NOVEL PURIFIED PLASMINOGEN ACTIVATOR, PROCESS FOR ITS PRODUCTION AND THROMBOLYTIC COMPOSITION CONTAINING IT

Abstract: A plasminogen activator obtained from a tissue cultured liquor of human normal tissue derived cells, e.g., human embryonic derived cells, human placenta derived cells etc., and having the following properties: a) molecular weight measured by gel filtration: 63,000 ± 10,000, b) isoelectric point : 7.0 - 8.5, c) affinity to fibrin: present, d) affinity to concanavalin A: present, e) optimum pH: 7 - 9.5, and f) stability : does not lose activity at 60°C for 10 hours but loses about 5% of activity at pH 2-3 at 98°C for a minute; a process for the production of the aforesaid plasminogen activator; and a thrombolytic composition containing the aforesaid plasminogen activator as an active ingredient are disclosed.

Description: Field of the Invention This invention relates to a novel purified plasminogen activator, a process for the production thereof and a thrombolytic composition containing it as an active ingredient. More specifically, relates to a plasminogen activator obtainable from a tissue cultured liquor of human normal tissue derived cells, a process for obtaining the activator in substantially pure form by separation and purification thrombolytic composition containing the activator. Background of the invention At present, urokinase separated and purified from urine or cultured liquor of kidney cells and streptokinase collected from cultured liquor of Streptococci are plasminogen activators employed in practical use as thrombolytic agents. However, since urokinase and streptokinase possess poor affinity to fibrin, it is frequently necessary to administer them in large amounts in order to obtain the required effect on treatment. When large doses are administered, side effects such as gastro-internal hemorrhage are manifested. Under such circumstances, a thrombolytic agent having thrombolyzing activity when administered in a small dose and having only low level of side effects such as causing a gastro-internal hemorrhage has been eagerly sought. In recent years, a plasminogen activator separated and purified from a tissue cultured liquor of human melanoma cells has been proposed to serve similar purposes (see Japanese Patent Application (OPI) No. 28009/1982). However, since tumor cells are used as a starting material and these are problems with antigenicity and carcinogenicity, such a plasminogen activator cannot be presented for practical use Also from FR-A-2,454,809 a plasminogen activator is known which, however, reacts with antiurokinase antibody. The substance of WO-A-81/01417 derived from urine or culture medium employing a fibrin containing adsorptive matrix. It is mentioned that the disclosed substance is a precursser of urokinase. It, therefore, shows cross reactivity with antiurokinase antibodies. In Biochem. Biophys. Acta 1982, 717(2), 327-336, a further substance is disclosed, which derived from kidney tissue. It was identified as another type of plasminogen activator having an apparent molecular weight of 54000. Biochem. Biophys. Acta 1979, 140-153, describes a plasminogen activator isolated from uterine tissue showing a molecular weight of 64000 as determined by gel filtration. This product, however, would not be separated by ion exchange chromatography nor by affinity chromatography. It had to be purified by employing chelated Zn²⁻ containing materials. The present inventors have discovered, as the result of their study on tissue cultured liquors of human normal tissue derived cells, that a substance having plasminogen activator activity different from urokinase is contained therein, and they are successfully separated and purified it, thereby having accomplished this invention. The plasminogen activator of this invention is separated from a tissue cultured liquor of normal tissue derived cells which are selected from cells derived from human embryonic lungs and thus does not have the above-described drawbacks of the plasminogen activator derived from melanoma cells. Summary of the invention Accordingly, an object of this invention is to provide a novel purified plasminogen activator having high affinity to fibrin and exhibiting a thrombolytic effect when administered in a small dose. Another object of this invention