JP2707273B2 - Thrombolytic agent - Google Patents

Description

The present invention relates to an in vivo stable staphylokinase that selectively dissolves fibrin, which is effective for treating, for example, myocardial infarction, cerebral infarction, arteriosclerosis, and the like. The present invention relates to a thrombolytic agent containing, as an active ingredient.

[Prior art] Urokinase (UK) and streptokinase (hereinafter, SK) conventionally used for thrombolytic therapy are plasminogen in circulating blood (liquid phase), and thus are the original purpose. In addition to fibrin degradation in the thrombus (solid phase), it also degrades fibrinogen in the circulating blood, causing side effects such as bleeding (tPA and Pro-UK:
Matsuo Osamu, Interdisciplinary Project, 1986).

In addition, these substances are usually administered directly to a thrombus site using a catheter or the like because of their non-specificity, requiring considerable medical equipment.

Therefore, unlike conventional thrombolytic agents that also degrade fibrinogen in blood, based on a completely new idea,
Development of a thrombolytic agent that selectively degrades fibrin has been desired.

Recently, tissue plasminogen activator (tPA), pro-urokinase (Pro-UK)
Was discovered and attracted attention as a thrombolytic agent having fibrin specificity.

[Problems to be Solved by the Invention] However, both tPA and Pro-UK have a short half-life in vivo and are rapidly inhibited by plasminogen activator inhibitor (PAI). (Collen, D. et al .: Trombos. Haemostas., 52; 24
-26, '84), with less success than initially expected (Sherry, S .: New England J. Med .; 313; 1014-1).
7, '85).

The fibrin specificity and blood stability of staphylokinase, which is the active ingredient of the thrombolytic agent of the present application, have not been investigated at all, and have been considered to be substances having the same action as streptokinase, It was not considered or used for thrombolytic therapy.

Therefore, the present invention, after elucidating the mechanism of action of staphylokinase, has few side effects such as bleeding containing the staphylokinase as a main component, has excellent blood stability, is hardly susceptible to inhibition by PAI, And to obtain a thrombolytic agent whose activity is maintained over a long period of time.

The thrombolytic agent according to the present invention [Means for Solving the Problems], and staphylokinase, plasminogen or plasmin, alpha ₂ - in which an active ingredient and plasmin inhibitor.

Furthermore, staphylokinase (SAK) of the present invention forms a complex with plasminogen, and this complex acts as a plasminogen activator (Kowalska-Loty, B. & Zakrzewski). , K.: Acta Bi
ocim. Pol., 22; 327-39, '75).

In this regard, the study of the present inventors has shown that the action mechanism of SAK is similar to that of streptokinase (hereinafter, SK), but the properties are different in some respects. In other words, the enzyme action of SAK is strongly enhanced in the presence of fibrin. That is, SAK has high fibrin specificity. However, in SK, they have no effect.

SAK- plasminogen complex, alpha ₂ - plasmin inhibitor (hereinafter, alpha ₂ -PI) is subjected to inhibition by, SAK reaction on fibrin, less susceptible to inhibition by alpha ₂ -PI, therefore, fibrin degradation It is expected that the reaction will selectively proceed to (thrombolysis). In contrast, the reaction of the SK-plasminogen complex is not inhibited by α ₂ -PI. For this reason, fibrinogen in the circulating blood is also decomposed, and side effects such as bleeding have occurred.

SAK is not easily degraded by plasmin and has good stability in plasma.

The molecular weight of SAK is about 15,000 (Nucleic Acids Research, 1
1, p.7679-93, '83), which is about 1/3 of SK, it is considered to have good penetration into thrombus.

It is considered that as a result of these properties interacting, fibrin specificity is expressed.

[Effects of the Invention] As will be described in detail in the following examples, the present invention
Is higher in fibrin specificity than SK, and SA generated in the bloodstream
K- plasminogen (or plasmin) complex is rapidly inhibited by alpha ₂ -PI, but plasminogen is not activated, the plasminogen (or plasmin) complexes inhibition of SAK bound to fibrin Plasminogen is activated and becomes plasmin,
Because of the action mechanism that thrombus dissolves, it has been clarified that it can be used as a thrombolytic agent with less side effects such as bleeding.

