Nothing Special   »   [go: up one dir, main page]

WO2005030172A1 - Compositions antimicrobiennes en nanoemulsion et methodes associees - Google Patents

Compositions antimicrobiennes en nanoemulsion et methodes associees Download PDF

Info

Publication number
WO2005030172A1
WO2005030172A1 PCT/US2004/031346 US2004031346W WO2005030172A1 WO 2005030172 A1 WO2005030172 A1 WO 2005030172A1 US 2004031346 W US2004031346 W US 2004031346W WO 2005030172 A1 WO2005030172 A1 WO 2005030172A1
Authority
WO
WIPO (PCT)
Prior art keywords
vol
present
bctp
oil
compositions
Prior art date
Application number
PCT/US2004/031346
Other languages
English (en)
Inventor
James R Baker, Jr.
Original Assignee
The Regents Of The University And Methods
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University And Methods filed Critical The Regents Of The University And Methods
Publication of WO2005030172A1 publication Critical patent/WO2005030172A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
    • A01N25/04—Dispersions, emulsions, suspoemulsions, suspension concentrates or gels

Definitions

  • the present invention relates to compositions and methods for decreasing the infectivity, morbidity, and rate of mortality associated with a variety of pathogens.
  • the present invention also relates to methods and compositions for decontaminating areas, samples, solutions, and foodstuffs colonized or otherwise infected by pathogens and microorganisms.
  • Pathogens such as bacteria, fungi, viruses, and bacterial spores are responsible for a plethora of human and animal ills, as well as contamination of food and biological and environmental samples.
  • the first step in microbial infections of animals is generally attachment or colonization of skin or mucus membranes, followed by subsequent invasion and dissemination of the infectious microbe.
  • the portals of entry of pathogenic bacteria are predominantly the skin and mucus membranes.
  • bacteria of the Bacillus genus form stable spores that resist harsh conditions and extreme temperatures. Contamination of farmlands with B. anthracis leads to a fatal disease in domestic, agricultural, and wild animals (See e.g., Dragon and Rennie, Can. Vet. J.
  • Human infection with this organism usually results from contact with infected animals or infected animal products (See e.g., Welkos et al., Infect. Immun, 51 :795 [1986]).
  • Human clinical syndromes include a pulmonary form that has a rapid onset and is frequently fatal.
  • the gastrointestinal and cutaneous forms of anthrax although less rapid, can result in fatalities unless treated aggressively (See e.g., Franz et al, JAMA 278:399 [1997]; and Pile et al, Arch. Intern. Med. 158:429 [1998]).
  • Bacillus anthracis infection in humans is no longer common due to effective animal controls that include vaccines, antibiotics and appropriate disposal of infected livestock.
  • animal anthrax infection still represents a significant problem due to the difficulty in decontamination of land and farms.
  • human infection brought about by warfare and/or terrorist activities. While an anthrax vaccine is available (See e.g., Ivins et al, Vaccine 13:1779
  • Bacillus cereus is a common pathogen. It is involved in food borne diseases due to the ability of the spores to survive cooking procedures. It is also associated with local sepsis and wound and systemic infection (See e.g., Drobniewski, Clin. Micro. Rev. 6:324 [1993]). Many bacteria readily develop resistance to antibiotics.
  • bacteria that develop resistance include Staphylococcus that often cause fatal infections, Pmumococci that cause pneumonia and meningitis; Salmonella and E. coli that cause diarrhea; and Enlerococci that cause blood-stream, surgical wound and urinary tract infections (See e.g., Berkelman et. al, J. Infcet. Dis. 170(2):272 [1994]).
  • Influenza A virus is a common respirator pathogen that is widely used as a model system to test anti-viral agents in vitro (See e.g., Karaivanova and Spiro, Biochem. J. 329:511 [1998]; Mammen et al, J. Med. Chem. 38:4179 [1995]; and Huang et al, FEBS
  • HA hemagglutinin
  • NA neuraminidase
  • the present invention relates to compositions and methods for decreasing the infectivity, morbidity, and rate of mortality associated with a variety of pathogens.
  • the present invention also relates to methods and compositions for decontaminating areas, samples, solutions, and foodstuffs colonized or otherwise infected by pathogens and microorganisms.
  • Certain embodiments of the present compositions are nontoxic and may be safely ingested by humans and other animals. Additionally, certain embodiments of the present invention are chemically stable and non-staining.
  • the present invention provides compositions and methods suitable for treating animals, including humans, exposed to pathogens or the threat of pathogens.
  • the animal is contacted with effective amounts of the compositions prior to exposure to pathogenic organisms. In other embodiments, the animal is contacted with effective amounts of the compositions after exposure to pathogenic organisms.
  • the present invention contemplates both the prevention and treatment of microbiological infections.
  • the present invention provides compositions and methods suitable for decontaminating solutions and surfaces, including organic and inorganic samples that are exposed to pathogens or suspected of containing pathogens.
  • the compositions are used as additives to prevent the growth of harmful or undesired microorganisms in biological and environmental samples.
  • the emulsion further comprises a solvent.
  • the solvent comprises an organic phosphate solvent .
  • the organic phosphate-based solvent comprises dialkyl phosphates or trialkyl phosphates (e.g., tributyl phosphate).
  • the emulsion further comprises an alcohol.
  • the solvent is provided in the oil phase of the composition.
  • compositions of the present invention further comprise one or more surfactants or detergents.
  • the surfactant is a non-anionic detergent.
  • the non-anionic detergent is a polysorbate surfactant.
  • the non-anionic detergent is a polyoxyethylene ether.
  • Surfactants that find use in the present invention include, but are not limited to surfactants such as the TWEEN, TRITON, and TYLOXAPOL families of compounds.
  • the compositions of the present invention further comprise one or more cationic halogen containing compounds, including but not limited to, cetylpyridinium chloride.
  • compositions of the present invention further comprise one or more compounds that promote or enhance the germination ("germination enhancers") of certain microorganism, and in particular the spore form of certain bacteria.
  • Germination enhancers contemplated for formulation with the inventive compositions include, but are not limited to, L-alanine, Inosine, CaCl 2 , and NH 4 C1, and the like, hi still further embodiments, the compositions of the present invention further comprise one or more compounds that increase the interaction (“interaction enhancers") of the composition with microorganisms (e.g., chelating agents like ethylenediaminetetraacetic acid, or ethylenebis(oxyethylenenitrilo)tetraacetic acid in a buffer).
  • interaction enhancers e.g., chelating agents like ethylenediaminetetraacetic acid, or ethylenebis(oxyethylenenitrilo)tetraacetic acid in a buffer.
  • the formulations further comprise coloring or flavoring agents (e.g., dyes and peppermint oil).
  • the composition further comprises an emulsifying agent 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 invention feature oil-in-water emulsion compositions that may readily be diluted with water to a desired concentration without impairing their anti-pathogenic properties.
  • oil-in-water emulsions can also contain other lipid structures, such as small lipid vesicles (e.g., lipid spheres that often consist of several substantially concentric lipid bilayers separated from each other by layers of aqueous phase), micelles (e.g., amphiphilic molecules in small clusters of 50-200 molecules arranged so that the polar head groups face outward toward the aqueous phase and the apolar tails are sequestered inward away from the aqueous phase), or lamellar phases (lipid dispersions in which each particle consists of parallel amphiphilic bilayers separated by thin Films of water).
  • small lipid vesicles e.g., lipid spheres that often consist of several substantially concentric lipid bilayers separated from each other by layers of aqueous phase
  • micelles e.g., amphiphilic molecules in small clusters of 50-200 molecules arranged so that the polar head groups face outward toward the aqueous phase and the
  • SLPs surfactant lipid preparations
  • SLPs are minimally toxic to mucous membranes and are believed to be metabolized within the small intestine (See e.g., Hamouda et al, J. Infect. Disease 180:1939 [1998]). SLPs are non-corrosive to plastics and metals in contrast to disinfectants such as bleach. As such, formulations of the present invention based on SLPs are contemplated to be particularly useful against bacteria, fungi, viruses and other pathogenic entities.
  • Certain embodiments of the present invention contemplate methods for decreasing the infectivity of microorganisms (e.g., pathogenic agents) comprising contacting the pathogen with a composition comprising an oil-in-water emulsion.
  • the emulsion is in the form of an oil phase distributed in an aqueous phase with a surfactant, the oil phase includes an organic phosphate based solvent and a carrier oil.
  • two or more distinct emulsions are exposed to the pathogen.
  • the emulsions are fusigenic and/or lysogenic.
  • the oil phase used in the method comprises a non- phosphate based solvent (e.g., an alcohol).
  • the contacting is performed for a time sufficient to kill the pathogenic agent or to inhibit the growth of the agent.
  • the present invention provides a method of decontaminating an environmental surface harboring harmful or undesired pathogens.
  • the pathogenic agent is associated with an environmental surface and the method comprises contacting the environmental surface with an amount of the composition sufficient for decontaminating the surface. While it may be so desired, decontamination need not result in total elimination of the pathogen.
  • the compositions and methods further comprise dyes, paints, and other marking and identification compounds to as to ensure that a treated surface has been sufficiently treated with the compositions of the present invention. hi certain embodiments, an animal is treated internally with a composition of the present invention.
  • the contacting is via intradermal, subcutaneous, intramuscular or intraperitoneal injection. In other embodiments, the contacting is via oral, nasal, buccal, rectal, vaginal or topical administration.
  • the compositions When the present compositions are administered as pharmaceuticals, it is contemplated that the compositions further comprise pharmaceutically acceptable adjutants, excipients, stabilizers, diluents, and the like.
  • the present invention contemplates compositions further comprising additional pharmaceutically acceptable bioactive molecules (e.g., antibodies, antibiotics, means for nucleic acid transfection, vitamins, minerals, co-factors, etc.).
  • the present invention provides a composition comprising an oil-in-water emulsion, said oil-in-water emulsion comprising a discontinuous oil phase distributed in an aqueous phase, a first component comprising an alcohol or glycerol, and a second component comprising a surfactant or a halogen- containing compound.
  • the aqueous phase can comprise any type of aqueous phase including, but not limited to, water (e.g., diH 2 0, distilled water, tap water) and solutions (e.g., phosphate buffered saline solution).
  • the oil phase can comprise any type of oil including, but not limited to, plant oils (e.g., soybean oil, avocado oil, flaxseed oil, coconut oil, cottonseed oil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil, and sunflower oil), animal oils (e.g., fish oil), flavor oil, water insoluble vitamins, mineral oil, and motor oil.
  • plant oils e.g., soybean oil, avocado oil, flaxseed oil, coconut oil, cottonseed oil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil, and sunflower oil
  • animal oils e.g., fish oil
  • flavor oil water insoluble vitamins, mineral oil, and motor oil.
  • the oil phase comprises 30-90 vol% of the oil-in-water emulsion (i.e., constitutes 30-90% of the total volume of the final emulsion), more preferably 50-80%>.
  • the alcohol is ethanol or methanol.
  • the surfactant is a polysorbate surfactant (e.g., TWEEN 20, TWEEN 40, TWEEN 60, and TWEEN 80), a pheoxypolyethoxyethanol (e.g., TRITON X-100, X-301, X-165, X-102, and X-200, and TYLOXAPOL) or sodium dodecyl sulfate.
  • the halogen-containing compound comprises a cetylpyridinium halides, cetyltrimethylammonium halides, cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides, cetyltributylphosphonium halides, dodecyltrimethylammonium halides, tetradecyltrimethylammonium halides, cetylpyridinium chloride, cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide, cetyltrimethylammonium bromide, cetyidimethylethylammonium bromide, cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide, or tetrad ecyltrimethyl
  • the emulsions may further comprise third, fourth, fifth, etc. components.
  • an additional component is a surfactant (e.g., a second surfactant), a germination enhancer, a phosphate based solvent (e.g., tributyl phosphate), a neutramingen, L-alanine, ammonium chloride, trypticase soy broth, yeast extract, L- ascorbic acid, lecithin, p-hyroxybenzoic acid methyl ester, sodium thiosulate, sodium citrate, inosine, sodium hyroxide, dextrose, and polyethylene glycol (e.g., PEG 200, PEG 2000, etc.).
  • a surfactant e.g., a second surfactant
  • a germination enhancer e.g., tributyl phosphate
  • a neutramingen e.g., L-alanine, ammonium chloride, tryptica
  • the present invention also provides non-toxic, non-irritant, a composition comprising an oil-in-water emulsion, said oil-in-water emulsion comprising a quaternary ammonium compound, wherein said oil-in-water emulsion is antimicrobial against bacteria, virus, fungi, and spores.
  • the oil-in-water emulsion has no detectable toxicity to plants or animals (e.g., to humans).
  • the oil-in-water emulsion causes no detectable irritation to plants or animals (e.g., to humans).
  • the oil-in-water emulsion further comprises any of the components described above.
  • Quaternary ammonium compounds include, but are not limited to, N-alkyldimethyl benzyl ammonium saccharinate, 1,3,5- Triazine-l,3,5(2H,4H,6H)-triethanol; 1-Decanaminium, N-decyl-N, N-dimethyl-, chloride (or) Didecyl dimethyl ammonium chloride; 2-(2-(p- (Diisobuyl)cresosxy)ethoxy)ehyl dimethyl benzyl ammonium chloride; 2-(2-(p- (Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzyl ammonium chloride; alkyl 1 or 3 benzyl- l-(2-hydroxethyl)-2-imidazolinium chloride; alkyl bis(2-hydroxyethyl) benzyl ammonium chloride; alkyl demethyl benzyl ammonium chloride; al
  • the present invention also provides methods of making each of the emulsions disclosed herein.
  • the present invention provides a method of making a oil- in-water emulsion comprising emulsifying a mixture, said mixture comprising an oil, an aqueous solution, a first component comprising an alcohol or glycerol, and a second component comprising a surfactant or a halogen-containing compound.
  • the present invention further provides methods for protecting (e.g.
  • the area comprises a solid surface (e.g., a medical device), a solution, the surface of an organism (e.g., an external or internal portion of a human), or a food product.
  • the present invention also provides methods for modifying any of the emulsions described herein, comprising: providing the emulsion and adding or removing a component from the emulsion to produce a modified emulsion.
  • the method further comprises the step of testing the modified emulsion in a biological assay (e.g., an antimicrobrial assay to dete ⁇ nine the effectiveness of the emulsion at reducing the amount of microorganisms associated with a treated area).
  • a biological assay e.g., an antimicrobrial assay to dete ⁇ nine the effectiveness of the emulsion at reducing the amount of microorganisms associated with a treated area.
  • the present invention also contemplates methods of using such modified emulsion in commerce.
  • the method further comprises the step of advertising the sale of the modified emulsion and/or selling the modified emulsion.
  • the present invention also provides systems comprising a delivery system (e.g., a container, dispenser, packaging etc.) containing any of the oil-in-water emulsions described herein.
  • a delivery system e.g., a container, dispenser, packaging etc.
  • the present invention further comprises a system comprising a material in contact with any of the oil-in-water emulsions described herein.
  • the present invention is not limited by the nature of the material in contact with the emulsion.
  • materials include, but are not limited to, medical devices, solutions, food products, cleaning products, motor oils, creams, and biological materials (e.g., human tissues).
  • the present invention also provides a composition comprising an antimicrobial oil-in-water nanoemulsion, said antimicrobial oil-in-water nanoemulsion comprising a solvent and a surfactant, wherein the antimicrobial oil-in-water nanoemulsion kills or disables Corona viruses when in contact with the Corona viruses.
  • the Corona virus is SARS-CoV.
  • the composition further comprises an ingredient selected from the group consisting of steroids, antiviral agents, and steroid-antiviral agent combinations.
  • the steroids are corticosteroids.
  • the antiviral agent is ribavirin.
  • the steroid-antiviral combination comprises corticosteroids and ribavirin.
  • the solvent is selected from the group consisting of an alcohol and glycerol.
  • the alcohol is ethanol.
  • the glycerol is polyethylene glycol.
  • the surfactant is a non-ionic detergent.
  • the non-ionic detergent is polysorbate.
  • the present invention also provides a method of treating SARS, comprising providing an antimicrobial oil-in-water nanoemulsion, the antimicrobial oil-in-water nanoemulsion comprises a solvent and a surfactant, wherein the antimicrobial oil-in- water nanoemulsion kills or disables Corona viruses when in contact with the Corona viruses, and a subject suffering from or suspected of suffering from SARS, and administering the antimicrobial oil-in-water nanoemulsion to the subject.
  • the Corona virus is the SARS-CoV virus.
  • the antimicrobial oil-in-water nanoemulsion further comprises an ingredient selected from the group consisting of steroids, antiviral agents, and steroid-antiviral agent combinations.
  • the steroids are corticosteroids.
  • the antiviral agent is ribavirin, In still further embodiments, the steroid- antiviral combination comprises corticosteroids and ribavirin.
  • the solvent is selected from the group consisting of an alcohol and glycerol, In some embodiments, the alcohol is ethanol. In other embodiments, the glycerol is polyethylene glycol. In still other embodiments, the surfactant is a non-ionic detergent. In yet other embodiments, the non-ionic detergent is polysorbate.
  • FIG. 1 illustrates the bactericidal efficacy of an emulsion of the present invention on B. cereus spores.
  • Figure 2A- Figure 2C illustrate bacterial smears showing the bactericidal efficacy £>f an emulsion of the present invention on B. cereus spores.
  • Figure 3 illustrates the sporicidal activity of different dilutions of an emulsion of the present invention on different B. anthracis spores.
  • Figure 4 illustrates a comparison of the sporicidal activity of an emulsion of the present invention and bleach over time
  • Figure 5 illustrates a comparison of the sporicidal activity of an emulsion of the present invention and bleach over time.
  • Figure 6 illustrates the sporicidal activity of different dilutions of an emulsion of the present invention in media on different B. anthracis spores.
  • Figure 7 illustrates the time course for the sporicidal activity of an emulsion of the present invention against B. anthracis from Del Rio, TX.
  • Figure 8 depicts an electron micrograph ofE. coli (10,000X).
  • Figure 9 depicts an electron micrograph of E. coli treated with BCTP (10,000X).
  • Figure 10 depicts an electron micrograph of E.
  • Figure 1 1 depicts an electron micrograph of Vibrio cholerae (25,000X).
  • Figure 12 depicts an electron micrograph of Vibrio cholerae treated with W 8 o8P (25,000X).
  • Figure 13 depicts an electron micrograph of Vibrio cholerae treated with BCTP (25,000X).
  • Figure 14 depicts an electron micrograph of Vibrio cholerae treated with X 8 W 60 PC (25,000X).
  • Figure 15 illustrates the effect of BCTP, W 80 8P and X 8 W 60 PC on influenza A activity.
  • Figure 16 illustrates the sporicidal activity of BCTP against 4 different Bacillus species compared to that of X 8 W6oPC against 2 Bacillus species.
  • BCTP showed a significant sporicidal activity after 4 hours of treatment against Bacillus cereus, Bacillus circulans, and Bacillus megaterium spores, but not against Bacillus subtilis spores.
