Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
  • A61K9/7007—Drug-containing films, membranes or sheets

Definitions

• the present invention relates generally to methods and compositions for managing wounds, including surface wounds or skin lesions of a human or animal patient.
• the present invention further relates to methods useful in treating wounds infected by microorganisms.
• the present invention also relates to methods of promoting the repair of wounds of mouth and the skin surfaces.
• the outermost layers of the skin form a physical barrier that protects an animal from microbial invasion and the establishment of opportunistic infections. Injuries to the skin, for instance an abrasion, incision, laceration, a burn from thermal,
• a breach in the skin combined with a compromised immune system means
• burn patients are highly susceptible to opportunistic infections. Of about seventy thousand burn victims per year serious enough to require hospitalization, as many as ten thousand will die, usually from a nosocomial infection. Many burn survivors will
• ATLANTA 382820v1 suffer permanent disfigurement from the tissue damage that accompanies microbial infections. Greater treatment needs and prolonged care of bums patients in the hospital also mean that infections represent a significant financial drain. Long-term recuperative costs can also be significant. 5 Ulcers are exposed surface lesions of the skin or a mucoid layer such as the lining of the mouth, where inflamed and necrotic tissue sloughs off. This exposed tissue is also highly susceptible to opportunistic microbial invasion. In this instance, the primary infected site is localized and best treated by a topical application of an antimicrobial agent, sometimes supplemented with systemic antibiotic administration.
• tissue damage is not usually as great as for a bum, infected ulcers are discomforting to the patient, disfiguring and also life-threatening if leading to a systemic infection.
• the interval between wounding and skin repair depends on the location of the injury, the degree of damage, the apposition of the wound edges, the medical condition of the patient and the type and severity of wound contamination with foreign debris and microbes.
• Proliferating microbes cause additional and accelerated tissue damage through both direct (toxins and cellular damage) and indirect (edema and accumulation of pus) impairment of vascular supply. These changes further impair access of immune system components to the wound as well as reducing the clearance of necrotic debris and preventing systemically delivered
• CoUagenase and proteases that accumulate in association with degenerating inflammatory cells damage connective tissue proteins and further inhibit wound healing.
• Macrophages that enter a wound serve a phagocytic function as well as the critical role of stimulating the maturation of fibroblast that are ultimately responsible
• Epithelial cell proliferation and migration are necessary for re- epithelization. This process is enhanced in a moist environment that is well oxygenated by a viable blood supply.
• Initiating treatment in a timely manner is most important to reduce or prevent microbial colonization and additional pathogen-associated wound damage.
• Betadine will reduce the number of bacteria in a wound while others suggest no effect on microbial colonization In vivo data suggests that significant numbers of bacteria can survive in all noncytotoxic concentrations of Betadine solution and Nolvasan.
• 30 or antiseptics can result in the proliferation of multi-drug resistant bacteria within a hospital, an animal facility or among the animal population cared for by a veterinary facility.
• the present invention addresses the need to manage a wound, such as a bum, a
• the present invention provides methods and compositions for use in the methods for wound management that comprise contacting a wound of a patient with an effective amount of a therapeutic composition
• the antimicrobial agent(s) has increased antimicrobial activity because of the synergy with the chelating agent and maintenance of the treated area at a pH suitable for sustained antibiotic activity.
• compositions of the present invention are, therefore, useful in counteracting or preventing an infection or will be more effective against infections caused by drug- resistant strains of microbes.
• the Vitamin E promotes tissue repair, thereby reducing the likelihood of opportunistic infection, improving wound healing and reducing pain sensation.
• the wound management methods of the present invention may further include delivering an effective amount of the therapeutic composition by contacting the wound with a medical dressing having an effective amount of the composition deposited thereon.
• the present invention further provides methods suitable for delivering the therapeutic composition, comprising an antimicrobial agent, a chelating agent and a buffer and a surfactant to an oral wound.
• Figure 2 IsoboloGram, illustrating the combined effect of EDTA and neomycin (in 10 50 mM Tris) on Pseudomonas aeruginosa.
