FR2522266A1 - PROCESS FOR THE PREPARATION OF A FIBRINOLYTICALLY ACTIVE AGENT AND AGENT OBTAINED - Google Patents

Abstract

THE INVENTION RELATES TO A PROCESS FOR PREPARING A FIBRINOLYTICALLY ACTIVE AGENT. ACCORDING TO THE INVENTION, THE EARTHWORM TISSUES BELONGING TO THE LUMBRICIDE FAMILY ARE EXTRACTED WITH AN AQUEOUS EXTRACTING AGENT TO GIVE AN AQUEOUS EXTRACT SOLUTION CONTAINING THE ACTIVE INGREDIENT, AND THIS SOLUTION IS CONCENTRATED OR DEHYDRATED; THE ATTACHED DRAWING SHOWS THE FIBRINOLYTIC ACTIVITY OF VARIOUS COMPOUNDS. THE INVENTION APPLIES IN PARTICULAR TO MEDICINE.

Description

The present invention relates to a novel fibrinolytic agent

  The invention relates more particularly to a novel fibrinolytically active agent extracted from earthworm and to the process for its preparation by the extraction of earthworms with a

aqueous extractant.

  In recent years, attention has been directed by physicians and pharmacists to various types of

  diseases due to blood clotting that occurs -

  in many cases of adults in the prime age and geriatric age Several of the well-known diseases of this type are, for example, myocardial infarction, cerebral thrombosis, disseminated intravascular coagulation syndrome. and the like, and as is well known, urokinase of human origin is used as a therapeutic drug in this case.

and streptokinase.

  However, these drugs are not quite satisfactory from several points of view. For example, human urokinase is prepared from

  of human urine as raw material, so the supply of

  ture, is limited by the availability of this material

  first in large volumes Streptokinase is defective

  because of its antigenicity. Moreover, these drugs

  Therefore, the patient undergoing treatment inevitably suffers from severe pain. Therefore, it has long been desirable to develop a new fibrinolytically active agent that can be used as a therapeutic drug of the above-mentioned diseases without the problems posed by conventional drugs. Indeed, one of the recent problems in pharmacy has been to develop a novel fibrinolytically active agent free from limitations due to the availability of the raw material in large quantities and which can be administered to the patient not by instillation but by other means, advantageously

  Orally, without causing him pain.

  The present invention therefore relates to a novel fibrinolytically active agent useful as a therapeutic drug for diseases due to blood coagulation, free of the availability limits of the raw material in large quantities and suitable for non-instillation administration with the Objective above, the inventors undertook intensive research with a wide variety of natural resources for the desired effective ingredient and came to the discovery that worm tissues contain such a substance at a good grade, which has pipe

  in accomplishing the present invention.

  Thus, the fibrinolytically active agent according to the invention is a material extracted from earthworms belonging to the family of hybrids with an agent.

  aqueous extraction, preferably with subsequent purification

  The process according to the invention for the preparation of such a fibrinolytically active agent consists in dispersing finely ground tissue of earthworms.

  in an aqueous extraction agent to extract the

  effective in the extraction agent, separating the solution of the extract containing the effective ingredient from the insoluble material and concentrating or dehydrating

  the effective ingredient contained in the extracted solution.

  The above-obtained concentrate or dehydrated material is, although still crude, effective as it is, but it is preferred that the crude product be further purified by suitable means such as adsorption, fractional precipitation with a polar organic solvent. , desalination, ultrafiltration, ion exchange chromatography, gel filtration, affinity chromatography, chromatography

hydrophobic and the like.

  The invention will be better understood, and other purposes, features, details and advantages thereof

  will appear more clearly in the description

  explanatory text which will follow with reference to the appended schematic drawings given solely by way of example illustrating several embodiments of the invention and in which: FIG. 1 is a graph illustrating the

  condition of separation of the fibrinolytic ingredient

  active ingredient according to the invention in the iso-

  electric, the number of fractions being indicated on the abscissa axis (each of 2.5 ml of volume), on the ordinate is indicated the fibrinolytic activity, mm 2 before dialysis (curve III) and after dialysis (curve IV ), at b is indicated the optical density at 750 nm (curve II) and at c is indicated p H (curve I); figure 2 is a graph illustrating the increases in peripheral blood fibrinolytic activity of three patients suffering from hypertension, the time after oral administration, in hours, being indicated on the abscissa axis and the fibrinolytic activity in fractions of erglobulin, in mm being indicated on the ordinate axis; Figure 3 shows the fibrinolytic activity and

  the optical density of the fractions obtained in the fraction-

  by column chromatography of the worm extract of Example 14, the number of fractions (each 20 ml volume) being indicated on the abscissa axis, fibrinolytic activity, mm 2 / l pl (curve I) has the optical density at 280 nm (curve II) in b and the conductivity in m S (curve III) in c; and FIGS. 4a and 4b show the dissolution time of fibroblast and the fibrinolytic activity, respectively, on the ordinate axis, as a function of time after oral administration, on the abscissa axis, respectively, of peripheral blood. men who have received oral administration of the fibrinolytically active agents obtained in Example 14, in a certain time

(see example 16).