is to provide a process for the production of said plasminogen activator by separation and purification thereof. A further object of this invention is to provide a novel thrombolytic composition containing the thus obtained plasminogen activator as an active ingredient and use thereof. This invention provides (1) a purified plasminogen activator obtainable by separation and purification from a tissue cultured liquor of human normal tissue derived cells which are selected from cells derived from human embryonic lungs and having the following properties: a) molecular weight measured by gel filtration: 63,000 ± 10,000 b) isoelectric point: 7.0-8.5, c) affinity to fibrin: present, d) affinity to concanavalin A: present, e) optimum pH: 7-9.5, and f) stability: does not lose activity at 60°C for 10 hours but loses about 5% of activity at pH 2-3 at 98°C for a minute, g) antiurokinase antibody: no reaction, h) separation and purification with column chromatography utilizing fibrin-bonded agarose (fibrin Sepharose®): possible, i) separation and purification with column chromatography utilizing carboxymethyl group-bonded agarose (CM Sepharose®): possible. This invention also provides (2) a process for the production of the aforesaid plasminogen activator which is characterized by separating a fraction containing a plasminogen activator from a tissue cultured liquor of human normal tissue derived cells which are selected from cells derived from human embryonic lungs and purifying it; and (3) a thrombolytic composition which contains the aforesaid plasminogen activator as an active ingredient. Brief description of the drawings Fig. 1 of the Drawing is a graph showing the optimum pH region for the substance of this invention and Fig. 2 of the Drawing is a graph plotting the concentration against degree of thrombolysis for urokinase and the plasminogen activator substance of this invention, respectively. Detailed description of the invention and preferred embodiments The plasminogen activator of this invention can be produced by separating and purifying it from a tissue cultured liquor using human normal tissue derived cells such as cells derived from human embryonic lungs in an appropriate growth medium. The tissue cultured liquor of human normal tissue derived cells employed in this invention may be any of those obtained by cultivating cells capable of producing the plasminogen activator in various suitable culture media, and examples thereof include such culture media as those described in Japanese Patent Application (OPI) Nos. 107510/1979, 107511/1979, 19001/1980 and 139323/1980, and Japanese Patent Publication No. 5159/1982. More specifically, the plasminogen activator of this invention can be produced by contacting the cells which are proliferated in a conventional manner for cultivation of animal cells (e.g., as described in Tissue Culture Methods and Applications , edited by P. F. Kruse et al, Academic Press, New York, San Francisco, London (1973)) with a nutrient solution containing carbon sources, nitrogen sources and optionally inorganic salts and/or other additives such as amino acids, vitamins, peptides, hormones, saccharides and organic acids. Usually, the production of the plasminogen activator is performed using at least 0.2 ml of the nutrient solution per 100,000 cells at a temperature of from 25 to 40°C, preferably from 35 to 38°C. During the production, the pH of the nutrient solution is adjusted to 6 to 8, preferably 7.0 to 7.4. The period required for the production is usually 4 to 30 days, but may exceed 30 days. Since the speed of production gradually decreases in the later stage of production, the period which provides the best efficiency is chosen for commercial production. Representative cultivating methods are illustrated in Reference Examples 1 and 2 described hereinbelow. The overall method for separating and purifying the plasminogen activator from the cultured liquor involves steps conventionally employed in protein chemistry, for example, adsorption using carriers, ion exchange, fractional precipitation, gel filtration, electrophoresis, various types of affinity chromatography especially those using specific antibodies, etc. There can, for example, be used a fibrin Sepharose column chromatography utilizing a fibrin-bonded