Synergistic antibacterial activity of medium polarity oils in combination with antibacterial agents on bacterial biofilms

Abstract

The compositions of the present invention comprise at least one medium polarity oil and at least one antibacterial agent, the combination of which produces a synergistic antibacterial effect against bacterial biofilms. Methods are disclosed for the reduction of bacteria in and/or elimination of bacterial biofilms on biological and non-biological surfaces, as well as methods for the treatment of wounds, skin lesions, mucous membrane lesions, and other biological surfaces infected or contaminated with bacterial biofilms.

Claims

1. A method of treating a wound, mucous membrane lesion, or skin lesion infected or contaminated with a bacterial biofilm, the method comprising topically administering to the wound, mucous membrane lesion, or skin lesion a composition consisting essentially of a pharmaceutical carrier and a sole anti-bacterial agent, wherein the anti-bacterial agent consists of a combination of a medium polarity oil having an octanol-water partition coefficient (log P) of 0.5 to 2.0 and at least one iodine compound, wherein the medium polarity oil is benzyl alcohol or propyl gallate and is at a concentration of 4.5% w/w to 5.5% w/w, wherein the iodine compound is cadexomer-iodine at a concentration of 40% w/w to 60% w/w, and wherein the combination consisting of the medium polarity oil and the iodine compound produces a synergistic antibacterial effect against bacteria in the biofilm.

2. The method of claim 1, wherein the pharmaceutical carrier is a lotion, solution, suspension, liquid, emulsion, cream, gel, ringing gel, ointment, paste, aerosol spray, aerosol foam, non-aerosol spray, non-aerosol foam, film, or sheet.

3. The method of claim 1, wherein the bacterial biofilm is a gram-positive bacterial biofilm.

4. The method of claim 3, wherein the gram-positive bacterium is Staphylococcus aureus or methicillin resistant Staphylococcus aureus (MRSA).

5. The method of claim 1, wherein the bacterial biofilm is a gram-negative bacterial biofilm.

6. The method of claim 5, wherein the gram-negative bacterial biofilm is Pseudomonas aeruginosa.

7. The method of claim 1, wherein the wound is a chronic wound.

8. The method of claim 7, wherein the chronic wound is a diabetic foot ulcer, venous ulcer, arterial ulcer, decubitus ulcer, stasis ulcer, pressure ulcer, or burn.

9. The method of claim 1, wherein the mucous membrane lesion or skin lesion is a blister, ulceration, abrasion, wart, scrape, or infection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the log reduction of bacteria in an in-vitro S. aureus biofilm model after treatment with various oils of various polarity vs. moist control.

(2) FIG. 2 shows the comparison of the retention times of the reference standards to the retention time of CAPMUL MCM to calculate the log P value of CAPMUL MCM as per ASTM method.

(3) FIG. 3 shows the log reduction of bacteria in the P. aeruginosa biofilm model after treatment with the treatment formulations from Example 2 (cadexomer iodine based formulations) vs. moist control.

(4) FIG. 4 shows the log reduction of bacteria in the P. aeruginosa biofilm model after treatment with the treatment formulations from Example 2 (silver chloride based formulations) vs. moist control.

(5) FIG. 5 shows the log reduction of bacteria in the P. aeruginosa biofilm model after treatment with the treatment formulations from Example 2 (other silver based formulations) vs. moist control.

(6) FIG. 6 shows the log reduction of bacteria in the P. aeruginosa biofilm model after treatment with the treatment formulations from Example 2 (povidone-iodine based formulations) vs. moist control.

(7) FIG. 7 shows the log reduction of bacteria in the P. aeruginosa biofilm model after treatment with the treatment formulations from Example 2 (gentamicin based formulations) vs. moist control.

(8) FIG. 8 shows the log reduction of bacteria in the P. aeruginosa biofilm model after treatment with the treatment formulations from Example 2 (colistin based formulations) vs. moist control.

DETAILED DESCRIPTION OF THE INVENTION

(9) The present invention relates to methods and compositions useful for the reduction of bacteria in and/or elimination of bacterial biofilms on surfaces. In particular, the present invention provides compositions which exhibit activity against bacterial biofilms, and methods of using the same to treat biological and non-biological surfaces infected or contaminated with bacterial biofilms by reducing bacteria in or eliminating the bacterial biofilm. In one aspect, the present invention relates to methods and compositions useful for the treatment of wounds, skin lesions, mucous membrane lesions, and other biological surfaces infected or contaminated with bacterial biofilms. In another aspect, the present invention relates to methods and compositions useful for the reduction of bacteria in and/or elimination of bacterial biofilms on non-biological surfaces such as medical devices.

(10) The compositions of the present invention comprise at least one medium polarity oil and at least one antibacterial agent. Surprisingly, the combination of at least one medium polarity oil and at least one antibacterial agent produces a synergistic antibacterial effect against bacterial biofilms. Stated another way, the total antibacterial activity against bacterial biofilms of the combination of the two components, i.e., the medium polarity oil plus the antibacterial agent, is greater than the sum of the antibacterial activity against biofilms of each component when measured separately.

I. COMPOSITIONS

(11) The compositions of the present invention comprise at least one medium polarity oil and at least one antibacterial agent, both of which are described below in a non-limiting manner. The concentrations of the at least one medium polarity oil and the at least one antibacterial agent in the compositions are at an amount that exhibits synergistic antibacterial activity against bacterial biofilms.

