US 200700.92547A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0092547 A1 Birnbaum et al. (43) Pub. Date: Apr. 26, 2007

(54) METHOD FOR (22) Filed: Oct. 2, 2006 TREATING/CONTROLLING/KILLING FUNG AND BACTERIA Related U.S. Application Data (76) Inventors: Jay E. Birnbaum, Montville, NJ (US); (60) Provisional application No. 60/729,624, filed on Oct. Thomas Blake, Budd Lake, NJ (US); 24, 2005. Mahmoud Ghannoum, Hudson, OH (US); Steven Vallespir, Princeton, NJ Publication Classification (US); Mark David Antonacci, Randolph, NJ (US); Michael Patrick (51) Int. Cl. Ryan, Mendham, NJ (US) AOIN 25/00 (2006.01) AOIN 65/00 (2006.01) Correspondence Address: (52) U.S. Cl...... 424/405; 424/750: 424/769: SONNENSCHEN NATH & ROSENTHAL LLP 424f778 P.O. BOX O6108O WACKER DRIVE STATION, SEARS TOWER (57) ABSTRACT CHICAGO, IL 60606-1080 (US) This present teaching describes how to treat substrates with novel compositions in order to limit fungi, dermatophytes, (21) Appl. No.: 11/541,822 yeasts, and bacteria thereon. Patent Application Publication Apr. 26, 2007 Sheet 1 of 10 US 2007/0092547 A1

Patent Application Publication Apr. 26, 2007 Sheet 2 of 10 US 2007/0092547 A1

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Figure 3 Patent Application Publication Apr. 26, 2007 Sheet 4 of 10 US 2007/0092547 A1

(A) s 1 S. 1 S 1F s 1 N 1F 3 1 9 1

Leather Pretreatment Figure 4 Patent Application Publication Apr. 26, 2007 Sheet 5 of 10 US 2007/0092547 A1

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Figure 6 Patent Application Publication Apr. 26, 2007 Sheet 7 of 10 US 2007/0092547 A1

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O d' y e . Fig. 10Aa enectorpretreatment21 s of insoles with (A) 0.01%S. e. 3% tea tree oil, or (C) 0.01% tolnaftate +3% tea tree oil on T. rubrun

r i. a. Fig. 10B. Effect of post-treatment: of infe nsoles with (A) 0.01% tolnaftate, (B) 3% tea tree oil, or (C) 0.01% tolnaftate + 3% tea tree oil on T. rubrunt growth. US 2007/0092547 A1 Apr. 26, 2007

METHOD FOR particularly useful as it can include several compounds TREATING/CONTROLLING/KILLING FUNG AND which have different activities which can act synergistically. BACTERA A mixture is applied 'simultaneously, namely all com pounds in the mixture are applied at the same time. CROSS-REFERENCE TO RELATED 0010. In a further aspect the invention includes a method APPLICATIONS for treating a substrate with an antifungal compound via a 0001. This application claims priority from U.S. Provi delivery method. sional Application Ser. No. 60/729,624 filed on Oct. 24. 0011. The inventors have also discovered that certain 2005, which is incorporated herein by reference in its antifungal compounds have antibacterial, as well as antifun entirety. gal, activity. The use of these compounds in treatment either INCORPORATION-BY-REFERENCE OF alone or in combination with other antifungal compounds MATERIAL SUBMITTED ON A COMPACT will lead to inhibition of bacterial and fungal growth on a DISC substrate. 0012. In another aspect the current invention includes a 0002) Not Applicable. method of decreasing the LD50 (Lethal Dose 50) of a compound with antifungal properties by combining said FIELD antifungal with a second compound wherein said second 0003. The present teachings relate to methods for treat compound may, or may not be, a naturally occurring anti ing/preventing fungi and bacteria on Substrates. fungal compound, synthetic, semi-synthetic, pro-drug, salt, etc. INTRODUCTION 0013 These and other features, aspects and advantages of 0004 Fungi are responsible for a broad range of diseases the present teachings will become better understood with of the epidermis of people and animals, including compan reference to a following description, examples and appended ion animals and pets. claims. 0005 The current invention involves a method, and com positions for the prevention and reduction of fungal diseases DRAWINGS in man and animals, including companion animals and pets 0014 FIG. 1. Pre-treatment assay: (A) Active agents by treating a substrate with at least one antifungal com showed a clearance Zone (arrow) around the biopsy disc, pound. Such treatment may lead to decontamination of the while (B) inactive agents showed fungal growth around the substrate. disc. Post treatment assay: (C) Discs treated with active agents showed no fungal growth. (D) Inactive agents SUMMARY showed fungal growth on discs. 0006 The present teachings include methods for treating 0.015 FIG. 2. (A) CVS Double Air Foam Insole, (B) Odor a Substrate that, directly or indirectly, contacts an epidermis eater insoles, (C) CVS Odor Stop Insoles, (D) Dr Scholls including: a) treating the Substrate with a first antifungal or Air Pillow Insoles. (E) Control. Dr. Scholls insoles did not antibacterial compound, and/or b) treating the Substrate with inhibit fungal growth. a second antifungal or antibacterial compound, and/or treat ing the Substrate with a third antifungal or antibacterial 0016 FIG. 3. Effect of 30% isopropanol on Trichophyton compound. mentagrophytes growth on (A) leather and (B) Dr. Scholls insole. Isopropanol did not inhibit fungal growth. 0007 Provided herein are newly discovered properties of compounds which include antibacterial, antifungal, and 0017 FIG. 4. Effect of pretreatment of insoles (A) or sporicidal properties. Also novel are the combinations of leather (B) biopsy discs with different agents on growth of compounds which lead to unexpected results in the treat dermatophytes. Zone diameter indicates Zone of clearance. ment and pre-treatment of Substrates against common fungi, dermatophytes, spores, and bacteria. The inventors show for 0018 FIG. 5. Effect of pretreatment of insoles with (A) the first time that combining naturally occurring fungicides 1% terbinafine. (B) 1% tolnaftate, or (C) 1% tea tree oil with known fungicides leads to unexpectedly good results 0.019 FIG. 6. Effect of acetone on the activity of tolnaf and they also show that uses of naturally occurring com tate against dermatophyte growth. (A) Growth of T. menta pounds (including fungicidal compounds), and for the first grophytes on insole disc pretreated with (A) acetone or (B) time, expands their utility against bacteria. 4% tolnaftate (w/v., prepared in acetone). (C) Activity of 4% tolnaftate (dissolved in acetone) on already established 0008 Provided herein are also compounds which exhibit contamination of T. mentagrophytes. (no fungal regrowth both antifungal and antibacterial properties. Such a com pound, or mixture, is particularly useful as it creates a was observed). treatment, or pre-treatment, that gives both fungal disinfec 0020 FIG. 7. Effect of post-infection treatment of insole tant and deodorant qualities to Substrates (including but not (A) or leather (B) biopsy discs with different agents on limited to shoes). dermatophyte growth. Zone diameter indicates Zone of 0009. In a further aspect of the method the antifungal growth. Treatment with 30% isopropanol served as vehicle compound(s) are applied in a single application (e.g. as a control. mixture) or in separate applications that are done serially, 0021 FIG. 8. Scanning electron microscopy (SEM) simultaneously, and some mixture thereof. A mixture is analyses of insoles infected with T. mentagrophytes. Mag US 2007/0092547 A1 Apr. 26, 2007 nification x2000 for all panels. Bar represents 20 uM for kill/inhibit the growth of fungal spores, for example, panels A through F, while it represents 10 uM for the any Substance that possesses a sporistatic (inhibitory) post-infected treated discs (Panels G-I). or sporicidal (killing) activity. See definition of Spori 0022 FIG. 9. Scanning electron microscopy (SEM) cidal compound below. analyses of leather biopsies infected with T. mentagrophytes. 0.031) Throughout this document the term ANTIFUN Magnification x2000; bar-20 lum. GAL COMPOUND will be an all encompassing term referring to any Substance (synthetic, semisynthetic, 0023 FIG. 10. FIG. 10A. Effect of pretreatment of salt, pro-drug, natural, etc.) with antifungal activity, insoles with (A) 0.01% tolnaftate. (B) 3% tea tree oil, or (C) including, inhibitory, killing, static, cidal, sporistatic or 0.01% tolnaftate +3% tea tree oil on T. rubrum growth on sporicidal activity. These compounds can in turn be shoe insoles. FIG. 10B. Effect of post-treatment of infected mixed with, for example, other antifungal compounds, insoles with (A) 0.01% tolnaftate. (B) 3% tea tree oil, or (C) detergents, and/or inactive ingredients to create formu 0.01% tolnaftate +3% tea tree oil on T. rubrum growth. lations. DETAILED DESCRIPTION 0032) SPORE: A spore is a fungus in its dormant, pro Abbreviations and Definitions tected State. It has a small, usually single-celled reproductive body that is highly resistant to desiccation and heat and is 0024 BOTANICAL: Abotanical is a compound isolated capable of growing into a new organism, produced espe from a plant. Botanical antifungal compounds can be iso cially by certain bacteria, fungi, algae, and non-flowering lated from, for example, Ocimum basilicum (Basil), Cinna plants. momum aromaticum var. Cassia (Cinnamon), Cedrus libani (Cedar of Lebanon), any Cedrus spp., Chamaemelum nobile 0033 SPORICIDAL COMPOUND: a substance that (Chamomile), Cymbopogon nardus (Citronella), Syzygium either inhibits the growth of, increase the susceptibility of aromaticum (Clove & clove bud), Cuminum cyminum and/or destroys fungal spores. These can be synthetic or (Cumin), Foeniculum vulgare (Fennel), Melaleuca altenfo naturally occurring. Activating spores allows fungicides that lia (Tea Tree), Mentha x piperita (Peppermint), Mentha only kill or inactivate actively growing fungi to kill those spicata (Spearmint), Curcuna longa (Tumeric), Cymbo spores activated. This can be used, for example, in a mixture pogon citratus (Lemongrass), Santalum album (Sandal wherein a chemical(s) that activates growth is mixed with a wood), as well as other compounds isolated from plants that chemical fungicide(s). It is also possible to use at least an have antifungal properties. activating compound alone, followed by at least a fungicide, serially. Activating spores is a method known in the art for 0025 NATURAL ANTIFUNGAL COMPOUND: A bacterial spores, for example in U.S. Pat. No. 6,656,919, natural antifungal compound (or naturally occurring anti which is herein incorporated by reference. fungal compound) is a compound isolated from a botanical Source (see botanical antifungal compound) or other natu 0034) BACTERICIDAL COMPOUND: a substance that rally occurring source (e.g. saliva, amphibian skin, inverte either inhibits the growth of, increases the susceptibility of brates (e.g. worms)). These compounds can be proteins and/or destroys bacteria or bacteria spores. These can be (enzymes) or other products produced by animals or plants. synthetic or naturally occurring. Activating spores allows bactericides that only kill or inactivate actively growing 0026 FUNGUS: Any of numerous eukaryotic organisms bacteria to kill those spores activated. This can be used, for of the kingdom Fungi, which lack chlorophyll and vascular example, in a mixture wherein a chemical (s) that activates tissue and range in form from a single cell (e.g., yeast) to a growth is mixed with a chemical fungicide(s). It is also body of mass branched filamentous hyphae that often pro possible to use at least an activating compound alone, duce specialized fruiting bodies and pseudohyphae. The followed by at least a fungicide, serially. Activating spores kingdom includes, but is not limited to, the yeasts, filamen is a method known in the art for bacterial spores, for tous molds, dermatophytes, Smuts, and mushrooms. example in U.S. Pat. No. 6,656,919, which is herein incor 0027. ANTIFUNGAL COMPOUND is defined as any porated by reference. chemical or substance that has the ability to inhibit the 0035 EPIDERMIS: The outer, protective, nonvascular growth of fungi, and/or kill fungal cells/spores. Compound layer of the skin of vertebrates, covering the dermis, it serves as used throughout this application includes salts and pro as the major barrier function of skin and is devoted to drugs of the compound. production of a cornified layer of the skin. Epidermally derived structures include hair (and fur), claws, nails, and 0028) Included in the definition of ANTIFUNGAL hooves. COMPOUNDS are substances that possess static (e.g. inhibitory) activity (FUNGISTATIC COMPOUNDS) 0036 TREATING: Treating a substrate means to contact and/or cidal (e.g. killing) activity (FUNGICIDAL the substrate with a substance. This can include, but is not COMPOUNDS) against fungal cells (vegetative and limited to, the delivery methods discussed below. A liquid or spore structures). powder can include, for example, at least one fungicide. The treatment can result in disinfecting the Surface, but need not 0029. Also included in the definition of ANTIFUN completely disinfect the surface. Pretreatment means to treat GAL COMPOUND is any substance that is synthetic, the substrate (or the materials used to create the substrate) semisynthetic or natural in origin that possesses anti prior to its use by the end user (e.g. consumer or producer fungal activity as defined. of products or materials). 0030. Also included in the definition of ANTIFUN 0037. MINIMAL INHIBITORY CONCENTRATION GAL COMPOUND is any substance that can destroy/ (MIC): Minimal inhibitory Concentration (MIC) is US 2007/0092547 A1 Apr. 26, 2007

