JPH0578259A - Modified type t-pa-containing composition - Google Patents

Abstract

PURPOSE:To stabilize a modified type human tissue plasminogen activating factor (modified type t-PA) by blending albumin with the modified type t-PA. CONSTITUTION:A stabilized modified type t-PA-containing composition containing a modified type t-PA, albumin and preferably a human blood serum albumin. The concentration of the modified type t-PA in the composition is preferably >=0.1mg/ml and the concentration of the albumin is preferably 0.1-5%, especially preferably 0.5-2%. (i) A modified type t-PA in which the F region and G region of t-PA are deficient and Gly at the 183-position and Ser at the 186-position are respectively substituted with Ser and Thr, (ii) a modified type t-PA in which the F region and G region of the t-PA are deficient and Ser at the 119-position is substituted with Met, (iii) a modified type t-PA in which the F region, G region and K2 region of the t-PA are deficient and Ser at the 119-position is substituted with Met, (iv) a modified type t-PA in which Cys at the 84-position of the t-PA is substituted with Ser, etc., are cited as the modified type t-PA.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention comprises a modified t-PA-containing composition containing modified human tissue plasminogen activator (hereinafter referred to as modified t-PA) and albumin, and albumin as a main component. The present invention relates to a stabilizer for the modified t-PA.

[0002]

BACKGROUND OF THE INVENTION Human tissue plasminogen activator (tissue-type plasminogen activator; hereinafter referred to as t-type plasminogen activator).
PA) has fibrin affinity and activates plasminogen bound to fibrin to lyse thrombus very efficiently. Attention has been paid. Furthermore, in recent years, research on modified t-PA in which the structure of this t-PA has been modified using genetic engineering techniques has been actively conducted, and compared with t-PA, in vivo
Excellent modified molecules having enhanced biological activity by prolonging the blood half-life in E. coli have been found.

[0003]

The modified t-PA is a glycoprotein, but the glycoprotein is generally unstable to heat, and the modified t-PA is no exception. Stabilization technology is essential for developing such substances as pharmaceuticals. An object of the present invention is to provide a modified t-PA composition, which is a glycoprotein having low stability, as a stabilized pharmaceutical product.

Conventionally, as a method for stabilizing t-PA, arginine (The Journal of Biolog-ical Chemistry, 25
4 , 1998 (1979)), gelatin (Biochemistry, 8 , 79. (196
9)) Many other methods are known. However, research on modified t-PA is still in its infancy, and there are few findings that clarify the stabilization method.

[0005]

The inventors of the present invention have proposed a modified t
-The stabilization of PA was investigated using various substances such as sugars and proteins. As a result, they found that albumin significantly stabilized the modified t-PA, and completed the present invention.

The composition of the present invention will be described in detail below.
The modified t-PA used in the present invention is an improved version of t-PA or an enhanced biological activity of t-PA, which achieves the stabilizing effect of the present invention. Any modified t-PA may be used as long as it is present. Examples of such modified t-PA include Japanese Patent Application Laid-Open No. 63-272020 and Japanese Patent Application No. 1-31943, which are related to the application of the present applicant.
Examples thereof include modified t-PA shown in No. 8 and the like, but are not limited thereto. That is, in the modified t-PA of the present invention, the F region and G region of t-PA were deleted, and
Mutants t-PA and t-PA in which Ser at position 186 and Ser at position 186 were respectively replaced by Ser and Thr were deleted, and Ser at position 119 was deleted.
Mutants in which Met was replaced by Met, the F region, G region and K2 region of t-PA were deleted, and 1
Mutation t-PA in which Ser at position 19 is replaced with Met, or mutation t in which Cys at position 84 of t-PA is replaced with Ser
-PA and the like are also included.

Although various kinds of albumin used as a stabilizer can be used, human serum albumin is particularly preferable when the present composition is used as a medicine.

The composition of the present invention contains modified t-PA, albumin, and if necessary various additives, and the composition is dissolved in water (purified water) under normal sterilization to prepare modified t-PA. The liquid preparation or this is further dried by a drying means such as freeze-drying to give a dissolution-type preparation at the time of use.

Here, the concentration of modified t-PA is at least about 0.1 mg / ml or more, while the concentration of albumin is preferably 0.1-5%, more preferably 0.5-2%.

On the other hand, as additives to be added as necessary, amines such as monoethanolamine, diethanolamine, triethanolamine, ethylenediamine and salts thereof, amino acids such as arginine and amino acid lower alkyl esters such as glycine ethyl ester, etc. Solubilizing agent for modified t-PA, non-reducing sugar such as sucrose, trehalose, sorbitol, xylit, mannitol, gelatin, etc., modified t-PA stabilizing agent other than albumin,
Tris-HCl buffer, phosphoric acid, carbonic acid, boric acid, citric acid,
Buffering agents that can be used as buffer solutions such as barbituric acid and amino acids, isotonic agents composed of electrolytes of inorganic salts such as sodium chloride, potassium chloride, sodium monohydrogen phosphate and sodium dihydrogen phosphate, polyoxygen Polyoxyethylene sorbitan fatty acid esters such as ethylene sorbitan monostearate and polyoxyethylene sorbitan monopalmitate, sorbitan monostearate, sorbitan fatty acid esters such as sorbitan sesquioleate, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene condensation Surfactants for things,
Examples include excipients such as cellulose, methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxymethyl ethyl cellulose and the like.