(12) The compositions of the invention can also include an acceptable carrier such as a carrier suitable for topical products or a carrier suitable for application to a non-biological surface, such as a medical device. The carrier may also be a pharmaceutical carrier suitable for application to biological surfaces including topical surfaces. The compositions of the present invention can comprise carriers suitable for topical treatment of skin, mucous membranes, and wounds. Non-limiting examples of carriers include lotions, solutions, suspensions, liquids, emulsions, creams, gels, ringing gels, ointments, pastes, aerosol sprays, aerosol foams, non-aerosol sprays, non-aerosol foams, films, powders, and sheets. The compositions can be impregnated in gauzes, bandages, or other wound dressing materials. Non-limiting examples of carriers suitable for topical treatment of skin, mucous membranes and wounds include those disclosed in U.S. Pat. No. 6,399,092, herein incorporated by reference. Other examples of carriers suitable for topical treatment of skin, mucous membranes, and wounds include polyethylene glycol ointments. Especially suitable carriers include ringing gels, which are viscous microemulsions that are generally transparent, and exhibit a ringing phenomenon when excited to mechanical vibrations. Ringing gels can be O/W or W/O. Ringing gels are inherently viscous and do not need the addition of thickening agents in order to provide a viscous composition. Formulations of ringing gels are known in the art. An example of a formulation of a ringing gel carrier comprises water, a glyceryl ester such as CAPMUL® MCM, and a poloxamer such as poloxamer-407. Ringing gels are especially suitable for topical applications, such as to wounds or skin, in that the viscous composition will not run out of a wound or run off the skin when applied. An additional benefit of the use of ringing gel carriers for the present invention is that since thickening agents are not needed, there is less chance of ingredients interfering with the synergistic activity of the composition and less chance for irritation when applied to biological surfaces such as skin or wounds. Viscosity values for ringing gel carriers of the present invention can be at least 6250 cps, or at least 12,500 cps, or at least 25,000 cps, or at least 50,000 cps, or at least 60,000 cps, or at least 75,000 cps, or 6250 cps to 125,000 cps, or 12,500 cps to 125,000 cps, or 25,000 cps to 125,000 cps, or 50,000 cps to 125,000 cps, or 60,000 cps to 125,000 cps, or 75,000 cps to 125,000 cps when measured with a Brookfield viscometer with a small sample adapter, spindle #14, 6R chamber, at room temperature (22°-25 ° C.), at 10 rpm for 1 minute.

(13) The compositions of the invention may further comprise functional ingredients suitable for use in compositions for application to biological surfaces or non-biological surfaces. Non-limiting examples include absorbents, super absorbents, antibacterial agents, antioxidants, binders, buffering agents, bulking agents, chelating agents, colorants, biocides, deodorant agents, emulsion stabilizers, film formers, fragrance ingredients, humectants, lytic agents, enzymatic agents, opacifying agents, oxidizing agents, pH adjusters, plasticizers, preservatives, reducing agents, emollient skin conditioning agents, humectant skin conditioning agents, moisturizers, surfactants, emulsifying agents, cleansing agents, foaming agents, hydrotopes, solvents, suspending agents, viscosity control agents (rheology modifiers), viscosity increasing agents (thickeners), and propellants. Listings and monographs of suitable functional ingredients are disclosed in McCutcheon's Vol. 1 Emulsifiers & Detergents, and Vol. 2 Functional Materials, 2001, herein incorporated by reference.

(14) The compositions of the invention can further comprise pharmaceutically active ingredients, cosmetically active ingredients, and vulnerary agents suitable for topical use. The compositions can be sterile or preserved with preservatives. In some embodiments, the compositions do not include C9-C12 aliphatic alcohols. In some embodiments, the compositions do not include organic acids. In some embodiments, the compositions do not include glyceryl monolaurate.

(15) The compositions of the present invention may be packaged in any suitable package configuration. Non-limiting examples include bottles, lotion pumps, toddles, tubes, jars, non-aerosol pump sprayers, aerosol containers, pouches, and packets. The packages may be configured for single-use or multiple-use administration.

(16) A. Medium Polarity Oils

(17) The compositions of the invention comprise at least one medium polarity oil with an octanol-water partition coefficient (log P) of 0.5 to 2.0. In some embodiments the octanol-water partition coefficient (log P) is 0.7 to 1.9. In other embodiments, the octanol-water partition coefficient (log P) is 0.7 to 1.8. The octanol-water partition coefficient, “K.sub.ow”, also represented as “P”, is the ratio of the equilibrium molar concentration of a chemical in n-octanol and water, in dilute solution at a given temperature. The value is usually expressed as the decadic logarithm of this coefficient represented as, “log K.sub.ow”, also represented as “log P”. The octanol-water partition coefficient is a measure of the hydrophobicity and hydrophilicity of a substance. Non-polar (hydrophobic) compounds have a high log P whereas polar (hydrophilic) compounds have a low log P. Medium polarity compounds have a log P in between non-polar and polar compounds. For the purposes of the present invention, medium polarity compounds have a log P of 0.5 to 2.0, or 0.7 to 1.9, or 0.7 to 1.8. For the purposes of the present invention, the term “medium polarity oil” is used interchangeably with the term “medium polarity compound,” and means a compound that is liquid or solid at room temperature having a log P of 0.5 to 2.0, or 0.7 to 1.9, or 0.7 to 1.8.

(18) Many methods exist for determining the octanol-water partition coefficient of a substance. However, for purposes of the present invention, the experimental determination of an octanol-water partition coefficient value utilizes a reverse phase (RP) HPLC method. One such method is described in the ASTM Standard Test Method for Partition Coefficient (N-Octanol/Water) Estimation by Liquid Chromatography, Designation E 1147-92 (Reapproved 2005), herein incorporated by reference. The methodology of the ASTM method is as follows. The test substance (solute) is injected onto a liquid chromatograph column containing a solid-phase support onto which a commercially available long-chain hydrocarbon (for example C8 or C18) has been bonded. Chemicals injected onto such a column move along it by partitioning between the mobile phase and the stationary hydrocarbon phase. A methanol/water solvent system is typically used to elute the solute which is subsequently analyzed using an ultraviolet/visible absorption detector, refractive index detector, electro-chemical detector, or other appropriate detector. If the test substance is not amenable to detection by the available LC detectors, the analyst may collect fractions of the column effluent and analyze for the test substance using gas chromatography, liquid scintillation, or other appropriate technique. The K.sub.ow of the test compound is estimated from a linear regression equation developed from a plot of log (t.sub.R−t.sub.o) versus log K.sub.ow, using data determined in a calibration step that involves injecting into the chromatograph a mixture of reference chemicals. A calibration graph of log (t.sub.R−t.sub.o) versus log K.sub.ow is developed for a number of reference compounds (typically between 5 and 10) which are structurally similar to the test chemical. Lists of values of measured log K.sub.ow are available for many chemicals. If data on the partition coefficients of structurally related compounds are not available, a more general calibration graph can be developed using other reference compounds. The reference compound or test chemical retention time (t.sub.R) is the time from sample injection to maximum concentration (peak height) of eluted reference compound or test chemical. The internal standard retention time (t.sub.o) is the time from sample injection to the maximum concentration (peak height) of the eluted internal standard. The normalized retention time for each unknown is t.sub.R−t.sub.o. The results are calculated and reported as follows. Using the plot of log (t.sub.R−t.sub.o) versus log K.sub.ow for the reference compounds, compute the linear regression equation of the form log K.sub.ow=a log (t.sub.R−t.sub.o)+b, where a and b are the slope and intercept, respectively. From the standard curve or regression equation, calculate an estimated log K.sub.ow for the test compound corresponding to the measured log (t.sub.R−t.sub.o). Report the standard curve of log (t.sub.R−t.sub.o) versus log K.sub.ow for each buffered or unbuffered eluent, or report the regression equation in the form of log K.sub.ow=a log (t.sub.R−t.sub.o)+b. In some embodiments, the octanol-water partition coefficient (log P) is experimentally determined by the ASTM Standard Test Method for Partition Coefficient (N-Octanol/Water) Estimation by Liquid Chromatography, Designation E 1147-92.

