JPH06206829A - Method for dissolving tissue plasminogen activator - Google Patents

Abstract

PURPOSE:To highly raise the solubility of a tissue plasminogen activator (t-PA) by using a solvent system containing arginine or an acid-added salt thereof. CONSTITUTION:The concentration of a t-PA in its aqueous solution is raised to >=0.2mg/ml by incorporating the solution with 1-500 (pref. 5-200)mM of arginine or an acid-added salt thereof. It is more preferable that 0.02-2.0 (pref. 0.1-1.0) M of a neutral salt, esp. sodium chloride, be made to coexist in said solution. With the above method, an aqueous solution of t-PA at high concentrations can be prepared; thereby, a small volume of vessel for pharmaceuticals (e.g. a vial, ampule) can be packed with a relatively large quantity of t-PA, enabling a therapeutically effective thrombolytic agent to be manufactured. And, when an arginine-contg. solvent is used in the purification process for t-PA such as dialysis, filtration or chromatography (esp. gel chromatography), t-PA can be purified in its highly concentrated state, resulting in its efficient purification.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing a thrombolytic agent based on a method for dissolving tissue plasminogen activator.

[0002]

2. Description of the Related Art Heretofore, as a thrombolytic agent, urokinase separated and purified from urine or cultured kidney cells has been developed, and streptokinase extracted from β-streptococcus has been put to practical use. However, since urokinase has a low affinity for thrombus, a large amount of urokinase is required to obtain a desired therapeutic effect, and in the case of systemic administration, bleeding due to destruction of coagulation factors by a large amount of plasmin in the bloodstream is required. I was worried. In this regard, in human or animal tissue,
Tissue plasminogen activator (hereinafter referred to as t-PA) found in cell culture medium or tumor cell culture medium derived from those tissues has a higher affinity for thrombus than urokinase and further has thrombolytic ability. Since it is also excellent, the desired therapeutic effect can be obtained with a small amount of administration, and it is expected as a new thrombolytic agent. In addition, recently, attempts have been made to produce t-PA by genetic engineering means, which has spurred future expectations.

[0003]

The present inventors have found that t-P
A has been purified and a method for producing a thrombolytic agent containing this as an active ingredient has been investigated. However, it became clear that the solubility of t-PA decreased as the degree of purification progressed, which was a major obstacle to formulation.

[0004]

The present inventors have found that t-PA
As a result of diligent research to solve the solubility problem of
It was found that the solubility of A was significantly increased by using a solvent system containing arginine or an acid addition salt thereof, and the present invention was completed.

The present invention is a method for producing a thrombolytic agent based on the method for dissolving t-PA, which comprises arginine or an acid addition salt thereof in order to increase the solubility of t-PA.

The arginine used in the present invention may be in any of D-form, L-form or racemic form, and may be an acid addition salt thereof, for example, hydrochloride (hereinafter, unless otherwise specified). , Arginine includes all of these). The amount of arginine required to increase the solubility of t-PA is 1 mM or more and 500 mM or less, preferably 5 mM to 200 mM. Experimental example 1 below
As shown in, even if it is 1000 mM, it is effective, but since the solubility of t-PA commensurate with it is not observed, 500 mM or less is suitable for practical use. Further, it is more preferable to use a neutral salt, particularly sodium chloride, in combination with arginine at a concentration of 0.02M to 2.0M, preferably 0.1 to 1.0M. The t-PA solution containing arginine or arginine and a neutral salt such as sodium chloride is preferably maintained at a pH of 2 to 12, preferably 6 to 11 with a buffer solution such as sodium phosphate.

The thrombolytic agent produced by the present invention may contain at least arginine in addition to the main component t-PA, and if necessary, other components such as albumin, mannitol, gelatin and chloride. It is optional to include excipients, stabilizers and the like which are generally used in the formulation of sodium and the like.

