US 2010O226983A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0226983 A1 Sutcliffe et al. (43) Pub. Date: Sep. 9, 2010

(54) COMPOSITIONS FOR TREATMENT AND (22) Filed: Jan. 28, 2010 PREVENTION OF ACNE, METHODS OF MAKING THE COMPOSITIONS, AND Related U.S. Application Data METHODS OF USE THEREOF (60) Provisional application No. 61/147,960, filed on Jan. 28, 2009. (75) Inventors: Joyce A. Sutcliffe, West Newton, MA (US); Susan M. Ciotti, Ann Publication Classification Arbor, MI (US); James R. Baker, (51) Int. Cl. JR. Ann Arbor, MI (US) A 6LX 9/07 (2006.01) A6II 3L/235 (2006.01) Correspondence Address: A613/60 (2006.01) FOLEY AND LARDNER LLP A63L/045 (2006.01) SUTESOO (52) U.S. Cl...... 424/484: 514/544: 514/159: 514/724 3OOOK STREET NW WASHINGTON, DC 20007 (US) (57) ABSTRACT The present invention relates to methods for treating and (73) Assignee: Nanobio Corporation preventing acne or P. acnes infection in a subject comprising topically administering to the Subject in need thereof an anti (21) Appl. No.: 12/6S6,421 acne nanoemulsion composition. Patent Application Publication Sep. 9, 2010 Sheet 1 of 8 US 2010/0226983 A1

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COMPOSITIONS FORTREATMENT AND and control acne, and acne related problems such as sebum PREVENTION OF ACNE, METHODS OF production. Herbal medicines are also used to treat acne and MAKING THE COMPOSITIONS, AND include Tea Tree Oil red clover, lavender, leaves of straw METHODS OF USE THEREOF berry, chaste tree berry extract, burdock root, dandelion leaves, milk thistle, papaya enzymes, burdock and dandelion, CROSS-REFERENCE TO RELATED eucalyptus, thyme, witch hazel, sage oil, camphor, cineole, APPLICATION roSmarinic acid and tannins in the sage oil. These various treatments for acne may have only temporary effects, and 0001. This application claims priority from U.S. Provi may cause drug-resistance or other undesirable side effects, sional Patent Application No. 61/147,960, filed Jan. 28, 2009. Such as allergy, skin redness, or skin hypersensitivity. The contents of that application is incorporated herein by 0006 Orally administered drugs are generally more effec reference in its entirety. tive than topically applied drugs, but because they act sys FIELD OF THE INVENTION temically rather than locally, the side effects of orally admin istered drugs can limit their use. 0002 The present disclosure relates to compositions and methods for preventing, and/or treating acne or killing, and/or C. Background Regarding Nanoemulsions inhibiting the growth of Propionibacterium acnes. The method comprises topically administering to a subject in need 0007 Prior teachings related to nanoemulsions are thereof a nanoemulsion composition having anti-acne prop described in U.S. Pat. No. 6,015,832, which is directed to erties. methods of inactivating a Gram-positive bacteria, a bacterial spore, or a Gram-negative bacteria. The methods comprise BACKGROUND OF THE INVENTION contacting the Gram-positive bacteria, bacterial spore, or Gram-negative bacteria with a bacteria-inactivating (or bac A. Acne and P. acnes Infection terial-spore inactivating) emulsion. U.S. Pat. No. 6,506,803 is 0003) Acne is a chronic inflammatory disease affecting directed to methods of killing or neutralizing microbial more than 85% ofteenagers, and continuing into adulthood in agents (e.g., bacteria, Virus, spores, fungus, on or in humans Some populations. Some individuals suffer from acne into using an emulsion. U.S. Pat. No. 6,559,189 is directed to their thirties, forties and beyond. Acne is most frequently methods for decontaminating a sample (human, animal, food, found on the face and upper neck, but also found on the chest, medical device, etc.) comprising contacting the sample with a back, shoulders and upper arms. Acne lesions can develop nanoemulsion. The nanoemulsion, when contacted with bac into comedo, papule, pustule, lupus, nodule, or scars. terial, virus, fungi, protozoa, or spores, kills or disables the 0004 Acne is a disease of pilosebaceous units in the skin. pathogens. The antimicrobial nanoemulsion comprises an oil, Although the cause of acne is not fully understood, some quaternary ammonium compound, one of ethanol/glycerol/ factors have been linked to acne. Such as genetic history, PEG, a surfactant, and water. U.S. Pat. No. 6,635,676 is hormone level, skin inflammation, etc. In acne, excessive directed to two different compositions and methods of decon sebum production occurs in the sebaceous gland. This causes taminating samples by treating a sample with either of the hyperkeratinization of the hair follicle and prevents normal compositions. Composition 1 comprises an emulsion that is shedding of the follicular keratinocytes. This results in antimicrobial against bacteria, virus, fungi, protozoa, and/or obstruction of the hair follicle and Subsequent accumulation spores. The emulsions comprise an oil and a quaternary of lipids and cellular debris in the blocked hair follicle. Colo ammonium compound. U.S. Pat. No. 7,314,624 is directed to nization of an anaerobic gram-positive bacterium, Propioni methods of inducing an immune response to an immunogen bacterium species., e.g., Propionibacterium acnes, occurs in comprising treating a Subject via a mucosal Surface with a the blocked follicle. This bacteria is present on most human combination of an immunogen and a nanoemulsion. The skin and lives on fatty acids in the pilosebaceous unit. Infec nanoemulsion comprises oil, ethanol, a Surfactant, a quater tion of the hair follicle results in inflammation. Inflammation nary ammonium compound, and distilled water. US-2005 is further enhanced by rupture of the hair follicle and release 0208083-A1 and US-2006-025 1684-A1 are directed to of lipids, bacteria, and fatty acids into the dermis. nanoemulsions having droplets with preferred sizes. US-2007-0054834-A1 is directed to compositions compris B. Conventional Treatment for Acne ing quaternary ammonium halides and methods of using the same to treat infectious conditions. The quaternary ammo 0005 Conventional treatment for acne includes topical or nium compound may be provided as part of an emulsion. oral administration of bactericidals, benzoyl peroxide, tri Finally, US-2007-003.6831-A1 is directed to nanoemulsions closan bekeratolytics, e.g., Salicylic acid, and chlorhexidine, comprising an anti-inflammatory agent. acitretin, alcloxa, aldioxa, allantoin, dibenzothiophene, 0008. There is a need in the art for improved treatment etarotent, etretinate, motretinide, nordihydroguaiaretic acid, options for patients affected by acne. Specifically, there is a podofilox, podophyllum resin, resorcinalm resorcinol need in the art for an effective topical agent to treat and monoacetate, Sumarotene, tetroquinone, retinoids, e.g., tret prevent acne and/or infection by P. acnes. The present inven inoin, isotretinoin, adapalene and tazarotene, antibiotics, e.g., tion satisfies these needs. erythromycin, clindamycin, tetracycline, minocycline, doxy cycline, hormones, e.g., estrogen, and progesterone, and SUMMARY OF THE INVENTION combination products, e.g., Stievamycin, Murad R, Benza clin R) and Benzamycin R. Other anti-acne ingredients include 0009. The present invention provides methods and com Ascorbyl Tetraisopalmitate, Dipotassium Glycyrrhizinate, positions for treating and/or preventing acne and/or infection Ascorbyl Tetraisopalmitate, Niacinamide, alpha bisabolol. by P. acnes in a subject comprising administering a All of these ingredients have properties that help to reduce nanoemulsion topically to the Subject. The nanoemulsion US 2010/0226983 A1 Sep. 9, 2010

comprises droplets having an average diameter of less than 0015 Inclusion of a second antibiotic into the nanoemul about 3 microns, and the nanoemulsion droplets comprise an sion should reduce the potential for resistance development aqueous phase, at least one oil, at least one surfactant, and at towards either the nanoemulsion or added antibiotic. The least one organic solvent. nanoemulsion may further comprise anti-comdeogenic, anti 0010 Surprisingly, it was discovered that the topically inflammatory, keratolytics, sebum Supressors as disclosed in applied nanoemulsions have potent cidal activity against P PCT publication No. WO/01/56556A2. One skilled in the art acnes and Synergy with other agents commonly used to treat will understand that any suitable or desirable second active acne. The composition of the invention allows for targeted agent useful in treating acne can be incorporated into the delivery into the pilosebaceous unit, the site of acne patho nanoemulsion of this invention. genesis. This is significant, as a topically applied, and there 0016 Preferably, the nanoemulsions for topical adminis fore local, site-specific activity, is highly preferable over an tration are in the form of any pharmaceutically acceptable orally administered, and therefore systemic activity. More dosage form, including but not limited to, ointments, creams, over, the nanoemulsions are able to enhance delivery, and thus emulsions, lotions, gels, liquids, bioadhesive gels, sprays, effectiveness, of other topical anti-acne agents incorporated shampoos, aerosols, pastes, foams, Sunscreens, capsules, into the nanoemulsion, thereby enhancing the efficacy and microcapsules, or in the form of an article or carrier. Such as reducing the detrimental side effects of the other anti-acne a bandage, insert, Syringe-like applicator, pessary, powder, agents. talc or other Solid, shampoo, cleanser (leave on and wash off 0011. In certain embodiments of the invention, the product), and agents that favor penetration within piloseba nanoemulsion can have an increased viscosity to aid in per ceous unit, the epidermis, the dermis and keratin layers. The meation of the nanoemulsion into the dermis and epidermis. nanoemulsion is capable of effectively treating, preventing, 0012. In other embodiments of the invention, the and/or curing acne, without being systemically absorbed and nanoemulsion at the time of topical application is at room without significantly irritating the skin. temperature or warmer. 0017. The foregoing general description and following 0013 The nanoemulsion comprises droplets having an brief description of the drawings and the detailed description average particle size of less than about 3 microns, and the are exemplary and explanatory and are intended to provide nanoemulsion comprises water, at least one oil, at least one further explanation of the invention as claimed. Other objects, Surfactant, and at least one organic solvent. In one embodi advantages, and novel features will be readily apparent to ment of the invention, the Surfactant present in the nanoemul those skilled in the art from the following detailed description sion is a cationic surfactant. In another embodiment of the of the invention. invention, the nanoemulsion further comprises a chelating agent. In one embodiment of the invention, nanoemulsions BRIEF DESCRIPTION OF THE DRAWINGS from the present invention, or those derived from the nanoemulsions of the present invention, are diluted. The 0018 FIG. 1 illustrates the cross-section view of the diluted samples can then be tested to determine if they main pilosebaceous unit in human cadaver skin and hamster ear tain the desired functionality, Such as Surfactant concentra after application of nanoemulsion plus fluorescein tion, stability, particle size, and/or anti-infectious activity 0019 FIG. 2 shows in vitro skin permeation of nanoemul (e.g., antimicrobial activity against P. acnes). sion formulations into the epidermal layer of pig abdominal 0014. In some embodiments, a second anti-acne agent is skin at 24 hours after a single topical application of 100 incorporated into the nanoemulsion to achieve improved ul/cm. delivery, efficacy and or tolerability of the second anti-acne 0020 FIG. 3 shows in vitro permeation of nanoemulsion agent. Preferably, the second anti-acne agent is selected from formulations in pig abdominal skin at 12 and 24 hours after a the group consisting of benzoyl peroxide, Salicylic acid, aci single topical application of 100 ul/cm. tretin, alcloxa, aldioxa, allantoin, dibenzothiophene, etaro 0021 FIG. 4 shows the in vitro MBC of a nanoemulsion tent, etretinate, motretinide, nordihydroguaiaretic acid, podo (NB-003) with and without (+/-) the presence of 25% sebum. filox, podophyllum resin, resorcinalm resorcinol The figure shows that the MBC of the nanoemulsion rises monoacetate, Sumarotene, tetroquinone, triclosan, chlorhexi 500-fold in the presence of sebum, unless additional EDTA is dine, aZelaic acid, hydrocortisone, sodium hyaluronate, Sul added to the formulation. fur, urea, retinoids or retinoid derivatives, e.g., tretinoin, isotretinoin, antibiotics, e.g., erythromycin, clindamycin, tet 0022 FIG. 5 shows the effect the concentration of a racycline, minocycline, doxycycline, meclocycline, hor nanoemulsion has on the particle size and Viscosity of the mones, e.g., estrogen, and progesterone, adapalene and taZ nanoemulsion. With a decrease in concentration of the active, arotene and combination products, e.g., Stievamycin, Viscosity (cP) declines (triangles), whereas the particle size MuradR), Benzaclin R) and Benzamycin R, and any combina remains constant (bars). tion thereof. Other anti-acne ingredients include Ascorbyl 0023 FIG. 6 shows the results of a permeation study uti Tetraisopalmitate, Dipotassium Glycyrrhizinate, Ascorbyl lizing pigskin epidermis with 5 skin sections (n=5) following Tetraisopalmitate, Niacinamide, and alpha bisabolol. All of administration of a nanoemulsion (NB-003) twice daily these skin care ingredients have properties that help to reduce (BID). Higher viscosity (greater than 1000 cps) nanoemul and control acne, and acne-related problems such as sebum sions (e.g., 0.8% NB-003) were found to enhance permeation production. Herbal medicines are also used to treat acne and of the nanoemulsion into the epidermis. include Tea Tree Oil red clover, lavender, leaves of straw 0024 FIG. 7 shows the results of a permeation study uti berry, chaste tree berry extract, burdock root, dandelion lizing pig skin dermis with 5 skin sections (n=5) following leaves, milk thistle, papaya enzymes, burdock and dandelion, administration of a nanoemulsion (NB-003) twice daily eucalyptus, thyme, witch hazel, sage oil, camphor, cineole, (BID). Higher viscosity (greater than 1000 cps) nanoemul roSmarinic acid and tannins in the sage oil. sions (e.g., 0.8% NB-003) were found to deliver three times US 2010/0226983 A1 Sep. 9, 2010

the amount of the surfactant, cetylpyridinium chloride (CPC) to the invention without any additional EDTA showed a 32 to to the dermis as compared to a lower viscosity nanoemulsion 64 fold increase in the presence of 25% artificial sebum. In (e.g., 0.25% NB-003). addition, MBCs of a nanoemulsion according to the invention 0025 FIG. 8 shows the effect of storage temperature of a showed 256 fold increases in the presence of sebum. The nanoemulsion (e.g., NB-003) on the in vitro activity of the addition of 10-20 mM of EDTA decreased the MICs and nanoemulsion against Pacnes in the presence of sebum. MBCs of a nanoemulsion according to the invention to equal or lesser than the test concentrations. DETAILED DESCRIPTION OF THE PREFERRED 0030 The nanoemulsions comprise droplets having an EMBODIMENTS average diameter of less than about 3 microns, and the 0026. The present disclosure provides methods and com nanoemulsions comprise an aqueous phase, at least one oil, at positions for treating, preventing, and/or curing acne and/or least one surfactant or detergent, and at least one organic infection by P. acnes in a Subject comprising administering solvent. In one embodiment of the invention, the surfactant topically or to the Subject a nanoemulsion. The nanoemulsion present in the nanoemulsion is a cationic Surfactant. More comprises droplets having an average diameter of less than than one surfactant or detergent can be present in the about 3 microns, and the nanoemulsion droplets comprise an nanoemulsions of the invention, and the second Surfactant can aqueous phase, at least one oil, at least one surfactant, and at be the same type (i.e., two cationic Surfactants) or the second least one organic solvent. The delivery of nanoemulsions is or third etc. Surfactant can be different from the first. For targeted to the site of acne pathogenesis. i.e., the piloseba example, the nanoemulsions can comprise a cationic Surfac ceous unit. See FIG. 1. tant in combination with a non-ionic Surfactant. In another 0027 Propionibacterium acnes, a gram-positive, non embodiment of the invention, the nanoemulsion further com spore forming, anaerobic bacillus, is one of the primary fac prises a chelating agent. The organic solvent and the aqueous tors involved in the pathogenesis of acne Vulgaris. It is the phase of the invention can be a non-phosphate based solvent. predominant microorganism of the pilosebaceous glands of 0031. In some embodiments, a second anti-acne agent is human skin, with up to 10 million viable organisms isolated also incorporated into the nanoemulsion to achieve improved from a single sebaceous unit. Although aerotolerant, Pacnes delivery, efficacy and/or tolerability of the added anti-acne typically grows in the anaerobic environment of the infrain agent. Examples of suitable topical anti-acne agents include, fundibulum, where it releases lipases and digests local accu but are not limited to, benzoyl peroxide, salicylic acid, aci mulations of the skin, oil and sebum. Sebaceous glands pro tretin, alcloxa, aldioxa, allantoin, dibenzothiophene, etaro duce an oily sebum that is primarily composed of waxes, tent, etretinate, motretinide, nordihydroguaiaretic acid, podo triglycerides, and free fatty acids. (Lu et al., “Comparison of filox, podophyllum resin, resorcinalm resorcinol artificial sebum with human and hamster sebum samples.” monoacetate, Sumarotene, tetroquinone, tetracycline, doxy Int. J. Pharm. (Epub date, Oct. 22, 2008); Valiveti et al., cycline, minocycline, meclocycline erythromycin, clindamy “Diffusion properties of model compounds in artificial cin, azelaic acid, hydrocortisone, Sodium hyaluronate, Sulfur, sebum.” Int. J. Pharm., 345:88-94 (2007); and Valiveti et al., urea, dapsone, adapalene, tretinoin, retinoids and retinoid “Investigation of drug partition property in artificial sebum. derived compounds. Other anti-acne ingredients include Int. J. Pharm., 346:10-16 (2008).) Studies described herein Ascorbyl Tetraisopalmitate, Dipotassium Glycyrrhizinate, have shown that nanoemulsion droplets of the compositions Ascorbyl Tetraisopalmitate, Niacinamide, alpha bisabolol. described herein (NB-00X nanodroplets) are concentrated in All of these skin care ingredients have properties that help to the pilosebaceous unit where Pacnes migrates to enjoy a rich reduce and control acne, and acne related problems such as source of food (sebum) and a preferred anaerobic environ sebum production. Examples of acne herbal medicines ment. (Ciotti et al., “Novel nanoemulsion NB-001 permeates include, but are not limited to, Tea Tree Oil red clover, lav skin by the follucular route. Abstr. 48th Intersci. Conf. on ender, leaves of strawberry, chaste tree berry extract, burdock Antimicrob. Agents Chemother., abstr. A-1898 (2008).) root, dandelion leaves, milk thistle, papaya enzymes, burdock 0028. One effect that impacts acne prevention and/or treat and dandelion, eucalyptus, thyme, witch hazel, Sage oil, cam ment is the reduction of P acnes. Specifically, this anti-acne phor, cineole, roSmarinic acid and tannins in the sage oil. effect can be expressed in vitro as minimum inhibitory con 0032. The nanoemulsions comprise high energy nanom centration (MIC) and minimum bactericidal concentration eter-sized droplets that permeate into the pilosebaceous unit (MBC) values, of a nanoemulsion of the invention and com where they kill or inhibit the growth of P. acnes. Droplets pared to the effect of other anti-acne drugs currently used for having a suitable particle size can permeate skin pores and the treatment of acne, on different strains of P. acnes. Surpris into the pilosebaceous unit, but can be excluded by tight ingly, the comparison shows that the nanoemulsions of the junctions between epithelial cells and thus do not disrupt invention are active against P. acnes, including antibiotic tissue matrices or enter blood vessels. This minimizes skin resistant strains. The minimum inhibitory concentrations irritation and systemic absorption, but yet provides for a (MIC) and minimum bactericidal concentrations (MBCoo) composition which is highly topically bioavailable in the for 90% of the isolates were 0.5 g/ml/2.0 ug/ml for NB-00X pilosebaceous unit, epidermal and dermal tissues without and 1 g/ml/2 ug/ml for NB-00X gel, respectively. Greater causing disruption to the normal epithelial matrix. than 50% of the isolates were resistant to erythromycin and 0033. In one embodiment of the invention, the nanoemul clindamycin; 44% of the isolates were resistant to tetracy sion comprises: (a) an aqueous phase; (b) about 1% oil to cline. If the MBC/MIC ratio is the agent is bactericidal; if about 80% oil; (c) about 0.1% organic solvent to about 50% >4, the agent is bacteriostatic. organic solvent; (d) about 0.001% surfactant or detergent to 0029. Example 5 below details the efficacy of a nanoemul about 10% surfactant or detergent; (e) about 0.0005% to sion according to the invention against Propionibacterium about 1.0% of a chelating agent; or (0 any combination acnes in the presence of artificial sebum. In particular, as thereof. In another embodiment of the invention, the shown in Example 5, the MICs of a nanoemulsion according nanoemulsion comprises: (a) about 10% oil to about 80% oil; US 2010/0226983 A1 Sep. 9, 2010

