A bioactive formulation

Field of the invention

The invention pertains to a bioactive formulation, which is used for wound healing and in cosmetics. The invention comprises of two or more growth factors; a delivery system; at least selected from an antioxidant, an immunomodulatory agent; and optionally an anti-microbial agent. The invention also pertains to methods for preparing and using the bioactive wound healing formulation and the pharmaceutical products in which the formulation may be used.

Background and Prior art

Wound healing is a well orchestrated complex series of events normally resulting in the restoration of structural integrity and functionality of the damaged tissue. Treatment strategies to enhance the blood flow, relieve pressure and control infections are already available in the prior art. However, in case of chronic wounds the healing process is impeded, thus causing a delay in restoration of the damaged tissues.

A wound is an easy entry point for invasion by pathogenic microbial flora. The bacterial colonization hampers normal wound healing by changing the wound microenvironment and making it unfavourable to the wound healing process. Following invasion, the microbes release various proteases that can destroy the growth factors produced by the host rendering the wound microenvironment highly unfavourable for healing. Furthermore, a wound increases the probability of invasion by certain opportunistic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. As an attempt to correct the microenvironment, broad spectrum antibacterial agents such as Silver sulfadiazine have been extensively used in the past. Also, several antioxidants such as lactoferrin have been effectively used to treat chronic wounds caused due to diabetes.

It is a known fact that a wide range of human polypeptide growth factors have been instrumental in promoting the growth of normal human cells. It has also been found in the past that each of these growth factors function differently and contribute to healing of chronic wounds at different stages. These polypeptides are of varied molecular weights and are mostly basic in nature. They are stable and exhibit growth stimulating activity on various epithelial and epidermal tissues. They transmit signals between cells that promote cell proliferation and are thus instrumental in synthesis of granulation tissues. Research on animal models showed enhanced soft tissue repair while human trials showed healing of chronic cutaneous ulcers in the presence of growth factors. However, clinical trial data generated using single growth factors for treatment of cutaneous wounds showed less effective healing. Moreover, the 5 recombinant growth factors are very expensive and are less stable due to which they have been proved to be less effective in the wound healing process. Thus, there is a need for a more stable and cost effective substitute for recombinant growth factors that can be used in wound healing formulations.

One other vital bio molecule that has been instrumental in wound healing is 10 Collagen. Collagen is a major protein of connective tissue and has been widely used for topical applications including cosmetic applications as about 70% of the skin component is collagen. Collagen has also been widely utilized as a scaffold for cell proliferation in various soft and hard tissues and has previously been used In wound dressings. Collagen implants are highly biocompatible, biodegradable, and are capable of promoting connective tissue deposition, angiogenesis, reepithelialisation, and fibroplasia. Procedures for rendering xenogeneic collagen substantially non-immunogenic are also available in the prior art.

Though one or more of these above mentioned ingredients have been used in formulations for wound healing in the past, these have not been very effective in treating of chronic wounds. Therefore, there is a need for improved wound healing formulations and efficient low cost delivery systems which allow good adhesion to the biological tissues for sustained delivery of the formulations.

Also, there is a need for developing a topically applicable formulation product which participates in the rapid wound healing process by providing the essential components of wound healing cascade and provide results comparable to the currently used treatments.

The present invention provides a wound healing formulation comprising of combination of growth factors extracted from natural sources like milk and formulated with effective delivery system. The formulation of the present invention further exerts marked additive and synergistic effects of multiple growth factors from natural extracts that are more stable than the recombinant derived growth factors.

Objects of the Invention

1. The primary object of the invention is to provide a bioactive formulation comprising of at least two growth factors and at least one of an antioxidant and an immunomodulatory agent and optionally an anti-microbial agent with a delivery system.

2. Another object of the invention is to provide a wound healing formulation comprising of a combination of at least two growth factors derived from a natural source and/or obtained in a recombinant manner.

3. A further object of the invention is to provide a wound healing formulation which is cost effective.

4. Another object of the invention is to provide a wound healing formulation comprising of combination of growth factors extracted from natural sources, more specifically from milk or whey.

5. A further object of the invention is to provide a wound healing formulation that can effectively heal chronic wounds including but not limited to diabetes related chronic wounds, venus ulcers, arterial ulcers and pressure ulcers.

6. A further object of the invention is to provide an effective wound healing formulation which can be administered topically.

7. A further object of the invention is to provide a bioactive formulation for use in cosmetics comprising of at least two growth factors and at least one of an antioxidant and an immunomodulatory agent with a delivery system.

