US3256157A - Agents having a fibrinolytic activity and being derived from molds and a process of making and using same - Google Patents

Description

June 1966 A. P. TRUANT ETAL 3, 6, 57

AGENTS HAVING A FIBRINOLYTIC ACTIVITY AND BEING DERIVED FROM MOLDS AND A PROCESS OF MAKING AND USING SAME Filed Feb. 20, 1959 2 Sheets-Sheet l Tij. l.

2 3 7 8 9 l 11 1213 Is Mws/Yunee llv KAyus Maw-Luvs; //v MICEONS Z 3 4 S 6 8 ID I! I2 I3 416 I00 Mus/vanes v Aquas W MKM AGE YT J1me 1966 A. P. TRUANT ETAL 3, 56, 57

AGENTS HAVING A FIBRINOLYTIC ACTIVITY AND BEING DERIVED FROM MOLDS AND A PROCESS OF MAKING AND USING SAME Filed Feb. 20, 1959 2 Sheets-Sheet 2 Mwszmem //v MICfO YS 3 S 6 7 8 9 I0 I! I2 13 145/6 52 4500 4000 am Z000 I900 I700 I600 I400 I500 1200 1100 I000 900 'am no 6117 IN V EN TORS r cao P. T uA/vr Fflrz G. Noeosrycn MWMWOL AGE VT United States Patent 3 256,157 AGENTS HAVING A FIBRINOLYTIC ACTIVITY AND BEING DERIVED FROM MOLDS AND A PROCESS OF MAKING AND USING SAME Aldo Peter Truant and Fritz George Norstrom, Worcester, Mass., assignors to Astra Pharmaceutical Products, Inc., Worcester, Mass., a corporation of New York Filed Feb. 20, 1959, Ser. No. 794,729 7 Claims. (Cl. l95--62) The present invention relates to agents having fibrinolytic activity and more particularly to agents having fibrinolytic activity and being derived from molds which agents have a selective fibrinolytic activity at the site of a blood clot without substantially affecting other blood and plasma constituents, and to a process of making and using same.

As is known, the clotting of blood will occur in the intact vascular system. If a clot does develop, the problem of thrombosis may arise and the resulting thrombus may lead to infarction and, as a result thereof, to necrosis of the tissue supplied by the affected blood vessel. A blood clot or embolus forced by the blood current from a thrombosed vessel into a smaller one or formed in a coronary artery may cause obstruction of vital areas of the body and often death.

A number of agents have been used in therapy to prevent an extension of the blood clot or to cause its dissolution. Heparin or other anticoagulants, for instance, have been administered for their property of prolonging the clotting time of blood which facilitates clot resolution and prevents extension of an existing intravascular clot.

Other agents, such as the enzyme streptokinase, have an activating elfect on the precursor of the fibrinolytic enzyme profibrinolysin which causes cleavage of fibrin, i.e. the material comprising the blood clot, into soluble polypeptides. An agent of similar activity is streptodornase. The enzyme trypsin has also been used for the lysis of fibrin and plasma clots, likewise the enzyme plasmin obtained from human blood.

These known agents, however, have a number of disadvantages.

(l) Heparin and coumarins do not directly alI'ect an existing blood clot and they do not limit the damage to the afiected vascular tissue.

(2) Trypsin, in doses insufiicient to induce clot lysis, causes an acceleration of the clotting mechanism and intravascular coagulation. There is no satisfactory means available to determine a priori the dose necessary for fibrinolytic activity.

(3) Streptokinase, although not antigenic, possesses a pyrogenic action.

(4) Plasmin is expensive and has an antigenic action.

(5) Both trypsin and streptokinase measurably affect other clotting and plasma constituents. Therefore, these known agentshave found only limited application in therapy.

It is one object of the present invention to provide new and valuable fibrinolytic agents which difier in their chemical constitution from that of other known fibrinolytic agents, do not substantially affect the other blood and plasma constituents, and have substantially no antigenic and/ or pyrogenic activity.

, Another object of the present invention is to provide a starting material for the preparation of such new and valuable fibrinolytic agents which material is readily available and permits large scale production of such agents.

A further object of the present invention is to provide a simple and effective process of producing such new and valuable fibrinolytic agents.

Still another object of the present invention is to .providesimple and elfective means of purifying and con- 3,256,157 Patented June 14, 1966 ice centrating such fibrinolytic agents and to produce products with a surprisingly high degree of activity.

