CA1198672A - Fibrinolytically active agent and a method for the preparation thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A disclosure is given of the discovery that tissues of earthworms belonging to the family of Lumbricidae contain certain fibrinolytically active ingredients in good contents and the invention provides a method for the preparation of a therapeutically useful medicament having a fibrinolytic activity from the tissues of earthworms.
The method comprises extracting the earthworm tissues with an aqueous extractant to give an extract solution followed by concentration or dehydration, preferably, accompanied by either preceding or succeeding purification of the active ingredients.

Description

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A FIBRINOLYTICALLY ACT~VE AGENT AND A METHOD F3R
THE PREPARATION THEREOF

BACKGROUND OF THE IMVENTION
The present invention relates to a novel fibrinoly-tically active agent and a method for the preparationthereof. ~ore particularly, the invention ~elates to a novel fibrinolytically active agent e~tracted from earth-worms as well as the method for the preparation thereof by the extraction of earthworms with an aqueous extractant.
1~ In recent years, attention is directed ~y the practi-tioners of medicine and pharmaceutics to various types of the deseases due -to -the coagulation of blood occurring in many cases of prime and aged adults from the standpoint of geria-trics. Several of the well known deseases of such a type are, for example, myocardina] infarction, cerebral thrombosis, syndrome of disseminated intravascular coagu-lation and the like and, as i5 well known, urokinase of man origin and streptokinase are used as a therapeu-tic medicament therefor.
These medicaments are, however, not quite satisfactory in several respects. For example, urokinase of man origin is prepared from human urine as the starting material so that the supply thereof is limited by the availability of this starting material in large volumes. Streptokinase is defective due to the antigenicity. In addition, both of these medicaments must be used by instillation so that the patient under treatment suffers great pains unavoidably.
Therefore, it has been long desired to develop a novel fibrinolytically active agent usable as a therapeutic medicament of the above mentioned deseases without the problems in the conventional medicam~nts therefor. That is, one of the recent problems in the pharmaceutics has been to develop a novel fibrinolytically active agent free from the limitation by the availability o-E the starting material in large quantities and capable of being ~mi ni s-trated to the patient not by the instillation but by other .

means, desirably orally~ without giving pains to the patient.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a novel fibrinolytically active agent useful as a therapeutic medicament for th~ diseases due to the coagula-tion of blood free from the limi~ation in the availability of the starting material in large quantities and suitable for administration not by instillation. With the above object, the inventors have conducted extensive investi-gations with a variety of natural resources for the desired effective ingredient and arrived at a discovery that tissues of earthworms contain such a substance in a good content leading to the completion of the present inventionO
Accordins to the invention there is provided a method for the preparation of a fibrinolytically active agent which comprises: (a) extracting the tissues of earthworms belonging to the family of Lumbricidae with an aqueous extractant having a pH in the range of 5 to 10 to give an aqueous extract solution containing the active ingredients, and (b~ concentrating or dehydrating the aqueous extract solution, Thus, the fibrinolytically active agent of the present invention is an extracted material from earthworms belonging to the family of Lumbricidae with an aqueous extractant having a p~ in the range of 5 to 10, preferably followed by purification, and then concentration and dehydration and the method of th~ present invention for the preparation of such a fibrinoly~ically active agent preferably comprises dis-persing finely ground tissues of earthworms in an aqueous extractant to extract the effective ingredient into the extractant, separating the extract solution containing the effective ingredient from the insoluble matter and concentrating or dehydrating the effective ingredient contained in the extract solution.

~198G72 - 2a -The above obtained concentrate or dehydrated material is, although it is still in a crude state, effective as such but it is preferable that the crude product is further purified by a suitable means such as adsorption, fractional S precipitation with a polar organic solvent, salting-out, ultrafiltration, ion-exchange chromatography, gel filtration, affinity chromatography, hydrophobic chromatography and the like.

