| Title of Invention | "HAEMOSTATIC AGENT AND METHOD OF PREPARING THE SAME" |
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| Abstract | A haemostatic agent comprising a chemically modified chitin having multifunctional haemostatic properties and pharmaceutical composition thereof, wherein modified chitin is highly water swellable and sticky thereby adhering to the wound causing clotting of blood and healing of wound. |
| Full Text | Field of Invention: This invention relates to a haemostatic agent, the process for preparing the same having haemostatic and anti-inflammatory properties. Background of the Invention: Control of bleeding is essential and critical in cases of acute wounds. Excessive bleeding has been a leading cause of death in a battle zone and other situations like accidents. Medicine has long been interested for a surface haemostatic agent that will act independently of normal clotting mechanisms and help subsequently in improving the healing of wounds. Effective haemostatic agents are also required for patients suffering from diabetes or undergoing extensive cortisone treatment, as they show extremely slow rates of healing of any wound [USP- 3903268, USP- 3,911,116]. Since stopping of bleeding is highly desirable and has immense medical and psychological bearing, a number of patents have been filed related to haemostatic agents and wound healing compositions. US 5,773,033 relates to an autologous fibrinogen incorporated in chitosan having strong haemostatic properties. The autologous fibrinogen was incorporated with a view to reduce the risk of disease transmission. The above art has a disadvantage in that the fibrinogen has to be obtained from the patients own blood. Thus, the haemostatic adhesive agent has to be prepared fresh every time before use, by drawing blood from individuals. The other disadvantage of this art is that plasma is to be separated from the blood and chemically treated with ammonium sulfate to get the required fibrinogen. Still further disadvantage of this invention is that the fibrinogen has to be coated on to the chitosan. Another disadvantage of this invention is that platelets are to be additionally added to the composition. Another disadvantage of the above art is that in case of the acute injury, fibrinogen has to be isolated from the pooled blood which carries the risk of disease transmission. As a result, it has to be purified to reduce the risk of transmission of any disease before it can be the part of the wound healing system. US 5,510,102 discloses an autologous platelet-rich plasma concentrate and a biocompatible polymer selected from alginates, poly l-amino acids, chitosan and chitin. The major disadvantage of this art is that the plasma is to be obtained from patents own blood to avoid undesirable immunogenic reactions. The other disadvantage is that the platelets are to be concentrated before incorporating in the composition and thus sufficient amount of blood has to be drawn. US 5,292,363 discusses tissue bonding and sealing composition containing naturally occurring peptides, synthetic peptides and serum proteins selected from albumin, a - globumin, B -globumin and y -globumin and thrombin along with glucosaminoglycan. The art has a disadvantage in that the tissue bonding and sealing requires an external energy source like Laser to activate bonding. The other disadvantage of this art is that the external sources are to be standardized to be able to provide required amount of energy. US 3,903,268 and US 3,911,116 relate to chitin and chitin derivatives that act as haemostatic agent for promoting wound healing. The art uses chitin of fungal origin. The major disadvantage of this art is that the fungi have to be cultured in brain-heart- infusion (BHI) and sabouraud's broth (SAB), which is time consuming and requires aseptic environment. Yet another disadvantage of the above art is that the culture has to be killed by placing under C02 environment at 127° C for 3hrs, which requires proper facility and time to execute the work. US 4,394,373 discloses method of achieving haemeostasis in wound, vascular grafts, and vascular patches by applying chitosan. The disadvantage of the present art is that it recommends the preparation of chitosan solution in acetic acid ( 2g /1000ml) which when applied to the open wound will be irritating. Another disadvantage of this art is that the pH of the solution remains between 4 and 5, and will presents a hostile environment to the wound. It is well know that acetic acid is toxic to biological cells. US 4,532,134 relates to method of achieving hemostasis by placing in contact with said wound an effective amount of chitosan acetate or chitosan hydrochloride. The disadvantage of this art is that the preferred haemostatic chitosan solution is prepared by dissolving chitosan in distilled water along with glacial acetic acid or hydrochloric acid. Both these acids are