US 2008022O103A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0220103 A1 Birnbaum et al. (43) Pub. Date: Sep. 11, 2008

(54) METHOD FOR Publication Classification TREATING/CONTROLLING/KILLING FUNG (51) Int. Cl. AND BACTERIA ON LIVING ANIMALS A636/00 (2006.01) A 6LX 36/899 (2006.01) (76) Inventors: Jay Birnbaum, Montville, NJ (US); A636/534 (2006.01) Thomas Blake, Budd Lake, NJ A6IR 36/6 (2006.01) (US); Mahmoud Ghannoum, A636/736 (2006.01) Hudson, OH (US); Steven A63L/045 (2006.01) Vallespir, Park Ridge, NJ (US) A63L/05 (2006.01) A63L/35 (2006.01) Correspondence Address: A63L/II (2006.01) SONNENSCHEN NATH & ROSENTHAL LLP A63L/92 (2006.01) P.O. BOX 061080, WACKER DRIVE STATION, A6II 3/474 (2006.01) SEARS TOWER A63L/35 (2006.01) CHICAGO, IL 60606-1080 (US) A6II 3/165. (2006.01) A6II 3/442 (2006.01) (21) Appl. No.: 12/047,992 A6IP3 L/10 (2006.01) (52) U.S. Cl...... 424/735; 424/725; 424/747: 514/739; (22) Filed: Mar. 13, 2008 514/731: 514/729; 514/456; 514/699; 514/570; 514/701: 514/399; 514/655; 514/599; 514/345 Related U.S. Application Data (63) Continuation-in-part of application No. 1 1/541,822, (57) ABSTRACT filed on Oct. 2, 2006. Provided is a method of treating a fungal infection on an animal epidermis, nail or hair, or in an orifice of an animal. (60) Provisional application No. 60/729,624, filed on Oct. The method comprises contacting the fungus infection with a 24, 2005. composition comprising an antifungal botanical. Patent Application Publication Sep. 11, 2008 Sheet 1 of 10 US 2008/022O103 A1

Patent Application Publication Sep. 11, 2008 Sheet 2 of 10 US 2008/022O103 A1

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Patent Application Publication Sep. 11, 2008 Sheet 7 of 10 US 2008/022O103 A1

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S SS S& ::$ Fig. 10A. Effect of pretreatment of insoles with (A) 0.01% tolnaftate, (B) 3% tea tree oil, or (C) 0.01% tolnaftate +3% tea tree oil on T. rubrum growth on shoe insoles.

Fig. 10B. Effect of post- ment tolnaftate, (B) 3% tea tree oil, or (C) 0.01% tolnaftate +3% tea tree oil on T. rubrunt growth.

Figure 10 US 2008/0220 1 03 A1 Sep. 11, 2008

METHOD FOR 0014 Secondary infections that can worsen diaper rash TREATING/CONTROLLING/KILLING FUNG include fungal organisms (for example yeasts of the genus AND BACTERA. ON LIVING ANIMALS Candida). 0015 The above fungi, as well as many other fungi, can CROSS-REFERENCE TO RELATED cause disease in pets and companion animals. The present APPLICATIONS teaching is inclusive of Substrates that contact animals directly or indirectly. Examples of organisms that cause dis 0001. This application is a continuation-in-part of U.S. ease in animals include Malassezia firfir: Epidermophyton patent application Ser. No. 1 1/541,822, filed Oct. 2, 2006, floccosur, Trichophyton mentagrophytes, Trichophyton which claims the benefit of U.S. Provisional Application No. rubrum, Trichophyton tonsurans, Trichophyton equinum, 60/729,624 filed Oct. 24, 2005, both of which are incorpo Dermatophilus congolensis, Microsporum Canis, rated herein by reference in their entirety. Microsporu audouinii, Microsporum gypseum, Malassezia BACKGROUND OF THE INVENTION ovale, Pseudallescheria, Scopulariopsis, Scedosporium, and Candida albicans. 0002 (1) Field of the Invention 0003. The present teachings relate to methods for treating/ BRIEF SUMMARY OF THE INVENTION preventing animal diseases and odors associated with fungi and bacteria on Surfaces or orifices of animals, including the 0016. The inventors have discovered that some botanicals, skin, hair, nails, mouth, nose and vagina of the animal. singly or combined with other antifungal agents, are effective 0004 (2) Description of the Related Art treatments for fungal diseases on Surfaces or orifices of ani 0005 Fungal diseases are some of the most common mals, including the skin, hair, nails, mouth, nose and vagina affecting mammals, and include some of the most common of the animal. The invention is thus directed to a method of infections in man. In humans these include, but are not limited treating a fungal infection on an animal epidermis, nail or tO: hair, or in an orifice of an animal. The method comprises 0006 Tinea corporis—(“ringworm of the body'). This contacting the fungus infection with a composition compris infection causes Small, red spots that grow into large rings ing an antifungal botanical. almost anywhere on the arms, legs, chest, or back. 0007 Tinea pedis fungal infection of the feet. Typically, BRIEF DESCRIPTION OF THE SEVERAL the skin between the toes (interdigital tinea pedis or "Athlete's VIEWS OF THE DRAWINGS foot) or on the bottom and sides of the foot (plantar or “moccasin type' tinea pedis) may be involved. Other areas of 0017 FIG. 1 is photographs of Petri plates showing assays the foot may be involved. for antifungal activity. Panels A and B show a pre-treatment 0008 Onychomycosis—fungal infection of the nail. The assay. In Panel A, active agents showed a clearance Zone most prevalent type is the DSO or Distal Subungual Onycho (arrow) around the biopsy disc, while (Panel B) inactive mycosis. Other types are White Superficial Onychomycosis, agents showed fungal growth around the disc. Panels Cand D Proximal Subungual Onychomycosis, Candidal Onychomy show a post-treatment assay. In Panel C, discs treated with cosis, and Total Dystrophic Onychomycosis. These can be active agents showed no fungal growth. In Panel D inactive caused by various fungi (esp. dermatophytes tinea unguium) agents showed fungal growth on discs. and yeast, including Candida albicans. 0018 FIG. 2 is photographs of Petri plates showing fungal 0009 Dandruff, which is the excessive shedding (exfolia growth assays using (A) CVS Double Air Foam Insoles, (B) tion) of the epidermis of the scalp. A fungus may cause, or Odor eater insoles, (C) CVS Odor Stop Insoles, (D) Dr aggravate, the condition. Scholl's Air Pillow Insoles, (E) Control. None of these com 0010 Tinea cruris: When the fungus grows in the moist, mercial insoles inhibited fungal growth. warm area of the groin, the rash is called tinea cruris. The 0019 FIG. 3 is photographs of Petri plates showing the common name for this infection is “jock itch.” effect of 30% isopropanol on Trichophyton mentagrophytes 0011 Tinea capitis, often called “ringworm of the scalp'. growth on (A) leather and (B) Dr. Scholl's insole. Isopropanol where the hair and scalp is affected, causes itchy, red areas, did not inhibit fungal growth. usually on the head. The hair is often destroyed, leaving bald 0020 FIG. 4 is graphs showing the effect of pretreatment patches. This tinea infection is most common in children, of insoles (A) or leather (B) biopsy discs with different agents although a carrier state has been reported in adults. on growth of dermatophytes. Zone diameter indicates Zone of 0012 Vaginal yeast infections, often caused by an over clearance. growth of a fungus that is a normal vaginal inhabitant, usually 0021 FIG. 5 is photographs of Petri plates showing the Candida albicans and Candida glabrata. effect of pretreatment of insoles with (A) 1% terbinafine. (B) 0013 The list above providing but a few of the most com 1% tolnaftate, or (C) 1% tea tree oil. mon of a long list of Such diseases in one mammal. Many 0022 FIG. 6 is photographs of Petri plates showing the diseases caused by fungi have been identified, and also effect of acetone on the activity of tolnaftate against dermato include Such common disease as oral thrush and diaper rash, phyte growth. Panels A and B shows the growth of T. menta often caused by members of the Candida genus. Fungi are grophytes on insole disc pretreated with (A) acetone or (B) often a complicating factor in diabetic and obese patients. In 4% tolnaftate (w/v. prepared in acetone). Panel C shows the addition, disease inhumans is caused by other fungi including activity of 4% tolnaftate (dissolved in acetone) on already but not limited to those from the genus Aspergillus, Blasto established contamination of T. mentagrophytes. (no fungal myces, Coccidioides, Cryptococcus, Histoplasma, Paracoc regrowth was observed). cidioides, Sporothrix, and at least three genera of Zygo 0023 FIG. 7 is graphs showing the effect of post-infection mycetes, as well as those mentioned below under animals. treatment of insole (A) or leather (B) biopsy discs with dif US 2008/0220 1 03 A1 Sep. 11, 2008 ferent agents on dermatophyte growth. Zone diameter indi natural in origin that possesses antifungal activity as defined. cates Zone of growth. Treatment with 30% isopropanol served "Antifungal compound also includes any Substance that can as vehicle control. destroy/kill/inhibit the growth of fungal spores, for example, 0024 FIG. 8 shows scanning electron microscopy (SEM) any Substance that possesses a sporistatic (inhibitory) or images of insoles infected with T. mentagrophytes. Magnifi sporicidal (killing) activity. See definition of Sporicidal com cation 2000x for all panels. Bar represents 20 uM for panels pound below. Thus, throughout this document the term Anti A through F, while it represents 100 M for the post-infected fungal compound is an all encompassing term referring to any treated discs (Panels G-I). Substance (synthetic, semisynthetic, salt, pro-drug, natural, 0025 FIG.9 shows scanning electron microscopy (SEM) etc.) with antifungal activity, including, inhibitory, killing, images of leather biopsies infected with T mentagrophytes. static, cidal, sporistatic or sporicidal activity. These com Magnification 2000x; bar-20 lum. pounds can in turn be mixed with, for example, other antifun 0026 FIG. 10 is photographs of Petri plates showing gal compounds, detergents, and/or inactive ingredients to assays for antifungal activity. The top panels (FIG. 10A) create formulation/s. shows the effect of pretreatment of insoles with (A) 0.01% 0031. As used herein, a SPORE is a fungus in its dormant, tolnaftate, (B)3%tea tree oil, or (C) 0.01% tolnaftate-3% tea protected State. It has a Small, usually single-celled reproduc tree oil, on T. rubrum growth on shoe insoles. The lower tive body that is highly resistant to desiccation and heat and is panels (FIG. 10B) shows the effect of post-treatment of capable of growing into a new organism (vegetative state), infected insoles with (A) 0.01% tolnaftate. (B)3% tea tree oil, produced especially by certain bacteria, fungi, algae, and or (C) 0.01% tolnaftate--3% tea tree oil on T. rubrum growth. non-flowering plants. 0032. As used herein, a SPORICIDAL COMPOUND is a DETAILED DESCRIPTION OF THE INVENTION substance that either inhibits the growth of, increase the sus ceptibility of and/or destroys fungal spores. These can be Abbreviations and Definitions synthetic, semisynthetic, or naturally occurring. Activating 0027. As used herein, a BOTANICAL is a compound iso spores allows fungicides that only kill or inhibit actively lated from a plant. Botanical antifungal compounds can be growing fungi to kill those spores activated. This can be used, isolated from, for example, Ocimum basilicum (Basil), Cin for example, in a mixture wherein a chemical(s) that activates namomum aromaticum var. Cassia (Cinnamon), Cedrus growth is mixed with a chemical fungicide(s). It is also pos libani (Cedar of Lebanon), any Cedrus spp., Chamaemelum sible to use at least an activating compound alone, followed nobile (Chamomile), Cymbopogonnardus (Citronella), Syzy by at least a fungicide, serially. Activating spores is a method gium aromaticu (Clove & clove bud), Cuminum cyminum known in the art for bacterial spores, for example in U.S. Pat. (Cumin), Foeniculum vulgare (Fennel), Melaleuca alternfo No. 6,656,919, which is herein incorporated by reference. lia (Tea Tree), Mentha x piperita (Peppermint), Mentha spi 0033. As used herein, a BACTERICIDAL COMPOUND cata (Spearmint), Curcuma longa (Tumeric), Cymbopogon is a substance that either inhibits the growth of increases the citratus (Lemongrass), Santalum album (Sandalwood), as susceptibility of and/or destroys bacteria or bacteria spores. well as other compounds or ingredients, such as, but not These can be synthetic, semisynthetic, or naturally occurring. limited to, eugenol, isolated from plants that have antifungal Activating spores allows bactericides that only kill or inhibit and/or antibacterial properties. actively growing bacteria to kill those spores activated. This 0028. As used herein, a NATURAL ANTIFUNGAL can be used, for example, in a mixture wherein a chemical (s) COMPOUND (or naturally occurring antifungal compound) that activates growth is mixed with a chemical bactericide(s). is a compound isolated from a botanical Source (see botanical It is also possible to use at least an activating compound alone, antifungal compound) or other naturally occurring Source followed by at least a bactericide, serially. Activating spores is e.g. mammalian cells, including but not limited to, neutro a method known in the art for bacterial spores, for example in phils, or body fluid, e.g., saliva, amphibian skin, invertebrates U.S. Pat. No. 6,656,919, which is herein incorporated by (e.g. worms). These compounds can be proteins (e.g., reference. enzymes), polysaccharides, Small organic molecules, or other 0034. As used herein, an EPIDERMIS is the outer, protec products produced by animals or plants. tive, nonvascular layer of the skin of vertebrates, covering the 0029. A FUNGUS is any of numerous eukaryotic organ dermis, it serves as the major barrier function of skin and is isms of the kingdom Fungi (mycota), which lack chlorophyll devoted to production of a cornified layer of the skin. Epider and vascular tissue and range in form from a single cell (e.g., mally derived structures include hair (and fur), claws, nails, yeast) to a body of mass branched filamentous hyphae that and hooves. often produce specialized fruiting bodies and pseudohyphae. 0035 Treating an animal epidermis, nail, hair or orifice, The kingdom includes, but is not limited to, the yeasts, fila means to contact, expose or apply a Substance to the epider mentous molds, dermatophytes, Smuts, and mushrooms. mis, nail, hair or orifice. This can include, but is not limited to, 0030. As used herein, an ANTIFUNGALCOMPOUND is the delivery methods discussed below. A cream, ointment, defined herein as any chemical or Substance that has the gel, liquid, Solution, foam, powder, paste, gum, lacquer, ability to inhibit the growth of fungi, and/or killfungal cells or shampoo, Suspension, fog, spray, aerosol, pump spray, wipe spores. Compound as used throughout this application or sponge, or any otherformulation, can include, for example, includes salts and pro-drugs of the compound. Included in the at least one fungicide. An effective treatment leads to the definition of antifungal compound is any Substance that pos reduction in the presence of the infecting organism, an inhi sess static (e.g. inhibitory) activity (FUNGISTATIC COM bition in growth of the infecting organism, and/or a reduction POUNDS) and/or cidal (e.g. killing) activity (FUNGICIDAL in the signs and/or symptoms of the infection, and need not COMPOUNDS) against fungal cells (vegetative and spore necessarily lead to eradication of the infection. structures). Also included in the definition of antifungal com 0.036 MINIMAL INHIBITORY CONCENTRATION pound is any Substance that is synthetic, semisynthetic or (MIC) is described, for instance, in Clin Infect Dis. 1997 US 2008/0220 1 03 A1 Sep. 11, 2008

