Title of Invention

A METHOD OF MAKING TOPICAL PHARMACEUTICAL PRE[ARATIONS HAVING COMBINED HAEMOSTATIC WOUND HEALING ANTISEPTIC

Abstract A water-soluble haemostatic preparation comprising as active ingredients a fractionated Snake venom from the Bothrops species and an antiseptic agent and a method of making the same.
Full Text



The present invention relates to a method of making a topical pharmaceutical preparation having combined haemostatic, wound healing and antiseptic properties.
Particularly, the present invention to medical and veterinary preparations, specifically to haemostatic and wound healing preparations for stopping capillary haemorrhages and enhancing wound healing in topical injuries, said preparations additionally having antiseptic activity.
The present trend of the modem health care world is to come out with newer and altemative venues of combating health disorder and minimising suffering. Cost effectiveness is also the prime concem of the new products being developed.
A wound has been defined as "the disruption of anatomic and functional continuity of living tissue". It is caused by physical, chemical, thermal, electrical and microbial insult to the tissue. Wound healing has many events / phases such as haemostasis, inflammation, granulation, collagenation, contraction, epitheliazation and scar remodelling. All these phases except the scar remodelling run concurrently and independently of each other.
Wounds and topical injuries are common to everybody irrespective of their age, gender, physical and biological build, profession etc. Generally such wounds and injuries lead to bleeding, which if left unchecked may lead to profuse blood loss, fatigue and sometimes may even prove fatal.
Haemostasis, the arrest of bleeding from an injured blood vessel requires the combined activity of vascular, platelet and plasma factors, counter-balanced by regulatory mechanisms to limit the accumulation of platelets and fibrin in the area of injury. Haemostasis thus is a very crucial process at times, for saving precious lives.

Wounds are visible results of individual cell death or damage. Healing of wounds is a physiological process wherein the damaged tissues and organs undergo intensive repair mechanisms to recover and restore back their normal functional state.
Haemostasis, the arrest of bleeding from an injured blood vessel requires the combined activity of vascular, platelet, and plasma factors, counterbalanced by regulatory mechanisms to limit the accumulation of platelets and fibrin in the area of injury.
The three main phases of haemostasis are:
> Vascular phase
> Platelet phase
> Coagulation phase
Vascular Phase:
The most immediate consequence of injury to small vessels is a reduction of blood flow as a result of vasoconstriction and extravasation of blood. Vasoconstriction tends to reduce markedly the blood flow through an injured area. The escape of blood into normal tissue is limited by extra vascular supporting tissue, and the increased tissue pressure ("tamponade") tends to collapse venules and capillaries, which may then rapidly cohere and even become obliterated. The ends of elastic vessels may retract into deeper tissues, and small puncture injuries are immediately sealed because of the elasticity of the skin. These virtually instantaneous phenomena are quickly supplemented by the events of the platelet phase.

Platelet Phase:
Within seconds after injury, the platelets begin to adhere to the surface of the injured vessel (adhesion). This is the result of a specific biochemical interaction between the platelets and certain subendothelial structures, including collagen fibers, elastin, and the basement membrane. The process of platelet adhesion initiates a complex secretory phenomenon, termed the 'release reaction*. This involves the mechanical contraction of the platelet, a marked changed in its shape, and the extrusion from storage organelles of numerous biologically active substances, including ATP, ADP, 5-Hydroxytryptamine (serotonin), and various enzymes.
Platelets then begin to adhere to one another (aggregation), a process, which is specifically triggered by ADP. This becomes self-perpetuating as additional ADP is released from aggregated platelets, and rapidly produces a plug or thrombus composed of irreversibly aggregated platelets.
In small injuries, the formation of a platelet thrombus alone may suffice to arrest bleeding, and in larger injuries, it may provide "temporary" haemostasis. "Permanent" haemostasis depends on the formation of a firm, impermeable fibrin thrombus as a result of the process of blood coagulation.
Coagulation Phase:
Blood coagulation is the process by which fluid blood is converted into a coagulum or a clot.
Blood coagulation is initiated by two distinct processes, namely, contact activation (intrinsic pathway) and the action of certain lipo-proteins ( extrinsic pathway) released fi'om injured tissues (tissue thromboplastins).

Factor X is a glycoprotein, which plays a central role in blood coagulation. Factor X isactivated by a sequence of reaction beginning with contact activation and involving factors XII, XI, IX and VIII. This is termed as the 'intrinsic pathway*. A functionally identical prothrombinase can be produced in a matter of seconds by tissue thromboplastins. This involves a sequence of reactions termed as 'extrinsic pathway* which, in addition to factors X and V requires only factor VIL Thus, only the two processes mentioned above i.e. contact activation and tissue thromboplastin initiate blood coagulation. It proceeds initially via two separate pathways i.e. the tissue activated extrinsic pathway and the contact activated intrinsic pathway. Later steps leading to the formation of fibrin proceed via a common pathway, requiring factors IX, V, PF-3, prothrombin and fibrinogen.
The final step in the coagulation phase, the thrombin-fibrinogen reaction, involves the transformation of fibrinogen into fibrin, which is the physical basis of all blood clots. This occurs in three separate steps; viz., (i) the enzymatic proteolysis of fibrinogen by thrombin, which removes four peptides (fibrinopeptides), (ii) the formation of a visible but unstable fibrin polymer (soluble fibrin), and finally (iii) the formation of a stable fibrin polymer (insoluble fibrin) as the result of the action of factor XIII (fibrin stabilising factor). Structurally, fibrin resembles the proteins of muscle and skin and provides an extremely strong and stable framework for the "permanent" haemostatic plug.
Conventional methods of treating wounds include wound cleaning, drying, covering; application of direct pressure to stop bleeding or in severe cases manual closures of gaping wounds with the aid of sutures, staples or tapes. In addition, topical or oral antibiotics is recommended. Only in cases of severe uncontrollable bleeding, a haemostatic agent is used.