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BRIEF DESCRIPTION OF THE DRAWING
FIGURE 1 is a graph illustrating the condition of separation of the inventive fibrinolytically active in~redient in the isoelectric focusing.
FIGURE 2 is a graph illustrating the increases in the fibrinolytic activity of the peripheral bloods of three patients of hypertension.
FIGURE 3 shows the fibrinolytic activity and the optical density of the fractions obtained in the column-chromatographic fractionation of the earthworm extract in Example 14.
FIGU~ES 4a and 4b show the euglobulin dissolving time and the fibrinolytic activity, respectively, of the peripheral blood of men orally administrated with the fibrinolytically active agents obtained in Example 14 in the lapse of time (see Example 16).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
As is known, earthworms belonging to the family of Lumbricidae have been utilized from time imm~m~rlal in the oriental coun-tries as a kind of legendary medicine for an anodyne, antipyretic, diuretic and the like. However, nothing is disclosed or suggested in the prior art liter-atures on the possibility that the tissues of earthworms may contain certain fibrinolytically active ingredient capable of exhibiting the desired effect of increasing the fibrinolytic activity of the peripheral blood of man when it is administrated to a patient, in particular, orally.
Therefore, it was quite unexpected that the extracted material rom earthworms with an aqueous extractant may exhibit such a fibrinolytic activity.
In th ollowing, the procedure for the preparation of the fibrinolytically active agent from earthworms is described in detail.
The starting material is obtained from the tissues of earthworms and fresh bodies of living earthworms and the bodies of earthworms from which the entrails have been removed as well as the entrails themselves are all suitable f as the starting material. The zoological kind of -the earthworms is not particularly limitative within the family of Lumbricidae and earthworms of any kind are substantially equally suitable including, for example, Lumbricus rubellus, Lumbricus terrestris, Eisenia foetida and the like. The earthworm bodies are used as a homoge-nate by finely grinding. It is a convenient way that tne tissues of earthworms are dried in advance by heating, vacuum-drying or freeze-drying and pulverized into a fine powder which may be used as such or after defatting. The most preferable starting material is a freeze-dried powder or fresh earthworm tissues with or without defatting.
The aqueous extractant, with which the finely ground tissues of earthworms are extracted, should have a p~ in the range from 5 to 10 or, preferably, from 6 to 8. The aqueous ex-tractant suitable for the purpose is not limited to pure water but may include physiological saline solu-tions, buffer solutions and undermentioned prepared salt solutions which may further contain a small amount of a polar organic solvent miscible with water such as methyl alcohol, ethyl alcohol, propyl alcohol, acetone, diethyl ether, dio~ane and the like. The most preferable aqueous extractants are the physiological saline solutions and buffer solutions having a pH of 5 to 10 or, preferably, from 6 to 8. Phosphate, acetate, borate, citrate and tris/hydrochloric acid buffer solutions are equally suitable. The above-mentioned prepared salt solution is a dilute aqueous solution prepared with admixture of a water-soluble organic or inorganic acid, such as hydro-chloric f sulfuric, phosphoric, acetic, lactic, citric andsuccinic acids, and an alkali, such as hydroxides and carbonates of an alkali metal and ammonia, to have a pH of 5 to 10 or, preferably, 6 to 8. The suitable proportion of the aqueous extractant to the starting material is in the range from 1 to 100 times by weight or, preferably, from 5 to 30 times by weight based on the dry weight of the starting material. The extraction is performed at a temperature not higher than 60 C or, preferably, in the i7;~

range from 5 to 40 C Eor a sufficient time up to 500 days or, preferably, from 30 minutes to about 30 days.
The extraction of the finely ground tissues of earth-worms with the aqueous extractant is performed by agitating or shaking the slurried mixture or by passing the aqueous extractant -through a bed of the powdered starting material.
It is preferable that the ear-thworm tissues are homogenized by use of a homogenizer, blender, ultrasonic disintegrator, pressurizing ce]l destroyer, grinder or the like machine into a homogenate, i.e. an aqueous suspension of the cell constituents, in order to destroy the cells of the earth-worm tissues before the slurried aqueous mixture is incubated to effect extraction.
When the extraction of the effective ingredients in the earthworm tissues is completed, the slurried aqueous mixture is filtered and the clear filtrate, i.e. the extract solution, is, optionally as combined ~ith the washings of the residue in the above filtration and kept for a suitable time at a suitable temperature, concentrated by a suitable known method such as evaporation with heating or under reduced pressure and/or ultrafiltration or dehydrated by evaporation of the aqueous solvent under reduced pressure or by freeze-drying into a solid material.
It is preferable that a small amount of an antiseptic or ~5 preservative is added to the aqueous slurried mixture of the earthworm tissues under incubation or the aqueous extract solution under the processing for concentration or dehydration~ In this respect of preventing denatura-tion, the addition of the above mentioned polar organic solvent to the aqueous extractant is eEfective in addi-tion to the effect of enhancing or accelerating the extraction of the effective ingredients thereby. The concentration of the organic solvent in the aqueous extractant should be determined depending on the kind of the solvent and other parameters although the concentration of the organic solvent should not exceed 50% by volume in the aqueous extractant.

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The above obtained aqueous extract solution followed by concentration or dehydration contains the desired fibrinolytically active ingredients in a crude state so that purification thereof should preferably follow. The purificatlon or fractionation of the effective ingredients is undertaken with the concentrated aqueous extra~t solu-tion as such or with the dehydrated material as dissolved in a small volume of water. The method of purification may be a conventional purification method for high poly-meric substances in gèneral. Following is a descriptionof several methods for the purification or fractionation of the fibrinolytically active ingredients obtained in the above.
The methods applicable to the purification or frac-tionation of the effective ingredients in a crude stateas in an aqueous solution include treatments by use of an adsorbent or polar organic solvent, salting-out, ultra-filtration, ion-exchange chromatography, gel filtration, affinity chromatography, hydrophobic chromatography and the like. Either one of the above mentioned methods may be sufficient in some cases but it is usual that two or more of the above methods are utilized in combina~ion in a suitable order to remove the undesirable impurities.
The adsorbent utilizable here is exemplified by active charcoal, acid clay, activated clay and synthetic resin-based adsorbent, for example, sold under a tradename of Amberlite XAD. The polar organic solvent used for the fractional precipitation of the effective ingredients is exemplified by methyl alcohol, ethyl alcohol, propyl alcohol, acetone, diethyl ether, dioxane and the like, of which ethyl alcohol, acetone and propyl alcohol are preferred.
The salt suîtable for the salting-out is exemplified by ammonium sulfate, sodium sulfate, magnesium sulfate, potassium phosphate, sodium chloride, potassium chloride, sodium citrate and the like, of which ammonium sulfate is preferred. The ion exchanger suitable for the ion exchange chromatography is exemplified by those based on '7~