February; 24(2):235-47. Tests for antifungal activity include cin, validamycin, azoxystrobin, dimoxystrobin, fluoxas MIC and MFC (Minimum Fungicidal Concentration) assays. trobin, kresoxim-methyl, metominostrobin, orysastrobin, These assays are used to determine the Smallest amount of picoxystrobin, pyraclostrobin, trifloxystrobin, biphenyl, drug or compound needed to inhibit (MIC) or kill (MFC) the chlorodinitronaphthalene, chloroneb, chlorothalonil, cresol, fungus. dicloran, hexachlorobenzene, pentachlorophenol, quin 0037 Examples of antifungal compounds can be selected toZene, sodium pentachlorophenoxide, tecnaZene, benomyl. from the following chemical classes, or chemicals below, or carbendazim, chlorfenazole, cypendazole, debacarb, naturally occurring compounds: aliphatic nitrogen com fuberidazole, mecarbinzid, rabenzazole, thiabendazole, pounds, amide compounds, acylamino acid compounds, ally furophanate, thiophanate, thiophanate-methyl, bentaluron, lamine compounds, anilide compounds, benzanilide com chlobenthiazone, TCMTB, bithionol, dichlorophen, diphe pounds, benzylamine compounds, furanilide compounds, nylamine, benthiavalicarb, furophanate, iprovalicarb, pro Sulfonanilide compounds, benzamide compounds, furamide pamocarb, thiophanate, thiophanate-methyl, benomyl, car compounds, phenylsulfamide compounds, Sulfonamide com bendazim, cypendazole, debacarb, mecarbinzid, pounds, valinamide compounds, antibiotic compounds, stro diethofencarb, climbazole, imazalil, Oxpoconazole, prochlo bilurin compounds, aromatic compounds, benzimidazole raz, triflumizole, imidazole compounds, azaconazole, bro compounds, benzimidazole precursor compounds, ben muconazole, cyproconazole, diclobutraZol, difenoconazole, Zothiazole compounds, bridged diphenyl compounds, car diniconazole, diniconazole-M, epoxiconazole, etaconazole, bamate compounds, benzimidazolylcarbamate compounds, fenbuconazole, fluguinconazole, flusilazole, flutriafol, fur carbanilate compounds, conazole compounds, conazole conazole, furconazole-cis, hexaconazole, imibenconazole, compounds (imidazoles), conazole compounds (triazoles), ipconazole, metconazole, myclobutanil, penconazole, propi copper compounds, dicarboximide compounds, dichlorophe conazole, prothioconazole, quinconazole, Simeconazole, nyl dicarboximide compounds, phthalimide compounds, tebuconazole, tetraconazole, triadimefon, triadimenol, triti dinitrophenol compounds, dithiocarbamate compounds, conazole, uniconazole, uniconazole-P. triazole compounds, cyclic dithiocarbamate compounds, polymeric dithiocarbam Bordeaux mixture, Burgundy mixture, Cheshunt mixture, ate compounds, imidazole compounds, inorganic com copper acetate, copper carbonate, basic, copper hydroxide, pounds, mercury compounds, inorganic mercury com copper naphthenate, copper oleate, copper oxychloride, cop pounds, organomercury compounds, morpholine per Sulfate, copper Sulfate, basic, copper Zinc chromate, compounds, organophosphorus compounds, organotin com cufraneb, cuprobam, cuprous oxide, mancopper, oxine cop pounds, Oxathin compounds, oxazole compounds, polyene per, famoxadone, fluoroimide, chlozolinate, dichlozoline, compounds, polysulfide compounds, pyrazole compounds, iprodione, isovaledione, mycloZolin, procymidone, VincloZo pyridine compounds, pyrimidine compounds, pyrrole com lin, captafol, captan, ditalimfos, folpet, thiochlorfemphim, pounds, quinoline compounds, quinone compounds, qui binapacryl, dinobuton, dinocap, dinocap-4, dinocap-6, dinoc noxaline compounds, thiocarbamate compounds, thiazole ton, dinopenton, dinosulfon, dinoterbon, DNOC, azithiram, compounds, thiophene compounds, triazine compounds, tria carbamorph, cufraneb, cuprobam, disulfiram, ferbam, Zole compounds, and urea compounds. metam, nabam, tecoram, thiram, Ziram, dazomet, etem, mil 0038 Antifungal compounds include the specific com neb, mancopper, mancoZeb, maneb, metiram, polycarbam pounds albaconazole, amorolfine (dimethylmorpholine), ate, propineb, Zineb, cyaZofamid, fenamidone, fenapanil, amphotericin B, including lipid formulations of amphotericin glyodin, iprodione, isovaledione, pefurazoate, triaZOxide, B Such as AmBisome and Abelcet, anidulafungin bifonazole, conazole compounds (imidazoles), potassium azide, potas butenafine, butoconazole, caspofungin, clioquinol, ciclo sium thiocyanate, sodium azide, Sulfur, copper compounds, piroX olamine, clotrimazole, econazole, fluconazole, griseof inorganic mercury compounds, mercuric chloride, mercuric ulvin, haloprogen, iodochlorhydroxyquine, itraconazole, oxide, mercurous chloride, (3-ethoxypropyl)mercury bro ketoconazole, miconazole, naftifine, oxiconazole, povidone mide, ethylmercury acetate, ethylmercury bromide, ethylm iodine sertaconazole, Sulconazole, terbinafine, terconazole, ercury chloride, ethylmercury 2,3-dihydroxypropyl mercap tioconazole, tolnaftate, undecylenic acid and its salts (cal tide, ethylmercury phosphate, N-(ethylmercury)-p- cium, copper, and Zinc), Voriconazole, the Sodium or Zinc toluenesulphonanilide, hydrargaphen, salts of proprionic acid, butylamine, cymoxanil, dodicin, 2-methoxyethylmercury chloride, methylmercury benzoate, dodine, guaZatine, iminoctadine, carpropamid, chlorani methylmercury dicyandiamide, methylmercury pentachlo formethan, cyflufenamid, diclocymet, ethaboxam, fenoxaniil, rophenoxide, 8-phenylmercurioxyquinoline, phenylmercuri flumetover, furametpyr, mandipropamid, penthiopyrad, urea, phenylmercury acetate, phenylmercury chloride, phe prochloraz, quinaZamid, silthiofam, triforine, benalaxyl, nylmercury derivative of pyrocatechol, phenylmercury benalaxyl-M, furalaxyl, metalaxyl, metalaxyl-M, micafun nitrate, phenylmercury Salicylate, thiomersal, tolylmercury gin, pefurazoate, benalaxyl, benalaxyl-M, boscalid, car acetate, aldimorph, benzamorf, carbamorph, dimethomorph, boxin, fenhexamid, metalaxyl, metalaxyl-M, metSulfovax, dodemorph, fenpropimorph, flumorph, tridemorph, ampro ofurace, oxadixyl, oxycarboxin, pyracarbolid, posaconazole, pylfos, ditalimfos, edifenphos, fosetyl, hexylthiofos, AN2690 (Anacor Pharmaceuticals), AN2718 (Anacor), thi iprobenfos, phosdiphen, pyrazophos, tolclofoS-methyl, tri fluzamide, tiadinil, benodanil, flutolanil, mebenil, mepronil, amiphos, decafentin, fentin, tributyltin oxide, carboxin, oxy salicylanilide, tecloftalam, fenfuram, furalaxyl, furcarbanil, carboxin, chloZolinate, dichloZoline, draZOXolon, famoxa methfuroxam, flusulfamide, benzohydroxamic acid, fluopi done, hymexaZol, metaZOXolon, mycloZolin, oxadixyl, collide, tioxymid, trichlamide, Zarilamid, Zoxamide, cycla VincloZolin, barium polysulfide, calcium polysulfide, potas furamid, furmecyclox, dichlofluanid, tolylfluanid, amisul sium polysulfide, Sodium polysulfide, furametpyr, penthiopy brom, cyaZofamid, benthiavalicarb, iprovalicarb, rad, boScalid, buthiobate, dipyrithione, fluaZinam, fluopi aureofungin, blasticidin-S, cycloheximide, griseofulvin, collide, pyridinitril, pyrifenox, pyroxychlor, pyroxyfur, kasugamycin, natamycin, polyoxins, polyoxorim, streptomy bupirimate, cyprodinil, diflumetorim, dimethirimol, ethiri US 2008/0220 1 03 A1 Sep. 11, 2008