A wide array of disinfectants and haemostatic preparations are available in the market, each performing its specific function. There is no preparation, which comprises all the properties of a potent haemostatic, a precursor of wound healing process or tissue repair mechanisms and an antiseptic rolled together in a single formulation.
Thus, there exists a dire need to come out with such a preparation, endowed with all the above-mentioned properties - a haemostatic and wound healer with an antiseptic like action, that would bring about instant hemostasis and rapid healing.
Haemocoagulase Injection under the brand name 'Botropase' , manufactured by the applicant for the past three decades is already existing in the market. It is a haemostatic used in various conditions of haemorrhages. In a groundbreaking study done in 1990, Botropase has been tested for its wound-healing efficacy. The results of the study has come out in the form of an article in the Indian Journal of Surgery, May 1990, Vol. 52, No. 5, Pg. 218 - 221. The abstract of the paper is as follows:-
"In view of the importance of blood coagulation in wound healing, Botropase, a fractionated snake venom used as systemic haemocoagulant to arrest bleeding is studied on different wound models in albino rats. Physical, biochemical and histological evaluation revealed that Botropase promotes reparative process, procoagulation effect and other enzymatic actions of Botropase is responsible for augmentation of healing".
Later in 1991, S.L. Udupa et.al. studied the "Effects of Botropase on wound healing" in rats. The results of the study led to the conclusion that in addition to

arresting bleeding, Botropase promotes wound healing and the systemic use of Botropase in surgical processes and various coagulopathies was advocated.
The effectiveness of fractionated snake venom (Botropase) and Aminocaproic acid (Haemocid) which are systemic haemocoagulants, used alone or in combination was studied on wound healing efficacy in female albino rats by Arun K. Sharma et.al. From this study, it is very clear that Botropase is more effective in wound healing when compared with Haemocid and a combination of both is much more effective than individual drugs alone.
These studies have clearly indicated that Botropase is suitable in haemostasis as it is not only effective in stopping bleeding, but also acts as a synergiser in wound healing.
As of now, many Doctors have been using Botropase injection for local application by breaking open an ampoule (and diluting the contents of the ampoule with approximately 10 ml of saline) and dabbing it on the wounded surface with a clean tampon. This prompted the applicants to develop a new formulation that would help thousands of doctors and people to control bleeding in open wounds easily, immediately and effectively thereby stopping undue blood loss.
This invention seeks to provide such a formulation for topical application.
The most important object of the present invention is to provide a new effective local haemostatic, which coagulates blood proteins and thus produces a rapid haemostatic effect in both normal and disordered blood coagulation systems.
Another object of the present invention is to provide such a haemostatic along with wound healing property and one that will have a longer shelf life.

It is an object of the present invention to provide a haemostatic preparation along with wound healing property that also affords antimicrobial protection and is ready to use.
Another object of the present invention is to provide a simpler and more controllable preparation method and a simplified sterilisation process.
Another object of the present invention is to provide a preparation which is easy to administer on open wounds by medical professionals and users.
Other objects of the present invention will be clear from the detailed description thereof
In the first embodiment, according to this invention there is provided a water-soluble haemostatic preparation, which also acts as a precursor for wound healing comprising as active ingredients, a haemocoagulant enzyme, isolated from the venom of Bothrops atrox or Bothrops jararaca and an antiseptic agent cum preservative.
The haemocoagulant in the preparation is an enzyme complex termed as "Haemocoagulase", isolated from the venom of Bothrops atrox or Bothrops jararaca. The active principle of Haemocoagulase comprises Batroxobin (a Thrombin like enzyme) and Factor X activator (possessing Thromboplastin like activity). Both Batroxobin and Factor X activator act synergistically to hasten the clot formation.
Blood coagulation is a prelude for wound healing. Fibrin plays a vital role in wound repair and maximisation of healing occurs with fibrin glue. Haemocoagulase helps in the formation of fibrin from fibrinogen and also

activates Factor XIII, which promotes the formation of fibrin polymer. Thus, Haemocoagulase promotes wound healing by reinforcing fibrin glue.
The haemostatic agent in the preparation is therefore the enzyme 'Haemocoagulase', fractionated from the venom of snakes belonging to the species of Bothrops, particularly from Bothrops atrox or Bothrops jararaca. Since the active ingredient is an enzyme, its activity is expressed in terms of Coagulation Unit (CU) or NIH (Thrombin) Unit (in comparison with standard Human Thrombin). The concentration of Haemocoagulase in the present invention ranges fi'om about 0.05 to 2.0 CU/ml or NIH (Thrombin) unit/ml, particularly from 0.1 to 0.5 CU/ml or NIH (Thrombin) unit/ml and preferably 0.2 CU/ml or NIH (Thrombin) unit/ml.
The antiseptic agent used as preservative in the present invention is selected from the class of Biguanides or quatemary ammonium compounds.
Typically the preservative from the class of Biguanide is Chlorhexidine Gluconate solution IP (20 %w/v) in the concentration range of 0.05 to 0.25% v/v (0.01 to 0.05 % w/v of Chlorhexidine Gluconate salt) and preferably 0.1 % v/v(0.02% w/v of Chlorhexidine gluconate salt).
Typically the preservative fi^om the class of QAC is Cetrimide in the concentration of 0.005% to 0.02 % w/v and preferably 0.0125 % w/v.
The pH of the haemostatic preparation of the invention is adjusted in the range between 4 and 9, preferably between about 6 to 8, and particularly a pH that substantially equals human blood physiological pH.
In an embodiment, the present invention relates to a method for the production of a haemostatic, wound healing and an antiseptic preparation, comprising Haemocoagulase, as a haemostatic and a precursor of wound healing and an

antiseptic agent as preservative selected from the class of Biguanides or Quaternary Ammonium compounds (QAC) comprising the steps of
(a) dissolving in water a haemostatic agent - Haemocoagulase
(b) introducing an antiseptic agent selected from the class of biguanides or quatemary ammonium compounds, or a combination of both
(c) adjusting the pH of the solution (b) to a pre-determined range using a suitable base.
(d) filter sterilising the solution obtained in step (c) and filling the same in sterile containers aseptically.
In another embodiment, the present invention relates to a method for the production of a water soluble haemostatic, wound healing and an antiseptic preparation comprising of Haemocoagulase, and an antiseptic agent as preservative selected from the class of biguanides or quatemary ammonium compounds comprising the steps of
(a) Dissolving in water an antiseptic agent selected from the class of biguanides or quatemary ammonium compounds, or in combination.
(b) introducing a haemostatic agent - Haemocoagulase
(c) Adjusting the pH of the solution (b) to a pre-determined range using a suitable base.
(d) Filter sterilising the solution obtained in step (c) and filling the same in sterile containers aseptically.
In yet another aspect, the invention relates to a method of treating a haemorrhaging patient by applying to the haemorrhage source, a composition of the invention or a composition produced by the method of the invention.
Detailed description of the invention:

The present invention relates to a preparation having combined haemostatic, wound healing and antiseptic properties, which has storage stability and long shelf life.
The haemostatic agent comprised in the preparation of the invention is an enzyme Haemocoagulase isolated from the venom of Bothrops atrox or Bothrops jararaca. The active principle of Haemocoagulase comprises of Batroxobin and Factor X activator. Batroxobin is a serine protease, which cleaves fibrinopeptides A from fibrinogen. The other component of Haemocoagulase is Factor X activator, which induces the formation of prothrombin and eventually forms Thrombin. Thrombin in tum plays a key role in bringing about the clotting process. Both Batroxobin and Factor X activator act synergistically to hasten the clot formation.
Thus, Haemocoagulase having thrombin like enzyme acts on Fibrinogen in the last steps of the coagulation process to bring about haemostasis efficiently. Irrespective of the coagulation cascade, Haemocoagulase actually acts in the culmination step of the coagulation process. The concentration of Haemocoagulase in the present invention ranges from about 0.05 to 2.0 CU/ml or NIH (Thrombin) unit/ml, particularly from 0,1 to 0.5 CU/ml or NIH (Thrombin) unit/ml and preferably 0.2 CU/ml or NIH (Thrombin) unit/ml. The action of Haemocoagulase is shown in figure 1 of the accompanying drawings.
Wounds are physical injuries of the skin and underlying structures resulting from mechanical, trauma, bums, chemical injury or microbial insult. Wound healing mechanisms are essentially the same regardless of the cause of the damage. The steps involved are:

1) Immediate haemostatic processes involving the formation of a platelet plug and fibrin clot.
2) Early granulation and re-epithelialization phase, platelet derived growth factors (PDGF) stimulate fibroblasts to produce granulation tissue, comprising a collagen matrix well supplied with capillary vessels, and growth of epidermal cells leading to re- epithelialization of the wound surface.
3) The final dermal repair and remodelling phase during which collagen matrix undergoes strengthening and there is a reduction in vascularity.
Various factors influence efficient wound healing such as adequate supplies of nutrients (especially Vitamin C and Zinc) and oxygen. A good supply of blood is essential. Clinical infection by environmental microbes leads to tissue damage and thereby delays healing. Process of wound repair requires many cellular and acellular factors like platelets and growth factors, which also influence healing. Thus, the patient's age, systemic conditions, concomitant drugs, nutritional status and congenital deficiencies all influence the healing rate.
Local wound management includes cleaning, exudate removal and prevention of microbial contamination. All wounds are colonised by microbes to some extent and a microbial infection delays the healing process. Hence, there is a prime requirement to eradicate any microbial contamination.
Conventional EMS guidelines recommends or advocates the management of wounds through control of bleeding. Thus, blood coagulation is a pre-requisite for wound healing. Wound healing is a therefore a complex and dynamic physiological process where damaged cellular structures and tissue layers are restored back to their normal active state.

The human adult wound healing process comprises of four distinct phases:-
(a) Haemostasis
(b) Inflammatory phase
(c) Proliferative phase
(d) Remodelling phase
These major phases include complex and co-ordinated series of events like chemotaxis, phagocytosis, neocoUagenesis, collagen degradation, and collagen remodelling, followed by angiogenesis, epithelialization and production of new glycosaminoglycans (GAGs) and proteoglycans. The culmination of these biological processes result in the replacement of normal skin structures with fibroblastic mediated scar tissue.
Sequence of events in wound healing:
1) Initial phase-Haemostasis:
Bleeding triggers vasoconstriction. This is followed by the adherence of platelets to damaged endothelium and the discharge of ADP. This promotes clumping of thrombocytes, which dams the wound. This triggers the coagulation cascade, which is completed by the cleavage of Fibrinogen into fibrin. Fibrin provides the structural support for cellular constituents of inflammation.
2) Second Phase - Inflammation:
This phase is initiated by the release of numerous cytokines by platelets. Alpha-granules liberate platelet-derived growth factors (PDGF), platelet factor IV and transforming growth factor beta (TGF-p). The dense bodies found in thrombocytes, release vasoactive amines like Histamine and Serotonin.

TGF-P facilitates the migration of Polymorphonuclear leuckocytes (PMN) from surrounding blood vessels into the wound. PMNs engorge the wound and cleanse the wound of its debris.
Other chemotactic agents including Fibroblastic growth factor (FGF), Transforming growth factors (TGF-b and TGF-a), PDGF and Plasma activated complements C3a and C5a (anaphylactic toxins) are released into the wound. They are sequestered by macrophages or interred within the scab or eschar.
Macrophages, are the monocytes that exude from the vessels and continue the wound cleaning process for a few more days. The macrophages co-ordinate the multiplication of endothelial cells with the sprouting of new blood vessels, duplication of smooth muscle cells and the creation of milieu by the Fibroblast. They also produce various growth factors, such as TGFs, Cytokines, Interleukin-1, Tumour necrosis factor and PDGF.
3) Third Phase - Granulation:
Granulation consists of several sub phases - Fibroplasia, Matrix deposition, angiogenesis and re-epithelialization. Fibroblasts migrate into the wounds and lay down new collagen of the sub types I and IIL The GAGs, which include Heparin sulfate, hyaluronic acid, chondroitin sulfate and keratan sulfate, along with fibronectin produced by fibroblasts, suffuse the wound. Proteoglycans are GAGs that are bound covalently to a protein core and contribute to matrix deposition.
Angiogenesis is the development of blood vessels, which requires extracellular matrix and basement membrane degradation followed by migration, mitosis, and maturation of endothelial cells. Basic FGF and vascular endothelial growth factors modulate Angiogenesis.

Re-epithelialization commences with the migration of cells from the periphery of the wound and adnexal structures. Peripheral cells divide and form a thin epithelial cell layer that bridges the wound.
4) Fourth Phase - Remodelling:
Right from the initial phase, the wound undergoes constant alterations known as - remodelling, wherein collagen is degraded and deposited in an equilibrium producing fashion. Collagen deposition in normal wound healing reaches a peak by the third week of the creation of wound. Proliferation of Myofibroblasts brings about wound contraction. PDGF is chemotactic for fibroblasts and along with TGF-p is a potent modulator of fibroblastic mitosis, leading to prolific collagen fibrin construction. The maximal tensile strength of the wound is achieved by the 12 week and the ultimate resultant scar has only 4/5^" or 80% the tensile strength of the original skin that it has replaced.
Thus, the process of wound healing constitutes an array of interrelated and concomitant events, culminating in the development of scar tissue to replace the tissue that has been injured or lost.
Blood coagulation is the prelude for wound healing and in absence of the former all the subsequent steps wouldn't proceed. This is where Haemocoagulase manifests its action. Haemocoagulase converts Fibrinogen into fibrin and also activates Factor XIIL Factor Xllla catalyses the cross-linking of fibronectin and fibrin. The fibrin - fibronectin - collagen network serves as a scaffold for granulocyte migration and this in turn brings about the fibroproliferative phase.
Enhanced fibrin deposition is one of the mechanisms that increase connective tissue formation, which hastens wound healing. Thus, haemocoagulase promotes wound healing by reinforcing the fibrin glue.

