JPH05967A - Tissual plasminogen activator-containing pharmaceutical composition - Google Patents

Abstract

PURPOSE:To obtain a pharmaceutical composition, containing a tissual plasminogen activator or its modified substance, a specific amine and a nonreducing sugar, improved in solubility within the neutral pH region and capable of exhibiting excellent stability. CONSTITUTION:A tissual plasminogen activator-containing pharmaceutical is obtained by including a tissual plasminogen activator (hereinafter abbreviated to t-PA) or its modified substance (hereinafter abbreviated to modified type t-PA) and an amine expressed by the formula [X(CH2)1]mNHn[X is OH or amino; (1) is 1-4; (m) is 1-3; (n) is 0-2 and a number having the relation of (3-m)] or its salt and a nonreducing sugar as active ingredients. The t-PA and the modified type t-PA are preferably used at 0.3-3mg/ml concentration and the amine is preferably used at 0.1-0.5M concentration. The nonreducing sugar is used at 3-10% concentration. Monoethanolamine is exemplified as the amine and sucrose is exemplified as the nonreducing sugar.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a tissue plasminogen activator (hereinafter referred to as t-PA) or a modified form thereof (hereinafter referred to as modified t-PA), a specific amine or a salt thereof, and a non-reducing sugar. Containing t-PA and modified t-
It relates to a pharmaceutical composition of PA.

[0002]

2. Description of the Related Art Unlike streptokinase and urokinase, which are already known to have a thrombolytic effect, t-PA has an affinity for fibrin and is concentrated at the thrombus site and bound to fibrin. It is attracting attention as a thrombolytic agent with few side effects such as bleeding tendency because it activates gen to generate plasmin and efficiently dissolves thrombus. In recent years, t-PA has been improved for the purpose of improving the drawbacks of natural t-PA, such as a short half-life in blood in vitro, or enhancing its biological activity. Research and development of modified t-PA having a modified structure is also active.

Natural t-PA, t-PA produced by a genetic engineering technique and t-PA obtained by modifying this are glycoproteins, which are not suitable for oral administration and are exclusively formulated as injections. Consideration is in progress. However, t-PA
The modified t-PA is poorly soluble and unstable in water, which makes it difficult to formulate a dosage form to be dissolved and administered in water. Therefore, in clinical use of t-PA and modified t-PA, there is a demand for development of a preparation having improved solubility and stability of these substances.

Conventionally, as a method of improving the solubility of t-PA in water, a method of adjusting the liquidity of the solution to pH 3 to 5 and dissolving it is known (JP-A-62-26233).
No. 62-36332). In addition, by adding an amino acid such as arginine hydrochloride or its salt,
A technique for stabilizing soluble modified t-PA is also known (JP-A-64-42442).

[0005]

However, the method of dissolving by lowering the liquidity of the solution is t-PA in the solution state.
Was rapidly degraded and inactivated, making it difficult to use clinically. On the other hand, the present invention, in clinical use of t-PA and modified t-PA, does not use amino acids or salts thereof, and is in a neutral pH range in which these substances are kept relatively stable, The purpose of the invention is to improve its solubility and to provide a stable pharmaceutical composition.

[0006] Therefore, as a result of diligent studies on the solubilization of t-PA and modified t-PA, the present inventors did not show clinically usable solubilities of tris (hydroxyethyl) aminomethane and the like. However, it was surprisingly found that the amine represented by the following general formula (I) or a salt thereof has a solubility suitable for clinical use. [X (CH ₂ ) l)] mNHn (I) (wherein the symbols have the following meanings: X: a hydroxyl group or an amino group; an integer of 1: 1 to 4; an integer of 1: 1 to 3; n: A number that is 0, 1 or 2 and has a relationship of 3-m, provided that when X is an amino group, m represents 2 and n represents 1.)

[0007] The present inventors have further investigated the stabilization, and generally used t-PA or saccharides such as t-PA, which is generally used as a stabilizer in the preparation of glycoproteins, such as lactic acid. When it was added to a composition of modified t-PA and an amine represented by the general formula (I) or a salt thereof, it turned brown or black over time, and t
It was found that the titers of -PA and modified t-PA also markedly decrease with the coloring thereof. Therefore, as a result of further research for the purpose of developing a pharmaceutical composition of t-PA or modified t-PA without such difficulties, the present inventors have found that the addition of a non-reducing sugar typified by sucrose to the same sugar results in addition of lactose. It was found that it is possible to provide t-PA and modified t-PA solubilized preparations which have markedly improved stability without the coloring and reduction in potency observed in reducing sugars such as The invention was completed.

