JP2715104B2 - Thrombin dry preparation - Google Patents

Description

The present invention relates to a stabilized dried thrombin preparation.
More particularly, it relates to a stable dry thrombin preparation comprising thrombin, a saccharide, a basic amino acid and an organic acid salt.

[Problems to be solved by the prior art and the invention]

Thrombin acts on fibrinogen to produce fibrin, thereby producing a blood clotting effect. For this reason,
Thrombin is clinically used as a local hemostat in the surgical field.

Currently, commercially available thrombin preparations are freeze-dried products, and are administered topically at the time of use as they are or dissolved in an appropriate solvent.

This thrombin is rarely administered orally. That is, in the case of upper gastrointestinal bleeding, it is administered as a thrombin solution.

By the way, as a stabilizer in the thrombin dry preparation, mannitol, sodium citrate (current thrombin preparation), albumin (JP-A-56-39782),
Aprotinin, gelatin (JP-A-56-68607), E
DTA et al. (JP-A-57-18985), collagen (JP-A
No. 60-112721) is known. However, in fact, satisfactory stabilizing effects have not always been obtained with these stabilizers.

Accordingly, it is an object of the present invention to provide a stable dried thrombin formulation.

[Means for solving the problem]

In order to solve the above-mentioned problems, the present inventors have studied stabilizing agents that contribute to the stabilization of a thrombin dry preparation, and as a result, by using a saccharide, a basic amino acid and an organic acid salt together, Can be remarkably improved, and further, the thrombin-dried preparation using the stabilizer is excellent in re-dissolvability, and the thrombin-containing solution is dried by the stabilizer (particularly freeze-drying). The present inventors have found that the stabilization at the time of performing is promoted, and completed the present invention.

The present invention relates to the use of a stabilizer comprising a saccharide, a basic amino acid and an organic acid salt for stabilizing a thrombin dry preparation.

The thrombin in the present invention may be any as long as it has biological activity or physiological activity as thrombin, and for example, thrombin obtained by fractionating plasma proteins or obtained by genetic engineering may be used. That is, for example, those prepared by allowing thromboplastin, snake venom, and the like to act on prothrombin purified from human or bovine plasma in the presence of Ca ²⁺ can be used. Alternatively, a commercially available product listed in a pharmacopeia may be used.

Also, as thrombin, its specific activity is 100-1000
It is preferable to use a protein of about unit / mg protein.

The thrombin dried preparation of the present invention is produced, for example, by drying a thrombin-containing aqueous solution as described later. In the thrombin-containing aqueous solution, the concentration of thrombin is 500 to 10,000 units / ml (particularly 1000 to 300 units).
0 unit / ml), and the pH of the solution is 5 to 10
(Especially 5.6 to 7.6) are preferred.

Preferred saccharides used as a stabilizer in the present invention include monosaccharides (glucose, mannose, etc.), disaccharides (maltose, sucrose, lactose, etc.), and sugar alcohols (sorbitol, mannitol, xylitol, etc.). And preferably disaccharide, more preferably sucrose.

The amount of sugar added is, for example, 1 to 20 w / v% (preferably 2 to 10 w / v%) per 500 to 5000 units / ml of thrombin.

Examples of the basic amino acid include arginine, lysine, histidine and the like, and particularly preferably arginine.

The amount of basic amino acid added is thrombin 500-500
1 to 10 w / v% per 0 unit / ml (preferably 1 to 5 w / v%)
It is about.

As the organic acid salt, a carboxylate having 2 to 8 carbon atoms is preferable. Such organic acid salts include fatty acid salts (eg, salts of acetic acid, propionic acid, butyric acid, valeric acid, etc.), oxyacid salts (salts of lactic acid, malic acid, tartaric acid, citric acid, etc.), dicarboxylic acid salts (eg, Oxalic acid, malonic acid,
Salts, such as succinic acid), and particularly preferably oxyacid salts, especially citrate.

Examples of the salt in the organic acid salt include an alkaline earth metal salt (such as a sodium salt and a potassium salt) and an alkali metal salt (such as a calcium salt).

The addition amount of the organic acid salt is about 0.001 to 1M (preferably 0.01 to 1M) per 500 to 5000 units / ml of thrombin.

A particularly preferred composition of the dry preparation of the present invention is, for example, about 20 to 200 mg of saccharides, about 10 to 100 mg of basic amino acids, and about 10 to 100 mg of organic acid salts per 10,000 units of thrombin.

As a method for preparing the dry preparation of the present invention, a method of drying a purified thrombin solution by a known means, specifically, dialysis, sterilization filtration, dispensing as necessary, and drying (particularly freeze-drying) It is manufactured by performing.

If desired, the thrombin may be subjected to a heat treatment.

The stabilizer of the present invention is preferably added before drying, and thereby exerts a stabilizing effect on the decomposition of thrombin by drying, especially by freeze-drying.