mol, fenarimol, ferimZone, mepanipyrim, nuarimol, Examples of orifices include a mouth, nose, ear, vagina, anus, pyrimethanil, triarimol, fenpiclonil, fludioxonil, fluoroimide, and urethra. Also included are artificially created orifices such ethoxyquin, halacrinate, 8-hydroxyquinoline Sulfate, quinac as fistulas. etol, quinoxyfen, benquinox, chloranil, dichlone, dithianon, 0043. In some aspects of these embodiments, the orifice is chinomethionat, chlorquinox, thioquinox, ethaboxam, etridi a mouth or nose. In other aspects the orifice is a vagina. azole, metsulfovax, octhillinone, thiabendazole, thiadifluor, 0044. In other embodiments, the fungal infection is on an thifluzamide, methasulfocarb, prothiocarb, ethaboxam, silth epidermis. iofam, anilazine, amisulbrom, bitertanol, fluotrimazole, tri 0045. In additional embodiments, the fungal infection is aZbutil, conazole compounds (triazoles), bentaluron, pency on a nail. curon, quinaZamid, acilbenzolar, acy petacs, allyl alcohol, 0046. In some of the invention methods, the fungal infec benzalkonium chloride, benzamacril, bethoxazin, carvone, tion causes or aggravates tinea corporis, tinea pedis, tinea chloropicrin, DBCP dehydroacetic acid, diclomezine, cruris, tinea unguium, tinea capitis, dandruff, a vaginal yeast diethyl pyrocarbonate, fenaminosulf, fenitropan, fenpropi infection, or diaper rash. 0047. In some embodiments, the fungal infection is of a din, formaldehyde, furfural, hexachlorobutadiene, Malassezia firfir, a Epidermophyton floccosum, a Tricho iodomethane, isoprothiolane, methyl bromide, methyl phyton, a Dermatophilus Congolensis, a Microsporum, a isothiocyanate, metrafenone, nitrostyrene, nitrothal-isopro Malassezia ovale, an Aspergillus, a Blastomyces, a Candida, pyl, OCH, 2-phenylphenol, phthalide, piperalin, probena a Coccidioides, a Cryptococcus, a Histoplasma, a Paracoc Zole, produinazid, pyroquilon, sodium orthophenylphenox cidioides, a Sporothrix, a Zygomycetes, a Pseudallescheria, a ide, Spiroxamine, Sultropen, thicyofen, tricyclazole, Scedosporum, or a Scopulariopsis. iodophor, silver, Nystatin, amphotericin B, griseofulvin, and 0048. In some aspects of these methods, the antifungal Zinc naphthenate. botanical is an essential oil. Preferred essential oils are clove 0039. The inventors have discovered that fungal infections bud oil, lemongrass oil, Sandalwood oil, spearmint oil, car on an animal epidermis, nail or hair, or in an orifice of an cacrol, thymol, a cardamom extract, caraway oil, a coriander animal can be effectively treated with antifungal botanicals. extract, linalool, almond oil, and tea tree oil. More preferably, Thus, provided herein are newly discovered properties of the essential oil is clove bud oil or lemongrass oil. Most compounds which include antifungal, sporicidal and antibac preferably, the essential oil comprises eugenol, terpinen-4-ol. terial properties. Also novel are the combinations of com cineole, cuminaldehyde, cinnamic acid, or perillaldehyde. pounds which provide unexpected results in the treatment and 0049 Combining agents can have a number of potential pre-treatment of the epidermis, nail, hair and orifices against benefits, including: (a) extending and broadening the spec common fungi and bacteria. The inventors show for the first trum of activity of the individual agents used, (b) increase the time that combining naturally occurring fungicides with antimicrobial potency of individual compounds, (c) reduce known synthetic and semisynthetic fungicides leads to unex the development of resistance, (d) treat resistant strains (e) pectedly good results on Substrates, including leather. The reduce the concentration used for at least one treatmentagent, same results are now expected with living epidermis, nail or and/or (f) have anti-sporicidal activity with or without acti hair, or in an orifice. The Examples they also show that uses of Vating the spores. The data in the Examples herein shows that naturally occurring fungicidal compounds can be expanded using a combination of the synthetic, semisynthetic, and natu to be utilized against bacteria and resistant microorganisms. It ral products will achieve these objectives. is now concluded by the inventors that: 1) essential oils (espe 0050 For example, since miconazole, unlike terbinafine cially lemongrass and clove bud oils, but not limited to them) and tolnaftate, possesses antibacterial activity, combining it can be used singly as natural products to inhibit microorgan with either agent will expand the spectrum of antimicrobial isms that infect epidermis, hair, nails, and orifices, and 2) activity of an antifungal to cover dermatophytes, yeasts, and combining essential oils with a synthetic or semisynthetic bacteria. In addition, because miconazole is a static agent antifungal compound will provide a broad spectrum activity. against fungi, combining it with either terbinafine or tolnaf In addition to treating common microorganisms, the methods tate, which we showed have fungal sporicidal activity, will of the invention can be employed to treat drug resistant micro expand the killing activity of the combination. These combi organisms such as terbinafine resistant Trichophyton rubrum nations are provided as examples and one of skill in the art can and multi-drug resistant Candida, as well as allow the use of deduce from these teachings other effective combinations lower concentrations of synthetic agents when combined that can work synergistically. with essential oils. The current method provides an effective 0051. The compositions used in the methods of the present means for preventing and treating fungal infection of the invention can thus also comprise a second antifungal com epidermis, hair, nails and orifices. pound. In some embodiments, the second antifungal com 0040 Having shown that antifungal compounds possess pound is a synthetic or a semisynthetic antifungal compound. potent anti-dermatophyte activity, the inventors also showed 0052. Where the second antifungal compound is a syn the activity of these agents against dermatophytes, other fungi thetic antifungal compound, preferred synthetic antifungal and bacteria using bioassays. See Examples. compounds are miconazole, terbinafine, tolnaftate or ciclo 0041. The present invention is thus directed to a method of piroX. Where the second antifungal compound is a semisyn treating a fungal infection on an animal epidermis, nail or thetic antifungal compound, the preferred semisynthetic anti hair, or in an orifice of an animal. The method comprises fungal compound is an echinocandin. contacting the fungus infection with a composition compris 0053. In other embodiments, the second antifungal com ing an antifungal botanical. pound is a botanical. Preferably, the botanical is an essential 0042. In some embodiments, the fungal infection is in an oil, as described above. orifice. As used herein, an orifice is an opening to a cavity or 0054 The invention method can be used on any animal a passage of the body of a live animal (including a human). including a human, a nonhuman vertebrate Such as a bird, or US 2008/0220 1 03 A1 Sep. 11, 2008

