Indian Patents. 218729:"A NOVEL THROMBOLYTIC ENZYME"

Title of Invention	"A NOVEL THROMBOLYTIC ENZYME"
Abstract	This invention relates to a novel thrombolytic enzyme named Thrombinase having a molecular weight of 31,700 Daltons, useful for dissolving blood clots.

Full Text	FIELD OF INVENTION This invention relates to novel thrombolytic enzyme . The thrombolytic enzyme of the prenet invention is named as Thrombinase, The novel enzyme is prepared in high purity and is useful for dissolving blood clots . The enzymes of the present invention dissolves the blood clot much faster than the currently available thrombolytic agents like Streptokinase, Urokinase In our co pending application no 2671Del98 we have described and claimed a process for the preparation of the said novel enzyme . Thjs application is divided out of the application no under Section 16 of the Patents Act 70 BACKGROUND OF INVENTION Thrombolytic enzymes ike Streptokinase, Urokinase, dissolves the blood clot Tissue Plasminogen Activator and hence has advantages for the treatment of cerebral thrombosis, myocardial infarction, deep vein thrombosis and in the prevention of post surgical adhesion. U.S.patent no.5,434,059 dated July 18, 1995 describes a process for the preparation of a thrombolytic enzyme, namely thrombinase with the improvement of yield by modification of the fermentation medium and increase in the purity by modification of the downstream processing methods. Such a thrombolytic enzyme has a molecular weight of 18,500. OBJECTS OF THE INVENTION The main objective of this invention is to provide a novel Thrombiolytic enzyme named as Thrombinase which dissolves the blood clot much faster than the currently available thrombolytic agents like Streptokinase, Urokinase Another object of this invention is to provide a new enzyme named as Thrombinase having a better purity in comparison to that of the known art. DESCRIPTION OF INVENTION In our co pending application no 2671 DEL 98 we have described and claimed a process for the preparation of the said novel enzyme . The process comprises culturing the filtrate of Bacillus sphaericus serotype H5a 5b in the culture medium consisting of 0.03 to 1.5% of yeast extract with one or more of constituents: 0.2 to 1.5% peptone, 1 to 1.6% sodium acetate, 0.3 to 0.5% beef extract, 0.2 to 0.5% sodium chloride, 0.5 to 1% soyapeptone, and 0.68% ammonium sulphate. The process comprises steps of cell removal by cross flow filtration using 0.22u filter, subjecting the cell supernatant thus obtained to two step ultra filtration using 1,00,000 MW (Molecular Weight) cut off membrane followed by ultra filtration of the filtrate thus obtained using 10,000 MW cut off membrane, rejecting the filtrate, salting out the retentate with ammonium sulphate, subjecting it to dialysis, reprecipitating it using ice-cold acetone, reconstituting in buffer, decolorizing by using modified CDR (Cell Debris Remover) treatment, dialyzing, lyophilizing and purification firstly by ion exchange chromatography followed by gel filtration chromatography, dialyzing the fraction showing fibrinolytic activity and lyophilizing to obtain purified thrombinase having a molecular weight of 31,700. Table 1 gives the various media along with their composition used for the fermentation of Bacillus sphaericus and the yields of Thrombinase obtained. TABLE-1 Several media compositions were tried for optimization of thrombinase yield. These include completely synthetic media, synthetic media with different percentages of yeast extract and complex media. Growth was very poor in completely synthetic medium unless it is supplemented with yeast extract. Table 1 gives a few media which show good results in shake flask: (Table Remove) SA = Sodium Acetate. YE = Yeast Extract. BE = Beef Extract. AS = Ammonium Sulphate * All media had trace elements in the following composition: CaCI2 - 0.013%, MgCI2 - .01%, MnS04 - 0.028%, ZnS04 - 0.0005%, FeS04 - 0.0001%, CuS04 -0.0001%). * pH of the media was 7.2 As will be apparent from the Table 1, the media composition comprises 0.03% to 1.5% of yeast extract, with or without each of 0.2 to 1% of peptone, 1 to 1.6% of sodium acetate, 0.3 to 0.5% of beef extract and 0.5% of sodium chloride. The pH of the media was maintained at pH 7.2. Reference is now made to Fig.l of the accompanying drawings which illustrates the flow diagram of the process. The fermentation broth is subjected to a process of purification consisting of a plurality steps. The first step A is that of micro filtration and wherein the fermented broth is passed through a membrane filter of for example, 0.22u. The second step of purification consists in the step of ultra filtration B of the filtrate Fl to remove particles having a molecular weight of more than 1,00,000. The retentate R from such a step is used for activity check and may be recycled. The filtrate F2 is subjected to a second step of ultra filtration C to have a retentate Rl with a molecular weight of 10,000 to 1,00,000. The filtrate F3 is rejected. The retentate Rl is subjected to the step of cell removal by precipitation D with ammonium sulphate having a concentration of up to 40% and preferably 20 to 40%. The precipitate PI is then subjected to the step of dialysis E followed by acetone precipitation. The precipitated enzyme is reconstituted in 0.01 to 8.0 M pH and further precipitated G with cold acetone at the ratio of 1:1 to 1:1.5 (v/v). Such a step reduces the volume and contaminants. The Precipitate P2 is subjected to a step of decolourization H. The precipitate obtained by acetone precipitation was reconstituted in minimum volume of buffer and subjected to decolourization using cell debris remover (CDR), Whatman. Briefly, a bed of CDR is made using a buchner funnel and the crude enzyme was layered over it and vacuum applied to the receiving flask. This is followed by elution with this Tris 0.01M, pH 8.0 containing 0.1M NaCI (2-3 bed volumes of buffer) to elute the fibrinolytic enzyme bound to the CDR. This method results in less than 6% activity loss. Hence the present method describes a process for the increase in the yield of Thrombinase by increasing the yield by this modified CDR treatment. TREATMENT ACTIVITY (IU) LOSS % Before CDR treatment CDR treatment by reported Method CDR treatment by present Method 2,36,24,000 3,51,000 2,21,50,200 98% 6% The colourless aqueous filtrate F3 is passed through an express ion exchanger I and then subjected to the step J of dialysis against distilled water for removal of the salt followed by the step K of solubilization and lyophilization L. Thus, the CDR treated material was first subjected to ion exchange chromatography. The CDR treated material was first loaded onto the column and eluted first with 0.01M containing 0.1 M NaCI and subsequently with Tris containing 0.5M NaCI. The fibrinolytic enzyme eluted with Iris containing 0.1 M NaCI which was dialysed and lyophilized. The lyophilized fraction was further purified by the step M of gel filtration chromatography using for example Sephacryl S-200. Elution was carried out using Tris buffer 0.01 M, pH 8.0 containing 0.1 M NaCI. The fraction showing fibrinolytic activity was dialyzed and lyophilized to yield pure Thrombinase. PRODUCT CHARACTERISATION The molecular weight of Thrombinase was found to be 31,700 as determined by mass spectral analysis, which is different to the molecular weight of 18,500 by gel filtration of the known art. The amino acid composition of Thrombinase has been determined. The effect of Thrombinase on the osmotic fragility of human RBCs have been determined by incubating the RBCs at 100 and 1000 times the therapeutic level (30000 and 300000 IU per milliliter of blood) for 30 minutes followed by evaluation of osmotic fragility of the RBCs at varying concentrations of sodium chloride (0%-0.85%). Such tests indicate no changes in the osmotic fragility pattern over the entire range of sodium chloride and hence indicating that Thrombinase does not affect the RBCs. The tests on the effect of Thrombinase on different protein substrates like casein, fibrinogen and bovine serum albumin shows that Thrombinase acts preferentially on fibrin with very little activity on other protein substrates. SUBSTRATE ACTIVITY (IU) Casein 1431 Fibrinogen 634 Bovine Serum Albumin 919 Fibrin Clot 170,000 WE CLAIM 1. A novel thrombolytic enzyme named Thrombinase having a molecular weight of 31,700 Daltons, useful for dissolving blood clots. 2 A novel thrombolytic enzyme named Thrombinase having a molecular weight of 31,700 Daltons, useful for dissolving blood clots as substantially described.