• Figure 4 Growth of Staphylococcus aureus on Mueller Hinton agar when treated alone or with combinations of EDTA, water, and neomycin. 15 Figure 5. Growth of Pseudomonas aeruginosa on Mueller Hinton agar when treated alone or with combinations of EDTA, water, and neomycin.
• Figure 6 Growth of Enterococcus faecalis on M Enterococcus agar when treated alone or with combinations of EDTA, water, and neomycin.
• the present invention addresses the need to manage a wound, such as a bum, a lesion, an incision or a laceration of the skin, or a lesion of the oral mucosa, of an animal or human patient to promote wound repair and reduce the likelihood of infection.
• a wound such as a bum, a lesion, an incision or a laceration of the skin, or a lesion of the oral mucosa, of an animal or human patient to promote wound repair and reduce the likelihood of infection.
• the present invention provides methods and compositions for wound
• the methods comprising contacting a wound of a patient with an effective amount of a therapeutic composition comprising a pharmaceutically acceptable chelating agent, a pharmaceutically acceptable pH buffering agent, an
• the antimicrobial agent(s) has increased antimicrobial activity because of the synergy with the chelating agent and maintenance of the treated area at a pH suitable for sustained antibiotic activity.
• the antimicrobial agent can, therefore, be used in 5 effective doses that are less than would be required for the same level of antimicrobial activity in the absence of the chelator.
• the compositions of the present invention are, therefore, useful in counteracting or preventing an infection or will be more effective against infections caused by drug-resistant strains of microbes.
• Vitamin E promotes tissue repair, thereby reducing the likelihood of
• the wound management methods of the present invention may further include delivering an effective amount of the therapeutic composition by contacting the wound with a medical dressing having an effective amount of the composition deposited thereon.
• the present invention further provides methods for delivery of a therapeutic agent
• composition comprising an antimicrobial agent, a chelating agent and a buffer and a surfactant for repairing a wound and inhibiting a microbial colonization of the oral mucosa, .
• compositions described herein are an amount that results in wound repair and/or a reduction in pain sensation at the site of a treated wound and the inhibition or prevention of microbial invasion of the treated wound.
• wound refers to a lesion or open wound that can expose underlying epidermal, dermal, muscular or adipoidal tissue to the air.
• Wounds include, but are not limited to, a puncture wound, an incision, a laceration, a penetrating wound, a perforating wound, a tunnel wound and the like. Wounds also include open wounds that have been sutured or otherwise mechanically closed but
• lesion and "surface lesion” as used herein refer to a circumscribed area of pathologically altered tissue, an injury or wound.
• Primary lesions are the immediate result of the pathological condition and include, but are not limited to, cuts, abrasions, vesicles, blebs, bullae chancres, pustules, tubercles or any other such 5 condition of the skin or a surface of the mouth, nose, anus or any other orifice of the body of a human or animal, or to the surface layers of the eye including the conjunctiva and cornea., or secondary lesions that later develop from a primary lesion and includes, but is not limited to, fissures and ulcers and other wounds.
• wound management refers to therapeutic methods that induce 10 and/or promote repair of a wound including, but not limited to, arresting tissue damage such as necrotization, promoting tissue growth and repair, reduction or elimination of an established microbial infection of the wound and prevention of new or additional microbial infection or colonization. The term may further include reducing or eliminating the sensation of pain attributable to a wound. 15
• wound healing and “wound repair” refer to a process involving tissue growth that partially or totally closes a wound, repairs a breach in the dermis or epidermis and partially or totally restores the barrier properties of the skin or the repair of the surface layers of the eye including the conjunctiva and cornea.
• microbial infection refers to any pathological
• microbial infection may include any systemic infection that is amenable to inhibition by application of the antimicrobial compositions of the present invention.
• bum refers to tissue injury of the skin caused by 25 thermal, chemical, or radiation exposure or abrasive friction.
• a bum may be a "first- degree bum” with superficial damage to the outer cornified layer, a “second-degree bum” with damage extends down into the epidermal layer of cells but is not of sufficient extent that regeneration of the skin is prevented, or a "third-degree bum” where the injury extends below the dermis to the underlying tissue and wherein repair 30 of the skin is not possible without grafting.