the hydrophilic polysaccharides such as cellulose, dextran, agarose and the like, of which diethylaminoethylcellulose (hereinafter referred to as DEA~-cellulose), triethylamino-ethylcellulose (hereinafter referred to as TEAE-cellulose), aminoethylcellulose (hereinafter referred to as AE-cellu-lose), carboxymethylcellulose (hereinafter referred to as CM-cellulose), phosphocellulose (hereinafter referred to as P-celiulose), phosphomethylcellulose (hereinafter referred to as PPM-cellulose), diethylaminoethylcellulofine (here-inafter referred to as DEAE-cellulofine) and the like are preferred.
The ion exchange chromatography may be carried out also by use of a conventional ion exchange resin including weakly acidic cation exchange resins such as those sold under trade~arks of Amberlite IRC-50, Amberlite IRC-75, Amberlite IRC-84, Dowex CCR-2 and the like and weakly basic anion exchange resins such as those under trademarks Amberlite IR-4B, Amberlite IR-45, Amberlite IRA-40Q, Dowex 3 and the like.
The gel filtration may be performed by use of those sold under trademarks of Sephadex, Sephalose, Biogel, Toyopearl Ultragel, Cellulofine and the like. The station-ary phase used in the affinity chromatography may be formed with various adsorbents such as Sephalose and Toyopearl as the carrier. Further, the hydrophobic chromatography can be carried out by use of an adsorbent such as those with a hydrophilic polysaccharide, e.g. agarose and cellu-lose as the base to which hydrophobic groups have been introduced ~y use of an aliphatic, alicyclic or aromatic compound having 2 to 20 carbon atoms in a molecule and having an amino group.
Following schemes 1 to 16 are several preferable examples of the processes of the inventive method given in the form of a flow chart.

Scheme 1~

¦earthworm tissues ¦

aqueous extractant ~ with pH 6-8 ¦incubation ¦

filtration or ¦washout ¦ ~~ centrifugal separation ., aqueous extractant ~, residue with pH 6-8 l extract solution ~washingS ¦

combination of extract solution and washings ~_ ultrafiltration or vacuum evaporation ~r ¦concentrated solution¦

vacuum-, freeze-~ or flash-drying crude fibrinolytic substance Scheme 2 ¦earthworm tissues ¦

aqueous extractant ~ with pH 6-8 ¦extraction and/or incubation ¦

fil-tration or ¦washout¦ c centrifugal separation aqueous ex-tractant ~ residue ¦
with pH 6-8 extract solution ¦washings ¦ l combination of extract solution and washings I

¦incubation ¦

_ ultrafiltration or vacuum evaporation ¦concentrated solution ¦

c vacuum-, freeze~
or flash-drying crude fibrinolytic substance Scheme 3. Scheme 4.

concentrated concen-trated solution in solution in Scheme 1 or 2 Scheme 1 or 2 polar polar -- organic ~- - organic solvent solvent ¦ precipitates ¦ ¦ precipltates ¦

¦ dissolution ¦ ¦ dissolution ¦

¦ ion exchange ¦ ¦ gel filtration ¦

fractionated ¦ ion exchange material `1/ purified ¦ precipitates ¦ fibrinolytic substance gel filtration or affinity chromatography purified fibrinolytic substance I~L 1~ ~ ~ IY ~d Scheme~5. Scheme 6.

concentrated concentrated solution in solution in Scheme 1 or 2 Scheme 1 or 2 ~ salting-out ¦ ~ salting-out ¦

v v ¦precipitates ¦ ¦ precipitates ¦

¦ dialysis ¦ ¦dialysis ¦

¦ion exchange ¦ ¦ gel filtration ¦

¦gel filtration ¦ affinity I chromatography ~/ I
purified fibrinolytic purified substance fibrinolytic substance Scheme 7. Scheme 8.

dehydrated crude dehydrated crude fibrinolytic - fibrinolytic substance in substance in Scheme 1 or 2 Scheme 1 or 2 c aqueous aqueous solvent ~~ solvent ¦solution ¦ ~solution ¦

polar ~ salting-out ¦
- organic solvent V ¦ precipitates ¦
¦precipikates ¦

1, ¦ solution ¦
¦solution ¦
`I' dialysis and/or gel filtration ion exchange and/or gel filtra-tion ¦ion exchange ¦
.
fractionated material fractionated I material ~b i purified fibrinolytic purified substance fibrinolytic substance Scheme 9. Scheme 10.

learthworm tissues ¦
fracti.onated material in Scheme 7 or ~
aqueous ~ solvent v ~/
affinity homogenization chromatographyand incubation u filtration or c centrifugal purified separation fibrinolytic substance ~I
¦extract solution¦

adsorption treatment ~' hydrophobic chromatography purified fibrinolytic substance Scheme 11.Scheme 12.

conce~trated concen~rated solution in solution in Scheme l or 2 Scheme l or 2 decolorization decolorization ~, \, adsorption with ion exchange synthetic adsorbent purified fibrinolytic purified substance fibrinolytic substance '7~2 Scheme 13. Scheme 14.