(19) Alternatively, for purposes of the present invention, the octanol-water partition coefficient values (log P) may be obtained from the “preferred” or “good” values defined and listed in Exploring QSAR, Vol. 1 Fundamentals and Applications in Chemistry and Biology, and Vol. 2, Hydrophobic, Electronic, and Steric Constants, Corwin Hansch, ACS Professional Reference Book, 1995, herein incorporated by reference.

(20) The inventors determined experimentally that medium polarity oils. i.e., oils with a log P of 0.5 to 2.0, exhibited some antibacterial activity against biofilms, whereas non-polar and polar oils generally did not exhibit very much antibacterial activity against biofilms. The log reduction of bacteria in an in-vitro S. aureus biofilm model after treatment with various oils of various polarity vs. moist control is shown in FIG. 1. As can be seen in FIG. 1, the compounds having a log P outside the range of 0.5 to 2.0 did not exhibit very much antimicrobial activity against biofilms compared to the compounds having a log P within the range of 0.5 to 2.0. The compounds having a log P outside the range of 0.5 to 2.0 include glyceryl triacetate, methyl benzoate, diethyl acetate, MONOMULS® (glyceryl monolaurate), isopropyl myristate, and oleic acid. Compounds having a log P within the range of 0.5 to 2.0 include resorcinol, anisyl alcohol, benzoic acid, benzyl alcohol, ethyl acetate, ethyl gallate, phenoxyethanol, phenyethanol, propyl gallate, and glyceryl caprylate/caprate, and are shown in Table 1 below.

(21) In some embodiments, the medium polarity oils are esters. Esters are the covalent compounds formed between acids and alcohols. In other embodiments, the medium polarity oils are fatty acid esters which are compounds formed between fatty acids and alcohols. In still other embodiments, the medium polarity oils are glyceryl esters. Glyceryl esters are primarily fatty acid mono-, di-, and/or tri-glycerides. One such glyceryl ester is glyceryl caprylate/caprate. Glyceryl caprylate/caprate is available from the Abitec Company under the trade name CAPMUL® MCM, NF and from Sasol Olefins & Surfactants GmbH under the trade name IMWITOR 742. Glyceryl caprylate/caprate is also known by its synonyms: caprylic/capric glycerides (INCI name); mono- and di-glycerides (NF name); glycerol monocaprylocaprate; medium chain mono- & diglycerides; glycerides C8-10 mono- di- tri-; and glyceryl mono- & dicaprylo/caprate. Glyceryl caprylate/caprate has an octanol-water partition coefficient value of 1.21 as determined experimentally by the ASTM method mentioned above. Glyceryl caprylate/caprate is a mono-diglyceride of medium chain fatty acids (mainly caprylic and capric). It is a mixture of monoacylglycerols, mainly mono-O-octanoylglycerol and mono-O-decanolyglycerol, containing variable quantities of di- and triacylglycerols. It is obtained by direct esterification of glycerol with caprylic (octanoic) and capric (decanoic) acids. CAPMUL MCM, NF and IMWITOR 742 meet the requirements of the USP/NF under the NF monograph for “mono- and di-glycerides.”

(22) TABLE-US-00001 TABLE 1 Medium Polarity Oil Log P value Resorcinol 0.80* Anisyl Alcohol 1.10* Benzoic Acid 1.87* Benzyl Alcohol 1.10* Ethyl Acetate 0.73* Ethyl Gallate 1.30* Phenyl ethanol 1.42* Phenoxyethanol 1.16* Propyl Gallate 1.80* Glyceryl Caprate/Caprylate  1.21** *value obtained from Exploring QSAR, Vol. 2 **value determined experimentally by ASTM method