agarose, a CM Sepharose column chromatography utilizing a carboxymethyl group-bonded agarose, a ligand-exchange chromatography utilizing a zinc chelate agarose, a lectin column chromatography utilizing a concanavalin A-bonded agarose, an affinity chromatography using antibodies specific to the plasminogen activator of this invention, and a gel filtration method utilising crosslinked dextran particles. They may be employed either alone or in combination. One example of a specific overall method for separating and purifying the plasminogen activator involves adding ammonium sulfate to a tissue cultured liquor or a concentrated cultured liquor, separating the formed precipitates, dissolving them in an ammonium thiocyanate solution containing sodium chloride, passing the solution through an anti-urokinase Ig-G Sepharose column and adsorbing the solution onto a fibrin Sepharose column. Thereafter, an eluate obtained by using arginine as an eluent is further passed through an anti-urokinase Ig-G Sepharose column, and freeze dried. The thus obtained powder is dissolved in water and is then gel filtered using Sephadex G-150 (registered trademark by Pharmacia Co.) to obtain the intended plasminogen activator. There is no doubt that the present substance is a plasminogen activator because it does not dissolve plasminogen-free fibrin, but dissolves plasminogen-containing fibrin. The physical and chemical properties of the plasminogen activator of this invention thus obtained are described below. The titer measurement was carried out by the following procedures (this also applies to the experiments described hereinbelow). Using an agar (fibrin-added plate prepared by using 95% clotable fibrinogen (plasminogen content: about 50 casein units/g clotable protein) as a starting material, the measurement was carried out by a plate method employing urokinase as the standard. A solution of the substance of this invention was diluted with a 0.067 M tris-HCI buffer (pH 8.0) containing 1% gelatin, 0.1 M sodium chloride and 0.1% sodium azide and the concentration of the substance of this invention exhibiting the lyzing zone same as that of 10 IU/ml of urokinase on the fibrin plate was designated as 10 IU/ml. a) Molecular weight 63000 ± 10000 This was measured by gel filtration using Sephadex G-150 equilibrated with a 0.01 M phosphate buffer (pH 7.0) containing 1.5 M sodium chloride, 0.1 M EDTA, 0.1 M arginine and 0.1% Tween 80 (registered trademark by Kao Atlas). When measured by SDS (sodium dodecyl sulfate) electrophoresis (nonreduction), it was about 70,000. b) Isoelectric point: 7.0-8.5 This was measured by isoelectric point electrophoresis using an ampholyte at about 0°C. c) Affinity to fibrin 20 µl of the substance of this invention (500 U/ml) was added to 950 µl of physiological saline containing 0.2% of plasminogen-free fibrinogen, and then 50 µl of thrombin (30 U/ml) was added thereto. The resulting solution was allowed to stand for 1 hour at room temperature, whereby fibrin was formed. The fibrin was separated, dehydrated and washed with physiological saline. By extracting the substance of this invention from the fibrin with 1 ml of a 2 M ammonium thiocyanate solution, it was found that about 70% of the substance was incorporated in the fibrin. For comparison, when urokinase (500 U/ml) was used, it was not incorporated in the fibrin at all. d) Affinity to concanavalin A When 2 ml of the substance of this invention (30 U/ml) was dissolved in physiological saline, absorbed onto a column (0.5 × 4 cm) of concanavalin A Sepharose (manufactured by Pharmacia Co.), and washed with a 1 M sodium chloride solution, nearly 100% of the substance was adsorbed. e) Optimum pH: 7-9.5 To 50 µl of the substance of this invention dissolved in physiological saline were added 50 µl of plasminogen (8 CU/ml) dissolved in physiological saline containing 10% glycerin and 100 µl of one of a 0.05 M citrate buffer (pH 5.0 or 6.0) containing 0.10 M sodium chloride, a phosphate buffer (pH 6.0, 7.0 or 8.0) or a glycine-sodium hydroxide buffer (pH 8.0, 9.0, 10.0 or 11.0) (i.e., seven buffers, each at a different pH of 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or 11.0), and each was preincubated at 37°C for 30 minutes. Thereafter, 500 µl of Boc-Glu-Lys-Lys-MCA (Peptide Institute, Inc.) dissolved