JPH0521551B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel plasminogen activator, a method for producing the same, and a thrombolytic agent containing the same as an active ingredient. More specifically, the double-stranded plasminogen activator collected from the tissue culture medium of human normal tissue-derived cells, the method for separating and purifying the same, and the pharmaceutical use of the same as a thrombolytic agent containing it as an active ingredient. Regarding. Today, as plasminogen activators, urokinase isolated and purified from urine or cultured kidney cells, and streptokinase collected from streptococci are in practical use as thrombolytic agents. However, since these have poor affinity for fibrin, large doses are often administered in order to obtain the necessary therapeutic effect, and it is known that side effects such as internal bleeding occur. That is, the plasmin produced in the circulating blood by these
They bind to plasmin inhibitors in the blood and are quickly deactivated, so in order to achieve therapeutic effects, it is necessary to administer large amounts of these to generate plasmin that exceeds the amount of plasmin inhibitors in the blood. However, when a large amount of plasmin is produced, it degrades fibrinogen and causes the side effect of bleeding tendency. On the other hand, if fibrin has a high affinity and plasmin can be generated on fibrin, fibrin can be degraded in small amounts without being affected by plasmin inhibitors in the circulating blood, and fibrinogen in the circulating blood can be degraded. The effect of decomposing is also weakened.
Under these circumstances, there is a demand for a thrombolytic agent that has high fibrin affinity, is used in small amounts, has high thrombolytic activity, and has few side effects. Previously, the present inventors searched tissue culture fluids of cells derived from various human normal tissues and found that they contained a substance with plasminogen activator activity different from urokinase. He succeeded in separating and purifying the substance and filed a patent application (Japanese Patent Application Laid-Open No. 59-51220). Subsequently, as a result of various studies on this substance that has plasminogen activator activity, it was discovered that this substance is a mixture of multiple proteins, and that they contain single-chain and double-chain plasminogen. The present invention was achieved based on the discovery that it consists of an activator. An object of the present invention is to provide a novel double-stranded plasminogen activator that has high fibrin affinity and is effective in small amounts. Another object is to provide a method for separating and purifying and producing the double-stranded plasminogen activator, and a novel thrombolytic agent containing the obtained double-stranded plasminogen activator as an active ingredient. That's true. For this purpose, the present inventors have developed a method for selectively producing double-stranded plasminogen activators, i.e., two plasminogen activators in a mixed state with single-stranded or double-stranded plasminogen activators. The present invention was completed by studying the method of converting into a chain plasminogen activator and the properties of a double-chain plasminogen activator. The double-chain plasminogen activator of the present invention can be used in cells derived from normal human tissues, such as human fetal kidney, intestine, lung, heart, ureter, skin, foreskin, and whole fetus, in a suitable growth medium. cells, cells derived from human placenta, or human kidney, intestine, lung, thyroid,
More preferable cells, such as cells derived from the heart, ureter, and skin, are obtained by separating a fraction having plasminogen activator activity from a tissue culture solution using cells derived from human fetal kidney, human fetal lung, or human fetal foreskin. This is then subjected to enzyme treatment or 10 to 80
It can be produced by incubating at a temperature of 5 to 10 days, preferably at 20 to 40°C, followed by separation and purification. In other words, these cells can be cultured using conventional culture methods used for culturing animal cells, such as "Tissue Culture Methods and Applications."
(PKKruse and MKPatterson Academic
Press New York San Francisco London
1973), the substances of the invention can be produced by contacting them with a solution containing a carbon source, a nitrogen source and, if necessary, inorganic salts or/and other additives. . Examples of additives to be allowed to coexist include amino acids, vitamins, peptides, sugars, and organic acids. Production of the substance of the present invention is usually carried out in 10 cells.
25°C to 40°C using 0.2ml or more of culture solution per 10,000 cells.
°C, preferably in the temperature range of 35 °C to 38 °C, from 6.0 to
It is carried out in the PH range of 8.0, preferably 7.0-7.4.
The production time is usually 4 to 30 days, but it is possible to exceed 30 days. Since the production rate of the substance of the present invention gradually slows down in the latter half of production, in the case of industrial production, the most efficient number of days is selected. Separation and purification methods from the culture solution include methods commonly used in protein chemistry, such as adsorption with carriers, ion exchange methods, fractional precipitation, gel filtration, electrophoresis, and various affinity chromatographies, especially specific methods. Methods using antibodies are preferable, and examples include fibrin-Sepharose column chromatography using fibrin-bound Sepharose, CM Sepharose column chromatography using carboxymethyl group-bonded Sepharose, and lysine-bound Sepharose column chromatography. lysine sepharose column chromatography using conjugated pharose, ligand exchange chromatography using zinc chelate cepharose, lectin column chromatography using cepharose conjugated with concanavalin A, and specific chromatography with the substance of the present invention. Examples include antibody affinity chromatography using bound antibodies, gel filtration using cross-linked dextran particles,
These can be used alone or in combination. The thus obtained plasminogen activator can be used not only as a medical thrombolytic agent but also as a drug to prevent the formation of blood clots by binding it to materials such as artificial blood vessels and artificial organs.
