JPS6055488B2 - A therapeutic composition having fibrinolytic activity, a therapeutic agent containing the same as a main component, and a method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a composition having a fibrinolytic action on mammals, which is obtained from the venom of a viper classified into the taxonomic genus Habu, and a treatment using this composition as a main ingredient. The present invention relates to drugs for use and methods for producing them.

As is well known, various types of thrombi and emboli are deeply involved in life-threatening lesions.

The object of the present invention is to provide a novel composition having a fibrinolytic action that effectively acts on such lesions, a therapeutic agent containing the composition as a main ingredient, and a method for producing the same. Habu toxin is often feared as causing fatal damage to humans and animals, but the present inventors have investigated the above-mentioned Habu toxin, which contains various proteinaceous toxic factors, in the blood coagulation system and fibrinolytic system. While conducting research on the effect of habu toxin on blood platelet function, etc., we discovered that crude habu toxin obtained by subjecting it to certain treatments had a remarkable fibrinolytic effect, and after further investigation, we found that , arrived at the present invention.

The gist of the invention is to dissolve the dry powder of crude venom in distilled water or physiological saline or in a buffer in the range of pH 6-8, and then to perform gel filtration with a molecular sieve effect, such as Sephadexl (trade name, Sweden). Ph
The crude hub poison solution was purified by column chromatography using a buffer solution consisting of an appropriate electrolyte with a pH in the range of 6 to 8 as a solvent and a column packing material of "Armacia Co., Ltd." (hereinafter the same shall apply) as a column packing material. A fraction with fibrinolytic activity is collected. Next, since this fraction still contains a considerable amount of other components, its fibrinolytic activity was purified by dialysis against distilled water and column chromatography using a cation exchanger as a packing material. A novel composition with fibrinolytic action obtained by separating the strongest fraction, average. The present invention also provides therapeutic agents which are effective mainly for thromboembolic diseases and which contain the composition as a main ingredient. The present invention will be explained in detail below.

The crude toxin used as a raw material in the present invention is taxonomically classified as belonging to the family CrOtaIidae and the genus Tr.
imeresurus). It refers to toxins obtained from snakes that belong to this category, such as Habu (T.
rimeresurusflavOviridis),
Tokara Hub (TrimeresurustOkareI)
iSiS), Taiwan hub (Trimeresurusmu)
crOsquamatus), Trimer
esurus-0kinavensis), Sakishima habu (TrimereSUrL] Selegans), Blue habu (Trimeresurusstejnegsi)
), Kumejimahab (Timeresurustinka)
mi), Yamahabu (TrimeresurusmOti)
cOla), Kikuchihab (Trimeresurusg)
I'Acills) etc. One or two of these poisonous snakes
Dry powder of crude venom obtained from seeds or more is mixed with a suitable solvent such as distilled water, physiological saline, or PH.
Dissolve in a buffer solution ranging from 6 to 8. Next, this crude hub poison solution was used as a column packing material for Sephadex (G-7).
5 or G-100), and then PH6
Four fractions JPI, P■, P■, and P■ are obtained by elution with a buffer consisting of an appropriate electrolyte of about 8 to 80%.
For each of these fractions, a fraction P■ having fibrinolytic activity is obtained by a standard fibrin plate method.

Since this P■ fraction still contains a considerable amount of other components, in order to remove this, the unpurified P■ fraction is first dialyzed against distilled water, and the dialyzed solution is freeze-dried.

Next, this freeze-dried fraction P■ having fibrinolytic activity is dissolved in a buffer solution of around PI]6, such as sodium acetate buffer, and heated at a low temperature to CM-, which is a cation exchanger.
It is adsorbed onto a column packed with cellulose, etc., and eluted by gradually increasing the salt concentration in the buffer solution.The fraction with the strongest fibrinolytic activity is collected and lyophilized to produce the desired fibrinolytic effect. obtain a composition having

Buffers used include sodium citrate buffer,
Examples include acetate buffer, phosphate buffer, and the like.