Still another object of the present invention is to provide a simple and efiective method of testing the starting material for its fibrinolytic activity, of determiningthe activity of the intermediate and final products obtained in the course of the manufacture of the new fibrinolytic agent, and of standardizing pharmaceutical preparations for clinical use containing such an agent.

Other objects of the present invention and advantageous features thereof will become apparent as the description proceeds.

In principle, the present invention consists in isolating fibrinolytic agents from cultures of specific molds that are capable of producing such agents. a

Of a large number of molds, the following four strains which have proved to be valuable starting material for the purpose of the present invention were supplied for the isolation and purification of the fibrinolytic agents according to the present invention.

Two strains of the species Aspergillus oryzae, which are available to the public from the Quartermaster Research and Engineering Center, United States Army, at Natick, Mass., under the depository numbers B-82i and B-1273, are capable of producing especially largeamounts of the new fibrinolytic agents. One strain of the species Aspergillus flavus, also available to the public from said Quartermaster Research and Engineering Center under the depository number B-4m, and one strain of the genus Absidia, species Absidia coerulea, also available to the public from said Quartermaster Research and Engineering Center under the depository number D-10l,- have also proved to produce said new fibrinolytic agents. It is understood, of course, that although these specific strains have proved to be especially suitable for the production of the new fibrinolytic agents, the present invention is not limited thereto. Any other strain of molds which, when tested as will be described hereinafter, shows selective vfibrinolytic activity, may also be used as starting material.

The cultures investigated were transferred to solid Sabourauds medium. The Sabourauds dextrose medium contained Neopeptone g 10 Bacto-dextrose g 40 Bacto-agar g 15 Water, to cc-.. 1000 After cultivating the molds on said solid medium at 30 37 C. for five to ten days, the resulting inoculum was transferred to a liquid mediumprepared from Sucrose g 7.2

Dextrose g 3.6

MgSO (crystals) g 1.23 1 0 g 13.69

KNO3 g 2 and H 0, to cc 1000 Aliquots of cc. were transferred to Erlenmeyers flasks of 250 cc. capacity. A sample of the Sabouraud culture, approximately 1 cm. in diameter, was transferred to each flask. Cultures were incubated at 29-30 C.

After a specific interval of time, the content of the flasks,

The precipitate is then dried in a vacuum at 37 C. According to the activity of the precipitate amounts between 0.5 mg. and 10.0 mg. of the dried material are suspended in 1 cc. each of distilled :water, saline solution, or phosphate buffer solution of a pH of 5.5, the total volume of the suspension being one tenth of the volume of the original filtrate. The resulting suspensions or, respectively, solutions are assayed by two methods:

1) Fresh platelet-poor human or bovine plasma in a volume of 0.2 cc. which did not show retraction four hours after clotting, were mixed in a test tube with 0.2 cc. of saline solution, 0.2 cc. of the above described suspension or solution of the active agent, and 0.2 cc. of thrombin solution containing 100 NIH units/ cc. After verifying that clots had formed normally, the test tubes were observed for lysis hourly for 6 hours whereby they were kept in a water bath at 37 C. If, on addition of 0.2 cc. of the suspension or solution of the precipitate, a clot does not occur, the suspension or solution must be further diluted. All molds which on cultivation on the above given culture medium yield extracts showing clot lysing activity in the above given test were selected for cultivation on a large scale and for production of the new fibrinolytic agents. It may be mentioned that this test is not Very sensitive. This is due to the pH-value of the mixture being about 6.87.2. At such a pH-value the fibrinolytic activity of the active agent is considerably inhibited. Consequently, the concentration of the active agent in the above described suspension or solution must be increased, if the test results are negative.

(2) The following second test method is more suitable for detecting fibrinolytic activity in molds andfor determining the activity of fibrinolytic agents. According to this method modified Astrup-Muellertz fibrin plates are prepared by rapidly adding 1 cc. of bovine thrombin solution containing 100 NIH units/ cc. to 10 cc. each of an 0.2% and a 1% solution of bovine or human fibrinogen. After rapidly mixing said solutions, the mixture is poured into petri dishes. for minutes. Thereafter a drop of 0.2 cc. of the mold culture filtrate or, respectively, of the above described suspensions or solutions are deposited on each plate by means of a pipette. After incubation in a thermostat at 37 C. up to 180 minutes, the degree of lysis is judged by measuring the area of clearing and digestion of the fibrin plate.

