JP2627950B2 - Plasminogen activator and thrombolytic agent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a plasminogen activator, which is a factor that works specifically on plasminogen and converts it into active plasmin. The present invention relates to a staphylokinase known as plasminogen activator produced by Staphylococcus aureus and a thrombolytic agent effective for treating thrombotic diseases.

[Prior Art] Conventionally, as a thrombolytic agent, urokinase (hereinafter referred to as UK), which is mainly synthesized in the kidney and excreted in urine, and streptokinase (hereinafter referred to as SK) which is a metabolite of β-hemolytic streptococcus. ) And other plasminogen activators have been used clinically.

Among them, SK is cheaper than UK and widely used in Europe and the United States, but it is a protein derived from bacteria, so its antigenicity is a problem ("Coagulation and Fibrinolysis" by Tamotsu Matsuda, Chugai Medical) In recent years, attempts to administer SK in the form of a complex with plasminogen have attracted attention in the clinical field (Japanese Patent Application Nos. 48-40921 and 53-52606). ). One of the objectives is to avoid the inhibitory action at high concentrations, and the other is to mask the antigenic site of SK by binding to plasminogen to reduce the antigenicity. With success. Attempts have also been made to acylate the active center of the SK-plasminogen complex (see,
No. 56-78590), which aims to increase fibrin specificity by deacylation at the site of thrombus, but also has the effect of preventing autolysis of the complex.

On the other hand, in recent years, mass production of staphylokinase (hereinafter referred to as SAK) by Escherichia coli has been made possible by genetic engineering techniques (Japanese Patent Application Laid-Open No. 58-67181). SAK, like SK, binds to plasminogen to form a complex, which is presumed to function as a plasminogen activator, but actually synthesizes and isolates the complex and confirms its activity There have been no reports. In addition, SAK is different from SK in amino acid sequence and antigenicity, and has a molecular weight of 1/3 or less of SK (Nucleic Acids Research (Nucleic
Asiehs Research) 11, p. 7679-93 (1983)). The difference in the mechanism of action between SAK and SK and its effectiveness have already been filed by the present inventors (Japanese Patent Application No. 63-090252).

FIG. 3 is an explanatory diagram schematically showing the difference in the reaction mechanism between SAK and SK elucidated by the present inventors. In the figure, SA
K is staphylokinase, SK is streptokinase, α
₂ -PI is α ₂ - plasmin inhibitor, Fn fibrin, Plg is plasminogen, Plm shows plasmin.

That is, when SAK is administered alone, it immediately binds to plasminogen in the bloodstream to form a SAK-plasminogen (or plasmin) complex. The resulting SAK
The plasminogen (or plasmin) complex is α
_2- Inhibited quickly by PI and the SAK-plasminogen complex is not activated, but the complex of plasminogen (or plasmin) bound to fibrin and SAK is not inhibited and plasminogen is activated Plasmin and the thrombus dissolves. On the other hand, in the case of SK alone occurred bloodstream SK- plasminogen (or plasmin) complexes without being inhibited by alpha ₂ -PI, as a result, fibrinogen (Figure not only fibrin occurs a large amount of plasmin (Not shown) are also decomposed. Therefore, fibrinogen degradation is much higher than thrombus solubility, and bleeding tendencies are more frequent.

[Problems to be Solved by the Invention] As described above, SAK is an excellent thrombolytic agent having fewer side effects as compared with SK. However, when the fibrinolytic activity of SAK is measured by the fibrin plate method, it takes 30 minutes before the effect appears. A lag time of a certain degree is observed, and an effect of suppressing the degradation of fibrin is also observed at a high concentration.

In ordinary thrombolytic therapy, urgency is required, and it is necessary to set an optimal dose. In this regard, it takes time to develop the effect, and when the inhibitory effect at a high concentration is not actually used in the treatment and the effect is not obtained, it is determined whether the dose is excessive or insufficient. This is one of the drawbacks of SAK.

Thus, the present inventors have made intensive efforts to obtain a thrombolytic agent which has solved the above-mentioned disadvantages of SAK.

[Means for Solving the Problems] The plasminogen activator according to the present invention is a staphylokinase-plasminogen (or plasmin) complex.

A thrombolytic agent according to another invention contains a staphylokinase-plasminogen (or plasmin) complex as an active ingredient.

In addition, plasminogen activator is
It is an equimolar complex of staphylokinase and plasminogen preformed in vitro.