  • X 8 6oPC in 4 hours, showed more effective killing against B. cereus and also had a sporicidal activity against B. subtilis which was resistant to BCTP.
  • Figure 17 illustrates the time course of the nanoemulsion sporicidal activity against Bacillus cereus. Incubation with BCTP diluted 1:100 resulted in 95% killing in 4 hours. Incubation with X 8 W 6 oPC diluted 1 : 1000 resulted in 95% killing in only 30 minutes.
  • Figure 18 depicts electron micrographs of Bacillus cereus spores pre- and post- treatment with BCTP. Note, the uniform density in the cortex and the well-defined spore coat before treatment with BCTP. Spores after 4 hours of BCTP treatment show disruption in both the spore coat and the cortex with loss of core components.
  • Figure 19 illustrates the effects of germination inhibition and stimulation on the sporicidal activity of BCTP diluted 1 :100. BCTP sporicidal activity was delayed in the presence of 10 mM D-alanine (germination inhibition), and accelerated in the presence of 50 M L-alanine and 50 M Inosine (germination stimulation).
  • Figure 20A- Figure 20F depict gross and histologic photographs of animals injected subcutaneously with different combinations of BCTP and B. cereus spores.
  • Figure 20A and Figure 20B illustrate animals that were injected with BCTP alone at a dilution of 1 :10. There was no gross tissue damage and histology showed no inflammation.
  • Figure 20C and Figure 20D illustrate animals that were injected with 4xl 0 7 Bacillus cereus spores alone subcutaneously. A large necrotic area resulted with an average area of 1.68 cm 2 . Histology of this area showed essentially complete tissue necrosis of the epidermis and dermis including subcutaneous fat and muscle.
  • Figure 20E and Figure 20F depict mice that were injected with 4xl0 7 Bacillus spores which had been immediately premixed with the BCTP nanoemulsion at final dilution 1 :10. These animals showed minimal skin lesions with average area 0.02 cm (an approximate 98% reduction from those lesions resulting from an untreated infection with spores). Histology in Figure 20F indicates some inflammation, however most of the cellular structures in the epidermis and dermis were intact. All histopathology is shown at 4X magnification.
  • Figure 21A- Figure 21F depict gross and histological photographs of animals with experimental wounds infected with Bacillus cereus spores.
  • Figure 21 A and Figure 21B depict mice with experimental wounds that were infected with 2.5xl0 7 Bacillus cereus spores but not treated. Histological examination of these wounds indicated extensive necrosis and a marked inflammatory response.
  • Figure 21C and Figure 2 ID depict mice with wounds that were infected with 2.5x10 7 Bacillus cereus spores and irrigated 1 hour later with saline. By 48 hours, there were large necrotic areas surrounding the wounds with an average area of 4.86 cm 2 . In addition, 80% of the animals in this group died as a result of the infection. Histology of these lesions indicated total necrosis of the dermis and subdermis and large numbers of vegetative Bacillus organisms.
  • Figure 21E and Figure 2 IF depict mice with wounds that were infected with 2.5x10 7 Bacillus cereus spores and irrigated 1 hour later with a 1 :10 dilution of BCTP. There were small areas of necrosis adjacent to the wounds (0.06 cm 2 ) which was reduced 98% compared to animals receiving spores and saline irrigation. In addition, only 20% of animals died from these wounds. Histology of these lesions showed no evidence of vegetative Bacillus illustrates several particular embodiments the various emulsions of the present invention.
  • Figure 22 illustrates the inhibition of influenza A infection by surfactant lipid preparations.
  • Figure 22A represents BCTP, W 80 8P, SS, and NN;
  • FIG. 22B BCTP and SS.
  • FIG. 23 illustrates the efficacy of BCTP as an anti-influenza agent as compared to TRITON X-100.
  • Influenza A virus was treated with BCTP, tri(n-butyl)phosphate/TRITON X-100/soybean oil (TTO), TRITON X-100/soybean oil (TO), and TRITON X-100 (T) alone for 30 min.
  • the concentration of TRITON X-100 was the same in all preparations used for treatment.
  • FIG. 24 shows that BCTP does not affect adenovirus infectivity.
  • Adenoviral vector AD.RSV ntlacZ
  • FIG. 25 illustrates the structures of influenza A and adenovirus viewed with electron microscopy.
  • Viruses were either untreated or incubated with BCTP at 1 : 100 dilution for 15 an .60 min at room temperature and were subjected to electron microscopy fixation procedure as described in the Examples.
  • Figure 25A illustrates the influenza A virus untreated
  • Figure 25B illustrates influenza A virus incubated with BCTP for 15 min
  • Figure 25C illustrates the adenovirus untreated
  • Figure 25D illustrates the adenovirus incubated with BCTP for 60 min.
  • magnification 200,000x. The bar represents 200nm.
  • Figure 26 illustrates the antibacterial properties of 1% and 10% BCTP.
  • FIG. 27 illustrates the antiviral properties of 10% and 1% BCTP as assessed by plaque reduction assays.
  • Figure 28 illustrates exemplary organisms that are target for the emulsions of the present invention.
  • Figure 29 illustrates several particular embodiments of the various emulsion compositions invention and certain uses for the emulsions.
  • Figure 30 illustrates several particular embodiments of the various emulsion compositions invention and certain uses for the emulsions.
  • Figure 31 schematically depicts various generalized formulations and uses of certain embodiments of the present invention.
  • Figure 31 A shows the log reduction of E.
  • FIG. 3 IB shows log reduction of 5. globigii spores by various nanoemulsions of the present invention for 10%, 1% and 0.10% dilutions of the nanoemulsion.
  • Figure 31C shows log reduction of influenza A (pfu/ml) by various nanoemulsions of the present invention for 10%, 1% and 0.10% dilutions of the nanoemulsion.
  • Figure 32 shows a graph of the log reduction of S. typhimurium treated with an emulsion of the present invention in the presence of EDTA at 40°C.
  • Figure 33 shows a graph of the log reduction of S.
  • Figure 34 shows the lytic effect of an emulsion of the present invention compared to the lytic effect of its non-emulsified ingredients.
  • Figure 35 shows the log reduction of Mycobacteria fortuitum by an emulsion of the present invention at room temperature and 37°C.
  • Figure 36 shows data for the decontamination of a surface using an emulsion of the present invention.
  • microorganism refers to microscopic organisms and taxonomically related macroscopic organisms within the categories of algae, bacteria, fungi (including lichens), protozoa, viruses, and subviral agents.
  • the term microorganism encompasses both those organisms that are in and of themselves pathogenic to another organism (e.g., animals, including humans, and plants) and those organisms that produce agents that are pathogenic to another organism, while the organism itself is not directly pathogenic or infective to the other organism.
  • pathogen refers to an organism, including microorganisms, that causes disease in another organism (e.g., animals and plants) by directly infecting the other organism, or by producing agents that causes disease in another organism (e.g., bacteria that produce pathogenic toxins and the like).
  • disease refers to a deviation from the condition regarded as normal or average for members of a species, and which is detrimental to an affected individual under conditions that are not inimical to the majority of individuals of that species (e.g., diarrhea, nausea, fever, pain, and inflammation etc). A disease may be caused or result from contact by microorganisms and/or pathogens.
  • host refers to organisms to be treated by the compositions of the present invention, Such organisms include organisms that are exposed to, or suspected of being exposed to, one or more pathogens. Such organisms also include organisms to be treated so as to prevent undesired exposure to pathogens. Organisms include, but are not limited to animals (e.g., humans, domesticated animal species, wild animals) and plants, As used herein, the term “inactivating,” and grammatical equivalents, means having the ability to kill, eliminate or reduce the capacity of a pathogen to infect and/or cause a pathological responses in a host.
  • fusigenic is intended to refer to an emulsion that is capable of fusing with the membrane of a microbial agent (e.g., a bacterium or bacterial spore).
  • a microbial agent e.g., a bacterium or bacterial spore.
  • fusigenic emulsions include, but are not limited to, W 80 8P described in U.S. Pat. Nos. 5,618,840; 5,547,677; and 5,549,901 and NP9 described in U.S. Pat. No. 5,700,679, each of which is herein inco ⁇ orated by reference in their entireties.
  • NP9 is a branched poly(oxy-l,2 ethaneolyl),alpha-(4-nonylphenal)-omega-hydroxy-surfactant. While not being limited to the following, NP9 and other surfactants that may be useful in the present invention are described in Table 1 of U.S. Patent 5,662,957, herein inco ⁇ orated by reference in its entirety.
  • the te ⁇ n "lysogenic" refers to an emulsion that is capable of disrupting the membrane of a microbial agent (e.g., a bacterium or bacterial spore).
  • An exemplary lysogenic composition is BCTP.
  • the presence of both a lysogenic and a fusigenic agent in the same composition produces an enhanced inactivating effect than either agent alone.
  • Methods and compositions using this improved antimicrobial composition are described in detail herein.
  • the term "emulsion,” as used herein, includes classic oil-in-water dispersions or droplets, as well as other lipid structures that can form as a result of hydrophobic forces that 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.
  • nanoemulsion refers to oil-in-water dispersions comprising small lipid structures.
  • the nanoemulsion comprise an oil phase having droplets with a mean particle size of approximately 0.1 or less to 5 microns (e.g., 0.1 to 1.0).
  • emulsion and nanoemulsion are often used herein, interchangeably, to refer to the nanoemulsions of the present invention.
  • the terms "contacted” and “exposed,” refers to bringing one or more of the compositions of the present invention into contact with a pathogen or a sample to be protected against pathogens such that the compositions of the present invention may inactivate the microorganism or pathogenic agents, if present.
  • the present invention contemplates that the disclosed compositions are contacted to the pathogens or microbial agents in sufficient volumes and/or concentrations to inactivate the pathogens or microbial agents.
  • 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.
  • cationic surfactant refers to a surfactant with a cationic head group.
  • anionic surfactant refers to a surfactant with an anionic head group.
  • HLB Index Number refers to an index for correlating the chemical structure of surfactant molecules with their surface activity. The HLB Index Number may be calculated by a variety of empirical formulas as described by Meyers, (Meyers, Surfactant Science and Technology, VCH Publishers Inc., New York, pp. 231-245 [1992]), inco ⁇ orated herein by reference.
  • the HLB Index Number of a surfactant is the HLB Index Number assigned to that surfactant in McCutcheon's Volume 1 : Emulsifiers and Detergents North American Edition, 1996 (inco ⁇ orated herein by reference).
  • the HLB Index Number ranges from 0 to about 70 or more for commercial surfactants. Hydrophilic surfactants with high solubility in water and solubilizing properties are at the high end of the scale, while surfactants with low solubility in water which are good solubilizers of water in oils are at the low end of the scale.
  • the term “germination enhancers” describe compounds that act to enhance the germination of certain strains of bacteria (e.g., L-amino acids [L-alanine], CaCl 2 , Inosine, etc).
  • the term “interaction enhancers” describes compounds that act to enhance the interaction of an emulsion with the cell wall of a bacteria (e.g., a Gram negative bacteria).
  • Contemplated interaction enhancers include but are not limited to chelating agents (e.g., ethylenediaminetetraacetic acid [EDTA], ethylenebis(oxyethylenenitrilo)tetraacetic acid [EGTA], and the like) and certain biological agents (e.g., bovine serum albumin [BSA] and the like).
  • chelating agents e.g., ethylenediaminetetraacetic acid [EDTA], ethylenebis(oxyethylenenitrilo)tetraacetic acid [EGTA], and the like
  • BSA bovine serum albumin
  • buffer or “buffering agents” refer to materials which when added to a solution, cause the solution to resist changes in pH.
  • reducing agent and “electron donor” refer to a material that donates electrons to a second material to reduce the oxidation state of one or more of the second material's atoms.
  • monovalent salt refers to any salt in which the metal (e.g., Na, K, or
  • Li has a net 1+ charge in solution (i.e., one more proton than electron).
  • divalent salt refers to any salt in which a metal (e.g., Mg, Ca, or Sr) has a net 2+ charge in solution.
  • 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.
  • solution refers to an aqueous or non-aqueous mixture.
  • the term "therapeutic agent,” refers to compositions that decrease the infectivity, morbidity, or onset of mortality in a host contacted by a pathogenic microorganism or that prevent infectivity, morbidity, or onset of mortality in a host contacted by a pathogenic microorganism.
  • Such agents may additionally comprise pharmaceutically acceptable compounds (e.g., adjutants, excipients, stabilizers, diluents, and the like).
  • the therapeutic agents of the present invention are administered in the fo ⁇ ri of topical emulsions, injectable compositions, ingestable solutions, and the like.
  • the form When the route is topical, the form may be, for example, a cream, ointment, salve or spray.
  • pharmaceutically acceptable or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse allergic or immunological reactions when administered to a host (e.g., an animal or a human).
  • the compositions of the present invention may be formulated for horticultural or agricultural use. Such formulations include dips, sprays, seed dressings, stem injections, sprays, and mists.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, wetting agents (e.g., sodium lauryl sulfate), isotonic and abso ⁇ tion delaying agents, disintrigrants (e.g., potato starch or sodium starch glycolate), and the like.
  • topically refers to application of the compositions of the present invention to the surface of the skin and mucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory, or nasal mucosa, and other tissues and cells which line hollow organs or body cavities).
  • topically active agents refers to compositions of the present invention that elicit pharmacological responses at the site of application (contact) to a host.
  • systemically active drugs is used broadly to indicate a substance or composition which will produce a pharmacological response at a site remote from the point of application or entry into a subject.
  • medical devices includes any material or device that is used on, in, or through a patient's body in the course of medical treatment (e.g., for a disease or injury). Medical devices include, but are not limited to, such items as medical implants, wound care devices, drug delivery devices, and body cavity and personal protection devices.
  • the medical implants include, but are not limited to, urinary catheters, intravascular catheters, dialysis shunts, wound drain tubes, skin sutures, vascular grafts, implantable meshes, intraocular devices, heart valves, and the like.
  • Wound care devices include, but are not limited to, general wound dressings, biologic graft materials, tape closures and dressings, and surgical incise drapes.
  • Drug delivery devices include, but are not limited to, needles, drug delivery skin patches, drug delivery mucosal patches and medical sponges.
  • Body cavity and personal protection devices include, but are not limited to, tampons, sponges, surgical and. examination gloves, and toothbrushes.
  • birth control devices include, but are not limited to, inter uterin devices (IUDs), diaphragms, and condoms.
  • IUDs inter uterin devices
  • the term “purified” or “to purify” refers to the removal of contaminants or undesired compounds from a sample or composition.
  • the term “substantially purified” refers to the removal of from about 70 to 90%, up to 100%, of the contaminants or undesired compounds from a sample or composition.
  • the term “surface” is used in its broadest sense.
  • the term refers to the outermost boundaries of an organism or inanimate object (e.g., vehicles, buildings, and food processing equipment, etc.) that are capable of being contacted by the compositions of the present invention (e.g., for animals: the skin, hair, and fur, etc., and for plants: the leaves, stems, flowering parts, and fruiting bodies, etc.).
  • the term also refers to the inner membranes and surfaces of animals and plants (e.g., for animals: the digestive tract, vascular tissues, and the like, and for plants: the vascular tissues, etc.) capable of being contacted by compositions by any of a number of transdermal delivery routes (e.g., injection, ingestion, transdermal delivery, inhalation, and the like).
  • sample is used in its broadest sense. In one sense it can refer to animal cells or tissues. In another sense, it is meant to include a specimen or culture obtained from any source, such as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention.
  • the present invention comprises compositions and methods for decreasing the infectivity, morbidity, and rate of mortality associated with a variety of microbial and pathogenic organisms.
  • the present invention also relates to methods and compositions for decontaminating areas colonized or otherwise infected by pathogenic organisms.
  • the present invention relates to methods and compositions for decreasing the infectivity of pathogenic organisms in foodstuffs.
  • decreased pathogenic organism infectivity, morbidity, and mortality is accomplished by contacting the pathogenic organism with an oil-in-water composition comprising an aqueous phase, and oil phase, an at least one other compound.
  • compositions of the present invention are non-toxic, non-irritant, and non-corrosive, while possessing potency against a broad spectrum of microorganisms, including bacteria, fungi, viruses, and spores, Certain illustrative embodiments of the present . invention are described below. The present invention is not limited to these specific embodiments. The description is provided in the following sections: I) Exemplary Compositions; IT) Exemplary Formulation Techniques; III) Properties and Activities; IV) Uses; and V) Specific Examples.
  • the emulsions of the present invention comprise (i) an aqueous phase; (ii) an oil phase; and at least one additional compound.
  • these additional compounds are admixed into either the aqueous or oil phases of the composition.
  • these additional compounds are admixed into a composition of previously emulsified oil and aqueous phases.
  • one or more additional compounds are admixed into an existing emulsion composition immediately prior to its use.
  • one or more additional compounds are admixed into an existing emulsion composition prior to the compositions immediate use.
  • Additional compounds suitable for use in the compositions of the present invention include but are not limited to one or more organic, and more particularly, organic phosphate based solvents, surfactants and detergents, cationic halogen containing compounds, germination enhancers, interaction enhancers, food additives (e.g., flavorings, sweetners, bulking agents, and the like) and pharmaceutically acceptable compounds.
  • organic phosphate based solvents e.g., ethylene glycol, g., g., g., g., g., peppermint, adiol, germination enhancers, interaction enhancers, food additives (e.g., flavorings, sweetners, bulking agents, and the like) and pharmaceutically acceptable compounds.
  • food additives e.g., flavorings, sweetners, bulking agents, and the like
  • pharmaceutically acceptable compounds e.g., exemplary embodiments of the various compounds contemplated for use in the compositions of the present invention are presented below
  • the aqueous phase comprises water at a pH of about 4 to 10, preferably about 6 to 8.
  • the pH is preferably 6 to 8.
  • the water is preferably deionized (hereinafter "DJH 2 O") or distilled.
  • the aqueous phase comprises phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the oil phase (e.g., carrier oil) of the emulsion of the present invention comprises 30-90, preferably 60-80, and more preferably 60-70, vol. % of oil, based on the total volume of the emulsion, although higher and lower amounts are contemplated.
  • suitable oils include, but are not limited to, soybean oil, avocado oil, flaxseed oil, coconut oil, cottonseed oil, squalene oil, olive oil, canola oil, com oil, rapeseed oil, safflower oil, sunflower oil, pine oil (e.g., 15%), Olestra oil, fish oils, flavor oils, water insoluble vitamins and mixtures thereof.
  • soybean oil is used.
  • the oil phase is preferably distributed throughout the aqueous phase as droplets having a mean particle size in the range from about 1-2 microns, more preferably from 0.2 to 0.8, and most preferably about 0.8 microns. In other embodiments, the aqueous phase can be distributed in the oil phase. In some preferred embodiments, very small droplet sizes are utilized (e.g., less than 0.5 microns) to produce stable nanoemulsion compositions. It is contemplated that small droplet comprosition also provide clear solutions, which may find desired use in certain product types. In some embodiments, the oil phase comprises 3-15, preferably 5-10 vol.