described, for instance, in Clin Infect Dis. 1997 February; thyl, metominostrobin, orysastrobin, picoxystrobin, pyra 24(2): 235-47. Tests for antifungal activity are the MIC clostrobin, trifloxystrobin, biphenyl, chlorodinitronaphtha (Minimum Inhibitory Concentration) and MFC (Minimum lene, chloroneb, chlorothalonil, cresol, dicloran, Fungicidal Concentration) assays. These assays are used to hexachlorobenzene, pentachlorophenol, quintoZene, Sodium determine the smallest amount of drug needed to inhibit pentachlorophenoxide, tecnaZene, benomyl, carbendazim, (MIC) or kill (MFC) the fungus. chlorfenazole, cypendazole, debacarb, fuberidazole, 0038 ANTIFUNGAL COMPOUNDS: Examples of mecarbinzid, rabenzazole, thiabendazole, furophanate, antifungal compounds can be selected from the following thiophanate, thiophanate-methyl, bentaluron, chlobenthiaz chemical classes, or chemicals below, or naturally occurring one, TCMTB, bithionol, dichlorophen, diphenylamine, compounds: aliphatic nitrogen compounds, amide com benthiavalicarb, furophanate, iprovalicarb, propamocarb, pounds, acylamino acid compounds, allylamine compounds, thiophanate, thiophanate-methyl, benomyl, carbendazim, anilide compounds, benzanilide compounds, benzylamine cypendazole, debacarb, mecarbinzid, diethofencarb, climba compounds, furanilide compounds, Sulfonanilide com Zole, imazalil, Oxpoconazole, prochloraz, triflumizole, imi pounds, benzamide compounds, furamide compounds, phe dazole compounds, azaconazole, bromuconazole, cypro nylsulfamide compounds, Sulfonamide compounds, valina conazole, diclobutraZol, difenoconazole, diniconazole, mide compounds, antibiotic compounds, strobilurin diniconazole-M, epoxiconazole, etaconazole, fenbucona compounds, aromatic compounds, benzimidazole com Zole, fluguinconazole, flusilaZole, flutriafol, furconazole, pounds, benzimidazole precursor compounds, benzothiazole furconazole-cis, hexaconazole, imibenconazole, ipconazole, compounds, bridged diphenyl compounds, carbamate com metconazole, myclobutanil, penconazole, propiconazole, pounds, benzimidazolylcarbamate compounds, carbanilate prothioconazole, quinconazole, Simeconazole, tebucona compounds, conazole compounds, conazole compounds Zole, tetraconazole, triadimefon, triadimenol, triticonazole, (imidazoles), conazole compounds (triazoles), copper com uniconazole, uniconazole-P. triazole compounds, Bordeaux pounds, dicarboximide compounds, dichlorophenyl dicar mixture, Burgundy mixture, Cheshunt mixture, copper boximide compounds, phthalimide compounds, dinitrophe acetate, copper carbonate, basic, copper hydroxide, copper nol compounds, dithiocarbamate compounds, cyclic naphthenate, copper oleate, copper oxychloride, copper Sul dithiocarbamate compounds, polymeric dithiocarbamate fate, copper Sulfate, basic, copper Zinc chromate, cufraneb, compounds, imidazole compounds, inorganic compounds, cuprobam, cuprous oxide, mancopper, oxine copper, mercury compounds, inorganic mercury compounds, orga famoxadone, fluoroimide, chloZolinate, dichloZoline, iprodi nomercury compounds, morpholine compounds, organo one, isovaledione, myclozolin, procymidone, vinclozolin, phosphorus compounds, organotin compounds, oxathiin captafol, captan, ditalimfos, folpet, thiochlorfenphim, bina compounds, oxazole compounds, polyene compounds, pacryl, dinobuton, dinocap, dinocap-4, dinocap-6, dinocton, polysulfide compounds, pyrazole compounds, pyridine com dinopenton, dinosulfon, dinoterbon, DNOC, azithiram, car pounds, pyrimidine compounds, pyrrole compounds, quino bamorph, cufraneb, cuprobam, disulfiram, ferbam, metam, line compounds, quinone compounds, quinoxaline com nabam, tecoram, thiram, Ziram, dazomet, etem, milineb, pounds, thiocarbamate compounds, thiazole compounds, mancopper, mancoZeb, maneb, metiram, polycarbamate, thiophene compounds, triazine compounds, triazole com propineb, Zineb, cyaZofamid, fenamidone, fenapanil, glyo pounds, and urea compounds. din, iprodione, isovaledione, pefurazoate, triaZoxide, cona 0039). ANTIFUNGAL COMPOUNDS include the spe Zole compounds (imidazoles), potassium azide, potassium cific compounds amorolfine (dimethylmorpholine), bifona thiocyanate, sodium azide, Sulfur, copper compounds, inor Zole, butenafine, butoconazole, clioquinol, ciclopiroX ola ganic mercury compounds, mercuric chloride, mercuric mine, clotrimazole, econazole, fluconazole, griseofulvin, oxide, mercurous chloride, (3-ethoxypropyl)mercury bro haloprogen, iodochlorhydroxyquine, itraconazole, keto mide, ethylmercury acetate, ethylmercury bromide, ethylm conazole, miconazole, naftifine, oxiconazole, povidone-io ercury chloride, ethylmercury 2,3-dihydroxypropyl mercap dine sertaconazole, Sulconazole, terbinafine, terconazole, tide, ethylmercury phosphate, N-(ethylmercury)-p- tioconazole, tolnaftate, undecylenic acid and its salts (cal toluenesulphonanilide, hydrargaphen, cium, copper, and Zinc), Voriconazole, the Sodium or Zinc 2-methoxyethylmercury chloride, methylmercury benzoate, salts of proprionic acid, butylamine, cymoxanil, dodicin, methylmercury dicyandiamide, methylmercury pentachlo dodine, guaZatine, iminoctadine, carpropamid, chlorani rophenoxide, 8-phenylmercurioxyquinoline, phenylmercuri formethan, cyflufenamid, diclocymet, ethaboxam, fenoxa urea, phenylmercury acetate, phenylmercury chloride, phe nil, flumetover, furametpyr, mandipropamid, penthiopyrad, nylmercury derivative of pyrocatechol, phenylmercury prochloraz, quinazamid, silthiofam, triforine, benalaxyl, nitrate, phenylmercury Salicylate, thiomersal, tolylmercury benalaxyl-M, furalaxyl, metalaxyl, metalaxyl-M, pefura acetate, aldimorph, benzamorf, carbamorph, dimethomorph, Zoate, benalaxyl, benalaxyl-M, boScalid, carboxin, fenhexa dodemorph, fenpropimorph, flumorph, tridemorph, ampro mid, metalaxyl, metalaxyl-M, metSulfovax, ofurace, oxa pylfos, ditalimfos, edifenphos, fosetyl, hexylthiofos, dixyl, oxycarboxin, pyracarbolid, thifluZamide, tiadinil, iprobenfos, phosdiphen, pyrazophos, tolclofoSmethyl, tri benodanil, flutolanil, mebenil, mepronil, salicylanilide, amiphos, decafentin, fentin, tributyltin oxide, carboxin, oxy tecloftalam, fenfuram, furalaxyl, furcarbanil, methfuroxam, carboxin, chloZolinate, dichloZoline, draZOXolon, famoxa flusulfamide, benzohydroxamic acid, fluopicolide, done, hymexaZol, metaZOXolon, mycloZolin, oxadixyl, tioxymid, trichlamide, Zarilamid, Zoxamide, cyclafuramid, VincloZolin, barium polysulfide, calcium polysulfide, potas furmecyclox, dichlofluanid, tolylfluanid, amisulbrom, cya sium polysulfide, Sodium polysulfide, furametpyr, penthi Zofamid, benthiavalicarb, iprovalicarb, aureofungin, blasti opyrad, boscalid, buthiobate, dipyrithione, fluazinam, flu cidin-S, cycloheximide, griseofulvin, kasugamycin, natamy opicolide, pyridinitril, pyrifenox, pyroxychlor, pyroxyfur, cin, polyoxins, polyoxorim, streptomycin, validamycin, bupirimate, cyprodinil, diflumetorim, dimethirimol, ethiri azoxystrobin, dimoxystrobin, fluoxastrobin, kresoxim-me mol, fenarimol, ferimZone, mepanipyrim, nuarimol, US 2007/0092547 A1 Apr. 26, 2007 pyrimethanil, triarimol, fenpiclonil, fludioxonil, fluoroim 0047 Animal and kennel items including animal bed ide, ethoxyquin, halacrinate, 8-hydroxyquinoline Sulfate, ding including pet bedding, livestock bedding (includ quinacetol, quinoxyfen, benquinox, chloranil, dichlone, ing straw). Pet bedding can be selected from the dithianon, chinomethionat, chlorquinox, thioquinox, following list, but any animal is contemplated includ ethaboxam, etridiazole, metSulfovax, octhillinone, thia ing a dog, cat, pig, bird, and reptile. Livestock bedding bendazole, thiadifluor, thifluzamide, methasulfocarb, pro can be selected from the group consisting of bedding thiocarb, ethaboxam, silthiofam, anilazine, amisulbrom, bit for bovinas, equinas, pigs, sheep, goats and birds. This ertanol, fluotrimazole, triaZbutil, conazole compounds list should not be construed as limiting, as any bedding (triazoles), bentaluron, pencycuron, quinaZamid, aciben is contemplated for treatment in the present invention. Zolar, acypetacs, allyl alcohol, benzalkonium chloride, ben Zamacril, bethoxazin, carvone, chloropicrin, DBCP dehy 0048. The substrate can be selected from the group droacetic acid, diclomeZine, diethyl pyrocarbonate, consisting of articles worn or otherwise in contact with fenaminosulf, fenitropan, fenpropidin, formaldehyde, fur animals. These items include but are not limited to fural, hexachlorobutadiene, iodomethane, isoprothiolane, leashes, fomites, bridles, halters, horseshoes, animal methyl bromide, methyl isothiocyanate, metrafenone, apparel, and all other Substrates and articles that nitrostyrene, nitrothal-isopropyl, OCH, 2-phenylphenol, directly or indirectly contact an animal's epidermis. phthalide, piperalin, probenazole, produinazid, pyroquilon, 0049. The substrate can also be a human or animal Sodium orthophenylphenoxide, Spiroxamine, Sultropen, thi grooming device selected from the group consisting of cyofen, tricyclazole, iodophor, silver, Nystatin, amphoteri combs, brushes, picks, razors, and cutters. This list cin B, griseofulvin, and Zinc naphthenate. should not be construed as limiting, as any grooming 0040 SUBSTRATES: The invention provides for treat device is contemplated for treatment in the present ments of Substrates. Substrates include fomites, including invention. but not limited to: 0050 All substrates, including those above, that come 0041 Foot apparel, including shoes (including but not in direct or indirect contact with the epidermis of an limited to Sneakers, running shoes etc., boots, Sandals, animal are part of this invention. moccasins, slippers, etc), materials inserted within the 0051 DELIVERY METHODS: The following delivery shoe (including insoles, orthotics, linings, etc.) or other methods are included in this invention, but the list should be “foot coverings' (including Socks, stockings, etc). construed as limiting: Included are all shoes made from different types of materials, including leather, other animal skins, wood 0.052 Aerosol and wood derivatives, fabric, or other material (natural, synthetic, or semisynthetic) Subject to fungal contami 0053 Spray nation. 0054 Fog 0042. Other substrates where squames/skin cells or 0.055 Powder hair or nails or the keratin protein of these structures are found, including those places where people take off or 0056 Wipes put on shoes or other footwear or other apparel or 0057. Insertion where the skin of humans (ex. feet) or other mammals who harbor fungal organisms may come into contact. 0.058 Impregnation of the substrate with the antifungal 0043. A floor, or covering thereof, including carpet, compound tile, mat, etc. found in homes, hospitals, gyms, Swim 0059 Each delivery system can be used either prior to ming pools, saunas, airports or other public places. contamination with the fungus, or post contamination. 0044) Apparel which is worn on or comes in direct or indirect contact with skin including pants, shirt, gloves, DETAILED DESCRIPTION underwear, diapers, coats, and hats. This list should not 0060) Fungal diseases are some of the most common be construed as limiting, as any apparel is contemplated affecting mammals, and include Some of the most common for treatment in the present invention. infections in man. In humans these include, but are not 0045 Bedding, including but not limited to, bedding limited to: Selected from the group consisting of sheets, blankets, 0061 a) Tinea corporis—(“ringworm of the body'). This pillow, pillow cases, mattresses, and bedsprings. This infection causes Small, red spots that grow into large rings list should not be construed as limiting, as any bedding almost anywhere on the arms, legs, chest, or back. is contemplated for treatment in the present invention. Bedding includes any item that contacts the skin, 0062 b) Tinea pedis fungal infection of the feet. Typi directly or indirectly, in a bed. cally, the skin between the toes (interdigital tinea pedis or 0046. Furniture, including but not limited to, substrates “Athlete's foot') or on the bottom and sides of the foot Selected from the group consisting of a couch, a chair, (plantar or “moccasin type' tinea pedis) may be involved. a bed, or any piece of furniture covered in any material Other areas of the foot may be involved. The infections may (including but not limited to leather, fabric, vinyl, spread to the toenails (tinea unguium or onychomycosis) carpets, mats, etc.) Subject to fungal contamination. where it causes the toenails to become thick and crumbly. It This list should not be construed as limiting, as any can also spread to the hands and fingernails. furniture is contemplated for treatment in the present 0063 c) When the fungus grows in the moist, warm area invention. of the groin, the rash is called tinea cruris. The common US 2007/0092547 A1 Apr. 26, 2007