• ATLANTA 382820v1 includes, but is not limited to, callous ulcers, chronic leg ulcers, decubitus, denture ulcers of the oral mucosa, traumatic ulcers of the mouth, infections stomatitis of the mouth and any type of secondary lesion that is a breach of the cornified and the epidermal layer of the skin or the mucosal surface of the mouth.
• antiimicrobial agent refers to the compounds and combinations thereof, including bacteristatic or bactericidal compositions or agents, that may be administered to an animal or human and which inhibit the proliferation of a microbial infection.
• the administered compound or combination of compounds will elicit, at most, a temporary detrimental effect on the health of the recipient human or animal.
• chelating agent refers to any organic or inorganic
• pH buffering agent refers to any pharmaceutically acceptable organic or inorganic compound or combination of compounds that will maintain the pH of an antibiotic-containing solution within 0.5 pH units of a selected pH value.
• carrier refers to any pharmaceutically acceptable solvent of antibiotics, chelating agents and pH buffering agents that will allow a therapeutic composition to be administered directly to a wound of the skin or to the oral mucosa.
• the carrier will also allow a composition to be applied to a medical dressing for application to such a wound.
• solvent as, but not limited to, water, saline, physiological saline, ointments, creams, oil-water emulsions, gels, or any other solvent or combination of solvents and compounds known to one of skill in the art that is pharmaceutically and physiologically acceptable to the recipient human or animal.
• carrier is understood not to include surfactants such as detergents, non-ionic surfactants such as
• surfactant refers to any detergent or other pharmaceutically acceptable non-ionic compound that lowers surface tension of an aqueous solution.
• One aspect of the present invention provides methods for wound management 5 wherein a wound of a human or animal patient is contacted with an effective amount a therapeutic composition comprising a chelating agent, a buffer, an antimicrobial agent, Vitamin E, a surfactant and a carrier. More than one antimicrobial agent may be used to inhibit the proliferation of a single invasive organism, or a mixed population of invasive organisms. The antimicrobial agent(s) should be selected after
• compositions of the present invention administered to a wound results in accelerated wound repair with reduced or no sepsis, as described in Examples 5-14 below. Even with wounds that penetrated the dermal layer, there is reduced pain
• tissue repair restrict opportunistic infections that would otherwise prolong the period of wound healing, increase the extent of the wound or even develop to threaten the life of the infected patient.
• the wound Before applying the therapeutic composition to the patient, the wound can be any suitable material.
• the wound may be prewashed before the application of the therapeutic composition using a composition comprising a chelating
• the composition comprises about 8 mM EDTA and 20 M Tris with between about 2% and about 30% v/v of cocamidopropyl betaine
• compositions used in the methods of wound management herein described may be applied to a wound by any number of methods including as a lavage where the wound is washed or irrigated.
• a lavage where the wound is washed or irrigated.
• the wound is washed or irrigated.
• ATLANTA 382820v1 compositions are absorbed onto the surface of the fibers of a wound dressing before or during the treatment, ensuring that while the wound is ventilated it is still subject to contact with the therapeutic compositions for a prolonged period.
• the treated 5 wound is in an oral mucosal surface of the patient.
• the therapeutic composition can be applied as a mouth wash or rinse or in combination with a dressing that may be secured over the wound.
• the therapeutic compositions of the invention are ophthalmic compositions suitable for administering to the surface of an eye for the repair or healing of a wound to the conjunctiva or
• the therapeutic compositions of the present invention may also be used as a bath for the total or partial immersion of a human or animal for the treatment of multiple skin lesions such as for managing or burnt foot, or hand, or large wound, of a human or animal.
• compositions of the present methods may be selected from ethylenediamenetetracetic acid (EDTA), triethylene tetramine dihydrochloride (TRIEN), ethylene glycol-bis (beta-aminoethyl ether)-N, N, N ⁇ N'-tetracetic acid (EGTA), diethylenetriamin- pentaacetic acid (DPTA), triethylenetetramine hexaacetic acid (TTG), deferoxamine, Dimercaprol, edetate calcium disodium, zinc citrate, penicilamine succimer and
• the chelating agent when delivered to a wound of a human or animal patient will have a concentration between from about 1 M to about 250 mM, more preferably from
• the chelating agent is EDTA having a concentration of about 8 mM.
• compositions of the present invention also include a pharmaceutically acceptable pH buffering agent that preferably will maintain the pH
• a pH buffering agent may be selected from, but is not limited to, Tris (hydroxymethyl) aminomethane (tromethaprim; TRIZMA
• the pH of the antimicrobial composition in solution is about 8.0.
• the buffering agent when delivered to a wound, has an effective dose of between about 5 mM and about 5 250 mM, more preferably between about 5 mM and about 100 mM, most preferably between about 10 mM and about 100 mM. In a preferred embodiment the buffer agent has a concentration of about 20 mM.
• compositions of the present invention may also comprise at least one antimicrobial agent.
• compositions of the present invention may be any opportunistic infection of a wound by a bacterium, or a multiple infection of more than one species of bacteria.
• Microbial species that may cause infections inhibited by the methods of the present invention include fungi and bacterial species that may cause infections of a bum, lesion, oral mucosal lesion or other wound of a human or animal including, but are
• Aerobacter aerongenes Aeromonas spp., Bacillus spp., Bordetella spp, Campylobacter spp., Chlamydia spp., Corynebacterium spp., Desulfovibrio spp., Escherichia coli, enteropathogenic E. coli, Enterotoxin-producing E coli, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophiia, Leptospira spp., Mycobacterium tuberculosis, M. bovis, Neisseria gonorrhoeae, N meningitidis,
• Nocardia spp. Proteus mirabilis, P vulgaris, Pseudomonas aeruginosa, Rhodococcus equi, Salmonella enteridis, S. typhimurium, S. typhosa, Shigella sonnei, S dysenterae, Staphylococcus aureus, Staph. epidermidis, Streptococcus anginosus, S. mutans, Vibrio cholerae, Yersinia pestis, Y. pseudotuberculosis, Actinomycetes spp., and Streptomyces spp.
• the action of the antimicrobial agent can be either bacteriostatic wherein the antibiotic arrests the proliferation of, but does not necessarily kill, the microorganism or the activity of the antibiotic can be bacteriocidal and kill the organism or a combination of activities.
• Antibiotics suitable for use in the wound management methods of the present invention include, but are not limited to, ⁇ -lactams (penicillins).
• tetracyclines include, but are not limited to, immunocycline, chlortetracycline, oxytetracycline, demeclocycline, 5 methacycline, doxycycline and minocycline and the like.
• aminoglycoside antibiotics include, but are not limited to, gentamicin, amikacin and neomycin and the like.
• a chelating agent may be used and the amount of the antimicrobial composition may be adjusted accordingly so as to inhibit growth of the microorganism.
• concentrations and amounts of the antimicrobial agent and chelating agent may be adjusted to levels that are physiologically accepted by the exposed tissue of the injury or lesion and effective against the microbial infection of the skin injury or skin lesion.
• the concentration of the antibiotic is in the range of about 0.04 mg/ml to about 25 mg/ml and the concentration of the chelating agent in the carrier is in the range of about 0.1 mM to about 100.0 mM.
• the antibiotic is a penicillin, an aminoglycoside, a vancomycin, a chloramphenicol, an erythromycin, a tetracycline, gentamicin,
• the antibiotic is tetracycline.
• the antibiotic is neomycin.
• the antibiotic is amikacin.
• the antibiotic is gentamicin.
• a combination of antibiotics may be used depending upon the antibiotic resistance profiles of the microbial population of the wound.
• the therapeutic compositions for use in the methods of wound management 5 also comprise a surfactant that can useful in cleaning a wound or contributing to bactericidal activity of the administered compositions.
• Suitable surfactants include, but are not limited to, phospholipids such as lecithin, including soy lecithin and detergents.
• the surfactant selected for application to a wound or skin surface is mild and not lead to extensive irritation or promote further tissue damage to
• Suitable nonionic surfactants which can be used are, for example: fatty alcohol ethoxylates (alkylpolyethylene glycols); alkylphenol polyethylene glycols; alkyl mercaptan polyethylene glycols; fatty amine ethoxylates (alkylaminopolyethylene glycols); fatty acid ethoxylates (acylpolyethylene glycols); polypropylene glycol
• ethoxylates Pluronic
• fatty acid alkylolamides fatty acid amide polyethylene glycols
• alkyl polyglycosides N-alkyl-, N-alkoxypolyhydroxy fatty acid amide, in particular N-methyl-fatty acid glucamide, sucrose esters; sorbitol esters,and esters of sorbitol polyglycol ethers.
• a preferred surfactant is polypropylene glycol ethoxylates with a preferred concentration of between about 5% wt % and about 25% wt %.
• the 20 most preferred surfactant is Pluronic F-127 (Poloxamer 407).
• the surfactant comprises lecithin with or without the addition of Pluronic F-127, the Pluronic F-127 being between about 2 and about 20 wt % for increasing the viscosity or gelling of the compositions.
• the therapeutic compositions further include Vitamin E that will promote Vitamin E.
• tissue growth and repair and reduce the pain experienced at the site of the skin injury.
• tissue repair not only is discomfort to the patient reduced, but there may be less scar tissue formation and hence less permanent disfiguring of the patient.
• faster healing of a skin injury or lesion is useful for reducing the likelihood of a nosocomial infection and the problems associated therewith.
• Vitamin E when delivered to a wound of a patient by the methods of the present invention, has a concentration of between about 20 IU/ml and 500 IU/ml, preferably between about 50 IU/ml and 500 IU/ml, more preferably between about 100 IU/ml
• the Vitamin E has a concentration of about 100 IU/ml. In another embodiment, the concentration is between about 325 IU/ml and about 360 IU/ml..
• the therapeutic compositions for use in the methods of the invention 5 preferably include a pharmaceutically acceptable carrier that provides the medium in which are dissolved or suspended the constituents of the compositions.
• Suitable carriers include any aqueous medium, oil, emulsion, ointment and the like that will allow the therapeutic compositions to be delivered to the target wound without increasing damage to the tissues of the wound.
• compositions of the invention can be prepared as precursor solutions, or as sterile powders or concentrates that are useful for the extemporaneous preparation of the administered compositions.
• the compositions may further include a preservative to extend the shelf- life of the composition.
• a particularly useful preservative is scorbic acid, preferably
• a preferred amount of the preservative is between about 0.1 wt % to about 5 wt %. A more preferred amount is about 0.2 wt %.
• Medical dressings suitable for use in the methods of the present invention for contacting a wound with the therapeutic compositions can be any material that is biologically acceptable and suitable for placing over any wound such as a bum, or a
• the support may be a woven or non-woven fabric of synthetic or non- synthetic fibers, or any combination thereof.
• the dressing may also comprise a support, such as a polymer foam, a natural or man-made sponge, a gel or a membrane that may absorb or have disposed thereon, a therapeutic composition.
• KYTM sodium carboxymethylcellulose 7H 4F (Hercules, Inc., Wilmington, DE)
• a film, a natural or synthetic polymer, or a rigid or malleable material that is known to one of ordinary skill in the art as being acceptable for insertion in the mouth of a human or animal, and which will place an antimicrobial composition according to
• the support is a gauze.
• the gauze may be absorbent and can be wetted with an antimicrobial
• the gauze may be impregnated with the therapeutic composition and then dried. This allows the impregnated 5 dressing to be stored for later use, or to avoid excessively dampening an injured area.
• a therapeutic composition is absorbed on the surface of the support material of the medical dressing. The composition may be applied to the surface by wetting the surface with a solution of the composition and drying the support to deposit the composition thereon.
• concentration of the composition that is effective for promoting wound repair and/or against the proliferation of a microorganism may be attained when the dressing is wetted by the patient's body.
• compositions comprising about 2 to about 50 wt % of a pharmaceutically acceptable chelating
• the surfactant is a detergent such as, but not limited to, a mild detergent that will avoid irritation to the surface of the skin or other tissue to which the therapeutic composition is applied.
• a mild detergent such as, but not limited to, a mild detergent that will avoid irritation to the surface of the skin or other tissue to which the therapeutic composition is applied.
• one suitable detergent is cocamidopropyl betaine, but it is contemplated that any detergent known to those of
• the detergent is about 3 to about 33 wt % of a therapeutic composition.
• the surfactant is lecithin.