It may be mentioned that this test method is more sensitive than the above mentioned first test method. The reason for this is that the pH-value of the filtrate as well as of the suspensions or, respectively, solutions is at the optimum pH-value of activity of the fibrinolytic agent and that, therefore, activity can be detected much more easily than with the first test method in which the filtrate or solutions or suspensions are mixed with the test medium having a pH value of 6.8-7.2 and, thus, are not at the optimum.

In the second method, although the pH-value of the plate is similar to that of the test medium of the above mentioned first method, namely at 6.8-7.2, the suspension or solution to be tested is not mixed with the test medium but acts thereon at one point only, namely at the place where the drop is deposited. Therefore, the pH-value of the mold culture filtrate which is between about 5.0 and 5.5, depending upon the conditions of cultivation, and the pH-value of the suspension or solution which is at about 5 .5 are capable of exerting their activating effect and are not neutralized on deposition on the plates. A pH-value of 5.5 has proved to be the optimum pH-value of activity of the'new fibrinolytic agents according to the present invention.

The following Table I shows the lytic activity of an alcoholic precipitate of an active mold filtrate prepared as described above. 0.2 cc. of the suspension having a pH of 5.5 were tested according to the above mentioned second test method. The diameter size of clearing is deter- The plates are then heated at 85 C.

TABLE I.ASSAY OF FIBRINOLYTIC AGENT BY THE FIBRIN PLATE METHOD Diameter (111.111.) of Clearing Time (min.) Amount of Agent/cc.

This table clearly shows that there is a linear relationship between the activity and amount of fibrinolytic agent. It follows that the second assay method is susceptible to quantitation. Therefore, this method permits quantitative determination of the fibrinolytic activity of a mold filtrate in a reliable manner.

For the production of the new fibrinolytic agents active fungi, as they can be selected and distinguished from inactive fungi, are cultivated on suitable culture media. Culture media containing a substantial amount of phosphates have a favorable effect on the amount of fibrinolytic agent produced by the fungus. It is, of course, understood that the culture medium contains a source of carbon, such as starches, dextrin, dextrose, lactose, sucrose, and other carbohydrates; a source of nitrogen such as ammonium salts, nitrates, or organic nitrogen sources such as peptones, wheat bran; mineral salts which supply potassium, sodium, magnesium, nitrate, phosphate, sulfate ions; and trace elements, such as iron; vitamins, especially the vitamins of the B-group; and other mold growth promoting substances. It has been found that the presence of large amounts of phosphates increases the yield. Amounts of potassium monophosphate of at least 1% and preferably of 1.2% to 1.5% of the culture medium have provide to be especially effective.

The following Table II clearly shows that a high phosphate content in the culture medium has a favorable effect on the yield of fibrinolytic agent. In said table the amount of potassium monophosphate is given in g. per liter of culture medium. The pH given in said table is the pH of the culture filtrate after cultivation for 13 days at a temperature of 25 C. The weight of the mycelium is given in g. after drying at 100 C. The last column shows the weight of the dried alcohol precipitate. It is clearly evident that the yield of precipitate decreases considerably with the decrease in phosphate concentration, although the amount of mycelium remains relatively constant. The activity of all four precipitated fibrinolytic agents on a dry weight'base is about the same so that the increase in yield on cultivation with a large amount of potassium monophosphate represents a factual increase in the amount of fibrinolytic agent produced.

TABLE II.-INFLUENCE OF PHOSPHATE CONTENT IN CULTURE MEDIUM ON YIELD OF FIBRINOLY'IIC AGENT pH of culture Weight of Weight of Amount of KH3PO4, g./l. medium mycelium dried alcohol after 13 days in g. precipitate achieved within 3 to 15 days depending upon cultivation temperature and other conditions.

The following Table III shows the effect of the cultivation temperature upon yield and activity of the fibrinolytic agent produced. The same mold culture was cultivated at two difierent temperatures, namely at 30 C. and at 37 C. The fibrinolytic agent was precipitated by the tannic acid method described hereinafter. The yield of precipitate is determined after 2, 4, 6 and 9 days. The activity of the precipitates was determined according to the above mentioned secondmethod. Furthermore, the activity/yield coefiicient is determined by multiplying the activity with the yield.