Note that the above operation can be performed at room temperature, but at low temperature, for example, 5°C.
It is best to do it before and after. The thus obtained composition having fibrinolytic action can be dissolved as it is in physiological saline to make an injection, but if necessary, it may be subjected to appropriate treatment for use as a drug. be done.

Next, the physical properties of this composition are that it is soluble in distilled water, the presence of hexose and deoxyribonucleic acid is not observed, and the molecular weight estimated by Sephadex (G-100) gel filtration method and desk electrophoresis is 2. It is estimated that this substance has absorption in the ultraviolet region and has a maximum wavelength of 280 nm.
It was hot. The pharmacological effect of the composition of the present application was assayed using Invit
As a general method using rO, fibrin plate dissolution area measurement was investigated. The principle of this measurement is to observe the fibrinolytic action of a specimen by measuring the area of fibrin plate dissolution due to the fibrinolytic action of the specimen on the fibrin plate as a substrate. In this application, the fibrin plate used does not contain plasminogen (a precursor of plasmin).

First, even when the present composition alone acts on fibrin plates, no dissolution of the fibrin plates is observed, and therefore the present composition has no plasmin effect.

Next, when a mixed solution of purified plasminogen and this composition is applied to a fibrin plate, the protein concentration of this composition is 430.
Fibrinolytic activity of 27.5% and 44.6% was observed at pVIm1 and 1170 μYlml, respectively, compared to the control (injected with a solution containing only plasminogen). (See Figure 1) For confirmation, when this composition was applied to a standard fibrin plate, clear dissolution of the plate was observed, and therefore, the mechanism of action of this composition can be concluded to be an activator-like action. As shown in the above pharmacological test results, the novel composition with fibrinolytic action disclosed in this application is expected to have clinical effects in the following cases through its effect of activating plasminogen to convert it to plasmin and dissolving fibrin. Fibrin, which is generally converted from fibrinogen by enzymes, is
It is one of the important causes of thrombosis and embolism. The composition of the present application can prevent peripheral arteriovenous thrombosis, pulmonary embolism, coronary artery occlusion, myocardial infarction, cerebrovascular occlusion, retinal artery thrombosis, vitreous hemorrhage, anterior chamber hemorrhage, etc. by the above-mentioned actions. It is also expected to have therapeutic effects.

In addition, it is expected to have a combined effect against cancer when used in combination with anticancer drugs, and can also be used as an anticoagulant during blood transfusions, to prevent embolization at suture lines in vascular surgery, and to maintain long-term function of arteriovenous shunts in hemodialysis. Expected to be effective.

The acute toxicity test of this composition was carried out in DdN male mice (body weight 20
±3y) When this composition was intravenously injected using 10 animals per group, the LD5O value after 7 days was measured using the Litchfield-Wilcoxon method (Litchfield & WillcOxOn method).
3smethOd), it was 10TfL9'K9.

When this fibrinolytic composition is applied therapeutically, intravenous injection or local perfusion can be considered as the administration method, and the dosage varies depending on the disease and administration method, as well as the patient's age, weight, and symptoms. It varies depending on the weight, etc., but the usual dose for adults is 0.2 once.
~80W! Adequate therapeutic effects can be expected by administration of 9.

Examples and pharmacological experimental examples, as well as reference pharmacological experimental examples for the highly purified P■ fraction are listed below.

Example Hub (TrimeresurusFlavOviri)
Freeze-dried crude poison 3y from disHallOwell was added to 15m of 0.02M (mol) borate buffer (PH7.5) at room temperature.
1 and passed through a 5x90d column packed with Sephadex (G-75) at an elution rate of 60ml/1 at 5°C.
At this time, four fractions of PI, P■, P■, and P■ (see Figure 3) are obtained by elution with 0.02M borate buffer (PH 7.5).