(23) The concentrations of the medium polarity oil and the antibacterial agent components in the compositions are at amounts that exhibit synergistic antibacterial activity against bacterial biofilms. The concentration of the medium polarity oil in the compositions can vary with different oils, but generally can be 1 to 50% w/w, or 1 to 40% w/w, or 1 to 30% w/w, or 1 to 25% w/w, or 1 to 20% w/w, or 1 to 10% w/w, or 5 to 10% w/w, or 5 to 11% w/w, or 5 to 12% w/w, or 5 to 15% w/w, or 5 to 25% w/w, or 5 to 30% w/w, or 5 to 40%, w/w, or 5 to 50% w/w, or 6 to 50% w/w, or 6 to 40% w/w, or 6 to 30% w/w, or 6 to 25% w/w, or 6 to 20% w/w, or 6 to 15% w/w, or 6 to 12% w/w, or 6 to 11% w/w, or 6 to 10% w/w, or 7 to 50% w/w, or 7 to 40% w/w, or 7 to 30% w/w, or 7 to 25% w/w, or 7 to 20% w/w, or 7 to 15% w/w, or 7 to 12% w/w, or 7 to 11% w/w, or 7 to 10% w/w, or 8 to 50% w/w, or 8 to 40% w/w, or 8 to 30% w/w, or 8 to 25% w/w, or 8 to 20% w/w, or 8 to 15% w/w, or 8 to 12% w/w, or 8 to 11% w/w, or 8 to 10% w/w, or 9 to 50% w/w, or 9 to 40% w/w, or 9 to 30% w/w, or 9 to 25% w/w, or 9 to 20% w/w, or 9 to 15% w/w, or 9 to 12% w/w, or 9 to 11% w/w, or 9 to 10% w/w, or at least 5% w/w, or at least 6% w/w, or at least 7% w/w, or at least 8% w/w, or at least 9% w/w, or at least 10% w/w. In some embodiments, the medium polarity oil is an ester, fatty acid ester, or glyceryl ester and the concentration in the composition is 6 to 50% w/w, or 6 to 40% w/w, or 6 to 30% w/w, or 6 to 25% w/w, or 6 to 20% w/w, or 6 to 15% w/w, or 6 to 12% w/w, or 6 to 11% w/w, or 6 to 10% w/w, or 7 to 50% w/w, or 7 to 40% w/w, or 7 to 30% w/w, or 7 to 25% w/w, or 7 to 20% w/w, or 7 to 15% w/w, or 7 to 12% w/w, or 7 to 11% w/w, or 7 to 10% w/w, or 8 to 50% w/w, or 8 to 40% w/w, or 8 to 30% w/w, or 8 to 25% w/w, or 8 to 20% w/w, or 8 to 15% w/w, or 8 to 12% w/w, or 8 to 11% w/w, or 8 to 10% w/w, or 9 to 50% w/w, or 9 to 40% w/w, or 9 to 30% w/w, or 9 to 25% w/w, or 9 to 20% w/w, or 9 to 15% w/w, or 9 to 12% w/w, or 9 to 11% w/w, or 9 to 10% w/w, or at least 6% w/w, or at least 7% w/w, or at least 8% w/w, or at least 9% w/w, or at least 10% w/w. In some embodiments, the medium polarity oil is glyceryl caprate/caprylate and the concentration in the composition is 6 to 50% w/w, or 6 to 40% w/w, or 6 to 30% w/w, or 6 to 25% w/w, or 6 to 20% w/w, or 6 to 15% w/w, or 6 to 12% w/w, or 6 to 11% w/w, or 6 to 10% w/w, or 7 to 50% w/w, or 7 to 40% w/w, or 7 to 30% w/w, or 7 to 25% w/w, or 7 to 20% w/w, or 7 to 15% w/w, or 7 to 12% w/w, or 7 to 11% w/w, or 7 to 10% w/w, or 8 to 50% w/w, or 8 to 40% w/w, or 8 to 30% w/w, or 8 to 25% w/w, or 8 to 20% w/w, or 8 to 15% w/w, or 8 to 12% w/w, or 8 to 11% w/w, or 8 to 10% w/w, or 9 to 50% w/w, or 9 to 40% w/w, or 9 to 30% w/w, or 9 to 25% w/w, or 9 to 20% w/w, or 9 to 15% w/w, or 9 to 12% w/w, or 9 to 11% w/w, or 9 to 10% w/w, or at least 6% w/w, or at least 7% w/w, or at least 8% w/w, or at least 9% w/w, or at least 10% w/w.

(24) B. Antibacterial Agents

(25) The compositions of the invention comprise at least one antibacterial agent. Various antibacterial agents are suitable for use with the present invention. Suitable antibacterial agents include silver compounds such as the following non-limiting examples: elemental silver, silver nanoparticles, silver zeolite, silver sulfadiazine, ionized silver, and silver salts such as silver chloride and silver nitrate. Other suitable antibacterial agents include iodine compounds such as the following non-limiting examples: iodine, tincture of iodine, Lugol's iodine solution, iodides, iodine topical solution, iodine complexed with phosphate ester of alkylaryloxy polyethylene, iodoquinol, undecoylium chloride-iodine, nonylphenoxypolyethanol-iodine complex, and iodophors such as povidone-iodine (PVP-iodine), polyvinyl alcohol-iodine, polyvinyl oxazolidone-iodine, polyvinyl imidazole-iodine, polyvinyl morpholone-iodine, and polyvinyl caprolactam-iodine, nonylphenolethoxylate-iodine, soluble starch-iodine, betacyclodextrin-iodine, polyoxyethylenepolyoxypropylene condensate-iodine, ethoxylated linear alcohol-iodine, and cadexomer-iodine. Additional non-limiting examples of suitable antibacterial agents include: quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, methylbenzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, cetrimonium, cetrimide, dofanium chloride, tetraethylammonium bromide, didecyldimethylammonium chloride and domiphen bromide; chlorine containing compounds such as sodium hypochlorite, calcium hypochlorite, and chlorine dioxide; hydrogen peroxide; benzoic acid and its salts; benzoyl peroxide; benzyl alcohol; bispyrithione salts; boric acid; camphorated metacresol; camphorated phenol; chlorobutanol; cloflucarban; dapsone; dehydroacetic acid and its salts; ethyl alcohol; hexachlorophene; hexitidine; hexylresorcinol; hydroxybenzoic acid and its salts; isopropyl alcohol; mafenide acetate; magnesium pyrithione; merbromin; mercufenol chloride; methylparaben; metronidazole and its derivatives; mupirocin and its salts; nitrofurazone; n-Propanol; organic peroxides; p-chloro-m-xylenol; phenol; phenoxyethanol; phenyl alcohol; phenyl ethyl alcohol; selenium sulfide; sodium oxychlorosene; sodium sulfacetamide; sorbic acid and its salts; sulfur; tetrachlorosalicylanilide; thymol; tribromsalan; triclocarbon; triclosan; and zinc pyrithione.

(26) Antibiotics and antibacterial peptides are also suitable antibacterial agents. Suitable antibiotics include polypeptide antibiotics, examples of which are colistin (polymyxin E), colistin A (polymyxin E1), colistin B (polymyxin E2), colistin sulfate, colistimethate sodium, actinomycin, bacitracin, and polymyxin B. Other suitable antibiotics include aminoglycoside antibiotics, examples of which are gentamicin, gentamicin sulfate, neomycin, kanamycin, and tobramycin. Other suitable antibiotics include glycopeptide antibiotics, examples of which are vancomycin, teicoplanin, telavancin, ramoplanin, decaplanin, and bleomycin. Other suitable antibiotics include macrolide antibiotics, examples of which are azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, spiramycin, and troleandomycin. Other suitable antibiotics include mupirocin, calcium mupirocin, and retapamulin.

(27) In some embodiments, the antibacterial agent is an iodine compound. In other embodiments, the iodine compound is an iodophor. In still other embodiments, the iodophor is cadexomer-iodine or povidone-iodine. In some embodiments, the cadexomer-iodine is at a concentration in the composition of 40 to 60% w/w, or 40 to 50% w/w, or 45 to 55% w/w, or 50 to 60% w/w, or about 50% w/w. In some embodiments, the povidone-iodine is at a concentration in the compositions of 1 to 25% w/w, or 1 to 20% w/w, or 1 to 15% w/w, or 3 to 15% w/w, or 5 to 10% w/w, or about 5% w/w, or about 10% w/w. In some embodiments, the antibacterial agent is a silver compound. In other embodiments, the silver compound is silver sulfadiazine, silver nitrate, or silver chloride. In some embodiments, the silver sulfadiazine is at a concentration in the composition of 0.1 to 10% w/w, or 0.1 to 5% w/w, or 0.1 to 2% w/w, or 0.1 to 1.5%, or 0.5 to 5% w/w, or 0.5 to 2% w/w, or 0.5 to 1.5% w/w, or 0.5 to 1% w/w, or 0.1 to 1% w/w, or 1 to 5% w/w, or about 0.5% w/w, or about 1% w/w. In some embodiments, the silver nitrate is at a concentration in the composition of 0.1 to 10% w/w, or 0.1 to 5% w/w, or 0.1 to 2% w/w, or 0.1 to 1.5%, or 0.5 to 1% w/w, or 0.5 to 5% w/w, or 0.5 to 2% w/w, or 0.5 to 1.5% w/w, or 0.1 to 1% w/w, or 1 to 5% w/w, or about 0.5% w/w, or about 1% w/w. In some embodiments, the silver chloride is at a concentration in the composition of 0.1 to 10% w/w, or 0.1 to 5% w/w, or 0.1 to 2% w/w, or 0.1 to 1.5%, or 0.5 to 1% w/w, or 0.5 to 5% w/w, or 0.5 to 2% w/w, or 0.5 to 1.5% w/w, or 0.1 to 1% w/w, or 1 to 5% w/w, or about 0.5% w/w, or about 1% w/w. In some embodiments, the antibacterial agent is an antibiotic. In other embodiments, the antibiotic is an aminoglycoside antibiotic. In still other embodiments, the aminoglycoside antibiotic is gentamicin or gentamicin sulfate. In some embodiments, the gentamicin or gentamicin sulfate is at a concentration in the composition of 0.1 to 10% w/w, or 0.1 to 5% w/w, or 0.1 to 2% w/w, or 0.1 to 1% w/w, or 0.5 to 5% w/w, or 0.5 to 2% w/w, or 0.5 to 1% w/w, or 0.5 to 0.7% w/w, or 0.7 to 1% w/w, or about 0.7% w/w. In other embodiments, the antibiotic is a polypeptide antibiotic. In still other embodiments, the polypeptide antibiotic is colistin or colistin sulfate. In some embodiments, the colistin or colistin sulfate is at a concentration in the composition of 0.01 to 5% w/w, or 0.01 to 2% w/w, or 0.01 to 1% w/w, or 0.01 to 0.5% w/w, or 0.01 to 0.2% w/w, or 0.05 to 1% w/w, or 0.05 to 0.5% w/w, or 0.05 to 0.2% w/w, or 0.05 to 0.15% w/w or about 0.1% w/w. In some embodiments, the antibacterial agent is not chlorhexidine gluconate.

(28) The concentrations of the medium polarity oil and the antibacterial agent components in the compositions are at amounts that exhibit synergistic antibacterial activity against bacterial biofilms. The concentration of the antibacterial agent in the compositions can vary with different antibacterial agents, but generally can be 0.01 to 75% w/w, or 0.01 to 60% w/w, or 0.01 to 50% w/w, or any range or number therein (e.g., at least 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, and up to 75 wt. %).

(29) C. Manufacture

(30) The compositions of the invention may be manufactured by methods and equipment known in the art for manufacture of topical products and products designed for application to non-biological surfaces, such as medical devices. Such methods include, but are not limited to the use of mechanical mixers including LIGHTNIN propeller mixers; COWLES dissolvers; SILVERSON dispersers; counter-rotating side-scrapping mixers; homogenizers and dispersers, including in-line or in-tank rotor-stator homogenizers; and mills, including 3-roll mills, ointment mills, or rotor-stator mills. “All-in-one” vacuum mixing systems that have a rotating side-scrapping mixer plus an in-tank homogenizer may also be used. Such mixers include, but are not limited to OLSA mixers, FRYMA-KORUMA mixers, and LEE TRI-MIX TURBO-SHEAR kettles. The compositions of the invention can be manufactured from small laboratory scale batches to full-scale production batches.

II. BACTERIAL BIOFILMS

(31) The compositions of the invention are suitable for the reduction of bacteria in and/or elimination of both gram-positive and gram-negative bacterial biofilms. Non-limiting examples of gram-positive bacteria include Staphylococcus spp., such as Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), and Staphylococcus epidermidis; Streptococcus spp, such as Streptococcus pneumonia; Bacillus spp.; Listeria monocytogenes; enterococci spp.; and lactic acid bacteria, such as Lactobacillus plantarum and Lactococcus lactis. Non-limiting examples of gram-negative bacteria include Pseudomonas spp., such as Pseudomonas aeruginosa; and Escherichia coli.

(32) A. In-Vitro Biofilm Model

(33) An in-vitro biofilm model was used to evaluate the biofilm efficacy of the formulations of the invention against bacterial biofilms. Bacteria are spotted onto a collagen matrix resting on a filter on a blood agar plate and incubated to allow biofilm formation. The model mimics in-vivo wound biofilms in that nutrients are provided from below the biofilm while topical treatments are applied at the air interface above. This in-vitro model and methodology is disclosed in the poster presentation, A Versatile In Vitro Biofilm Model Using Two Wound Pathogens to Screen Formulations, Van der Kar, et al., presented at the 2010 Wound Healing Society Annual Meeting, Poster BRC09, on Apr. 18, 2010 in Orlando, Fla., and is herein incorporated by reference. Further in-vitro biofilm models and methodologies are disclosed in the following publications all of which are herein incorporated by reference: Penetration of Rifampin through Staphylococcus epidermidis Biofilms, Zheng, et al., Antimicrobial Agents and Chemotherapy, March 2002, p. 900-903; Oxygen Limitation Contributes to Antibiotic Tolerance of Pseudomonas aeruginosa in Biofilms, Borriello et al., Antimicrobial Agents and Chemotherapy, July 2004, p. 2659-2664; and Heterogeneity in Pseudomonas aeruginosa Biofilms Includes Expression of Ribosome Hibernation Factors in the Antibiotic-Tolerant Subpopulation and Hypoxia-Induced Stress Response in the Metabolically Active Population, Williamson et al., Journal of Bacteriology, February 2012, p. 2062-2073.