  As is known, earthworms belonging to the family of earthworms have been used since time immemorial, in eastern countries as a kind of

  legendary drug for an innocuous and anti-diuretic

  However, nothing is revealed or suggested in the literature according to the prior art concerning the possibility that the tissues of earthworms may contain a fibrinolytically active ingredient.

  capable of having the desired effect of increasing the activity

  Thus, it has been quite unexpected that the material extracts earthworms with an aqueous extractant, for example, when it is administered to a patient, in particular, orally. present such

fibrinolytic activity.

  In what follows, the process for the preparation of the fibrinolytic agent will be described in detail.

active from earthworms.

  The raw material is obtained from earthworms tissues and earthworms fresh bodies alive and the bodies of earthworms where the bowels have been removed as well as the bowels themselves are all suitable as raw material. zoology of earthworms is not particularly limiting in the family of earthworms and earthworms

  any kind are substantially equally appropriate

  For example, Lumbricus rubellus, Lumbricus terrestris, Eisenia foetida and the like The bodies of earthworms

  are used as a homogenate by finely grinding them.

  It is easy for earthworm tissues to be dried in advance by heating them, drying them under vacuum or freeze-drying them and then being sprayed into a fine powder that can be used as is or after it has been defatted. most preferred is a lyophilized powder of fresh tissue

  of earthworms with or without degreasing.

  The aqueous extraction agent, with which the finely ground earthworm tissues are extracted, must have a pH between 5 and 10 or preferably between 6 and 8. The aqueous extractant suitable for this purpose is is not limited to pure water but may include physiological saline solutions, buffer solutions and salt solutions hereunder mentioned 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, dioxane and the like Aqueous extractants

  most preferred are physiological saline solutions

  and buffer solutions having a pH of from 5 to 10 or preferably from 6 to 8. Buffer solutions of phosphate, acetate, borate, citrate and tris / hydrochloric acid are also suitable. The salt solution prepared above is a dilute aqueous solution prepared with a mixture of a water-soluble organic or inorganic acid such as hydrochloric, sulfuric, phosphoric, acetic, lactic, citric and succinic acid 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 appropriate proportion of the aqueous extractant relative to the raw material is between 1 and 100 times as much. weight or preferably between 5 and 30 times by weight in

  based on the dry weight of the raw material The extrac-

  The reaction is carried out at a temperature of not more than 600 ° C or preferably between 5 ° and 400 ° C for a sufficient time up to 500 days or preferably

from 30 minutes to about 30 days.

  Extraction of the finely ground woven fabrics of earthworms with the aqueous extractant is accomplished by stirring or shaking the mixture into a slurry or by passing the aqueous extractant through a bed of the raw material. Powder It is preferred that the earthworm tissues be homogenized using a homogenizer, mixer, ultrasonic disintegrator, pressurized cell destroyer, mill or the like to a homogenate, i.e., in an aqueous suspension of the constituents cells to destroy tissue cells of the earthworms before the aqueous slurry mixture is injected for

perform the extraction.

  When the extraction of the effective ingredients in the earthworm tissues is accomplished, the aqueous slurry mixture is filtered and the filtrate clear, i.e. the solution of the extract is optionally

  combined with the washes of the residue in the filtra-

  above and maintained for a suitable time at a suitable temperature, concentrated by a known method suitable as evaporation with heating under reduced pressure and / or ultrafiltration or dehydration by evaporation of the aqueous solvent under reduced pressure or freeze drying to a solid material. It is preferred that a small amount of an antiseptic or preservative be added to the aqueous slurry mixture of the earthworm tissues in incubation or to the solution of the aqueous extract during concentration or dehydration treatment to prevent denaturation. the addition of the aforementioned polar organic solvent to the aqueous extractant is effective in addition to the effect of improving or accelerating the extraction of the effective ingredients. The concentration of the organic solvent in the agent aqueous extraction must be determined by solvent and other parameters, although the concentration The organic solvent should not exceed 50% by volume in the

aqueous extraction.

  The solution of the above aqueous extract obtained with subsequent concentration or dehydration contains the desired fibrinolytically active ingredients in the state.

  raw, so their purification should preferably follow.