The production of the thrombolytic agent of the present invention is carried out using purified t
-PA is dissolved in a solvent containing arginine, then sterile filtered and filled in an ampoule, a vial or the like, but may be lyophilized if desired. Further, instead of dissolving t-PA in a solvent containing arginine, t-PA and a predetermined amount of arginine are weighed and, if desired, dissolved in a suitable solvent together with other components, or as a mixture thereof, You may manufacture by filling in a proper container.
Distilled water for injection as a solvent used here, physiological saline,
0.01M-0.1M phosphate buffer etc. can be mentioned. As a dosage form of the preparation, it is usually advantageous to use an injection, but any dosage form may be used as long as the thrombolytic ability of t-PA can be maintained. The human dose of the thrombolytic agent produced by the present invention is generally 20,000 to 50,000 IU / Kg, but it may be appropriately increased or decreased depending on the symptoms.

As described above, when formulation is carried out using a solvent containing arginine, t-PA can be made into a high-concentration solution, and therefore, a small-capacity formulation container such as a vial or ampoule. And the like can be filled with a large amount of t-PA and a therapeutically effective thrombolytic agent can be prepared.

Incidentally, dialysis in the purification process of t-PA,
When a solvent containing arginine is used in a purification step such as filtration or chromatography, particularly a step for carrying out gel chromatography, t-PA can be maintained at a high concentration for purification, and therefore, efficient. Purification can be performed. Therefore, the inclusion of arginine in the t-PA solution is extremely useful when formulating t-PA, and is also extremely useful for efficiently producing high-purity t-PA on an industrial scale.

Next, the present invention will be described in more detail with reference to experimental examples and examples, but the present invention is not limited to these examples. In addition, in the following Experimental Examples and Examples, purification was carried out from a human melanoma cell culture medium and a culture medium of Chinese hamster Ovari (hereinafter, referred to as CHO) cells into which the t-PA gene was transferred by a genetic engineering means. Although t-PA has been described, t-PA obtained from human or animal tissue, t-PA purified from cell culture solution derived from human or animal tissue, or t-PA obtained by genetic engineering means other than the above, that is, , T-PA obtained from a microorganism such as E. coli, Bacillus subtilis, yeast, etc. into which the t-PA gene has been transferred or a t-PA gene has been transferred.

The activity of t-PA was determined by measuring 95% coagulated fibrinogen (plasmin-gen-containing about 50 casein units /
The fibrin plate method was performed using t-PA (WHO certified product) as a standard product.

(Production Example 1 of t-PA) Colene (Col
len) et al. (The Journal of Biological Chemistry (The J. Biol. Che.
m. ) 256 (13), 7035-7041, 198.
Approximately 60 L of the crude t-PA culture solution produced using human melanoma-derived cells according to 1) was added to Rijken.
Et al. (Thrombosis and Hemostasis (Thr
omb. Haemostas. ) 48 (3), 294-
296, 1982), and the purification operation was performed. That is, the culture solution was subjected to Zn-chelate chromatography.
After purification with (13.5 × 17.5 cm), Con A
Sepharose chromatography (5 × 30 cm) was performed. 0.4M α-from ConA Sepharose column
T-PA eluted with a 2.0 M potassium thiocyanate solution containing D-methyl mannoside was concentrated with polyethylene glycol, and then concentrated with 0.01 M phosphate buffer (pH 7.5) containing 0.25 M arginine hydrochloride. Gel filtration was carried out with a column (7.5 × 90 cm) packed with equilibrated Sephaacrylic S200 (Pharmacia) for purification t
-PA was obtained. The specific activity of 2.
5 × 10 ⁵ IU / mg, 18 million IU of purified t-PA
Got The obtained t-PA was dialyzed against distilled water at 4 ° C. overnight, lyophilized, and used in Experimental Examples and Examples described later.