dehydrated crude dehydrated crude fibrinolytic fibrinolytic substance in substance in Scheme 1 or 2 Scheme 1 or 2 aqueous ~ aqueous ~ solvent solvent decolor- ~ .decolor-ization ization ultrafiltration ultrafiltration purified dïalysis or fibrinoly-tic gel filtr.ation substance purified fibrinolytic substance 7~

~6 Scheme 15. Scheme 1~

concen-trated concentrated solution in solution in Scheme 1 or 2 Scheme 1 or 2 ¦ decolorization ¦adsorpt~on with synthetic adsorbent polar organic solvent6~ salting-out ¦

¦ precipltates ¦¦ precipitates ¦

¦ dissolution ¦¦ dissolution ¦

¦ ultrafiltration ¦¦ ultrafiltration ¦

hydrophobic ¦ ion exchange ¦
chromatography purified purified fibrinolytic fibrinolytic substance substance ~L¢~7~

Following is a description of a preferred embodiment of the inventive method for the preparation of a fibrino-lytically active agent from earthworms given in further detail as well as the characterization of the thus obtained products~

I. Preparation of the fibrinolytically active agents A defatted powder of freeze-dried earthworms is dispersed in lQ times by weight of a 50 mM phosphate buffer solution having a pH of 7.0 and subjected to incubation at 37 C for 200 hours to extract the fibrinolytically active ingredients into the aqueous phase. The aqueous extract solution is concentrated by ultrafiltration and the concentrated extract solution is admixed with a sufficient volume of ethyl alcohol to fractionally precipitate the effective ingredients. The precipitates are dissolved in distilled water and fractionated by use of a DEAE-cellulose into three fibrinolytically active fractions called hereinafter F-I, F-II and F-III, respectively.
Each of the fractions F-I and F-II is subjected to salting-out with ammonium sulfate followed by the trea-tment with Sephadex G-75 and freeze-drying of the active frac-tion. The third fraction F-III is subjected to desalting as such ollowed by freeze-drying to give a purified product.

II. Characteristic properties common to fractions F-~to F-~III and the mPthod for activity determination (l) Activity. each of them has an activity to solubilize fibrin.

(2) Substrate specificity: each oE them has a strong activity to decompose ~ibrin~

(3) Optimum pH and stabilizing pH: the optimum pH of the fibrinolytic substance is about 8-10 while the stabilizing pH is about 5-10.

(4) Activity determination fibrinogen is dissolved in a 0.17M borate buffer solution having a pH of 7.8 and containing 0.01M sodium chloride in such a concentration 7~

that the concentration of the coagulable protein is 0.15%
by weight and 10 ml of the solution are taken in a steril-ized glass dish of 80 mm diameter with admixture of 0.5 ml of a 20 units/ml solution of thrombin followed by standing for 1 hour at room temperature with the dish covered.
The standard fibrin plate test is performed by dropping 0.03 ml of the above prepaSed test solution on a standard fibrin plate prepared with 10 ml of a 0.15~
fibrinogen solution and, after keeping for 10 minutes with a filter paper inserted under the glass cover, placing it in a thermostat at 37 C to be kept there for 18 hours to effect the reaction. The fibrinolytic activity is expressed by the product in mm2 of the lengths of the major and minor axes of the area on the standard fibrin plate formed by dissolving.

(5) Stability: at least 92% of the residual activity is obtained with the fibrinolytically active ingredients of the invention in an aqueous solution having a pH of 7.5 or 9.0 ater 30 minutes at 50 C.
t6) Inhibitors: the activity of the fibrinolytic substance is inhibited by aprotinin (Trasylol, a ~rademark of Baeyer Co.), tranexamic acid (Transamine, a trademark of Dai~ichi Seiyaku Co.) and soybean trypsin inhibitor ~available from Miles Laboratories, Inc.) and serum.
(7) Fibrinolytic activity: the fibrinolytically active agent of the invention has an activity of plasminogen activation so that fibrin is solubilized indirectly in addition to the direct reaction therewith to effect solu-bilization. Further, similar activity is also exhibited by an extract solution prepared by the incubation at 37 C for 20 days of a homogenate of 300 g of a freeze-dried powder of earthworm tissues in 3 liters of a physiological saline solution or the freeze-dried material of the extract solutio~. ~
In an isoelectric focusing undertaken with an extract solution obtained in the above described manner after filtration, the pH gradient curve and the optical density curve at 750 nm by use of the copper-Folin reagent of the ,, ~.~

-fractions each in a 2.5 ml volume were as shown in FIGURE
1 by the curves I and II, respectively. These results indicate the presence of proteinous or quasi-proteinous substances having the isoelectric points at pH values of 1.5, 3.4 and 5.6.
FIGURE 1 also inc~udes -the results of the determina-tion of the fibrinolytic activity of the fractions in the isoelectric focusing of the above obtained extract solution as filtered or after dialysis by the curves III and IV, respectively. These results indicate the presence of a highly fibrinolytic substance in the fractions of the extract solution ~ithout dialysis obtained at and near the isoelectric point of pH 3.4 and the presence of fibrinolyt-ically active substances having somewhat lower activity than above in the fractions obtained at and near the isoelectric points of pH 1.5 and pH 4Ø In contrast thereto, the fractions obtained at and near the isoelectric point of pH 5.6 have no fibrinolytic activity. On the other hand, the dialysis of the extract solution has an cO effect to remove the fibrinolytically active substance appearing in the fractions of the extract solution before the dialysis obtained at and near the isoelectric point of pH 1.5 while the activity of the fractions obtained at and near the isoelectric point of pH 3.4 is retained even after the dialysis. Thus it is concluded from the results shown in FIGURE 1 that the active ingredient has an isoelectric point at about pH 3.4 while the substance having an isoelectric point of about pH 5.6 is irrelevant to the fibrinolytic activity of the extracted material from earthworms.