  The purification or fractionation of the effective ingredients is undertaken with the concentrated aqueous solution of the extract as such or with the dehydrated material as dissolved in a small volume of water.

  purification may be a conventional process of purification

  cation for polymeric substances in general

  will give below a description of several methods

  purification or fractionation of ingredients

  fibrinolytically active agents obtained above.

  Methods that can be applied to the purification or fractionation of the effective ingredients in a crude state as in an aqueous solution include treatments using an adsorbent or an organic solvent

  polarization, desalination, ultrafiltration,

  Ion exchange, gel filtration, affinity chromatography, hydrophobic chromatography and the like Each of the above methods may be sufficient in some cases but it is customary to use two or more in combination in one order suitable for removing non-impurities

desirable.

  The adsorbent agent that can be used here is represented by activated charcoal, acidic clay, activated clay and an adsorbent agent based on synthetic resin, for example sold under the trade name Amberlite XAD. The polar organic solvent used for Fractional precipitation of the effective ingredients is methyl alcohol, ethyl alcohol, propyl alcohol, acetone, diethyl ether, dioxane and the like, of which alcohol is preferred.

  ethyl, acetone and propyl alcohol.

  The appropriate salt for desalination is

  ammonium sulphate, sodium sulphate, magnesium sulphate, potassium phosphate, sodium chloride, potassium chloride, sodium citrate and the like, of which ammonium sulphate is preferred. Suitable ion exchangers for ion exchange chromatography are those based on hydrophilic polysaccharides such as cellulose, dextran, agarose and the like, of which diethylaminoethylcellulose (hereinafter referred to as DEAE-cellulose) is preferred. ), triethylaminoethylcellulose (hereinafter referred to as TEAE-cellulose), aminoethylcellulose (hereinafter referred to as AE-cellulose), carboxymethylcellulose (hereinafter referred to as CM-cellulose) and phosphocellulose (hereinafter referred to as P-cellulose). , phosphomethylcellulose

  (hereinafter referred to as PPM-cellulose), diethylaminoethyl-

  cellulofine (hereinafter referred to as DEAE-cellulofine) and the like. Ion exchange chromatography can also be carried out using a conventional ion exchange resin comprising weakly acidic cation exchange resins such as those sold under the trade names Amberlite IRC-50, Amberlite IRC-75, Amberlite IRC-84, Dowex CCR-2 and analogues and weakly basic anion exchange resins such as those sold under the trade names Amberlite IR-4B, Amberlite IR-45, Amberlite IRA-400,

Dowex 3 and the like.

  Gel filtration may be accomplished using those sold under the trade names Sephadex, Sephalose, Biogel, Toyopearl Ultragel, Cellulofine and the like. The stationary phase used in affinity chromatography may be formed of various adsorbents such as Sephalose and Toyopearl.

  As a vehicle, hydrographic chromatography is also

  phobe can be carried out using an adsorbent such as those with a hydrophilic polysaccharide, such as agarose and cellulose as a base o hydrophobic groups were introduced using an aliphatic, alicyclic or aromatic compound having 2 to carbon atoms in a molecule and having a

amino group.

  The following diagrams 1 to 16 give several preferable examples of the process processes

  according to the invention given in the form of a flowchart.

Figure 1 combination of the

solution of the extract e-

  washers drying under vacuum> by freeze-drying or flashing Figure 2 aqueous extractant at pH 6-8

combination of the -

  solution of the extract and washings extractive agent, water-soluble in the form of a polar organic solvent Scheme 4 Diatomic substance Diagram 5 Scheme 6 Scheme 8 Scheme 7 Dehydrated fibrinolytic substance of Scheme 1 or 2 solvent polar organic ion exchange and / or gel filtratic / Scheme 9 fractionated material of Scheme 7 or 8 ichroma ographiel 1-arafni-it I Scheme 10 purified fibrinolytic substance Scheme 11 Scheme 12 concentrated solution of Scheme 1 or 2 decolorization Synthetic adsorbent adsorbtion ubstance ibrinolytic ubstance Diagram 13 Diagram 14 dehydrated crude fibrinolytic substance of Scheme 1 or 2 Dehydrated fibrinolytic substance of Scheme 1 or 2 Sch 6 ma 15 S Scheme 16 organic solvantr purified fibrinolytic substance A description will be given below of a preferred embodiment of the process according to the invention for the preparation of a fibrinolytically active agent from ve rs of

  in more detail, as well as the characteris-

  products thus obtained. Preparation of fibrinolytically active agents A defatted powder of freeze-dried earthworms is dispersed in 10 times by weight a solution of a 50 mM phosphate buffer having a pH of 7.0 and is incubated at 37 ° C. for 200 hours to extract the fibrinolytically active ingredients in the aqueous phase The aqueous solution of the extract is concentrated by ultrafiltration and the concentrated extracted solution is mixed with a sufficient volume of ethyl alcohol to precipitate in fractions, the effective ingredients The precipitates are dissolved in distilled water and fractionated using DEAEcellulose in three fibrinolytically activated fractions

  hereinafter F-I, F-II and F-III, respectively.

