NANOEMULSION COMPOSITIONS HAVING ENHANCED PERMEABILITY

FIELD OF THE APPLICATION

The present application is directed to nanoemulsion compositions administered topically, mucosally, vaginally, or intranasally having enhanced permeability.

BACKGROUND OF THE INVENTION

Nanoemulsions have been used as topical antimicrobial formulations as well as vaccine adjuvants. Prior teachings related to nanoemulsions are described in, for example, U.S. Pat. Nos. 6,015,832; 6,506,803; 6,559,189; 6,635,676; and 7,314,624. Other exemplary nanoemulsion related patents and patent publications include but are not limited to U.S. Pat. Nos. 6,635,676; 7,655,252; 7,767,216; 8,226,965; 8,232,320; 8,236,335; 8,668,911; 8,703,164; 8,747,872; 8,771,731; 8,877,208; 8,962,026; 9,131,680; 9,144,606; 9,259,407; 9,415,006; 9,492,525; 9,561,271; 9,839,685; 9,974,844; 9,801,842; WO 2017/023751; WO 2017/196979; WO 2017/201390; US 2013-0052235; US 2016-0074504; US 2017-0007689; US 2017-0007694; US 2017-0007690; and US 2018-0071380.

There exists a need to develop nanoemulsion compositions having enhanced permeability into human and animal skin and mucosal tissues, and the ability to enhance delivery of therapeutic and active agents into skin and mucosal tissues. The present disclosure satisfies these needs.

SUMMARY OF INVENTION

In one aspect, a composition for topical, transdermal, mucosal (e.g. intranasal, ocular, buccal, vaginal) or oral application or administration is provided. The composition comprises an oil-in-water nanoemulsion, and the nanoemulsion comprises: (a) an aqueous phase; (b) at least one oil; (c) at least one quaternary ammonium compound; and (d) at least one nonionic surfactant. The composition can additionally and optionally comprise an active/therapeutic agent. In addition, (i) the droplets of the nanoemulsion have a mean droplet size of less than about 1 micron; (ii) the nanoemulsion is diluted resulting in a formulation of about 0.5% to about 60% nanoemulsion; (iii) the concentration ratio of the quaternary ammonium compound to nonionic surfactant is about 5:1 to about 1:27; and (iv) the nanoemulsion enhances delivery of the quaternary ammonium compound (and/or additional active/therapeutic agent) into tissue by at least about 25% as compared to a solution with the same concentration of the same quaternary ammonium compound but lacking a nanoemulsion, and as compared to a nanoemulsion with a concentration ratio of the same quaternary ammonium compound to the same nonionic surfactant outside of the range from about 5:1 to about 1:27. In another aspect of the disclosure, the quaternary ammonium compound is a cationic surfactant or is part of a zwitterionic surfactant. Optionally the nanoemulsion can comprise at least one organic solvent.

In one aspect, a composition for topical, transdermal, mucosal (e.g. intranasal, ocular, buccal, vaginal), or oral application or administration is provided, the composition comprising an oil-in-water nanoemulsion, the nanoemulsion comprising (a) an aqueous phase; (b) at least one oil; (c) at least one quaternary ammonium compound; and (d) at least one nonionic surfactant. The composition can additionally and optionally comprise an active/therapeutic agent. For the composition, (i) the droplets of the nanoemulsion have a mean droplet size of less than about 1 micron; (ii) the nanoemulsion is diluted resulting in a formulation of about 0.5% to about 60% nanoemulsion; (iii) the viscosity of the nanoemulsion is less than about 1000 cp; and (iv) the nanoemulsion enhances delivery of the quaternary ammonium compound into tissue by at least about 25% as compared to a solution with the same concentration of the same quaternary ammonium compound but lacking a nanoemulsion, and as compared to a nanoemulsion with a viscosity greater than about 1000 cp. In another aspect of the disclosure, the quaternary ammonium compound is a cationic surfactant or is part of a zwitterionic surfactant. Optionally the nanoemulsion can comprise at least one organic solvent.

In one aspect, a composition for topical, transdermal, mucosal (e.g. intranasal, ocular, buccal, vaginal) or oral application or administration is provided, the composition comprising an oil-in-water nanoemulsion, the nanoemulsion comprising: (a) an aqueous phase; (b) at least one oil; (c) at least one quaternary ammonium compound; and (d) at least one nonionic surfactant. The composition can additionally and optionally comprise an active/therapeutic agent. For the composition, (i) the droplets of the nanoemulsion have a mean droplet size of less than about 1 micron; (ii) the nanoemulsion is diluted resulting in a formulation of about 0.5% to about 60% nanoemulsion; (iii) the zeta potential of the nanoemulsion is greater than about 20 mV; and (iv) the nanoemulsion enhances delivery of the quaternary ammonium compound into tissue by at least about 25% as compared to a solution with the same concentration of the same quaternary ammonium compound but lacking a nanoemulsion, and as compared to a nanoemulsion with a zeta potential less than about 20 mV. In another aspect of the disclosure, the quaternary ammonium compound is a cationic surfactant or is part of a zwitterionic surfactant. Optionally the nanoemulsion can comprise at least one organic solvent.

In one aspect, a composition for topical, transdermal, mucosal (e.g. intranasal, ocular, buccal, vaginal) or oral application or administration is provided, the composition comprising an oil-in-water nanoemulsion, the nanoemulsion comprising: (a) an aqueous phase; (b) at least one oil; (c) at least one quaternary ammonium compound; and (d) at least one nonionic surfactant. The composition can additionally and optionally comprise an active/therapeutic agent. For the composition, (i) the droplets of the nanoemulsion have a mean droplet size of less than about 1 micron; (ii) the nanoemulsion is diluted resulting in a formulation of about 0.5% to about 60% nanoemulsion; (iii) at least about 33% of the quaternary ammonium compound is entrapped in the oil phase of the nanoemulsion and at least about 0.2% of the weight of the oil phase of the nanoemulsion is attributed to the quaternary ammonium compound; and (iv) the nanoemulsion enhances delivery of the quaternary ammonium compound into tissue by at least about 25% as compared to a solution with the same concentration of the same quaternary ammonium compound but lacking a nanoemulsion, and as compared to a nanoemulsion with less than about 0.2% of the weight of the oil phase of the nanoemulsion attributed to the quaternary ammonium compound. In another aspect of the disclosure, the quaternary ammonium compound is a cationic surfactant or is part of a zwitterionic surfactant. Optionally the nanoemulsion can comprise at least one organic solvent.

In one aspect, a composition for topical, transdermal, mucosal (e.g. intranasal, ocular, buccal, vaginal) or oral application or administration is provided, the composition comprising an oil-in-water nanoemulsion, the nanoemulsion comprising: (a) an aqueous phase; (b) at least one oil; (c) at least one quaternary ammonium compound; and (d) at least one nonionic surfactant. The composition can additionally and optionally comprise an active/therapeutic agent. For the composition, (i) the droplets of the nanoemulsion have a mean droplet size of less than about 1 micron; (ii) the nanoemulsion is diluted resulting in a formulation of about 0.5% to about 60% nanoemulsion; (iii) the mean droplet size of the nanoemulsion does not change by more than about 10% after centrifuging the nanoemulsion at a speed of 200,000 rpm for one hour; and (iv) the nanoemulsion enhances delivery of the quaternary ammonium compound into tissue by at least about 25% as compared to a solution with the same concentration of the same quaternary ammonium compound but lacking a nanoemulsion, and as compared to a nanoemulsion with a mean droplet size that changes by more than about 10% after centrifuging the nanoemulsion at a speed of 200,000 rpm for one hour. In another aspect of the disclosure, the quaternary ammonium compound is a cationic surfactant or is part of a zwitterionic surfactant. Optionally the nanoemulsion can comprise at least one organic solvent.

For all of the compositions described herein, after a single application or administration of the composition to the dermis, epidermis, mucosa, and/or squamous epithelium: (a) the composition delivers at least about 25% more of quaternary ammonium compound (and/or additional active/therapeutic agent) to the epidermis; and/or (b) the composition delivers at least about 25% more of the quaternary ammonium compound (and/or additional active/therapeutic agent) to the dermis; (c) the composition delivers at least about 25% more of the quaternary ammonium compound (and/or additional active/therapeutic agent) to the mucosa; and/or (d) the composition delivers at least about 25% more of the quaternary ammonium compound (and/or additional active/therapeutic agent) to the squamous epithelium, all as compared to a composition comprising the same quaternary ammonium compound (and/or additional active/therapeutic agent) at the same concentration but lacking a nanoemulsion, measured at any suitable time period after administration or application.

In other embodiments, after a single application or administration of the composition topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal), or orally the composition delivers at least about 25%, at least about 50%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, at least about 325%, at least about 350%, at least about 375%, at least about 400%, at least about 425%, at least about 450%, at least about 475%, or at least about 500% more of the quaternary ammonium compound (and/or additional active/therapeutic agent) to the epidermis, dermis, mucosa, and/or squamous epithelium as compared to a composition comprising the same quaternary ammonium compound (and/or additional active/therapeutic agent) at the same concentration but lacking a nanoemulsion, and applied using the same method, measured at any suitable time point after application or administration.

In some embodiments, after a single application or administration of the composition topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal), or orally the composition delivers at least about 1.25×, at least about 1.5×, at least about 1.75×, at least about 2×, at least about 2.25×, at least about 2.5×, at least about 2.75×, at least about 3×, at least about 3.25×, at least about 3.5×, at least about 3.75×, at least about 4×, at least about 5×, at least about 6×, at least about 7×, at least about 8×, at least about 9×, or at least about 10× more of the quaternary ammonium compound (and/or additional active/therapeutic agent) to the epidermis, dermis, mucosa, and/or squamous epithelium as compared to a composition comprising the same quaternary ammonium compound (and/or additional active/therapeutic agent) at the same concentration but lacking a nanoemulsion, and applied using the same method, measured at any suitable time point after application or administration.

For all of the compositions described herein, after a single administration or application of the composition: (a) the composition has a longer residence time at the site of application as compared to a composition comprising the same quaternary ammonium compound (and/or additional active/therapeutic agent) at the same concentration but lacking a nanoemulsion, wherein the longer residence time is determined by comparing the amount of the quaternary ammonium compound (and/or additional active/therapeutic agent) present at the site of application for the nanoemulsion composition as compared to the non-nanoemulsion composition, measured at any suitable time period after administration or application; and/or (b) the composition delivers at least about 25% more, at least about 50% more, at least about 75% more, at least about 100% more, at least about 125% more, at least about 150% more, at least about 175% more, or at least about 200% more of the quaternary ammonium compound (and/or additional active/therapeutic agent) to the epidermis, dermis, nasal tissue, mucosa, and/or squamous epithelium as compared to a composition comprising the same quaternary ammonium compound (and/or additional active/therapeutic agent) at the same concentration but lacking a nanoemulsion, measured at any suitable time period after administration or application. In other aspects of the compositions described herein, the longer residence time can be an increase of about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, or about 200%.

For all of the compositions described herein, when the composition is applied to skin, nasal tissue, mucosa, and/or squamous epithelium, then the composition can result in increased skin, nasal tissue, mucosa, and/or squamous epithelium hydration as compared to a composition comprising the same quaternary ammonium compound (and/or additional active/therapeutic agent) at the same concentration but lacking a nanoemulsion, measured at any suitable time period after application. Optionally, the increase in skin, nasal tissue, mucosa, and/or squamous epithelium hydration can be about 25%, about 50%, about 75%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, about 325%, about 350%, about 375%, about 400%, about 425%, about 450%, about 475%, about 500%, about 525%, about 550%, about 575%, about 600%, about 625%, about 650%, about 675%, about 700%, about 725%, about 750%, about 775%, about 800%, about 825%, about 850%, about 875%, about 900%, about 925%, about 950%, about 975%, or about 1000%.

In one aspect, all of the compositions described herein are non-toxic in humans and animals. In another embodiment, the composition is not systemically toxic to the subject. In some embodiments, the composition is non-toxic in humans and animals. In yet a further embodiment, the composition produces minimal or no inflammation upon administration or application.

In another aspect, all of the compositions described herein are thermostable. In yet another aspect, all of the compositions described herein are (a) stable for at least 3 months at 50° C.; and/or (b) stable for at least 3 months at 40° C.; and/or (c) stable for at least 3 months at 25° C.; and/or (d) the composition is stable for at least 3 months at 5° C.; and/or (e) the composition is stable at 5° C. for up to at least 60 months; and/or (f) the composition is stable at 50° C. for up to at least 12 months.

In another aspect, the compositions described herein have broad spectrum antimicrobial activity; and/or the composition kills at least about 99.9% of gram positive and gram negative bacteria following a 60 second exposure using the ASTM E2315-16 Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure. For example, the gram positive bacteria can be selected from the group consisting of Staphylococcus, Enterococcus, Methicillin-resistant Staphylococcus aureus (MRSA), and Community Associated-MRSA (CA-MRSA); and/or the gram negative bacteria can be selected from the group consisting of Pseudomonas, Serratia, Acinetobacter, and Klebsiella.

Further, the compositions described herein can be effective in killing microorganisms when applied topically, intranasally, mucosally, vaginally, and/or via the squamous epithelium, wherein the microorganism is selected from the group consisting of: (a) a microorganism population derived from a bacteria, a fungus, a protozoa, a virus, or any combination thereof, (b) bacteria comprising vegetative bacteria, bacterial spore, or a combination thereof, (c) bacteria comprising Gram negative bacteria, Gram positive bacteria, an acid fast bacilli, or a combination thereof; (d) bacteria comprising Bacillus anthracis, Bacillus cereus, Bacillus circulans, Bacillus megaterium, Bacillus subtilis, Clostridium botulinum, Clostridium tetani, Clostridium perfringens, Haemophilus influenzae, Neisseria gonorrhoeae, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Vibrio cholerae, Staphylococcus aureus, Yersinia species, Gardnerella vaginalis, Mobiluncus species, Mycoplasma hominis, Salmonella species, Shigellae species, Pseudomonas species, Escherichia species, Klebsiella species, Proteus species, Enterobacter species, Serratia species, Moraxella species, Legionella species, Bordetella species, Helicobacter species, Arthobacter species, Micrococcus species, Listeria species, Corynebacteria species, Planococcus species, Nocardia species, Rhodococcus species, Mycobacteria species, Acinetobacter species, Staphylococcus species, Enterococcus species, Methicillin-resistant Staphylococcus aureus (MRSA), and Community Associated-MRSA (CA-MRSA), Chlamydia species, and any combination thereof, (e) virus belonging to a family selected from the group consisting of Orthomyxoviridae, Retroviridae, African Swine Fever Viruses, Papovaviridae, Hepadnaviridae, Coronaviridae, Flaviviridae, Togaviridae, Picornaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae; (f) Orthomyxovirdae virus which is influenza virus, herpes simplex, herpes zoster, sendai virus, sindbis virus, pox virus, small pox virus, vaccinia virus, influenza virus, seasonal flu virus, or pandemic flu virus; (g) Ebolavirus; (h) Respiratory syncytial virus (RSV); (i) Rotavirus; (j) Norovirus; (k) flavivirus which is zika virus, West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, and insect-specific flaviviruses; (l) Coronavirus which is Middle East Respiratory Syndrome Coronavirus (MERS-CoV); (m) Retroviridae which is human immunodeficiency virus, west nile virus, hanta virus, or human papilloma virus; (n) fungus which is a yeast or a filamentous fungus; (o) filamentous fungus which is Aspergillus species or a dermatophyte; (p) dermatophyte which is Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum, or Epidermophyton floccosum; and (q) fungus comprising Cladosporium, Fusarium, Alternaria, Curvularia, Aspergillus, Penicillium, Candida.

In some embodiments, the ratio of the concentration of the quaternary ammonium compound (or optionally a cationic compound) to nonionic surfactant is: (a) about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24, about 1:25, about 1:26, or about 1:27; (b) about 4:1 to about 1:27; (c) about 1:2, about 1:5, about 1:9, about 1:14, or about 1:18; (d) about 1:2 to about 1:18; and/or (e) about 1:5 to about 1:14.

In some embodiments, the nonionic surfactant is Generally Recognized as Safe (GRAS) by the US Food and Drug Administration. In some embodiments, the nonionic surfactant is a polysorbate, a poloxamer, or a combination thereof. In some embodiments, the nonionic surfactant is (a) selected from the group consisting of polysorbate 20 (TWEEN 20), poloxamer 407, or any combination thereof, (b) selected from the group consisting of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, or any combination thereof, (c) selected from the group consisting of poloxamer 407, poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, Poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, poloxamer 407, poloxamer 105 Benzoate, poloxamer 182 Dibenzoate, or any combination thereof, (d) selected from the group consisting of an ethoxylated surfactant, an alcohol ethoxylated, an alkyl phenol ethoxylated, a fatty acid ethoxylated, a monoalkaolamide ethoxylated, a sorbitan ester ethoxylated, a fatty amino ethoxylated, an ethylene oxide-propylene oxide copolymer, Bis(polyethylene glycol bis[imidazoyl carbonyl]), nonoxynol-9, Bis(polyethylene glycol bis[imidazoyl carbonyl]), Brij® 35, Brij® 56, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor® EL, Decaethylene glycol monododecyl ether, N-Decanoyl-N-methylglucamine, n-Decyl alpha-D-glucopyranoside, Decyl beta-D-maltopyranoside, n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside, n-Dodecyl beta-D-maltoside, n-Dodecyl beta-D-maltoside, Heptaethylene glycol monodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethylene glycol monotetradecyl ether, n-Hexadecyl beta-D-maltoside, Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-630, Methyl-6-O—(N-heptylcarbamoyl)-alpha-D-glucopyranoside, Nonaethylene glycol monododecyl ether, N— N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl-beta-D-glucopyranoside, Pentaethylene glycol monodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol monohexyl ether, Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctyl ether, Polyethylene glycol diglycidyl ether, Polyethylene glycol ether W-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate, Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillaja bark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85, Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tergitol, Tergitol, Type TMN-10, Tergitol, Type TMN-6, Tetradecyl-beta-D-maltoside, Tetraethylene glycol monodecyl ether, Tetraethylene glycol monododecyl ether, Tetraethylene glycol monotetradecyl ether, Triethylene glycol monodecyl ether, Triethylene glycol monododecyl ether, Triethylene glycol monohexadecyl ether, Triethylene glycol monooctyl ether, Triethylene glycol monotetradecyl ether, Triton CF-21, Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, Triton X-15, Triton X-151, Triton X-200, Triton X-207, Triton® X-114, Triton® X-165, Triton® X-305, Triton® X-405, Triton® X-45, Triton® X-705-70, Tyloxapol, n-Undecyl beta-D-glucopyranoside, semi-synthetic derivatives thereof, or any combinations thereof, or (e) any combination thereof.

In some embodiments, the quaternary ammonium compound is monographed by the US FDA as an antiseptic for topical use. In some embodiments, the monographed quaternary ammonium compound is benzalkonium chloride (BZK), cetylpyridimium chloride (CPC), benzethonium chloride (BEC), dioctadecyl dimethyl ammonium chloride (DODAC), or octenidine dihydrochloride (OCT).

In some embodiments, the quaternary ammonium compound is benzalkonium chloride (BZK). In some embodiments, the BZK is present in a concentration of: (a) from about 0.05% to about 0.40%; (b) from about 0.05% to about 0.20%; (c) from about 0.10% to about 0.20%; or (d) from about 0.10% to about 0.15%. In some embodiments, the BZK is present in a concentration of about 0.13%.

In some embodiments, the quaternary ammonium compound is cetylpyridimium chloride (CPC). In some embodiments, the CPC is present in a concentration of: (a) from about 0.05% to about 0.40%; (b) from about 0.05% to about 0.20%; (c) from about 0.15% to about 0.30%; or (d) from about 0.08% to about 0.15%. In some embodiments, the CPC is present in a concentration of about 0.20% or about 0.10%.

In some embodiments, the quaternary ammonium compound is benzethonium chloride (BEC). In some embodiments, the BEC is present in a concentration of: (a) from about 0.05% to about 1%; or (b) from about 0.10% to about 0.30%. In some embodiments, the BEC is present in a concentration of about 0.20%.

In some embodiments, the quaternary ammonium compound is dioctadecyl dimethyl ammonium chloride (DODAC). In some embodiments, the DODAC is present in a concentration of: (a) from about 0.05% to about 1%; or (b) from about 0.10% to about 0.40%. In some embodiments, the DODAC is present in a concentration of about 0.20%.

In some embodiments, the quaternary ammonium compound is octenidine dihydrochloride (OCT). In some embodiments, the OCT is present in a concentration of: (a) from about 0.05% to about 1%; or (b) from about 0.10% to about 0.40%. In some embodiments, the OCT is present in a concentration of about 0.20%.

In some embodiments, the quaternary ammonium compound is part of a zwitterionic surfactant.

In some embodiments, the oil is an animal oil, plant oil, or a vegetable oil. In some embodiments, the oil comprises soybean oil, mineral oil, avocado oil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil, sunflower oil, fish oils, flavor oils, cinnamon bark, coconut oil, cottonseed oil, flaxseed oil, pine needle oil, silicon oil, essential oils, water insoluble vitamins, or a combination thereof. In some embodiments, the oil comprises soybean oil.

In some embodiments, the composition additionally comprises an organic solvent which can be: (a) a C₁-C₁₂alcohol, diol, or triol, a dialkyl phosphate, a trialkyl phosphate, or a combination thereof; or (b) ethanol, methanol, isopropyl alcohol, glycerol, medium chain triglycerides, diethyl ether, ethyl acetate, acetone, dimethyl sulfoxide (DMSO), acetic acid, n-butanol, butylene glycol, perfumers alcohol, isopropanol, n-propanol, formic acid, propylene glycol, glycerol, sorbitol, industrial methylated spirit, triacetin, hexane, benzene, toluene, diethyl ether, chloroform, 1,4-dioxane, tetrahydrofuran, dichloromethane, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, formic acid, a semi-synthetic derivative thereof, or a combination thereof. In some embodiments the solvent is ethanol.

In some embodiments, the composition further comprises a chelating agent. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(p-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), or a combination thereof. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

In some embodiments, the composition comprises: (a) BZK at a concentration of about 0.13%; (b) poloxamer 407; (c) glycerol; (d) soybean oil; (e) EDTA; and (e) water.

In some embodiments, the nanoemulsion comprises droplets having an average particle size diameter of: (a) less than about 1000 nm; (b) about 150 nm to about 800 nm; or (c) about 300 nm to about 600 nm.

In some embodiments, the composition further comprises a therapeutic or active agent. In some embodiments, the therapeutic agent is (a) an antimicrobial agent; an antiviral agent; an antifungal agent; vitamin; homeopathic agent; anti-inflammatory agent; keratolytic agent; antipruritic agent; pain medicine; steroid; anti-acne drug; macromolecule; small molecule; small, lipophilic, low-dose drug; protein; peptide; or an antigen; and/or (b) is recognized as being suitable for transdermal, intranasal, mucosal, vaginal, or topical administration or application; and/or (c) has low oral bioavailability but is suitable for nasal administration when formulated into a nanoemulsion; and/or (d) is a lipophilic agent having poor water solubility; and/or (e) present within a nanoemulsion is formulated for transdermal or intranasal administration, where the therapeutic agent when not present in a nanoemulsion is conventionally given via oral, IV or IM administration due to the desire for fast onset of action or because of the difficulty in obtaining suitable bioavailability with other modes of administration; and/or (f) present within a nanoemulsion formulated for topical administration, where the therapeutic agent when not present in a nanoemulsion is conventionally applied via topical administration but does not achieve optimal delivery of the therapeutic agent into the epidermis, dermis, nasal tissue, mucosa, and/or squamous epithelium; and/or (g) selected from the group consisting of a penicillin, a cephalosporin, cycloserine, vancomycin, bacitracin, miconazole, ketoconazole, clotrimazole, polymyxin, colistimethate, nystatin, amphotericin B, chloramphenicol, a tetracycline, erythromycin, clindamycin, an aminoglycoside, a rifamycin, a quinolone, trimethoprim, a sulfonamide, zidovudine, gangcyclovir, vidarabine, acyclovir, poly(hexamethylene biguanide), terbinafine, and a combination thereof; and/or (h) an anti-inflammatory agent which is a steroid or a non-steroidal anti-inflammatory drug; and/or (i) an anti-inflammatory agent which is a steroid which is selected from the group consisting of clobetasol, halobetasol, halcinonide, amcinonide, betamethasone, desoximetasone, diflucortolone, fluocinolone, fluocinonide, mometasone, clobetasone, desonide, hydrocortisone, prednicarbate, triamcinolone, and a pharmaceutically acceptable derivative thereof, and/or (j) an anti-inflammatory agent which is a non-steroidal anti-inflammatory drug selected from the group consisting of aceclofenac, aspirin, celecoxib, clonixin, dexibup6fen, dexketoprofen, diclofenac, diflunisal, droxicam, etodolac, etoricoxib, fenoprofen, flufenamic acid, flurbiprofen, ibuprofen, indomethacin, isoxicam, ketoprofen, ketorolac, licofelone, lornoxicam, loxoprofen, lumiracoxib, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, oxaprozin, parecoxib, phenylbutazone, piroxicam, rofecoxib, salsalate, sulindac, tenoxicam, tolfenamic acid, tolmetin, or valdecoxib.

In some embodiments, the therapeutic agent is present in a concentration of: (a) from about 0.01% to about 10%; (b) from about 0.01% to about 1%; (c) from about 0.01% to about 0.75%; or (d) from about 0.1% to about 0.5% (per dose). For an antigen, the amount present can be from about 1 to 250 μg/per dose.