  • % of an organic solvent based on the total volu ⁇ ie of the emulsion, although higher and lower amounts are contemplated. While the present invention is not limited to any particular mechanism, it is contemplated that the organic phosphate-based solvents employed in the emulsions serve to remove or disrupt the lipids in the membranes of the pathogens. Thus, any solvent that removes the sterols or phospholipids in the microbial membranes finds use in the emulsions of the present invention. Suitable organic solvents include, but are not limited to, organic phosphate based solvents or alcohols.
  • the organic phosphate based solvents include, but are not limited to, dialkyl- and trialkyl phosphates (e.g., tri-n-butyl phosphate [TBP]) in any combination.
  • TBP tri-n-butyl phosphate
  • a particularly preferred trialkyl phosphate in certain embodiments comprises tri-n-butyl phosphate, which is a plasticizer.
  • each alkyl group of the di- or trialkyl phosphate has from one to ten or more carbon atoms, more preferably two to eight carbon atoms.
  • the present invention also contemplates that each alkyl group of the di- or trialkyl phosphate may or may not be identical to one another.
  • mixtures of different dialkyl and trialkyl phosphates can be employed.
  • solvents include, but are not limited to, methanol, ethanol, propanol and octanol.
  • the alcohol is ethanol.
  • the oil phase, and any additional compounds provided in the oil phase may further be sterile and pyrogen free.
  • compositions of the present invention further comprise one or more surfactants or detergents (e.g., from about 3 to 15 %, and preferably about 10%, although higher and lower amounts are contemplated). While the present invention is not limited to any particular mechanism, and an understanding of the mechanism is not required to practice the present invention, it is contemplated that surfactants, when present in the compositions, help to stabilize the compositions. Both non-ionic (non- anionic) and ionic surfactants are contemplated. Additionally, surfactants from the BRIJ family of surfactants find use in the compositions of the present invention. The surfactant can be provided in either the aqueous or the oil phase.
  • Surfactants suitable for use with the emulsions include a variety of anionic and nonionic surfactants, as well as other emulsifying compounds that are capable of promoting the formation of oil-in-water emulsions.
  • emulsifying compounds are relatively hydrophilic, and blends of emulsifying compounds can be used to achieve the necessary qualities.
  • nonionic surfactants have advantages over ionic emulsifiers in that they are substantially more compatible with a broad pH range and often form more stable emulsions than do ionic (e.g., soap-type) emulsifiers.
  • the compositions of the present invention comprises one or more non-ionic surfactants such as a polysorbate surfactants (e.g., polyoxyethylene ethers), polysorbate detergents, pheoxypolyethoxyethanols, and the like.
  • a polysorbate surfactants e.g., polyoxyethylene ethers
  • polysorbate detergents include, but are not limited to, TWEEN 20, TWEEN 40, TWEEN 60, TWEEN 80, etc.
  • TWEEN 60 polyoxyethylenesorbitan monostearate
  • TWEEN 40 and TWEEN 80 comprise polysorbates that are used as emulsifiers in a number of pharmaceutical compositions.
  • these compounds are also used as co-components with adjuvants.
  • TWEEN surfactants also appear to have virucidal effects on lipid-enveloped viruses (See e.g., Eriksson et al, Blood Coagulation and Fibtinolysis 5 (Suppl. 3):S37-S44 [1994]).
  • pheoxypolyethoxyethanols, and polymers thereof, useful in the present invention include, but are not limited to, TRITON (e.g., X-100, X-301, X-165, X-102, X-200), and TYLOXAPOL.
  • TPJTON X-100 is a strong non-ionic detergent and dispersing agent widely used to extract lipids and proteins from biological structures. It also has virucidal effect against broad spectrum of enveloped viruses (See e.g., Maha and Igarashi, Southeast Asian J. Trop. Med. Pub. Health 28:718 [1997]; and Portocala et al, Virologie 27:261 [1976]). Due to this anti-viral activity, it is employed to inactivate viral pathogens in fresh frozen human plasma (See e.g., Horowitz et al, Blood 79:826 [1992]).
  • the surfactants TRITON X-100 t-octylphenoxypolyethoxyethanol
  • TYLOXAPOL t-octylphenoxypolyethoxyethanol
  • spe ⁇ nicides e.g., Nonoxynol-9
  • Additional surfactants and detergents useful in the compositions of the present invention may be ascertained from reference works (e.g., McCutheon's Volume 1 : Emulsions and Detergents - North American Edition, 2000).
  • compositions that comprise a surfactant and an organic solvent are useful for inactivating enveloped viruses and Gram positive bacteria.
  • compositions of the present invention further comprise a cationic halogen containing compound (e.g., from about 0.5 to 1.0 wt. % or more, based on the total weight of the emulsion, although higher and lower amounts are contemplated).
  • a cationic halogen containing compound e.g., from about 0.5 to 1.0 wt. % or more, based on the total weight of the emulsion, although higher and lower amounts are contemplated.
  • the cationic halogen-containing compound is preferably premixed with the oil phase; however, it should be understood that the cationic halogen-containing compound may be provided in combination with the emulsion composition in a distinct formulation.
  • Suitable halogen containing compounds may be selected, for example, from compounds comprising chloride, fluoride, bromide and iodide ions.
  • suitable cationic halogen containing compounds include, but are not limited to, cetylpyridinium halides, cetyltrimethylammonium halides, cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides, cetyltributylphosphonium halides, dodecyltrimethylammonium halides, or tetradecyltrimethylammonium halides.
  • suitable cationic halogen containing compounds comprise, but are not limited to, cetylpyridinium chloride (CPC), cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide (CPB), cetyltrimethylammonium bromide (CTAB), cetyidimethylethylammonium bromide, cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide, and tetrad ecyltrimethylammonium bromide.
  • CPC cetylpyridinium chloride
  • CPC cetyltrimethylammonium chloride
  • cetylbenzyldimethylammonium chloride cetylpyridinium bromide
  • CAB cetyltrimethylammonium bromide
  • cetyltributylphosphonium bromide cetyidimethylethylammonium bromide
  • the cationic halogen containing compound is CPC, although the compositions of the present invention are not limited to formulation with an particular cationic containing compound.
  • addition of 1.0 % wt. or more of a cationic containing compound to the emulsion compositions of the present invention provides a composition that is useful in inactivating enveloped viruses, Gram positive bacteria, Gram negative bacteria and fungi.
  • the compositions further comprise one or more germination enhancing compounds (e.g., from about 1 mM to 15 mM, and more preferably from about 5 mM to 10 mM, although higher and lower amounts are contemplated).
  • the germination enhancing compound is provided in the aqueous phase prior to formation of the emulsion.
  • the present invention contemplates that when germination enhancers are added to the disclosed compositions the sporicidal properties of the compositions are enhanced.
  • the present invention further contemplates that such germination enhancers initiate sporicidal activity near neutral pH (between pH 6 - 8, and preferably 7),
  • neutral pH emulsions can be obtained, for example, by diluting with phosphate buffer saline (PBS) or by preparations of neutral emulsions.
  • PBS phosphate buffer saline
  • the sporicidal activity of the compositions preferentially occurs when the spores initiate germination.
  • the emulsions of the present invention have sporicidal activity. While the present invention is not limited to any particular mechanism, it is believed that the fusigenic component of the emulsions acts to initiate germination and before reversion to the vegetative form is complete the lysogenic component of the emulsion acts to lyse the newly germinating spore. These components of the emulsion thus act in concert to leave the spore susceptible to disruption by the emulsions. The addition of germination enhancer further facilitates the anti-sporicidal activity of the emulsions of the present invention, for example, by speeding up the rate at which the sporicidal activity occurs.
  • Gennination of bacterial endospores and fungal spores is associated with increased metabolism and decreased resistance to heat and chemical reactants. For germination to occur, the spore must sense that the environment is adequate to support vegetation and reproduction.
  • the amino acid L-alanine stimulates bacterial spore germination (See e.g., Hills, J. Gen. Micro. 4:38 [1950]; and Halvorson and Church, Bacteriol Rev. 21 :112 [1957]).
  • L-alanine and L-proline have also been reported to initiate fungal spore germination (Yanagita, Arch Mikrobiol 26:329 [1957]).
  • Simple ⁇ -amino acids such as glycine and L-alanine, occupy a central position in metabolism.
  • Transamination or deamination of ⁇ -amino acids yields the glycogenic or ketogenic carbohydrates and the nitrogen needed for metabolism and growth.
  • transamination or deamination of L-alanine yields pyruvate which is the end product of glyco lytic metabolism (Embden-Meyerhof-Parnas Pathway).
  • Oxidation of pyruvate by pyruvate dehydrogenase complex yields acetyl-CoA, NADH, H + , and C0 2 .
  • Acetyl-CoA is the initiator substrate for the tricarboxylic acid cycle (Kreb's Cycle) which in turns feeds the mitochondrial electron transport chain. Acetyl-CoA is also the ultimate carbon source for fatty acid synthesis as well as for sterol synthesis. Simple ⁇ -amino acids can provide the nitrogen, C0 2 , glycogenic and or- ketogenic equivalents required for - germination and the metabolic activity that follows.
  • suitable germination enhancing agents of the invention include, but are not limited to, ⁇ -amino acids comprising glycine and the L-enantiomers of alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof. Additional information on the effects of amino acids on germination may be found in U.S. Pat. No. 5,510,104, herein inco ⁇ orated by reference in its entirety.
  • a mixture of glucose, fructose, asparagine, sodium chloride (NaCl), ammonium chloride (NH 4 C1), calcium chloride (CaCl 2 ) and potassium chloride (KCI) also may be used.
  • the formulation comprises the germination enhancers L-alanine, CaCl 2 , Inosine and NH 4 C1.
  • the compositions further comprise one or more common forms of growth media (e.g., trypticase soy broth, and the like) that additionally may or may not itself comprise germination enhancers and buffers.
  • a candidate germination enhancer should meet two criteria for inclusion in the compositions of the present invention: it should be capable of being associated with the emulsions of the present invention and it should increase the rate of germination of a target spore in the when inco ⁇ orated in the emulsions of the present invention.
  • One skilled in the art can determine whether a particular agent has the desired function of acting as an germination enhancer by applying such an agent in combination with the compositions of the present invention to a target and comparing the inactivation of the target when contacted by the admixture with inactivation of like targets by the composition of the present invention without the agent.
  • Any agent that increases gennination, and thereby decrease or inhibits the growth of the organisms is considered a suitable enhancer for use in the present invention.
  • addition of a gennination enhancer (or growth medium) to a neutral emulsion composition produces a composition that is useful in treating bacterial spores in addition to enveloped viruses, Gram negative bacteria, and Gram positive bacteria.
  • the present invention provides antimicrobial compositions, including compositions that do not comprise emulsion or nanoemulsions, that comprise a germination enhancer.
  • germination enhancers may be added to any other material (e.g., commercial disinfectants, solutions, etc.) to promote germination and increase the ability of a composition to kill or neutralize spores as compared to the acitivty of the composition in the absence of the germination enhancer(s).
  • compositions of the present invention comprise one or more compounds capable of increasing the interaction of the compositions (i.e., "interaction enhancer") with target pathogens (e.g., the cell wall of Gram negative bacteria such as Vibrio, Salmonella, Shigella and Pseudomonas).
  • target pathogens e.g., the cell wall of Gram negative bacteria such as Vibrio, Salmonella, Shigella and Pseudomonas.
  • the interaction enhancer is preferably premixed with the oil phase; however, in other embodiments the interaction enhancer is provided in combination with the compositions after emulsification.
  • the interaction enhancer is a chelating agent (e.g., ethylenediaminetetraacetic acid [EDTA] or ethylenebis(oxyethylenenitrilo)tetraacetic acid [EGTA] in a buffer [e.g., tris buffer]).
  • chelating agents are merely exemplary interaction enhancing compounds. Indeed, other agents that increase the interaction of the compositions of the present invention with microbial agents and/or pathogens are contemplated.
  • the interaction enhancer is at a concentration of about 50 to about 250 M, although higher and lower amounts are contemplated.
  • an interaction enhancer Any agent that increases the interaction and thereby decrease or inhibits the growth of the bacteria in comparison to that parameter in its absence is considered an interaction enhancer.
  • the addition of an interaction enhancer to the compositions of the present invention produces a composition that is useful in treating enveloped viruses, _some Gram positive bacteria and some Gram negative bacteria. . . .
  • the nanoemulsion composition comprise one or more additional components to provide a desired property or functionality to the nanoemulsions. These components may be inco ⁇ orated into the aqueous phase or the oil phase of the nanoemulsions and may be added prior to or following emulsification.
  • the nanoemulsions further comprise phenols (e.g., triclosan, phenyl phenol), acidifying agents (e.g., citric acid [e.g., 1.5-6%], acetic acid, lemon juice), alkylating agents (e.g., sodium hydroxide [e.g., 0.3%]), buffers (e.g., citrate buffer, acetate buffer, and other buffers useful to maintain a specific pH), and halogens (e.g., polyvinylpyrrolidone, sodium hypochlorite, hydrogen peroxide).
  • phenols e.g., triclosan, phenyl phenol
  • acidifying agents e.g., citric acid [e.g., 1.5-6%], acetic acid, lemon juice
  • alkylating agents e.g., sodium hydroxide [e.g., 0.3%]
  • buffers e.g., citrate buffer, acetate buffer, and other buffers useful to maintain a specific
  • the pathogen inactivating oil-in-water emulsions of the present invention can be formed using classic emulsion forming techniques.
  • the oil phase is mixed with the aqueous phase under relatively high shear forces (e.g., using high hydraulic and mechanical forces) to obtain an oil-in-water nanoemulsion.
  • the emulsion is formed by blending the oil phase with an aqueous phase on a volume-to-volume basis ranging from about 1 :9 to 5:1, preferably about 5:1 to 3:1, most preferably 4:1, oil phase to aqueous phase.
  • 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, NH). Methods of producing such emulsions are described in U.S. Pat. Nos. 5,103,497 and 4,895,452, herein inco ⁇ orated by reference in their entireties.
  • the compositions used in the methods of the present invention comprise droplets of an oily discontinuous phase dispersed in an aqueous continuous phase, such as water.
  • compositions of the present invention are stable, and do not decompose even after long storage periods (e.g., one or more years).
  • Certain compositions of the present invention are non-toxic and safe when swallowed, inhaled, or contacted to the skin of a host. This is in contrast to many chemical microbicides, which are known irritants. Additionally, in some embodiments, the compositions are also non-toxic to plants.
  • the compositions of the present invention can be produced in large quantities and are stable for many months at a broad range of temperatures. Undiluted, they tend to have the texture of a semi-solid cream and can be applied topically by hand or mixed with water.
  • the emulsion may be in the form of lipid structures including, but not limited to, unilamellar, multilamellar, and paucliamellar lipid vesicles, micelles, and lamellar phases.
  • Some embodiments of the present invention employ an oil phase containing ethanol.
  • the emulsions of the present invention contain (i) an aqueous phase and (ii) an oil phase containing ethanol as the organic solvent and optionally a germination enhancer, and (iii) TYLOXAPOL as the surfactant (preferably 2-5%, more preferably 3%),
  • This formulation is highly efficacious against microbes and is also non-irritating and non-toxic to mammalian users (and can thus be contacted with mucosal membranes).
  • the emulsions of the present invention comprise a first emulsion emulsified within a second emulsion, wherein (a) the first emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and an organic solvent; and (iii) a surfactant; and (b) the second emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and a cationic containing compound; and (iii) a surfactant.
  • BCTP comprises a water-in oil nanoemulsion, in which the oil phase was made from soybean oil, tri-n-butyl phosphate, and TRITON X-100 in 80% water.
  • X 8 WeoPC comprises a mixture of equal volumes of BCTP with W 80 8P.
  • W 80 8P is a liposome-like compound made of glycerol monostearate, refined oya sterols (e.g., GENEROL sterols), TWEEN 60, soybean oil, a cationic ion halogen-containing CPC and peppermint oil.
  • the GENEROL family are a group of a polyethoxylated soya sterols (Henkel Co ⁇ oration, Ambler, Pennsylvania). Emulsion formulations are given in Table 1 for certain embodiments of the present invention.
  • compositions listed above are only exemplary and those of skill in the art will be able to alter the amounts of the components to arrive at a nanoemulsion composition suitable for the pu ⁇ oses of the present invention.
  • the ratio of oil phase to water as well as the individual oil carrier, surfactant CPC and organic phosphate buffer, components of each composition may vary.
  • certain compositions comprising BCTP have a water to oil ratio of 4:1, it is understood that the BCTP may be formulated to have more or less of a water phase. For example, in some embodiments, there is 3, 4, 5, 6, 1, 8, 9, 10, or more parts of the water phase to each part of the oil phase. The same holds true for the W 8o 8P formulation.
  • the ratio of Tri(N-butyl)phosphate:TRITON X-100:soybean oil also may be varied.
  • Table 1 lists specific amounts of glycerol monooleate, polysorbate 60, GENEROL 122, cetylpyridinium chloride, and carrier oil for W 80 8P, these are merely exemplary.
  • An emulsion that has the properties of W 8 o8P may be formulated that has different concentrations of each of these components or indeed different components that will fulfill the same function.
  • the emulsion may have between about 80 to about lOOg of glycerol monooleate in the initial oil phase.
  • the emulsion may have between about 15 to about 30 g polysorbate 60 in the initial oil phase.
  • the composition may comprise between about 20 to about 30 g of a GENEROL sterol, in the initial oil phase.
  • the nanoemulsions structure of the certain embodiments of the emulsions of the present invention may play a role in their biocidal activity as well as contributing to the non-toxicity of these emulsions.
  • the active component in BCTP, TRITON-X100 shows less biocidal activity against virus at concentrations equivalent to 11% BCTP. Adding the oil phase to the detergent and solvent markedly reduces the toxicity of these agents in tissue culture at the same concentrations.
  • the nanoemulsion enhances the interaction of its components with the pathogens thereby facilitating the inactivation of the pathogen and reducing the toxicity of the individual components. It should be noted that when all the components of BCTP are combined in one composition but are not in a nanoemulsion structure, the mixture is not as effective as an antimicrobial as when the components are in a nanoemulsion structure. Numerous additional embodiments presented in classes of formulations with like compositions are presented below. The effect of a number of these compositions as antipathogenic materials is provided in Figure 31. The following compositions recite various ratios and mixtures of active components.
  • the inventive formulation comprise from about 3 to 8 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of cetylpyridinium chloride (CPC), about 60 to 70 vol. % oil (e.g., soybean oil), about 15 to 25 vol. % of aqueous phase (e.g., DiH 2 0 or PBS), and in some formulations less than about 1 vol. % of IN NaOH.
  • Some of these embodiments comprise PBS.
  • one embodiment of the present invention comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 64 vol. % of soybean oil, and about 24 vol. % of DiH 2 0 (designated herein as Y3EC).
  • Another similar embodiment comprises about 3.5 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, and about 1 vol. % of CPC, about 64 vol.
  • Yet another embodiment comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 0.067 vol. % of IN NaOH, such that the pH of the formulation is about 7.1, about 64 vol. % of soybean oil, and about 23.93 vol. % of DiH 2 O (designated herein as Y3EC pH 7.1). Still another embodiment comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 0.67 vol.