(b) about 1% organic solvent to about 50% organic solvent; increasing the concentration of the nanoemulsion, the Viscos (c) at least one non-ionic Surfactant present in an amount of ity of the nanoemulsion can be increased to provide improved about 0.1% to about 10%; (d) at least one cationic agent therapeutic effectiveness. Examples of methods of increasing present in an amount of about 0.01% to about 2%; (e) about the Viscosity of a nanoemulsion according to the invention 0.0005% to about 1.0% of a chelating agent; or (O any com including increasing the concentration of the nanoemulsion bination thereof. (e.g., increasing CPC concentration), or adding a thickening 0034. In yet another embodiment of the invention, the agent or gelling agent to the formulation (see e.g., FIGS. 2 and nanoemulsion additionally includes at least one suitable or desirable active agent useful in treating acne. The exemplary 3). active agents for treating acne are benzoyl peroxide, salicylic 0041. Thus, in one embodiment of the invention, the acid and retinoids. The active agent can be present in a thera nanoemulsion has a viscosity of greater than about 12 centi peutically effective amount, such as from about 0.001% up to poise (cP), greater than about 15 cl, greater than about 20 cp. about 99%, about 0.01% up to about 95%, about 0.1% up to greater than about 25 cF. greater than about 30 cp greater than about 90%, about 3% up to about 80%, about 5% up to about about 35 cB. greater than about 40 cp greater than about 45 cB. 60%, about 10% up to about 50%, or any combination thereof greater than about 50 cp greater than about 55 cl, greater than (e.g., about 3% up to about 10%). about 60 cp, greater than about 65 cF. greater than about 70cP. 0035. The quantities of each component present in the greater than about 75 cl, greater than about 80 cp greater than nanoemulsion refer to a therapeutic nanoemulsion, and not to about 85 cB. greater than about 90 cp greater than about 95 cB. a nanoemulsion to be tested in vitro. This is significant, as greater than about 100 cp greater than about 150 cB. greater nanoemulsions tested in vitro. Such as the nanoemulsions than about 200 cp, greater than about 300 cp, greater than described in the examples, generally have lower concentra about 400 cp greater than about 500 cp, greater than about tions of oil, organic solvent, Surfactant or detergent, and (if 600 cB. greater than about 700 cP. greater than about 800 cB. present) chelating agent than that present in a nanoemulsion greater than about 900 cp, greater than about 1000 cp greater intended for therapeutic use, e.g., topical use. This is because than about 1500 cp greater than about 2000 cB. greater than in vitro microbiology studies do not require the nanoemulsion about 2500 cp, greater than about 3000 cp greater than about droplets to traverse the skin or other barriers. For topical use, 3500 cB. greater than about 4000 cP greater than about 4500 the concentrations of the components must be higher to result cP greater than about 5000 cF. greater than about 5500 cB. in therapeutic levels of nanoemulsion. However, the relative quantities of each component used in a nanoemulsion tested greater than about 6000 cp, greater than about 7000 cp greater in vitro are applicable to a nanoemulsion to be used therapeu than about 8000 cB. greater than about 9000 cP greater than tically and, therefore, in vitro quantities can be scaled up to about 10,000 cp greater than about 15,000 cp greater than prepare a therapeutic composition, and in vitro data may well about 20,000 cp greater than about 30,000 cp greater than be predictive of topical application Success. about 40,000 cp greater than about 50,000 cp greater than 0036 Viscosity about 60,000 cB. greater than about 70,000 cB. greater than 0037 Examples 6 and 7 below demonstrate that increas about 80,000 cB. greater than about 90,000 cB. greater than ing the viscosity of the nanoemulsion can enhance perme about 100,000 cB. greater than about 150,000 cB. greater than ation of the nanoemulsion into the skin, thereby producing a about 200,000 cB. greater than about 250,000 cB, or up to nanoemulsion more effective in killing bacteria or other about 259,300 cP. organisms. 0042 Temperature 0038 FIG. 5 shows the relationship between the particle 0043. As described in Example 8, one tactic that can size (nm), concentration of active (%), and viscosity of a increase the effectiveness of a nanoemulsion according to the nanoemulsion. The particle size does not change upon dilu invention in treating acne is ensuring that the nanoemulsion is tion of a nanoemulsion; however viscosity significantly at room temperature or warmer prior to application. The decreases as a function of the decrease in particle concentra results of Example 8, depicted in FIG. 8, show that cooling the tions. Thus, embodiment of the invention encompass using nanoemulsion decreases the effectiveness of the nanoemul dilutions of a nanoemulsion. Table 14 (below) shows the sion in killing P acnes. Conversely, nanoemulsions at room effect dilution of a nanoemulsion has on the concentration of temperature and warmed to 37° C. showed an increased effec the active (CPC), viscosity, and particle size. tiveness in killing Pacnes. The nanoemulsion warmed to 37 0039 FIGS. 2, 3, 6 and 7 show the results for epidermis C. showed an initial greater effectiveness in killing Pacnes as and dermis permeation, respectively. Higher viscosity compared to the room temperature nanoemulsion, with this nanoemulsions were found to increase the permeation of the increase in effectiveness diminishing about 15 minutes after nanoemulsion into the epidermis (FIGS. 2, 3 and FIG. 6) and application. dermis (FIGS. 3 and 7). 0040. More particularly, as shown in FIGS. 6 and 7, lower 0044 Thus, in another embodiment of the invention, concentration nanoemulsions, e.g., 0.25% to 0.30%, are encompassed are methods of treating acne comprising appli effective in penetrating the skin. Slightly higher or lower cation of a nanoemulsion according to the invention, wherein concentrations are also effective. However, at a concentration the nanoemulsion is at room temperature (e.g., 20 to 25°C.). of 0.5%, permeation significantly declined. Surprisingly, In another embodiment of the invention, encompassed are higher concentrations such as 0.8% or more showed a dra methods of treating acne comprising application of a matic increase in permeation due to the increased viscosity of nanoemulsion according to the invention, wherein the the composition. It is theorized that the increase in Viscosity nanoemulsion has been warmed prior to application. For inhibits or limits the evaporation of water from the skin after example, the nanoemulsion can be warmed prior to applica application of the emulsion, thus preventing the crystalliza tion to a temperature selected from the group consisting of tion of the active from the nanoemulsion. As an alternative to about 30° C. or warmer, about 31° C. or warmer, about 32° C. US 2010/0226983 A1 Sep. 9, 2010

or warmer, about 33°C. or warmer, about 34°C. or warmer, 0055 As used herein, the term “topically’ refers to appli about 35°C. or warmer, about 36°C. or warmer, about 37° C. cation of the compositions of the present invention to the or Warmer, Surface of the skin and tissues.

A. DEFINITIONS B. STABILITY OF THE NANOEMULSIONS OF 0045. The present invention is described herein using sev THE INVENTION eral definitions, as set forth below and throughout the appli cation. 0056. The nanoemulsions of the invention are stable at 0046. As used herein, “about will be understood by per about 40° C. and about 75% relative humidity for a time sons of ordinary skill in the art and will vary to some extent period of at least up to about 1 month, at least up to about 3 depending upon the context in which it is used. If there are months, at least up to about 6 months, at least up to about 12 uses of the term which are not clear to persons of ordinary months, at least up to about 18 months, at least up to about 2 skill in the art given the context in which it is used, “about years, at least up to about 2.5 years, or at least up to about 3 will mean up to plus or minus 10% of the particular term. years. 0047. The terms “buffer” or “buffering agents” refer to 0057. In another embodiment of the invention, the materials which when added to a solution, cause the Solution nanoemulsions of the invention are stable at about 25°C. and to resist changes in pH. about 60% relative humidity for a time period of at least up to 0048. The terms “chelator” or “chelating agent” refer to about 1 month, at least up to about 3 months, at least up to any materials having more than one atom with a lone pair of about 6 months, at least up to about 12 months, at least up to electrons that are available to bond to a metalion. about 18 months, at least up to about 2 years, at least up to 0049. The term “dilution refers to dilution of the about 2.5 years, or at least up to about 3 years, at least up to nanoemulsions of the present invention or those derived from about 3.5 years, at least up to about 4 years, at least up to about the nanoemulsions of the present invention using, for 4.5 years, or at least up to about 5 years. example, an aqueous system comprised of PBS or water (Such 0058. Further, the nanoemulsions of the invention are as diHO), or other water soluble components, to the desired stable at about 4°C. for a time period of at least up to about 1 final concentration. month, at least up to about 3 months, at least up to about 6 0050. The term “nanoemulsion,” as used herein, includes months, at least up to about 12 months, at least up to about 18 dispersions or droplets, as well as other lipid structures that months, at least up to about 2 years, at least up to about 2.5 can form as a result of hydrophobic forces that drive apolar years, at least up to about 3 years, at least up to about 3.5 residues (i.e., long hydrocarbon chains) away from water and years, at least up to about 4 years, at least up to about 4.5 drive polar head groups toward water, when a water immis years, at least up to about 5 years, at least up to about 5.5 cible oily phase is mixed with an aqueous phase. These other years, at least up to about 6 years, at least up to about 6.5 lipid structures include, but are not limited to, unilamellar, years, or at least up to about 7 years. paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. The droplets have an average diameterofless C. NANOEMULSIONS than about 3 microns. 0051. The terms “pharmaceutically acceptable' or “phar 0059. The term “nanoemulsion', as defined herein, refers macologically acceptable, as used herein, refer to composi to a dispersion or droplet or any other lipid structure. Typical tions that do not substantially produce adverse allergic or lipid structures contemplated in the invention include, but are immunological reactions when administered to a host (e.g., not limited to, unilamellar, paucilamellar and multilamellar an animal or a human). Such formulations include any phar lipid vesicles, micelles and lamellar phases. maceutically acceptable dosage form. As used herein, "phar 0060. The nanoemulsion of the present invention com maceutically acceptable carrier includes any and all sol prises droplets having an average diameter size of less than Vents, dispersion media, coatings, wetting agents (e.g., about 3 microns, less than about 2500 nm, less than about Sodium lauryl Sulfate), isotonic and absorption delaying 2000 nm, less than about 1500 nm, less than about 1000 nm, agents, disintegrants (e.g., potato starch or Sodium starch less than about 950 nm, less than about 900 nm, less than glycolate), and the like. about 850 nm, less thanabout 800 nm, less than about 750 nm, 0052. The term “stable' when referring to a “stable less than about 700 nm, less than about 650 nm, less than nanoemulsion' means that the nanoemulsion retains its struc about 600 nm, less thanabout 550 nm, less than about 500 nm, ture as an emulsion. A desired nanoemulsion structure, for less than about 450 nm, less than about 400 nm, less than example, may be characterized by a desired size range, mac about 350 nm, less thanabout 300 nm, less than about 250 nm, roscopic observations of emulsion science (is there one or less than about 200 nm, less than about 150 nm, or any more layers visible, is there visible precipitate), pH, and a combination thereof. In one embodiment, the droplets have stable concentration of one or more the components. an average diameter size greater than about 125 nm and at 0053. The term “subject' as used herein refers to organ least 400 nm. In another embodiment, the droplets have an isms to be treated by the compositions of the present inven average diameter of 180 nm. tion. Such organisms include animals (domesticated animal 0061 1. Aqueous Phase species, wild animals), and humans. 0062. The aqueous phase can comprise any type of aque 0054 The term “surfactant” refers to any molecule having ous phase including, but not limited to, water (e.g., H2O, both a polar headgroup, which energetically prefers Solvation distilled water, tap water) and Solutions (e.g., phosphate by water, and a hydrophobic tail which is not well solvated by buffered saline (PBS) solution). In certain embodiments, the water. The term "cationic surfactant” refers to a surfactant aqueous phase comprises water at a pH of about 4 to 10, with a cationic head group. The term “anionic Surfactant” preferably about 6 to 8. The water can be deionized (herein refers to a surfactant with an anionic head group. after “DiHO). In some embodiments the aqueous phase US 2010/0226983 A1 Sep. 9, 2010

comprises phosphate-buffered saline (PBS). The aqueous Valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isos phase may further be sterile and pyrogen free. tearyl alcohol, semi-synthetic derivatives thereof, and any 0063. 2. Organic Solvents combinations thereof. 0064 Organic solvents in the nanoemulsions of the inven 0069. The oil may further comprise a silicone component, tion include, but are not limited to, C-C alcohol, diol, triol, Such as a volatile silicone component, which can be the sole dialkyl phosphate, tri-alkyl phosphate, such as tri-n-butyl oil in the silicone component or can be combined with other phosphate, semi-synthetic derivatives thereof, and combina silicone and non-silicone, Volatile and non-volatile oils. Suit tions thereof. In one aspect of the invention, the organic able silicone components include, but are not limited to, Solvent is an alcohol chosen from a nonpolar solvent, a polar methylphenylpolysiloxane, simethicone, dimethicone, phe Solvent, a protic solvent, or an aprotic solvent. nyltrimethicone (or an organomodified version thereof), 0065 Suitable organic solvents for the nanoemulsion alkylated derivatives of polymeric silicones, cetyl dimethi include, but are not limited to, ethanol, methanol, isopropyl cone, lauryl trimethicone, hydroxylated derivatives of poly alcohol, glycerol, medium chain triglycerides, diethyl ether, meric silicones, such as dimethiconol, Volatile silicone oils, ethyl acetate, acetone, dimethyl sulfoxide (DMSO), acetic cyclic and linear silicones, cyclomethicone, derivatives of acid, n-butanol, butylene glycol, perfumers alcohols, isopro cyclomethicone, hexamethylcyclotrisiloxane, octamethylcy panol, n-propanol, formic acid, propylene glycols, glycerol, clotetrasiloxane, decamethylcyclopentasiloxane, Volatile lin Sorbitol, industrial methylated spirit, triacetin, hexane, ben ear dimethylpolysiloxanes, isohexadecane, isoeicosane, Zene, toluene, diethyl ether, chloroform, 1,4-dixoane, tet isotetracosane, polyisobutene, isooctane, isododecane, semi rahydrofuran, dichloromethane, acetone, acetonitrile, dim synthetic derivatives thereof, and combinations thereof. ethylformamide, dimethyl sulfoxide, formic acid, semi 0070 The volatile oil can be the organic solvent, or the synthetic derivatives thereof, and any combination thereof. Volatile oil can be present in addition to an organic solvent. 0.066 3. Oil Phase Suitable volatile oils include, but are not limited to, a terpene, 0067. The oil in the nanoemulsion of the invention can be monoterpene, sesquiterpene, carminative, aZulene, menthol, any cosmetically or pharmaceutically acceptable oil. The oil camphor, thujone, thymol, nerol, linalool, limonene, can be volatile or non-volatile, and may be chosen from geraniol, perillyl alcohol, nerolidol, farnesol, ylangene, bis animal oil, vegetable oil, natural oil, synthetic oil, hydrocar abolol, farmesene, ascaridole, chenopodium oil, citronellal, bon oils, silicone oils, semi-synthetic derivatives thereof, and citral, citronellol, chamaZulene, yarrow, guaiaZulene, chamo combinations thereof. mile, semi-synthetic derivatives, or combinations thereof. 0068 Suitable oils include, but are not limited to, mineral (0071. In one aspect of the invention, the volatile oil in the oil, squalene oil, flavor oils, silicon oil, essential oils, water silicone component is different than the oil in the oil phase. insoluble vitamins, Isopropyl stearate. Butyl stearate, Octyl (0072 4. Surfactants/Detergent palmitate, Cetyl palmitate, Tridecyl behenate, Diisopropyl 0073. The surfactant or detergent in the nanoemulsion of adipate, Dioctyl sebacate, Menthyl anthranhilate, Cetyl the invention can be a pharmaceutically acceptable ionic Sur octanoate, Octyl salicylate, Isopropyl myristate, neopentyl factant, a pharmaceutically acceptable nonionic Surfactant, a glycol dicarpate cetols, Ceraphyls(R), Decyl oleate, diisopro pharmaceutically acceptable cationic Surfactant, a pharma pyl adipate, C2-alkyl lactates, Cetyl lactate, Lauryl lactate, ceutically acceptable anionic Surfactant, or a pharmaceuti IsoStearyl neopentanoate, Myristyl lactate, Isocetyl Stearoyl cally acceptable Zwitterionic Surfactant. stearate, Octyldodecyl stearoyl stearate, Hydrocarbon oils, 0074 Exemplary useful surfactants are described in Isoparaffin, Fluid paraffins, Isododecane, Petrolatum, Argan Applied Surfactants: Principles and Applications. Tharwat F. oil, Canola oil, Chile oil, Coconut oil, corn oil, Cottonseed oil, Tadros, Copyright 82005 WILEY-VCHVerlag GmbH & Co. Flaxseed oil, Grape seed oil, Mustard oil, Olive oil, Palm oil, KGaA, Weinheim ISBN: 3-527-30629-3), which is specifi Palm kernel oil, Peanut oil, Pine seed oil, Poppy seed oil, cally incorporated by reference. Pumpkin seed oil, Rice bran oil, Safflower oil, Tea oil, Truffle 0075. Further, the surfactant can be a pharmaceutically oil,Vegetable oil, Apricot (kernel) oil, Jojoba oil (simmondsia acceptable ionic polymeric Surfactant, a pharmaceutically chinensis seed oil), Grapeseed oil, Macadamia oil. Wheat acceptable nonionic polymeric Surfactant, a pharmaceuti germ oil, Almond oil, Rapeseed oil, Gourd oil, Soybean oil, cally acceptable cationic polymeric Surfactant, a pharmaceu Sesame oil, Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, tically acceptable anionic polymeric Surfactant, or a pharma Bois oil, Kuki nut oil, Avocado oil, Walnut oil, Fish oil, berry ceutically acceptable Zwitterionic polymeric Surfactant. oil, allspice oil, juniper oil, seed oil, almond seed oil, anise Examples of polymeric Surfactants include, but are not lim seed oil, celery seed oil, cumin seed oil, nutmeg seed oil, leaf ited to, a graft copolymer of a poly(methyl methacrylate) oil, basil leafoil, bay leafoil, cinnamon leafoil, common sage backbone with multiple (at least one) polyethylene oxide leaf oil, eucalyptus leaf oil, lemon grass leaf oil, melaleuca (PEO) side chain, polyhydroxystearic acid, an alkoxylated leaf oil, oregano leaf oil, patchouli leaf oil, peppermint leaf alkyl phenol formaldehyde condensate, a polyalkylene glycol oil, pine needle oil, rosemary leaf oil, spearmint leaf oil, tea modified polyester with fatty acid hydrophobes, a polyester, tree leaf oil, thyme leaf oil, wintergreen leaf oil, flower oil, semi-synthetic derivatives thereof, or combinations thereof. chamomile oil, clary sage oil, clove oil, geranium flower oil, 0076 Surface active agents or surfactants, are amphip hyssop flower oil, jasmine flower oil, lavender flower oil, athic molecules that consist of a non-polar hydrophobic por manuka flower oil, Marhoram flower oil, orange flower oil, tion, usually a straight or branched hydrocarbon or fluorocar rose flower oil, ylang-ylang flower oil, Bark oil, cassia Bark bon chain containing 8-18 carbon atoms, attached to a polar oil, cinnamon bark oil, Sassafras Bark oil, Wood oil, camphor or ionic hydrophilic portion. The hydrophilic portion can be wood oil, cedar wood oil, rosewood oil, Sandalwood oil), nonionic, ionic or Zwitterionic. The hydrocarbon chain inter rhizome (ginger) wood oil, resin oil, frankincense oil, myrrh acts weakly with the water molecules in an aqueous environ oil, peel oil, bergamot peel oil, grapefruit peel oil, lemon peel ment, whereas the polar orionic head group interacts strongly oil, lime peel oil, orange peel oil, tangerine peel oil, root oil, with water molecules via dipole or ion-dipole interactions. US 2010/0226983 A1 Sep. 9, 2010