In some embodiments, when the composition further comprises a therapeutic or active agent, after a single application of the composition topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal), or orally, the composition delivers a greater amount of therapeutic agent to the dermis, epidermis, mucosa, and/or squamous epithelium as compared to a composition comprising the same therapeutic agent at the same concentration but lacking a nanoemulsion, and applied using the same method, measured at any suitable time point after application. For example, in some embodiments, after a single application of the composition to skin, mucosa, or squamous epithelium, the composition delivers (i) at least about 25% more of the therapeutic or active agent to the epidermis, and/or (ii) at least about 25% more of the therapeutic or active agent to the dermis, and/or (iii) at least about 25% more of the therapeutic or active agent to the nasal tissue, and/or (iv) at least about 25% more of the therapeutic or active agent to the mucosa, and/or (v) at least about 25% more of the therapeutic or active agent to the squamous epithelium, and/or (vi) at least about 25% more of the therapeutic or active agent to the systemic circulation following patch, microneedle, transdermal or intranasal application, and/or (vii) at least about 25% more of the therapeutic or active agent to the central nervous system following patch, microneedle, transdermal or intranasal application, all as compared to a composition comprising the same therapeutic or active agent at the same concentration but lacking a nanoemulsion, and applied or delivered using the same method, measured at any suitable time point after administration or application.

In one embodiment, after a single administration or application of the composition, the composition delivers at least about 25% more, at least about 50% more, at least about 75% more, at least about 100% more, at least about 125% more, at least about 150% more, at least about 175% more, or at least about 200% more of the therapeutic agent to the epidermis, dermis, mucosa, and/or squamous epithelium, as compared to a composition comprising the same therapeutic agent at the same concentration but lacking a nanoemulsion, measured at any suitable time period after administration or application.

In some embodiments, when the composition further comprises a therapeutic or active agent, after a single application or administration of the composition topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal), or orally, the composition delivers at least about 25%, at least about 50%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, at least about 325%, at least about 350%, at least about 375%, at least about 400%, at least about 425%, at least about 450%, at least about 475%, or at least about 500% more of the therapeutic agent to the dermis, epidermis, mucosa and/or squamous epithelium as compared to a composition comprising the same therapeutic agent at the same concentration but lacking a nanoemulsion, and applied using the same method, measured at any suitable time point after application or administration.

In some embodiments, when the composition further comprises a therapeutic or active agent, after a single application or administration of the composition topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal), or orally, the composition has a longer residence time at the site of application or administration as compared to a composition comprising the same therapeutic agent at the same concentration but lacking a nanoemulsion, and applied using the same method, measured at any suitable time point after application. The longer residence time can be determined by comparing the amount of the therapeutic agent present at the site of application or administration for the nanoemulsion composition as compared to the non-nanoemulsion composition, measured at any suitable time point after application. The longer residence time at the site of application can be, for example, an increase of about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, or about 200%, as compared to the residence time of the same therapeutic agent, present at the same concentration, and applied using the same method, measured at any suitable time point after application or administration.

For any embodiment where a non-nanoemulsion formulation is compared to a nanoemulsion formulation, measurements can be taken at any suitable time point minutes or hours after application or administration, such as at about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours after application or administration.

In some embodiments, the composition enters the epidermis, dermis, squamous epithelium, or a combination thereof. In some embodiments, the composition permeates into the epidermis, dermis, mucosa, and/or squamous epithelium via the follicular route using skin pores and hair follicles. In some embodiments, the composition diffuses through the skin, skin pores, mucosa, squamous epithelium, nail, scalp, hair follicles, lateral or proximal folds, nail, hyponichium, or any combination thereof.

In some embodiments, the composition is formulated into a dosage form selected from the group consisting of oral, topical, buccal, sublingual, nasal, inhalation, rectal, or suppository dosage form; and/or a dosage form selected from the group consisting of a liquid, lotion, cream, dry powder/talc, ointment, salve, spray, aerosol, tablet, syrup, capsule, thin film, drops, or transdermal patch; and/or a liquid dosage form, solid dosage form, or semisolid dosage form.

In some embodiments, the composition is formulated into a vaccine or immunotherapy treatment.

In one aspect, the compositions described herein can be autoclaved, and optionally the composition retains its structural and/or chemical integrity following autoclaving.

Also provided herein in one aspect is a dermal patch or wipe impregnated or saturated with or incorporating any composition described herein. In some embodiments, the wipe dispenses a greater amount of the quaternary ammonium compound and/or therapeutic/active agent to an application site, as compared to a wipe impregnated or saturated with or incorporating a composition comprising the same quaternary ammonium compound and/or therapeutic/active agent at the same concentration but lacking a nanoemulsion. Measurements can be taken at any suitable time point hours after application, such as at about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours after application.

In some embodiments, the wipe dispenses about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200% more of the quaternary ammonium compound and/or therapeutic/active agent to an application site, as compared to a wipe impregnated or saturated with or incorporating a composition comprising the same quaternary ammonium compound and/or therapeutic/active agent at the same concentration but lacking a nanoemulsion.

In other embodiments provided herein is a nasal swab saturated or impregnated with or incorporating any composition described herein. The swab can also be packaged in a kit with a container comprising a composition described herein, with the swab being exposed to the nanoemulsion prior to use. Such swabs are useful for sanitizing or reducing bacteria present in or on nasal mucosa, which is beneficial in controlling infections, such as in hospital settings.

In some embodiments, the composition, wipe, or swab can be autoclaved. In another embodiment, following autoclaving the composition (either alone or embedded in the wipe or swab) retains its structural and chemical integrity, meaning that there is no significant particle size growth and none of the component compound are degraded.

In another embodiment, encompassed is a dry eye formulation comprising a composition as described herein. In yet another embodiment, the eye drop formulation can further comprise a cyclosporine or another therapeutic agent used to treat eye conditions.

In another embodiment, encompassed is an ontological (ear) formulation comprising a composition described herein.

Finally, in yet another embodiment encompassed is a formulation for treating vaginitis, comprising a composition described herein.

Further, provided in one aspect is a method of reducing or killing a microorganism population in a human or animal subject thereof, comprising topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal) or orally administering or applying to the human or animal subject any one of the compositions, wipes or swabs described herein. In one aspect of this embodiment, the composition enters the epidermis, dermis, nasal tissue, mucosa, squamous epithelium, or any combination thereof. In another aspect, the composition permeates into the epidermis, dermis, nasal tissue, mucosa, and/or squamous epithelium via the follicular route using skin pores and hair follicles. In a further aspect, the composition diffuses through the skin, skin pores, nail, nasal tissue, mucosa, squamous epithelium, scalp, hair follicles, lateral or proximal folds, nail, hyponichium, or any combination thereof.

Also encompassed is a method of decontaminating a surface comprising applying a composition, dermal patch or wipe, or swab described herein, wherein the method comprises applying the composition, dermal patch or wipe, or swab to the surface.

Also encompassed is a method of delivering an active agent to a subject, comprising administering a composition according to the disclosure, wherein the composition comprises at least one therapeutic agent as described herein. In one embodiment, the therapeutic agent is not an antimicrobial agent. The composition can be delivered, for example, topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal) or orally. In another embodiment, the composition enters the epidermis, dermis, nasal tissue, mucosa, squamous epithelium, or any combination thereof. In yet another embodiment, the composition permeates into the epidermis, dermis, nasal tissue, mucosa, and/or squamous epithelium via the follicular route using skin pores and hair follicles. Further, the composition can diffuse through the skin, skin pores, nail, nasal tissue, mucosa, squamous epithelium, scalp, hair follicles, lateral or proximal folds, nail, hyponichium, or any combination thereof.

In another embodiment, encompassed is a method of delivering an antigen, protein, or peptide to a subject, comprising administering a composition according to the invention to a subject, wherein the composition comprises at least one antigen, protein, or peptide.

In one embodiment, the composition delivers at least about 25% more of the antigen, protein or peptide to the epidermis, dermis, nasal tissue, mucosa, and/or squamous epithelium as compared to a composition comprising the same antigen, protein or peptide at the same concentration but lacking a nanoemulsion, measured at any suitable time period after administration or application. In one embodiment, administration of the composition results in a protective immune response. For example, the protective immune response can comprises a Th1 immune response, a Th2 immune response, a Th17 immune response, or any combination thereof. In another embodiment, the composition is not systemically toxic to the subject. In yet a further embodiment, the composition produces minimal or no inflammation upon administration.

The antigen can be, for example, recombinant, whole virus, isolated, or a protein, killed pathogen, or isolated fragment thereof. In addition, in another embodiment the antigen can be (a) a viral protein or antigen from a virus belonging to a family selected from the group consisting of Orthomyxoviridae, Retroviridae, African Swine Fever Viruses, Papovaviridae, Hepadnaviridae, Coronaviridae, Flaviviridae, Togaviridae, Picornaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae; (b) a viral protein or antigen from an orthomyxovirdae virus which is influenza virus, herpes simplex, herpes zoster, sendai virus, sindbis virus, pox virus, small pox virus, vaccinia virus, influenza virus, seasonal flu virus, or pandemic flu virus; (c) a viral protein or antigen from Ebolavirus; (d) a viral protein or antigen from Respiratory syncytial virus (RSV); (e) a viral protein or antigen from Rotavirus; (f) a viral protein or antigen from Norovirus; (g) a viral protein or antigen from flavivirus which is zika virus, West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, and insect-specific flaviviruses; (h) a viral protein or antigen from Coronavirus which is Middle East Respiratory Syndrome Coronavirus (MERS-CoV); (i) a viral protein or antigen from Retroviridae which is human immunodeficiency virus, west nile virus, hanta virus, or human papilloma virus; (j) a bacterial protein or antigen; and/or (k) a fungal protein or antigen; and/or (l) a food allergen protein or antigen; and/or (m) an aero allergen protein or antigen; and/or (n) a cancer protein or antigen.

In one embodiment, the composition comprising at least one antigen, protein, or peptide can be administered orally, topically, intranasally, vaginally, mucosally, or via transdermal administration.

The foregoing general description and following brief description of the drawings and the detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosed as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the disclosed.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a diagram of an in-vitro diffusion cell apparatus.

FIG. 2 shows epidermal levels of BZK (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulations (0.13% BZK) with surfactant blend ratios 1:5 and 1:9 and Purell® Foam (0.13% BZK).

FIG. 3 shows dermal levels of BZK (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulations (0.13% BZK) with surfactant blend ratios 1:5 and 1:9 and Purell® Foam.

FIG. 4 shows the epidermal levels of BZK (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulations (0.13% BZK) with different surfactant blend ratios (5:1, 2:1, 1:1, 1:2, 1:5, 1:9, 1:14, 1:18, and 1:27) and Purell® Foam (0.13% BZK).

FIG. 5 shows the dermal levels of BZK (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulations (0.13% BZK) with different surfactant blend ratios (5:1, 2:1, 1:1, 1:2, 1:5, 1:9, 1:14, 1:18, and 1:27) and Purell® Foam (0.13% BZK).

FIG. 6 shows the log killing of NE-2 (surfactant blend ratio: 1:5; 0.13% BZK) microorganisms following one-minute exposure.

FIG. 7 shows skin hydration study results of NE-1 (surfactant blend ratio: 1:5; 0.13% BZK) and Purell® Foam (0.13% BZK).

FIG. 8 shows the % of BZK dispensed from the wipe (spunlace washcloth) with aqueous BZK (0.13% BZK), NE-1 (surfactant blend ratio: 1:9; 0.13% BZK), and Purell® Foam (0.13% BZK) at the following time points: initial, 2 hours and 5 days.

FIG. 9 shows the % of BZK dispensed from the wipe (airlaid washcloth) with aqueous BZK (0.13% BZK), NE-1 (surfactant blend ratio: 1:9; 0.13% BZK), and Purell® Foam (0.13% BZK) at the following time points: initial, 2 hours and 5 days.

FIG. 10 shows a diagram of the mucin coated Transwell® membrane in a 24 well plate.

FIG. 13 shows the serum levels of Compound A following one administration with the commercially available intranasal product of Compound A (0.50% Compound A) and the NE-2 and NE-4 formulations (surfactant blend ratios: 1:5 and 1:2) with 0.50% of Compound A.

FIG. 15 shows the dermal levels of terbinafine (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulation (surfactant ratio of 1:9 with 1% terbinafine) with Lamisil AT® (1% terbinafine).

FIG. 16 shows the epidermal levels of miconazole (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of the NE-1 formulation (surfactant ratio of 1:12 with 2% miconazole) with Monistat® (2% miconazole).

FIG. 17 shows the dermal levels of miconazole (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulation (surfactant ratio of 1:12 with 2% miconazole) with Monistat® (2% miconazole).

FIG. 18 shows the epidermal levels of salicylic acid (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of the NE-1 formulation (surfactant ratio of 1:12 with 1% and 2% salicylic acid) with Dermarest® (3% salicylic acid).

FIG. 19 shows the epidermal levels of hydrocortisone (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of the NE-1 formulation (surfactant ratio of 1:9 with 1% hydrocortisone) with Cortizone-10® (1% hydrocortisone).

FIG. 20 shows the dermal levels of hydrocortisone (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulation (surfactant ratio of 1:9 with 1% hydrocortisone) with Cortizone-10® (1% hydrocortisone).

FIG. 21 shows the epidermal levels of adapalene (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of the NE-1 formulation (surfactant ratio of 1:9 with 0.1% adapalene) with Differin® (0.1% adapalene).

FIG. 22 shows the dermal levels of adapalene (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulation (surfactant ratio of 1:9 with 0.1% adapalene) with Differin® (0.1% adapalene).

FIG. 23 shows the epidermal levels of peanut proteins Ara h2, Ara h1, Ara h3, and Ara hX (μg/g tissue) in human abdominal skin following one application (occluded dose of 100 μl/cm², measured at 18 hours) of the NE-1 formulation (surfactant ratio of 1:6 with 0.1% peanut protein) with an aqueous formulation (0.1% peanut protein).

FIG. 24 shows the dermal levels of peanut proteins Ara h2, Ara h1, Ara h3, and Ara hX (μg/g tissue) in human abdominal skin following one application (occluded dose of 100 μl/cm², measured at 18 hours) of NE-1 formulation (surfactant ratio of 1:6), NE-2 formulation (surfactant ratio of 1:6), and NE-3 formulation (surfactant ratio of 1:9) with 0.1% peanut protein with aqueous formulation (0.1% peanut protein).

FIG. 25 shows the epidermal levels of BEC (μg/g tissue) in human abdominal skin following one application (single dose of 100 μl/cm², measured at 24 hours) of the NE formulation (surfactant ratio of 1:6 with 0.2% BEC) with an aqueous formulation (0.2% BEC), New-Skin® spray (0.2% BEC), and CVS Liquid Bandage (0.2% BEC).

FIG. 26 shows the dermal levels of BEC (μg/g tissue) in human abdominal skin following one application (single dose of 100 μl/cm², measured at 24 hours) of the NE formulation (surfactant ratio of 1:6 with 0.2% BEC) with an aqueous formulation (0.2% BEC), New-Skin® spray (0.2% BEC), and CVS Liquid Bandage (0.2% BEC).

FIG. 27 shows the epidermal levels of PCMX (μg/g tissue) in human abdominal skin following one application (single dose of 100 μl/cm², measured at 24 hours) of the NE formulation (surfactant ratio of 1:6 with 3.0% PCMX) with an 70% ethanol formulation (3% PCMX).

FIG. 28 shows the dermal levels of PCMX (μg/g tissue) in human abdominal skin following one application (single dose of 100 μl/cm², measured at 24 hours) of the NE formulation (surfactant ratio of 1:6 with 3.0% PCMX) with an 70% ethanol formulation (3% PCMX).

FIG. 30 shows the dermal levels of chlorhexidine (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulation (surfactant ratio of 1:9 with 2% chlorhexidine) with a 70% IPA solution containing 2% chlorhexidine.

FIG. 31 shows epidermal permeability results for nanoemulsion formulations of various nanoemulsion concentrations (0.5%, 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, and 60%) and Purell® Foam (0.13% BZK).

FIG. 32 shows dermal permeability results for nanoemulsion formulations of various nanoemulsion concentrations (0.5%, 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, and 60%) and Purell® Foam (0.13% BZK).

FIG. 33 shows epidermal permeability results for nanoemulsion formulations relative to their viscosity (1.33 cp, 1.36 cp, 1.37 cp, 1.39 cp, 1.52 cp, 2.06 cp, 3.32 cp, 6.08 cp, and 261 cp) and Purell® Foam (0.13% BZK).

FIG. 34 shows dermal permeability results for nanoemulsion formulations relative to their viscosity (1.33 cp, 1.36 cp, 1.37 cp, 1.39 cp, 1.52 ep, 2.06 cp, 3.32 ep, 6.08 cp, and 261 cp) and Purell® Foam (0.13% BZK).

FIG. 35 shows epidermal permeability results for nanoemulsion formulations relative to their zeta potential (75.2 mV, 47.6 mV, 34.7 mV, 34.8 mV, 28.3 mV, 27.8 mV, 27.0 mV, 27.4 mV) and Purell® Foam (0.13% BZK).

FIG. 36 shows dermal permeability results for nanoemulsion formulations relative to their zeta potential (75.2 mV, 47.6 mV, 34.7 mV, 34.8 mV, 28.3 mV, 27.8 mV, 27.0 mV, 27.4 mV) and Purell® Foam (0.13% BZK).

FIG. 37 shows epidermal permeability results for nanoemulsion formulations relative to their entrapment of the quaternary ammonium salt (19.86%, 11.04%, 2.85%, 1.48%, 0.86%, 0.57%, 0.32%, 0.26%, 0.21%) and Purell® Foam (0.13% BZK).

FIG. 38 shows dermal permeability results for nanoemulsion formulations relative to their entrapment of the quaternary ammonium salt (19.86%, 11.04%, 2.85%, 1.48%, 0.86%, 0.57%, 0.32%, 0.26%, 0.21%) and Purell® Foam (0.13% BZK).

FIG. 39 shows epidermal permeability results for nanoemulsion formulations of the disclosure relative to the formulation's stability as measured by the percent (%) change in mean droplet size following prolonged centrifugation (0.2%, 2.0%, 0.5%, 1.8%, 2.9%, 2.2%, 5.4%, 0.2%, 0.5%) and Purell® Foam (0.13% BZK).

FIG. 40 shows dermal permeability results for nanoemulsion formulations of the disclosure relative to the formulation's stability as measured by the percent (%) change in mean droplet size following prolonged centrifugation (0.2%, 2.0%, 0.5%, 1.8%, 2.9%, 2.2%, 5.4%, 0.2%, 0.5%) and Purell® Foam (0.13% BZK).

FIG. 41 shows epidermal levels of lidocaine delivered by Salonpas patch (left), nanoemulsion (NB liquid, center), and nanoemulsion patch (NB patch, right).

FIG. 42 shows dermal levels of lidocaine delivered by Salonpas patch (left), nanoemulsion (NB liquid, center), and nanoemulsion patch (NB patch, right).

FIG. 43 shows levels of transdermal lidocaine delivered to the receptor by Salonpas patch (left), nanoemulsion (NB liquid, center), and nanoemulsion patch (NB patch, right).

FIG. 44 shows images depicting nanoemulsion sample after centrifugation. Image taken under normal lighting conditions (left) and corresponding negative image (right).

DETAILED DESCRIPTION
I. Overview

The present invention is directed to the surprising discovery that nanoemulsion compositions comprising a combination of a quaternary ammonium compound (which may be a cationic surfactant or part of a zwitterionic surfactant) and a non-ionic surfactant, and having a narrow range of a ratio of the quaternary ammonium compound to the non-ionic surfactant, have significant and dramatic increased permeation when applied or administered topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal), or orally.

The significant and dramatic permeation can be compared to nanoemulsions having quaternary ammonium compound/non-ionic surfactant ratios outside the narrow range disclosed herein, or as compared to permeation of the quaternary ammonium compound present at the same concentration and applied in the same manner, but in the absence of a nanoemulsion (e.g., using the quaternary ammonium compound as a marker for measuring permeation). Permeation can be measured at any suitable time period following application.

Applicant's data clearly and unequivocally details the surprising and significant results observed with the claimed narrow range of a surfactant blend ratio. Specifically, Example 2 shows that in a comparison of a non-nanoemulsion formulation having 0.13% BKC (Purell® Foam) with nanoemulsion (NE) formulations having 0.13% BZK and surfactant blend ratios of 1:5 and 1:9, the amount of BZK delivered into human abdominal skin epidermal tissue was almost 600% higher for the nanoemulsion formulation having a 1:9 surfactant blend ratio as compared to the non-nanoemulsion formulation (6642 ng BZK/gram tissue, as compared to 953 ng BZK/gram tissue for the Purell® Foam). See also FIGS. 2 (epidermis) and 3 (dermis), showing graphs of levels of BZK (μg/g tissue) following application of one dose of 100 μl/cm³measured 24 hours after application. More specifically, after one application of 0.13% NE formulations to human skin, the nanoemulsion formulation delivered almost 4 to 7 times more BZK into the epidermis as compared to a marketed 0.13% Purell® Foam (FIG. 2). Additionally, with respect to the dermis levels, the nanoemulsion formulation delivered 3 to 4 times more BZK as compared to the marketed product, Purell® Foam, indicating that the BZK was able to penetrate into the deeper dermal levels of the skin from the nanoemulsion formulations (FIG. 3).

As clearly depicted in FIGS. 2 and 3, nanoemulsions having representative surfactant ratios of 1:5 and 1:9 showed dramatic and significantly greater permeation (amount of BZK (ng)/tissue weight (g)) as compared to a non-nanoemulsion formulation having the same quantity of BZK.

A clear bell curve of permeation vs. surfactant blend ratio is depicted in FIGS. 4 and 5, demonstrating that nanoemulsions having a preferred surfactant blend ratio show dramatic and significant increased permeation in the epidermis (FIG. 4) and dermis (FIG. 5) as compared to non-nanoemulsion formulations of the same quaternary ammonium compound at the same concentration (Purell® Foam), and as compared to nanoemulsion formulations having surfactant blend ratios outside the claimed range of about 5:1 up to about 1:27. Outside the claimed surfactant blend ratio, the amount of drug in the epidermis (FIG. 4) and dermis (FIG. 5) is dramatically less. The impact of the claimed narrow range of surfactant blend ratios on permeation was not known prior to the present invention.

This enhanced permeability allows for the nanoemulsion compositions described herein to deliver more of the quaternary ammonium compound to the site of application, as well as any additional therapeutic agent present in the nanoemulsion, and to also have a longer residence time at the site of application as compared to non-nanoemulsion compositions containing the same quaternary ammonium compound present at the same concentration.

The nanoemulsion compositions described herein can also comprise a therapeutic agent suitable for topical, mucosal, vaginal, or intranasal delivery. The enhanced permeability of the nanoemulsions described herein allows for the nanoemulsion compositions to deliver more of the therapeutic agent to site of application, and to also have a longer residence time of the therapeutic agent at the site of application, as compared to non-nanoemulsion compositions containing the same therapeutic agent at the same concentration. The site of application can be, for example, mucosa, the dermis, epidermis, skin, and/or squamous epithelium (the nasal vestibule is completely lined by squamous epithelium), or for orally administered dosage forms the site of absorption can be the stomach tissue or GI tract, depending upon the dosage form design.

For example, as graphically depicted in FIG. 11, the permeation of a representative model therapeutic agent Compound A was significantly greater when present in a nanoemulsion formulation as compared to a non-nanoemulsion formulation, having the same drug concentration. In particular, the commercial product of Compound A, having a drug concentration of 50% present in non-nanoemulsion formulation, showed a cumulative concentration of Compound A (μg/mL) at 6 hours following application of about 325 μg/mL, in contrast to a concentration of about 730 μg/mL for the nanoemulsion having a surfactant ratio of 1:9 and a drug concentration of 50%, an increase in drug permeation of about 125%.

Similarly, Examples 8 and 9 show in vitro and in vivo data, respectively, for a nanoemulsion having a model Compound A incorporated within the nanoemulsion. In vitro all of the nanoemulsion formulations resulted in significantly greater serum levels of Compound A (μg/mL)—all greater than about 3500 μg/mL—as compared to the conventional, non-nanoemulsion formulation having the same compound at the same concentration; e.g., about 2750 μg/mL—a difference of about 30% (FIG. 13). The results from Example 9 demonstrate that greater mucin penetration of Compound A incorporated in a nanoemulsion measured in vitro directly correlates with Compound A penetration in the nasal epithelium in vivo when animals are intranasally treated with the NE-Compound A formulations, and leads to greater systemic drug delivery as compared to the commercially available product containing the same concentration of Compound A.

These results show that nanoemulsion formulations having a preferred surfactant blend ratio significantly enhance the systemic absorption of a representative incorporated therapeutic agent (Compound A) in vivo as compared to a non-nanoemulsion commercial product having the same active at the same concentration. Also demonstrated is that a significantly lower amount of a therapeutic agent can be administered with any one of the nanoemulsion compositions described herein to achieve systemic absorption equivalent or greater than a non-nanoemulsion composition having the same therapeutic agent.

These results show that nanoemulsion formulations having a preferred surfactant blend ratio significantly enhance the permeation of a component therapeutic agent.

Mucosal and transdermal delivery have a variety of advantages as compared to an oral route of administration. In particular, it is used when there is a significant first-pass effect of the liver that can prematurely metabolize drugs. Transdermal delivery also has advantages over hypodermic injections, which are painful, generate dangerous medical waste and pose the risk of disease transmission by needle re-use, especially in developing countries. In addition, transdermal systems are non-invasive and can be self-administered. They can provide release for long periods of time (up to one week). They also improve patient compliance.