III. METHODS OF USE AND TREATMENT

(34) The compositions of the invention are useful for the reduction of bacteria in and/or elimination of bacterial biofilms on biological and non-biological surfaces, and are also useful for treatment of wounds, skin lesions, mucous membrane lesions, and other biological surfaces infected or contaminated with bacterial biofilms.

(35) A. Biological Surfaces

(36) The compositions of the invention are useful for reducing bacteria in and/or eliminating a bacterial biofilm on a biological surface by administering the compositions to the biological surface. Non-limiting examples of biological surfaces include wounds (including chronic and acute wounds), skin lesions, skin, mucous membranes, mucous membrane lesions, internal organs, body cavity, oral cavity, bone tissue, muscle tissue, nerve tissue, ocular tissue, urinary tract tissue, lung and trachea tissue, sinus tissue, ear tissue, dental tissue, gum tissue, nasal tissue, vascular tissue, cardiac tissue, epithelium, and epithelial lesions, and peritoneal tissue. Non-limiting examples of chronic wounds include diabetic foot ulcers, venous ulcers, arterial ulcers, decubitus ulcers, stasis ulcers, pressure ulcers, and burns. Non-limiting examples of acute wounds include cuts and surgical wounds. Non-limiting examples of skin lesions and mucous membrane lesions include blisters, ulcers, abrasions, warts, scrapes, and skin and mucosal infections such as staph or MRSA infections. Examples of skin lesions and mucous membrane lesions are disclosed in “Description of Skin Lesions”, MacNeal, Robert J., the on-line Merck Manual Professional Version, March 2013, http://www.merckmanuals.com/professional/dermatologic-disorders/approach-to-the-dermatologic-patient/description-of-skin-lesions herein incorporated by reference. Skin lesions can appear on the epidermis, lips, ear canal, scalp, cuticle, nail bed, or genitalia. Mucous membrane lesions can appear on the oral mucosa, nasal mucosa, penile and vaginal mucosa, or anus.

(37) B. Topical Treatment of Wounds

(38) The compositions of the invention are useful for the treatment of wounds, including chronic wounds and acute wounds, infected or contaminated with bacterial biofilms, by topically administering the compositions to the wound. Non-limiting examples of chronic wounds include diabetic foot ulcers, venous ulcers, arterial ulcers, decubitus ulcers, stasis ulcers, pressure ulcers, and burns. Non-limiting examples of acute wounds include cuts and surgical wounds.

(39) C. Topical Treatment of Skin Lesions and Mucous Membrane Lesions

(40) The compositions of the invention are useful for the treatment of skin lesions or mucous membrane lesions infected or contaminated with bacterial biofilms by topically administering the compositions to the skin lesion or mucous membrane lesions. Non-limiting examples of skin lesions and mucous membrane lesions include blisters, ulcerations, abrasions, warts, scrapes, and skin and mucosal infections such as staph or MRSA infections. Skin lesions can appear on the epidermis, lips, ear canal, scalp, cuticle, nail bed, or genitalia. Mucous membrane lesions can appear on the oral mucosa, nasal mucosa, penile and vaginal mucosa, or anus.

(41) D. Treatment of Other Biological Surfaces

(42) The compositions of the invention are useful for the treatment of other biological surfaces infected or contaminated with bacterial biofilms by administering the compositions to the biological surface. Non-limiting examples of other biological surfaces include internal organs, body cavity, oral cavity, bone tissue, muscle tissue, nerve tissue, ocular tissue, urinary tract tissue, lung and trachea tissue, sinus tissue, ear tissue, dental tissue, gum tissue, nasal tissue, vascular tissue, cardiac tissue, epithelium, and epithelial lesions, and peritoneal tissue.

(43) E. Non-Biological Surfaces

(44) The compositions of the invention are useful for reducing bacteria in and/or eliminating a bacterial biofilm on a non-biological surface, such as a medical device, by administering the compositions to the non-biological surface. Non-limiting examples of medical devices include urinary tract prostheses; urinary tract catheters, peritoneal membrane catheters, peritoneal dialysis catheters, indwelling catheters for hemodialysis and for chronic administration of chemotherapeutic agents (Hickman catheters); cardiac implants such as pacemakers, prosthetic heart valves, ventricular assist devices, and synthetic vascular grafts and stents; prostheses; percutaneous sutures; and tracheal and ventilator tubing. The surface of an article of manufacturing, including medical devices, can be coated with the compositions of the inventions prior to the presence of a bacterial biofilm in order to prevent the formation of bacterial biofilms; or can be coated after the presence of a bacterial biofilm on the surface in order to reduce bacteria in and/or eliminate the bacterial biofilm on the surface.

IV. EXAMPLES

A. Example 1

Determination of Octanol-Water Partition Coefficient for CAPMUL MCM by ASTM Method

(45) 1. Prepared reference standard samples of compounds with known log P values shown in Table 2 at concentrations of approximately 200 mg/L in methanol.

(46) 2. Prepared test sample of CAPMUL MCM at a concentration of approximately 200 mg/mL in methanol.

(47) 3. Ran reference and test samples on HPLC using the parameters shown in Table 3.

(48) 4. Compared the retention times of the reference standards to the retention time of CAPMUL MCM to calculate the log P value of CAPMUL MCM as per ASTM method.

(49) The retention times of the reference substances are shown in Table 2 and plotted in FIG. 2. The retention time of CAPMUL MCM is 3.011 giving a log P of 1.21 for CAPMUL MCM.