  Each of the fractions F-I and F-II is subjected to desalination with ammonium sulphate, followed by treatment with Sephadex G-75 and lyophilization of the active fraction. The third fraction F-III is subjected to desalination as it is, followed by freeze-drying.

to give a purified product.

  II Characteristic properties common to fractions F-I to F-I It and method of determining activity (1) Activity: each of them has activity

to solubilize fibrin.

  (2) Specificity of the substrate: each of them

  has a strong activity to break down fibrin.

  (3) optimal pH and stabilizing pH: the optimum pH of the fibrinolytic substance is of the order of 8-10

  while the stabilization p H is of the order of 5-10.

  (4) Determination of activity: The fibrinogen is dissolved in a 0.17 M borate buffer solution having a pH of 7.8 and containing 0.01 M sodium chloride in a concentration such that the concentration of the coagulable protein is 0.15% by weight and 10 ml of the solution is taken in a sterile glass dish 80 mm in diameter with a mixture of 0.5 ml of a 20 units / ml thrombin solution. with then rest for 1 hour at room temperature with the

covered cup.

  Testing of the standard fibrin plate is accomplished by dropping 0.03 ml of the above test solution prepared on a standard fibrin plate prepared with 10 ml of a 0.15% fibrinogen solution and after having maintained for 10 minutes with a filter paper inserted under the glass lid, placing it in a thermostat at 37 C to be maintained for 18 hours to carry out the reaction The fibrinolytic activity is expressed by the product in mm of lengths of the major and minor axes of the surface on the

  standard fibrin plate formed by dissolution.

  (5) Stability: at least 92% of the residual activity is obtained with the fibrinolytically active ingredients according to the invention in an aqueous solution having

  a pH of 7.5 or 9.0 after 30 minutes at 50 ° C.

  (6) Inhibitors: The activity of the fibrinolytic substance is inhibited by aprotinin (Trasylol, Baeyer Co product), tranexamic acid (Transamine, product of Dai-ichi Seiyaku Co) and trypsin inhibitor soybean (marketed by Miles Laboratories,

Inc) and serum.

  (7) Fibrinolytic activity: the fibrinolytic agent

  active ingredient according to the invention has an activating activity.

  Thus, fibrin is solubilized indirectly in addition to the direct reaction thereof to effect solubilization. Furthermore, a similar activity is also presented by an extract solution prepared by incubation at 37 ° C. for one day. homogenate of 300 g lyophilized powder of worm tissues in 3 liters of physiological saline solution or the freeze-dried material of

the solution of the extract.

  In an isoelectric focusing carried out with an extract solution obtained in the manner described above after filtration, the gradient curve of p H and the optical density curve at 750 nm using the copper-Folin reagent fractions each in a volume of 2.5 ml are as shown in Figure 1 by curves I and II, respectively These results

  indicate the presence of protein substances or virtually

  proteins having the isoelectric points to the values

p H 1.5, 3.4 and 5.6.

  FIG. 1 also shows the results of the determination of the fibrinolytic activity of the fractions in the isoelectric focusing of the solution of the above extract obtained as filtered or after dialysis by curves III and IV, respectively. presence of a very fibrinolytic substance in the fractions of the solution of the extract without dialysis at or near the isoelectric point at p H 3,4 and the presence of fibrinolytically active substances having a somewhat lower activity than above. above in the fractions obtained at or near the isoelectric points of p H 1.5 and p H 4.0 Contrary

  to this, the fractions obtained at or near the iso-

  Electrical p H 5.6 have no fibrinolytic activity.

  Moreover, the dialysis of the extract solution has an effect to remove the fibrinolytically active substance appearing in the fractions of the solution of

  the extract before dialysis at or near the iso-

  The activity of the fractions obtained at or near the isoelectric point of pH 3.4 is maintained even after the dialysis. Thus, it can be concluded from FIG. active at an isoelectric point at about pH 3.4 while the substance having an isoelectric point of the order of pH 5.6 is not related to fibrinolytic activity

  material extracted from earthworms.

