Even after being left to stand under severe conditions, the preparation neither becomes turbid nor generates filamentous substances when dissolved in an aqueous solvent, and retains its activity after the dissolution. Further it has such an excellent long-term stability after being lyophilized that it is more safe as a medical preparation than the conventional ones and is practicable.

Description

Description Plasminogen dry preparation

Technical field

The present invention relates to an improvement in a plasminogen preparation. More specifically, the present invention relates to a plasminogen dry preparation in which the solubility of the plasminogen dry preparation in water is improved. Background art

Plasminogen is activated by perokinase and the like to become plasmin, which dissolves fibrin and causes fibrinolysis. Plasminogen is used not only for the study of fibrinolysis phenomena, but also in clinical applications as a fibrinolytic therapeutic agent (thrombotic agent) in recent years.

In a plasminogen preparation, plasminogen itself is a precursor enzyme, and is stable unless plasmin or the like is not contained as a contaminant in the preparation. Plasminogen has poor solubility and dissolution in water by making it into a dry preparation.c.Applicant must first add a dry preparation of plasminogen to A plasminogen preparation to which sugar or amino acid is added as a solubilizing agent for suppressing the generation has been proposed (Japanese Patent Application Laid-Open No. Sho 62-153324).

It was found that by subjecting the plasminogen-containing composition to severe conditions (for example, treatment for virus inactivation), the solubility and dissolution of the dried preparation deteriorated.

For this reason, we have proposed an improved dry formulation using a combination of albumin, sugar and amino acid. (Japanese Unexamined Patent Publication No. 2-96653)

An object of the present invention is to provide a dry plasminogen preparation which has good solubility and dissolution in water of dried plasminogen even under severe conditions. Disclosure of the invention

The first invention of the present invention is a dried plasminogen preparation comprising plasminogen and a nonionic surfactant.

The second invention of the present invention is a dried plasminogen preparation comprising plasminogen, sucrose, amino acid and albumin.

The plasminogen dry preparation in the present invention refers to a preparation in a substantially dried state, and may contain a certain amount of water, for example, to the extent that it can be made into a powder.

The dried plasminogen preparation of the present invention contains dried plasminogen as a main component. Examples of the dried plasminogen include a powdered form, particularly, lyophilized plasminogen. Such a dried plasminogen is produced by drying a plasminogen-containing liquid by a known means, particularly by freeze-drying.

The plasminogen-containing solution is not particularly limited.For example, serum III, plasma, ascites, placental extract, placental tissue extract, and corn fraction III of the low-temperature alcohol fractionation method of animals including humans As described above, a plasminogen purified from a plasminogen-containing fraction by a widely known method, a solution containing plasminogen obtained by genetic engineering, and the like can be mentioned.

Examples of the plasminogen of the present invention include a methionyl type (the N-terminal of which is methionine), a glutamyl type (the N-terminal of which is glutamic acid), and a lysyl type (the N-terminal of which is lysine). Further, a mixture thereof may be used.

Preferred is lysyl monoplasminogen.

Representative methods for purifying plasminogen include, for example, the method described in Japanese Patent Application Laid-Open No. 55-159392 and the purification method using lysine-sepharose (Science, 170, 1095, 1970). It can be exemplified as a simple method.

It is preferable that the plasminogen preparation has been treated for virus inactivation. Examples of the virus inactivation treatment include a method of heating a liquid composition of plasminogen at 50 to 100 ° C for 5 to 30 hours, and a method of heating a dry composition at 50 to 100 ° C for 10 to 1 hour. 50 hours heating method, surfactant contact method, other chemicals such as trialkyl phosphate contact method and treatment Combinations are included.

Hereinafter, the plasminogen dry preparation according to the first invention of the present invention will be described. The solubilizer used in the first invention is a nonionic surfactant. Examples of the nonionic surfactant include polyoxyethylene sorbitan fatty acid ester (twin type), polyoxyethylene-polyoxypropylene copolymer (pull nick type) and the like.

Examples of the former fatty acids include stearic acid, panolemitic acid, oleic acid, lauric acid, stearic acid and the like. In addition, the latter has a molecular weight of about 2000 to 2000.

It is more preferable to add a saccharide and an amino acid as a solubilizing agent in addition to the nonionic surfactant.