TABLE III.-INFLUENCE F TEMPERATURE ON YIELD OF FIBRINOLYTIC AGENT AND ITS ACTIVITY This table clearly shows that at the higher cultivation temperature of 37 C. the highest activity/yield coefficient is obtained between the 4th and the 6th day while at the lower cultivation, temperature of 30 C. it takes at least six days to produce a satisfactory activity/yield coefiicient.

Cultivation can be carried out at a temperature between about 22 C. and about 37 C. The preferred cultivation temperature is between about 28 C. and about 32 C. Substantially no activity was found on cultivation below 20 C. and above 40 C. The fibrinolytic agents may be obtained by both surface or submerged cultivation.

The preferably inoculum is grown on a solid culture medium preferably on Sabourauds dextrose medium, until abundant sporulation is observed. Such Well sporulated inoculum is then transferred to the liquid culture medium mentioned hereinabove. Three to four inoculums of the size of about 3 cm. in diametereach are transferred to a 5 1. bottle, containing 1,000 cc. of said liquid culture medium. Of course, smaller or larger volumes of culture medium may also be used.

trations above 70%, cause a certain decrease in activity.

The alcohol precipitate contains the active agent. This Preferably such an amount of tannic acid is added that Working up of the culture medium in order to concentrate the fibrinolytic agent may be effected in different ways whereby after each step of isolation the fibrinolytic activity of the resulting intermediate solution and products is determined in order to find out whether a specific step actually causes concentration of the active agent.

One method of producing solutions of the fibrinolytic agents according to the present invention follows closely the method described for testing molds for their fibrinolytic activity. According to said process, the liquid culture medium which has a pH between 5.2 and 5.8 is filtered or centrifuged to remove the mycelium. Preferably filtration is effected through Whatman No. 2 paper. Two parts by volume of 95% ethanol precooled, preferably to 20 C.,-are added to one part by volume of the filtrate precooled to 010 C. The mixture is kept overnight in the refrigerator below 0 C. or is cooled to 20 C. and centrifuged after standing for half an hour. It was found that best results are obtained when using two parts by volume of alcohol whereby the mixture has an alcohol concentration between about 60% and about 65% Lower alcohol concentrations'than about 40% do not cause precipitation. Higher alcohol concentrations, i.e. concenthe concentration thereof in the filtrate begins with 25 mg. of tannic acid per cc. of culture filtrate and increases with the amount of fibrinolytic agent in the bath. Tannic acid is preferably added in aqueous 10% solution (100 mg. per cc.). Tannic acid'precipitation is carried out at about 0 C. The resulting tannic acid precipitate is about 20 times as active as the alcohol precipitate on a dry weight base. Tannic acid, thus, has a greater selective precipitating effect on the fibrinolytic agent than alcohol since only minimum amounts of salts are trapped in the precipitate.

The following Table IV shows the effect of varying amounts of tannic acid added to the mold filtrate. In order to carry out precipitation under exactly the same conditions, the pI-I-value of the filtrate was adjusted to a pH of 5.5 by the addition of 5 N sodium hydroxide solution before the tannic acid was added. It is evident from this table that there is a relatively insignificant difference between the various amounts of tannic acid used, provided that an amount of about 0.25 mg. of tannic acid per cc. of culture filtrate is used. The yield is given in this table in mg. per 200 cc. of'filtrate. The activity is determined according to the above mentioned second test method after min. and is expressed in percent change in the diameter of the clearing as compared with the diameter at zero time of application of the test solution to the plate. The activity/yield coefficient is expressed as the yield multiplied by the activity percent change. The table clearly shows that best results are achieved with amounts of about 0.75 mg. to 1.0 mg. of tannic acid per cc. of filtrate.

TABLE IV.YIELD 0F FIBRINOLYTIC AGENT IN RELA- TION TO THE AMOUNT OF TANNIC ACID ADDED TO THE CULTURE FILTRATE The tannic acid precipitate is obtained by centrifuging and decanting the supernatant liquid. The precipitate is washed with anhydrous acetone or any other tannic acid solvent to remove the tannic acid. The remaining active precipitate is then dried under vacuum. The resulting precipitates of active agent, whether obtained by tannic acid precipitation or by alcohol precipitation may be further purified in order to remove insoluble reactive material and contaminating soluble salts. Purification is preferably elfected by resuspending the precipitate in water, separating the insoluble material, di-alyzing the solution against water, lyophilizing the dialyzed solution.