In addition, SAK has a blood stability of plasminogen such as SK.
It is superior to the activator, and by analogy with the mechanism of action at α ₂ -PI, it is unlikely to be inhibited by the other PAI in the thrombus portion, and its activity is maintained in vivo for a long period of time.

Therefore, it is possible to always increase the fibrinolytic activity in blood, and it is considered that it is also effective for chronic thrombotic diseases.

Furthermore, SAK has a smaller molecular weight than other conventionally used thrombolytic agents, and is advantageous in terms of permeability into thrombus.

For the above reasons, using SAK for thrombolytic therapy has an effect not seen with conventional thrombolytic agents.

[Examples] 1. Analysis of reaction mechanism of SAK FIG. 1 is an explanatory diagram schematically showing the difference in the action mechanism of SAK and SK analyzed by the present inventors. In the figure, SAK indicates staphylokinase, SK indicates streptokinase, α ₂ -PI indicates α ₂ -plasmin inhibitor, Fn indicates fibrin, Plg indicates plasminogen, and Plm indicates plasmin.

That is, the SAK-plasminogen (or plasmin) complex formed in the bloodstream is rapidly inhibited by α ₂ -PI and plasminogen is not activated, but plasminogen (or plasmin) bound to fibrin The complex of SAK and SAK is not inhibited, plasminogen is activated to plasmin, and the thrombus dissolves. On the other hand, in the case of SK, the SK-plasminogen (or plasmin) complex formed in the bloodstream is not inhibited by α ₂ -PI, and as a result, a large amount of plasmin is generated in the bloodstream,
Therefore, fibrinogen (not shown) is decomposed.
At this time, if there is a thrombus in a part of the circulatory system, a part of plasmin dissolves the thrombus (fibrin). But,
Fibrinogen degradation is much higher than thrombus solubility, and bleeding tendencies are more frequent.

 Hereinafter, the analysis of the mechanism of action of SAK will be described in order.

1-1. Examination of fibrin specificity by closed circulation model As a method of examining the fibrin specificity of a thrombolytic agent (plasminogen activator), a closed circulation system developed by Matsuo et al. Matsuo, O.et al.:Thrombos.Res.,24;347-58,'81)
There is.

Therefore, the closed circulation model (model close to the living body)
The fibrin specificity of SAK was examined.

[Method] (1) ¹²⁵ I-labeling of fibrinogen Human-fibrinogen (Plasminogen Free, manufactured by Sigma) subjected to depraminogen treatment is labeled with ¹²⁵ I by chloramine T method, and the labeled fibrinogen is purified by gel filtration. did. As a result, about 3.5 × 10 ⁶
1.5 ml of ¹²⁵ I-fibrinogen at cpm / ml were obtained.

(2) Preparation of ¹²⁵ I-labeled thrombus A solution of 20 μl of ¹²⁵ I-fibrinogen solution was added to 1 ml of human plasma.
After adding 100 μl of a 25 mM potassium chloride (CaCl ₂ ) solution, 50 μl of a thrombin solution (100 U / ml; Sigma), which is an enzyme for converting fibrinogen into fibrin, was added to prepare a ¹²⁵ I-labeled thrombus. .

(3) Thrombus dissolution experiment using closed circulation model Fig. 2A is an explanatory diagram showing the configuration of the closed circulation experiment device, Fig. 2B
The figure is an explanatory view showing the configuration of the main part of FIG. 2A. In the drawings, the same reference numerals indicate the same.

In the figure, human-plasma 1 (total volume 21 ml) was added at 1.95 ml / min.
Lower chamber 3, peristaltic pump 4, upper chamber 5 connected by silicon tube 2 at a flow rate of
The labeled thrombus 6 was placed in the upper chamber 5. 1 ml of the test sample 7 (SAK or SK) is dropped into the lower chamber 3, and the remaining 5 ml is a peristaltic pump 8 (flow rate).
0.04 ml / min). The reaction system was kept at 37 ° C., and circulating plasma was collected from the lower chamber 3 every hour, and used for measurement of radioactivity and plasma components.

(4) Measurement of the degree of fibrin degradation The radioactivity in plasma was measured with a gamma counter, the radioactivity in the whole circulating plasma was calculated from the value, and the degree of fibrin degradation was calculated as a percentage of the radioactivity of the labeled thrombus used. %).

