Title of Invention

"A PROCESS FOR THE PREPARATION OF A NOVEL FIBRIN POWDER FOR MEDICAL APPLICATIONS"

Abstract

A process for the preparation of a novel fibrin powder for medical applications which comprises i) purifying, bleaching and masticating crude fibrin in a known manner to form a paste, ii) graft copolymerising the said fibrin present in the paste, with an acrylic monomer in presence of redox initiator and a known crosslinker at a temperature of around 40-65°C over a period of 2-4 hrs., iii) optionally reacting with drugs containing NH2, OH, COOH groups to be coupled to the graft copolymer, as formed in step (ii), then drying the resulting copolymer, iv) pulverizing the dried copolymer, as formed in step(iii), to a particle size of 75-750 microns, v) sterilizing the powder, as formed in step (iv), by conventional gamma irradiation.

Full Text

The present invention relates to a process for the preparation of a novel fibrin powder for medical applications. The fibrin powder finds potential use as a dressing aid in the treatment of various wounds of different nature, which include external injury, cut wounds, burn wounds and even ulcers in animals and human being. The fibrin powder, applied on a wound, not only prevents prevents any infection on the wound, but also stops further loss of electrolytes from the wound by acting as a haemostatic agent. Efficacy of wound-healing of the powder increases manifold when antiinfiamatory drugs, bactericides and even antibiotics such as terramicin,gentamicin and cephalaxin are incorporated into it. As reported by Styler ( Biochemistry, W.H.Freeman & Co., New York,1988),fibrin monomers spontaneously assemble into ordered fibrous arrays called fibrin.Bleeding from any wound in an animal body stops automatically due to the natural process of blood clotting, which is caused by the conversion of fibrinogen, present in blood, into fibrous netlike fibrin in presence of thrombin, a proteolytic enzyme. It has also been confirmed by Bergel ( Deut. Med. Wochsch.,35, 663, 1909) and Piechotta et al ( Aesthetiic plast.surg,17,263,1983), that fibrin possesses good haemostatic and wound healing properties. Although the net like structure of natural fibrin film arrests the blood cells, which cannot come out, serum still oozes out along with electrolyte and thereby causes microbial infection. It is therefore necessary to dress the wound externally to accelerate the healing process. Management of deep wounds normally involves pouring ofantibiotic powder or fibrin powder followed by a dressing by different materials like hand-aid, sys pur derm, nylon velour, polyester urethane, collagen sheet, fibrin sheet etc. Powder is sometimes poured into deep wounds like stab marks or leprosy ulcers as fillers without any covering of a sheet. The main drawback of antibiotic powder dressing is that the powder contains some carriers along with the actual antibiotic material and compatibility of this carrier material with the animal body is obviously one of the important criteria of great concern. Apart from these, keeping in mind the ever-growing concern for hygienic products, global attention has recently been shifting towards less-hazardous natural products. Raw blood is defibrinated by vigorous churning for the purpose of isolating various biochemicals like haemoglobin.The crude fibrin, separated from raw blood during the preocess of defibrination, does not produce good film. Even after drying, this fibrin cannot be used for wound dressing due to its britttle nature. This crude fibrin is either rejected usually as waste or used sometimes as plant-fertiliser. Laki ( Fibrinogen, Marcel Dekker,Inc, New York, 1968) mentioned that fibrin could be isolated from fibrinogen and be made in the form of sponge, film, powder, fibrin glue etc. Fibrinogen is isolated from blood using centrifugation in combination with either cryoprepcipitation or other precipitating agents like ethanol, ammomium sulfate or polyethylene glycol. Concentrated solution of isolated fibrinogen is diluted and mixed with thrombin in aqueous medium to form fibrin suspension. This mixture is dried and pulverized to produce natural fibrin powder, which is used for dressing of wound. The main drawback of this system is that fibrin being a protein,is susceptible to decay by proteolytic attack.Thus shelf life of this natural fibrin powder is obviously very low,because crude protein is used as such in this process. For the same reason, it requires frequent changes of dressing and lingers the healing process, when used for wound dressing. Another drawback of this natural fibrin powder is that thrombin is not readily available and it adds to the cost of preparation of the film. Yet