  • the formulation comprises about 8% TYLOXAPOL, about 8% ethanol, about 1 vol. % of CPC, and about 64 vol. % of soybean oil, and about 19 vol. % of DiH 2 0 (designated herein as Y8EC).
  • a further embodiment comprises about 8 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol, % of CPC, about 64 vol. % of soybean oil, and about 19 vol. % of lx PBS (designated herein as Y8EC PBS).
  • the inventive formulations comprise about 8 vol. % of ethanol, and about 1 vol. % of CPC, and about 64 vol. % of oil (e.g., soybean oil), and about 27 vol. % of aqueous phase (e.g., DiH 2 0 or PBS) (designated herein as EC).
  • some embodiments comprise from about 8 vol.
  • the inventive formulation comprise from about 1 to 2 vol. % of TRITON X-100, from about 1 to 2 vol. % of TYLOXAPOL, from about 7 to 8 vol. % of ethanol, about 1 vol. % of cetylpyridinium chloride (CPC), about 64 to 57.6 vol.
  • one embodiment of the present invention comprises about 2 vol. % of TRITON X-100, about 2 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % CPC, about 64 vol. % of soybean oil, and about 23 vol.
  • the formulation comprises about 1.8 vol. % of TRITON X-100, about 1.8 vol. % of TYLOXAPOL, about 7.2 vol. % of ethanol, about 0.9 vol. % of CPC, about 5 mM L-alanine/Inosine, and about 10 mM ammonium chloride, about 57.6 vol. % of soybean oil, and the remainder of lx PBS (designated herein as 90% X2Y2EC/GE).
  • the formulations comprise from about 5 vol. % of TWEEN 80, from about 8 vol. % of ethanol, from about 1 vol. % of CPC, about 64 vol.
  • the formulations comprise from about 5 vol. % of TWEEN 20, from about 8 vol. % of ethanol, from about 1 vol. % of CPC, about 64 vol. % of oil (e.g., soybean oil), and about 22 vol. % of DiH 2 0 (designated herein as W 2 o5EC).
  • the formulations comprise from about 2 to 8 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 60 to 70 vol.
  • the present invention contemplates formulations comprising about 2 vol. % of TRITON X-100, about 8 vol, % of ethanol, about 64 vol. % of soybean oil, and about 26 vol. %> of DiH 2 0 (designated herein as X2E).
  • the formulations comprise about 3 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 25 vol. % of DiH 2 0 (designated herein as X3E).
  • the formulations comprise about 4 vol. % Triton of X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 24 vol. % of DiH 2 0 (designated herein as X4E). In yet other embodiments, the formulations comprise about 5 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 23 vol. % of DiH 2 0 (designated herein as X5E). Another embodiment of the present invention comprises about 6 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 22 vol.
  • the formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 20 vol. % of DiH 2 O (designated herein as X8E). In still further embodiments of the present invention, the formulations comprise about 8 vol. %> of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of olive oil, and about 20 vol. %> of DiH 2 O (designated herein as X8E O). In yet another embodiment comprises 8 vol. % of TRITON X-100, about 8 vol.
  • the formulations comprise from about 1 to 2 vol. % of TRITON X-100, from about 1 to 2 vol. % of TYLOXAPOL, from about 6 to 8 vol. % TBP, from about 0.5 to 1.0 vol. % of CPC, from about 60 to 70 vol. % of oil (e.g., soybean), and about 1 to 35 vol. % of aqueous phase (e.g., DiH 2 0 or PBS). Additionally, certain of these formulations may comprise from about 1 to 5 vol.
  • the fo ⁇ nula comprises a casein hydrolysate (e.g., Neutramigen, or Progestimil, and the like).
  • the inventive fo ⁇ nulations further comprise from about 0.1 to 1.0 vol. % of sodium thiosulfate, and from about 0.1 to 1.0 vol. % of sodium citrate.
  • PBS phosphate buffered saline
  • one embodiment comprises about 2 vol. % of TRITON X-100, about 2 vol. % TYLOXAPOL, about 8 vol. % TBP, about 1 vol. % of CPC, about 64 vol. % of soybean oil, and about 23 vol. % of DiH 2 0 (designated herein as X2Y2EC).
  • the inventive formulation comprises about 2 vol. % of TRITON X-100, about 2 vol. % TYLOXAPOL, about 8 vol. % TBP, about 1 vol.
  • the formulations comprise about 1.7 vol. % TRITON X-100, about 1.7 vol, % TYLOXAPOL, about 6.8 vol. % TBP, about 0.85% CPC, about 29.2% NEUTRAMIGEN, about 54.4 vol. % of soybean oil, and about 4.9 vol. % of DiH 2 0 (designated herein as 85%> X2Y2PC/baby).
  • the formulations comprise about 1.8 vol. % of TRITON X-100, about 1.8 vol. % of TYLOXAPOL, about 7.2 vol. % of TBP, about 0.9 vol. % of CPC, about 5mM L- alanine/Inosine, about lOmM ammonium chloride, about 57,6 vol. % of soybean oil, and the remainder vol. % of O.lx PBS (designated herein as 90% X2Y2 PC/GE).
  • the formulations comprise about 1.8 vol. % of TRITON X-100, about 1.8 vol. % of TYLOXAPOL, about 7.2 vol. % TBP, about 0.9 vol.
  • the formulations comprise about 1.8 vol. % TRITON X-100, about 1.8 vol. % TYLOXAPOL, about 7.2 vol. % TBP, about 0,9 vol. % CPC, about 1 vol. % yeast extract, about 57.6 vol. % of soybean oil, and about 29.7 vol. % of DiH 2 0 (designated herein as 90% X2Y2PC/YE).
  • the inventive formulations comprise about 3 vol. % of TYLOXAPOL, about 8 vol. % of TBP, and about 1 vol. % of CPC, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 30 vol. % of aqueous phase (e.g., DiH 2 0 or PBS).
  • the inventive formulations comprise about 3 vol. % of TYLOXAPOL, about 8 vol. % of TBP, and about 1 vol. % of CPC, about 64 vol. % of soybean, and about 24 vol. % of DiH 2 0 (designated herein as Y3PC).
  • the inventive formulations comprise from about 4 to 8 vol. % of TRITON X-100, from about 5 to 8 vol. % of TBP, about 30 to 70 vol. % of oil (e.g., soybean or olive oil), and about 0 to 30 vol. % of aqueous phase (e.g., DiH 2 0 or PBS). Additionally, certain of these embodiments further comprise about 1 vol. % of CPC, about 1 vol. % of benzalkonium chloride, about 1 vol. % cetylyridinium bromide, about 1 vol.
  • the inventive formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 20 vol. % of DiH 2 0 (designated herein as X8P).
  • the inventive formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1% of CPC, about 64 vol. % of soybean oil, and about 19 vol.
  • the formulations comprise about 8 vol. % TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 50 vol. % of soybean oil, and about 33 vol. % of DiH 2 0 (designated herein as ATB-X1001).
  • the formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 2 vol. % of CPC, about 50 vol. % of soybean oil, and about 32 vol. % of DiH 2 0 (designated herein as ATB-X002).
  • Another embodiment of the present invention comprises about 4 vol.
  • % TRITON X-100 about 4 vol. % of TBP, about 0.5 vol. % of CPC, about 32 vol. % of soybean oil, and about 59.5 vol. % of DiH 2 O (designated herein as 50% X8PC).
  • Still another related embodiment comprises about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 0.5 vol. % CPC, about 64 vol. % of soybean oil, and about 19.5 vol. % of DiH 2 O (designated herein as X8PC1/2).
  • the inventive fo ⁇ nulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 2 vol.
  • the inventive formulations comprise about 8 vol. % of TRITON X-100, about 8% of TBP, about 1% of benzalkonium chloride, about 50 vol. % of soybean oil, and about 33 vol. % of DiH 2 0 (designated herein as X8P BC).
  • the formulation comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of cetyl yridinium bromide, about 50 vol. %> of soybean oil, and about 33 vol.
  • the formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of cetyldimethyletylammonium bromide, about 50 vol. % of soybean oil, and about 33 vol. % of DiH 2 0 (designated herein as X8P CTAB).
  • the present invention comprises about 8 vol. % of TRITON X- 100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 500 M EDTA, about 64 vol. % of soybean oil, and about 15.8 vol, % DiH 2 0 (designated herein as X8PC EDTA).
  • Additional similar embodiments comprise 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 10 mM ammonium chloride, about 5mM Inosine, about 5mM L-alanine, about 64 vol. % of soybean oil, and about 19 vol. % of DiH 2 0 or PBS (designated herein as X8PC GE ⁇ x ).
  • the inventive formulations further comprise about 5 vol. %> of TRITON X-100, about 5% of TBP, about 1 vol. % of CPC, about 40 vol. % of soybean oil, and about 49 vol.
  • the inventive formulations comprise about 2 vol. % TRITON X-100, about 6 vol. % TYLOXAPOL, about 8 vol. % ethanol, about 64 vol. % of soybean oil, and about 20 vol. % of DiH 2 0 (designated herein as X2Y6E).
  • the formulations comprise about 8 vol. % of TRITON X-100, and about 8 vol. % of glycerol, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 25 vol.
  • % of aqueous phase e.g., DiH 2 0 or PBS.
  • Certain related embodiments further comprise about 1 vol. % L- ascorbic acid.
  • one particular embodiment comprises about 8 vol. % of TRITON X-100, about 8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 20 vol. % of DiH 2 0 (designated herein as X8G).
  • the inventive formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of glycerol, about 1 vol. % of L-ascorbic acid, about 64 vol. % of soybean oil, and about 19 vol.
  • the inventive formulations comprise about 8 vol. % of TRITON X-100, from about 0.5 to 0.8 vol. % of TWEEN 60, from about 0.5 to 2,0 vol. % of CPC, about 8 vol. % of TBP, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 25 vol. % of aqueous phase (e.g., DiH 2 0 or PBS).
  • the formulations comprise about 8 vol. % of TRITON X-100, about 0.70 vol. % of TWEEN 60, about 1 vol.
  • Another related embodiment comprises about 8 vol. % of TRITON X-100, about 0.71 vol. % of TWEEN 60, about 1 vol. % of CPC, about 8 vol. % of TBP, about 64 vol. %> of soybean oil, and about 18.29 vol. % of DiH 2 0 (designated herein as W6O 0 . 7 X8PC).
  • the inventive fo ⁇ nulations comprise from about 8 vol. % of TRITON X-100, about 0.7 vol.
  • the present invention comprises about 8 vol. % of TRITON X-100, about 0.71 vol. % of TWEEN 60, about 2 vol. % of CPC, about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 17.3 vol. % of DiH 2 0.
  • the formulations comprise about 0.71 vol, % of TWEEN 60, about 1 vol. % of CPC, about 8 vol.
  • the inventive formulations comprise about 2 vol. % of dioctyl sulfosuccinate, either about 8 vol. % of glycerol, or about 8 vol. % TBP, in addition to, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 20 to 30 vol. % of aqueous phase (e.g., DiH 2 0 or PBS).
  • oil e.g., soybean or olive oil
  • aqueous phase e.g., DiH 2 0 or PBS
  • one embodiment of the present invention comprises about 2 vol.
  • the inventive formulations comprise about 2 vol, % of dioctyl sulfosuccinate, and about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 26 vol. % of DiH 2 0 (designated herein as D2P).
  • the inventive formulations comprise about 8 to 10 vol. % of glycerol, and about 1 to 10 vol. % of CPC, about 50 to 70 vol.
  • compositions further comprise about 1 vol. % of L-ascorbic acid.
  • one particular embodiment comprises about 8 vol. % of glycerol, about 1 vol. % of CPC, about 64 vol. % of soybean oil, and about 27 vol. % of DiH 2 0 (designated herein as GC).
  • An additional related embodiment comprises about 10 vol, % of glycerol, about 10 vol. % of CPC, about 60 vol. % of soybean oil, and about 20 vol.
  • the inventive formulations comprise about 10 vol. % of glycerol, about 1 vol. % of CPC, about 1 vol. % of L- ascorbic acid, about 64 vol. % of soybean or oil, and about 24 vol. % of DiH 2 0 (designated herein as GCV C ).
  • the inventive formulations comprise about 8 to 10 vol. % of glycerol, about 8 to 10 vol. % of SDS, about 50 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 30 vol.
  • compositions further comprise about 1 vol. % of lecithin, and about 1 vol. % of p-Hydroxybenzoic acid methyl ester.
  • exemplary embodiments of such formulations comprise about 8 vol. % SDS, 8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 20 vol. % of DiH 2 0 (designated herein as S8G).
  • a related formulation comprises about 8 vol. % of glycerol, about 8 vol. % of SDS, about 1 vol, % of lecithin, about 1 vol.
  • the inventive formulations comprise about 4 vol. % of TWEEN 80, about 4 vol. % of TYLOXAPOL, about 1 vol. % of CPC, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 19 vol. % of DiH 2 0 (designated herein as W 80 4Y4EC). In some embodiments of the present invention, the inventive formulations comprise about 0.01 vol. % of CPC, about 0.08 vol.
  • the inventive formulations comprise about 8 vol. % of sodium lauryl sulfate, and about 8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 20 vol. % of DiH 2 0 (designated herein as SLS8G).
  • a candidate composition made of 4.5% sodium thiosulfate, 0.5% sodium citrate, 10% n-butanol, 64% soybean oil, and 21% DiH 2 0 did not form an emulsion.
  • the candidate emulsion should form a stable emulsion.
  • An emulsion is stable if it remains in emulsion form for a sufficient period to allow its intended use. For example, for emulsions that are to be stored, shipped, etc., it may be desired that the composition remain in emulsion form for months to years. Typical emulsions that are relatively unstable, will lose their form within a day.
  • a candidate composition made of 8% 1-butanol, 5% Tween 10, 1% CPC, 64% soybean oil, and 22% DiH 2 0 did not form a stable emulsion.
  • the following candidate emulsions were shown to be stable using the methods described herein: 0.08% Triton X-100, 0.08% Glycerol, 0.01% Cetylpyridinium Chloride, 99% Butter, and 0.83% diH 2 0 (designated herein as 1% X8GC Butter); 0.8% Triton X-100, 0.8% Glycerol, 0.1% Cetylpyridinium Chloride, 6.4% Soybean Oil, 1.9% diH 2 0, and 90% Butter (designated herein as 10% X8GC Butter); 2% W 20 5EC, 1% Natrosol 250L NF, and 97% diH 2 0 (designated herein as 2% W 20 5EC L GEL); 1% Cetylpyridinium Chloride, 5% Tween 20, 8%
  • nanoemulsions of the present invention are stable for over a week, over a month, or over a year.
  • the candidate emulsion should have efficacy for its intended use.
  • an anti-bacterial emulsion should kill or disable bacteria to a detectable level or to a preferred kill level (e.g., 1 log, 2 log, 3 log, 4 log, . . . reduction).
  • a preferred kill level e.g., 1 log, 2 log, 3 log, 4 log, . . . reduction.
  • certain emulsions of the present invention have efficacy against specific microorganisms, but not against others. Using the methods described herein, one is capable of determining the suitability of a particular candidate emulsion against the desired microorganism.
  • a candidate composition made of 1% ammonium chloride, 5%o Tween 20, 8% ethanol, 64% soybean oil, and 22% DiH 2 0 was shown not to be an effective emulsion.
  • the nanoemulsions are nontoxic (e.g., to humans, plants, or animals), non-i ⁇ itant (e.g., to humans, plants, or animals), and non-corrosive (e.g., to humans, plants, or animals or the environment), while possessing potency against a broad range of microorganisms including bacteria, fungi, viruses, and spores.
  • non-toxic nanoemulsions comprise surfactant lipid preparations (SLPs) for use as broad-spectrum antimicrobial agents that are effective against bacteria and their spores, enveloped viruses, and fungi.
  • SLPs surfactant lipid preparations
  • these SLPs comprises a mixture of oils, detergents, solvents, and cationic halogen-containing compounds in addition to several ions that enhance their biocidal activities.
  • Non-toxic nanoemulsions include, but are not limited to: detergents (e.g., TRITON X-100 [5-15%] or other members of the TRITON family, TWEEN 60 [0.5-2%] or other members of the TWEEN family, or TYLOXAPOL [1- 10%]); solvents (e.g., tributyl phosphate [5-15%]); alcohols (e.g., ethanol [5-15%] or glycerol [5-15%]); oils (e.g., soybean oil [40-70%]); cationic halogen-containing compounds (e.g., cetylpyridinium chloride [0.5-2%], cetylpyridinium bromide [0.5-2%]), or cetyldimethylethyl am
  • Emulsions are prepared, for example, by mixing in a high shear mixer for 3-10 minutes.
  • the emulsions may or may not be heated before mixing at 82°C for 1 hour.
  • Quaternary ammonium compounds for use in the present include, but are not limited to, N-alkyldimethyl benzyl ammonium saccharinate; 1,3,5-Triazine- l,3,5(2H,4H,6H)-triethanol; 1-Decanaminium, N-decyl-N, N-dimethyl-, chloride (or) Didecyl dimethyl ammonium chloride; 2-(2-(p-(Diisobuyl)cresosxy)ethoxy)ehyl dimethyl benzyl ammonium chloride; 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzyl ammonium chloride; alkyl 1 or 3 benzyl- l-(
  • the preferred non-toxic nanoemulsions are characterized by the following: they are approximately 200-800 nm in diameter, although both larger and smaller diameter nanoemulsions are contemplated; the charge depends on the ingredients; they are stable for relatively long periods of time (e.g., up to two years), with preservation of their biocidal activity; they are non-i ⁇ itant and non-toxic compared to their individual components due, at least in part, to their oil contents that markedly reduce the toxicity of the detergents and the solvents; they are effective at concentrations as low as 0.1%; they have antimicrobial activity against most vegetative bacteria (including Gram-positive and Gram-negative organisms), fungi, and enveloped and nonenveloped viruses in 15 minutes (e.g., 99.99% killing); and they have sporicidal activity in 1-4 hours (e.g., 99.99% killing) when produced with germination enhancers.
  • compositions of the present invention possess a range of beneficial activities and properties.
  • a number of the exemplary beneficial properties and activities are set forth below: A) Microbicidal and Microbistatic Activity; B) Sporicidial and
  • the nanoemulsions of the present invention have broad spectrum killing activities, whereby they kill or disable, two or more of: 1) bacteria (gram positive and gram negative), 2) viruses, 3) fungi, and 4) spores (e.g., 1 log, 2 log, 3 log, 4 log, . . . reduction).
  • A. Microbicidal and Microbistatic Activity The methods of the present invention can be used to rapidly inactivate bacteria.
  • the compositions are particularly effective at inactivating Gram positive bacteria.
  • the inactivation of bacteria occurs after about five to ten minutes.
  • bacteria may be contacted with an emulsion according to the present invention and will be inactivated in a rapid and efficient manner. It is expected that the period of time between the contacting and inactivation may be as little as 5-10 minutes or less where the bacteria is directly exposed to the emulsion.
  • the inactivation may occur over a longer period of time including, but not limited to, 5, 10, 15, 20, 25, 30, 60 minutes post application.