name for this infection is “jock itch.' Tinea cruris generally prior to washing can lead to a reduction in the passage of one occurs in men, especially if they often wear athletic equip infective unit from one piece of apparel to another. ment. 0072 The method provides for treating a substrate. This 0064 d) Tinea capitis, which is called “ringworm of the treatment involves contacting a substrate with the fungicidal Scalp' causes itchy, red areas, usually on the head. The hair compound in any manner (delivery methods are provided). is often destroyed, leaving bald patches. This tinea infection The substrate can be selected from any substrate that directly is most common in children. or indirectly contacts an epidermis. For example, items that contact an epidermis directly include horse bedding contact 0065 e) Dandruff, which is the excessive shedding (exfo ing the horse hoof, or dog bedding contacting the hair liation) of the epidermis of the scalp. A fungus may cause, protruding through the epidermis of the dog. Substrates that or aggravate, the condition. directly or indirectly contact a human epidermis can be 0.066 The list above providing but a few of the most widely varied as discussed in the definition of substrate. common of a long list of Such diseases in one mammal. 0073. In a further aspect the method includes a first and Many diseases caused by fungi have been identified, and a second antifungal compound that are applied in a single also include Such common disease as oral thrush and diaper application or in separate applications. Specific antifungal rash. Fungi are often a complicating factor in diabetic and compounds are not limited to any particular type or class of obese patients. In addition, disease in humans is caused by antifungal compounds. Although specific antifungal com other fungi including but not limited to those from the genus pounds are discussed herein these are only presented as Aspergillus, Blastomyces, Candida, Coccidioides, Crypto examples and should not be construed as limiting. coccus, Histoplasma, Paracoccidioides, Sporothrix, and at least three genera of Zygomycetes, as well as those men 0074 Direct contact of the epidermis includes all apparel tioned below under animals. that directly contacts the epidermis. 0075 A further aspect includes antifungal compounds 0067 Secondary infections that can worsen diaper rash that can be used in the method wherein the antifungal include fungal organisms (for example yeasts of the genus compound is selected from the group of known antifungal Candida). compounds, or classes of compounds including naturally 0068. In pets and companion animals the above fungi, as occurring compounds (including botanicals). well as many other fungi, can cause disease. The present 0076. The above lists should not be construed as limiting teaching is inclusive of Substrates that contact animals as any and all antifungal compounds are contemplated in the directly or indirectly. Examples of organisms that cause present invention. disease in animals include Malassezia firfiur; Epidermophy ton floccosum, Trichophyton mentagrophytes, Trichopyton 0077. A further aspect includes antifungal compounds in rubrum, Trichophyton tonsurans, Trichophyton equinum, the method wherein at least one of the first and second Dermatophilus Congolensisl, Microsporum canis, antifungal compound is a naturally occurring antifungal Microsporum audouini, Microsporum gypsium, Malassezia compound. ovale, Pseudalescheria, Scopulariopsis and Candida albi 0078. In addition, the inventors have shown for the first CCS. time that certain antifungal compounds have antibacterial 0069. The present teachings include methods for treating properties. This is particularly useful when treating certain a Substrate that, directly or indirectly, contacts an epidermis substrates that are susceptible to growth by both types of including: a) treating a substrate with a first antifungal organisms. compound, and b) treating a Substrate with a second anti 0079. In another aspect, a method for pre-treating a fungal compound. This treatment can occur at any time and Substrate that, directly or indirectly, contacts an epidermis includes treatment during manufacture of the Substrate or the comprising treating the Substrate with an antifungal com material that makes up the Substrate, as well as treatment pound prior to use by the products end user is provided. prior to or after contamination with a fungus or bacteria. This can be done through any delivery method. 0070 Decontamination of the apparel of individuals can 0080 Combining agents has been suggested to have a reduce fungal contact with the epidermis. This in turn can number of potential benefits, including: (a) extending and reduce initial fungal contamination rate, and reduce re broadening the spectrum of activity of the individual agents contamination rates for affected individuals. In the case of used, (b) increase the antimicrobial potency of individual Athlete's foot, treatment of the feet and the apparel can compounds, (c) reduce the development of resistance, (d) reduce the re-contamination rate and result in a more endur treat resistant strains and (e) reduce the concentration used ing cure of Athlete's Foot. This same paradigm is true for for at least one treatment agent, and (f) have anti-sporicidal many fungal contaminations. For example, white line dis activity with or without activating the spores. The data in the ease in horses (often caused by a fungal contamination of the Examples herein shows that using a combination of the hoof) can be passed through bedding, and recontamination synthetic, semi synthetic, and natural products will achieve of the same horse, or another, from bedding and/or the stall these objectives. in which they are kept. Treatment of substrates with anti fungal compounds could lead to decreased rates of contami 0081 For example, since miconazole, unlike terbinafine nation/infection and re-contamination/re-infection. and tolnaftate, possesses antibacterial activity, combining it with either agent will expand the spectrum of activity of 0071. It is also true that treatment prior to contamination disinfectant to cover dermatophytes, yeasts, and bacteria. In can reduce contamination rates, and treatment of apparel addition, because miconazole is a static agent against fungi, US 2007/0092547 A1 Apr. 26, 2007