Provided in one aspect is a composition for topical, transdermal, mucosal (e.g. intranasal, ocular, buccal, vaginal), or oral administration or application comprising an oil-in-water nanoemulsion, the nanoemulsion comprising an aqueous phase, at least one pharmaceutically acceptable oil, at least one pharmaceutically acceptable organic solvent, at least one pharmaceutically acceptable quaternary ammonium compound selected from the group consisting of benzalkonium chloride (BZK), cetylpyridimium chloride (CPC), benzethonium chloride (BEC), dioctadecyl dimethyl ammonium chloride (DODAC), and octenidine dihydrochloride (OCT); and (e) at least one pharmaceutically acceptable nonionic surfactant. In some embodiments, the droplets of the nanoemulsion have a mean or average droplet size of less than about 1 micron. In some embodiments, the concentration ratio of the quaternary ammonium compound to nonionic surfactant is from about 5: about 1 to about 1: about 27.

These nanoemulsion compositions are useful for a variety of applications, including topical, transdermal (e.g. intranasal, ocular, buccal, vaginal), and oral applications. In the instance where the nanoemulsion composition is applied to the skin, nasal tissue, mucosa, and/or squameous epithelium, the enhanced permeability also results in increased skin, mucosa, and/or squamous epithelium hydration. For example, the increase in skin, mucosa, and/or squamous epithelium hydration can be about 25%, about 50%, about 75%, about 100%, about 125%, about 150%, about 175%, or about 200%, as compared to the skin, mucosa, and/or squamous epithelium hydration prior to application of the nanoemulsion.

In particular, Example 6 and FIG. 7 detail data showing that a nanoemulsion having a surfactant blend ratio of 1:5 and 0.13% BZK shows significant and dramatically improved hydration as compared to a non-nanoemulsion formulation comprising the same quaternary ammonium compound at the same concentration (Purell® Foam (0.13% BZK)). These results demonstrate that single application of a nanoemulsion according to the invention resulted in a significant and sustained increase in skin hydration.

Furthermore, in some embodiments, the nanoemulsions described herein with a specific surfactant blend ratio exhibit surprising and unexpected long-term stability even at high temperatures. In particular, Example 5 details data demonstrating that a nanoemulsion having a surfactant blend ratio of 1:5 was stable for 1 month even at the most extreme storage condition of 50° C. (122° F.). Additional data (not shown) demonstrates that nanoemulsions according to the invention, including nanoemulsions comprising an incorporated therapeutic agent, are stable for at least 3 months at up to 50° C., up to 12 months at 50° C., and up to 60 months at 5° C. This is highly unexpected. At severely high temperatures, emulsions are prone to rapid destabilization within a few hours to a couple of days. This data demonstrates that the tested formulations will offer key advantages for use in extremely high temperature climates. This is particularly desirable for therapeutics to be used in developing countries where refrigeration is not readily available.

Further, in some embodiments, the nanoemulsion compositions described herein have broad spectrum antimicrobial activity. Antimicrobial nanoemulsions have been shown to have broad antimicrobial activity against bacteria, enveloped viruses, and fungi at concentrations that are nontoxic in animals. These nanoemulsions function by fusing with lipid bilayers of cell membranes and destabilizing the lipid membrane of the pathogen; hence their antimicrobial activity. The antimicrobial activity of nanoemulsions is nonspecific, unlike that of antibiotics, thus allowing broad-spectrum activity while limiting the capacity for the generation of resistance.

II. Nanoemulsion Compositions

A nanoemulsion is a composition comprising an aqueous phase, at least one oil, and at least one organic solvent. The term “emulsion” refers to, without limitation, any oil-in-water dispersions or droplets, including lipid structures that can form as a result of hydrophobic forces that drive apolar residues (e.g., long hydrocarbon chains) away from water and polar head groups toward water, when a water immiscible phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases.

In one embodiment, the nanoemulsion comprises droplets having an average or mean particle size diameter of less than about 1000 nm, less than about 950 nm, less than about 900 nm, less than about 850 nm, less than about 800 nm, less than about 750 nm, less than about 700 nm, less than about 650 nm, less than about 600 nm, less than about 550 nm, less than about 500 nm, less than about 450 nm, less than about 400 nm, less than about 350 nm, less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, or less than about 100 nm. In another embodiment, the nanoemulsion comprises droplets having an average or mean particle size diameter of less than about 1000 nm. In another embodiment, the nanoemulsion comprises droplets having an average or mean particle size diameter of about 250 nm to about 1000 nm.

The nanoemulsion composition described herein comprises an aqueous phase, at least one oil, at least one organic solvent, at least one quaternary ammonium compound selected from the group consisting of benzalkonium chloride (BZK), cetylpyridimium chloride (CPC), benzethonium chloride (BEC), dioctadecyl dimethyl ammonium chloride (DODAC), and octenidine dihydrochloride (OCT), and at least one nonionic surfactant.

Throughout this disclosure, the compositions of the invention utilize a quaternary ammonium compound, which optionally can be a cationic surfactant or part of a zwitterionic surfactant. The present disclosure however is not limited to the use of cationic surfactants, and the genus of quaternary ammonium compounds is broader than “cationic surfactants.”

In some embodiments, the nanoemulsion composition described herein comprises BZK at a concentration of about 0.13%, poloxamer 407, soybean oil, EDTA, and water.

A. Aqueous Phase

The nanoemulsion composition comprises an aqueous phase. The aqueous phase may be any type of aqueous phase including, but not limited to, water (e.g., H₂O, distilled water, tap water), solutions (e.g., phosphate-buffered saline (PBS) solution), or any combination thereof. In some embodiments, the aqueous phase comprises water at a pH of about 4 to about 10, preferably about 6 to about 8. In some embodiments, the aqueous phase is deionized. In some embodiments, the aqueous is purified. In some embodiments, the aqueous phase is sterile and/or pyrogen free. In some embodiments, the aqueous phase is present in a concentration that is greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95%. In some embodiments, the aqueous phase is present in a concentration that is from about 50% to about 99%.

B. Oil

The nanoemulsion compositions described herein comprise at least one oil. The oil in the nanoemulsion composition described herein may be any cosmetically or pharmaceutically acceptable oil. The oil may be volatile or non-volatile, and may be chosen from animal oil, plant oil, vegetable oil, natural oil, synthetic oil, hydrocarbon oils, silicone oils, semi-synthetic derivatives thereof, and combinations thereof. In some embodiments, the oil is an animal oil, plant oil, or a vegetable oil. In some embodiments, the oil is present in a concentration that is equal to or less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1%. In some embodiments, the oil is present in a concentration that is from about 1% to about 30%.

Suitable oils include, but are not limited to, mineral oil, squalene oil, flavor oils, silicon oil, essential oils, water insoluble vitamins, isopropyl stearate, butyl stearate, octyl palmitate, cetyl palmitate, tridecyl behenate, diisopropyl adipate, dioctyl sebacate, menthyl anthranhilate, cetyl octanoate, octyl salicylate, isopropyl myristate, neopentyl glycol dicarpate cetols, Ceraphyls®, decyl oleate, diisopropyl adipate, C₁₂-15 alkyl lactates, cetyl lactate, lauryl lactate, isostearyl neopentanoate, myristyl lactate, isocetyl stearoyl stearate, octyldodecyl stearoyl stearate, hydrocarbon oils, isoparaffin, fluid paraffins, isododecane, petrolatum, argan oil, canola oil, chile oil, coconut oil, corn oil, cottonseed oil, flaxseed oil, grape seed oil, mustard oil, olive oil, palm oil, palm kernel oil, peanut oil, pine seed oil, poppy seed oil, pumpkin seed oil, rice bran oil, safflower oil, tea oil, truffle oil, vegetable oil, apricot (kernel) oil, jojoba oil (Simmondsia chinensis seed oil), grapeseed oil, macadamia oil, wheat germ oil, almond oil, rapeseed oil, gourd oil, soybean oil, sesame oil, hazelnut oil, maize oil, sunflower oil, hemp oil, bois oil, kuki nut oil, avocado oil, walnut oil, fish oil, berry oil, allspice oil, juniper oil, seed oil, almond seed oil, anise seed oil, celery seed oil, cumin seed oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil, cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemon grass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli leaf oil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmint leaf oil, tea tree leaf oil, thyme leaf oil, wintergreen leaf oil, flower oil, chamomile oil, clary sage oil, clove oil, geranium flower oil, hyssop flower oil, jasmine flower oil, lavender flower oil, manuka flower oil, Marhoram flower oil, orange flower oil, rose flower oil, ylang-ylang flower oil, Bark oil, cassia Bark oil, cinnamon bark oil, sassafras bark oil, wood oil, camphor wood oil, cedar wood oil, rosewood oil, sandalwood oil), rhizome (ginger) wood oil, resin oil, frankincense oil, myrrh oil, peel oil, bergamot peel oil, grapefruit peel oil, lemon peel oil, lime peel oil, orange peel oil, tangerine peel oil, root oil, valerian oil, oleic acid, linoleic acid, oleyl alcohol, isostearyl alcohol, semi-synthetic derivatives thereof, and any combinations thereof.

In some embodiments, the oil comprises soybean oil, avocado oil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil, sunflower oil, fish oils, cinnamon bark, coconut oil, cottonseed oil, flaxseed oil, pine needle oil, silicon oil, mineral oil, essential oil, flavor oils, water insoluble vitamins, and combinations comprising one or more of the foregoing oils. In some embodiments, the oil comprises soybean oil.

C. Organic Solvent

The nanoemulsions described herein can optionally comprise at least one organic solvent. Organic solvents contemplated for use include but are not limited to C₁-C₁₂alcohols, diols, triols, or a combination thereof. Organic phosphate solvents, alcohols and combinations thereof are also contemplated for use as organic solvents. Suitable organic phosphate solvents include, but are not limited to, dialkyl and trialkyl phosphates having one to ten carbon atoms, more preferably two to eight carbon atoms. The alkyl groups of the di- or trialkyl phosphate can all the same or the alkyl groups can be different. In one embodiment, the trialkyl phosphate is tri-n-butyl phosphate. In some embodiments, the organic solvent comprises a C₁-C₁₂alcohol, diol, or triol, a dialkyl phosphate, a trialkyl phosphate, or a combination thereof. In some embodiments, the organic solvent is present in a concentration that is less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, less than about 0.5%, less than about 0.1%. In some embodiments, the organic solvent is present in a concentration that is from about 0.1% to about 5%.

Suitable organic solvents for the nanoemulsion include, but are not limited to, ethanol, methanol, isopropyl alcohol, glycerol, medium chain triglycerides, diethyl ether, ethyl acetate, acetone, dimethyl sulfoxide (DMSO), acetic acid, n-butanol, butylene glycol, perfumers alcohols, isopropanol, n-propanol, formic acid, propylene glycols, glycerol, sorbitol, industrial methylated spirit, triacetin, hexane, benzene, toluene, diethyl ether, chloroform, 1,4-dixoane, tetrahydrofuran, dichloromethane, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, formic acid, semi-synthetic derivatives thereof, and a combination thereof.

D. Quaternary Ammonium Compound

The quaternary ammonium compound may be benzalkonium chloride (BZK), cetylpyridinium chloride (CPC), benzethonium chloride (BEC), dioctadecyl dimethyl ammonium chloride (DODAC) and/or octenidine dihydrochloride (OCT). In some embodiments, the quaternary ammonium compound is a cationic surfactant or is part of a zwitterionic surfactant.

If BZK is present as the quaternary ammonium compound, then the BZK is present at a concentration of from about 0.05% to about 5.0%, or any amount in-between these two amounts. In some embodiments, the BZK is present at a concentration of from about 0.05% to about 0.40%. In some embodiments, the BZK is present at a concentration of from about 0.05% to about 0.20%. In some embodiments, the BZK is present at a concentration of from about 0.10% to about 0.20%. In some embodiments, the BZK is present at a concentration of from about 0.10% to about 0.15%. In some embodiments, the BZK is present at a concentration of about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.30%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39%, or about 0.40%. In some embodiments, the BZK is present at a concentration of 0.13%.

In one embodiment, the quaternary ammonium compound is monographed by the US FDA as an antiseptic for topical use. The monographed quaternary ammonium compound can be BZK.

If cetylpyridinium chloride (CPC) is present as the quaternary ammonium compound, then the CPC is present at a concentration of from about 0.05% to about 5.0%, or any amount in-between these two amounts. In some embodiments, the CPC is present at a concentration of from about 0.05% to about 0.40%. In some embodiments, the CPC is present at a concentration of from about 0.05% to about 0.20%. In some embodiments, the CPC is present at a concentration of from about 0.15% to about 0.30%. In some embodiments, the CPC is present at a concentration of from about 0.08% to about 0.15%. In some embodiments, the CPC is present at a concentration of about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.30%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39%, or about 0.40%. In some embodiments, the CPC is present at a concentration of 0.10%. In some embodiments, the CPC is present at a concentration of 0.20%.

If benzethonium chloride (BEC) is present as the quaternary ammonium compound, then the BEC is present at a concentration of from about 0.05% to about 5.0%, or any amount in-between these two amounts. In some embodiments, the BEC is present in a concentration of: (a) from about 0.05% to about 1%; or (b) from about 0.10% to about 0.30%. In some embodiments, the BEC is present at a concentration of about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, or about 0.30%. In some embodiments, the BEC is present in a concentration of about 0.2%.

If dioctadecyl dimethyl ammonium chloride (DODAC) is present as the quaternary ammonium compound, then the DODAC is present at a concentration of from about 0.05% to about 5.0%, or any amount in-between these two amounts. In some embodiments, the DODAC is present in a concentration of: (a) from about 0.05% to about 1%; or (b) from about 0.10% to about 0.40%. In some embodiments, the DODAC is present at a concentration of about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.30%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39%, or about 0.40%. In some embodiments, the DODAC is present in a concentration of about 0.2%.

If octenidine dihydrochloride (OCT) is present as the quaternary ammonium compound, then the OCT is present at a concentration of from about 0.05% to about 5.0%, or any amount in-between these two amounts. In some embodiments, the OCT is present in a concentration of: (a) from about 0.05% to about 1%; or (b) from about 0.10% to about 0.40%. In some embodiments, the OCT is present at a concentration of about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.30%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39%, or about 0.40%. In some embodiments, the OCT is present in a concentration of about 0.2%.

E. Nonionic Surfactant

The nonionic surfactants described herein are Generally Recognized as Safe (GRAS) by the US Food and Drug Administration. Exemplary useful surfactants are described in Applied Surfactants: Principles and Applications, Tharwat F. Tadros (Copyright 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30629-3), which is specifically incorporated by reference.

Suitable nonionic surfactants include polysorbate surfactants (i.e., polyoxyethylene ethers), poloxamers, or a combination thereof. Examples of polysorbate detergents include the following sold under the tradenames: TWEEN® 20, TWEEN® 21, TWEEN® 40, TWEEN® 60, TWEEN® 61, TWEEN® 65, TWEEN® 80, TWEEN® 81, and TWEEN® 85. Poloxamers are polymers made of a block of polyoxyethylene, followed by a block of polyoxypropylene, followed by a block of polyoxyethylene. The average number of units of polyoxyethylene and polyoxypropylene varies based on the number associated with the polymer. For example, the smallest polymer, Poloxamer 101, consists of a block with an average of 2 units of polyoxyethylene, a block with an average of 16 units of polyoxypropylene, followed by a block with an average of 2 units of polyoxyethylene. Examples of poloxamers include, but are not limited to, Poloxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122, Poloxamer 123, Poloxamer 124, Poloxamer 181, Poloxamer 182, Poloxamer 183, Poloxamer 184, Poloxamer 185, Poloxamer 188, Poloxamer 212, Poloxamer 215, Poloxamer 217, Poloxamer 231, Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407, Poloxamer 105 Benzoate, and Poloxamer 182 Dibenzoate. In some embodiments, the nonionic surfactant is polysorbate 20 (TWEEN® 20), poloxamer 407, or a combination thereof.

Nonionic surfactants can also include, but are not limited to, an ethoxylated surfactant, an alcohol ethoxylated, an alkyl phenol ethoxylated, a fatty acid ethoxylated, a monoalkaolamide ethoxylated, a sorbitan ester ethoxylated, a fatty amino ethoxylated, an ethylene oxide-propylene oxide copolymer, Bis(polyethylene glycol bis[imidazoyl carbonyl]), nonoxynol-9, Bis(polyethylene glycol bis[imidazoyl carbonyl]), Brij®35, Brij® 56, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor® EL, Decaethylene glycol monododecyl ether, N-Decanoyl-N-methylglucamine, n-Decyl alpha-D-glucopyranoside, Decyl beta-D-maltopyranoside, n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside, n-Dodecyl beta-D-maltoside, n-Dodecyl beta-D-maltoside, Heptaethylene glycol monodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethylene glycol monotetradecyl ether, n-Hexadecyl beta-D-maltoside, Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-630, Methyl-6-O—(N-heptylcarbamoyl)-alpha-D-glucopyranoside, Nonaethylene glycol monododecyl ether, N— N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl-beta-D-glucopyranoside, Pentaethylene glycol monodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol monohexyl ether, Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctyl ether, Polyethylene glycol diglycidyl ether, Polyethylene glycol ether W-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate, Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillaja bark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85, Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tergitol, Tergitol, Type TMN-10, Tergitol, Type TMN-6, Tetradecyl-beta-D-maltoside, Tetraethylene glycol monodecyl ether, Tetraethylene glycol monododecyl ether, Tetraethylene glycol monotetradecyl ether, Triethylene glycol monodecyl ether, Triethylene glycol monododecyl ether, Triethylene glycol monohexadecyl ether, Triethylene glycol monooctyl ether, Triethylene glycol monotetradecyl ether, Triton CF-21, Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, Triton X-15, Triton X-151, Triton X-200, Triton X-207, Triton® X-114, Triton® X-165, Triton® X-305, Triton® X-405, Triton® X-45, Triton® X-705-70, TWEEN® 20, TWEEN® 21, TWEEN® 40, TWEEN® 60, TWEEN® 61, TWEEN® 65, TWEEN® 80, TWEEN® 81, TWEEN® 85, Tyloxapol, n-Undecyl beta-D-glucopyranoside, semi-synthetic derivatives thereof, or any combinations thereof.

F. Ratio of Quaternary Ammonium Compound to Nonionic Surfactant

This disclosure recognizes that the nanoemulsion compositions with certain concentration ratios of quaternary ammonium compound to nonionic surfactant provide greater delivery of the quaternary ammonium compound (or an additional active agent present in the composition) to the site of application and/or increased skin hydration when the nanoemulsions are applied to the skin as compared to non-nanoemulsion compositions comprising the same quaternary ammonium compound (or additional active agent). The ratio of the concentration of the quaternary ammonium compound to nonionic surfactant is about 5:1 to about 1:27. In some embodiments, the ratio of the concentration of the quaternary ammonium compound to nonionic surfactant is selected from the group consisting of about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24, about 1:25, about 1:26, and about 1:27. In some embodiments, the ratio of the concentration of the quaternary ammonium compound to the nonionic surfactant is from about 4:1 to about 1:27. In some embodiments, the ratio of the concentration of the quaternary ammonium compound to the nonionic surfactant is selected from the group consisting of about 1:2, about 1:5, about 1:9, about 1:14, and about 1:18. In certain embodiments, the concentration of the quaternary ammonium compound to the nonionic surfactant is about 1:2 to about 1:18.

G. Therapeutic Agents

The nanoemulsion compositions described herein may further comprise one or more active or therapeutic agents suitable for topical, transdermal, mucosal (e.g., (e.g. intranasal, ocular, buccal, vaginal), or oral administration. The Examples below describe incorporation of a model therapeutic agent, Compound A, demonstrating the effectiveness of incorporating additional therapeutic agents in a nanoemulsion formulation. Compound A is a high molecular weight compound, thereby demonstrating that low molecular weight compounds can also successfully be incorporated in a nanoemulsion formulation.

In some embodiments, the therapeutic agent is a vitamin, anti-inflammatory agent, keratolytic agent (opens pores—e.g., retinoid), homeopathic agent (e.g., eucalyptic globulus), antigen (e.g., for vaccines), antipruritic agent (anti-itch agent, e.g., cortisone), pain medicine, antimicrobial agent, antiviral agent, antifungal agent; steroid, anti-acne drug retinoid, retinol, etc.), macromolecules and vaccines, such as insulin, parathyroid hormone and influenza vaccine; or small, lipophilic, low-dose drugs.

In other embodiments, the therapeutic agent is recognized as being suitable for transdermal, intranasal, mucosal, vaginal, or topical administration or application. In some embodiments, the therapeutic agent (a) has low oral bioavailability but is suitable for nasal administration when formulated into a nanoemulsion; (b) is a lipophilic agent having poor water solubility; (c) present within a nanoemulsion is formulated for transdermal or intranasal administration, where the therapeutic agent when not present in a nanoemulsion is conventionally given via oral, IV, or IM administration due to the desire for fast onset of action or because of the difficulty in obtaining suitable bioavailability with other modes of administration (e.g., for pain medicine, anti-seizure medicine, allergy medicine, anti-migraine medicine, etc.); (d) is a small, lipophilic, low-dose drug; (e) is a small molecule; (f) is a protein; (g) is a peptide; and/or (h) is present within a nanoemulsion formulated for topical administration, where the therapeutic agent when not present in a nanoemulsion is conventionally applied via topical administration but does not achieve optimal delivery of the therapeutic agent into the epidermis, dermis, nasal tissue, mucosa, and/or squamous epithelium.

Suitable therapeutic agents include but are not limited to a penicillin, a cephalosporin, cycloserine, vancomycin, bacitracin, miconazole, ketoconazole, clotrimazole, polymyxin, colistimethate, nystatin, amphotericin B, chloramphenicol, a tetracycline, erythromycin, clindamycin, an aminoglycoside, a rifamycin, a quinolone, trimethoprim, a sulfonamide, zidovudine, gangcyclovir, vidarabine, acyclovir, poly(hexamethylene biguanide), terbinafine, and a combination thereof.

Several US monographs identify active agents approved for certain uses. Monographs referenced herein are specifically incorporated by reference.

Cosmetic/OTC products that are governed by an FDA monograph include, for example: (1) Anti-acne products—This monograph describes 40 different ingredients that can be used for anti-acne. The rule was finalized in 1990 although there was some additional review in 2010 relating to benzoyl peroxide and 2017 Adapalene was added to the monograph. (2) Toothpaste & anti-cavity products—This monograph gives a list of over 20 ingredients that can be used to fight cavities. The final rule was issued in 1995. (3) Topical anti-fungal—Products that are topically applied to places that need anti-fungal effects (diaper rash, feet, etc). The final rule was originally passed in 1993. (4) Anti-microbial products—There is a long list of ingredients that can be used for topical anti-microbial products. For most of the antimicrobial ingredients, the final rule has not yet been issued. (5) Antiperspirant—This monograph is for products that are designed to stop sweating. The final monograph was originally issued in 2003 and lists 26 active ingredients. (6) Astringents—These are classified as skin protectants. The final rule was originally issued in 2003. (7) Corn & Callus removers. (8) Dandruff products—The final monograph was issued in 1991 & revised in 1992. (9) Hair growth/hair loss—The final monograph for these types of products was issued in 1989 and includes nothing that works. However, in 1994, Minoxidil was switched from a prescription drug to an OTC. It remains the only non-prescription option. (10) Nail biting products—There is a monograph for products that are designed to stop people from biting their nails. The final monograph was issued in 1993. (11) Psoriasis—These products are designed to treat the condition of psoriasis. The tentative monograph was issued in 1986 and has yet to be finalized. Only a couple of active ingredients are allowed including Coal Tar and Salicylic acid. (12) Skin bleaching—Skin lightening products are OTCs in the US. The tentative final monograph was issued in 1982 but it has yet to be finalized. There are only 2 active ingredients acceptable for skin lightening. (13) Sunscreen—a final monograph on this topic was issued in 2011. (14) Topical analgesic—These products find a wide variety of applications and cover products such as those designed for diaper rash, cold sore treatments, poison ivy treatments, and others. (15) Wart remover—Products that are used to remove warts. The final monograph was issued in 1990 but updated in 1994. Thirteen active ingredients are listed.

Topical Analgesic (Monograph): See the monograph of list of active agents, Federal Register, 58(88): 27636-27644, and in particular pp 27641-27643 (May 10, 1993), 21 CFR 310 et seq., § 310.545 Drug products containing certain active Ingredients offered over-the counter (OTC) for certain uses.

Topical Antiseptics (Monograph): See US Federal Register, 82(243): 60480 (Table 3) (Dec. 20, 2017), which lists the following active agents monographed for topical antiseptic use: alcohol, benzalkonium chloride, benzethonium chloride, chloroxylenol (para-chloro-meta-xylenol; PCMX), cloflucarban, fluorosalan, hexylresorcinol, methylbenzethonium chloride, phenol, secondary amyltricresols, sodium oxychlorosene, triclocarban, triclosan, isopropyl alcohol, and mercufenol chloride.

Topical keratolytic agents (also for acne): salicylic acid (monographed). Salicylic acid is a topical keratolytic agent. It is used to remove excess keratin in hyperkeratotic skin disorders such as common and plantar warts, psoriasis, seborrheic dermatitis, calluses, and corns. Salicylic acid also is used to treat acne. Salicylic acid works by causing desquamation of the horny layer of skin.

Topical antimicrobial drug products for OTC human use, Topical Acne Drug Products. (a) Benzoyl peroxide, 2.5 to 10%; (b) Resorcinol, 2%, when combined with sulfur in accordance with 21 CFR 333.320(a); (c) Resorcinol monoacetate, 3%, when combined with sulfur in accordance with 21 CFR 333.320(b); (d) Salicylic acid, 0.5 to 2%; (e) Sulfur, 3 to 10%; (f) Sulfur, 3 to 8%, when combined with resorcinol or resorcinol monoacetate. See Guidance for Industry, Topical Acne Drug Products for Over-the-Counter Human Use Revision of Labeling and Classification of Benzoyl Peroxide as Safe and Effective, US Dept. of HHS, FDA, CDER (June 2011 OTC).