  • the inactivation may take two, three, four, five or six hours to occur.
  • the compositions and methods of the invention can also rapidly inactivate certain Gram negative bacteria, hi some embodiments, the bacteria inactivating emulsions are premixed with a compound that increases the interaction of the emulsion by the cell wall.
  • the use of these enhancers in the compositions of the present invention is discussed herein below. It should be noted that certain emulsions especially those comprising enhancers are effective against certain Gram positive and negative bacteria and may be administered orally where they will come in contact with necessary gut bacteria.
  • the present invention has shown that the emulsions of the present invention have potent, selective biocidal activity with minimal toxicity against vegetative bacteria.
  • BCTP was highly effective against B. cereus, B. circulans and B. megaterium, C. perfringens, H. in ⁇ uenzae, N. gonorrhoeae, S. agalactiae, S. pneumonia, S. pyogenes and V. cholerae classical and Eltor (FIG. 26). This inactivation starts immediately on contact and is complete within 15 to 30 minutes for most of the susceptible microorganisms.
  • Figure 31 A shows the effectiveness of a number of exemplary nanoemulsions of the present invention against E. coli.
  • the present invention has demonstrated that the emulsions of the present invention have sporicidal activity. Without being bound to any theory (an understanding of the mechanism is not necessary to practice the present invention, and the present invention is not limited to any particular mechanism), it is proposed the that the sporicidal ability of these emulsions occurs through initiation of ge ⁇ nination without complete reversion to the vegetative form leaving the spore susceptible to disruption by the emulsions. The initiation of ge ⁇ nination could be mediated by the action of the emulsion or its components. The results of electron microscopy studies show disruption of the spore coat and cortex with disintegration of the core contents following BCTP treatment.
  • Sporicidal activity appears to be mediated by both the TRITON X-100 and tri-n-butyl phosphate components since nanoemulsions lacking either component are inactive in vivo.
  • This unique action of the emulsions which is similar in efficiency to 1% bleach, is interesting because Bacillus spores are generally resistant to most disinfectants including many commonly used detergents (Russell, Clin. Micro. 3;99 [1990]).
  • the present invention demonstrates that mixing BCTP with B. cereus spores before injecting into mice prevented the pathological effect of 5. cereus. Further, the present invention shows that BCTP treatment of simulated wounds contaminated with B. oereus spores markedly reduced the risk of infection and mortality in mice.
  • X 8 W 6 oPC diluted 1:1000 had more sporicidal activity against B. anthracis, B. cereus, and B. subtilis and had an onset of action in less than 30 minutes.
  • mixing BCTP with B. cereus before subcutaneous injection or wound irrigation with BCTP 1 hour following spore inoculation resulted in over 98% reduction in skin lesion size. Mortality was reduced 4-fold in the latter experiment.
  • the present compositions are stable, easily dispersed, non-irritant and nontoxic compared to the other available sporicidal agents.
  • the bacteria-inactivating oil-in-water emulsions used in the methods of the present invention can be used to inactivate a variety of bacteria and bacterial spores upon contact.
  • the presently disclosed emulsions can be used to inactivate
  • Clostridium e.g., C. botulinum and C. tetani.
  • the methods of the present invention may be particularly useful in inactivating certain biological warfare agents (e.g., B. anthracis).
  • the formulations of the present invention also find use in combating C. perftingens, H. in ⁇ uenzae, N. gonorrhoeae, S. agalactiae, S. pneumonia, S. pyogenes and V. cholerae classical and Eltor (FIG. 26).
  • BCTP contains TRITON X-100 while SS and W 80 8P contain TWEEN 60, and NN contained nonoxynol-9 surfactant.
  • Each is a non-ionic surfactant, but differs in its chemistry and biological characteristics.
  • Nonoxynol-9 has strong spermicidal activity and it is widely used as a component of vaginally delivered contraceptive products (Lee, 1996), It has been claimed to have virucidal effect against enveloped viruses (Hermonat et al, 1992; Zeitlin et al, 1997). However, nanoxynol-9 has not been shown to be effective against nonenveloped viruses (Hermonat et al, 1992).
  • Figure 3 IB shows the effectiveness of a number of exemplary nanoemulsions of the present invention against B. globigii spores.
  • the nanoemulsion compositions of the present invention have anti-viral properties.
  • the effect of these emulsions on viral agents was monitored using plaque reduction assay (PRA), cellular enzyme-linked immunosorbent assay (ELISA), ⁇ -galactosidase assay, and electron microscopy (EM) and the cellular toxicity of lipid preparations was assessed using a (4,5-dimethylthiazole-2-yl)-2,5 diphenyltetrazolium (MTT) staining assay (Mosmann 1983).
  • PRA plaque reduction assay
  • ELISA cellular enzyme-linked immunosorbent assay
  • EM electron microscopy
  • BCTP and SS at dilution 1 : 10 reduced virus infectivity over 95%.
  • Two other emulsions showed only intermediate effects on the virus reducing infectivity by approximately 40% at dilution 1 : 10.
  • BCTP was the most potent preparation and showed undiminished virucidal effect even at dilution 1 :100.
  • Kinetic studies showed that 5 min incubation of virus with BCTP at 1 : 10 dilution completely abolished its infectivity.
  • TRITON X-100 an active compound of BCTP, at dilution 1 :5000 only partially inhibited the infectivity of virus as compared to BCTP, indicating that the nanoemulsion itself contributes to the anti-viral efficacy.
  • the nanoemulsions of the present invention are used in conjunction with a low pH buffer. Such nanoemulsions find use as rapid killers of viruses (e.g., rhinovirus or other picornaviruses),
  • Figure 31C shows the effectiveness of a number of exemplary nanoemulsions of the present invention against influenza A.
  • Common agents of fungal infections include various species of the genii Candida and Aspergillus, and types thereof, as well as others. While external fungus infections can be relatively minor, systemic fungal infections can give rise to serious medical consequences.
  • 1 % BCTP has a greater than 92% fungistatic activity when applied to Candida albicans.
  • Candida was grown at 37 ⁇ C overnight.
  • Fungistatic effect 1 - # of treated cells- Initial # of cells x 100 # of untreated cells- Initial # of cells
  • nanoemulsions of the present invention find use in combatting infections such as athletes foot, candidosis and other acute or systemic fungal infections.
  • compositions disclosed herein A) Pharmaceuticals and Therapeutics; B) Decontamination and Sterilization; C) Food Preparation; D) Kits, as well as a description of methods and systems for the E) Modification, Preparation, and Delivery of the compositions of the present invention; F) Livestock Udder Maintenance; G) Mold Remediation Uses; and H) Additional Examples.
  • A. Pharmaceuticals and Therapeutics The present invention contemplates formulations that may be employed in pharmaceutical and therapeutic compositions and applications suitable for combatting and/or treating microbial infections.
  • compositions may be employed to reduce infection, kill microbes, inhibit microbial growth or otherwise abrogate the deleterious effects of microbial infection.
  • the compositions can be administered in any effective pharmaceutically acceptable form to warm blooded animals, including human and animal subjects. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention include but are not limited to oral, nasal, buccal, rectal, vaginal, topical or nasal spray or in any other form effective to deliver active compositions of the present invention to a site of microorganism infection, hi preferred embodiments, the route of administration is designed to obtain direct contact of the compositions with the infecting microorganisms.
  • administration may be by orthotopic, intradermal, subcutaneous, intramuscular or intraperitoneal injection.
  • the compositions may also be administered to subjects parenterally or intraperitonealy. Such compositions would normally be administered as pharmaceutically acceptable compositions.
  • the pharmaceutically acceptable carrier may 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 employed in pharmaceutical compositions for topical administration.
  • compositions in which the emulsions are formulated for oral or topical administration include liquid capsules, and suppositories.
  • the compositions may be admixed with one or more substantially inert diluent (e.g., sucrose, lactose, or starch, and the like) and may additionally comprise lubricating agents, buffering agents, enteric coatings, and other components well known to those skilled in the art.
  • the compositions of the invention may be specifically designed for in vitro applications, such as disinfecting or sterilization of medical instruments and devices, contact lenses and the like, particularly when the devices or lenses are intended to be used in contact with a patient or wearer.
  • the compositions may be used to cleanse and decontaminate medical and surgical instruments and supplies prior to contacting a subject. Additionally, the compositions may be used to post-operatively, or after any invasive procedure, to help minimize the occu ⁇ ence of post operative infections. In especially preferred embodiments, the compositions are administered to subjects with compromised or ineffective immunological defenses (e.g., the elderly and the very young, burn and trauma victims, and those infected with HIV and the like).
  • the compositions may be conveniently provided in the form of a liquid, foam, paste or gel and may be provided with emulsifiers, surfactants, buffering agents, wetting agents, preservatives, metal ions, antibiotics and other components commonly found in compositions of this type.
  • the compositions are used in association with organ or artifical tissue transplantation or maintenance.
  • the composition may be used on the surface of a transplanted organ to sterilize the organ.
  • the compositions may also be used in conjuction with organ preservation or storage solutions (e.g., VIASPAN, Barr Laboratories).
  • the compositions may be impregnated into abso ⁇ tive materials, such as sutures, bandages, and gauze, or coated onto the surface of solid phase materials, such as surgical staples, zippers and catheters to deliver the compositions to a site for the prevention of microbial infection.
  • abso ⁇ tive materials such as sutures, bandages, and gauze
  • solid phase materials such as surgical staples, zippers and catheters to deliver the compositions to a site for the prevention of microbial infection.
  • Other delivery systems of this type will be readily apparent to those skilled in the art.
  • the compositions can be used in the personal health care industry in deodorants, soaps, acne/dermatophyte treatment agents, treatments for halitosis, treatments for vaginal yeast infections, and the like.
  • the compositions can also be used to treat other internal and external microbial infections (e.g., influenza, H. simplex, toe-nail fungus, etc.).
  • the emulsions can be formulated with therapeutic carriers as described above.
  • the nanoemulsions of the present invention are fo ⁇ uulated into gels, wherein the gels are applied topically.
  • the antimicrobial compositions and methods of the present invention also include a variety of combination therapies.
  • the nanoemulsion compositions of the present invention are used as a delivery system for another agent (e.g., a pharmaceutical agent).
  • the agent has antimicrobial properties.
  • the nanoemulsions of the present invention increase the antimicrobial effect, compared to the delivery of the agent in the absence of the nanoemulsions of the present invention.
  • the nanoemulsions are used without another antimicrobial agent (i.e., the nanoemulsion itself is the only antimicrobial portion of the composition).
  • antimicrobial agents currently available for use in treating bacterial, fungal and viral infections.
  • the skilled artisan is referred to Goodman & Gilman's "The Pharmacological Basis of Therapeutics" Eds. Hardman et al, 9th Edition, Pub.
  • these agents include agents that inhibit cell wall synthesis (e.g., penicillins, cephalosporins, cycloserine, vancomycin, bacitracin); and the imidazole antifungal agents (e.g., miconazole, ketoconazole and clotrimazole); agents that act directly to disrupt the cell membrane of the microorganism (e.g., detergents such as polmyxin and colistimethate and the antifungals nystatin and amphotericin B); agents that affect the ribosomal subunits to inhibit protein synthesis (e.g., chloramphenicol, the tetracyclines, erthromycin and clindamycin); agents that alter protein synthesis and lead to cell death (e.g., aminoglycosides); agents that affect nucleic acid metabolism (e.g., the rifamycins and the
  • compositions and any enhancing agents in the compositions may be varied so as to obtain amounts of emulsion and enhancing agents at the site of treatment that are effective in killing vegetative as well as sporular microorganisms and neutralizing their toxic products. Accordingly, the selected amounts will depend on the nature and site for treatment, the desired response, the desired duration of biocidal action and other factors.
  • the emulsion compositions of the invention will comprise at least 0.001% to 100%, preferably 0.01 to 90%, of emulsion per ml of liquid composition. It is envisioned that viral infections may be treated using between about 0.01% to 100% of emulsion per ml of liquid composition.
  • Bacterial infections may be attacked with compositions comprising between about 0.001% to about 100% of emulsion per ml of liquid composition. Spores can be killed by emulsions comprising from about 0.001% to about 100% of emulsion per ml of liquid composition. These are merely exemplary ranges. It is envisioned that the formulations may comprise abo ⁇ t 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.025%, about 0.05%, about 0.075%, about 0. 1 %, about 0.25%, about 0.5%, about 1.0%, about 2.5%, about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 20%, about 25%, about
  • the present invention contemplates compositions and methods that find use as environmental decontamination agents and for treatment of casualties in both military and terrorist attack.
  • pathogens including vegetative bacteria and enveloped viruses
  • vegetative bacteria and enveloped viruses See e.g., Chatlyyne et al, "A lipid emulsion with effective virucidal activity against HIV-l and other common viruses," Foundation for Retrovirology and Humna Health, 3rd Conference on retroviruses and Opportunistic Infections, Washington, DC, U.S.A.
  • compositions of the present invention can be rapidly produced in large quantities and are stable for many months at a broad range of temperatures. These properties provide a flexibility that is useful for a broad range of decontamination applications. For example, certain fo ⁇ nulations of the present invention are especially effective at destroying many of the bacterial spores and agents used in biological warfare. In this regard, the compositions and methods of the present are useful in decontaminating personnel and materials contaminated by biological warfare agents. Solutions of present compositions may be sprayed directly onto contaminated materials or personnel from ground based, or aerial spraying systems.
  • the present invention contemplates that an effective amount of composition be contacted to contaminated materials or personnel such that decontamination occurs.
  • personal decontamination kits can be supplied to military or civilians likely to become contaminated with biological agents.
  • pathogens including vegetative bacteria and enveloped viruses (See e.g., Chatlyyne et al, "A lipid emulsion with effective virucidal activity against HIV-l and other common viruses," Foundation for Retrovirology and Humna Health, 3rd Conference on retroviruses and Opportunistic Infections, Washington, DC, U.S.A.
  • the present compositions particularly well suited for use as general decontamination agents before a specific pathogen is identified.
  • certain embodiments of the present invention specifically contemplate the use of the present compositions in disinfectants and detergents to decontaminate soil, machinery, vehicles and other equipment, and waterways that may have been subject to an undesired pathogen.
  • Such decontamination procedures may involve simple application of the formulation in the form of a liquid spray or may require a more rigorous regimen.
  • the present emulsions can be used to treat crops for various plant viruses (in place of or for use with conventional antibiotics).
  • Nanoemulsions may also be used to decontaminate farm animals, animal pens, surrounding surfaces, and animal carcasess to eliminate, for example, noneveloped virus of hoof and mouth disease.
  • the formulations also find use in household detergents for general disinfectant pu ⁇ oses.
  • some embodiments of the present invention can be used to prevent contamination of food with bacteria or fungi (e.g., non-toxic compositions). This can be done either in the food preparation process, or by addition to the food as an additive, disinfectant, or preservative.
  • the inventive emulsions are preferably used on hard surfaces in liquid form. Accordingly, the foregoing components are admixed with one or more aqueous carrier liquids.
  • aqueous carrier is not critical. However, it should be safe and it should be chemically compatible with the inventive emulsions.
  • the aqueous carrier liquid comprises solvents commonly used in hard surface cleaning compositions. Such solvents should be compatible with the inventive emulsions and should be chemically stable at the pH of the emulsions. They should also have good filming/residue properties. Solvents for use in hard surface cleaners are described, for example, in U.S. Pat. No. 5,108,660, herein inco ⁇ orated by reference in its entirety.
  • the aqueous carrier is water or a miscible mixture of alcohol and water. The alcohol can be used to adjust the viscosity of the compositions.
  • the alcohols are preferably C2 -C4 alcohols.
  • ethanol is employed.
  • the aqueous carrier liquid is water or a water-ethanol mixture containing from about 0 to about 50% ethanol.
  • the present invention also embodies non-liquid compositions. These non-liquid compositions can be in granular, powder or gel forms, preferably in granular forms. Optionally, some compositions contain auxiliary materials that augment cleaning and aesthetics so long as they do not interfere with the activity of the inventive emulsions.
  • the compositions can optionally comprise a non-interfering auxiliary surfactant. A wide variety of organic, water-soluble surfactants can optionally be employed.
  • auxiliary surfactant depends on the desires of the user with regard to the intended pu ⁇ ose of the compositions and the commercial availability of the surfactant.
  • Other optional additives such as perfumes, brighteners, enzymes, colorants, and the like can be employed in the compositions to enhance aesthetics and/or cleaning performance.
  • Detergent builders can also be employed in the compositions. Detergent builders sequester calcium and magnesium hardness ions that might otherwise bind with and render less effective the auxiliary surfactants or co-surfactants. Detergent builders are especially useful when auxiliary surfactants or co-surfactants are employed, and are even more useful when the compositions are diluted prior to use with exceptionally hard tap water e.g., above about 12 grains/gallon.
  • the composition further comprise, suds suppressors.
  • the compositions preferably comprise a sufficient amount of a suds suppressor to prevent excessive sudsing when contacting the compositions to hard surfaces. Suds suppressors are especially useful in formulations for no-rinse application of the composition.
  • the suds suppressor can be provided by known and conventional means. Selection of the suds suppressor depends on its ability to formulate in the compositions, and the residue and cleaning profile of the compositions. The suds suppressor must be chemically compatible with the components in the compositions, it must be functional at the pH range described herein, and it should not leave a visible residue on cleaned surfaces.
  • Low-foaming co-surfactants can be used as suds suppressor to mediate the suds profile in the compositions. Co-surfactant concentrations between about 1 part and about 3% > are no ⁇ nally sufficient.
  • suitable co-surfactants for use herein include block copolymers (e.g., PLURONIC and TETRONIC gels [polyethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) polymer gels, BASF Company, Parispany, NJ]) and alkylated (e.g., ethoxylated/propoxylated) primary and secondary alcohols (e.g., TERIGTOL [Union Carbide, Danbury, CT]; POLY-TERGENTO [Olin Co ⁇ oration, Norwalk, CT]).
  • block copolymers e.g., PLURONIC and TETRONIC gels [polyethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)
  • the optional suds suppressor preferably comprises a silicone-based material. These materials are effective as suds suppressors at very low concentrations. At low concentrations, the silicone-based suds suppressor is less likely to interfere with the cleaning performance of the compositions.
  • An example of suitable silicone-based suds suppressors for use in the compositions is Dow Corning DSE. These optional but preferred silicone-based suds suppressors can be inco ⁇ orated into the composition by known and conventional means.
  • the compositions may be used by health care workers, or any persons contacting persons or areas with microbial infections, for their personal health-safety and decontamination needs.
  • inventive emulsions can be formulated into sprays for hospital and household uses such as cleaning and disinfecting medical devices and patient rooms, household appliances, kitchen and bath surfaces, etc.
  • the compositions may be used by sanitation and environmental services workers, food processing and agricultural workers and laboratory personnel when these individuals are likely to contact infectious biological agents.
  • the compositions may be used by travelers and persons contacting ares likely to harbor infectious and pathological agents.
  • compositions described herein may be employed in the food processing and preparation industries in preventing and treating food contaminated with food born bacteria, fungi .and toxins.