combining it with either terbinafine or tolnaftate, which we thoroughness of coverage, etc. These can also be used to showed have fungal sporicidal activity, will expand the cause the penetration of a spore by an antifungal or anti killing activity of the combination. These combinations are bacterial compound. Excipients can also be used to cause the provided as examples and one of skill in the art can deduce spore to end dormancy and begin germination, thus making from these teachings other effective combinations that can the spore more susceptible to the compounds. work Synergistically. 0086) The composition comprising the antifungal or anti 0082 In addition, we describe compositions that limit bacterial compound can also include a compound to increase growth of odor causing bacteria, and the bacteria that cause adherence to the Substrate. Increasing adherence to the cellulitis. The addition of these compounds to a treatment or substrate can increase the length of time for which the pre-treatment composition will lead to advantageous syner compound remains in contact with the Substrate. gistic effects including limiting foot odor and cellulitis while treating fungal contamination. In a preferred embodiment 0087 Aspects of the present teachings may be further the composition contains a mixture of synthetic antifungal understood in light of the following examples, which should compounds and naturally occurring antifungal compounds not be construed as limiting the Scope of the present teach (e.g. terbinafine or tolnaftate and lemongrass oil or terbin ings. afine or tolnaftate lemongrass oil.). The inventors also have determined that the use of bactericidal compounds alone EXAMPLES (including naturally occurring compounds, including but not limited to clove bud, lemongrass, and Sandalwood oils) to Example 1 treat bacteria alone would also be advantageous (e.g. treat ing or pre-treating Substrates with bactericidal compounds). Examples of Antifungal Compounds that Function The advantages of treating with bactericidal compounds in the Invention include, but are not limited to, decreased odor from the substrate. 0088. The treatment in this example consists of at least two antifungal compounds. Typical compounds are listed by 0.083 Provided herein are newly discovered properties of their general class, chemical or otherwise. Concentrations compounds which include antibacterial, antifungal, and pertain to the class. They are stated as an overall range. One sporicidal properties. Also novel are the combinations of would select at least one compound from each group of compounds which provide unexpected results in the treat antifungal compounds described above or below and create ment and pre-treatment of Substrates against common fungi, a mixture of the two or more compounds. All percents (dermatophytes, yeasts, etc.) and bacteria. The inventors indicate weight/volume. show for the first time that combining naturally occurring fungicides with known fungicides leads to unexpectedly 0089 IMIDAZOLES (0.01-10%): bifoconazole, buto good results and they also show that uses of naturally conazole, clotrimazole, econazole, fluconazole, itra occurring fungicidal compounds, and for the first time, conazole, ketoconazole, miconazole, oxiconazole, expands their utility against bacteria. It was concluded by Saperconazole, Sertaconazole, Sulconazole, tercona the inventors that: 1) essential oils (especially lemongrass Zole, tioconazole, Voriconazole, ioloconazole and clove bud oils) can be used singly as natural products to inhibit microorganisms that infect Substrates and 2) com 0090 ALLYLAMINES AND BENZYLAMINES bining essential oils with a synthetic antifungal compound (0.001-10%; 0.05-5%): butenafine, naftifine, terbin will provide a broad spectrum activity. In addition to treating afine. common microorganisms, they can be used to treat drug 0.091 POLYENES (0.01-10%: 0.5-5%): amphotericin resistant microorganisms such as terbinafine resistant Tri B, candidicin, filipin, fungimycin, nystatin. chophyton rubrum and multi-drug resistant Candida, as well as allow the use of lower concentrations of synthetic agents 0092 MISCELLANEOUS SYNTHETIC ANTIFUN when combined with essential oils. Our method identified GAL COMPOUNDS (for example at 0.05-25%): amo disinfectant methods and regimens that have potent antifun rolfine (demethymorpholine), cicloproX olamine, halo gal and antibacterial activity and provides an effective progen, clioquinol, tolnaftate, undecylenic acid, means for preventing and treating fungal contamination of hydantoin, chlordantoin, pyrrolnitrin, salicylic acid, substrates (i.e. all substrates described herein), including but ticlatone, triacetin, griseofulvin, Zinc pyrithione. not limited to, shoes. 0093) DISINFECTANTS (for example at 0.001-20%): 0084 Having shown that antifungal compounds possess copper sulfate, Gentian Violet, betadyne/povidone potent anti-dermatophyte activity, the inventors also showed iodine, colloidal silver, Zinc. the activity of these agents against dermatophytes using bioassays. These results show that treatment, and pretreat 0094 BOTANICALS (for example at 0.01-10%): ment, of Substrates can lead to novel, unexpected results and Basil (Oncinum basilicum), Cassia (Cinnamomum provide manufacturers and consumers with novel techniques aromaticum var. Cassia), Cedrus wood oil (Cedrus and compositions for controlling bacteria, fungi, dermato libani or Cedrus spp). Chamomile (Chamaemelum phytes and other unwanted microorganisms on Substrates nobile), Citronella (Cymbopogon nardus), Clove (including, but not limited to, shoes). (Syzgium aromaticum), Cumin (Cuminum cyminum), Fennel (Foeniculum vulgare), Menth The/Mint (Men 0085. The use of particular excipients (detergents, oils, thThe X piperita/Menth The spicata), Tea Tree Oil etc.) can also function in the invention to increase the (Melaleuca alternfolia), Tumeric leaf oil (CurcumThe penetration of the substrate, the rate of penetration, the longa), Lemongrass Oil (Cymbopogon citratus). US 2007/0092547 A1 Apr. 26, 2007

Example 2 animals, thus preventing contamination and re-contamina Application Methods for Mixtures of Antifungal tion of their coat, skin, nails, hoofs, and similar structures by Compounds that means. 0.095. It is known, for example, that Athlete's foot, once 0101 The diseases include superficial dermatological cured by appropriate antifungal compounds on the skin, contaminations such as ringworm, rain rot, muck itch, girth reoccurs when the feet are re-infected by the same contami itch, white line, and thrush. Articles worn by animals that are nated footwear. The inventors have shown that treatment of substrates for treatment include, but are not limited to, shoe materials with antifungal compounds and combinations leashes, bridles, cinches, saddles, blankets, booties, fomites, of antifungal compounds can lead to unexpectedly good and horseshoes. results in treatment of Athlete's foot. This treatment can be 0102) A typical use of this invention is to decontaminate either a treatment of the substrate in contact with the foot the underside of saddles or saddle blankets, thus preventing prior to contamination (or use) or post contamination with contamination and re-contamination of equine ringworm. fungus or bacteria. In a preferred embodiment at least one of Another expected use of the invention is to decontaminate the natural oils describes herein as effective in treatment of the straw. This treatment will to prevent contamination and bacteria is used in the composition in order to limit fungal re-contamination by the myriad disease-causing fungi which growth while at the same time limiting foot/shoe odor and dwell within. cellulitis. 0096. In addition treatment of various other substrates Example 6 can also help break the cycle of Athlete's foot infections (infected feet, treated feet, reinfection of feet by untreated/ Evaluation of the Activity of Synthetic Antifungal contaminated footwear or other Substrates) including treat Compounds and Natural Substances Against ment of flooring. Microorganisms Infecting Shoes Using In Vitro and Example 3 Shoe and Insole Biopsy Disc Assays 0103) The shoe disinfecting activities of the following Treatment for Military Apparel compounds were studied: terbinafine, tolnaftate, micona 0097. A typical use of the invention will be to disinfect Zole, Cedrus oil, and tea tree oil, clover bud oil, lemongrass military socks, combat boots, and/or other apparel thus oil, sandalwood oil and spearmint oil. breaking the cycle of Athlete's foot re-contamination by contaminated footwear and clothing. The net effect will be 0104. In Vitro Susceptibility Testing a soldier relatively free from the itching and discomfort of that disease and/or other fungal contaminations. In this 0105 Determination of Minimum Inhibitory Concentra application boots, socks, and/or apparel is sprayed or soaked tion (MIC) and Minimum Fungicidal Concentration (MFC) in antifungal compounds in at least a antifungal compounds 0106 Minimum Inhibitory Concentration (MIC): Mini solution, or created from substrates previously treated at the mum inhibitory concentrations of synthetic and natural producer or manufacturer. All types and users of footwear products against dermatophytes were determined using a and clothing are contemplated as users of this invention, but modification of the Clinical Laboratory Standards Institute military clothing, footwear, and other apparatus are particu (CLSI, formerly National Committee of Clinical Laboratory larly prone to carry contamination since they are often worn Standards, NCCLS) M38A standard method for dermato for long periods. And as such are embodiments of the phytes developed at the Center for Medical Mycology (1) invention. while MIC of these agents against Candida species were Example 4 determined using the CLSI M27-A2 methodology (2). The method used to determine the MIC against bacteria was Treatment for Flooring and Rugs/Mats based on the CLSI document M7-A7 (3). 0098. A typical use of the invention will be to decon 0.107 For dermatophytes, serial dilutions of terbinafine, taminate the rugs commonly found near Swimming pools or tolnaftate were prepared in a range of 0.004-2 ug/ml, while in gyms, locker rooms (e.g. near locker room showers) and for miconazole concentrations ranged between 0.015-8 yoga classes. Since fungi thrive in warm, wet places, the ug/ml. Finally, for essential oils, the concentrations tested rugs can be cleared of the infectious organisms that cause were between 0.03-16 ug/ml. The only exception was tea ringworm and Athlete's foot. These substrates will be treated tree oil where dilutions were prepared in a range of 0.0078-4 using fungicidal compositions of the invention after use has ug/ml. The MIC was read at 4 days and defined as the lowest begun or prior to being put in place (e.g. at the manufacturer) concentration of an agent to inhibit 80% of fungal growth as in order to limit the growth of fungi on them. compared to the growth control (Table 2). 0099. The treatment of substrates on/in the flooring are 0.108 To determine the MIC of agents against Candida also contemplated at places such as gyms, security check species, serial dilutions of terbinafine and tolnaftate were points in airports, and other places where people regularly prepared in a range of 0.125-64 ug/ml, miconazole in a range remove their shoes. of 0.03-16 lug/ml and tea tree oil had a range between Example 5 0.125-4 ug/ml. The remaining essential oils were prepared in a dilution range of 0.03-16 lug/ml. For Candida the MIC was Treatment for Animal Substrates read at 24 hours and defined as the lowest concentration to 0100. The disinfectant kills or disables disease-causing inhibit 50% of fungal growth as compared to the growth fungi and fungus-like organisms in or on articles worn by control (Table 4). US 2007/0092547 A1 Apr. 26, 2007

