JPH0565232A - Use of staphylokinase for preparation of pharmaceutical composition for treating arterial thrombosis - Google Patents

Abstract

PURPOSE: To obtain a pharmaceutical composition that is particularly useful as a thrombolysis for lysis of platelet-rich clots, does not cause antibody induction by repeated dosing and is used for treatment of arterial thrombosis, particularly for myocardial infarction. CONSTITUTION: This thrombolytic composition contains staphylokinase, preferably a recombinant staphylokinase as an active ingredient, in a pharmaceutically effective amount and is used for the treatment of arterial thrombosis, especially, myocardial infarction. This active ingredient is particularly useful as a thrombolytic agent for the lysis of platelet-rich clots and did not induce antibody induction and other side-effects upon repeated administration. The concentration of the active ingredient can be widely changed from 0.1-100% depending the character of the diseases and the administration modes. The dose of the active ingredient can be altered from 0.05-10mg per kg of body weight.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the use of staphylokinase for the preparation of a pharmaceutical composition for treating arterial thrombosis. More particularly, it relates to the use of staphylokinase for the preparation of a pharmaceutical composition for treating myocardial infarction.

[0002]

BACKGROUND OF THE INVENTION Thrombotic complications of cardiovascular disease are a major cause of death and helplessness, and therefore thrombolysis is life threatening such as myocardial infarction, cerebrovascular thrombosis and venous thromboembolism. Positively influences the outcome of the disease. A thrombolytic agent is a plasminogen activator that converts the inactive preenzyme of the fibrinolytic system in blood, plapuminogen, to the proteolytic enzyme plasmin. Plasmin dissolves fibrin in blood clots, but also disrupts the normal components of the hemostatic system, leading to a so-called dissolved state. However, physiological fibrinolysis, plasminogen activator, fibrin, plasmin (Ogen) and alpha ₂ - is fibrin-oriented as a result of specific molecular interactions between antiplasmin.

Recently, one of the thrombolytic agents used for therapy is streptokinase. Streptokinase is β
- is a M r45,000 protein that is secreted by hemolytic streptococcus. It is used in the treatment of thrombolysis, but its administration is associated with widespread systemic fibrinogen weakness. Its efficacy on coronary thrombolysis in patients with evolving acute myocardial infarction is limited, with about 50% coronary artery recanalization within 90 minutes. Exposure to streptokinase induces an allergic reaction in about 5% of treated patients, consistently inducing specific antibody formation, which prevents its reuse within months. In addition, streptokinase antibodies promote platelet activation and thromboxane production, which limits their thrombolytic capacity.

[0004]

[Objectives and effects of the invention] Staphylokinase is an effective thrombolytic agent for platelet-rich blood clots found in patients with developing platelet-rich arterial thrombotic disease, including developing acute myocardial infarction. Things have now been found. Experiments in baboons and dogs have shown that pre-existing antibodies have not been demonstrated against staphylokinase nor have they been induced to neutralize antibodies following intravenous administration.

In the hamster pulmonary embolism model, staphylokinase has been shown to be more effective against clots containing 1,500,000 platelets per μl than streptokinase. Furthermore, in dogs, side recanalization with staphylokinase was shown to be faster, more frequent, and more persistent than with streptokinase.

Thus, the present invention shows that staphylokinase is another practical thrombolytic agent for streptokinase, without the undesirable side effects of the latter.

Pharmaceutical compositions containing staphylokinase as active ingredient for treating arterial thrombosis in human or veterinary practice are in the form of powders or solutions, and may be intravenous, intraarterial or intramuscular. Can be used for administration. Such compositions include pharmaceutically acceptable excipients with neutral properties of the active compound (e.g., aqueous or non-aqueous solvents, stabilizers, emulsifiers, detergents,
Confused with additives) and more optionally dyes (e.g., mixed,
It can be prepared by dissolving. The concentration of active ingredient in the therapeutic composition depends on the nature of the disease and the mode of administration and can vary between 0.1% and 100%. Furthermore, the dose of active ingredient to be administered is 0.
It can vary between 05 mg and 10 mg.

The invention is illustrated in more detail in the following examples, which are not intended to limit the scope of the invention.