Another use is as a diagnostic agent for thrombosis, etc. The tissue culture medium of human normal tissue-derived cells used in the present invention can be obtained by culturing cells capable of producing plasminogen activator in various culture mediums.
Publication No. 107510, Japanese Unexamined Patent Publication No. 107511, Japanese Unexamined Patent Publication No. 107510
Examples include culture solutions described in JP-A-55-19001, JP-A-55-139323, and JP-A-57-5159. For typical culture methods, see Reference Examples 1 and 2.
For example: From the plasminogen activator mixture from the culture medium or from the single chain plasminogen activator to the double chain plasminogen of the present invention.
Activator conversion can be carried out at any step of the separation and purification, using enzyme treatment methods or simply 10-80
This can be achieved by incubating at a temperature of 0.degree. C., more preferably 20 to 40.degree. Enzyme treatment methods include direct enzyme action,
Alternatively, methods used in ordinary enzymatic reactions, such as a method of contacting with an immobilized enzyme, can be used. Examples of the enzyme include plasmin, kallikrein, and activated blood coagulation factor and factor F. As the immobilization carrier, carriers commonly used for enzyme immobilization, such as Sepharose, can be used. One example of a specific method for separating and purifying the plasminogen activator of the present invention is to pass a tissue culture medium or concentrated culture medium through a plasmin sepharose column, add ammonium sulfate, and separate the resulting precipitate. , dissolved in a rhodan ammonium solution to which sodium chloride has been added, passed through an anti-urokinase Ig-G Sepharose column, and adsorbed onto a Fiprin Sepharose column. The eluate obtained by using arginine as an elution solvent is further passed through an anti-urokinase Ig-G sepharose column, and then lyophilized and concentrated. The desired double-stranded plasminogen activator is obtained by gel-filtering the concentrated solution using Cephadex G-150 (registered trademark of Pharmacia). This substance is clearly a plasminogen activator because it does not dissolve fibrin that does not contain plasminogen, but dissolves fibrin that contains plasminogen. The physicochemical properties of the double-stranded plasminogen activator of the present invention thus obtained will be explained below. The titer was measured using the following method (the same applies to the following experiments). An agar-added fibrin plate prepared from 95% coagulated fibrinogen (plasminogen content: approximately 50 casein units/g coagulated protein) was used as a raw material. The measurement was carried out using the plate method using urokinase as a standard. A 0.067M solution of the substance of the present invention containing 1% gelatin, 0.1M sodium chloride and 0.1% sodium nitride
The solution of the substance of the present invention was diluted with Tris-HCl buffer (PH8.0) to give a concentration of 10 U/ml, which showed the same dissolution window as 10 IU/ml urokinase on a fibrin plate. a Molecular weight: 63000±10000 Sephadex G-150 equilibrated with 0.01M phosphate buffer (PH7.0) containing 1.5M sodium chloride, 0.1MEDTA, 0.1M arginine and 0.1% Twin 80 (Kao Atlas registered trademark). It was measured using the gel filtration method used. b Isoelectric point: 7.0 to 8.5 The isoelectric point was fractionated and measured by an isoelectric focusing method using amphotolite. c Affinity for fibrin: Add 20μ of the substance of the present invention (500U/ml) to 950μ of plasminogen-free fibrinogen solution (0.2%) dissolved in physiological saline, and further add 50μ of throbin (30U/ml). and leave it at room temperature for 1 hour. The resulting fibrin is collected and dehydrated,
Wash with saline. The substance of the present invention in fibrin is extracted with 1 ml of 2M ammonium thiocyanate solution. As a result, the substance of the present invention has approximately 70%
% was incorporated into the fibrin clot. On the other hand, when uroginase (500 IU/ml) was used as a control, no uptake was observed. d Affinity for concanavalin A: 2 ml of the substance of the present invention (30 U/ml) was dissolved in physiological saline and adsorbed on a column (0.5 x 4 cm) of concanavalin A-Sepharose (manufactured by Pharmacia).