(Production Example 2 of t-PA) Penn
ica et al.'s method (Nature 301 )
(20), 214-221, 1983) according to t-P
A gene is prepared and the method of Kaufman and Sharp is described (Journal of Molecular Biology (J. Mol. Biol.) 1
59 , 601-621, 1982).
After transferring the PA gene into CHO cells, 40 L of crude t-PA culture medium produced using the CHO cells was obtained. This crude t-PA culture solution was purified in the same manner as in t-PA Production Example 1, and the specific activity was 1.7 × 10 ⁵ IU / mg, 12 million IU.
Of purified t-PA was obtained.

Experimental Example 1 The effect of arginine concentration on the solubility of t-PA was investigated. Purified t-obtained in Production Example 1
Each 5 mg of PA was weighed and dissolved in 0.5 ml of a solution having the arginine concentration shown in Table 1 to obtain t-P in the solution.
The activity of A was measured to examine the solubility of t-PA. t-P
When A was not completely dissolved and precipitation occurred, the supernatant activity was measured. The results are shown in Table 1 and FIG.

[0016]

[Table 1]

(Experimental Example 2) The influence of the type of arginine on the solubility of t-PA was examined. 1 mg of t-PA purified in Production Examples 1 and 2 was dissolved in 0.5 ml of a solution containing 25 mM of various arginines shown in Table 2, and the activity of t-PA in the solution was measured in the same manner as in Experimental Example 1. The results are shown in Table 2.
It was shown to.

[0018]

[Table 2]

Experimental Example 3 The solubility in a solvent system containing L-arginine hydrochloride and sodium chloride was examined. The purified t-PA5 obtained in Production Example 1 was added to 0.5 ml of a solution containing L-arginine hydrochloride and sodium chloride and having its pH adjusted.
mg or 1 mg was dissolved and the t-PA activity of the solution was measured to examine the solubility of t-PA. The results are shown in Table 3 and Table 4.
It was shown to.

[0020]

[Table 3]

[0021]

[Table 4]

(Experimental Example 4) Toxicity test The preparations prepared in Examples 1 and 2 described below were dissolved in physiological saline or distilled water for injection to give 5 mice and guinea pigs.
After administration of 0,000 IU / Kg, no abnormality was observed.
Further, the same preparation of 200,000 IU / Kg was administered to rabbits and a pyrogen test was conducted, but all the preparations were negative.

As is clear from the above experimental examples, arginine is useful for increasing the solubility of t-PA.

[0024]

【Example】

(Example 1) t-PA 5,000,000 IU L-arginine hydrochloride 21 mg sodium phosphate 173.9 mg purified gelatin 100.0 mg The above components were dissolved in 10 ml of distilled water for injection. After aseptic filtration, 1.0 ml each was filled in a vial and freeze-dried to prepare a thrombolytic agent.

(Example 2) t-PA 5,000,000 IU L-arginine hydrochloride 52.5 mg sodium phosphate 173.9 mg sodium chloride 64.3 mg human serum albumin 20 mg The above components were weighed. A thrombolytic agent was obtained and prepared in the same manner as in Example 1.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the relationship between the solubility of t-PA and the concentration of arginine.

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[Procedure amendment]

[Submission date] October 2, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Title: Dissolution method of tissue plasminogen activator

[Claims]

Detailed Description of the Invention

[0001]

The present invention relates to] is relates to the dissolution method of Kumi繊plasminogen activator. In detail
Is a tissue plasminogen activator that excludes other proteins.
The concentration of water in an aqueous solution of at least 0.2 mg / ml
The present invention relates to the above method.

[0002]

2. Description of the Related Art Heretofore, as a thrombolytic agent, urokinase separated and purified from urine or cultured kidney cells has been developed, and streptokinase extracted from β-streptococcus has been put to practical use. However, since urokinase has a low affinity for thrombus, a large amount of urokinase is required to obtain a desired therapeutic effect, and in the case of systemic administration, bleeding due to destruction of coagulation factors by a large amount of plasmin in the bloodstream is required. I was worried. In this regard, in human or animal tissue,
Tissue plasminogen activator (hereinafter referred to as t-PA) found in cell culture medium or tumor cell culture medium derived from those tissues has a higher affinity for thrombus than urokinase and further has thrombolytic ability. Since it is also excellent, the desired therapeutic effect can be obtained with a small amount of administration, and it is expected as a new thrombolytic agent. In addition, recently, attempts have been made to produce t-PA by genetic engineering means, which has spurred future expectations.