a non-human mammal. Examples of nonhuman vertebrates 0062. The separate antifungal compound can also be a include any farm animal, for example a cow, a pig, a chicken, botanical. Preferably, the botanical is an essential oil. Most or a horse, or a companion animal Such as a dog, a cat, a preferably, the botanical comprises eugenol, terpinen-4-ol. hamster, a gerbil, a guinea pig, amouse, a rat, a potbellied pig, cineole, cuminaldehyde, cinnamic acid, or perillaldehyde. a ferret, or a caged bird. 0063. In addition, the compositions of the invention limit 0055. The methods of the present invention are not nar growth of odor causing bacteria, and the bacteria that cause rowly limited to any particular method of contacting the com cellulitis. positions to the fungus infection. In some embodiments, the 0064 Preferred embodiments of the invention are composition is applied directly on the fungal infected tissue. described in the following examples. Other embodiments In other embodiments, the composition is applied to an article within the scope of the claims herein will be apparent to one that comes in contact with the fungus infection. Preferred skilled in the art from consideration of the specification or examples of Such articles include, but are not limited to, practice of the invention as disclosed herein. It is intended that clothing, a towel, a comb, a brush, a diaper, human bedding, the specification, together with the examples, be considered or animal bedding. exemplary only, with the scope and spirit of the invention 0056. The compositions used in the invention methods are being indicated by the claims, which follow the examples. not limited to any particular formulation, provided the for mulation is pharmaceutically acceptable. By “pharmaceuti EXAMPLES cally acceptable' it is meant a material that: (i) is compatible Example 1 with the other ingredients of the composition without render Examples of Antifungal Compounds that Function in ing the composition unsuitable for its intended purpose, and the Invention (ii) is suitable for use with subjects as provided herein without 0065. The treatment in this example consists of at least two undue adverse side effects (such as toxicity, irritation, and antifungal compounds. Examples of typical compounds are allergic response). Side effects are “undue' when their risk listed by their general class, chemical or otherwise. Concen outweighs the benefit provided by the composition. Non trations pertain to the class. They are stated as an overall limiting examples of pharmaceutically acceptable carriers range. One would select at least one compound from each include, without limitation, any of the standard pharmaceu group of antifungal compounds described above or below and tical carriers such as phosphate buffered saline Solutions, create a mixture of the two or more compounds. All percents water, emulsions such as oil/water emulsions, microemul indicate weight/volume. sions, nanoemulsions, and the like. 0066. Imidazoles (0.01-10%): albaconazole, bifocona 0057 The use of particular excipients (detergents, oils, Zole, butoconazole, clotrimazole, econazole, fluconazole, enzymes, etc.) can also function in the invention to increase itraconazole, ketoconazole, miconazole, oxiconazole, posa the penetration of the substrate, the rate of penetration, the conazole, Saperconazole, AN 2690, Sertaconazole, Sulcona thoroughness of coverage, etc. These can also be used to Zole, terconazole, tioconazole, Voriconazole, luliconazole. cause the penetration of a spore or epidermis, hair or nails, or 0067 Allylamines And Benzylamines (0.001-10%; 0.05 tissues of an orifice by an antifungal or antibacterial com 5%): butenafine, naftifine, terbinafine. pound. Excipients can also be used to cause the spore to end 0068 Polyenes (0.01-10%; 0.5-5%): amphotericin Band dormancy and begin germination, thus making the spore its lipid preparations, candidicin, filipin, fungimycin, nysta more Susceptible to the antifungal compound(s). tin. 0058. The composition comprising the antifungal or anti 0069 Miscellaneous Synthetic Antifungal Compounds bacterial compound can also include a compound to increase (for example at 0.05-25%): amorolfine (demethymorpho adherence to the epidermis, hair, nails or orifice. Increasing line), cicloproX olamine, haloprogen, clioquinol, tolnaftate, adherence can increase the length of time for which the com undecylenic acid, hydantoin, chlordantoin, pyrrolnitrin, Sali pound remains in contact with the skin, hair and nails cylic acid, ticlatone, triacetin, griseofulvin, Zinc pyrithione. 0059. The above-described compounds can thus be for (0070 Disinfectants (for example at 0.001-20%): copper mulated without undue experimentation for administration to sulfate, Gentian Violet, betadyne/povidone iodine, colloidal a mammal, including humans, as appropriate for the particu silver, zinc. lar application. Additionally, proper dosages of the composi (0071. Botanicals (for example at 0.01-10%): Basil (Onci tions can be determined without undue experimentation using mum basilicum), Cassia (Cinnamomum aromaticum var. cas standard dose-response protocols. sia), Cedrus wood oil (Cedrus libani or Cedrus spp.), Cha 0060. Non-limiting examples of forms of the composi momile (Chamaemelum nobile), Citronella (Cymbopogon tions include a cream, ointment, gel, liquid, Solution, foam, nardus), Clove (Syzgium aromaticum), Cumin (Cuminum powder, paste, gum, lacquer, shampoo, Suspension, fog, Cyminum), Fennel (Foeniculum vulgare), Mint (Mentha xpip spray, aerosol, pump spray, wipe or sponge. erita/Mentha spicata), Tea Tree Oil (Melaleuca alternfolia), 0061 The methods of the present invention can further Tumeric leaf oil (Curcuna longa), Lemongrass Oil (Cymbo comprise contacting the fungal infection with a second com pogon citratus), and ingredients isolated from these botani position. In these embodiments, the second composition cals. comprises a separate antifungal compound. In some embodi ments, the separate antifungal compound is a synthetic or Example 2 semisynthetic antifungal compound. Where the separate anti Evaluation of the Activity of Synthetic Antifungal fungal compound is a synthetic antifungal compound, pre Compounds and Natural Substances Against Micro ferred separate antifungal compounds are miconazole, terbin organisms Infecting Shoes. Using In Vitro and Shoe afine, tolnaftate or ciclopirox. Where the separate antifungal and Insole Biopsy Disc Assays compound is a semisynthetic antifungal compound, the pre 0072 The shoe disinfecting activities of the following ferred second antifungal compound is an echinocandin. compounds were studied: terbinafine, tolnaftate, miconazole, US 2008/0220 1 03 A1 Sep. 11, 2008