The non-reducing sugar can also be used as an excipient. These additives are appropriately selected, and the same kind may be contained in two or more kinds. In addition, when the composition of the present invention is to be dissolved before use, a solubilizing agent or the like can be added to the solution, or the solution can be used as a buffer solution.

[0012]

INDUSTRIAL APPLICABILITY The composition of the present invention remarkably stabilizes modified t-PA, and is useful as a composition which can provide a clinically usable preparation of modified t-PA. The improved stability of the modified t-PA according to the present invention was confirmed by the following test method.

Experimental Example 1 Various freeze-dried products were prepared from the freeze-dried products of Example 1 and the following comparative examples.
Temperature, the appearance and solubility are observed, and the residual titer ratio is checked.
Stability was determined by the Clot Lysis method.
In addition, the comparative example was prepared by the following formulation according to the method of Example 1.
did. Comparative Example 1 Modified t-PA 0.5 mg / ml sodium phosphate / 2 sodium salt 0.1M (pH 7.2) sodium chloride 0.9% glycine 1% Tween 80 0.01%  4 ml / Vial Comparative Example 2 Modified t-PA 0.5 mg / ml Sodium citrate 0.1 M Maltose 5% Tween 80 0.01% Hydrochloric acid pH to 7.2  4ml / Vial In addition, Tween 80 is manufactured by Kao Atlas Co., Ltd., Polyoki
Trade name of ethylene higher fatty acid ester
Same). In addition, modified t-PA is disclosed in International Publication 89/038.
The refined TPA [VI] described in No. 74
Using. Hereinafter, this modified t-PA is abbreviated as TPA.

[0014]

[Table 1]

In the table, Initial represents an initial condition, W represents a week, and M represents a month (the same applies hereinafter). Experimental Example 2 The freeze-dried product of Comparative Example 3 below prepared in the same manner as in Example 2 and Example 2 was allowed to stand under various temperature conditions, and Experimental Example 1
The stability was investigated in the same manner as in. Comparative Example 3 TPA 0.5 mg / ml phosphoric acid 0.025 M triethanolamine 0.05 M sucrose 5% sodium chloride 5% Tween 80 0.01% 4 ml / Vial (pH 7.2) The results are shown in Table 2.

[0016]

[Table 2]

As shown in Example 1, a modified t-P
By adding human serum albumin to A and lyophilizing it, it was possible to prepare a highly stable preparation. The high stability is clear by comparison with comparative examples using glycine and maltose as stabilizers. Further, as is clear from Example 2, human serum albumin also showed a high stabilizing effect in the preparation to which triethanolamine was added as a solubilizing agent.

[0018]

EXAMPLES The present invention will be described in more detail with reference to the following examples. Example 1 TPA 0.5 mg / ml sodium phosphate / 2 sodium salt 0.1 (pH 7.2) sodium chloride 0.8% human serum albumin 1% Tween 80 0.01% A 0.1 M phosphate buffer solution (pH 7.2) containing 0.9% sodium chloride and 0.01% Tween 80 containing 4 ml / Vial TPA at a concentration of 0.5 mg / ml was prepared, and human serum was added to the solution. After adding albumin to a concentration of 1%, it was dispensed into glass vials in 4 ml portions, and a freeze-dried product that was dissolved before use was manufactured using a freeze dryer manufactured by Kyowa Vacuum Technology Co., Ltd.

Example 2 TPA 0.5 mg / ml phosphoric acid 0.025 M triethanolamine 0.05 M human serum albumin 1% Tween 80 0.01%
 0.2 M Trieta containing 4 ml / Vial (pH 7.2) TPA at a concentration of 2 mg / ml.
0.1M containing Nolamine and 0.01% Tween 80
Prepare a phosphate buffer (pH 7.2) and add 1 m of the solution.
1, followed by 200 μl of 20% human serum albumin, 0.
2.8 ml of 01% Tween 80 solution in a glass vial
And then use a freeze dryer manufactured by Kyowa Vacuum Technology Co., Ltd.
Then, a freeze-dried product that was dissolved before use was produced.

Continued Front Page (72) Inventor Satoru Nakamura 368-2 Mikana, Yaizu City, Shizuoka Prefecture Yamanouchi Pharmaceutical Co., Ltd.

Claims (2)

[Claims] 1. A modified human tissue plasminogen activator-containing composition comprising a modified human tissue plasminogen activator and albumin. 2. A stabilizer for modified human tissue plasminogen activator, which comprises albumin as a main component.