Examples of the saccharide include monosaccharides (such as glucose and galactose), disaccharides (such as sucrose and lactose), and sugar alcohols (such as mannitol and sorbitol). Preferred saccharides are disaccharides, and sucrose is particularly preferable. It is.

Examples of the amino acids include neutral amino acids (such as glycine and alanine), acidic amino acids (such as glutamic acid and aspartic acid), and basic amino acids (such as arginine and lysine). Preferably, lysine is used.

The dried plasminogen preparation of the present invention is usually prepared by adding a solubilizer to a plasminogen solution and subjecting it to a known drying treatment.

In the present invention, the compounding amount of the solubilizer is an amount sufficient to suppress the generation of a filamentous substance when dissolving the plasminogen dry preparation of the present invention in water or the like, and specifically, It is as follows for each component.

The amount of the nonionic surfactant to be used is about 0.01 to 0.1% (W / V), more preferably 0.01 to 0.4, based on 100 to 500 CU / ml of plasminogen. % (W / V).

The amount of saccharide and amino acid to be used is preferably about 0.005% (W / V) to 10% (W / V), respectively, based on 100 to 500 CUZml of plasminogen. Or about 0.1 to 5% (WZV). The preferred weight ratio of the saccharide [especially, sucrose] (A) to the basic amino acid [especially, lysine] (B), that is, BZA is 0.001 to 0.2, and most preferably 0%. . 0

1 to 0.1.

Within the above-mentioned range of the amount of the dissolving agent to be added, the balance between the stability of the formulation, water solubility, dissolution and formulation is the best.

Next, the plasminogen dry preparation according to the second invention of the present invention will be described. The lysing agents used in the second invention are sucrose, amino acids and albumin. The amino acid used in the second invention is preferably the same as the amino acid used in the first invention.

Albumin is preferably human-derived albumin due to the problem of antigenicity, and there is no particular limitation as long as it is purified for medical use. Its purity is preferably such that at least 80% of albumin is analyzed by electrophoresis. Methods for obtaining human-derived albumin include the ethanol fractionation method (Japanese Patent Publication No. 47-2869, Japanese Patent Publication No. 35-52997), and heating in the presence of an organic acid. Methods (Japanese Patent Publication No. Sho 43-1640, Japanese Patent Publication No. Sho 51-40132) are exemplified. In particular, those obtained by heat-treating albumin (preferably heat-treating at 60 ° C for about 10 hours) and inactivating virus such as hepatitis virus are used.

The amount of sucrose and amino acid used in the present invention is about the same as the amount used in the first invention, and the amount of albumin used is 0 to 500 to 500 CU Zml of plasminogen. It is about 0.5 to 5% (WZV), more preferably about 0.25 to 1% (W / V).

Further, it is preferred that the pH of the solution of the dried plasminogen preparation of the present invention before drying is 7.2 to 7.8.

The plasminogen dry preparation of the present invention is administered after being dissolved in an aqueous solvent such as physiological saline or distilled water for injection before use. For administration, it is generally administered intraarterially or intravenously. For example, for treatment of thrombosis, 100 to 300 CU of plasminogen is administered at a time. In addition, an effective amount of excipients and the like may be added to the preparation of the present invention in accordance with customary techniques for pharmaceutical production. BEST MODE FOR CARRYING OUT THE INVENTION

Purification of plasminogen solution

Fraction obtained by cold ethanol fractionation of corn ΙΙ + ペースト

0 units Suspend in Tris-HCl buffer (H8.3) containing aprotinin of Znil and 0.1 M sodium chloride, agitate for a short time, then add 5% ammonium sulfate.'Stir and separate supernatant by centrifugation did. Further, 30% ammonium sulfate was added to the supernatant, stirred, and the precipitate was separated by centrifugation. This precipitate was suspended in a 0.9% glycine solution (pH 7.2) containing 0.9% sodium chloride and subjected to the method of Deutsch, DG et al. [Science, 170, 1095, (1970)]. Inject into a lysine-sepharose column as described above, adsorb plasminogen, and then add 1 M sodium chloride containing the impure protein.

After washing with 9% glycine solution (pH 7.2), the adsorbed plasminogen was eluted using a solvent containing 0.2Με-aminocaproic acid and 0.9% sodium chloride (ρΗ7.0). .