When washed with 1M sodium chloride solution, almost
100% adsorbed. e Optimum pH: 7 to 9.5 10 to 50μ of the present substance dissolved in physiological saline.
50μ of plasminogen (8CU/ml) dissolved in saline containing % glycerin and 0.05M citrate buffer containing 0.10M sodium chloride (PH5.0,
6.0), phosphate buffer (PH6.0, 7.0, 8.0) or glycine-sodium hydroxide buffer (PH8.0, 9.0,
10.0, 11.0) (PH5.0, 6.0, 7.0, 8.0, 9.0, 10.0,
11.0) in 100μ aliquots and pre-incubate at 37℃ for 30 minutes. Next, Boc-Glu-Lys dissolved in 0.15M Tris-HCl buffer (PH8.0)
After adding 500μ of -Lys-MCA and further incubating at 37°C for 15 minutes, 1 ml of acetic acid was added to stop the reaction, and the aminomethylcoumarin produced was measured by fluorescence to determine the ultimate pH. f Stability: The substance of the present invention (100 U/ml) dissolved in physiological saline containing 0.02% Tween 80 was subjected to heat treatment at 55°C for 3 hours, but no decrease in activity was observed. g Hydrolysis activity for various synthetic substrates: 50μ of the substance of the present invention (100U/ml) was mixed with 0.05M Tris-HCl buffer (PH) containing 0.1M sodium chloride.
8.0) Add 0.1mM of various substrates dissolved in 450μ,
Incubate for 15 minutes at 37°C. The reaction was stopped by adding 0.5 ml of 20% acetic acid, and the aminomethylcoumarin produced was measured at an excitation wavelength of 370 nm, a slit width of 5 nm, a fluorescence wavelength of 460 nm, and a slit width of 5 nm to determine the water-dissolving activity. The results are shown in Table 1.

[Table] ※※ -:＜2%
h Effects of various protease inhibitors 50μ of the substance of the present invention (100U/ml), 50μ of solutions of various proteinase inhibitors, and 300μ of 0.05M Tris-HCl buffer (PH8.0) containing 0.1M sodium chloride.
Add and react at 37°C for 5 minutes. Next, for the substance of the present invention, 0.1mM Boc-Phe-Ser-Arg
-Add 100μ of MCA (Peptide Institute) and 0.1mM Glt-Gly-Arg-MCA (Peptide Institute) for urokinase, and add
Let it react for a minute. Then add 0.5ml of 20% acetic acid,
The reaction was stopped, and the aminomethylcoumarin produced was measured using an excitation wavelength of 370 nm, a slit width of 2 nm, a fluorescence wavelength of 460 nm, and a slit width of 2 nm to determine the water-splitting activity. The concentration of the protease inhibitor that inhibits the activity of the substance of the present invention by 50% (IC ₅₀ ) was determined, and the results are shown in Table 2.

[Table] i Behavior in response to reduction treatment The substance of the present invention (0.5 mg protein/ml) was mixed in equal amounts with 2% sodium dodecyl sulfate, 2% β-mercaptoethanol, and 40% glycerin in Tris-HCl buffer (PH 6.8). , heat treatment at 100°C for 5 minutes. This solution was treated with BDS-
It was subjected to polyacrylamide gel electrophoresis. It was confirmed that the substance of the present invention was resolved into two bands with molecular weights of about 33,000 and about 35,000. The molecular weight was determined using a calibration kit (manufactured by Pharmacia Fine Chemicals). j Activity enhancement by fipurin Plasminogen (10CU/ml) solvent 50μ,
The substance of the present invention 50μ, 0.1mM Boc-Val-Leu-
Lys-MCA100μ and thrombin (2U/ml)
100μ of the solution is sequentially added to 200μ of 0.05M Tris-HCl buffer (PH7.5) containing 0.1M sodium chloride in which 0.05% fipurinogen is dissolved, and the mixture is reacted at 25°C for 1 hour. The reaction was stopped with 500μ of 20% acetic acid, and the water-splitting activity was determined in the same manner as in test g. Note that a comparison was made with a case in which no fibrinogen was added. The results are shown in Table 3.