Example 1 Thrombolysis of Platelet-rich and Enriched Human Plasma Clots in a Hamster Pulmonary Embolism Model Evaluation of the thrombolytic ability of staphylokinase and streptokinase against platelet-rich human plasma clots in vivo was performed by Stassen, Fibrinolysis 4 such as JM
This was performed using a pulmonary embolism model in heparinized hamsters as described in (Supplement 2), 15-21, 1990. Platelet-rich clots were prepared from fresh platelet-rich human plasma (platelet count 3 × 10 ⁵ / μl) or ¹²⁵
Prepared from platelet enriched human plasma (platelet count 1.5 × 10 ⁶ / μl) containing traces of I-labeled fibrinogen. Platelet rich human plasma was obtained from a 10 ml sample of fresh blood collected on acid-citrate-dextrose by centrifugation at 100 g for 10 minutes. Platelet-rich supernatant plasma
(About 3 ml) was removed and centrifuged at 500 g for 2 minutes to obtain a platelet pellet. Platelet pellets were obtained from blood resuspended in 200 μl platelet poor ACD plasma and centrifuged at 1,000 g for 5 minutes. The platelet count of the resuspended platelet pellet was measured and 300,000 / μl (platelet-rich plasma) or 1,500
The platelet count was adjusted to 000 (platelet-enriched plasma).

50 μl ¹²⁵ I-fibrin labeled human platelet rich or platelet enriched plasma clots were generated in vitro as described above and injected into the jugular vein of heparinized, outbred hamsters. Staphylokinase or streptokinase was infused intravenously over 60 minutes and lysis was measured 30 minutes after the end of infusion as the difference between the radioactivity initially incorporated in the clot and the residual radioactivity in the lung and heart. did. Fibrinogen and α ₂ -antiplasmin levels in plasma from blood samples taken at the end of the experiment were determined by Claus A., Acta Haematology, 1
7, 237-246. 1957; and Eddie Jay et al., Progress in Chemical Fibrinosis and Thrombosis, Davidson Jay F. et al.
Volume 3, Raven Press, New York, 315-
322, 1978.

The thrombolytic ability of the reagents in the hamster venous thrombosis model (percent clot lysis vs. dose of compound administered per kg body weight) was determined as follows. Percent lysis vs. dose value in mg / kg is based on the statistical program Graffit (Elysax, Middlesex, UK)
S-shaped function changed to an index using This program allows the following parameters to be measured.
Maximum lysis achieved in percent (C); dose in mg compound administered per kg body weight with the highest ratio of clot lysis (b); in percent lysis per mg compound administered per kg body weight. Maximum rate of clot dissolution (Z = a
c / 1 ・ eb). These parameters were obtained as the mean ± SEM, and the significance of the differences between these parameters was determined using Student's t-test. Relative potency parameters measured on a weight basis were 45,000 for streptokinase and 1550 for staphylokinase.
The b-values of staphylokinase; their relatives of streptokinase must be multiplied by factor 3 and the Z-value divided by factor 3 to convert to molar amounts, which is based on a molecular weight of 0.

The relative thrombolytic capacity of the compounds (percent dissolution per mg compound administered per kg body weight) was also demonstrated in the following manner. Individual dose-response data (percent lysis vs. dose in mg / kg) were corrected for background lysis,
The corrected data matched the linear regression line that was run through. Significance of differences between the regression linear slopes was measured using Student's test. The results are summarized in Table 1.
For comparison, the results obtained with platelet-poor human plasma clots are also included. These results will be reported elsewhere in detail (Linen et al., Thrombosis & Haemostasis, in submission). Table 1 shows that in hamsters with platelet-rich human plasma clots in the pulmonary artery, 25 experiments with saline infusion showed 18 ± 2 percent (mean ± SE) at 90 minutes.
Values were given for spontaneous dissolution of G). At the end of the experiment fibrinogen levels were 160 ± 13 percent of baseline and α ₂ -antiplasmin levels were 95 ± 5 percent. With staphylokinase, the lysis was 38 at 27 μg / kg 90 minutes after the start of infusion in the group of 3 hamsters.
Increased from ± 17 percent to 75 ± 9 percent at 90 μg / kg. At a dose of 27 μg / kg, streptokinase resulted in 34 ± 4 percent dissolution in 90 minutes, while 8
A concentration of 0 μg / kg produced 90 ± 1 percent lysis. In hamsters with platelet-enriched human plasma in the pulmonary artery, 5 experiments with saline were 19 ± 4 at 190 minutes.
Values were given for spontaneous dissolution in percent (mean ± SEM). Fibrinogen level is 110 ± 15 of the baseline
In percentage, α ₂ -antiplasmin is 140 ± of baseline.
It was 15 percent. With staphylokinase, lysis at 90 minutes after the start of infusion increased from 23 ± 3 percent at 27 μg / kg to 88 ± 1 percent at 160 μg / kg. Streptokinase 3 at a dose of 80 μg / kg
There was 0 ± 4 percent lysis and a dose of 500 μg / kg resulted in 69 ± 11 percent lysis. Fibrinogen and alpha ₂ - antiplasmin levels after injection of staphylokinase and streptokinase, it did not drop significantly.