[0003]

The present inventors have found that t-P
A has been purified and a method for producing a thrombolytic agent containing this as an active ingredient has been investigated. However, it became clear that the solubility of t-PA decreased as the degree of purification progressed, which was a major obstacle to formulation.

[0004]

The present inventors have found that t-PA
As a result of diligent research to solve the solubility problem of
It was found that the solubility of A was significantly increased by using a solvent system containing arginine or an acid addition salt thereof, and the present invention was completed.

The present invention comprises an effective amount of arginine or an acid addition salt thereof in order to increase the solubility of t-PA.
It relates to methods for dissolving t-PA characterized by that
is there.

The arginine used in the present invention may be in any of D-form, L-form or racemic form, and may be an acid addition salt thereof, for example, hydrochloride (hereinafter, unless otherwise specified). , Arginine includes all of these). The amount of arginine required to increase the solubility of t-PA is 1 mM or more and 500 mM or less, preferably 5 mM to 200 mM. Experimental example 1 below
As shown in, even if it is 1000 mM, it is effective, but since the solubility of t-PA commensurate with it is not observed, 500 mM or less is suitable for practical use. Further, it is more preferable to use a neutral salt, particularly sodium chloride, in combination with arginine at a concentration of 0.02M to 2.0M, preferably 0.1 to 1.0M. The t-PA solution containing arginine or arginine and a neutral salt such as sodium chloride is preferably maintained at a pH of 2 to 12, preferably 6 to 11 with a buffer solution such as sodium phosphate.

[0007] Thrombolytic agents produced by the dissolution method t-PA of the present invention, other t-PA which is a main component, at least <br/>, need only contain arginine, optionally ,
Other components, such as albumin, mannitol, gelatin, sodium chloride, etc., which are generally used in formulation, excipients, stabilizers, etc. , phosphoric acid, citric acid, etc. are optional.

The production of a thrombolytic agent by the method for dissolving t-PA of the present invention is carried out by dissolving purified t-PA in a solvent containing arginine, then aseptically filtering and filling in ampoules, vials and the like. May be lyophilized if desired. Further, instead of dissolving t-PA in a solvent containing arginine, t-PA and a predetermined amount of arginine are weighed and, if desired, dissolved in a suitable solvent together with other components, or
Alternatively, the mixture may be prepared by filling a suitable container. Examples of the solvent used here include distilled water for injection, physiological saline, 0.01 M to 0.1 M phosphate buffer, and the like. As a dosage form of the preparation, it is usually advantageous to use an injection, but any dosage form may be used as long as the thrombolytic ability of t-PA can be maintained. The human dose of the thrombolytic agent produced by the method for dissolving t-PA of the present invention is generally 20,000 to 50,000 IU / Kg, but it may be appropriately increased or decreased depending on the symptoms.

As described above, if the solubility of t-PA is increased by using a solvent containing arginine, it becomes possible to prepare a high-concentration solution of t-PA. Large amount of t-P in containers such as vials and ampoules
It becomes possible to fill A, and a thrombolytic agent which is therapeutically effective can be prepared.

Incidentally, dialysis in the purification process of t-PA,
When a solvent containing arginine is used in a purification step such as filtration or chromatography, particularly a step for carrying out gel chromatography, t-PA can be maintained at a high concentration for purification, and therefore, efficient. Purification can be performed. Therefore, the inclusion of arginine in the t-PA solution is extremely useful when formulating t-PA, and is also extremely useful for efficiently producing high-purity t-PA on an industrial scale.