[Table] Based on the above properties, the double-chain plasminogen activator of the present invention is a novel substance different from urokinase derived from human urine or renal cell tissue culture. Next, FIG. 1 shows the results of measuring the thrombolytic agent of the double-stranded plasminogen activator of the present invention by the Chandra loop method [Quart. Exp. Physiol. 46, 1 (1961)]. Incidentally, fresh human blood was used, and the blood clot formation time was 30 minutes, and the blood clot dissolution time was 4 hours. As a result, it was confirmed that the thrombolytic activity of the double-chain plasminogen activator of the present invention was 30 times stronger than that of Urokinaza. Therefore, the plasminogen activator of the present invention is extremely useful as a thrombolytic agent that can produce a strong thrombolytic effect with a small amount of administration. The substance of the present invention can be administered intravascularly, particularly intravenously at the site of thrombus occurrence, and is usually preferably administered intravenously.The dosage varies depending on the patient's condition, but is in the range of 50 to 1,000,000 units per day. It can be administered with As for the method of intravenous administration, administration by injection is preferable, and it can also be administered after being dissolved in an infusion or the like. Formulation example 1 Inventive substance 12,000 units Purified gelatin 20 mg Mannitol 100 mg Sodium chloride 7.8 mg Sodium phosphate 15.4 mg Dissolve the above ingredients in 2 ml of distilled water for injection, place in a sterile vial, and stand at -30°C to -40°C for 2 hours. Freeze, primary dry at -30℃+20℃, vacuum degree 0.05 to 0.1 Torr for 35 hours, then 30℃, vacuum degree 0.01 to 0.05
A vial for injection was prepared by secondary drying at Torr for 5 hours. Before use, this product is dissolved in 500 ml of physiological saline or glucose injection and injected intravenously. Formulation Example 2 Inventive substance 6000 units Albumin 5 mg Mannitol 25 mg Sodium chloride 1.95 mg Sodium phosphate 3.85 mg An injection vial was produced in the same manner as Formulation Example 1 using the above ingredients. Reference example 1 Human fetal kidney cells were implanted in a 100 mm plastic container at a density of 7 x 10 ⁴ cells/ml, and incubated at 37°C.
10 as a growth medium in air containing 5% carbon dioxide.
Add 10 ml of medium MEM containing % fetal bovine serum, allow sufficient growth, wash with physiological saline, and add 20 ml of production medium consisting of medium 199 containing serum-free 0.5% lactalbumin hydrolyzate and 0.8% fumaric acid. Change it to a new one. After holding for 7 days, the culture medium is replaced with fresh production medium and the culture solution containing the substance of the present invention is collected. Reference example 2 Human fetal lung cells in a 500ml spinner flask
They were implanted at a density of 10 ⁵ cells/ml with a concentration of 2.5 mg/ml Cytodex (bead carrier for cell culture, registered trademark of Pharmacia), and grown at 37°C in air containing 5% carbon dioxide with 10% growth medium. Add 300 ml of medium MEM containing fetal bovine serum and turn at 60 rpm.
Culture in suspension while stirring at a rotation speed of . After culturing for 8 days and allowing the cells to proliferate sufficiently, wash the bead carrier to which the cells have adhered with physiological saline and add serum-free medium containing 0.5% lactalbumin hydrolyzate.
199 Change to 300 ml and culture while stirring at 60 rpm. The culture medium containing the substance of the present invention is collected every 5 days while replacing the medium. Example 1 The single-chain plasminogen activator isolated and purified by culturing in Reference Example 1 in the presence of aprotinin was added to 1% human serum albumin and
Dissolve in 0.01M phosphate buffer (PH7.4) containing 0.14M sodium chloride and adjust to 250U/ml. Add 300μ of plasmin solution ⁽ Note ¹⁾ to 200μ of this solution,
React at ℃. After a certain period of time, add 200μ of aprotinin buffer solution to neutralize plasmin activity.
Stop the reaction. Using 70μ of this solution, 0.1mM synthetic substrate Boc
The hydrolysis activity of -Phe-Ser-Arg-MCA ⁽ Note ²⁾ was measured. The titer of the substance of the present invention before and after treatment was measured.
The results are shown in Figure 2. As a result of plasmin treatment,
100% of the inventive substance was obtained. Furthermore, no decrease in activity was observed before and after plasmin treatment. In addition, as a result of subjecting the reaction solution reacted for 20 minutes or more to the reduction treatment using β-mercaptoethanol shown in section (), 100% decomposition was achieved. This shows that plasmin treatment completely converts it into double-stranded plasminogen activator. Note 1 A commercially available plasmin solution (manufactured by Midori Juji Co., Ltd.) was treated with an anti-urokinase Ig-G sepharose column and a lysine sepharose column to prepare a urokinase-free plasmin solution, which was diluted with 0.04 CU of the above phosphate buffer. It was used after being diluted to /ml. Note 2 The synthetic substrate Boc-Phe-Ser-Arg-MCA is
It is hardly hydrolyzed by single-chain plasminogen activator. Example 2 A solution of 3000 U/ml of single-stranded and double-stranded plasminogen activator obtained by concentrating the culture solution produced in Reference Example 1 was mixed with 0.15 M sodium chloride.