Dose-response agreement with an exponentially altered sigmoidal function is summarized in Table 1 by Z (maximal rate of clot lysis), b (dose at which maximum lysis occurs) and c (maximum reached). Value). The ability of staphylokinase to clot into human plasma clots did not change with increasing platelet count. Although streptokinase's ability to platelet-poor and platelet-rich clots was similar to that of staphylokinase (by weight), streptokinase had a clearly reduced reactivity to platelet-enriched plasma clots. The results are also shown in Figure 1, which shows that platelet-rich plasma clots (3
Staphylokinase (●) in hamsters with pulmonary embolism (panel A) or in platelet-rich human plasma clots (1,500,000 platelets / μl) (panel A). ) And streptokinase
The thrombolytic ability of (■) (percent dissolution per mg / kg compound injected) is shown. Data represent mean ± SEM as summarized in Table 1, and function Matches A linear regression analysis of the data confirmed a significantly reduced thrombolytic ability of streptokinase,
No staphylokinase for platelet-enriched plasma clots (data not shown).

Example 2 Thrombolysis in dogs with mixed femoral vein clot and platelet-enriched femoral artery abduction graft Thrombus disruption model for femoral thrombolysis is Jung Yi Kay et al., Circulation 79, 920-928, 1989 and modified from the rabbit femoral abduction graft thrombolysis model. 0.2 for adult mongrel dogs weighing 10-20 kg
5 mg / kg fluanisone was given intramuscularly, intubated if necessary and artificially aerated, and anesthetized with 30 mg / kg intravenous sodium pentobarbital followed by a 60 mg bolus. Cefacidal (1 g) was given intravenously. A catheter was inserted into the left jugular vein for blood sampling and the brachial vein for infusion in dogs. The left carotid artery was exposed and separated. Then, a 3 cm piece of the left carotid artery was cut, inverted, and immersed in physiological saline. The left carotid artery was then cannulated for continuous blood pressure monitoring. The right femoral artery was exposed in the groin and contacted when a side branch was present. The baseline blood flow in the right femoral artery was measured with an electromagnetic flow probe (Medellat Durn, Belgium). Next, a piece of 7-0 nylon (DG atraumatic) was turned over from the left carotid artery.
(Trademark) American Cyanamid Company, Danbury, Conn.) Was inserted into a transfemoral artery transversally through an anastomosis using a 12-16 interrupted suture system. The microvascular clamp closing the proximal and end ends of the transected artery was then released. The inside-out section was subjected to thrombus formation, and the stability of the occluded thrombus was monitored for 1 hour. The surgical procedure was performed under aseptic conditions. The recanalization time taken as the time from disclosure of thrombolytic or placebo infusion to reflow of occluded graft sections was measured with an electromagnetic flow probe. Blood flow was recorded continuously for 3 hours to demonstrate reocclusion or stable reflow.

Venous clots were generated in the left femoral vein as described by Kolininger See et al., Klin Invest, 69, 573-580, 1982. Briefly, the femoral vein was separated between the inguinal ligament and the end bifurcation, and all lateral branches were carefully connected except for the main cannulated myocutaneous branch. After introduction of the wool system into the lumen, a 4 cm section of the femoral vein was separated between two vein clamps, emptied and rinsed with saline via a side branch catheter. The sections were then filled with a mixture of trace amounts of ¹²⁵ I-labeled human fibrinogen, 0.6-1.2 ml of fresh blood and 2 units of thrombin solution. The venous clamp was opened after the right femoral artery clot had stabilized for 60 minutes and then opened and the infusion protocol started approximately 1 minute later. The amount of radioisotope in a venous clot was calculated by subtracting the radioisotope absorbed on the cotton swab placed around the vein segment from the original amount of ¹²⁵ I injected into the isolated vein segment, The radioactive cord flowing out from the thrombus into the bloodstream was measured for 1 minute after removing the clamp. 2 hours after starting the injection of thrombolytic agent or placebo,
Thrombus-forming sections of the femoral vein were joined on both sides, removed, and their radioisotope content was measured. The degree of clot lysis was measured as the residual radioactivity in the venous section and expressed as a percentage of the radioactivity originally contained in the clot. The isotope recovery balance (extravascular distribution multiplied by 3) was expressed by comparing the sum of total blood radioactivity at the end of the experiment with the radioactivity in the recovered thrombus originally present in the clot. It was