• composition further comprises
• the embodiments may also include from about 2 to about 50 wt % of Vitamin E and/or from about 2 to about 98 wt % of a pharmaceutically acceptable carrier.
• the therapeutic compositions include a preservative that will increase the shelf-life of the compositions.
• a typical preservative is scorbic acid, or the salts thereof.
• the chelating agent is selected from the group consisting of ethylenediamenetetracetic acid (EDTA), triethylene tetramine dihydrochloride (TRIEN), ethylene glycol-bis (beta-aminoethyl ether)-N,
• compositions can further comprise 1 to 20 wt
• an anti-inflammatory agent such as, but not limited to dexamethasone.
• the antimicrobial agent(s) that may be included in the various embodiments of the compositions include, but are not limited to, a ⁇ -lactam, an aminoglycoside, a vancomycin, a bacitracin, a macrolide, an erythromycin, a lincosamide, a 10 chloramphenicol, a tetracycline, a gentamicin, an amphotericin, a cefazolin, a clindamycin, a mupirocin, a nalidixic acid, a sulfonamide and trimethoprim, a streptomycin, a rifampicin, a metronidazole, a quinolone, a novobiocin, a polymixin and a gramicidin.
• the antibiotic s selected from the group consisting of a ⁇ -lactam, an aminoglycoside, a vancomycin, a chloramphenicol, an 15 erythromycin, a tetracycline, gentamicin, nalidixic acid and a streptomycin.
• the antimicrobial agent is oxytetracycline.
• the antimicrobial agent is amikacin.
• the antimicrobial agent is neomycin.
• compositions of the present invention may also include a carrier, as described 20 above, for dissolving or suspending the components of the therapeutic composition.
• the pharmaceutically acceptable pH buffering agent can be Tris (hydroxymethyl) aminomethane (TRIZMA Base) which, when dissolved in a carrier will have a .concentration of between about 5 mM and about 250 mM, preferably between about 5 mM and about lOOmM, more 25 preferably between about 10 mM and about 100 mM. In a most preferred embodiment, theconcentration of the buffering agent is about 20 mM.
• Tris (hydroxymethyl) aminomethane TriZMA Base
• the Vitamin E when dissolved in a carrier has a concentration of between about 20 IU/ml and 500 IU/ml, preferably between about 50 IU/ml and 500 IU/ml, more preferably yet between about
• Vitamin 1 30 100 IU/ml and about 500 IU/ml. In one embodiment the concentration of the Vitamin
• Vitamin E is between about 325 IU/ml and about 360 IU/ml.
• the Vitamin E may be prepared by dissolving in hydrous lanolin as a carrier in a volume ratio from about
• the pharmaceutically acceptable carrier is non-allergenic.
• kits that comprise therapeutic compositions as described above, or the components to prepare the compositions, and packaging 5 that includes instructions on how to prepare and use the compositions to manage a wound and promote healing thereof.
• One embodiment of the invention therefore, comprises a vessel containing a pharmaceutically acceptable chelating agent, a pharmaceutically acceptable buffering agent suitable for maintaining the pH of the site of a treated wound, a pharmaceutically acceptable antimicrobial agent, Vitamin E,
• the packaging material comprises instructions directing the use of the kit for preparing the therapeutic composition of the present invention and delivering the composition to a wound or to the mouth of a human or animal to accelerate healing of a wound.
• the kit may further comprise separate vessels containing Vitamin E and/or surfactant,
• Results were plotted as isobolograms for the determination of antagonistic, neutral or additive, or synergistic effects.
• FICs of the two test solutions were plotted individually on the x-axis and y-axis to determine the 10 effect of combining the two test solutions on bacterial growth.
• a line that curves away from the zero point and the coordinates indicates antagonism.
• a straight line indicates neutral or additive effects. Lines that curves toward the zero point and the coordinates are indicative of synergism if there is at least a 4-fold decrease in the MIC of each compound, when used in combination, as compared with the MIC of each test 15 compound alone.
• FIC index is equal to the sum of the values of FIC for the individual drugs:
• An FIC index greater than 1.0 indicates an antagonistic interaction
• an FIC index of 1.0 indicates addition
• an FIC index of less than or equal to 0.5 indicates 25 synergism between the two test agents.