known to be toxic to cells. The other disadvantage of this art is that the solution is to be stored at 4 degree Centigrade. Yet another disadvantage of this art is that the haemostatic chitosan fiber mats are prepared by placing an appropriate quantity of sterile solution in sterile silicone coated tubes, crystallizing the solution at minus 60 degree Centigrade and freeze drying. This requires elaborate instrumentation. Another disadvantage of this art is that chitosan haemostatic powder is prepared by long process of converting solution into mat form and then grinding the mats with sterile mortar and pestle. The powder thus may contain some amounts of acetic and or hydrochloric acids which are toxic substances for cells and irritants to open wounds. US 6,806,260 discloses methods of preparing functional derivatives of chitosan like lactose, maltose and melibiose-substituted chitosan. The art describes first functionalization, second functionalization, and third functionalization of chiosan. The disadvantage of this art is that the chitosan derivates are prepared by multi-stepped chemical reactions which are tedious. The other disadvantage of this art is that the chemicals used for dispersing chitosan are costly [Tetramethylethylenediamine (TEMED), Carbodiimide (EDC) and iodinated lactose]. Yet, another drawback of this art is that it uses a cross- linking agent which requires photo-irradiation for cross-linking of the second functionalization of chitosan. There are number of patents [US 6,923,961, , US 6,890,344, US 6,967,261, US 897,348, US 7,008,392] which deals with the derivatized carboxypolysaccharides (CPS) films incorporating polyethylene oxide (PEG) for drug delivery, for anti-adhesion preparations and promoting haemostasis; Preparation of various haemostasis pad and bandages incorporating 'acid-sensitive' species, carboxylic-oxidized cellulose hemostats ; Cationic biopolymer of glucosamine as the haemostatic agent ; layered bandage incorporating poly(ethyleneoxide)-based compound along with chitosan. The art contained in US 7,019,191 describes methods of making haemostatic wound dressing comprising a fibrous fabric surface (knitted oxidized Rayon fabric) and thrombin, fibrinogen or fibrin. The limitation of this art is that oxidized polysaccharides (carboxylic and aldehyde-oxidized cellulose) are to be prepared in a per-fluorocarbon solvent. The other disadvantage of this art is that the fabric has to be washed with solvents like carbon tetrachloride and isopropyl alcohol. The other drawback of this art is that the base fabric has to be constructed in the desired woven or non-woven pattern. The other drawback of this art is that the fabric has to be exposed to 4 degree C to about 90 degree C and to a range of humidity for a protracted time frame. While surgical biological glues (obtained from bovine thrombin) having haemostatic action have been mentioned [J. Ped. Sur. 24:867 (1998); Am. J. Surg, 161: 479 (1991); Bri. J. Plast Surg. 42: 54(1989); Ann.Thorac Surg. 50:143(1990), J. Trauma, 31, 408 (1991)], their toxicity and possible anaphylactic reaction are of great concerns. Fatal reactions to the use of fibrin glue in deep hepatic wound is described in J. Trauma, 31, 408 (1991). The fatal reaction believed to be caused by activation and conversion of fibrinogen into a fibrin using a bovine thrombin resulting in anaphylactic shock leading to death. Therefore, use of exogenous thrombin is eliminated. In the previous arts, to make Chitosan water soluble, it has been dissolved in dilute acetic acid or dilute hydrochloric acid medium and haemostatic compositions have been prepared in this acid soluble form in number of patent literatures mentioned above. There is an urgent need firstly of an effective haemostatic agent that can be used in such a form (eg. Powder) that can be applied directly on the wound particularly onto deep wounds. Secondly, the agent should have sticking property and can immediately adhere on the wound surface after its application. Particularly, for a wound with oozing blood, it should swell immediately (after the application) by absorbing the water content of the blood and stick to the entire surface of the wound in order to be retained as a barrier against the positive pressure of the blood. Such moisture absorbing sticky haemostatic substance will act not only as haemostatic agent but also as an accelerated wound healing agent, providing required moist environment to the wound. It is also desirable that a haemostatic composition should also act as an anti- inflammatory agent to reduce the pain that accompanies a wound. Objects of the present Invention: The object of the present invention is to provide a haemostatic agent for achieving haemostasis even in the absence of the normal auto clotting mechanism. A further object of the invention is to provide a method of achieving haemostasis through the use of a homeostatic