That is, the present invention provides t-PA or a modified t-PA, a t-PA or a modified t-PA, an amine represented by the general formula (I) or a salt thereof, and a non-reducing sugar. The t-PA pharmaceutical composition constitutes the invention.
Hereinafter, the pharmaceutical composition of the present invention will be described in detail. In the present invention, t-PA means, in addition to human-derived natural t-PA,
It also includes t-PA obtained by genetic engineering techniques. In the present invention, the modified t-PA improves the difficulty of t-PA or enhances the biological activity of t-PA, etc., and achieves the purpose of stabilizing solubilization of the present invention. Any modified t-PA may be used as long as it does. Examples of such modified t-PA include Japanese Patent Application No. 63-272020 and Japanese Patent Application No.
Examples thereof include modified t-PA shown in No. 19438 and the like, but are not limited thereto. That is, the modified t-PA of the present invention lacks the F region and G region of natural t-PA, and has a mutation t in which Gly at position 183 and Ser at position 186 are substituted with Ser and Thr, respectively.
-PA, the F region and G region of native t-PA are missing,
Mutant t-PA in which Ser at position 119 is replaced with Met,
Mutant t-P in which F region, G region and K ₂ region of native t-PA are deleted and Ser at position 119 is replaced with Met
A and mutant t-PA in which Cys at position 84 of natural t-PA is replaced with Ser are also included.

The specific amine used in the present invention is
Any of the amines represented by the general formula (I) can enhance the solubility of t-PA and modified t-PA in water at neutral pH, but among them, monoethanolamine, diethanolamine and triethanolamine (Hereinafter referred to as ethanolamines) is preferable. The salts of this specific amine include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,
Mineral acids such as nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, tartaric acid, carbonic acid, picric acid,
Examples thereof include acid addition salts with organic acids such as methanesulfonic acid and ethanesulfonic acid, salts with amino acids such as glutamic acid and aspartic acid, and ammonium salts.

The non-reducing sugar used in the present invention may be any sugar as long as it does not have a reducing end, that is, a free reducing group, and does not exhibit reducing properties. The reducing ends of monosaccharides such as sucrose and trehalose are the same. Examples include oligosaccharides of disaccharides or more that have been bound by glycerides and have lost reducibility, sugar alcohols such as sorbit, xylit, and mannitol and polysaccharides that do not have a reducing end. Among them, sucrose is a stable point. Is most suitable.

The concentration of t-PA or modified t-PA is at least about 0.1 mg / ml or more, preferably 0.3 mg.
/ Ml to 3 mg / ml, and the concentration of the specific amine or its salt is 0.01 to 1.0 M, preferably 0.1 to 0.5 M
And the concentration of non-reducing sugar is about 1-40%, preferably 3
Used at 10% to 10% respectively.

To prepare the pharmaceutical composition of the present invention, usually, t-PA or modified t-PA under sterilization, an amine or a salt thereof, a non-reducing sugar and, if necessary, other pharmaceutical bases are added. Then, this is dissolved in water (purified water) to obtain a liquid agent, or this liquid is applied with a drying means such as freeze-drying, and dried to give a dissolution-type preparation at the time of use.

[0013] The formulation bases added as necessary here include buffers such as Tris-hydrochloric acid buffer, phosphoric acid, carbonic acid, boric acid, citric acid, barbituric acid and amino acids, sodium chloride, and chloride. Isotonic agents such as electrolytes of inorganic salts such as potassium, sodium monohydrogen phosphate and sodium dihydrogen phosphate, polyoxyethylene fatty acid esters such as polyoxyethylene sorbitan monostearate and polyoxyethylene sorbitan monopalmitate, sorbitan Monostearate, sorbitan fatty acid ester such as sorbitan sesquioleate, polyoxyethylene hydrogenated castor oil, surfactant such as polyoxyethylene polyoxypropylene condensate, cellulose, methyl cellulose, ethyl cellulose, hydroxymethyl cellulose,
Excipients such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxymethyl ethyl cellulose and the like can be mentioned. The non-reducing sugar such as sucrose can also be used as an excipient. These additives may be appropriately selected and the same kind may be used in two or more kinds. In addition,
When the preparation is a dissolution-type preparation at the time of use, a solubilizing agent may be added to the dissolution solution or the dissolution solution may be used as a buffer solution.