FIG. 3 is a diagram showing the time-dependent change in the degree of fibrin degradation at each concentration of SAK and SK, in which the ordinate represents the degree of fibrin degradation, the abscissa represents time, ● represents 0.625 μg / ml, and Δ represents 1.25.
μg / ml, △: 2.5 μg / ml, ▲: 5.0 μg / ml, ▼: 10 μg / m
l, ★, 20 μg / ml, ×, 40 μg / ml, *, 50 μg / ml SAK or SK concentration.

FIG. 4 is a diagram showing the concentration dependence of the fibrin degradation degree, in which the time (vertical axis) at which fibrin degradation of 50% is observed is plotted against the concentration (horizontal axis). In the figure, ● indicates SAK and ○ indicates SK.

(5) Quantification of plasma fibrinogen After adding 200 μl of barbital buffer (pH 7.75) containing 50 mM calcium chloride (CaCl ₂ ) to 50 μl of test plasma,
50 μl of the mixture was dispensed into a 96-well titer plate.
50 μl of a thrombin solution (100 U / ml; Sigma) was added,
After reacting at 37 ° C. for 10 minutes, the absorbance at a wavelength of 405 nm was measured. In addition, the fibrinogen concentration (%) is the absorbance of water (not coagulated) added with water instead of thrombin as 0%, and the coagulated normal plasma is 100%,
It was determined by proportional calculation.

FIG. 5 is a diagram showing the time-dependent change in the amount of fibrinogen at each concentration of SAK and SK, the ordinate indicates the amount of fibrinogen in plasma (%), the abscissa indicates time, and ● indicates 0.625 μg.
/ ml, △ is 1.25μg / ml, △ is 2.5μg / ml, ▲ is 5.0μg / m
l, ▼, 10 μg / ml, ★, 20 μg / ml SAK or SK concentration.

FIG. 6 is a diagram showing a comparison of fibrin specificity between 2.5 μg / ml SAK and 10 μg / ml SK, in which the vertical axis represents the degree of fibrin degradation (%) (left axis), (○) and the amount of fibrinogen (%).
(Right axis) (△), horizontal axis shows time.

FIG. 7 is a graph showing the concentration dependence of the thrombolytic properties, the vertical axis represents the thrombolytic properties after 6 hours, and the horizontal axis represents SAK.
(●) and SK (○) are shown.

 In addition, the thrombolytic property is represented by the following equation.

Thrombolytic property = Fibrin lysis amount / Fibrinogen amount (6) Determination of plasma plasminogen 36 μl of test plasma diluted 40-fold with 50 mM Tris-HCl buffer (pH 7.4) was placed in a 96-well titer plate, and urokinase ( (5000 IU / ml) was added and incubated at 37 ° C for 15 minutes to generate plasmin. Further, 10 μl of synthetic chromogenic substrate CBS33.08 (7.5 mM; manufactured by Stago) was added, and
And then incubate for 15 minutes with 2% citric acid solution for 20 minutes.
The reaction was stopped by adding 0 μl, and the absorbance at a wavelength of 405 nm was measured.

In addition, plasminogen concentration (%) is normal plasma (100
%) And 4-fold diluted plasma (25%) were measured in the same manner, and determined by proportional calculation.

FIG. 8 is a diagram showing the time-dependent changes in the amount of plasminogen at each concentration of SAK and SK. The ordinate shows the percentage of plasminogen in plasma, and the abscissa shows time.

(7) plasma α ₂ - plasmin inhibitor (α ₂ -PI)
28 μl of test plasma diluted 50-fold with 50 mM Tris-HCl buffer (pH 7.4) containing 120 mM monomethylamine hydrochloride was placed in a 96-well titer plate, and plasmin (16.7 μg / ml)
Incubating 22μl added 37 ° C. for 15 minutes, plasma alpha ₂ -
A complex with PI was formed. Furthermore, synthetic chromogenic substrate CBS33.
After adding 10 μl of 08 (7.5 mM; manufactured by Stago) and incubating at 37 ° C. for 15 minutes, 180 μl of 2% citric acid solution was added to stop the reaction, and the absorbance at a wavelength of 405 nm was measured.