Full Text

FIELD OF INVENTION
This invention relates to novel thrombolytic enzyme . The thrombolytic enzyme of the prenet invention is named as Thrombinase, The novel enzyme is prepared in high purity and is useful for dissolving blood clots . The enzymes of the present invention dissolves the blood clot much faster than the currently available thrombolytic agents like Streptokinase, Urokinase
In our co pending application no 2671Del98 we have described and claimed a process for the preparation of the said novel enzyme . Thjs application is divided out of the application no under Section 16 of the Patents Act 70
BACKGROUND OF INVENTION
Thrombolytic enzymes ike Streptokinase, Urokinase, dissolves the blood clot Tissue Plasminogen Activator and hence has advantages for the treatment of cerebral thrombosis, myocardial infarction, deep vein thrombosis and in the prevention of post surgical adhesion.
U.S.patent no.5,434,059 dated July 18, 1995 describes a process for the preparation of a thrombolytic enzyme, namely thrombinase with the improvement of yield by modification of the fermentation medium and increase in the purity by modification of the downstream processing methods. Such a thrombolytic enzyme has a molecular weight of 18,500.
OBJECTS OF THE INVENTION
The main objective of this invention is to provide a novel Thrombiolytic enzyme named as Thrombinase which dissolves the blood clot much faster than the currently available thrombolytic agents like Streptokinase, Urokinase
Another object of this invention is to provide a new enzyme named as Thrombinase having a better purity in comparison to that of the known art.
DESCRIPTION OF INVENTION
In our co pending application no 2671 DEL 98 we have described and claimed a process for
the preparation of the said novel enzyme . The process comprises culturing the filtrate of