Most wounds are infected with microbes, which cause delay in wound healing and may even lead to further physiological complications. Thus it is imperative to eradicate microbial growth completely from the wound. Hence an antiseptic agent was incorporated in the present preparation to serve the aforementioned purposes.
The main objective of the present invention is to bring about instant hemostasis and enhance wound healing along with preventing microbial infection of the wound. The present preparation was developed with the purpose of having a combined effect of all the above mentioned three properties, in a single preparation, which the presently available products lack.
The main active ingredient of the preparation, that serves as a Haemostatic and a precursor of wound healing is an enzyme fraction isolated from the venom of Bothrops atrox or Bothrops jararaca termed "Haemocoagulase". Since Haemocoagulase has a thrombin-like enzyme acting on fibrinogen, it's potency is determined using fibrinogen as substrate and is expressed in "Coagulation Unit (CU)" or "NIH (Thrombin) unit".
It is a normal human tendency to be apprehensive while using any product obtained from poisonous substances such as venom. Once the Haemocoagulase enzyme is isolated from the venom, it is subjected to series of analyses in order to make sure that there are no toxic components in it.
The three main toxic substances that are present in the venom are Phospholipase, Phosphotase, and L-Aminoacid Oxidase. After the isolation of the enzyme, the isolate is checked for the absence / inactivation of the toxic substances by well-established assay procedures.

The Haemocoagulase thus isolated has been subjected to bacterial endotoxin
tests and has been found to be well within the prescribed limits. Apart from these, toxicity tests of Haemocoagulase was carried out on Pigeons (which are very sensitive to the serpentine toxin) and Mice. Haemocoagulase has been found to be free from all these toxins.
All these rigorous toxicity profiles vouch for the atoxicity of the enzyme and thus prove its safety in incorporating it as an active principle in the pharmaceutical formulations.
Moreover, Botropase injection, comprising of Haemocoagulase for intravenous / intramuscular injection / local application, manufactured and marketed by us since last 35 years has never been reported for toxic, which is an ample proof of its safety.
Several experiments were conducted to study the effectiveness of Haemocoagulase on wound healing, wherein the evaluation was done physically, biochemically and histologically by testing the granuloma tissue formed on dead space wound, determination of the tensile strength, lauryl oxidase activity, collagen and mucopolysaccharidal contents, hydroxyproline content of dry granuloma.
The results indicated that Haemocoagulase promotes the first phase of wound healing haemostasis and the phase of fibroblastic proliferation, which initiates collagen cross-linking and increases the tensile strength. The enhanced fibrin depositions isone of the mechanisms that increase connective tissue formation and thus promotes wound healing.
In all the studies of wound healing efficacy of Haemocoagulase, the method of introducing the drug is similar i.e., through intra-peritoneal. But the amount of

the sample injected varies between 0.1 to 0.4 ml. It is well known that Botropase contains 1 CU/ml, therefore the amount of enzyme varied was 0,1 CU to 0.4 CU. The percentage gain in tensile strength of the wound at various doses were analysed and the following data were obtained.
The antiseptic agent use in the preparation is selected from the class of biguanide is an N,N substituted Biguanide , Chlorhexidine in the form of gluconate. Chlorhexidine belongs to the functional category of disinfectant, antimicrobial preservative and antiseptic. Chlorhexidine and its salts exhibit antimicrobial activity against most microorganisms. The optimum microbial activity is in the pH range of 5.0 to 7.0. The normal minimum inhibitory concentration of Chlorhexidine gluconate is in the range of 1 to 10 µg/ml for gram positive bacteria and 1-15 |ig/ml for gram negative bacteria.
MIC values of Chlorhexidine for some of the microorganisms are as follows.
1. Bacillus species 1.0-3.0µg/ml.
2. Clostridium species 1.8-70 µg/ml.
3. Corynebacterium species 5.0 - 10µg/ml.
4. Staphylococcus species 0.5 - 6µg/ml.
5. Streptococcus faecalis 2000 - 5000 µg/ml.
6. Streptococcus species 0.1-7 µg/ml.
7. Escherichia coli 2.5 - 7.5 |ig/ml.
8. Klebsiella species 1.5 - 12,5µg/ml.
9. Proteus species 3-100 µg/ml. 10.Pseudomonas species 3-60µg/ml.
11 .Serratia marcescens 3-75µg/ml.
12. Salmonella species 1.6 - 15µg/ml.
13.Aspergillus species 75 - 500 µg/ml.
14,Candida albicans 7-15 |ig/mL
15Microsporum species 12-18 µg/ml.
16.Penicillium species 150 - 200µg/ml.
17 .Saccharomyces species 50 - 125µg/ml.
18.Trichophyton species 2.5 - 14 µg/ml.

Chlorhexidine is used as disinfectants and its salts are used as antimicrobial preservatives. As excipients, Chlorhexidine salts are mainly used for preservation of eye-drops at a concentration of 0.01% w/v. solutions containing between 0.002 to 0.006% w/v. Chlorhexidine gluconate is used for the disinfection of hydrophilic contact lenses. Salts is used in topical antiseptic creams, mouthwashes, and dental gels, also used as constituents of medicated dressing, dusting powders, and sprays. 0.05% aqueous solution is used to treat wounds, 0.02% solution is used in bladder irrigation and also to store sterile instruments. Chlorhexidine Gluconate Solution with Cetrimide is used as skin disinfectants in hospital nursery against Streptococcal and Staphylococcal infections.
Chlorhexidine gluconate has the following toxicity value:
a) LD50 (mouse, IV): 0.01 g/kg.
b) LD50 (mouse, oral): 1.26 g/kg.
c) LD50 (mouse, SC): 1.14 g/kg.
d) LD50 (rat, IV): 0.02 g/kg.
e) LD50 (rat, oral): 2.0 g/kg.
f) LD50 (rat, SC): 3.32 g/kg.
Chlorhexidine salts are included in non-parenteral and parenteral medicines licensed in the UK.
Chlorhexidine Gluconate Solution IP in the present invention is used in the range of 0.05 to 0.25% v/v (0.01 to 0.05 % w/v of Chlorhexidine Gluconate salt) and preferably 0.1 % v/v.