III. In vivo activity test of the inventive fibrinolytic substance In the use of the inventive fibrinolytically active agent as a therapeutic medicament for human patient by oral admirlistration, the crude or partially purified products at any intermediate stages of purification in the above given schemes for purification may be used although, needless to say, the highly purified final product is the most preferable form from the standpoint of exhibiting excellent effectiveness as a therapeutic medicamen-t by oral administration.
The purified product obtained in the following Example l was orally administrated to three male patients of 60, 73 and 59 years old suffering from arteriosclerosis each in a dose of 600 mg as freeze-dried and the peripheral blood of each pa-tient was taken periodically at 1 hour intervals, for which the fibrinolytic activity in mm2 was examined by the euglobulin fractionation to give the results shown in FIGURE 2 by the curves I, II and III for the above three patients, respectively. The white circles and black circles on each of the curves correspond to complete and incomplete solubilization of fibrin, respec-tively. The conclusion derived from these results is that the fibrinolytic activity in the peripheral blood begins to increase 2 hours after administration and reaches the maximum value 4 to 6 hours after administration of the inventive fibrinolytically active agent.

Example l.
An aqueous slurry of 1 kg of a finely powdered, freeze-dried tissues of earthworms of the species Lumbricus rubellus after defatting with acetone dispersed in lO liters of a 50 mM phosphate buffer solution having a pH of 7~0 was agitated for lO0 hours at 37 C to effect extraction of the water-soluble ingredients followed by filtration. The residue from the filtration was washed with 3 liters of the same buffer solution and the washings were combined with the filtrate to give a total volume of 12.8 liters of a clear extract solution. The fibrinolytic activity o~ this extract solution was 490 mm2/ml after lO times dilution with distilled water.
The above obtained extract solution was concentrated by ultrafiltration to give a volume of 1.75 liters of the concentrated extract solution having a fibrinolytic

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activity of 550 mm2/ml after 60 times dilution. The effective ingredients contained in this concentrated extract solution was fractionally precipitated by first adding 1.75 liters of e-thyl alcohol to the extract solution and then by adding 7.0 liters of ethyl alcohol to the filtrate from the first step precipitation. The precipi-tates collected from the above two-step precipitation were combined and dissolved in l.l liters of the same buffer solution. The thus obtained solution had a fibrinolytic activity of 694 mm /ml after 60 times dilution. The solution was further subjected to chromatographic frac-tionation by use of DEAE-Sephalose (a product by Pharmacia Co.) into 3 fractions of F-I, F-II and F-III. Each of the ~ractions F-I and F-II was subjected to salting out by 60% saturation with ammonium sulfate ~ollowed by the treatment with Sephadex G-75 and freeze-drying to give 625 mg of a dehydrated powdery material having a fibrinolytic activity of 12,300 mm2/mg and 665 mg of a powdery material having a fibrinolytic activity of 10,700 mm2/mg from the fractions F-I and F-II, respectively. The third fraction F-III was, after desalting and concentration, freeze-dried to give 1200 mg of a powdery material ~aving a fibrinolytic activity of 11,500 mm2/mg.

Example 2.
Live earthworms of the same species as in Example 1 weighing 84 g were added to a physiological saline solution to give a total volume of 300 ml and ground into a homoge-neous suspension, which was incubated for 100 hours at 37 C followed by centrifugal separation into an extract solution and insoluble residue~ The residue was washed with 150 ml of the physiological saline solution and the washings were combined with the extract solu-tion to give a total volume of 400 ml of the combined solution having a fibrinolytic activity of 375 mm2/ml after lO times dilution. The dehydrated material obtained from this combined solution by freeze-drying had a fibrinolytic activity o~ 142 mm2/mg.

Example 3.
Live earthworms weighing g4 g were added -to 500 ml of distilled water containing 0.3 g of phenol and ground into a homogeneous suspension which was incubated for 76 hours at 30 C followed by filtration to remove the insoluble residue from the aqueous extract solution. The residue was washed with 200 ml of distilled water and the washings were combined with the extract solution to give a total volume of 650 ml of the combined solution ha~ing a fibrino-lytic activity of 220 mm2/ml after 10 times dilution~

Example 4.
~ive earthworms weighing 84 g were added to an aqueous mixture of 400 ml of distilled water and 30 ml of ethyl alcohol and ground into a homogeneous suspension, which was incuba~ed for 240 hours at 25 C followed by centrif-ugal separation to give a clear extract solution having a fibrinolytic activity of 350 mm2/ml a~ter 10 times dilution.