Example 1

The effect of adding lysine and sucrose to the plasminogen preparation was examined. Purified plasminogen (inactive is 8. lxl 0 ⁶ mg per ^{1. 9 5 X 1 0 8 cu} ) a 0.2% lysine, 5% sucrose, 0.4 5% sodium chloride containing 5 0 mM Li down The solution was dialyzed against an acid buffer (PH 7.2) and the appearance of the solution was observed. After dialysis, the solution was frozen at 130 ° C or less for at least one week, and the appearance of the solution when thawed was observed.

Example 2

The effect of PH on plasminogen preparations was investigated. Prepare a plasminogen solution of pH 6.4, 7.2, and 7.8 consisting of 0.4% resin, 5% sucrose, and 50 mM phosphate buffer, and incubate at 45 ° C for 3 hours. After one session, the appearance and residual activity of each solution were confirmed. The residual activity was measured by the synthetic substrate method according to the method of Kato et al. [J. Biochem., 88, 183, (1980)].

Table 2

(100% immediately before the incubation)

Example 3

The effect of adding sugar on the plasminogen preparation was examined. After adding 0.2% lysine and 5% sucrose or mannitol (final concentration) to the plasminogen solution and freeze-drying 61 ° C for 7 hours Dry heating → 37 ° C for 8 weeks The sample was redissolved in distilled water, and after 30 minutes, the appearance, residual activity, and polymer content were confirmed. The residual activity was measured in the same manner as in Example 2, and the polymer content was measured by the HPLC method.

Three

(Remaining activity rate is 100% before freeze-drying) Example 4

The effect of the stabilizer on the plasminogen preparation was investigated. Add a certain amount of stabilizer to a plasminogen solution consisting of 0.4% lysine, 5.0% sucrose, 50 mM sodium phosphate, and pH 7.2 to 7.8. Left for 2 weeks at ° C. After redissolving in distilled water, the appearance, residual activity ratio and polymer content were confirmed one hour later. Table 4 shows the results of using a stabilizer as a comparative example, and Table 5 shows the results of using a nonionic surfactant and albumin of the present invention as stabilizers.

Table 4

Additive Appearance Polymer Remaining

Content Activity Rate Type Final concentration Color Opaque Insoluble substance

Additive Slightly white turbid particulate Z-fiber 0.51% 107% citric acid 0.4% Slightly white turbid particle 0.70% 72% Sodium 0.2 Slightly white turbid particle 0.47 124

0.1 Slightly white granular 0.48

Hydrochloric acid 0.4% Slightly white turbid Particulate Z fibrous 0.32% 107% Sodium 0.2 Slightly white turbid Particulate Z fibrous 0.37

0.1 Slightly white turbid Particle / fibrous 0.46 111 Caprylic acid 133 mg / ml Slightly white turbid particle 1.37% 82% Sodium 66 Slightly white turbid particle 0.96 94

33 Slightly white turbid particles 0.73 90 Maltose 5.0% Yellow clear particles 2.58%

2.5 Yellow clear particle 1.43 76%

Additive Appearance Polymer Remaining

Content Activity Final concentration Color Opaque Insoluble substance

Additive Slightly white Cloudy Particulate fibrous 0.04% 95% Twin 0.04% Colorless and clear 0.25%

80 0 02 colorless clear Not observed 0.23 93%

0.01 Colorless clear Not observed 0.221 97 Poloxamer 0.04% Colorless clear Not observed 0.41%

188 0.02 Colorless clear Not observed 0.47 99%

0.01 Colorless clear Not observed 0.57 110

1.0% colorless clear Not observed 0.09% 91% albumin 0.5 Colorless clear Not observed 0.159

0.25 Colorless clear No observation 0.19 No addition * Colorless clear No observation 0.04% 1 10%

(Remaining activity rate is 100% before freeze-drying)

*: When redissolved in 0.02% Tween 80 instead of distilled water Industrial applicability

The plasminogen dry preparation of the present invention suppresses the production of filamentous substances when dissolved in water, and retains its activity even after dissolution. In addition, it has excellent stability during lyophilization, especially long-term stability, making it more safe and practical for use as a pharmaceutical formulation. Therefore, the preparation of the present invention can more effectively treat, for example, various thrombosis.

Claims

The scope of the claims

1. A dried plasminogen preparation containing plasminogen and a nonionic surfactant.

2. A plasminogen dry preparation containing plasminogen, sucrose, amino acids and albumin.

3. The dried plasminogen preparation according to claim 1 or 2, wherein the pH of the solution before drying is 7.2 to 7.8.