Based on the nature of the hydrophilic group, Surfactants are mide ethoxylated, a Sorbitan ester ethoxylated, a fatty amino classified into anionic, cationic, Zwitterionic, nonionic and ethoxylated, an ethylene oxide-propylene oxide copolymer, polymeric Surfactants. Bis(polyethylene glycol bisimidazoyl carbonyl), nonox 0.077 Suitable surfactants include, but are not limited to, ynol-9, Bis(polyethylene glycol bisimidazoyl carbonyl), ethoxylated nonylphenol comprising 9 to 10 units of ethyl BrijR35, BrijR 56, BrijR 72, Brij.R. 76, Brij(R92V, BrijR 97, eneglycol, ethoxylated undecanol comprising 8 units of eth Brij(R) 58P. Cremophor(R) EL, Decaethylene glycol mon yleneglycol, polyoxyethylene (20) Sorbitan monolaurate, ododecyl ether, N-Decanoyl-N-methylglucamine, n-Decyl polyoxyethylene (20) Sorbitan monopalmitate, polyoxyeth alpha-D-glucopyranoside, Decyl beta-D-maltopyranoside, ylene (20) sorbitan monostearate, polyoxyethylene (20) sor n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D- bitan monooleate, Sorbitan monolaurate, Sorbitan mono maltoside, n-Dodecyl beta-D-maltoside, n-Dodecyl beta-D- palmitate, Sorbitan monostearate, Sorbitan monooleate, maltoside, Heptaethylene glycol monodecyl ether, Heptaeth ethoxylated hydrogenated ricin oils, Sodium laurylsulfate, a ylene glycol monododecyl ether, Heptaethylene glycol diblock copolymer of ethyleneoxyde and propyleneoxyde, monotetradecyl ether, n-Hexadecyl beta-D-maltoside, Hexa Ethylene Oxide-Propylene Oxide Block Copolymers, and ethylene glycol monododecyl ether, Hexaethylene glycol tetra-functional block copolymers based on ethylene oxide monohexadecyl ether, Hexaethylene glycol monooctadecyl and propylene oxide, Glyceryl monoesters, Glyceryl caprate, ether, Hexaethylene glycol monotetradecyl ether, Igepal Glyceryl caprylate, Glyceryl cocate, Glyceryl erucate, Glyc CA-630, Igepal CA-630, Methyl-6-O-(N-heptylcarbamoyl)- eryl hydroxysterate, Glyceryl isostearate, Glyceryl lanolate, alpha-D-glucopyranoside. Nonaethylene glycol monodode Glyceryl laurate, Glyceryllinolate, Glyceryl myristate, Glyc cyl ether, N-N-Nonanoyl-N-methylglucamine, Octaethylene eryl oleate, Glyceryl PABA, Glyceryl palmitate, Glyceryl glycol monodecyl ether, Octaethylene glycol monododecyl ricinoleate, Glyceryl Stearate, Glyceryl thighlycolate, Glyc ether, Octaethylene glycol monohexadecyl ether, Octaethyl eryl dilaurate, Glyceryl dioleate, Glyceryl dimyristate, Glyc ene glycol monooctadecyl ether, Octaethylene glycol eryl disterate, Glyceryl sesuioleate, Glyceryl Stearate lactate, monotetradecyl ether, Octyl-beta-D-glucopyranoside, Penta Polyoxyethylene cetyl/stearyl ether, Polyoxyethylene cho ethylene glycol monodecyl ether, Pentaethylene glycol mon lesterol ether, Polyoxyethylene laurate or dilaurate, Polyoxy ododecyl ether, Pentaethylene glycol monohexadecyl ether, ethylene Stearate or distearate, polyoxyethylene fatty ethers, Pentaethylene glycol monohexyl ether, Pentaethylene glycol Polyoxyethylene lauryl ether, Polyoxyethylene stearyl ether, monooctadecyl ether, Pentaethylene glycol monooctyl ether, polyoxyethylene myristyl ether, a steroid, Cholesterol, Beta Polyethylene glycol diglycidyl ether, Polyethylene glycol sitosterol, Bisabolol, fatty acid esters of alcohols, isopropyl ether W-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethyl myristate, Aliphati-isopropyl n-butyrate, Isopropyl n-hex ene 100 stearate, Polyoxyethylene 20 isohexadecyl ether, anoate, Isopropyl n-decanoate, Isoproppyl palmitate, Octyl Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate, dodecyl myristate, alkoxylated alcohols, alkoxylated acids, Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate, alkoxylated amides, alkoxylated Sugar derivatives, alkoxy Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene lated derivatives of natural oils and waxes, polyoxyethylene 25 propylene glycol Stearate, Saponin from Quillaja bark, polyoxypropylene block copolymers, nonoxynol-14, PEG-8 SpanR 20, Span R 40, Span R. 60, Span(R) 65, Span R 80, laurate, PEG-6 Cocoamide, PEG-20 methylglucose ses SpanR 85, Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, quistearate, PEG40 lanolin, PEG-40 castor oil, PEG-40 Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type hydrogenated castor oil, polyoxyethylene fatty ethers, glyc 15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, eryl diesters, polyoxyethylene Stearyl ether, polyoxyethylene Type NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Ter myristyl ether, and polyoxyethylene lauryl ether, glyceryl gitol, Tergitol, Type TMN-10, Tergitol, Type TMN-6, Tet dilaurate, glyceryl dimyState, glyceryl distearate, semi-syn radecyl-beta-D-maltoside, Tetraethylene glycol monodecyl thetic derivatives thereof, or mixtures thereof. ether, Tetraethylene glycol monododecyl ether, Tetraethylene 0078. Additional suitable surfactants include, but are not glycol monotetradecyl ether, Triethylene glycol monodecyl limited to, non-ionic lipids, such as glyceryl laurate, glyceryl ether, Triethylene glycol monododecyl ether, Triethylene gly myristate, glyceryl dilaurate, glyceryl dimyristate, semi-syn col monohexadecyl ether, Triethylene glycol monooctyl thetic derivatives thereof, and mixtures thereof. ether, Triethylene glycol monotetradecyl ether, Triton CF-21, 0079. In additional embodiments, the surfactant is a poly Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, oxyethylene fatty ether having a polyoxyethylene head group Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, ranging from about 2 to about 100 groups, or an alkoxylated Triton X-15, Triton X-151, Triton X-200, Triton X-207, Tri alcohol having the structure Rs (OCHCH.), OH, ton RX-114, Triton RX-165, Triton RX-305, Triton RX-405, wherein Rs is a branched or unbranched alkyl group having Triton(R) X-45, Triton(R) X-705-70, TWEENR) 20, TWEENR) from about 6 to about 22 carbonatoms and y is between about 21, TWEENR 40, TWEENR 60, TWEENR 61, TWEENR) 4 and about 100, and preferably, between about 10 and about 65, TWEENR 80, TWEENR 81, TWEENR 85, Tyloxapol, 100. Preferably, the alkoxylated alcohol is the species n-Undecyl beta-D-glucopyranoside, semi-synthetic deriva wherein Rs is a lauryl group andy has an average value of 23. tives thereof, or combinations thereof. 0080. In a different embodiment, the surfactant is an I0082 In addition, the nonionic surfactant can be a polox alkoxylated alcohol which is an ethoxylated derivative of amer. Poloxamers are polymers made of a block of polyoxy lanolin alcohol. Preferably, the ethoxylated derivative of ethylene, followed by a block of polyoxypropylene, followed lanolin alcohol is laneth-10, which is the polyethylene glycol by a block of polyoxyethylene. The average number of units ether of lanolin alcohol with an average ethoxylation value of of polyoxyethylene and polyoxypropylene varies based on 10. the number associated with the polymer. For example, the 0081. Nonionic surfactants include, but are not limited to, smallest polymer, Poloxamer 101, consists of a block with an an ethoxylated Surfactant, an alcohol ethoxylated, an alkyl average of 2 units of polyoxyethylene, a block with an aver phenol ethoxylated, a fatty acid ethoxylated, a monoalkaola age of 16 units of polyoxypropylene, followed by a block with US 2010/0226983 A1 Sep. 9, 2010 an average of 2 units of polyoxyethylene. Poloxamers range Alkyl dimethylbenzyl ammonium chloride (C12-16), Alkyl from colorless liquids and pastes to white solids. In cosmetics dimethylbenzyl ammonium chloride (C12-18), dialkyl dim and personal care products, Poloxamers are used in the for ethylbenzyl ammonium chloride, Alkyl dimethyl dimethy mulation of skin cleansers, bath products, shampoos, hair benzyl ammonium chloride, Alkyl dimethyl ethyl ammonium conditioners, mouthwashes, eye makeup remover and other bromide (90% C14, 5% C16, 5% C12), Alkyl dimethyl ethyl skin and hair products. Examples of Poloxamers include, but ammonium bromide (mixed alkyland alkenyl groups as in the are not limited to, Poloxamer 101, Poloxamer 105, Polox fatty acids of soybean oil), Alkyl dimethyl ethylbenzyl amer 108, Poloxamer 122, Poloxamer 123, Poloxamer 124, ammonium chloride, Alkyl dimethyl ethylbenzyl ammonium Poloxamer 181, Poloxamer 182, Poloxamer 183, Poloxamer chloride (60% C14), Alkyl dimethyl isopropylbenzyl ammo 184, Poloxamer 185, Poloxamer 188, Poloxamer 212, Polox nium chloride (50% C12, 30% C14, 17% C16, 3% C18), amer 215, Poloxamer 217, Poloxamer 231, Poloxamer 234, Alkyl trimethyl ammonium chloride (58% C18, 40% C16, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 1% C14, 1% C12), Alkyl trimethyl ammonium chloride (90% 282, Poloxamer 284, Poloxamer 288, Poloxamer 331, Polox C18, 10% C16), Alkyldimethyl(ethylbenzyl) ammonium amer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, chloride (C12-18), Di-(C8-10)-alkyl dimethyl ammonium Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer chlorides, Dialkyl dimethyl ammonium chloride, Dialkyl 407, Poloxamer 105 Benzoate, and Poloxamer 182 Diben methylbenzyl ammonium chloride, Didecyl dimethylammo ZOate. nium chloride, Diisodecyl dimethyl ammonium chloride, 0.083 Suitable cationic surfactants include, but are not Dioctyl dimethyl ammonium chloride, Dodecyl bis(2-hy limited to, a quarternary ammonium compound, an alkyl droxyethyl) octyl hydrogen ammonium chloride, Dodecyl trimethyl ammonium chloride compound, a dialkyl dimethyl dimethyl benzyl ammonium chloride, Dodecylcarbamoyl ammonium chloride compound, a cationic halogen-contain methyl dimethyl benzyl ammonium chloride, Heptadecyl ing compound. Such as cetylpyridinium chloride, Benzalko hydroxyethylimidazolinium chloride, Hexahydro-1,3,5-tris nium chloride, Benzalkonium chloride, Benzyldimethyl (2-hydroxyethyl)-s-triazine, Myristalkonium chloride (and) hexadecylammonium chloride, Quat RNIUM 14, N,N-Dimethyl-2-hydroxypropylammo Benzyldimethyltetradecylammonium chloride, Benzyldode nium chloride polymer, n-Tetradecyl dimethylbenzyl ammo cyldimethylammonium bromide, Benzyltrimethylammo nium chloride monohydrate, Octyl decyl dimethyl ammo nium tetrachloroiodate, Dimethyldioctadecylammonium nium chloride, Octyl dodecyl dimethyl ammonium chloride, bromide, Dodecylethyldimethylammonium bromide, Dode Octyphenoxyethoxyethyl dimethylbenzyl ammonium chlo cyltrimethylammonium bromide, Dodecyltrimethylammo ride, Oxydiethylenebis(alkyl dimethyl ammonium chloride), nium bromide, Ethylhexadecyldimethylammonium bromide, Trimethoxysily propyl dimethyl octadecyl ammonium chlo Girard's reagent T. Hexadecyltrimethylammonium bromide, ride, Trimethoxysilyl quats, Trimethyl dodecylbenzyl ammo Hexadecyltrimethylammonium bromide, N,N',N'-Polyoxy nium chloride, semi-synthetic derivatives thereof, and com ethylene(10)-N-tallow-1,3-diaminopropane, Thonzonium binations thereof. bromide, Trimethyl(tetradecyl)ammonium bromide, 1,3,5- 0084 Exemplary cationic halogen-containing compounds Triazine-1,3,5(2H4H.6H)-triethanol, 1-Decanaminium, include, but are not limited to, cetylpyridinium halides, cetyl N-decyl-N,N-dimethyl-, chloride, Didecyl dimethyl ammo trimethylammonium halides, cetyldimethylethylammonium nium chloride, 2-(2-(p-(Diisobutyl)cresosxy)ethoxy)ethyl halides, cetyldimethylbenzylammonium halides, cetyltribu dimethyl benzyl ammonium chloride, 2-(2-(p-(Diisobutyl) tylphosphonium halides, dodecyltrimethylammonium phenoxy)ethoxy)ethyl dimethylbenzyl ammonium chloride, halides, or tetradecyltrimethylammonium halides. In some Alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazolinium particular embodiments, Suitable cationic halogen containing chloride, Alkyl bis(2-hydroxyethyl)benzyl ammonium chlo compounds comprise, but are not limited to, cetylpyridinium ride, Alkyl demethylbenzyl ammonium chloride, Alkyl dim chloride (CPC), cetyltrimethylammonium chloride, cetyl ethyl 3,4-dichlorobenzyl ammonium chloride (100% C12), benzyldimethylammonium chloride, cetylpyridinium bro Alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride mide (CPB), cetyltrimethylammonium bromide (CTAB), (50% C14, 40% C12, 10% C16), Alkyl dimethyl 3,4-dichlo cetylidimethylethylammonium bromide, cetyltributylphos robenzyl ammonium chloride (55% C14, 23% C12, 20% phonium bromide, dodecyltrimethylammonium bromide, C16), Alkyl dimethylbenzyl ammonium chloride, Alkyl dim and tetradecyltrimethylammonium bromide. In particularly ethyl benzyl ammonium chloride (100% C14), Alkyl dim preferred embodiments, the cationic halogen containing ethyl benzyl ammonium chloride (100% C16), Alkyl dim compound is CPC, although the compositions of the present ethyl benzyl ammonium chloride (41% C14, 28% C12), invention are not limited to formulation with a particular Alkyl dimethylbenzyl ammonium chloride (47% C12, 18% cationic containing compound. C14), Alkyl dimethylbenzyl ammonium chloride (55% C16, 0085 Suitable anionic surfactants include, but are not lim 20% C14), Alkyl dimethylbenzyl ammonium chloride (58% ited to, a carboxylate, a Sulphate, a Sulphonate, a phosphate, C14, 28% C16), Alkyl dimethylbenzyl ammonium chloride chenodeoxycholic acid, chenodeoxycholic acid sodium salt, (60% C14, 25% C12), Alkyl dimethyl benzyl ammonium cholic acid, ox or sheep bile, Dehydrocholic acid, Deoxy chloride (61% C11, 23% C14), Alkyl dimethylbenzyl ammo cholic acid, Deoxycholic acid, Deoxycholic acid methyl nium chloride (61% C12, 23% C14), Alkyl dimethylbenzyl ester, Digitonin, Digitoxigenin, N,N-Dimethyldodecylamine ammonium chloride (65% C12, 25% C14), Alkyl dimethyl N-oxide, Docusate sodium salt, Glycochenodeoxycholic acid benzyl ammonium chloride (67% C12, 24% C14), Alkyl Sodium salt, Glycocholic acid hydrate, synthetic, Glyco dimethylbenzyl ammonium chloride (67% C12, 25% C14), cholic acid sodium salt hydrate, synthetic, Glycodeoxycholic Alkyl dimethyl benzyl ammonium chloride (90% C14, 5% acid monohydrate, Glycodeoxycholic acid sodium salt, Gly C12), Alkyl dimethylbenzyl ammonium chloride (93% C14, colithocholic acid 3-sulfate disodium salt, Glycolithocholic 4% C12), Alkyl dimethylbenzyl ammonium chloride (95% acid ethyl ester, N-Lauroylsarcosine Sodium salt, N-Lauroyl C16, 5% C18), Alkyl didecyl dimethyl ammonium chloride, sarcosine Solution, N-Lauroylsarcosine solution, Lithium US 2010/0226983 A1 Sep. 9, 2010