0109 For bacterial species, the medium used to evaluate aim was to develop an assay that has utility in determining the antifungal and antibacterial activity of antifungal agents the activity of different agents to prevent (through pre and essential oils were RPMI 1640 (Hardy Diagnostics Santa treatment) and treat (through post-treatment) contamination Maria, Calif.) and Mueller-Hinton (Oxoid Ltd., Basingstoke, on shoes. The first step in the bioassay development was to Hampshire, England), respectively. Serial dilutions of identify optimal insole and leather material that represent miconazole, terbinafine, and tolnaftate were prepared in a substrates used in shoes and that do not inhibit fungi by range of 0.125-64 ug/ml and serial dilutions of tea tree oil themselves. To select the optimal shoe insole, discs mea were prepared in a range of 0.0078-4 ug/ml, while those for Suring 8 mm were cut using a Dermal Biopsy punch (Miltex, the rest of the essential oils were prepared in a range Bethpage, N.Y.) from four commercially available shoe 0.031-16 lug/ml. The MIC was read at 16 hand defined as the insoles (CVS odor stop insoles, Dr Scholls air pillow lowest concentration to inhibit 80% of bacterial growth insoles which claim antifungal activity, odor eater insoles, compared to the growth control (Table 5). and CVS double air foam insoles). These biopsy discs were placed on T. mentagrophytes seeded PDA plates. T. menta 0110 Minimum fungicidal concentration (MFC): The grophytes was used as a typical organism and is represen minimum fungicidal concentrations of various agents were tative of an entire class of fungi that grows on/in shoes and determined using the technique described earlier by Canton other substrates. The ability of insole biopsy discs from et al. (4). In this method, fungal conidia were collected existing products to inhibit dermatophytes, following incu following growth on potato dextrose agar (PDA) plates and bation for 7 days at 35° C., was determined. Our data were used to inoculate 96-well plates containing different showed that three of the insoles (CVS Odor Stop, Odor concentrations of agents. Following incubation at 35°C. for Eater, and CVS Double Air Foam) had a minimal antifungal 4 days (for dermatophytes) or 24 hours (for yeast), wells activity (FIG. 2A-C) while Dr Scholls insole did not inhibit showing no visible growth were cultured to determine the T. mentagrophytes at all (FIG. 2D). Similar approach was MFC (defined as the lowest concentration of a given agent used to determine whether biopsy discs from the leather hide that kills 99-100% of fungal conidia or spores). The MFC we obtained inhibit fungal growth. Our data showed that the value represents the level of the agent at which spores or leather material did not have any antifungal activity by itself conidia were killed. (FIG.2E). Therefore, Dr Scholl's insole and the leather hide 0111 Evaluation of the activity of combination of anti were used as Substrates in Subsequent experiments. fungal agents and essential oils against microorganisms 0.117) In our bioassay, we decided to use isopropanol as infecting shoes: a vehicle to dissolve the various disinfectants. We selected 0112 Combining agents has been suggested to have a this solvent because it is a common solvent used in different number of potential benefits, including: (a) extending and preparations marketed for the treatment of tinea pedis. We broadening the spectrum of activity of the individual agents next performed experiments to identify a concentration of used, (b) increase the antimicrobial effectiveness of indi isopropanol that did not inhibit fungal growth by itself. The vidual compounds, (c) reduce the development of resistance, ability of three different concentrations (30%, 50%, and (d) treat resistant strains and (e) reduce the concentration 100%) of isopropanol to inhibit dermatophyte growth was used for at least one treatment agent, and (f) have sporicidal tested. Our data showed that 30% isopropanol was the activity (5). optimal concentration at which the vehicle did not inhibit fungal growth on the insoles and leather surface (FIG. Bioassay 3A-B). In some experiments, because tolnaftate does not 0113. The shoe substrate used in this study was Dr dissolve very well in isopropanol, we performed additional SchollsC) air pillow insoles. We selected this substrate to experiments using acetone as a vehicle. use in our bioassay because we showed earlier that this 0118 Based on the above experiments, our disc biopsy insole has no inhibitory activity against dermatophytes (see assay employed Dr. Scholls insole and leather discs as the below), and is representative of the type of material used in optimal Substrates representing materials used in shoes, and manufacturing shoe insoles. 30% isopropanol as the optimal vehicle to dissolve the 0114) To evaluate the ability of the agents to prevent and agents to be tested in pre-treatment and post-treatment treat fungal contamination of insoles and leather, we deter studies. mined their activity against the dermatophyte T. mentagro 0119) Evaluation of the Ability of Various Agents to phytes, and developed novel insole/leather biopsy assays. T. Prevent and Treat Fungal Shoe Contamination. mentagrophytes was used as the model Strain in our bioassay studies because this fungus is a major cause of tinea pedis 0120) Two types of disc biopsy assays were used to and onychomycosis. Unlike T. rubrum, which is often iden evaluate the ability of different synthetic and natural sub tified as the causative organism in these diseases but is a stances to disinfect shoe material: (a) Pre-treatment assay: poor producer of spores/conidia, T. mentagrophytes, in addi Where discs were pre-treated with antifungals first and then tion to being an etiological agent of these diseases, produces infected with T. mentagrophytes, and (b) Post-treatment conidia reproducibly and therefore, is amenable for use in a assay: where discs were first infected with T. mentagro bioassay. It is expected that activity in this assay against T. phytes, then treated with drugs. These assays will reveal the mentagrophytes will be indicative of activity against T. ability of different agents to prevent and treat shoe fungal rubrum and other dermatophytes. contamination, respectively. 0115 Development of a Shoe Bioassay. 0121 Pre-treatment assays: To evaluate the ability of different agents to prevent fungal contaminations of shoes, 0116. To evaluate the shoe disinfecting ability of various PDA plates were prepared on which 10 T. mentagrophytes agents, it was necessary to develop a bioassay method. Our cells were evenly spread. Next, discs from insoles and US 2007/0092547 A1 Apr. 26, 2007

leather were treated as follows (with either agent or control Results vehicle): discs were pretreated with a single spray, spraying for 15 second or 30 second. Other discs were immersed in 0125) In Vitro Susceptibility Testing agent or vehicle for 30 min. Following this treatment, discs 0.126 Minimum Inhibitory Concentration (MIC) and were air-dried by placing them in a Petri plate for 1 min. Minimum Fungicidal These dried discs were then placed (drug side down) on the 0127 Concentration (MFC) seeded PDA plates. Plates were then incubated for 4 days at 30° C. Following incubation, fungal growth was recorded. 0.128 Evaluation of the inhibitory activity of various agents showed that these agents were effective in inhibition Active agents showed a clearance Zone around the biopsy of dermatophytes, yeasts and bacteria to varying degrees. disc (FIG. 1A, arrow), while inactive agents showed fungal Data from these MIC/MFC studies are summarized in Table growth all around the disc (FIG. 1B). Diameter of the 1 (for details of the MIC/MFC data, please see Tables 2-5) clearance Zone (CZD) was measured. The relative activity of Summary of the antifungal and antibacterial activity of different agents and control were assessed. In this assay, different synthetic and natural products tested is Summarized active agents had higher CZD than inactive or less active below. OS. Antimicrobial Activity of Synthetic Agents: 0122) Post-treatment assays: To evaluate the ability of 0129. Terbinafine: Our results showed that terbinafine various agents to treat infected shoes, PDA plates were was highly active against all isolates of the three dermato prepared on which 10 T. mentagrophytes cells were evenly phytes genera tested where low MIC values were noted spread on their surface. Next, untreated biopsy discs were (MIC range=0.008-0.06 ug/mL). In addition, this agent was placed on these PDA plates and incubated for 4 days at 30° able to kill spores of these dermatophytes as demonstrated C. Incubating the biopsy discs in this manner allowed the by low MFC values (MFC range was between 0.03-0.125 ug/mL). Evaluation of the anti-yeast activity of terbinafine fungi to invade the discs. Infected discs were picked and showed that this agent possesses high activity against all C. post-treated with different agents by spraying. Post-treated parapsilosis isolates tested (MIC values for all isolates was discs were allowed to air dry and were then placed on fresh, 0.25 ug/mL). In this regard, C. parapsilosis is a known skin uninoculated PDA plates and incubated for 4 additional days normal flora inhabitant. Our data showed that terbinafine at 30° C. Incubation of the discs under these conditions was less active against C. albicans compared to C. parap allows any fungi that are not killed by the sprayed agent to silosis with one to three fold higher MIC values against the grow. In other words, agents that are effective in the treat majority of isolates tested relative to C. parapsilosis (MIC ment of shoe material will not show any fungal growth values for 5 strains ranged between 0.5 and-2 g/mL). around the disc biopsy (FIG. 1C). In contrast, discs treated Interestingly, terbinafine exhibited no effect against one C. with ineffective agents will show fungal growth emanating albicans strain (strain 8280 where the MIC was >64 g/mL). from them (FIG. 1D). Diameter of the growth Zone (GZD) In contrast to the high activity of terbinafine seen against was determined as a measure of the activity of the agent dermatophytes and yeast strains, this agent did not show any tested. In this assay, inactive or less active agents had higher antibacterial activity against all bacterial strains examined GZD than active agents, while active agents did not show (MICd64 ug/mL for all strains tested). any fungal growth (GZD=0). 0.130 Tolnaftate: Evaluation of the antifungal activity of tolnaftate showed that this agent is highly active against the 0123 Scanning Electron Microscopy (SEM). dermatophytes tested both in fungal inhibition (MIC range against all dermatophytes tested was 0.008-0.125 ug/mL) 0124 Scanning electron microscopy (SEM) was used to and spore killing (MFC range was 0.06-0.125 ug/mL). monitor the ability of agents to eradicate fungal growth on Tolnaftate inhibitory and sporicidal activity was similar to shoe insoles or leather biopsy discs. Pre- and post-treated terbinafine or slightly (one dilution) higher. Evaluation of discs were processed for SEM by the method of Chandra et the anti-yeast activity of tolnaftate showed that this agent al. as described earlier (6). One set of discs was used as a has a reduced activity against yeast compared to terbinafine. control in which no drug pre- or post-treatment was per Elevated MICs for tolnaftate was observed against all C. formed. In addition, one set of biopsy discs was used as albicans strains tested (MIC value for all strains was 264 blank where no fungal cells or drug were added. Following ug/mL). While activity of tolnaftate against C. parapsilosis treatment, discs were fixed with 2% glutaraldehyde for 2 h, was strain-dependent with one strain (#7629) showing low and then washed with sodium cacodylate buffer (three times MIC (0.5 lug/mL), while the other isolates exhibited rela for 10 minutes each). The discs were then treated with 1% tively high MIC values (MIC=8-16 lug/mL). Susceptibility osmium tetraoxide (for 1 h at 4°C.) followed by a series of testing of bacteria to tolnaftate showed that this agent had no washing with sodium cacodylate buffer, followed by a two S. aureus antibacterial activity (MIC for all strains tested times washing with distilled water. Next, the discs were was >64 g/mL), while possessing some strain-dependent treated with 1% tannic acid washed three times with distilled activity against S. epidermidis strains: two strains had MIC water, and followed by 1% uranyl acetate with two water values of 2 ug/mL, while the remaining four exhibited MICs washings. The samples were then dehydrated through a ranging between 16 and >64 g/mL. series of ethanol solutions (range from 25% (vol/vol) etha 0131 Miconazole: Susceptibility testing of dermato nol in distilled water to absolute ethanol). Prepared samples phytes against miconazole showed that this agent possesses were then sputter coated with Au/Pd (60/40) and viewed a potent antifungal activity against T. mentagrophytes, T. with Amray 1000B scanning electron microscope. rubrum, and E. floccosum with MIC values ranging from US 2007/0092547 A1 Apr. 26, 2007