(50) TABLE-US-00002 TABLE 2 log Reference retention Substance log P time Anisyl alcohol 1.1 0.5008 Phenoxyethanol 1.16 0.5161 Diethyl phthalate 2.42 0.5944 Benzyl cinnamate 4.06 0.8331 Benzyl salicylate 4.31 0.8719 Dibutyl sebacate 6.3 1.2709

(51) TABLE-US-00003 TABLE 3 Mobile Phase Solvent A 40:60 ACN/H2O Mobile Phase Solvent B 70:30 ACN/H2O Detector 249 nm Injection 20 microliters Flow Rate 1 mL/min Column Luna C18(2) 5 micron, 100 angstroms, 250 × 4.6 mm Gradient: 0-15 min 100% A 15-15:50 Increase to 100% B 15:50-20 100% B 20-25 100% A Temperature 22° C.

B. Example 2

Formulations

(52) Various formulations were prepared and are shown in tables 4-9 below.

(53) Cadexomer iodine based formulations are shown in Table 4.

(54) TABLE-US-00004 TABLE 4 Cadexomer Iodine Based Formulations Formula Cadexomer Cadexomer Cadexomer Cadexomer/ Cadexomer Cadexomer Iodine Iodine/ Iodine/ Cadexomer 10% oil Iodine/ Iodine Component (% w/w) Control 5% oil 10% oil Control control 2.5% oil 1% oil PEG-400 38 34.3 30.4 41 30.4 36 37.2 PEG-4000 10 9 8 10.7 8 9.5 9.8 Poloxamer 184 2.1 1.8 1.6 2.2 1.6 1.9 1.9 CAPMUL MCM NF — 5 10 — 10 2.5 1 Cadexomer Iodine 50 50 50 — — 50 50 Cadexomer Base — — — 46 50 — —

(55) Procedure (for concentration of each ingredient, see Table 4.): Mixed all ingredients except cadexomer iodine and/or cadexomer base at 70° C. until uniform. Added cadexomer iodine or cadexomer base and mixed until uniform. Cooled to room temperature (RT) while mixing.

(56) Silver chloride based formulations are shown in Table 5.

(57) TABLE-US-00005 TABLE 5 Silver Chloride Based Formulations Component Formula (% w/w) Ag alone Oil alone Ag + Oil HEC 250 HX (Aqualon) 11 11 11 Silver Chloride 1 — 1 CAPMUL MCM — 10 10 Phenoxyethanol 0.7 0.7 0.7 PEG 600 39 35 34 PEG 400 39 35 34 PEG 3350 4 4 4 ARISTOFLEX AVC 1 1 1 Glycerin 4 4 4

(58) Procedure (for concentration of each ingredient, see Table 5.): Homogenized PEG 600, PEG 400, PEG 3350, Glycerin, ARISTOFLEX AVC, CAPMUL MCM (if present), and Silver Chloride (if present) at high temperature using a Silverson homogenizer for 1 minute at 8000 rpm. Cooled the mixture to 50° C. and added HEC 250 HX while mixing. Continued mixing until the temperature was less than 35° C.

(59) Other silver based formulations are shown in Table 6.

(60) TABLE-US-00006 TABLE 6 Other Silver Based Formulations Formula 1% AgNO3/ 1% AgCl/ 1% SSD/ 1% 1% 1% Component (% w/w) 9% oil 9% oil 9% oil AgNO3 AgCl SSD Placebo Poloxamer 407 6 6 6 6 6 6 7 Glycerin 2 2 2 2 2 2 2 Stearyl Alcohol 3.8 3.8 3.8 3.8 3.7 3.7 3.0 Polysorbate 60 3.7 3.7 3.7 3.8 3.7 3.7 3.2 CAPMUL MCM NF 9 9 9 — — — 9 Isopropyl Myristate 4.6 4.6 4.6 13.7 13.7 13.7 4.6 Silver Nitrate 1 — — 1 — — — Silver Chloride — 1 — — 1 — — Silver Sulfadiazine — — 1 — — 1 — PHOSPHOLIPON 90G 2.3 2.3 2.3 2.3 2.3 2.3 2.3 DI Water qs ad 100 qs ad 100 qs ad 100 qs ad 100 qs ad 100 qs ad 100 qs ad 100

(61) Procedure (for concentration of each ingredient, see Table 6.): Poloxamer 407, glycerin and water were mixed until dissolved at RT to form a water phase. Stearyl alcohol, polysorbate 60, isopropyl myristate, PHOSPHOLIPON G and CAPMUL MCM (if present) were mixed at 70° C. until clear to form an oil phase. The water phase and oil phase were combined and mixed at 70° C. for 2 hours and then cooled to RT while mixing. An active phase was made with water (10% w/w) and silver nitrate or silver chloride or silver sulfadiazine (SSD). The active phase (except for placebo) was then was added to the batch and mixed until uniform.

(62) Povidone-iodine based formulations are shown in Table 7.

(63) TABLE-US-00007 TABLE 7 Povidone-Iodine Based Formulations Formula 5% Component Placebo 5% PVI + (% w/w) plus oil PVI 10% oil Poloxamer-407 15.0 15.1 14.6 Propylene Glycol 5.0 5.0 5.4 Povidone-Iodine — 5.0 4.9 CAPMUL MCM NF 10.0 — 10.1 DI Water qs ad 100 qs ad 100 qs ad 100

(64) Procedure (for concentration of each ingredient, see Table 7.): Poloxamer-407 and propylene glycol were dissolved in water. Povidone Iodine and/or CAPMUL MCM were added while mixing and mixed until homogenous. The formulations containing povidone-iodine were brown solutions

(65) Gentamicin based formulations are shown in Table 8.

(66) TABLE-US-00008 TABLE 8 Gentamicin Based Formulations Formula 0.7% Component Placebo 0.7% GENTA + (% w/w) plus oil GENTA 10% oil Poloxamer-407 15.0 14.8 15.0 Propylene Glycol 5.0 5.0 5.0 Gentamicin Sulfate — 0.7 0.7 CAPMUL MCM NF 10.0 — 10.8 DI Water qs ad 100 qs ad 100 qs ad 100

(67) Procedure (for concentration of each ingredient, see Table 8.): Poloxamer-407 and propylene glycol were dissolved in water. Gentamicin sulfate and/or CAPMUL MCM were added while mixing and mixed until homogenous. The formulations containing CAPMUL MCM were thick, ringing gels. The viscosity of the ringing gel formulation “0.7% GENTA+10% oil” was 89,000 cps as measured using a Brookfield RV viscometer with a small sample adapter, spindle #14, at room temperature (22°-25 ° C.), at 10 rpm for 1 minute.