Cedrus oil, and tea tree oil, clove bud oil, lemongrass oil, reduce the concentration used for at least one treatmentagent, sandalwood oil and spearmint oil. and (f) have sporicidal activity. In Vitro Susceptibility Testing Bioassay 0073 Minimum Inhibitory Concentration (MIC): Mini 0080. The shoe substrate used in this study was Dr mum inhibitory concentrations of synthetic and semisyn Scholl's Cc air pillow insoles. This substrate was used in our thetic and natural products against dermatophytes were deter bioassay because this insole has no inhibitory activity against mined using a modification of the Clinical Laboratory dermatophytes (see below), and is representative of the type Standards Institute (CLSI, formerly National Committee of of material used in manufacturing shoe insoles. Clinical Laboratory Standards, NCCLS) M38A standard I0081. To evaluate the ability of the agents to prevent and method for dermatophytes, while MIC of these agents against treat fungal contamination of insoles and leather, we deter Candida species were determined using the CLSI M27-A2 mined their activity against the dermatophyte T. mentagro methodology. The method used to determine the MIC against phytes, and developed a novel insole/leather biopsy assay. T. bacteria was based on the CLSI document M7-A7. mentagrophytes was used as the model strain in our bioassay 0074 For dermatophytes, serial dilutions of terbinafine, studies because this fungus is a major cause oftinea pedis and tolnaftate were prepared in a range of 0.004-2 g/ml, while onychomycosis. Unlike T. rubrum, which is often identified for miconazole concentrations ranged between 0.015-8 as the causative organism in these diseases but is a poor ug/ml. Finally, for essential oils, the concentrations tested producer of spores/conidia, T. mentagrophytes, in addition to were between 0.03-16 g/ml. The only exception was tea tree being an etiological agent of these diseases, produces conidia oil where dilutions were prepared in a range of 0.0078-4 reproducibly and therefore, is amenable for use in a bioassay. ug/ml. The MIC was read at 4 days post inoculation and It is expected that activity in this assay against T. mentagro defined as the lowest concentration of an agent to inhibit 80% phytes will be indicative of activity against T. rubrum and of fungal growth as compared to the growth control (Table 2). other dermatophytes. 0075 To determine the MIC of agents against Candida I0082 To evaluate the shoe disinfecting ability of various species, serial dilutions of terbinafine and tolnaftate were agents, a bioassay was developed that measured the activity prepared in a range of 0.125-64 ug/ml, miconazole in a range of various agents in preventing (through pre-treatment) and of 0.03-16 g/ml and tea tree oil had a range between 0.125-4 treating (through post-treatment) contamination on shoes. ug/ml. The remaining essential oils were prepared in a dilu The first step in the bioassay development was to identify tion range of 0.03-16 g/ml. For Candida the MIC was read at optimal insole and leather material that represent Substrates 24 hours and defined as the lowest concentration to inhibit used in shoes and that do not inhibit fungi by themselves. To 50% of fungal growth as compared to the growth control select the optimal shoe insole, discs measuring 8 mm were cut (Table 4). using a Dermal Biopsy punch (Miltex, Bethpage, N.Y.) from 0076 For bacterial species, the medium used to evaluate four commercially available shoe insoles (CVS odor stop the antibacterial activity of agents and essential oils was insoles, Dr Scholl's airpillow insoles which claimantifungal Mueller-Hinton (Oxoid Ltd., Basingstoke, Hampshire, activity, odor eater insoles, and CVS double air foam England). Serial dilutions of miconazole, terbinafine, and insoles). These biopsy discs were placed on T. mentagro tolnaftate were prepared in a range of 0.125-64 ug/ml and phytes seeded PDA plates. T. mentagrophytes was used as a serial dilutions of tea tree oil were prepared in a range of typical organism and is representative of an entire class of 0.0078-4 ug/ml, while those for the rest of the essential oils fungi that grows on/in shoes and other Substrates. The ability were prepared in a range 0.031-16 lug/ml. The MIC was read of insole biopsy discs from existing products to inhibit der at 16 hand defined as the lowest concentration to inhibit 80% matophytes, following incubation for 7 days at 35° C., was of bacterial growth compared to the growth control (Table 5). determined (FIG. 1). Three of the insoles (CVS Odor Stop, 0077 Minimum fungicidal concentration (MFC): The Odor Eater, and CVS Double Air Foam) had a minimal anti minimum fungicidal concentrations of various agents were fungal activity (FIG. 2A-C) while Dr Scholl's insole did not determined using the technique described by Canton et al. inhibit T. mentagrophytes at all (FIG.2D). A similar approach (Antimicrob Agents Chemother. 2004 8:2477-82). In that was used to determine whether biopsy discs from a leather method, fungal conidia were collected following growth on hide inhibit fungal growth. As shown in FIG. 2E, the leather potato dextrose agar (PDA) plates and were used to inoculate material did not have any antifungal activity by itself. There 96-well plates containing different concentrations of agents. fore, Dr Scholl's insole and the leather hide were used as Following incubation at 35°C. for 4 days (for dermatophytes) Substrates in Subsequent experiments. or 24 hours (for yeast), wells showing no visible growth were I0083. In our bioassay, we used isopropanol as a vehicle to cultured to determine the MFC (defined as the lowest con dissolve the various disinfectants/antifungals. Isopropanol centration of a given agent that kills>99.999% of fungal was selected because it is a common solvent used in different conidia or spores). The MFC value represents the level of the preparations marketed for the treatment of tinea pedis. We agent at which spores or conidia were killed. next performed experiments to identify a concentration of 0078 Evaluation of the activity of combination of antifun isopropanol that did not inhibit fungal growth by itself. The gal agents and essential oils against microorganisms infecting ability of three different concentrations (30%, 50%, and Substrates, including skin, hair, and nails. 100%) of isopropanol to inhibit dermatophyte growth was 0079 Combining agents has a number of potential ben tested. As shown in FIGS. 3A-B, 30% isopropanol was the efits, including: (a) extending and broadening the spectrum of optimal concentration at which the vehicle did not inhibit activity of the individual agents used, (b) increase the antimi fungal growth on the insoles and leather Surface. Because crobial effectiveness of individual compounds, (c) reduce the tolnaftate does not dissolve very well in isopropanol, we development of resistance, (d) treat resistant strains, (e) performed additional experiments using acetone as a vehicle. US 2008/0220 1 03 A1 Sep. 11, 2008

0084. Based on the above experiments, our disc biopsy biopsy discs was used as blank where no fungal cells or drug assay employed Dr. Scholl's insole and leather discs as the were added. Following treatment, discs were fixed with 2% optimal Substrates representing materials used in shoes, and glutaraldehyde for 2 h, and then washed with sodium cacody 30% isopropanol as the optimal vehicle to dissolve the agents late buffer (three times for 10 minutes each). The discs were to be tested in pre-treatment and post-treatment studies. then treated with 1% osmium tetraoxide (for 1 h at 4°C.) followed by a series of washing with sodium cacodylate Evaluation of the Ability of Various Agents to Prevent and buffer, followed by a two times washing with distilled water. Treat Fungal Shoe Contamination. Next, the discs were treated with 1% tannic acid washed three times with distilled water, and followed by 1% uranyl acetate 0085. Two types of disc biopsy assays were used to evalu with two water washings. The samples were then dehydrated ate the ability of different synthetic and natural substances to through a series of ethanol solutions (range from 25% (vol/ disinfect shoe material: (a) Pre-treatment assay: where discs Vol) ethanol in distilled water to absolute ethanol). Prepared were pre-treated with antifungals first and then infected with samples were then sputter coated with Au/Pd (60/40) and T. mentagrophytes, and (b) Post-treatment assay: where discs viewed with Amray 1000B scanning electron microscope. were first infected with T. mentagrophytes, then treated with drugs. These assays reveal the ability of different agents to Results prevent and treat shoe fungal contamination, respectively. I0086 Pre-treatment assays: To evaluate the ability of dif I0089 Minimum Inhibitory Concentration (MIC) and ferent agents to prevent fungal contaminations of shoes, PDA Minimum Fungicidal Concentration (MFC): plates were prepared on which 10 T. mentagrophytes cells (0090. Evaluation of the inhibitory activity of various were evenly spread. Next, discs from insoles and leather were agents showed that these agents were effective in inhibition of treated as follows (with either agent or control vehicle): discs dermatophytes, yeasts and bacteria to varying degrees. Data were pretreated with a single spray, spraying for 15 second or from these MIC/MFC studies are summarized in Table 1 (for 30 second. Other discs were immersed in agent or vehicle for details of the MIC/MFC data, see Tables 2-5). Summary of 30 min. Following this treatment, discs were air-dried by the antifungal and antibacterial activity of different synthetic placing them in a Petri plate for 1 min. These dried discs were and natural products tested is Summarized below. then placed (drug side down) on the seeded PDA plates. Antimicrobial activity of Synthetic Agents: Plates were then incubated for 4 days at 30° C. Following 0091 Terbinafine: Our results showed that terbinafine was incubation, fungal growth was recorded. Active agents highly active against all isolates of the three dermatophytes showed a clearance Zone around the biopsy disc (FIG. 1A, genera tested where low MIC values were noted (MIC arrow), while inactive agents showed fungal growth all range=0.008-0.06 ug/mL). In addition, this agent was able to around the disc (FIG. 1B). Diameter of the clearance Zone kill spores of these dermatophytes as demonstrated by low (CZD) was measured. The relative activity of different agents MFC values (MFC range was between 0.03-0.125 g/mL). and control were assessed. In this assay, active agents had Evaluation of the anti-yeast activity of terbinafine showed higher CZD than inactive or less active ones. that this agent possesses high activity against all C. parapsi 0087 Post-treatment assays: To evaluate the ability of losis isolates tested (MIC values for all isolates was 0.25 various agents to treat infected shoes, PDA plates were pre ug/mL). In this regard, C. parapsilosis is a known skin normal pared on which 104 T. mentagrophytes cells were evenly flora inhabitant. Our data showed that terbinafine was less spread on their surface. Next, untreated biopsy discs were active against C. albicans compared to C. parapsilosis with placed on these PDA plates and incubated for 4 days at 30°C. one to three fold higher MIC values against the majority of Incubating the biopsy discs in this manner allowed the fungi isolates tested relative to C. parapsilosis (MIC values for 5 to invade the discs. Infected discs were picked and post strains ranged between 0.5 and -2 ug/mL). Interestingly, treated with different agents by spraying. Post-treated discs terbinafine exhibited no effect against one C. albicans strain were allowed to air dry and were then placed on fresh, unin (strain 8280 where the MIC was 264 ug/mL). In contrast to oculated PDA plates and incubated for 4 additional days at the high activity of terbinafine seen against dermatophytes 30°C. Incubation of the discs under these conditions allows and yeast Strains, this agent did not show any antibacterial any fungi that are not killed by the sprayed agent to grow. In activity against all bacterial strains examined (MIC >64 other words, agents that are effective in the treatment of shoe ug/mL for all strains tested). material will not show any fungal growth around the disc 0092 Tolnaftate: Evaluation of the antifungal activity of biopsy (FIG. 1C). In contrast, discs treated with ineffective tolnaftate showed that this agent is highly active against the agents will show fungal growth emanating from them (FIG. dermatophytes tested both in fungal inhibition (MIC range 1D). Diameter of the growth Zone (GZD) was determined as againstall dermatophytes tested was 0.008-0.125 g/mL) and a measure of the activity of the agent tested. In this assay, spore killing (MFC range was 0.06-0.125 ug/mL). Tolnaftate inactive or less active agents had higher GZD than active inhibitory and sporicidal activity was similar to terbinafine or agents, while active agents did not show any fungal growth slightly (one dilution) higher. Evaluation of the anti-yeast activity of tolnaftate showed that this agent has a reduced (GZD=0). activity against yeast compared to terbinafine. Elevated MICs Scanning Electron Microscopy (SEM). for tolnaftate was observed against all C. albicans strains tested (MIC value for all strains was >64 g/mL). While 0088 Scanning electron microscopy (SEM) was used to activity of tolnaftate against C. parapsilosis was strain-de monitor the ability of agents to eradicate fungal growth on pendent with one strain (#7629) showing low MIC (0.5 shoe insoles or leather biopsy discs. Pre- and post-treated ug/mL), while the other isolates exhibited relatively high discs were processed for SEM by the method of Chandra et al. MIC values (MIC=8-16 lug/mL). Susceptibility testing of One set of discs was used as a control in which no drug pre bacteria to tolnaftate showed that this agent had no S. aureus or post-treatment was performed. In addition, one set of antibacterial activity (MIC for all strains tested was >64 US 2008/0220 1 03 A1 Sep. 11, 2008