  • such compositions may be employed to reduce or inhibit microbial growth or otherwise abrogate the deleterious effects of microbial contamination of food.
  • the emulsion compositions are applied in food industry acceptable forms such as additives, preservatives or seasonings.
  • accepted in the food industry refers to compositions that do not substantially produce adverse, or allergic reactions when taken orally by humans or animals.
  • accepted in food industry media includes any and all solvents, dispersion substances, any and all spices and herbs and their extracts.
  • compositions may be specifically designed for applications such as disinfecting or sterilization food industry devices, equipment, and areas where food is processed, packaged and stored.
  • compositions may be conveniently provided in the form of a liquid or foam, and may be provided with emulsifiers, surfactants, buffering agents, wetting agents, preservatives, and other components commonly found in compositions of this type.
  • the compositions are applied to produce or agricultural products prior to or during transportation of those goods.
  • Compositions of the invention may be impregnated into abso ⁇ tive materials commonly used in packaging material for the prevention of food contamination during transport and storage (e.g., cardboard or paper packaging). Other delivery systems of this type will be readily apparent to those skilled in the art.
  • compositions of the invention may be varied so as to obtain appropriate concentrations of emulsion and enhancing agents to effectively prevent or inhibit food contamination caused by food born microbes and their toxic products. Accordingly, the selected concentrations will depend on the nature of the food product, packaging, storage procedure and other factors.
  • the emulsion compositions of the invention will comprise at least 0.001 % to about 90% of emulsion in liquid composition, It is envisioned that the formulations may comprise about 0.001 %, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.025%, about 0.05%, about 0.075%, about 0.1 %, about 0.25%, about 0.5%, about 1.0%, about 2.5%, about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% of emulsion per ml of liquid composition.
  • emulsions can be used as disinfectants and detergents to decontaminate and prevent microbial infection of food, soil and water, machinery and other equipment, and animals.
  • inventive emulsions can be used by the food industry to prevent contamination. For example, inclusion of the emulsion within the food product itself would be effective in killing bacteria that may have been accidentally contaminated meat or poultry. This could also allow the industry to use a potentially broader spectrum of food products and reduce costs. Certain embodiments of the present invention can also be used in the beverage industry.
  • the inventive emulsions could be included in juice products to prevent growth of certain fungi, which cause contamination and lead to production of mycotoxins, which are dangerous to consumers.
  • the inventive emulsions Through the addition of small amounts of the inventive emulsions, the most common fungal contaminants in fruit juice were prevented. This effect was achieved with as little as one part in 10,000 of the emulsion (an amount which did not alter the flavor or the composition of the juice product).
  • contamination of productes such are GATORADE by organisms such as Byssochlamys fulva are presented with the use of the nanoemulsions of the present invention.
  • the inventive emulsions can be used to essentially remove infectious agents on machinery and other equipment.
  • the emulsions can be used to eliminate contaminations in meat processing plants, particularly of organisms such as Listeria monocytogenes and Salmonellae microorganisms, by cleaning slaughterhouses or food packaging facilities on a continual basis with the emulsion.
  • the person responsible for administration will, in any event, determine the appropriate dose for individual application.
  • said above application should meet general safety and purity standards as required by the FDA office.
  • kits In other embodiments of the present invention, the methods and compositions, or components of the methods and compositions may be formulated in a single formulation, or may be separated into separate formulations for later mixing during use, as may be desired for a particular application. Such components may advantageously be placed in kits for use against microbial infections, decontaminating instruments and the like. In some embodiments, such kits contain all the essential materials and reagents required for the delivery of the formulations of the present invention to the site of their intended action. In some embodiments, intended for in vivo use, the methods and compositions of the present invention may be fo ⁇ nulated into a single or separate pharmaceutically acceptable syringeable compositions.
  • the container means may itself be an inhalant, syringe, pipette, eye dropper, or other like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, injected into an animal, or even applied to and mixed with the other components of the kit.
  • the kits of the present invention also typically include a means for containing the vials in close confinement for commercial sale (e.g., injection or blow-molded plastic containers into which the desired vials are retained). Irrespective of the number or type of containers, the kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/ad ministration or placement of the ultimate complex composition within the body of an animal.
  • an instrument may be an inhalant, syringe and antiseptic wipe, pipette, forceps, measured spoon, eyedropper or any such medically approved delivery vehicle.
  • the present invention further provides a variety of methods and systems for the modification of the nanoemulsions of the present invention, the inco ⁇ oration of the nanoemulsions into other products, packaging and delivery of the compositions of the present invention, and methods for reducing the costs associated with the use or handling of materials or samples that might be contaminated with microorganisms.
  • the following description is intended to simply provide some examples of the modification, preparation, and delivery of the compositions of the present invention. Those skilled in the art will appreciate variations of such methods.
  • the present invention provides methods for improving or altering the nanoemulsions described herein. Such methods include, for example, taking a nanoemulsion described herein and changing one or more components of the nanoemulsion.
  • nanoemulsions of the present invention are diluted.
  • the diluted samples can then be tested to determine if they maintain the desired functionality.
  • the nanoemulsions of the present invention, or those derived from the nanoemulsions of the present invention are pass through a quality control (QC) and/or quality assurance (QA) procedure to confirm the suitability of the nanoemulsion for sale or delivery to a user or retailer.
  • QC quality control
  • QA quality assurance
  • the nanoemulsions of the present invention are added to another product to add or improve anti-microbial capabilities of the product or to test a suspected or provide a perceived improved anti-microbial capability to the product (i.e., it is contemplated that the addition of a nanoemulsion of the present invention into a product is within the scope of the present invention regardless of whether it has a detectable, or any, antimicrobial capabilities).
  • the nanoemulsions of the present invention are added to cleaning or disinfectant materials (e.g., household cleaning agents).
  • the nanoemulsions are added to medical or first aid materials.
  • the nanoemulsions may be added to (or used directly as) sterilization agents and wound care products.
  • the nanoemulsions are added to industrial products.
  • the nanoemulsions are added to motor oils to prevent or reduce, for example, fungal contamination.
  • effective, stable emulsion can even be synthesized using motor oil as the oil component (e.g., W 2 o5GC Mobil 1).
  • the nanoemulsions are added to food products.
  • the nanoemulsions can be added to beverages to prevent the growth of unwanted organisms in the beverage.
  • the nanoemulsion of the present invention, whether alone, or in conjunction with other materials can be provided in many different types of containers and delivery systems.
  • the nanoemulsions are provided in a cream or other solid or semi-solid form.
  • the emulsions of the present invention may be inco ⁇ orated into hyrdogel formulations while maintaining antimicrobial capabilities.
  • the use of the emulsions in hydrogel provides a number of useful features.
  • hydrogels can be prepared in semi-solid structures of desired sizes and shapes. This allows, for example, the insertion of the hydrogel materials into tubes or other passageways to create antimicrobrial filters (i.e., materials passed through the hydrogel are decontaminated by the emulsions of the present invention).
  • the nanoemulsions can be delivered (e.g., to user or customers) in any suitable container.
  • Container can be used that provide one or more single use or multi-use dosages of the nanoemulsion for the desired application.
  • the nanoemulsions are provided in a suspension or liquid form.
  • Such nanoemulsions can be delivered in any suitable container including spray bottles (e.g., pressurized spray bottles).
  • spray bottles e.g., pressurized spray bottles.
  • large volumes (e.g., tens to thousands of liters) of nanoemulsion may be provided in a single container configured appropriately to allow distribution or use of the nanoemulsion.
  • Such containers may be used in conjunction with large-scale manufacturing facilities.
  • nanoemulsions of the present invention are used in conjunction with an existing business practice to reduce the costs associated with or improve the safety of the operation of the business practice.
  • the use of the nanoemulsions of the present invention can reduce costs associated with the use or handling of materials or samples that might be contaminated with microorganisms.
  • the nanoemulsions of the present invention are used to improve safety or reduce the costs associated with the medical industries.
  • the nanoemulsions find use as cheap and efficient sterilization agents for use on medical materials (e.g., surface that come in contact with animals, people, or biological samples) or with patients (e.g., internally or externally).
  • the present invention provides non-toxic nanoemulsions.
  • nanoemulsions are provided herein that include ingredients that are currently approved by the appropriate regulatory agencies (e.g., FDA, USD A, etc.) for use in medical, agriculture, and food applications.
  • methods are provided herein for the generation of additional nanoemulsions with the desired functionality that can be composed entirely of non-toxic and approved substances.
  • the nanoemulsions of the present invention can be used in applications without - incurring having to undergo the time consuming and expensive process of gaining regulatory approval. Indeed, the emulsions can be less toxic than the sum of their individual components.
  • X8PC was tested to compare the lytic effect of the emulsion on sheep red blood cells tested on blood agar plates as compared to the lytic effect of mixtures of the non-emulsified ingredients.
  • the data is present in Figure 34.
  • the two black bars in Figure 34 show the lytic effect of the X8PC nanoemulsion compared to the lytic effect of a non-emulsified mixture of all the ingredients.
  • Livestock Udder Maintenance Livestock animals (e.g., cow, sheep, goat) have a mammary gland system which may become infected leading to undesired dairy production. Udder (e.g., mammary gland) health is central to profitable milk production.
  • mastitis is defined as an inflammation of the udder of the livestock animal.
  • mastitis is an abnormality in the dairy production such as, for example, flakes, clots, and a watery or other unusual appearance.
  • Mastitis may be caused, for example, by bacteria and their toxins. Numerous forms of mastitis exist. Udder infection occurs when the udder cavity is invaded by microorganisms that cause udder inflammation. Subclinical mastitis results in undesired somatic cell counts in the milk. Clinical mastitis results in presence of leukocytes (white blood cells) in the milk. Mild clinical mastitis involves abnormality in the milk such as flakes, clots, and a watery or other unusual appearance.
  • mastitis Severe clinical mastitis involves a hot, hard sensitive udder that is quite painful to the livestock animal, along with an absence of dairy production. Chronic mastitis is a persistent udder infection results in hard lumps in the udder from the "walling off of bacteria and the forming of connective tissue.
  • the primary cause of mastitis in livestock animals is well-recognized groups of microorganisms (e.g., Streptococcus organisms, Staphylococcus organisms, Pasteurella organisms and coliforms, Escherichia coli, Enterobacter organisms, Klebsiella organisms, Staphylococcus organisms, Streptococcus organisms, Micrococcus organisms).
  • Yeast and fungus also frequently infect the udder.
  • One of the most important keys to controlling mastitis in livestock animals is good management practice. The incidence of mastitis is greater in closely confined herds. Microorganisms thrive in dark, wet, warm bedding. When livestock animals lay down to rest, the bacteria in dirty bedding can easily enter the teat when the udder is full of milk. Microorganisms can enter the teat canal.
  • the present invention is contemplated in the maintenance of livestock udder health.
  • the present invention is contemplated in the treatment of livestock mastitis.
  • the present invention is applied to the udder as a topical agent (e.g., spray, lotion, solution).
  • the present invention is used to disinfect the end of a livestock teat, In further prefe ⁇ ed embodiments, the present invention may be infused through a teat canal. In other prefe ⁇ ed embodiments, the present invention is used in the maintenance of livestock udder or treatment or mastitis in combination with the disinfecting of a livestock teat end with alcohol. In other prefe ⁇ ed embodiments, the present invention is used in combination with the disinfecting of a livestock teat end with alcohol and the infusing of a tube of mastitis antibiotic through the teat canal.
  • the present invention is used in combination with antibiotic (e.g., penicillin, dihydrostreptomycin, dexamethasone) therapy in the treatment of mastitis.
  • antibiotic e.g., penicillin, dihydrostreptomycin, dexamethasone
  • the present invention may be used in combination with antihistamine therapy in the treatment ofmastitis.
  • compositions described herein may be employed in the remediation of molds (e.g., acremonium, alternaria, arthrinium, ascospores, aspergillus, aspergillus niger, aspergillus fumigatus, basidiospores, beauveria, botrytis , chaetomium, chrysonilia, cladosporium, curvularia, dematiaceous mold , drechslera / bipolaris, epicoccum, exophiala, fusarium, geotrichum, gliocladium, memnoniella, mucor, myxomycete / rust / smut, paecilomyces, penicillium, phoma, pithomyces, rhizopus, scopulariopsis, stachybotrys, stachybotrys chartarum, stemphylium, syncephalastrum
  • Mold is a leading biological pollutant generated in indoor settings. Molds are a menace to an individual's health, and mold remediation costs millions of dollars each year. Indoor molds cause severe allergic reactions such as watery eyes, runny nose and sneezing, nasal congestion and fatigue, especially to individuals with a mold allergy.
  • the present invention is used in the cleaning, prevention, and remediation of molds. It is further contemplated that the present invention may assume various forms (e.g., gas, liquid, solid) for the cleaning, prevention and remediation of mold.
  • the prevention invention is combined with cleansing agents (e.g., chlorine, bleach, chlorine bleach) in the cleaning, prevention, and remediation of molds.
  • the prevention is combined with air duct cleaning vacuums in the cleaning, prevention, and remediation of molds.
  • the present invention is combined with vacuums (e.g., wet vacuums, gas vacuums, dry vacuums) in the cleaning, prevention, and remediation of molds.
  • the present invention is combined with HEPA filtering systems in the cleaning, preventing, and remediation of molds.
  • the present invention is combined with a dehumidifying agent (e.g., industrial dehumidifier) in the cleaning, preventing, and remediation of molds.
  • a dehumidifying agent e.g., industrial dehumidifier
  • nanoemulsions of the present invention may be used alone, or in conjunction with other materials and products to perfo ⁇ n specific desired functions.
  • the nanoemulsions may be added to or formulated with health care products, hygiene products, and household products to prevent contamination of the products and/or to add or improve anti-microbial properties of the products, hi some embodiments, the functional components of the products are included in the aqueous phase or oil phase of the nanoemulsion (e.g., any of the nanoemulsion compositions described above). Components may be added prior to, during, or following emulsification.
  • formulations and uses include (ingredients and concentrations are illustrative; modifications may be made as appropriate or desired): acne treatment (e.g., 0.10% adapalene, 20% azelaic acid, 2.5-20% benzoyl peroxide, 1% clindamycin, 1.5-2% erythromycin, 0.05% isotetrinoin, 1% meclocycline, 4% nicotinamide, 1-3% resorcinol, 0.5-5% salicylic acid, 0.5-5% sulfur, 6% sulfurated lime [dilute 1 :10], 2.2 mg/ml tetracycline hydrochloride, and 0.025-0.1% tretinoin); deep pore purifying astringent (Witch Hazel); antacids (e.g., ⁇ 600 mg/5 ml alumina [aluminum hydroxide], aluminum carbonate, aluminium phosphate, ⁇ 850 mg/5 ml calcium carbonate, 540 mg/5 ml magal
  • resorcinol resorcinol + sulfur 2% + 5-8%
  • candidiasis e.g., 2% butoconazole, 1% ciclopirox, 1 - 10% clotrimazole, clotrimazole and betamethasone 1 %> and 0.05%, 150 mg/dose econazole, 2% ketoconozole, 500 mg and 100,000 Units metronidazole and nystatin, 2 - 5% miconazole, 100,000 Units / Gram nystatin, 100,000 Units and 1 mg / gram nystatin and triamcinolone, 1% sulconazole, 0.4 - 0.8 % terconazole, 6.50%) tioconazole); antifungus products (e.g., 3% clioquinol, 1% haloprogin, 1% naftifme, 1% tolnaftate, 1% terbinafine, 1% oxiconazole); Tinea vers
  • the present invention provides antimicrobial oil-in- water nanoemulsions having one or more of a first component comprising a solvent (e.g., ethanol, glycerol, polyethylene glycol, isopropanol), a second component comprising a halogen-containing compound (e.g., benzethonium chloride, methylbenzethonium chloride, N-alkyldimethyl benzylammonium chloride), alkyldimethyl-3,4-dichlorobenzyl ammonium chloride, cetypyridinium chloride), a third component comprising a surfactant (e.g., TWEEN-20, TRITON X-100, SDS, Poloxamer, sodium lauryl sulfate), and a fourth component (e.g., an addition surfactant, methanol, EDTA, tribuyl phosphate, tyloxapol, 2-phenylphenol, sodium chloride, triso
  • a solvent
  • the present invention may be used in killing/disabling corona viral contaminated surfaces (e.g., topical applications), and/or related diseases (e.g., severe acute respiratory syndrome (SARS), common cold).
  • SARS severe acute respiratory syndrome
  • the present invention may be used in killing/disabling SARS.
  • SARS may be caused by, for example, the SARS-associated coronavirus (SARS- CoV).
  • SARS- CoV SARS-associated coronavirus
  • the present invention is combined with steroids (e.g., corticosteroids, corticosteroids with ribavirin) in the treatment of SARS.
  • the present invention is combined with antiviral agents (e.g., ribavirin, ribavirin in conjunction with steroids, antiviral agents that inhibit SARS viral protease) in the treatment of SARS.
  • antiviral agents e.g., ribavirin, ribavirin in conjunction with steroids, antiviral agents that inhibit SARS viral protease
  • the present invention is combined with interferon in the treatment of SARS.
  • the present invention is combined with a vaccine (e.g., SARS-CoV vaccine) in the treatment of SARS.
  • the present invention may be used in treating tinea pedis.
  • Tinea pedis may be caused, for example, by Tinea rubrum, Tinea mentagrophytes, Tinea mentagrophytes var interdigitale, Scytalidium hyalinum, Scytalidium dimidiatum, Candida albicans and Epidermophyton ⁇ occosum.
  • the present invention is combined with topical imidiazoles (e.g., clotrimazole, ketoconazole, miconazole, oxiconazole) in the treatment of tinea pedis.
  • topical pyridones e.g., ciclopirox
  • the present invention is combined with topical allylamines (e.g., naftifme, terbinafme) in the treatment of tinea pedis.
  • topical benzylamines e.g., butenafme
  • oral antimycotics e.g., itraconazole, terbinafme, fluconazole
  • the present invention is combined with dermatological agents (e.g., aluminum acetate solution, ammonium lactate lotion, drying powders) in the treatment of tinea pedis.
  • dermatological agents e.g., aluminum acetate solution, ammonium lactate lotion, drying powders
  • the present invention may be used in treating tinea manuum.
  • Tinea manuum may be caused, for example, by Tinea rubrum, Tinea mentagrophytes, Tinea mentagrophytes var interdigilale, Scytalidium hyalinum, Scytalidium dimidiatum, Candida albicans and Epidermophyton ⁇ occosum.
  • the present invention is combined with topical imidiazoles (e.g., clotrimazole, ketoconazole, miconazole, oxiconazole) in the treatment of tinea manuum.
  • topical imidiazoles e.g., clotrimazole, ketoconazole, miconazole, oxiconazole
  • topical pyridones e.g., ciclopirox
  • topical allylamines e.g., naftifme, terbinafme
  • the present invention is combined with topical benzylamines (e.g., butenafine) in the treatment of tinea manuum.
  • the present invention is combined with oral antimycotics (e.g., itraconazole, terbinafme, fluconazole) in the treatment of tinea manuum.