Bacillus sphaericus serotype H5a 5b in the culture medium consisting of 0.03 to 1.5% of yeast extract with one or more of constituents: 0.2 to 1.5% peptone, 1 to 1.6% sodium acetate, 0.3 to 0.5% beef extract, 0.2 to 0.5% sodium chloride, 0.5 to 1% soyapeptone, and 0.68% ammonium sulphate.
The process comprises steps of cell removal by cross flow filtration using 0.22u filter, subjecting the cell supernatant thus obtained to two step ultra filtration using 1,00,000 MW (Molecular Weight) cut off membrane followed by ultra filtration of the filtrate thus obtained using 10,000 MW cut off membrane, rejecting the filtrate, salting out the retentate with ammonium sulphate, subjecting it to dialysis, reprecipitating it using ice-cold acetone, reconstituting in buffer, decolorizing by using modified CDR (Cell Debris Remover) treatment, dialyzing, lyophilizing and purification firstly by ion exchange chromatography followed by gel filtration chromatography, dialyzing the fraction showing fibrinolytic activity and lyophilizing to obtain purified thrombinase having a molecular weight of 31,700.
Table 1 gives the various media along with their composition used for the fermentation of Bacillus sphaericus and the yields of Thrombinase obtained.
TABLE-1
Several media compositions were tried for optimization of thrombinase yield. These include completely synthetic media, synthetic media with different percentages of yeast extract and complex media. Growth was very poor in completely synthetic medium unless it is supplemented with yeast extract. Table 1 gives a few media which show good results in shake flask:
(Table Remove)
SA = Sodium Acetate. YE = Yeast Extract. BE = Beef Extract. AS = Ammonium Sulphate
* All media had trace elements in the following composition:
CaCI2 - 0.013%, MgCI2 - .01%, MnS04 - 0.028%, ZnS04 - 0.0005%, FeS04 - 0.0001%, CuS04 -0.0001%).
* pH of the media was 7.2
As will be apparent from the Table 1, the media composition comprises 0.03% to 1.5% of yeast extract, with or without each of 0.2 to 1% of peptone, 1 to 1.6% of sodium acetate, 0.3 to 0.5% of beef extract and 0.5% of sodium chloride. The pH of the media was maintained at pH 7.2.
Reference is now made to Fig.l of the accompanying drawings which illustrates the flow diagram of the process. The fermentation broth is subjected to a process of purification consisting of a plurality steps. The first step A is that of micro filtration and wherein the fermented broth is passed through a membrane filter of for example, 0.22u. The second step of purification consists in the step of ultra filtration B of the filtrate Fl to remove particles having a molecular weight of more than 1,00,000. The retentate R from such a step is used for activity check and may be recycled. The filtrate F2 is subjected to a second step of ultra filtration C to have a retentate Rl with a molecular weight of 10,000 to 1,00,000. The filtrate F3 is rejected.
The retentate Rl is subjected to the step of cell removal by precipitation D with ammonium sulphate having a concentration of up to 40% and preferably 20 to 40%.
The precipitate PI is then subjected to the step of dialysis E followed by acetone precipitation. The precipitated enzyme is reconstituted in 0.01 to 8.0 M pH and further precipitated G with cold acetone at the ratio of 1:1 to 1:1.5 (v/v). Such a step reduces the volume and contaminants.
The Precipitate P2 is subjected to a step of decolourization H. The precipitate obtained by acetone precipitation was reconstituted in minimum volume of buffer and subjected to decolourization using cell debris remover (CDR), Whatman. Briefly, a bed of CDR is made using a buchner funnel and the crude enzyme was layered over it and vacuum applied to the receiving flask. This is followed by elution with this Tris 0.01M, pH 8.0 containing 0.1M NaCI (2-3 bed volumes of buffer) to elute the fibrinolytic enzyme bound to the CDR. This method results in less than 6% activity loss. Hence the present method describes a process for the increase in the yield of Thrombinase by increasing the yield by this modified CDR treatment.