Alpha hydroxy acids: FDA has approved the first over-the-counter (OTC) retinoid treatment for acne in 2016. Adapalene (Differin Gel 0.1%, Galderma Laboratories, LP) is applied once daily to the affected skin in patients aged 12 years and older.

Finally a list of OTC active ingredients and monograph status/identification can be found on the US FDA website, e.g., at https://www.fda.gov/downloads/AboutFDA/CentersOffices/CDER/UCM135688.pdf (downloaded on Nov. 9, 2018), the contents of which are specifically incorporated by reference.

In some embodiments, the therapeutic agent is an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is a steroid or a non-steroidal anti-inflammatory drug. Suitable steroids include, but are not limited to, clobetasol, halobetasol, halcinonide, amcinonide, betamethasone, desoximetasone, diflucortolone, fluocinolone, fluocinonide, mometasone, clobetasone, desonide, hydrocortisone, prednicarbate, triamcinolone, and a pharmaceutically acceptable derivative thereof. Examples of pharmaceutically acceptable derivatives include, but are not limited to, propionate, valerate, acetonide, acetate, butyrate, and furoate. Examples of suitable non-steroidal anti-inflammatory drugs include aceclofenac, aspirin, celecoxib, clonixin, dexibuprofen, dexketoprofen, diclofenac, diflunisal, droxicam, etodolac, etoricoxib, fenoprofen, flufenamic acid, flurbiprofen, ibuprofen, indomethacin, isoxicam, ketoprofen, ketorolac, licofelone, lornoxicam, loxoprofen, lumiracoxib, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, oxaprozin, parecoxib, phenylbutazone, piroxicam, rofecoxib, salsalate, sulindac, tenoxicam, tolfenamic acid, tolmetin, and valdecoxib.

In some embodiments, the therapeutic agent is present in a concentration of from about 0.01% to about 10%; from about 0.01% to about 1%; from about 0.01% to about 0.75%; and from about 0.1% to about 0.5%. In some embodiments, the therapeutic agent is present in a concentration of from about 0.01%, about 0.02%, about 0.05%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. For an antigen, the amount present can be from about 1 to about 250 μg/per dose.

In some embodiments, when the composition further comprises a therapeutic or active agent, after a single application or administration of the composition topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal) or orally, the composition delivers at least about 25%, at least about 50%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, at least about 325%, at least about 350%, at least about 375%, at least about 400%, at least about 425%, at least about 450%, at least about 475%, or at least about 500% more of the therapeutic agent to the dermis, epidermis, mucosa, and/or squamous epithelium as compared to a composition comprising the same therapeutic agent at the same concentration but lacking a nanoemulsion, and applied using the same method, measured at any suitable time point after application or administration.

In some embodiments, when the composition further comprises a therapeutic or active agent, after a single application or administration of the composition topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal) or orally, the composition has a longer residence time at the site of application or administration as compared to a composition comprising the same therapeutic agent at the same concentration but lacking a nanoemulsion, and applied using the same method, measured at any suitable time point after application. The longer residence time can be determined by comparing the amount of the therapeutic agent present at the site of application or administration for the nanoemulsion composition as compared to the non-nanoemulsion composition, measured at any suitable time point after application. The longer residence time at the site of application can be, for example, an increase of about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, or about 200%, as compared to the residence time of the same quaternary ammonium compound, present at the same concentration, and applied using the same method, measured at any suitable time point after application or administration.

H. Additional Ingredients

Additional compounds suitable for use in the disclosed methods or compositions include, but are not limited to, one or more solvents, such as an organic phosphate-based solvent, bulking agents, coloring agents, pharmaceutically acceptable carriers, a preservative, pH adjuster, buffer, chelating agent, an auxiliary surfactant, a suds suppressor, a detergent builder, etc. The additional compounds can be admixed into a previously formulated composition, or the additional compounds can be added to the original mixture to be further formulated. In certain of these embodiments, one or more additional compounds are admixed into an existing disclosed composition immediately prior to its use.

Suitable preservatives in the disclosed composition include, but are not limited to, cetylpyridinium chloride, benzalkonium chloride, benzyl alcohol, chlorhexidine, imidazolidinyl urea, phenol, potassium sorbate, benzoic acid, bronopol, chlorocresol, paraben esters, phenoxyethanol, sorbic acid, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, sodium ascorbate, sodium metabisulphite, citric acid, edetic acid, semi-synthetic derivatives thereof, and combinations thereof. Other suitable preservatives include, but are not limited to, benzyl alcohol, chlorhexidine (bis (p-chlorophenyldiguanido) hexane), chlorphenesin (3-(-4-chloropheoxy)-propane-1,2-diol), Kathon CG (methyl and methylchloroisothiazolinone), parabens (methyl, ethyl, propyl, butyl hydrobenzoates), phenoxyethanol (2-phenoxyethanol), sorbic acid (potassium sorbate, sorbic acid), Phenonip (phenoxyethanol, methyl, ethyl, butyl, propyl parabens), Phenoroc (phenoxyethanol 0.73%, methyl paraben 0.2%, propyl paraben 0.07%), Liquipar Oil (isopropyl, isobutyl, butylparabens), Liquipar PE (70% phenoxyethanol, 30% liquipar oil), Nipaguard MPA (benzyl alcohol (70%), methyl & propyl parabens), Nipaguard MPS (propylene glycol, methyl & propyl parabens), Nipasept (methyl, ethyl and propyl parabens), Nipastat (methyl, butyl, ethyl and propyel parabens), Elestab 388 (phenoxyethanol in propylene glycol plus chlorphenesin and methylparaben), and Killitol (7.5% chlorphenesin and 7.5% methyl parabens).

Suitable pH adjusters include, but are not limited to, diethyanolamine, lactic acid, monoethanolamine, triethylanolamine, sodium hydroxide, sodium phosphate, semi-synthetic derivatives thereof, and combinations thereof.

Suitable buffers include pharmaceutically acceptable buffering agents. Examples of buffering agents are disclosed in U.S. Patent Publication No. 2010/0226983

In addition, the disclosed composition can comprise a chelating agent. In one embodiment of the disclosed, the chelating agent is present in an amount of about 0.0005% to about 1%. Examples of chelating agents include, but are not limited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(R-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), phytic acid, polyphosphoric acid, citric acid, gluconic acid, acetic acid, lactic acid, dimercaprol, or any combination thereof. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid.

Suitable auxiliary surfactants include compounds that enhance the properties of a nanoemulsion composition. The choice of auxiliary surfactant depends on the desire of the user with regard to the intended purpose of the composition and the commercial availability of the surfactant. In one embodiment, the auxiliary surfactant is an organic, water-soluble surfactant.

Suitable suds suppressors are low-foaming co-surfactants that prevents excessive sudsing during employment of the compositions on hard surfaces. Suds suppressors are also useful in formulations for no-rinse application of the composition. Concentrations of about 0.5 vol % to about 5 vol % are generally effective. Selection of a suds suppressor depends on its ability to formulate in a nanoemulsion composition and the residue as well as the cleaning profile of the composition. The suds suppressor should be chemically compatible with the components in a nanoemulsion composition and functional at the pH of a given composition. In one embodiment the suds suppressor or composition containing a suds suppressor does not leave a visible residue on surfaces on which a composition is applied.

Low-foaming co-surfactants can be used as a suds suppressor to mediate the suds profile in a nanoemulsion composition. Examples of suitable suds suppressors include block copolymers, alkylated primary and secondary alcohols, and silicone-based materials. Exemplary block co-polymers include, e.g., Pluronic® and Tetronic® (BASF Company). Alkylated alcohols include those which are ethoxylated and propoxylated, such as, tergitol (Union Carbide) or Poly-Tergent® (Olin Corp.). Silicone-based materials include DSE (Dow Corning).

Suitable detergent builders include compounds that sequester calcium and magnesium ions that might otherwise bind with and render less effective the auxiliary surfactants or co-surfactants. Detergent builders are particularly useful when auxiliary surfactants are used, and when the compositions are diluted prior to use with hard tap water, especially water having a hardness of, above about 12 grains/gallon.

The disclosed methods and compositions can comprise one or more emulsifying agents to aid in the formation of emulsions. Emulsifying agents include compounds that aggregate at the oil/water interface to form a kind of continuous membrane that prevents direct contact between two adjacent droplets. Certain embodiments of the present disclosure feature nanoemulsion compositions that may readily be diluted with water or another aqueous phase to a desired concentration without impairing their desired properties.

I. Viscosity

As noted herein, in one aspect of the disclosure, a composition is provided for topical, transdermal, mucosal (e.g. intranasal, ocular, buccal, vaginal) or oral application or administration. The composition comprises an oil-in-water nanoemulsion, the nanoemulsion comprising: (a) an aqueous phase; (b) at least one oil; (c) at least one quaternary ammonium compound; and (d) at least one nonionic surfactant; wherein the droplets of the nanoemulsion have a mean droplet size of less than about 1 micron; and wherein (i) the nanoemulsion is diluted resulting in a formulation of about 0.5% to about 60% nanoemulsion; (ii) the viscosity of the nanoemulsion is less than about 1000 cp; and (iii) the nanoemulsion enhances delivery of the quaternary ammonium compound into tissue by at least about 25% as compared to a solution with the same concentration of quaternary ammonium compound but lacking a nanoemulsion and as compared to a nanoemulsion with a viscosity greater than about 1000 cp. In another aspect of the disclosure, the quaternary ammonium compound is a cationic surfactant or is part of a zwitterionic surfactant.

In some embodiments, the nanoemulsion compositions described herein have a viscosity of less than about 1000 cP. In some embodiments, the nanoemulsion compositions described herein have a viscosity of less than about 900, less than about 800, less than about 700, less than about 600, less than about 500, less than about 400, less than about 300, less than about 275, less than about 250, less than about 225, less than about 200, less than about 100, less than about 75, less than about 50, less than about 25, less than about 20, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, less than about 2, or less than about 1.5 cP. Optionally the viscosity is greater than 0.

In some embodiments, the viscosity is from about 1 cP to about 1000 cP; or from about 1.2 cP to about 275 cP.

In some aspects, nanoemulsions described herein enhance delivery of the quaternary ammonium compound (and/or additional active/therapeutic agent) into tissue by at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as compared to a solution with the same concentration of quaternary ammonium compound but lacking a nanoemulsion and as compared to a nanoemulsion with a viscosity greater than the referenced viscosity (e.g., greater than about 1000, greater than about 900, greater than about 800, . . . greater than about 300, greater than about 275 cP . . . , or greater than any other viscosity amount described herein).

J. Zeta Potential

As noted herein, in one aspect of the disclosure, a composition is provided for topical, transdermal, mucosal (e.g. intranasal, ocular, buccal, vaginal) or oral application or administration, the composition comprising an oil-in-water nanoemulsion, the nanoemulsion comprising: (a) an aqueous phase; (b) at least one oil; (c) at least one quaternary ammonium compound; and (d) at least one nonionic surfactant; wherein (i) the droplets of the nanoemulsion have a mean droplet size of less than about 1 micron; (ii) the nanoemulsion is diluted resulting in a formulation of about 0.5% to about 60% nanoemulsion; (iii) the zeta potential of the nanoemulsion is greater than about 20 mV; and (iv) the nanoemulsion enhances delivery of the quaternary ammonium compound (and/or additional active/therapeutic agent) into tissue by at least about 25% as compared to a solution with the same concentration of quaternary ammonium compound but lacking a nanoemulsion and as compared to a nanoemulsion with a zeta potential of less than about 20 mV. In another aspect of the disclosure, the quaternary ammonium compound is a cationic surfactant or is part of a zwitterionic surfactant.

Zeta potential is a scientific term for electrokinetic potential in colloidal dispersions. The usual units are volts (V) or millivolts (mV). From a theoretical viewpoint, the zeta potential is the electric potential in the interfacial double layer (DL) at the location of the slipping plane relative to a point in the bulk fluid away from the interface. In other words, zeta potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle.

In some embodiments, the nanoemulsion has a zeta potential from about 20 mV to about 40 mV; from about 40 mV to about 60 mV; from about 60 mV to about 80 mV; or from about 80 mV to about 100 mV. In other embodiments, the nanoemulsion has a zeta potential of greater than or equal to about 20 mV, about 25 mV, about 30 mV, about 35 mV, about 40 mV, about 45 mV, about 50 mV, about 55 mV, about 60 mV, about 65 mV, about 70 mV, about 75 mV, about 80 mV, about 85 mV, about 90 mV, about 95 mV, or greater than or equal to about 100 mV.

In some aspects, nanoemulsions described herein enhance delivery of the quaternary ammonium compound (and/or additional active/therapeutic agent) into tissue by at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as compared to a solution with the same concentration of quaternary ammonium compound but lacking a nanoemulsion and as compared to a nanoemulsion with a zeta potential less than the referenced zeta potential (e.g., less than 20 mV, less than about 30 mV, or less than any other zeta potential amount described herein for the described nanoemulsions).

K. Entrapment of Quaternary Ammonium Compound by Oil Phase

As noted herein, in one aspect of the disclosure, a composition is provided for topical, transdermal, mucosal (e.g. intranasal, ocular, buccal, vaginal) or oral application or administration, the composition comprising an oil-in-water nanoemulsion, the nanoemulsion comprising: (a) an aqueous phase; (b) at least one oil; (c) at least one quaternary ammonium compound; and (d) at least one nonionic surfactant; wherein (i) the droplets of the nanoemulsion have a mean droplet size of less than about 1 micron; (ii) the nanoemulsion is diluted resulting in a formulation of about 0.5% to about 60% nanoemulsion; (iii) at least about 33% of the quaternary ammonium compound is entrapped in the oil phase of the nanoemulsion and at least about 0.2% of the weight of the oil phase of the nanoemulsion is attributed to entrapment of the quaternary ammonium compound; and (iv) the nanoemulsion enhances delivery of the quaternary ammonium compound (and/or additional active/therapeutic agent) into tissue by at least about 25% as compared to a solution with the same concentration of quaternary ammonium compound but lacking a nanoemulsion and as compared to a nanoemulsion with less than about 0.2% of the weight of the oil phase of the nanoemulsion attributed to entrapment of the quaternary ammonium compound. In another aspect of the disclosure, the quaternary ammonium compound is a cationic surfactant or is part of a zwitterionic surfactant.

In some embodiments, (a) at least about 33% of the quaternary ammonium compound is entrapped in the oil phase of the nanoemulsion; (b) at least about 0.2% of the weight of the oil phase of the nanoemulsion is attributed to the quaternary ammonium compound; or (c) the composition satisfies both (a) and (b).

In some embodiments, at least about 0.20%, at least about 0.21%, at least about 0.22%, at least about 0.23%, at least about 0.24%, at least about 0.25%, at least about 0.26%, at least about 0.27%, at least about 0.28%, at least about 0.29%, at least about 0.30%, at least about 0.35%, at least about 0.40%, at least about 0.45%, at least about 0.50%, at least about 0.55%, at least about 0.60%, at least about 0.65%, at least about 0.70%, at least about 0.75%, at least about 0.80%, at least about 0.85%, at least about 0.90%, at least about 0.95%, at least about 1.00%, at least about 1.25%, at least about 1.40%, at least about 1.50%, at least about 20.00%, at least about 20.50%, at least about 20.75%, at least about 20.85%, at least about 30.00%, at least about 40.00%, at least about 5.00%, at least about 60.00%, at least about 70.00%, at least about 80.00%, at least about 90.00%, at least about 10.00%, at least about 11.00%, at least about 12.00%, at least about 13.00%, at least about 14.00%, at least about 15.00%, at least about 16.00%, at least about 17.00%, at least about 18.00%, at least about 19.00%, at least about 20.00%, or up to about 25% of the weight of the oil phase of the nanoemulsion is attributed to entrapment of the quaternary ammonium compound.

In some embodiments, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% of the quaternary ammonium compound is entrapped in the oil phase of the nanoemulsion.

In some embodiments, any combination of the percentage of the quaternary ammonium compound entrapped in the oil phase of the nanoemulsion described herein (e.g., about 33%, about 35%, etc.) can be combined with any percentage of the weight of the oil phase of the nanoemulsion attributed to entrapment of the quaternary ammonium compound described herein (e.g., at least about 0.2% up to about 25%).

In some embodiments, (a) at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, or at least about 50% of the quaternary ammonium compound is entrapped in the oil phase of the nanoemulsion; (b) at least about 0.20% of the weight of the oil phase of the nanoemulsion is attributed to entrapment of the quaternary ammonium compound; or (c) the composition satisfies both (a) and (b).

In some embodiments, (a) at least about 70% of the quaternary ammonium compound is entrapped in the oil phase of the nanoemulsion; (b) at least about 0.2% of the weight of the oil phase of the nanoemulsion is attributed to entrapment of the quaternary ammonium compound; or (c) the composition satisfies both (a) and (b). In some embodiments, (a) at least about 90% of the quaternary ammonium compound is entrapped in the oil phase of the nanoemulsion; (b) at least about 0.2% of the weight of the oil phase of the nanoemulsion is attributed to entrapment of the quaternary ammonium compound; or (c) the composition satisfies both (a) and (b).

In some embodiments, (a) at least about 33% of the quaternary ammonium compound is entrapped in the oil phase of the nanoemulsion; (b) at least about 0.4% of the weight of the oil phase of the nanoemulsion is attributed to entrapment of the quaternary ammonium compound; or (c) the composition satisfies both (a) and (b). In some embodiments, (a) at least about 33% of the quaternary ammonium compound is entrapped in the oil phase of the nanoemulsion; (b) at least about 0.6% of the weight of the oil phase of the nanoemulsion is attributed to entrapment of the quaternary ammonium compound; or (c) the composition satisfies both (a) and (b). In some embodiments, (a) at least about 33% of the quaternary ammonium compound is entrapped in the oil phase of the nanoemulsion; (b) at least about 0.8% of the weight of the oil phase of the nanoemulsion is attributed to entrapment of the quaternary ammonium compound; or (c) the composition satisfies both (a) and (b). In some embodiments, (a) at least about 33% of the quaternary ammonium compound is entrapped in the oil phase of the nanoemulsion; (b) at least about 1.0% of the weight of the oil phase of the nanoemulsion is attributed to entrapment of the quaternary ammonium compound; or (c) the composition satisfies both (a) and (b).

In some aspects, nanoemulsions described herein enhance delivery of the quaternary ammonium compound (and/or additional active/therapeutic agent) into tissue by at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as compared to a solution with the same concentration of quaternary ammonium compound but lacking a nanoemulsion and as compared to a nanoemulsion with a less than about 0.20% of the weight of the oil phase of the nanoemulsion attributed to entrapment of the quaternary ammonium compound.

L. Average or Mean Particle Size Diameter and Stability Thereof

The nanoemulsion compositions described herein have droplets having an average or mean particle size diameter of about 250 nm to about 1000 nm. In some embodiments, the droplets have an average or mean particle size diameter of about 250 nm to about 600 nm. In some embodiments, the droplets have an average or mean particle size diameter of about 300 nm to about 600 nm. In some embodiments, the droplets have an average or mean particle size diameter of about 150 nm or less, about 200 nm or less, about 250 nm or less, about 260 nm or less, about 270 nm or less, about 280 nm or less, about 290 nm or less, about 300 nm or less, about 310 nm or less, about 320 nm or less, about 330 nm or less, about 340 nm or less, about 350 nm or less, about 360 nm or less, about 370 nm or less, about 380 nm or less, about 390 nm or less, about 400 nm or less, about 410 nm or less, about 420 nm or less, about 430 nm or less, about 440 nm or less, about 450 nm or less, about 460 nm or less, about 470 nm or less, about 480 nm or less, about 490 nm or less, about 500 nm or less, about 510 nm or less, about 520 nm or less, about 530 nm or less, about 540 nm or less, about 550 nm or less, about 560 nm or less, about 570 nm or less, about 580 nm or less, about 590 nm or less, or about 600 nm or less.

In some embodiments, the mean droplet size of the nanoemulsion does not change by more than about 10% after centrifuging the nanoemulsion at a speed of about 200,000 rpm for about one hour. In other embodiments, the mean droplet size of the nanoemulsion does not change by more than about 9%, more than about 8%, more than about 7%, more than about 6%, more than about 5%, more than about 4%, more than about 3%, more than about 2%, more than about 1%, more than about 0.9%, more than about 0.8%, more than about 0.7%, more than about 0.6%, more than about 0.5%, more than about 0.4%, more than about 0.3%, or more than about 0.2%, after centrifuging the nanoemulsion at a speed of about 200,000 rpm for about one hour.

In some aspects, nanoemulsions described herein enhance delivery of the quaternary ammonium compound (and/or additional active/therapeutic agent) into tissue by at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as compared to a solution with the same concentration of quaternary ammonium compound but lacking a nanoemulsion and as compared to a nanoemulsion with a change in mean droplet size, following centrifuging the nanoemulsion at a speed of about 200,000 rpm for about one hour, of greater than about 10%.

M. Stability of Nanoemulsion Compositions

The nanoemulsion compositions described herein are stable. In certain embodiments, the nanoemulsion compositions herein demonstrate stability even under storage conditions at high temperatures (e.g., about 50° C.). In some embodiments, the nanoemulsion compositions described herein are thermostable. In some embodiments, the compositions are stable for at least about 1 month, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 12, at least about 24, at least about 30, at least about 36, at least about 42, at least about 48, at least about 54, or at least about 60 months at about 5° C., about 25° C., about 40° C., and/or about 50° C. In some embodiments, the compositions are stable for at least about 3 months at about 5° C., about 25° C., about 40° C., and/or about 50° C. In some embodiments, the compositions are stable for at least about 60 months at 5° C. In other embodiments the compositions are stable for at least about 12 months at 50° C.

Further, because the nanoemulsion compositions of the invention are highly thermostable, the nanoemulsion compositions can be autoclaved without losing the structural or chemical integrity of the compositions. This is desirable as sterile formulations may be preferable for some disease indications and/or patient populations.

In one embodiment, stability of a nanoemulsion according to the invention is measured by a lack of a substantial increase in average particle size over time and/or upon exposure to elevated temperatures. A “lack of a substantial increase in average particle size” of a nanoemulsion can mean a particle size growth of less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 3%. The period of time over which stability is measured can be any suitable period of time, such as about 1 month, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 12, at least about 24, at least about 30, at least about 36, at least about 42, at least about 48, at least about 54, or at least about 60 months.

In yet another embodiment, stability is measured by the ability of the composition upon exposure to elevated temperatures, and/or prolonged storage, to exhibit minimal particle aggregation formation and/or retain at an at least 80% label claim of an active agent and/or of the quaternary ammonium compound present in the nanoemulsion. Time points for measurement can be as described above. Other label claim thresholds can be about 85%, about 90%, or about 95% (see e.g. the methodology of Example 5).

N. Antimicrobial Activity

The nanoemulsion compositions described herein have broad spectrum antimicrobial activity. In some embodiments, the composition is non-toxic in human and animals. In some embodiments, the composition kills at least about 99.9% of gram positive and gram negative bacteria following a 60 second exposure using the ASTM E2315-16 Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure.

In some embodiments, the gram positive bacteria are selected from the group consisting of Staphylococcus, Enterococcus, Methicillin-resistant Staphylococcus aureus (MRSA), and Community Associated-MRSA (CA-MRSA). In some embodiments, the gram negative bacteria are selected from the group consisting of Pseudomonas, Serratia, Acinetobacter, and Klebsiella.

In some embodiments, the compositions are effective in killing and/or inactivating a microorganism population derived from a bacteria, a fungus, a protozoa, a virus, or any combination thereof.

In some embodiments, the compositions are effective in killing and/or inactivating bacteria comprising vegetative bacteria, bacterial spore, or a combination thereof. In some embodiments, the composition is effective in killing and/or inactivating bacteria comprising Gram negative bacteria, Gram positive bacteria, an acid fast bacilli, or a combination thereof. Gram negative bacteria include, for example and without limitation, Vibrio, Salmonella, Shigella, Pseudomonas, Escherichia, Klebsiella, Proteus, Enterobacter, Serratia, Moraxella, Legionella, Bordetella, Gardnerella, Haemophilus, Neisseria, Brucella, Yersinia, Pasteurella, Bacteroids, Mobiluncus, and Helicobacter. Gram positive bacteria include, for example, and without limitation, Bacillus, Clostridium, Arthrobacter, Micrococcus, Staphylococcus, Streptococcus, Listeria, Corynebacteria, Planococcus, Mycobacterium, Nocardia, Rhodococcus, and acid fast Bacilli such as Mycobacterium.

In some embodiments, the composition is effective in killing and/or inactivating bacteria comprising Bacillus anthracis, Bacillus cereus, Bacillus circulans, Bacillus megaterium, Bacillus subtilis, Clostridium botulinum, Clostridium tetani, Clostridium perfringens, Haemophilus influenzae, Neisseria gonorrhoeae, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Vibrio cholerae, Staphylococcus aureus, Yersinia species, Gardnerella vaginalis, Mobiluncus species, Mycoplasma hominis, Salmonella species, Shigellae species, Pseudomonas species, Escherichia species, Klebsiella species, Proteus species, Enterobacter species, Serratia species, Moraxella species, Legionella species, Bordetella species, Helicobacter species, Arthobacter species, Micrococcus species, Listeria species, Corynebacteria species, Planococcus species, Nocardia species, Rhodococcus species, Mycobacteria species, Chlamydia species, Acinetobacter species, Staphylococcus species, Enterococcus species, and any combination thereof.

