DE69323179T2 - New thrombolytic agent, process for its preparation and its use for the production of a thrombolytic drug - Google Patents

Description

The invention relates to a method for producing thrombinase. Thrombinase is a thrombus-dissolving agent that contains a fibrinolytic enzyme that is advantageously used for the treatment of cerebral thrombosis, myocardial infarction, thrombosis of deep vessels and for the prevention of postoperative adhesions.

Streptokinase (from streptococci), urokinase (from human urine), and tissue plasminogen activator (TPA, from human tissue culture) are the main thrombolytics currently known. Streptokinase-aspirin combinations produce significant improvement even when treatment is started 24 hours after a heart attack. Prourokinase, also known as renal plasminogen activator, is designed to dissolve blood clots without interfering with the body's normal blood clotting process. A clot-dissolving product from Beecham Laboratories, an isolated plasminogen streptokinase activator complex, brand name Eminase, claims to reduce the death rate in heart attack victims by 50%. Genentech's TPA (Activase) is also highly effective at dissolving blood clots. Urokinase, streptokinase and TPA, the first plasminogen activators available for clinical use, activate plasminogen to active plasmin. The actual clot dissolution is accomplished by active plasmin. However, the action of plasmin is not limited to lysis of fibrin present in a thrombus; plasminogen also attacks fibrinogen and other clotting factors, which can lead to severe bleeding. The fibrinolytic efficacy and specificity of these agents are greatly enhanced by covalently binding them to a fibrin-specific antibody. This type of targeting of the fibrinolytic agent to the site of a thrombus opens the possibility of using direct-acting fibrinolytic enzymes instead of plasminogen activators.

It is an object of this invention to propose a new process for the production of thrombinase.

It is a further object of this invention to propose such an improved process using material currently discarded as waste in the known fermentation process of Bacillus sphaericus serotypes H5a and H5b.

It is still another object of this invention to provide such a process in which the waste or the material to be disposed of in the above conventional process for producing useful chemicals is used as a by-product in an economical manner so that the known main fermentation itself becomes more economical and productive.

It is therefore a further object of this invention to provide an improved process for the production of biopesticides.

In the known fermentations of Bacillus sphaericus serotypes H5a and H5b, a cell-free culture filtrate is obtained. This is generally discarded and thus treated as waste.

We have turned our attention to an investigation of the filtrate, which is currently being discarded, to find out whether any valuable chemical is present in it and whether such chemical, if any, can be economically recovered so as to obtain a by-product and thus make the main production of the biopesticide economical.

We investigated the effect of this filtrate on various protein sources, e.g. casein and bovine serum albumin, mainly with regard to protease activity. It was observed that it could liquefy blood clots. Further investigations showed that it did not affect red blood cells. and only acted on the fibrin. This confirmed our above observations and that it is an enzyme, specifically a protease.

We have therefore turned our attention to the preparation of a useful agent from this cell-free filtrate.

It is known from Agricultural and Biological Chemistry, Vol. 41, No. 5, 1977, Tokyo, pp. 745-754, that a culture filtrate of a strain of Bacillus sphaericus can contain a protease. The protease described in the said publication has maximum caseinolytic activities around pH 9.0 to 9.3, and its molecular weight was estimated to be 27,000 by gel chromatography.

That some other types of bacteria of the genus Bacillus produce enzymes with plasmin-type activities is known from two Russian publications according to Chemical Abstracts, Vol. 114, No. 21, Abstract No. 203257 and Vol. 106, No. 9, Abstract No. 64089.

From the nature of the protease we identified, based on known activities and published literature, we were able to conclude that this enzyme (the protease) could be used to produce a useful thrombolytic agent.

Various studies were carried out using different sources of cell-free filtrate and we were able to develop a simple physicochemical procedure for the preparation of the thrombolytic agent.

Generally speaking, our process involves the processing of the culture filtrate obtained from the main process of conventional submerged fermentation of Bacillus Sphaericus serotype H5a and H5b in simple physicochemical steps that include ultrafiltration of the cell-free filtrate, concentration of the same and salting out of all proteins from the said cell-free filtrate using ammonium sulfate and decolorization of the protein in liquid medium.

The recovered decolorized protein can be further dialyzed and lyophilized to obtain a crude powder that can be subjected to further purification.

The general process steps are illustrated in the flow diagram attached as Fig. 1. The invention will now be described in more detail with reference to the following examples:

It should be noted that salting out using ammonium sulfate only serves to change the solubility of the macromolecules of the filtrate.

We present below a table showing the details of the filtrate before it was subjected to protein salting and after it was subjected to protein salting.

The protein salted out from the filtrate was transferred to aqueous solution and then decolorized using a commercially available modified cellulose, such as the cell debris removers sold by Whatman or a DE 52 ion exchange resin from the same manufacturer.

Suitable other forms of decolorizing agents can be investigated.

Dialysis and lyophilization

These two steps are carried out as follows: The precipitate obtained after salting out is dissolved in water, placed in tubular dialysis membrane bags with a molecular weight cut-off of 10,000 and dialyzed overnight against distilled water.

Purification of the crude powder obtained after lyophilization

The resulting raw powder is subjected to a two-stage purification.