dodecyl sulfate, Lithium dodecyl sulfate, Lithium dodecyl a concentration of about 0.3% to about 4%. In yet another Sulfate, Lugol Solution, Niaproof 4. Type 4.1-Octanesulfonic embodiment of the invention, the nanoemulsion comprises a acid sodium salt, Sodium 1-butanesulfonate, Sodium 1-de cationic surfactant present in a concentration of about 0.01% canesulfonate, Sodium 1-decanesulfonate, Sodium 1-dode to about 2%, in combination with a nonionic Surfactant. canesulfonate, Sodium 1-heptanesulfonate anhydrous, I0089 5. Active Agents Sodium 1-heptanesulfonate anhydrous, Sodium 1-nonane 0090. Optionally, a second anti-acne agent is incorporated Sulfonate, Sodium 1-propanesulfonate monohydrate, Sodium into the nanoemulsion to achieve better efficacy, tolerability 2-bromoethanesulfonate, Sodium cholate hydrate, Sodium and/or synergistic antimicrobial activity effect in sebum. choleate, Sodium deoxycholate, Sodium deoxycholate Preferably, the second anti-acne agent is benzoyl peroxide monohydrate, Sodium dodecyl sulfate, Sodium hexane salicylic acid, or a retinoid. However, any active agent useful Sulfonate anhydrous, Sodium octyl sulfate, Sodium pentane in treating acne can be incorporated into the nanoemulsion. Sulfonate anhydrous, Sodium taurocholate, Taurochenode 0091 Exemplary topical anti-acne agents include, but are oxycholic acid sodium salt, Taurodeoxycholic acid sodium not limited to, benzoyl peroxide, Salicylic acid, acitretin, salt monohydrate, Taurohyodeoxycholic acid sodium salt alcloxa, aldioxa, allantoin, dibenzothiophene, etarotent, hydrate, Taurolithocholic acid 3-sulfate disodium salt, Taur etretinate, motretinide, nordihydroguaiaretic acid, podofilox, oursodeoxycholic acid sodium salt, Trizma(R) dodecyl sulfate, podophyllum resin, resorcinalm resorcinol monoacetate, TWEENR 80, Ursodeoxycholic acid, semi-synthetic deriva Sumarotene, tetroquinone, adapalene, tretinoin, erythromy tives thereof, and combinations thereof. cin, clindamycin, azelaic acid, hydrocortisone, Sodium hyalu 0.086 Suitable Zwitterionic surfactants include, but are not ronate, Sulfur, urea, meclocycline, dapsone, retinoids and ret limited to, an N-alkyl betaine, laurylamindo propyl dimethyl inoid derivatives. Other anti-acne ingredients include betaine, an alkyl dimethyl glycinate, an N-alkyl amino pro Ascorbyl Tetraisopalmitate, Dipotassium Glycyrrhizinate, pionate, CHAPS, minimum 98% (TLC), CHAPS, minimum Ascorbyl Tetraisopalmitate, Niacinamide, alpha bisabolol 98% (TLC), CHAPS, for electrophoresis, minimum 98% can also be included in the nanoemulsion of this invention. All (TLC), CHAPSO, minimum 98%, CHAPSO, CHAPSO, for of these skin care ingredients have properties that help to electrophoresis, 3-(Decyldimethylammonio)propane reduce and control acne, and acne related problems such as Sulfonate inner salt, 3-Dodecyldimethylammonio)propane sebum production. Sulfonate inner salt, 3-(Dodecyldimethylammonio)propane 0092 Additional anti-acne agents include acne herbal sulfonate inner salt, 3-(N,N-Dimethylmyristylammonio) medicines, such as Tea Tree Oil red clover, lavender, leaves of propanesulfonate, 3-(N,N-Dimethyloctadecylammonio) strawberry, chaste tree berry extract, burdock root, dandelion propanesulfonate, 3-(N,N-Dimethyloctylammonio) leaves, milk thistle, papaya enzymes, burdock and dandelion, propanesulfonate inner salt, 3-(N.N- eucalyptus, thyme, witch hazel, sage oil, camphor, cineole, Dimethylpalmitylammonio)propanesulfonate, semi roSmarinic acid and tannins in the sage oil. synthetic derivatives thereof, and combinations thereof. (0093. 6. Additional Ingredients 0087. In some embodiments, the nanoemulsion comprises 0094. Additional compounds suitable for use in the a cationic Surfactant, which can be cetylpyridinium chloride. nanoemulsions of the invention include but are not limited to In other embodiments of the invention, the nanoemulsion one or more solvents, such as an organic phosphate-based comprises a cationic Surfactant, and the concentration of the Solvent, bulking agents, coloring agents, pharmaceutically cationic Surfactant is less than about 5.0% and greater than acceptable excipients, a preservative, pH adjuster, buffer, about 0.001%. In yet another embodiment of the invention, chelating agent, etc. The additional compounds can be the nanoemulsion comprises a cationic Surfactant, and the admixed into a previously emulsified nanoemulsion, or the concentration of the cationic Surfactant is selected from the additional compounds can be added to the original mixture to group consisting of less than about 5%, less than about 4.5%, be emulsified. In certain of these embodiments, one or more less than about 4.0%, less than about 3.5%, less than about additional compounds are admixed into an existing 3.0%, less than about 2.5%, less than about 2.0%, less than nanoemulsion composition immediately prior to its use. about 1.5%, less than about 1.0%, less than about 0.90%, less 0.095 Suitable preservatives in the nanoemulsions of the than about 0.80%, less than about 0.70%, less than about invention include, but are not limited to, cetylpyridinium 0.60%, less than about 0.50%, less than about 0.40%, less chloride, benzalkonium chloride, benzyl alcohol, chlorhexi than about 0.30%, less than about 0.20%, or less than about dine, imidazolidinyl urea, phenol, potassium Sorbate, benzoic 0.10%. Further, the concentration of the cationic agent in the acid, bronopol, chlorocresol, paraben esters, phenoxyetha nanoemulsion is greater than about 0.002%, greater than nol, Sorbic acid, alpha-tocophernol, ascorbic acid, ascorbyl about 0.003%, greater than about 0.004%, greater than about palmitate, butylated hydroxyanisole, butylated hydroxytolu 0.005%, greater than about 0.006%, greater than about ene, Sodium ascorbate, Sodium metabisulphite, citric acid, 0.007%, greater than about 0.008%, greater than about edetic acid, semi-synthetic derivatives thereof, and combina 0.009%, greater than about 0.010%, or greater than about tions thereof. 0.001%. In one embodiment, the concentration of the cationic 0096. The nanoemulsion may further comprise at least one agent in the nanoemulsion is less than about 5.0% and greater pH adjuster. Suitable pH adjusters in the nanoemulsion of the than about 0.001%. invention include, but are not limited to, diethyanolamine, 0088. In another embodiment of the invention, the lactic acid, monoethanolamine, triethylanolamine, sodium nanoemulsion comprises at least one cationic Surfactant and hydroxide, Sodium phosphate, semi-synthetic derivatives at least one non-cationic Surfactant. The non-cationic Surfac thereof, and combinations thereof. tant is a nonionic Surfactant, Such as a polysorbate (Tween), 0097. In addition, the nanoemulsion can comprise a such as polysorbate 80 or polysorbate 20. In one embodiment, chelating agent. In one embodiment of the invention, the the non-ionic Surfactant is present in a concentration of about chelating agent is present in an amount of about 0.0005% to 0.05% to about 7.0%, or the non-ionic surfactant is present in about 1.0%. Examples of chelating agents include, but are not US 2010/0226983 A1 Sep. 9, 2010 limited to, ethylenediamine, ethylenediaminetetraacetic acid (autoclaved), Phosphate buffered saline, washing buffer for (EDTA), and dimercaprol, and a preferred chelating agent is peroxidase conjugates in Western Blotting, 10x concentrate, ethylenediaminetetraacetic acid. piperazine, anhydrous, 299.0% (T), Potassium D-tartrate 0098. The nanoemulsion can comprise a buffering agent, monobasic, 299.0% (T), Potassium acetate solution, for Such as a pharmaceutically acceptable buffering agent. molecular biology, Potassium acetate Solution, for molecular Examples of buffering agents include, but are not limited to, biology, 5 M in HO, Potassium acetate solution, for molecu 2-Amino-2-methyl-1,3-propanediol, 299.5% (NT), lar biology, ~1 M in HO, Potassium acetate, 299.0% (NT), 2-Amino-2-methyl-1-propanol, 299.0% (GC), L-(+)-Tar Potassium acetate, for luminescence, 299.0% (NT), Potas taric acid, 299.5% (T), ACES, 299.5% (T), ADA, 299.0% sium acetate, for molecular biology, 299.0% (NT), Potas (T), Acetic acid, 299.5% (GC/T), Acetic acid, for lumines sium bicarbonate, 299.5% (T), Potassium carbonate, anhy cence, 299.5% (GC/T), Ammonium acetate solution, for drous, 299.0% (T), Potassium chloride, 299.5% (AT), molecular biology, ~5 M in H2O, Ammonium acetate, for Potassium citrate monobasic, 299.0% (dried material, NT), luminescence, 299.0% (calc. on dry substance, T), Ammo Potassium citrate tribasic solution, 1 M in HO, Potassium nium bicarbonate, 299.5% (T), Ammonium citrate dibasic, formate solution, 14 Min HO, Potassium formate, 299.5% 299.0% (T), Ammonium formate solution, 10 M in H.O. (NT), Potassium oxalate monohydrate, 299.0% (RT), Potas Ammonium formate, 299.0% (calc. based on dry substance, sium phosphate dibasic, anhydrous, 299.0% (T), Potassium NT), Ammonium oxalate monohydrate, 299.5% (RT), phosphate dibasic, for luminescence, anhydrous, 299.0% Ammonium phosphate dibasic solution, 2.5 M in H.O. (T), Potassium phosphate dibasic, for molecular biology, Ammonium phosphate dibasic, 299.0% (T), Ammonium anhydrous, 299.0% (T), Potassium phosphate monobasic, phosphate monobasic solution, 2.5 M in H2O, Ammonium anhydrous, 299.5% (T), Potassium phosphate monobasic, phosphate monobasic, 299.5% (T), Ammonium sodium for molecular biology, anhydrous, 299.5% (T), Potassium phosphate dibasic tetrahydrate, 299.5% (NT), Ammonium phosphate tribasic monohydrate, 295% (T), Potassium sulfate solution, for molecular biology, 3.2 MinH2O, Ammo phthalate monobasic, 299.5% (T), Potassium sodium tartrate nium tartrate dibasic solution, 2 Min HO (colorless solution solution, 1.5 M in H2O, Potassium sodium tartrate tetrahy at 20° C.), Ammonium tartrate dibasic, 299.5% (T), BES drate, 299.5% (NT), Potassium tetraborate tetrahydrate, buffered saline, for molecular biology, 2x concentrate, BES. 299.0% (T), Potassium tetraoxalate dihydrate, 299.5% 299.5% (T), BES, for molecular biology, 299.5% (T), (RT), Propionic acid solution, 1.0 M in HO, STE buffer BICINE buffer Solution, for molecular biology, 1 M in HO, solution, for molecular biology, pH 7.8, STET buffer solu BICINE, 299.5% (T), BIS-TRIS, 299.0% (NT), Bicarbon tion, for molecular biology, pH 8.0, Sodium 5,5-diethylbar ate buffer solution, >0.1 MNaCO >0.2 MNaHCO, Boric biturate, 299.5% (NT), Sodium acetate solution, for molecu acid, 299.5% (T), Boric acid, for molecular biology, 299. lar biology, ~3M in H2O, Sodium acetate trihydrate, 299.5% 5% (T), CAPS, 299.0% (TLC), CHES, 299.5% (T), Cal (NT), Sodium acetate, anhydrous, 299.0% (NT), Sodium cium acetate hydrate, 299.0% (calc. on dried material, KT). acetate, for luminescence, anhydrous, 299.0% (NT), Sodium Calcium carbonate, precipitated, 299.0% (KT), Calcium cit acetate, for molecular biology, anhydrous, 299.0% (NT), rate tribasic tetrahydrate, 298.0% (calc. on dry substance, Sodium bicarbonate, 299.5% (T), Sodium bitartrate mono KT), Citrate Concentrated Solution, for molecular biology, 1 hydrate, 299.0% (T), Sodium carbonate decahydrate, 299. M in HO, Citric acid, anhydrous, 299.5% (T), Citric acid, 5%. (T), Sodium carbonate, anhydrous, 299.5% (calc. on dry for luminescence, anhydrous, 299.5% (T), Diethanolamine, Substance, T), Sodium citrate monobasic, anhydrous, 299. 299.5% (GC), EPPS, 299.0% (T), Ethylenediaminetet 5%. (T), Sodium citrate tribasic dihydrate, 299.0% (NT), raacetic acid disodium salt dihydrate, for molecular biology, Sodium citrate tribasic dihydrate, for luminescence, 299.0% 299.0% (T), Formic acid solution, 1.0 M in HO, Gly-Gly (NT), Sodium citrate tribasic dihydrate, for molecular biol Gly, 299.0% (NT), Gly-Gly, 299.5% (NT), Glycine, 299. ogy, 299.5% (NT), Sodium formate solution, 8 M in H2O, 0% (NT), Glycine, for luminescence, 299.0% (NT), Glycine, Sodium oxalate, 299.5% (RT), Sodium phosphate dibasic for molecular biology, 299.0% (NT), HEPES buffered dihydrate, 299.0% (T), Sodium phosphate dibasic dihydrate, saline, for molecular biology, 2x concentrate, HEPES, 299. for luminescence, 299.0% (T), Sodium phosphate dibasic 5%. (T), HEPES, for molecular biology, 299.5% (T), Imida dihydrate, for molecular biology, 299.0% (T), Sodium phos zole buffer Solution, 1 M in HO, Imidazole, 299.5% (GC), phate dibasic dodecahydrate, 299.0% (T), Sodium phos Imidazole, for luminescence, 299.5% (GC), Imidazole, for phate dibasic solution, 0.5 M in H2O, Sodium phosphate molecular biology, 299.5% (GC), Lipoprotein Refolding dibasic, anhydrous, 299.5% (T), Sodium phosphate dibasic, Buffer, Lithium acetate dihydrate, 299.0% (NT), Lithium for molecular biology, 299.5% (T), Sodium phosphate citrate tribasic tetrahydrate, 299.5% (NT), MES hydrate, monobasic dihydrate, 299.0% (T), Sodium phosphate 299.5% (T), MES monohydrate, for luminescence, 299.5% monobasic dihydrate, for molecular biology, 299.0% (T), (T), MES solution, for molecular biology, 0.5 M in H.O. Sodium phosphate monobasic monohydrate, for molecular MOPS,299.5% (T), MOPS, for luminescence, 299.5% (T), biology, 299.5% (T), Sodium phosphate monobasic solu MOPS, for molecular biology, 299.5% (T), Magnesium tion, 5 M in HO, Sodium pyrophosphate dibasic, 299.0% acetate solution, for molecular biology, ~1 M in H.O. Mag (T), Sodium pyrophosphate tetrabasic decahydrate, 299.5% nesium acetate tetrahydrate, 299.0% (KT). Magnesium cit (T), Sodium tartrate dibasic dihydrate, 299.0% (NT), rate tribasic nonahydrate, 298.0% (calc. based on dry sub Sodium tartrate dibasic solution, 1.5 M in HO (colorless stance, KT), Magnesium formate solution, 0.5 M in H.O. solution at 20°C.), Sodium tetraborate decahydrate, 299.5% Magnesium phosphate dibasic trihydrate, 298.0% (KT). (T), TAPS, 299.5% (T), TES, 299.5% (calc. based on dry Neutralization solution for the in-situ hybridization for in-situ substance. T), TM buffer solution, for molecular biology, pH hybridization, for molecular biology, Oxalic acid dihydrate, 7.4, TNT buffer solution, for molecular biology, pH 8.0, TRIS 299.5% (RT), PIPES, 299.5% (T), PIPES, for molecular Glycine buffer solution, 10x concentrate, TRIS acetate biology, 299.5% (T), Phosphate buffered saline, solution EDTA buffer solution, for molecular biology, TRIS buffered US 2010/0226983 A1 Sep. 9, 2010

saline, 10x concentrate, TRIS glycine SDS buffer solution, are not limited to, alcohols such as ethanol, triglycerides and for electrophoresis, 10x concentrate, TRIS phosphate-EDTA aloe compositions. The amount of the penetration-enhancing buffer solution, for molecular biology, concentrate, 10x con agent may comprise from about 0.5% to about 40% by weight centrate, Tricine, 299.5% (NT), Triethanolamine, 299.5% of the formulation. (GC), Triethylamine, 299.5% (GC), Triethylammonium 0105. In some embodiments, the formulation for delivery acetate buffer, volatile buffer, ~1.0M in HO, Triethylammo via a “patch” comprising a therapeutically effective amount nium phosphate solution, volatile buffer, ~1.0 M in H2O, of the nanoemulsion is envisioned. As used herein a “patch' Trimethylammonium acetate solution, volatile buffer, ~1.0M comprises at least a topical formulation and a covering layer, such that the patch can be placed over the area to be treated. in HO. Trimethylammonium phosphate solution, volatile Preferably, the patch is designed to maximize delivery buffer, ~1 M in H2O, Tris-EDTA buffer solution, for molecu through the stratum corneum and into the epidermis or der lar biology, concentrate, 100x concentrate, Tris-EDTA buffer mis, while minimizing absorption into the circulatory system, solution, for molecular biology, pH 7.4, Tris-EDTA buffer and little to no skin irritation, reducing lag time, promoting solution, for molecular biology, pH 8.0, Trizma(R) acetate, uniform absorption, and reducing mechanical rub-off and 299.0% (NT), Trizma R base, 299.8% (T), Trizma(R) base, dehydration. 299.8% (T), Trizma(R) base, for luminescence, 299.8% (T), 010.6 Adhesives for use with the drug-in-adhesive type Trizma(R) base, for molecular biology, 299.8% (T), Trizma(R) patches are well known in the art. Suitable adhesive include, carbonate, 298.5% (T), Trizma Rhydrochloride buffer solu but are not limited to, polyisobutylenes, silicones, and acryl tion, for molecular biology, pH 7.2. Trizma Rhydrochloride ics. These adhesives can function under a wide range of buffer solution, for molecular biology, pH 7.4, Trizma(R) conditions, such as, high and low humidity, bathing, Sweating hydrochloride buffer solution, for molecular biology, pH 7.6, etc. Preferably the adhesive is a composition based on natural Trizma(R) hydrochloride buffer solution, for molecular biol or synthetic rubber, a polyacrylate Such as, polybutylacrylate, ogy, pH 8.0, Trizma(R) hydrochloride, 299.0% (AT), polymethylacrylate, poly-2-ethylhexyl acrylate; polyviny Trizma(R) hydrochloride, for luminescence, 299.0% (AT), lacetate; polydimethylsiloxane; or and hydrogels (e.g., high Trizma(R) hydrochloride, for molecular biology, 299.0% molecular weight polyvinylpyrrolidone and oligomeric poly (AT), and Trizma(R) maleate, 299.5% (NT). ethylene oxide). The most preferred adhesive is a pressure 0099. The nanoemulsion can comprise one or more emul sensitive acrylic adhesive, for example DurotakR adhesives Sifying agents to aid in the formation of emulsions. Emulsi (e.g., DurotakR 2052, National Starch and Chemicals). The fying agents include compounds that aggregate at the oil/ adhesive may contain a thickener, such as a silica thickener water interface to form a kind of continuous membrane that (e.g., Aerosil, Degussa, Ridgefield Park, N.J.) or a crosslinker prevents direct contact between two adjacent droplets. Cer such as aluminumacetylacetonate. tain embodiments of the present invention feature nanoemul 0.107 Suitable release liners include but are not limited to sions that may readily be diluted with water to a desired occlusive, opaque, or clear polyester films with a thin coating concentration without impairing their anti-fungal orantiyeast of pressure sensitive release liner (e.g., silicone-fluorsilicone, properties. and perfluorcarbon based polymers. 0.108 Backing films may be occlusive or permeable and D. PHARMACEUTICAL COMPOSITIONS are derived from synthetic polymers like polyolefin oils poly 0100. The nanoemulsions of the invention may be formu ester, polyethylene, polyvinylidine chloride, and polyure lated into pharmaceutical compositions that comprise the thane or from natural materials like cotton, wool, etc. Occlu nanoemulsion in a therapeutically effective amount and Suit sive backing films, such as synthetic polyesters, result in able, pharmaceutically-acceptable excipients for topical hydration of the outer layers of the stratum corneum while administration to a human Subject in need thereof. Such non-occlusive backings allow the area to breath (i.e., promote excipients are well known in the art. water vapor transmission from the skin surface). More pref 0101 By the phrase “therapeutically effective amount' it erably the backing film is an occlusive polyolefin foil (Alevo, is meant any amount of the nanoemulsion that is effective in Dreieich, Germany). The polyolefin foil is preferably about preventing and/or treating acne. One possible way to treat 0.6 to about 1 mm thick. acne is by killing or inhibiting the growth of Pacnes, causing 0109 The shape of the patch can be flat or three-dimen P. acnes to lose pathogenicity, or any combination thereof. sional, round, oval, square, and have concave or convex outer 0102 Topical administration includes administration to shapes, or the patch or bandage can also be segmented by the the skin, including surface of the hair follicle and piloseba user into corresponding shapes with or without additional ceous unit. auxiliary means. 0103 Pharmaceutically acceptable dosage forms for topi 0110. The nanoemulsions of the invention can be applied cal administration include, but are not limited to, ointments, and/or delivered utilizing electrophoretic delivery/electro creams, liquids, emulsions, lotions, gels, bioadhesive gels, phoresis. Such transdermal methods, which comprise apply aerosols, pastes, foams, Sunscreens, or in the form of an ing an electrical current, are well known in the art. article or carrier, Such as abandage, insert, Syringe-like appli 0111. The pharmaceutical compositions for topical cator, pessary, powder, talc or other Solid, cleanser (leave on administration may be applied in a single administration or in and wash off product), and agents that favor penetration multiple administrations. The pharmaceutical compositions within the pilosebaceous gland. are topically applied for at least once a week, at least twice a 0104. The pharmaceutical compositions may be formu week, at least once a day, at least twice a day, multiple times lated for immediate release, Sustained release, controlled daily, multiple times weekly, biweekly, at least once a month, release, delayed release, or any combinations thereof, into the or any combination thereof. The pharmaceutical composi epidermis or dermis, with no systemic absorption. In some tions are topically applied for a period of time of about one embodiments, the formulations may comprise a penetration month, about two months, about three months, about four enhancing agent for enhancing penetration of the nanoemul months, about five months, about six months, about seven sion through the stratum corneum and into the epidermis or months, about eight months, about nine months, about ten dermis. Suitable penetration-enhancing agents include, but months, about eleven months, about one year, about 1.5 years, US 2010/0226983 A1 Sep. 9, 2010 about 2 years, about 2.5 years, about 3 years, about 3.5 years, oil is mixed with the aqueous phase under relatively high about 4 years, about 4.5 years, and about 5 years. Between shear forces (e.g., using high hydraulic and mechanical applications, the application area may be washed to remove forces) to obtain a nanoemulsion comprising oil droplets any residual nanoemulsion. having an average diameterofless than about 1000 nm. Some 0112 Preferably, the pharmaceutical compositions are embodiments of the invention employ a nanoemulsion having applied to the skin area in an amount of from about 0.001 an oil phase comprising an alcohol Such as ethanol. The oil mL/cm to about 5.0 mL/cm. An exemplary application and aqueous phases can be blended using any apparatus amount and area is about 0.2 mL/cm, although any amount capable of producing shear forces sufficient to form an emul from 0.001 mL/cm up to about 5.0 mL/cm can be applied. Sion, such as French Presses or high shear mixers (e.g., FDA Following topical administration, the nanoemulsion may be approved high shear mixers are available, for example, from occluded or semi-occluded. Occlusion or semi-occlusion Admix, Inc., Manchester, N.H.). Methods of producing such may be performed by overlaying abandage, polyoleofin film, emulsions are described in U.S. Pat. Nos. 5,103,497 and impermeable barrier, or semi-impermeable barrier to the topi 4,895,452, herein incorporated by reference in their entire cal preparation. Preferably, after application, the treated area ties. 0116. In an exemplary embodiment, the nanoemulsions is covered with a dressing. used in the methods of the invention comprise droplets of an E. EXEMPLARY NANOEMULSIONS oily discontinuous phase dispersed in an aqueous continuous phase, such as water. The nanoemulsions of the invention are 0113. Several exemplary nanoemulsions are described stable, and do not decompose even after long storage periods. below, although the methods of the invention are not limited Certain nanoemulsions of the invention are non-toxic and to the use of such nanoemulsions. The components and quan safe when swallowed, inhaled, or contacted to the skin of a tity of each can be varied as described herein in the prepara Subject. tion of other nanoemulsions. Unless otherwise noted, all con 0117 The compositions of the invention can be produced centrations are expressed in terms of% w/w. in large quantities and are stable for many months at a broad

TABLE 1. Exemplary Therapeutically Effective Nanoemulsions Soybean Tween 20 CPC 96. EDTA HO Form. (CPC 9% w/v) oil 96 % Ethanol % (mg/mL) % (mM) % Formulation #1: 0.50% 31.4 2.96 3.37 0.53 (5) 0.037 (1) 61.70 Formulation #2; 0.25% 15.7 1.48 1.68 0.27 (2.5) 0.0185 (0.5) 80.85 Formulation #3: 1.0% 62.79 S.92 6.73 1.068 (10) 0.075 (2) 23:42 Formulation #4; 0.3% 18.84 1.78 2.02 0.320 (3) 0.0224 (0.6) 77.03 Formulation #5; 0.1% 6.28 O.S9 O.67 0.107 (1) 0.0075 (0.2) 92.34

0114 Several additional exemplary nanoemulsions are range of temperatures. The nanoemulsion can have textures/ described below. For therapeutic topical use on a subject, the consistencies ranging from that of a semi-solid cream to that concentrations of each component would be increased, as of a thin lotion and can be applied topically by hand and described above. sprayed onto a surface. As stated above, at least a portion of

TABLE 2 Exemplary Nanoemulsions Form. Soybean Tween 20 CPC 96 EDTA H2O (CPCw/v 96) oil 96 % Ethanol % (ig/mL) 96 (uM) % Formulation O.OSO 0.00474 0.00538 0.00085 (8) 5.96 x 10 (1.6) 99.94 #6; 0.0008% Formulation O.O2S 0.00237 0.00269 0.00043 (4) 2.98 x 100.8) 99.97 #7; 0.0004% Formulation O.O13 0.00118 0.00135 0.00021 (2) 1.49 x 10 (0.4) 99.98 #8; 0.0002%

F. METHODS OF MANUFACTURE the emulsion may be in the form of lipid structures including, but not limited to, unilamellar, multilamellar, and paucliamel 0115 The nanoemulsions of the invention can be formed lar lipid vesicles, micelles, and lamellar phases. using classic emulsion forming techniques. See e.g., U.S. 0118. The present invention contemplates that many 2004/0043041. See also the method of manufacturing variations of the described nanoemulsions will be useful in nanoemulsions described in U.S. Pat. Nos. 6,559,189, 6,506, the methods of the present invention. To determine if a can 803, 6,635,676, 6,015,832, and U.S. Patent Publication Nos. didate nanoemulsion is suitable for use with the present 2004.0043041, 20050208083, 2006025 1684, and invention, three criteria are analyzed. Using the methods and 2007003.6831, and WO 05/030172, all of which are specifi standards described herein, candidate emulsions can be easily cally incorporated by reference. In an exemplary method, the tested to determine if they are suitable. First, the desired US 2010/0226983 A1 Sep. 9, 2010 ingredients are prepared using the methods described herein, manufacturing method. The two phases (aqueous phase and to determine if a nanoemulsion can beformed. If a nanoemul oil phase) are combined together and processed to yield an sion cannot be formed, the candidate is rejected. Second, the emulsion. The emulsion is further processed to achieve the candidate nanoemulsion should form a stable emulsion. A desired particle size. For the gel formulation, a thickening nanoemulsion is stable if it remains in an emulsion form for a agent, such as Klucel can be added to the nanoemulsion. For sufficient period to allow its intended use. For example, for example, Klucel is dissolved in water or any aqueous solvent nanoemulsions that are to be stored, shipped, etc., it may be and added to the nanoemulsion to achieve the desired con desired that the nanoemulsion remain in emulsion form for centration. months to years. Typical nanoemulsions that are relatively unstable, will lose their form within a day. Third, the candi Example 2 date nanoemulsion should have efficacy for its intended use. For example, the emulsions of the invention should kill or The Nanoemulsions have Potent Activity Against P disable Propionibacterium species in vitro or reduce inflam CaS mation and/or non-inflammatory lesions in humans. To deter 0.124 Nanoemulsions according to the invention were mine the potency of a particular candidate nanoemulsion tested in in vitro to determine the minimum inhibitory con against P. acnes, MICs are determined under Standardized centration (MIC) and minimum bactericidal concentration conditions (National Committee for Clinical Laboratory (MBC) against 16 clinical isolates of P. acnes, some of which Standards, Methods for Antimicrobial Susceptibility Testing have defined ribosomally-based resistance mechanisms to of Anaerobic Bacteria, 7" ed.:” Approved Standard M11-A7. erythromycin, clindamycin and/or tetracycline. The National Committee for Clinical Laboratory Standards, nanoemulsions (“NB-00X) comprised, in an aqueous Wayne, Pa. (2007)). medium, Soybean oil, Tween 20R) as a nonionic Surfactant, 0119) Alternatively, P. acnes can be exposed to the ethanol, cetylpyridinium chloride (CPC) as a cationic surfac nanoemulsion for one or more time periods in a side-by-side tant, EDTA, and water, and optionally, a thickening agent for experiment with an appropriate control sample (e.g., a nega the gel formulation.