another limitation of this natural powder is that coupling of other drug is not possible for the betterment of the functionality of the product. The main objective of the present invention is to provide a process for the preparation of a novel fibrin powder for medical applications, which obvitiioo the drawbacks stated above. Another objective of the present invention is to utilise the crude fibrin,which is otherwise discarded as waste, thereby generating wealth out of waste. Yet another objective of the present invention is to prepare a product which is capable of incorporating drugs like bactericides and antibiotics such as terramicin, gentamicin, cephalexin for effecting better efficacy of the treatment. Still another objective of the present invention is to prepare modified fibrin with better longevity on wound,so that the number of dressings can be reduced. Accordingly the present invention provides a process for the preparation of a novel fibrin powder for medical applications which comprises i) purifying, bleaching and masticating crude fibrin in a known manner to form a paste, ii) graft copolymerising the said fibrin present in the paste, with an acrylic monomer in presence of redox initiator and a known crosslinker at a temperature of around 40-65°C over a period of 2-4 hrs., iii) optionally reacting with drugs containing NH2 OH, COOH groups to be coupled to the graft copolymer, as formed in step (ii), then drying the resulting copolymer. iv) pulverizing the dried copolymer, as formed in step (iii), to a particle size of 75- 750 microns, v) sterilising the powder, as formed in step (iv), by conventional gamma irradiation. In an embodiment of the present invention, 2-5% by weight, of a metallic salt of an organic acid on the weight of crude fibrin is used for purification of fibrin. In another embodiment of the present invention, 2-5% w/w uf crosslinking agents on the dry fibrin film is used for the graft copolymerisation. In yet another embodiment of the present invention, drugs containing -NH2, -OH, - COOH groups can be coupled to the graft copolymer. In still another embodiment of the present invention, the copolymer emulsion is dried to a moisture content of 8-10%. Crude fibrin obtained after defibrinating raw blood, is used as the starting material for the present invention. It is washed conventionally in 3-5 changes of plain water and 2-5% w/w, of a matallic salt of an organic acid, dissolved in 20-50 times of volumes of water, to remove blood clots. Washed fibrin is then suspended in 80-150% w/v of water and is bleached with 10-30% v/v, of a conventional bleaching agent at a pH in the range of 7.5-10, adjusted by a conventional alkali, over a period of 10-15 hrs.The colour of the fibrin, at this stage, turns white. The bleached fibrin is then washed conventionally in 200-300% w/v of plain water for 10-20 mins and thenground in a conventional way to form a paste. The ground paste is suspended in 6-10 times w/v of water and is polymerised with 20- 40% by weight of an acrylic monomer on dry film weight in presence of 2-5 times w/w, of redox initiator,2-5% w/w of crosslinking agents on the dry fibrin film at a temperature in the range of 40-60°C over a period of 30-180 mins. 10% by weight of a drag containing -NH2, -OH, -COOH groups on fibrin weight is optionally treated with the reaction mixture with constant stirring for preparing drag-coupled fibrin-polyacrylate graft copolymer. The reaction is continued for a further period of 20-40 mins. Completion of polymerisation reaction is ascertained by conventional titration method. The resulting graft copolymer,obtained in the form of emulsion, is dried conventionally at a temperature in the range of 40-70°C over a period of 3-8 hrs to 8-10% moisture content. The dried copolymer is is treated with 3-5 times w/v of an organic solvent to extract the homopolymers present in the sheet over a period of 2-5 hrs. The resulting sheet is sterilised by exposing the same to gamma irradiation in the range of 1-3 M rads and is stored in 90-95% solution of an aqueous organic solvent. Thus this process of invention provides a simple new method of utilising crude fibrin for making powder of better efficacy, stability and shelf-life. The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention. EXAMPLE-1 500 gms of crude fibrin containing blood clots was collected from the slaughter house for the present experiment. It was washed thoroughly under running water and then treated with 25gms of sodium acetate,dissolved in 1 lit of water to remove the blood clots/blood stains from the fibrin. Blood clot free fibrin was then put in 750 ml of water in a plastic tub. pH of the bath was adjusted to 8.5 by adding a few drops of 0.1 N sodium hydroxide solution. 