agent, having blood clotting, and anti-inflammatory properties. A further object of the invention is to provide a method of achieving haemostasis in open wounds and areas to be sutured. A still further object of the invention is to provide a method of attaining haemostasis with suppressed inflammation. A still further object of the invention is to provide a haemostatic composite which is a powder having blood clotting and anti-inflammatory properties and can be applied to open wounds. Summary of the Invention: In order to achieve the said objective the present invention provides a haemostatic agent comprising a chemically modified chitin having multifunctional haemostatic properties and pharmaceutical composition thereof, wherein modified chitin is highly water swellable and sticky thereby adhering to the wound causing clotting of blood and healing of wound. Further the invention provides the method of preparing modified chitin powder from chitin comprises of the following steps: a) reacting chitin with 47 - 50% aqueous caustic soda solution in a ratio 1-20 to 1-40 (weight/volume) at 40 - 90°C for 2 - 7 hours under stirring condition, b) filtering the reacted chitin, washing the reacted mass with 5% aqueous caustic soda solution, squeezing to take out maximum liquid, c) reacting the squeezed mass of step(b) by dispersing it in isopropyl alcohol in a ratio with respect to chitin used in step (a) as 1:15 to 1:30, stirring the mass keeping the temperature at 50 to 70°C, d) adding monochloroacetic acid (MCA) solution, taking 0.1 to 0.9 parts MCA per one part chitin over a period of 1 to 5 hours maintaining pH of the reaction medium at 8 ± 0.5, e) agitating the reaction mixture for 0.5-2hours, f) cooling at 25-30°C, filtering the reaction mass, washing it thoroughly with a mixture of isopropyl alcohol - water squeezing, air drying and grinding to fine powder to obtain water swellable powder. Detailed Description of the Invention: Carboxymethylation of chitin is carried out by alkalizing and partial deacetylation of chitin in small flakes form. The starting raw material i.e. chitin is obtained from India Sea Foods, Cochin, India and it has following properties: Colour: White Ash content: 0.54%, Moisture content: 1.2% Bulk density: 0.21 gm/cc, E. Coli: not detected Coliform: not detected Salmonella: not detected Heavy metal content (in ppm) Copper: 15.0 Zinc: 6.0 Cadmium: The synthesis of modified chitin powder from chitin comprises of the following steps: a) reacting chitin with aqueous caustic soda solution of 47 - 50% concentration in a ratio 1 -20 to 1 -40 (weight/volume) at 40 - 90° C for a period of 2 - 7 hours under stirring condition, b) filtering the reacted chitin, washing the reacted mass with 5% aqueous caustic soda solution, squeezing to take out maximum liquid, c) reacting the squeezed mass of step(b) by dispersing it in isopropyl alcohol in a ratio with respect to chitin used in step (a) as 1:15 to 1:30, stirring the mass keeping the temperature at 50 to 70°C, adding monochloro acetic acid (MCA) solution [prepared by dissolving solid MCA in isopropyl alcohol in a ratio 1:5 to 1:15 (wt. by volume)], taking 0.1 to 0.9 parts MCA per one part chitin over a period of 1 to 5 hours maintaining pH of the reaction medium at 8 ± 0.5 (if required, by addition of 10% caustic soda solution), continuing the reaction for an hour more after the completion of addition of MCA solution, d) cooling at 25-30°C, filtering the reaction mass, washing it thoroughly with isopropyl alcohol - water mixture (90:10, volume by volume), squeezing, air drying and grinding to fine powder (so that it passes 100 mesh sieve) to obtain water swellable powder. Evaluation of Water absorption capacity: The powder product was then evaluated for its water absorption capacity adopting the same process practiced in case of hydrogel (zhag et al, J. Polym Sci. Pol. Chem. 1999, 37, 455) i.e. by taking one gram dry powder, allowing to swell completely in excess of distilled water for 30 min and then taking out all unabsorbed water. The calculation of the percent of absorption is done as per the given equation: 100 x(Wf-W,)/ W, where, Wf is the weight of the hydrogel after swelling and W,- is the initial weight of the hydrogel before swelling. Evaluation of swelling characteristics: The sticky character of the swelled powder product was assessed by placing 1 mg of powder on a glass slide, placing 2 drops of water on it, observing the time required for absorbing the water drop, and it's sticking property by physically attempting to scrap the swelled mass from the glass surface. The composite haemostatic agent was made by intimate mixing of the two components namely modified chitin and anti-inflammatory agent in powder form at different proportions. Various compositions were made and the preferred compositions are those containing modified chitin powder - 90 % to 50% and aleovera - 50% to 10%. Only few