Cetrimide is the other antiseptic agent chosen in our present preparation. Cetrimide is a quaternary ammonium antiseptic with actions and uses typical of cationic surfactants.
Cetrimide has bactericidal activity against gram positive bacteria and at higher concentrations against some gram-negative bacteria. It has variable anti-ftmgal activity and is effective against some viruses. It is ineffective against bacterial spores.
MIC values of Cetrimide for some of the microorganisms are as follows.
1. Escherichia coli 30 |j.g/mL
2. Pseudomonas aeruginosa 300 |ig/ml.
3. Staphylococcus aureus 10 |ig/ml.
Cetrimide is most effective in neutral or slightly alkaline solution.
The bactericidal activity is reduced in acid media and alcohols enhance the bactericidal activity.
Aqueous solutions of Cetrimide is sterilised by autoclaving.
Cetrimide is used in Cosmetics and Pharmaceutical formulations as an antimicrobial preservative.
Cetrimide is used in eye-drops at a concentration of 0.005% w/v. In topical solution, 0.1 to 1.0% w/v. Cetrimide is used to cleanse skin, wounds, bums etc.
A mixture of Cetrimide with Chlorhexidine is preferred to Cetrimide alone.
0.5 to 1.0% Cetrimide solution is used as a scolicide to irrigate hydatid cysts during surgery.
Cetrimide has the following toxicity value:

(a)LD5o (guinea pig, SC): 100 mg/kg. (b)LD5o (mouse, IP): 106 mg/kg. (c)LD5o (mouse, IV): 32 mg/kg. (d)LD5o (rabbit, IP): 125 mg/kg. (e)LD5o (rabbit, SC): 125 mg/kg. (f) LD50 (rat, IV): 44 mg/kg. (g)LD5o (rat, oral): 410 mg/kg.
In the present invention Cetrimide is used in the range of 0.005 % to0.02 % w/v and preferably at 0.0125%.
The pH of the haemostatic preparation of the invention is between pH 4.0 to 9.0, particularly between 6.0 to 7.5 and preferably pH 7.0
The hemostatic preparations of the invention is in liquid form, particularly aqueous solutions having long shelf life. The solution is clear, colourless, odourless sterile solution filled aseptically in a LDPE or LUPOLEN sterile container.
The liquid is directly incorporated in bandages or the like, or applied directly to a wound or to body tissues. The possibility of directly applying the preparations of the invention to wounds and body tissues, for example during operation, is one of its major advantages.
The present invention thus provides a haemostatic material having a strong hemostatic and bactericidal action that produces no known side effects in the course of its application.
The present invention relates to a method for the production of a water soluble haemostatic, wound healing and an antiseptic preparation, comprising of

Haemocoagulase in the concentration of 0,05 to 2.0 CU/ml or NIH (Thrombin) Unit/ml, particularly from 0.1 to 0.5 CU/ml or NIH (Thrombin) Unit/ml and preferably 0.2 CU/ml or NIH (Thrombin) Unit/ml and the antiseptic agent as preservative selected from the class of biguanides or quaternary ammonium compounds comprising the steps of
(a) dissolving in water a haemostatic agent - Haemocoagulase in the concentration of 0.05 to 2.0 CU/ml or NIH (Thrombin) Unit/ml particularly from 0.1 to 0.5 CU/ml or NIH (Thrombin) Unit/ml and preferably 0.2 CU/ml or NIH (Thrombin) Unit/ml,
(b) introducing an antiseptic agent selected from the class of biguanides or quaternary ammonium compounds, or a combination of both.
(c) adjusting the pH of the solution (b) to a pre-determined range using a suitable base.
The method of invention may further comprise of steps to filter sterilise the solution obtained in step (c) through 0.2 micron membrane filter of type Cellulose acetate using 0.8 micron membrane filter of type Cellulose acetate, as pre-filter and filling the same in sterile containers aseptically.
In another embodiment, the present invention relates to a method for the production of a water soluble haemostatic, wound healing and an antiseptic preparation comprising of Haemocoagulase in the concentration of 0.05 to 2.0 CU/ml or NIH (Thrombin) Unit/ml, particularly from 0.1 to 0.5 CU/ml or NIH (Thrombin) Unit/ml and preferably 0.2 CU/ml or NIH (Thrombin) Unit/ml and the antiseptic agent as preservative selected from the class of biguanides or quaternary ammonium compounds comprising the steps of

V
(a) dissolving in water an antiseptic agent selected from the class of biguanides or quatemary ammonium compounds, or in combination.
(b) introducing a haemostatic agent - Haemocoagulase in the concentration of 0.05 to 2.0 CU/ml or NIH (Thrombin) Unit/ml particularly from 0.1 to 0.5 CU/ml or NIH (Thrombin) Unit/ml and preferably 0.2 CU/ml or NIH (Thrombin) Unit/ml,
(c) adjusting the pH of the solution (b) to a pre-determined range using a suitable base.
The method of invention may further comprise of steps to filter sterilise the solution obtained in step (c) through 0.2 micron membrane filter of type Cellulose acetate using 0.8 micron membrane filter of type Cellulose acetate, as pre-filter and filling the same in sterile containers aseptically.
The method of invention is described in detail in the following examples.
In a further aspect, the invention relates to a method of treating haemorrhage in a patient in need of such treatment, comprising of application to the haemorrhage source, a composition of the invention.
These and other aspects of the invention will be described in more detail on hand of the following Examples, which are descriptive only and do not in any sense limit the invention, which is only defined by the appended claims.

EXAMPLES
£xatnple-l: Hemostatic preparation with Chlorhexidine Gluconate Solution IP.
Preparation-!: Preparation of Haemostatic with wound healing and antiseptic property of 0.05 CU/ml and 0.05 % v/v of Chlorhexidine Gluconate solution IP
1. Preparation of bulk Solution:
Manufacturing of the bulk solution is undertaken in a controlled area conforming to class 10000 standards.
a) Freshly collected about 9.0 It. of Water for Injection IP into a clean stainless steel vessel and cooled to room temperature.
b) Added 5 ml of Chlorhexidine Gluconate solution IP to Water for Injection.
c) Added 500 CU or NIH (Thrombin) unit of Haemocoagulase powder to the above solution and mixed thoroughly to dissolve. Adjusted the pH to 6.95 using O.IN Sodium Hydroxide solution IP (freshly prepared in Water for Injection).
d) After adjustment of pH, made up the final volume to 10.0 litres with freshly collected and cooled Water for Injection IP.
2. Aseptic filtration:
The membrane filtration is carried out in class 100 standard controlled and confirmed area.
(a) Checked the membrane filter for integrity testing.