  III In Vivo Activity Assay of the Fibrinolytic Substance According to the Invention In the use of the fibrinolytically active agent according to the invention as a therapeutic drug for a human patient by oral administration, the raw or partially purified products in any intermediate stage in the above-mentioned schemes for purification can be used but it is needless to say that the highly purified final product is the most preferably formed by the fact that it exhibits quite excellent efficiency as a therapeutic drug by administration

oral.

  The purified product obtained in Example 1 which follows was orally administered to three male patients, aged 73 and 59, suffering from arteriosclerosis, each at a dose of 600 mg in freeze-dried form, and the peripheral blood of each patient was taken periodically at intervals of one hour, for which the activity

  fibrinolytic in 1 mm 2 was examined by fractional

  The white circles and black circles on each of the curves correspond to a complete and incomplete solubilization of the I / II / III curve for the above three patients, respectively. The conclusion derived from these results is that the fibrinolytic activity in the peripheral-free begins to increase 2 hours after administration for

  reach the maximum value 4 to 6 hours after

  treatment of the fibrinolytically active agent according to the invention.

Example 1

  An aqueous slurry of 1 kg of freeze-dried and finely powdered tissues of earthworms of the species Lumbricus rubellus, after degreasing with acetone, dispersed in 10 liters of a 50 mM solution of phosphate buffers at a pH of of 7.0, was shaken during

  hours at 370 C to extract the ines-

  The residue of the filtration was washed with 3 liters of the same solution of tampors and the washings were combined with the filtrate to give a total volume of 12.8 liters of a solution of water. The clear extract The fibrinolytic activity of this extract solution was 490 mm 2 / ml after 10-fold dilution with distilled water. The above extract solution obtained was concentrated by ultrafiltration to give a volume of 1.75 liters of the concentrated extract solution having a fibrinolytic activity of 550 mm 2 / ml after 60-fold dilution. The effective ingredients contained in this concentrated extract solution was precipitated by fractionation, first adding 1.75 liters of ethyl alcohol to the extract solution and then adding 7.0 liters of ethyl alcohol to the filtrate of the precipitation of the first The precipitates collected at the two-step precipitation above were combined and dissolved in 1.1 liters of the same buffer solution. The solution thus obtained had a fibrinolytic activity of 694 mm / ml after 60-fold dilution. The solution was again submitted to a

  Chromatographic fractionation using DEAE-

  Sephalose (product of Pharmacia Co) in three fractions of F1, F-II and FIII Each of the IF and F-II fractions was subjected to 60% saturation desalting with ammonium sulfate followed by treatment with Sephadex G- 75 and lyophilization to give 625 mg of a dehydrated powder material having a fibrinolytic activity of 12,300 mm 2 / mg and 665 mg of a powder material having a fibrinolytic activity of 10,700 mm 2 / mg, from fractions FI and F The third fraction F-III was freeze-dried, after desalting and concentration, to give 1200 mg of a powder material having

  fibrinolytic activity of 11 500 me / mg.

Example 2

  Live earthworms of the same species as in Example 1, weighing 84 g, were added to physiological saline to give a total volume of 300 ml and milled to a homogeneous suspension, which was incubated

  for 100 hours at 370 C with subsequent separation

  in an extract solution and an insoluble residue.

  The residue was washed with 150 ml of physiological saline and the washings were combined with the extract solution to give a total volume of 400 ml of the combined solution having fibrinolytic activity of 375 mm 2 / ml after dilution at once The dehydrated material obtained from this solution

  freeze-drying combined with fibrinogen-

lytic of 142 mm 2 / mg.

Example 3

  Live earthworms weighing 84 g were added to 500 ml of distilled water containing 0.3 g of phenol and ground in a homogeneous suspension which was incubated for 76 hours at 300 ° C with subsequent filtration. remove the insoluble residue from the solution of the aqueous extract 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 solution combined having an activity

  fibrinolytic of 220 mm 2 / ml after 10-fold dilution.

  Example 4 Live earthworms weighing 84 g were added to an aqueous mixture of 400 ml of distilled water and ml of ethyl alcohol and ground into a homogeneous suspension, which was incubated for 240 hours at 250 ° C. with then centrifugal separation to give a clear extract solution having a fibrinolytic activity of

  350 mm 2 / ml after dilution to 10 times.

Example 5

  An aqueous suspension was prepared by mixing 50 g of vacuum-dried earthworm powder with 400 ml of physiological saline and 100 ml of a salt solution having a pH of 6.5, prepared with a 1.8% aqueous solution of phosphoric acid and 1.8% of ammonia water and the suspension was incubated for 100 hours at 380 ° C., followed by filtration to give a solution of the clear extract having a fibrinolytic activity of 725 mm 2 / ml after a 10-fold dilution corresponding to an activity of 72.5 mmi 2 / mg of

the dry powder of earthworms.