In some embodiments, the compositions are effective in killing and/or inactivating a microorganism population is derived from a virus. Viruses include those of families Baculoviridae, Herpesviridae, Iridoviridae, Poxviridae, “African Swine Fever Viruses,” Adenoviridae, Caulimoviridae, Myoviridae, Phycodnaviridae, Tectiviridae, Papovaviridae, Circoviridae, Parvoviridae, Hepadnaviridae, Cystoviridae, Birnaviridae, Reoviridae, Coronaviridae, Flaviviridae, Togaviridae, “Arterivirus,” Astroviridae, Caliciviridae, Picornaviridae, Potyviridae, Retroviridae, Orthomyxoviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Arenaviridae, Retroviridae, and Bunyaviridae.

In some embodiments, the composition is effective in killing a virus belonging to a family selected from the group consisting of Orthomyxoviridae, Retroviridae, African Swine Fever Viruses, Papovaviridae, Hepadnaviridae, Coronaviridae, Flaviviridae, Togaviridae, Picornaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae. In some embodiments, the composition is effective in killing the Orthomyxovirdae virus, which include influenza virus, herpes simplex, herpes zoster, sendai virus, sindbis virus, pox virus, small pox virus, vaccinia virus, influenza virus, seasonal flu virus, or pandemic flu virus.

In some embodiments, the composition is effective in killing Retroviridae, which includes human immunodeficiency virus, west nile virus, hanta virus, and human papilloma virus. In some embodiments, the composition is effective in killing Ebolavirus, Respiratory syncytial virus (RSV), rotavirus, norovirus, and/or flavivirus which is zika virus, West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, and insect-specific flaviviruses.

In some embodiments, the compositions are effective in killing a microorganism population is derived from a fungus. Suitable fungi include yeast or a filamentous fungus. Examples of yeast include but are not limited to various species of Candida (e.g., Candida albicans). In some embodiments, the filamentous fungus is Aspergillus species or a dermatophyte. In some embodiments, the dermatophyte is Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Microsporum gypseum, or Epidermophyton floccosum. In some embodiments, the fungus comprises Cladosporium, Fusarium, Alternaria, Curvularia, Aspergillus, Penicillium, Candida, or a combination thereof.

O. Quaternary Ammonium Compound Delivery

In some embodiments, after a single application of the composition, the composition delivers at least 25% more of the quaternary ammonium compound to the epidermis, and/or at least 25% more of the quaternary ammonium compound to the dermis, and/or at least 25% more of the quaternary ammonium compound to the mucosa, and/or at least 25% more of the quaternary ammonium compound to the squamous epithelium as compared to a composition comprising the same quaternary ammonium compound at the same concentration but lacking a nanoemulsion, measured at any suitable time point after application, such as 24 hours after application.

In some embodiments, after a single application of the composition, the composition has a longer residence time at the site of application as compared to a composition comprising the same quaternary ammonium compound at the same concentration but lacking a nanoemulsion, wherein the longer residence time is determined by comparing the amount of the quaternary ammonium compound present at the site of application for the nanoemulsion composition as compared to the non-nanoemulsion composition.

In some embodiments, after a single application of the composition, the composition delivers at least about 1.25×, at least about 1.5×, at least about 1.75×, at least about 2×, at least about 2.25×, at least about 2.5×, at least about 2.75×, at least about 3×, at least about 3.25×, at least about 3.5×, at least about 3.75×, at least about 4×, at least about 5×, at least about 6×, at least about 7×, at least about 8×, at least about 9×, or at least about 10× more of the quaternary ammonium compound to the epidermis, dermis, mucosa, and/or squamous epithelium as compared to a composition comprising the same quaternary ammonium compound at the same concentration but lacking a nanoemulsion.

In some embodiments, after a single application of the composition, the composition delivers at least about 25%, at least about 50%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, at least about 325%, at least about 350%, at least about 375%, at least about 400%, at least about 425%, at least about 450%, at least about 475%, or at least about 500% more of the quaternary ammonium compound to the epidermis, dermis, mucosa, and/or squamous epithelium as compared to a composition comprising the same quaternary ammonium compound at the same concentration but lacking a nanoemulsion. In some embodiments, after a single application of the composition, the composition delivers from about 25% to about 500% more of the quaternary ammonium compound to the epidermis, dermis, mucosa, and/or squamous epithelium as compared to a composition comprising the same quaternary ammonium compound at the same concentration but lacking a nanoemulsion.

In some embodiments, when the composition is applied to skin, mucosa and/or squamous epithelium, the composition results in increased skin, mucosa and/or squamous epithelium hydration as compared to a composition comprising the same quaternary ammonium compound at the same concentration but lacking a nanoemulsion.

In some embodiments, the increase in skin, mucosa and/or squamous epithelium hydration is from about 50% to about 1000%. In some embodiments, the increase in skin, mucosa and/or squamous epithelium hydration is about 50%, about 75%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, about 325%, about 350%, about 375%, about 400%, about 425%, about 450%, about 475%, about 500%, about 525%, about 550%, about 575%, about 600%, about 625%, about 650%, about 675%, about 700%, about 725%, about 750%, about 775%, about 800%, about 825%, about 850%, about 875%, about 900%, about 925%, about 950%, about 975%, or about 1000%.

III. Pharmaceutical Compositions

The nanoemulsions of the present disclosure may be formulated into pharmaceutical compositions that are administered in a therapeutically effective amount to a subject and may further comprise one or more suitable, pharmaceutically-acceptable excipients, additives, or preservatives. Suitable excipients, additives, and/or preservatives are well known in the art. In some embodiments, the nanoemulsions described herein are formulated as a vaccine.

Suitable pharmaceutically acceptable excipients or pharmaceutically acceptable carriers, may include solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like, and combinations comprising one or more of the foregoing carriers as described, for instance, in Remington's Pharmaceutical Sciences, 15th Ed. Easton: Mack Publishing Co. pp. 1405-1412 and 1461-1487 (1975), and The National Formulary XIV 14th Ed., Washington: American Pharmaceutical Association (1975). Suitable carriers include, but are not limited to, calcium carbonate, carboxymethylcellulose, cellulose, citric acid, dextrate, dextrose, ethyl alcohol, glucose, hydroxymethylcellulose, lactose, magnesium stearate, maltodextrin, mannitol, microcrystalline cellulose, oleate, polyethylene glycols, potassium diphosphate, potassium phosphate, saccharose, sodium diphosphate, sodium phosphate, sorbitol, starch, stearic acid and its salts, sucrose, talc, vegetable oils, water, and combinations comprising one or more of the foregoing carriers. Except insofar as any conventional media or agent is incompatible with the emulsions of the present invention, their use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

For topical applications, pharmaceutically acceptable carriers can take the form of a liquid, cream, foam, lotion, or gel, and may additionally comprise organic solvents, emulsifiers, gelling agents, moisturizers, stabilizers, surfactants, wetting agents, preservatives, time release agents, and minor amounts of humectants, sequestering agents, dyes, perfumes, and other components commonly used in pharmaceutical compositions for topical and mucosal administration.

By the phrase “therapeutically effective amount” it is meant any amount of the composition that is effective in killing or inhibiting the growth of any one of the microorganisms described herein.

Topical administration includes administration to the skin, mucosa, and squamous epithelium, including surface of the hair follicle and pilosebaceous unit. In some embodiments, the composition enters the epidermis, dermis, mucosa, squamous epithelium, or any combination thereof. In some embodiments, the composition permeates into the epidermis and dermis via the follicular route using skin pores and hair follicles. In some embodiments, the composition diffuses through the skin, skin pores, nail, scalp, hair follicles, lateral or proximal folds, nail, hyponichium, or a combination thereof.

Pharmaceutically acceptable dosage forms for administration include, but are not limited to, ointments, creams, liquids, emulsions, lotions, gels, bioadhesive gels, aerosols, pastes, foams, sunscreens, or in the form of an article or carrier, such as a bandage, insert, syringe-like applicator, pessary, powder, talc or other solid, cleanser (leave on and wash off product), and agents that favor penetration within the pilosebaceous gland. In some embodiments, composition is administered in the form of a liquid, lotion, cream, ointment, salve, or spray.

The pharmaceutical compositions may be formulated for immediate release, sustained release, controlled release, delayed release, or any combinations thereof, into the epidermis or dermis, with no systemic absorption. In some embodiments, the formulations may comprise a penetration-enhancing agent for enhancing penetration of the nanoemulsion through the stratum corneum and into the epidermis or dermis. Suitable penetration-enhancing agents include, but are not limited to, alcohols such as ethanol, triglycerides and aloe compositions. The amount of the penetration-enhancing agent may comprise from about 0.5% to about 40% by weight of the formulation.

In some embodiments, the formulation for delivery via a “patch” comprising a therapeutically effective amount of the nanoemulsion is envisioned. As used herein a “patch” comprises at least a topical formulation and a covering layer, such that the patch can be placed over the area to be treated. Preferably, the patch is designed to maximize delivery through the stratum corneum and into the epidermis or dermis, while minimizing absorption into the circulatory system, and little to no skin irritation, reducing lag time, promoting uniform absorption, and reducing mechanical rub-off and dehydration.

Adhesives for use with the drug-in-adhesive type patches are well known in the art. Suitable adhesive include, but are not limited to, polyisobutylenes, silicones, and acrylics. These adhesives can function under a wide range of conditions, such as, high and low humidity, bathing, sweating etc. Preferably the adhesive is a composition based on natural or synthetic rubber; a polyacrylate such as, polybutylacrylate, polymethylacrylate, poly-2-ethylhexyl acrylate; polyvinylacetate; polydimethylsiloxane; or and hydrogels (e.g., high molecular weight polyvinylpyrrolidone and oligomeric polyethylene oxide). The most preferred adhesive is a pressure sensitive acrylic adhesive, for example Durotak® adhesives (e.g., Durotak® 2052, National Starch and Chemicals). The adhesive may comprise a thickener, such as a silica thickener (e.g., Aerosil, Degussa, Ridgefield Park, N.J.) or a crosslinker such as aluminumacetylacetonate.

Suitable release liners include but are not limited to occlusive, opaque, or clear polyester films with a thin coating of pressure sensitive release liner (e.g., silicone-fluorsilicone, and perfluorcarbon based polymers.

Backing films may be occlusive or permeable and are derived from synthetic polymers like polyolefin oils polyester, polyethylene, polyvinylidine chloride, and polyurethane or from natural materials like cotton, wool, etc. Occlusive backing films, such as synthetic polyesters, result in hydration of the outer layers of the stratum corneum while non-occlusive backings allow the area to breath (i.e., promote water vapor transmission from the skin surface). More preferably the backing film is an occlusive polyolefin foil (Alevo, Dreieich, Germany). The polyolefin foil is preferably about 0.6 to about 1 mm thick.

The shape of the patch can be flat or three-dimensional, round, oval, square, and have concave or convex outer shapes, or the patch or bandage can also be segmented by the user into corresponding shapes with or without additional auxiliary means.

The pharmaceutical compositions may be applied in a single administration or in multiple administrations. The pharmaceutical compositions can be applied for any suitable time period, such as 1× or multiples times per day. The compositions can be applied for at least once a week, at least twice a week, at least once a day, at least twice a day, multiple times daily, multiple times weekly, biweekly, at least once a month, or any combination thereof. The pharmaceutical compositions are applied for a period of time of about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about ten months, about eleven months, about one year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, and about 5 years. Between applications, the application area may be washed to remove any residual nanoemulsion.

Preferably, the pharmaceutical compositions are applied to the skin area in an amount of from about 0.001 mL/cm²to about 5.0 mL/cm². An exemplary application amount and area is about 0.2 mL/cm², although any amount from 0.001 mL/cm²up to about 5.0 mL/cm²can be applied. Following topical administration, the nanoemulsion may be occluded or semi-occluded. Occlusion or semi-occlusion may be performed by overlaying a bandage, polyoleofin film, impermeable barrier, or semi-impermeable barrier to the topical preparation. Preferably, after application, the treated area is covered with a dressing.

In some embodiments, the compositions described herein are formulated for mucosal delivery, for example by contacting any one of the compositions described herein to a nasal mucosal epithelium, a bronchial or pulmonary mucosal epithelium, an oral, gastric, intestinal or rectal mucosal epithelium, or a vaginal mucosal epithelium. In some embodiments, the compositions described herein are formulated for intranasal delivery, (e.g., nasal mucosal delivery or intranasal mucosal delivery).

IV. Dermal Patches, Wipes and Swabs

Also provided herein in one aspect is a dermal patch or wipe impregnated or saturated with or incorporating any one of the nanoemulsions described herein. In some embodiments, the wipe dispenses a greater amount of the quaternary ammonium compound and/or incorporated active or therapeutic agent to an application site, as compared to a wipe impregnated or saturated with or incorporating a composition comprising the same quaternary ammonium compound and/or incorporated active or therapeutic agent at the same concentration but lacking a nanoemulsion.

In some embodiments, the wipe dispenses about 20% to about 100% more of the quaternary ammonium compound and/or incorporated active or therapeutic agent to an application site, as compared to a wipe impregnated or saturated with or incorporating a composition comprising the same quaternary ammonium compound and/or incorporated active or therapeutic agent at the same concentration but lacking a nanoemulsion. In some embodiments, the wipe dispenses about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% more of the quaternary ammonium compound and/or incorporated active or therapeutic agent to an application site, as compared to a wipe impregnated or saturated with or incorporating a composition comprising the same quaternary ammonium compound and/or incorporated active or therapeutic agent at the same concentration but lacking a nanoemulsion.

As detailed in Example 7, a comparison of a wipe saturated with a non-nanoemulsion formulation and compared to a wipe saturated with a nanoemulsion formulation revealed that the nanoemulsion-saturated wipe released much more of the component active agent (e.g., the cationic active agent). It is theorized that the active agent in the non-nanoemulsion formulation binds to the fibers or compounds in the wipe, preventing a significant portion of the active agent from being deposited on the surface or skin where the wipe is applied. This lack of active agent deposition is undesirable, as the result is a reduced effectiveness—e.g., a reduced effectiveness in antimicrobial activity when the wipe is used for disinfection.

In another embodiment, encompassed is a nasal swab, dropper, or spray for use with any nanoemulsion composition described herein. The nasal swab, dropper, or spray can be impregnated or saturated with or incorporating the any nanoemulsion composition described herein, or the nasal swab, dropper, or spray can be packaged in a kit with a container comprising a nanoemulsion composition described herein, with the swab being exposed to the nanoemulsion prior to use. Such swabs are useful to prevent and/or minimize infections in hospital settings.

Antibiotic-resistant infections, especially Methicillin-resistant Staphylococcus aureus (MRSA) are becoming common in U.S. hospitals. Surprisingly, up to 85% of staph infections are caused by a patient's own bacteria. Three in 10 Americans carry staph bacteria in their noses, where the germs live benignly unless they are allowed to enter the body through an open wound like a surgical incision. If such a patient touches his or her nose and then the surgical site, the bacteria can wreak havoc.

Recently a research team at the University of Iowa published a study outlining a simple, three-step plan to cut MRSA infection rates by up to 71% and infections from a broader class of gram-positive bacteria by up to 59%. Schweizer et al., “Effectiveness of a bundled intervention of decolonization and prophylaxis to decrease Gram positive surgical site infections after cardiac or orthopedic surgery: systemic review and meta-analysis,” BMJ, 346:f2743 (2013).

Based on their review of 39 studies of infection prevention strategies in U.S. hospitals, the research team recommends that doctors swab patients' noses before surgery to test for MRSA bacteria. If the patient has MRSA bacteria naturally living in his or her nose, apply an antibiotic nose ointment in the days before surgery. During the procedure, doctors should give those patients an antibiotic that targets MRSA and give all other patients a more general antibiotic.

Thus, in another embodiment of the invention, encompassed are swabs useful for treating nasal passages, such as prior to surgery, to prevent, reduce the risk, or minimize bacterial infections, including MRSA infections. Current commercial products, such as Mupirocin, SinoFrech Nasal and Sinus Care, do not kill all or 99.9% of bacteria, which is in contrast to the nanoemulsions of the invention. Further, because the nanoemulsions of the present invention are non-irritating, the swab can be applied high up in the nasal cavity, thereby eradicating more sources of bacteria and resulting in greater antimicrobial effectiveness.

A nasal spray comprising a nanoemulsion according to the invention can also be used to treat and/or prevent bacterial infections originating in the nasal cavities, such as infections that could result from the presence of bacteria in a patient's nasal cavities prior to surgery. Moreover, both the nasal swab, dropper, and spray are hydrating, as hydration is a feature of the nanoemulsions described herein. Thus, the swab and spray will hydrate the nasal mucosa, as well as be antimicrobial.

V. Methods

The compositions disclosed herein are useful in methods of reducing or killing a microorganism population, and/or inactivating (e.g., fully inactivating) in or on a human subject thereof, comprising topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal) or orally administering or applying to the human subject a composition, wipe, or swab as described herein.

In some embodiments, the composition or wipe enters the epidermis, dermis, mucosa, squamous epithelium, or any combination thereof. In some embodiments, the composition, wipe, and/or swab permeates into the epidermis, dermis, mucosa, and/or squamous epithelium via the follicular route using skin pores and hair follicles. In some embodiments, the composition, wipe, and/or swab diffuses through the skin, skin pores, nail, scalp, hair follicles, lateral or proximal folds, nail, hyponichium, or a combination thereof.

One benefit of the nanoemulsions, wipes and swabs described herein is that use of the compositions, wipes and/or swabs does not result or produce drug-resistant microorganisms. This is because the mechanism of action in killing the microorganisms does not result in drug-resistant microorganisms. In particular, nanoemulsions lyse pathogens upon contact, thereby overcoming existing resistance mechanisms. The appearance of drug-resistant (DR) bacteria in the community is a crucial development, and is associated with increased morbidity, mortality, healthcare costs, and antibiotic use.

Surface decontamination: In another embodiment, encompassed are methods of decontaminating surfaces using the nanoemulsions described herein. Such a method comprises applying the composition, dermal patch or wipe, and/or swab described herein to a surface requiring decontamination. The surface can be, for example, any hard or porous surface, including clothing. One benefit of the compositions, patches, wipes, and swabs of the invention is that the nanoemulsion has a longer residence time at the site of application as compared to non-nanoemulsion formulations, such as Purell®. This means that the compositions, patches, wipes and swabs have greater antimicrobial effectiveness when applied to surfaces, such as surgical tools, surfaces that may be exposed to wounds (e.g., in an ambulance, hospital setting, or military setting). The compositions are also low cost, and thus can be liberally used to decrease the risk of infection where and when appropriate.

Eye treatment: In yet another embodiment, the nanoemulsion compositions of the invention can be used as a hydrating eye product for dry eye. Dry eye syndrome is a chronic and typically progressive condition. Depending on its cause and severity, it may not be completely curable. The increased permeation and hydration of the nanoemulsions according to the invention would be highly preferable over existing commercial products. Further, such a composition could additional comprise a therapeutic agent, such as a cyclosporine, which is frequently used in treating dry eye. Examples of current non-nanoemulsion product used to treat dry eye are Restasis® (cyclosporine emulsion) and Xiidra® (lifitegrast solution), but both of these products are known to be associated with eye irritation. In contrast, the nanoemulsions of the invention are non-irritating as well as moisturizing.

Ear infection treatment: In yet another embodiment, the nanoemulsion compositions of the invention can be used in formulations for treating ear infections—e.g., for humans or animals. External otitis is an acute infection of the ear canal skin typically caused by bacteria (Pseudomonas is most common). Symptoms include pain, discharge, and hearing loss if the ear canal has swollen shut. The increased permeation and hydration of the nanoemulsions according to the invention would be highly preferable over existing commercial products. Further, some ear infections are resistant to treatment as it is theorized that the bacteria or fungi causing the infection reside in a biofilm. Akyildiz et al., “Bacterial Biofilm Formation in the Middle-Ear Mucosa of Chronic Otitis Media Patients,” Indian J. Otolaryngol. Head Neck Surg., 65(Suppl. 3):557-561 (2013). Biofilms are matrix-enclosed microbial aggregates that adhere to a biological or nonbiological surface. Biofilms are often responsible for chronic illness and hospital-acquired (nosocomial) infections. In most cases, biofilm-related infections are not responsive to conventional antimicrobials and persistently reoccur. One benefit of the nanoemulsions described herein as they function to disrupt biofilms, and effectively kill component microorganisms present in the biofilm.

Further, in yet another embodiment the nanoemulsion compositions of the invention can be used in vaginal formulations. Three common types of vaginal infections (vaginitis) are yeast infections, bacterial vaginosis (BV), and trichomoniasis. Nanoemulsion formulations according to the invention are useful in treating all of these types of infections. In addition, the increased permeation and hydration of the nanoemulsions according to the invention would be highly preferable over existing commercial products used to treat vaginal infections.

Delivery of therapeutic agents: Also encompassed is a method of delivering an active agent to a subject, comprising administering a composition according to the invention, wherein the composition comprises at least one therapeutic agent as described herein. In one embodiment, the therapeutic agent is not an antimicrobial agent. The composition can be delivered, for example, topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal) or oral administration. In another embodiment, the composition enters the epidermis, dermis, mucosa, squamous epithelium, or any combination thereof. In yet another embodiment, the composition permeates into the epidermis, dermis, mucosa, and/or squamous epithelium via the follicular route using skin pores and hair follicles. Further, the composition can diffuse through the skin, skin pores, nail, mucosa, squamous epithelium, scalp, hair follicles, lateral or proximal folds, nail, hyponichium, or any combination thereof.

Vaccines: Additionally, the nanoemulsion compositions described herein can be combined with at least one antigen to produce a vaccine formulation. The at least one antigen is preferably present in an amount of about at least 1 up to about 250 μg/dose. Any antigen can be combined with a nanoemulsion adjuvant to produce a vaccine. Such vaccines can be administered via any pharmaceutically acceptable means, including oral, nasal, topical, mucosal, IP, IV, IM, etc.

For example, encompassed is a method of delivering an antigen, protein, or peptide to a subject, comprising administering a composition according to the invention to a subject, wherein the composition comprises at least one antigen, protein, or peptide. In one embodiment, administration of the composition results in a protective immune response. For example, the protective immune response can comprises a Th1 immune response, a Th2 immune response, a Th17 immune response, or any combination thereof. In another embodiment, the composition is not systemically toxic to the subject. In yet a further embodiment, the composition produces minimal or no inflammation upon administration. The antigen can be, for example, a protein, whole virus, killed pathogen, or isolated fragment thereof. In addition, in another embodiument the antigen can be selected from the group consisting of: (a) a viral antigen from a virus belonging to a family selected from the group consisting of Orthomyxoviridae, Retroviridae, African Swine Fever Viruses, Papovaviridae, Hepadnaviridae, Coronaviridae, Flaviviridae, Togaviridae, Picornaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae; (b) a viral protein or antigen from an orthomyxovirdae virus which is influenza virus, herpes simplex, herpes zoster, sendai virus, sindbis virus, pox virus, small pox virus, vaccinia virus, influenza virus, seasonal flu virus, or pandemic flu virus; (c) a viral protein or antigen from Ebolavirus; (d) a viral protein or antigen from Respiratory syncytial virus (RSV); (e) a viral protein or antigen from Rotavirus; (f) a viral protein or antigen from Norovirus; (g) a viral protein or antigen from flavivirus which is zika virus, West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, and insect-specific flaviviruses; (h) a viral protein or antigen from Coronavirus which is Middle East Respiratory Syndrome Coronavirus (MERS-CoV); or (i) a viral protein or antigen from Retroviridae which is human immunodeficiency virus, west nile virus, hanta virus, or human papilloma virus. In one embodiment, the composition comprising at least one antigen can be administered topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal) or orally.

Methods of Manufacture: The nanoemulsions of the invention can be formed using classic emulsion forming techniques. See e.g., U.S. 2004/0043041. In an exemplary method, the oil is mixed with the aqueous phase under relatively high shear forces (e.g., using high hydraulic and mechanical forces) to obtain a nanoemulsion comprising oil droplets having an average diameter of less than about 1000 nm. Some embodiments of the invention employ a nanoemulsion having an oil phase comprising an alcohol such as ethanol. The oil and aqueous phases can be blended using any apparatus capable of producing shear forces sufficient to form an emulsion, such as French Presses or high shear mixers (e.g., FDA approved high shear mixers are available, for example, from Admix, Inc., Manchester, N.H.). Methods of producing such emulsions are described in U.S. Pat. Nos. 5,103,497 and 4,895,452, herein incorporated by reference in their entireties.

In an exemplary embodiment, the nanoemulsions used in the methods of the invention comprise droplets of an oily discontinuous phase dispersed in an aqueous continuous phase, such as water or PBS. The nanoemulsions of the invention are stable, and do not deteriorate even after long storage periods. Certain nanoemulsions of the invention are non-toxic and safe when swallowed, inhaled, or contacted to the skin of a subject.

The compositions of the invention can be produced in large quantities and are stable for many months at a broad range of temperatures. The nanoemulsion can have textures ranging from that of a semi-solid cream to that of a thin lotion, to that of a liquid and can be applied topically, transdermally, mucosally (e.g. intranasal, ocular, buccal, vaginal) or orally by any pharmaceutically acceptable method as stated above, e.g., by hand, or nasal drops/spray, or via any other pharmaceutically acceptable method.

The present invention contemplates that many variations of the described nanoemulsions will be useful in the methods of the present invention. To determine if a candidate nanoemulsion is suitable for use with the present invention, three criteria are analyzed. Using the methods and standards described herein, candidate emulsions can be easily tested to determine if they are suitable. First, the desired ingredients are prepared using the methods described herein, to determine if a nanoemulsion can be formed. If a nanoemulsion cannot be formed, the candidate is rejected. Second, the candidate nanoemulsion should form a stable emulsion. A nanoemulsion is stable if it remains in emulsion form for a sufficient period to allow its intended use. For example, for nanoemulsions that are to be stored, shipped, etc., it may be desired that the nanoemulsion remain in emulsion form for months to years. Typical nanoemulsions that are relatively unstable, will lose their form within a day. Third, the candidate nanoemulsion should have efficacy for its intended use. The nanoemulsion of the invention can be provided in many different types of containers and delivery systems.