One unit of activity of the fibrinolytic agent is expressed as the amount of activity contained inone mg. of dry material which causes fragmentation of the fibrin plate prepared as described hereinabove, to a diameter of 1 cm. after 60 minutes'at 37 C., when applied to such a plate in an amount of 0.1' cc. of an 0.1% aqueous solution. For instance, the tannic acid precipitates correspond to 2 to 4 units/mg.

The resulting fibrinolytic agents, depending on their degree of purity, contain variable amounts of nitrogen. However, the conventional tests performed for nitrogen, such as Ninhydrin, Micro-Kjeldahl associated with Nesslerization, and Tyrosin tests, have produced a typical color reaction, and consequently, the total amount of nitrogen cannot be reliably reported.

Infrared spectra of relatively pure fibrinolytic substances have been made. The attached drawings show the following infrared spectra:

FIGURE 1 illustrates the infrared spectrum obtained from a dialyzed mold culture filtrate.

FIGURE 2 illustrates the infrared spectrum of a mold culture filtrate precipitated with ethanol as described hereinabove and dialyzed.

FIGURE 3 illustrates the infrared spectrum from a mold culture filtrate precipitated with tannic acid and dialyzed.

It will be noted from said spectra that the following bands are common to the three preparations:

Near 3.0 microns, indicating the presence of an OH- group or an NH group or a combination thereof.

Near 3.45 microns, indicating that the substance is an organic material containing carbon and hydrogen.

Near 6.05 microns, indicating a carbon adjacent to a nitrogen.

Near 9.4- microns, indicating a carbon-oxygen bond, and from its intensity, it would indicate a highly hydroxylated compound.

It will be noted that in the spectrum of FIGURE 3, an additional band may be clearly seen at 6.5 microns, indicating a secondary carbon to nitrogen vibration. However, it must be pointed out that this spectrum was obtained with material utilizing tannic acid as a precipitant. At this wave length, aromatic compounds containing phenolic groups also absorb, and such a phenolic group is present in tannic acid. Since the tannic acid precipitate has a much greater fibrinolytic activity, this band may also be related to the fibrinolytic property of the product.

FIGURE 4 shows the ultraviolet spectrum for the same substance as was used in FIGURE 3. It shows an absorption at 280 millimicrons which is characteristic of a phenolic group. Whether such a phenolic group is a component of the fibrinolytic agent itself or whether it is due to tannic acid or the presence of a protein containing the phenolic tyrosin which also absorbs at 280 millimicrons, will have to be established.

By means of dialysis, utilizing a cellulose membrane with an average pore size of 24 Angstroms, it is possible to further purify and concentrate the active agent.

On suspending the dry alcohol precipitate in distilled water, the pH-value of the resulting supernatant solution is between 5.0 and 5.7 at a concentration of 1 mg. to mg./cc., while the tannic acid precipitate has a pH between 5.9 and 6.1. The tannic acid precipitate forms with small amounts of water a viscous sticky paste. The active agent is soluble in water in amounts of 0.5 mg. to 5.0 mg. per cc. 10 mg. of the precipitate are insoluble in ether, ethyl acetate, acetone, and generally in lipid solvents.

The precipitates were tested for their antigenic and pyrogenic elfects and were found to be substantially free of such efiects.

' Experiments with dogs in' which a venous blood clot was produced, showed that intravenous injection of 0.250 mg. to 1.0 g. of the tannic acid-precipitated agents suspended in saline solutions caused gradual lysis of the clot and reversal of the biochemical changes in the blood clotting mechanisms which accompany the formation and propagation of the blood clot at the level of thrombosis. A very important additional efiect of the new agent is its anti-inflammatory effect at the site of the clot. Furthermore, when an experimental clot is formed, it will ordinarily continue to propagate, especially if there is an alteration of the endothelial lining. However, under treatment with the fibrinolytic agents according to the present invention, propagation does not continue, even when the dosage is inadequate to cause complete disappearance of the clot. It was also noted that, under treatment, bleeding occurred at the site of incision from small vessels which had previously spontaneously thrombosed without ligatures.

The following examples serve to illustrate the present invention without, however, limiting the same thereto.

Example 1.Preparati0n of an inoculum of Aspergillus oryzae strain No. B-1273 From a spore culture of Aspergillus oryzae strain No. -B1273 the solid Sabourauds medium is streaked, using a 1 cm. loop and allowed to stand at 37 C. until the mycelium has developed and an abundant quantity of spores has been formed. Usually five to six days are required. A deep green color is noted and is an indication of satisfactory sporulation for inoculation.