• the organisms of this study were isolated from human bum patients. They included strains of methicillin resistant Staphylococcus aureus, and vancomycin
• MLB Mueller-Hinton agar
• the EDTA-Tris treatment solutions were prepared from a stock solution containing 0.5 mols/1 sodium EDTA and 1.0 mols/1 Tris-HCl, pH 8.0.
• the treatment solutions contained 5mM sodium EDTA and 50 mM Tris-HCl with or without of 5 neomycin sulfate 1 mg / ml.
• Antibiotic resistance profiles were determined by the disc diffusion method on
• MHA nalidixic acid
• MICs Minimal Inhibitory Concentrations
• MHCs Minimal Bactericidal Concentrations
• EDTA-Tris and neomycin were determined by the broth- dilution microtiter method in MHB or BHI according to the method of B lair et al., Manual of Clinical Microbiology, p.307 (pub: Am. Soc. Microbiol Williams and 15 Wilkins, Baltimore 1970), incorporated herein by reference in its entirety.
• Example 3 In vitro effect of EDTA-Tris and neomycin on Enterococcus faecalis, Pseudomonas aerueinosa, and Staphylococcus aureus
• the multipoint contactors were sterilized by autoclaving. To collect samples, a multipoint contactor was touched to an overnight bacterial culture grown on 2xNA as described above. Replicate plates were then inoculated by lightly pressing the needles bearing the test bacteria onto either MHA plates, BA plates or EA plates for Ps. aeruginosa, Staph. aureus and Ent. faecalis respectively.
• Example 4 The antibiotic resistance profiles. MIC and MBC values for test strains of Staph. aureus, Ps. aerusinosa, and Ent. faecalis
• the antibiotic resistance profiles and MIC values for test strains of Staph. aureus, Ps. aeruginosa, and Ent. faecalis are shown on Table 1. 10 Table 1. Antibiotic resistance profiles of Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis .
• MIC Minimal Inhibitory Concentration
• the MBC values for EDTA and neomycin were decreased by at least 75% for
• MBC Minimal Bactericidal Concentrations (MBC), of Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis reacted with EDTA (mM) and neomycin (mg/ml) in 50 mM Tris.
• the MBC values for EDTA and neomycin when combined were decreased by 50% as compared to the bactericidal 10 effect of each when individually administered.
• MBC values for EDTA and neomycin when in combination were decreased 99.2% compared to when EDTA or neomycin were individually administered.
• MBC values of EDTA and neomycin were both reduced 75% compared to when EDTA and neomycin were
• ATLANTA 382820V 1 Example 6: Treatment of a skin burn of a dog and antimicrobial protection of graft donor sites by Vitamin E with EDTA-Tris and antibiotic
• the dog was given initial 5 emergency treatment for 5 days and the burned area cultured for microbial infection, identifying: ⁇ -hemolytic E. coli, Klebsiella oxytoca, Proteus sp., and Enterococcus sp.
• the dog was administered cefazolin systemically and the burned area cleared of tissue debris and wetted with a solution of EDTA-Tris and neomycin daily.
• the bum area was free of the four bacteria after 3 days of systemic and topical EDTA-Tris- 10 neomycin therapy.
• neomycin was replaced with amikacin.
• the dog received an autologous skin graft approximately three weeks after the bum incident and the donor site treated with EDTA-Tris-amikacin and 100 IU of Vitamin E. The dog was discharged from veterinarian hospital care two weeks later.
• Example 7 Treatment of microbially infected skin and oral lesions
• a composition comprising EDTA, Tris and neomycin in KYTM gel carrier was applied to skin ulcers of a turtle, a snake and a frog. Infection was reduced until eliminated, and the treated animals fully healed of their injuries and infections.
• the mouth was swabbed with a cotton-tipped swab twice daily for a week with a solution containing 5 mM EDTA, 50 mM Tris, and 2 mg/ml neomycin. After the first week, the mouth and gums were swabbed twice weekly for a further month. Following clearance of the infection from the animal's mouth, there was no recurrence for at least one year. A similar human oral lesion also responded to this
• mouthwashes also containing EDTA, Tris and neomycin, as above, were used to treat and heal infections stomatitis of the oral cavities of iguanas and snakes.
