DESC:FIELD OF INVENTION
The present invention relates to pharmaceutical compositions for treatment of hypertrophic scars. More specifically, the present invention relates to pharmaceutical compositions comprising Guanidine compound or its pharmaceutically acceptable salt, ester, prodrug. The invention also relates to method of preparation of such pharmaceutical compositions and treatment of hypertrophic scars.
BACKGROUND
Scar is a skin fibrotic condition that can be caused by minor insults to skin, such as acne or ear piercing, or by severe injuries such as burns. It has been reported that Scar is mainly formed due to excess deposition of collagen.
A hypertrophic scar is a thickened, wide, often raised scar that develops where skin is injured. Scars often forms during the wound healing process. A hypertrophic scar is the result of an abnormal response to a trauma or injury.
In certain people, body cells called myofibroblasts overexpress collagen during healing. This can happen simply as a result of a person’s skin type and healing tendencies. More commonly, overproduction of collagen occurs when a wound is infected or inflamed, under a great deal of tension or motion (such as in injuries over a joint), or left to heal without stitches.
The scars are a frequent complication of burn injuries, but can also form after piercings, cuts, or even acne. Hypertrophic scars are similar to keloid scars but tend to be milder and do not grow beyond the boundaries of the original skin injury.
The scars aren’t dangerous or life-threatening. They can be itchy and painful, but more often are simply a cosmetic issue. Some people seek treatment to minimize the appearance of the scar. There isn’t an officially established treatment regimen for hypertrophic scars, but a variety of treatments can help get rid of the scar more quickly.
Mechanical tension on a wound has been identified as a leading cause for hypertrophic scar formation.
When a normal wound heals, the body produces new collagen fibers at a rate which balances the breakdown of old collagen. Hypertrophic scars are red and thick and may be itchy or painful. They do not extend beyond the boundary of the original wound, but may continue to thicken for up to six months. They usually improve over one or two years, but may cause distress due to their appearance or the intensity of the itching; they can also restrict movement if they are located close to a joint. Some people have an inherited tendency to this type of scarring, for example, those with Ehlers–Danlos syndrome, classic type. It is not possible to completely prevent hypertrophic scars, so those with a history of them should inform their doctor or surgeon if they need surgery. Scar therapies may speed up the process of change from a hypertrophic scar to a flatter, paler one.
Excessive scarring can dramatically affect a patient’s quality of life, both physically and psychologically by causing pruritus, pain and contractures. A total of 100 million patients develop scars in the developed world alone each year as a result of 55 million elective operations and 25 million operations after trauma. The market for scar treatment products is anticipated to grow from an estimated US$ 16,048.7 million in 2015 to US$ 31,904.8 million by 2022 at CAGR of 10.3% during the forecast period.
Excess scar formation secondary to traumatic or surgical injuries can have devastating consequences, ranging from body disfigurement to organ dysfunction. Hypertrophic scars and keloids are skin fibrotic conditions that can be caused by minor insults to skin, such as acne or ear piercing, or by severe injuries such as burns. Differences between keloids, hypertrophic scars and normal scars include distinct scar appearance, histologic morphology and cellular function in response to growth factors. Recent advances in our understanding of the wound healing process reveal possible causes for hypertrophic scars and keloids. This information might assist in the development of efficacious treatment for hypertrophic scar and keloid formation (Tai-LanTuan etal, Molecular Medicine Today, 4(1), 19-24, January 1998.)
Before treating a hypertrophic scar, it’s important to differentiate it from a similar type of scar called a keloid. Keloid scars are smooth, hard, benign growths that also form when scar tissue grows excessively. Even a doctor may have difficulty telling the difference between a hypertrophic scar and a keloid, but it’s important to distinguish them because the treatment may be different.
In general, hypertrophic scars; are raised, but rarely more than 4 millimeters above the skin; are red or pink in color; can develop anywhere on the body. On the other hand, keloids usually are raised more than 4 millimeters from the skin; grow beyond the boundaries of the original incision or wound; are pink to purple in color; evolve and grow over time; form on the earlobes, shoulders, cheeks, and chest above the sternum. Both scars tend to occur more commonly in darker skin types.
The following preventive measures are suggested cleaning and caring for the wound properly, such as by applying wound dressings to prevent infection, using silicone sheeting after a surgery; injecting a corticosteroid after a surgery.
Granulation tissue begins to form in the wound site and fills the site approximately five days after wound induction. Granulation tissue contains new collagen, fibroblasts, new blood vessels and inflammatory cells, especially macrophages (E. Rubin and J. L. Farber, Pathology, Lippincott, publ., pp. 85-95 (1994)). After seven to ten days, the wound has regained only 10% of the tissue's original strength.
Secondary healing causes a greater inflammatory response and more granulation tissue is formed. In addition, contraction of the wound, resulting from contraction of the fibroblasts of the granulation tissue, brings the edges of the wound together to speed the healing process, but sometimes contributes to disfiguring and debilitating scars. Additionally, excessive deposition of extracellular matrix leads to the formation of keloids, or hypertrophic scars, which are irregularly-shaped, elevated scars that tend toward progressive enlargement.
Angiogenesis is generally believed to be a necessary feature of repair (Kovacs, E. et al., Fibrogenic cytokines and connective tissue production, FASEB J., 8:854-861 (1994). Numerous growth factors and cytokines, secreted first by platelets in response to coagulation and then by macrophages in response to hypoxia and lactic acidosis, stimulate angiogenesis (Shah, M. et al., The Lancet, 339:213-214 (1992)). Angiogenesis generally becomes visible at a microscopic level about four days after injury but begins two or three days earlier when new capillaries sprout out of preexisting venules and grow toward the injury in response to chemoattractants released by platelets and macrophages. In primarily closed wounds, sprouting vessels soon meet counterparts migrating from the other side of the wound and blood flow across the wound is reestablished. In unclosed wounds, or those not well closed, the new capillaries fuse only with neighbors migrating in the same direction, and a large amount of granulation tissue is formed instead.
In normal wound healing, the tissue surrounding a wound undergoes a degree of hypoxia and a concomitant increase in secretion of vascular endothelial growth factor, or VEGF, typically occurring one to two days following injury (Brown, L. F. et al. Expression of VPF (VEGF) by epidermal keratinocytes during wound healing, J. Exp. Med., 176:1375-79 (1992)). VEGF stimulates the rapid proliferation of blood vessel endothelial cells which results in the formation of densely sprouting capillaries. This rapid hypoxia-induced, VEGF-driven capillary formation stimulates infiltration of inflammatory cells and leads eventually to scarring.
While inflammation causes scarring, inflammation is also beneficial. Inflammatory cells release growth signals and lytic enzymes that are very important for repair. In fact, patients who receive anti-inflammatory agents often experience impaired healing due to inadequate inflammation at the site of a wound.
An important aspect of wound repair is the time involved. The rate at which a wound heals has implications for the prevention of infection and improvement of the overall health of the patient. Rapid, even healing without excessive contraction is a desirable result from a medical and cosmetic standpoint.
Furthermore, it is a recognized clinical phenomenon that surgery in a tumor patient may lead to tumor progression if the site of the surgical incision is in proximity to the site of the tumor. In addition, the surgical incisions show high susceptibility to metastatic implantation. (Murthy et al., Cancer, 64:2035-2044 (1989); Murthy et al., Cancer, 68:1724-1730 (1991); Schackert, H. K. et al., Int. J. Cancer, 44:177-81 (1989)). The stimulatory effect of wounds on tumors is manifested as accelerated growth of residual tumor near the site of surgical intervention, as well as an increased probability of metastatic implantation at the site of surgery. Furthermore, wounds located at the site of a tumor regularly fail to heal (Gatenby, R. A. et al., Suppression of wound healing in tumor bearing animals, Cancer Research, 50:7997-8001 (1990)). Persistent wounds that continuously accelerate tumor progression may be a frequent side effect of surgical interventions associated with cancer therapy. Therefore, deciding whether to operate on a tumor patient is often a difficult decision in which the benefits of surgery must be compared to the risks of worsening a cancer patient's overall condition.
The Guanidine hydrochloride was found to be potential agent for topical treatment of hypertropic scars (Narasimha Murthy et al, AAPS Annual meeting and Exposition, Chicago, (2012)).
US Patent No. 4,163,800 assigned to Procter and Gamble Co covers dermatological combination composition of benzoyl peroxide along with compound reducing skin irritation, wherein such compound were selected from the group consisting of guanidine hydrochloride, guanidine sulfate, guanidine carbonate, and guanidine phosphate.
Chinese application CN105535015 assigned to Shanxi Haibo Beima Bio-Tech Co Ltd covers polyhexamethylene guanidine hydrochloride antipruritic spray water composition.
Commercially following treatment are available for the treatment of scars.
1. Silicon gel dressings: Silicone sheets are reported to work by increasing the temperature, hydration, and perhaps the oxygen tension of the occluded scar, causing it to soften and flatten. It is not effective after complete formation of scar.
2. Mederma: The main ingredient of Mederma cream is extract of allium cepa. However, there is no reports supporting its activity in hypertrophic scars.
3. Corticosteroid treatments: Corticosteroid injections are considered a first-line treatment for hypertrophic scars. Injecting a steroid into the scar every six weeks may help flatten and soften the scar. There’s a limit to the number of times this can be performed, however, as the steroids may also weaken normal tissue around the scar.
4. Laser therapy: Laser therapy is more effective in newly formed scars than in older scars. The lasers work by burning and flattening elevated scars. They also target the red and pink pigments in the scars to lighten them.
5. Bleomycin: Bleomycin is a metabolite of a strain of soil bacteria. It’s shown promising results when injected directly into a hypertrophic scar. It may help improve the scar appearance and relieve itching and pain. More clinical data is needed to confirm its efficacy.
6. Cryotherapy: In cryotherapy, a doctor or dermatologist freezes the scar tissue with liquid nitrogen to help flatten it. Cryotherapy has been shown to be successful, safe, nontoxic, and well-tolerated in a number of small studies.
7. Surgery: After waiting at least a year, a hypertrophic scar can be excised, or cut out, and closed again with stitches. This treatment tries to re-heal the injury while eliminating the issues that may have caused the scar in the first place, such as infection, inflammation, or tension.
8. Pressure and massage: One of the cheapest and most effective ways to help heal the scar is to apply pressure and massage to the area. Bandages or tape can be used to apply pressure. Over time, it can help weaken the scar tissue and improve the appearance of the scar.
9. Bio Oil: Bio Oil is marketed as a treatment for all different types of scars. It can be purchased at many beauty supply stores. Clinical trials for Bio Oil showed positive results. However, the sample sizes were small. More research is needed to confirm that Bio Oil can effectively reduce the appearance of hypertrophic scars.
All the treatments have some or the other disadvantage like Laser Treatment, Cryotherapy and Surgery are costly treatment options whereas others like pressure and massage may be painful and do not completely treat scar. The corticosteriods and belomycin use has their own limitations and disadvantage being steroid and antimicrobial. Thus, there is a need to develop new and improved topical composition for treatment of scars especially hypertrophic scar.
OBJECTIVE OF INVENTION
An object of the present invention is to provide a composition for treatment of scar.
Yet another object of the invention is to provide a topical composition for treatment of scar.
Another object of the present invention is to provide a composition for treatment of hypertrophic scar.
Another object of the present invention is to provide the topical composition for treatment of hypertrophic scar.
Another object of the present invention is providing the method for preparation of topical composition.
Another object of the present invention is to provide the topical composition of Guanidine for treatment of scar.
Yet another object of the present invention is to provide the topical composition of Guanidine for treatment of hypertrophic scar.
Another object of the present invention is to provide the method for preparation of topical composition of Guanidine.
SUMMARY OF THE INVENTION
One of the embodiment of the present invention provides topical composition for treating a patient having a wound.
Another embodiment of the present invention provides topical composition of Guanidine or its pharmaceutically acceptable salt, to minimize scarring and to accelerate wound healing.
In a preferred feature, the present invention provides a pharmaceutical composition comprising: Guanidine compound or its pharmaceutically acceptable salt, ester, prodrug; and one or more pharmaceutically acceptable excipients.
In another preferred feature, the present invention provides a pharmaceutical composition consisting essentially of: Guanidine compound or its pharmaceutically acceptable salt, ester, prodrug; and one or more pharmaceutically acceptable excipients.
In yet another preferred feature, the present invention provides a pharmaceutical composition consisting of: Guanidine compound or its pharmaceutically acceptable salt, ester, prodrug; and one or more pharmaceutically acceptable excipients.
Another embodiment of the invention is method of manufacture of topical composition for treatment of scar.
Another embodiment of the present invention is method of manufacture of topical composition of Guanidine for treatment of scar.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 Shows process of denaturation of Collagen.
FIG. 2 Shows Percentage of Collagen denatured using Guanidine HCl at various concentration.
FIG. 3 DSC Thermogram showing denaturation of collagen by guanidine hydrochloride.
FIG. 4 UV Spectra showing denaturation of collagen by guanidine hydrochloride.
FIG. 5 Circular Dichroism Spectra showing denaturation of collagen by guanidine hydrochloride.
FIG.6 Shows In-vitro cell line studies indicating inhibition of collagen by Guanidine HCl.
FIG. 7 Shows Graph indicating transport of Guanidine HCl across human scar skin.
FIG. 8 Shows Graph for Cell Proliferation Assay
FIG. 9 Shows Collagen I and III Content
FIG. 10 Shows Collagen I and III Content
FIG. 11 Shows Morphology of scar
FIG. 12 Shows Morphology of scar
DETAILED DESCRIPTION OF THE INVENTION
Before describing the present invention, it is to be understood that this invention is not limited to specific pharmacologically active carriers, formulation types, treatments, so forth, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
The inventors of this invention have surprisingly found that Guanidine or its pharmaceutically acceptable salt, especially Guanidine Hydrochloride facilitates wound healing by promoting rapid healing with minimal scarring. Guanidine presumably produces these beneficial effects by inhibiting collagen expression that contributes to the scarring associated with wound healing. This mechanism of action also contributes to rapid, even healing of wounds in tumor patients, thus reducing the likelihood of tumor proliferation or metastatic implantation at the site of surgery or other wounding.
DEFINITIONS:
The term "therapeutically effective amount" or "effective amount" is used herein to denote any amount of a topical formulation which will cause a substantial improvement in a disease condition when applied to the affected area. A single application can be sufficient, or the formulation can be applied repeatedly over a period of time. The amount will vary with the condition being treated, the stage of advancement of the condition, and the type and concentration of formulation applied. In the present invention, Guanidine or its pharmaceutically acceptable salts is present in the concentration range of about 0.01% to about 20 %, preferably from about 0.5 % to about 15 %, more preferably from about 0.1 % to about 10 % w/w of the total composition.
The terms "drug" and "pharmaceutical" are also used interchangeably to refer to a pharmacologically active substance or composition.
The term "topical composition" or "topical formulation" means a composition in which the drug may be placed for direct application to a skin surface and from which an effective amount of the drug is released. Such formulations may include but are not limited to plasters, ointment, paste, cream, solution, suspension, emulsion, lotion, liniment, jelly, gel, poultice, foam, collodion, paints, powder, transdermal patch, and any combination thereof or any other dosage form suitable for topical application and the like. In some aspects, such formulations may be applied to the skin in an unoccluded form with/without additional backing, structures or devices.
The term "skin" or "skin surface" is meant to include the outer skin of a subject comprising one or more of epidermal layers to which a drug composition may be administered.
The term “wound” of skin as used herein means an injury to living tissue caused by a cut, blow, or other impact, typically one in which the skin is cut or broken and has general meaning as defined in common medical practice incorporated herein via such reference.
The term "treating" or "treatment" of a state, disorder or condition as used herein means treating scar formed.
The term “body weight” is used colloquially and in the biological and medical sciences to refer to a person's mass or weight. Body weight is measured in kilograms.
The term “pharmaceutically acceptable salt” as used herein is meant to include salts of the compounds of the invention which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the alts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 66: 1-19 (1977)). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compounds in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents. In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds or complexes described herein readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment.
Guanidine is found in urine as normal product of a protein metabolism. Guanidine Hydrochloride (GHCL) is crystalline powder freely soluble in water and phosphate buffered saline and alcohol and has melting point of 180°C-185°C. The structure of Guanidine Hydrochloride is as depicted below

Guanidine is approved by US FDA for the reduction of the symptoms of muscle weakness and easy fatigability associated with the myasthenic syndrome of Eaton-Lambert. It is not indicated for treating myasthenia gravis. The Eaton-Lambert syndrome is ordinarily differentiated from myasthenia gravis by the usual association of the syndrome with small cell carcinoma of the lung, but myography may be necessary to make the diagnosis, wherein Guanidine hydrochloride appears to acts by enhancing the release of acetylcholine following a nerve impulse. It also appears to slow the rates of depolarization and repolarization of muscle cell membranes. Guanidine hydrochloride is a strong chaotropic agent and is one of the strongest denaturants used in physiochemical studies of protein folding to disrupt the hydrogen binding in the protein molecule. The inventors of this invention have postulated that this property of denaturation by Guanidine can be used in treatment of scars.
A method of preparation of a Guanidine hydrochloride is provided in article Synlett, 2014, 25, 1302-1306 (K. Tsubokura, T. Iwata, M. Taichi, A. Kurbangalieva, K. Fukase, Y. Nakao, K. Tanaka) using efficient guanylation of various amines with cyanamide proceeds in the presence of catalytic amounts of scandium(III) triflate under mild conditions in water without using pre activated guanylation reagents and J. Org. Chem., 72, 6763-6767, 2007 (Q. Li, S. Wang, S. Zhou, G. Yang, X. Zhu, Y. Liu) by guanylation of both aromatic and secondary amines with a high activity under mild conditions using simple lanthanide amides as highly efficient catalyst; these catalysts are compatible with a wide range of solvents and substrates.
Kerl H. et al. in Formation and treatment of pathologic scars--clinical and micromorphologic investigations, Z Hautkr. 1981 Mar 1; 56(5):282-300 discloses use of results of treatment with Calmurid and Calmurid-HC in patients with hypertrophic scars and keloids of various causes. The article further evaluates results of treating hypertrophic scars with urea preparations.
One aspect of the present invention is to provide topical composition for treating a patient having a wound by administering Guanidine or its pharmaceutically acceptable salt thereof, specifically Guanidine hydrochloride, in an amount sufficient to reduce scarring and/or to accelerate wound healing. Determination of the reduction of scarring and/or the acceleration of healing may be performed by a variety of methods including, but not limited to, visual observation, measurement of vessel density at the site of the wound, e.g., by magnetic resonance imaging, measurement of the amount and/or rate at which granulation tissue is formed, and measurement of skin tensile strength at the site of the wound.
The Guanidine or its pharmaceutically acceptable salts are preferably combined with a pharmaceutically acceptable carrier and administered to a patient topically at the site of scar or wound. The carrier is preferably one that is compatible with Guanidine or its pharmaceutically acceptable salts to form a topical composition.
The amount of Guanidine or its pharmaceutically acceptable salts that is administered to a patient to reduce scarring or accelerate the rate of wound healing is an amount that is sufficient to denature the collagen at wound site or scar site to reduce the amount or rate of granulation tissue formation at a wound site, or that is sufficient to reduce vascularization, and particularly vessel density, at a wound site. A preferred dosage range is 1 to 100 µg/kg body weight. A more preferred dosage range, however, is 1 µg/kg to 50 µg/kg body weight, and most preferred is a dosage in the range of 1 µg/kg to 25 µg/kg. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the age, body weight, general health, sex, diet, and severity of the wound. The amount of Guanidine hydrochloride present in dosage form is selected from about 0.01% to about 20 %, preferably from about 0.5 % to about 15 %, more preferably from about 0.1 % to about 10 %. The topical dosage form is applied one to four times daily preferably once daily for a 2-6 month preferably for 3-4 months.
Guanidine or its pharmaceutical salts treatment inhibits scarring and accelerates healing of wounds in various types of tissues and is appropriate for wounds of various depths. Scarring at the level of the epidermis as well as internal scarring is reduced by the method of the present invention. A scar as used herein is an irregularity of the skin or other tissue formed from connective tissue replacement of tissue, especially tissue damaged by a wound process. An adhesion may also be considered a scar in this context. A wound as used herein is injury or damage wherein the skin or other tissue is adversely affected, particularly wherein the skin or other tissue is torn, pierced, cut, or broken.
Additionally, the present invention may be used to treat reperfusion injuries due to surgical wounds or traumatized wounds. Specifically, restenosis, wherein scarring within a blood vessel leads to stricture of the vessel lumen, is advantageously treated by the method of the present invention. Further, it may be used in minimizing the likelihood of formation of adhesions, such as the type which sometimes occurs as a result of abdominal surgery. Clearly, the method may be used in specific types of surgery, such as plastic or cosmetic surgery, implantation, reconstruction, transplantation, bypass operations, and balloon angioplasty where improved wound healing is critical and scarring is particularly problematic.
As stated above, the stimulatory effect of wounds on tumors is a recognized clinical phenomenon that is manifested as accelerated growth of the residual tumor near the site of surgical intervention, as well as an increased probability of metastatic implantation at the site of surgery. The present invention also includes administration of Guanidine or its pharmaceutically acceptable salts to surgery patients with tumors in order to facilitate wound healing and to minimize metastatic implantation and tumor proliferation. Treatment for wound healing in surgery patients with tumors includes prevention or reduction of the likelihood of occurrence, and reduction of any tumor that has moved to the wound site.
Clearly, administration of Guanidine or its pharmaceutically acceptable salts pre-surgery to reduce scarring and accelerate wound healing has great utility and will favorably impact the medical community. Similarly, the administration of Guanidine or its pharmaceutically acceptable salts to a patient having a wound resulting from unexpected injury has great utility in facilitating healing of the wound.
Without limitation to a particular theory, it can be hypothesized that Guanidine or its pharmaceutically acceptable salts, and specifically Guanidine hydrochloride, plays an important role in allowing wounds to heal at an accelerated rate and with minimal scarring because it interferes with the excess deposition of Collagen that results in tissue granulation and scarring, but not with physiological blood vessel repair processes which are necessary for the wound to heal. Collagen is believed to perform a major role in restoring strength and remodeling scar tissue, the precise way in which the collagen molecule is involved is still unclear (JOHN W. MADDEN, M.D., ERLE E. PEACOCK, JR., M.D., Ann. Surg. 174 (3), 511-518, Sep 1971.) The most important biological mechanisms involved in wound healing, the main agents that modify the healing process and the physiological and pharmacological role of collagen. Putative mechanisms of collagen in wound repair are described with particular emphasis on haemostatic effect, interaction with platelets and fibronectin, properties of increasing fluid exudate and its cellular component (macrophages) and the "scaffold" role for fibroblastic proliferation. Experimental and clinical data clearly suggest that the potential use of collagen in wound repair and its main therapeutical applications: treatment chronic leg ulcers and pressure sores, burns, urological surgery, gynaecological surgery, dentistry and oral surgery, reconstructive surgery, abdominal and vascular surgery, orthopaedy (Mian M, Beghè F, Mian E, International Journal of Tissue Reactions, 14 (1-9)). Because Guanidine or its pharmaceutical acceptable salt allows denaturation of excess of Collagen, it is an extremely useful compound for the beneficial treatment of wounds.
The denaturation of Collagen was studied in-vitro and was measured by various analytical techniques including but not limited to Differential Scanning Calorimetry (DSC), Ultraviolet (UV) Spectroscopy, Circular Dichroism (CD).
Another aspect of the present invention is topical composition for treatment of hypertrophic scar. The topical dosage forms include but are not limited to gel, solution, creams, lotion, foam, aerosol, transdermal patch, dispersion, emulsion mousse, patch, pomade, powder, pump spray, solid, solution, stick or towelette. Suitable emulsions include oil-in-water, water-in-oil, and water-in-silicone emulsions. The compositions described herein may comprise, consist or consist essentially of the specified ingredients or specific ones thereof. Further, it will be understood that the formulations of the invention may be widely varied, as regards the absolute amounts and relative proportions thereof, in relation to specific examples, and illustrative compositions.
According to another aspect of invention the topical formulation comprises active along with surfactant-emulsifier, one or more solvent(s) and/or pro-penetrating agent(s), for the active agent; one or more gelling agent(s), antioxidants, emollients, moisturiser, humectants, preservatives, pH adjusting agent, chelating agents, and water.
In a preferred embodiment, the process of the present invention also includes adding to the mixture an additional component or additional components that are suitable for rendering the composition of the present invention more cosmetically or aesthetically acceptable or to provide the composition with additional usage benefits. The addition can be done at any stage although it is generally most simple to add additional components before or during the heating of the mixture.
The gelling agent according to the present invention are but not limited to acacia, alginic acid, bentonite, Carbopols (now known as carbomers), carboxymethyl cellulose, ethylcellulose, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, magnesium aluminum silicate (Veegum), methyl cellulose, hydroxylpropyl methyl cellulose, poloxamers (Pluronics), polyvinyl alcohol, sodium alginate, tragacanth, and xanthan gum.
Additional components include any pharmaceutically acceptable or cosmetically acceptable ingredients such as those found in the The Cosmetic, Toiletry and Fragrance Association of New Zealand (CTFA)’s International Cosmetic Ingredient Dictionary and Handbook, 8th edition, edited by Wenninger and Canterbery (The Cosmetic, Toiletry, and Fragrance Association, Inc., Washington, D.C., 2000). Representative examples include but are not limited to anti-irritants, anti-oxidants, buffering agents, bulking agents, colorants, deodorants, dermatological active ingredients, diluents, emollients, humectants, moisturizers, occlusive agents, penetration enhancers and skin penetration enhancers, perfuming agents, pH adjusting agents, preservatives, protectants, softeners, solubilizers, stearically stabilizing substances, sun screening agents, sunless tanning agents, surfactants and vitamins.
Suitable anti-irritants that can be used in preparing a composition of the present invention include, but are not limited to, steroidal and non-steroidal anti-inflammatory agents or other materials such as aloe vera, chamomile, alpha-bisabolol, cola nitida extract, green tea extract, tea tree oil, licorice extract, allantoin, caffeine or other xanthines, glycyrrhizic acid and its derivatives. Suitable anti-oxidants that can be used in preparing a composition of the present invention include, but are not limited to, Sodium metabisulfite, ascorbic acid (vitamin C) and its salts, ascorbyl esters of fatty acids, ascorbic acid derivatives (e.g., magnesium ascorbyl phosphate, sodium ascorbyl phosphate, ascorbyl sorbate), tocopherol (vitamin E), tocopherol sorbate, tocopherol acetate, tocopheryl acetate other esters of tocopherol, Butylated hydroxy toluene, butylated hydroxy benzoic acids and their salts, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (commercially available under the tradename Trolox®), gallic acid and its alkyl esters, especially propyl gallate, uric acid and its salts and alkyl esters, sorbic acid and its salts, lipoic acid, amines (e.g., N,N-diethylhydroxylamine, amino-guanidine), sulfhydryl compounds (e.g., glutathione), dihydroxy fumaric acid and its salts, lycine pidolate, arginine pilolate, nordihydroguaiaretic acid, bioflavonoids, curcumin, lysine, methionine, proline, superoxide dismutase, silymarin, tea extracts, grape skin/seed extracts, melanin, and rosemary extracts. Suitable emulsifier includes non-ionic surfactants like the polysorbates, which are mixtures of partial esters of sorbitol and its mono- and dianhydrides, typically condensed with approximately 20 mol of ethylene oxide; polyethyoxylated alkyl ethers and esters, in which the alkyl chain can be either saturated, unsaturated, branched or linear; polyethoxylated alkyl phenols, in which the hydrophobic group normally octyl or nonylphenol; and poloxamers, polyoxyethylene-polyoxypropylene block copolmyers, in which the polyoxypropylene chain acts as the hydrophobic moiety. Some commercially available non-ionic surfactants are Brij 99, Brij 78, polyoxyl 40 stearate and polysorbate 80. Brij 99 and Brij 78 are polyethylene glycol (PEG), fatty alcohol ethers. Polyoxyl 40 stearate is a mixture of mono and distearate esters of polyoxyethylene and of free polyoxyethylene. Polysorbate 80 is polyoxyethylene (20) sorbitan mono-oleate. Oil/water emulsifiers include, for example, stearyl alcohol, polyoxyethylene stearates (such as, for example, "Myrj"), polyoxyethylene polyoxylpropylene glycol, complex emulsifiers (e.g., "Amphoterin"), and sorbitan fatty acid esters (such as, for example, "Span") or carboxyvinyl polymers (e.g., "Carbopol"). Glyceryl Stearate (and) PEG 100 Stearate, Glycerol monostearate, Polyoxyl 20 cetostearyl ether, sorbitan stearate, stearic acid. Co-emulsifier selected from the group consisting of: cetyl alcohol, stearyl alcohol and mixtures thereof. Suitable pro-penetrating agent or co-solvents includes but not limited to polyol ethers include diethylene glycol monoethyl ether (ethoxydiglycol) (Transcutol®, Gattefosse Corporation, Paramus, N.J.), propylene glycol, polyethylene glycol (PEG) like PEG 400.
Suitable dermatological inactive ingredients that can be used in preparing a composition of the present invention include, but are not limited to, jojoba oil and aromatic oils such as methyl salicylate, wintergreen, peppermint oil, bay oil, eucalyptus oil and citrus oils, as well as ammonium phenolsulfonate, bismuth subgallate, zinc phenolsulfonate and zinc salicylate.
Suitable emollients that can be used in preparing a composition of the present invention include, but are not limited to, ammonium lactate, dodecane, squalane, cholesterol, Cetostearyl alcohol, White Soft Paraffin, glycerol, White beeswax, Lanolin alcohol, isohexadecane, isononyl isononanoate, PPG Ethers, petrolatum, lanolin, dimethicone, Light liquid paraffin, mineral oil, white mineral oil, polyethylene glycols, safflower oil, castor oil, coconut oil, cottonseed oil, palm kernel oil, palm oil, peanut oil, soybean oil, polyol carboxylic acid esters, derivatives thereof and mixtures thereof. Suitable humectants and moisturizers that can be used in preparing a composition of the present invention include, but are not limited to ammonium lactate, dimethicone, urea, guanidine, glycolic acid and glycolate salts (e.g. ammonium salt and quaternary alkyl ammonium salt), aloe vera in any of its variety of forms (e.g., aloe vera gel), allantoin, urazole, polyhydroxy alcohols such as sorbitol, glycerol, glycerin, tocopherol acetate, hexanetriol, propylene glycol, butylene glycol, hexylene glycol and the like, polyethylene glycols, sugars and starches, sugar and starch derivatives (e.g., alkoxylated glucose), hyaluronic acid, lactamide monoethanolamine, acetamide monoethanolamine and any combination thereof. Suitable occlusive agents that can be used in preparing a composition of the present invention include, but are not limited to, petrolatum, mineral oil, beeswax, silicone oil, lanolin and oil-soluble lanolin derivatives, saturated and unsaturated fatty alcohols such as behenyl alcohol, hydrocarbons such as squalane, and various animal and vegetable oils such as almond oil, peanut oil, wheat germ oil, linseed oil, jojoba oil, oil of apricot pits, walnuts, palm nuts, pistachio nuts, sesame seeds, rapeseed, cade oil, corn oil, peach pit oil, poppy seed oil, pine oil, castor oil, soybean oil, avocado oil, safflower oil, coconut oil, hazelnut oil, olive oil, grape seed oil and sunflower seed oil. Suitable penetration enhancers that can be used in preparing a composition of the present invention include, but are not limited to, dimethylsulfoxide (DMSO), dimethyl formamide (DMF), allantoin, urazole, N,N-dimethylacetamide (DMA), decylmethylsulfoxide (C10 MSO), polyethylene glycol monolaurate (PEGML), propylene glycol (PG), propylene glycol monolaurate (PGML), glycerol monolaurate (GML), polyethylene glycol (PEG) 400, lecithin, the 1-substituted azacycloheptan-2-ones, particularly 1-n-dodecylcyclazacycloheptan-2-one, alcohols, and the like. Suitable penetration enhancers include vegetable oils including, for example, safflower oil, cottonseed oil and corn oil. Suitable pH adjusting agents that can be used in preparing a composition of the present invention include, but are not limited to, one or more adipic acids, triethanolamine, Sodium dihydrogen phosphate dihydrate, glycines, citric acids, calcium hydroxides, magnesium aluminometasilicates, buffers or any combinations thereof. Suitable preservatives that can be used in preparing a composition of the present invention include, but are not limited to, one or more alkanols, disodium EDTA (ethylenediamine tetraacetate), EDTA salts, EDTA fatty acid conjugates, isothiazolinone, parabens such as methylparaben and propylparaben, sorbates, urea derivatives such as diazolindinyl urea, or any combinations thereof. Suitable softeners that can be used in preparing a composition of the present invention include, but are not limited to, ammonium lactate, dodecane, squalane, cholesterol paraffin, isohexadecane, isononyl isononanoate, PPG Ethers, petrolatum, lanolin, safflower oil, castor oil, coconut oil, cottonseed oil, palm kernel oil, palm oil, peanut oil, soybean oil, polyol carboxylic acid esters, derivatives thereof and mixtures thereof. Suitable solubilizers that can be used in preparing a composition of the present invention include, but are not limited to, complex-forming solubilizers such as citric acid, ethylenediamine-tetraacetate, diethylene glycol monoethyl ether, sodium meta-phosphate, succinic acid, urea, cyclodextrin, polyvinylpyrrolidone, diethylammonium-ortho-benzoate, and micelle-forming solubilizers such as Tweens and Spans, e.g., Tween 80. Other solubilizers that are usable for the compositions of the present invention are, for example, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene n-alkyl ethers, n-alkyl amine n-oxides, poloxamers, organic solvents, phospholipids and cyclodextrines. Suitable stearically stabilizing substances that can be used in preparing a composition of the present invention include, but are not limited to, poloxamers and poloxamines (block copolymers of polyoxyethylene and polyoxypropylene), ethoxylated esters of sorbitan fatty acids, in particular polysorbates (such as Polysorbate 80 or Tween 80), ethoxylated mono- and diglycerides, ethoxylated lipids, ethoxylated fatty alcohols or fatty acids, and charged ionic stabilizers and peptizing agents, such as diacetyl phosphates, phosphatidyl glycerol, as well as saturated and unsaturated fatty acids, sodium cholate, sodium glycocholate, sodium taurocholate or mixtures thereof, amino acids or peptizing agents such as sodium citrate (Lucks Int. J. Pharmaceutics, 63: 183-188(1990)). Chelating agents that can be used in preparing a composition of the present invention include, but are not limited to, Disodium edetate, HBED (N,N'-bis(2-hydroxbenzyl)ethylenediamine-N—N-diacetic acid), HBPD (N,N'-Bis(2-hydroxybenyzyl)propylene-1,3-diamine-N,N'-diacetic acid. Suitable surfactants that can be used in preparing a composition of the present invention include, but are not limited to, polyethylene glycol hexadecyl ether, ethoxylated esters of sorbitan fatty acids, block polymers and block copolymers (such as poloxamers and poloxamines), polyglycerol ethers and polyglycerol esters, lecithins of various origins (such as egg lecithin or soya lecithin), chemically modified lecithins (such as hydrogenated lecithins), as well as phospholipids and sphingolipids, mixtures of lecithins with phospholipids, sterols (such as cholesterol and its derivatives, specifically stigmasterol), esters and ethers of sugars or of sugar alcohols with fatty acids or fatty alcohols (such as saccharose monostearate, white beeswax).
Suitable perfumes that can be used in preparing a composition of the present invention include, but are not limited to lavender, raspberry, Pineapple Lily, Rose, Vanilla, Grapefruit, Peppermint Oil, sweet orange, jasmine.
The compositions of the invention are be administered topically. In general, the invention is included in a cosmetically acceptable vehicle. Examples of cosmetically acceptable vehicles suitable for all embodiments of the present invention include, but are not limited to, water, glycerin, various alcohols such as ethanol, propyl alcohol, vegetable oil, white mineral oil, mineral oil, silicone oils, fatty ethers, fatty esters, fatty alcohols, glycols, polyglycols, Light liquid paraffin, lanolin alcohol, Sorbitan stearate, stearic acid or any combinations thereof. Methods of formulation are well known in the art and are disclosed, for example, in Remington's Pharmaceutical Sciences, Gennaro, Mack Publishing Co., Easton Pa., 1990, which is incorporated herein by reference
Another aspect of the present invention is an article of manufacture, such as a kit, and a method for making the article of manufacture. The article includes a pharmaceutical composition comprising a Guanidine or its pharmaceutically acceptable salts, and particularly Guanidine hydrochloride, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be placed in a suitable container, as is well known in the art. Also included are instructions for treatment of patients according to the methods of the present invention.
It will be understood that various modifications may be made to the embodiments disclosed herein. Therefore, the description should not be construed as limiting, but merely as exemplifications of preferred embodiments. For example, the functions described above and implemented as the best mode for operating the present invention are for illustration purposes only. Other arrangements and methods may be implemented by those skilled in the art without departing from the scope and spirit of this invention.

EXAMPLES

Example 1: Ointment Composition
Table 1: Ointment Composition
Sr. No. Ingredient Concentration (% w/w)
1 Guanidine hydrochloride 5.00
2 Mineral oil 6.00
3 Cetostearyl alcohol 7.20
4 Polyoxyl 20 cetostearyl ether 1.80
5 White Soft Paraffin 15.00
6 Glycerol 5.00
7 Purified water q.s. to 100.0

Manufacturing Process: The water quantity was divided into two part, one part was used for preparation of aqueous phase while other part was used for preparation of drug solution
i) Preparation of oil phase: The weighted quantity of mineral oil, cetostearyl alcohol, Polyoxyl 20 cetostearyl ether and white soft paraffin were melted together and heated to 65-70 oC in a water bath. The molten mixture was stirred at 600 ± 100 rpm to get a clear phase.
ii) Preparation of aqueous phase: The purified water was heated in main vessel (SS Vessel) to 65-70oC. The weighed quantity of glycerol was then added to the said vessel under stirring.
iii) Emulsification: The molten oil phase was added to main vessel containing aqueous phase under vigorous stirring at 1000 ± 100 rpm for 30 min at 65-70 oC followed by slow stirring till temperature reaches 37oC.
iv) Preparation of drug solution: The weighted quantity of Guanidine hydrochloride was dissolved in part of purified water under stirring at room temperature.
v) Ointment Preparation: The drug solution was added to emulsified mixture of step (iii) in main vessel at 37oC under stirring at 500 ± 100 rpm. The ointment thus formed was further stirred for 20-25 min. The weight of bulk was adjusted using purified water and contents of vessel were stirred for 10 min.

Example 2: Cream Composition
Table 2: Cream Composition
Sr. No. Ingredients A
(% w/w) B
(% w/w) C
(% w/w)
1 Guanidine hydrochloride 3.00 5.00 7.50
2 Glyceryl Stearate (and) PEG 100 Stearate 1.25 1.25 1.25
3 Butylated hydroxy toluene 0.10 0.10 0.10
4 Cetostearyl alcohol 2.50 2.50 2.50
5 White beeswax 3.00 3.00 3.00
6 Dimethicone 350 1.00 1.00 1.00
7 Disodium edetate 0.10 0.10 0.10
8 Glycerin 1.00 1.00 1.00
9 Light liquid paraffin 3.50 3.50 3.50
10 Methylparaben 0.18 0.18 0.18
11 Lanolin alcohol 2.00 2.00 2.00
12 Propylparaben 0.05 0.05 0.05
13 Propylene glycol 6.00 6.00 6.00
14 Sorbitan stearate 1.25 1.25 1.25
15 Stearic acid 0.40 0.40 0.40
16 Tocopheryl Acetate 1.00 1.00 1.00
17 Triethanolamine 0.10 0.10 0.10
18 White soft paraffin 1.00 1.00 1.00
19 Purified water q.s. to 100.0 q.s. to 100.0 q.s. to 100.0

Manufacturing Process: The purified water was divided into three portion, one portion was used in preparation of aqueous phase, and other one was used in preparation of drug solution and third portion was used in preparation of triethanolamine solution.
i) Preparation of oil phase: The weighed quantities of Glyceryl Stearate (and) PEG 100 Stearate, cetostearyl alcohol, white beeswax, light liquid paraffin, lanolin alcohol, sorbitan stearate, stearic acid, tocopheryl acetate, and white soft paraffin were taken in an SS vessel and the contents were heated to 75±3ºC under stirring at 500 ± 100 rpm until contents were completely dissolved. Further batch quantity of butylated hydroxy toluene, methylparaben, propylPropyl was added was added to the SS vessel under stirring at 500 ± 100 rpm. The temperature was maintained up to 75± 3 ºC and mixing was continued at 500 ± 100 rpm until all ingredients were melted and a clear phase was obtained.
ii) Preparation of Aqueous phase: Purified water was taken in the main SS vessel and heated to 75± 3 ºC. The temperature was maintained throughout the phase. Disodium EDTA and glycerin was added under stirring at 500 ± 100 rpm till complete dissolution followed by addition of weighed quantity of propylene glycol until clear solution was obtained.
iii) Preparation of Triethanolamine phase Purified water was taken in an SS vessel and triethanolamine was added under stirring at room temperature with SS spatula to get a clear solution.
iv) Emulsification step: The oil phase was slowly transferred at 75± 3 ºC to the main vessel under anchor stirring at 800 ± 100, Further the weighed amount of Dimethicone was added the said vessel under stirring at 800 ± 100 rpm and stirring was continued for 10 mins. The contents were homogenized for 30 min at 3500 ± 200 rpm at 75± 3oC. The triethanolamine solution was added under homogenization and homogenization was continued for 10 mins at 3500 ± 200 rpm at 75± 3oC. The contents of vessel were allowed to cool to 37 ºC under anchor stirring.
v) Preparation of Guanidine Hydrochloride phase and addition to main vessel. Purified water was taken in an SS vessel. The weighed amount of Guanidine hydrochloride was added under manual stirring using an SS spatula until complete dissolution at room temperature.
vi) Preparation of Cream: The drug phase thus prepared was added to contents of main vessel kept at 37 ºC under stirring at 500 ± 100 rpm. Stirring was continued for about 30 min. The weight of the bulk was adjusted with purified water and contents were stirred for 15 min to obtain cream composition.
Observation: the stability of batch A and C is reported in Table No 3 & Table No 4

Table 3: Stability data of composition ‘A’ containing 3% w/w of Guanidine hydrochloride.
Condition Initial 1 Month 3 Month 6 Month 9 Month
25°C/60% RH 30°C/65% RH 40°C/75% RH 25°C/60% RH 30°C/65% RH 40°C/75% RH 25°C/60% RH 30°C/65% RH 40°C/75% RH 25°C/60% RH 30°C/65% RH
Description WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC*
pH 5.62 5.61 5.59 5.55 5.58 5.43 5.42 5.53 5.50 5.39 5.47 5.44
Viscosity (cP) 589.70 650.20 709.10 670.40 611.70 634.60 725.70 585.60 604.70 703.50 525.90 635.30
Microscopy
(µ) D10=1.27 1.331 1.331 1.384 1.337 1.337 1.337 1.337 1.474 1.282 1.174 1.309
D50=1.87 2.076 1.981 2.076 2.085 2.026 2.026 2.165 2.457 2.026 1.876 1.975
D90=3.13 3.609 3.460 3.425 3.845 3.544 3.625 4.423 5.074 3.743 3.386 3.809
Assay of Guanidine HCl 95.60 103.80 104.50 103.80 96.10 97.60 95.30 101.00 104.70 104.70 100.10 105.10
WSHC*: White Smooth Homogenous Cream

Table 4: Stability data of composition ‘C’ containing 7.5% w/w of Guanidine hydrochloride.
Condition Initial 1 Month 3 Month 6 Month 9 Month
25°C/60% RH 30°C/65% RH 40°C/75% RH 25°C/60% RH 40°C/75% RH 25°C/60% RH 40°C/75% RH 25°C/60% RH
Description WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC*
pH 5.48 5.50 5.51 5.50 5.57 5.39 5.43 5.30 5.38
Viscosity (cP) 588.10 616.10 610.10 736.40 650.20 646.80 616.50 706.80 579.00
Microscopy
(µ) D10=1.31 1.33 1.33 1.33 1.33 1.33 1.33 1.33 1.17
D50=1.80 2.01 2.07 2.01 2.02 2.08 1.99 2.07 1.86
D90=2.91 3.52 3.46 3.34 3.41 3.62 3.63 4.05 3.33
Assay of Guanidine HCl 100.60 99.00 107.30 107.40 98.10 101.00 102.00 97.80 98.90

WSHC*: White Smooth Homogenous Cream
Result: The composition developed were found to be stable over varied temperature & RH, indicating that developed composition can be taken up for further evaluation such as animal study, toxicity etc.

Example 3: Cream Composition
Table 5: Cream Composition
Sr. No. Ingredient Concentration (% w/w)
1 Guanidine hydrochloride 5.00
2 Glyceryl Stearate (and) PEG 100 Stearate 1.25
3 Butylated hydroxy toluene 0.10
4 Cetostearyl alcohol 2.50
5 White beeswax 3.00
6 Dimethicone 350 1.00
7 Disodium edetate 0.10
8 Glycerin 1.00
9 Light liquid paraffin 3.50
10 Perfume 0.05
11 Methylparaben 0.18
12 Lanolin alcohol 2.00
13 Propylparaben 0.05
14 Propylene glycol 6.00
15 Sorbitan stearate 1.25
16 Stearic acid 0.40
17 Tocopheryl Acetate 1.00
18 Triethanolamine 0.10
19 White soft paraffin 1.00
20 Purified water q.s. to 100.0

Manufacturing Process: The 5% cream composition was prepared using process described in example 2; except that perfume was added under stirring at 500 ± 100 rpm at 37 ºC.
Observation: The stability data is depicted in Table No 6
Table 6: The stability data of composition of example 3.
Condition Initial 1 Month 3 Month 6 Month 9 Month
25°C/60% RH 30°C/65% RH 40°C/75% RH 25°C/60% RH 30°C/65% RH 40°C/75% RH 25°C/60% RH 40°C/75% RH 25°C/60% RH
Description WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC* WSHC*
pH 5.71 5.34 5.56 5.58 5.52 5.56 5.50 5.50 5.36 5.44
Viscosity (cP) 679.20 698.00 699.20 745.10 622.20 680.00 623.20 642.20 663.00 698.50
Microscopy(µ) D10=1.27 1.33 1.33 1.33 1.33 1.33 1.31 1.184 1.18 1.32
D50=1.80 2.076 1.95 2.01 2.01 2.01 1.98 1.77 1.77 2.14
D90=3.13 3.589 3.31 3.56 3.46 3.52 3.53 3.08 3.26 4.11
Assay of Guanidine HCl 95.40 105.90 105.70 105.90 96.80 95.20 95.30 95.80 95.10 101.60

WSHC*: White Smooth Homogenous Cream
Result: The composition developed was found to be stable over varied temperature & RH, indicating that developed composition can be taken up for further evaluation such as animal study, toxicity etc.

Example 4: Cream Composition
Table 7: Cream Composition
Sr. No. Ingredient Concentration (% w/w)
1 Guanidine hydrochloride 5.00
2 White mineral oil 10.00
3 White soft paraffin 20.00
4 Polyoxyl 20 cetostearyl ether 2.00
5 Cetostearyl alcohol 8.00
6 Methylparaben 0.08
7 Propylparaben 0.04
8 Disodium EDTA 0.01
9 Sodium dihydrogen phosphate dehydrate 0.27
10 Sodium metabisulfite 0.10
11 Xanthan gum 0.20
12 Purified water q.s. to 100.0

Manufacturing Process: The purified water was divided into three portion.
i) Preparation of oil phase: The weighed quantities of white mineral oil, white soft paraffin, and Polyoxyl 20 cetostearyl ether and cetostearyl alcohol were weighed in main SS vessel and the contents were heated to 60-65ºC. The mixture was stirred at 900 ± 100 rpm till complete dissolution. The weighted quantity of methylparaben and propylparaben were added under stirring till complete solubilization. The temperature was maintained up to 60-65ºC and stirring was continued at 500 ± 100 rpm for 10 min.
ii) Preparation of Aqueous phase: Purified water was heated to 60-62ºC in an SS vessel, weighted quantity of xanthan gum was added to SS vessel under stirring 500 rpm to form vortex. The stirring was continued for 30 min.
iii) Emulsification step: The aqueous phase was added to the oil phase in the main vessel under stirring at 1500 ± 100 rpm. After complete addition of the aqueous phase, the stirring speed was increased to 2000 ± 100 rpm and stirring was continued for 15 min at 60-62ºC. In another SS vessel purified water was heated to 60-62ºC. The weighed amount of disodium EDTA, sodium dihydrogen phosphate dihydrate and sodium metabisulfite were added under stirring to said vessel until a clear solution was obtained. The solution thus obtained was added to the contents of main vessel at 60-62ºC under stirring followed by homogenization for 30 min at 3000 ± 200 rpm. The contents were then anchor stirred until temperature of 37ºC was obtained.
iv) Preparation of guanidine hydrochloride phase and addition to main vessel. The weighted quantity of guanidine hydrochloride was added under stirring at room temperature to purified water in SS vessel until drug solution was obtained. The drug solution thus obtained was added to the main vessel at 37ºC under stirring. The contents of vessel were stirred for 30 min. The weight of the bulk was adjusted with purified water to and contents were further stirred for 10 min to obtain cream composition.

Example 5: Gel Composition
Table 8: Gel Composition
Sr. No. Ingredient Concentration (% w/w)
1 Guanidine hydrochloride 5.00
2 Hydroxyl propyl methyl cellulose 1.1
3 Xanthan gum 1.1
4 Purified water q.s. to 100.0

Manufacturing Process:
i) Preparation of Gel phase: Purified water was heated to 60 ºC in SS Vessel using a water bath. The weighted quantities of hydroxyl propyl methyl cellulose and xanthan gum were added under stirring at 150 rpm. The stirring was continued for 30 min at 60ºC to ensure lump free dispersion. The dispersion was allowed to soak for another 30 min. The gel phase was allowed to cool to room temperature.
ii) Preparation of Guanidine hydrochloride solution: Purified water was taken in an SS vessel and stirring was initiated at 500 rpm. Guanidine hydrochloride was added at room temperature to purified water under stirring at 500 rpm in SS vessel until drug solution was obtained.
iii) Gel Preparation: The Guanidine hydrochloride solution prepared above was added to the gel phase under stirring. The contents were stirred for 5 min to gel uniform gel. The weight of the bulk was adjusted with purified water and stirred further for 10 min to get final gel composition.

Example 6: Gel Composition
Table 9: Gel Composition
Sr. No. Ingredient Concentration (% w/w) Concentration (% w/w)
A B
1 Guanidine hydrochloride 5.00 5.00
2 Hydroxyl propyl methyl cellulose 20.00 10.00
3 Purified water q.s. to 100.0 q.s. to 100.0

Manufacturing Process:
i) The weighed quantity of the guanidine hydrochloride was added under stirring to purified water in SS vessel at 400 ± 100 rpm at room temperature until drug solution was obtained.
ii) The weighted quantity of hydroxyl propyl methyl cellulose was added to solution of step (i) at room temperature under stirring at 400 ± 200 rpm. Stirring was continued until lump free phase was observed. The volume was made using purified water followed by stirring until gel composition was formed.

Example 7: Gel Composition
Table 10: Gel Composition
Sr. No. Ingredient Concentration (% w/w)
1 Guanidine hydrochloride 5.00
2 Hydroxyl propyl methyl cellulose 1.10
3 Xanthan gum 1.10
4 Propylene glycol 10.0
5 PEG 400 2.0
6 Dimethicone 1.0
7 Methylparaben 0.08
8 Sodium metabisulfite 0.10
9 Disodium EDTA 0.01
10 Allantoin 0.20
11 Diethylene glycol monoethyl ether 10.0
12 Poloxamer 407 0.2
13 Purified water q.s to 100.0

Manufacturing Process:
i) Preparation of Gel phase: The purified water was heated at 60-65°C in SS Vessel using water bath. The weighed amount of disodium EDTA and Poloxamer 407 were added to the said heated water under stirring at 500 ± 100 rpm until complete solubilization was obtained, followed by addition of weighed quantity of hydroxyl propyl methyl cellulose and xanthan gum. The stirring was continued for 30 min further maintaining temperature of 60-65°C to obtain lump free phase. The gel phase was allowed to soak for another 30min at 60-65°C.
ii) Preparation of Propylene Glycol solution: The weighed quantity of propylene glycol was heated at 60 °C in SS Vessel followed by addition of weighed quantity of methylparaben under stirring at 500± 100 rpm.
iii) Preparation of Guanidine Hydrochloride solution: The purified water was heated at 60°C in SS Vessel using water bath followed by addition of weighed quantity of sodium metabisulfite under stirring at 500 ± 100 rpm until completely solubilized was obtained. The weighed quantity of allantoin was added under stirring till clear solution was obtained. The solution was cooled to room temperature followed by addition of weighed quantity of Guanidine hydrochloride was further added under stirring till clear solution was obtained.
iv) Gel Preparation: The propylene glycol solution formed in step (ii) was added to gel phase of step (i) at 60-65°C. The contents were allowed to cool to room temperature under stirring. The weighed quantity of Transcutol P, PEG 400, and Dimethicone was added under stirring. The Guanidine hydrochloride solution of step (iii) was further added under stirring at room temperature under stirring at 500 ± 100 rpm. The stirring was continued for 30 min at room temperature. The volume was made using purified water and solution was further stirred for 15 min to obtain gel composition.

Example 8: In vitro studies to investigate Collagen Denaturation
The human collagen solution in concentration of 1 µg/ml was used to investigate collagen denaturation by Guanidine Hydrochloride. The Gaunidine hydrochloride in concentration of 1 % w/v, 5 % w/v and 10 % w/v was added to above human collagen solution. The total amount of desaturated collagen denatured was estimated by performing ELISA. The denaturation was further confirmed by using analytical method a) DSC, b) UV spectroscopy as well as by c) Circular Dichroism.
Conclusion: The in-vitro study showed that Guanidine hydrochloride is effective in denaturing the collagenase as confirmed by ELISA test. FIG. 1 shows process of denaturation of Collagen. The percentage of collagen denatured at varied Guanidine hydrochloride is depicted in FIG. 2. The results of DSC, UV and Circular Dichroism is depicted in FIG 3, FIG 4 and FIG 5 respectively which confirms denaturation of collagen by Guanidine hydrochloride.

Example 9: Inhibition of Collagen Expression by Guanidine hydrochloride
The effect of Guanidine hydrochloride on denaturation of Collagen was tested using Human dermal fibroblast cell line, cultured in the serum free culture media (EMEM). A fibroblast cell was incubated with transforming growth factor (TGF) at concentration of 200 ng/ml to induce expression of Collagen for 24 hours. After incubation 0.5 ml of Guanidine hydrochloride solution was added at various different concentrations to each well containing 1 ml of culture media and was further incubated for 24 hours. Following the incubation the amount of Collagen was estimated using Sirius Red Collagen Estimation Kit. The results are depicted in FIG. 6.
Conclusion: Guanidine hydrochloride has the ability to denature the collagen as well as suppress the overexpression of collagen.

Example 10: Study of Transportation of Guanidine hydrochloride across Scar Tissue
The transportation of Guanidine hydrochloride was studied using Franz diffusion cells for 8 hours. The receiver compartment fluid (PBS, pH 7.2) was stirred at 600 rpm. The different concentrations of Guanidine hydrochloride solution was placed in donor compartment. The receiver compartment fluid was sampled every 2 hours interval and the guanidine hydrochloride was quantitated by HPLC.
Conclusion: The transport flux of Guanidine hydrochloride across the full thickness scar skin was found to be 2.3±0.03 µg/cm²/h. The result is depicted in FIG. 7.

Example 11: Cell Proliferation Assay
The cell proliferation assay (MTS) was conducted to confirm the dermal safety of Guanidine hydrochloride. MTS was performed on the human dermal Fibroblast cells at concentration of 200 µl/well in culture media well, treated by different concentrations of Guanidine hydrochloride solution for 24 hours concentration of 50 µl/well.
Conclusion: The amounts used in the assay were many fold higher than the actual amounts the cells were exposed to in-vivo due to slow permeation and rapid clearance of guanidine hydrochloride. Therefore, Guanidine hydrochloride up to 1% exposure level was quite safer for topical use. The result is depicted in FIG. 8.

Example 12: Anti-Scar Study of Guanidine hydrochloride formulations
The in-vivo study of Guanidine hydrochloride was performed in Liveon Biolabs Pvt. Ltd. and Invivo Biosciences using rabbit ear model (New Zealand white). Rabbits were anesthetized with ketamine (22.5mg/kg) and xylazine (2.5mg/kg). Two full thickness defects (involving epidermis, dermis, and perichondrium) were created on each ear, using an 8mm biopsy punch. The wounds were developed at least 1cm from midline to prevent injuries of central artery and marginal ear vein. Removal of perichondrium was ensured under microscopic magnification. Haemostasis was achieved through manual pressure. The wound was allowed to heal and form a hypertrophic scar. After the scar was completely matured (Typically 3-4 weeks), the treatment with test product and placebo was started. Post formation of scar; test product (Test item ‘A’ 3%, Test item ‘B’ 5% and Test item ‘C’ 7.5%) and placebo containing 15 mg/cm2 of product (Test and Placebo) was applied on said scar for twice a day until there was significant improvement in scar perceived visually.
After completion of treatment period; the part of scar was excised using biopsy punch. The collagen I and III was analyzed by ELISA method. The collagen content I and III are depicted FIG.9 and 10. Additionally the morphology of the scar was photographed on different days until there was significant improved perceived visually. The morphology of the scar was photographed and are depicted in FIG. 11 and FIG. 12.
Conclusion: Considering results obtained from both the labs the levels of Collagen I and Collagen III in the treated groups were found to be significantly lower (p < 0.05) compared to the placebo and untreated groups. Treatment with Test items was found to decrease the collagen I and III content, though significant differences (p > 0.05) was not observed between the treated groups. Overall, from the morphological observation of the wound and the collagen content, it is evident that the test item B is effective in treating the hypertrophic scar. It can be concluded that Test item B having strength of 5% and beyond could be effectively used treat the scar tissue.
,CLAIMS:1. A pharmaceutical composition comprising
a) Guanidine compound or its pharmaceutically acceptable salt, ester, prodrug; and
b) one or more pharmaceutically acceptable excipients.
2. The pharmaceutical composition as claimed in claim 1, wherein the composition is a topical dosage form.

3. The pharmaceutical composition as claimed in claim 2, wherein the topical dosage form is in the form of solution, ointment, suspension, cream, gel, paste, powder and tincture.

4. The pharmaceutical composition as claimed in claim 1, wherein the Guanidine compound is its hydrochloride salt.

5. The pharmaceutical composition as claimed in claim 4, wherein guanidine hydrochloride is present in an amount of from 1.00 to 20.00 wt. %.

6. The pharmaceutical composition as claimed in any of claims 1-5, wherein the composition further comprises a lipid, an occlusive agent, an emollient, an anti-oxidant, a humectant and moisturizer, a chelating agent, a thickening agent, a pH adjusting agent, a surfactant and emulsifier, a preservative, colouring agent and opacifying agent, perfume, polymer, penetration enhancer and a solvent.

7. A composition for treating scar comprising guanidine compound or its pharmaceutically acceptable salt, ester, prodrug and a lipid, an occlusive agent, an emollient, an anti-oxidant, a humectant and moisturizer, a chelating agent, a thickening agent, a pH adjusting agent, a surfactant and emulsifier, a preservative, colouring agent and opacifying agent, perfume, polymer, penetration enhancer and a solvent.
8. The pharmaceutical composition as claimed in any of claims 1-7, wherein the composition optionally comprises an additional active.