Example 2.--Cultivation of Aspergillus oryzae strain No. B-1273 Using the spores of the culture according to Example 1, three 2 cm. loops are transferred, under sterile conditions, to 5 1. bottles, each containing 1,000 cc. of the liquid culture medium referred to in col. 2 hereinabove and having a pH of 4.5. Cultivation is effected at 29 C. until sporulation sets in which is usually the case after a period of about 6 days to 10 days. The pH is now about 5.4. Spo-rulation is readily ascertained by the occurrence of a yellowish or light brownish-green color on the surface of the mycelium.

Cultivation at 22-25 C. requires about 7 days to 14 days before sporulation sets in, while cultivation at 34- 37 C. will result in beginning sporulation after 3 days to 6 days.

Example 3.Precipitati0n of fibrinolytic agent by means of alcohol 15.7 1. of the culture filtrate obtained on filtration of cultures prepared by cultivation at 25 C. for 7 days which are pre-cooled to about 5 C., are precipitated by the addition of 31.5 1. of ethanol, pre-cooled to 20 C. The mixture is allowed to stand at about 18 C. to 20 C. for 48 hours. The precipitate is filtered by suction at the same low temperature. The filtered precipitate is dried in a vacuum at 3740 C. for about 36 hours. About 74 g. of dry fibrinolytic agent are obtained.

Example 4.Precipitati0n of fibrinolytic agent by means of tannic acid 14.5 1. of the mold culture filtrate obtained on filtration of cultures prepared by cultivation for 7 days at 25 C. and having a pH of 5.1, which are pre-cooled to about 5 C. are precipitated by the admixture of 7.0 g. of tannic acid dissolved in cc. of water. The precipitate is allowed to completely settle out by standing at 4 C. for 48 hours. A substantial amount of the supernatant liquid is removed and the remainder, with the precipitate, is centrifuged. The centrifuged precipitate is stirred with 12 cc. of anhydrous acetone at -2() C. 48 cc. of acetone are admixed and the mixture is allowed to stand at -20 C. The precipitate is filtered on Whatman 50 paper, and washed with 30 cc. of acetone to remove the acetone soluble fractions. The Washed precipitate is dried'in a vacuum. About 0.45 g. of dried fibrinolytic material are obtained.

Example 5 .Purification of the alcohol precipitate The alcohol precipitate obtained according to Example 3 is suspended in 740 cc. of distilled water while stirring.

9 The mixture is centrifuged and the supernatant liquid is adjusted to a pH of 5.5 by the addition of5 N sodium hydroxide solution. To this is added 1.5 mg. of tannic acid in finely divided form per cc. of said supernatant liquid, and the mixture is permitted to stand at C. for 24 hours. The resulting precipitate is worked up in the same manner as described hereinabove in Example 4. The precipitate is Washed with acetone as described in Example 4 and the washed material is dried at room temperature in a vacuum. 0.280 g. of dried fibrinolytic agent are obtained.

Example 6 Example 7 Of the material obtained according to Example 280 mg. are resu-spended in 56 cc. of distilled water and the resulting suspension is worked up in the same manner as described in Example 6, resulting in a yield of about 12 mg. of highly fibrinolytic agent.

Of course, many changes and variations in the preparation of the inoculum, in the cultivation of the fibrinolytically active molds, in the isolation of-the fibrinolytic agents from the mold culture filtrates, in the purification of said agents, in their administration in human and veterinary therapy, and the like may be made by those skilled in the art in accordance with the principles set forth herein and in the claims annexed hereto.

We claim:

1. In a process of producing a fibrinolytic agent not substantially affecting other blood and plasma constituents, the steps which comprise cultivating, on a liquid culture medium of a high phosphate ion content, a mold having fibrinolytic activity at a temperature between 22 C. and 37 C. until sporulation sets in, said mold being selected from the group consisting of Aspergillus oryz ae B82i, Aspergillus oryzae Bl273, Aspergz'llus flavus B4m, and Absidia coerulea D-101, discontinuing cultivation, and separating the mycelium from the liquid culture medium containing the active agent.

2. The process according to claim 1, wherein the liquid culture medium contains at least 1% of potassium monophosphate.

3. The process according to claim 1, wherein the pH- value of the liquid culture medium is initially about 4.4 to 4.5 and, at the time sporulation sets in, has increased to about 5.2 to 5.8.