0.06 to 0.125 g/mL. Compared to terbinafine and tolnaftate, 0.137 C.1.2. Activity Against Yeast: miconazole had a slightly lower activity. Moreover, unlike these agents, miconazole was static against dermatophytes. 0138 Next we tested the ability of these oils to inhibit (MFC of miconazole against all T. mentagrophytes isolates yeast (C. albicans and C. parapsilosis). As can be seen in and the majority of T. rubrum and E. floccosum isolates Table 8, four of the essential natural oils (clover bud, tested was 28 ug/mL). Our data show that miconazole lemongrass, spearmint, and tea tree oils) were active against possesses a modest anti-yeast activity. In general, the MIC these clinically important fungi, with MIC range between values of miconazole against both C. albicans and C. 0.063-0.5 lug/mL. The only exception was sandalwood oil, parapsilosis were higher than those obtained for terbinafine. which had an MIC of 4 to >16 ug/mL (Table 3). These C. albicans showed some strain-dependent Susceptibility results suggested that sandalwood oil exhibited no inhibitory against miconazole, with an MIC=1-2 g/mL for four iso activity against Candida species and strains tested. lates, 16 Jug/mL for another and 216 g/mL for the remain 0.139 C.1.3. Activity against Bacteria: ing albicans strain (8280). MIC values of miconazole against C. parapsilosis were also strain dependent (MICs 0140 We next tested the in vitro activity of the essential ranging from 4 to 216 g/mL). In contrast to terbinafine and natural oils against: (a) odor-producing (Micrococcus and tolnaftate (which had no activity against bacteria), micona Corynebacteria) bacteria, and (b) Staphylococcus aureus (a Zole was active against both S. aureus and S. epidermidis major cause of cellulitis). As seen in Table 9, clove bud, isolates tested (MIC values against all Staphylococcus iso lemongrass, and sandalwood oils were active against the lates were between 0.5 and 2 ug/mL). odor-producing bacteria tested (MIC=0.25-2 ug/mL), while spearmint and tea tree oils did not show in vitro activity 0132 Cedrus oil: Antifungal susceptibility testing of (MIC=2-8 ug/mL). Furthermore, clove bud, lemongrass, and Cedrus oil showed that this natural oil possessed acceptable sandalwood oils showed some activity (MIC=0.25-8 ug/mL) antifungal activity against dermatophytes in vitro with MIC against Staphylococcus. Moreover, lemongrass tended to ranging between 0.5 and 2 g/mL. In addition, Cedrus oil have one to two dilutions lower MIC than clove and san exhibited species-dependent cidality: MFC against T. men dalwood oils, indicating it is more active. In contrast, tagrophytes was noticeably higher (MFC=4-16 ug/mL) than spearmint and tea tree oil did not show noticeable activity against E. floccosum, and T. rubrum isolates (MFC=0.5-4 against any of the pathogenic bacterial isolates tested (MIC= ug/mL). Results are detailed in Table 1. 8-32 g/mL). These studies showed that clove bud and 0133) Tea Tree Oil: Antifungal susceptibility testing of lemongrass had the broadest antimicrobial activity com dermatophytes against tea tree oil showed that this natural pared to the other essential oils and are viable candidate for product is highly active in inhibiting and spore killing of use as natural products to treat shoes. these fungi (MICs=0.125-0.4, while MFCs were =0.25 to 0.141. D. 1. Evaluation of the Activity of Combination of >4 g/mL against all dermatophytes tested). Moreover, the Antifungal Agents and Essential Oils Against Microorgan MIC and MFC values of tea tree oil against dermatophytes isms Infecting Shoes: were lower than those noted for Cedrus oil. A majority of the yeast isolates were resistant to tea tree oil (MICd4 g/mL). 0.142 To assess the potential for using antifungal syn Interestingly, one C. albicans isolates (8280) was suscep thetic agents (e.g. terbinafine, tolnaftate and miconazole) tible to tea tree oil, although the same isolate was resistant and essential oils (e.g. clove bud, lemongrass, Sandalwood, to terbinafine, tolnaftate, and miconazole (with an MIC of spearmint and tea tree oil) in combination, we evaluated the 64, >64, and >16 ug/mL, respectively). This finding is very ability of essential oils to inhibit terbinafine-resistant T. interesting because it indicates that combining tea tree oil rubrum and C. albicans strain (strain number MRL 8280) with any of the three agents may provide enhanced shoe that exhibits multi-resistance to terbinafine, miconazole and disinfecting activity, Suggesting that adding tea tree oil to tolnaftate. As shown in Table 10, all the terbinafine-resistant any of the antifungals may provide a broad coverage against T. rubrum isolates tested were susceptible to the essential resistant isolates (MICd4 ug/mL). The possibility of com natural oils, with an MIC range of 0.031 to 0.25 g/mL. The bining tea tree oil with different agents against this resistant most potent oil was lemongrass which showed very low fungus was evaluated (see below). The bacterial strains MICs against these Trichophyton isolates. tested were not susceptible to tea tree oil. Results are 0.143 Similarly, the essential oils were effective in inhib detailed in Table 1. iting the multi-resistant C. albicans strain. The most effec tive essential oil in inhibiting this resistant strain was Results lemongrass (see Table 11). 0134) Antimicrobial activity of all effective essential oils against dermatophytes known to grow on feet, causing tinea D. CONCLUSIONS pedis, yeasts known to cause nail infection, and bacteria that 0144) Based on these data it is concluded that: 1) essen can cause foot infection or generate unpleasant odor. tial oils (especially lemongrass and clove bud oils) can be 0135 C.1.2. Activity Against Dermatophytes: used singly as natural products to inhibit microorganisms that infect shoes. 2) combining essential oils with a synthetic 0136. In these studies we evaluated the activity of essen antifungal provides a broad spectrum activity, treats terbin tial oils against dermatophytes, yeast and bacteria. Table 7 afine resistant Trichophyton rubrum, and multi-drug resis presents a Summary of the anti-dermatophyte activity of tant Candida, as well as allows the use of lower concentra essential oils. As can be seen, the five essential oils tested tions of synthetic agents when combined with essential oils. exhibited potent antifungal activity against dermatophytes Our method identified disinfectant “systems” that have with MICs ranging between 0.125 and 0.5 lug/mL. potent antifungal and antibacterial activity and provides an US 2007/0092547 A1 Apr. 26, 2007