  • the present invention is combined with dermatological agents (e.g., aluminum acetate solution, ammonium lactate lotion, drying powders) in the treatment of tinea manuum.
  • the present invention may be used in treating tinea cruris.
  • Tinea cruris may be caused, for example, by Tinea rubrum, E floccosum, T mentagrophytes, and T verrucosum.
  • the present invention is combined with antifungal agents (e.g., terbinafme, butenafine, clotrimazole, miconazole, ketoconazole, econazole, naftifme, tolnaftate, haloprogin, ciclopirox, itraconazole, sulconazole, friseofulvin) in the treatment of tinea cruris.
  • the present invention may be used in treating tinea capitis. Tinea capitis may be caused, for example, by Trichophyton schoenleinii, Trichophyton violaceum, Microsporum gypsum, Trichophyton mentagrophytes,
  • Trichophyton tonsurans Pityrosporum orbiculare (Pityrosporum ovale), Microsporum
  • the present invention is combined with antifungal agents (e.g., griseofulvin, itraconazole, ketoconazole, fluconazole, terbinafine) in the treatment of tinea capitis.
  • antifungal agents e.g., griseofulvin, itraconazole, ketoconazole, fluconazole, terbinafine
  • the present invention may be used in treating tinea co ⁇ oris. Tinea co ⁇ oris may be caused, for example, by Trichophyton rubrum, Microsporum canis, Trichophyton mentagrophytes, Trichophyton tons rans and Trichophyton concentricum.
  • the present invention is combined with antifungal agents (e.g., ketoconazole, naftifiine, itraconazole, fluconazole, terbinafine) in the treatment of tinea co ⁇ oris.
  • antifungal agents e.g., ketoconazole, naftifiine, itraconazole, fluconazole, terbinafine
  • the present invention may be used in treating tinea barbae.
  • Tinea barbae may be caused, for example, by Trichophyton mentagrophytes var granulosum, Trichophyton verrucosum, Microsporum canis, Trichophyton mentagrophytes var erinacei, Trichophyton rubrum, Trichophyton violaceum, Trichophyton megninii, and Trichophyton schoenleinii.
  • the present invention is combined with antifungal agents (e.g., griseofulvin, terbinafme, itraconazole, fluconazole) in the treatment of tinea barbae.
  • antifungal agents e.g., griseofulvin, terbinafme, itraconazole, fluconazole
  • the present invention may be used in treating tinea faciale. Tinea faciale may be caused, for example, by Trichophyton schoenleinii, Trichophyton violaceum, Microsporum gypsum, Trichophyton mentagrophytes,
  • the present invention is combined with antifungal agents (e.g., ketoconazole, clotrimazole, econazole, miconazole, terbinafine, naftifine, griseofulvin, itraconazole, fluconazole) in the treatment of tinea faciale.
  • antifungal agents e.g., ketoconazole, clotrimazole, econazole, miconazole, terbinafine, naftifine, griseofulvin, itraconazole, fluconazole
  • the present invention may be used in treating tinea unguium (e.g., onychomycosis).
  • Tinea unguium may be caused, for example, by Trichophyton schoenleinii, Trichophyton violaceum, Microsporum gypsum, Trichophyton mentagrophytes, Trichophyton tonsurans, Pityrosporum orbiculare (Pityrosporum ovale), Microsporum audouinii, Microsporum canis, Microsporum ferrugineum, Epidermophyton floccosum, Candida albicans, Trichophyton concentricum, and Trichophyton rubrum.
  • the present invention is combined with antifungal agents (e.g., terbinafine, itraconazole, fluconazole) in the treatment of tinea unguium.
  • antifungal agents e.g., terbinafine, itraconazole, fluconazole
  • the present invention may be used in treating Shingles
  • Shingles may be caused, for example, by varicella-zoster virus.
  • the present invention is combined with antiviral medications (e.g., acyclovir, valacyclovir, famciclovir) in the treatment of Shingles.
  • antiviral medications e.g., acyclovir, valacyclovir, famciclovir
  • the present invention is combined with cortisone steroids in the treatment of Shingles.
  • the present invention may be used in treating he ⁇ es simplex related illnesses (e.g., acute he ⁇ etic gingivostomatitis, acute he ⁇ etic pharyngotonsillitis, he ⁇ es labialis, genital he ⁇ es, primary genital he ⁇ es, recu ⁇ ent genital he ⁇ es, subclinical genital he ⁇ es).
  • He ⁇ es simplex related illnesses may be caused, for example, by he ⁇ es simplex I virus, and he ⁇ es simplex II virus.
  • the present invention is combined with antiviral medication (e.g., acyclovir, valacyclovir, famciclovir) in the treatment of he ⁇ es simplex related illnesses.
  • antiviral medication e.g., acyclovir, valacyclovir, famciclovir
  • the present invention may be used in treating human papillomavirus related illnesses (e.g., common warts, plantar warts, flat warts, Butcher's warts, mosaic warts, ungula squamous cell carcinoma, epidermodysplasia verruciformis, nonwarty skin lesions, respiratory papillomatosis, squamous cell carcinoma of the lung, laryngeal papilloma, laryngeal carcinoma, maxillary sinus papilloma, squamous cell carcinoma of the sinuses, conjunctival papillomas, conjunctival carcinoma, oral focal
  • Human papillomavirus related illnesses may be caused, for example, by the human papilloma viruses (HPV) (e.g., HPV 1, 2, 4, 26, 27, 29, 41, 57, 65).
  • HPV human papilloma viruses
  • the present invention is combined with keratolytics (e.g., popophyllum resin, podofilox, 5-fluorouracil) in the treatment of human papilloma virus related illnesses.
  • keratolytics e.g., popophyllum resin, podofilox, 5-fluorouracil
  • the present invention is combined with immune response modifiers (e.g., interferon alfa-n3, imiquimod) in the treatment of human papillomavirus related illnesses.
  • the present invention may be used in treating vaginitis
  • Vaginitis may be caused, for example, by Candida albicans, Trichomonas vaginalis, bacteria, Escherichia coli, shigella sonnei, and Enterobius vermicularis.
  • the present invention is combined with antifungal agents (e.g., clotrimazole, fluconazole).
  • the present invention is combined with antibiotics (e.g., ceftriaxone, erythromycin, clindamycin, metronidazole, cefoxitin, triple sulfa, azithromycin) in the treatment of vaginitis
  • antibiotics e.g., ceftriaxone, erythromycin, clindamycin, metronidazole, cefoxitin, triple sulfa, azithromycin
  • the present invention may be combined with estrogen (e.g., conjugated estrogens) in the treatment of vaginitis.
  • the present invention may be used in treating vaginosis
  • Vaginosis may be caused, for example, by bacterial infection, Candida albicans, Trichophyton schoenleinii, Trichophyton violaceum, Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton concentricum, and Trichophyton rubrum.
  • antifungal agents e.g., clotrimazole, fluconazole.
  • the present invention is combined with antibiotics (e.g., ceftriaxone, erythromycin, clindamycin, metronidazole, cefoxitin, triple sulfa, azithromycin) in the treatment of vaginosis.
  • antibiotics e.g., ceftriaxone, erythromycin, clindamycin, metronidazole, cefoxitin, triple sulfa, azithromycin
  • the present invention may be combined with estrogen (e.g., conjugated estrogens) in the treatment of vaginosis.
  • the present invention may be used in treating sexually transmitted diseases (e.g., chlamydia, genital he ⁇ es, gono ⁇ hea, syphilis, chancroid, HIV, genital warts).
  • Sexually transmitted diseases may be caused, for example, by bacterial infection.
  • the present invention may be combined with antifungal agents (e.g., clotrimazole, fluconazole) in the treatment of sexually transmitted diseases.
  • antifungal agents e.g., clotrimazole, fluconazole
  • antibiotics e.g., ceftriaxone, erythromycin, clindamycin, metronidazole, cefoxitin, triple sulfa, azithromycin
  • EXAMPLE 1 Methods of Formulating Emulsions
  • the emulsion is produced as follows: an oil phase is made by blending organic solvent, oil, and surfactant and then heating the resulting mixture at 37-90°C for up to one hour.
  • the emulsion is formed either with a reciprocating syringe instrumentation or Silverson high sheer mixer.
  • the water phase is added to the oil phase and mixed for 1-30 minutes, preferably for 5 minutes.
  • the volatile ingredients are added along with the aqueous phase, _ .
  • the emulsion was formed as follows: an oil phase was made by blending tri-butyl phosphate, soybean oil, and a surfactant (e.g., TRITON X-100) and then heating the resulting mixture at 86°C for one hour. An emulsion was then produced by injecting water into the oil phase at a volume/volume ratio of one part oil phase to four parts water.
  • the emulsion can be produced manually, with reciprocating syringe instrumentation, or with batch or continuous flow instrumentation. Methods of producing these emulsions are well l nown to those of skill in the art and are described in e.g., U.S. Pat. Nos.
  • Table 2 shows the proportions of each component, the pH, and the size of the emulsion as measured on a Coulter LS 130 laser sizing instrument equipped with a circulating water bath.
  • the emulsions of the present invention are highly stable. Indeed, emulsions were produced as described above and allowed to stand overnight at room temperature in sealed 50 to 1000 mL polypropylene tubes. The emulsions were then monitored for signs of separation. Emulsions that showed no signs of separation were considered “stable.” Stable emulsions were then monitored over 1 year and were found to maintain stability. Emulsions were again produced as described above and allowed to stand overnight at -20°C in sealed 50 mL polypropylene tubes. The emulsions were then monitored for signs of separation. Emulsions that showed no signs of separation were considered “stable.” The BCTP and BCTP 0.1, emulsions have been found to be substantially unchanged after storage at room temperature for at least 24 months.
  • EXAMPLE 2 Characterization Of An Exemplary Bacteria-inactivating Emulsion Of The Present Invention As An Emulsified Liposome Formed In Lipid Droplets
  • a bacteria inactivating emulsion of the present invention designated X 8 WeoPC, was formed by mixing a lipid-containing oil-in-water emulsion with BCTP.
  • a lipid-containing oil-in-water emulsion having glycerol monooleate (GMO) as the primary lipid and cetylpyridinium chloride (CPC) as a positive charge producing agent (refe ⁇ ed to herein as GMO/CPC lipid emulsion or "W 80 8P") and BCTP were mixed in a 1 :1 (volume to volume) ratio.
  • GMO glycerol monooleate
  • CPC cetylpyridinium chloride
  • BCTP positive charge producing agent
  • U.S. Pat. No. 5,547,677 (herein inco ⁇ orated by reference in its entirety), describes the GMO/CPC lipid emulsion and other related lipid emulsions that may be combined with BCTP to provide the bacteria-inactivating oil-in-water emulsions of the present invention.
  • the emulsions of the present invention were mixed with various bacteria for 10 minutes and then plated on standard microbiological media at varying dilutions. Colony counts were then compared to untreated cultures to determine the percent of bacteria killed by the treatment. Table 3 summarizes the results of the experiment.
  • EDTA ethylenediamine-tetraacetic acid
  • Bacillus cereus (B. cereus, ATCC #14579) was utilized as a model system for Bacillus anthracis.
  • BCTP diluted preparations to study the bactericidal effect of the compounds of the present invention on the vegetative form (actively growing) of 5. cereus were performed.
  • Treatment in medium for 10 minutes at 37°C was evaluated, As summarized in Table 6, the BCTP emulsion is efficacious against the vegetative form of 5. cereus.
  • a 10 minute exposure with this preparation is sufficient for virtually complete killing of vegetative forms of B. cereus at all concentrations tested including dilutions as high as 1 : 100.
  • the spore form of B. anthracis is one of the most likely organisms to be used as a biological weapon. Spores are well known to be highly resistant to most disinfectants. As describe above, effective killing of spores usually requires the use of toxic and irritating chemicals such as formaldehyde or sodium hypochlorite (i.e., bleach). The same experiment was therefore performed with the spore form of B. cereus. As shown in Table 7, treatment in both medium for 10 minutes at 37°C was not sufficient to kill B. cereus spores.
  • CFU/ml The number of colony forming units per milliliter (CFU/ml) was quantitated after 0,5, 1, 2, 4, 6, and 8 hours. As shown in FIG. 1, CFU/ml in the untreated control increased over the first 4 hours of incubation and then reached a plateau. Bacterial smears prepared at time zero,, 1, 2, 4 and 6 hours, and stained for spore structures, revealed that by 2 hours no . spore structures remained (FIGS. 2A-2C). Thus, 100%> germination of spores occurred in the untreated control by the 2 hour time point. In the spore preparation treated with BCTP, CFU/ml showed no increase over the first 2 hours and then declined rapidly over the time period from 2-4 hours.
  • EXAMPLE 6 In Vivo Bactericidal Efficacy Study Animal studies were prefo ⁇ ned to demonstrate the protective and therapeutic effect of the inventive emulsions in vivo. Bacillus cereus infection in experimental animals has been used previously as a model system for the study of anthrax (Burdon and Wende, 1960; Burdon et al, 1967; Lamanna and Jones, 1963), The disease syndrome induced in animals experimentally infected with B. cereusis in some respects similar to anthrax (Drobniewski, 1993; Fritz " et al, 1995). The inventive emulsions were mixed with B. cereus spores before injecting into mice.
  • I ⁇ igation of experimentally infected wounds with saline did not result in any apparent benefit.
  • Irrigation of wounds infected with B. cereus spores with inventive emulsion showed substantial benefit, resulting in a consistent 98% reduction in the lesion size from 4.86 cm 2 to 0.06 cm 2 .
  • This reduction in lesion size was accompanied by a three-fold reduction in mortality (60% to 20%) when compared to experimental animals receiving either no treatment or saline i ⁇ igation. Histology of these lesions showed no evidence of vegetative Bacillus organisms and minimal disruption of the epidermis (Hamouda et al, 1999).
  • CD-I mice were injected with inventive emulsion diluted 1 : 10 in saline as a control and did not exhibit signs of distress or inflammatory reaction, either in gross or histological analysis.
  • inventive emulsion diluted 1 : 10 in saline as a control and did not exhibit signs of distress or inflammatory reaction, either in gross or histological analysis.
  • a suspension of 4x10 7 B. cereus spores was mixed with saline or with inventive emulsion at a final dilution of 1 : 10 and then immediately injected subcutaneously into the back of CD-I mice.
  • Mice that were infected subcutaneously with B. cereus spores without inventive emulsion developed severe edema at 6-8 hours.
  • Mucous membrane Intranasal toxicity was preformed in mice by installation of 25 ⁇ L of 4% of the nanoemulsion per nare. No clinical or histopathological changes were observed in these mice.
  • Oral toxicity testing in rats was performed by gavaging up to 8 mL per kg of 25% nanoemulsion. The rats did not lose weight or show signs of toxicity either clinically or histopathologically. There were no observed changes in the gut bacterial flora as a result of oral administration of the emulsions.
  • Bacillus cereus was passed three times on blood agar (TSA with 5% sheep blood, REMEL). B.
  • the B. cereus suspension was divided into two tubes. An equal volume of sterile saline was added to one tube and mixed 0.1 cc of the B. cereus suspension/saline was injected subcutaneously into 5 CD-I mice. An equal volume of BCTP (diluted 1 :5 in sterile saline) was added to one tube and mixed, giving a final dilution of BCTP at 1 :10. The 5. cereus suspension/BCTP was incubated at 37°C for 10 minutes while being mixed 0.1 cc of the B. cereus suspension BCTP was injected subcutaneously into 5 CD-I mice.
  • TAB trypticase soy broth
  • Bacillus cereus was grown on Nutrient Agar (Difco) with 0.1% Yeast Extract (Difco) and 50 ⁇ g/ml MnS0 for induction of spore formation.
  • the plate was scraped and suspended in sterile 50% ethanol and incubated at room temperature for 2 hours with agitation in order to lyse remaining vegetative bacteria.
  • the suspension was centrifuged at 2,500 x g for 20 minutes and the supernatant discarded.
  • the pellet was resuspended in diH 2 O, centrifuged at 2,500 X g for 20 minutes, and the supernatant discarded.
  • the spore suspension was divided.
  • the pellet was resuspended in TSB. 0.1 cc of the B.
  • Bacillus cereus was grown on Nutrient Agar (Difco) with 0.1% Yeast Extract (Difco) and 50 (g/ml MnS0 4 for induction of spore formation). The plate was scraped and suspended in sterile 50% ethanol and incubated at room temperature for 2 hours with agitation in order to lyse remaining vegetative bacteria. The suspension was centrifuged at 2,500 X g for 20 minutes and the supernatant discarded. The pellet was resuspended in distilled H 2 0, centrifuged at 2,500 X g for 20 minutes, and the supernatant discarded. The pellet was resuspended in TSB. The B. cereus spore suspension was divided into three tubes.
  • B. cereus Re-isolation of B. cereus was attempted from skin lesions, blood, liver, and spleen (Table 13), Skin lesions were cleansed with betadine followed by 70% sterile isopropyl alcohol. An incision was made at the margin of the lesion and swabbed. The chest was cleansed with betadine followed by 70% sterile isopropyl alcohol. Blood was drawn by cardiac puncture. The abdomen was cleansed with betadine followed by 70% sterile isopropyl alcohol. The skin and abdominal muscles were opened with separate sterile instruments. Samples of liver and spleen were removed using separate sterile instruments. Liver and spleen samples were passed briefly through a flame and cut using sterile instruments. The freshly exposed surface was used for culture, BHI agar (Difco) was inoculated and incubated aerobically at 37°C overnight.
  • BHI agar Difco
  • CD-I mice were injected subcutaneously with 0.1 cc of the compounds of the present invention and observed for 4 days for signs of inflammation and/or necrosis. Dilutions of the compounds were made in sterile saline. Tissue samples from mice were preserved in 10% neutral buffered formalin for histopathologic examination. Samples of skin and muscle (from mice which were injected with undiluted compounds) sent for histological review were reported to show indications of tissue necrosis. Tissue samples from mice which were injected with diluted compounds were not histologically examined. Tables 14 and 15 show the results of two individual experiments.
  • Guinea pigs were injected intramuscularly (in both hind legs) with 1.0 cc of compounds of the present invention per site and observed for 4 days for signs of inflammation and/or necrosis. Dilutions of the compounds were made in sterile saline. Tissue samples from guinea pigs were preserved in 10% neutral buffered formalin for histological examination. Tissue samples were not histologically examined.
  • EXAMPLE 8 In Vivo Toxicity Study II
  • One group of Sprague-Dawley rats each consisting of five males and five females were placed in individual cages and acclimated for five days before dosing. Rats were dosed daily for 14 days. On day 0-13, for 14 consecutive days each rat in Group 1 received by gavage three milliliters of BCTP, 1 : 100 concentration, respectively. The three-milliliter volume was detemiined to be the maximum allowable oral dose for rats. Prior to dosing on Day 0 and Day 7, each rat was weighed. Thereafter rats were weighed weekly for the duration of the study. Animals were observed daily for sickness or mortality.
  • General techniques for toxicity testing include dermal irritation testing, eye i ⁇ itation testing, subcutaneous test, intramuscular tests, open wound irrigation, intranasal tests, and oral tests.