TABLE 3 Compositions of the Nanoemulsions (NB-00X) and Nanoemulsion Gels (NB-Gel). The percentages are wit/wt, unless otherwise noted. Lot Soybean Tween 20 Ethanol CPC EDTA Klucel Water Formulation i oil 96 % % % (w/v) % % % 89-16-09A 6.279 O.S92 0.679 0.107 O.OO74 O 92.34 O.3% NB-OOX X-1160 18.837 1.776 2.037 0.32O O.O22 O 77.01 0.1% NB-Gel 89-16-09C 6.279 O.S92 20.679 0.107 O.OO74 196 92.34 0.3% NB-Gel 89-7025 18.837 1.776 22.037 0.32O O.O22 196 77.01 tive control Such as water) and determining if, and to what 0.125. The nanoemulsions were tested at 10 different con degree, the nanoemulsion kills or disables P. acnes. centrations, as two-fold serial dilutions from 0.0064% 0120. The nanoemulsion of the invention can be provided NB-00X (equivalent to 64 pug CPC/ml) to 0.0000125% in many different types of containers and delivery systems. NB-00X (equivalent to 0.125 ug CPC/ml). Each dilution con For example, in some embodiments of the invention, the tained varying concentrations of soybean oil. Tween 20R, nanoemulsions are provided in a cream or other solid or ethanol, CPC, and EDTA. Combination products were also semi-solid form. The nanoemulsions of the invention may be evaluated; stock emulsions containing NB-00X gel (3 mg incorporated into hydrogel formulations. CPC/ml)+2% salicyclic acid or NB-00X gel--0.5% benzoyl 0121 The nanoemulsions can be delivered (e.g., to a sub peroxide (BPO) were serially diluted two-fold and each con ject or customers) in any suitable container. Suitable contain centration was tested against 16 P. acnes isolates. In general, ers can be used that provide one or more single use or multi the standard methodology was followed for MIC and MBC use dosages of the nanoemulsion for the desired application. determination. In some embodiments of the invention, the nanoemulsions are 0.126 The MIC (minimum inhibitor concentration) and provided in a suspension or liquid form. Such nanoemulsions MBC (minimum bactericidal concentration) values for the can be delivered in any suitable container including spray nanoemulsions were compared to the MIC and MBC values bottles (e.g., pressurized spray bottles). of anti-acne drugs currently in use: erythromycin, clindamy G. EXAMPLES cin, tetracycline, benzoyl peroxide and Salicylic acid. 0122) The invention is further described by reference to I0127. A. Source of Drugs and P. acnes Isolates the following examples, which are provided for illustration I0128 NB-00X (liquid formulation), lot X1151 and only. The invention is not limited to the examples, but rather NB-00X gel, lot X1158, were prepared at concentrations of includes all variations that are evident from the teachings 6000 g/ml and 3000 ug/ml respectively. These lots were provided herein. All publicly available documents referenced prepared at NanoBio Corporation from NB-PO-004-FP herein, including but not limited to U.S. patents, are specifi manufactured at Contract Pharmaceutical Laboratories cally incorporated by reference. (CPL), Buffalo, N.Y., USA. Placebo lots X1161 and X1162 Example 1 (placebo for NB-00X gel, contains thickening agent and addi tional solvent) were prepared from lotA0494 manufactured at Preparation of Nanoemulsions NanoBio Corporation. Since nanoemulsions are not a single 0123. These emulsions are produced by mixing a water small molecule, their relative activity can be expressed in immiscible oil phase into an aqueous phase with a proprietary terms of the concentration of cationic Surfactant present. US 2010/0226983 A1 Sep. 9, 2010

Thus, the antibacterial activity of NB-00X formulations is system (Misubishi, No. 10-01) and a dry anaerobic indicator expressed in microgram CPC per ml. NB-00X gel (lot strip (BBL, Becton, Dickinson & Co. #271051). MICs were X1158) contained a thickening agent in addition to the com read visually using a 96-well plate reader fitted with a mag ponents of NB-00X. Combination products were made as stock emulsions containing NB-00X gel (3 mg CPC/ml)+2% nifying mirror (Biodesign of New York). Because of the opac salicyclic acid or NB-00X gel--0.5% benzoyl peroxide ity of benzoyl peroxide, 20 ul of Cell Titer Blue (alamar blue (BPO). from Promega G8080) was added after 48 hrs; the plates were 0129. Comparator compounds, erythromycin, clindamy incubated for an additional hour prior to reading. Colony cin, tetracycline and chlorhexidine were purchased from forming units were counted after 72 h of incubation to ensure Sigma Chemicals, USP Fluka and Aldrich as catalog num that the initial inocula were between 2-5x10 cfu/ml. bers E0774, 1136002, 87128, and 282227 respectively. Sali cylic acid was purchased from J. T. Baker as VWR Interna 0.138. The minimal bactericidal concentrations (MBC) for tional catalog number 0300-01. BPO in the form of Invisible P. acnes were determined by plating 10 ul from the well Acne cream containing 10% BPO was purchased from Meijer determined to be the MIC plus 4 wells above the MIC on Distribution Inc. (Grand Rapids, Mich.). blood-supplemented Mueller-Hinton agar plate. Inoculated 0130. The source of bacterial strains was mainly Basilea petri plates were incubated for 72 hat 35°C. under anaerobic Pharmaceutica, AG, Basel, Switzerland (Heller, S. L. Kellen conditions. The MBC was calculated as the concentration of berger and S. Shapiro, 2007, Antipropionibacterial activity of drug that gave 23-log reduction from the initial inoculum BAL19403, A Novel Macrollide Antibiotic, J. Antimicrob. concentration. Chemother. 51: 1956-1961). The majority of these isolates had defined resistance mechanisms to erythromycin, clinda I0139 MICs for NB-00X or NB-00X gel (formulation mycin and/or tetracycline. The resistance mechanisms were modified to include a thickening agent and additional solvent) mutations in either the 16S or 23S rRNA of the small or large ranged from 0.25-1.0 ug/ml and MBCs ranged from 0.5-4 ribosomal subunit conferring tetracycline O ug/ml (Table 4). The MIC and MBC values were 0.5 erythromycin-tclindamycin resistance, respectively, or resis ug/ml and 2.0 g/ml for NB-00X and 1 lug/ml and 2 ug/ml for tance was conferred by an erm(X) methylase that dimethy NB-00X gel, respectively. Greater than 50% of the isolates lates residue A2058 in 23S rRNA, conferring high level eryth were resistant to erythromycin and clindamycin; 44% of the romycin and clindamycin resistance. Three isolates were isolates were resistant to tetracycline. However, multidrug obtained from the American Type Culture Collection resistant isolates were equally susceptible to either formula (ATCC), Manassas, Va., USA. 0131 B. Preparation of Drug Concentrations tion of NB-00X. Neither placebo had any microbiological 0132 Weighing of drugs and potency calculations were activity. NB-00X was bactericidal against all the isolates, done as prescribed by Clinical and Laboratory Standards including strains that were erythromycin-, clindamycin- and/ Institute (1). Dimethyl sulfoxide (DMSO) was used to pre or tetracycline-resistant. The MICs and MBCs of chlorhexi pare stock Solutions of the water-insoluble compound tetra dine for all the strains were at or below the lowest tested level cycline at 100x concentrations. Stock solutions of erythro of 10 ug/ml (equivalent to 0.001% chlorhexidine). mycin and clindamycin were prepared in sterile deionized 0140 Since NB-00X is a nanoemulsion and is preferen water (DI water) at a 100x of the highest test concentration. tially taken up by the transfollicular route (Ciotti et al., Stock solutions of chlorhexidine and NB-00X were prepared “Novel nanoemulsion NB-001 permeates skin by the follicu at a 4x concentration in DI water. lar route.” Abstr. 45" Intersci. Conf. Antimicrob. Agents 0.133 C. Preparation of 96 Well Drug Plates Chemother., abstr. A-1898 (2008)), incorporation of another 0134) To prepare intermediate concentrations, 100x stock anti-acne drug into the nanodroplets could be used to effec solutions were serially diluted 1:1 using DMSO or DI water. tively deliver these additional agents to the site of infection. Final concentrations were made by 1:50 or 1:1 dilutions in Thus, we looked at the microbiological activity of NB-00X Wilkin Chalgren media (1) to give 2x of the test concentra tions, with final DMSO concentrations at 1%. 50 ul of these gel formulated with either benzoyl peroxide or salicyclic acid drug concentrations were transferred to 96-well plates using and compared the MICs and MBCs of the combination prod multi-channel pipettes. ucts to benzoyl peroxide or salicyclic acid alone. Since nei 0135. D. Determination of MIC and MBC ther BPO or salicyclic acid were highly potent (MICso values 0.136 Pacnes strains grown on sheep blood agar for 24-48 of 50 and 1000 g/ml, respectively), the antimicrobial activity hrs at 35°C. were used as the sources of inocula for suscep seen with the combination products NB-00X--BPO or tibility studies as per Clinical and Laboratory Standards Insti NB-00X--salicyclic acid reflected the intrinsic activity of tute. A bacterial suspension with turbidity equivalent to a 0.5 NB-00X, with MIC values of 0.5 lug/ml for either combi McFarland standard was diluted to 1:75 in saline or Wilkins nation and MBCoo values of 4 and 2 ug/ml, respectively. Chalgren broth (GLP Corporation) to give >10 cfu/ml in each well after inoculation. Within 15 minutes, each well 0141 NB-00X has relevant microbiological and bacteri (except the negative growth controls) of the microtiter tray cidal activity against a collection of recent clinical isolates of containing the serial dilutions of test compounds received 50 P. acnes, including multidrug-resistant Strains. Comparator ul of inoculum, resulting into a log dilution of both drug and drugs that have been used to treat acne-erythromycin, clin bug in each well. Verification of the colony-forming units in damycin, tetracycline, benzoyl peroxide and salicyclic the inoculum was performed by diluting the adjusted inocu acid—were much less effective. Combinations of the lum preparation to 10' and plating 100 ulon blood agarplate. nanoemulsion NB-00X with BPO or salicyclic acid were as 0.137 Microtiter and blood agar plates were incubated at effective as NB-00Xalone. However, given the transfollicular 35-37° C. for 48 h in a 7.0 L AnaeroPack Jar (Mitsubishi gas route of NB-00X (2), additional acne agents could be deliv chemical; No. 50-70) fitted with an anaerobic gas generating ered more effectively to the site of infection. US 2010/0226983 A1 Sep. 9, 2010

TABLE 4

MICoo/MBCoo Values Against 16 P. acnes Isolates MICs/MBCso MICoo/MBCoo Range Value Value

Compound MIC MBC MICso MBCso MICoo MBCoo

NB-OOX O.25-1 O5-2 O.S 2 O.S 2 NB-00X gel O.5-1 1-4 O.S 2 1 2 Erythromycin s:0.25->128 >4->128 2 >64 >128 >128 Clindamycin sO.125->64 >2->64 2 >64 >64 >64 Tetracycline sO.O63->32 >1->32 1 >16 >32 >32 Chlorhexidine s10 s10 s10 s10 s10 s10 Benzoyl peroxide s50-100 100-400 50 2OO 50 200 Salicylic acid SOO-1OOO 2OOO->2OOO 1000 2OOO 1000 2OOO NB-00X placebo >64 >64 >64 >64 >64 >64 NB-00X gel >64 >64 >64 >64 >64 >64 placebo NB-OOX-BPO O.5-1 2-4 O.S 4 O.S 4 NB-00X + SA O.25-1 O5-2 O.S 2 O.S 2

NB-00X gel (3 mg CPC/ml) + 0.5% BPO NB-00X gel (3 mg CPC/ml) + 2% salicyclic acid

Example 3 The Nanoemulsions have Potent Activity Against P TABLE 6 acnes in the Presence of Sebum Composition of artificial sebum. 0142 Nanoemulsions according to the invention were tested in in vitro antibacterial assays in the presence of 50% Amt artilificial C1al sebSebum to determinet ine the eminimum mini inhibi 1tory concen- Ingredient Manufacturer iLot wiw% (g)200 for g tration (MIC) and minimum bactericidal concentration (MBC) against 16 clinical isolates of Pacnes. The nanoemul- Oleic acid Aldrich 10529CH 14 2.80 sions (“NB-002) comprised, in an aqueous medium, Soy- Palmitoleic acid MP Biomedical 8855J 5 10.00 Squalene MP Biomedical 75O1F 15 30.00 bean oil, Tween 20R aS a O1O1C surfactant, ethanol, Olive oil Spectrum XAO813 10 SO.OO cetylpyridinium chloride (CPC) as a cationic surfactant, (C16-18) EDTA, and water.

TABLE 5 Composition of NB-00X formulations

Soybean Tween 20 CPC 96 EDTA Klucel Water Formulation Lotti oil 96 % Ethanol % (w/v) 90 % %

O.1% NB-OOX 89-16-09A 6.279 O.S92 0.679 0.107 O.OO74 O 92.34 O.3% NB-OOX X-1160 18.837 1776 2.037 0.32O O.O22 O 77.01 0.1% NB-Gel 89-16-09C 6.279 O.S92 20.679 0.107 O.OO74 19 92.34 O.3% NB-Ge. 89-7025 18.837 1776 22.037 0.32O O.O22 196 77.01

0143. The source of drugs and isolates were the same as in Example 2. 100X drug stocks were prepared as in Example 2. TABLE 6-continued 0144 A. Preparation of Artificial Sebum M 0145 Artificial- Y - sebum was prepared by adding the entire Composition of artificial sebum. ingredients given in the Table 6 and heating at 60° C. in a L f (SR water bath, with intermittent stirring until all solids melted Ingredient Manufacturer i Ot WFW% 200g) Tor g resulting in to a clear yellow liquid (Valiveti et al., “Diffusion Cottonseed oil Spectrum XC1142 25 SO.OO properties of model compounds in artificial sebum. Int. J. (C16-18) Pharm., 345:88-94 (2007)). US 2010/0226983 A1 Sep. 9, 2010 16

TABLE 6-continued TABLE 7

Composition of artificial sebum. Susceptiblity of 16 Pacnes isolates in the presence of 50% artificial sebum Amt Lot wiw (g) for MICValues Ingredient Manufacturer i % 200 g (ig/ml) Cholesterol JT Baker E33H12 1.2 2.40 Cholesterol oleate Gantaur (Cath21130) 12373 2.4 4.8O Active Substance MICoo MBCoo Palmitic acid Calbiochem DOOO13930 5 10.00 Spermaceti wax Aqua Solutions 3O46O1 15 30.00 Erythromycin >128 >128 Paraffin wax Sigma O6007DE 10 2O.OO Clindamycin >64 >64 (mp 58-62 C.) Tetracycline 32 >32 Coconut oil Aldrich AO162449 10 2O.OO Chlorhexidine 78 12SO (C12-16) NB-OOX 128 1024 NB-00X gel 128 1024 0146 B. Preparation of Drug Plates NB-00X/Benzoyl peroxide gel 128 2S6 0147 To prepare intermediate concentrations, 100x stock NB-00X/Salicylic acid gel 128 2S6 solutions were serially diluted 1:1 using DMSO or DI water. Benzoyl peroxide 400 1600 Final concentrations were made by 1:50 dilutions in Wilkin Salicylic acid 1OOO 2OOO Chalgren media to give 2x of the final test concentrations, with final DMSO concentrations at 1%. 50 ul of these drug concentrations were transferred to 96-well plates using multi Example 4 channel pipettes. 0148 Sebum was prewarmed to 50° C. and each well Skin Permeation Studies received 45 ul of sebum. After ten minutes at 35° C., five microliters of a Pacnes culture at 10 colony-forming units/ ml was added to each well. Plates were incubated and MICs 0151. The purpose of this example was to evaluate the in and MBCs determined as described in Example 2. vitro absorption into the epidermis and dermis of nanoemul 0149 C. Results sions according to the invention. Pig skin was used as an 0150 NB-00X was compared to NB-00X geland the com animal model. binations of 0.3% NB-00X gel (3 mg CPC/ml)+0.5% benzoyl 0152 A. In Vitro Skin Model peroxide (BPO) or NB-00X gel-2% salicylic acid (SA). The MIC and MBC values for NB-00X formulations and com O153 The in vitro skin model has proven to be a valuable parators against sixteen isolates of P. acnes in the presence of tool for the study of percutaneous absorption of topically 50% sebum are shown in Table 7. NB-00X was bactericidal applied compounds. The model uses excised skin mounted in for all strains of P. acnes with MICs/MBC values of 0.5/2 specially designed diffusion chambers that allow the skin to ug/ml in the absence of sebum (Table 4). The MIC/MBC be maintained at a temperature and humidity that match typi values in the presence of 50% sebum increased to 128/1024 cal in vivo conditions. (Franz, T.J., “Percutaneous absorption: ug/ml (Table 7). A reduction in the MBC for NB-00X on the relevance of invitro data.J. Invest. Dermatol., 64:190 occurred when BPO or SA was integrated into the formula 195 (1975).) A finite dose of formulation is applied to the tion, resulting in a MICs/MBC of 128/256 ug/ml in the epidermis, and outer Surface of the skin and compound presence of 50% sebum. The MIC/MBC values of SA absorption is measured by monitoring its rate of appearance (1000/2000 ug/ml) were not significantly impacted by the in the receptor solution bathing the dermal surface of the skin. presence of sebum, but the MICs/MBC values of BPO Data defining total absorption, rate of absorption, as well as increased eight-fold in the presence of sebum (400/1600 skin content can be accurately determined in this model. The ug/ml) (Tables 4 and 7). The addition of sebum also did not method has historic precedent for accurately predicting in impact the microbiological activities of erythromycin, clin vivo percutaneous absorption kinetics. (Franz TJ, “The finite damycin and tetracycline, at least up to the concentrations dose technique as a valid in vitro model for the study of tested (Tables 4 and 7). The MIC of chlorhexidine in the percutaneous absorption in man. In: Skin. Drug Application presence of sebum increased at least eight-fold in the pres and Evaluation of Environmental Hazards, Current Problems ence of sebum and the MBCs increased at least 125-fold in Dermatology, vol. 7, Simon et al. (Eds) (Basel, Switzer (Tables 4 and 7). land, S. Karger, 1978, pp 58-68.) US 2010/0226983 A1 Sep. 9, 2010 17

0154 B. Nanoemulsions Used in the Study

TABLE 8 Composition of the Formulations Soybean Tween 20 CPC 96. EDTA Klucel Water Formulation Lotti oil 96 % Ethanol % (w/v) 90 % % O.1% NB-OO1 89-16-09A 6.279 O.S92 0.679 O.107 O.OO74 O 92.34 O.3% NB-001 X-1160 18.837 1776 2.037 O.32O O.O22 O 77.01 0.1% NB-Gel 89-16-09C 6.279 O.S92 20.679 O.107 O.OO74 19 92.34 O.3% NB-Ge. 89-7025 18.837 1776 22.037 O.32O O.O22 196 77.01