of these compositions are mentioned in the examples given, but these are not the only compositions possible. These are given as illustrations only and many more compositions are possible to prepare in the light of descriptions disclosed here. Those versed in the art can easily vary the composition and combinations. The swellable modified chitin was evaluated for anti-mutagenic properties, skin irritancy and haemostatic behavior. Preparation of modified chitin .powders will now be explained with the help od following examples (Example -I - IV). However, the scope of the invention should not be limited to these examples as the person skilled in the art can easily vary the composition and combinations. Example -I 20 gm chitin was reacted with caustic soda taking it in 600 ml 48% caustic soda solution and stirring the same for 4 hr keeping the temperature of the reaction mass at 80°C. The reaction mass was then filtered, washed with 5% caustic soda solution squeezed and dispersed in 500 ml isopropyl alcohol. The reaction with MCA was carried out by adding 2 gm MCA taking in 30 ml isopropyl alcohol, isolating the product washed dried and grinded. The product was found to absorb distilled water 10 times of its own weight; the absorption is relatively slow and form non sticky gel. Example - II: The powdered chitin based product was prepared adopting the same process as described in Example I but taking 5 gm MCA taken in 60 ml isopropyl alcohol. The powder was found to absorb distilled water 13 times of its own weight with moderate absorption rate and form relatively less sticky gel. Example -III The powdered chitin based product was prepared adopting the same process as described in Example I but taking 10 gm MCA taken in 100 ml isopropyl alcohol. The powder was found to absorb distilled water 20 times of its own weight with fast absorption of water and forms sticky gel. Example -IV The powdered chitin based product was prepared adopting the same process as described in Example I but taking 15 gm MCA taken in 120 ml isopropyl alcohol. The powder was found to become nearly soluble and remains sticky on addition of couple of drops of water. Example -V The powdered product prepared as per example III was evaluated for anti-mutagenic properties and skin irritancy. Following table shows the non-mutagenic nature of swellable chitosan. Mutagenicitv of Swellable Chitin in Ames Salmonella / microsome Mutagenicitv (Table Removed) The skin irritancy test carried out on rabbit pinna showed that the product is nonirritant. The composition of few composite haemostatic agents and their evaluation are described in the following examples. Three sets of experiments were conducted for the composite haemostatic agent on human blood of different volunteers and two sets of experiments were done on rabbits in order to study the haemostatic effect. EXAMPLE - VI A 5 ml human blood (0+ group) was drawn. 1ml of blood each was placed on three eppendroff tubes at 25 °C containing 10 mg of composite haemostatic agent (modified chitin powder of example III and Aleovera taken in 4:1 ratio). Remaining two drops (1 ml each) were used as control. Three observers measured the time taken by the blood to clot. The average clotting time of composite haemostatic agent treated blood was 45 Sec, with a variability of ± 05 Sec. The untreated blood showed an average of 11 minutes clotting time with a variability of ± 20 Sec. Thus the clotting time of composite haemostatic agent treated blood, was reduced by almost ten times. EXAMPLE -VII: A 5 ml human blood (A+ group) was drawn from another volunteer. 1ml of blood each was placed in three eppendroff tube at 25 °C containing 10 mg of haemostatic agent (modified chitin powder of example III and Aleovera taken in 2:1 ratio). Remaining two eppendroff tubes containing 1 ml each blood, were used as control. Time taken by the blood to clot was measured by three observers. The average clotting time of composite haemostatic agent treated blood was 40 Sec, with a variability of ± 08 Sec. The untreated blood drops showed an average of 10 minutes and 30 Sec with a variability of ± 20 Sec. Thus the clotting time of composite haemostatic agent treated blood drops was reduced by ten folds. EXAMPLE -VIII: A 5 ml human blood (0+ group) was drawn from a volunteer. 