[Action] The delay in the time to onset of the effect (see Fig. 1) occurs after the binding of SAK to plasminogen, which alters the plasminogen conformation and is necessary for the expression of protease activity. (Acta Biochimica Polonica)
22, P329-31 (1975)).

In addition, the inhibitory effect at high concentrations (see Fig. 2) is that most of plasminogen (or plasmin) in blood
It is considered that unbound plasmin having fibrinolytic activity is depleted as a result.

These problems can be solved by previously mixing SAK with plasminogen and incubating for a certain period of time. That is, by this operation, the molar ratio = 1: 1 SAK
-A plasminogen (or plasmin) complex is obtained, which already has protease activity and can be used as it is as a plasminogen activator. Further, in this case, since all SAKs exist as a complex with plasminogen (or plasmin), they do not bind to plasminogen (plasmin) in the blood any more and are not suppressed even at a high concentration.
Although the SK-plasminogen (plasmin) complex is susceptible to self-decomposition, the SAK-plasminogen (plasmin) complex is less susceptible to self-decomposition and is stable in its original form for a long time.

The SAK-plasminogen (plasmin) complex has a plasminogen activating effect. That is, plasminogen is limitedly decomposed and converted to plasmin. Plasmin degrades fibrin in the thrombus, resulting in thrombolysis. Therefore SAK-plasminogen (plasmin)
The complex is effective for treating various thrombotic diseases including thrombosis, as well as urokinase (UK) and streptokinase (SK).

[Examples] Example (1) Antigenicity test of SAK and SK Rabbits were immunized to prepare an antibody (polyclonal) against SAK, and a cross test with SK was performed by the Ouchterlony method.

As a result, no sedimentation line was formed between the anti-SAK antibody and SK, indicating that both proteins had different antigenicities.

Example (2) Formation of SAK-plasminogen (plasmin) complex: 2 ml of human plasminogen solution (Seikagaku Corporation, 0.6 mg / ml) dissolved in 0.1 M sodium phosphate buffer (pH 7.4)
Of a staphylokinase solution (2.4 mg / ml concentration) dissolved in the same buffer so that the molar ratio becomes 1: 1.
Was added and incubated at room temperature for 1 hour. By this operation, a SAK-plasminogen (plasmin) complex having an active center was formed. This is super loin (Supe
rose) 12H / R 10/30 column (FPLC system) for gel filtration. The SAK-plasminogen complex was eluted in the polymer region, and was separated and purified from SAK not bound to plasminogen.

Example (3) Time-dependent change in fibrinolysis by SAK-plasminogen (plasmin) complex: The fibrinolytic activity of the complex obtained in Example (2) was determined by standard fibrin plating method ("Coagulation and fibrinolysis" by Yasushi Matsuda) Author, Chugai Medical Journal, P201-202). That is, 80 mg of human fibrinogen (manufactured by Mold, grade L) was added to 20 ml of physiological saline (pH 7.4 with a barbital-acetate buffer).
And insolubles were removed by centrifugation.
8 ml of this solution was placed in a flat petri dish having a diameter of 8 cm, and 0.3 ml of human-thrombin (3.3 IU / ml) was added to coagulate fibrinogen. A sample was dropped on the fibrin plate thus prepared using a micropipette, and the mixture was incubated at 37 ° C. to observe the dissolution of fibrin.

FIG. 1 is an explanatory view showing the change over time of a fibrin plate. In the figure, A is SAK 0.2μM alone, B is SAK 0.2μ
Complex of M + plasminogen 0.2 μM, C is SK 0.2 μM
M alone and D indicate a complex of 0.2 μM of SK + 0.2 μM of plasminogen, and 5 μ of a 5 μl dropped fibrin plate.
Minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes later are shown.

As shown in FIG. 1, when SAK alone (A) was used,
At the minute, lytic spots were finally observed, whereas the SAK-plasminogen complex (B) showed lytic activity already at 15 minutes, and was more clear at 30 minutes, indicating immediate efficacy.

On the other hand, SK alone (C) and SK-plasminogen complex (D) both showed plaques at 30 minutes, and showed almost the same activity.

Thus, it was clarified that the lag time was shortened by forming SAK as a complex with plasminogen, and that SAK became a plasminogen activator with an immediate effect equal to or higher than that of SK.

Example (4) Concentration change of fibrin dissolution by SAK-plasminogen (plasmin) complex: The dissolution reaction by SAK, SK alone and the concentration change of plasminogen-SAK, plasminogen-SK complex were compared. In the figure, A is a complex of SAK alone, B is a complex of SAK + plasminogen equimolar, C is a complex of SK alone, D is a complex of SK + plasminogen equimolar, 0.04 μM, 0.2 μM, 1.0 μM,
For each concentration of 5.0 μM, the state after 5 μ drops and 120 minutes is shown.