Received an intravenous bolus injection of heparin
(100 u / kg per hour for 3 hours) All animals started 10 minutes prior to the infusion of the right femoral artery clamp and thrombolytics before removal of aspirin (5 mg / kg). 16-32 μg in 13 dogs with stable arterial occlusion for 60 minutes
/ Kg staphylokinase or 250 to 500 μg / k
Arterial occlusion persisted over the observation period in 5 control dogs given aspirin and heparin but not thrombolytics. Injection of staphylokinase at a dose of 32 μg / kg in 3 dogs and 62 μg / kg in 1 dog was 3
A time to recanalization of 6 ± 9 minutes was associated with regurgitation in all dogs, which was a sustained release in all dogs. Infusion of staphylokinase at a dose of 16 μg / kg in 3 dogs resulted in recanalization within 30 ± 3 minutes, followed by brief reocclusion and reflow in 2 animals. 5 with 3 dogs
Infusion of streptokinase at a dose of 00 μg / kg
Two dogs were associated with regurgitation, which was followed by a sustained release in one dog, while three dogs produced regurgitation within 97 ± 3 minutes at a dose of 250 μg / kg, which Was followed by circulatory reocclusion and reflow in all animals. Statistical analysis of femoral artery blood flow and venous clot lysis data showed no differences between the two groups receiving staphylokinase or the two groups receiving streptokinase. Therefore, the data obtained at the two doses for each dog were combined for subsequent comparison. The time to reflow was significantly longer in the streptokinase group than in the staphylokinase group (p = 0.006). The Kruskal-Wallis analysis with open states defined in the sequence of persistent occlusion (PO), circulatory reocclusion (CR) after the first recanalization and persistent open (PP) after the first reflow was combined. The injection of streptokinase was subjected to the difference between staphylokinase and bound streptokinase groups. Another 5 dogs receiving heparin and aspirin but not thrombolytics were controlled.

A rectal control heating pad was used to maintain a body temperature of 37.5 ° C. during the experiment. Blood gas volume was checked routinely. In some dogs selected for further study, all catheters were withdrawn one hour after injection and the wound was stitched. When spontaneous breathing returned, the tracheal tube was removed and the animals returned to their cages. 500 ml of 0.9% to maintain hydration
The NaCl solution and 100 ml of 5% glucose were injected before closing the wound.

Mold bleeding time was measured before and 6
It was measured after 0 and 120 minutes. Bleeding time incisions were made using an automatic spring-loaded device (Synplate II, General Diagnostics, Morris Plains, New Jersey) with the front foot surface of the forefoot applied. The area of the incision site was washed, shaved and dried before the first bleeding time run. Blood samples were collected with citric acid (final concentration 0.11 M) before the start of the infusion protocol, 10, 30, 60 and 120 minutes and 24 hours after the start. These samples contained platelet counts and ADPs (about 2 individually titrated).
It was used to measure ex vivo platelet aggregation induced by 0 μM and the combination of epinephrine (about 10 μM) and arachidonic acid (0.5 mM). Plasma was obtained for immediate determination of the fibrinogen level, alpha ₂ - stored frozen for subsequent determination of antiplasmin and activated partial thromboplastin time. Platelet count is an automatic cell counter
(Sequoier Turner Corporation Mountain View, California). Platelet aggregation was performed within 1 hour after blood sampling using a standard platelet aggregometer (Chronolog Corporation, Harbortown, PA). Fibrinogen and α ₂ -antiplasmin have been described by Stassen, JM et al., Fibrinolysis 4 (Appendix 2), 15-21, 199.
0 and activated partial thromboplasmin time was performed by routine laboratory assay.

Significant differences in arterial open differences between groups were compared using Fisher's exact test for absolute data or Student's t-test for paired or unpaired values. The Ilskar-Wallis non-parametric analysis of variance was as follows: 0 = persistent occlusion, 1 = circulatory reocclusion and reflow followed by initial recanalization to 2 = persistent opening followed by first passage, as measured by an electromagnetic blood flow probe. It was performed in the order of variable and open femoral artery. This form of variability analysis was chosen because of the open-state variable non-Gaussian distribution.