O155 C. PigSkin 0166. At the twelve hour time point, the gel formulation 0156 Full thickness, abdominal skin (~1000 um thick delivered two-fold higher levels of CPC into the epidermis, ness) from 5.4 month old male Hanford Swine (S/N 5353) was indicating a fast rate of delivery. The dermal levels were used in permeation studies and obtained from Sinclair similar (See FIG. 3). Research Center, Inc., Auxvasse, Mo. The subcutaneous fat 0167. In summary, the present invention provides a was removed using a scalpel and the skin was stored in alu nanoemulsion for treating acne. Since the mechanism of the minum foil pouches at -70° C. until use. At time of use, the nanoemulsion is physical via membrane destabilization, it is skin was thawed by placing the sealed pouch in 30°C. water unlikely to induce resistance to the nanoemulsion. for approximately five minutes. Thawed skin was removed (0168 Greater than 50% of the Pacnes isolates were resis from the pouch and cut into circular discs (30 mm diameter) tant to erythromycin and clindamycin. 44% of the isolates to fit between the donor and receiversides of the permeation were resistant to tetracycline. However, single or multi-drug chambers. resistant isolates were equally susceptible to either NB-00X (O157 D. Franz Diffusion Cell Methodology: Conditions, or NB-00X gel. Neither NB-00X placebo had any microbio Parameters, Procedure logical activity. NB-00X was bactericidal against all the iso 0158 Percutaneous absorption was measured using the in lates, including isolates that were erythromycin-clindamy vitro cadaver skin finite dose technique. Thirty mm of swine cin- and/or tetracycline-resistant. In the absence of artificial skin was placed onto the surface of each cell. Each receptor sebum under anaerobic conditions, NB-00X has MIC/ compartment was filled with distilled water, pH 7 and the MBC values of 0.5/2 lug/ml. Benzoyl peroxide and salicy donor compartment was left open to ambient laboratory con clic acid had MICs/MBC values of 50/200 lug/ml and ditions. The receptor compartment spout was covered with a 1000/2000 ug/ml, respectively. screw cap to minimize evaporation of the receptor Solution. 0169 NB-00X has relevant anti-microbiological and bac All cells were mounted in a diffusion apparatus in which the tericidal activity against a collection of recent clinical isolates receptor solution was maintained at 37°C. The receptor com of P acnes, including multidrug-resistant strains. Compara partment was maintained at 34.5°C. in a water bath and was tor drugs that have been used to treat acne. Such as erythro stirred with a magnetic stirrer. mycin, clindamycin, tetracycline, benzoyl peroxide and Sali 0159. The skin was equilibrated before applying 113 uL of cyclic acid were much less effective comparing to the each test article onto the skin Surface. nanoemulsion of the invention. 0160 E. Sampling (Receptor Sampling, Epidermis, Der (0170 The MICs/MBC values in the presence of 50% mis, Surface Swabs) sebum increased to 128/1024 ug/ml. A reduction in the 0161 Twenty-four hours after application of the first dose, MBC for NB-00X occurred when BPO or SA was inte the Surface of the dosing area was rinsed with ethanol Solution grated into the formulation, resulting in a MICoo/MBCoo of and Swabbed independently to remove all residual formula 128/256 ug/ml. This result Suggests a synergy between tion from the skin Surface. Receptor Solution was also NB-00X and benzoyl peroxide or salicylic acid. sampled at 24 hours from the receptor of each cell and filtered into vials. Example 5 0162 Skin samples were collected as described above: weights of the epidermal and dermal samples were obtained. Effect of EDTA on the Efficacy of NB003 and Other The epidermal and dermal tissues were extracted with abso Emulsions Against P. acnes in the Presence and lute ethanol, Sonicated, and filtered and assayed using HPLC. Absence of Artificial Sebum 0163 F. Epidermal and Dermal Calculations 0164. The amount of CPC that permeated into the epider 0171 Background and Purpose of Study: As noted above, mis, dermis and the receptor compartment was determined by Propionibacterium acnes, a gram-positive, non-spore form HPLC. A standard concentration of CPC was generated and ing, anaerobic bacillus, is one of the primary factors involved used to determine the concentration of CPC in the dosing in the pathogenesis of acne Vulgaris. It is the predominant area. The levels of CPC in each skin area are represented as microorganism of the pilosebaceous glands of human skin, the amount per wet tissue weight (ug/grams)--the standard with up to 10 million viable organisms isolated from a single deviation. sebaceous unit. Although aerotolerant, P. acnes typically 0.165. The results of CPC permeation studies are shown in grows in the anaerobic environment of the infrainfundibulum, FIGS. 2 and 3. There was an increase in the delivery of the where it releases lipases and digests local accumulations of CPC marker to the epidermis and dermis with the 0.3% the skin, oil and sebum. Sebaceous glands produce an oily NB-00X as compared to the 0.1% NB-001X formulation, as sebum that is primarily composed of waxes, triglycerides, and expected. The gels for the 0.1% NB-00X and 0.3% NB-00X free fatty acids. Previous studies have shown that NB-00X did not hinder delivery. The amount of CPC found in the nanodroplets are concentrated in the pilosebaceous unit receptor compartment at 24 hours was below the level of where P acnes migrates to enjoy a rich source of food (se detection (5ug/ml) for all the formulations. bum) and a preferred anaerobic environment. Purpose of this US 2010/0226983 A1 Sep. 9, 2010 study was to evaluate the efficacy to nanoemulsion against propionibacterium acnes in the presence of artificial sebum. TABLE 10-continued 0172. The efficacy of the nanoemulsions with varying concentrations of added ethylenediaminetetraacetic acid Composition of artificial sebum (EDTA) was evaluated using broth microdilution standard method prescribed by Clinical and Laboratory Standard Insti Wendorf Amt (g) for tute (CLSI). (National Committee for Clinical Laboratory Ingredient Manufacturer Catalogue # wiw % 200 g Standards, “Methods for Antimicrobial Susceptibility Test Cottonseed oil Spectrum CO145 25 SO.O ing of Anaerobic Bacteria.” 7" ed., Approved Standard M11 Cholesterol JT Baker 676-OS 1.2 2.40 A7 (National Committee for Clinical Laboratory Standards, Cholesterol oleate Aldrich 372935 2.4 4.8O Palmitic acid VWR 8O108-2S2 S.O 1O.O Wayne, Pa., 2007.) Minimum inhibitory concentration (MIC) Spermaceti wax VWR (Aqua 101226-030 15 3O.O and minimum bactericidal concentration (MBCs) of different Solutions) emulsions was determined in the presence and absence of Paraffin wax (mp Aldrich 327212 10 2O.O 25% artificial sebum. 58-62 C.) (0173 Materials and Methods Coconut oil Aldrich C1758 10 2O.O 0.174 Source of drugs and isolates: Emulsions tested in this study were NB-003, 10% W5 GBA2ED, and 50% S8GC. Each of these compositions is described in the table (0176 Preparation of 96-well drug plates with different below (the composition of the neat, undiluted NB-003 for concentration of EDTA. Stock solutions of drugs were pre mulation is given in the table below). pared at 4x of first test concentration in sterile deionized water (DI water). Intermediate dilutions were prepared by 1:1 TABLE 9 serial dilutions from Stock. Final concentrations were made by 1:1 dilutions of intermediate concentrations in 2x Wilkin Nanoemulsions Tested Chalgren media to give 2x of the final test concentrations. 50 Nanoemulsion Components Weight% ul of final dilutions were placed in 96 well plates. Different concentrations of EDTA were added to 96 well plates. To 10% W5GBA2ED EDTA, USP O.OO7 achieve 5 mM-20 mM of EDTA/well, 5ul to 20 ul of 100 mM (wfw %) BTC 824 0.4 EDTA stock solution was added to each well. For 1 mM to 5 Sterile Distilled 91.71 Water mM concentration of EDTA? well, stock solution of 100 mM Tween 20 O.S92 was diluted 20 mM and 5 ul-25ul of diluted stock was added Glycerol 1.008 into each well. Prior to inoculation, 25% of sebum was added Soybean Oil 6.279 50% S8GC CPC 0.535.9% to appropriate plates. To obtain 25% of sebum concentration, Distilled Water 60 25ul of artificial sebum kept at 60°C. was pipetted into each Glycerol 4% well. SDS 4% 0177 Determination of MICs and MBCs. P. acnes strains Soybean Oil 31.5 grown on sheep blood agar for 24-48 hrs at 35°C. were used NB-003 (neat) Distilled Water 23.42 CPC 1.07 as the sources of inocula for Susceptibility studies as per EDTA O.O7 CLSI. A bacterial suspension with a turbidity equivalent to a Tween 20 S.92 0.5 McFarland standard was diluted to 1:10 to 1:50 in sterile Ethanol 6.73 saline. 5 ul to 50 uL of the adjusted inocula was added into Soybean oil 62.79 each well to give ~10 cfu/ml after inoculation. Verification of the colony-forming units in the inoculum was performed by diluting the adjusted inoculum preparation to 10' and plating Seven of the clinical isolates of P. acnes (PAC-004 to 100 ul on blood agar plate. PAC010) used in this study were obtained from Basilea Phar 0.178 Microtiter and blood agar plates were incubated at maceutica, AG, Basel, Switzerland. The majority of these 35-37° C. for 48 h in a 7.0 L AnaeroPack Jar (Mitsubishi gas isolates had defined resistance mechanisms to erythromycin, chemical; No. 50-70) fitted with an anaerobic gas generating clindamycin, and/or tetracycline. Isolate numbers PAC-001 system (Misubishi, No. 10-01) and a dry anaerobic indicator to PAC-003 were obtained from American Type Culture col strip (BBL. Becton, Dickinson & Co.). MICs were read visu lection (ATCC) (Manassas, Va.). ally using a 96-well plate reader fitted with a magnifying 0175 Preparation of artificial sebum. Artificial sebum was mirror (Biodesign of New York). Colony-forming units were prepared by adding the entire ingredients given in the Table counted after 72 h of incubation to ensure that the initial 10 and heating at 60° C. in a water bath, with intermittent inocula were between 2-5x10" cfu/ml. stirring until all solids melted to a clear yellow liquid. (Lu et (0179 The minimal bactericidal concentrations (MBC) for al., “Comparison of artificial sebum with human and hamster P. acnes were determined by plating 10 ul from the well sebum samples.” Int. J. Pharm., Epub date, Oct. 22, 2008.) representing the MIC plus 4 wells above the MIC on blood agar plates. TABLE 10 0180 Inoculated petri plates were incubated for 72 hat 35° C. under anaerobic conditions. The MBC was calculated Composition of artificial sebum as the concentration of drug that gave 23-log reduction from Wendorf Amt (g) for the initial inoculum concentration. Ingredient Manufacturer Catalogue # wfw % 200 g 0181 Results Oleic acid Aldrich 364S25 1.4 2.80 0182. As shown in Table 11, the MIC of NB-003 without Palmitoleic acid VWR (Acros) 200020-298 S.O 1O.O any EDTA ranged from 0.25 to 0.5ug/mL. In the presence of Squalene VWR (MP 102948 15 3O.O 25% sebum, the MIC range increased to 16-32 ug/mL. With Biomedical) addition of 1 mM to 20mMofEDTA, the MIC in the presence Olive oil Spectrum OL130 10 SO.O and absence of sebum decreased to 2tested concentration of 0.063 and 1 ug/ml, respectively. US 2010/0226983 A1 Sep. 9, 2010 19

TABLE 11 MIC of NB-003 emulsions in the presence and absence of artificial sebum

O.S9/o O.S90 O.S90 O.S9, NBOO3 - NBOO3- NBOO3- NBOO3 - 20 mM 10 mM 5 mM 1 mM

No 25% No 25% No 25% No 25% No 25% PAC# sebum sebum sebum sebum sebum sebum sebum sebum sebum sebum

PAC- sO.063 ND sO.063 ND s0.063 s s0.063 s O.S 32 OO1 PAC- sO.063 ND sO.063 ND s0.063 s s0.063 s O.25 16 OO2 PAC- sO.063 ND sO.063 ND s0.063 s s0.063 s O.25 32 OO3 PAC- sO.063 ND sO.063 ND s0.063 s s0.063 s O.S 32 OO)4 PAC- sO.063 ND sO.063 ND s0.063 s s0.063 s O.25 16 005 PAC- sO.063 ND sO.063 ND s0.063 s s0.063 s O.S 16 OO6 PAC- sO.063 ND sO.063 ND s0.063 s s0.063 s O.S 16 OO7 PAC- sO.063 ND sO.063 ND s0.063 s s0.063 s O.S 16 O08 PAC- sO.063 ND sO.063 ND s0.063 s s0.063 s O.S 16 O09 PAC- sO.063 ND sO.063 ND s0.063 s s0.063 s O.25 16 O10 MIC sO.063 ND sO.063 ND s0.063 s s0.063 s O.25-O.S 16-32 range MIC 50 sO.063 ND sO.063 ND s0.063 s s0.063 s O.S 16 MIC 90 s.0.063 ND sO.063 ND s0.063 s s0.063 s O.S 32

0183 MBCs data of NB-003 with varying concentration of EDTA is presented in Table 12. A review of this table shows that addition of 25% sebum increased the MBCS to 128->256 fold. The addition of EDTA decreases the MBCs in the pres ence of sebum. At a concentration of 10 and 20 mM of EDTA, the MBCs for all isolates were reduced to stested concentra tion.

TABLE 12 MBCs of NB003 emulsions in the presence and absence of artificial sebum

O.S9/o O.S90 O.S90 NBOO3 - NBOO3- O.S90 NBOO3 - 20 mM 10 mM NBOO3 - 5 mM 1 mM EDTA? well EDTA? well EDTA? well EDTA? well O.S90 NBOO3

No 25% No 25% No 25% No 25% No 25% PAC# sebum sebum sebum sebum sebum sebum sebum sebum sebum sebum

PAC sO.063 s1 sO.063 s1 sO.063 <1 2 >16 2 256 OO1 PAC sO.063 s1 sO.063 s1 O.12S 1 2 16 2 512 OO2 PAC sO.063 s1 sO.063 s1 O.S 4 1 32 2 512 OO3 PAC sO.063 s1 sO.063 s1 O.25 <1 >1 >16 2 512 OO)4 PAC sO.063 s1 sO.063 s1 sO.063 4 >1 16 2 256 005 PAC sO.063 s1 sO.063 s1 O.25 <1 1 >16 2 256 OO6 PAC sO.063 s1 sO.063 s1 O.12S <1 >1 >16 1 >512 OO7 US 2010/0226983 A1 Sep. 9, 2010 20

TABLE 12-continued MBCs of NB003 emulsions in the presence and absence of artificial sebum

O.S9/o O.S90 O.S90 NBOO3 - NBOO3- O.S90 NBOO3 - 20 mM 10 mM NBOO3 - 5 mM 1 mM EDTA? well EDTA? well EDTA? well EDTA? well O.S90 NBOO3

No 25% No 25% No 25% No 25% No 25% PAC# sebum sebum sebum sebum sebum sebum sebum sebum sebum sebum

PAC- sO.063 s1 sO.063 s1 O.12S <1 >1 >16 2 256 O08 PAC- sO.063 s1 sO.063 s1 O.S <1 >1 >16 2 512 O09 PAC- sO.063 s1 sO.063 s1 O.12S <1 >1 >16 2 128 O10 MBC sO.063 s1 sO.063 s1 sO.O63-0.5 <1-4 1->1 16->16 1-2 128->512 Range MBC sO.063 s1 sO.063 s1 O.12S <1 >1 >16 2 256 50 MBC90 s.0.063 s1 sO.063 s1 O.S 4 2 >16 2 512

0184 Table 13 shows the MICs and MBCs of Benzalko- 0187 FIG. 5 shows the relationship between the particle nium Chloride and SDS emulsions with addition of 20 mM size (nm), concentration of active (%), and Viscosity of a EDTA to test concentrations. The trend of reduced MICs and nanoemulsion. The particle size does not change upon dilu MBCs with addition of EDTA is continued. tion of a nanoemulsion; however viscosity significantly decreases as a function of the decrease in particle concentra TABLE 13 tions. Table 14 shows the effect dilution of a nanoemulsion has on the concentration of the active (CPC), viscosity, and MICs and MBCs of Selected Nanoemulsions particle size. MIC MBC TABLE 1.4 Without With Without With Drug sebum sebum sebum sebum NB-001 Process Optimization - Dilution 10% O.S 62.5 1 62.5 Percentage of Theoretical CPC Particle W5GBA-2OED Concentrated Potency Viscosity Size 10% sO.125 s2 sO.125 s2 NB-001 (% wt v) (cP) (nm) W5GBAED + 20 mM 100% 1.O 259,300 181 EDTA? well 80% O.8 3200 179 50% S8GC O.S 8 2 16 60% O6 11.5 181 50% S8GC- sO.063 sO.063 s1 SO% O.S 11.5 18O 20 mM 40% 0.4 7.5 178 EDTA? well 30% O.3 6.5 179 20% O.2 4.5 181 10% O.1 2.5 18O 0185. Conclusion: The MICs of a nanoemulsion accord ing to the invention (e.g., NB-003) without any additional EDTA showed a 32 to 64 fold increase in the presence of 25% artificial sebum. MBCs of a nanoemulsion according to the Example 7 invention (e.g., NB-003) showed 256 fold increases in the Viscosity and Permeation presence of sebum. The addition of 10-20 mM of EDTA decreased the MICs and MBCs of a nanoemulsion according 0188 The purpose of this example was to evaluate the to the invention (e.g., NB-003) to equal or lesser the test effect viscosity of a nanoemulsion has on the permeation of concentrations. See also FIG. 4, which shows the in vitro the active into the dermis and epidermis. MBC of a nanoemulsion (NB-003) with and without (+/-) the 0189 A permeation study was conducted using the proto presence of ethylenediaminetetraacetic acid (EDTA). The col described in Example 4 with five skin sections (n=5). Four figure shows that the MBC of the nanoemulsion rises 500 different concentrations of nanoemulsion (see Table 14) were fold in the presence of sebum, unless additional EDTA is tested: 0.25%, 0.30%, 0.50% and 0.80%. FIGS. 6 and 7 show added to the formulation. the results for epidermis and dermis permeation, respectively. Example 6 Specifically, FIG. 6 shows the results of the permeation study utilizing pig skin epidermis with 5 skin sections (n=5) fol Viscosity lowing administration of a nanoemulsion (NB-003) twice 0186 The purpose of this example was to evaluate the daily (BID). Higher viscosity (greater than 1000 cps) effect of concentration of a nanoemulsion has on the Viscosity nanoemulsions (e.g. 0.8% NB-003) were found to have of the nanoemulsion. greater permeation of the nanoemulsion into the epidermis. US 2010/0226983 A1 Sep. 9, 2010

(0190. Similarly, FIG. 7 shows the results of a permeation greater than about 95 cp, greater than about 100 cp, greater study utilizing pig skin epidermis with 5 skin sections (n=5) than about 150 cp, greater than about 200 cp, greater than following administration of a nanoemulsion (NB-003) twice about 300 cp greater than about 400 cp, greater than about daily (BID). Higher viscosity (greater than 1000 cps) 500 cB. greater than about 600 cP. greater than about 700 cB. nanoemulsions (e.g. 0.8% NB-003) were found to deliver greater than about 800 cp greater than about 900 cB. greater three times the amount of the Surfactant, cetylpyridinium than about 1000 cp greater than about 1500 cB. greater than chloride (CPC) to the dermis as compared to a lower viscosity about 2000 cp, greater than about 2500 cp greater than about nanoemulsion (e.g., 0.25% NB-003). 3000 cB. greater than about 3500 cP greater than about 4000 0191 Thus, increasing the viscosity of a nanoemulsion cP greater than about 4500 cp, greater than about 5000 cp. can increase the permeation of the nanoemulsion into the greater than about 5500 cp, greater than about 6000 cp greater dermis and epidermis, thereby producing a composition more than about 7000 cp greater than about 8000 cB. greater than effective in killing bacteria or other organisms. about 9000 cp, greater than about 10,000 cp, greater than about 15,000 cp greater than about 20,000 cp greater than Example 8 about 30,000 cp greater than about 40,000 cp greater than about 50,000 cB. greater than about 60,000 cB. greater than Effect of Temperature on Nanoemulsion Effective about 70,000 cB. greater than about 80,000 cB. greater than SS about 90,000 cB. greater than about 100,000 cB. greater than 0.192 The purpose of this example was to evaluate the about 150,000 cB. greater than about 200,000 cB. greater than effect of the temperature of the nanoemulsion on the efficacy about 250,000 cB, or up to about 259,300 cF. of the nanoemulsion against P. acnes. 4. The method of claim 1, wherein the nanoemulsion is at (0193 The effectiveness of the nanoemulsion (NB-003) in room temperature at the time of administration. killing P acnes over time at the three different temperatures 5. The method of claim 1, wherein prior to application the was evaluated: 5° C. room temperature, and 37° C. The nanoemulsion is warmed to a temperature selected from the nanoemulsion was tested in the presence of 25% serum. group consisting of about 30°C. or warmer, about 31° C. or 0194 The results, depicted in FIG. 8, show that cooling warmer, about 32° C. or warmer, about 33° C. or warmer, the nanoemulsion decreases the effectiveness of the about 34°C. or warmer, about 35° C. or warmer, about 36°C. nanoemulsion in killing Pacnes. Conversely, nanoemulsions or warmer, and about 37° C. at room temperature and warmed to 37° C. showed an 6. The method of claim 1, wherein: increased effectiveness in killing Pacnes. The nanoemulsion (a) the nanoemulsion droplets have an average diameter warmed to 37° C. showed an initial greater effectiveness in selected from the group consisting of less than about 950 killing P. acnes as compared to the room temperature nm, less than about 900 nm, less than about 850 nm, less nanoemulsion, with this increase in effectiveness diminishing than about 800 nm, less than about 750 nm, less than about 15 minutes after application. These results suggest that about 700 nm, less than about 650 nm, less than about one tactic that may increase the effectiveness of a nanoemul 600 nm, less than about 550 nm, less than about 500 nm, sion according to the invention in treating acne is ensuring less than about 450 nm, less than about 400 nm, less than that the nanoemulsion is at room temperature or warmer prior about 350 nm, less than about 300 nm, less than about to application. 250 nm, less than about 200 nm, less than about 150 nm, 0.195. It will be apparent to those skilled in the art that less than about 100 nm, greater than about 50 nm, greater various modifications and variations can be made in the meth than about 70 nm, greater than about 125 nm, and any ods and compositions of the present invention without depart combination thereof ing from the spirit or scope of the invention. Thus, it is (b) the nanoemulsion droplets have an average diameter intended that the present invention cover the modifications greater than about 125 nm andless than about 450 nmi; or and variations of this invention provided they come within the (c) any combination thereof. Scope of the appended claims and their equivalents. 7. The method of claim 1, wherein the topical application is What is claimed is: to any Superficial skin structure. 1. A method of killing Pacnes, in a subject in need thereof 8. The method of claim 1, wherein the nanoemulsion fur comprising administering topically to the Subject a ther comprises a chelating agent. nanoemulsion, wherein: 9. The method of claim 8, wherein the chelating agent: (a) the nanoemulsion comprises droplets having an average (a) is present in an amount of about 0.0005% to about diameter of less than about 3 microns; and 1.0%: (b) the nanoemulsion droplets comprise an oil phase with (b) is selected from the group consisting of ethylenedi at least one oil, an aqueous phase comprising at least one amine, ethylenediaminetetraacetic acid, and dimerca Surfactant, at least one organic solvent, and water. prol; or 2. The method of claim 1, wherein the nanoemulsion drop (c) any combination thereof. lets target the pilosebaceous gland. 10. The method of claim 1, wherein the nanoemulsion 3. The method of claim 1, wherein the nanoemulsion has a comprises: Viscosity selected from the group consisting of greater than (a) an aqueous phase; about 12 centipoise (cP), greater than about 15 cl, greater (b) about 1% oil to about 80% oil; than about 20 cp greater than about 25 cF. greater than about (c) about 0.1% organic solvent to about 50% organic sol 30 cp, greater than about 35 cF. greater than about 40 cp. vent; greater than about 45 cF. greater than about 50 cp greater than (d) at least one surfactant present in an amount of about about 55 cB. greater than about 60 cp greater than about 65 cB. 0.001% surfactant to about 10% surfactant; greater than about 70 cp, greater than about 75 cl, greater than (e) about 0.0005% to about 1.0% of a chelating agent; or about 80 cp, greater than about 85 cF. greater than about 90 cp. (f) any combination thereof. US 2010/0226983 A1 Sep. 9, 2010 22