Example 6

  An aqueous suspension was prepared by dispersing 50 g of freeze-dried earthworm powder in a mixture of 250 ml of a dilute salt solution having a pH of 6.3, prepared with a 2% solution of aqueous acetic acid and a 2% solution of aqueous sodium hydroxide, ml of a physiological saline solution and 50 ml of distilled water with addition of 0.5 g of sodium azide and the suspension was incubated for 72 hours at 37.degree. C then with filtration to give a clear solution of the extract having a fibrinolytic activity of 460 mmi 2 / ml

after dilution to 10 times.

  EXAMPLE 7 An aqueous suspension was prepared by dispersing a lyophilized earthworm freeze-dried powder in a mixture of 200 ml of a solution of acetate buffers having a pH of 7.0, 200 ml of borate buffer solution having the same pH value as above, ml of distilled water, 10 ml of propyl alcohol and 10 ml of dioxane, and the suspension was incubated for 240 hours at 320 ° C. with subsequent filtration for give a clear extract solution with a fibrinolytic activity of

  772 mm 2 / ml after dilution to 10 times.

Example 8

  An aqueous suspension was prepared by dispersing degreased lyophilized worm body powder with the entrails removed in an aqueous mixture of 250 ml of a dilute salt solution having a pH of 6.8 prepared with an acidic mixture. aqueous solution containing 1.8% phosphoric acid and 3.5% 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 251 C then with suction filtration to give a clear extract solution having a fibrinolytic activity of

  600 mm 2 / ml after dilution to 10 times.

Example 9

  An aqueous suspension was prepared by dispersing dehydrated powder of dehydrated earthworms by flash drying at a high temperature in an aqueous mixture of 50 ml of a phosphate buffer solution having a pH of 6.4 and 50 ml of a solution of citrate buffers having a pH of 6.5 and the suspension was incubated for 7 hours at 370 ° C., followed by filtration to give a clear extract solution. The residue was washed with saline solution The combined solution was mixed with 0.1 g of sodium azide and further incubated for 1 hour. The combined solution was combined with the extract solution above to give a total volume of 120 ml of the combined solution. hours at 371C The resulting solution had a fibrinolytic activity of 280 mm 2 / ml after dilution

to 10 times.

Example 10

  An aqueous suspension was prepared by dispersing a lyophilized earthworm freeze-dried powder in an aqueous mixture of 50 ml of a phosphate buffer solution having a pH of 7.4 and 50 ml of a saline solution. physiological and the suspension was stirred for 4 hours at 220 ° C to extract the water-soluble ingredients in the aqueous solution followed by centrifugal separation to give a clear extract solution. The fibrinolytic activity of this solution was after mixing 0.07 g of sodium azide and incubating for 5 hours at 370 ° C., 190 mm 2 / ml after

10-fold dilution.

Example 11

  Live earthworms weighing 10 g were added to an aqueous mixture of 70 ml of a solution of acetate buffers having a pH of 7.0 and 30 ml of distilled water, and ground into a homogeneous aqueous suspension which was stirred for 2 hours at 200 ° C to extract the water-soluble ingredients in the aqueous solution followed by centrifugal separation to give a clear extract solution The residue was washed with water the water and the washings were combined with the extract solution to give a total volume of 180 ml of the combined solution having fibrinolytic activity, after mixing 0.1 of sodium azide and incubating for 7 hours at 371 C,

mm 2 / ml after dilution to 10 times.

Example 12

  An aqueous suspension prepared by dispersing 1 kg of freeze-dried earthworm powder in 10 liters of an aqueous solution containing 0.1% by weight of sodium benzoate and 0.9% by weight of sodium chloride was stirred for 96 hours. hours at 320 C to extract the

  ingredients soluble in water with subsequent filtration.

  The residue from the above filtration was washed with 3 liters of the same aqueous sodium benzoate and sodium chloride solution as above, and the washings were combined with the filtrate to give 12.5 liters of water. clear extract solution having a fibrinolytic activity of 480 mm 2 / ml after

10-fold dilution.

  The extract solution thus obtained was concentrated to a total volume of 0.5 liter by ultrafiltration and the concentrated solution was subjected to fractional precipitation by first adding 0.5 liter of ethyl alcohol to obtain precipitates and then adding another volume of ethyl alcohol in the filtrate from the first precipitation to give a final ethyl alcohol concentration of 80% to obtain an additional amount of precipitates The precipitates obtained in the two-step precipitation above were combined and washed with ethyl alcohol followed by drying under vacuum to give 40.5 g of a powder

  dry having a fibrinolytic activity of 1 285 mm 2 / mg.