Dissolve in 0.02M Tris-HCl buffer. This solution 2
ml and 1 ml of plasmin-cephalose ⁽ note ⁾ ,
React at 25°C for 5 hours. After the reaction, the plasmin-cephalose was removed, and the double-stranded plasminogen activator content was measured from the hydrolysis activity for the synthetic substrate and the results of the reduction treatment in the same manner as in Conversion Example (1). As a result, plasmin-cephalose treatment resulted in 100% double-stranded plasminogen.
It was confirmed that it was converted to an activator. (Note) Plasmin and cepharose were prepared using the following method. 1 ml of human plasminogen (100 CU/ml) is mixed with 0.1 ml of urokinase sepharose (100 IU/ml), reacted overnight at room temperature, and converted to plasmin. After removing urokinase cephalose from this plasmin solution, it is reacted with cepharose activated with Promcyan (BrCN) to produce plasmin cepharose. When we measured the activity of this using a synthetic substrate (Boc-Glu-Lys-Lys-MCA), we found that it was approximately 0.5 CU per ml of resin.
activity. Example 3 Ammonium sulfate is added to cell culture solution 3.6 derived from human fetal foreskin to 60% saturation and stirred at 0°C for 2 hours.
The resulting precipitate was collected by centrifugation at 5000 rpm for 30 minutes, and added with 0.15M sodium chloride and 0.1% Twin 80.
The solution is dissolved in 0.02M Tris-HCl buffer (PH 7.5) containing 0.02M Tris-HCl buffer (PH7.5), and dialyzed overnight at 4°C against a solution having the same composition as this solution. Next, after incubating at 25°C for one week, this was loaded onto a zinc sepharose column (2.6φ
×6cm), 50mM imidazole,
Contains 0.5M Sodium Chloride and 0.1% Twin 80
Elute with 0.05M Tris-HCl buffer (PH7.5).
The volume of the obtained eluate was 156 ml, the activity of the substance of the present invention was 173 U/ml, and the specific activity was 850 U/A280. The resulting solution was diluted 10 times with 0.02M Tris-HCl buffer (PH7.6) containing 0.1% Twin 80, and a lysine sepharose column ( 1.6 φ Elute. The volume of the obtained eluate was 20.5 ml, the activity of the substance of the present invention was 103 U/ml, and the specific activity was 21000 U/A280. This material was subjected to the reduction treatment shown in () and SDS
As a result of measuring the content of the present invention substance before and after treatment by electrophoresis, it was found that after treatment at 25℃ for one week, almost
It can be seen that 100% of the substance of the present invention was obtained.

[Brief explanation of the drawing]

Figure 1 is a graph showing the relationship between the concentration of the substance of the present invention and thrombolysis rate, and Figure 2 is a graph showing the relationship between plasmin treatment time and synthetic substrate degrading activity and fibrin degrading activity of single-chain plasminogen activator. It is a graph.

Claims (1)

[Scope of Claims] 1. A double-stranded plasminogen activator having the following properties, which was collected from the tissue culture medium of cells derived from human normal tissue. a Molecular weight: 63000±10000 b Isoelectric point: 7.0-8.5 c Affinity for fibrin: Yesd Affinity for concanavalin A: Yese Optimum pH: 7-9.5 f Stability: Stable after 3 hours treatment at 55℃g Behavior in response to reduction treatment: Degradation h Hydrolytic activity towards synthetic substrates: As shown in Table 1 2 Fractions containing plasminogen activator are separated from the tissue culture fluid of human normal tissue-derived cells, and this is treated with enzymes or A method for producing a double-stranded plasminogen activator having the following properties, which comprises incubating at 10 to 80°C and then purifying it. a Molecular weight: 63000±10000 b Isoelectric point: 7.0-8.5 c Affinity for fibrin: Yesd Affinity for concanavalin A: Yese Optimum pH: 7-9.5 f Stability: Stable after 3 hours treatment at 55℃g Behavior against reduction treatment: Degradation h Hydrolytic activity against synthetic substrates: As shown in Table 1 3 A thrombolytic agent containing a double-stranded plasminogen activator having the following properties as an active ingredient. a Molecular weight: 63000±10000 b Isoelectric point: 7.0-8.5 c Affinity for fibrin: Yesd Affinity for concanavalin A: Yese Optimum pH: 7-9.5 f Stability: Stable after 3 hours treatment at 55℃g Behavior against reduction treatment: Decomposition h Hydrolysis activity against synthetic substrates: As shown in Table 1