effective means for preventing and treating fungal contami already established on shoe insoles, we determined the effect nations of shoes and other Substrates. of post-treating infected insoles with these agents on their 0145 Having shown that terbinafine, tolnaftate, and ability to clear the established fungal contamination using essential oils possess potent anti-dermatophyte activity our post-treatment shoe biopsy disc assay developed (see against microorganisms that colonize and infect the foot above). As shown in FIG. 7A (and Table 6), while the using in vitro Susceptibility assays, we next investigated the vehicle control failed to treat already established fungal activity of these agents against dermatophytes using the shoe contamination as evidenced by the presence of fungal disc bioassay we developed, and SEM techniques. regrowth (GZD=33 mm of growth), terbinafine completely 0146 Effect of Pretreatment of Shoe Insoles and Leather eradicated established contamination on insoles (GZD=0 Surfaces with Synthetic and Natural Products on Preventing mm). Tolnaftate (both 1% and 2%) were also effective in Dermatophyte Shoe Contamination clearing the contamination of insole, although some minimal 0147 Pretreatment of Insoles regrowth was observed (GZD=8 mm and 10 mm, respec 0148. To determine the ability of terbinafine, tolnaftate, tively). In contrast, tea tree oil was not able to treat the and tea tree oil to prevent shoe contamination we used the contamination present on insole biopsies (GZD=33 mm). pretreatment insole biopsy bioassay method described O155 Post Contamination Treatment of Leather above. As shown in FIG. 4 (and Table 11, representative images in FIG. 5), pretreatment of insoles with terbinafine 0156 Next, we determined whether terbinafine, tolnaf 1% solution resulted in complete inhibition of fungal growth tate, or tea tree oil can treat T. mentagrophytes contamina (CZD=85 mm, fungal inhibition reached to the edge of the tion already established on leather biopsy discs. As shown in Petri dish) compared to vehicle control (CZD=0 mm). This FIG. 7B (and Table 6), terbinafine completely cleared the complete inhibition was observed even when the insoles established contamination on leather disc (GZD=0 mm). discs were pretreated with a single spray. Pre-treatment of Moreover, tolnaftate (1% and 2%) also reduced contamina biopsy discs with tolnaftate (1% and 2%) also inhibited tion of leather (GZD=11 mm for both concentrations). Tea fungal growth (CZD=25 mm and 11 mm, respectively) tree oil induced very minimal inhibition of fungal growth on compared to vehicle control, albeit to a lesser extent than the infected leather biopsies compared to vehicle control terbinafine. Because tolnaftate dissolves better in acetone, (GZD=26 mm versus 33 mm). These data clearly demon we repeated Some experiments using acetone as a vehicle. strate that post treatment of insoles and leather with terbin Our data showed that tolna?tate has a potent preventive afine and tolnaftate is an effective way for treating infected activity against dermatophytes infecting shoes (FIG. 5). shoes. Increasing the concentration of tolnaftate to 3% and 4% increased the activity. In contrast, 1% tea tree oil had no Effect of Combination of Tolnaftate and Tea Tree Oil on inhibitory prevention effect (CZD=0 mm). Taken together, Preventing Dermatophyte Shoe Contamination these data show that pretreatment of shoe insoles and leather 0157 To determine whether using combination of syn material with terbinafine or tolnaftate is an effective way to thetic compounds and essential oils will allow the use of low prevent fungal contamination of shoes. Importantly, these concentrations of synthetic drugs, we tested the ability of agents were superior to the marketed Dr. Scholl's brand in combination of 0.01% tolnaftate and 3% tea tree oil to preventing fungal contamination of insoles. prevent shoe contamination using the pre- and post-treat 0149 Pretreatment of Leather ment insole bioassays described above. 0150. To determine whether pretreatment of leather biopsy discs with terbinafine, tolnaftate, or tea tree oil can 0158 As shown in FIG. 10, pretreatment of insoles with prevent growth of T. mentagrophytes, we tested their activity 0.01% terbinafine or 3% tea tree oil singly did not result in using the bioassay method described above. As shown in any inhibition of fungal growth. In contrast, the combination FIG. 4B, pretreatment of leather disc with vehicle did not of 0.01% terbinafine and 3% tea tree oil induced a noticeable result in any inhibition (CZD=0 mm), while terbinafine inhibition of fungal growth (FIG. 10C). pretreatment resulted in complete inhibition of fungal 0159 Furthermore, while 0.01% tolnaftate or 3% tea tree growth (CZD=85 mm, also see Table 6). Pretreatment of oil did not prevent growth of fungus on insole after post leather biopsies with 1% tolnaftate resulted in inhibition of treatment (FIG. 10A (A)-(B), post-contamination treatment fungal growth (CZD=16 mm, FIG. 4B). However, pretreat with the combination of these agents reduced fungal growth ment of leather disc with 1% tea tree alone oil did not inhibit (FIG. 10A (C). fungal growth (CZD=0 mm). 0151. These data indicate that pre-treatment of leather 0.160 These data show that combining tolnaftate and tea material with terbinafine or tolnaftate is an effective way for tree oil will allow the use of low concentration of tolnaftate preventing fungal contamination of the leather used in to prevent and treat shoe contamination. shoes. 0.161 Scanning Electron Microscopy Analyses 0152 Effect of Post-Contamination Treatment with Syn thetic and Natural Products on Eradication of Pre-Estab 0162 SEM of Pre-Treated and Post-Treated Insoles lished Dermatophyte Contamination on Insoles and Leather 0.163 To determine the effect of synthetic and natural Surfaces products (pre- and post-contamination treatments) on the 0153. Ability of Agents to Treat Insoles Infected with ability of T. mentagrophytes to grow on insole biopsies, we Dermatophytes performed SEM analysis. As shown in FIG. 8, pretreatment 0154) To determine the ability of terbinafine, tolnaftate, of insole with the vehicle had no effect on fungal growth or tea tree oil to treat T. mentagrophytes contamination (FIG. 8C), while terbinafine and tolnaftate completely eradi US 2007/0092547 A1 Apr. 26, 2007 cated fungal growth (FIG. 8D.E; no fungal elements were 0173 Terbinafine and tolnaftate were able to kill der seen). However, and similar to the bioassay studies, pre matophytes fungal spores that may be present in shoes, treatment with tea tree oil reduced the fungal growth on while miconazole is static against dermatophyte spores. insoles but did not eliminate it from the biopsy disc (FIG. 8F). Post-contamination treatment of insole with terbinafine 0.174 Terbinafine and miconazole were also effective resulted in complete clearance of fungal growth (FIG. 8G). against the yeast Candida species, but in a strain- and while treatment with tolnaftate was minimally effective, as species-dependent manner. shown by the presence of several filaments (FIG. 8H). In contrast, post-contamination treatment with tea tree oil had 0.175 Miconazole exhibited activity against bacterial no activity against T mentagrophytes (FIG. 8I). species, while tolnaftate exhibited strain-dependent inhibi 0164. These data show that pre- and post-treatment with tion of S. epidermidis. terbinafine is highly effective in eradicating fungal elements 0176 Among the natural products tested (tea tree oil, from shoe material infected with T. mentagrophytes. Addi Cedrus oil, clove bud, lemongrass oil, Sandalwood Oil, tionally, tolnaftate was effective in eradicating fungal ele Spearmint Oil). ments only if insole biopsies were pretreated with this agent. 0165 Taken together, these data indicate that pre- and 0177 3. Our findings show that: post-treatment of insoles with either terbinafine or tolnaftate 0.178 Essential oils have a broad antimicrobial activity is effective in preventing the fungal colonization of, and covering dermatophytes, yeast and bacteria that infect shoes. treatment of already existing, fungal growth on insoles. The most active essential oils were lemongrass followed by 0166 Our data also demonstrate that combining tolnaf clove bud. tate and tea tree oil will allow the use of low concentration of tolnaftate to prevent and treat shoe contamination 0.179 Essential oils possess inhibitory activity against bacteria that produces unpleasant odors and Staphylococcus 0167 SEM Analysis of Pre-Treated Leather Biopsy Discs aureus (a major cause of cellulitis). 0168 We used SEM analyses to determine the effect of terbinafine, tolnaftate and tea tree oil pretreatment on their 0180. The essential natural oils have potent in vitro ability to prevent T. mentagrophytes growth on leather activity against terbinafine-resistant dermatophytes, as well biopsies. As shown in FIG. 9, pretreatment of leather biopsy as multi-resistant C. albicans. Lemongrass possesses the disc with terbinafine completely eradicated fungal growth, most potent activity in this regard. where no fungal elements were seen (FIG.9D), compared to untreated leather disc (FIG. 9B) or vehicle-treated disc (FIG. 0181. In bioassay studies, terbinafine and tolnaftate pre 9C), where massive fungal elements can be seen invading treatment were able to inhibit fungi on insoles and leather the leather material. Pretreatment with tolnaftate appeared to shoe biopsies compared to vehicle control. Terbinafine was reduce the fungal density on leather discs, but did not the most active pre-treatment agent. completely eradicate dermatophyte growth (FIG.9E). Discs pre-treated with tea tree oil did not show any effect on fungal 0182 Terbinafine post-treatment was able to treat estab growth (FIG. 9F), and were similar in appearance to the lished fungal contamination on shoe biopsy discs. Although discs pretreated with isopropanol, the vehicle control. tolnaftate showed antifungal activity as a post-treatment 0169 Taken together, these results revealed that pre agent, its activity was less than that of terbinafine. Tea tree treatment of leather with terbinafine was highly effective in oil was ineffective as a post-treatment agent. preventing and eradicating fungal elements from leather material infected with T. mentagrophytes, while tolnaftate 0183 Combining a synthetic antifungal agent with an was minimally effective. In contrast, tea tree oil was inef essential oil allows the use of low doses of the synthetic fective in eradicating contamination of leather discs. Post antifungal to prevent and treat infected shoe insole. contamination treatment of leather with terbinafine, tolnaf 0.184 Our data indicate that combining agents is likely to tate, and tea tree oil is currently being performed. provide benefit by expanding the spectrum of activity of a 0170 Our findings show that: disinfectant through the inhibition of resistant fungal strains. 0171 Among the synthetic agents tested (terbinafine, 0185. In conclusion, the invented disinfectant has potent tolnaftate, miconazole): antifungal and antibacterial activity and provides an effec 0172 The most active agent inhibiting dermatophytes tive means for preventing and treating fungal contamina was terbinafine, followed by tolnaftate and miconazole. tions of shoes and other Substrates.

TABLE 1. Range of MICs (ig/ml) and MFCs (ig/ml) of Terbinafine, Tolnaftate, Miconazole, Tea Tree Oil and Cedrus Oil against Dermatophytes, Yeasts and Bacteria Terbinafine Tolnaftate Miconazole Tea tree oil Cedrus oil Organism (g/ml) (ig/ml) (g/ml) (g/ml) (Lig/ml) All Dermatophytes

MIC Range O.O15 O.12S O.O15-0.25 O.0625- 0.25 0.25-10 MFC Range O.O3-0.125 0.06-1.O OS->8.O O.125->2 OS-8 US 2007/0092547 A1 Apr. 26, 2007 13

TABLE 1-continued Range of MICs (ig/ml) and MFCs (ig/ml) of Terbinafine, Tolnaftate, Miconazole, Tea Tree Oil and Cedrus Oil against Dermatophytes, Yeasts and Bacteria

Terbinafine Tolnaftate Miconazole Tea tree oil Cedrus oil Organism (g/ml) (ig/ml) (g/ml) (ig/ml) (g/ml)

All Yeasts

MIC range O.25->64 OS->64 1->16 O.125->2 ND MFC range OS-2 ND: All Bacteria

MIC range >64 2->64 OS-2 >4 ND: MFC range ND:

*ND - not determined

0186

TABLE 2 Minimum Inhibitory Concentration (MIC, Ig/mL) and Minimum Fungicidal Concentration (MFC, ugmL) of Terbinafine, Tolnaftate, and Miconazole Against Dermatophytes

MICONAZOLE

Organism MIC MFC E. floccosum

1666 1798 1925 1926 1961 2 5 216S MIC Range (n = 6) MICso MICoo T. rubrum

1967 2098 2246 8063 8071 8092 s MIC Range (n = 6) 1-8 MICso 8 MICoo T. mentagrophytes

1720 2124 2125 2126 2127 2128 MIC Range (n = 6) MICso MICoo All dermatophytes

MIC Range (n = 18) O.O15- 0.25 MICso O.12S MICoo O.25 *ND = Not Determined

US 2007/0092547 A1 Apr. 26, 2007

0189)

TABLE 5 Minimum Inhibitory Concentration (MIC, Ig/mL) of Terbinafine, Tolna?tate, Miconazole, and Tea Tree Oil against Staphylococcus species.

TEA TERBINAFINE TOLNAFTATE MICONAZOLE TREE OIL STRAIN MIC MIC MIC MIC

S. atiretts

93 NON-VLABLE NON-VIABLE NON-VIABLE NON-VIABLE

734 >64 >64 2 >4 847O >64 >64 2 >4 MIC Range (n = 6) >64 >64 2 >4 MICso >64 >64 2 >4 MICoo >64 >64 2 >4 S. epidermidis

8472 >64 2 O.S >4 8473 >64 16 1 >4 8474 >64 2 O.S >4 8475 >64 >64 1 >4 8476 >64 >64 1 >4 8477 >64 >64 1 >4 MIC Range (n = 6) >64 2->64 OS-1 >4 MICso >64 16 1 >4 MICoo >64 >64 1 >4 All bacteria

MIC Range (n = 12) >64 2->64 OS-2 >4 MICso >64 >64 1 >4 MICoo >64 >64 2 >4

0190.

TABLE 6

Effect of pretreatment and post-contamination treatment of leather and insole biopsy discs with different agents on growth of T. mentagrophytes.