ug/mL), while possessing some strain-dependent activity potent antifungal activity against dermatophytes with MICs against S. epidermidis strains: two strains had MIC values of ranging between 0.125 and 0.5 g/mL. 2 ug/mL, while the remaining four exhibited MICs ranging between 16 and >64 g/mL. Activity Against Yeast: 0093 Miconazole: Susceptibility testing of dermato phytes against miconazole showed that this agent possesses a (0098 Next we tested the ability of these oils to inhibit potent antifungal activity against T. mentagrophytes, T. yeast (C. albicans and C. parapsilosis). As can be seen in rubrum, and E. floccosum with MIC values ranging from 0.06 Table 8, four of the essential natural oils (clover bud, lemon to 0.125 ug/mL. Compared to terbinafine and tolnaftate, grass, spearmint, and tea tree oils) were active against these miconazole had a slightly lower activity. Moreover, unlike clinically important fungi, with MIC range between 0.063 these agents, miconazole was static against dermatophytes 0.5 ug/mL. The only exception was sandalwood oil, which (MFC of miconazole against all T. mentagrophytes isolates had an MIC of 4 to >16 ug/mL (Table 3). These results and the majority of T. rubrum and E. floccosum isolates tested suggested that sandalwood oil exhibited no inhibitory activity was 28 ug/mL). Our data show that miconazole possesses a against Candida species and strains tested. modest anti-yeast activity. In general, the MIC values of miconazole against both C. albicans and C. parapsilosis were Activity Against Bacteria: higher than those obtained for terbinafine. C. albicans (0099. We next tested the in vitro activity of the essential showed some strain-dependent Susceptibility against micona natural oils against: (a) odor-producing (Micrococcus and Zole, with an MIC=1-2 ug/mL for four isolates, 16 ug/mL for Corynebacteria) bacteria, and (b) Staphylococcus aureus (a another and >16 ug/mL for the remaining albicans strain major cause of cellulitis). As seen in Table 9, clove bud, (8280). MIC values of miconazole against C. parapsilosis lemongrass, and sandalwood oils were active against the were also strain dependent (MICs ranging from 4 to > 16 odor-producing bacteria tested (MIC=0.25-2 ug/mL), while ug/mL). In contrast to terbinafine and tolnaftate (which had spearmint and tea tree oils did not show in vitro activity no activity against bacteria), miconazole was active against (MIC=2-8 ug/mL). Furthermore, clove bud, lemongrass, and both S. aureus and S. epidermidis isolates tested (MIC values sandalwood oils showed some activity (MIC=0.25-8 ug/mL) against all Staphylococcus isolates were between 0.5 and 2 against Staphylococcus. Moreover, lemongrass tended to Lig/mL). have one to two dilutions lower MIC than clove and sandal 0094 Cedrus oil: Antifungal susceptibility testing of wood oils, indicating it is more active. In contrast, spearmint cedrus oil showed that this natural oil possessed acceptable and tea tree oil did not show noticeable activity against any of antifungal activity against dermatophytes in vitro with MIC the pathogenic bacterial isolates tested (MIC=8-32 g/mL). ranging between 0.5 and 2 g/mL. In addition, cedrus oil These studies showed that clove bud and lemongrass had the exhibited species-dependent cidality: MFC against T. menta broadest antimicrobial activity compared to the other essen grophytes was noticeably higher (MFC-4-16 lug/mL) than tial oils and are viable candidates for use as natural products against E. floccosum, and T. rubrum isolates (MFC=0.5-4 to prevent and treat tinea pedis, onychomycosis, and skin ug/mL). Results are detailed in Table 1. infections. 0095 Tea Tree Oil: Antifungal susceptibility testing of dermatophytes against tea tree oil showed that this natural Evaluation of the Activity of Combination of Antifungal product is highly active in inhibiting and spore killing of these Agents and Essential Oils Against Microorganisms Causing fungi (MIC range=0.125-0.4, while MFCs were =0.25 to >4 Superficial Fungal Infections, Including but not Limited to ug/mL against all dermatophytes tested). Moreover, the MIC Tinea Pedis and Tinea Unguium/Onychomycosis. and MFC values of tea tree oil against dermatophytes were lower than those noted for cedrus oil. A majority of the yeast 0100. To assess the potential for using antifungal synthetic isolates were resistant to tea tree oil (with an MIC>4 g/mL). agents (e.g. terbinafine, tolnaftate and miconazole) and Interestingly, one C. albicans isolates (8280) was susceptible essential oils (e.g. clove bud, lemongrass, Sandalwood, spear to tea tree oil, although the same isolate was resistant to mint and tea tree oil) in combination, we evaluated the ability terbinafine, tolnaftate, and miconazole (with an MIC of 64. ofessential oils to inhibitterbinafine-resistant T. rubrum and >64, and >16 ug/mL, respectively). This finding is very inter C. albicans strain (strain number MRL 8280) that exhibits esting because it indicates that combining tea tree oil with any multi-resistance to terbinafine, miconazole and tolnaftate. As of the three agents may provide enhanced antifungal activity, shown in Table 10, all the terbinafine-resistant T. rubrum Suggesting that adding tea tree oil to any of the antifungals isolates tested were susceptible to the essential natural oils, may provide a broad coverage against resistant isolates with an MIC range of 0.031 to 0.25 ug/mL. The most potent (MICd4 ug/mL). The possibility of combining tea tree oil oil was lemongrass which showed very low MICs against with different agents against this resistant fungus was evalu these Trichophyton isolates. ated (see below). The bacterial strains tested were not suscep 0101 Similarly, the essential oils were effective in inhib tible to tea tree oil. Results are detailed in Table 1. iting the multi-resistant C. albicans strain. The most effective 0096 Antimicrobial activity of all effective essential oils essential oil in inhibiting this resistant strain was lemongrass against dermatophytes known to grow on skin, hair, and nails, (see Table 11). causing tinea pedis and tinea ungunium/onychomycosis, yeasts known to cause nail and cutaneous infections, and CONCLUSIONS bacteria that can cause infection or generate unpleasant non disease odor. 0102 Based on these data it is concluded that: 1) essential oils (especially lemongrass and clove bud oils) can be used Activity Against Dermatophytes: singly as natural products to inhibit microorganisms that infect the skin, hair and nails; and 2) combining essential oils 0097. In these studies we evaluated the activity of essential with a synthetic or semisynthetic antifungal provides abroad oils against dermatophytes, yeast and bacteria. Table 7 pre spectrum activity, treats terbinafine resistant Trichophyton sents a Summary of the anti-dermatophyte activity of essential rubrum, multi-drug resistant Candida, and odor causing bac oils. As can be seen, the five essential oils tested exhibited teria, as well as allows the use of lower concentrations of US 2008/0220 1 03 A1 Sep. 11, 2008 synthetic agents when combined with essential oils. Our treatment shoe biopsy disc assay developed (see above). As method identified antimicrobial “systems” that have potent shown in FIG. 7A (and Table 6), while the vehicle control antifungal and antibacterial activity and provides an effective failed to treat already established fungal contamination as means for preventing and treating fungal infections. evidenced by the presence of fungal regrowth (GZD-33 mm 0103 Having shown that terbinafine, tolnaftate, and of growth), terbinafine completely eradicated established essential oils possess potent anti-dermatophyte activity contamination on insoles (GZD=0 mm). Tolnaftate (both 1% against microorganisms that colonize and infect the foot and 2%) were also effective in clearing the contamination of using in vitro Susceptibility assays, we next investigated the insole, although some minimal regrowth was observed activity of these agents against dermatophytes using the shoe (GZD-8 mm and 10 mm, respectively). In contrast, tea tree disc bioassay we developed, and SEM techniques. oil was not able to treat the contamination present on insole Effect of Pretreatment of Shoe Insoles and Leather Surfaces biopsies (GZD-33 mm). with Synthetic and Natural Products on Preventing Dermato phyte Shoe Contamination Post Contamination Treatment of Leather Pretreatment of Insoles 0.108 Next, we determined whether terbinafine, tolnaf tate, or tea tree oil can treat T. mentagrophytes contamination 0104. To determine the ability of terbinafine, tolnaftate, already established on leather biopsy discs. As shown in FIG. and tea tree oil to prevent shoe contamination we used the 7B (and Table 6), terbinafine completely cleared the estab pretreatment insole biopsy bioassay method described above. lished contamination on leather disc (GZD=0 mm). More As shown in FIG. 4 (and Table 11, representative images in over, tolnaftate (1% and 2%) also reduced contamination of FIG. 5), pretreatment of insoles with terbinafine 1% solution leather (GZD=11 mm for both concentrations). Tea tree oil resulted in complete inhibition of fungal growth (CZD-85 induced very minimal inhibition of fungal growth on the mm, fungal inhibition reached to the edge of the Petri dish) infected leather biopsies compared to vehicle control compared to vehicle control (CZD=0 mm). This complete (GZD-26 mm versus 33 mm). These data clearly demon inhibition was observed even when the insoles discs were strate that post treatment of insoles and leather with terbin pretreated with a single spray. Pre-treatment of biopsy discs afine and tolnaftate is an effective way for treating infected with tolnaftate (1% and 2%) also inhibited fungal growth shoes. (CZD-25 mm and 11 mm, respectively) compared to vehicle control, albeit to a lesser extent than terbinafine. Because Effect of Combination of Tolnaftate and Tea Tree Oil on tolnaftate dissolves better in acetone, we repeated some Preventing Dermatophyte Shoe Contamination experiments using acetone as a vehicle. Our data showed that tolnaftate has a potent preventive activity against dermato 0109 To determine whether using combination of syn phytes infecting shoes (FIG. 5). Increasing the concentration thetic compounds and essential oils will allow the use of low of tolnaftate to 3% and 4% increased the activity. In contrast, concentrations of synthetic drugs, we tested the ability of 1% tea tree oil had no inhibitory prevention effect (CZD-0 combination of 0.01% tolnaftate and 3% tea tree oil to prevent mm). Taken together, these data show that pretreatment of shoe contamination using the pre- and post-treatment insole shoe insoles and leather material with terbinafine ortolinaftate bioassays described above. is an effective way to prevent fungal contamination of shoes. 0110. As shown in FIG. 10, pretreatment of insoles with Importantly, these agents were Superior to the marketed Dr. 0.01% terbinafine or 3% tea tree oil singly did not result in any Scholl's brand in preventing fungal contamination of insoles. inhibition of fungal growth. In contrast, the combination of 0.01% terbinafine and 3% tea tree oil induced a noticeable Pretreatment of Leather inhibition of fungal growth (FIG. 10C). 0111. Furthermore, while 0.01% tolnaftate or 3% tea tree 0105 To determine whether pretreatment of leather oil did not prevent growth of fungus on insole after post biopsy discs with terbinafine, tolnaftate, or tea tree oil can prevent growth of T. mentagrophytes, we tested their activity treatment (FIG. 10A (A)-(B), post-contamination treatment using the bioassay method described above. As shown in FIG. with the combination of these agents reduced fungal growth 4B, pretreatment of leather disc with vehicle did not result in (FIG. 10A (C). any inhibition (CZD=0 mm), while terbinafine pretreatment 0112 These data show that combining tolnaftate and tea resulted in complete inhibition of fungal growth (CZD-85 tree oil will allow the use of low concentration of tolnaftate to mm, also see Table 6). Pretreatment of leather biopsies with prevent and treat shoe contamination. 1% tolnaftate resulted in inhibition of fungal growth Scanning Electron Microscopy Analyses (CZD=16 mm, FIG. 4B). However, pretreatment of leather disc with 1% tea tree oil alone did not inhibit fungal growth 0113 To determine the effect of synthetic and natural (CZD=0 mm). products (pre- and post-contamination treatments) on the 0106 These data indicate that pre-treatment of leather ability of T mentagrophytes to grow on insole biopsies, we material with terbinafine or tolnaftate is an effective way for performed SEM analysis. As shown in FIG. 8, pretreatment of preventing fungal contamination of the leather used in shoes. insole with the vehicle had no effect on fungal growth (FIG. Effect of Post-Contamination Treatment with Synthetic and 8C), while terbinafine and tolnaftate completely eradicated Natural Products on Eradication of Pre-Established Dermato fungal growth (FIGS. 8D.E; no fungal elements were seen). phyte Contamination on Insoles and Leather Surfaces However, and similar to the bioassay studies, pretreatment 0107 To determine the ability ofterbinafine, tolnaftate, or with tea tree oil reduced the fungal growth on insoles but did tea tree oil to treat T. mentagrophytes contamination already not eliminate it from the biopsy disc (FIG.8F). Post-contami established on shoe insoles, we determined the effect of post nation treatment of insole with terbinafine resulted in com treating infected insoles with these agents on their ability to plete clearance of fungal growth (FIG. 8G), while treatment clear the established fungal contamination using our post with tolnaftate was minimally effective, as shown by the US 2008/0220 1 03 A1 Sep. 11, 2008