Example 5.
An aqueous suspension was prepared by a~mixi ng 50 g of a powder of vacuum-dried earthworms with 400 ml of a physiological saline solution and 100 ml of a salt solution having a pH of 6.5 as pr~pared with a 1.8% aqueous solution of phosphoric acid and a 1.8% ammonia water and the sus-pension was incubated for 100 hours at 38 C followed by iltration to give a clear extract solution having a fibrinolytic activity of 725 mm2/ml after 10 times dilution corresponding to an activity of 72.5 mm2/mg of the dry powder of earthworms.

Example 6.
~ n aqueous suspension was prepared by dispersing 50 g of a powder of freeze~dried earthworms in a mixture of 250 ml of a dilute salt solution having a pH of 6.3 as prepared with a 2% aqueous a~etic acid solution and a 2% aqueous sodium hydroxide solution, 200 ml of a physiological saline ~9~

solution and 50 ml of~ distilled water with admixture of 0.5 g of sodium azide and the suspension was incubated for 72 hours at 37 C followed by filtration to give a clear extract solution having a fibrinolytic activity of 460 mm2/ml after 10 times dilution.

Example 7.
An aqueous suspension was prepar~d by dispersing 50 g of a defatted powder of freeze-dried earthworms into a mixture of 200 ml of an acetate buffer solution having a pH of 7.0, 200 ml of a borate buffer solution having the same value of p~ as above, 100 ml of distilled water, 10 ml of propyl alcohol and 10 ml of dioxane and the suspen-sion was incubated for 2~0 hours at 32 C followed by filtration to give a clear extract solution having a fibrinolytic acitivity of 772 mm2/ml after 10 times dilution.

Example 8.
An aqueous suspension was prepared by dispersing 50 g of a defatted powder of freeze-dried earthworm bodies with the entrails removed into an aqueous mixture of 250 ml of a dilute salt solution having a pH of 6.8 as prepared with an aqueous acid mixture containing 1.8% of phosphoric acid and 3.5% of hydrogen chloride and a 2N aqueous solution of potassium hydroxide, 225 ml of a physiological saline solution and 25 ml of acetone and the suspension was incubated for 96 hours at 25 C followed by filtration with suction to give a clear extract solution having a fibrinolytic activity of 600 mm2/ml after lG times ~ilution.

Example 9.
An aqueous suspension was prepared by dispersing 10 g of a defatted powder of earthworms dehydrated by high-temperature flash drying into an a~ueous mixture of 50 ml of a phosphate buffer solution having a pH of 6.~ and 50 ml of a citrate buffer solution having a pH of 6.5 and the suspension was incubated for 7 hours at 37 C ~ollowed by filtration to give a clear extract solution. The residue was washed with a physiological saline solution and the washings were combinecL with the above extract solution -to give a total volume of 120 ml of the combined solution. This combined extract solution was admixed with 0.1 g of sodium azide and further incubated for 10 hours at 37 C. The resultant solution had a fibrinolytic activity of 280 mm2/ml after 10 times dilution.

Example 10.
An aqueous suspension was prepared by dispersing 10 g of a defatted powder of freeze-dried earthworms into an aqueous mixture of 50 ml of a phosphate buffer solution having a p~I of 7.4 and 50 ml of a physiological saline solution and the suspension was agitated for ~ hours at 22 C to effect extraction of the water-soluble ingredients into the aqueous solution followed by centrifugal separa-tion to give a clear extract solution. The fibrinolytic activity of this extract solution was, after admixture of 0.07 g o~ sodium azide and incubation for 5 hours at 37 C, 190 mm~/ml after 10 times dilution.

Example 11.
Live earthworms weighiny 10 g were added to an aqueous mixture of 70 ml of an aceta~e buffer solution having a pH of 7.0 and 30 ml of distilled water and ground into a homogeneous aqueous suspension which was agitated for 2 hours at 20 C to e~fect extraction of the water-soluble ingredients into the aqueous solution followed by centrif-ugal separation to give a clear extract solution. The residue was washed with water and the washings were combined with the extract solution to give a total volume of 180 ~1 of -the combined solution having a ~ibrinolytic activity of, after admixture of 0.1 g of sodium azide and incuba-tion ~or 7 hours at 37 C, 40 mm2/ml after 10 times dilution.

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~ 25 -Example 12.
An aqueous suspension prepared by dispersing 1 kg of a freeze-dried powder of earthworms in 10 liters of an aqueous solution containing 0.1% by weight of sodium benzoate and 0.9% by weight of sodium chloride was agitated for 96 hours at 32 C to extract the water-soluble ingre-dients followed by filtration. The residue from the above filtration was washed with 3 liters of the same aqueous solution of sodium benzoate and sodium chloride as above and the washings were combined with the filtrate to give 12.5 liters of a clear extract solution having a fibrino-lytic activity of 490 mm2/ml after 10 times dilution.
The thus obtained extract solution was concentrated to a total volume of 0.5 liter by ultrafiltratio.n and the concentrated solu~ion was subjected to fractional precipi-tation by first adding O.S liter of ethyl alcohol thereto to obtain precipitates and then by adding a furt~er volume o~ ethyl alcohol to the filtrate from the first precipita-tion to give a final ethyl alcohol concentration of 80%
so that an additional amount of precipitates was obtained.
The precipitates obtained in the above two-step precipita-tion were combined and washed with ethyl alcohol fol.iowed by vacuum-drying to give 40.5 g of a dry powder having a fibrinolytic activity of 1285 mm2/mg.
The above obtained powder was dissol~ed in 1 liter of a lOmM phosphate buffer solution having a pH of 8.0 and the solution was passed through a column filled with a hexyl-Sepharose prepared by the reaction of hexylamine with agarose ac-t.ivate~ with epichl.orohydrin (Sepharose, trademark, a product by Pharmacia Fine Chemicals Co.) to have the active ingredients adsorbed thereon. After washing with -the same buffer solution as above, elution of the column was undertaken with the same buffer solution as above but containing sodium chloride in a ~oncentration of 0.25M
as the eluant to give 1 liter of an eluate solution.
The eluate solution was, after dialysis, dehydrated by freeze-dryin~ to give 5.75 g of a dehydrated material having a fibrinolytic activity of 7241 mm2/mg.