The nanoemulsions can be delivered (e.g., to a subject or customers) in any suitable container. Suitable containers can be used that provide one or more single use or multi-use dosages of the nanoemulsion for the desired application. In some embodiments of the invention, the nanoemulsions are provided in a suspension or liquid form. Such nanoemulsions can be delivered in any suitable container including spray bottles and any suitable pressurized spray device. Such spray bottles may be suitable for example for delivering the nanoemulsions intranasally or via inhalation.

VI. Definitions

The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. Any suitable materials and/or methodologies known to those of ordinary skill in the art can be utilized in carrying out the methods described herein.

As used in the description of the invention and the appended claims, the singular forms “a”, “an” and “the” are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless the context clearly indicates otherwise. Also, as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

“Administration” can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, the disease being treated and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art.

The terms “buffer” or “buffering agents” refer to materials which when added to a solution, cause the solution to resist changes in pH.

A used herein, “quaternary ammonium compound” refers to a compound containing an ammonium moiety. The ammonium moiety may include four bonds to a positively charged nitrogen atom.

As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method. “Consisting of” shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).

The terms “chelator” or “chelating agent” refer to any materials having more than one atom with a lone pair of electrons that are available to bond to a metal ion.

As used herein, the term “intranasal(ly)” refers to application of the compositions of the present disclosure to the surface of the skin and mucosal cells and tissues of the nasal passages, e.g., nasal mucosa, sinus cavity, nasal turbinates, or other tissues and cells which line the nasal passages.

As used herein, the term “microorganism” refers to without limitation, bacteria, viruses, bacterial spores, molds, fungi, and the like. Also included are biological microorganisms that are capable of producing an undesirable effect upon a host animal, and includes, for example, without limitation, bacteria, viruses, bacterial spores, molds, fungi, and the like. This includes all such biological microorganisms, regardless of their origin or of their method of production

The term “nanoemulsion,” as used herein, includes small oil-in-water dispersions or droplets, as well as other lipid structures which can form as a result of hydrophobic forces which drive apolar residues (i.e., long hydrocarbon chains) away from water and drive polar head groups toward water, when a water immiscible oily phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. The present disclosure contemplates that one skilled in the art will appreciate this distinction when necessary for understanding the specific embodiments herein disclosed.

The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse allergic or adverse immunological reactions when administered to a host (e.g., an animal or a human). Such formulations include any pharmaceutically acceptable dosage form. Examples of such pharmaceutically acceptable dosage forms include, but are not limited to, dips, sprays, seed dressings, stem injections, lyophilized dosage forms, sprays, and mists. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, wetting agents (e.g., sodium lauryl sulfate), isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like.

As used herein, the term “topical(ly)” refers to application of the compositions of the present disclosure to the surface of the skin, mucosal, and squamous epithelium cells and tissues (e.g., buccal, lingual, sublingual, masticatory, respiratory or nasal mucosa, nasal turbinates and other tissues and cells which line hollow organs or body cavities). As used herein “topical(ly)” is in reference to application to the surface of the skin.

As used herein “subject,” “patient,” or “individual” refers to any subject, patient, or individual, and the terms are used interchangeably herein. In this regard, the terms “subject,” “patient,” and “individual” includes mammals, and, in particular humans. When used in conjunction with “in need thereof,” the term “subject,” “patient,” or “individual” intends any subject, patient, or individual having or at risk for a specified symptom or disorder.

The term “stable” when referring to a “stable nanoemulsion” means that the nanoemulsion retains its structure as an emulsion. A desired nanoemulsion structure, for example, may be characterized by a desired size range, macroscopic observations of emulsion science (is there one or more layers visible, is there visible precipitate), pH, and a stable concentration of one or more the components.

The term “surfactant” refers to any molecule having both a polar head group, which energetically prefers solvation by water, and a hydrophobic tail which is not well solvated by water. The term “cationic surfactant” refers to a surfactant with a cationic head group.

As used herein, the phrase “therapeutically effective” or “effective” in context of a “dose” or “amount” means a dose or amount that provides the specific pharmacological effect for which the compound or compounds are being administered. It is emphasized that a therapeutically effective amount will not always be effective in achieving the intended effect in a given subject, even though such dose is deemed to be a therapeutically effective amount by those of skill in the art. For convenience only, exemplary dosages are provided herein. Those skilled in the art can adjust such amounts in accordance with the methods disclosed herein to treat a specific subject suffering from a specified symptom or disorder. The therapeutically effective amount may vary based on the route of administration and dosage form.

The terms “treatment,” “treating,” or any variation thereof includes reducing, ameliorating, or eliminating (i) one or more specified symptoms and/or (ii) one or more symptoms or effects of a specified disorder. The terms “prevention,” “preventing,” or any variation thereof includes reducing, ameliorating, or eliminating the risk of developing (i) one or more specified symptoms and/or (ii) one or more symptoms or effects of a specified disorder.

The disclosed is further described by reference to the following examples, which are provided for illustration only. The disclosed is not limited to the examples, but rather includes all variations that are evident from the teachings provided herein. All publicly available documents referenced herein, including but not limited to U.S. patents, are specifically incorporated by reference.

EXAMPLES
Example 1—Nanoemulsion Test Formulations

The purpose of this example was to prepare several test nanoemulsions having different surfactant blend ratios.

The nanoemulsion test formulations comprised 0.13% BZK or 0.10% CPC, and were made using conventional homogenization techniques. The compositions of the BZK or CPC formulations are listed in Tables 1, 2, and 3 as NE-1, NE-2, and NE-3 formulations, respectively.

To manufacture the nanoemulsion, the water soluble ingredients are first dissolved in water. The oil is then added and the mixture is mixed using high shear homogenization and/or microfluidization until a viscous white emulsion is formed. The emulsion may be further diluted with water to yield the desired concentration of emulsion or quaternary ammonium compound.

Nanoemulsions used in this study are oil-in-water (o/w) emulsions with mean droplet diameters of 300-600 nm. BZK or CPC resides at the interface between the oil and water phases. The hydrophobic tail of the surfactant distributes in the oil core and its polar head group resides in the water phase.

The nanoemulsions described herein are made from surfactants approved for human consumption and common food substances and are ‘Generally Recognized as Safe’ (GRAS) by the FDA. These emulsions are produced by mixing a water-immiscible oil phase into an aqueous phase. The two phases (aqueous phase and oil phase) are combined and processed to yield an emulsion. The emulsion is further processed to achieve the desired particle size.

TABLE 1

NE-1 formulations with 0.13% BZK.

NE-1
NE-1
NE-1
NE-1
NE-1
NE-1

(Surfactant
(Surfactant
(Surfactant
(Surfactant
(Surfactant
(Surfactant

Formulation
Blend
Blend
Blend
Blend
Blend
Blend

Excipients
Ratio: 1:2)
Ratio: 1:5)
Ratio: 1:9)
Ratio: 1:14)
Ratio: 1:18)
Ratio: 1:27)

Purified
95.744
91.805
83.929
76.047
68.2
58.458

Water

BZK
0.13
0.13
0.13
0.13
0.13
0.13

Poloxamer
0.296
0.592
1.184
1.776
2.368
3.552

407

Glycerol
0.504
1.008
2.016
3.024
4.032
6.048

Soybean
3.139
6.279
12.558
18.837
25.116
37.674

Oil

EDTA
0.186
0.186
0.186
0.186
0.186
0.186

Total
100%
100%
100%
100%
100%
100%

The above percentages are wt/wt, unless otherwise noted.

TABLE 2

NE-2 formulations with 0.13% BZK.

NE-2
NE-2

Formulation
(Surfactant Blend
(Surfactant Blend

Excipients
Ratio: 1:5)
Ratio: 1:9)

Purified Water
91.805
83.929

BZK
0.13
0.13

Tween 20
0.592
1.184

Glycerol
1.008
2.016

Soybean Oil
6.279
12.558

EDTA
0.186
0.186

Total
100%
100%

The above percentages are wt/wt, unless otherwise noted.

TABLE 3

NE-3 formulations with 0.10% CPC.

NE-3
NE-3

Formulation
(Surfactant Blend
(Surfactant Blend

Excipients
Ratio: 1:6)
Ratio: 1:12)

Purified Water
91.835
83.956

CPC
0.1
0.1

Poloxamer 407
0.592
1.184

Glycerol
1.008
2.016

Soybean Oil
6.279
12.558

EDTA
0.186
0.186

Total
100%
100%

The above percentages are wt/wt, unless otherwise noted.

Example 2—Permeation Study

The goal of this study was to investigate the permeation of benzalkonium chloride (BZK) from various different nanoemulsions via human skin in-vitro permeation studies.

Nanoemulsions comprising 0.13% BZK were topically applied to dermatomed cadaver human skin in a Franz diffusion cell chamber and compared against each other and against a marketed non-nanoemulsion product comprising the same concentration of BZK, 0.13% (Purell® Foam). Permeation was measured by HPLC in the epidermis and dermis 24 hours after a single topical dose.

The in vitro human cadaver skin model has proven to be a valuable tool for the study of percutaneous absorption of topically applied compounds. The model uses human cadaver skin mounted in specially designed diffusion chambers that allow the skin to be maintained at a temperature and humidity that match typical in vivo conditions. A finite dose of formulation is applied to the epidermal layer, e.g., the outer surface of the skin, and compound absorption is measured by monitoring the compound's rate of appearance in the receptor solution bathing the dermal surface of the skin. Data defining total absorption, rate of absorption, as well as skin content can be accurately determined in this model. The method has historic precedent for accurately predicting in vivo percutaneous absorption kinetics. Franz, T J, “Percutaneous absorption: on the relevance of in vitro data,” J. Invest. Dermatol., 64:190-195 (1975).

Cryopreserved, dermatomed human cadaver abdominal skin from a 67-year-old Caucasian female donor was used in permeation studies and obtained from Science Care (Phoenix, Ariz.) organ donor bank. Cadaver skin was stored in aluminum foil pouches at −70° C. until use. At the time of use, the skin was thawed by placing the sealed pouch in 37° C. water for approximately five minutes. Thawed skin was removed from the pouch and cut into circular discs (30 mm diameter) to fit between the donor and receiver sides of the permeation chambers.

Percutaneous absorption was measured using the in-vitro cadaver skin finite dose technique. Franz et al., “The finite dose technique as a valid in vitro model for the study of percutaneous absorption in man,” In Skin: Drug Application and Evaluation of Environmental Hazards, Current Problems in Dermatology, vol. 7, G, edited by Simon et al., pp 58-68 (Basel, Switzerland, S. Karger, 1978). The receptor compartment was filled with 7.0 mL of distilled water, comprising 10% (v/v) ethanol in water, and was placed in the donor compartment and left open to ambient laboratory conditions. The receptor compartment spout was covered with a Teflon screw cap to minimize evaporation of the receptor solution. Correctly-sized human abdominal skin was placed onto the opening on the permeation cell. All cells were individually clamped with a clamp-support and placed in a heating bath which was maintained at 37° C. by a circulating water bath on the outside of the cells. The receptor compartment was maintained at 37° C. with the water bath and magnetic stirring. The surface temperature of the skin was appropriately 32° C. as determined by an JR surface temperature probe. The illustration and parameters for the diffusion study are shown in FIG. 1 and Table 4.

TABLE 4

Parameters for the human skin study

using diffusion cell methodology.

Apparatus
Diffusion cell apparatus

Membrane
Human Abdominal Skin Lot#

09-03010, female (Caucasian)

Replicates
5

Duration
24
hours

Dosing Surface Area
1.13
cm²

Dose
113
μL

Dose per Surface Area
100
μL/cm²

Dosing Frequency
QD, Once

Test Formulations
0.13% NE-1; 013% BZK NE-2

0.13% BZK in Purell ® Foam

Concentrations
0.13% BZK

Cell Volume
7.0
mL

Receptor Solution
Distilled water, pH 7 with

10% (v/v) ethanol in water

Receptor Sampling Volume
2
mL

Receptor Sampling Time
24
hours

Extraction Solvent
200 proof Ethanol

Surface Wash
1 mL rinse with 70% ethanol/water

solution, 4 times with

cotton swabs dipped in 70%

ethanol/water solution

Assay Method
HPLC

Samples Collected
Surface wash, epidermis, dermis,

and receptor samples

The skin was equilibrated for a period of 30 minutes before applying a 113 μL dose (over a dosing area of 1.13 cm²) of the test formulations onto the epidermal surface of the donor chamber of the diffusion cells using a positive displacement pipette. The exposed dosing epidermal surface area was 1.13 cm². Twenty-four hours after the application of the first dose, the surface of the skin was rinsed with 1 ml of 70% ethanol/water solution and then cleaned with a 70% ethanol-soaked cotton swab, four times. Following alcohol swabbing, the donor cap was removed, and the skin was removed from the apparatus. The epidermis was removed from the dermis via a scraping method and placed in a tarred scintillation vial. A punch biopsy was taken through the dermis and placed in a tarred scintillation vial. Weights of dermis and epidermis were recorded. The epidermal and dermal tissues were extracted with a 200 proof ethanol solution, sonicated for 30 minutes, filtered through a 25 mm, 0.45 μm PTFE membrane syringe filter into HPLC vials and assayed using HPLC. The excess skin portion was placed in scintillation vial with the surface swabs. One mL of the receptor solution was also sampled at 24 hours from the receptor of each cell and filtered through a 0.45 μm PTFE (25 mm) membrane syringe filter. The filtrates were collected in HPLC snap cap vials.

An assay of BZK, extracted from human skin samples, was determined accordingly. This determination was performed on a HPLC equipped with UV detector set at 254 nm. The HPLC column, reverse phase, used was Phenomenex, Luna CN, 250×4 mm, 5 μm at 55° C. The mobile phase composition was acetate buffer and acetonitrile (ACN) in the ratio of 48:52 in isocratic mode. The method was qualified for linearity and for specificity. Experimental conditions are tabulated below in Table 5.

TABLE 5

Experimental conditions for HPLC analysis of BZK

samples extracted from human skin samples.

HPLC System
LC System: Shimadzu LC-20AT

Software: LC Solutions

Communications Bus Module:

Shimadzu CBM-20A

UV-VIS Detector: Shimadzu SPD-20AV

Column Oven: CTO-20AC

Mobile Phase
Acetate Buffer:ACN (48:52)

(v/v or v/v/v)

Column
Phenomenix, Luna 5μ, CN,

100 Å, 250 × 4 mm

Detector Wavelength
254 nm

Column Temperature
30° C.

Injection Volume
100 μL

Flow Rate
2 mL/min

Run Time
15 minutes

Bracketing Standard
160 μg/mL

ACN = Acetonitrile

The amount of BZK that permeated into the epidermis, dermis, and the receptor compartment (at 24 hours after first dose) was determined by HPLC. The concentration of BZK in the dosing area was determined with respect to a standard preparation. The level of BZK each skin area is represented as the amount per wet tissue weight (ng/grams)±the standard deviation. The number of replicas used in the calculation was 5 for each formulation.

The amount of BZK delivered into the human abdominal skin epidermal tissue was the highest with NE-2 (Surfactant Blend Ratio 1:9), with 6642 ng BZK/gram tissue, as compared to 953 ng BZK/gram tissue for the Purell® Foam with the same percentage of 0.13% BZK (0.13%) in each formulation, e.g., equivalent to a 597% increase in permeation with the nanoemulsion formulation having a 1:9 surfactant blend ratio. Similarly, the nanoemulsion having a 1:5 surfactant blend ratio showed an about 300% increase in permeation as compared to the non-nanoemulsion formulation (Purell® Foam).

After one application of 0.13% NE formulations to human skin, this formulation delivered almost 4 to 7 times more BZK into the epidermis as compared to a marketed 0.13% Purell® Foam. With respect to the dermis levels, the nanoemulsion formulation delivered 3 to 4 times more BZK as compared to the marketed product, Purell® Foam, indicating the BZK was able to penetrate into the deeper dermal levels of the skin from the nanoemulsion formulations. There were no detectable levels of BZK in the receptor for any of the formulations tested. Table 6 summarizes these results. FIG. 2 graphically shows the epidermal levels of BZK (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours), and FIG. 3 shows the dermal levels of BZK (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours).

As clearly depicted in FIGS. 2 and 3, nanoemulsions having surfactant ratios of 1:5 and 1:9 showed dramatic and significantly greater permeation (amount of BZK (ng)/tissue weight (g)) as compared to a non-nanoemulsion formulation having the same quantity of BZK.

TABLE 6

Percutaneous absorption of BZK into human skin over

24 hours from a single topical application.

Purell Foam
NE 1 - 1:5 Ratio
NE 1 - 1:9 Ratio

Formulation
(0.13% BZK)
(0.13% BZK)
(0.13% BZK)

Amount
μg/g
μg/g
μg/g

Epidermis
953 ± 235
3794 ± 525
6642 ± 1554

Dermis
20 ± 4
54 ± 16
77 ± 10

Receptor
0
0
0

Number of
4
4
4

Replica

Epidermal and dermal human skin summary (amount of BZK (ng) per surface area (cm²): mean of replicates ± SD; amount of BZK (μg) per weight tissue (g): mean of replicates ± SD).

Example 3—Expanded Ex Vivo Skin Permeation Study

Following the ex vivo skin permeation study outlined in Example 2, the following 0.13% BZK NE-1 formulations were evaluated against the Purell® Foam using the same methodology of Example 2:

TABLE 7

0.13% BZK NE-1 Formulations Tested

NE-1 Ratio (0.13% BZK)

5:1

2:1

1:1

1:2

1:5 (repeated

from Example 2)

1:9 (repeated

from Example 2)

1:14

1:18

1:27

1:36

1:46

FIG. 4 graphically shows the epidermal levels of BZK (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of the different NE-1 formulations with different surfactant blend ratios and Purell® Foam. FIG. 5 shows the dermal levels of BZK (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of different NE-1 formulations with different surfactant blend ratios and Purell® Foam.

The results were significant and unexpected, with a clear bell curve regarding permeation vs surfactant blend ratio demonstrating that a narrow range of a surfactant blend ratio shows dramatic increased permeation. Outside the claimed surfactant blend ratio of about 5: about 1 and ranging up to about 1: about 27, the amount of drug in the epidermis (FIG. 4) and dermis (FIG. 5) is dramatically less. The impact of the claimed narrow range of surfactant blend ratios on permeation was not known prior to the present invention.

Example 4—Time Kill Study

The purpose of this example was to evaluate the antimicrobial properties of the nanoemulsions according to the invention.

The antimicrobial activity of the nanoemulsion formulations described were assessed according to the procedures described in ASTM E2315-16—Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure.

Using the method described in the Standard Guide, a sample of the test formulation was inoculated with a suspension of a test organism. At the exposure (contact) time, an aliquot was removed, neutralized in BPB+ and plated onto TSA agar to be quantitatively assayed for surviving test organisms. The plates were incubated for 24 hours and the survivors were enumerated. Plate counts were converted into log 10 format and compared to an initial starting population to determine log reduction.

Table 8 shows the in vitro 60 second time kill studies for each of the nanoemulsion formulations indicated (P407=Poloxamer 407; TW20=Tween 20). The results indicate that formulation changes did not impact killing and that each of the tested formulations completely killed all of the organism tested. Additionally, FIG. 6 shows that the NE-2 (surfactant blend ratio: 1:5) demonstrates rapid killing (60 second exposure time) of gram+, gram− bacteria.

TABLE 8

Log killing of selected microorganisms following one-minute exposure to each formulation.

NE-1
NE-1
NE-1
NE-2
NE-3

(Surfactant
(Surfactant
(Surfactant
(Surfactant
(Surfactant

Purell ®
Blend
Blend
Blend
Blend
Blend

Foam
Ratio: 1:2)
Ratio: 1:5)
Ratio: 1:9)
Ratio: 1:5)
Ratio: 1:6)

Formulation

Quaternary
0.13% BZK
0.13% BZK
0.13% BZK
0.13% BZK
0.13% BZK
0.10% CPC

ammonium

compound %

Nonionic
—
0.30% P407
0.59% P407
1.18% P407
0.59% TW20
0.59% P407

Surfactant %

Surfactant
—
1:2
1:5
1:9
1:5
1:6

Blend Ratio

60 Second

Log Killing*

Gram-

Positive

Bacteria:

CA-MRSA
>6.30
>6.30
>6.30
>6.30
>6.30
>6.30

(USA 300)

Enterococcus

>5.44
>5.44
>5.44
>5.44
>5.44
>5.44

faecium

(#51559)

Staphylococcus

>6.39
>6.39
>6.39
>6.39
>6.39
>6.39

epidermidis

(#12228)

Gram-

Negative

Bacteria:

Acinetobacter

>6.77
>6.77
>6.77
>6.77
>6.77
>6.77

baumannii

(#19606)

Serratia

>7.95
>7.95
>7.95
>7.95
>7.95
>7.95

marescens

(#14756)

Klebsiella

>5.09
>5.09
>5.09
>5.09
>5.09
>5.09

pneumoniae

(#13883)

*a greater than symbol (>) indicates that 100% of the bacteria sample was killed.

Example 5—High Temperature Stability

The purpose of this example was to demonstrate the stability at high temperatures of nanoemulsions having a preferred surfactant blend ratio.

Stability at extremely high temperatures (e.g. 50° C.; 122° F.) in robust packaging components (e.g. PET plastic bottles with sprayers, not glass vials) would provide significant advantages for extremely hot climates.

NE-2 (Surfactant Blend Ratio: 1:5; 0.13% BZK) was produced at a 4 kg scale and placed on stability at 5° C., 25° C., 40° C., and 50° C. (122° F.). Table 9 shows that NE-2 (Surfactant Blend Ratio: 1:5; 0.13% BZK) is stable for 1 month even at the most extreme storage condition of 50° C. (122° F.). This is highly unexpected. At severely high temperatures, emulsions are prone to rapid destabilization within a few hours to a couple of days. This data demonstrates that the nanoemulsion formulations having the claimed surfactant blend ratio will offer key advantages for use in extremely high temperature climates.

The BZK Potency was determined with RP-HPLC, as described previously (e.g. permeation section). The appearance was determined via a visual assessment of color, creaming, settling and phase separation with predetermined acceptance criteria. The particle size and polydispersity index (PdI) of the sample were measured by dynamic light scattering using photon correlation spectroscopy with a Malvern Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK), according to SOP #208.01 version 1: Particle Sizing (Malvern). All measurements were carried out at 25° C. after appropriate dilution with double distilled 0.22 μm filtered water.

TABLE 9

Summary of NE-2 (Surfactant Blend Ratio: 1:9) stability summary.

BZK Potency
Mean Particle
Polydispersity
Viscosity

Stability
Time
Appearance
pH
(90-110%
Size (nm)
Index
(cP)

Condition
(months)
(Pass/Fail)
(3-6)
Label Claim)
(250-500 nm)
(<0.25)
(cP > 1.0)

Lot X-2112: 20% NE-2 (0.13% BZK)

Stored in PET Slim Line Cylinder Bottles with Fine mist sprayers

Initial
0
Pass
4.73
99.3
315.4 ±
0.151 ±
2.13

0.2
0.058

5° C./41° F.
1
Pass
4.64
101.5
320.8 ±
0.195 ±
2.12

4.1
0.016

25° C./77° F.
1
Pass
4.57
100.4
326.9 ±
0.190 ±
2.17

5.5
0.004

40° C./104° F.
1
Pass
4.55
101.2
337.6 ±
0.220 ±
2.17

3.5
0.004

50° C./122° F.
1
Pass
4.55
103.7
335.7 ±
0.193 ±
2.20

6.2
0.003

5° C./41° F.
3
Pass
4.65
103.1
291.4 ±
0.121 ±
2.09

4.2
0.008

25° C./77° F.
3
Pass
4.58
98.9
314.9 ±
0.158 ±
2.23

1.2
0.024

40° C./104° F.
3
Pass
4.51
100.0
319.3 ±
0.143 ±
2.24

4.0
0.033

50° C./122° F.
3
Pass
3.46
99.6
320.0 ±
0.185 ±
2.35

3.1
0.016

The data shows that no significant particle growth was observed at higher temperatures, demonstrating the stability of the formulation.

Rapid killing of pathogen demonstrated above coupled with stability at extremely high temperatures (shown in Table 9) makes this technology an ideal fit for extremely high temperature climates.

Example 6—In Vivo Skin Hydration Study

The purpose of this example was to evaluate the effect on skin hydration of nanoemulsions having a preferred surfactant blend ratio.

Two skin areas were tested in vivo, which were the human forearm and backarm. Two test formulations were tested: NE-1 (surfactant blend ratio: 1:5; 0.13% BZK) and Purell® Foam (0.13% BZK). 1 mL of each formulation was applied with rubbing for twenty seconds. Skin hydration was measured 5 times with a Delfin Moisture meter at 10, 20, 30, 60, and 180 minutes after application, with lower readings indicate lower skin hydration levels.

FIG. 7 shows skin hydration study results of NE-1 (surfactant blend ratio: 1:5; 0.13% BZK) and Purell® Foam (0.13% BZK), with the figure clearly and unequivocally showing significant and dramatically improved hydration with nanoemulsion formulations according to the invention as compared to a non-nanoemulsion formulation comprising the same quaternary ammonium compound at the same concentration. These results demonstrate that single application of NE-1 resulted in a significant and sustained increase in skin hydration.

Example 7—Wipe Dispensing Study

The objective of this study was to compare the NE formulations comprising BZK described herein to other products comprising the same amount of BZK but lacking a nanoemulsion. Two different wipe materials were tested: spunlace washcloth and airlaid washcloth. Three test formulations comprising the same amount of BZK were tested: (i) an aqueous solution of 0.13% BZK; (ii) NE-1 (surfactant blend ratio: 1:9; 0.13% BZK); and (iii) Purell® Foam (0.13% BZK). The wipes were saturated with consistent volumes of each tested formulation and the amount of BZK dispensed was measured at the following three time points—initial, 2 hours and 5 days.