The composition of the present invention comprises t-PA and modified t-PA.
It has a high stability in the neutral pH range, which makes it relatively stable, and exhibits excellent stability, so it can be used clinically.
It is useful as a pharmaceutical composition of -PA and modified t-PA. The improvement in solubility and stability of t-PA, modified t-PA, and modified t-PA by the composition of the present invention was confirmed by the following experimental method.

Experimental Example 1 (Solubility Test) A freeze-dried product of natural t-PA was weighed, and 0.01% of Tween 80 (trade name, manufactured by Kao Atlas, polyoxyethylene higher fatty acid ester, hereinafter referred to as Tween 80) was used. 50 mM phosphate buffer solution having a pH of 7.0, which is referred to as (referred to), was added and shaken well. This was centrifuged and the supernatant was collected to measure the protein concentration. Natural type t thus obtained
-PA solubility was 0.06 mg / ml. While maintaining the pH at 7.0, adjust the concentration of phosphoric acid to 100 mM, 500
Even in the case of mM, natural t-PA in each solution
The solubility was low at 0.13 mg / ml and 0.25 mg / ml. Next, tris (hydroxyethyl)
Aminomethane, ethylenediamine, and monoethanolamine are each included, and the pH is adjusted to 7.0 with phosphoric acid.
Using a buffer containing 1% Tween 80, the natural t-type
Similar experiments were performed on PA. The result is shown in FIG. From FIG. 1, it is clear that the amines and salts thereof described in the present invention have the effect of increasing the solubility of natural tPA. On the other hand, modified t-PA (International Publication No. 89/038
A purified version of the improved TPA [VI] of No. 74. Below TP
A) had a solubility of 0.24 mg / ml in a 50 mM phosphate buffer having a pH of 7.0, but in a phosphate buffer containing 0.2 M monoethanol, precipitation was performed at a concentration of 4.0 mg / ml or more. It was kept stable without waking.

Experimental Example 2 (Solubility Test) Using natural t-PA, the same experiment as in Experimental Example 1 was conducted using diethanolamine and triethanolamine. The experimental results are shown in FIG. As shown in FIG. 2, the solubilizing effect of natural t-PA by ethanolamines was the same for the three kinds of ethanolamines of mono-, di-, and tri-. This also applies to the modified t-PA,
It was kept as a clear solution at a concentration of 2.0 mg / ml or more in a phosphate buffer containing 0.2 M or more of mono- or diethanolamine.

Experimental Example 3 (Manufacturing Test) Based on the knowledge obtained from Experimental Examples 1 and 2, a freeze-dried product having the following composition was produced. The display of TPA shows the modified tPA of Example 1. Treatment 1 TPA 2 mg / ml Phosphoric acid 0.1 M Triethanolamine 0.2 M Mannitol 10% Tween 80 0.01% Treatment 2 TPA 5 mg / ml Phosphoric acid 0.3 M Triethanolamine 0.5 M Hydroxyethylcellulose 10% Tween 80 0.01% When these lyophilized products were redissolved in water for injection, the TPA concentration in the solution was 2 to 5 mg / ml, but a clear liquid was obtained. It was revealed from this example that a lyophilized product that gives a high concentration of t-PA when used can be produced.

Experimental Example 4 (Stability Test) The stability of freeze-drying of Examples and the following Comparative Examples was examined by leaving them under various temperature conditions. Comparative Example 1 A lyophilized product of a diethanolamine-lactose addition system was obtained in the same manner as in Example 1 except that a solution containing 5% lactose was used instead of 5% sucrose. Comparative Example 2 A triethanolamine-lactose-added freeze-dried product was obtained in the same manner as in Example 2 except that 5% lactose was used instead of 5% sucrose. (1) Stabilizing effect of diethanolamine-sucrose The freeze-dried product of Example 1 and the freeze-dried product of Comparative Example 1 were allowed to stand under various temperature conditions, and the stability was examined. (2) Stabilizing effect of triethanolamine-sucrose The freeze-dried product of Example 2 and the freeze-dried product of Comparative Example 2 were allowed to stand under various temperature conditions and the stability was examined. Show. In the table, W means week, M means month, and Initial means initial condition.