TREATMENT

ACTIVITY (IU)

LOSS %

Before CDR treatment
CDR treatment by reported Method
CDR treatment by present Method

2,36,24,000 3,51,000
2,21,50,200

98% 6%

The colourless aqueous filtrate F3 is passed through an express ion exchanger I and then subjected to the step J of dialysis against distilled water for removal of the salt followed by the step K of solubilization and lyophilization L. Thus, the CDR treated material was first subjected to ion exchange chromatography. The CDR treated material was first loaded onto the column and eluted first with 0.01M containing 0.1 M NaCI and subsequently with Tris containing 0.5M NaCI. The fibrinolytic enzyme eluted with Iris containing 0.1 M NaCI which was dialysed and lyophilized.
The lyophilized fraction was further purified by the step M of gel filtration chromatography using for example Sephacryl S-200. Elution was carried out using Tris buffer 0.01 M, pH 8.0 containing
0.1 M NaCI. The fraction showing fibrinolytic activity was dialyzed and lyophilized to yield pure Thrombinase.
PRODUCT CHARACTERISATION
The molecular weight of Thrombinase was found to be 31,700 as determined by mass spectral analysis, which is different to the molecular weight of 18,500 by gel filtration of the known art. The amino acid composition of Thrombinase has been determined.
The effect of Thrombinase on the osmotic fragility of human RBCs have been determined by incubating the RBCs at 100 and 1000 times the therapeutic level (30000 and 300000 IU per milliliter of blood) for 30 minutes followed by evaluation of osmotic fragility of the RBCs at varying concentrations of sodium chloride (0%-0.85%). Such tests indicate no changes in the osmotic fragility pattern over the entire range of sodium chloride and hence indicating that Thrombinase does not affect the RBCs.
The tests on the effect of Thrombinase on different protein substrates like casein, fibrinogen and bovine serum albumin shows that Thrombinase acts preferentially on fibrin with very little activity on other protein substrates.
SUBSTRATE ACTIVITY (IU)
Casein 1431
Fibrinogen 634
Bovine Serum Albumin 919
Fibrin Clot 170,000

WE CLAIM
1. A novel thrombolytic enzyme named Thrombinase having a molecular weight of 31,700 Daltons, useful for dissolving blood clots.
2 A novel thrombolytic enzyme named Thrombinase having a molecular weight of 31,700 Daltons, useful for dissolving blood clots as substantially described.

Documents:

52-DEL-2004-Abstract-(12-03-2008).pdf

52-DEL-2004-Abstract-01-04-2008.pdf

52-del-2004-abstract.pdf

52-DEL-2004-Claims-(12-03-2008).pdf

52-DEL-2004-Claims-01-04-2008.pdf

52-del-2004-claims.pdf

52-DEL-2004-Correspondence-Others-(12-03-2008).pdf

52-DEL-2004-Correspondence-Others-01-04-2008.pdf

52-del-2004-correspondence-others.pdf

52-del-2004-correspondence-po.pdf

52-DEL-2004-Description (Complete)-(12-03-2008).pdf

52-del-2004-description (complete).pdf

52-DEL-2004-Description (Complete)01-04-2008.pdf

52-del-2004-drawings.pdf

52-del-2004-form-1.pdf

52-del-2004-form-18.pdf

52-DEL-2004-Form-2-(12-03-2008).pdf

52-del-2004-form-2.pdf

52-DEL-2004-Form-3-(12-03-2008).pdf

52-DEL-2004-Form-5-(12-03-2008).pdf

52-del-2004-gpa.pdf

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Patent Number

218729

Indian Patent Application Number

52/DEL/2004

PG Journal Number

24/2008

Publication Date

13-Jun-2008

Grant Date

10-Apr-2008

Date of Filing

12-Jan-2004

Name of Patentee

NATIONAL RESEARCH DEVELOPMENT CORPORATION

Applicant Address

GOVT. OF INDIA ENTERPRISE 020-22 ZAMROODPUR COMMUNITY CENTER, KAILASH COLONY EXTENSION, NEW DELHI 110048

Inventors:

#	Inventor's Name	Inventor's Address
1	CHIVUKULA SEKHAR	MALADI RESEARCH CENTRE, 52 JAWAHARLAL NEHRU ROAD, EKKATTUTHANGAL, CHENNAI, TAMIL NADU 600097, INDIA.
2	PERURMADOM RAMAIYER MAHADEVAN	MALADI RESEARCH CENTRE, 52 JAWAHARLAL NEHRU ROAD, EKKATTUTHANGAL, CHENNAI, TAMIL NADU 600097, INDIA.

PCT International Classification Number

A61K30/36

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA