JPH0637398B2 - Thrombolytic agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel thrombolytic agents.

Thrombosis is often caused by the formation of blood clots in blood vessels during surgery, labor, trauma or infectious diseases, and various attempts have been made to eliminate this risk. . Administration of anticoagulants such as heparin and coumarin derivatives is one example. Another example is the use of thrombolytic agents. The thrombolytic agent has a function of moving blood clots generated in thrombosis from blood vessels by dissolution.

The clot is composed of fibrin formed from fibrinogen by the action of the enzyme thrombin. On the other hand, normal blood contains plasminogen, and when this plasminogen acts on plasmin, it dissolves fibrin and decomposes it by enzymatic action. Although the circulating blood contains all the ingredients necessary to degrade and remove the blood clots that immediately form in the blood vessels, in reality, the body's potential for autothrombolysis is:
It is difficult to satisfy such a purpose sufficiently. This is due to an insufficient concentration of plasminogen activator in blood. Therefore, administration of a thrombolytic agent from the outside of the body is a powerful treatment method for thrombosis.

Today, urokinase, which is a plasminogen activator isolated from urine or cultured kidney cells, and streptokinase, which is a plasminogen activator obtained from Streptococcus, are used as thrombolytic agents. However, both urokinase and streptokinase are different from normal plasminogen activator taken from blood, and in particular, because they do not have a specific affinity for fibrin, urokinase in the treatment of thrombosis. And the results of using streptokinase are not always satisfactory in all respects. Although a relatively large amount of administration is required to obtain the desired effect, the large amount administration causes dangerous side effects such as fibrinogen lysis or internal bleeding. Therefore, there is a strong demand for the development of a drug which can be produced on a relatively large scale and has a high thrombolytic activity with few side effects.

The present invention and the like have a strong relationship with normal plasminogen activator collected from blood, and as a result of searching for plasminogen activator having a large thrombolytic activity in the treatment of thrombosis, human We have succeeded in separating and purifying a novel plasminogen activator from kidney tissue, and have already proposed this substance and its production method. The novel plasminogen activator according to the above proposal is a tissue plasminogen activator separated and purified from human kidney. Kwaan et al. (F
ed.Proc. 24 , 387 (1965)) is also studying human kidney tissue plasminogen activator. Kucinski
Et al., Bernik et al., Barlow et al., Åsted et al. Or Lewis' subsequent studies showed that the plasminogen activator obtained from such human kidney tissue cultures was immunologically and physicochemically identical to urokinase. Now that the side effects of urokinase have been revealed, plasminogen activator was isolated and purified from tissues such as human kidney, and the courage to use it as a thrombolytic agent was lost. However, surprisingly, the present inventors succeeded in separating and purifying tissue plasminogen activator having completely different properties from urokinase from human kidney, and proceeded to investigate this novel activator from all viewpoints. Among them, a strong thrombolytic effect was found, and the present invention was completed.

The tissue plasminogen activator, which is a component of the thrombolytic agent according to the present invention, has the following features.

(1) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis method showing one protein band and apparent molecular weight of about 70,000 ± 5,000.

(2) The pI of the main band by isoelectric focusing is in the range of 7-9.

(3) It has an immunological property that it is not adsorbed by anti-urokinase IgG-sepharose affinity chromatography.

(4) The substance of the following group A has a hydrolytic activity and the substance of group B has no activity.

Group A: HD-Valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride and HD
-Isoleucil-L-prolyl-L-algin-p-nitroanilide dihydrochloride.

Group B: Boc-L-valyl-L-prolyl-L-arginine-4-methylcoumaryl-7-amide, carbobenzoxy-L-phenylalanyl-L-arginine-4
-Methylcoumaryl-7-amide, L-prolyl-L-phenylalanyl-L-arginine-4-methylcoumaryl-7-amide, glutarylglycyl-L-arginine-
4-Methylcoumaryl-7-amide.

(5) 1-2 M NH ₄ SCN (pH from human kidney tissue)
It must be extractable or perfused in 7.4) and can be purified in the presence of 0.1% Tween 80 or the like.

(6) Substantially all of it is adsorbed on the fibrin-seypharose column and is eluted by 2M NH ₄ SCN.

(7) Stable in a pH 7.4 buffer solution at 4 ° C for 2 weeks.

The tissue plasminogen activator, which is a component of the thrombolytic agent according to the present invention, is produced, for example, as follows.

Extracted from human kidney in the presence of ammonium thiocyanate by a conventional method, and then the extract was a column holding an ion exchanger, a column in which L-arginine or an arginine derivative was bound to a carrier, and a column in which hemagglutinin was bound to a carrier. It can be obtained by purifying by passing through a column selected from columns holding a carrier having a molecular sieving ability or a plurality of columns in which these are combined.