The amount (%) of α ₂ -PI is 25 as in the case of plasminogen.
The values of% and 100% were measured and determined by proportional calculation.

FIG. 9 is a diagram showing the time course of the plasma α ₂ -PI amount at each concentration of SAK and SK, the vertical axis shows the α ₂ -PI amount (%) in plasma, and the horizontal axis shows time.

[Results and Discussion] As shown in FIG. 3, SAK hardly decomposed fibrin at 0.625 μg / ml, but showed concentration-dependent degradation at a concentration of 1.25 μg / ml or more, and showed almost the maximum at 10 μg / ml. It exhibited fibrinolytic activity. On the other hand, SK showed fibrin degradation at a concentration of 1.25 μg / ml or more, and had the highest fibrin degradation activity at 20 μg / ml. As described above, in both cases, fibrin degradation was observed in a concentration-dependent manner, but the specific activity was strong in SAK, for example, SAK5 μg / ml and SK20 μg / ml were equivalent.

FIG. 4 shows that SAK degrades fibrin in a shorter amount and in a shorter time than SK, confirming the effectiveness of SAK.

In addition, a clear difference was observed between the two reaction curves. In other words, it was found that, with SK, the change with time was linear, whereas with SAK, fibrin degradation was hardly observed at the beginning of the reaction, but once the degradation occurred, the reaction proceeded rapidly thereafter.

Next, the movement of fibrinogen in circulating plasma will be described. As shown in FIG. 5, fibrinogen degradation was remarkable in SK, while it was mild in SAK, especially 2.5%.
In the case of μg / ml, fibrin is 100% after 6 hours.
Degraded but fibrinogen remains 85%,
Strong fibrin specificity was shown for SAK.

FIG. 6 clearly shows that SAK has stronger fibrin degradation and weaker fibrinogen degradation than SK.

Also, in FIG. 7, SK showed a low value of 1 or less at all concentrations, whereas SAK showed a high thrombolytic property in the range of 1.25 to 5 μg / ml, and in particular, 2.5 μg / ml
It was shown to be a high value of 6.6.

Even with thrombolytic agents such as t-PA and pro-UK, thrombolytic properties rarely exceed 3, indicating that SAK is a substance with particularly excellent fibrin specificity.

FIGS. 8 and 9 show that the decrease in plasminogen and α ₂ -PI is weaker in SAK than in SK, and the activation of plasminogen in circulating plasma is mild.

As described above, closed circulation model (model close to living body)
Showed that SAK dissolves plasma soul (artificial thrombus) at a lower concentration than SK, and also showed less degradation of fibrinogen in circulating plasma and higher thrombolytic properties. The decrease in plasma plasminogen and α ₂ -PI was also weak with SAK, confirming that the plasminogen activation reaction in plasma was mild.

1-2. Investigation of fibrin specificity by adding thrombin to human plasma Thrombin was added to human plasma to convert fibrinogen into fibrin, and the plasminogen activation reaction was analyzed.

[Method] 0.1M Tris-HCl buffer (pH
7.5) Take 150 µl, add 20 µl of human plasma (Ci-Trol, manufactured by Midori Cross) and 10 µl of synthetic chromogenic substrate S-2251 (5 mg / ml; mold Vitram), which is a substrate of plasmin, and add human- Thrombin solution (1U / ml; Sigma)
Alternatively, add 10 μl of water as a control,
Alternatively, the reaction was started with 10 μl of the SK solution. Reaction is 37
C., and the change in absorbance at a wavelength of 405 nm was measured.

FIG. 10 shows SAK (10, 20, 40 μg / ml) and SK (2.5, 5, 10 μg / ml).
/ ml) is a diagram showing the plasminogen activation reaction in the presence and absence of thrombin, wherein the vertical axis represents the change in absorbance at a wavelength of 405 nm, the horizontal axis represents time (min), SA
In the K diagram, ○ and ● indicate SAK concentration of 10 μg / ml, △ and ▲ indicate 20 μg
g / ml, □ and △ are 40 μg / ml, in the SK diagram, ○ and ● are SK concentrations of 2.5 μg / ml, Δ and ▲ are 5 μg / ml, □ and Δ are 10 μg / ml,
△, △, □ indicate the absence of thrombin, and ●, ▲, ■ indicate the presence of thrombin.