The dry preparation of the present invention is further prepared as powders, granules, tablets, capsules, topical preparations, and external preparations by a usual preparation technique, and is packaged in a unit.

Generally, the amount of thrombin contained in one unit
About 500 to 20,000 units.

The efficacy of the dry preparation of the present invention includes bleeding from small blood vessels, capillaries and parenchymal organs that are difficult to stop by normal ligation (for example, bleeding due to trauma, bleeding during surgery,
(E.g., osseous bleeding, bladder bleeding, bleeding after tooth extraction, epistaxis, and bleeding from the upper gastrointestinal tract).

In addition, the dosage and administration of the dry preparation of the present invention is usually sprayed or irrigated with a solution (50 to 1000 units / ml as thrombin) dissolved in physiological saline on the bleeding site, or withdrawn as powder. An example is shown below. In the treatment of upper gastrointestinal bleeding, a solution (200 to 400 units / ml as thrombin) dissolved in an appropriate buffer is orally administered. The dose may be appropriately increased or decreased depending on the site and degree of bleeding.

〔effect〕

ADVANTAGE OF THE INVENTION According to this invention, the thrombin at the time of freeze-drying and at the time of preservation | save of a formulation is rich, and the thrombin dried formulation excellent in the solubility at the time of use can be provided.

〔Example〕

EXAMPLES The present invention is described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited thereto.

Example 1 From normal human plasma, barium chloride adsorption method and DEAE-Sephadex column chromatography method [Baja, S. et al. P. Et al., Journal of Biological Chemistry (Bajaj, SP, et al., J. Biol. Chem.) 248, 77
29) Prothrombin was purified by 1973), and thromboplastin prepared from human placenta was added to this prothrombin.
Human plasma and calcium chloride solution were added, and the mixture was converted to thrombin to obtain crude thrombin (thrombin activity per 10 mg protein: 10 units). This crude thrombin was subjected to SP-Sephadex column chromatography [Randblad, R .; L., Biochemistry (Lundblad, R.
L., Biochemistry), 10 , 2501 (1971)].
After concentrating the purified thrombin, a 100 mM citrate buffer (p
Dialysis against H7.0) and thrombin solution (3500 units / ml, 1
thrombin activity per mg protein).

65 g of sucrose and 27 g of arginine are added per 45 ml of this thrombin solution (final concentration is 1500 units / m of thrombin)
l, sucrose 65 w / v%, arginine 27 w / v%) and the pH was adjusted to 5.9 with sodium hydroxide. After heat-treating this solution at 60 ° C. for 20 hours, it was dialyzed against 0.1 M citrate buffer (pH 6.7). This solution is concentrated and filtered, and then a thrombin liquid composition (0.1 M citrate buffer containing 3000 units / ml of thrombin, 7 w / v% of sucrose, 4.7 w / v% of arginine, pH 6.7) 105
ml was prepared.

 The solution was further sterilized, filtered, aliquoted and freeze-dried.

This thrombin dried preparation was stored at 5 ° C. for one month,
As a result of tests on liquid (appearance), thrombin activity, cellulose acetate membrane electrophoresis, and gel filtration, no remarkable change was observed, and it was found that thrombin was stable.

Example 2 The extraction residue of corn fractions II + III by the cold ethanol method was extracted with physiological saline, and the obtained extract was operated in the same manner as in Example 1 to obtain a thrombin dried preparation.

Example 3 Composition of dried thrombin preparation Thrombin 10,000 units Sucrose 120 mg Arginine 60 mg Sodium citrate 60 mg Experimental example 1 Thrombin / 0.1 M citrate buffer (pH 6.7) prepared according to Example 1 (thrombin concentration 3200 units / ml) ), Various heat stabilizers described in Table 1 were added, heat treatment and freeze drying were carried out in the same manner as in Example 1, and the remaining thrombin activity was measured. Table 1 shows the results.

The thrombin activity is determined by confirming the coagulation with a fibrometer (manufactured by BBL), and the unit is calculated by assuming that 2 units are necessary for coagulating 1 ml of human plasma in 15 seconds.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Kazuo Takechi 2-1180-1 Invitation Otani, Hirakata City, Osaka Prefecture Inside the Midori Cross Research Institute, Inc. (72) Inventor Yoshiro Iga 2-1180 Invitation Otani, Hirakata City, Osaka Prefecture (1) Inside the Midori Cross Research Laboratory, Inc. (56) References JP-A-62-81327 (JP, A) JP-A-57-39849 (JP, A) JP-A-56-39782 (JP, A) JP 62-106028 (JP, A) JP-A-57-116018 (JP, A)

Claims (2)

(57) [Claims] 1. A dry thrombin preparation comprising thrombin, a saccharide, a basic amino acid and an organic acid salt. 2. The thrombin dry preparation according to claim 1, wherein the basic amino acid is arginine.