(68) Colistin based formulations are shown in Table 9.

(69) TABLE-US-00009 TABLE 9 Colistin Based Formulations Formula 0.1% 0.1% Colistin Component Placebo Colistin Sulfate + (% w/w) plus oil Sulfate 10% oil Poloxamer-407 15.0 15.0 15.0 Propylene Glycol 5.0 5.0 5.1 Colistin Sulfate — 0.1 0.1 CAPMUL MCM NF 10.0 — 10.0 DI Water qs ad 100 qs ad 100 qs ad 100

(70) Procedure (for concentration of each ingredient, see Table 9.): Poloxamer-407 and propylene glycol were dissolved in water. Colistin sulfate and/or CAPMUL MCM were added while mixing and mixed until homogenous. The formulations containing CAPMUL MCM were thick, ringing gels.

C. Example 3

In-Vitro P. aeruginosa Biofilm Model with Various Treatment Formulations

(71) P. aeruginosa ATCC 27312 was grown overnight on tryptic soy agar (TSA) at 37° C. The next day, a single colony was picked and passed into tryptic soy broth (TSB), then grown at 37° C. overnight with shaking (150 rpm). The overnight culture was diluted to ˜1.5×10.sup.8 cfu/mL in PBS (inoculum). Tryptic soy agar with 5% sheep blood (TSAB) plates were prepared with six 13 mm black 0.2 micron TEFLON filters. Each 13 mm filter had a single 4 mm collagen plug placed in the center and was then inoculated with 3 μL of inoculum placed on the center of the plug (13 mm filter plus inoculated plug=colony biofilm assembly or CBFA). The CBFA plates were transferred to a 37° C. incubator and incubated for 24 hours. At the end of the incubation, growth was sampled and treatment with the test formulations of Example 2 was started. The test formulations for the cadexomer iodine based formulations (Table 4) were mixed 50/50 (weight/volume) with PBS and applied (200 μL) to PBS moistened (200 μL) 13 mm TELFA non-adherent dressing squares. The other test formulations (gel and liquid formulations from Tables 5-9) were applied directly to the PBS moistened TEFLA squares (the liquid formulations were mixed for 10 seconds prior to the application). The treatments were applied with the formulation directly in contact with the biofilm (TELFA on top) and gently tamped down to ensure contact with the biofilm (moist control was TELFA only). The treated CBFA plates were transferred to the 37° C. incubator and incubated for 24 hours. At the end of the incubation, the treated CBFA were recovered into 5 mL DE Broth PBS and vortexed at 2500 rpm for 2 minutes to knock off the treatments and resuspend any surviving bacteria. The recoveries were serially diluted (1:10 eight point dilution series in PBS broth) and 10 volumes spotted on Charcoal Agar plates (the plates serve to neutralize any active treatments). The plates were allowed to dry and incubated at 37° C. overnight with colony counts determined the next day. Counts were converted to colony forming units per milliliter and transformed to log values. Efficacy was determined by subtracting the log cfu/mL value of a treatment from the moist control to generate a log reduction value in addition to direct comparison of recovered log cfu/mL.

(72) The results of the log reduction of bacteria in the biofilm model after treatment with the treatment formulations from Example 2 (cadexomer iodine based formulations—Table 4) vs. moist control are shown in FIG. 3. The results of the log reduction of bacteria in the biofilm model after treatment with the treatment formulations from Example 2 (silver chloride based formulations—Table 5) vs. moist control are shown in FIG. 4. Results of the log reduction of bacteria in the biofilm model after treatment with the treatment formulations from Example 2 (other silver based formulations—Table 6) vs. moist control are shown in FIG. 5. The results of the log reduction of bacteria in the biofilm model after treatment with the treatment formulations from Example 2 (povidone-iodine based formulations—Table 7) vs. moist control are shown in FIG. 6. The results of the log reduction of bacteria in the biofilm model after treatment with the treatment formulations from Example 2 (gentamicin based formulations—Table 8) vs. moist control are shown in FIG. 7. The results of the log reduction of bacteria in the biofilm model after treatment with the treatment formulations from Example 2 (colistin based formulations—Table 9) vs. moist control are shown in FIG. 8.

(73) The results in FIG. 5 show the cumulative log reduction effect of the “1% AgCl” formulation plus the “Placebo” formulation was less than the log reduction effect of the “1%AgCl/9% oil” formulation; the cumulative log reduction effect of the “1% AgNO” formulation plus the “Placebo” formulation was less than the log reduction effect of the “1% AgNO/9% oil” formulation; and the cumulative log reduction effect of the “1% SSD” formulation plus the “Placebo” formulation was less than the log reduction effect of the “1% SSD/9% oil” formulation. These results indicate synergistic antibacterial activity by the combination of the antibacterial agent and the medium polarity oil.

(74) The results in FIG. 4 show the cumulative log reduction effect of the “Ag alone” formulation plus the “Oil alone” formulation is less than the log reduction effect of the “Ag+Oil” formulation at 24 hours indicating synergistic antibacterial activity by the combination of the antibacterial agent and the medium polarity oil.

(75) The results in FIG. 3 show the cumulative log reduction effect of the “Cadexomer 10% oil control” formulation plus the “Cadexomer Iodine Control” formulation is less than the log reduction effect of the “Cadexomer Iodine 10% oil” formulation indicating synergistic antibacterial activity by the combination of the antibacterial agent and the medium polarity oil.

(76) The results in FIG. 6 show the cumulative log reduction effect of the “Placebo plus oil” formulation plus the “5% PVI” formulation is less than the log reduction effect of the “5% PVI+10% oil” formulation indicating synergistic antibacterial activity by the combination of the antibacterial agent and the medium polarity oil.

(77) The results in FIG. 7 show the cumulative log reduction effect of the “Placebo plus oil” formulation plus the “0.7% GENTA” formulation is less than the log reduction effect of the “0.7% GENTA+10% oil” formulation indicating synergistic antibacterial activity by the combination of the antibacterial agent and the medium polarity oil.

(78) The results in FIG. 8 show the cumulative log reduction effect of the “Placebo plus oil” formulation plus the “0.1% colistin sulfate” formulation is less than the log reduction effect of the “0.1% colistin sulfate+10% oil” formulation indicating synergistic antibacterial activity by the combination of the antibacterial agent and the medium polarity oil.