FIG. 11 is a diagram showing the plasminogen activation reaction in the presence and absence of thrombin at each concentration of SAK and SK after 40 minutes of the reaction, where the vertical axis indicates the change in absorbance at a wavelength of 405 nm after 40 minutes of the reaction. , The horizontal axis represents SAK and SK concentrations (μg / ml), ○ and ● represent SAK, □ and Δ represent SK, ○ and □ represent the absence of thrombin, and ● and Δ represent the presence of thrombin.

[Results and discussion] As shown in Figs.
The plasminogen activation reaction by K was significantly enhanced. On the other hand, the SK reaction is also enhanced by thrombin, but SA
It is weaker than K. For example, when comparing SAK40 μg / ml and SK10 μg / ml, the activity in the presence of thrombin is almost the same, but the SK activity in the absence is about 10 times that of SAK.

As described above, SAK activity was significantly enhanced by thrombin (that is, in the presence of fibrin) as compared with SK, indicating high fibrin specificity.

1-3. Alpha ₂ SAK in the presence of fibrin by enhancing alpha ₂ -PI and fibrinogen SAK reaction in -PI presence and S
The effect of K was measured to determine the effect of fibrin formation by thrombin.

[Method] Take 10 μl of SAK or SK solution in a small test tube and
After adding 10 μl of a thrombin solution (20 units / ml; manufactured by Sigma), 140 μl of 50 mM Tris-HCl buffer (pH 7.4, 0.01% Tween 80 added), human-fibrinogen solution (3.0
mg / ml; Plasminogen Free, Sigma) 10 μl, human
10 μl of plasminogen solution (0.15 mg / ml; Glutamyl-type, manufactured by Mold Vitram) and human-α ₂ -PI solution (0.06 mg
/ ml; manufactured by Plotogen) and synthetic chromogenic substrate S-2251
180 μl of a mixed solution (10 μm; manufactured by Mold Vitram) was added, and the reaction was started. After incubation at 37 ° C. for 30 minutes, the reaction was stopped by adding 50 μl of 8% citric acid solution,
200 μl of the mixture was placed in a 96-well titer plate, and the change in absorbance at a wavelength of 405 nm was measured with a titer plate spectrophotometer.

The reaction solution gels by adding thrombin,
The citric acid solution was added and dissolved by shaking.

Figure 12 is alpha ₂ -PI thrombin presence of SAK and SK each concentration of the absence is a diagram showing the plasminogen activation reaction in the absence, Fig. 13 in the presence of alpha ₂ -PI SAK
FIG. 3 is a diagram showing a plasminogen activation reaction in the presence and absence of thrombin at various concentrations of SK and SK. In the figure, the vertical axis indicates the change in absorbance at a wavelength of 405 nm after 30 minutes of the reaction, the horizontal axis indicates the concentration of each of SAK and SK (μg / ml), ○ and □ indicate the absence of thrombin, and ● and Δ indicate the presence of thrombin.

[Results and Discussion] As shown in FIG. 12, in the absence of α ₂ -PI, SA
The activities of K and SK were hardly affected by the addition of thrombin, and it was found that under these reaction conditions, both had no specificity for fibrin.

However, in the circumstances the presence of alpha ₂ -PI as shown in FIG. 13 changes completely, although somewhat weaker than in the absence of SK in alpha ₂ -PI, the same order of activity was observed, by thrombin added 2
A recovery of about 3 times the activity was observed. In contrast, SAK
Showed no activity even at a concentration of 5.0 μg / ml in the absence of thrombin, and sufficient activity was observed at 0.5 μg / ml in the absence of α ₂ -PI.

Further, as a more important change, under these conditions, the SAK activity was significantly enhanced by the addition of thrombin as compared to SK.For example, in the case of 5.0 μg / ml, the change in absorbance without thrombin was zero. On the other hand, the addition of thrombin showed a high value of 0.93.

Thus SAK has no fibrin specificity in the presence non-alpha ₂ -PI, alpha ₂ is the first time the fact that express fibrin specificity in -PI presence, strong alpha ₂ -PI the SAK fibrin-specific expression Indicates that they are involved.