in a 0.15 M tris-HCI buffer (pH 8.0) was added, and after incubation at 37°C for 15 minutes, the reaction was terminated by adding 1 ml of acetic acid, and the formed aminomethylcoumarin was measured by fluorometry to determine the optimum pH. The results of these measurements are shown in Fig. 1. f) Stability (1) To the substance of this invention dissolved in physiolological saline (100 U/ml) was added human serum albumin at a proportion of 1 to 10 mg/ml, and a virulying treatment at 60°C for 10 hours (see Gellis S. S. et al, J. Clin. Invest ., 27 239 (1943)) was carried out. A reduction in activity was not observed. (2) A glycine hydrochloride buffer containing the substance of this invention (100 v/ml) having a pH of 2-3 was prepared, and then a virulyzing treatment at 98°C for a minute (see Krugman S. et al., J. Inf. Dis., 122 432 (1970)) was carried out, deactivation by about 5% was observed. g) Hydrolysis activity on various organic substrates To 50 µl of either the substance of this invention (100 U/ml) or urokinase (120 IU/ml) was added 0.1 mM of each of various substrates dissolved in 450 µl of a 0.05 M tris-HCI buffer (pH 8.0) containing 0.1 M sodium chloride, and each reaction was effected at 37°C for 15 minutes. The reaction was terminated by adding 0.5 ml of 20% acetic acid, and then measurement for formed aminomethylcoumarin was carried out at an excitation wavelength of 370 nm, a slip width of 5 nm, an emission wavelength of 460 nm and a slit width of 5 nm to determine the hydrolysis activity. The results are shown in Table 1. h) Influence of various proteinase inhibitors To 50 µl of either the substance of this invention (100 U/ml) or urokinase (100 IU/ml) were added 50 µl of one of a number of solutions of various proteinase inhibitors and 300 µl of a 0.05 M tris-HCI buffer (pH 8.0) containing 0.1 M sodium chloride, and each reaction was effected at 37°C for 5 minutes. Thereafter, 100 µl of either of 0. 1 mM of Boc-Phe-Ser-Arg-MCA (Peptide Institute, Inc.) for the substance of this invention or 0.1 mM of Glt-Gly-Arg-MCA (Peptide Institute, Inc.) for the urokinase was added, and each reaction was effected at 37°C for 60 minutes. Thereafter, 0.5 ml of 20% acetic acid was added to terminate the reaction, which was then measured for the formed aminomethylcoumarin at an excitation wavelength of 370 nm, a slit width of 2 nm, an emission wavelength of 460 nm and a slit width of 2 nm to determine the hydrolysis activity. The concentrations (IC₅₀) of the proteinase inhibitors which inhibit 50% of the activity of the substance of this invention and urokinase were determined and the results are shown in Table 2. Further, the substance of this invention and urokinase were completely inhibited by diisopropyl fluorophosphate (DFP) which is an inhibitor for serine proteinase. i) Enhancement of activity with fibrin To 200 µl of a 0.05 M tris-HCI buffer (pH 7.5) containing 0.05% offibrinogen and 0.1 M sodium chloride were added subsequently 50 µl of a plasminogen solution (10 CU/ml), 50 µl of either the substance of this invention (5 U/ml) or urokinase 15 IU/ml), 100 µl of a 0.1 mM Boc-Val-Leu-Lys-MCA solution and 100 µl of a thrombin solution (2 U/ml), and the reaction was carried out at 25°C for 1 hour. The reaction was terminat by adding 500 µl of 20% acetic acid, and then hydrolysis activity was measured in the same manner as in the test (g) above. Further, the same procedure as above was repeated except fibrinogen was not added. The results are shown in Table 3. As evident from the various properties described above, the plasminogen activator of this invention is a novel purified substance different from urokinase derived from human urine or a tissue cultured liquor of kidney cells. Thereafter, the thrombolytic activity of the plasminogen activator of this invention was measured by the Chandler's loop method ( Quart. J. Exp. Physiol., 46, 1 (1961)). The degree of thrombosis as compared with that of urokinase is shown in Fig. 2. The blood used was human fresh blood, the thrombus forming time was 30 minutes, and the thrombolysis time was 4 hours. In Fig. 2, curve 1 is for the substance of this invention and curve 2 is for urokinase. As a result, it was confirmed that the thrombolytic activity of the plasminogen activator of this invention