As shown in FIG. 2, when SAK and SK are simple (A, C), when the concentration is high (5 μM), the dissolving spots have a snake-like pattern, and the dissolution reaction is suppressed in the central part (that is, the high concentration part). It was shown that. On the other hand, the complex (B, D) with plasminogen did not inhibit the dissolution reaction and was dissolved up to the center.

At low concentration (0.04 μM), SAK alone (A)
Although the lytic activity is weak, the plasminogen complex (B) shows lytic activity almost equal to the high concentration, and especially in the case of SAK, the complex with plasminogen is more effective in a wider concentration range. It has been shown. On the other hand, SK alone (C) showed clear plaques at low concentrations even at a low concentration, and no expansion of the effective range due to the plasminogen complex was observed.

Example (5) Acute toxicity test of SAK; Using mice (28 to 35 g of 10 ICR males),
As a result of a toxicity test in which 1 mg / kg of AK was intravenously administered, no mice died.

As described above, as shown in Example (3), the pre-incubation of SAK with plasminogen reduced the lag time and became more effective than SK. Further, by mixing with plasminogen at a molar ratio of 1: 1 as shown in Example (4), the inhibitory action at high concentrations was lost, and at low concentrations, the activity was enhanced, and the activity was wider than that of SAK alone. It was confirmed that it was effective in the concentration range. Therefore, it was clarified that when SAK was used as a thrombolytic agent in a form pre-incubated with plasminogen, it could be used as a drug that was immediate-acting, effective locally and could be administered in a wide concentration range. In addition, these properties, in vivo (in vivo)
It is considered that this is reflected also in.

By the way, attempts to administer SK in the form of a complex with plasminogen have been made in the clinical field, one of the purposes of which is to avoid the inhibitory action at high concentrations, and It is intended to mask the antigenic site of SK by binding to plasminogen to reduce antigenicity. Therefore, in the case of the SAK-plasminogen complex as well, a decrease in antigenicity can be expected.

In addition, it is known that when SK is incubated with plasminogen (molar ratio = about 1: 1), it is rapidly degraded, and plasminogen (plasmin) also undergoes autolysis gradually, and it is known that it cannot be administered as it is. I have. Accordingly, attempts have been made to acylate the active center of the SK-plasminogen complex for the purpose of increasing fibrin specificity by being deacylated at the thrombus site and preventing autolysis of the complex. (JP-A-56-78590).
However, the activity of the SAK-plasminogen complex according to the present invention does not decrease at all when incubated at 37 ° C. for 24 hours, and plasminogen (plasmin) also becomes less susceptible to autolysis due to binding to SAK. The data show that SAK is sufficiently stable even without active site acylation.

Thus, the SAK-plasminogen complex is advantageous not only in overcoming the disadvantages of SAK but also in terms of stability and the like as compared with the SK-plasminogen complex.

In addition, thrombolysis in dogs has been observed by administering 0.002 mg of SAK, and if this value is roughly estimated in humans, 0.1 to
A dose of 0.2 mg is considered sufficient. This amount is equivalent to about 1 mg of plasminogen on a molar basis.

[Effects of the Invention] As described above, the SAK-plasminogen (plasmin) complex of the present invention is an active plasminogen activator in its intact form, and therefore has a more immediate effect than using SAK alone. Becomes In addition, since all SAKs exist as a complex with plasminogen (plasmin), they do not inhibit fibrinolysis even when used at a high concentration. Furthermore, the complex of SAK and plasminogen (plasmin) is more stable than SK-plasminogen complex and less likely to undergo autolysis. Further, since many of the antigenic sites of SAK are masked, a decrease in antigenicity can be expected as in SK.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing the results of the time-dependent change in the fibrinolysis of the SAK-plasminogen (plasmin) complex, and FIG. 2 shows the results of the fibrinolysis concentration dependence of the SAK-plasminogen (plasmin) complex. FIG. 3 is an explanatory view schematically showing the difference in the reaction mechanism between SAK and SK.

Claims (2)

(57) [Claims] 1. A plasminogen activator, which is a staphylokinase-plasminogen (or plasmin) complex. 2. A thrombolytic agent comprising a staphylokinase-plasminogen (or plasmin) complex as an active ingredient.