FIG. 8 shows the percent (%) of BZK dispensed from the wipe (spunlace washcloth) with aqueous BZK (0.13% BZK), NE-1 (surfactant blend ratio: 1:9; 0.13% BZK), and Purell® Foam (0.13% BZK) at the following time points: initial, 2 hours and 5 days. The results graphically depicted in the figure show that the aqueous BZK and Purell® Foam formulations had significantly less compound (BSK) dispensed from the wipe as compared to the nanoemulsion formulation. This result is significant, as the goal of a wipe-dispensed product is to dispense as much drug as possible. Retention of drug in a wipe is contrary to the goal of drug dispension.

In particular, FIG. 8 (spunlace washcloth) shows that the nanoemulsion formulation dispensed over 95% of the BZK label claim from the wipe at each of the tested time points, with over a 110% measurement at 5 days. In contrast, the aqueous BZK formulation had a high BZK % label claim of 60% at the initial time point, and the Purell® Foam formulation had a high of an initial % BZK label claim of about 73%, also at the initial time point.

FIG. 9 (airlaid washcloth) shows the % of BZK dispensed from the wipe with aqueous BZK (0.13% BZK), NE-1 (surfactant blend ratio: 1:9; 0.13% BZK), and Purell® Foam (0.13% BZK) at the following time points: initial, 2 hours and 5 days. The data shown in the figure demonstrates that the nanoemulsion formulation dispensed about 85% of the % BZK label claim at the initial and 2 hour test points, and about 95% of the % BZK label claim at 5 days. In contrast, the aqueous BZK formulation had a high of about a 35% of the % BZK label claim at the initial test point, with the percentage decreasing at the 2 hour and 5 day test points. Similarly, the Purell® Foam formulation had a high of just over 40% of the % BZK label claim at the initial time point, with decreasing amounts at the 2 hour (35%) and 5 day (20%) time points.

These results demonstrate that the wipes comprising the nanoemulsion formulations with preferred surfactant blend ratios significantly dispensed more BZK than non-emulsion formulations of the same active (BZK) present at the same concentration (0.13%).

Example 8—In Vitro Mucin Permeation Study

The objective of this study was to compare the in vitro permeation of Compound A, a therapeutic compound, across a mucin layer (as a surrogate for the nasal mucous) using a commercially available intranasal product and the nanoemulsion emulsion formulations described herein.

Porcine stomach mucin type III (a mixture of different mucins) and HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) were purchased from Sigma-Aldrich (St. Louis, Mo.). Transwell® membranes (6.5 mm diameter inserts, 3.0 μm pore size in polycarbonate membrane) were purchase from Corning Incorporated (Kennebunk Me.). 24 well plates were purchased from VWR (Radnor, Pa.).

Porcine gastric mucin type III was rehydrated at 10 mg/mL in 1 mM HEPES, pH 7 at 25° C. for 30 minutes. Transwell® membranes were coated with 10 mg/mL mucin in 1 mM HEPES, pH 7 overnight at 37° C. hanging in a lower buffer reservoir (1 mM HEPES, pH 7). Mucin coated Transwell® membranes were moved to a fresh reservoir containing 600 μL of fresh 1 mM HEPES buffer, pH 7, at 37° C. 100 μL of NE-1 (surfactant blend ratio of 1:9)+Compound A (0.25% or 0.5%) or a commercial product containing Compound A (0.5%) was added to the top of each Transwell® membrane (as shown in FIG. 10) and incubated at 37° C.

At pre-determined timepoints, the lower buffer reservoir solution was removed and replaced with 600 μL of fresh buffer. Compound A was measured by RP-HPLC analysis in reservoir samples. Each formulation was tested in triplicate.

TABLE 10

NE-1 formulations with Compound A.

NE-1
NE-1

Formulation
(Surfactant Blend Ratio:
(Surfactant Blend Ratio:

Excipients
1:9; 0.50% Compound A)
1:9; 0.25% Compound A)

Buffer
88.414
83.664

BZK
0.12
0.12

Poloxamer 407
1.184
1.184

Glycerol
2.224
2.224

Soybean Oil
12.558
12.558

EDTA
0.5
0.25

Total
100%
100%

*Buffer contains 0.4% sodium citrate and 0.15% citric acid in purified water.

The above percentages are wt/wt, unless otherwise noted.

FIG. 11 shows the results of the in vitro mucin permeation studies of Compound A with the commercially available intranasal product of Compound A (0.50% Compound A) and the NE-1 (surfactant blend ratio: 1:9) with 0.50% and 0.25% of Compound A. As graphically depicted in FIG. 11, the permeation of Compound A was greater when present in a nanoemulsion formulation as compared to a non-nanoemulsion formulation. In particular, the commercial product of Compound A, having a drug concentration of 50%, showed a cumulative concentration of compound A (μg/mL) at 6 hours following application of about 325 μg/mL, in contrast to a concentration of about 730 μg/mL for the nanoemulsion having a surfactant ratio of 1:9 and a drug concentration of 50%, an increase in drug permeation of 125%.

These results show that nanoemulsion formulations having a preferred surfactant blend ratio significantly enhance the permeation of a component therapeutic agent.

Example 9—In Vivo Rat Study

The objective of this study was to compare the serum levels of Compound A following intranasal administration of a commercially available intranasal product and nanoemulsion formulations described herein.

Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington, Mass.; Source; Stock #400) and were 6 weeks old upon arrival. Rats were housed in specific pathogen-free conditions. All procedures were approved by the University Committee on the Use and Care of Animals (UCUCA) at the University of Michigan (ULAM IVAC #: IV1060). Animals were housed in ventilated racks, 3 rats per cage. The in-life duration of the study included 50 μL intranasal administration (25 μL per nare) of each test formulation to three separate rats, timed bleeds, and euthanasia of the animals. The intranasal administration was performed under brief anesthesia.

The test formulations included: (1) a commercial product with 0.5% Compound A (a representative therapeutic agent) or (2) nanoemulsion formulated with either 0.25% or 0.5% Compound A (NE-2 with surfactant blend ratio of 1:2, 1:5, 1:9, and NE-4 with surfactant blend ratio of 1:2 and 1:5).

Blood was collected pre-dose at 72 hours, and then bled at 4 hours, 24 hours and 48 hours week postdose. Blood collection was approximately 1.0 mL in volume and allowed for sufficient serum to allow for analyze and measure of Compound A. Animals were monitored daily by IVAC and husbandry staff and any observations recorded on data sheets. Animals were monitored closely for reactions to test articles. There were no significant reactions that occurred as defined by the University Committee on Care and Use of Animals (UCUCA) humane endpoint guidelines. Upon euthanasia, animals were bled via cardiac puncture; with blood provided for analysis of Compound A.

TABLE 11

NE-2 and NE-4 formulations with Compound A.

NE-2
NE-2
NE-2
NE-2
NE-4
NE-4

(Surfactant
(Surfactant
(Surfactant
(Surfactant
(Surfactant
(Surfactant

Blend Ratio:
Blend Ratio:
Blend Ratio:
Blend Ratio:
Blend Ratio:
Blend Ratio:

Formulation
1:2; 0.50%
1:5; 0.50%
1:5; 0.25%
1:9; 0.50%
1:2; 0.50%
1:5; 0.50%

Excipients
Compound A)
Compound A)
Compound A)
Compound A)
Compound A)
Compound A)

Buffer*
95.389
91.397
91.647
83.414
95.608
91.836

BZK
0.12
0.12
0.12
0.12
0.12
0.12

Tween 80
0.296
0.592
0.592
1.184
0.296
0.592

Glycerol
0.556
1.112
1.112
2.224
—
—

Ethanol
—
—
—
—
0.3365
0.673

Soybean
3.1395
6.279
6.279
12.558
3.1395
6.279

Oil

EDTA
0.5
0.5
0.25
0.5
0.5
0.5

Total
100%
100%
100%
100%
100%
100%

*Buffer contains 0.4% sodium citrate and 0.15% citric acid in purified water.

The above percentages are wt/wt, unless otherwise noted.

Compound A concentration in rat serum was determined using a competitive enzyme linked immunoassay performed by chemiluminescence at Texas A&M Veterinary Medical Diagnostic Laboratory (College Station, Tex.). Briefly, a ICN Pharmaceuticals SimulTRAC-SNB kit uses purified intrinsic factor. The R SimulTRAC-SNB is used for the simultaneous quantitative determination of Compound A in serum. This assay did not require boiling and utilizes both 57Cobalt and 125Iodine. In competitive protein binding, the binder should have an equal affinity for the standard and the substance which is present in the rat serum sample. The unlabeled Compound A competes with its labeled species for the limited number of available binding sites on its specific binder, thus reducing the amount of labeled Compound A bound. Therefore, the level of radioactivity bound is inversely related to the concentration in the rat serum sample or standard.

FIG. 12 shows the % increase in serum levels of Compound A following intranasal administration with the commercially available intranasal product of Compound A (0.50% Compound A) and the NE-2 (surfactant blend ratios: 1:9, 1:5, and 1:2) and NE-4 formulations (surfactant blend ratios: 1:5 and 1:2) with 0.50% or 0.25% of Compound A. In particular and as shown in FIG. 12, the non-nanoemulsion product of Compound A had a 50 percent (%) increase in serum levels of Compound A at 24 hours vs baseline. This is in contrast to increases of up to 150% for a nanoemulsion having the same drug concentration and a surfactant ratio of 1:5. Most surprisingly, a nanoemulsion having a surfactant ratio of 1:5 and half the quantity ofdrug, e.g., 0.25% concentration, showed a 100% increase in serum levels of the drug—a doubling of the increase shown with that observed for the commercial non-nanoemulsion product having twice as much drug (50% drug concentration).

These results show that nanoemulsion formulations having preferred surfactant ratios delivered significant amounts of an incorporated therapeutic agent when administered intranasally, as all of the tested nanoemulsion formulations resulted in an increase in serum levels of the drug of over 100%.

FIG. 13 shows the serum levels of Compound A following one intranasal administration with the commercially available intranasal product of Compound A (0.50% Compound A) and the NE-2 and NE-4 formulations (surfactant blend ratios: 1:5 and 1:2) with 0.50% of Compound A. All of the nanoemulsion formulations resulted in significantly greater serum levels of Compound A (μg/mL)—all greater than about 3500 μg/mL—as compared to the conventional, non-nanoemulsion formulation—about 2750 μg/mL—a difference of about 30%.

The results from Examples 8 and 9 taken together demonstrate that greater mucin penetration of Compound A measured in vitro directly correlates with Compound A penetration in the nasal epithelium in vivo when animals are intranasally treated with the NE-Compound A formulations and leads to greater systemic drug delivery as compared to the commercially available product containing the same concentration of Compound A.

These results show that the nanoemulsion formulations when administered intranasally significantly enhanced the systemic absorption of a representative incorporated therapeutic agent (Compound A) in vivo as compared to a non-nanoemulsion commercial product having the same active at the same concentration. Also demonstrated is that a significantly lower level of Compound A can be administered with an intranasal formulation with any one of the nanoemulsion compositions described herein to achieve systemic absorption equivalent or greater than the commercial product. Similar results are expected with other active agents that are formulated with the any one of the nanoemulsion compositions described herein for intranasal use.

Example 10

The purpose of this example was to evaluate the antimicrobial effectiveness of a nanoemulsion according to the invention on human skin.

The nanoemulsion tested had a surfactant ratio of 1:9 and a BZK amount of 0.13% (NE-1 from Table 1, supra). The positive control was 3M Skin and Nasal Antiseptic Povidone-Iodine Solution 5% (w/w) USP REF 192401 Lot 0006461182 (Exp 2020-06-21) (St Paul, Minn.). The negative control was PBS (1×).

Materials and Reagents: (1) Human abdominal skin, dermatomed 700-1000 μm (Science Care, Aurora, Colo.). Donor Information: C111551, Sex: Female, Age: 45, Wt.: 170, Race: Caucasian, Negative/Non-reactive for HsAG, HCV, HIV; (2) 70% (v/v) Alcohol (Ethyl alcohol, 200 proof-Absolute Anhydrous (no denaturants) USP Grade Pharmco-Apper, Brookfield, Conn.; (3) Sterile Water for Injection, Rocky Mountain Biologicals, West Jordan, Utah); (4) 6 mm biopsy punch sterile (Sklar Instruments, West Chester, Pa.); (5) Scalpel sterile (Integra, Life Sciences, York, N.Y.); (6) RPMI Medium 1640 (1×) (Gibco, Life Technologies, Grand Island, N.Y.); (7) Human serum off the clot Type AB (PAA Laboratories, Dartmouth, Mass.); (8) 0.4 μm pore size cell culture inserts sterile, count 24 (Corning Inc., Durham, N.C.); (9) 6-well cell culture plates sterile, count 4 (Corning Inc., Durham, N.C.); (10) 48-well cell culture plates sterile, count 1 (Corning Inc., Durham, N.C.); (11) S. aureus (USA300 Methicillin-Resistant Staphylococcus aureus (MRSA), clinical isolates) (University of Dentistry and Medicine of New Jersey); (12) TSA (Tryptic Soy Agar) plates (IPM Scientific, Inc., Sykesville, Md.). PBS (1×) (Corning Inc., Durham, N.C.); (13) Butterfield's Buffer (Hardy Diagnostics, Santa Maria, Calif.); (14) T Shaped spreader sterile (Coran Diagnostics Inc, Murrieta, Calif.); (15) Microplate Shaker (VWR, Radnor, Pa.); (16) Incubator Water Jacketed, C02 (Therma Scientific Forma, Grand Island, N.Y.); and (17) Pipettes with sterile tips.

Procedure: Skin Preparation: Each test formulation was done in triplicate. Decolonization of normal flora was achieved by drying the surface of the specimen and swabbing the area with 70% alcohol twice for 30 seconds. 24 explants of uniform size were obtained using a sterile 6-mm biopsy punch on the skin donor. The skin surface area was ˜28.27 mm².

12 tissue explants were placed in a 50 mL sterile conical tube and washed with 15 mL of RPMI 1640 (antibiotics-free) medium for 1 minute with gentle swirling. The skin explants were then placed stratum corneum side up on a 0.4 μm cell culture insert in a 6-well plate with 1 mL of RPMI1640 (antibiotics-free) medium. 12 tissue explants were placed in a 50 mL sterile conical tube and washed with 15 mL RPMI 1640 (antibiotics-free) medium plus 2% human serum for 1 minute with gentle swirling. The skin explants were placed stratum corneum side up on a 0.4 μm cell culture insert in a 6-well plate with 1 mL RPMI1640 (antibiotics-free) medium. 1.2 mL/well of the appropriate medium (e.g. RPMI 1640 (antibiotics-free) medium+/−2% (v/v) human serum was placed into 6-well plate and placed in an incubator at 37° C. and 7% CO₂.

S. aureus Bacteria: S. aureus was inoculated into a TSA plate and incubated overnight at 37° C. and 7% CO₂. A single colony of S. aureus was chosen from the TSA plate and resuspended in RPMI 1640 (antibiotics-free) medium to a concentration of approximately 5×10⁸CFU/mL to be used as the inoculum.

Infection of Skin Explants: 2 μL of S. aureus inoculum were applied onto the stratum corneum side of each piece of skin (1×10⁶CFU/tissue disc). Incubated for 2 hours at 37° C. and 7% CO₂.

Topical Application of Test Formulations to Skin Explant: After S. aureus infection, 50 μL of each test formulation was applied on top of skin surface of three skin explants with a pipette. After 30 seconds, another 50 μL of the test formulation was applied for a total dosing volume of 100 μL. Incubated for 1 hour at 37° C. and 7% CO₂. Wash Skin Explants: 1 mL of PBS (1×) was applied in each insert to wash the tissue for 10 seconds, while swirling the plate gently to wash the tissues. 1 mL wash was removed from each insert and discarded. Incubate Skin Extracts: incubation was continued for 1 hour at 37° C. and 7% CO₂.

Neutralize & Recover (Bacterial (CFU) Enumeration): The infected skin explants were removed from each cell insert and transferred to a 48-well plate containing 250 μL Butterfield's Buffer (neutralization medium) per well. The 48-well plate containing skin explants was placed on a Microplate Shaker for 4 minutes at 500 rpm. The suspension was removed and serially diluted 4 times in PBS and then spread onto TSA plates using a T-shaped sterile spreader. The TSA plates were incubated for 48 hours at 37° C. and 7% CO₂. The colonies were then counted, with the results shown in Table 12 below.

Skin explants infected with MSRA and then treated with the nanoemulsion test formulation showed a significant log reduction of >5.1 as compared to the negative control, PBS. The nanoemulsion formulation showed the same log reduction as compared to the positive control, 3M Skin and Nasal antiseptic containing 5% Povidone Iodine.

TABLE 12

Test Formulations

Nano-
3M Nasal
PBS

RPMI
emulsion
Antiseptic
(1X) Neg-

Log CFU/Log
Medium
(0.13%
(5% Povidone-
ative

Reduction
Tested
BZK)
Iodine)
control

Log CFU recovered
With 2%
<0.4
<0.4
5.5

Log Reduction
(v/v)
>5.1
>5.1
NA

Human

Serum

Log CFU recovered
Without
<0.4
<0.4
5.5

Log Reduction
Serum
>5.1
>5.1
NA

Example 11—Additional Ex Vivo Skin Permeation Study with Topical Agents

The purpose of this example was to evaluate the delivery of several topical agents with a nanoemulsion according to the invention using the ex vivo skin permeation study outlined in Example 2 and the actives for each study were analyzed according the experimental conditions show in Tables 13-15.

TABLE 13

Experimental conditions for HPLC analysis of actives extracted from human skin samples.

Benzethonium
Terbinafine
Miconazole

Actives
chloride (BEC)
Hydrochloride
Nitrate
Hydrocortisone

HPLC System
LC System: Shimadzu LC-20AT
LC System: Waters

Software: LC Solutions
Software: Empower

Communications Bus
Detector: 2497 Dual λ Absorbance Detector

Module: Shimadzu CBM-20A
Separation Module: Waters 2695

UV-VIS Detector:

Shimadzu SPD-20AV

Column Oven: CTO-20AC

Mobile Phase
Acetate Buffer:ACN
PO4 Buffer:MeOH:THF
Acetate Buffer:ACN:MeOH
ACN:Water

(v/v or v/v/v)
(48:52)
(52:40:8)
(2:3:5)
(40:60)

Column
Phenomenex, Luna 5μ,
Agilent, Zorbax
Waters Symmetry
Waters Symmetry

CN, 100 Å,
300 SB C-18,
C8 5μ,
C18 5 μm,

250 × 4 mm
150 × 4.6 mm,
3.9 × 150 mm
3.9 × 150 mm

3.5 μm

Detector
215
nm
220
nm
230
nm
254
nm

Wavelength

Column
30°
C.
35°
C.
25°
C.
25°
C.

Temperature

Injection
100
μL
20
μL
20
μL
20
μL

Volume

Flow Rate
2
mL/min
1
mL/min
1
mL/min
1
mL/minutes

Run Time
12
minutes
10
minutes
10
minutes
10
minutes

Standard
50
μg/mL
12.5
μg/mL
60
μg/mL
12
μg/mL

TABLE 14

Experimental conditions for HPLC analysis of actives extracted from human skin samples.

Chlorhexidine

Actives
Salicylic Acid
Adapalene
PCMX
Gluconate

HPLC System
LC System: Waters

Software: Empower

Detector: 2497 Dual λ Absorbance Detector

Separation Module: Waters 2695

Mobile Phase
Water:MeOH:HAc
ACN:THF:TFA:Water
ACN:Water:H3PO4
PO4 Buffer:ACN

(v/v or v/v/v)
(60:40:1)
(350:430:0.3:220)
(100:100:0.2)
(70:30)

Column
Thermo Hypersil ODS
Thermo Hypersil
Waters Symmetry
Waters Symmetry

5 μm,
ODS 5 μm,
C18 5 μm,
C18 5 μm,

4.6 × 100 mm
4.6 × 250 mm
3.9 × 150 mm
3.9 × 150 mm

Detector
234
nm
235
nm
280
nm
239
nm

Wavelength

Column
35°
C.
45°
C.
25°
C.
40°
C.

Temperature

Injection
20
μL
20
μL
50
μL
10
μL

Volume

Flow Rate
0.7
mL/minutes
1
mL/minutes
1
mL/minutes
1
mL/minutes

Run Time
10
minutes
10
minutes
10
minutes
6
minutes

Standard
60
μg/mL
40
μg/mL
100
μg/mL
40
μg/mL

PO₄Buffer = Phosphate Buffer

ACN = Acetonitrile

MeOH = Methanol

HAc = Acetic Acid

THF = Tetrahydrofuran

H₃PO₄= Phosphoric Acid

TABLE 15

Experimental conditions for HPLC analysis

Agent
Peanut Extract

HPLC System
LC System: Waters

Software: Empower

Detector: 2497 Dual λ Absorbance Detector

Separation Module: Waters 2695

Mobile Phase
A: 0.1% TFA in Water

B: 100% Acetonitrile

Column
Waters Symmetry C18 5 μm, 3.9 × 150 mm

Detector Wavelength
280
nm

Column Temperature
25°
C.

Injection Volume
20
μL

Flow Rate
1.5
mL/min

Run Time
26
minutes

Standard
NA

Terbinafine Delivery: The nanoemulsion tested had a surfactant ratio of 1:9 and a terbinafine amount of 1.0% as shown in the below table. This nanoemulsion was evaluated against the Lamisil AT® (100 terbinafine) using the same methodology of Example 2:

TABLE 16

NE formulations with Terbinafine.

NE-1

Formulation
(Surfactant Blend Ratio:

Excipients
1:9; 1% Terbinafine)

Water
76.3972

Terbinafine Hydrochloride
1.0

BZK
0.13

Poloxamer 407
1.184

Glycerol
2.016

Soybean Oil
12.558

Ethanol
6.70

EDTA
0.0148

Total
100%

FIG. 14 shows the epidermal levels of terbinafine (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of the NE-1 formulation (surfactant ratio of 1:9 with 1% terbinafine) with Lamisil AT® (1% terbinafine). FIG. 15 shows the dermal levels of terbinafine (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulation (surfactant ratio of 1:9 with 1% terbinafine) with Lamisil AT® (1% terbinafine).

As clearly depicted in FIGS. 14 and 15, the nanoemulsions having surfactant ratios of 1:9 showed dramatic and significantly greater permeation (amount of terbinafine (μg)/tissue weight (g)) as compared to a non-nanoemulsion formulation having the same quantity of terbinafine.

Miconazole Delivery: The nanoemulsion tested had a surfactant ratio of 1:12 and a miconazole amount of 2.0% as shown in the below table. This nanoemulsion was evaluated against the Monistat® (2% miconazole) using the same methodology of Example 2:

TABLE 17

NE formulations with Miconazole.

NE-1

Formulation
(Surfactant Blend Ratio:

Excipients
1:12; 2% Miconazole)

Water
75.4272

Miconazole Nitrate
2.0

BZK
0.10

Poloxamer 407
1.184

Glycerol
2.016

Soybean Oil
12.558

Ethanol
6.70

EDTA
0.0148

Total
100%

FIG. 16 graphically shows the epidermal levels of miconazole (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of the NE-1 formulation (surfactant ratio of 1:12 with 2% miconazole) with Monistat® (2% miconazole). FIG. 17 shows the dermal levels of miconazole (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulation (surfactant ratio of 1:12 with 2% miconazole) with Monistat® (2% miconazole).

As clearly depicted in FIGS. 16 and 17, the nanoemulsion having surfactant ratio of 1:12 showed dramatic and significantly greater permeation (amount of miconazole (μg)/tissue weight (g)) as compared to a non-nanoemulsion formulation having the same quantity of miconazole.

Salicyclic Acid Delivery: The nanoemulsions tested had a surfactant ratio of 1:12 and a salicylic acid amounts of 1.0% and 2.0% as shown in the below table. These nanoemulsions was evaluated against the Dermarest® (3% salicylic acid) using the same methodology of Example 2:

TABLE 18

NE formulations with Salicylic Acid.

NE-1
NE-1

(Surfactant Blend
(Surfactant Blend

Formulation
Ratio: 1:12;
Ratio: 1:12;

Excipients
1% Salicylic Acid)
2% Salicylic Acid)

Water
76.4272
75.4272

Salicylic Acid
1.0
2.0

BZK
0.1
0.1

Poloxamer 407
1.184
1.184

Glycerol
2.016
2.016

Soybean Oil
12.558
12.558

Ethanol
6.7
6.7

EDTA
0.0148
0.0148

Total
100%
100%

FIG. 18 graphically shows the epidermal levels of salicylic acid (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of the NE-1 formulation (surfactant ratio of 1:12 with 1% and 2% salicylic acid) with Dermarest® (3% salicylic acid).

As clearly depicted in FIG. 18, the nanoemulsions having surfactant ratio of 1:12 showed dramatic and significantly greater permeation (amount of salicylic acid (μg)/tissue weight (g)) as compared to a non-nanoemulsion formulation having the greater quantity of salicylic acid.

Hydrocortisone Delivery: The nanoemulsion tested had a surfactant ratio of 1:9 and a hydrocortisone amount of 1.0% as shown in the below table. This nanoemulsion was evaluated against the Cortizone-10® (1% hydrocortisone) using the same methodology of Example 2:

TABLE 19

NE formulations with Hydrocortisone.