Statement of the invention

The disclosed invention relates to a bioactive formulation comprising of combinations of two or more growth factors formulated with effective delivery systems.

According to the one of the embodiments of the invention, the bioactive formulation for wound healing and for use in cosmetics comprises of a combination of two or more growth factors formulated with effective delivery systems.

According to the one of the embodiments of the invention, the bioactive formulation comprises of a combination of at least two growth factors or a fragment thereof; at least one of an immunomodulatory agent and antioxidant; and an anti-microbial agent; wherein, the application of the bioactive formulation promotes wound healing.

According to the one of the embodiments of the invention, the bioactive formulation comprises of a combination of at least two growth factors or a fragment thereof; at least one of an immunomodulatory agent and antioxidant; wherein, the application of the bioactive formulation is used for cosmetics.

Another embodiment of the invention describes a bioactive formulation comprising of a combination of at least two growth factors selected from a group consisting of IGF-I, IGF-II, PDGF, EOF, acidic FGF, FGF, TGF-p, HGF and bioactive fragments or mimetics thereof, at least one of an immunomodulatory agent and an antioxidant and optionally an anti- microbial agent; the said growth factors are isolated from natural sources including but not limited to milk, whey or milk derivatives.

Another embodiment of the invention describes a bioactive formulation comprising of a combination of at least two growth factors selected from a group consisting of IGF-I, IGF-II. PDGF, EGF, acidic FGF, FGF, TGF-p, HGF and bioactive fragments or mimetics thereof, at least one of an immunomodulatory agent and an antioxidant and optionally an anti- microbial agent; wherein, the said growth factors are isolated from natural sources, wherein the growth factors may be formulated with Hyaluronic acid.

One other embodiment of the invention describes a bioactive formulation comprising of a combination of at least two growth factors, at least one of an immunomodulatory agent and an antioxidant and optionally an anti-microbial agent; wherein, the antimicrobial agent is selected from a group consisting of therapeutic antibodies, toxins, antibiotics, anti-viral agents, anti inflammatory agents, a peptides and peptidomimetic molecules.

Another embodiment of the invention describes a bioactive formulation comprising of a combination of at least two growth factors, at least one of an immunomodulatory agent and an antioxidant and optionally an anti-microbial agent; wherein, the antimicrobial agent is selected from a group consisting of silver sulfadiazine, povidine iodine, ofloxacin, metronidazole, ciprofloxacin, Levofloxacin, Clindamycin, CeftriaxonCi Cefotetan, Zosyn and derivatives thereof

One further embodiment of the invention describes a bioactive formulation comprising of a combination of at least two growth factors, at least one of an immunomodulatory agent and an antioxidant and optionally an anti-microbial agent; wherein, the antioxidant is selected from a group consisting of glutathione, ascorbic acid, tocopherol and butylated hydroxyl anisole.

In another embodiment of the invention, the bioactive formulation may be adsorbed on a non-aqueous matrix wherein the non-aqueous matrix comprises of collagen, Hyaluronic acid, gelatine and dextran. The matrix shall be of a defined density and uniform porosity thus enabling promotion of connective tissue deposition, reepithelization and blood vessel formation in the wounds of varying etiology.

These and other embodiments of the invention herein will be better appreciated and understood when considered in conjunction with the following description. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

Detailed description

The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and adapt for various applications such specific embodiments without departing from the generic concept and therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the appended claims.

Wound healing involves many processes including inflammation, fibroplasia, epithelialisation, angiogenesis, and formation and contraction of connective tissue. As we better understand the molecular control of wound healing, therapeutic possibilities become apparent.

The present invention discloses a bioactive formulation comprising of a combination of growth factors formulated with effective delivery systems which is not only effective in healing chronic wounds and useful in cosmetics, but is also cost effective.

The disclosed invention comprises of a bioactive formulation for wound healing comprising of a combination of at least two growth factors or a fragment thereof; at least one antimicrobial agent; at least one antioxidant; at least one immunomodulatory agent; at least one formulation delivery system further comprising of at least one polymer, preservative, healing promoting agent or chemical species; wherein, the bioactive compound formulation is for promoting wound healing. The bioactive formulation may be adsorbed on a non-aqueous matrix wherein the non-aqueous matrix may be collagen. Hyaluronic acid, gelatine and dextran.