Femoral artery blood flow measurements after insertion of the everted carotid artery section into the transverse femoral artery are summarized in Table 2. Blood flow was observed in all 18 dogs after the tube clamp was released.
It recovered and stabilized at 20-32% of baseline. Transplant occlusion lasting 60 minutes occurred within 30 minutes in all dogs. Heart rate and blood pressure that produced a p-value of 0.016 did not differ significantly between groups (data not shown).

The femoral vein clot lysis and isotope recovery are also summarized in Table 2. In 5 control dogs given aspirin and heparin but not thrombolytics, the range of overt clot lysis was 23 ± 6% with a calculated isotope recovery equilibrium of 86 ± 6%. In groups given 16 or 32 μg / kg staphylokinase, the extent of clot lysis was 71 ± 6 and 81 ± 6 in groups given 250 or 500 μg / kg streptokinase, respectively.
79 ± 10 and 92 ± 3%, respectively, compared to% lysis. The difference between the staphylokinase and streptokinase groups was not significant (p = 0.20). The calculated isotope recovery equilibrium ranged between 86 and 108% in all groups.

In aggregates, the results obtained with staphylokinase and streptokinase in dogs show that the thrombolytic ability of staphylokinase is at least 5 times higher than that of streptokinase on a molar basis. However, importantly, staphylokinase had a significantly higher potency against platelet-rich arterial thrombus than streptokinase at doses that were equi-effective for venous clot lysis. The template bleeding time measured before injection in study dogs averaged 2 to 13 minutes. Bleeding time was not significantly prolonged with staphylokinase or streptokinase. If anything, deplete fibrinogen
It was emitted more or less with streptokinase, which sparing with fibrinogen than with depleting staphylokinase. ADP-induced ex vivo platelet aggregation was unchanged with aspirin and heparin and was slowly compromised with streptokinase and staphylokinase.
Platelet aggregation by the combination of epinephrine and arachidonic acid was completely or almost completely inhibited as a result of aspirin injection (data not shown). Platelet counts did not change significantly in any group. Table 3 shows the template bleeding time, ADP / induced platelet aggregation, platelet count and fibrinogen amount (g / l). FIG. 2 is a schematic representation of femoral artery reflow and reocclusion status in dogs that were everted with a femoral artery graft everted, followed by staphylokinase (SAK), streptokinase (SK) or saline (control). is there. Values shown between parentheses indicate percent venous clot lysis.

Example 3 In vitro staphylokinase / streptokinase assay in plasma 3.1. Human and Baboon Plasma Staphylokinase / streptokinase test performed as described by Amari Ay et al., Thrombosis et diateris haemollika, 9, 175-188, 1963, in vitro applied to human or baboon plasma Was done. Increasing the concentration of staphylokinase or streptokinase (200 to 1 of streptokinase
0.00000 units / ml or 0.2 of staphylokinase
To 350 μl (50 μl volume containing 1,000 μg / ml)
l Immediately add thrombin (50 NI
H units / ml) and 100 μl of a mixture containing CaCl ₂ (25 mM) were added.

In plasma samples obtained from eight healthy human volunteers, the concentration of staphylokinase required to produce a clot lysis time of 20 minutes was 0.78 to 4.4.
1.8 ± 1.1 μg / ml plasma with individual values up to μg / ml
(Mean ± SD). Comparative values for streptokinase were 0.50 ± 0.36 μg / ml (mean ± SD) with individual values ranging from 0.10 to 1.0 μg / ml.
In 14 plasma samples obtained from patients with coronary artery disease,
These values are 1.6 ± 0. for staphylokinase.
6 μg / ml (1.0 to 2.0 μg / ml) and 0.42 ± 0.28 μg / ml (0.14 to 1.0 μg / ml) for streptokinase. 20 in baboon plasma
The concentration of staphylokinase required to lyse the clot in minutes was 2.
2.0 ± 1. With individual values ranging from 0 to 4.7 μg / ml.
It was 0 μg / ml (mean ± SD: n = 6). For streptokinase, the corresponding value was 68 ± 11 μg / ml (mean ± SD; n = 9) with individual values ranging from 53 to 93 μg / ml. The high resistance of baboon plasma to streptokinase persisted after absorption of protein A-Sepharose pooled plasma. Furthermore, addition of baboon plasma to human plasma (1: 1
The volume: volume ratio did not significantly change the reactivity of human plasma to streptokinase. This indicates that the high resistance of plasma from animals not previously treated with this drug to streptokinase is not due to the presence of inhibitors, but to the endogenous activation of baboon plasma fibrinolytic system in vitro by streptokinase. It is due to resistance.