4. In a process of producing a fibrinolytic agent not substantially afiecting other blood and plasma constituents, the steps which comprise cultivating, on a liquid culture medium of a high phosphate ion content, a mold having fibrinolytic activity at a temperature between about 22 C. and 37 C. until sporulation sets in, said mold being selected from the group consisting of Aspergillus oryzae B82i, Aspergillus oryzae Bl273, Asp'ergillus flavus B-4m, and Absidza coerulea D-lOl, separating the mycelium from the liquid culture medium, adding alcohol to said liquid culture medium at a temperature not substantially exceeding 0 C. in an amount suflicient to produce an alcohol concentration of at least 60%, allowing the mixture to stand until precipitation is completed, and separating the precipitate containing the fibrinolytic agent.

5. In a process of producing a fibrinolytic agent not substantially affecting other blood and plasma constituents,

the steps which comprise cultivating, on a liquid culture medium of a high phosphate ion content, a mold having fibrinolytic activity at a temperature between 22 C. and 37 C. until sporulation sets in, said mold being selected from the group consisting of Aspergillus oryzae B82i, Aspergillus oryzae-B-1273, Aspergillus flavus B-4m, and Absz'a'ia coerulea D101, separating themycelium from the liquid culture medium, adding tannic acid to said liquid culture medium at about 0 C. in an amount sufilcient to cause substantially complete precipitation of the fibrinolytic agent, and separating the precipitate containing the fibrinolytic agent. i

6. In a process of producing a fibrinolytic agent not substantially affecting other blood and plasma constituents, the steps which comprise cultivating, on a liquid culture medium of a high phosphate ion content, a mold having fibrinolytic activity at a temperature between 22 C. and 37 C. until sporulation sets in, said mold being selected from the group consisting of Aspergillus oryzae B82i, Aspergillus oryzae Bl273, Aspergillus flavus B4m, and Absidia coerulea D-101, separating the mycelium from the liquid culture medium, adding tannic acid to said liquid culture medium at about 0 C. in an amount sufficient to cause substantially complete precipitation of the fibrinolytic agent, separating the precipitate containing the fibrinolytic agent, stirring the precipitate with anhydrous acetone, filtering the mixture, removing the acetone, repeating said stirring with acetone, filtering, and removing the acetone until substantially all the acetone soluble material has been removed, drying the acetone-treated precipitate, suspending said precipitate in water, removing insoluble matter by centrifuging, and dialyzing the supernatant liquid against running water at about 10 C. until substantially all dialyzable material has been removed.

- 7. The fibrinolytic agent obtained from cultures of molds being selected from the group consisting of Aspergillus oryzae B82i, Aspergillus oryzae Bl273, Aspergillus flavus B4m, and Absidia coerulea D101, said agent not substantially aifecting other blood and plasma constituents, being soluble in water, insoluble in ether, ethyl acetate, and acetone, the pH-value of its aqueous solution being between 5.2 and 6.1, aqueous solutions of said fibrinolytic agent losing their fibrinolytic activity on adjusting their pH-value above a pH of 6.5 and below a pH of 3.0 when standing, the dry fibrinolytic agent being inactivated on standing at a temperature of above 75 C., the infrared spectrum of the alcohol-precipitated fibrinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, and near 9.4' microns, and the infrared spectrum of the tannic acid-precipitated fibrinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, near 6.5 microns, and near 9.4 microns, the ultraviolet absorption spectrum of said tannic acid-precipitated fibrinolytic agent showing an absorption at 280 millimicrons, the fibrinolytic agent not being dialyzable against Water through a cellulose membrane with an average pore size of 24 Angstroms, the. agent being substantially non-pyrogenic and nonantigenic, its aqueous solution causing lysis of thrombinclotted platelet-poor human and bovine plasma and of thrombin-clotted human and bovine fibrinogen plates.

7 References Cited by the Examiner UNITED STATES PATENTS 2,716,084 8/ 1955 Carlson et al. 66 2,753,291 7/1956 Mowat et al 195-66 2,795,529 6/ 1957 Album et al. 16773 2,857,316 10/1958 Grimm 195-6 2,857,317 10/ 1958 Grimm 195 6 FOREIGN PATENTS 566,369 11/ 1958 Canada.

(Other references on following page) 3,2 56,1 5 7 1 1 1 2 OTHER REFERENCES Yoshida et 21.: Free. of the Int. Sym. on Enzyme Chem, Australian Journal of Biological Sciences, vol. 6 (1195 3 Tokyo 1957 504-509 pp. 410-462, pp. 411, 418, 428-432 and 447-451 par- LOUIS MON ACELL, Primary Examinerticularly relied upon.