11. The method of claim 1, wherein the nanoemulsion (c) is selected from the group consisting of animal oil, comprises: Vegetable oil, natural oil, synthetic oil, hydrocarbon oils, (a) an aqueous phase; silicone oils, and semi-synthetic derivatives thereof; (b) about 5% oil to about 80% oil; (d) is selected from the group consisting of mineral oil, (c) about 0.1% organic solvent to about 10% organic sol squalene oil, flavor oils, silicon oil, essential oils, water insoluble vitamins, Isopropyl Stearate. Butyl stearate, Vent; Octyl palmitate, Cetyl palmitate, Tridecyl behenate, (d) at least one non-ionic surfactant present in an amount of Diisopropyl adipate, Dioctyl sebacate, Menthyl anthra about 0.1% to about 10%: nhilate, Cetyl octanoate, Octyl salicylate, Isopropyl (e) at least one cationic agent present in an amount of about myristate, neopentyl glycol dicarpate cetols, Cera 0.01% to about 2%; phyls-R, Decyl oleate, diisopropyl adipate, Cls alkyl (f) about 0.0005% to about 1.0% of a chelating agent; or lactates, Cetyl lactate. Lauryl lactate. Isostearyl neopen (g) any combination thereof. tanoate, Myristyl lactate. Isocetyl stearoyl stearate. 12. The method of claim 1, wherein: Octyldodecyl stearoyl stearate, Hydrocarbon oils, Iso (a) the nanoemulsion is stable at about 40° C. and about paraffin, Fluid paraffins, Isododecane, Petrolatum, 75% relative humidity for a time period selected from Argan oil, Canola oil, Chile oil, Coconut oil, corn oil, the group consisting of up to about 1 month, up to about Cottonseed oil, Flaxseed oil, Grape seed oil, Mustard 3 months, up to about 6 months, up to about 12 months, oil, Olive oil, Palm oil, Palm kernel oil, Peanut oil, Pine up to about 18 months, up to about 2 years, up to about seed oil, Poppy seed oil, Pumpkin seed oil, Rice bran oil, 2.5 years, and up to about 3 years; Safflower oil, Tea oil, Truffle oil, Vegetable oil, Apricot (b) the nanoemulsion is stable at about 25° C. and about (kernel) oil, Jojoba oil (simmondsia chinensis seed oil), 60% relative humidity for a time period selected from Grapeseed oil. Macadamia oil. Wheat germ oil. Almond the group consisting of up to about 1 month, up to about oil. Rapeseed oil, Gourd oil, Soybean oil, Sesame oil, 3 months, up to about 6 months, up to about 12 months, Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, Bois up to about 18 months, up to about 2 years, up to about oil, Kuki nut oil, Avocado oil, Walnut oil, Fish oil, berry 2.5 years, up to about 3 years, up to about 3.5 years, up oil, allspice oil, juniper oil, seed oil, almond seed oil, to about 4 years, up to about 4.5 years, and up to about 5 anise seed oil, celery seed oil, cumin seed oil, nutmeg years; seed oil, leafoil, basil leafoil, bay leafoil, cinnamon leaf (c) the nanoemulsion is stable at about 4° C. for a time oil, common sage leaf oil, eucalyptus leaf oil, lemon period selected from the group consisting of up to about grass leaf oil, melaleuca leaf oil, oregano leaf oil, 1 month, up to about 3 months, up to about 6 months, up patchouli leaf oil, peppermint leaf oil, pine needle oil, to about 12 months, up to about 18 months, up to about rosemary leaf oil, spearmint leaf oil, tea tree leaf oil, 2 years, up to about 2.5 years, up to about 3 years, up to thyme leaf oil, wintergreen leaf oil, flower oil, chamo about 3.5 years, up to about 4 years, up to about 4.5 mile oil, clary sage oil, clove oil, geranium flower oil. years, up to about 5 years, up to about 5.5 years, up to hyssop flower oil, jasmine flower oil, lavenderflower oil, about 6 years, up to about 6.5 years, and up to about 7 manuka flower oil, Marhoram flower oil, orange flower years; or oil, rose flower oil, ylang-ylang flower oil, Bark oil, (d) any combination thereof. cassia Bark oil, cinnamon bark oil, Sassafras Bark oil, Wood oil, camphor wood oil, cedar wood oil, rosewood 13. The method of claim 1, wherein the organic solvent: oil, sandalwood oil), rhizome (ginger) wood oil, resin (a) is selected from the group consisting of C-C alcohol, oil, frankincense oil, myrrh oil, peel oil, bergamot peel diol, triol, dialkyl phosphate, tri-alkyl phosphate, semi oil, grapefruit peel oil, lemon peel oil, lime peel oil, synthetic derivatives thereof, and combinations thereof; orange peel oil, tangerine peel oil, root oil, Valerian oil, (b) is an alcohol which is selected from the group consist Oleic acid, Linoleic acid, Oleyl alcohol, Isostearyl alco ing of a nonpolar solvent, a polar solvent, a protic sol hol, semi-synthetic derivatives thereof, and combina Vent, and an aprotic solvent; tions thereof; or (c) is selected from the group consisting of ethanol, metha (e) any combination thereof. nol, isopropyl alcohol, glycerol, medium chain triglyc 16. The method of claim 1, further comprising a silicone erides, diethyl ether, ethyl acetate, acetone, dimethyl component. Sulfoxide (DMSO), acetic acid, n-butanol, butylene gly col, perfumers alcohols, isopropanol, n-propanol, for 17. The method of claim 16, wherein the silicone compo mic acid, propylene glycols, glycerol, sorbitol, indus nent comprises at least one volatile silicone oil, wherein: trial methylated spirit, triacetin, hexane, benzene, (a) the volatile silicone oil can be the sole oil in the silicone toluene, diethyl ether, chloroform, 1,4-dixoane, tetrahy component or it can be combined with other silicone and drofuran, dichloromethane, acetone, acetonitrile, dim non-silicone oils, and wherein the other oils can be vola ethylformamide, dimethyl sulfoxide, formic acid, semi tile or non-volatile; Synthetic derivatives thereof, and any combination (b) the volatile oil used in the silicone component is differ thereof; or ent than the oil in the oil phase: (c) the silicone component is selected from the group con (d) any combination thereof. sisting of methylphenylpolysiloxane, simethicone, 14. The method of claim 13, wherein the tri-alkyl phos dimethicone, phenyltrimethicone (or an organomodified phate is tri-n-butyl phosphate. version thereof), alkylated derivatives of polymeric sili 15. The method of claim 1, wherein the oil: cones, cetyl dimethicone, lauryl trimethicone, hydroxy (a) is any cosmetically or pharmaceutically acceptable oil: lated derivatives of polymeric silicones, such as dime (b) is non-volatile; thiconol, Volatile silicone oils, cyclic and linear US 2010/0226983 A1 Sep. 9, 2010 23

silicones, cyclomethicone, derivatives of cyclomethi palmitate, Glyceryl ricinoleate, Glyceryl Stearate, Glyc cone, hexamethylcyclotrisiloxane, octamethylcyclotet eryl thighlycolate, Glyceryl dilaurate, Glyceryl dioleate, rasiloxane, decamethylcyclopentasiloxane, Volatile lin Glyceryl dimyristate, Glyceryl disterate, Glyceryl sesui ear dimethylpolysiloxanes, isohexadecane, isoeicosane, oleate, Glyceryl Stearate lactate, Polyoxyethylene cetyl/ isotetracosane, polyisobutene, isooctane, isododecane, stearyl ether, Polyoxyethylene cholesterol ether, Poly semi-synthetic derivatives thereof, and combinations oxyethylene laurate or dilaurate, Polyoxyethylene thereof; or Stearate or distearate, polyoxyethylene fatty ethers, (d) any combination thereof. Polyoxyethylene lauryl ether, Polyoxyethylene stearyl 18. The method of claim 1, wherein the nanoemulsion ether, polyoxyethylene myristyl ether, a steroid, Choles comprises a volatile oil, wherein: terol, Betasitosterol, Bisabolol, fatty acid esters of alco (a) the volatile oil can be the organic solvent, or the volatile hols, isopropyl myristate, Aliphati-isopropyl n-butyrate, oil can be present in addition to an organic solvent; Isopropyl n-hexanoate, Isopropyl n-decanoate, Isoprop (b) the Volatile oil is a terpene, monoterpene, sesquiter pyl palmitate, Octyldodecyl myristate, alkoxylated alco pene, carminative, azulene, semi-synthetic derivatives hols, alkoxylated acids, alkoxylated amides, alkoxy thereof, or combinations thereof; lated sugar derivatives, alkoxylated derivatives of (c) the Volatile oil is selected from the group consisting of natural oils and waxes, polyoxyethylene polyoxypropy a terpene, monoterpene, sesquiterpene, carminative, lene block copolymers, nonoxynol-14, PEG-8 laurate, aZulene, menthol, camphor, thujone, thymol, nerol, lina PEG-6 Cocoamide, PEG-20 methylglucose sesquistear lool, limonene, geraniol, perillyl alcohol, nerolidol, far ate, PEG40 lanolin, PEG-40 castor oil, PEG-40 hydro neSol, ylangene, bisabolol, farmesene, ascaridole, che genated castor oil, polyoxyethylene fatty ethers, glyc nopodium oil, citronellal, citral, citronellol, eryl diesters, polyoxyethylene stearyl ether, chamaZulene, yarrow, guaiaZulene, chamomile, semi- polyoxyethylene myristyl ether, and polyoxyethylene synthetic derivatives thereof, and combinations thereof; lauryl ether, glyceryl dilaurate, glyceryl dimyState, glyc O eryl distearate, semi-synthetic derivatives thereof, and (d) any combination thereof. mixtures thereof; 19. The method of claim 1, wherein the surfactant is: (e) a non-ionic lipid selected from the group consisting of (a) a pharmaceutically acceptable ionic Surfactant, a phar glyceryl laurate, glyceryl myristate, glyceryl dilaurate, maceutically acceptable nonionic Surfactant, a pharma glyceryl dimyristate, semi-synthetic derivatives thereof, ceutically acceptable cationic surfactant, a pharmaceu and mixtures thereof tically acceptable ionic Surfactant, a pharmaceutically (f) a polyoxyethylene fatty ether having a polyoxyethylene acceptable anionic Surfactant, or a pharmaceutically head group ranging from about 2 to about 100 groups; acceptable Zwitterionic Surfactant; (g) an alkoxylated alcohol having the structure shown in (b) a pharmaceutically acceptable ionic polymeric Surfac formula I below: tant, a pharmaceutically acceptable nonionic polymeric Rs (OCH2CH2), OH Formula I Surfactant, a pharmaceutically acceptable cationic poly wherein Rs is a branched or unbranched alkyl group having meric Surfactant, a pharmaceutically acceptable anionic from about 6 to about 22 carbon atoms and y is between polymeric Surfactant, or a pharmaceutically acceptable about 4 and about 100, and preferably, between about 10 Zwitterionic polymeric Surfactant; and about 100: (c) a polymeric Surfactant which is selected from the group (h) analkoxylated alcohol which is an ethoxylated deriva consisting of a graft copolymer of a poly(methyl meth tive of lanolin alcohol; acrylate) backbone with at least one polyethylene oxide (i) is nonionic and is selected from the group consisting of (PEO) side chain, polyhydroxystearic acid, an alkoxy nonoxynol-9, lated alkyl phenol formaldehyde condensate, a poly an ethoxylated Surfactant, alkylene glycol modified polyester with fatty acid an alcohol ethoxylated, hydrophobes, a polyester, semi-synthetic derivatives an alkyl phenol ethoxylated, thereof, and combinations thereof; a fatty acid ethoxylated, (d) selected from the group consisting of ethoxylated non a monoalkaolamide ethoxylated, ylphenol comprising 9 to 10 units of ethyleneglycol, a sorbitan ester ethoxylated, ethoxylated undecanol comprising 8 units of ethyleneg a fatty amino ethoxylated, lycol, polyoxyethylene (20) Sorbitan monolaurate, poly an ethylene oxide-propylene oxide copolymer, oxyethylene (20) Sorbitan monopalmitate, polyoxyeth Bis(polyethylene glycol bisimidazoyl carbonyl), ylene (20) sorbitan monostearate, polyoxyethylene (20) Sorbitan monooleate, Sorbitan monolaurate, Sorbitan E. ... monopalmitate, Sorbitan monostearate, Sorbitan B 1) (R) 7 2. monooleate, ethoxylated hydrogenated ricin oils, ry s sodium laurylsulfate, a diblock copolymer of ethyl- BrijR 76, eneoxyde and propyleneoxyde, Ethylene Oxide-Propy- BrjR 92V, lene Oxide Block Copolymers, and tetra-functional BrijR 97, block copolymers based on ethylene oxide and propy- BrijR 58P. lene oxide, Glyceryl monoesters, Glyceryl caprate, Cremophor(R) EL, Glyceryl caprylate, Glyceryl cocate, Glyceryl erucate, Decaethylene glycol monododecyl ether, Glyceryl hydroxysterate, Glyceryl isostearate, Glyceryl N-Decanoyl-N-methylglucamine, lanolate, Glyceryl laurate, Glyceryl linolate, Glyceryl n-Decyl alpha-D-glucopyranoside, myristate, Glyceryl oleate, Glyceryl PABA, Glyceryl Decyl beta-D-maltopyranoside, US 2010/0226983 A1 Sep. 9, 2010 24

n-Dodecanoyl-N-methylglucamide, Triethylene glycol monododecyl ether, n-Dodecyl alpha-D-maltoside, Triethylene glycol monohexadecyl ether, n-Dodecyl beta-D-maltoside, Triethylene glycol monooctyl ether, Heptaethylene glycol monodecyl ether, Triethylene glycol monotetradecyl ether, Heptaethylene glycol monotetradecyl ether, Triton CF-21, Heptaethylene glycol monododecyl ether, Triton CF-32, n-Hexadecyl beta-D-maltoside, Triton DF-12, Hexaethylene glycol monododecyl ether, Triton DF-16, Hexaethylene glycol monohexadecyl ether, Triton GR-5M, Hexaethylene glycol monooctadecyl ether, Triton QS-15, Hexaethylene glycol monotetradecyl ether, Triton QS-44, Igepal CA-630, Triton X-100, Methyl-6-O-(N-heptylcarbamoyl)-alpha-D-glucopyrano Triton X-102, side, Triton X-15, Nonaethylene glycol monododecyl ether, Triton X-151, N-Nonanoyl-N-methylglucamine, Triton X-200, Octaethylene glycol monodecyl ether, Triton X-207, Octaethylene glycol monododecyl ether, Triton X-114, Octaethylene glycol monohexadecyl ether, Triton X-165, Octaethylene glycol monooctadecyl ether, Triton X-305, Octaethylene glycol monotetradecyl ether, Triton X-405, Octyl-beta-D-glucopyranoside, Triton X-45, Pentaethylene glycol monodecyl ether, Triton X-705-70, Pentaethylene glycol monododecyl ether, TWEENR) 20, Pentaethylene glycol monohexadecyl ether, TWEENR 21, Pentaethylene glycol monohexyl ether, TWEENR 40, Pentaethylene glycol monooctadecyl ether, TWEENR 60, Pentaethylene glycol monooctyl ether, TWEENR 61, Polyethylene glycol diglycidyl ether, TWEENR) 65, Polyethylene glycol ether W-1, TWEENR 80, Polyoxyethylene 10 tridecyl ether, TWEENR 81, Polyoxyethylene 100 stearate, TWEENR 85, Polyoxyethylene 20 isohexadecyl ether, Tyloxapol Polyoxyethylene 20 oleyl ether, n-Undecyl beta-D-glucopyranoside, Polyoxyethylene 40 stearate, Poloxamer 101, Polyoxyethylene 50 stearate, Poloxamer 105, Polyoxyethylene 8 stearate, Poloxamer 108, Polyoxyethylene bis(imidazolyl carbonyl), Poloxamer 122, Polyoxyethylene 25 propylene glycol stearate, Poloxamer 123, Saponin from Quillaja bark, Poloxamer 124, Span R) 20, Poloxamer 181, Span R 40, Poloxamer 182, Span R. 60, Poloxamer 183, Span R) 65, Poloxamer 184, Span R 80, Poloxamer 185, Span R 85, Poloxamer 188, Tergitol, Poloxamer 212, Tergitol, Type 15-S-12, Poloxamer 215, Tergitol, Type 15-S-30, Poloxamer 217, Tergitol, Type 15-S-5, Poloxamer 231, Tergitol, Type 15-S-7, Poloxamer 234, Tergitol, Type 15-S-9, Poloxamer 235, Tergitol, Type NP-10, Poloxamer 237, Tergitol, Type NP-4, Poloxamer 238, Tergitol, Type NP-40, Poloxamer 282, Tergitol, Type NP-7, Poloxamer 284, Tergitol, Type NP-9, Poloxamer 288, Tergitol, Type TMN-10, Poloxamer 331, Tergitol, Type TMN-6, Poloxamer 333, Tetradecyl-beta-D-maltoside, Poloxamer 334, Tetraethylene glycol monodecyl ether, Poloxamer 335, Tetraethylene glycol monododecyl ether, Poloxamer 338, Tetraethylene glycol monotetradecyl ether, Poloxamer 401, Triethylene glycol monodecyl ether, Poloxamer 402, US 2010/0226983 A1 Sep. 9, 2010 25