25 ml of hydrogen peroxide was then added with constant stirring for 10 hrs. after which colour of the fibrin turned to white. The bleached white fibrin was removed from the bleaching bath, washed thoroughly with cold running water and ground with the help of mixer to form a paste. 10 gm of the above paste was suspended in 100 ml of water in a 500 ml conical flask fitted with a two necked adapter and 100 ml of water was added to it. 0.25 gms of Sodium metabisulphite and an equal amount of potassium persulphate were added to the above mixture with constant stirring and the reaction mixture was heated to a temperature of 55°C. Then 3 ml of hydroxyethyl methacrylate was added drop by drop to the solution with stirring,which was continued for a further period of 45 mins.Then 0.08 gm of glutaraldehyde was added to the bath with constant stirring for 5 mins. Temperature was maintained at 55°C throughout the reaction. After 60 mins, butylamine titrartion confirmed the absence of free hydroxyethyl methacrylate and hence completion of the polymerisation reaction. The resulting fibrin- polyhydroxyethyl methacrylate graft copolymer was taken out from the flask and kept in 100 ml of plain water in a beaker. After a period of 1 hr, water was decanted and the wet copolymer was treated with 100 ml of acetone for 3 hrs with continuous shaking. The resulting material was dried at 55°C for 3 hrs in a hot air oven and was then pulverized in a bone mill to a particle size of 750 microns. The powder was exposed to gamma irradation at 1.5 M rads. The fibrin-polyhydroxyethyl methacrylate graft copolymer powder was finally stored in polythene cover. EXAMPLE-2 250 gms of crude fibrin containing blood clots was collected from the slaughter house for the present experiment. It was washed thoroughly under running water and then treated with 15gms of potassium acetate,dissolved in 500 ml of water to remove the blood clots/blood stains from the fibrin. Blood clot free fibrin was then put in 500 ml of water in a plastic tub. pH of the bath was adjusted to 8 by adding a few drops of 0.1 N potassium hydroxide solution. 15 ml of sodium peroxide was then added with constant stirring for 8 hrs, after which colour of the fibrin turned to white. The bleached white fibrin was removed from the bleaching bath, washed thoroughly with cold running water and ground with the help of mixer to foim a paste. 10 gm of the above paste was suspended in 100 ml of water in a 500 ml conical flask fitted with a two necked adapter and 100 ml of water was added to it. pH was adjusted to 3 by using 0.1 N hydrochloric acid. 0.2 gms of Sodium bisulphite and an equal amount o ammonium persulphate were added to the above mixture with constant stirring and the reaction mixture was heated to a temperature of 65°C. Then 4 ml of methyl methacrylate was added drop by drop to the solution with stirring,which was continued for a further period ol 45 mins. Then 0.1 gm of basic chromium sulphate, dissolved in 2 ml of water, was added to the bath with constant stirring for 5 mins. Temperature was maintained at 65°C throughout the reaction. After 60 mins, butylamine titrartion confirmed the absence of free methyl methacrylate and hence completion of the polymerisation reaction. The resulting fibrin- polymethyl methacrylate graft copolymer was taken out from the flask and kept in 100 ml of plain water in a beaker. After a period of 1 hr, water was decanted and the wet copolymer was treated with 100 ml of acetone for 3 hrs with continuous shaking The resulting material was dried at 60°C for 3 hrs in a hot air oven and then was pulverized in a bone mill to a particle size of 750 microns. The powder was exposed to gamma irradation at 2 M rads. The fibrin-polymethyl methacrylate graft copolymer powder was finally stored in polythene cover. EXAMPLE-3 1000 gms of crude fibrin containing blood clots was collected from the slaughter house for the present experiment. It was washed thoroughly under running water and then treated with 50 gms of sodium acetate,dissolved in 2 lit of water to remove the blood clots/blood stains from the fibrin. Blood clot free fibrin was then put in 1500 ml of water in a plastic tub. pH of the bath was adjusted to 8 by adding a few drops of 0.1 N sodium hydroxide solution. 50 ml of sodium peroxide was then added with constant stirring for 8 hrs, after which colour of the fibrin turned to white. The bleached white fibrin was removed from the bleaching bath, washed thoroughly with cold running water and ground with the help of mixer to form a paste. 