1-4. Alpha ₂ -PI influence of SAK and SK on plasmin inhibition reactions 1-3. Chapter to SAK fibrin-specific expression observed in alpha ₂ -PI involve more detail analysis for purposes of the alpha The effects of SAK and SK on plasmin inhibition of _2- PI were investigated.

[Method] In a 96-well assay plate, a human-plasmin solution (0.05
CU / ml; Sigma) (50 μl), add 10 μl of a SAK or SK solution (0 to 50 μg / ml each), leave the mixture at room temperature for 5 minutes, and then use α ₂ -PI (0 to 5 I.U./ml). ; Protogen) (10 μl) and 50 mM Tris-HCl buffer (pH 7.4) 1
A mixed solution (total 130 μl) of 20 μl and 10 μl of a synthetic chromogenic substrate S-2251 (10 mM; manufactured by Mold Vitram) was added to start the reaction. The reaction was carried out at 37 ° C., and the change in absorbance at a wavelength of 405 nm after 30 minutes was measured with a spectrophotometer for an assay plate.

Percent inhibition of alpha ₂ -PI not added when the plasmin activity A, activity and B upon addition, was calculated according to the formula shown below.

Inhibition rate (%) = (AB) / A × 100 FIG. 14 shows SA when α ₂ -PI concentration was fixed (5 IU / ml).
FIG. 2 is a diagram showing the inhibition rate of plasmin activity when the concentrations of K and SK are changed. In the figure, the ordinate represents the inhibition rate (%), the abscissa represents the concentration of SAK and SK (μg / ml), ○ represents SAK,
● indicates SK.

FIG. 15 is a diagram showing the inhibition rate of plasmin activity when the concentration of α ₂ -PI is changed. In the figure, the ordinate indicates the inhibition rate (%), the abscissa indicates the α ₂ -PI concentration (IU / ml), は indicates control, ■ indicates SAK (concentration 50 μg / ml), △ indicates SK (concentration 50 μg)
/ ml).

[Results and Discussion] As can be seen in FIG. 14, there is a 80% inhibition at the time of the addition of _{α 2 -PI (5I.U./ml), SAK} , inhibition of Increasing the SK concentration plasmin activity The rate decreases and plasmin activity recovers. It can be seen that the addition of 50 μg / ml of SK hardly inhibits the recovery, whereas the recovery of plasmin activity is weak in SAK.

FIG. 15 shows that the opposite concentrations of SAK and SK are constant (50 μg).
/ ml), and shows the inhibition rate of plasmin activity when α ₂ -PI concentration is changed. When SK was added, no inhibition by α ₂ -PI was observed, whereas SAK showed α ₂ -PI concentration Was confirmed, and the results in FIG. 14 were confirmed.

There are some reports that SK suppresses the α ₂ -PI plasmin activity inhibition reaction (SACe
derholm-williams et al, Eur.J.Biochem.100, P.125-13
2, '79), there is no research report examining the relationship between SAK and α ₂ -PI.

Although the mechanism of the recovery of plasmin activity by SK is not clear, it is likely that SK binds to the L (B) chain of plasmin and sterically hinders the binding of α ₂ -PI to the active center. In that respect, the molecular weight of SAK is about 1/3 of SK,
Like SK, it binds to the L (B) chain, but appears to have less steric hindrance.

alpha ₂ -PI is plasmin are inhibitors of proteinaceous with specificity, but rapidly inhibits plasmin generated in the bloodstream, plasmin bound to fibrin lysine binding site (hereinafter, LBS) is saturated Α _2- via LBS
Unable to bind to PI, resulting in no inhibition (B.Wi
man et al, Biocim. Biophys. Acta, 579, 142-154, '79
other).

Because of this property, one of the roles of α ₂ -PI is
There is a control function of inhibiting plasmin generated in the bloodstream without inhibiting plasmin generated on the thrombus, and as a result, selectively degrading fibrin.

Therefore, as described in the action mechanism shown in FIG.
It was found that SAK acquired fibrin specificity consistent with the control function of α ₂ -PI.