Poloxamer 403, Alkyl dimethyl benzyl ammonium chloride (67% C12, Poloxamer 407, 24% C14), Poloxamer 105 Benzoate, Alkyl dimethyl benzyl ammonium chloride (67% C12, Poloxamer 182 Dibenzoate, 25% C14), semi-synthetic derivatives thereof, and combinations Alkyl dimethylbenzyl ammonium chloride (90% C14.5% thereof; C12), () the Surfactant is cationic and is selected from the group Alkyl dimethylbenzyl ammonium chloride (93%. C14.4% consisting of C12), a quarternary ammonium compound, Alkyl dimethylbenzyl ammonium chloride (95% C16, 5% an alkyl trimethyl ammonium chloride compound, C18), a dialkyl dimethyl ammonium chloride compound, Alkyl didecyl dimethyl ammonium chloride, Benzalkonium chloride, Alkyl dimethylbenzyl ammonium chloride (C12-16), Benzyldimethylhexadecylammonium chloride, Alkyl dimethylbenzyl ammonium chloride (C12-18), Benzyldimethyltetradecylammonium chloride, dialkyl dimethylbenzyl ammonium chloride, Benzyldodecyldimethylammonium bromide, Alkyl dimethyl dimethybenzyl ammonium chloride, Benzyltrimethylammonium tetrachloroiodate, Alkyl dimethyl ethyl ammonium bromide (90% C14, 5% cetylpyridinium chloride, C16, 5% C12), Dimethyldioctadecylammonium bromide, Alkyl dimethylethyl ammonium bromide (mixed alkyland Dodecylethyldimethylammonium bromide, alkenyl groups as in the fatty acids of soybean oil), Dodecyltrimethylammonium bromide, Alkyl dimethyl ethylbenzyl ammonium chloride, Ethylhexadecyldimethylammonium bromide, Alkyl dimethyl ethylbenzyl ammonium chloride (60% Girard's reagent T. C14), Hexadecyltrimethylammonium bromide, Alkyl dimethyl isopropylbenzyl ammonium chloride (50% N,N',N'-Polyoxyethylene(10)-N-tallow-1,3-diaminopro C12, 30% C14, 17% C16, 3% C18), pane, Alkyl trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1% C12), Thonzonium bromide, Alkyltrimethylammonium chloride (90% C18, 10% C16), Trimethyl(tetradecyl)ammonium bromide, Alkyldimethyl(ethylbenzyl) ammonium chloride (C12 1,3,5-Triazine-1,3,5(2H4H.6H)-triethanol, 18), 1-Decanaminium, N-decyl-N,N-dimethyl-, chloride, Di-(C8-10)-alkyl dimethyl ammonium chlorides, Didecyl dimethyl ammonium chloride, Dialkyl dimethyl ammonium chloride, 2-(2-(p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl ben Dialkyl methylbenzyl ammonium chloride, Zyl ammonium chloride, Didecyl dimethyl ammonium chloride, 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl ben Diisodecyl dimethyl ammonium chloride, Zyl ammonium chloride, Dioctyl dimethyl ammonium chloride, Alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazolinium Dodecyl bis(2-hydroxyethyl) octyl hydrogen ammonium chloride, chloride, Alkyl bis(2-hydroxyethyl)benzyl ammonium chloride, Dodecyl dimethylbenzyl ammonium chloride, Alkyl demethylbenzyl ammonium chloride, Dodecylcarbamoyl methyl dimethyl benzyl ammonium Alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride chloride, (100% C12), Heptadecyl hydroxyethylimidazolinium chloride, Alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, (50% C14, 40% C12, 10% C16), Myristalkonium chloride (and) Quat RNIUM 14, Alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride N,N-Dimethyl-2-hydroxypropylammonium chloride (55% C14, 23% C12, 20% C16), polymer, Alkyl dimethylbenzyl ammonium chloride, n-Tetradecyl dimethylbenzyl ammonium chloride mono Alkyl dimethylbenzyl ammonium chloride (100% C14), hydrate, Alkyl dimethylbenzyl ammonium chloride (100% C16), Octyl decyl dimethyl ammonium chloride, Alkyl dimethyl benzyl ammonium chloride (41% C14, Octyl dodecyl dimethyl ammonium chloride, 28% C12), Octyphenoxyethoxyethyl dimethyl benzyl ammonium Alkyl dimethyl benzyl ammonium chloride (47% C12, chloride, 18% C14), Oxydiethylenebis(alkyl dimethyl ammonium chloride), Alkyl dimethyl benzyl ammonium chloride (55% C16, Trimethoxysily propyl dimethyl octadecyl ammonium 20% C14), chloride, Alkyl dimethyl benzyl ammonium chloride (58% C14, Trimethoxysilyl quats, 28% C16), Trimethyl dodecylbenzyl ammonium chloride, Alkyl dimethyl benzyl ammonium chloride (60% C14, semi-synthetic derivatives thereof, and combinations 25% C12), thereof; Alkyl dimethyl benzyl ammonium chloride (61% C11, (k) the Surfactant is anionic and is selected from the group 23% C14), consisting of Alkyl dimethyl benzyl ammonium chloride (61% C12, a carboxylate, 23% C14), a Sulphate, Alkyl dimethyl benzyl ammonium chloride (65% C12, a Sulphonate, 25% C14), a phosphate, US 2010/0226983 A1 Sep. 9, 2010 26

Chenodeoxycholic acid, 3-(N,N-Dimethylmyristylammonio)propanesulfonate Chenodeoxycholic acid sodium salt, inner salt, Cholic acid, ox or sheep bile, 3-(N,N-Dimethyloctadecylammonio)propanesulfonate, Dehydrocholic acid, 3-(N,N-Dimethyloctylammonio)propanesulfonate inner Deoxycholic acid, salt, Deoxycholic acid methyl ester, 3-(N,N-Dimethylpalmitylammonio)propanesulfonate, Digitonin, semi-synthetic derivatives thereof, and combinations Digitoxigenin, thereof; or N,N-Dimethyldodecylamine N-oxide, (m) any combination thereof. Docusate sodium salt, 20. The method of claim 19, wherein: Glycochenodeoxycholic acid sodium salt, (a) the alkoxylated alcohol is the species wherein Rs is a Glycocholic acid hydrate, synthetic, lauryl group and y has an average value of 23; Glycocholic acid sodium salt hydrate, synthetic, (b) the ethoxylated derivative of lanolin alcohol is laneth Glycodeoxycholic acid monohydrate, 10, which is the polyethylene glycol ether of lanolin Glycodeoxycholic acid sodium salt, alcohol with an average ethoxylation value of 10. Glycolithocholic acid 3-sulfate disodium salt, 21. The method of claim 1, wherein the nanoemulsion Glycolithocholic acid ethyl ester, comprises at least one cationic Surfactant. N-Lauroylsarcosine Sodium salt, 22. The method of claim 1, wherein the nanoemulsion N-Lauroylsarcosine Solution, comprises a cationic Surfactant which is cetylpyridinium Lithium dodecyl sulfate, chloride. Lugol Solution, 23. The method of claim 1, wherein the nanoemulsion Niaproof 4, Type 4, comprises a cationic Surfactant, and wherein: 1-Octanesulfonic acid sodium salt, (a) the concentration of the cationic Surfactant is less than Sodium 1-butanesulfonate, about 5.0% and greater than about 0.001%; Sodium 1-decanesulfonate, (b) the concentration of the cationic surfactant is selected Sodium 1-dodecanesulfonate, from the group consisting of less than about 5%, less Sodium 1-heptanesulfonate anhydrous, than about 4.5%, less than about 4.0%, less than about Sodium 1-nonanesulfonate, 3.5%, less than about 3.0%, less than about 2.5%, less Sodium 1-propanesulfonate monohydrate, than about 2.0%, less than about 1.5%, less than about Sodium 2-bromoethanesulfonate, 1.0%, less than about 0.90%, less than about 0.80%, less Sodium cholate hydrate, thanabout 0.70%, less than about 0.60%, less than about Sodium choleate, 0.50%, less than about 0.40%, less than about 0.30%, Sodium deoxycholate, less than about 0.20%, less than about 0.10%, greater Sodium deoxycholate monohydrate, than about 0.002%, greater than about 0.003%, greater Sodium dodecyl sulfate, than about 0.004%, greater than about 0.005%, greater than about 0.006%, greater than about 0.007%, greater Sodium hexanesulfonate anhydrous, than about 0.008%, greater than about 0.009%, greater Sodium octyl sulfate, than about 0.010%, and greater than about 0.001%; or Sodium pentanesulfonate anhydrous, (c) any combination thereof. Sodium taurocholate, 24. The method of claim 1, wherein the nanoemulsion Taurochenodeoxycholic acid sodium salt, comprises at least one cationic Surfactant and at least one Taurodeoxycholic acid sodium salt monohydrate, non-cationic Surfactant. Taurohyodeoxycholic acid sodium salt hydrate, 25. The method of claim 24, wherein: Taurolithocholic acid 3-sulfate disodium salt, (a) the non-cationic Surfactant is a nonionic Surfactant; TaurourSodeoxycholic acid sodium salt, (b) the non-cationic Surfactant is a nonionic Surfactant Trizma R) dodecyl sulfate, which is a polysorbate; Ursodeoxycholic acid, (c) the non-cationic Surfactant is a nonionic Surfactant semi-synthetic derivatives thereof, and combinations which is polysorbate 20 or polysorbate 80 or polysorbate thereof; 60; (1) the surfactant is Zwitterionic and is selected from the (d) the non-cationic Surfactant is a nonionic Surfactant and group consisting of the non-ionic Surfactant is present in a concentration of an N-alkyl betaine, about 0.05% to about 7.0%: lauryl amindo propyl dimethyl betaine, (e) the non-cationic Surfactant is a nonionic Surfactant and an alkyl dimethylglycinate, the non-ionic Surfactant is present in a concentration of an N-alkyl amino propionate, about 0.5% to about 4%; or CHAPS, minimum 98%, (f) any combination thereof. CHAPS, minimum 98%, 26. The method of claim 1, wherein the nanoemulsion CHAPS, for electrophoresis, minimum 98%, comprises a cationic Surfactant present in a concentration of CHAPSO, minimum 98%, about 0.5% to about 2%, in combination with a nonionic CHAPSO, Surfactant. CHAPSO, for electrophoresis, 27. The method of claim 1, wherein the nanoemulsion 3-(Decyldimethylammonio)propanesulfonate inner salt, further comprises: 3-(Dodecyldimethylammonio)propanesulfonate inner (a) at least one preservative; salt, (b) at least one a pH adjuster; US 2010/0226983 A1 Sep. 9, 2010 27

(c) at least pharmaceutically acceptable buffer; or Calcium citrate tribasic tetrahydrate, 298.0% (calc. on dry (d) any combination thereof. Substance, KT). 28. The method of claim 27, wherein: Citrate Concentrated Solution, for molecular biology, 1 M (a) the preservative is selected from the group consisting of in H.O. cetylpyridinium chloride, benzalkonium chloride, ben Citric acid, anhydrous, 299.5% (T), Zyl alcohol, chlorhexidine, imidazolidinyl urea, phenol, Citric acid, for luminescence, anhydrous, 299.5% (T), potassium Sorbate, benzoic acid, bronopol, chlorocre Diethanolamine, 299.5% (GC), Sol, paraben esters, phenoxyethanol, Sorbic Acid, alpha EPPS, 299.0% (T), tocophernol, ascorbic acid, ascorbyl palmitate, buty Ethylenediaminetetraacetic acid disodium salt dihydrate, lated hydroxyanisole, butylated hydroxytoluene, for molecular biology, 299.0% (T), Sodium ascorbate, sodium metabisulphite, citric acid, Formic acid solution, 1.0 M in H.O. edetic acid, semi-synthetic derivatives thereof, and com Gly-Gly-Gly, 299.0% (NT), binations thereof Gly-Gly, 299.5% (NT), (b) the pH adjuster is selected from the group consisting of Glycine, 299.0% (NT), diethyanolamine, lactic acid, monoethanolamine, tri Glycine, for luminescence, 299.0% (NT), ethylanolamine, Sodium hydroxide, Sodium phosphate, Glycine, for molecular biology, 299.0% (NT), semi-synthetic derivatives thereof, and combinations HEPES buffered saline, for molecular biology, 2x concen thereof; trate, (c) the buffer is selected from the group consisting of HEPES, 299.5% (T), 2-Amino-2-methyl-1,3-propanediol. 299.5% (NT), HEPES, for molecular biology, 299.5% (T), 2-Amino-2-methyl-1-propanol. 299.0% (GC), Imidazole buffer Solution, 1 M in H2O, L-(+)-Tartaric acid, 299.5% (T), Imidazole, 299.5% (GC), ACES, 299.5% (T), Imidazole, for luminescence, 299.5% (GC), ADA, 299.0% (T), Imidazole, for molecular biology, 299.5% (GC), Acetic acid, 299.5% (GC/T), Lipoprotein Refolding Buffer, Acetic acid, for luminescence, 299.5% (GC/T), Lithium acetate dihydrate, 299.0% (NT), Ammonium acetate solution, for molecular biology, ~5 M Lithium citrate tribasic tetrahydrate, 299.5% (NT), in H2O, MES hydrate, 299.5% (T), Ammonium acetate, for luminescence, 299.0% (calc. on MES monohydrate, for luminescence, 299.5% (T), dry Substance. T), MES solution, for molecular biology, 0.5M in H.O. Ammonium bicarbonate, 299.5% (T), MOPS, 299.5% (T), Ammonium citrate dibasic, 299.0% (T), MOPS, for luminescence, 299.5% (T), Ammonium formate solution, 10 Min H.O. MOPS, for molecular biology, 299.5% (T), Ammonium formate, 299.0% (calc. based on dry sub Magnesium acetate solution, for molecular biology, ~1 M stance, NT), in H2O, Ammonium oxalate monohydrate, 299.5% (RT), Magnesium acetate tetrahydrate, 299.0% (KT). Ammonium phosphate dibasic solution, 2.5 M in H.O. Magnesium citrate tribasic nonahydrate, 298.0% (calc. Ammonium phosphate dibasic, 299.0% (T), based on dry substance, KT), Ammonium phosphate monobasic solution, 2.5M in H.O. Magnesium formate solution, 0.5 M in HO, Ammonium phosphate monobasic, 299.5% (T), Magnesium phosphate dibasic trihydrate, 298.0% (KT). Ammonium Sodium phosphate dibasic tetrahydrate, 299. Neutralization solution for the in-situ hybridization for 5% (NT), in-situ hybridization, for molecular biology, Ammonium sulfate solution, for molecular biology, 3.2M Oxalic acid dihydrate, 299.5% (RT), in H.O. PIPES, 299.5% (T), Ammonium tartrate dibasic solution, 2 M in HO (color PIPES, for molecular biology, 299.5% (T), less solution at 20°C.), Phosphate buffered saline, solution (autoclaved), Ammonium tartrate dibasic, 299.5% (T), Phosphate buffered saline, washing buffer for peroxidase BES buffered saline, for molecular biology, 2x concen conjugates in Western Blotting, 10x concentrate, trate, piperazine, anhydrous, 299.0% (T), BES, 299.5% (T), Potassium D-tartrate monobasic, 299.0% (T), BES, for molecular biology, 299.5% (T), Potassium acetate Solution, for molecular biology, BICINE buffer Solution, for molecular biology, 1 M in Potassium acetate solution, for molecular biology, 5 M in HO, HO, BICINE, 299.5% (T), Potassium acetate solution, for molecular biology, ~1 Min BIS-TRIS, 299.0% (NT), HO, Bicarbonate buffer solution, >0.1 M NaCO >0.2 M Potassium acetate, 299.0% (NT), NaHCO, Potassium acetate, for luminescence, 299.0% (NT), Boric acid, 299.5% (T), Potassium acetate, for molecular biology, 299.0% (NT), Boric acid, for molecular biology, 299.5% (T), Potassium bicarbonate, 299.5% (T), CAPS, 299.0% (TLC), Potassium carbonate, anhydrous, 299.0% (T), CHES, 299.5% (T), Potassium chloride, 299.5% (AT), Calcium acetate hydrate, 299.0% (calc. on dried material, Potassium citrate monobasic, 299.0% (dried material, KT), NT), Calcium carbonate, precipitated, 299.0% (KT). Potassium citrate tribasic solution, 1 M in H2O, US 2010/0226983 A1 Sep. 9, 2010 28

Potassium formate solution, 14 Min H2O, Sodium tetraborate decahydrate, 299.5% (T), Potassium formate, 299.5% (NT), TAPS, 299.5% (T), Potassium oxalate monohydrate, 299.0% (RT), TES, 299.5% (calc. based on dry substance. T), Potassium phosphate dibasic, anhydrous, 299.0% (T), TM buffer solution, for molecular biology, pH 7.4, Potassium phosphate dibasic, for luminescence, anhy TNT buffer solution, for molecular biology, pH 8.0, drous, 299.0% (T), TRIS Glycine buffer solution, 10x concentrate, Potassium phosphate dibasic, for molecular biology, anhy TRIS acetate-EDTA buffer solution, for molecular biol drous, 299.0% (T), Ogy, Potassium phosphate monobasic, anhydrous, 299.5% (T), Potassium phosphate monobasic, for molecular biology, TRIS buffered saline, 10x concentrate, anhydrous, 299.5% (T), TRIS glycine SDS buffer solution, for electrophoresis, 10x Potassium phosphate tribasic monohydrate, 5% (T), concentrate, Potassium phthalate monobasic, 299.5% (T), TRIS phosphate-EDTA buffer solution, for molecular biol Potassium sodium tartrate solution, 1.5M in H2O, ogy, concentrate, 10x concentrate, Potassium sodium tartrate tetrahydrate, 299.5% (NT), Tricine, 299.5% (NT), Potassium tetraborate tetrahydrate, 299.0% (T), Triethanolamine, 299.5% (GC), Potassium tetraoxalate dihydrate, 299.5% (RT), Triethylamine, 299.5% (GC), Propionic acid solution, 1.0 M in H.O. Triethylammonium acetate buffer, volatile buffer, ~1.0 M STE buffer solution, for molecular biology, pH 7.8, in H2O, STET buffer solution, for molecular biology, pH 8.0, Triethylammonium phosphate solution, volatile buffer, Sodium 5,5-diethylbarbiturate, 299.5% (NT), -1.0 M in HO, Sodium acetate solution, for molecular biology, ~3 M in Trimethylammonium acetate solution, volatile buffer, ~1.0 HO, M in HO, Sodium acetate trihydrate, 299.5% (NT), Trimethylammonium phosphate solution, volatile buffer, Sodium acetate, anhydrous, 299.0% (NT), -1 M in HO, Sodium acetate, for luminescence, anhydrous, 299.0% Tris-EDTA buffer solution, for molecular biology, concen (NT), Sodium acetate, for molecular biology, anhydrous, 299. trate, 100x concentrate, 0% (NT), Tris-EDTA buffer solution, for molecular biology, pH 7.4, Sodium bicarbonate, 299.5% (T), Tris-EDTA buffer solution, for molecular biology, pH 8.0, Sodium bitartrate monohydrate, 299.0% (T), Trizma Racetate, 299.0% (NT), Sodium carbonate decahydrate, 299.5% (T), Trizma(R) base, 299.8% (T), Sodium carbonate, anhydrous, 299.5% (calc. on dry sub Trizma(R) base, 299.8% (T), stance, T), Trizma(R) base, for luminescence, 299.8% (T), Sodium citrate monobasic, anhydrous, 299.5% (T), Trizma(R) base, for molecular biology, 299.8% (T), Sodium citrate tribasic dihydrate, 299.0% (NT), Trizma(R) carbonate, 298.5% (T), Sodium citrate tribasic dihydrate, for luminescence, 299. Trizma(R) hydrochloride buffer solution, for molecular 0% (NT), biology, pH 7.2. Sodium citrate tribasic dihydrate, for molecular biology, Trizma(R) hydrochloride buffer solution, for molecular 299.5% (NT), biology, pH 7.4, Sodium formate solution, 8 M in H2O, Sodium oxalate, 299.5% (RT), Trizma(R) hydrochloride buffer solution, for molecular Sodium phosphate dibasic dihydrate, 299.0% (T), biology, pH 7.6, Sodium phosphate dibasic dihydrate, for luminescence, Trizma(R) hydrochloride buffer solution, for molecular 299.0% (T), biology, pH 8.0, Sodium phosphate dibasic dihydrate, for molecular biol Trizma Rhydrochloride, 2999.0% (AT), ogy, 299.0% (T), Trizma Rhydrochloride, for luminescence, 299.0% (AT), Sodium phosphate dibasic dodecahydrate, 299.0% (T), Trizma(R) hydrochloride, for molecular biology, 299.0% Sodium phosphate dibasic solution, 0.5 M in H.O. (AT), and Sodium phosphate dibasic, anhydrous, 99.5% (T), Trizma(R) maleate, 299.5% (NT); or Sodium phosphate dibasic, for molecular biology, 299.5% (d) any combination thereof. (T), 29. The method of claim 1, wherein the water is present in Sodium phosphate monobasic dihydrate, 299.0% (T), Phosphate Buffered Saline (PBS). Sodium phosphate monobasic dihydrate, for molecular 30. The method of claim 1, wherein the nanoemulsion is biology, 299.0% (T), topically applied: Sodium phosphate monobasic monohydrate, for molecular (a) in a single administration; biology, 299.5% (T), (b) for at least once a week, at least twice a week, at least Sodium phosphate monobasic solution, 5 M in H.O. once a day, at least twice a day, multiple times daily, Sodium pyrophosphate dibasic, 299.0% (T), multiple times weekly, biweekly, at least once a month, Sodium pyrophosphate tetrabasic decahydrate, 299.5% or any combination thereof; (T), (c) for a period of time selected from the group consisting Sodium tartrate dibasic dihydrate, 299.0% (NT), of about one week, about two weeks, about three weeks, Sodium tartrate dibasic solution, 1.5M in H2O (colorless about one month, about two months, about three months, solution at 20°C.), about four months, about five months, about six months, US 2010/0226983 A1 Sep. 9, 2010 29

about seven months, about eight months, about nine 32. The method of claim 1, wherein the nanoemulsion is a months, about ten months, about eleven months, about controlled release formulation, Sustained release formula one year, about 1.5 years, about 2 years, about 2.5 years, tion, immediate release formulation, or any combination about 3 years, about 3.5 years, about 4 years, about 4.5 thereof. years, and about 5 years; 33. The method of claim 1, wherein the nanoemulsion (d) followed by washing the application area to remove any further comprises at least one anti-acne agent. residual nanoemulsion; or 34. The method of claim 33, wherein the anti-acne agent is (e) any combination thereof. selected from the group consisting of benzoyl peroxide, Sali 31. The method of claim 1, wherein the nanoemulsion cylic acid and retinoid. droplets enter the pilosebaeous gland (unit), hair follicle, epidermis, dermis, or a combination thereof. c c c c c