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6~7~

Example 13.
The procedure down to the fractional precipi-tation with ethyl alcohol followed by vacuum-drying was substan-tially the same as in Example 12 above and 47 g of the dried powder having a fibrinolytic activity of 1100 mm2/mg were dissolved in 1 liter of a 20mM phosphate buffer solution having a pH of 7Ø This solu-tion was passed through a column filled with an ETI (albumen trypsin inhibitor)-Sepharose prepared by combining an albumen trypsin inhibi-tor (a product b~ Sigma Co.) to agarose activated with epichlorohydrin to have the active ingre-dients adsorbed thereon. After washing first with the same buffer solution as above and then with a O.lM acetate bufer solution having a pH of 5.0, elution of the column was undertaken with the same acetate buffer solution as above but containing sodium chloride and alginine in concantrations oE lM and 0.5M, respectively, as the eluant to give 0~8 liter of an eluate solution.
The eluate solution was, af-ter dialysis, dehydrated by freeze-drying to give 255 mg of a dehydrated material haviny a fibrinolytic activity of 70,960 mm2/mg.

Example 14.
An aqueous dispersion of 1 kg of a powdex of freeze-dxied earthworms in 10 liters of an aqueous salt ~olutioncontaining 0.1% of sodium benzoate and 0.9% of sodium chloride was agita-ted for 72 hours at 30 C to effect extraction of the water-soluble ingredients into the aqueous solu~ion followed by filtration to give a clear e~tract solution. The residue was washed with 3 liters of the same salt solution as above and the washings were combined wlth the extract solution to give a total volume of 13 liters of the clear combined solution having a fibrinolytic activity of 450 mm2/ml after 10 times dilution.
The above obtained aqueous solution was concentrated by ul~rafiltxation into a liquid volume of 0.71 litex and then admixed with equal volume of ethyl alcohol to precipitate solid material which was collected by filtra-tion. The filtrate was further admixed with ethyl alcohol to give a final concentration of e-thyl alcohol of 80% to give further precipitates which were collected and washed with ethyl alcohol followed by vacuum-drying into a dry powder. The total yield of the dry powdery products in the above two-step precipitation was 42 g and the fihrino-lytic activity thereof was 1322 mm2/mg.
The above obtained powdery product was dissolved in 1000 ml of distilled water and subjected to column-chromatographic fractionation by use of an adsorbent of DEAE-Cellulofine (a product by Chisso Co.) to give three fractions F-I, F-II and F-III. FIGURE 3 gives the results of the fibrinolytic activity in mm2 and the optical density at 280 nm of the eluate fractions each in a 20 ml volume obtained in the above mentioned column chromatography by the curves I and II, respectively. The broken line in FIGURE 3 indicates the concentration of sodium chloride in the eluate fractions given by the electric conductivity in m mho.
Each of the fractions F-I to F-III was subjected to a treatment of salting-out by ~0~ saturation with ammonium sulfate and the precipitates were dissolved in a small volume of a 10 mM phosphate buffer solution having a pH
of 8Ø The solution was successively subjected to gel filtration with Sephacryl S-200 and desalting concentration by ultrafiltration followed by freeze-drying ~o give 629 mg, 879 mg or 1070 mg of the dehydrated product having a fibrinolytic activity of 13,780 mm2/mg, 9,290 mm2/mg or 17,620 mm2/mg rom the fractions F-I, F-II and F-III, respectively.
The plasminogen activator activity was examined for each of the above obtained dehydrated products. Thus, the dehydrated product was dissolved in water in a con-centration o~ 0.1 mg/ml and 20 ~1 of this solution wereadmixed with 10 ~1 of ~he plasminogen having an activity of 5 units/ml ~a product by Si~ma Co.~ and 30 ~1 of a 0.17M
borate buf~ex solution having a pH of 7.8 and containing 16i~

0.01M sodium chloride and, after standing for 10 minutes at 37 C, 0.03 ml of the mixed solution was dropped vertically on to a fibrin plate free of plasminogen (a product by Miles Laboratories, Inc.). The area in mm2 of the dissolved portion was determined on the plate after 18 hours of the reaction at 37 C. When the above obtained area was taken as A and the corresponding area in mm2 obtained by use of 10 ~1 of the 0.17~ borate buffer solution in place of -the plasminogen was taken as B, then the plasminogen activator activity is expressed by A-B.
The values of the thus determined plasminogen activator activity were 2025 mm2/mg, 1721 mm2/mg and 12~3 mm2/mg for the dehydra-ted products obtained from the fractions F-I, F-II and F-III, respectively.