(b)The bulk solution is filtered aseptically, through a 0.22 micron cellulose acetate membrane filter using 0.8 µ cellulose acetate membrane as prefilter into a clean aseptic filling vessel, under the influence of Nitrogen pressure at 10 to 15PSL
(c) Re-checked the integrity of filter after filtration.
3. Aseptic Filling :
Filling and sealing is carried out in class 100 standard controlled and confirmed area.
a) Ensured that the filling machine and the surrounding area is cleaned and free from extraneous matter.
b) Aligned clean tubings, syringes in place and adjusted the fill volume to 10 ml with the help of a calibrated measuring cylinder.
c) Started filling the solution into white Lupolen or LDPE bottles.
d) Closed the bottles with block nozzles and caps.
e) Labelled the bottles and packed them into cartons.
f) Finally, packed the cartons into a shipper.
Preparation - 2: Preparation of Haemostatic with wound healing and antiseptic property of 0.2 CU/ml and 0.1 % v/v of Chlorhexidine Gluconate solution IP:
(a) Freshly collected about 9.0 Its. of Water for Injection IP into a clean stainless steel vessel and cooled to room temperature.

(b) Added 10 ml of Chlorhexidine Gluconate solution IP to Water for Injection.
(c) Added 2000 CU or NIH (Thrombin) unit of Haemocoagulase powder to the above solution and mixed thoroughly to dissolve. Adjusted the pH to 7.0 with O.IN Sodium Hydroxide solution IP (freshly prepared in Water for Injection).
(d) After adjustment of pH, made up the final volume to 10.0 Its. with fi'eshly collected and cooled Water for Injection IP.
(e) Aseptically filtered and filled the solution as mentioned in example land preparation - 1.
Preparation - 3: Preparation of Haemostatic with wound healing and antiseptic property of 1.0 CU/ml and 0.15 % v/v of Chlorhexidine Gluconate solution IP:
(a) Freshly collected about 9.0 It. of Water for Injection IP into a clean stainless steel vessel and cooled to room temperature.
(b) Added 15 ml of Chlorhexidine Gluconate solution IP to Water for Injection.
(c) Added 10000 CU or NIH (Thrombin) unit of Haemocoagulase powder and mixed thoroughly to dissolve. Adjusted the pH to 7.10 with O.IN Sodium Hydroxide solution IP (freshly prepared in Water for Injection).
(d) After adjustment of pH, made up the final volume to 10.0 Itrs. with freshly collected and cooled Water for Injection IP.
(e) Aseptically filtered and filled the solution as mentioned in example 1 and preparation - 1.

Preparation - 4: Preparation of Haemostatic with wound
antiseptic property of 1.5 CU/ml and 0.15 Vo v/v of Chlorhexidine Gluconate solution IP
(a) Freshly collected about 9.0 It. of Water for Injection IP into a clean stainless steel vessel and cooled to room temperature.
(b) Added 15000 CU or NIH (Thrombin) unit of haemocoagulase powder to water for injection and mixed thoroughly to dissolve.
(c) Added 15 ml of Chlorhexidine Gluconate solution IP to the above solution.
(d)Adjusted the pH to 6.8 with 0.1N Sodium Hydroxide solution IP (freshly prepared in Water for Injection).
(e) After adjustment of pH, made up the final volume to 10.0 Itrs. with freshly collected and cooled Water for Injection IP.
(f) Aseptically filtered and filled the solution as mentioned in example land preparation - 1.
Preparation - 5; Preparation of Haemostatic with wound healing and antiseptic property of 2.0 CU/ml and 0.25% v/v of Chlorhexidine Gluconate solution IP:
(a) Freshly collected about 9.0 It. of Water for Injection IP into a clean stainless steel vessel and cooled to room temperature.
(b) Added 20000 CU or NIH (Thrombin) unit of Haemocoagulase powder to water for injection and mixed thoroughly to dissolve.
(c) Added 25 ml of Chlorhexidine Gluconate solution IP to the above solution.

(d) Adjusted the pH to 7.07 with 0.1N Sodium Hydroxide solution IP (freshly prepared in Water for Injection).
(e) After adjustment of pH, made up the final volume to 10.0 Itrs. with freshly collected and cooled Water for Injection IP.
(f) Aseptically filtered and filled the solution as mentioned in example land preparation - 1.
Example - 2: Hemostatic preparation with Cetrimide.
Preparation - 6: Preparation of Haemostatic with wound healing and antiseptic property of 0.2 CU/ml and 0.0125% w/v. of Cetrimide:
(a) Freshly collected about 9.0 It. of Water for Injection IP into a clean stainless steel vessel and cooled to room temperature.
(b) Dissolved 1.25 g. of Cetrimide in Water for Injection IP.
(c) Added 2000 CU or NIH (Thrombin) unit of Haemocoagulase powder to the above solution and mixed thoroughly to dissolve. Adjusted the pH to 6.75 with 0.1N Sodium Hydroxide solution IP (freshly prepared in Water for Injection).
(d) After adjustment of pH, made up the final volume to 10.0 Itrs. with freshly collected and cooled Water for Injection IP.
(e) Aseptically filtered and filled the solution as mentioned in example land preparation - 1.
Preparation - 7; Preparation of Haemostatic with wound healing and antiseptic property of 0.5 CU/ml and 0.005% w/v. of Cetrimide:

(a) Freshly collected about 9.0 It. of Water for Injection IP into a clean stainless steel vessel and cooled to room temperature.
(b)Dissolved 0.5 g. of Cetrimide in Water for Injection IP.
(c) Added 5000 CU or NIH (Thrombin) unit of Haemocoagulase powder to the above solution and mixed thoroughly to dissolve. Adjusted the pH to 6.59 with 0.1N Sodium Hydroxide solution IP (freshly prepared in Water for Injection).
(d) After adjustment of pH, made up the final volume to 10.0 Itrs. with freshly collected and cooled Water for Injection IP.
(e) Aseptically filtered and filled the solution as mentioned in example land preparation - 1.
preparation - 8: Preparation of Haemostatic with wound healing and antiseptic property of 1.5 CU/ml and 0.025% w/v. of Cetrimide:
(a) Freshly collected about 9.0 It. of Water for Injection IP into a clean stainless steel vessel and cooled to room temperature.
(b) Added 15000 CU or NIH (Thrombin) unit of Haemocoagulase powder to water for injection and mixed thoroughly to dissolve.
(c) Added 2.5 g. of Cetrimide to the above solution.
(c) Adjusted the pH to 6.82 with 0.1N Sodium Hydroxide solution IP (freshly prepared in Water for Injection).