was 30 times as strong as that of urokinase. Therefore, the plasminogen activator of this invention is extremely useful as a thrombolytic agent which provides a strong thromoolytic effect upon administration of a small dose. The substance of this invention is preferably administered intravenously, and the dose, although varying depending on the condition of the patient, may be in the range of 200-1,000,000 units per day. The method for intravenous administration is preferably by injection, or it may be administered by dissolving in a transfusion medium etc. The substance of this invention can be formulated into e.g., an injectable preparation, for example, by mixing the substance with a conventional excipient for injection, a buffer (e.g., phosphates, sodium chloride, etc.), an isotonic agent, a filler (e.g., mannitol, dextran, cyclodextrin, etc.) a stabilizer (e.g., gelatin, albumin, sulfites, etc.) or the like, dissolving the mixture in distilled water for injection, and freeze-drying and/or vacuum-drying the solution to obtain a drug composition which is filled in a vial for injection. Other applications of the plasminogen activator, in addition to medical use as a thrombolytic agent, are for preventing the formation of a thrombus by, for example, combining it with materials such as artificial blood vessels, artificial organs etc., or as a diagnostic agent for thrombosis etc. Reference Example 1 Human embryonic lung cells were implanted to a 500-ml spinner flask at a density of 10⁵ cells/ml together with Cytodex I (bead carrier for cell culture, registered trademark by Pharmacia Co.) at a concentration of 2.5 mg/ml, and suspension cultured by using 300 ml of Medium MEM containing 10% fetal calf serum as a growth medium at 37°C in air containing 5% carbon dioxide, while stirring at a rotation of 60 rpm. After sufficient proliferation of the cells by their cultivation for 8 days, the bead carrier to which the cells had been adhered was washed with physiological saline, and the medium was replaced by 300 ml of serum-free Medium 199 containing 0.5% lactoalbumin hydrolysate and cultivation was continued for 25 days with stirring at a rotation of 60 rpm, while the medium was replaced on every fifth dry. Thus the cultured liquor containing the substance of this invention was recovered. Example 1 One liter of a human embryonic lung tissue cultured liquor as obtained by Reference Example 2 was passed through an anti-urokinase Ig-G Sepharose column, and then adsorbed onto a fibrin Sepharose column (1.5 × 10 cm). After washing sufficiently with a 0.5 M sodium chloride solution containing 0.1% Tween 80, a 0.5 M arginine solution containing 0.1% Tween 80 was used to elute and collect 50 ml of a fraction containing the activity of the substance of this invention. The activity of this solution was 62 U/ml and the specific activity was 950 U/A280. This solution was dialyzed against physiological saline containing 0.1% Tween 80, then adsorbed onto a concanavalin A Sepharose column (1 × 20 cm), and washed with a 0.01 M phosphate buffer (pH 7.0) containing 1 M sodium chloride and 0.1% Tween 80, followed by eluting the substance of this invention according to a linearly gradient elution method using the above buffer at initial stage while continuously changing the composition of buffer to a 0.01 M phosphate buffer (pH 7.0) containing 0.4 M methylmannoside, 2 M ammonium thiocyanate and 0.1% Tween 80. The obtained solution was 25 ml in liquid volume, and the activity was 98 U/ml and its specific activity was 5500 U/A280. After dialysis, the dialysate was concentrated by ultrafiltration and gel filtered by Sephadex G-150 to recover a 15 ml fraction containing activity. The activity was 135 U/ml and the specific activity was 12500 U/A280. Example 2 The purified substance of this invention was dissolved in physiological saline at the concentration of 1 mg/ml and mixed with Freund's Complete Adjuvant in the mixing ratio of 1/1 by volume. 