[0019]

[Table 1]

[0020]

[Table 2]

From Experimental Example 4, even in a preparation using diethanolamine or triethanolamine as a solubilizing agent for modified t-PA, lactose which is a reducing sugar when sucrose which is a non-reducing sugar is added. It is clear that it is dramatically stabilized as compared with the case of adding.

Experimental Example 5 (Recognition test for cause of coloring) Lactose, sucrose and dextran 40 were added to 5 each in 0.11M phosphate buffer pH 7.2) containing 0.2M diethanolamine and 0.01% Tween 80. The solution that had been dissolved to a concentration of 10% was aseptically filtered, then dispensed into a glass ampoule and sealed. As a control liquid, a filling product of a solution containing no sugar was also prepared. For each sample, the ampoule-filled product, in which a nitrogen-substituted group and a nitrogen-unsubstituted group were produced, was left at each temperature of 25 ° C, 40 ° C, and 50 ° C,
The results of measuring H, osmotic pressure ratio, and absorbance at 410 nm are shown in Tables 3 and 4. In addition, M in the table, WInitia
l has the same meaning as described above.

[0023]

[Table 3]

[0024]

[Table 4]

As is clear from the results of Experimental Example 5, the solution containing lactose and dextran 40 was colored over time, and the absorbance value at 410 nm increased. Also, in parallel with this, the pH decreased. In contrast, the solution containing sucrose showed no change. For all samples, no difference was observed between the nitrogen-substituted group and the non-substituted group. Therefore, the coloring reaction is a reaction between an amino acid or amine and a saccharide, not an oxidation reaction due to oxygen in the air, and a reaction with an amino acid or an amine when sucrose, which is a non-reducing sugar, is added as a sugar. It has become clear that does not happen.

[0026]

EXAMPLES The present invention will be described in more detail with reference to the following examples. The present invention is not limited to the description of the embodiments.

Example 1 0.11 M phosphate buffer (pH 7.0) containing 0.2 M diethanolamine, 5% sucrose, 5% sodium chloride and 0.01% Tween 80 containing TPA at a concentration of 0.5 mg / ml. 2) was dispensed into glass vials at a rate of 2 ml each, and then freeze-dried using a freeze dryer manufactured by Kyowa Vacuum Technology Co., Ltd. to obtain a freeze-dried preparation that was dissolved before use.

Example 2 0.1 M phosphate buffer (pH: 0.2 M triethanolamine, 5% sucrose, 5% sodium chloride and 0.01% Tween 80) containing TPA at a concentration of 0.5 mg / ml.
7.2) was prepared and a freeze-dried preparation that was dissolved before use was obtained in the same manner as in Example 1.

[Brief description of drawings]

FIG. 1 shows the experimental results of the solubilizing effect of specific amines on natural t-PA, and FIG. 2 shows the experimental results of the solubilizing effect of ethanolamines on natural t-PA.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location A61K 47/26 G 7329-4C J 7329-4C (72) Inventor Nakamura Victory Mikaname 368, Yaizu City, Shizuoka Prefecture -2 Yamanouchi Pharmaceutical Co., Ltd. Suruga Dormitory (72) Inventor Reiaki Ishikawa 2-30-4 Oiwa, Shizuoka City, Shizuoka Prefecture (72) Inventor Jun Sekino 1506-6 Mikana, Yaizu City, Shizuoka Prefecture Yaizuso Yamanouchi Pharmaceutical Co., Ltd.

Claims (3)

[Claims] 1. A tissue plasminogen activator or a modified tissue plasminogen activator, and a compound of the general formula (I) [X (CH ₂ ) l)] mNHn (I) (wherein the symbols have the following meanings): X: hydroxyl group or amino group, l: integer of 1 to 4, m: integer of 1 to 3, n: 0, 1 or 2 and a number having a relationship of 3-m, where X is amino. Group, m is 2 and n is 1), and a composition of a tissue plasminogen activator or a modified tissue plasminogen activator, which comprises an amine or a salt thereof represented by object. 2. The pharmaceutical composition according to claim 1, wherein the amine or a salt thereof is monoethanolamine, diethanolamine or triethanolamine, or a salt thereof. 3. The non-reducing sugar is sucrose.
The described pharmaceutical composition.