• Example 8 Vitamin E does not reduce the antibacterial efficacy of EDTA-Tris 30 and antibiotics
• Table 4 Effect of Vitamin E on the Efficacy of EDTA-Tris and Antibiotics when Tested against Pseudomonas aeruginosa.
• Example 9 Self-inflicted full thickness flap wound of labial mucosa
• the patient had a self-inflicted full thickness flap wound of labial mucosa. Based on previous similar wounds untreated, it was anticipated that severe swelling and pain would persist for 7 to 10 days. The wound was rinsed with tap water and immediately contacted with. 5 mM EDTA, 50 mM Tris and 1.0 mg/ml Neomycin in
• Example 10 Third degree gasoline burn in dog.
• a dog suffering fro third degree bums received an application of 5 mM
• Example 12 Management of a burn wound
• a first to second degree bum wound on the inner surface of the lower arm of 15 approximately 1 week duration was treated. While the bu was healing, it remained crusty and pruritic.
• Application of 35 mis of hydrous lanolin, 15 mis of 333 IU/ml Vitamin E, and 0.73 gms of EDTA, 0.6 gms of Tris dissolved in 2 mis of distilled water and 0.1 mg/ml of ampicillin resulted in cessation of pruritis within 15 minutes. Repeat application when the wound begin to itch resulted in similar cessation of 20 itching
• a composition containing 35 mis of hydrous lanolin, 15 mis of 333 IU/ml Vitamin E, and 0.73 gms of EDTA, 0.6 gms of Tris dissolved in 2 mis of distilled 25 water and 0.1 mg/ml of ampicillin was applied once to a small painful cut on a finger. After a single application, the pain was gone along with redness. The lesion healed quickly thereafter. The medication was applied to a 1 day old very painful toe lesion that typically are sore and red for several days. After 1 application, the pain was gone and the lesion rapidly healed.
• ATLANTA 382820v1 superficial myositis faced either amputation or euthanasia
• the wound was debrided, flushed with sterile saline and 3 gms EDTA, 2.4 gms Tris and 100 mg of ampicillin dissolved in a liter of distilled water. Initially, the wound was dressed with a wet bandage soaked in above solution.
• the wound was coated with an emulsion of 35 mis of hydrous lanolin, 15 mis of 333 IU/ml Vitamin E, and 0.73 gms of EDTA, 0.6 gms of Tris dissolved in 2 mis of distilled water and 0.1 mg/ml of ampicillin once per day (if a bandage was applied) or 3 to 4 times a day (no bandage was applied).
• the cat showed no discomfort even when the wound was left open, and a sufficient granulation bed was formed to allow surgical closure in two stages.
• Example 16 Stock formulations having increased Vitamin E compared to formulations with hydrous lanolin
• Vitamin E mixed tocopherols (333 - 352 IU/g) 50 gms
• Lecithin and sorbic acid were dispersed in Vitamin E oil; lecithin was 25 dissolved over a 12 - 24 hour period to form an oily solution to which could be added a lipid soluble antibiotic or other agent (s).
• the Pluronic F-127 and potassium sorbate were place in a volumetric flask; add sufficient purified water to make 100 ml of solution; immediately refrigerate to 5 keep pourable, since at room temperature a gel is formed.
• water soluble antibiotic, or other agent (s) may be added to produce the desired effect.
• Tris-EDTA may be added to the water at a concentration such that the final formulation administered to the patient was about 8 mM EDTA and 20 mM Tris.
• ratios of oleaginous and aqueous parts were prepared. For example, mixing 5 ml of each produced a soft creamy emulsion, with a low coefficient of spreading containing 176 IU/ml of Vitamin E, and 0.2% sorbic acid/potassium sorbate as a mold/yeast/fungus inhibitor (potassium sorbate is the potassium salt of sorbic acid; sorbic acid is used in oleaginous solutions, the salt in aqueous solutions).
• Lecithin as the surfactant in the above discussion was 50%; the effective concentration range for lecithin in the above formulations was 20-50 wt %; it could also be formulated to the lower to a range of about 2-50 wt %.

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