Therefore, the staphylokinase / streptokinase reactivity test for the detection of staphylokinase or streptokinase neutralizing antibodies in baboon plasma is
Modified with a mixture of 300 μl human plasma and 50 μl buffer and used baboon plasma, baboon plasma absorbed protein A-Sepharose or baboon IgG solution (protein A-Sepharose elution at acid pH). Plasma clot lysis time was measured and plotted against the concentration of staphylokinase vs streptokinase. From this curve, the plasminogen activator that produced complete clot lysis at 20 minutes was measured.
Antibody titer is the amount of compound required to dissolve a clot composed of 300 μl human and 50 μl buffer and those of human plasma and a mixture of baboon plasma, baboon plasma absorption protein A-Sepharose or baboon IgG solution in 20 minutes. Was defined as the difference in.

Neutralizing antibody titers were expressed in μg / ml or units per ml of the mixture added to human plasma. This other staphylokinase / streptokinase reactivity test was also applied to dog plasma obtained from animals receiving staphylokinase or streptokinase. Baseline neutralizing antibody titer was 39 ± 33 units streptokinase (mean ± SD, n = 8) per ml baboon plasma, whereas in the mixture of human plasma and baboon plasma or buffer against lysis by staphylokinase. No difference was found. In three baboons that were continuously assayed, the titer of the neutralizing antibody to streptokinase was 1 after the administration of streptokinase.
Increased to 1,300 ± 1,000 units / ml within 3 weeks, 3
740 ± 200 units after 6 weeks and 380 ± 1 after 6 weeks
Reduced to 10 units / ml. After administration of streptokinase,
The antibody titer of the plasma sample obtained in one week was 1,600 units / ml by adsorption with protein A-sepharose by total recovery of the inhibitory activity in the protein A-sepharose eluate.
To 10 units / ml. In these baboons, staphylokinase-neutralizing antibodies were assayed for 1 to 1 using the original reactivity test and the modified staphylokinase reactivity test by addition of baboon plasma or protein A-Sepharose eluate as quoted above. Not proven after four intravenous doses of 63 μg / kg staphylokinase at 6-week intervals.
The two bars on the left side of FIG. 3 (stripe and open, respectively) show jugular vein clot lysis following administration of staphylokinase (striped bar) and streptokinase (open bar) to baboons. The bar on the right shows the results of the extracorporeal loop with repeated administration. The antibody titers against staphylokinase (●) and streptokinase (■) are also shown.

3.2. Dog plasma In looped dog plasma, the concentration of staphylokinase required for dissolution of blood clot in 20 minutes was 0.17 μg / ml.
And the corresponding value for streptokinase is 47 units / ml.
Met. In another staphylokinase / streptokinase reactivity test, the streptokinase neutralizing activity of dog plasma was found to be 73 ± 10 units / ml (n-12). 500 μg / kg streptokinase was given 2
In one dog, the titer increased from 68 ± 4 units at baseline to 160 ± 10 units (p <0.001) after 9 days, while
No significant increase in streptokinase neutralizing antibody was seen at 9 days in the two dogs fed staphylokinase (75 ± 7 and 90 ± 14 units, respectively, p =).
0.31). Streptokinase neutralizing activity was found in the Ig fraction of dog plasma (protein A-sepharose eluate).
Not detected at baseline or after staphylokinase administration. However, the neutralizing activity induced within 9 days after administration of streptokinase was completely recovered in the Ig eluate. In this assay, staphylokinase-neutralizing activity was measured at baseline or at 3 in the Ig fraction of dog plasma.
Not shown 9 days after administration of 2 μg / kg staphylokinase.

Example 4 In Vivo Jugular Thrombosis and Extracorporeal Loop Thrombosis Model in Baboons 4.1. Jugular vein thrombosis model The in vivo thrombolytic properties of staphylokinase and streptokinase were compared in baboons (Papio hamtraeus). 0.2 ml ¹²⁵ I-fibrin-labeled autologous whole blood clot was added to Korn et al., Circulation 82; 1744-1753,
Separated jugular vein sections were generated using procedures for anesthesia and surgery as described in 1990. Plasminogen activator was administered by slow intravenous infusion of 10% over 5 minutes, followed by continuous infusion of the remaining 90% dose over 55 minutes. Thrombolysis was quantified 30 minutes after injection by measuring the radioactivity remaining in the jugular vein section. Separately, the rate of thrombolysis was continuously monitored by external gamma calculations as previously described by Stassen J. M. et al., Fibrinolysis 4 (Appendix 2), 15-21, 1990. Each baboon was used twice at 2 day intervals and was randomly given a first infusion with staphylokinase or streptokinase and a second infusion with the other drug. No failure of the surgical procedure occurred and the study consisted of all 9 baboons. In 4 baboons, the rate of spontaneous clot lysis was calculated in vitro gamma 90 minutes prior to injection of the thrombolytic agent to obtain background values.