1ml of blood each was placed on three eppendroff tubes at kept at 25 °C containing 10 mg of homeostatic agent (modified chitin powder of example III and Aleovera taken in 1:1 ratio). Remaining two eppendroff tubes containing (1ml each) blood drops were used as control. Three observers measured time taken by each blood sample to clot. The average clotting time of composite haemostatic agent treated drops was 33 Sec, with a variability of ± 10 Sec. The untreated blood drops showed an average of 09 minutes with a variability of ± 18 Sec. Thus the clotting time of haemostatic composition treated blood drops was reduced by more than ten times. The clotting-time of blood in the above three examples is not significantly different from one another indicating that all the combinations are very effective. EXAMPLE - IX: A small oozing wound of a size about 2 mm was made on both the sides of penna of Rabbit. Wound on one side was immediately treated with 70 mg of the composite haemostatic composition as described in Example -VI. The clotting of blood was formed in 50 Seconds. The unattended wound took 5 Minutes and 30 Seconds to form the clot. Thus the clotting of treated wound showed significant reduction in the clotting time ( >10 times). EXAMPLE -X: Two wounds, 1cm long and about 0.3 cm deep were made on both the sides of penna of Rabbit of 2.0 kg body weight. Approximately 150 mg of composite haemostatic composition as described in Example - VI, was applied immediately onto one of the wounds so as to cover the entire wound. The oozing of wound stopped within 30 seconds. The swelled mass remained adhered to the wound till the formation of epithelial layer. The unattended wound however continued to bleed till it was treated with the composite haemostatic composition. Results: The haemostatic agent showed highly effective blood clotting ability along with good sticking property due to controlled and almost instantaneous swelling and gel formation. The above examples show that the composite haemostatic agent which is a combination of two components is highly effective in reducing blood clotting duration and stopping of blood flow. Since the raw materials i.e. chitin and aleovera are produced/available from natural sources at cost competitive prices, and can be processed and applied as a single component, the developed homeostatic agent is easily affordable and can be used in case of injury. We Claim: 1. A haemostatic agent comprising a chemically modified chitin having multifunctional haemostatic properties and pharmaceutical composition thereof, wherein modified chitin is highly water swellable and sticky thereby adhering to the wound causing clotting of blood and healing of wound. 2. The method of preparing modified chitin powder from chitin comprises of the following steps: a) reacting chitin with 47 - 50% aqueous caustic soda solution in a ratio 1-20 to 1-40 (weight/volume) at 40 - 90°C for 2 - 7 hours under stirring condition, b) filtering the reacted chitin, washing the reacted mass with 5% aqueous caustic soda solution, squeezing to take out maximum liquid, c) reacting the squeezed mass of step(b) by dispersing it in isopropyl alcohol in a ratio with respect to chitin used in step (a) as 1:15 to 1:30, stirring the mass keeping the temperature at 50 to 70°C, d) adding monochloroacetic acid (MCA) solution, taking 0.1 to 0.9 parts MCA per one part chitin over a period of 1 to 5 hours maintaining pH of the reaction medium at 8 ± 0.5, e) agitating the reaction mixture for 0.5-2hours, f) cooling at 25-30°C, filtering the reaction mass, washing it thoroughly with a mixture of isopropyl alcohol and water, air drying and grinding to fine powder to obtain water swellable powder. 3. The method as claimed in claim 2 wherein monochloroacetic acid (MCA) solution is prepared by dissolving solid MCA in isopropyl alcohol in a ratio 1:5 to 1:15 (wt. by volume). 4. The method as claimed in claim 2 wherein optionally 10% caustic soda solution is added in step d) to maintain the pH. 5. The method as claimed in claim 2 wherein the mixture of isopropyl alcohol - water is 90:10 volume by volume. 6. A haemostatic agent comprising a chemically modified chitin as claimed in claim 1 wherein the haemostatic agent is topically administered to the wound. 7. A haemostatic agent comprising a chemically modified chitin substantially as herein described with reference to the foregoing examples. 8. The method of preparing modified chitin powder from chitin substantially as herein described with reference to the foregoing examples. |
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| Patent Number | 269975 | ||||||||||||||||||||||||
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| Indian Patent Application Number | 1110/DEL/2007 | ||||||||||||||||||||||||
| PG Journal Number | 48/2015 | ||||||||||||||||||||||||
| Publication Date | 27-Nov-2015 | ||||||||||||||||||||||||
| Grant Date | 22-Nov-2015 | ||||||||||||||||||||||||
| Date of Filing | 23-May-2007 | ||||||||||||||||||||||||
| Name of Patentee | THE DIRECTOR-GENERAL, DEFENCE RESEARCH & DEVELOPMENT ORGANISATION | ||||||||||||||||||||||||
| Applicant Address | ER & IPR,IPR GROUP, ROOM NO.348, B-WING, DRDO BHAVAN RAJAJI MARG, NEW DELHI-110 011,INDIA | ||||||||||||||||||||||||
Inventors:
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| PCT International Classification Number | A61K | ||||||||||||||||||||||||
| PCT International Application Number | N/A | ||||||||||||||||||||||||
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