The disclosed invention comprises of a bioactive formulation for use as a cosmetic agent comprising of a combination of at least two growth factors or a fragment thereof; at least one antioxidant; at least one immunomodulatory agent; at least one formulation delivery system further comprising of at least one polymer, preservative, healing promoting agent or chemical species; wherein, the bioactive compound formulation is for promoting wound healing. The bioactive formulation may be adsorbed on a non-aqueous matrix wherein the non-aqueous matrix may be collagen, Hyaluronic acid, gelatine and dextran.

Given below is a brief description of each of the ingredients which form a part of the disclosed bioactive formulation.

Growth Factors

'Growth factors' is a holistic term used to describe a variety of chemical messengers found in a wide range of tissues, which have a number of functions including the regulation of cell and tissue growth and development. Growth factors attract cells into the wound, stimulate their proliferation, and have profound influence on extracellular matrix deposition. Several growth factors have been proposed as ideal agents to promote wound healing.

Platelet derived growth factor (PDGF), insulin-like growth factor, transforming growth factor beta (TGF-p), transforming growth factor alpha, epidermal growth
factor (EGF), Hepatocyte growth factor (HGF), and fibroblast growth factor are polypeptides that have been described extensively in wound healing because of their ability to stimulate cell growth. PDGF is a cationic, heat-stable protein found in the granules of circulating platelets which is known to stimulate in vitro protein synthesis and collagen production by fibroblasts. It is also known to act as a mitogen and chemotactic agent for fibroblasts and smooth muscle cells.

EGF is believed to exert its effect through interactions with other growth factors and cells recruited to the wound site by other growth factors such as PDGF, FGF, TGF-p.

Although, EGF was shown to be useful for wounds like venus ulcers where re-epithelization is required, it was not suitable for wounds, such as, diabetic foot ulcers, that require extra cellular matrix formation & angiogenesis. Although the prior art teaches the use of FDGF to promote in vivo wound healing by increasing the amount of new collagen synthesized, studies showing the accelerated wound healing using PDGF alone or in combination with FGF and EGF is lacking.

Further, studies on wound healing using growth factors have been impeded due to lack of effective delivery systems capable of allowing the polypeptides and growth factors to reach the target cells and tissues.

For the enablement of the disclosed Invention, the growth factors may be selected from the group consisting of IGF-I, IGF-II, PDGF, EGF, acidic FGF, FGF, TGF-p, HGF and bioactive fragments or mimetics thereof. The growth factors may be isolated from natural sources and/or obtained in a recombinant manner. The growth factors may preferably be isolated from natural sources. The preferred natural source is milk, whey or milk derivatives. They may be also formulated with Hyaluronic acid.

Hyaluronic acid may also be used in internal wound healing such as healing of joints or in the anterior chamber of the eye, A 1% solution of sodium hyaluronate (molecular weight 4,000,000) could be formulated with the mixture of growth factors to obtain a concentration of 100 microgram to 2.5 micrograms/ml. The viscosity of the 1% HA solution was 44,000 cps. An HA/growth factors formulation could be a yet another formulation to stimulate re-endothelization in the anterior chamber of the eye and thus can be used to treat eye injuries,

The formulation promotes effective and enhanced wound healing activity on various types of wounds like diabetic, venus, arterial and pressure ulcers. The formulation without limiting the scope of the invention may also be used for treating post operative surgery wounds. It is also be used in various cosmetic preparations including but not limiting to the treatment of acne.

The growth factors used herein are comprised in the formulation. The formulation may be capable of cell signalling, cell proliferation and series of other events essential for an effective wound healing.

These growth factors may be isolated from natural sources such as milk and products
derived from milk such as whey and/or derived from recombinant sources. These isolates may be processed by non-denaturing methods without subjecting the milk to higher temperatures, low pH and other usual stressful denaturing processes involved in the whey manufacturing.

The bioactive growth factors may belong to the family of epidermal growth factor (EGF), transforming growth factor (TGF), fibroblast growth factors (FGF) and the insulin like growth factors (IGF-I and IGF-II).

"Peptide" as used herein refers to short polymers of amino acid molecule, and accordingly "polypeptide" refers to long polymers of amino acid molecules. "Proteins" are polypeptide molecules. The 'peptide" can also be obtained from a gene product, a purified or isolated product, an expression product, from fragmentation of protein(s) or a synthetic peptide.

The term "growth factor" refers to a naturally occurring polypeptide or protein capable of stimulating cellular proliferation and cellular differentiation. "Growth factors" are important for regulating a variety of cellular processes and may act as signalling molecules between cells. It should be understood that other biologically active fragments, growth factors or chemically synthesized derivatives of the growth factors may also be used without deviating from the invention.

Antimicrobial Agents

A wound is an easy entry point for the invasion by pathogenic microbial flora. Following invasion, the microbes release various proteases that can destroy the growth factors produced by the host rendering the wound microenvironment highly unfavourable for healing. The present invention therefore provides that the bioactive formulation may comprise of one or more antimicrobial agents. The antimicrobial agent may be therapeutic antibody, toxin, an antibiotic, anti-Viral or anti inflammatory agent, a peptide and peptidomimetic. The said antimicrobial agent may further be selected from the group consisting of silver sulfadiazine, Povidine Iodine, ofloxacin, ciprofloxacin, Levofloxacin, Clindamycin, Metronidazole, Ceftriaxone, Cefotetan, Zosyn and derivatives thereof The agent may also be selected from the list consisting of EPA's Registered Antimicrobial Products Registered with the EPA as Sterilizers. (Last updated on January 9,2009)

In particular the said antimicrobial agent may be silver sulfadiazine. The silver sulphadiazine concentration may be in the range of 20-100 microgram per gram of the bioactive formulation.

"Antimicrobial agents" as used herein refers to a substance capable of killing
microbes (microbicidal) or preventing the microbial colonization (microbistatic) or both. "Microbes" may refer to bacteria, fungi, or viruses. Certain Antibiotics may also be present in the formulation for preventing the growth of certain bacteria* s in the wound. The antibiotics may be selected from the group consisting of Aminoglycoside Antibiotics, Cephalosporins, Fluoroquinolone Antibiotics, Macrolide Antibiotics, Penicillins, Sulfonamides, Tetracycline Antibiotics and derivatives, salt forms and mixtures thereof.

Antioxidant

The bioactive formulation of the present invention may also comprise of at least one antioxidant selected from the group consisting of Butylated hydroxy anisole, Vitamins A, Vitamins C, Vitamins E, Beta-Carotene, Canthaxanthine, Co-Enzyme Q10 (ubiquinone/CoQ10), Selenium, Zinc, Manganese, L-Camosine, L-Arginine, Myricetin, Ascorbic acid, Tryptophan, Cysteine, Tyrosine and Homocysteine and their derivatives thereof.

Naturally occurring substances such as lactoferrin which contain antioxidants like glutathione may also be used. Lactoferrin also contributes to the formulation by acting as a preservative. Due to its dual ftinction, Lactoferrin is one of the preferred antioxidant in this invention.

An "antioxidant" as used herein refers to a molecule capable of slowing or preventing the oxidation of other molecules. Antioxidants may also have the capacity to control microbial growth and thus have the ability to contribute to the formulation in more than one ways.

Immunomodulatory agent

The bioactive formulation of the present invention may also comprise of at least one immunomodulatory agent selected from the group consisting of lactoferrin and talactoferrin,

Lactoferrin which is found in milk and some mucosal secretions is a protein that plays an important role in the development and modulation of the body's immune system. Lactofenin is also believed to have antibacterial, antiviral, antifungal, anti-inflammatory, antioxidant and immunomodulatory activities,

Talactoferrin is a recombinant form of human lactoferrin and also plays an important role in the development and modulation of the body's immune system.

Lactoferrin and Talactoferrin are used in the bioactive formulation in a range of 0.01% to 50%.

Formulation Delivery System

The bioactive formulation may further comprise at least one formulation delivery system comprising of at least one polymer, atleast one preservative, at least one 5 healing promoting agent and at least one chemical species.

Polyacrylic acid Gel based formulation

The said polymer may be selected from the group consisting of Hyaluronic Acid, Pluronic acid, cellulose, methyl cellulose, hydropropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, carbopol and/or derivatives thereof. In particular the at least one polymer may be carbopol. Carbopol may be hydrophilic capable of keeping the wound microenvironment moist.

Carbopol is being used in the controlled release solid dosage formulations since last four decades, Carbomers are very well suited to aqueous formulations of the topical dosage forms. Many commercial topical products available today have been formulated with these lands of polymers, as they provide the following numerous benefits to topical formulations:

Safe & Effective: Carbopol polymers have a long history of safe and effective use in topical gels, creams, lotions, and ointments. They are also supported by extensive toxicology studies.

Non-Sensitizing: Carbopol polymers have been shown to have extremely low irritancy properties and are non-sensitizing with repeat usage.

No Effect on the Biological Activity of the Drug: Carbopol polymers provide an excellent vehicle for drug delivery. Due to their extremely high molecular weight, they cannot penetrate the skin or affect the activity of the drug.

Excellent Thickening, Suspending, & Emulsification Properties for Topical Formulations Products with a wide range of viscosities and flow properties have been successfully formulated and commercialized. Carbopol polymers are used to permanently suspend the active ingredients in transdermal reservoirs as well as in topical gels and creams.

Polyacrylic acid gels (Carbopol) were prepared according to the present invention. Prefened grades of polyacrylic acid are those referred to as Carbopol 934 P and 940 at concentrations of 0.02-2.0%. Higher concentrations of polyacrylic acid lower the release rate of the EGF, Viscosities of polyacrylic acids are generally stable between pH 6-10, preferably in the pH range 6.5-7.5. The viscosity of the gels prepared ranged from about 5000,000 to about 550,000 cps.

Healing promoting agent

Zinc oxide has been shown to be an essential healing promoting agent in many biochemical reactions. Zinc has been reported to display antibacterial effects. Zinc oxide has also been shown to maintain a constant level of zinc when applied to wounds as compared to salts of zinc.

Zinc oxide may act as a healing promoting agent promoting re-epithelialisation and maintain constant levels of zinc in the wound envirotmient contributing to antibacterial activity. It may also promote cleansing of wounds, decrease deterioration and inflammation of existing wound and also correct the local zinc deficiency. Research has shown that patients with diabetes mellitus have zinc deficiencies attributing to impaired wound healing. Studies have established healing effect of zinc oxide in skin grafts.

Since zinc oxide can accelerate the healing of both chronic and acute wounds while increasing endogenous gene expression of insulin-like growth factor-1, the present invention provides for the inclusion of zinc oxide in the bioactive formulation. The inventors also propose that zinc oxide may act as a preservative for polypeptide growth factors comprised in the formulation of the present invention.

Optional Ingredients

Apart from the ingredients mentioned above, other optional ingredients such as preservatives, catalytic agents, and gelling agents among others also can be included in the formulation.

The preservatives may be selected from the group consisting of ethyl paraben, propyl paraben, butyl paraben, diazolidinyl urea, quatemium salts.

The chemical species may be selected from the group consisting of lysine, arginine, histidine. protamine, amino guanidine, zinc and magnesium.

The bioactive formulations for topical or incisional wound healing includes exemplary bases which includes gels (e.g., hydrogels), salves, lotions, creams, and the like, as are known to be useful in topical compositions. The gel or cream formulation polymer may be selected from the group consisting of vinyl polymers, polyoxyethylene-polyoxypropylene copolymers, polysaccharides, proteins, polyethylene oxide, acrylamide polymers, derivatives and/or salts thereof. It is understood that polyethylene oxide includes polyethylene glycol In the gel formulations for use in healing wounds in the anterior chamber of the eye, the polymers may be the same except that polyoxyethylene-polyoxypropylene copolymers or polyethylene oxide are not preferred. The bioactive formulation may be biodegradable. "Biodegradable" as used herein refers to breaking down of the formulation into harmless constituents that can be drained from or metabolized by the host. In the low viscosity, aqueous formulations for use in ophthalmic wound healing, the gel forming polymers may be the same as for topical or incisional wound healing, except that polyethylene oxide is not preferred to be used. The present invention also provides that the formulation delivery system exhibit bioadhesive properties. It may be able to stick to the skin for a longer period of time but upon removal, no residues are left on the skin.

The gels may be hydrogels. Hydrogels are three-dimensional, hydrophilic, polymeric networks capable of imbibing a large amount of water and water miscible solvents. The high solvent content of hydrogels can provide a great capacity to accommodate a high dose of drugs and good compliance compared to conventional dosage forms such as ointments, creams, gels and patches. Therefore, bioadhesive hydrogels formulated in proper dosage forms, e.g. plasters, will overcome the above mentioned problems associated with the conventional systems. Since the bioadhesive polymer molecules are cross-linked with an appropriate cross-linking agent in the matrix, they are not sticky but still maintain their bioadhesive properties. Further, hydrogels may have good compatibility with the skin.

Accordingly, the bioactive formulation comprising the composition described in the preceding description adsorbed on a non-aqueous matrix wherein, the non-aqueous matrix comprises of collagen, Hyaluronic acid, gelatine and dextran. Accordingly, the present invention provides a topical and transdermal bioactive compound delivery system comprising a non-aqueous matrix, the matrix comprising a water-sensitive bioactive compound. In particular, the invention provides a bioadhesive topical and transdermal bioactive compound delivery system comprising a non¬aqueous matrix, the matrix comprising a water-sensitive bioactive compound.

The matrix may be collagen matrix with defined density. It may also preferably have uniform porosity. The collagen matrix is capable of promoting connective tissue deposition, re-epithelization and blood vessel formation in the wounds of varying etiology while delivering the polypeptide growth factors in a sustained manner with increased contact rate at the wound site. Collagen may also act as a bioadhesive. Further, the above said growth factors may be formulated with appropriate protein stabilizing agents in the buffering system to provide the active physiological micro environment. The collagen may be in the form of collagen implants capable of sustained release of active substances. The use of collagen matrices in the formulation may provide several advantages over frequent administration for instance repeated injections of active substances alone. These advantages include (1) the ability to maintain the active substance at the treated site over a period of time following each administration, (2) optimal handling properties for the physician, (3) decreased trauma to the patient (e.g., 1-3 treatments per week instead of treatments daily or more), and (4) decreased treatment costs to the patient. Furthermore, the collagen composition of these matrices may provide an environment similar to host tissue, aiding in the wound healing response wherein the collagen is replaced by host tissue as the wound heals.

Hyaluronic acid is found in the synovial fluid of joints, vitreous body of the eye-ball, umbilical cord, blood vessels and cartilage. Because of its water retaining ability and protein linking ability it's being employed as a lubricant and shock absorbing agent. It's considered to be safe molecule when used inside the human body.

Therefore a formulation base with the combination of 1% sodium hyaluronate with the molecular weight of 4,000,000-4,500,000 can be formulated with whey derived growth factor extracts and 1% collagen along with silver sulfadiazine , The formulation prepared according to the present invention has demonstrated in the experimental studies to stimulate re-endotheliazation and faster curing of the injuries in the anterior chamber of the eye.

Procedure for preparation

The procedure for preparation of 1000 g of gel requires a wide mouthed container selected based on the amount of gel to be prepared. The container used have to be provided with a paddle type stirrer for the uniform mixing of the all the ingredients. For the 1000gms of the gel preparation one can use about 2.0 Lit volume containers.

To start with, all the excipients and preservatives have to be weighed as follows:

1. l.5gms of Methylparaben

2. 0.5gms of Propyl paraben

3. 50gms of Mannitol

4. 5ml of Glycerol

The above mentioned ingredients have to be mixed along with slow addition of WFI until all the solids get dispersed and dissolved completely.

After all the solids get dissolved, 20 g of carbopol have to be sieved through 40-50 mesh screen in to the solution and mixed slowly by stirring with the use of a paddle like mechanical stirrer. The carbopol should be allowed to swell. The solution has to be neutralized to pH7.0 by the addition of tri-ethanolamine.

The required amount of the swelled gel (approximately 900g) has to be sterilized by autoclaving.

Heat labile ingredients like Lysine and proteineous poly peptides have to be separately filtered. Sterlized Lysine (to give final cone., of 0.5%) containing the stock solution of Growth factor complex (to give final cone, of growth factor concentrate is about 0.5 mg/ml to 5 mg/ml) has to be slowly added and the contents have to be mixed gently to make a uniformly distributed active ingredients in the form of a gel. The above mentioned activity has to be most preferably carried out 15 under the LAF.

The gel has to be then placed in an autoclaved pressure vessel Using Nitrogen the gel has to be forced to flow out of the pressure vessel into 10ml syringes via a sterile piece of tubing.

It shall be noted that the process mentioned above is one of the preferred processes that can be adopted to enable the invention and shall not be considered to be limiting the invention.

Examples of the bioactive formulation within the scope of this invention are given below.

Specific examples of the Formulations:

All the examples of the formulation provided below may have the growth factors in the percentage range of 0.05 % to 1% if not specifically mentioned in the formulation.

Formulation 1

Methyl paraben 0.15%

Propyl paraben 0.05%

Mannitol 5%

Lysine HC1 0.52%

Glycerol 0.5%

Silver sulfadiazine 100 mg

All the ingredients are to be preferably made using a 2.0% Carbopol gel base.

Formulation 2
Methyl paraben 0.15%
Propyl paraben 0.5%
Mannitol 5%
Lysine HC1 0.52%
Glycerol 0.5%
Silver sulfadiazine 100mg

Formulation 3
Methyl paraben 0.15%
Propyl paraben 0.5%
Mannitol 5%
Lysine HC1 0.52%
Glycerol 0.5%
Povidone-Iodine 150 micrograms

Formulation 4

Methyl paraben 0.15%
Propyl paraben 0.5%
Trehalose 5%
Lysine HC1 0.52%
Glycerol 0.5%
Silver sulfa l00microgram

Formulation 5

Methyl paraben 0.15%
Propyl paraben 0.5%
Trehalose 5%
Lysine HC1 0.52%
Glycerol 0.5%
Povidine-Iodine 100mg

Formulation 6

Methyl paraben 0.15%
Propyl paraben 0.5%
Mannitol 5%
Lysine HC1 0.52%
Glycerol 0.5%
Silver sulfadiazine 100microgram
Sodium hyaluronate 0.5%

Formulation 7

Methyl paraben 0.15%
Propyl paraben 0.5%
Mannitol 5%
Lysine HC1 0.52%
Glycerol 0.5%
Silver sulfadiazine 100microgram
Zinc oxide 0.5%

Formulation 8

Methyl paraben 0.15%
Propyl paraben 0.5%
Trehalose 5%
Lysine HC1 0.52%
Glycerol 0.5%
Povidone-Iodine 100microgram
Zinc oxide 2%

Formulation 9

Methyl paraben 0.15%
Propyl paraben 0.5%
Trehalose 5%
Lysine HC1 0.52%
Glycerol 0.5%
Povidone-Iodine 100 micrograms
Zinc oxide 1%

Formulation 10

Growth factor concentrate 0.25%
Mannitol 5%
Lysine HC1 0.52%
Glycerol 0.5%
Silver sulfadiazine 100 microgram

Formulation 11

Growth factor concentrate 0.25%
Mannitol 5%
Lysine HC1 0.52%
Glycerol 0.5%
Silver sulfadiazine 100 microgram

Formulation 12

Growth factor concentrate 0.4%
Mannitol 5%
Lysine HC1 0.52%
Glycerol 0.5%
Silver sulfadiazine 100 microgram

Formulation 13

Growth factor concentrate 0.2%
Trehalose, 5%
Lysine HC1 0.52%
Glycerol 0.5%
Silver sulfadiazine 100 microgram

Formulations for internal wound healing

Methyl paraben 0.15%
Propyl paraben 0.5%
Mannitol 5%
Lysine HC1 0.52%
Glycerol 0.5%
Silver sulfadiazine 100microgram
Sodium hyaluronate 1%

What is claimed is:

1. A bioactive formulation comprising:

at least two growth factors;

at least one of an anti-oxidant and an immunomodulatory agent;

at least one formulation delivery system; and

optionally at least one anti-microbial agent.

2. The bioactive formulation for administering to a wound as claimed in claim 1, comprising:

at least two growth factors;

at least one of an anti-oxidant and an immunomodulatory agent;

at least one formulation delivery system; and

at least one anti-microbial agent.

3. The bioactive formulation as claimed in claim 2, wherein the growth factors are selected from the group consisting of IGF-I, IGF-II, PDGF, EOF, acidic FGF,
FGF, TGF-p, HGF and bioactive fragments or mimetics thereof.

4. The bioactive formulation as claimed in claim 2, wherein the growth factors are isolated from natural sources.

5. The bioactive formulation as claimed in claim 2, wherein the growth factors are isolated from a natural source selected from a group consisting of milk, whey and derivatives thereof.

6. The bioactive formulation as claimed in claim 2, wherein the growth factors are isolated from recombinant sources.

7. The bioactive formulation as claimed in claim 2, wherein the immunomodulatory agent is selected from the group consisting of lactoferrin and talactoferrin.

8. The bioactive formulation as claimed in claim 2, wherein the immunomodulatory agent is present in a range of 0.01% to 50%.

9. The bioactive formulation as claimed in claim 2, wherein the at least one antioxidant is selected from the group consisting of glutathione, ascorbic acid, tocopherol, Vitamins A, Beta-Carotene, Canthaxanthine, Co-Enzyme Q10 (ubiquinone/CoQlO), Selenium, Zinc, Manganese, L-Camosine, L-Arginine, Myricetin, Ascorbic acid, Tryptophan, Cysteine, Tyrosine, Homocysteine, butylated hydroxy anisole and derivatives thereof.

10, The bioactive formulation as claimed in claim 2, wherein the antimicrobial agent is selected from the group consisting of therapeutic antibodies, toxins, antibiotics, anti-virals, anti inflammatory agents, peptides, proteins and peptidomimetlcs.

11. The bioactive formulation as claimed in claim 2, wherein the antimicrobial agent is selected from the group consisting of silver sulfadiazine, povidine iodine, ofloxacin, metronidazole, ciprofloxacin, Levofloxacin, Clindamycin, Ceftriaxone, Cefotetan, Zosyn and derivatives thereof.

12. The bioactive formulation as claimed in claim 2, wherein the antimicrobial agent is silver sulfadiazine.

13. The bioactive formulation as claimed in claim 2, wherein the formulation delivery system comprises at least one of a polymer, a preservative, a healing promoting agent and a chemical species.

14. The bioactive formulation as claimed in claim 13, wherein the polymer is selected from the group consisting of Carbopol, Hyaluronic Acid, pluronic acid, cellulose, methyl cellulose, hydropropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and derivatives thereof.

15. The bioactive formulation as claimed in claim 13, wherein the preservative is selected from the group consisting of methyl paraben, propyl paraben, butyl
paraben, diazolidinyl urea and quatemium salts.

16. The bioactive formulation as claimed in claim 13, wherein the healing promoting agent is zinc oxide.

17. The bioactive formulation as claimed in claim 13. wherein the chemical species is selected from the group consisting of lysine, arginine, histidine, protamine, amino guanidine, zinc and magnesium.

18. The bioactive formulation as claimed in claim 2, wherein the formulation delivery system is a gel.

19. The bioactive formulation as claimed in claim 2, wherein the bioactive formulation is adsorbed on a non-aqueous matrix.

20. The bioactive formulation as claimed in claim 19, wherein the non-aqueous matrix is selected from the group consisting of collagen, Hyaluronic acid, gelatine and dextran.

21. The bioactive formulation as claimed in claim 2, wherein the formulation delivery system further comprises of liposomes.

22. The bioactive formulation as claimed in claim 2, wherein the formulation delivery system further comprises of nanoparticles.

23. The bioactive formulation for use in cosmetics as claimed in claim 1, comprising:

at least two growth factors;

at least one of an anti*oxidant and an immunomodulatory agent; and

at least one formulation delivery system;

24. The bioactive formulation as claimed in claim 23, wherein the growth factors are selected from the group consisting of IGF-I, IGF-II, PDGF, EOF, acidic FGF,
FGF, TGF-β HGF and bioactive fragments or mimetics thereof..

25. The bioactive formulation as claimed in claim 23, wherein the growth factors are isolated from natural sources.

26. The bioactive formulation as claimed in claim 23, wherein the growth factors are isolated from a natural source selected from the group consisting of milk, whey and derivatives thereof

27. The bioactive formulation as claimed in claim 23, wherein the growth factors are isolated from recombinant sources.

28. The bioactive formulation as claimed in claim 23, wherein the immunomodulatory agent is selected from the group consisting of lactoferrin and talactoferrin.

29. The bioactive formulation as claimed in claim 23, wherein the immunomodulatory agent is present in a range of 0.01% to 50%.

30. The bioactive formulation as claimed in claim 23, wherein the at least one antioxidant is selected from the group consisting of glutathione, ascorbic acid, tocopherol, Vitamins A, Beta-Carotene, Canthaxanthine, Co-Enzyme QIO (ubiquinone/CoQlO), Selenium, Zinc, Manganese, L-Camosine, L-Arginine, Myricetin, Ascorbic acid, Tryptophan, Cysteine, Tyrosine, Homocysteine, butylated hydroxy anisole and derivatives thereof.

31. The bioactive formulation as claimed in claim 23, wherein the formulation delivery system comprises at least one of a polymer, a preservative, a healing promoting agent and a chemical species.

32.The bioactive formulation as claimed in claim 31, wherein the polymer is selected from the group consisting of Carbopol, Hyaluronic Acid, pluronic acid, cellulose, methyl cellulose, hydropropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and derivatives thereof.

33. The bioactive formulation as claimed in claim 31, wherein the preservative is selected from the group consisting of methyl paraben, propyl paraben, butyl paraben, diazolidinyl urea and quatemium salts.

34. The bioactive formulation as claimed in claim 31, wherein the healing promoting agent is zinc oxide.

35. The bioactive formulation as claimed in claim 31, wherein the chemical species is selected from the group consisting of lysine, arginine, histidine, protamine, amino guanidine, zinc and magnesium,

36. The bioactive formulation as claimed in claim 23, wherein the formulation delivery system is a gel.

37. The bioactive formulation as claimed in claim 23, wherein the bioactive formulation is adsorbed on a non-aqueous matrix.

38. The bioactive formulation as claimed in claim 37, wherein the non-aqueous matrix is selected from the group consisting of collagen, Hyaluronic acid, gelatine and dextran,

39. The bioactive formulation as claimed in claim 23, wherein the formulation delivery system further comprises of liposomes.