presence of several filaments (FIG. 8H). In contrast, post (0123 Terbinafine and miconazole were also effective contamination treatment with tea tree oil had no activity against the yeast Candida species, but in a strain- and species against Timentagrophytes (FIG. 8I). dependent manner. 0114. These data show that pre- and post-treatment with 0.124 Miconazole exhibited activity against bacterial spe terbinafine is highly effective in eradicating fungal elements cies, while tolnaftate exhibited strain-dependent inhibition of from shoe material infected with T. mentagrophytes. Addi S. epidermidis. tionally, tolnaftate was effective in eradicating fungal ele 0.125 Among the natural products tested were tea tree oil, ments only if insole biopsies were pretreated with this agent. cedrus oil, clove bud, lemongrass oil, Sandalwood oil, and 0115 Taken together, these data indicate that pre- and spearmint oil. post-treatment of insoles with either terbinafine or tolnaftate 0.126 Essential oils have a broad antimicrobial activity is effective in preventing the fungal colonization of and treat covering dermatophytes, yeast and bacteria that infect the ment of already existing, fungal growth on insoles. skin, hair and nails. The most active essential oil was lemon 0116 Our data also demonstrate that combining tolnaftate grass followed by clove bud. and tea tree oil will allow the use of low concentration of I0127. Essential oils possess inhibitory activity against tolnaftate to prevent and treat shoe contamination bacteria that produces unpleasant and unacceptable odors and 0117 We also used SEM analyses to determine the effect Staphylococcus aureus (a major cause of cellulitis). ofterbinafine, tolnaftate and tea tree oil pretreatment on their I0128. The essential natural oils have potent in vitro activ ability to prevent T. mentagrophytes growth on leather biop ity against terbinafine-resistant dermatophytes, as well as sies. As shown in FIG. 9, pretreatment of leather biopsy disc multi-resistant C. albicans. Lemongrass possesses the most with terbinafine completely eradicated fungal growth, with potent activity in this regard. no fungal elements seen (FIG. 9D), compared to untreated I0129. In bioassay studies, terbinafine and tolnaftate pre leather disc (FIG. 9B) or vehicle-treated disc (FIG. 9C), treatment were able to inhibit fungi on insoles and leather where massive fungal elements can be seen invading the shoe biopsies compared to vehicle control. Terbinafine was leather material. Pretreatment with tolnaftate appeared to the most active pre-treatment agent. reduce the fungal density on leather discs, but did not com 0.130 Terbinafine post-treatment was able to treat estab pletely eradicate dermatophyte growth (FIG.9E). Discs pre lished fungal contamination on shoe biopsy discs. Although treated with tea tree oil did not show any effect on fungal tolnaftate showed antifungal activity as a post-treatment growth (FIG.9F), and were similar in appearance to the discs agent, its activity was less than that ofterbinafine. Tea tree oil pretreated with isopropanol, the vehicle control. was ineffective as a post-treatment agent. 0118 Taken together, these results revealed that pre-treat 0131 Combining a synthetic antifungal agent with an ment of leather with terbinafine was highly effective in pre essential oil allows the use of low doses of the synthetic venting and eradicating fungal elements from leather material antifungal to prevent and treat infections of the skin, nails, and infected with T. mentagrophytes, while tolnaftate was mini hair. mally effective. In contrast, tea tree oil was ineffective in I0132) Our data indicate that combining agents is likely to eradicating contamination of leather discs. provide benefit by expanding the spectrum of activity of an 0119. In summary, our findings show that: antifungal through the inhibition of resistant fungal Strains. 0120. Among the synthetic agents tested (terbinafine, I0133. In conclusion, the invented antimicrobial system tolnaftate, miconazole): has potent antifungal and antibacterial activity and provides 0121 The most active agent inhibiting dermatophytes was an effective means for preventing and treating fungal infec terbinafine, followed by tolnaftate and miconazole. tions of the skin, hair and nails. 0122 Terbinafine and tolnaftate were able to kill dermato I0134. In the following Tables, MICs and MIC are phyte fungal spores that may infect hair, nails, and skin, while defined as the minimal concentrations of a compound that can miconazole is static against dermatophyte spores. inhibit 50% and 90% of the tested organisms, respectively.

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 Teatree oil Cedrus oil Organism (ig/ml) (Lig/ml) (g/ml) (Lig/ml) (ig/ml) All Dermatophytes

MIC Range O.O15 O.12S O.O15-0.25 O.0625-0.25 0.25-1.0 MFC Range O.O3-0.12S O.O6-1.O O.5->8.O O.125->2 O.S.-8 All Yeasts

MIC range O.25->64 O5->64 1->16 O.125->2 ND: MFC range O5-2 ND: All Bacteria

MIC range >64 2->64 O5-2 >4 ND: MFC range ND:

*ND—not determined

US 2008/0220 1 03 A1 Sep. 11, 2008 12

TABLE 3-continued TABLE 4-continued Minimum Inhibitory Concentration (MIC, ugmL) and Minimum Inhibitory Concentration (MIC, Ig/mL) of Terbinafine, Minimum Fungicidal Concentration (MFC, Ig/mL) of Tolna?tate, Miconazole, and Tea Tree Oil against Candida species. Cedrus Oil and Tea Tree Oil against Dermatophytes Terbinafine Tolnaftate Miconazole Tea Tree Oil Cedrus Oil Tea Tree Oil Strain MIC MIC MIC MIC

Organism MIC MFC MIC MFC 2153 O.S >64 1 >4 828O >64 >64 >16 O.25 2127 O.S 8 O.25 4 8283 O.S >64 6 O.S 2128 O.S 4 O.25 4 8364 2 >64 2 >4 MIC Range (n = 6) O.25-2 4-16 O.125-0.2S 4->4 MIC Range O.5->64 64->64 1->16 O.25->4 MICso O.S 8 O.25 4 (n = 6) MICoo O.S 16 O.25 >4 MICso O.S >64 2 >4 All dermatophytes MICoo 2 >64 6 >4 C. parapsilosis MIC Range (n = 18) O.5-2 1-16 O.12S-O.S O.25->4 MICso O.S 2 O.25 2 7629 O.25 O.S 4 O.25 MICoo 1 8 O.S 4 76.68 O.25 8 6 >4 7672 O.25 8 8 >4 7995 O.25 8 4 >4 81.48 O.25 8 >16 2 TABLE 4 84.42 O.25 16 4 >4 MIC Range O.25-0.25 O.5-16 4->16 O.25->4 Minimum Inhibitory Concentration (MIC, ugmL) of Terbinafine, (n = 6) MICso O.25 8 4 >4 Tolna?tate, Miconazole, and Tea Tree Oil against Candida Species. MICoo O.25 8 6 >4 Terbinafine Tolnaftate Miconazole Tea Tree Oil All yeasts Strain MIC MIC MIC MIC MIC Range O.25->64 O.5->64 1->16 O.25->4 C. albicans (n = 12) MICso O.25 16 4 >4 1740 1 >64 1 >4 MICoo 2 >64 >16 >4 2108 0.5 64 2 >4

TABLE 5 Minimum Inhibitory Concentration (MIC, g/mL) of Terbinafine, Tolnaftate, Miconazole, and Tea Tree Oil against StaphyloCOCCuS Species.