  The powder obtained above was dissolved in 1 liter of a 10 mM phosphate buffer solution having a pH of 8.0 and the solution was passed through a column filled with hexyl-Sepharose prepared by the reaction of hexylamine with activated agarose with epichlorohydrin (Sepharose, product of Pharmacia Fine Chemicals Co) to have the active ingredients adsorbed After washing with the same buffer solution as above, the elution of the column was carried out with the same buffer solution as above but containing sodium chloride at a concentration of 0.25 Mc as eluent to give 1 liter of an eluate solution. The eluate solution was dehydrated, after dialysis, by lyophilization to give 5.75 g of a dehydrated material having fibrinolytic activity.

7 241 mm 2 / mg.

Example 13

  The process to fractional precipitation with ethyl alcohol and then vacuum drying was substantially the same as in Example 12 above and 47 g of the dried powder having a fibrinolytic activity of 1100 was dissolved. mm 2 / mg, in 1 liter of a

  20 mM phosphate buffer solution having a pH of 7.0.

  This solution was passed through a column

  filled with ETI (trypsin inhibitor of the albumen,) -

  Sepharose prepared by combining an albumen trypsin inhibitor (product of Sigma Co) with activated agarose with epichlorohydrin to have the active ingredients adsorbed After washing first with the same buffer solution as above then with a solution of 0.1M acetate buffers with a pH of 5.0, elution of the column was undertaken with the same acetate buffer solution as above but containing sodium and alginine at concentrations of 1 M and 0.5 M, respectively, as eluent to give 0.8 liter of a

eluate solution.

  The eluate solution was dehydrated, after dialysis, by lyophilization to give 255 mg of a dehydrated material having fibrinolytic activity

of 70 960 mm 2 / mg.

Example 14

  An aqueous dispersion of 1 kg of freeze-dried earthworm powder in 10 liters of an aqueous salt solution containing 0.1% sodium benzoate and 0.9% sodium chloride was stirred for 72 hours at room temperature. 300 C to extract the soluble ingredients

  in water in the aqueous solution and then filtered

  The residue was washed with 3 liters of the same salt solution as above and the washings were combined with the extract solution to give a total volume of 13 liters of water. the combined clear solution having a fibrinolytic activity of 450 mm 2 / ml after dilution

to 10 times.

  The aqueous solution obtained above was concentrated by ultrafiltration into a liquid volume of 0.71 liter and then mixed with an equal volume of ethyl alcohol to precipitate the solid material which was collected by filtration. The filtrate was further mixed with ethyl alcohol to give a final ethyl alcohol concentration of 80% to give other precipitates which were collected and washed with ethyl alcohol followed by vacuum drying to a dry powder The total yield of the products powdery and dry in the rush of the above steps was to

  42 g and their fibrinolytic activity was 1322 mm 2 / mg.

  The pulverulent product obtained above was dissolved in 1000 ml of distilled water and subjected to fractionation by column chromatography using a DEAE-Cellulofine adsorbent agent (product of Chisso

  Co.) to give three fractions F-I, F-II and F-III.

  Figure 3 gives the results of fibrinogen

  lytically in mm 2 and the optical density at 280 nm fractions of the eluate each to a volume of 20 ml obtained in the column chromatography above by curves I and II respectively The dashed line in Figure 3 indicates the concentration of sodium chloride in fractions of the eluate given by the electrical conductivity in m S Each of the fractions FI to F-III was subjected to a 60% saturation desalting treatment with ammonium sulfate and the precipitates were dissolved in a small volume of a 10 mM phosphate buffer solution having a pH of 8.0. The solution was subjected in succession to gel filtration with Sephacryl S-200 and at desalted concentration. by ultrafiltration followed by lyophilization to give 629 mg, 879 mg or

  1.070 mg of the dehydrated product having a fibrinogen

  1380 mm 2 / mg, 9 290 mm 2 / mg or 17 620 mm 2 / mg,

  respectively, from fractions F-I, F-II and F-III.

  The activity of the plasminogen activator was examined for each of the dehydrated products obtained above. Thus, the dehydrated product was dissolved in water at a concentration of 0.1 mg / ml and 20 / c was mixed. 1 of this solution to 10 '11 of plasminogen having an activity of 5 units / ml (product of Sigma Co) and 30 / of a solution of 0.17 M borate buffer having a p H of 7.8 and containing 0.01 M sodium chloride and, after standing for 10 minutes at 37 ° C., 0.03 ml of the mixed solution was dripped vertically onto a plasminogen-free fibrin plate (a product of Miles Laboratories, Inc) The area in mm 2 of the dissolved part was determined on the plate after 18 hours of reaction at 37 ° C. When the surface obtained above is taken for A and the corresponding surface in mm 2 that is gets using ml of 0.17 M borate buffer solution instead of the plasminogen is taken for B, then the plasminogen activator activity is expressed as AB. The values of the plasminogen activator activity so determined are 2.025 mm 2 / mg, 1721 mm 2 / mg and 1.283 mm 2 / mg. for dehydrated products obtained from