10 gm of the above paste was suspended in 100 ml of water in a 500 ml conical flask fitted with a two necked adapter and 100 ml of water was added to it. 0.5 gms of Sodium metabisulphite and an equal amount of ammonium persulphate were added to the above mixture with constant stirring and the reaction mixture was heated to a temperature of 60°C. Then 10 ml of hydroxyethyl methacrylate was added drop by drop to the solution with stirring,which was continued for a further period of 45 mins. Then 0.16 ml of glutaraldehyde was added to the bath with constant stirring for 5 mins. Temperature was maintained at 60°C throughout the reaction. After 60 mins, the reaction mixture was cooled to a temperature of 25°C and stirred for 30 mins. Then 100 gms of tetracycline was added to the leaction mixture with constant stirring and the temperature was maintained at 25°C. After 90 mins, butylamine titrartion confirmed the absence of free hydroxyethyl methacrylate and hence completion of the polymerisation reaction. The resulting fibrin- polyhydroxyethyl methacrylate graft copolymer, coupled with tetracycline, was taken out from the flask and kept in 100 ml of plain water in a beaker. After a period of 1 hr, water was decanted and the wet copolymer was treated with 100 ml of acetone for 3 hrs with continuous shaking. The resulting material was dried at 40OC for 5 hrs in a hot air oven and then was pulverized in a bone mill to a particle size of 500 microns.The powder was exposed to gamma irradation at 1 M rads. The tetracycline coupled fibrin-polyhydroxyethyl methacrylate graft copolymer powder was finally stored in polythene cover. The main advantages of the present invention are the following. 1. Crude fibrin, which is usually discarded as waste, is used as the raw material,thereby suggesting an economical method for the disposal of the same. 2. Grafting of biocompatible polymers onto fibrin improves the function of the product. 3. Since a hydrophilic and biocompatible monomer is graft-copolymerised with fibrin, the powder absorbs the wound exudate readily to keep the wound clean and dry, thereby preventing any possible infection. 4. This novel powder can be applied to deep wounds, stab wounds and ulcers upon which a sheet cannot be applied. 5. The powder may be stored for a longer time 6. Polymerisation reaction takes place at comparatively lower temperature, thereby making the temperature management much easier. 7. The powder is biocompatible and does not produce any irritation on skin. 8. It is very useful in veterinary surgery for amputation of horn,tail etc. 9. Antibiotics like gentamicin and tetracylin could be coupled to the grafted composite through the hydroxyl functional groups introduced by polyhydroxyethyl methacrylate. These graft copolymers coupied with antibiotics can be used in the case of above said infected tissue repair. We claim: 1. A process for the preparation of a novel fibrin powder for medical applications which comprises i) purifying, bleaching and masticating crude fibrin in a known manner to form a paste, ii) graft copolymerising the said fibrin present in the paste, with an acrylic monomer in presence of redox initiator and a known crosslinker at a temperature of around 40-65°C over a period of 2-4 hrs., iii) optionally reacting with drugs containing NH2 OH, COOH groups to be coupled to the graft copolymer, as formed in step (ii), then drying the resulting copolymer, as formed in step (V) iv) pulverizing the dried copolymer, as formed in step (iii), to a particle size of 75-750 microns, v) sterilising the powder, as formed in step (iv), by conventional gamma irradiation. 2. A process as claimed in claim 1 wherein the redox initiators used is selected from among potassium per sulphate, sodium bisulphite, ammonium per sulphate 3. A process as claimed in claims 1 and 2 wherein the crosslinkers used are selected from among glutaraldehyde, basic chromium sulphate. 4. A process as claimed in claims 1 to 3 wherein the acrylic monomers used are selected from hydroxyethyl methacrylate, Methyl Methacrylate. 5. A process as claimed in claims 1 to 4 wherein the bleaching agents used are hydrogen peroxide, sodium peroxide. 6. A process as claimed in claims 1 to 5 wherein the purification of the crude fibrin is carried over using metallic salt of an organic acid selected from sodium acetate and potassium acetate. 7. A process for the preparation of a novel fibrin powder as claimed in claim 1for medical applications substantially as herein described with reference to the examples.

Documents:

2596-del-1997-abstract.pdf

2596-del-1997-claims.pdf

2596-del-1997-complete specification granted.pdf

2596-del-1997-correspondence-others.pdf

2596-del-1997-correspondence-po.pdf

2596-del-1997-description (complete).pdf

2596-del-1997-form-1.pdf

2596-del-1997-form-2.pdf