Each fraction is assayed for fibrinolytic activity by a standard fibrin plate method to obtain a second fraction, the P■ fraction, which has this activity. The fraction having fibrinolytic activity is dialyzed against distilled water, and the dialyzed fluid is lyophilized. This one
00m9 with pH6. 1.5 x 40C77 packed with CM-cellulose dissolved in 40ml of 0.05M sodium acetate buffer at 5°C! The sample is adsorbed onto a column of 1, and eluted by gradually increasing the salt concentration of a sodium acetate buffer to 0.3M, and the fraction with the strongest fibrinolytic activity is collected and lyophilized.

The thus obtained composition with strong fibrinolytic action has the following physical properties.

That is, (a) it is soluble in water; (b) The molecular weight is estimated to be around 20,000 by Sephadex (G-100) gel filtration method and desk electrophoresis.

(c) Has absorption in the ultraviolet region, maximum wavelength is 280r)
It is m.

(See Figure 2) (d) Anthrone (AnthrOrl)
e) According to the method, it contains almost no hexoses.

(e) Deoxyribonucleic acid is not detected in the determination of deoxypentose using the diphenylamine method.

Experimental Example 1 In order to investigate the effect of the composition of the present invention on fibrinolytic phenomenon, a protein concentration of 0.8 was applied to a fibrin plate (manufactured by Kowa Co., Ltd., hereinafter the same).
Even if the present composition of m91m1 is allowed to act alone, it will not be dissolved.

Therefore, the composition of the present application does not have a plasmin effect. Experimental Example 2 A 5CUIm1 solution of purified plasminogen (manufactured by KABI) was prepared in physiological saline, and 50μe of this solution and this fraction composition were added to a 0.05M Tris-HCl buffer (PH7.
4) and adjusted the protein concentration to 110, 21, 43, 850 and 1170 μFlml, respectively.
The activator action of this fraction substance was investigated by mixing 5μ' of the substance with e and injecting 5μ' into the holes on the fibrin plate to allow it to act, and measuring the area of dissolution on the fibrin plate after standing for 3γC'C- time. did.

The fibrin plate used this time forms a lysis ring with a clear boundary depending on the fibrinolytic activity.The diameter of the ring is linearly related to the logarithm of the fibrinolytic activity (concentration), so by measuring the diameter, , the activator activity of this fraction composition was searched.

The experimental results are shown in Table 1 and the figure.

According to this, as shown in Table 1, the activator effect of this fraction composition was 430 and 1170μGlml compared to the control.
The activator activity was 27.5% and 44.6% at each protein concentration, respectively.
It was recognized that t. Experimental Example 3 Under ice-cooling, in a test tube, add 0.3ml of this composition solution (a solution prepared by dissolving this composition in Tris buffer and adjusting the protein concentration to 300μFlml)11 plasminogen (a solution prepared by adjusting purified human plasminogen to 15CTAur1it1ml) )
0.05 m1 and thrombin Ca (50 ur1 it-
Mix 0.1ml of 0.1M'ml) solution, add 0.05ml of a 2.6% solution of purified human fibrinogen, place in a 37°C bath to form a fibrin clot, and add the fibrinogen solution. The time taken for the fibrin clot to dissolve after the addition was measured.

As a result, the fibrin clot in the test tube to which the present composition solution was added was completely dissolved after 4 hours.

On the other hand, in a control using physiological saline instead of the present composition solution, the composition was almost completely dissolved after 10 hours. Experimental Example 4 A solution of the present composition (a solution prepared by dissolving the present composition in Tris buffer and adjusting the protein concentration to 300 μVIml) was applied to a standard fibrin plate, and the diameter of the dissolution ring after 2 m was measured.

The results are shown in Table 2. Reference Experiment Example 1 Even when only the unpurified P■ fraction with a protein concentration of 570 py1ml is applied to a fibrin plate (plasminogen-free fibrin plate, manufactured by Kowa Co., Ltd., the same applies hereafter), the fibrin plate is not dissolved.