Next, the present invention will be described in more detail with reference to experimental examples and examples, but the present invention is not limited to these examples. In addition, in the following Experimental Examples and Examples, t-PA purified from a human melanoma cell culture medium and a culture medium of Chinese hamster ovary (hereinafter, referred to as CHO) cells into which the t-PA gene was transferred by a genetic engineering means. As described above, t-PA obtained from human or animal tissue, t-PA purified from cell culture solution derived from human or animal tissue, or t-PA obtained by genetic engineering means other than the above, that is, t-PA. It may be a eukaryotic cell into which the gene has been transferred or t-PA or the like obtained from a microorganism such as Escherichia coli, Bacillus subtilis or yeast into which the t-PA gene has been transferred.

The activity of t-PA was determined by measuring 95% coagulated fibrinogen (plasminogen-containing about 50 casein units /
The fibrin plate method was performed using t-PA (WHO certified product) as a standard product.

(Production Example 1 of t-PA) Colene (Col
len) et al. (The Journal of Biological Chemistry (The J. Biol. Che
m. ) 256 (13), 7035-7041, 198.
Approximately 60 L of the crude t-PA culture solution produced by using human melanoma-derived cells according to 1) was added to Rijken.
Et al. (Thrombosis and Hemostasis (Thr
omb. Haemostas. ) 48 (3), 294-
296, 1982), and the purification operation was performed. That is, the culture solution was purified by Zn-chelate chromatography (13.5 × 17.5 cm), and then Con A
Sepharose chromatography (5 x 30 cm) was performed. 0.4M α-from ConA Sepharose column
T-PA eluted with a 2.0 M potassium thiocyanate solution containing D-methyl mannoside was concentrated with polyethylene glycol, and then equilibrated with 0.01 M phosphate buffer (pH 7.5) containing 0.25 M arginine hydrochloride. Gel filtration was performed with a column (7.5 × 90 cm) packed with the Sephaacrylic S200 (manufactured by Pharmacia) for purification t
-PA was obtained. The specific activity of 2.
5 × 10 ⁵ IU / mg, 18 million IU of purified t-PA
Got The obtained t-PA was dialyzed against distilled water at 4 ° C. overnight, lyophilized, and used in Experimental Examples and Examples described later.

(Production Example 2 of t-PA) Penn
ica et al. method (Nature 301 )
(20), 214-221, 1983) according to t-P
The A gene is prepared and the method of Kaufman and Sharp (Journal of Molecular Biology (J. Mol. Biol.) 1
59 , 601-621, 1982).
After transferring the PA gene into CHO cells, 40 L of crude t-PA culture medium produced using the CHO cells was obtained. This crude t-PA culture solution was purified in the same manner as in t-PA Production Example 1, and the specific activity was 1.7 × 10 ⁵ IU / mg, 12 million IU.
Of purified t-PA was obtained.

(Production Example 3 of t-PA) Colene (Col)
len) et al. (The Journal of Biology
Cal Chemistry (The J. Biol. Che
m. ) 256 (13), 7035-7041, 198.
Crude cells produced using human melanoma-derived cells according to 1)
About 60 L of t-PA culture solution was added to Rijken.
(Thrombosis and Hemocystus (Thr
omb Haemostas. ) 48 (3), 294-
296, 1982), and the purification operation was performed. That is, the culture solution was purified by Zn-chelate chromatography (13.5 × 17.5 cm), and then Con A
Sepharose chromatography (5 x 30 cm) was performed. 0.4 M α from ConA Sepharose column
T-PA eluted with 2.0 M potassium thiocyanate solution containing -D methyl mannoside was concentrated with polyethylene glycol, and then equilibrated with 0.01 M phosphate buffer (pH 7.5) containing 0.25 M arginine hydrochloride. Gel filtration was performed using a column (7.5 × 90 cm) packed with the modified Sephacryl S200 (Pharmacia). The eluted t-PA was concentrated with polyethylene glycol, and then Sephacryl S equilibrated with 0.01M phosphate buffer (pH 7.5) containing 0.25M arginine hydrochloride.
Again with a column packed with 200 (7.5 x 90 cm),
Gel filtration was performed to obtain purified t-PA. This series of purification
Depending on the process, the specific activity is 4.2 × 10 ⁵ IU / mg, 140
100,000 IU of purified t-PA was obtained. The obtained t-PA was 4
After dialysis against distilled water at ℃ overnight, lyophilize.
Used in the experimental examples and examples below.