The thrombolytic agent according to the present invention is, as will be apparent from the examples shown below, a tissue plasminogen
It should be noted that the activator is derived from normal cells. Plasminogen derived from malignant tumor cells
Activators are also known to have thrombolytic activity, but in view of the nature as a drug to be administered to humans, considering all the dangers of long-term or large-scale administration, the thrombolytic agent according to the present invention The safety of is already secured in its origin.

Further, unlike urokinase, tissue plasminogen activator, which is a component of the thrombolytic agent according to the present invention,
It has a specific affinity for fibrin that is similar to that of normal plasminogen activator taken from blood, and thus gives satisfactory results for the treatment of thrombosis.
In addition, there is no side effect such as internal bleeding or anaphylaxis / shock, and the thrombolytic agent according to the present invention is an extremely effective therapeutic agent.

The thrombolytic agent according to the present invention may be administered to a patient at an appropriate dose and has the potential of having little or no side effects and an effective thrombolytic power. Therefore, the thrombolytic agent of the present invention, deep vein thrombosis, pulmonary embolism, myocardial infarction, arterial occlusion,
It is used to treat acute and chronic thrombotic occlusions of different vascular beds, such as those encountered in extracorporeal circulation and arteriovenous closure.

The thrombolytic agent of the present invention will be specifically described below with reference to examples.

Example 1 Tissue plasminogen, which is a component of the thrombolytic agent of the present invention,
Separation and purification of activator 100 g of defatted powder of human kidney with acetone was added to pH 7.4.
500 ml of 2M ammonium thiocyanate buffered with 0.02M Tris HCl was added, and the mixture was stirred at 4 ° C for 10 hours.
The extract and the precipitate were separated by centrifugation (× 13,000 g, 30 minutes).

The extract was adjusted to pH 3.0 with 1N HCl, and the precipitate was collected by centrifugation (× 13,000 g, 30 minutes). This precipitate was further added to 500 ml of 1 mM EDTA, 0.3 M NiCl, 10 KIU / ml aprotinin, 5 mM iprosilone aminocaproic acid, 0.01%.
It was dissolved in 0.01 molar sodium phosphate buffer (pH 7.0) containing Tween 80 (buffer A). Then add 100 ml to this buffer
Fibrin celite of was added, and the mixture was stirred at 4 ° C. for 30 minutes and then centrifuged (× 3,000 g, 10 minutes). The precipitate was resuspended in 100 ml of buffer A, packed in a 4 × 10 cm column and washed with 500 ml of buffer A. Then 0.
Plasminogen activator was eluted with buffer A containing 4M arginine. The eluate was fractionated by 10 ml. The results are shown in FIG. 1 and Table 1. This purification method shows a high recovery rate of about 30%, and utilizes the strong affinity of the tissue plasminogen activator, which is a component of the thrombolytic agent of the present invention, with fibrin.

Example 2 Comparison of thrombolytic action of the thrombolytic agent of the present invention and urokinase Tissue plasminogen, which is a component of the thrombolytic agent of the present invention,
The thrombolytic action of activator and urokinase was compared by the Chandandra loop method. An experiment was performed using human blood obtained from a healthy volunteer boy. Exactly 5 ml of blood was collected, and 3.1% sodium citrate was added to 0.1%.
5 ml was added to prevent blood clotting and 50 μl fibrinogen labeled with ¹²⁵ I was added. After mixing well, 1.
5 ml aliquots were placed in a Tygon tube with a length of 28 cm and an inner diameter of 0.36 cm, and after adding 20 mM calcium chloride,
Both ends of the tube were connected to form a ring. The annular circulation tube was fixed to a turntable with a 23 degree gradient rotating at 17 rpm. After performing thrombus formation at room temperature (24-25 ° C.) for 1 hour or 24 hours, the tube was opened and a 10 μl sample was taken to measure the radioisotope. Then add plasminogen activator,
I closed both ends of the tube and turned it again on the turntable.

After a predetermined time, 10 μl of a blood sample was taken and the isotope was measured. The activator activity is more soluble than the thrombus. ^It was expressed as the amount of ¹²⁵ I in the blood.