Therefore, the unpurified P■ fraction has no plasmin effect.
Reference Experiment Example 2 A 5C (CaseinurliOIml solution) of purified plasminogen (manufactured by KAB■) was prepared in physiological saline, and 50μ' of this solution and 0.05M Tri of unpurified P■ fraction were prepared.
Dissolve in s-HCl buffer (PH 7.4) to a protein concentration of 2.
1 of the solutions obtained by adjusting the volumes to 80 and 570μFlml, respectively.
The activator action of the unpurified P2 fraction was investigated by mixing 5Pe with 00 μl and injecting it into a hole on a fibrin plate to allow it to act. After standing at 37° C. for 2 hours, the lysis area was measured.

The fibrin plates used here form well-circumscribed lytic rings depending on their fibrinolytic activity.

The diameter of the ring has a linear relationship with the logarithm of the fibrinolytic activity (concentration), so by measuring this, it is possible to
■Each activator activity of each fraction was searched. The experimental results are shown in Table 3.

According to this, the activator action was 280 and 570μ compared to the control (injected with a mixture of only a buffer solution containing a plasminogen solution and an unpurified P fraction).
Hml showed 7.5% and 18.0% activity at each protein concentration, and the activator activity was DOsedeper]De
nt.

[Brief explanation of drawings]

FIG. 1 is a graph showing the effect of the composition according to the present invention on plasminogen. FIG. 2 is an ultraviolet absorption spectrum of the composition according to the present invention. FIG. 3 is a diagram showing the analytical pattern of column chromatography of crude habu venom. 1...CO
ntrOl, 2, 3, 4, 5, 6 are the protein concentration, 1
10μFlml, 210μYlml, 430μFlml
, 850μ9177! The case of t and 1700 μy'ml is shown. 7... Average diameter, 8... Increase rate relative to control.

Claims (1)

[Scope of Claims] 1. Has an absorption maximum at 280 nm in the ultraviolet region, is soluble in water, and uses Sephadex (trade name, manufactured by Pharmacia, Sweden), G-100 gel filtration method; The molecular weight estimated by desk electrophoresis is around 20,000, the presence of hexose and deoxyribonucleic acid is hardly recognized, and it has a remarkable activating effect on fibrinolytic enzyme. Alternatively, a therapeutic composition characterized in that it is obtained by a method of separating a fibrinolytic active fraction obtained by elution using G-100 as a column packing material and a buffer consisting of an electrolyte having a pH of 6 to 8. thing. 2 It has an absorption maximum at 280 nm in the ultraviolet region, is soluble in water, has an estimated molecular weight of around 20,000 by Sephadex G-100 gel filtration method and desk electrophoresis, and has almost no hexose and deoxyribonucleic acid. Unable to do so.
It has a remarkable activating effect on fibrinolytic enzymes, and from crude habu venom solution, Cephadex G-75 or G-1
00 as a column packing material and a method of separating a fibrinolytic active fraction obtained by elution using a buffer consisting of an electrolyte with a pH of 6 to 8. Fibrinolytic active agent as an ingredient. 3 It has an absorption maximum at 280 nm in the ultraviolet region, is soluble in water, has an estimated molecular weight of around 20,000 by Sephadex G-100 gel filtration method and desk electrophoresis, and has almost no hexose and deoxyribonucleic acid. Unable to do so.
It has a remarkable activating effect on fibrinolytic enzymes, and from crude habu venom solution, Cephadex G-75 or G-1
00 as a column packing material and a method of separating a fibrinolytic active fraction obtained by elution using a buffer consisting of an electrolyte with a pH of 6 to 8. A therapeutic agent for thromboembolic disease as a component. 4 A crude habu venom solution with a pH of 6 to 8 is subjected to column chromatography using gel filtration with a molecular sieve effect, and the pH
1. A method for producing a therapeutic composition having fibrinolytic activity, which comprises eluting with a buffer consisting of 6 to 8 electrolytes, separating and purifying a fraction having fibrinolytic activity. 5. The purification operation converts the fraction having fibrinolytic activity into
The manufacturing method according to claim 4, which comprises the steps of dialysis against distilled water and column chromatography using a cation exchanger. 6 Sephadex G as a gel with molecular sieve effect
Claim 4 using -75 or G-100
5. A method for producing a platelet aggregation enhancement promoter according to item 5.