[0016] examined the effect of arginine concentration on the solubility of (Experimental Example 1) t-PA. Purified t-obtained in Production Example 1
Each 5 mg of PA was weighed and dissolved in 0.5 ml of a solution having the arginine concentration shown in Table 1 to obtain t-P in the solution.
The activity of A was measured to examine the solubility of t-PA. t-P
When A was not completely dissolved and precipitation occurred, the supernatant activity was measured. The results are shown in Table 1 and FIG. 1 mM
Arginine solution of t-PA 0.2 mg / ml or more
Solubility was obtained.

[0017]

[Table 1]

[0018] (Experimental Example 2) purified t-PA obtained in Preparation Example 3
The effect of the type of arginine on the solubility of arginine was investigated in Experimental Example
Same as above. As a result, the solubility of t-PA was actually reduced.
The same results as in Test Example 1 were obtained.

Experimental Example 3 The influence of the type of arginine on the solubility of t-PA was examined. 1 mg of t-PA purified in Production Examples 1 and 2 was dissolved in 0.5 ml of a solution containing 25 mM of various arginines shown in Table 2, and the activity of t-PA in the solution was measured in the same manner as in Experimental Example 1. The results are shown in Table 2.
It was shown to.

[0020]

[Table 2]

[0021] investigated the solubility in a solvent system comprising a (Experimental Example 4) L-arginine hydrochloride and sodium chloride. The purified t-PA5 obtained in Production Example 1 was added to 0.5 ml of a solution containing L-arginine hydrochloride and sodium chloride and having its pH adjusted.
mg or 1 mg was dissolved and the t-PA activity of the solution was measured to examine the solubility of t-PA. The results are shown in Table 3 and Table 4.
It was shown to.

[0022]

[Table 3]

[0023]

[Table 4]

[0024] (Experimental Example 5) was dissolved preparation produced by toxicity tests following Examples 1 and 2 in physiological saline or water for injection, 5 mice and guinea pigs
After administration of 0,000 IU / Kg, no abnormality was observed.
Further, the same preparation of 200,000 IU / Kg was administered to rabbits and a pyrogen test was conducted, but all the preparations were negative.

As is clear from the above experimental examples, arginine is useful for increasing the solubility of t-PA.

[0026]

【Example】

[0027] (Example 1) the t-PA 5,000,000 IU L- arginine hydrochloride 21 mg sodium 173.9 mg purified gelatin phosphate 100.0 mg The above components were dissolved in distilled water for injection 10ml After aseptic filtration, 1.0 ml each was filled in a vial and freeze-dried to prepare a thrombolytic agent.

[0028] (Example 2) t-PA 5,000,000 IU L- arginine hydrochloride 52.5 mg Sodium phosphate 173.9 mg sodium chloride 64.3 mg human serum albumin 20 mg The above components weigher A thrombolytic agent was obtained and prepared in the same manner as in Example 1.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the relationship between the solubility of t-PA and the concentration of arginine.

Claims (3)

[Claims] 1. A tissue plasminogen activator is dissolved in a medicinal solvent in the presence of at least arginine or an acid addition salt thereof to give a tissue plasminogen activator concentration of at least 0.1 mg / ml. And arginine or its acid addition salt 1 mM-5
A method for producing a thrombolytic agent, which comprises 00 mM. 2. The concentration of tissue plasminogen activator is at least 0.1 mg / ml, and
The method for producing a thrombolytic agent according to claim 1, which contains 5 mM to 200 mM of arginine or an acid addition salt thereof. 3. The thrombus according to claim 1, wherein the tissue plasminogen activator is dissolved in a medicinal solvent in the presence of at least arginine or an acid addition salt thereof and freeze-dried. A method for producing a dissolving agent.