In the first step, the powder is dissolved in water and subjected to ion exchange treatment in the following manner: The decolorized fraction was applied to a Q-Sepharose column and eluted in two steps using Tris-HCl buffer, pH 8.0 with a NaCl concentration of 0.1 M and 0.5 M, respectively. The unbound fraction eluted with a NaCl concentration of 0.1 M contained the fibrinolytic protease activity.

The second purification is carried out by gel filtration in the following way: The active fraction obtained after Q-Sepharose chromatography was further purified on a Sephacryl 5300 column. Elution was carried out with 0.01 M Tris-HCl buffer, pH 8. Two fractions were obtained, of which the second fraction contained the fibrinolytic enzyme.

Cleaning the raw powder

The types of ion exchangers for cleaning in the first stage must be specifically specified and the degree of purification can be indicated.

Second stage purification can be explained more precisely by specifying the gel filtration, gel types, gel specifications, stages and final purity achieved.

Product description

The product is a new thrombus dissolving agent containing a fibrinolytic enzyme (thrombinase) that was first isolated, identified and purified from Bacillus Sphaericus. This product has potential use in the treatment of thrombotic stroke, myocardial infarction, deep vessel thrombosis and in the prevention of postoperative adhesions. The preparation can be used as such or in combination with specific antibodies. Although the product is not a plasminogen activator, it has a very specific effect on fibrin clots. The fibrinolytic effect is greater than that of streptokinase and urokinase.

It shows optimal activity in the range of pH 7 to 9, and is stable within the pH range of 5 to 7.5. The molecular weight (gel filtration) was estimated to be about 18500. It is stable below 40ºC and retains 80% or more of its activity when left to stand for 24 hours at 30ºC in a pH 7.2 Tris-HCl buffer.

Biological activity Test for fibrinolytic efficacy

The fibrinolytic activity of the purified enzyme was evaluated in comparison with standard streptokinase according to the modified method of Astrup and Mullertz (1952).

Fibrinolytic potential was estimated by measuring the amount of liquefaction (zone diameter) produced by the enzyme on a fibrin clot of fibrinogen, plasminogen and thrombin at pH 7.5 and 37°C in 15 minutes. Streptokinase as a reference standard was diluted to contain 500 units/ml. Thrombinase was dissolved in a Tris-HCl buffer, pH 7.5, to contain 1 mg protein/ml. 20 and 40 µl amounts of streptokinase and test preparation were added as droplets to the surface of the fibrin clot and incubated at 37°C. The diameter of the digested zone was measured after 30 minutes of incubation. Table Summary of the purification of fibrinolytic protease from B. sphaericus

Claims (9)

1. Thrombolytic agent obtainable from a cell-free culture filtrate of fermentations of Bacillus sphaericus serotypes H5a and H5b and having an optimal enzymatic activity range of pH 7 to 9 and a molecular weight (gel filtration) of approximately 18500. 2. A process for the preparation of a new thrombolytic agent according to claim 1, which comprises - Obtaining a cell-free culture filtrate of the known culture broth from the fermentation of Bacillus Sphaericus serotype H5a and H5b, - subjecting said cell-free culture filtrate to an ultrafiltration stage in order to obtain a concentrated retentate, - followed by substantially complete salting out of the protein from said concentrate using a salting out agent such as ammonium sulphate, - Transferring the salted-out protein into an aqueous solution and decolorizing this solution using a modified cellulose, - dialyzing and lyophilizing the decolorized fraction to obtain a crude powder of said protein, followed by preparing a solution of said crude powder and subjecting it to chromatography to obtain a purified material, and - final lyophilization of the purified material to obtain the thrombolytic agent. 3. A process according to claim 2, wherein the modified cellulose is a commercially available product with the designation "DE52" sold by Whatman Biosystems Ltd., England. 4. A method according to claim 2 or 3, wherein the dialysis and the lyophilization of the cellulose-treated fraction is carried out by using a semipermeable membrane (cut-off limit: 10,000 Daltons) or a lyophilizer to obtain said crude protein powder. 5. A process according to any one of claims 2 to 4, wherein the chromatographic treatment is carried out in two stages, namely with a first ion exchange chromatography stage and a second gel filtration chromatography stage. 6. A process according to claim 5, wherein the first step ion exchange chromatography is carried out by subjecting a solution of the crude powder to Q-Sepharose (Pharmacia Ltd., Sweden) anion exchange chromatography using a 0.01 M Tris-HCl buffer, pH 8, containing 0.001 M CaCl₂ and eluted stepwise using the same buffer containing 0.1 M NaCl or 0.5 M NaCl. 7. A process according to claim 5 or 6, wherein the gel filtration chromatography in the second stage is carried out as gel filtration on Sephacryl S 3000 (Pharmacia Ltd., Sweden) using a 0.5 M Tris-HCl buffer, pH 7.5, containing 0.1 M NaCl. 8. A process according to any one of claims 2 to 7, wherein the final step is carried out by lyophilizing the dialyzed positive fractions after an additional gel filtration chromatography. 9. Use of a thrombolytic agent according to claim 1 or the product of the process according to any one of claims 2 to 8 for the manufacture of a medicament for dissolving thrombi.