(d) After adjustment of pH, made up the final volume to 10.0 Itrs. with freshly collected and cooled Water for Injection IP.
(e) Aseptically filtered and filled the solution as mentioned in example - land preparation - 1.
Example - 3: Hemostatic preparation in combination of Chlorhexidine Gluconate Solution IP and Cetrimide.
Preparation -9; Preparation of Haemostatic with wound healing and antiseptic property of 0.2 CU/ml, 0.1% v/v. Chlorhexidine Gluconate Solution IP and 0.0125%w/v. of Cetrimide:
(a) Freshly collected about 9.0 It. of Water for Injection IP into a clean stainless steel vessel and cooled to room temperature.
(b) Dissolved 10 ml. of Chlorhexidine Gluconate solution IP and 1.25 g. of Cetrimide in Water for Injection IP.
(c) Added 2000 CU or NIH (Thrombin) unit of Haemocoagulase to the above solution and mixed thoroughly to dissolve.
(d)Adjusted the pH to 6.97 with 0.1N Sodium Hydroxide solution IP (freshly prepared in Water for Injection).
(e) After adjustment of pH, made up the final volume to 10.0 Itrs. with freshly collected and cooled Water for Injection IP.
(f) Aseptically filtered and filled the solution as mentioned in example land preparation - 1.

Preparation - 10: Preparation of Haemostatic with wound healing and antiseptic property of 0.4 CU/ml, 0.15% v/v. Chlorhexidine Gluconate Solution IP and 0.005%w/v. of Cetrimide:
(a) Freshly collected about 9.0 It. of Water for Injection IP into a clean stainless steel vessel and cooled to room temperature.
(b) Added 4000 CU or NIH (Thrombin) units of Haemocoagulase powder to water for injection and mixed thoroughly to dissolve.
(c) Dissolved 15 ml. of Chlorhexidine Gluconate solution IP and 0.5 g. of Cetrimide in the above solution and mixed well.

(d) Adjusted the pH to 6.97 with 0.1N Sodium Hydroxide solution IP (freshly prepared in Water for Injection).
(e) After adjustment of pH, made up the final volume to 10.0 Itrs. with freshly collected and cooled Water for Injection IP.
(f) Aseptically filtered and filled the solution as mentioned in example land preparation - 1.
Example - 4: Study of Blood Clotting and Wound healing properties of the present invention on Rats.
The product obtained from example 1; preparation 2, i.e., Haemocoagulase 0.2 CU/ml and 0.1 % v/v. Chlorhexidine Gluconate solution IP, is tested for wound healing efficacy on rats. Matured Wistar Albino Rats were used for the experiment. They were individually housed and cared under suitable environmental conditions and fed with normal diet and water. Animals were maintained under laboratory conditions before experiment was carried out. The

animals were divided into two groups consisting of 2 animals in each group. One group is considered as control (untreated) for which no application of the sample is done and the other as Test (treated) for which the solution of present invention is applied.
The right or left side of the abdominal portion of the rat is shaved by using surface sterilised scissors under Chloroform anesthesia and cleaned with cotton. The incision is done by surface sterilised surgical blade. The length and opening of the wound is traced on to the butter paper.
Then, the wound is treated by adding 2 drops of the test sample on to the wound. The cotton is wetted with few drops of the test sample and placed over the incision wound and bandaged.
For the control, the wound is treated with only 70% alcohol and bandaged.
The application is repeated every day with test sample for test group and with 70% alcohol for control group.
The progressive decrease in the wound length and opening is monitored periodically by tracing the wound length and opening margin on a tracing paper. The weight of the animal is also monitored. The results are neatly tabulated and recorded.
Example - 5: Preservative efficacy test of the present invention:
Preservative efficacy study was conducted to determine the efficacy of the antiseptic used in the example 1; preparation 2, as described in the general test chapter of the U.S. Pharmacopoeia: Antimicrobial Preservatives-Effectiveness -51 (USP 23, p. 1681). The assay involves challenging the formulation with bacteria of three types (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa), a yeast {Candida albicans) and fungi {Aspergillus

niger). Then monitoring the microbial population periodically at intervals of 7 days by Plate Count method. An effective preservative would not allow any increase in the microbial population from the initial inoculum size. The results are neatly tabulated.
Example - 6: Bacterial Growth Inhibition:
Aseptically inoculated 0.1 ml of 24 hours Bacterial culture in Nutrient broth to Nutrient agar plates by pour plate technique. Aseptically prepared different dilutions of the preparation (of example 1; preparation 2), in the range of 0% to 100% v/v. Soaked the sterile filter discs in the different concentrations for 1 hour prior to use. Placed the presoaked filter discs separately on the middle of the inoculated plate and incubate at 37°C for 24 hours. Noted the zone of clearance around the discs and tabulate.

Example - 4: Test for wound healing in Mice:



Example - 15: Preservative efficacy test:



Example - 16: Bacterial Growth Inhibition.

From the above analysed data, it is clear that 0.2 CU is quite sufficient to bring about the desired action. Hence, the present preparation containing 0.2 CU/ml of Heamocoagulase is used to evaluate its wound-healing efficacy on Rats. As shown in the result of example 4, topical application of a few drops of

preparation on incision wound of test animals, the rate of wound healing is faster when compared to untreated control. The complete contraction or closure of the wound was earlier (4-8 days) in the treated animals as compared to control (10-11 days). No significant changes in the body weight of both control and treated animals were observed.
It is reported that Haemocoagulase initially manifests the primary step of wound healing mechanism - Haemostasis, by catalysing the conversion of fibrinogen to fibrin and also activating Factor XIII. Factor XIIIa catalyses the cross-linking of fibrin to bring about blood coagulation. Later it brings about the cross-linking of fibrin, fibronectin and collagen. The fibrin-fibronectin-collagen network serves as a template for the fibroproliferative phase, which completes the wound healing process. Thus, Haemocoagulase serves a dual role - that of potent haemostatic agent and that of a precursor of wound repair mechanism.
All wounds are infected by microbes to some extent, which delays the healing process. Microbial infection also leads to several other physiological complications. Hence it is a must to eradicate all such inordinate microbial infections from wounds. This purpose is served by the incorporation of an antimicrobial agent in the preparation. The preservative selected for any pharmaceutical preparation should be evaluated for its specific claims. Evaluation of preservatives is done by conducting preservative efficacy test as per the USP guidelines. The preservative efficacy test conducted for the preservative Chlorhexidine Gluconate Solution IP, showed the complete inhibition of pathogenic microbes indicating the effectiveness of the preservative.
From the example 6, it is clear that even in lower concentration, the preparation effectively inhibits the growth of pathogenic strains.