TERBINAFINE TOLNAFTATE MICONAZOLE TEATREE OIL SPECIES MIC MIC MIC MIC

S. airetts

93 NON-VLABLE 730 2 732 733 734 847O MIC Range (n = 6) MICso MICoo s S. epidermidis

8472 8473 16 8474 8475 8476 8477 MIC Range (n = 6) MICso 16 MICoo All bacteria MIC Range (n = 12) MICso MICoo US 2008/0220 1 03 A1 Sep. 11, 2008 13

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 Post Insoles contamination Leather 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 19%. To 196 TTO** 11 17 11 2O 290 TOI 19/o TTO 19 8 15 11 29. To 2% TTO 10 34 O 30 3%. To 196 TTO 25 11 2O 11 *CZD–clearance Zone diameter; GZD-growth Zone diameter. **To Tolnaftate: TTO tea tree oil.

TABLE 7 Activity of essential oils against dermatophytes Clove Lemongrass Sandalwood Spearmint Tea Genus Species Bud Oil Oil Oil Oil Tree 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 O.12S O.S O.25 O.25 O.25 Trichophyton mentagrophytes 0.125 0.25 0.25 0.25 0.25 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.125-0.125 O.25-0.5 O.25-O.S O.125-O.S. O.2S-O.S MICso O.12S O.25 O.25 O.25 O.S MICoo O.12S O.S O.S O.S O.S

TABLE 8 Activity of essential natural oils against yeast Isolates Clove Bud Lemongrass Sandalwood Spearmint Tea Genus Species 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 O.12S O.12S 4 O.S O.25 Candida parapsilosis O.12S O.12S >16 O.S O.25 Candida parapsilosis O.25 O.12S >=16 1 O.25 MIC Range O.125-0.5 OO63-0.25 4->16 O5-2 O.25-1 MICso O.12S O.12S >16 O.S O.25 MICoo O.S O.25 >16 2 1

TABLE 9 Activity of natural oils against (A) odor-causing and (B) pathogenic bacterial isolates Clove Bud Lemongrass Sandalwood Spearmint Tea Tree MRL Oil MIC Oil MIC Oil MIC Oil MIC Oil Number Organism (mg/mL) (mg/mL) (mg/mL) (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 initeus O.S O.S O.25 4 2 784 Micrococcus initeus O.S O.25 O.S 2 4 MIC Range (n = 4) O5-2 O.25-O.S O.25-0.5 2-8 2-8 US 2008/0220 1 03 A1 Sep. 11, 2008 14

TABLE 9-continued

Activity of natural oils against (A) odor-causing and (B) pathogenic bacterial isolates Clove Bud Lemongrass Sandalwood Spearmint Tea Tree MRL Oil MIC Oil MIC Oil MIC Oil MIC Oil Number Organism (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) MICso O.S O.25 O.25 4 2 MICoo 2 O.S O.S 8 8 (B) Pathogenic bacteria

730 S. attrelts 2 1 2 8 32 732 S. attrelts 1 1 O.25 8 NG 733 S. attrelts 1 O.S O.S 8 8 8470 S. attreus 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

TABLE 10 Activity of natural oils against terbinafine-resistant T. rubrum Isolates MRL Terbinafine Clove Bud Lemongrass Sandalwood Spearmint Tea Tree Organism Number MIC oil MIC oil MIC oil MIC oil MIC oil MIC

T. rubrum 666 16 O.25 O.063 2 2 4 T. rubrum 670 16 O.12S O.063 O.S O.S 1 T. rubrum 671 4 O.12S O.063 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. rubrun 1807 4. 0.125 O.O63 0.5 0.5 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 O.5-4 MICso 4 O.12S O.063 O.S O.S 2 MICoo 16 O.25 O.12S 2 2 4

0.137 As various changes could be made in the above TABLE 11 methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the Activity of essential oils against a multi-resistant above description and shown in the accompanying drawings strain of C. albicans (strain 8280). shall be interpreted as illustrative and not in a limiting sense. Essential Oil MIC (ig/mL) 0.138 All references cited in this specification are hereby incorporated by reference. The discussion of the references CloveLemongrass Bud Oil Oil O.O63O.12S hereinerein is intended merelylv tto summarizeize uneth assertionsrti maded Sandalwood Oil >16 by the authors and no admission is made that any reference Spearmint Oil O.S constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references. 0135 The detailed description set-forth above is provided to aid those skilled in the art in practicing the present inven What is claimed is: tion. However, the invention described and claimed herein is 1. A method of treating a fungal infection on an animal not to be limited in scope by the specific embodiments herein epidermis, nail or hair, or in an orifice of an animal, the disclosed because these embodiments are intended as illus method comprising contacting the fungus infection with a tration of several aspects of the invention. Any equivalent composition comprising an antifungal botanical. embodiments are intended to be within the scope of this 2. The method of claim 1, wherein the fungal infection is in invention. Indeed, various modifications of the invention in an orifice. addition to those shown and described herein will become apparent to those skilled in the art from the foregoing descrip 3. The method of claim 2, wherein the orifice is a mouth or tion which do not depart from the spirit or scope of the present OS. inventive discovery. Such modifications are also intended to 4. The method of claim 2, wherein the orifice is a vagina. fall within the scope of the appended claims. 5. The method of claim 1, wherein the fungal infection is on 0136. In view of the above, it will be seen that the several an epidermis. advantages of the invention are achieved and other advan 6. The method of claim 1, wherein the fungal infection is on tages attained. a nail. US 2008/0220 1 03 A1 Sep. 11, 2008

7. The method of claim 1, wherein the fungal infection 24. The method of claim 22, wherein the nonhuman verte causes or aggravates tinea corporis, tinea pedis, tinea cruris, brate is a mammal. tinea unguium?onychomycosis, tinea capitis, dandruff, or dia 25. The method of claim 22, wherein the nonhuman verte per rash. brate is a farm animal. 8. The method of claim 1, wherein the fungal infection is of 26. The method of claim 24, wherein the farm animal is a a Malassezia firfiur, a Epidermophyton floccosum, a Tricho cow, a pig, a chicken, or a horse. phyton, a Dermatophilus congolensis, a Microsporum, a 27. The method of claim 22, wherein the nonhuman verte brate is a companion animal. Malassezia ovale, an Aspergillus, a Blastomyces, a Candida, 28. The method of claim 27, wherein the companion ani a Coccidioides, a Cryptococcus, a Histoplasma, a Paracoc mal is a dog, a cat, a hamster, a gerbil, a guinea pig, a mouse, cidioides, a Sporothrix, a Zygomycetes, a Pseudallescheria, a a rat, a potbellied pig, a ferret, or a caged bird. Scedosporum or a Scopulariopsis. 29. The method of claim 1, wherein the composition is 9. The method of claim 1, wherein the antifungal botanical applied directly on the fungal infected tissue. is an essential oil. 30. The method of claim 1, wherein the composition is 10. The method of claim 9, wherein the essential oil is clove applied to an article that comes in contact with the fungus bud oil, lemongrass oil, Sandalwood oil, spearmint oil, car infection. cacrol, thymol, a cardamom extract, caraway oil, a coriander 31. The method of claim 30, wherein the article is clothing, extract, linalool, almond oil, or tea tree oil. a towel, a comb, a brush, a diaper, human bedding, a shoe, or 11. The method of claim 9, wherein the essential oil is clove animal bedding. bud oil or lemongrass oil. 32. The method of claim 1, wherein the composition is in 12. The method of claim 9, wherein the essential oil com the form of a cream, ointment, gel, liquid, Solution, foam, prises eugenol, terpinen-4-ol, cineole, cuminaldehyde, cin powder, paste, gum, lacquer, shampoo, Suspension, fog, namic acid, or perillaldehyde. spray, aerosol, pump spray, wipe or sponge. 13. The method of claim 1, wherein the composition fur 33. The method of claim 1, further comprising contacting ther comprises a second antifungal compound. the fungal infection with a second composition, wherein the 14. The method of claim 13, wherein the second antifungal second composition comprises a separate antifungal com compound is a synthetic or semisynthetic antifungal com pound. pound. 34. The method of claim 33, wherein the separate antifun 15. The method of claim 14, wherein the second antifungal gal compound is a synthetic or semisynthetic antifungal com compound is a synthetic antifungal compound. pound. 16. The method of claim 15, wherein the synthetic antifun 35. The method of claim 34, wherein the separate antifun gal compound is miconazole, terbinafine, tolnaftate or ciclo gal compound is a synthetic antifungal compound. piroX. 36. The method of claim 35, wherein the synthetic antifun 17. The method of claim 14, wherein the second antifungal gal compound is miconazole, terbinafine, tolnaftate or ciclo compound is a semisynthetic antifungal compound. piroX. 37. The method of claim 34, wherein the separate antifun 18. The method of claim 17, wherein the semisynthetic gal compound is a semisynthetic antifungal compound. antifungal compound is an echinocandin. 38. The method of claim 35, wherein the semisynthetic 19. The method of claim 13, wherein the second antifungal antifungal compound is an echinocandin. compound is a botanical. 39. The method of claim 33, wherein the separate antifun 20. The method of claim 19, wherein the botanical is an gal compound is a botanical. essential oil. 40. The method of claim 39, wherein the botanical is an 21. The method of claim 1, wherein the animal is a human. essential oil. 22. The method of claim 1, wherein the animal is a nonhu 41. The method of claim 40, wherein the essential oil is man vertebrate. clove bud oil or lemongrass oil. 23. The method of claim 22, wherein the nonhuman verte brate is a bird. c c c c c