2 ml of the resulting mixture was subcutaneously injected to a rabbit five times at an interval of one week, and an antiserum specific to the substance of this invention was obtained from the rabbit. The antiserum was purified by a Protein A Sepharose 4B column, and an anti-substance of this invention Ig-G was separated. This Ig-G was combined to CNBr-activated Sepharose 4B in a proportion of 1 mg per ml of gel to prepare Ig-G Sepharose. The above ingredients were dissolved in 2 ml of distilled water for injection, charged into a sterile vial, preliminarily freeze dried at -30°C to -40°C for 2 hours, then subjected to primary drying at -30°C to +20°C with a degree of vacuum of 0.05-0.1 Torr for 35 hours and to secondary drying at 30°C with a degree of vacuum of 0.01-0.05 Torr for 5 hours to prepare a vial for injection. On use, the above is dissolved in 500 ml of physiological saline or glucose solution for injection instilled intravenously. The above ingredients were used similarly as in Preparation Example 1 to prepare a vial for injection. As disclosed hereinbefore, the plasminogen activator of the present invention has a high affinity for fibrin so that the activator preferentially deposits on a thrombus composed of fibrin. Since plasminogen is converted into plasmin (which is capable of dissolving fibrin) on the surface of the thrombys due to the presence of the deposited plasminogen activator of this invention, the plasmin thus formed is less susceptible to being trapped by plasmin inhibitor present in the blood plasma as compared to the case using urokinase. Urokinase, which is not preferentially attracted to fibrin, primarily activates plasminogen present in the blood. The produced plasmin since it is present inblood plasma, is susceptible to being trapped. Accordingly, the plasminogen activator of the present invention can be utilized in relatively low dosages without causing side effects such as gastro-internal hemorrhage.

Claims: 1. A purified plasminogen activator obtainable by separation and purification from a tissue cultured liquor of human normal tissue derived cells which are selected from cells derived from human embryonic lungs and having the following properties: a) molecular weight measured by gel filtration: 63,000 ± 10,000, b) isoelectric point: 7.0-8.5, c) affinity to fibrin: present, d) affinity to concanavalin A: present, e) optimum pH: 7-9.5, and f) stability: does not lose activity at 60°C for 10 hours but loses about 5% of activity at pH 2-3 at 98°C for a minute, g) antiurokinase antibody: no reaction, h) separation and purification with column chromatography utilizing fibrin-bonded agarose (fibrinSepharos®): possible, i) separation and purification with column chromatography utilizing carboxymethyl group-bonded agarose (CM Sepharose®): possible. 2. A process for the production of a plasminogen activator having the following properties: a) molecular weight measured by gel filtration: 63,000 ± 10,000, b) isoelectric point: 7.0-8.5, c) affinity to fibrin: present, d) affinity to concanavalin A: present, e) optimum pH: 7-9.5, and f) stability: does not lose activity at 60°C for 10 hours but loses about 5% of activity at pH 2-3 at 98°C for a minute, g) antiurokinase antibody: no reaction, h) separation and purification with column chromatography utilizing fibrin-bonded agarose (fibrinSepharose®): possible, i) separation and purification with column chromatography utilizing carboxymethyl group-bonded agarose (CM Sepharose®): possible, which comprises separating a fraction containing the plasminogen activator from a tissue cultured liquor of human normal tissue derived cells which are selected from cells derived from human embryonic lungs and purifying it. 3. A process for the production of a plasminogen activator as claimed in Claim 2, wherein the tissue cultured liquid is obtained by cultivating the human normal tissue derived cells in at least 0.2 ml of a nutrient solution per 100,000 cells. 4. A process for the production of a plasminogen activator as claimed in Claim 3, wherein the culltivating step is performed at a temperature of 25 to 40°C and a pH of 6 to 8. 5. A process for the production of a plasminogen activator as claimed in Claim 4,, wherein the cultivation step is performed at a temperature of 35 to 38°C and a pH of 7.0 to 7.4. 6. A thrombolytic composition which contains a plasminogen activator according to claim 1.

Inventors: HASEGAWA, AKIO, MORI, TOSHIHITO, YOSHIZAKI, HIDEO
Applicants: ASAHI KASEI KOGYO KABUSHIKI KAISHA, KOWA COMPANY, LTD.
Application year: 1983
Main classification: C12N9
IPC codes: A61K38, C12N9, C12N5, A61P7, C12R1