  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 human blood plasma, 0.02 ml of aqueous solution at 250 mM calcium chloride and 0.02 ml

  of an aqueous solution of one of the fibrinolytic products

  at a varied concentration 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 set for the thrombo-Wellco test (manufactured by Wellcome Co) The results are shown in Table 1 in columns under the heading of Ca C 12 (+) for five different concentrations of 10-4 to 10-1 ng / ml The (+), (++), and (+++) matrices of the table indicate the formation of FDP from

  fibrin, the increase in the number of brands + corre-

  to the increase in FDP training while the (-) mark indicates the lack of FDP training in

  from fibrin for each concentration.

  On the other hand, the same test as above was repeated in the absence of calcium chloride, that is to say by using a physiological saline solution instead of the aqueous solution of calcium chloride, to find that it was not formed of FDP despite the fraction

  fibrinolytically active products and their concentration

  as shown by the columns of Table 1 under the heading of Ca C 12 (-). These results support the conclusion that the fibrinolytically active agent according to the invention has reactivity with fibrin and not

with fibrinogen.

TABLE 1

  Fraction F I F -I II oncentratio C Cl 1 of substance 2 of the (+) (-) fibrinolytic substance

104 ng / ml + +++ -

103 +++ +++ -

102 +++ +++ -

+ +++ + -

1 + +++ + -

-1 + ++ + -

_

Example 16

  Each of the purified and freeze-dried fractions of FI to F-III fractions of Example 14 was administered orally to healthy men at a dose of I / -g / kg body weight and peripheral blood of subjects. was taken periodically to give the fractions of emglobulin, with which measurements were undertaken in search of the complete dissolution time of euglobulin in hours, and the activity of dissolution of fibrin in mm 2 by the test of standard fibrin plate The results are illustrated on the

Figures 4a and 4b, respectively.

  As shown in FIG. 4a, the time for solubilization of euglobulin had remarkably decreased about 2 hours after oral administration of the purified fibrinolytically active agents obtained from fractions FI (curve I) and F-III (curve III) and the decrease in euglobulin solubilization time continued steadily. On the other hand, the time for solubilization of euglobulin began to decrease gradually about 6 hours after 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 for improving fibrinogen

  lytic of the peripheral blood of man during a

oral administration.

  Moreover, FIG. 4b indicates that, although with considerable differences between individuals, the fibrinolytic activity of the globulin fractions obtained by the peripheral blood was at most 2 to 7 hours after oral administration of the active agents obtained. from fractions FI to F-III (curves I to III respectively) to subjects and their fibrinolytic activity was maintained sustainably

  even 10 hours after administration.

Claims (9)

R E V E N D I C A T I 0 N S   1. Process for the preparation of a fibrinogen   lytically active, characterized in that it consists of:   (a) extract the tissues of earthworms belonging to   to the family of earthworms with an aqueous extractant to provide an aqueous extract solution containing the active ingredients, and (b) concentrate or dehydrate the aqueous extract solution. 2 Process according to claim 1, characterized in that the worm tissues subjected to the extraction   in step (a) are in the form of a lyophilized powder.   3. Method according to claim 1, characterized in that the aqueous extraction agent is a physiological saline solution or a buffer solution having a p H between 5 and 10.   4. Method according to claim 1, characterized in that the extraction in step (a) is performed at a temperature between 5 and 40 WC for at least 30 minutes.   Process according to claim 1, characterized in that the amount of the aqueous extractant is from 1 to 100 times by weight based on   the dry weight of worm tissues.   Process according to claim 1, characterized in that the aqueous extractant contains a polar organic solvent in admixture with it at a concentration   not to exceed 50% by volume.   7. Method according to claim 1, characterized in that it further comprises the purification step of the   active ingredients either before or after step (b).   8. Process according to claim 7, characterized in that the purification of the active ingredients is accomplished by a process selected from the group of processes comprising adsorption on and desorption of an adsorbent, fractional precipitation with a polar organic solvent, desalination, ultrafiltration, ion exchange chromatography, filtration on   gel, affinity chromatography and chromato-   Hydrophobic graph. 9. Fibrinolytically active agent, characterized in that it is prepared by a process comprising the extraction of worm tissues belonging to the family of earthworms with an aqueous extractant to give an extract solution and the concentration or dehydrating said extract solution.