The added ¹²⁵ I-fibrinogen was incorporated into the thrombus polymer with an efficiency of about 95-98%, but after 1 hour and 2
After 4 hours, there was a difference in the degree of cross-linking, and partially cross-linked (Partial cross linked = PXL) and almost completely (Totally cross linked = TXL) cross-linked were obtained. FIG. 2 shows the state of dissolution of tissue plasminogen activator and urokinase, which are components of the thrombolytic agent of the present invention, in PXL depending on the dose. In FIG. 2, (a) shows the case of activator, and (b) shows the case of urokinase. The thrombolytic agent plasminogen activator of the present invention has a very high efficiency as compared with urokinase. It shows that it dissolves well. After 10 hours of reaction, when the same amount of tissue plasminogen activator as the component of the thrombolytic agent of the present invention as in (a) was added to the system of (b), almost complete lysis was induced (Fig. 2 ( c)). In FIG. 2, each point shows the average value of three experimental values. When the activator concentration is 1 × 10 ⁻⁹ M, the activator of the present invention dissolves PXL almost completely after 20 hours. However, urokinase elutes only about 40% thereof. Therefore, when the activator of the present invention was further added to this system after 10 hours, almost complete release of ¹²⁵ I was caused, showing that the activator has a strong thrombolytic action. In addition, in an experiment using completely cross-linked thrombi, urokinase produced a 35% lysis of 2%.
As shown only after 0 hour, the tissue plasminogen activator, which is a component of the thrombolytic agent of the present invention, showed 90%.
% Dissolution was shown. On the other hand, when the activator was not injected at all as a control, ¹²⁵ I release was not detected at all, indicating that the dissolution of the thrombus depends on the activator.

Example 3 Action of Thrombolytic Agent According to the Present Invention on Thrombus of Various Animals Blood samples were collected from various animals in the same manner as in Example 2, and 1 hour later, crosslinked thrombi (PXL) were crosslinked in the plasma of the animals. Tissue plasminogen activator, which is a component of the thrombolytic agent, was activated to examine its lytic effect. In each experiment, the activator concentration was 20 IU / ml, and the dissolution state was measured by the release of ¹²⁵ I from the clot until 10 hours after the start of the reaction. In this system as well, back ground was almost the same as when no activator was injected, and only 10 to 3% was detected even after 10 hours. In the human system, in contrast, almost 96% was dissolved. The results of solubility measurement in humans and various animals are shown in FIG. In Figure 3, (a) is human, (b) is monkey, (c)
Shows a rabbit, (d) shows a dog, and (e) shows a rat. As shown in FIG. 3, although the degree of lysis varies depending on the animal, monkeys work as well as humans, but rats do not. This indicates that the thrombolytic agent according to the present invention is a human- or monkey-specific enzyme.

[Brief description of drawings]

FIG. 1 is an elution curve when tissue plasminogen activator, which is a component of the thrombolytic agent of the present invention, was eluted by the method shown in Example 1. FIG. 2 is a diagram comparing the thrombolytic action of the thrombolytic agent of the present invention and urokinase, wherein (a) is the thrombolytic agent of the present invention, (b) is urokinase, and (c) is urokinase. The cases where the thrombolytic agent of the present invention was added after the respective times are shown. FIG. 3 is a graph showing the thrombolytic effect of the thrombolytic agent of the present invention on human and various animal thrombi. In FIG. 3, (a) is a human, (b) is a monkey, (c) is a rabbit, (d) is a dog, and (e) is a rat.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References Japanese Patent Publication Sho 56-50953 (JP, B2) Blood and Vascular, 13 [3], (1982) Katsuo Iishi et al. 347-350 Biochimica et Biophysica Acta, 717 [2] (1982) P. 327 to 336 Biochimica et Biop hysica Acta, 621 (1980) P. 241 to 254 Biochimica et Biop hysica Acta, 580 (1979) P. 140 to 153 The Journal of Almost Biological. 7035 to 7041 The Journal of Bio logical Chemistry, 255 [8] (1980) P. 3665 to 3672, The Journal of Biological Chemistry, 254 [6] (1979) P. 1998-2003 Proceedings of The National Academia of Sciences of USA, 78 [7] (1981) P. 4265-4269

Claims (1)

[Claims] 1. A thrombolytic agent comprising a human kidney-derived tissue plasminogen activator having the following characteristics. (1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single protein band with an apparent molecular weight of about 70,000 ± 5,000. (2) The pI of the main band by isoelectric focusing is in the range of 7-9. (3) It has an immunological property that it is not adsorbed by anti-urokinase IgG-Sepharose affinity chromatography. (4) It has the property of showing hydrolysis activity to the following Group A substances and not showing activity to the Group B substances. Group A: HD-Valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride and HD
-Isoleucil-L-prolyl-L-arginine-p-
Nitroanilide dihydrochloride. Group B: Boc-L-valyl-L-prolyl-L-
Arginine-4-methylcoumaryl-7-amide, carbobenzoxy-L-phenylalanyl-L-arginine-
4-methylcoumaryl-7-amide, L-prolyl-L-
Phenylalanyl-L-arginine-4-methylcoumaryl-7-amide, glutarglycyl-L arginine-4
-Methylcoumaryl-7-amide. (5) 1-2 M NH ₄ SCN (PH
It must be extractable or perfused in 7.4) and can be purified in the presence of 0.1% Tween 80 or the like. (6) Substantially all of them are adsorbed on the fibrin-sepharose column and are eluted by 2M NH ₄ SCN. (7) Stable in a pH 7.4 buffer solution at 4 ° C. even after 2 weeks.