Blood samples were taken at 30, 60 and 9 before the start of the experiment.
It took 0 minutes. These samples radioactivity, fibrinogen and alpha ₂ - used in the measurement of anti-plasmin (data not shown). Isotope recovery equilibration was done by adding the amount of recovered isotope to the vein section and that in blood (multiplied by factor 3 to correct extravascular distribution) at the end of the experiment. Dose-response curves were measured following iv infusion of staphylokinase or streptokinase over 60 minutes in baboons with autologous whole blood clots induced in the jugular vein (not shown).
In 4 control experiments, the background lysis at 90 minutes was 3 ±, as measured by the continuous outer gamma calculation.
It was 1%. Systemic infusion of staphylokinase or streptokinase resulted in progressive dose-dependent clot lysis. The extent of clot lysis was 27 ± 1 at 32 μg / kg staphylokinase, as measured by ex vivo isotope recovery.
1% (n = 3), 68 ± 15 at a dose of 125 μg / kg
% (N = 3), compared with 37 ± 15% (n = 3) and 500 μg / kg of 125 μg / kg streptokinase
67 ± 9% (n = 3) with streptokinase. The isotope equilibrium ranged between 86 ± 10 and 97 ± 3% (data not shown), confirming that a significant portion of the clot was not lost due to embolization. Fibrinogen and α
_2- Antiplasmin levels were not reduced in any group. Active partial thromboplastin time was extended in all animals from baseline values of 58 ± 13 (mean ± SEM, n = 18) to> 3 minutes measured at 60 and 90 minutes after initiation of staphylokinase or streptokinase infusion. (Data not shown).

The continuous outer gamma calculation is a slow onset dissolution, acceleration during the second half of the infusion period and 3 at the end of the infusion.
Shown is a sigmoidal dissolution vs. time curve characterized by levelling-off during the 0 minute observation. The shapes of the curves obtained with staphylokinase and streptokinase at similar molar concentrations were very similar.

Fitting the dose-response data of clot lysis with exponentially deformed sigmoidal function at 90 minutes yielded values for b, z and c. The thrombolytic capacity of staphylokinase tended to be somewhat higher than that of streptokinase, as evidenced by low b-values and high z-values. With the ex-vivo isotope recovery method, the b value is
0.037 ± 0.008 and 0.11 ± 0.03 for staphylokinase and streptokinase, respectively
mg / kg (p = 0.024), z value is 1 per mg / kg,
500 ± 850 and 290 ± 120 percent dissolution (p =
It was 0.16). The corresponding value obtained by the external gamma calculation is 0.037 ± 0.01 for each b-value.
4 and 0.14 ± 0.05 mg / kg (p = 0.06), z-
The values were 1800 ± 2,100 and 170 ± 96 percent dissolution per mg / kg, respectively. However, due to the difference in molecular weight between staphylokinase and streptokinase, their thrombolytic abilities on a molar basis were virtually identical.

4.2. Extracorporeal Loop Thrombosis Model Staphylokinase or Streptokinase The thrombolytic properties of repeated infusion in vivo also show the combined 0.3 ml ¹²⁵ I-fibrin labeled autologous plasma clot and 0.3 ml ¹²⁵ I inserted into the extracorporeal arteriovenous loop. -Comparison in baboons with fibrin-labeled pooled baboon plasma clots (Papio hamadraeus). Therefore, the exposed anterior tibial artery was inserted with a 4 French catheter (Portex White, Hortex, Hithe, UK) and connected via two hypodermic syringes to the catheterized brachial vein. Blood flow through the extracorporeal loop was maintained at 10 ml / min with a peristaltic pump. Clots from pooled baboon plasma were introduced into the base syringe and clots from autologous plasma were introduced into the end syringe. Plasma clots consisted of 0.3 ml plasma in trace amounts (about 1.5 UCi) ¹²⁵ I-labeled human fibrinogen solution (Amersham, Buckinghamshire, UK), 0.07 ml bovine thrombin (1
It was prepared by mixing a mixture of 5 NIH units / ml) and 0.5M CaCl ₂ and incubated for 30 minutes at 37 ° C. 30 minutes before the start of infusion, 7.5 mg / kg Lidogrel
(A combined thromboxane synthase inhibitor and prostaglandin endoperoxide receptor antagonist) was administered as an intravenous bolus to prevent platelet deposition in the extracorporeal loop. Baboons are heparin (30
0 units / kg, then 60 units / hour throughout the experiment
(kg continuous injection) anticoagulated. Animals were randomly assigned to infuse with 63 μg / kg staphylokinase or 250 μg / kg streptokinase over 1 hour followed by a second infusion with another drug 30 minutes after the end of the infusion. The labeled plasma clot in the extracorporeal loop was replaced with fresh clot between the two infusions. Thrombolytics were given intravenously over 1 hour as a 10% bolus and 90% infusion.