That is, the SAK-plasminogen (or plasmin) complex formed in the bloodstream is rapidly inhibited by α ₂ -PI and plasminogen is not activated, but plasminogen (or plasmin) bound to fibrin The complex of SAK and SAK is not inhibited, plasminogen is activated and becomes plasmin, which dissolves thrombus. On the other hand, in the case of SK, the SK-plasminogen (or plasmin) complex formed in the bloodstream is not inhibited by α ₂ -PI, and as a result, a large amount of plasmin is generated and fibrinogen (not shown) is generated. It is thought to decompose.

Therefore, according to this mechanism of action (FIG. 1), the SAK reaction is inhibited by α ₂ -PI in the bloodstream, but the action is selectively advanced because α ₂ -PI does not easily act on fibrin. It was found that SAK had higher fibrin specificity than SK.

Therefore, since the SAK reaction but plasminogen is inhibited in the case of a free state, in which the lysine binding sites in the plasminogen bound to fibrin is saturated to have alpha ₂ -PI can not act (Wiman, B.et al.:Biochem.Biophys.Acta,
579; 142-54, '79), indicating that the SAK reaction proceeds selectively.

From the above, SAK has a higher fibrin specificity than SK and inhibits the degradation of fibrinogen in the circulating blood, so it can be used as a thrombolytic agent with less side effects such as bleeding, which had a problem with side effects such as bleeding. Was revealed.

2. Stability of SAK in plasma SAK or SK was added to human plasma, incubated at 37 ° C, a part of which was subjected to SDS-polyacrylamide gel electrophoresis, and fibrinolytic activity was examined by fibrin autography. .

As a result, the activity of SAK did not decrease even after incubation for 20 hours, but was completely eliminated by SK. Similar results were obtained when human-plasminogen was used instead of plasma, indicating the stability of SAK.

3. Toxicity test Using mice (ICR male 28-35g, 10 mice), SAK
As a result of the toxicity test in which 1 mg / kg was intravenously administered, no mice died.

As described above, according to the present invention, SAK has a blood stability of SK.
It is better than plasminogen activator such as α _2-
By analogy with the mechanism of action of PI, it is unlikely to be inhibited by other PAIs, and its activity is expected to be maintained for a long time in vivo. Therefore, the fibrinolytic activity in blood can always be increased, and it will be effective for chronic thrombotic diseases.

Furthermore, SAK has a smaller molecular weight than other conventionally used thrombolytic agents, and is advantageous in terms of permeability into thrombus.

Previously, SAK was used for the production of SAK, so the productivity was poor, and there was also a problem with contaminating toxins.
Easy mass production (Sako, T.: Eur.J.BIoche
m., 149; 557-63, '85). Therefore, it is more advantageous in price than other thrombolytic agents.

For the above reasons, the use of SAK for thrombolytic therapy has an effect not seen with conventional thrombolytic agents.

[Brief description of the drawings]

FIG. 1 is an explanatory view schematically showing the difference between the reaction mechanisms of SAK and SK, FIG. 2 is an explanatory view showing the configuration of a closed circulation experiment apparatus,
FIG. 3 is a diagram showing the time-dependent change in the degree of fibrin degradation, and FIG.
The figure shows a graph showing the concentration dependence of the degree of fibrin degradation, FIG. 5 shows a graph showing the change over time in the amount of fibrinogen, FIG. 6 shows a graph showing a comparison of fibrin specificity, and FIG. A graph showing concentration dependency, FIG. 8 is a graph showing a time-dependent change in the amount of plasminogen, FIG. 9 is a graph showing a time-dependent change in the amount of α ₂ -PI, FIG. 10 is the presence or absence of thrombin A diagram showing the plasminogen activation reaction below, FIG. 11 is a diagram showing the plasminogen activation reaction in the presence and absence of thrombin 40 minutes after the reaction, and FIG. 12 is a diagram showing α ₂ −
Diagram showing plasminogen activation reaction in the presence and absence of thrombin in the absence of PI, FIG. 13 shows plasminogen activation reaction in the presence and absence of thrombin in the presence of α ₂ -PI diagram showing, Fig. 14 graph showing the inhibition of plasmin activity in the case of changing the concentration of SAK and SK, the inhibition of plasmin activity as FIG. 15 with varying alpha ₂ -PI concentration FIG.

Claims (1)

(57) [Claims] 1. A thrombolytic agent comprising staphylokinase, plasminogen or plasmin, and α ₂ -plasmin inhibitor as active ingredients.