Hence, the antimicrobial agent used in our preparation not only prevents microbial contamination of the topical solution but also inhibits wound infection by pathogenic strains. Thus, also by preventing undue wound infection, it promotes wound healing.
Thus, the claims made by the present preparation that of being a potent haemostatic, a precursor of wound repair along with possessing antiseptic activity are all true and just.
From the results of the experiments and from the available literatures it is evident that Haemocoagulase is a potent haemostatic agent and also a promoter of wound healing. Salient features of Haemocoagulase:
> Rapidly converts fibrinogen into fibrin clot.
> Activates factor XIII, which brings about polymerization of fibrin monomer.
> Shortens the blood coagulation time.
> Stimulates platelet-fibrin interactions.
> Promotes wound healing by catalyzing haemostasis and reinforcing fibrin glue.
> Shortens the recalcification process of plasma.
> Increases the thromboplastin activity of plasma.
> Leaves the prothrombin concentration unaltered.
> Can be administered through IM, IV and local routes.
> well tolerated, non-toxic and time tested.
These features of Haemocoagulase enabled it to be selected as the best candidate to serve the objectives of the present invention.
The antiseptic agent incorporated in the present invention, not only preserves the topical preparation from microbial contamination, but also protects the wounds

from microbial infection, which would otherwise delay the healing process. Also by minimising the microbial infection, wound healing is promoted.
Thus the present preparation, comprising of Haemocoagulase and an antiseptic agent from the class of Biguanides or Quaternary ammonium compounds, serves all the fiinctions of a haemostatic, a precursor of wound repair mechanism and an antiseptic agent.
The present preparation brings about rapid haemostasis and healing thereby minimizing undue blood loss and prolonged suffering.
Its ease of administration makes it a sought after first aid medication which can be used by one and all for various kinds of bleeding injuries. -






We Claim:
1. A method for the production of a water-soluble haemostatic antiseptic preparation, comprising;
(a) Dissolving in water
a haemostatic agent - Haemocoagulase in the concentration of 0.05 to 2.0 CU/ml or NIH (Thrombin) Unit/ml particularly from 0.1 to 0.5 CU/ml or NIH (Thrombin) Unit/ml and an antiseptic agent selected from the class of Biguanides or Quatemary ammonium compounds, or a combination of both;
(b) Adjusting the pH of the solution (b) to a pre-determined range using a
suitable base.
2. The method as claimed in claim 1, wherein said fractionated venom is an enzyme Haemocoagulase isolated from the venom of Bothrops species.
3. The method as claimed in claim 1 or claim 2, wherein said Haemocoagulase enzyme is isolated from the venom of Bothrops atrox or Bothrops jararaca.
4. The method as claimed in any one of the claims 1 to 3, wherein the concentration of said Haemocoagulase is from about 0.05 CU/ml or NIH (Thrombin) Unit/ml to 2.0 CU/ml or NIH (Thrombin) Unit/ml.
5. The method as claimed in claim 4, wherein the concentration of said Haemocoagulase is from 0.1 CU/ml or NIH (Thrombin) Unit/ml to 0.5 CU/ml or NIH (Thrombin) Unit/ml,

6. The method as claimed in claim 1, wherein said antiseptic agent is selected from the class of Biguanides or Quatemary ammonium compounds or a combination of both.
7. The method as claimed in claim 6, wherein said antiseptic agent, selected from the class of Biguanides is Chlorhexidine Gluconate Solution IP.
8. The method as claimed in claim 7, containing from about 0.05%v/v to 0.25%v/v of Chlorhexidine Gluconate Solution IP.
9. The method as claimed in claim 6, wherein said antiseptic agent, selected from the class of Quatemary ammonium compounds is Cetrimide.
10.The method as claimed in claim 9, wherein the antiseptic agent is 0.005% w/v. to 0.02% w/v. of Cetrimide.
11.The method as claimed in claim 6, wherein said antiseptic agent is the combination of a Biguanide and a Quatemary ammonium compound.
12,The method as claimed in claim 11, wherein said Biguanide is Chlorhexidine Gluconate Solution IP and Quatemary ammonium compound is Cetrimide.
13.The method as claimed in claim 12, wherein the concentration of the said Biguanide - Chlorhexidine Gluconate Solution IP is about 0.05% to 0.25% v/v and said Quatemary ammonium compound - Cetrimide is about 0.005 to 0.02% w/v.

14.The method as claimed in any one of claims 1 to 13, in which the pH is adjusted between about 4.0 and about 9.0.
15.The method as claimed in any one of claims 1 to 13, in which the pH is adjusted between 6.0 to 8.0.
16.The method as claimed in any one of claims 1 to 13, in which the pH is adjusted to substantially equal to human blood physiological pH.
17.A method as claimed in claim 1 , further comprising the steps of filter sterilising the solution obtained in step (c) and filling the same in sterile containers aseptically.
18.The method as claimed in claim 17, wherein the filter sterilisation is done by passing the solution through 0.22µ Cellulose acetate filter membrane using 0.8µ Cellulose acetate filter membrane as prefilter.
19.The method as claimed in claim 18, wherein the filter sterilised solution is filled in sterile Lupolen or LDPE containers.


Documents:

410-che-2003-abstract.pdf

410-che-2003-claims duplicate.pdf

410-che-2003-claims original.pdf

410-che-2003-correspondnece-others.pdf

410-che-2003-correspondnece-po.pdf

410-che-2003-description(complete) duplicate.pdf

410-che-2003-description(complete) original.pdf

410-che-2003-drawings.pdf

410-che-2003-form 1.pdf

410-che-2003-form 19.pdf

410-che-2003-form 26.pdf

410-che-2003-form 3.pdf


Patent Number 196273
Indian Patent Application Number 410/CHE/2003
PG Journal Number 20/2006
Publication Date 19-May-2006
Grant Date 09-Jan-2006
Date of Filing 14-May-2003
Name of Patentee M/S. JAGDALE INDUSTRIES LIMITED
Applicant Address 27, BULL TEMPLE ROAD BASAVANGUDI, BANGALORE 560004
Inventors:
# Inventor's Name Inventor's Address
1 NAGARAJA RAO RADHAKRISHANARAO JAGDALE JAGDALE INDUSTRIES LIMITED, 27, BULL TEMPLE ROAD BASAVANGUDI, BANGALORE 560004
PCT International Classification Number A61L33/14
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA