DESC:Cognate Application
The patent application is a cognate application of Indian Patent Application Nos.: 201821028958 filed on August 01, 2018; 201821034451 filed on September 12, 2018 and 201821034592 filed on September 13, 2018.
Field of the Invention
The present invention relates to a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof, and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally Silver.
In particular, the present invention also relates to a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally Silver for treatment and/or prevention and/or prophylaxis of burn wound infection.
In particular, the present invention also relates to a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally Silver for treatment and/or prevention and/or prophylaxis of infectious diabetic wound.
In particular, the present invention also relates to a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally Silver for treatment and/or prevention and/or prophylaxis of infections such as Impetigo and secondary bacterial infection.
The invention also relates to a combination therapy of topical antibiotics for the treatment of burn wound infection. In particular the present invention relates to a combination therapy of topical antibiotics for prevention and/or prophylaxis of burn wound infection.
The invention also relates to a combination therapy of topical antibiotics for the treatment of infectious diabetic wound. In particular the present invention relates to a combination therapy of topical antibiotics for prevention and/or prophylaxis of infectious diabetic wound.
The invention also relates to a combination therapy of topical antibiotics for the treatment of infections such as Impetigo and secondary bacterial infection. In particular, the present invention relates to a combination therapy of topical antibiotics for prevention and/or prophylaxis of infections such as Impetigo and secondary bacterial infection.
Background
Burns are a common type of trauma across the world. The World Health Organization (WHO) estimates that annually, 11 million people suffer a burn that requires medical attention, while there are 180,000 burns deaths worldwide. The WHO classifies burns as the greatest burden in terms of morbidity including disability, disfigurement, stigma and rejection. Ninety per cent of burns occur within low or middle-income countries where the availability of specialist acute or long-term care for burns patients may be limited. Across all countries the cost of providing burns care remains high due to the need for multiple surgeries, intensive care, long term follow-up and rehabilitation. The WHO’s main aim is to prevent burns across the globe to reduce the economic and societal burden. Most burns occur within the home and are either preventable or their burden are manageable with burn education, first aid and care programs.
Burns are traumatic injuries usually caused by thermal events but may also be as a result of chemical, electrical or radiation exposure. The damaged area has an increase in capillary permeability allowing fluid and large molecules such as albumin to leak out of the circulation. This leads to significant fluid loss, especially if a large surface area is affected. The escape of large molecule compounds results in fluid loss as it creates an oncotic gradient favouring the tissues, meaning even more fluid leaves the circulation. Left untreated, this leads to significant dehydration and cardiovascular collapse.
The loss of the ability to regulate body temperature via the skin may precipitate significant hypothermia, contributing to the impaired immune response propagated by the high levels of endogenous steroids and the loss of the pathological barrier causing a high risk of sepsis in burns patients.
Types of Burn
Thermal burns: ‘Conventional’ burns occur as a product of the temperature gradient applied to the skin and the duration of that application. The larger the temperature gradient and the longer that this is applied leads to an increased severity of burn or burn over a larger surface area.
Friction burns: Heat is generated as a result of friction forces between the skin and other objects. In addition to this there is direct damage by the opposing surface causing mechanical disruption of the skin compounding the burn injury.
Chemical burns: There is an increasing incidence of the use of corrosive substances in violent acts. These so-called ‘acid attacks’ usually involve a corrosive substance (either acidic or alkali in nature) and have a predominance of causing injury to the head, neck and face. Chemical burns can also occur in the industrial setting where accidental spillage of a substance can affect any part of the body.
Electrical burns: Electrical burns usually occur from high voltages and the actual visible injury may be small. The electricity will follow the path of least resistance to earth and may do significant damage as it passes through the body.
Radiation burns: These occur as a result of exposure to electromagnetic or ionizing radiation. The most common example of this form is ‘sunburn’, which although is often widespread but is generally superficial in nature. Ionizing radiation burns occur more rarely but can affect those who work within the nuclear industry as well as patients receiving repeated or prolonged exposure to therapeutic radiation for diagnosis and treatment. All types of radiation burn increase the risk of malignancy. (Christopher Johnson, Management of burns. Surgery, 36(8), 435-440, (2018)).
Burns cause coagulative necrosis of the epidermis and underlying tissues, with the depth depending on the temperature to which the skin is exposed and the duration of exposure. The causes include injury from flame, hot liquids (scald), contact with hot or cold objects, chemical exposure, and conduction of electricity. The first three induce cellular damage primarily by the transfer of energy, inducing coagulative necrosis. Chemicals and electricity cause direct injury to cellular membranes in addition to the transfer of heat. Burn depth is classified into degree of injury in the epidermis, dermis, subcutaneous fat, and underlying structures.
First-degree burns are, by definition, injuries confined to the epidermis. These burns are painful, erythematous, and blanch to the touch with an intact epidermal barrier. Second degree burns are divided into two types: superficial (IIa) and deep (IIb). All second degree burns have some degree of dermal damage, and the division is based on the depth of injury into this structure. Superficial dermal burns are erythematous, painful, blanch to touch, and often blister. Examples include scald injuries from overheated bathtub water and flash flame burns from open carburettors. These wounds spontaneously re-epithelialize from retained epidermal structures in the rete ridges, hair follicles, and sweat glands in 7 to 14 days. After healing, these burns may have some slight skin discoloration over the long term. Deep dermal burns into the reticular dermis appear more pale and mottled, do not blanch to touch, but remain painful to pinprick. These burns heal in 14 to 35 days by re-epithelialization from hair follicles and sweat gland keratinocytes, often with severe scarring as a result of the loss of dermis. The other degrees of burns are more severe and usually require hospitalization.
Burn wound healing is a complex process with three sequential phases: inflammation, proliferation and tissue formation, and tissue remodelling. The phase of tissue remodelling extends for weeks and months after closure of the lesion, and the fibroblast is the cornerstone of remodelling of the dermis, vessels, and basement membrane. Usually, in second-degree burns only wound colonization is present, defined as a multiplication of bacteria causing a delay in wound healing, usually associated with an exacerbation of pain, but still with no overt host reaction. Several topical therapeutic options have been used to promote healing and to prevent bacterial colonization to turn into a wound infection, which delays healing. (Costagliola, M et al, Second-degree burns: a comparative, multicenter, randomized trial of hyaluronic acid plus silver sulfadiazine vs. silver sulfadiazine alone. current medical research and opinion, 21(8), 1235-1240, (2005)).
There are many serious problems associated with the care and treatment of topical burns. For example, the problems include dehydration due to loss of skin vapor barrier capability, direct thermal loss through burned skin and underlying epithelial tissues, and evaporative heat loss by leakage of water vapor, all of which problems must be controlled to maintain body homeostasis. Additionally, eschar and necrotic tissue must be removed and burn wound sepsis due to bacterial colonization and fungal invasion must also be controlled for successful burn management.
Burn wound infections are one of the most important and potentially serious complications that occur in the acute period following injury. Factors that have played a significant role in decreasing the overall fatality rates from burn wound infection and sepsis include the use of topical and prophylactic antibiotics and advances in infection control measures in modern burn.
Pathogenesis of Burn Wound Infection: Thermal destruction of the skin barrier and concomitant depression of local and systemic host cellular and humoral immune responses are pivotal factors contributing to infectious complications in patients with severe burns. The burn wound surface (in deep partial-thickness and in all full-thickness burns) is a protein-rich environment consisting of avascular necrotic tissue (eschar) that provides a favorable niche for microbial colonization and proliferation. The avascularity of the eschar results in impaired migration of host immune cells and restricts delivery of systemically administered antimicrobial agents to the area, while toxic substances released by eschar tissue impair local host immune.
Although burn wound surfaces are sterile immediately following thermal injury, these wounds eventually become colonized with microorganisms. The nature and extent of the thermal injury along with the types and amounts of microorganisms colonizing the burn wound appear to influence the future risk of an invasive wound infection. Gram-positive bacteria that survive the thermal insult, such as staphylococci located deep within sweat glands and hair follicles, heavily colonize the wound surface within the first 48 h unless topical antimicrobial agents are used. Eventually (after an average of 5 to 7 days), these wounds are subsequently colonized with other microbes, including gram-positive bacteria, gram-negative bacteria, and yeasts derived from the host's normal gastrointestinal and upper respiratory flora and/or from the hospital environment or that are transferred via a health care worker's hands.
Over the last several decades, gram-negative organisms have emerged as the most common etiologic agents of invasive infection by virtue of their large repertoire of virulence factors and antimicrobial resistance traits. If the patient's host defenses and therapeutic measures (including excision of necrotic tissue and wound closure) are inadequate or delayed, microbial invasion of viable tissue occurs, which is the hallmark of an invasive burn wound infection. (Church D et al. Burn wound infections. Clin Microbiol Rev, 19 (2), 403– 434, (2006)).
J.P. publication no. 2002514220 discloses local spray formulation for the burn treatment and microbial infection to humans or animals. This non-aerosol formulation contains an antimicrobial agent, i.e. Silver sulfadiazine in a state of being dispersed or dissolved in a cream or lotion based matrix which can be sprayed directly from a common trigger sprayer.
The comparative analysis of Framycetin and Silver sulphadiazine as separate arms and, according to their results Framycetin appears to be alternative to Silver sulphadiazine as a topical agent for major burn (Ahuja R, B et al. A prospective double-blinded comparative analysis of framycetin and silver sulphadiazine as topical agents for burns: A pilot study. Burns. 35 (5), 672-676, (2009)).
An antimicrobials like Alginic acid (alginate), Fusidic acid, Usnic acid,aminoglycosides (Framycetin) and Silver etc. can be used in prevention, prophylaxis and/or treatment of burn wound infection (Sevgi, M, et al. Topical antimicrobials for burn infections - an update. Recent Pat Antiinfect Drug Discov. 8 (3), 161–197, (2013)).
The list of common prophylactic treatments for burn wound infection is disclosed in Barajas-Nava LA et al. Antibiotic prophylaxis for preventing burn wound infection. Cochrane Database Syst Rev. Issue 6; Article No. CD008738, (2013)) as below:
• Topical application: offers broad-spectrum coverage and is most often used for middermal to full-thickness wounds with necrosis and in areas distant from mucous membranes. Topical antibiotics such as ointments and dressings are recommended for treatment of infected burn wounds.
• Systemic Administration: Systemic antibiotics given orally or parenterally (intravenously or via intramuscular injection) for treatment of infected burn wounds.
• Non-absorbable antibiotic prophylaxis: Selective intestinal decontamination with antibiotics.
• Local antibiotic prophylaxis: Antibiotics administered with aerosols by airway route.
The Infection of burn wounds is a serious problem as it can delay healing, increase scarring and invasive infection may result in the death of the patient. Antibiotic prophylaxis is one of several interventions that may prevent burn wound infection and protect the burned patient from invasive infections. The prior arts refer to the treatment of burn wound infection by way of monotherapy with either Fusidic acid or Framycetin or Silver. The art also provides treatment of burn wound infection in general by combination of Fusidic acid with Silver or Framycetin with Silver. However, none of them have been quite effective and there has been an unmet need to identify a medicament for an effective treatment for burn wound infection.
It is estimated that 1.5 to 2.0 % of the entire population of the world suffers from diabetes mellitus of some type. Diabetes mellitus is a chemical disorder of the human body primarily involving an inability of the body to properly utilize sugar and other chemical compounds in the metabolism of the body. It is characterized by an elevation in the concentration of sugar in the blood and also by the appearance of sugar in the urine.
In general terms, diabetes mellitus is classified into three types, namely, Type I (due to autoimmune b-cell destruction, usually leading to absolute insulin deficiency), Impaired Glucose Tolerance (IGT), Type II (due to a progressive loss of b-cell insulin secretion frequently on the background of insulin resistance), Gestational diabetes mellitus (GDM), and Specific types of diabetes due to other causes. In Type I diabetes, the beta cells in the pancreas, probably through an auto-immune reaction, cease producing insulin into the bloodstream of the person. Insulin is a chemical substance which is normally secreted into the bloodstream by beta cells within the pancreas. Insulin is vitally important to the person because it enables the person to properly utilize and consume sugar in the bloodstream as part of the metabolism process.
In IGT and Type II diabetes, the pancreas continues to produce insulin but, some or all of the insulin may fail to bind to the body's cell receptors and/or internalization of insulin in the cells is reduced. In such cases, there may be a sufficient level of insulin in the blood, but the ability of the cells to uptake glucose is reduced or non-existent because of reduced internalized insulin.
GDM diabetes is diagnosed in the second or third trimester of pregnancy that was not clearly overt diabetes prior to gestation.
Specific types of diabetes due to other causes, e.g., monogenic diabetes syndromes (such as neonatal diabetes and maturity-onset diabetes of the young [MODY]), diseases of the exocrine pancreas (such as cystic fibrosis and pancreatitis), and drug- or chemical induced diabetes (such as with glucocorticoid use, in the treatment of HIV/AIDS, or after organ transplantation).
Symptoms of diabetes can be slow-healing wounds, sores, or bruises, Dry and itchy skin, frequent or recurring skin, gum, bladder, or vaginal infection, headaches, increased urination, thirst, nausea, weight loss, fatigue and coma.
The American Diabetic Association (ADA) in its latest publication of standard of Medical Care in Diabetes, 2018 discloses “Diabetes may be diagnosed based on plasma glucose criteria, either the fasting plasma glucose (FPG) or the 2-h plasma glucose (2-h PG) value during a 75-g oral glucose tolerance test (OGTT), or A1C criteria.” The criteria for diagnosis is as given below;
FPG Test: FPG = 126 mg/dL (7.0 mmol/L).
Fasting is defined as no caloric intake for at least 8 hr.
OGTT Test: 2-hr PG = 200mg/dL
(11.1mmol/L) during OGTT. The test should be performed as described by the WHO, using a glucose load containing the equivalent of 75-g anhydrous glucose dissolved in water.
A1C criteria: A1C = 6.5% (48 mmol/mol).
In a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a random plasma glucose = 200 mg/dL (11.1 mmol/L).
Often the FPG Test OGTT Test and A1C are combined to interpret the diabetic condition.
Diabetes mellitus can be virtually harmless if controlled, but the state of abnormally high blood glucose levels associated with the condition can lead to some serious complications. If left uncontrolled for a long time, or if diabetic patients fail to adapt their lifestyles in order to manage the disease, they will have more difficulty preventing complications from occurring. A serious complication that diabetics may encounter are diabetic wounds.
Causes of Diabetic Wounds: The main concern with diabetic wounds is poor or delayed healing. Healing problems are caused by the peripheral arterial diseases and peripheral neuropathy that can occur with diabetes, wherein the small blood vessels in different parts of the body, especially in the extremities (hands and feet), grow narrower and reduce the blood circulation to those areas. A lack of circulation in the extremities can result in a reduced supply of oxygen and nutrients to the body tissue and nerves, which is necessary for healing. Over time, nerves in these areas may become damaged, decreasing the sensation of pain, temperature and touch, making patients vulnerable to injury.
Types of Diabetic Wounds: For a diabetic patient, every wound is a health concern and requires immediate attention. The most common two types are wounds of external origin and wounds of internal origin. Due to peripheral neuropathy, wounds of external origin, such as skin cuts, burns, bumps and bruises, may often go unnoticed by the diabetic patient. If external wounds go unnoticed for some time, delayed treatment can put the patient at risk for further complications. Wounds of internal origin, such as skin ulcers, ingrown toenails or calluses, can lead to the breakdown of skin and surrounding tissue, increasing the risk of bacterial infections.
Signs and Symptoms of Diabetic Wounds: Diabetic wounds may present with the following signs and symptoms: Chronic pain or completely painless, signs of inflammation (swelling, redness, heat, pain and loss of function), signs of infection (pus drainage, discharge, bad odour and dead tissue), new numbness and dullness (signs of nerve damage), fever and/or chills (signs of progressively worsening infection that can be limb-threatening or even life-threatening)
Treatment of Diabetic Wounds: The best treatment is prevention, since medical treatment for diabetic wounds provides limited help. If a wound occurs, treatment can include:
• Keeping all wounds clean and properly dressed.
• Antibiotics (for infected wounds or as a preventive measure for wounds at risk of getting infected)
• Surgical debridement (the dead or infected tissue is removed to allow the healthy tissue to heal and regenerate)
• Referral to a podiatrist or a wound care center (for patients with calluses, corns, hammertoes, bunions, toenail problems or chronic non-healing ulcers)
• Limb amputation (to save as much of a limb as possible when there is a serious infection)
Diabetic wound healing efficacy of two Ayurvedic topical creams in comparison with Framycetin sulfate cream and found that Ayurvedic formulation had better effect over Framycetin sulfate. (Nehete M et al. Comparative efficacy of two polyherbal creams with Framycetin sulfate on diabetic wound model in rats. J Ayurveda Integr Med. 7(2), 83-87 (2016)).
U. S. publication no. 20070283483 discloses sock with the Silver coated fibers for therapeutic uses in diabetic patients.
PCT publication no. 2003084534 discloses method of treating a diabetic foot infection in a mammal using oral, parenteral, or intravenous administration of a pharmaceutical formulation containing effective amount of drugs like flucloxacillin, cefalexin, metronidazole, amoxicillin and clavulanic acid, clindamycin, ciprofloxacin, Fusidic acid, and rifampicin. However the ‘534 application do not provide motivation for topical composition of said actives.
U. S. patent number 6,428,800 discloses the combination of bioactive glass, one or more topical antibiotics and wound or burn dressings. Topical antibiotic can be chloramphenicol, chlortetracycline, clindamycin, clioquinol, erythromycin, Framycetin, gramicidin, Fusidic acid, gentamicin, mafenide, mupiroicin, neomycin, polymyxin B, bacitracin, Silver sulfadiazine, tetracycline, chlortetracycline, or combinations thereof.
PCT publication no. 2011076216 discloses method of treating a wound using combination of acetic acid and at least one antibacterial compound. The application also discloses antibacterial compound such as xylitol, Framycetin, Fusidic acid, nitrofural, phenylmercuric nitrate, benzododecinium, triclosan, cetylpyridinium, aluminium chlorohydrate, povidone-iodine, clioquinol, benzalkonium, chlorhexidine, iodoform, hypochloric acid and Silver. Silver sulfadiazine has been proven to have a synergistic effect in combination with acetic acid.
U.S. patent no. 8,835,170 discloses compositions and methods useful for treating wounds and enhancing wound healing, particularly for diabetic wound healing; in which a therapeutically effective amount of self-organizing mesenchymal blastemal (SOMB) derived from adipose tissue derived stem cells in combination with one or more therapeutic agents selected from the group consisting of Granulocyte-Colony Stimulating Factor (GCSF), Interleukin-6 (IL6), Interleukin-8 (IL8), Interleukin-10 (IL10), Monocyte Chemotactic Protein-1 (MCP1), Monocyte Chemotactic Protein-2 (MCP2), Keratinocyte Growth Factor (KGF), Placental Growth Factor (PLGF), Matrix Metalloproteinase-1 (MMP1), Matrix
Metalloproteinase-9 (MMP9), Tissue Inhibitor Metallopeptidase 1 (TIMP1), and Tissue Inhibitor Metallopeptidase 2 (TIMP2) to treat said wound is administered to a subject in need. The ‘170 patent also discusses use of Silver salts like as chloride, bromide, iodide, and periodate as one of the antimicrobials to be used in combination with SOMB.
Topical preparations of antibiotics that can be used in the wound care include both topical antibacterials such as Silver sulfadiazine, Fusidic acid and metronidazole and topical antiseptics such as sodium chloride, chlorhexidine and povidone iodine. (R. S. Howell-Jones et al. A review of the microbiology, antibiotic usage and resistance in chronic skin wounds. J Antimicrob Chemother. 55(2), 143–149, (2005)).
Healing impairment of diabetic patients is still a serious clinical problem for physicians worldwide due to unclear etiology. Hence, impaired wound healing in diabetics has caught the attention of the world to help promote healing process and prevent the rising complications. A new era in wound healing research is required, involving new treatment strategies to deal with this emerging issue. One of these is the use of a combination of therapeutic agents intended for diabetic wound management and treatment.
The prior arts refer to the treatment of infectious diabetic wound by way of monotherapy with either Fusidic acid or Framycetin or Silver. The art also provides treatment of wound in general by combination of Fusidic acid with Silver or Framycetin with Silver. However, none of them have been quite effective and there has been an unmet need to identify a medicament for an effective treatment for infectious diabetic wound.
Bacteria such as staphylococcus can live harmlessly on many skin surfaces, but when the skin is punctured or broken for any reason, staphylococcus bacteria can enter the wound and cause infection. There are more than 30 species in the staphylococcus family of bacteria and they can cause different kinds of illnesses. Along with staphylococcus stain, streptococcus bacteria are also more common pathogen involved in various skin infections, most common are S. pyogenes, S. agalactiae, S. dysgalactiae, S. bovis, S. anginosus, S. sanguinis, S. suis, S. mitis, S. mutans, S. pneumoniae. But most staphylococcus infections are caused by the species Staphylococcus aureus (S. aureus) and Streptococcus pyogenes. Skin and soft-tissue infections are among the most common infections and may lead to serious local and systemic complications.
Bacterial infections occur frequently in lesions of eczema and atopic dermatitis. One of the common causes of skin and soft-tissue infections is the occurrence of secondary bacterial infection that complicates skin lesions. Secondary bacterial skin infections are common complications of primary dermatoses, primary nonbacterial skin infections, traumatic lesions, ulcers, cutaneous infestations, and other miscellaneous skin diseases. Despite the differences in the underlying cause and clinical presentation, overt secondary bacterial infection with Staphylococcus aureus and Streptococcus pyogenes, or both, is a common problem in patients with inflammatory skin diseases such as allergic contact dermatitis, atopic dermatitis, or psoriasis. Various host factors have been suggested to be the cause of secondarily infected dermatitis (SID), including the lack of expression of antimicrobial peptides on the skin, and the demonstration of increased adherence of Staphylococcus aureus to the skin of patients with atopic dermatitis, A serious consequence of atopic dermatitis, psoriasis, and allergic contact dermatitis is that the integrity of the skin barrier is compromised. Recent studies of patients with atopic dermatitis have shown that levels of both epidermal hydration and skin surface lipids are reduced, making the skin more susceptible to colonization with pathogens.
Antibacterial, whether topical or systemic, in combination, may produce a more rapid decrease in S. aureus colonization than topical antibacterial monotherapy due to its synergistic effect.
Sander Koning (BMJ. 2002 Jan 26; 324(7331): 203) et al provides comparative double blind randomised placebo-controlled trial for use of Fusidic acid cream in the treatment of impetigo.
Erwin M Brown (BMJ. 2002 Jun 8; 324(7350): 1394) et al provides editorial article indicating use of Fusidic acid with restraint in treatment of Impetigo.
Luciana Baptista Pereira (An Bras Dermatol. 2014 Mar-Apr; 89(2): 293–299) provides review on Impetigo, wherein the author acknowledges that Mupirocin and fusidic acid are the first-choice options and further states Fusidic acid is highly effective against S. aureus, with good penetration into cutaneous surface and high concentration at the site of infection. It is also effective to a lesser extent against Streptococcus and Propionibacterium acnes. Gram-negative bacilli are resistant to fusidic acid. It also further teaches commercially available formulation is a mixture of neomycin B and C, while framycetin used in Canada and several European countries, is composed of pure neomycin B. Neomycin sulfate is active mainly against aerobic Gram-negative bacteria (Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus vulgaris).
Rob Edge, Charlene Argáez in Topical antibiotics for impetigo: a review of the clinical effectiveness and guidelines. Ottawa: CADTH; 2017 Feb. (CADTH rapid response report: summary with critical appraisal) which provides review the existing clinical effectiveness evidence on the treatment of patients with impetigo with the topical antibiotics: polymyxin B sulfate-bacitracin (Polysporin ointment), polymyxin B sulfate-gramicidin (Polysporin cream), polymyxin B sulfate-bacitracin-gramicidin (Polysporin triple ointment), bacitracin (Bacitin ointment), mupirocin (Bactroban cream/ointment), silver sufadiazine (Flamazine cream), fusidic acid/fusidate sodium (Fucidin cream/ointment), and fusidic acid 2% with Hydrocortisone (Fucidin H), compared to each other, placebo or oral antibiotics.
Barry H. Long (Acta Derm Venereol 2008; Suppl 216: 14–20) provides literature for use of Fusidic acid in skin and soft tissue infections which includes primary as well as secondary infections.
Sahm, J Deane (Antimicrobial Agents and Chemotherapy, June 2013, 57: 4535-4346) et al teaches the in vitro activity of fusidic acid against important Staphylococcus aureus clones and resistance phenotypes. The findings of this study demonstrate that fusidic acid maintained potent in vitro activity against selected S. aureus strains, including those from Europe, with resistance to currently utilized agents and representatives of the most common clones currently encountered in the United States. This level of activity is consistent with the high level of activity found in generalized surveillance initiatives for U.S. S. aureus isolates and underscores the potential that this compound has for the management of acute bacterial skin and skin structure infections encountered in the United State.
US 6673783 assigned to Leo Pharmaceutical Products Ltd, provides compounds of Fusidic acid derivatives. The invention also provides combination of Fusidic acid derivatives with other therapeutically active components such as penicillins, cephalosporins, tetracyclines, rifamycins, erythromycins, linocomycin, clindamycin, fluoroquinolones and corticosteroids.
WO2007051468 assigned to Leo Pharma, provides the pharmaceutical composition comprising crystalline fusidic acid. The patent application also provides compositions further comprising another therapeutically active compound selected from the group consisting of antibiotics like penicillins, erythromycins or clindamycin.
H. Deng (Environ. Sci. Technol., 2016, 50 (16), pp 8840–8848) et al teaches combination of silver nanoparticles (AgNPs) and an antibiotic [ß-lactam (ampicillin and penicillin), quinolone (enoxacin), aminoglycoside (kanamycin and neomycin), and polykeptide (tetracycline)] can synergistically inhibit bacterial growth, especially against the drug-resistant bacteria Salmonella typhimurium. However, the article is silent on use of fusidic acid as well as framycetin in combination.
Danielle McShan (J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2015; 33(3): 369-84) et al tested Synergistic antibacterial activity of combined silver nanoparticles (AgNPs) with tetracycline (polykeptide), neomycin (aminoglycoside), and penicillin (ß- lactam) multi-drug resistant bacterium Salmonella typhimurium DT104. Dose-dependent inhibition of Salmonella typhimurium DT104 growth is observed for tetracycline-AgNPs and neomycin- AgNPs combination with IC50 of 0.07 µg/mL and 0.43 µg/mL, respectively.
Bahareh Rahimian Zarif (Open Journal of Ecology, 2016, 6, 452-459) et al provides investigation for synergistic effect of silver nanoparticles (AgNPs) with neomycin or gentamicin antibiotic on mastitis-causing Staphylococcus aureus.
WO2010119383 assigned to Apex Labs Private Ltd provides composition for treating bacterial skin infections, along with skin rejuvenation using framycetin sulphate cream and chitosan. The patent application also provides framycetin sulphate is preferred agent for treatment of bacterial dermatoses and pyodermes as impetigo, furunculosis etc.
KL Bairy (Int J Pharm Sci Bio 2010;1(2):100-102) et al compare the effect of sodium fusidate, framycetin and calcium mupirocin of Apex Laboratories Chennai, on experimentally induced burn wound healing in male Wistar rats and compare with available market preparation namely, Fucidin cream 2% (ranbaxy), T Bact cream (GSK) & Soframycin cream (Sanofi-aventis) as mono therapy arm and concluded that Sodium fusidate possess significant wound healing properties.
http://www.diseasesremedies.com/3-popular-impetigo-remedies-that-work/ lists colloidal silver as one of the active use in treatment of Impetigo.
The prior arts refer to the treatment of bacterial infection by way of monotherapy with either fusidic acid or framycetin or silver or in combination of fusidic acid with silver or framycetin with silver. However, none of them have been quite effective and there has been an unmet need to identify a medicament for an effective treatment for Impetigo or secondary bacterial infection.
Summary of the Invention
Accordingly, the present invention relates to a pharmaceutical composition comprising:
(a) fusidic acid or its pharmaceutically acceptable salt, ester, prodrug thereof; and
(b) framycetin or its pharmaceutically acceptable salt, ester, prodrug thereof.
In an embodiment, the pharmaceutical composition optionally comprises silver or its pharmaceutically acceptable salt, ester, prodrug thereof.
In another embodiment, the pharmaceutical composition comprises one or more with pharmaceutically acceptable excipients.
In yet another embodiment, the present invention relates to a topical composition. More particularly, the present invention relates to topical composition which can considerably enhance the preventive effect against infection and facilitate the treatment for impetigo and/or Secondary bacterial infection and/or infection in burn wound and/ or infection in diabetic wound in comparison to a conventional topical treatment and/or composition
Objective of the Invention
The primary object of the present invention is to provide a topical pharmaceutical composition comprising a combination of topical antibiotics for the treatment of burn wound infection.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof for the treatment of burn wound infection.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally Silver for treatment of burn wound infection.
Another primary object of the present invention is to provide a topical pharmaceutical composition comprising a combination of topical antibiotics for the prevention of burn wound infection.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof for prevention of burn wound infection.
Yet another object of the present invention is to provide a topical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally Silver for prevention of burn wound infection.
Another primary object of the present invention is to provide a topical pharmaceutical composition comprising a combination of topical antibiotics for the prophylaxis of burn wound infection.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof for prophylaxis of burn wound infection.
Yet another object of the present invention is to provide a topical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally Silver for prophylaxis of burn wound infection.
Yet another object of the present invention is to provide topical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally Silver to accelerate burn wound healing.
Yet another primary object of the present invention is to provide a topical pharmaceutical composition comprising a combination of topical antibiotics for the treatment of infectious diabetic wound.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, pro drug thereof, and Framycetin or pharmaceutically acceptable salt, ester, pro drug thereof, for the treatment of infectious diabetic wound.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, pro drug thereof, and Framycetin or pharmaceutically acceptable salt, ester, pro drug thereof, and optionally Silver for treatment of infectious diabetic wound.
Another primary object of the present invention is to provide a topical pharmaceutical composition comprising a combination of topical antibiotics for the prevention of infectious diabetic wound.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, pro drug thereof, and Framycetin or pharmaceutically acceptable salt, ester, pro drug thereof, for prevention of infectious diabetic wound.
Yet another object of the present invention is to provide topical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, pro drug thereof, and Framycetin or pharmaceutically acceptable salt, ester, pro drug thereof, and optionally Silver for prevention of infectious diabetic wound.
Yet another primary object of the present invention is to provide a topical pharmaceutical composition comprising a combination of topical antibiotics for the prophylaxis of infectious diabetic wound.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, pro drug thereof, and Framycetin or pharmaceutically acceptable salt, ester, pro drug thereof, for prophylaxis of infectious diabetic wound.
Another embodiment of the present invention provides topical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, pro drug thereof, and Framycetin or pharmaceutically acceptable salt, ester, pro drug thereof, and optionally Silver for prophylaxis of infectious diabetic wound.
Yet another object of the present invention is to provide topical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, pro drug thereof, and Framycetin or pharmaceutically acceptable salt, ester, pro drug thereof, and optionally Silver to accelerate diabetic wound healing.
Another primary object of the present invention is to provide a topical pharmaceutical composition comprising a combination of topical antibiotics for the treatment of bacterial infections.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of fusidic acid and framycetin for treatment of bacterial infections.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of fusidic acid and framycetin and optionally silver for treatment of bacterial infections.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of fusidic acid, framycetin and silver for treatment of bacterial infections.
Yet another primary object of the present invention is to provide a topical pharmaceutical composition comprising a combination of topical antibiotics for treatment of Impetigo.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of topical antibiotics for treatment of secondary bacterial infection.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of fusidic acid and framycetin for treatment of Impetigo.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising a combination of fusidic acid and framycetin optionally along with silver for treatment of Impetigo.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising combination of fusidic acid and framycetin and silver for treatment of Impetigo.
Yet another primary object of the present invention is to provide a topical pharmaceutical composition comprising combination of fusidic acid and framycetin for treatment of secondary bacterial infection.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising combination of fusidic acid and framycetin optionally along with silver for treatment of secondary bacterial infection.
Yet another object of the present invention is to provide a topical pharmaceutical composition comprising combination of fusidic acid and framycetin and silver for treatment of secondary bacterial infection.
Brief Description of the Drawings
Figure 1: Skin Infection Rating Scale Score for Impetigo.
Figure 2: Skin Infection Rating Scale Score for Secondary Bacterial Infection (Gram Positive)
Figure 3: Skin Infection Rating Scale Score for Secondary Bacterial Infection (Gram Negative)
Figure 4: Wound Size Reduction in patient with Secondary End Point Secondary Bacterial Infection (Gram Positive)
Figure 5: Percentage Decrease in Wound Size in patient with Secondary End Point Secondary Bacterial Infection (Gram Positive)
Figure 6: Wound Size Reduction in patient with Secondary End Point Secondary Bacterial Infection (Gram Negative)
Figure 7: Skin Induration reduction in patient with Impetigo
Figure 8: Skin Induration reduction in patient with Secondary Bacterial Infection (Gram Positive)
Figure 9: Skin Induration reduction in patient Secondary Bacterial Infection (Gram Negative)
Figure 10: MIC of Clostridium tertium
Figure 11: MIC of Enterococcus faecalis
Figure 12: MIC of Escherichia coli
Figure 13: MIC of Klebsiella pneumoniae
Figure 14: MIC of Pseudomonas aeruginosa
Figure 15: MIC of Proteus mirabilis
Figure 16: MIC of Streptococcus pyogenes
Figure 17: MIC of MRSA
Figure 18: MIC of MSSA
Detailed Description of the Invention
Before describing the present invention in detail, 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 present invention relates to a pharmaceutical composition comprising:
(a) fusidic acid or its pharmaceutically acceptable salt, ester, prodrug thereof; and
(b) framycetin or its pharmaceutically acceptable salt, ester, prodrug thereof.
In an embodiment, the pharmaceutical composition optionally comprises silver or its pharmaceutically acceptable salt, ester, prodrug thereof.
In an embodiment, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.
In another embodiment, the pharmaceutical composition comprises silver in the form of nanoparticles.
In another embodiment, the pharmaceutical composition comprises silver in the form of lunar silver.
In another embodiment, the pharmaceutical composition comprises Framycetin, wherein Framycetin is Framycetin sulphate.
Definitions
The term “prevention” as disclosed in this specification meaning action taken to decrease the chance of burn wounds and diabetic wounds from getting infected.
The term “prophylaxis” as disclosed in this specification meaning a measure taken to maintain health and prevent the spread of disease.
The terms prevention and prophylaxis are commonly interchanged.
The term "treating" or "treatment" of a state, disorder, disease or condition as used herein means treating burn wounds and diabetic wounds which is being infected; and treating Impetigo and any underlying causes of Impetigo and secondary bacterial infection.
The term “infectious diabetic wound” as disclosed in this specification meaning an infected wound, due to, either, gram positive or negative bacteria in a patient with HbA1c between 7 to 10.
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 un-occluded 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 “burn wound” of skin as used herein means an injury to living tissue caused by burn.
The term “burn injury” as disclosed in this specification is an injury to the skin or other organic tissue caused by thermal, chemical, electrical, radiation and friction.
The term “burn wound” and “burn injury” are commonly interchanged.
The term “wound” of skin is defined as loss or breaking of cellular, anatomical, or functional continuity of living tissues; 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 “induration” of skin as used herein means an increase in the fibrous elements in tissue commonly associated with inflammation and marked by loss of elasticity and pliability and has general meaning as defined in common medical practice incorporated via such reference.
The term “Secondary Bacterial Infection” of skin as used means an infection that occurs during or after treatment for another infection. Secondary bacterial skin infection is common clinical implication which is common in routine clinical practice. Secondary bacterial infection is characterized as skin infection that are common complications of primary dermatoses, primary nonbacterial skin infections, traumatic lesions, ulcers, cutaneous infestations, and other miscellaneous skin diseases. Most diagnoses are based on information obtained by patient history and physical examination. Secondary bacterial skin infections may be polymicrobial, commonly include the pathogens Staphylococcus aureus and Streptococcus pyogenes, and require proper antibiotic treatment.
Impetigo is characterized as skin infection caused by bacteria. It is usually caused by staphylococcal (staph) bacteria, but it can also be caused by streptococcal (strep) bacteria. It is most common in children between the ages of two and six. It usually starts when bacteria get into a break in the skin, such as a cut, scratch, or insect bite. The symptoms start with red or pimple-like sores surrounded by red skin. These sores can be anywhere, but usually they occur on your face, arms and legs. The sores fill with pus, then break open after a few days and form a thick crust. They are often itchy but scratching them can spread the sores. Impetigo can spread by contact with sores or nasal discharge from an infected person. It can be treated with antibiotics.
The term "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.
The terms "drug" and "pharmaceutical" are also used interchangeably to refer to a pharmacologically active substance or composition.
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 salts 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. Journal of Pharmaceutical Science, 66(1), 1-19, Jan 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.
The term "pharmaceutically acceptable carrier" or a "pharmaceutically acceptable salt/solvate or derivative" is meant a compound that is not biologically or otherwise undesirable, i.e., the compound may be incorporated into a topical formulation of the invention and administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained.
The term "carriers" or "vehicles" as used herein refer to pharmaceutically acceptable carrier materials suitable for topical drug administration. Carriers and vehicles useful herein include any such materials known in the art that are non-toxic and do not interact with other components of the composition in a deleterious manner.
"Fusidic acid" is a steroidal antibiotic, chemically related to cephalosporin P. It interferes with amino acid transfer from amino aminoacyls RNA to protein on the ribosomes inhibiting bacterial protein synthesis. It may be bacteriostatic or bactericidal depending upon the drug concentration. Fusidic acid is active against gram-positive bacteria, particularly staphylococci, with almost no activity against gram-negative organisms. It is also active against Streptococci (including Pneumococci) and Corynebacteria. At least 99% of oxacillin resistant staphylococcus aureus were inhibited at a concentration of 2 mg/L, 100% of oxacillin-sensitive staphylococcus aureus were inhibited at a concentration of 0.12 mg/L and 99% of staphylococcus epidermidis were inhibited at 4 mg/L. Other bacteria that demonstrated 90% of organisms inhibited at 2 mg/L or less included; Peptococcus species, Clostridium species Propionibacterium Danes, and most Bacteroides species. Fusidic acid 2% cream monotherapy or combination therapy (betamethasone, beclomethasone dipropionate, ketoconazole, and sodium fusidate) is effective in the treatment of secondary bacterial infections occurring in dermatitis, pyoderma, furuncle, eczema, burns, and psoriasis.
"Framycetin" is a component of neomycin that is produced by Streptomyces fradiae. On hydrolysis it yields neamine and neobiosamine B and is chemically related to Neomycin. Neomycin belongs to aminoglycoside class of antibiotics that contain two or more aminosugars connected by glycosidic bonds. Framycetin resistance is conferred by either one or two aminoglycoside phosphotransferase genes. A neo gene is commonly included in DNA plasmids used by molecular biologists to establish stable mammalian cell lines expressing cloned proteins in culture; many commercially available protein expression plasmids contain neo as a selectable marker. Non-transfected cells will eventually die off when the culture is treated with neomycin or similar antibiotic. Neomycin or kanamycin can be used for prokaryotes, but Geneticin (G418) is, in general, needed for eukaryotes. Framycetin binds to the 30S subunit of the ribosome and inhibits translation of proteins from mRNA. Neomycin exhibits a high binding affinity for phosphatidylinositol 4,5-bisphosphate (PIP2), which is a phospholipid component of cell membranes. Neomycin B is composed of four linked parts: D-neosamine, 2-deoxystreptamine (2-DOS), D-ribose, and L-neosamine.
Silver ions and Silver-based compounds are antimicrobial agents which are highly toxic to microorganisms showing strong bacteriocidal effects on as many as 16 species of bacteria. Thus, Silver ions, as an antibacterial component, have been used in the formulation of dental resin composites and ion exchange fibers and in coatings of medical devices. The Silver ions and Silver-based compounds can be any compound showing antimicrobial activity and not just limited to lunar Silver or nano-Silver.
The topical formulations of the present invention include those suitable for topical, transdermal, rectal and buccal (e.g., sub-lingual) administration etc. Preferably, the formulations of the present invention are administered topically and are provided in the form of semisolid dosage forms.
Suitable dosage forms include hydrous or anhydrous semisolids such as creams, ointments, gels, lotions or any other dosage form suitable for topical application and the like. The term "hydrous" as used herein means the presence of water in a concentration range of about 5% to 95% in the composition. The term "anhydrous" as used herein means the presence of water in a concentration range of less than 5% in the composition.
The present invention relates to a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof, and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally Silver for the treatment of burn wound infection. In particular, the present invention also relates to a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally Silver for prevention and/or prophylaxis of burn wound infection. The invention also relates to a combination therapy of topical antibiotics for the treatment of burn wound infection. In particular the present invention relates to a combination therapy of topical antibiotics for prevention and/or prophylaxis of burn wound infection.
In an embodiment, the present invention also relates to a method of treating burn wound infection using combination therapy of antimicrobials. In particular, the present invention relates to a method of treating burn wound infection using combination therapy of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally in combination with Silver.
In an embodiment, the present invention also relates to method of preventing burn wound infection using combination therapy of antimicrobials. In particular the present invention relates to method of preventing burn wound infection using combination therapy of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally in combination with Silver.
In an embodiment, the present invention also relates to method of prophylaxis of burn wound infection using combination therapy of antimicrobials. In particular the present invention relates to method of prophylaxis of burn wound infection using combination therapy of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof and optionally in combination with Silver.
The present invention relates to a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof, and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof, and optionally Silver for the treatment of infectious diabetic wound. In particular, the present invention also relates to a topical pharmaceutical composition comprising a combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof, and Framycetin or pharmaceutically acceptable salt, ester, pro drug thereof, and optionally Silver for prevention and/or prophylaxis of infectious diabetic wound.
In another embodiment, the present invention also relates to a combination therapy of topical antibiotics for the treatment of infectious diabetic wound. In particular the present invention relates to a combination therapy of topical antibiotics for prevention and/or prophylaxis of infectious diabetic wound.
In another embodiment, the present invention also relates to method of treating infectious diabetic wound using combination therapy of antimicrobials. In particular the present invention relates to method of treating infectious diabetic wound using combination therapy of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof, and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof, and optionally in combination with Silver.
In yet another embodiment, the present invention also relates to method of preventing infectious diabetic wound using combination therapy of antimicrobials. In particular the present invention relates to method of preventing infectious diabetic wound using combination therapy of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof, and Framycetin or pharmaceutically acceptable salt, ester, pro drug thereof, and optionally in combination with Silver.
In yet another embodiment, the present invention also relates to method of prophylaxis of infectious diabetic wound using combination therapy of antimicrobials. In particular the present invention relates to method of prophylaxis of infectious diabetic wound using combination therapy of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof, and Framycetin or pharmaceutically acceptable salt, ester, pro drug thereof, and optionally in combination with Silver.
One embodiment of the present invention provides a topical pharmaceutical composition comprising a combination of a therapeutically effective amount of an antibiotic agent such as fusidic acid and therapeutically effective amount of another antibiotic such as framycetin or any other pharmaceutically acceptable salts/solvates of framycetin and a pharmaceutically acceptable carrier thereof.
In an embodiment, the pharmaceutical composition comprises Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof in a concentration ranging from 0.05 % w/w – 5.0 % w/w of total composition.
In an embodiment, the pharmaceutical composition comprises Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof in a concentration ranging from 0.05 % w/w to 5.0 % w/w of total composition.
In an embodiment, the pharmaceutical composition optionally comprises Silver in a concentration ranging of 0.0001 % w/w to 0.1 % w/w of total composition.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of: a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; and c) a pharmaceutically acceptable carrier.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of: a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; c) optionally 0.0001 %w/w – 0.1 % w/w silver; and d) a pharmaceutically acceptable carrier.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of: a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; c) 0.0001 %w/w – 0.1 % w/w silver; and d) a pharmaceutically acceptable carrier.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of: a) 0.05 % w/w – 5 % w/w of fusidic acid; b) 0.05 % w/w to 5 % w/w of framycetin; and c) a pharmaceutically acceptable carrier for treatment of bacterial infections.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of: a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; c) optionally 0.0001 %w/w – 0.1 %w/w silver and d) a pharmaceutically acceptable carrier for bacterial infection.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of: a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; c) 0.0001 %w/w – 0.1 %w/w silver; and d) a pharmaceutically acceptable carrier for bacterial infection.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of: a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; and c) a pharmaceutically acceptable carrier for treatment of Impetigo.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of: a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; c) optionally 0.0001 % w/w – 0.1 % w/w silver; and d) a pharmaceutically acceptable carrier for treatment of Impetigo.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of combination of: a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; c) 0.0001 % w/w – 0.1 % w/w silver; and d) a pharmaceutically acceptable carrier for treatment of Impetigo.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of: a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; and c) a pharmaceutically acceptable carrier for treatment of secondary bacterial infection.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of: a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; c) optionally 0.0001 % w/w – 0.1 % w/w silver; and d) a pharmaceutically acceptable carrier for treatment of secondary bacterial infections.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of: a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; c) 0.0001 % w/w – 0.1 % w/w silver; and d) a pharmaceutically acceptable carrier for treatment of secondary bacterial infections.
Another embodiment of the present invention provides a topical pharmaceutical composition comprising the combination of therapeutically effective amount of a) 0.05 % w/w - 5%w/w of fusidic acid; b) 0.05% w/w to 5%w/w of framycetin; c) 0.00001 % w/w – 0.1 % w/w silver; and d) a pharmaceutically acceptable carrier for treatment of bacterial infections.
Another embodiment of the present invention provides the use of a topical pharmaceutical dosage form comprising the combination of therapeutically effective amount of antibiotic or any other pharmaceutically acceptable salts/solvates thereof useful in treatment of bacterial infection.
One embodiment of the present invention provides a topical pharmaceutical composition in a suitable dosage form comprising a combination of therapeutically effective amount of antibiotics, and pharmaceutically acceptable carrier. Suitable dosage forms include hydrous or anhydrous semisolids such as creams, gels, ointments, lotions and the like.
Another embodiment of the present invention provides the process for preparing topical pharmaceutical dosage forms comprising a combination of a) an effective amount of fusidic acid; and b) an effective amount of framycetin; and c) a pharmaceutically acceptable carrier thereof. The topical compositions of the present invention having the ratio of Fusidic acid and Framycetin ranging from 1:2.5 to 1:20.
Another embodiment of the present invention provides the process for preparing topical pharmaceutical dosage forms comprising a combination of a) an effective amount of fusidic acid; and b) an effective amount of framycetin; c) an effective amount of silver and d) a pharmaceutically acceptable carrier thereof. The topical compositions of the present invention having the ratio of Fusidic acid: Framycetin: Silver ranging from 1:1:20 to 20:20:1.
In an embodiment, the pharmaceutical composition of the present invention is in the form of a topical composition. Suitable dosage forms include hydrous or anhydrous semisolids such as creams, gels, ointments, lotions, pastes or any other dosage form suitable for topical application and the like.
In one of the embodiments, the topical pharmaceutical composition of the present invention may include suitable pharmaceutically acceptable carriers. The wide range of pharmaceutically acceptable carriers include but are not limited to emollients; emulsifying agents; emulsion stabilizers and viscosity builders; humectants; odorants; preservatives, antioxidants, and chemical stabilizers; solvents; thickening, stiffening, and suspending agents; buffers, neutralizing agents and agents to adjust pH; coloring agents; opacifiers and decoloring agents; pigments; antifoaming agents, skin feel modifiers and the like.
Exemplary emollients include octyldodecanol, caprylic/capric triglycerides, castor oil, ceteareth-20, ceteareth-30, cetostearyl alcohol, ceteth 20, cetostearyl alcohol, cetyl alcohol, cetyl stearyl alcohol, cocoa butter, diisopropyl adipate, glycerine, gyceryl monooleate, glyceryl monostearate, glyceryl stearate, isopropyl myristate, isopropyl palmitate, lanolin, lanolin alcohol, hydrogenated lanolin, liquid paraffins, linoleic acid, mineral oil, oleic acid, white petrolatum, polyethylene glycol, polyoxyethylene glycol fatty alcohol ethers, polyoxypropylene 15-stearyl ether, propylene glycol stearate, propylene glycol mono stearate squalane, steareth-2 or -100, stearic acid, stearyl alcohol, urea and the like. Exemplary emulsifying agents include Propylene glycol stearate, Polyethylene glycol hexadecyl ether, aluminium starch octenylsuccinate, ammonium hydroxide, amphoteric-9, beeswax, white beeswax, synthetic beeswax, carrageenan, carbomer 934, carbomer 934P, carbomer 940, ceteareth-20, ceteareth-30, cetearyl alcohol, ceteth 20, cetyl alcohol, cholesterol, cyclomethicone, diglycerides, dimethicone (e.g., dimethicone 350), disodium monooleamido sulfosuccinate, NF emulsifying wax, fatty acid pentaerythritol ester, glycerides, glyceryl monooleate, glyceryl monostearate, glyceryl stearate, lanolin, lanolin alcohol, hydrogenated lanolin, magnesium stearate, mineral oil, monoglycerides, polyethylene glycol, PEG stearate, polyethylene glycol 6000 distearate, polyethylene glycol 1000 monocetyl ether, polyethylene glycol monostearate, polyethylene glycol 400 monostearate, polyoxyethylene glycol fatty alcohol ethers, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polysorbates, PPG-26 oleate, propylene glycol stearate, quatemium-15, simethicone, sodium laureth sulfate, sodium lauryl sulfate, sorbitan esters, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan palmitate, sorbitan sesquioleate, steareth-2, steareth-100, stearic acid, stearyl alcohol, triethanolamine, trolamine and the like. Exemplary emulsion stabilizers and viscosity builders include carbomer 934, carbomer 934P, carbomer 940, cetearyl alcohol, cetostearyl alcohol, cetyl alcohol, cetyl stearyl alcohol, dextrin, diglycerides, disodium edetate, edetate disodium, glycerides, glyceryl monostearate, glyceryl stearate, hydroxypropyl cellulose, monoglycerides, plasticized hydrocarbon gel, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 1450, polyethylene glycol 8000, polyethylene glycols, propylene glycol stearate, stearyl alcohol and the like. Exemplary humectants include glycerine, propylene glycol, sorbitol, urea and the like. Exemplary odorants include hypoallergenic perfume, menthol and the like. Exemplary preservatives, antioxidants, and chemical stabilizers include alcohol, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, calcium acetate, castor oil, chlorocresol, Potassium sorbate, 4chloro-m-cresol, citric acid, disodium edetate, Dowicil 200 (Dow), edetate disodium, ethoxylated alcohol, ethyl alcohol, glycerine, Glydant Plus (Lonza), 1 ,2,6-hexanetriol, Kathon CG (Rohm & Haas), Liquid Germall Plus (ISP Sutton Labs), Liquipar (ISP Sutton Labs), methyl paraben, parabens, potassium sorbate, propyl gallate, propylene glycol, propyl paraben, sodium bisulfite, sodium citrate, sodium metabisulfite, sorbic acid, tannic acid, triglycerides of saturated fatty acids, Ucarcide (Union Carbide), Vitamin E, zinc stearate and the like. Exemplary solvents include alcohol, castor oil, diisopropyl adipate, ethoxylated alcohol, ethyl alcohol, fatty alcohol citrate, glycerine, 1,2,6-hexanetriol, hexylene glycol, isopropyl alcohol, isopropyl myristate, isopropyl palmitate, mineral oil, phosphoric acid, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 1450, polyethylene glycol 8000, polyethylene glycol 1000 monocetyl ether, polyethylene glycol monostearate, polyethylene glycol 400 monostearate, polyethylene glycols, polyoxyl 20 cetostearyl ether, polyoxypropylene 15-stearyl ether, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polysorbates, octyldodecanol, propylene carbonate, propylene glycol, purified water, and SD alcohol 40, triglycerides of saturated fatty acids and the like. Exemplary thickening, stiffening and suspending agents include aluminium stearate, beeswax, white beeswax, synthetic beeswax, carbomer 934, carbomer 934P, carbor?er 940, cetostearyl alcohol, cetyl alcohol, cetyl esters wax, dextrin, glyceryl monostearate, hydroxypropyl cellulose, kaolin, paraffin, white soft paraffin, petrolatum, white petrolatum polyethylene, propylene glycol stearate, starch, stearyl alcohol, wax, white wax, xanthan gum, bentonite and the like. Exemplary buffers, neutralizing agents and agents to adjust pH include phosphoric add, ammonium hydroxide, citric acid, diisopropanolamine, hydrochloric acid, lactic acid, monobasic sodium phosphate, sodium citrate, sodium hydroxide, sodium acid phosphate, triethanoiamine, trolamine and the like. Exemplary opacifiers/colorants include organic and/or inorganic compounds suitable for use and the like. Suitable examples include titanium dioxide, and pre- dispersed titanium dioxide. Exemplary antifoaming agents include cyclomethicone, dimethicone (e.g., dimethicone 350), simethicone and the like. Exemplary skin feel modifiers include Aluminium starch octenylsuccinate (gamma irradiated) and the like.
In yet another embodiment, the topical formulations of the present invention with Fusidic acid and Framycetin optionally in combination with Silver have been surprisingly found to be effective in the prevention and/ or prophylaxis and/ or treatment of burn wounds from getting infected; found to be effective in the prevention and/or the prophylaxis and/or the treatment of diabetic wounds; found to be effective in the treatment of impetigo and secondary bacterial infection.
It is 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: Topical composition containing fusidic acid, framycetin sulphate and silver
Table No 1. Topical Composition containing 0.67 % Fusidic Acid, 0.33 % Framycetin sulphate & 0.00067 % silver
Ingredients Concentration(%w/w)
Fusidic acid 0.67
Framycetin sulphate 0.33
Lunar Silver CS* 0.00067
White mineral oil 10.0
Polyethylene glycol hexadecyl ether 2.0
Cetostearyl alcohol 8.0
White soft paraffin 20.0
Methyl paraben 0.08
Propyl paraben 0.04
Disodium EDTA 0.01
Sodium acid phosphate 0.5
Sodium metabisulfite 0.1
Carrageenan 0.2
Purified water q.s.
*: Aqueous solution of lunar Silver equivalent to 0.00067 % Silver was used
Manufacturing Process:
i) Preparation of aqueous phase: Purified water was divided into three parts. One part was used for aqueous phase preparation, second part for Framycetin sulfate phase preparation and third part was used for rinsing Fusidic acid polybag. To first part of water, the batch quantity of Lunar Silver CS was added. This phase was heated to 60-62 °C. After temperature was attained, the batch quantity of carrageenan was added under stirring at 1500 ± 200 rpm and stirring was continued for 30 mins.
ii) Preparation of oil phase: The batch quantity of white mineral oil, polyethylene glycol hexadecyl ether, cetostearyl alcohol and white soft paraffin was loaded in SS vessel and heated to 65 °C. After all ingredients were melted and a clear phase was observed, the batch quantity of propyl and methyl paraben were added under stirring at 900 ± 200 rpm and stirred till clear phase was obtained.
iii) Emulsification step: Aqueous phase was added to oil phase under stirring at 65 °C at 1500 ± 200 rpm. The stirring speed was increased to 2000 ± 200 rpm and stirring was continued for 10 minutes. The emulsion was then subjected to homogenization at 3000± 200 rpm. To this the batch quantity of Fusidic acid was added under homogenization at 60-65 °C. Rinsing of the API polybag was done with a part of purified water. Homogenization was then continued for 30 minutes. After 30 minutes the SS vessel was removed from the water bath and homogenization was continued at room temperature to attain 45 °C for the bulk preparation.
iv) Framycetin sulfate phase preparation: To the remaining part of purified water sodium acid phosphate, sodium metabisulfite and disodium EDTA was added under stirring at 900 rpm at 45 °C. To this Framycetin sulfate was added and stirred till clear phase was obtained.
v) Addition of Framycetin sulfate phase to main vessel: Framycetin sulfate phase was added to the main vessel at 45 °C under homogenization at 3000 ± 200 rpm. The vessel was rinsed with purified water and added to the above phase and further homogenized for 30 minutes. Following homogenization, weight of the batch was noted. Amount of lost water was calculated and added to the cream and mixed well manually. The cream formulation was then anchor stirred for 10 minutes at 1000 ± 200 rpm at 5-6 °C. Weight of the batch was recorded after water compensation to calculate the percentage yield.
Example 2: Topical formulation containing 20 % aqueous lunar silver.
Table No 2. Topical Composition containing 20 % aqueous Lunar Silver
Ingredients Concentration(%w/w)
Fusidic acid 2.0
Framycetin sulphate 1.0
Lunar Silver CS* 20.0
White mineral oil 10.0
Polyethylene glycol hexadecyl ether 2.0
Cetostearyl alcohol 8.0
White soft paraffin 20.0
Methyl paraben 0.08
Propyl paraben 0.04
Disodium EDTA 0.01
Sodium acid phosphate 0.5
Sodium metabisulfite 0.1
Carrageenan 0.2
Purified water q.s. to 100
*: Aqueous solution of lunar silver was used equivalent to 0.005 % of silver.
Manufacturing Process: The composition of example 2 is prepared using process exemplified in example 1.
Stability: The composition of Example 2 was subjected to various stability condition and various parameters were evaluated. The stability data is represented in Table No 3.
Table No 3: Stability Data for composition of Example 2
Condition Initial 1 Month 3 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
Description Buff-smooth
Homoge-nous Buff-smooth
Homoge-nous Buff-smooth
Homoge-nous Buff-smooth
Homoge-nous Buff-smooth
Homoge-nous Buff-smooth
Homoge-nous Buff-smooth
Homoge-nous
Microscopy
D10 0.924 µm 0.974 µm 1.089 µm 0.974 µm 0.974 µm 1.089 µm 1.089 µm
D50 1.461 µm 1.461 µm 1.461 µm 1.378 µm 1.461 µm 1.541 µm 1.461 µm
D90 2.179 µm 1.973 µm 2.009 µm 1.961 µm 2.067 µm 2.146 µm 2.067 µm
Assay
Fusidic Acid 99.00 % 100.30 % 100.20 % 100.90 % 98.20 % 98.50 % 97.80 %
Framycetin sulphate 93.20 % 94.40 % 90.30 % 93.20 % 98.30 % 90.30% 93.20 %
Example 3: Topical composition containing 8 % aqueous lunar silver
Table No 4: Topical composition containing 8 % aqueous lunar silver
Ingredients Concentration(%w/w)
Fusidic acid 2.0
Framycetin sulphate 1.0
Lunar Silver CS* 8.0
White mineral oil 10.0
Polyethylene glycol hexadecyl ether 2.0
Cetostearyl alcohol 8.0
White soft paraffin 20.0
Methyl paraben 0.08
Propyl paraben 0.04
Disodium EDTA 0.01
Sodium acid phosphate 0.5
Sodium metabisulfite 0.1
Carrageenan 0.2
Purified water q.s. to 100
*: Aqueous solution of lunar silver was used which is equivalent to 0.002 % of silver
Manufacturing Process: The composition of example 3 is prepared using process exemplified in example 1 except for use of 8 % lunar silver in place of 20 % lunar silver.
Stability: The composition of Example 3 was subjected to various stability condition and various parameters were evaluated. The stability data is represented in Table No 5.
Table No 5: Stability Data for composition of Example 3
Condition Initial 3 Month 6 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
Description Buff-smooth homogenous Buff-smooth homogenous Buff-smooth homogenous Buff-smooth homogenous Buff-smooth homogenous Buff-smooth homogenous Buff-smooth homogenous
Microscopy
D10 0.898 µm 0.875 µm 0.875 µm 0.875 µm 0.789 µm 0.875 µm 0.875 µm
D50 1.461 µm 1.331 µm 1.276 µm 1.331 µm 1.238 µm 1.331 µm 1.313 µm
D90 2.465 µm 2.552 µm 2.357 µm 2.476 µm 2.495 µm 2.671 µm 2.635 µm
Assay
Fusidic acid 98.80 % 97.80 % 97.80 % 98.70 % 101.80 % 99.10 % 98.80 %
Framycetin sulphate 103.40 % 99.90 % 99.40 % 99.10 % 101.10 % 100.90 % 99.60 %
Example 4: Topical composition containing 4% aqueous Lunar silver
Table No 6: Topical composition containing 4% aqueous Lunar silver
Ingredients Concentration(%w/w)
Fusidic acid 1.0
Framycetin sulphate 0.5
Lunar Silver CS* 4.0
White mineral oil 10.0
Polyethylene glycol hexadecyl ether 2.0
Cetostearyl alcohol 8.0
White soft paraffin 20.0
Methyl paraben 0.08
Propyl paraben 0.04
Disodium EDTA 0.01
Sodium acid phosphate 0.5
Sodium metabisulfite 0.1
Carrageenan 0.2
Purified water q.s. to 100
*: Aqueous solution of lunar silver was used which is equivalent to 0.001 % of silver
Manufacturing Process: The composition of example 4 is prepared using process exemplified in example 1 except for use of 4 % lunar silver in place of 20 % lunar silver.

Example 5: Topical composition containing 2.664% aqueous Lunar silver
Table No 7: Topical composition containing 2.664% aqueous Lunar silver
Ingredients Concentration(%w/w)
Fusidic acid 0.66
Framycetin sulphate 0.33
Lunar Silver CS* 2.664
White mineral oil 10.0
Polyethylene glycol hexadecyl ether 2.0
Cetostearyl alcohol 8.0
White soft paraffin 20.0
Methyl paraben 0.08
Propyl paraben 0.04
Disodium EDTA 0.01
Sodium acid phosphate 0.5
Sodium metabisulfite 0.1
Carrageenan 0.2
Purified water q.s. to 100
*: Aqueous solution of lunar silver was used which is equivalent to 0.00067 % of silver
Manufacturing Process: The composition of example 5 is prepared using process exemplified in example 1 except for use of 2.664% lunar silver in place of 20 % lunar silver.
Example 6: Topical composition containing Fusidic Acid and Framycetin sulfate
Table No 8: Topical composition containing combination of Fusidic Acid & Framycetin Sulfate
Ingredients A (%w/w) B (%w/w)
Fusidic acid 2.0 2.0
Framycetin sulphate 1.5 2.0
Liquid paraffin 10.0 10.0
White soft paraffin 20.0 20.0
Polyethylene glycol hexadecyl ether 2.0 2.0
Cetostearyl alcohol 8.0 8.0
Methyl paraben 0.08 0.08
Propyl paraben 0.04 0.04
Disodium EDTA 0.01 0.01
Sodium acid phosphate 0.5 0.5
Sodium metabisulphite 0.1 0.1
Carrageenan 0.2 0.2
Purified water q.s. to 100 q.s. to 100
Manufacturing process: The compositions of example 6 is prepared using process described in example 1 except for use of silver in aqueous phase.

Example 7: Clinical Study to evaluate effectiveness of test formulation in prophylaxis of burn wound infection.
An open label single centre, observational study for evaluation of effectiveness of formulation developed as per example 1 for the prophylaxis of infection in patients with burn wound will be conducted at Department of Pharmacology, Grant Government Medical College and Sir J J group of Hospitals, Byculla, Mumbai, 400008. The study period will be of 14 days with patient visiting investigator on Day 0 (visit 2), Day -3 (visit 1), Day 7 (visit 3), Day 10 (visit 4) & Day 21. Patients are 1st screened and the on visit 1 i.e. at Day 3, all the patients that satisfied the inclusion criteria were enrolled in the study on visit 2; On Day 10 ± 2 days study was concluded & on Day 21 telephonic follow up with patients was done to assess overall study drug compliance.
Primary end point for Effectiveness will be determined to be “proportion of patients not contacting wound infection during the study treatment period and clinical cure rate” using parameters like skin infection rating scale, Clinician Global Impression of Improvement (CGI-I), and Patient Global Impression of Improvement (PGI-I). The safety will be evaluated based on number of patients reporting any adverse event. After completion of the study, investigator will follow-up with subjects telephonically (on day 21/one week after end of the study visit) regarding overall safety with regards to study drug. The standard known statistical method such as Chi-Square or Fisher’s exact test and two sample t-test will be applied to evaluate and report the results of study.
Observation: Total 20 patients were enrolled for this study of which 19 patients were males. None of the patient was discontinued from the treatment or withdrawn from the trial. Subjects were classified as “cured” by end of trial as to have a SIRS score of 0 for blistering, exudates/pus, crusting, itching/pain and score of 0 or 1 for all other signs and symptoms i.e erythema/inflammation. The Table No 9 indicates the cure rate in terms of frequency as well as percentage cure. Table No 10 indicates SIRS score for visits 1, 3 & 4 as median value.
Table No 9: Primary End Point (Cure Rate)
Frequency Percent
Cured 11 55.0
Not Cured 9 45.0
Total 20 100.0
Table No 10: Primary End Point (SIRS score in median)
Visit Median
Visit 1 9.00
Visit 3 5.00
Visit 4 1.00
The secondary end point of study was evaluated on parameters like Hyperpigmentation; Patient Global Impression of Improvement (PGI-I) and Clinician Global Impression of Improvement (CGI-I); the mean was evaluated for Visit 3 & Visit 4. Hyperpigmentation that was analysed using visual analogue scale (VAS). The value reflects the observed cure rate and condition of wound. Table No 11 indicates Median value of hyperpigmentation
Table No 11: Hyperpigmentation (SIRS score in median)
Visit Median
Visit 1 6.00
Visit 3 4.00
Visit 4 2.00
Both the PGI-I and CGI-I gradually decreased during the treatment period. The Table No 12 indicates mean values for PGI-I as well as CGI-I.
Table No 12: PGI-I & CGI-I (median)
Visit PGI-I CGI-I
Visit 3 3.00 3.00
Visit 4 1.00 1.00
Results: The study showed gradual reduction in SIRS score. The overall cure rate for study is 55 % indicating that the combination composition was able to prevent onset of infection in patients with burn wound. There was statistically significant reduction in hyperpigmentation VAS score; PGI-I & CGI-I indicating the triple combination composition was able to prevent onset of infection in patients with burn wound.
Example 8: Clinical Study to evaluate effectiveness of test formulation in treatment of diabetes wound
An open label single centre, observational study for evaluation of effectiveness of formulation developed as per example 1 in the diabetic patients with wound will being evaluated at Department of Pharmacology, Grant Government Medical College and Sir J J group of Hospitals, Byculla, Mumbai, 400008. The study period will be of 14 days with patient visiting investigator on Day 0 (visit 2), Day -3 (visit 1), Day 7, Day 14 & Day 21. Patients are 1st screened and the on visit 1 i.e. at Day 3, all the patients that satisfied the inclusion criteria were enrolled in the study on visit 2; On Day 14 ± 2 days study was concluded & on Day 21 telephonic follow up with patients was done to assess overall study drug compliance.
Primary end point for Effectiveness is determined to be “change in the wound size of the lesions seen in diabetic wound and clinical cure rate” using parameters like skin infection rating scale, Clinician Global Impression of Improvement (CGI-I), Patient Global Impression of Improvement (PGI-I), and Improvement in wound pain by using global pain scale and change in quantity of microbial cell culture. The safety will be evaluated based on number of patients reporting any adverse event. After completion of the study, investigator will follow-up with subjects telephonically (on day 21/one week after end of the study visit) regarding overall safety with regards to study drug. The standard known statistical method such as Chi-Square or Fisher’s exact test and two sample t-test will be applied to evaluate and report the results of study.
Observation: Total 20 patients were enrolled for this study. Of the 20 patients 18 patients were males. None of the patient was discontinued from the treatment or withdrawn from the trial. The clinical cure rate was primary end point. Subjects were classified as “cured” by end of trial as to have a SIRS score of 0 for blistering, exudates/pus, crusting, itching/pain and score of 0 or 1 for all other signs and symptoms i.e. erythema/inflammation. The Table No. 13 depicts the mean clinical cure rate frequency as well as percentage cure. Table No. 14 indicates SIRS score for visits 1, 3 & 4 as median value.
Table No 13: Clinical cure rate
Frequency Percent
Cured 9 45.0
Not cured 11 55.0
Total 20 100.0
Table No 14: SIRS score
Visit Median
Visit 1 11.0
Visit 3 6.0
Visit 4 2.0
The secondary end point was percentage decrease in wound size; change in microbial culture and Patient Global Impression of Improvement (PGI-I) and Clinician Global Impression of Improvement (CGI-I), the mean was evaluated for Visit 1, Visit 3 & Visit 4. The percentage decrease in wound size is determined using the data collected from patients during their visits & is depicted in Table No 15 as decrease in wound size.
Table No 15: decrease in wound size
Visit Mean SD
Visit 1 9.71 4.05
Visit 3 4.15 1.69
Visit 4 1.68 0.48
The change microbial culture is depicted as reduction in quantity of microbial cell culture & is tabulated in Table No 16.
Table No16: Quantity of Microbial Culture
Visit Mean SD
Visit 1 944.00 143.15
Visit 3 616.28 183.62
Visit 4 317.11 193.73
Both the PGI-I & CGI-I were found to gradually decrease during treatment. The PGI-I as well as CGI-I is tabulated in Table No 17 as median values at visit 3 & 4.
Table No 17: PGI-I & CGI-I (median)
Visit PGI-I CGI-I
Visit 3 3.00 3.00
Visit 4 2.00 2.00
Result: The study indicated gradual reduction in SIRS score in during the treatment duration. The overall cure rate for study is 45 % indicating that the combination composition was able to prevent onset of infection in patients with diabetic wound. There was statistically significant reduction in wound size, microbial culture count as well as PGI-I & CGI-I indicating the triple combination composition was able to prevent onset of infection in patients with diabetic wound.
Example 9: Efficacy and Safety Evaluation of test formulation:
An open label randomized comparative study for evaluation of efficacy and safety of formulation developed as per example 4 in comparison with commercially available monotherapy of fusidic Acid 2%, Framycetin 1% and silver 0.002% respectively in patient having impetigo and secondary bacteria skin infections is being evaluated at Department of Pharmacology, Grant Government Medical College and Sir J J group of Hospitals, Byculla, Mumbai – 400008. The study period is of 14 days with patient visiting investigator as Visit 1 (Day -3); Visit 2 (Day 0); Visit 3 (Day 7) & Visit 4 (Day 14). Patients diagnosed with impetigo and secondary bacterial infection were selected and randomized in respective treatment arm on day 0 visit.
Primary end point for Efficacy is determined to be “change in the wound size of the lesions seen in Secondary Skin bacterial infections and clinical cure rate” using parameters like skin infection rating scale, Clinician Global Impression of Improvement (CGI-I), Patient Global Impression of Improvement (PGI-I), reduction in skin induration and change in microbiological colony formation. The safety will be evaluated based on number of patients reporting any adverse event. After completion of the study, investigator will follow-up with subjects telephonically (on day 21/one week after end of the study visit) regarding overall safety with regards to study drug. The standard known statistical method will be applied to evaluate and report the results of study.
Observation: The study was designed to monitor clinical signs and symptoms such as SIRS Score, Skin Induration, CGI-1, PGI-I, SIRS Score, Wound Size. Overall Global Assessment of Efficacy: At the end of study, an overall assessment of the efficacy of the medication was performed by both the investigator and the patient. The SIRS Score obtained was subjected to Fisher’s Exact Test statistical testing. The result of SIRS score for Impetigo is reported in Table No 18 and results of same is depicted in figure 1.
Table No 18: SIRS Score for Impetigo
Treatment Arm Visit 1 Visit 3 Visit 4
FDC 6.60 3.60 0.30
Fusidic 6.80 2.80 0.10
Framycetin 5.60 4.20 2.10
Silver 6.80 3.80 1.00
SIRS score for Secondary Bacterial Infection (gram positive bacteria) is reported in Table No 19 and figure 2.
Table No 19: SIRS Score for Secondary Bacterial Infection (gram positive bacteria)
Treatment Arm Visit 1 Visit 3 Visit 4
FDC 7.10 3.20 0.50
Fusidic 6.20 4.50 1.40
Framycetin 6.90 5.00 3.00
Silver 6.80 4.60 0.50
The SIRS score for Secondary Bacterial Infection (gram negative bacteria) is reported in Table No 20 and figure 3.
Table No 20: SIRS score for Secondary Bacterial Infection (gram negative bacteria)
Treatment Arm Visit 1 Visit 3 Visit 4
FDC 6.50 4.40 2.20
Framycetin 6.80 5.50 3.60
Silver 7.10 4.90 2.90
Change in Wound Size of Lesion: The change in wound size of lesion was measured as one of the primary end point along with SIRS for secondary bacterial infection. The change in Wound Size of Lesion for Secondary bacterial infection (gram positive bacteria) are tabulated in Table No 21 and depicted in figure 4 & figure 5.
Table No 21: Change in Wound Size of Lesion for Secondary bacterial infection (gram positive bacteria)
Treatment Visit(Baseline) Visit 3 Visit 4 Change from Baseline
FDC N 8 8 8
Mean 9.50 5.25 2.00 -7.50
% reduction 78.33
Fucidic N 9 9 9
Mean 9.00 6.11 3.66 -5.33
% reduction 60.49
Framycetin N 9 9 9
Mean 10.44 7.66 5,77 -4.67
% reduction 45.31
Silver N 9 9 9
Mean 9.66 6.11 3.00 -6.66
% reduction 68.96
Framycetin Vs FDC 0.004
Fusidic Vs FDC 0.426
Framycetin Vs FDC 0.004
NS: Not Significant; S: Significant; *p-value calculated using ANOVA; Post hoc ANOVA using Dunnet’s Test
The results of change in Wound Size of Lesion for Secondary bacterial infection (gram negative bacteria) are tabulated in Table No 22 and depicted in figure 6.
Table No 22: Change in Wound Size of Lesion for Secondary bacterial infection (gram negative bacteria)
Treatment Visit 1 (Baseline) Visit 3 Visit 4 Change from Baseline
FDC N 10 10 10
Mean 9.50 6.70 4.10 -5.40
Framycetin N 10 10 10
Mean 9.30 7.30 5.40 -3.90
Silver N 10 10 10
Mean 9.80 6.90 4.90 -4.90
Framycetin Vs FDC 0.39 (NS)
Silver Vs FDC 0.54 (NS)
NS: Not Significant; S: Significant; *p-value calculated using ANOVA; Post hoc ANOVA using Dunnet’s Test
Skin Induration: The change in skin induration was considered as secondary end point for Impetigo as well as secondary bacterial infection. The change in skin induration in patients with Impetigo was tabulated in Table No 23 and depicted in figure 7.
Table No 23: Change in Skin Induration of Lesion in patient with Impetigo
Treatment Visit 1 (Baseline) Visit 3 Visit 4
FDC Mean 40.20 22.40 7.10
% Decrease 82.47
Fusidic Mean 41.70 25.10 13.90
% Decrease 65.75
Framycetin Mean 38.60 29.30 18.90
% Decrease 52.06
Silver Mean 33.80 24.50 14.10
% Decrease 59.04
The change in skin induration in patients with Secondary bacterial infection (gram positive) was tabulated in Table No 24 and depicted in figure 8.
Table No 24: Change in Skin Induration of Lesion in patient with Secondary Bacterial Infection (gram positive)
Treatment Visit 1 (Baseline) Visit 3 Visit 4
FDC Mean 40.70 25.40 11.70
% Decrease 71.60
Fusidic Mean 40.70 28.60 14.70
% Decrease 64.22
Framycetin Mean 38.60 32.60 25.40
% Decrease 34.19
Silver Mean 39.10 27.10 12.30
% Decrease 68.53
Maximum 52.0 41.0 35.0
The change in skin induration in patients with Secondary bacterial infection (gram negative) was tabulated in Table No 25 and depicted in figure 9.
Table No 25: Change in Skin Induration of Lesion in patient with Secondary bacterial infection (gram negative)
Treatment Visit 1 (Baseline) Visit 3 Visit 4
FDC Mean 39.10 26.90 16.50
% Decrease 57.70
Framycetin Mean 37.30 30.00 22.80
% Decrease 39.17
Silver Mean 39.90 29.00 19.00
% Decrease 51.53
Patient Global Impression Improvement (PGI-I): The PGI-I is also secondary end point for Impetigo as well as Secondary Bacterial Infection and is measured by evaluating scale given by patient under each treatment arm. The change in Patient Global Impression of Improvement in patient with Impetigo was tabulated in table 26.
Table No 26: Change in Patient Global Impression of Improvement in patient with Impetigo
Treatment N Median Minimum Maximum Range
FDC 10 -1.00 -2.0 0.0 2.0
Framycetin 10 -1.00 -1.0 0.0 1.0
Fusidic 10 -1.00 -1.0 -1.0 0.0
Silver 10 -1.00 -2.0 0.0 2.0
p-value* 0.473(NS)
NS: Not Significant. *p-value calculated with Median Test
The change in Patient Global Impression of Improvement in patient with Secondary Bacterial Infection (gram positive) was tabulated in table 27.
Table No 27: Change in Patient Global Impression of Improvement in patient with Secondary bacterial Infection (gram positive)
Treatment N Median Minimum Maximum Range
FDC 10 -1.00 -2.0 -1.0 1.0
Fusidic 10 -1.00 -2.0 0.0 2.0
Framycetin 10 -1.00 -1.0 0.0 1.0
Silver 10 -1.00 -2.0 -1.0 1.0
p-value* 0.551(NS)
NS: Not Significant.*p-value calculated with Median Test
The change in Patient Global Impression of Improvement in patient with Secondary Bacterial Infection (gram negative) was tabulated in table 28.
Table No 28 Change in Patient Global Impression of Improvement in patient with Secondary bacterial Infection (gram negative)
Treatment N Median Minimum Maximum Range
FDC 10 -1.00 -2.0 0.0 2.0
Framycetin 10 -1.00 -2.0 0.0 2.0
Silver 10 -1.00 -2.0 -1.0 1.0
p-value* 0.133(NS)
NS: Not Significant. *p-value calculated with Median Test
Clinician Global Impression Improvement (CGI-I): The CGI-I is also secondary end point for Impetigo as well as Secondary Bacterial Infection and is measured by evaluating scale given by clinician who is treating patients under each treatment arm. The change in Clinician Global Impression of Improvement in patient with Impetigo was tabulated in table no 29.
Table No 29: Change in Clinician Global Impression of Improvement in patient with Impetigo
Treatment N Median Minimum Maximum Range
FDC 10 -1.00 -1.0 -1.0 0.0
Fusidic 10 -1.00 -2.0 0.0 2.0
Framycetin 10 -1.00 -1.0 0.0 1.0
Silver 10 -1.00 -2.0 0.0 2.0
p-value* 0.473 (NS)
NS: Not Significant. *p-value calculated with Median Test
The change in Clinician Global Impression of Improvement in patient with Secondary Bacterial Infection (gram positive) was tabulated in table no 30.
Table No 30: Change in Clinician Global Impression of Improvement in patient with Secondary Bacterial Infection (gram positive)
Treatment N Median Minimum Maximum Range
FDC 10 -1.00 -2.0 -1.0 1.0
Fusidic 10 -1.00 -2.0 -1.0 1.0
Framycetin 10 -1.00 -2.0 0.0 2.0
Silver 10 -1.00 -2.0 -1.0 1.0
p-value* 0.097(NS)
NS: Not Significant. *p-value calculated with Median Test
The change in Clinician Global Impression of Improvement in patient with Secondary Bacterial Infection (gram negative) was tabulated in table no 31.
Table No 31: Change in Clinician Global Impression of Improvement in patient with Secondary Bacterial Infection (gram negative)
Treatment N Median Minimum Maximum Range
FDC 10 -1.00 -2.0 0.0 2.0
Framycetin 10 -1.00 -2.0 0.0 2.0
Silver 10 -1.00 -2.0 0.0 2.0
p-value* 0.749 (NS)
NS: Not Significant. *p-value calculated with Median Test
Result: The trial conducted in patients provides that the combination of Fusidic acid, Framycetin and Silver is not only efficacious in treatment of Impetigo and secondary infection including gram negative bacteria but is also safe for use in human at the concentration tested.
Example 10: Efficacy and Safety Evaluation of test formulation against mupirocin 2 % cream
An open label single centre, observational study for evaluation of effectiveness of formulation developed as per example 1 in the patients with infected traumatic skin lesion will being evaluated at Department of Pharmacology, Grant Government Medical College and Sir J J group of Hospitals, Byculla, Mumbai, 400008. . The study period will be of 14 days with patient visiting investigator on Visit 1 (Day -3); Visit 2 (Day 0); Visit 3 (Day 7) & Visit 4 (Day 14). ; On Day 10 ± 2 days study was concluded & on Day 17 telephonic follow up with patients was done to assess overall study drug compliance.
Primary end point for Effectiveness is determined to be “change in the wound size of the lesions seen in diabetic wound and clinical cure rate” using parameters like skin infection rating scale, Clinician Global Impression of Improvement (CGI-I), Patient Global Impression of Improvement (PGI-I), and Bacteriological cure rate. The safety will be evaluated based on number of patients reporting any adverse event. After completion of the study, investigator will follow-up with subjects telephonically (on day 17 after end of the study visit) regarding overall safety with regards to study drug. The standard known statistical method such as Chi-Square or Fisher’s exact test and two sample t-test will be applied to evaluate and report the results of study.
Observation: Total 20 patients were enrolled for this study & were divided into Test arm and comparator arm. Of the 20 patients 18 patients were males. None of the patient was discontinued from the treatment or withdrawn from the trial. The primary end point is clinical cure rate. Subjects were classified as “cured” by end of trial as to have a SIRS score of 0 for blistering, exudates/pus, crusting, itching/pain and score of 0 or 1 for all other signs and symptoms i.e. erythema/inflammation. The Table No 32 indicates cure rate as well as percentage cure (% Cure) of study. The SIRS score is depicted in Table No. 33.
Table No 32: Clinical cure rate
Observation Test arm Comparator arm
Frequency % Cure Frequency % Cure
Cured 8 80 8 80
Not Cured 2 20 2 20
Table No. 33 SIRS score
Group Visit 1 Visit 3 Visit 4
Test arm Mean 10.10 5.30 1.60
Median 9.00 5.00 0.50
Std. Deviation 1.524 1.947 2.633
Comparator arm Mean 9.10 5.50 1.90
Median 9.00 5.50 0.50
Std. Deviation 0.738 3.028 3.381
The secondary end point was percentage decrease in wound size; Bacteriological cure rate and Patient Global Impression of Improvement (PGI-I) and Clinician Global Impression of Improvement (CGI-I). The percentage decrease in wound size is determined using the data collected from patients during their visits & is depicted in Table No 34 as decrease in wound size.
Table No 34: Decrease in wound size
Group Mean SD
Test Arm 6.60 2.50
Comparator Arm 7.30 4.73
The gradual decrease in microbial culture was observed in both arms and is depicted as reduction in quantity of microbial cell culture & is tabulated in Table No 35
Table No 35: Quantity of Microbial Culture
Group Visit 1 Visit 3 Visit 4
Test arm Mean 960.40 559.30 223.00
Median 996.50 574.00 145.00
Std. Deviation 148.680 140.971 183.690
Comparator
Arm Mean 993.10 585.00 241.50
Median 961.00 548.50 130.50
Std. Deviation 161.652 190.468 253.980
Patient Global Impression of Improvement (PGI-I) and Clinician Global Impression of Improvement (CGI-I); the mean was evaluated for Visit 3 & Visit 4. The results are depicted in Table No. 36
Table No 36: PGI-I & CGI-I
Group PGI-I CGI-I
Visit 3 Visit 4 Visit 3 Visit 4
Test arm Mean 2.50 1.40 2.50 1.30
Median 2.50 1.00 2.50 1.00
Std. Deviation 0.527 0.843 0.527 0.675
Comparator
arm Mean 2.90 1.70 2.90 1.70
Median 3.00 1.50 3.00 1.50
Std. Deviation 0.316 0.823 0.316 0.823
Result: The gradual reduction in SIRS score was observed in both treatment arms; the cure frequency as well as percentage cure and SIRS score of triple combination using lower concentration of individual drugs was found to be same as that of Mupirocin Cream 2 %. The decrease in wound size was more in Mupirocin cream arm but was not statistically superior over test arm. The bacteriological cure rate for both the arms were found comparative with each other & showed almost same result.
Example 11. A study on in vitro synergistic effect of a novel combination cream against bacterial biofilms
The study to evaluate in vitro synergistic effect was planned in School of Life Sciences, MAHE, Manipal. The objective of this study is to determine Minimal inhibitory concentration & Minimal bactericidal concentration of novel combination cream compared with individual monotherapy against bacterial biofilms against microbes like Clostridium tertium, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pyogenes, Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA). The formulations that were evaluated were identified in Table no 37 as below.
Table No 37: Formulation coding
Formulation Active Agent
Formulation -1 (FA) Fusidic acid (FA)
Formulation -2 (FS) Framycetin Sulphate (FS)
Formulation -3 (LS) Lunar Silver CS (LS)
Formulation -4 (FDC) Triple combination of FA, FS & LS
Formulation -5 (Placebo) Placebo
The procedure followed for conducting microbiological assay study was as follow
1. Preparation of samples and standards Strain revival:
a) 10µl of cryopreserved stock of bacterial species (Clostridium tertium, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus and Staphylococcus epidermidis) was inoculated into 5ml of peptone water and kept in a shaker incubator at 37°C with 220rpm for 16 hours.
b) A loopful of the bacterial suspension was streaked onto Nutrient agar and MacConkey agar for 8 bacterial species (Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus and Staphylococcus epidermidis) while Clostridium tertium was streaked onto Brain heart infusion agar and the plates were incubated at 37°C for 16 hours.
2. Minimal inhibitory concentration & Minimal bactericidal concentration assay:
a) Mid log phase culture of the bacterial colony grown in peptone water was adjusted to 0.1 McFarland’s standard to get a uniform cell density. 100 µl of the adjusted cell suspension was seeded to each well of the 96-well microtiter plate.
b) The MIC value for 9 bacterial isolates against the 5 cream formulations at 5 different concentrations (50, 10, 1.0, 0.5 and 0.1 mg/ml) were tested. 20 µl of the cream dissolved in distilled water at different concentrations were added to the cell suspension in the well and mixed thoroughly. The final volume was made up to 200µl in each well by adding media to get the final concentration as given above. The experiment was performed in triplicates. The plate was incubated at 37°C for 16 hours.
c) 5 µl of the cell suspension was taken from one well of each concentration and added to 4995 µl of distilled water (1:1000 dilution) and mixed well.
d) 25 µl of this diluted suspension was taken and spread on a Mueller Hinton agar plate to determine MBC.
e) 5µl of cells were removed from the other wells also to maintain the volume. Spectrophotometric readings of the microtiter plate at 600 nm was taken to analyze the growth of cells in various concentrations of cream and graph was plotted (Wiegand et al., 2008).
3. Antibiofilm test:
a) 4 hours old cultures of 9 bacterial species (Clostridium tertium, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus and Staphylococcus epidermidis) were grown in TSB was taken for this experiment.
b) Bacterial culture was adjusted to 0.1 McFarland’s Standard (1 x108 CFU/ml)
c) Biofilm inhibition ability of the 5 cream formulations with 3 different concentrations was tested. The three concentrations were MIC, < MIC (Half the concentration of MIC), >MIC (Double the concentration of MIC).
d) 100 µl of the adjusted culture was seeded on the 96-well plate along with 80 µl media and incubated for an hour. 20µl of cream formulation to be tested was added and the total volume was made up to 200µl. For the control group instead of the cream formulation, 20µl of Milli Q was added.
e) 96-well plate was incubated at 37°C for 24 hours and the biofilm was quantified using crystal violet assay. The planktonic cells were removed and thereafter, 200µl of 0.1% crystal violet solution is added to stain the biofilm.
f) The plate was incubated for 20 min at room temperature and washed in distilled water to remove excess stain. The stained biofilm was solubilized with 150µl of 30% glacial acetic acid for 10 minutes at room temperature and the amount of biofilm formed was quantified by measuring OD at 540nm (Murali et al., 2014).
Observation: The MIC of test combination formulation along with individual mono compositions were evaluated against various microbes. The MIC of Clostridium tertium is tabulated in Table No 38 and figure 10; The MIC of Enterococcus faecalis is tabulated in Table No 39 and figure 11; The MIC of Escherichia coli is tabulated in Table No 40 and figure 12; The MIC of Klebsiella pneumoniae is tabulated in Table No 41 and figure 13; The MIC of Pseudomonas aeruginosa is tabulated in Table No 42 and figure 14; The MIC of Proteus mirabilis is tabulated in Table No 43 and figure 15; The MIC of Streptococcus pyogenes is tabulated in Table No 44 and figure 16; The MIC of MRSA is tabulated in Table No 45 and figure 17; The MIC of MSSA is tabulated in Table No 46 and figure 18.
Table No 38: MIC of Clostridium tertium
Test Formulation 0.1 mg/ml 0.5 mg/ml 1 mg/ml 10 mg/ml 50 mg/ml
Control 100 100 100 100 100
FA 49.10 44.17 43.00 40.44 35.56
FS 36.13 20.67 18.89 8.56 9.61
LS 62.85 48.56 31.95 6.85 8.01
FDC 100.80 50.12 52.70 31.87 19.39
Placebo 46.77 55.98 53.54 47.01 39.42
Table No 39: MIC of Enterococcus faecalis
Test Formulation 0.1 mg/ml 0.5 mg/ml 1 mg/ml 10 mg/ml 50 mg/ml
Control 100 100 100 100 100
FA 97.23 92.85 84.41 83.85 77.63
FS 100.12 97.94 82.44 75.96 72.91
LS 46.09 40.00 36.51 31.93 14.54
FDC 102.82 98.03 92.58 84.14 70.61
Placebo 33.07 18.49 11.40 6.33 5.36
Table No 40: MIC of Escherichia coli
Test Formulation 0.1 mg/ml 0.5 mg/ml 1 mg/ml 10 mg/ml 50 mg/ml
Control 100 100 100 100 100
FA 84.06 83.58 79.31 85.18 77.66
FS 96.45 96.92 97.05 96.12 94.80
LS 77.59 36.94 24.20 20.82 17.63
FDC 89.22 92.85 93.19 96.06 95.18
Placebo 54.07 33.88 5.16 3.60 3.97
Table No 41: MIC of Klebsiella pneumoniae
Test Formulation 0.1 mg/ml 0.5 mg/ml 1 mg/ml 10 mg/ml 50 mg/ml
Control 100 100 100 100 100
FA 87.33 81.18 76.29 76.13 75.15
FS 79.00 77.23 75.92 69.13 68.35
LS 58.28 29.65 27.43 25.83 13.27
FDC 80.66 80.07 68.70 66.00 53.59
Placebo 43.37 16.78 11.95 7.30 3.33
Table No 42: MIC of Pseudomonas aeruginosa
Test Formulation 0.1 mg/ml 0.5 mg/ml 1 mg/ml 10 mg/ml 50 mg/ml
Control 100 100 100 100 100
FA 74.86 67.17 67.61 64.56 64.66
FS 82.78 81.25 77.45 46.57 43.46
LS 85.32 65.66 56.46 39.53 30.76
FDC 73.64 66.90 59.30 58.65 55.68
Placebo 78.17 60.21 47.21 21.06 11.36
Table No 43: MIC of Proteus mirabilis
Test Formulation 0.1 mg/ml 0.5 mg/ml 1 mg/ml 10 mg/ml 50 mg/ml
Control 100 100 100 100 100
FA 89.67 85.79 84.33 82.52 79.88
FS 88.42 87.80 65.84 44.27 33.37
LS 80.01 53.48 51.94 45.32 23.78
FDC 89.43 91.44 82.03 75.85 70.36
Placebo 65.55 33.10 14.64 5.97 29.58
Table No 44: MIC of Streptococcus pyogenes
Test Formulation 0.1 mg/ml 0.5 mg/ml 1 mg/ml 10 mg/ml 50 mg/ml
Control 100 100 100 100 100
FA 75.43 67.28 58.68 57.30 61.14
FS 76.93 75.85 76.58 56.32 43.24
LS 69.49 41.49 36.12 33.08 33.37
FDC 74.78 57.08 43.37 38.55 38.98
Placebo 64.46 61.96 55.16 28.45 9.89
Table No 45: MIC of MRSA
Test Formulation 0.1 mg/ml 0.5 mg/ml 1 mg/ml 10 mg/ml 50 mg/ml
Control 100 100 100 100 100
FA 72.58 59.41 62.88 59.40 47.48
FS 57.46 42.03 38.88 42.98 17.43
LS 48.93 41.05 39.76 38.29 33.79
FDC 82.08 80.38 80.51 77.90 46.87
Placebo 39.15 30.56 22.94 16.74 42.30
Table No 46: MIC of MSSA
Test Formulation 0.1 mg/ml 0.5 mg/ml 1 mg/ml 10 mg/ml 50 mg/ml
Control 100 100 100 100 100
FA 76.98 75.31 72.12 65.14 59.52
FS 82.85 82.47 66.96 57.82 54.41
LS 81.38 62.07 54.53 52.06 47.00
FDC 84.99 83.93 77.45 67.52 53.18
Placebo 65.82 35.86 26.82 21.42 6.47
The Anti-biofilm assay for Clostridium tertium indicated that fusidic acid was only able to reduce the biofilm formed by significant level. Framycetin showed reduced biofilms at 50 mg/ml concentration. Lunar silver showed good effect and reduced the biofilm formed. The anti-biofilm assay of triple combination was found comparative against Framycetin only.
The Anti-biofilm assay for Escherichia coli showed that fusidic acid and lunar silver had no effect on biofilm formation. Framycetin showed little effect in reducing the biofilm levels at higher concentrations. Triple combination composition too also had very little effect against biofilm formation.
The Anti-biofilm assay for Klebsiella penumoniae showed that fusidic acid and lunar silver had no effect on biofilm formation. Framycetin showed little effect in reducing the biofilm levels. Triple combination composition too also had very little effect against biofilm formation.
The Anti-biofilm assay for Pseudomonas aeruginosa showed that fusidic acid and lunar silver had no effect on biofilm formation. Framycetin showed more than 50 % reduction in the biofilm levels at 10 mg/ml concerntration. Triple combination composition too also showed same effect as that of Framycetin against biofilm formation.
The Anti-biofilm assay results of Proteus mirabilis showed that fusidic acid and lunar silver had no effect on biofilm formation in Proteus mirabilis. Framycetin showed good reduction in biofilm levels at concentrations above 10 mg/ml. Triple combination composition showed good effect against biofilm formation only at concentration above 50 mg/ml.
The Anti-biofilm assay results of Streptococcus pyogenes showed that fusidic acid, Framycetin & lunar silver had no effect on biofilm formation. Triple combination showed some effect against biofilm formation at concentrations above 1 mg/ml.
The Anti-biofilm assay results of MRSA showed that Fusidic acid & lunar silver had no effect on biofilm formation. Framycetin showed good reduction in biofilm levels at concentrations above 10 mg/ml. Triple combination had some effect against biofilm formation in MRSA.
The Anti-biofilm assay of MSSA results showed that fusidic acid & lunar silver had no effect on biofilm formation in MSSA. Framycetin showed good reduction in biofilm levels at concentrations above 10 mg/ml. Triple combination had good effect against biofilm formation in MSSA at concentrations above 10 mg/ml.
Result: The MIC of Framycin & Lunar silver was found to be better for Clostridium tertium in comparison with other formulation, the triple combination composition was found to be less effective as compared to Framycin & Lunar silver.
The MIC of Lunar Silver was found to be better for Enterococcus faecalis in comparison with other formulation, the triple combination composition was found to be less effective as compared to Lunar silver. Surprisingly even placebo showed better activity against Enterococcus faecalis.
The MIC of Lunar Silver at concentration 0.5 mg/ml and above was found to be better for Escherichia coli in comparison with other formulation, the triple combination composition was found to be less effective as compared to Lunar silver. The MIC of Lunar Silver at concentration 0.5 mg/ml and above was found to be better for Klebsiella pneumoniae in comparison with other formulation, the triple combination composition was found to be less effective as compared to Lunar silver. Surprisingly even placebo showed better activity against Klebsiella pneumoniae.
The MIC of Lunar Silver at concentration 0.5 mg/ml and above was found to be better for Pseudomonas aeruginosa in comparison with other formulation, the triple combination composition was found to be less effective as compared to Lunar silver. Surprisingly even placebo at concentration of 50 mg/ml showed better activity against Pseudomonas aeruginosa.
The MIC of Lunar Silver at concentration 0.5 mg/ml and above was found to be better for Proteus mirabilis in comparison with other formulation, the triple combination composition was found to be less effective as compared to Lunar silver. Surprisingly, even placebo at concentration of 10 mg/ml showed better activity against Proteus mirabilis.
The MIC of triple combination was found comparative with other mono drugs for Streptococcus pyogenes in comparison with other formulation. Surprisingly even placebo at concentration of 50 mg/ml showed better activity against Streptococcus pyogenes.
The MIC of Framycetin at concentration 50 mg/ml and above was found to be better for MRSA in comparison with other formulation, the triple combination composition was found to be less effective as compared to framycetin. The MIC of triple combination was found comparative with other mono drugs for MSSA in comparison with other formulation.
The Anti-biofilm assay results indicated Triple combination composition exhibited good or comparative effects against Clostridium tertium, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pyogenes, MRSA & MSSA; whereas Triple combination composition had very little effect against Escherichia coli, Klebsiella penumoniae.
,CLAIMS:We Claim:
1. A Pharmaceutical composition comprising:
(a) Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof, and
(b) Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof.
2. The pharmaceutical composition as claimed in claim 1, wherein the composition optionally comprises silver.
3. The pharmaceutical composition as claimed in claim 2, wherein the silver is present in the form of nanoparticles.
4. The pharmaceutical composition as claimed in claim 2, wherein the silver is present in the form of lunar silver.
5. The pharmaceutical composition as claimed in claim 1, wherein Framycetin is Framycetin sulphate.
6. A pharmaceutical composition comprising combination of Fusidic acid or pharmaceutically acceptable salt, ester, prodrug thereof; and Framycetin or pharmaceutically acceptable salt, ester, prodrug thereof for topical use.
7. The pharmaceutical composition as claimed in claim 6, wherein the composition optionally comprises silver.
8. The pharmaceutical composition as claimed in any of claims 1-7, wherein the composition is a topical dosage form.
9. The pharmaceutical composition as claimed in claim 8, wherein the topical dosage form is in the form of lotion, solution, cream, ointment and paste.