NE-1

Formulation
(Surfactant Blend Ratio:

Excipients
1:9; 1% Hydrocortisone)

Water
76.3972

Hydrocortisone
1

BZK
0.13

Poloxamer 407
1.184

Glycerol
2.016

Soybean Oil
12.558

Ethanol
6.7

EDTA
0.0148

Total
100%

FIG. 19 graphically shows the epidermal levels of hydrocortisone (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of the NE-1 formulation (surfactant ratio of 1:9 with 1% hydrocortisone) with Cortizone-10® (1% hydrocortisone). FIG. 20 shows the dermal levels of hydrocortisone (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulation (surfactant ratio of 1:9 with 1% hydrocortisone) with Cortizone-10® (1% hydrocortisone).

As clearly depicted in FIGS. 19 and 20, the nanoemulsion having a surfactant ratio of 1:9 showed dramatic and significantly greater permeation (amount of hydrocortisone (μg)/tissue weight (g)) as compared to a non-nanoemulsion formulation having the same quantity of hydrocortisone.

Retinoid Delivery: The nanoemulsion tested had a surfactant ratio of 1:9 and a retinoid (adapalene) amount of 0.1% as shown in the below table. This nanoemulsion was evaluated against the Differin® Gel (0.1% adapalene) using the same methodology of Example 2:

TABLE 20

NE formulations with Adapalene.

NE-1

Formulation
(Surfactant Blend Ratio:

Excipients
1:9; 0.1% Adapalene)

Water
77.2972

Adapalene
0.1

BZK
0.13

Poloxamer 407
1.184

Glycerol
2.016

Soybean Oil
12.558

Ethanol
6.7

EDTA
0.0148

Total
100%

FIG. 21 graphically shows the epidermal levels of adapalene (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of the NE-1 formulation (surfactant ratio of 1:9 with 0.1% adapalene) with Differin® (0.1% adapalene). FIG. 22 shows the dermal levels of adapalene (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulation (surfactant ratio of 1:9 with 0.1% adapalene) with Differin® (0.1% adapalene).

As clearly depicted in FIGS. 21 and 22, the nanoemulsion having surfactant ratio of 1:9 showed dramatic and significantly greater permeation (amount of adapalene (μg)/tissue weight (g)) as compared to a non-nanoemulsion formulation having the same quantity of adapalene.

Topical Protein Delivery: The nanoemulsions tested had a surfactant ratio of 1:6 and 1:9 and a peanut extract protein amount of 0.1% as shown in the below table, where each of the following peanut proteins were used: Ara h2, Ara h1, Ara h3 and Ara hX. This nanoemulsion was evaluated against an aqueous formulation (0.1% peanut protein) using the same methodology of Example 2:

TABLE 21

NE-1, NE-2 and NE-3 formulations with Peanut Extract Protein.

NE-1
NE-2
NE-3

(Surfactant
(Surfactant
(Surfactant

Blend Ratio:
Blend Ratio:
Blend Ratio:

Formulation
1:6; 0.1% Peanut
1:6; 0.1% Peanut
1:9; 0.1% Peanut

Excipients
Extract Protein)*
Extract Protein)
Extract Protein)

Buffer
84.6
84.6
83.9972

(PBS 1X)

Peanut Extract
0.1
0.1
0.1

Protein*

CPC
0.212
—
—

DODAC
—
0.212
—

BZK
—
—
0.13

Tween 80
1.184
1.184
—

Poloxamer 407
—
—
1.184

Glycerol
—
—
2.016

Ethanol
1.346
1.346
—

Soybean Oil
12.558
12.558
12.558

EDTA
—
—
0.0148

Total
100%
100%
100%

*Peanut Extract Protein = one of the following: Ara h2, Ara h1, Ara h3, and Ara hX

FIG. 23 graphically shows the epidermal levels of peanut proteins Ara h2, Ara h1, Ara h3, and Ara hX (μg/g tissue) in human abdominal skin following one application (occluded dose of 100 μl/cm², measured at 18 hours) of the NE-1 formulation (surfactant ratio of 1:6 with 0.1% peanut protein) with an aqueous formulation (0.1% peanut protein). FIG. 24 shows the dermal levels of peanut proteins Ara h2, Ara h1, Ara h3, and Ara hX (μg/g tissue) in human abdominal skin following one application (occluded dose of 100 μl/cm², measured at 18 hours) of NE-1 formulation (surfactant ratio of 1:6), NE-2 formulation (surfactant ratio of 1:6), and NE-3 formulation (surfactant ratio of 1:9) using three different quaternary ammonium compounds and two different nonionic surfactants combined with 0.1% peanut protein) with aqueous formulation (0.1% peanut protein).

As clearly depicted in FIGS. 23 and 24, each of the nanoemulsions having surfactant ratios of 1:6 and 1:9 showed dramatic and significantly greater permeation (amount of peanut protein (μg)/tissue weight (g)) as compared to a non-nanoemulsion formulation having the same quantity of peanut protein. Furthermore, by interchanging the three quaternary ammonium compounds and two nonionic surfactants in the nanoemulsion formulations tested in FIG. 24 and still achieving significantly greater permeation in each case as compared to a non-nanoemulsion formulation, the importance of the concentration ratio of the quaternary ammonium compound to the nonionic surfactant as opposed to the specific surfactants used in each formulation is demonstrated.

Topical BEC Delivery: The nanoemulsion tested had a surfactant ratio of 1:6 and a BEC amount of 0.2% as shown in the below table. This nanoemulsion was evaluated against an aqueous formulation (0.2% BEC), New-Skin® spray (0.2% BEC), and CVS Liquid Bandage (0.2% BEC) using the same methodology of Example 2:

TABLE 22

NE formulations BEC.

NE

Formulation
(Surfactant Blend Ratio:

Excipients
1:6; 0.2% BEC)

Water
83.953

BEC
0.20

Poloxamer 407
1.184

Ethanol
1.346

Soybean Oil
12.558

EDTA
0.7588

Total
100%

FIG. 25 graphically shows the epidermal levels of BEC (μg/g tissue) in human abdominal skin following one application (single dose of 100 μl/cm², measured at 24 hours) of the NE formulation (surfactant ratio of 1:6 with 0.2% BEC) with an aqueous formulation (0.2% BEC), New-Skin® spray (0.2% BEC), and CVS Liquid Bandage (0.2% BEC). FIG. 26 graphically shows the dermal levels of BEC (μg/g tissue) in human abdominal skin following one application (single dose of 100 μl/cm², measured at 24 hours) of the NE formulation (surfactant ratio of 1:6 with 0.2% BEC) with an aqueous formulation (0.2% BEC), New-Skin® spray (0.2% BEC), and CVS Liquid Bandage (0.2% BEC).

As clearly depicted in FIGS. 25 and 26, the nanoemulsion having surfactant ratio of 1:6 showed dramatic and significantly greater permeation (amount of BEC (μg)/tissue weight (g)) as compared to a non-nanoemulsion formulation having the same quantity of BEC.

Topical Chloroxylenol (para-chloro-meta-xylenol; PCMX) Delivery: The nanoemulsion tested had a surfactant ratio of 1:6 and a PCMX amount of 3% as shown in the below table. This nanoemulsion was evaluated against an 70% ethanol formulation (3% PCMX) using the same methodology of Example 2:

TABLE 23

NE formulation with BEC and PCMX.

NE

Formulation
(Surfactant Blend Ratio:

Excipients
1:6; 3% PCMX)

Water
83.951

PCMX
3.0

BEC
0.2

Poloxamer 407
1.184

Ethanol
1.346

Soybean Oil
9.56

EDTA
0.7588

Total
100%

FIG. 27 graphically shows the epidermal levels of PCMX (μg/g tissue) in human abdominal skin following one application (single dose of 100 μl/cm², measured at 24 hours) of the NE formulation (surfactant ratio of 1:6 with 3.0% PCMX) with an 70% ethanol formulation (3% PCMX). FIG. 28 graphically shows the dermal levels of PCMX (μg/g tissue) in human abdominal skin following one application (single dose of 100 μl/cm², measured at 24 hours) of the NE formulation (surfactant ratio of 1:6 with 3.0% PCMX) with an 70% ethanol formulation (3% PCMX).

As clearly depicted in FIGS. 27 and 28, the nanoemulsion having surfactant ratio of 1:6 showed dramatic and significantly greater permeation (amount of PCMX (μg)/tissue weight (g)) as compared to a non-nanoemulsion formulation having the same quantity of PCMX.

Chlorhexidine Delivery: The nanoemulsion tested had a surfactant ratio of 1:9 and a chlorhexidine amount of 2.0% as shown in the below table. This nanoemulsion was evaluated against the 70% isopropanol (IPA) solution containing 2% chlorhexidine using the same methodology of Example 2.

TABLE 24

NE formulation with Chlorhexidine

NE-1

Formulation
(Surfactant Blend Ratio:

Excipients
1:9; 2% Chlorhexidine)

Water
81.911

Chlorhexidine Gluconate
2.0

BZK
0.13

Poloxamer 407
1.184

Glycerol
2.016

Soybean Oil
12.558

EDTA
0.201

Total
100%

FIG. 29 shows the epidermal levels of chlorhexidine (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of the NE-1 formulation (surfactant ratio of 1:9 with 2% chlorhexidine) with a 70% IPA solution containing 2% chlorhexidine. FIG. 30 shows the dermal levels of chlorhexidine (μg/g tissue) in human abdominal skin following one application (dose of 100 μl/cm², measured at 24 hours) of NE-1 formulation (surfactant ratio of 1:9 with 2% chlorhexidine) with a 70% IPA solution containing 2% chlorhexidine.

As clearly depicted in FIGS. 29 and 30, the nanoemulsions having surfactant ratios of 1:9 showed dramatic and significantly greater permeation (amount of chlorhexidine (μg)/tissue weight (g)) as compared to a non-nanoemulsion formulation having the same quantity of chlorhexidine.

Example 12—Determination of Viscosity of Samples

The purpose of this example was to measure the viscosity of different nanoemulsions and to correlate the viscosity with improved epidermal and dermal permeation of the component quaternary ammonium compound.

To determine the viscosity the nanoemulsion (NE) samples ranging from 0.5% NE to 100% NE, Brookfield Viscometers Models LV and RV (Brookfield Engineering Laboratories, Inc., USA) were used. Prior to taking the viscosity reading, the viscometers and NE samples were allowed come to 22.0±1° C. Each NE sample was placed in a BD Falcon™ 50 mL Conical Centrifuge Tube wide enough to properly cover the specified spindle. The tube containing the NE sample was placed under the spindle and centered to the immersion line. For NE samples 0.5% NE to 60% NE, a LV viscometer using an UL adaptor was used. The viscosity of each NE sample was measured at a property speed of either 100, 50 or 1 rpm. The viscosity (cP) readings were recorded. The 80% NE sample was measured using a LV viscometer using a LV2 spindle at a speed of 3 rpm. Due to tremendous increase in viscosity of the 100% NE sample, an RV viscosity with a F spindle at 100 rpm was used to determine the viscosity.

FIG. 33 shows epidermal permeability results, and FIG. 34 shows dermal permeability results, for nanoemulsion formulations of various nanoemulsion concentrations. Nanoemulsions falling within the preferred viscosity range of the present disclosure shown in the shaded box have significant and dramatic increased permeability as compared to the nanoemulsion formulations outside the viscosity range of the disclosure.

Example 13—Particle Size Analysis Polydispersity Index (PdI) and Zeta Potential

The purpose of this example was to measure the zeta potential of different nanoemulsions and to correlate the zeta potential with improved epidermal and dermal permeation of the component quaternary ammonium compound.

The mean particle size (Z-AVE), polydispersity index (PdI) and zeta potential were determined for samples by dynamic light scattering using photon correlation spectroscopy in a Malvern Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK). For particle size, the test sample of nanoemulsion diluted was to 1% final nanoemulsion concentration. For zeta potential, the test sample of nanoemulsion diluted was to 0.1% final nanoemulsion concentration. All measurements were carried out at 25° C. after appropriate dilution with double distilled 0.2 μm filtered water.

FIG. 35 shows epidermal permeability results, and FIG. 36 shows dermal permeability results, for nanoemulsion formulations within preferred zeta potential range and outside the scope of the disclosure relative to the formulation's zeta potential. Nanoemulsions of the disclosure shown in the shaded box show significant and dramatic increased permeability as compared to the nanoemulsion formulations outside the claimed zeta potential range.

Example 14—Centrifugation Study to Determine Quaternary Ammonium Compound Entrapment

The purpose of this example was to measure the amount of quaternary ammonium compound present in the oil phase of the NE, and to correlate the results with improved epidermal and dermal permeation of the component quaternary ammonium compound.

The amount of BZK in the external (aqueous phase) the nanoemulsion was determined. The experiment required separation of the nanoemulsion droplets from the external aqueous phase of the formulation using centrifugation while maintaining emulsion droplet structure (i.e. intact droplets in close proximity to each other) and not to cause coalescence (fusing of the droplets and then measuring the concentration of quaternary ammonium compound.

Approximately 5 grams of the nanoemulsion samples was placed into pre-weighed centrifuge tubes. The weight of nanoemulsion in each tube varied slightly to balance the centrifuge tubes in the rotor. The actual weight of the centrifuged emulsion was determined by the difference of the weight of the filled tube from the empty tube. The samples were centrifuged at 30,000 rpm for 30 minutes.

The nanoemulsion droplets concentrate at the top of the tube and the clear aqueous phase below the emulsion droplets. Images depicting nanoemulsion sample after centrifugation are shown in FIG. 44. Image taken under normal lighting conditions (left) and corresponding negative image (right). The negative image illustrates the clarity of the aqueous phase. A portion of the aqueous phase was removed from the tube with a 26-gauge needle and syringe without distributing the top layer of the nanoemulsion droplets. The sample of the aqueous phase was than assayed for BZK using RP-HPLC. The concentration of BZK in the extracted aqueous phase, the weight of the nanoemulsion in each tube and the percentage of the aqueous phase verses the oil phase was used to determine the entrapment of the quaternary ammonium compound in the oil phase of the nanoemulsion.

FIG. 37 shows epidermal permeability results, and FIG. 38 shows dermal permeability results, for nanoemulsion formulations falling within the disclosure and outside the scope of the disclosure relative to the formulation's entrapment of the quaternary ammonium compound in the oil phase of the nanoemulsion. Nanoemulsions falling within the disclosure are shown in the shaded box and show significant and dramatic increased permeability as compared to the nanoemulsion formulations outside the range of the disclosure (e.g., 80% and 100% nanoemulsion (NE)) and a current commercial formulation (Purell®).

Example 15—Centrifugation on Particle Size Stability of Nanoemulsion Formulations

The purpose of this example was to measure the stability of droplet size of various nanoemulsions following centrifugation, and to correlate the results with improved epidermal and dermal permeation of the component quaternary ammonium compound.

Nanoemulsion samples were placed under a very high centrifugal force and long duration to force the nanoemulsion droplets from the external aqueous phase to come near each other. If the interface of the nanoemulsion droplets is not strong, the droplets will coalescence (fusing of the droplets) and the mean particle size will be effected.

Approximately 5 grams of the nanoemulsion samples was placed into pre-weighed centrifuge tubes. The weight of nanoemulsion in each tube varied slightly in order to balance the centrifuge tubes in the rotor. The actual weight of the centrifuged emulsion is determined by the difference of the weight of the filled tube from the empty tube. The samples were centrifuged at 200,000 rpm for 1 hour. The emulsion droplets concentrate at the top of the tube and the clear aqueous phase below the emulsion droplets. Following centrifugation, droplets are re-distributed in the external aqueous phase by simple shaking and the particle size distribution determined using the Malvern Zetasizer. The mean particle size was determined before and after centrifugation as shown below in Table 25 and the % change in the mean was determined. A change of more than 10% was considered unstable.

TABLE 25

Mean particle size of nanoemulsion compositions

Mean Particle Size
Mean Particle Size

% NE
(Initial)
(After centrifugation)
% Change

0.5%
630.9 ± 2.8
631.0 ± 8.2
0.2

1%
201.7 ± 2.0
197.3 ± 4.9
2.0

2.5%
196.2 ± 1.8
195.0 ± 0.9
0.5

5%
213.6 ± 2.3
208.5 ± 2.7
1.8

10%
240.7 ± 3.2
233.4 ± 0.4
2.9

20%
317.8 ± 2.4
310.3 ± 1.6
2.2

30%
361.2 ± 5.0
382.3 ± 6.7
5.4

40%
423.2 ± 5.3
423.9 ± 13.2
0.2

60%
425.2 ± 4.9
423.0 ± 1.8
0.5

80%
412.8 ± 3.5
543.7 ± 5.6
23

100%
366.5 ± 1.1
432.6 ± 4.5
15

FIG. 39 shows epidermal permeability results, and FIG. 40 shows dermal permeability results, for nanoemulsion formulations falling within the disclosure and formulations outside the scope of the disclosure, relative to the formulation's stability (as measured by change in mean droplet size) following prolonged centrifugation. Nanoemulsions of the present disclosure are shown in the shaded box show significant and dramatic increased permeability as compared to the nanoemulsion formulations outside the claimed range (e.g., 80% and 100% nanoemulsion (NE)) and a current commercial formulation (Purell®).

The unexpected and dramatic cutaneous permeation properties of the nanoemulsions encompassed by the present invention are also demonstrated by studies measuring dermal permeation of nanoemulsion formulations for each of the five attributes examined in the Examples above. Figures for each of these attributes showing dermal permeability results for nanoemulsion formulations falling within the disclosure and outside the scope of the disclosure are shown in FIGS. 4, 5, and 31-40, and in each case, demonstrate that nanoemulsions outside the disclosed ranges show significant and dramatically reduced permeability.

Example 16—Lidocaine Permeation Study

Cryopreserved, dermatomed human cadaver male thigh skin from a donor was used in permeation studies and obtained from Science Care (Tucson, Ariz.) tissue organ donor bank. Cadaver skin was stored in aluminum foil pouches at −70° C. until use. At the time of use, the skin was thawed by placing the sealed pouch in 37° C. water for approximately five minutes. Thawed skin was removed from the pouch and cut into circular discs (30 mm diameter) to fit between the donor and receiver sides of the permeation chambers.

The receptor compartment was filled with 7.0 mL of distilled water, and was placed in the donor compartment. The receptor compartment spout was covered with a Teflon screw cap to minimize evaporation of the receptor solution. Correctly-sized human cadaver skin was placed onto the opening on the permeation cell. All cells were individually clamped with a clamp-support and placed in a heating bath which was maintained at 37° C. by a circulating water bath on the outside of the cells. The receptor compartment was maintained at 37° C. with the water bath and magnetic stirring. The surface temperature of the skin was appropriately 32° C. as determined by an IR surface temperature probe.

The test articles included the following: Salonpas Gel Patch with 4% Lidocaine, NDC #46581-830-06 (Hisamitsu, Japan), Salonpas Roll on Liquid with 4% Lidocaine, 10% Benzyl Alcohol, NDC #55328-901-03, (Hisamitsu, Japan), 20% NE with 0.13% BZK and 4% Lidocaine (non-occluded), 20% NE with 0.13% BZK and 4% Lidocaine (occluded). The composition of the NE is shown in Table 26.

TABLE 26

NE formulation with 0.13% BZK and 4% Lidocaine

Formulation
Percentage in NE (wt/wt)

Excipients
(Surfactant Blend Ratio: 1:9)

Purified Water
73.4

BZK
0.13

Poloxamer 407
1.184

Glycerol
2.016

Soybean Oil
12.558

EDTA
0.0148

Lidocaine
4.0

Ethanol
6.7

Total
100%

The skin was equilibrated for a period of 30 minutes before dosing. A 113 μL (over a dosing area of 1.13 cm²) dose of the liquid test formulations were topically applied onto the epidermal surface of the cadaver skin mounted on the donor chamber of the diffusion cells using a positive displacement pipette. Half of the cells with the NE formulation was left non-occluded and half were occluded with a parafilm film placed over the donor cap to stop any evaporation of the NE from the skin surface. With respect to the Salonpas Gel Patch, a piece of the patch was cut to fit a surface area of 1.13 cm²area and the donor cap was clamped into the cell.

At one and eight hours after the application of the topical dose, anything from the surface was removed (e.g. patch) and the surface of the skin was rinsed with 1 ml of 70% ethanol/water solution and then cleaned with a 70% ethanol-soaked cotton swab, four times. Following alcohol swabbing, the donor cap was removed, and the skin was removed from the apparatus. The epidermis was removed from the dermis via a scraping method and placed in a tared scintillation vial. A punch biopsy was taken through the dermis and placed in a tared scintillation vial. Weights of dermis and epidermis were recorded. The excess skin portion was placed in scintillation vial with the surface swabs.

Two mL of the receptor solution was also sampled at 8 hours from the receptor of each cell and filtered through a 0.45 μm PTFE (25 mm) membrane syringe filter. The filtrates were collected in HPLC snap cap vials.

Skin samples were then collected after removal of the diffusion chamber. Briefly, the epidermis was removed from the dermis in the dosing area via a scraping technique, placed in a tared vial and weighed. The epidermal and dermal tissues were extracted with a 200-proof ethanol solution, sonicated for 30 minutes, filtered through a 25 mm, 0.45 μm PTFE membrane syringe filter into HPLC vials and assayed using HPLC.

Assay of the active agent (Lidocaine) extracted from human skin samples was determined by BlueWillow Biologics, Ann Arbor, Mich. This determination was performed on HPLC equipped with UV detector. See Table 27, below for experimental HPLC conditions for Lidocaine.

The amount of active agent (Lidocaine) that permeated into the epidermis (at 1 and 8 hours, see FIG. 41), dermis (at 1 and 8 hours, see FIG. 42) and the receptor compartment (at 8 hours, see FIG. 43) was determined by HPLC. The levels of the active agent (Lidocaine) in each skin area are represented as the amount per wet tissue weight (μg/grams)±the standard deviation. The number of replicas used in the calculation was 4 or 5 for each formulation.

TABLE 27

Experimental conditions for HPLC analysis of actives extracted from human skin samples.

Benzalkonium
Benzethonium

Chloride
chloride
Terbinafine
Miconazole
Hydrocor-
Salicylic

Actives
(BZK)
(BEC)
Hydrochloride
Nitrate
tisone
Acid

HPLC
LC System: Shimadzu LC-20AT
LC System: Waters

System
Software: LC Solutions
Software: Empower

Communications Bus Module:
Detector: 2497 Dual λ Absorbance Detector

Shimadzu CBM-20A
Separation Module: Waters 2695

UV-VIS Detector: Shimadzu

SPD-20AV

Column Oven: CTO-20AC

Mobile Phase
Acetate Buffer:ACN
PO4
Acetate
ACN:Water
Water:MeOH:HAc

(v/v or v/v/v)
(48:52)
Buffer:MeOH:THF
Buffer:ACN:MeOH
(40:60)
(60:40:1)

(52:40:8)
(2:3:5)

Column
Phenomenex, Luna 5 μ, CN,
Agilent, Zorbax
Waters
Waters
Thermo

100 Å, 250 × 4 mm
300 SB C-18,
Symmetry
Symmetry
Hypersil

150 × 4.6 mm,
C8 5 μ, 3.9 ×
C18 5 μm,
ODS 5 μm,

3.5 μm
150 mm
3.9 × 150 mm
4.6 × 100 mm

Detector
254
nm
215
nm
220
nm
230
nm
254
nm
234
nm

Wavelength

Column
30°
C.
30°
C.
35°
C.
25°
C.
25°
C.
35°
C.

Temperature

Injection
100
μL
100
μL
20
μL
20
μL
20
μL
20
μL

Volume

Flow Rate
2
mL/min
2
mL/min
1
mL/min
1
mL/min
1
mL/min
0.7
mL/min

Run Time
15
minutes
12
minutes
10
minutes
10
minutes
10
minutes
10
minutes

Standard
160
μg/mL
50
μg/mL
12.5
μg/mL
50
μg/mL
12
μg/mL
60
μg/mL

Chlorhexidine

Actives
Adapalene
PCMX
Gluconate
Lidocaine

HPLC
LC System: Waters

System
Software: Empower

Detector: 2497 Dual λ Absorbance Detector

Separation Module: Waters 2695

Mobile Phase
ACN:THF:TFA:Water
ACN:Water:H3PO4
PO4
PO4

(v/v or v/v/v)
(350:430:0.3:220)
(100:100:0.2)
Buffer:ACN
Buffer:ACN

(70:30)
(50:50)

Column
Thermo Hypersil
Waters
Waters Symmetry
Waters

ODS 5 μm,
Symmetry C18
C18 5 μm,
Symmetry

4.6 × 250 mm
5 μm,
3.9 × 150 mm
C18 5 μm,

3.9 × 150 mm

3.9 × 150 mm

Detector
235
nm
280
nm
239
nm
210
nm

Wavelength

Column
45°
C.
25°
C.
40°
C.
25°
C.

Temperature

Injection
20
μL
50
μL
10
μL
10
μL

Volume

Flow Rate
1
mL/min
1
mL/min
1
mL/min
0.5
mL/min

Run Time
10
minutes
10
minutes
6
minutes
5
minutes

Standard
40
μg/mL
100
μg/mL
40
μg/mL
100
μg/mL

PO₄Buffer = Phosphate Buffer

ACN = Acetonitrile

MeOH = Methanol

HAc = Acetic Acid

THF = Tetrahydrofuran

H₃PO₄= Phosphoric Acid

While certain embodiments have been illustrated and described, it should be understood that changes and modifications can be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the following claims.

The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of” will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of” excludes any element not specified.

The present disclosure is not to be limited in terms of the particular embodiments described in this application. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and compositions within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, or compositions, which can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof, inclusive of the endpoints. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Other embodiments are set forth in the following claims

	Number	Date	Country
	62767966	Nov 2018	US
	62860089	Jun 2019	US

NANOEMULSION COMPOSITIONS HAVING ENHANCED PERMEABILITY

Information

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Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

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Provisional Applications (2)