Example 15.
The fibrinolytically active products obtained in Example 14 were examined for the reactivity with fibrin and fibrinogen. Thus, 0.18 ml of blood plasma of man, 0.02 ml of a 250 mM aqueous solution of calcium chloride and 0.02 ml of an aqueous solution of one of the fibrino-lytically active products in a varied concentration were mixed and the FDP (fibrin-decomposition peptide) produced by the reaction for 30 minutes at 37 C in the above mixture was determined by the latex coagulation test using a kit for the thrombo-wellco test (manufactured by Wellcome Co.). The results are shown in Table 1 in the columns under the heading of CaC12 (+) for 5 different concentratlons of 10~ to 10 1 ng/ml. The marks (+), (+~) and (~++) in the table in~icate the formation of FDP from fibrin, the increase of th~ number of the + marks corre-sponding to the increase in the formation of FDP, while the mark (-) indicates the absence~ OL formation of FDP
from fibrin for each concentration.
On the other hand, the sam~ test as above was repeated în the absence of calcium chloride, i.e. by the use of a physiological saline solution in place of the aqueous solution of calcium chloride, to find that no FDP was formed irrespective of the fraction of the fibrinolytically active products and the concentration thereof as is shown in the columns of Table 1 under the heading of CaCl2 (~).
These results support the conclu ion that the fibrinolyti-cally ac-tive agent of the invention has reactivity with fibrin but not with fibrinogen.

T a b l e ~ F - I F - II F - III
Concentration \
of fibrinolytic \ CaCl2 (+) ( ) (+) ( ) (+
substance 104 ng/ml - - + - ++~ -103 _ _ +++ _ +++
2 _ _ +++ _ ~++

1 0 + _ +++ _ +
+ _ ++~ _ +

+ _ ++ _ +

Example 16.
Each of the freeze-dried purified products of the fractions F-I to F-III in Exampoe 14 was orally adminis--trated to healthy men in a dose of l ~g/kg body weight and the peripheral blood of the subjects was taken periodically to give the euglobulin fractions, with which measurements were undertaken for the time of complete dissolution of euglobulin in hours and the fibrin dissolving activity in mm2 by the s andard fibrin plate test. The results are shown in FIGURES 4a and 4b, respectively.
As is shown in FIGRURE 4a, the time for the solubi-lization of euglobulin was remarkably decreased about 2

7~:

hours after the oral administration of the purified fibrinolytically active agents obtained from the fractions F-I (curve I) and F-III (curve III) and the decrease in the time of euglobulin solubilization continued sustainedly.
On the other hand, the time for the euglobulin solubili-zation began to gradually decrease about 6 hours after the oral administration of the fibrinolytically active agent obtained from the fraction F-II (curve II). These results support the conclusion that each of the purified fibrinolytically active agents obtained from the fractions F-I to F-III has an effect to enhance the fibrinolytic activity of ~he peripheral blood of man when administrated orally.
Further, FIGURE 4b indicates that, though with consid-1~ erable differences between individuals, the fibrinolytic activity of the euglobulin fractions obtained from the peripheral blood was maximum at 2 to 7 hours after the oral administration of the active agents obtained from the fractions F-I to F-III (curves I to II~, respec-tively) to the subjects and the fibrinolytic activity thereof was kept sustainedly even 10 hours after administration.

Claims (9)

Claims:

1. A method for the preparation of a fibrinolytically active agent which comprises:
(a) extracting the tissues of earthworms belonging to the family of Lumbricidae with an aqueous extractant having a pH in the range of 5 to 10 to give an aqueous extract solution containing the active ingredients, and (b) concentrating or dehydrating the aqueous extract solution.

2. The method as claimed in claim 1 wherein the tissues of earthworms subjected to the extraction in the step (a) are in the form of a freeze-dried powder.

3. The method as claimed in claim 1 wherein the aqueous extractant is a physiological saline solution or a buffer solution having a pH in the range from 5 to 10.

4. The method as claimed in claim 1 wherein the extrac-tion in the step (a) is performed at a temperature in the range from 5 to 40 °C for at least 30 minutes.

5. The method as claimed in claim 1 wherein the amount of the aqueous extractant is in the range from 1 to 100 times by weight based on the dry weight of the tissues of earthworms.

6. The method as claimed in claim 1 wherein the aqueous extractant contains a polar organic solvent admixed therewith in a concentration not to exceed 50% by volume.

7. The method as claimed in claim 1 which further comprises a step of purification of the active ingredients either before or after the step (b).

8. The method as claimed in claim 7 wherein the puri-fication of the active ingredients is performed by a procedure selected from the group of the methods including adsorption on and desorption from an adsorbent, fractional precipitation with a polar organic solvent, salting-out, ultrafiltration, ion-exchange chromatography, gel fil-tration, affinity chromatography and hydrophobic chromato-graphy.

9. A fibrinolytically active agent prepared by the method comprising extraction of tissues of earthworms belonging to the family of Lumbricidae with an aqueous extractant having a pH in the range of 5 to 10 to give an extract solution and concentration or dehydration of the extract solution.