Three baboons were administered with staphylokinase and streptokinase 6 weeks, 7 weeks, 1 week after the first administration.
Repeated for 4 weeks and 14 1/2 weeks. 63 μg / kg staphylokinase and 250 μg / kg in these animals
Streptokinase lysis was measured in plasma clots induced by extracorporeal arteriovenous loops (data not shown). 63 μg /
Lysis by kg staphylokinase 66 in jugular vein model
± 7% (mean ± SEM, n = 3) 6, after the first dose,
78 ± 6, 80 ± 5, 78 ± 1, and 7 in the extracorporeal loop measured at 7, 14 and 14 1/2 weeks, respectively.
It was 0 ± 9%. These values did not differ significantly. Dissolution by 250 μg / kg streptokinase in jugular vein model was 54 ± 5% (n = 3) 6, 7, 14
And 14 1/2 weeks later, in the extracorporeal loop, 2 respectively
4 ± 8, 29 ± 9, 57 ± 2 and 23 ± 7%.
The difference between baseline jugular vein clot lysis and in vitro clot lysis at 6 weeks was significant (p = 0.04). Between the 7th and 14th week,
Reactivity to streptokinase significantly recovered from 29 ± 9 to 57 ± 2% (p = 0.03) was similar to baseline jugular vein clot lysis values, but again 3 days after new streptokinase administration. 0.25 mg in 14 1/2 weeks
Clot lysis by / kg streptokinase was again significantly reduced (57 ± 2 to 23 ± 7% (p = 0.003)).

The four sets of bars on the right side of FIG. 3 show the results of plasma clot lysis experiments following repeated administration of staphylokinase (striped bar) and streptokinase (open bar) to baboons in an in vitro loop model. Staphylokinase
Antibody titers against (●) and streptokinase (□) are also shown. The figure very clearly shows the higher clot lysis capacity and lower antibody titer of staphylokinase compared to streptokinase. These data indicate that streptokinase administration in baboons induces neutralizing antibody formation by binding resistance to clot lysis by the new administration again, as observed in humans. Neutralizing antibodies were not shown in human or baboon plasma, no induction of neutralizing antibodies was shown with staphylokinase administration, and clot lysis with repeated administration of staphylokinase remained unchanged. The above example is a comparison to streptokinase, wherein staphylokinase is an effective plasminogen activator for platelet-rich blood clots such as those found in animal thrombotic disorders such as myocardial infarction, and It shows that it does not induce antibody production, and thus can be repeatedly administered.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Brief description of drawings]

1 shows the thrombolytic ability of staphylokinase and streptokinase in hamsters with pulmonary embolism.

FIG. 2 is a schematic representation of femoral reflow and reocclusion conditions in dogs administered staphylokinase or streptokinase.

FIG. 3 shows the results of jugular vein clot lysis following administration of staphylokinase and streptokinase to baboons, and in vitro loop plasma clot lysis experiments with repeated administration.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (71) Applicant 592051486 DESIRE JOSE COLLEN Belgium, Be-3020 Winxcele-Helent, Shionji Hitleran No. 20 (72) Inventor Desiree Jose Collen Belgium, Be-3020 Winksere-Helent, Shion Jichtleran No. 20 (72) Inventor Jiyan-Marie Starsen Be-3030 Wilsere, Belgium, Josef Raffautstraat No. 5 (72) Inventor Henri Roger・ Reinen Belgium, Beh-3020 Helent, Acacia Laan 50 No.

Claims (3)

[Claims] 1. Use of staphylokinase for the preparation of a pharmaceutical composition for treating arterial thrombosis. 2. Use of staphylokinase for the preparation of a pharmaceutical composition for treating arterial thrombosis, wherein the arterial thrombosis is myocardial infarction. 3. Use of staphylokinase according to any one of the preceding claims, wherein the staphylokinase is a recombinant staphylokinase.