Insoles Post- Leather Insoles contamination Leather Post-contamination Pretreatment Treatment Pretreatment Treatment Spray (CZD*, mm) (GZD*, mm) (CZD, mm) (GZD, mm)

30% Isopropanol O 33 O 33 1% Terbinafine 85 O 85 O 1% Tolnaftate 25 8 18 11 2% Tolnaftate 10 10 6 11 1% Tea Tree Oil O 33 O 26 196 Tol. 19, TTO** 11 17 11 2O 290 TOI 19/o TTO 19 8 15 11 290 TOI 296 TTO 10 34 O 3O 3%. To 196 TTO 25 11 2O 11

*CZD - clearance Zone diameter; GZD - growth Zone diameter. **Tol - Tolnaftate: TTO - tea tree oil. US 2007/0092547 A1 Apr. 26, 2007

0191)

TABLE 7 Activity of essential oils against dermatophytes Clove Lemongrass Sandalwood Spearmint Tea Tree Organism Species Bud Oil Oil Oil Oil Oil Epidermophyton fioccostin O.12S O.25 O.S O.S O.S Epidermophyton fioccostin O.12S O.25 O.25 O.12S O.S Trichophyton mentagrophytes 0.125 O.S O.25 O.25 O.25 Trichophyton mentagrophytes 0.125 O.25 O.25 O.25 O.25 Trichophyton rubrum O.12S O.25 O.S O.25 O.S Trichophyton rubrum O.12S O.25 O.S O.S O.S MIC Range O.125- 0.12S O.25- O.S O.25- O.S O.125 O.S 0.25- O.S MICso O.12S O.25 O.25 O.25 O.S MICoo O.12S O.S O.S O.S O.S

0192)

TABLE 8 Activity of essential natural oils against yeast Isolates Clove Lemongrass Sandalwood Spearmint Tea Organism Species Bud Oil Oil Oil Oil Tree Oil Candida Albicans O.12S O.O63 >16 O.S O.25 Candida Albicans O.12S O.25 >16 2 1 Candida Albicans O.S O.12S >16 2 1 Candida Parapsilosis 0.125 0.125 4 0.5 0.25 Candida Parapsilosis 0.125 O.12S >16 O.S O.25 Candida Parapsilosis 0.25 O.12S >=16 1 O.25 MIC Range O.125 O.S. O.O63-0.25 4->16 OS-2 O.25-1 MICso O.12S O.12S >16 O.S O.25 MICoo O.S O.25 >16 2 1

0193)

TABLE 9 Activity of natural oils against (A) odor-causing and (B) pathogenic bacterial isolates Clove Bud Lemongrass Sandalwood MRL Oil MIC Oil MIC Oil MIC Spearmint Oil Tea Tree Oil Number Organism (mg/mL) (mg/mL) (mg/mL) MIC (mg/mL) (mg/mL) (A) Odor causing bacteria

781 Corynebacterium sp. 2 O.25 O.S 8 8 782 Corynebacterium sp. 1 O.25 O.25 4 2 783 Micrococcus luteus O.S O.S O.25 4 2 784 Micrococcus luteus O.S O.25 O.S 2 4 MIC Range(n = 4) OS-2 O.25- O.S O.25- O.S 2-8 2-8 MICso O.S O.25 O.25 4 2 MICoo 2 O.S O.S 8 8 (B) Pathogenic bacteria

730 S. auretts 2 1 2 8 32 732 S. auretts 1 1 O.25 8 NG 733 S. auretts 1 O.S O.S 8 8 8470 S. auretts 2 1 2 16 32 MIC Range(n = 4) 1-2 OS-1 O.25-2 8-16 8-32 MICso 2 1 25 8 32 MICoo 2 1 >2 8 >32 US 2007/0092547 A1 Apr. 26, 2007

0194)

TABLE 10 Activity of natural oils against terbinafine-resistant T. rubrum isolates Terbinafine Clove Bud Lemongrass Sandalwood Spearmint Tea Tree oil Organism MRL Number MIC oil MIC oil MIC oil MIC oil MIC MIC T. rubrum 666 16 O.25 O.O63 2 2 4 T. rubrum 670 16 O.12S O.O63 O.S O.S 1 T. rubrum 671 4 O.12S O.O63 1 O.S 1 T. rubrum 1386 4 O.12S &=0.031 O.S O.25 4 T. rubrum 1806 4 O.12S &=0.031 O.S O.25 O.S T. rubrum 1807 4 O.12S O.O63 O.S O.S 4 T. rubrum 1808 16 O.12S O.25 O.S O.S 2 T. rubrum 1809 16 O.25 O.12S 2 2 4 T. rubrum 1810 4 O.O63 &=0.031 O.12S O.12S 2 T. rubrum 2499 4 O.12S O.12S 1 O.S 4 T. rubrum 2727 2 O.12S &=0.031 O.25 O.12S 1 MIC Range(n = 11) 2-16 O.O63-0.25 &=0.031-0.25 O.125-1 O.125-2 OS 4 MICso 4 O.12S O.O63 O.S O.S 2 MICoo 16 O.25 O.12S 2 2 4

0195 the Substrate with a second antifungal compound wherein the application process is selected from the group consisting TABLE 11 of serially, simultaneously, or separately. 2. The method of claim 1 wherein said first antifungal Activity of essential oils against a multi-resistant strain of C. albicans compound and said second antifungal compound are applied strain 8280). in a single application. Essential Oil MIC (ig/mL) 3. The method of claim 1 wherein said first antifungal Clove Bud Oil O.12S compound and said second antifungal compound are applied Lemongrass Oil O.O63 in separate applications. Sandalwood Oil >16 4. The method of claim 1 wherein at least a third anti Spearmint Oil O.S fungal compound is applied to the Substrate in an applica tion, wherein the application process is selected from the Other Embodiments group consisting of serially, simultaneously, or separately. 5. The method of claim 1 wherein the substrate is apparel. 0196. The detailed description set-forth above is pro vided to aid those skilled in the art in practicing the present 6. The method of claim 5 wherein said apparel is a shoe. invention. However, the invention described and claimed 7. The method of claim 5 wherein said apparel is a sock. herein is not to be limited in scope by the specific embodi 8. The method of claim 5 wherein said apparel is a pants, ments herein disclosed because these embodiments are shirt, glove, underwear, diapers, coat, and hat. intended as illustration of several aspects of the invention. 9. The method of claim 1 wherein said substrate is Any equivalent embodiments are intended to be within the bedding. Scope of this invention. Indeed, various modifications of the 10. The method of claim 9 wherein said bedding is invention in addition to those shown and described herein selected from a group consisting of sheets, blankets, pillows, will become apparent to those skilled in the art from the pillow cases, mattresses, and bedsprings. foregoing description which do not depart from the spirit or 11. The method of claim 1 wherein said substrate is scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended furniture. claims. 12. The method of claim 11 wherein said furniture is selected from a group consisting of a couch, a chair, a bed, 0.197 All publications, patents, patent applications and and a piece of furniture. other references cited in this application are incorporated 13. The method of claim 1 wherein said substrate is herein by reference in their entirety for all purposes to the selected from the group consisting of animal bedding, straw, same extent as if each individual publication, patent, patent grooming devices, stalls, and cages. application or other reference was specifically and individu ally indicated to be incorporated by reference in its entirety 14. The method of claim 13 wherein said animal bedding for all purposes. Citation of the reference herein shall not be is selected from a group consisting of pet bedding and construed as an admission that Such is prior art to the present livestock bedding. invention. 15. The method of claim 14 wherein said pet bedding is selected from a group consisting of bedding for a dog, cat, What is claimed is: pig, bird, and reptile. 1. A method for treating a substrate that, directly or 16. The method of claim 14 wherein said livestock indirectly, contacts an epidermis comprising: a) treating the bedding is selected from a group consisting of bedding for Substrate with a first antifungal compound, and b) treating bovinas, equinas, pigs, sheep, goats, and birds. US 2007/0092547 A1 Apr. 26, 2007

17. The method of claim 13 where the grooming devices 30. The method of claim 27 wherein said substrate is a are selected from the group consisting of combs, brushes, Sock. picks, razors, and cutters. 31. A method of decreasing the LD50 of a compound with 18. The method of claim 1 wherein said substrate is antifungal properties by combining said antifungal with a selected from articles worn or otherwise in contact with second compound wherein said second compound is a animals. botanical antifungal compounds. 19. The method of claim 18 wherein said article is 33. A method of claim 1 wherein the substrate is a shoe. selected from the group consisting of fomites, bridles, 34. A method of claim 1 wherein the substrate comprises halters, horseshoes, and apparel. the floor of hospital, gym, or airport. 20. The method of claim 1 wherein at least one of said first 35. A method of claim 1 wherein at least one antifungal antifungal compound and said second antifungal compound compound is selected for its ability to inhibit or kill spores. is selected from a class of known antifungal compounds. 36. A method of claim 35 wherein the antifungal com 21. The method of claim 1 wherein at least one of said first pound is naturally occurring. antifungal compound and second antifungal compound is a 37. A method of claim 36 wherein the antifungal com naturally occurring antifungal compound(s). pound is botanical. 22. The method of claim 21 wherein at least one of said 38. A method of claim 37 wherein the antifungal com first antifungal compound and second antifungal compound pound is selected from the group consisting of clove bud, is a botanical antifungal compound(s). lemongrass, and Sandalwood oils. 23. A method for treating the substrate that, directly or 39. A method of claim 38 wherein the substrate is shoes. indirectly, contacts an epidermis comprising: a) treating the 40. A method of claim 39 wherein the treatment is a Substrate with the first antifungal compound, and b) treating pre-treatment. the Substrate with at least a second antifungal compound, 42. A composition for treating shoes wherein the compo wherein at least one of said first antifungal compound and sition comprises about 0.01% tolnaftate and about 3% tea said second antifungal compound is applied via an aerosol. tree oil. 24. A method for treating a substrate that, directly or 43. A composition for treating a substrate wherein the indirectly, contacts an epidermis comprising: a) treating the composition comprises less than 1% tolnaftate and greater Substrate with a first antifungal compound, and b) treating than 1% tea tree oil. the Substrate with at least a second antifungal compound, 44. A method of claim 1 wherein at least one antifungal wherein at least one of said first antifungal compound and compound is a bactericidal compound. said second antifungal compound is applied via a delivery 45. A method of claim 44 wherein the bactericidal com method. pound is naturally occurring. 25. A method for delivering one or more antifungal 46. A method of claim 45 wherein the bactericidal com compound to a Substrate wherein said system applies at least pound is botanical. one of said first antifungal compound and said second 47. A method of claim 46 wherein the bactericidal com antifungal compound via a fog, aerosol, spray, powder, pound is clove bud, lemongrass, and sandalwood oils. wipe, insertion, or impregnation. 48. A method of applying an antifungal or antibacterial 26. A method of claim 25 wherein at least one antifungal compound or mixture of at least a antifungal or antibacterial compound is naturally occurring. compounds of the invention which increases the Suscepti 27. A method for pre-treating a substrate that, directly or bility of spores by causing spore germination or increasing indirectly, contacts an epidermis comprising treating the the penetration of the antifungal or antibacterial compound Substrate with one or more antifungal compound(s) prior to into the spore. use by the product’s end user. 49. A method of claim 1 wherein the composition com 28. A method of claim 27 wherein at least one antifungal prising the antifungal compound also includes a compound compound is naturally occurring. to increase adherence to the Substrate. 29. The method of claim 27 wherein said substrate is a shoe or shoe insole.