Proc. Int. Sym. Enzyme Chemistry, Tokyo, 1957, pp. 5 MORRIS WOLK, TOBIAS' LEVOW, ABRAHAM 482-485, 504 509 H. WINKELSTEIN, Exammers.

Stefani et 211.: Proceedings of the Society for Experi- F CACCIAPAGLIA, L, M, SHAPIRO, mental Biology and Medicine, vol. 99, pp. 504-507, 1958. Assistant Examiners,

Claims (2)

1. IN A PROCESS OF PRODUCING A FIBRINOLYTIC AGENT NOT SUBSTANTIALLY AFFECTING OTHER BLOOD AND PLASMA CONSTITUENTS, THE STEPS WHICH COMPRISE CULTIVATING, ON A LIQUID CULTURE MEDIUM OF A HIGH PHOSPHATE ION CONTENT, A MOLD HAVING FIBRINOLYTIC ACTIVITY AT A TEMPERATURE BETWEEN 22*C. AND 37*C. UNTIL SPORULATION SETS IN, SAID MOLD BEING SELECTED FROM THE GROUP CONSISTING OF ASPERGILLUS ORZAE B-82I, ASPERGILLUS ORYZAE B-1273, ASPERGILLUS FLAVUS B-4M, AND ABSIDIA COERULEA D-101, DISCONTINUING CULTIVATION, AND SEPARATING THE MYCELIUM FROM THE LIQUID CULTURE MEDIUM CONTAINING THE ACTIVE AGENT.

7. THE FIBRINOLYTIC AGENT OBTAINED FROM CULTURES OF MOLDS BEING SELECTED FROM THE GROUP CONSISTING OF ASPERGILLUS ORYZAE B-82I, ASPERGILLUS ORYZAE B-1273, ASPERGILLUS FLAVUS B-4M, AND ABSIDIA COERULEA D-101, SAID AGENT NOT SUBSTANTIALLY AFFECTING OTHER BLOOD AND PLASMA CONSTITUENTS, BEING SOLUBLE IN WATER, INSOLUBLE IN ETHER, ETHYL ACETATE, AND ACETONE, THE PH-VALUE OF ITS AQUEOUS SOLUTION BEING BETWEEN 5.2 AND 6.1, AQUEOUS SOLUTIONS OF SAID FIBRINOLYTIC AGENT LOSING THEIR FIBRINOLYTIC ACTIVITY ON ADJUSTING THEIR PH-VALUE ABOVE A PH OF 6.5 AND BELOW A PH OF 3.0 WHEN STANDING, THE DRY FIBRINOLYTIC AGENT BEING INACTIVIATED ON STANDING AT A TEMPERATURE OF ABOVE 75*C., THE INFRARED SPECTRUM OF THE ALCOHOL-PRECIPITATED FIBRINOLYTIC AGENT SHOWING ABSORPTION BANDS NEAR 3 MICRONS, NEAR 3.45 MICRONS, NEAR 6.05 MICRONS, AND NEAR 9.4 MICRONS, AND THE INFRARED SPECTRUM OF THE TANNIC ACID-PRECIPITATED FIBRINOLYTIC AGENT SHOWING ABSORPTION BANDS NEAR 3 MICRONS, NEAR 3.45 MICRONS, NEAR 6.05 MICRONS, NEAR 6.5 MICRONS, AND NEAR 9.4 MICRONS, THE ULTRAVIOLET ABSORPTION SPECTRUM OF SAID TANNIC ACID-PRECIPITATED FIBRINOLYTIC AGNET SHOWING AN ABSORPTION AT 280 MILLIMICRONS, THE FIBRINOLYTIC AGENT NOT BEING DIALYZABLE AGAINST WATER THROUGH A CELLULOSE MEMBRANE WITH AN AVERAGE PORE SIZE OF 24 ANGSTROMS, THE AGENT BEING SUBSTANTIALLY NON-PYROGENIC AND NONANTIGENIC, ITS AQUEOUS SOLUTION CAUSING LYSIS OF THROMBINCLOTTED PLATELET-POOR HUMAN AND BOVINE PLASMA AND OF THROMBIN-CLOTTED HUMAN AND BOVINE FIBRINOGEN PLATES.

Images