  • Dermal tests can be conducted on rabbits wherein 0.5 ml of 10% emulsion is applied to the skin or rabbits for four hours. The skin reaction is recorded for up to 72 hours.
  • a Draize scale is used to score the irritation.
  • For eye irritation testing 0.1 ml of 10% emulsion is applied to the eye of rabbits and the eye reaction is recorded for up to 72 hours.
  • a Draize scale is used to score the irritation.
  • Subcutaneous and intramuscular tests inject 0.1 ml of 10% emulsion in mice.
  • mice Two ml of 10% emulsion is applied in an open wound i ⁇ igation test using mice. For intranasal testing, 0.25 m/naris of 2-4% emulsion are applied to mice. For oral testing, 4 ml/kg/day of 10% emulsion are given orally for 1 week or 8 ml/kg of 100%o emulsion is given in a single dose.
  • Example 2 provides an insight into a proposed the mechanisms of action of the emulsions of the present invention and to show their sporicidal activity. This mechanism is not intended to limit the scope of the invention an understanding of the mechanism is not necessary to practice the present invention, and the present invention is not limited to any particular mechanism.
  • the effect of a GMO/CPC lipid emulsion ("W 8 o8P") and BCTP on E. coli was examined. W 8 o8P killed the E. coli (in deionized H 2 O) but BCTP was ineffective against this organism, FIG. 8 shows the control and FIG. 9 shows the E. coli treated with BCTP. As shown in FIG. 9, the BCTP treated E. coli look. normal, with defined structure and intact lipid membranes.
  • FIG. 9 shows the BCTP treated E. coli look. normal, with defined structure and intact lipid membranes.
  • the W 80 8P treated Vibrio cholerae again shows swelling and changes in the interior of the organism, but the cells remain intact.
  • the BCTP treated Vibrio cholerae (FIG. 13) are completely lysed with only cellular debris remaining.
  • X 8 W 6 oPC (FIG. 14) showed a combination of effects, where some of the organisms are swelled but intact and some are lysed. This clearly suggests that BCTP, W 80 8P and X 8 W 6 oPC work by different mechanisms.
  • a third comparative study was perfo ⁇ ned to evaluate efficacy of the emulsions at various concentrations.
  • X 8 W 6 oPC is more effective as a biocide at lower concentrations (higher dilutions) in bacteria sensitive to either W 80 8P or BCTP.
  • six other bacteria that are resistant to W 8 ⁇ 8P and BCTP are all susceptible to X 8 W 6 oPC. This difference in activity is also seen when comparing W 80 8P and BCTP and X 8 W 60 PC in influenza infectivity assays.
  • both BCTP and X 8 W 6 rjPC are effective at a 1:10 and 1:100 dilutions and additionally, X 8 W ⁇ oPC is effective at the lowest concentration, 1 : 1,000 dilution.
  • W 8u 8P has little activity even at 1:10 dilution, suggesting that it is not an effective treatment for this enveloped organism.
  • X 8 W ⁇ 5 oPC kills yeast species that are not killed by either W 80 8P or BCTP,
  • EXAMPLE 11 Further Evidence Of The Sporicidal Activity of the Nanoemulsion against Bacillus Species
  • the present Example provides the results of additional investigations of the ability of particular embodiments of the emulsions of the present invention to inactivate different Bacillus spores. The methods and results from these studies are outlined below.
  • Surfactant lipid preparations BCTP, a water-in-oil nanoemulsion, in which the oil phase was made from soybean oil, tri-n-butyl phosphate, and TRITON X-100 in 80% water.
  • X 8 W 60 PC was prepared by mixing equal volumes of BCTP with W 80 8P which is a liposome-like compound made of glycerol monostearate, refined Soya sterols, TWEEN 60, soybean oil, a cationic ion halogen-containing CPC and peppermint oil.
  • Spore preparation For induction of spore formation, Bacillus cereus (ATTC 14579), B. circulars (ATC 4513), B. megaterium (ATCC 14581), and 5. subtilis (ATCC 1 1774) were grown for a week at 37°C on NAYEMn agar (Nutrient Agar with 0.1% Yeast Extract and 5 mg/1 MnS0 ).
  • the plates were scraped and the bacteria spores suspended in sterile 50% ethanol and incubated at room temperature (27°C) for 2 hours with agitation in order to lyse the remaining vegetative bacteria.
  • the suspension was centrifuged at 2,500 X g for 20 minutes and the pellet washed twice in cold diH 2 O.
  • the spore pellet was resuspended in trypticase soy broth (TSB) and used immediately for experiments.
  • TTB trypticase soy broth
  • spores were resuspended in TSB. 1 ml of spore suspension containing 2x10 6 spores (final concentration 10 6 spores/ml) was mixed with 1 ml of BCTP or X 8 W 6 oPC (at 2X final concentration in diH 2 O) in a test tube. The tubes were incubated in a tube rotator at 37°C for four hours. After treatment, the suspensions were diluted 10-fold in diH 2 O.
  • cereus spores (at a final concentration 10 6 spores/ml) were suspended in TSB with either the germination inhibitor D-alanine (at final concentration of 1 M) or with the germination stimulator L-alanine + inosine (at final concentration of 50 M each) (Titball and Manchee, 1987; Foster and Johnston., 1990; Shibata et al, 1976) and then immediately mixed with BCTP (at a final dilution of 1 : 100) and incubated for variable interval. Then the mixtures were serially diluted, plated and incubated overnight. The next day the plates were counted and percentage sporicidal activity was calculated.
  • In vivo sporicidal activity Two animal models were developed; in the first B. cereus spores (suspended in sterile saline) were mixed with an equal volume of BCTP at a final dilution of 1 :10. As a control, the same B. cereus spore suspension was mixed with an equal volume of sterile saline. 100 ⁇ l of the suspensions containing 4xl0 7 spores was then immediately injected subcutaneously into CD-I mice. In the second model, a simulated wound was created by making an incision in the skin of the back of the mice. The skin was separated from the underlying muscle by blunt dissection.
  • the "pocket" was inoculated with 200 1 containing 2.5xl0 7 spores (in saline) and closed using wound clips. One hour later, the clips were removed and the wound irrigated with either 2 ml of sterile saline or with 2 ml of BCTP ( 1 : 10 in sterile saline). The wounds were then closed using wound clips. The animals were observed for clinical signs. Gross and histopathology were performed when the animals were euthanized 5 days later. The wound size was calculated by the following formula: Vi a x Vi b x where a and b are two pe ⁇ endicular diameters of the wound.
  • BCTP In vitro sporicidal activity: To assess the sporicidal activity of BCTP, spores from four species of Bacillus genus, B. cereus, B. circulans, B. megatetium, and B. subtilis were tested. BCTP at 1 :100 dilution showed over 91%) sporicidal activity against B. cereus and B. megaterium in 4 hours (FIG, 16). B. circulans was less sensitive to BCTP showing 80% reduction in spore count, while B. subtilis appeared resistant to BCTP in 4 hours. A comparison of the sporicidal effect of BCTP (at dilutions of 1 : 10 and 1 :100) on B.
  • cereus sporicidal time course A time course was performed to analyze the sporicidal activity of BCTP diluted 1:100 and X 8 W 60 PC diluted 1 :1000 against B. cereus over an eight hour period. Incubation of BCTP diluted 1 :100 with B. cereus spores resulted in a 77% reduction in the number of viable spores in one hour and a 95% reduction after 4 hours. Again, X 8 W ⁇ oPC diluted 1 :1000 was more effective than BCTP 1 :100 and resulted in about 95% reduction in count after 30 minutes (FIG. 17). BCTP B.
  • anthracis sporicidal activity Following initial in vitro experiments, BCTP sporicidal activity was tested against two virulent strains of B. anthracis (Ames and Vollum IB). It was found that BCTP at a 1 :100 final dilution inco ⁇ orated into growth medium completely inhibited the growth of lxlO 5 B. anthracis spores. Also, 4 hours incubation with BCTP at dilutions up to 1 : 1000 with either the Ames or the Vollum 1 B spores resulted in over 91% sporicidal activity when the mixtures were incubated at RT, and over 96%> sporicidal activity when the mixtures were incubated at 37°C (Table 19).
  • BCTP sporicidal activity against 2 different strains of Bacillus anthracis spores as detemiined by colony reduction assay (%> killing). BCTP at dilutions up to 1 : 1000 effectively killed > 91 % of both spore strains in 4 hours at either 27 or 37°C; conditions that differed markedly in the extent of spore ge ⁇ nination. Sporicidal activity was consistent at spore concentrations up to lxl0 6 /ml.
  • Xs ⁇ oPC B. anthracis sporicidal activity Since X 8 W 6 oPC was effective at higher dilutions and against more species of Bacillus spores than BCTP, it was tested against 4 different strains of 5. anthracis at dilutions up to 1:10,000 at RT to prevent germination. X 8 W 60 PC showed peak killing between 86% and 99.9%> at 1 :1000 dilution (Table 20). Table 20: X 8 W ⁇ oPC sporicidal activity against 4 different strains of B. anthracis representing different clinical isolates. The spores were treated with XgWeoPC at different dilutions in RT to reduce germination. There as no significant killing at low dilutions. The maximum sporicidal effect was observed at 1:1000 dilution.
  • Electron microscopy examination of the spores Investigations were carried out using B. cereus because it is the most closely related to B. anthracis. Transmission electron microscopy examination of the B. cereus spores treated with BCTP diluted 1 : 100 in TSB for four hours revealed physical damage to the B. cereus spores, including extensive disruption of the spore coat and cortex with distortion and loss of density in the core (FIG. 18).
  • Germination stimulation and inhibition To investigate the effect of initiation of germination on the sporicidal effect of BCTP on Bacillus spores, the germination inhibitors D-alanine (Titball and Manchee, 1987; Foster and Johnston, 1990), and germination simulators L-alanine and inosine (Shibata et al, 1976) were incubated with the spores and BCTP for 1 hour. The sporicidal effect of BCTP was delayed in the presence of 10 mM D-alanine and accelerated in the presence of 50 ⁇ M L-alanine and 50 ⁇ M inosine (FIG. 19).
  • CD-I mice injected with BCTP diluted 1 : 10 in saline as a control did not exhibit signs of distress or inflammatory reaction, either in gross or histological analysis (FIG. 20A, FIG. 20B).
  • a suspension of 4x10 7 B. cereus spores was mixed with saline or with BCTP at a final dilution of 1 : 10 and then immediately injected subcutaneously into the back of CD-I mice. Mice which were infected subcutaneously with B. cereus spores without BCTP developed severe edema at 6-8 hours.
  • FIG. 20C This was followed by a gray, necrotic area surrounding the injection site at 18-24 hours, with severe sloughing of the skin present by 48 hours, leaving a dry, red-colored lesion (FIG. 20C, FIG. 20D).
  • Simultaneous injection of spores and BCTP resulted in a greater than 98% reduction in the size of the necrotic lesion from 1.68 cm 2 to 0.02 cm 2 when the spores were premixed with BCTP. This was associated with minimal edema or inflammation (FIG. 20E, FIG. 20F).
  • a 1 cm skin wound was infected with 2.5x10 7 B. cereus spores then closed without any further treatment (FIG. 21 A, FIG.21 B).
  • Enveloped viruses are of great concern as pathogens. They spread rapidly and are capable of surviving out of a host for extended periods. Influenza A virus was chosen because it is a well accepted model to test anti-viral agents (Karaivanova and Spiro, 1998; Mammen et al, 1995; Huang et al, 1991). Influenza is a clinically important respiratory pathogen that is highly contagious and responsible for severe pandemic disease (Mulder and Hers, 1972).
  • SLPs Surfactant lipid preparations
  • the SLPs were then formed by injecting water or 1 % bismuth in water (SS) into the oil phase at a volume/volume ratio using a reciprocating syringe pump.
  • Viruses Influenza virus A/AA/6/60 (Hedocher et al, 1996) was kindly provided by Dr. Hunein F. Maassab (School of Public Health, University of Michigan). Influenza A vims was propagated in the allantoic cavities of fertilized pathogen-free hen eggs (SPAFAS, Norwich, CT) using standard methods (Ba ⁇ ett and Inglis, 1985). Vims stock was kept in aliquots (10 8 pfu/ml) of infectious allantoic fluids at -80°C.
  • Adenoviral vector (AD.RSV ntlacZ) was provided by Vector Core Facility (University of Michigan Medical Center, Ann Arbor, MI) and was kept in aliquots (10 12 pfu ml at -80°C).
  • the vector is based on a human adenoviral (serotype 5) genomic backbone deleted of the nucleotide sequence spanning E1A and E1B and a portion of E3 region. This impairs the ability of the vims to replicate or transform nonpermissive cells. It carries the Eschetichia coli LacZ gene, encoding, ⁇ -galactosidase, under control of the promoter from the Rouse sarcoma virus long terminal repeat (RSV-LTR).
  • RSV-LTR Rouse sarcoma virus long terminal repeat
  • nt nuclear targeting
  • the 293 cells express the transforming gene of adenovirus 5 and therefore restore the ability of Ad.RSV ntlacZ vector to replicate in the host cell (Graham et al, 1977).
  • Cell maintenance media MDCK cells were maintained in Eagle's minimal essential medium with Earle's salts, 2 mM L-glutamine, and 1.5 g/1 sodium bicarbonate (Mediatech, Inc., Hemdon, VA) containing 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT).
  • the medium was supplemented with 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 100 U penicillin/ml and streptomycin 100 ⁇ g/ml (Life Technologies, Gaithersburg, MD).
  • the 293 cells were maintained in Dulbecco's modified Eagle medium (Mediatech, Inc., Hemdon, VA), containing 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate. It also contained 100 U penicillin/ml and streptomycin 100 ⁇ g/ml (Life Technologies, Gaithersburg, MD) and was supplemented with 10% FBS (Hyclone Laboratories, Logan, UT).
  • Influenza A infection medium was the MDCK cell maintenance medium (without FBS) supplemented with 3.0 ⁇ g/ml of tolylsulfonyl 5 phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Worthington Biochemical Co ⁇ oration, Lakewood, NJ). Adenovims infection medium was 293) cell maintenance medium with a reduced concentration of serum (2% FBS).
  • Influenza A overlay medium Overlay medium consisted of equal amounts of 2x infection medium and 1.6% SEAKEM ME agarose (FMC BioProducts, Rockland, MD).
  • Staining agarose overlay medium consisted of agarose overlay medium plus 0.01 % neutral red solution (Life Technologies, Gaithersburg, MD) without TPCK-treated trypsin.
  • influenza A vims-SLP treatments and controls were diluted in infection medium to contain 30-100 pfu/250 1. Confluent cell monolayers were inoculated in triplicate on 3 plates and incubated at 37°C/5% CO 2
  • In situ cellular enzyme-linked immunosorbent assay To detect and quantitate viral proteins in MDCK cells infected with influenza A vims, the in situ cellular ELISA was optimized. Briefly, 2xl0 4 MDCK cells in 100 ⁇ l complete medium were added to flat-bottom 96-well microtitre plates and incubated overnight. On the next
  • the culture medium was removed and cells were washed with serum free maintenance medium.
  • One hundred ⁇ l of viral inoculum was added to the wells and incubated for 1 hour.
  • the viral inoculum was removed and replaced with 100 ⁇ l of MDCK cell maintained medium plus 2% FBS.
  • the infected MDCK cells were incubated for an additional 24 h.
  • the cells were washed once with PBS and fixed with ice cold ethanol: acetone mixture (1 :1 ) and stored at -20°C.
  • the wells of fixed cells were washed with PBS and blocked with 1 % dry milk in PBS for 30 min. at 37°C.
  • ⁇ -galactosidase assay was performed on cell extracts as described elsewhere (Lim, 1989), Briefly, 293 cells were seeded on 96-well "U"- bottom tissue culture plates at approximately 4xl0 4 cells/well and incubated overnight at 37°C/5%>CO 2 in maintenance medium.
  • DPBS Dulbecco's phosphate buffered saline
  • Adenovims stock was diluted in infection medium to a concentration of 5x10 7 pfu mi and mixed with different concentrations of BCTP as described below.
  • vims was diluted with infection medium to a concentration of IxlO 4 pf ⁇ /mi and overlaid on 293 cells.
  • Cells were incubated at 37 0 C/5%> CO 2 for 5 days, after which the plates were centrifuged, the medium was removed and the cells were washed three times with PBS without Ca++ and Mg++.
  • the PBS was aspirated and 100 ⁇ l of 1 x Reporter Lysis Buffer (Promega, Madison, WI) was placed in each well.
  • 1 x Reporter Lysis Buffer Promega, Madison, WI
  • plates were frozen and thawed three times and the ⁇ -galactosidase assay was performed following the instruction provided by the vendor of ⁇ -galactosidase (Promega, Madison, WI) with some modifications.
  • Five microliters of cell extract was transfened to a 96-well flat bottom plate and mixed with 45 ⁇ l of lx Reporter Lysis Buffer (1 : 10).
  • 2x assay buffer 120 mM Na 2 HPO 4 , 80 mM NaH 2 P0 , 2 mM MgCl 2 , 100 mM ⁇ -mercaptoethanol, 1.33 mg/ml ONPG (Sigma, St. Louis, MO) were added and mixed with the cell extract. The plates were incubated at RT until a faint yellow color developed. At that time the reaction was stopped by adding 100 (1 of 1 M sodium bicarbonate. Plates were read at a wavelength of 420 nm in an ELISA microplate reader. A standard, consisting of (u/ ⁇ l ⁇ -galactosidase (Sigma, St.
  • the dilutions of the mixture of vims and SLPs applied in susceptibility testing were made to be at least one order of magnitude higher than the safe concentration of SLP assessed.
  • Approximately lxlO 8 pfu of either influenza A or adenovims were incubated with lipid preparation at final concentrations of 1:10, 1 : 100, and 1 : 1000 for different time periods as indicated in results on a shaker. After incubation, serial dilutions of the SLP/vims mixture were made in proper infection media and overlaid on MDCK (influenza A) or 293 (adenovirus) cells to perform PRA, cellular ELISA or ⁇ -galactosidase assays as described above.
  • Influenza A virus was semi-purified from allantoic fluid - by passing through a 30% sucrose cushion prepared with GTNE (glycine 200 mM, Tris-HCI 10 mM (pH 8.8), NaCl 100 mM, and EDTA 1 mM) using ultra centrifugation (Beckman rotor SW 28 Ti, at 20,000 ⁇ m for 16 hours). Pelleted vims was reconstituted in GTNE. Ten microliters of respective samples (adenovims, influenza vims, adenovims + BCTP, influenza vims + BCTP) were incubated for 15 and 60 min, then placed on parlodian coated 200 mesh copper grids for 2 min.
  • GTNE glycine 200 mM, Tris-HCI 10 mM (pH 8.8), NaCl 100 mM, and EDTA 1 mM
  • BCTP and SS exhibited over 95% inhibition of influenza A infection at a 1 :10 dilution.
  • NN and W 80 8P showed only an intermediate effect on influenza A virus, reducing infection by approximately 40%.
  • BCTP's virucidal effect was undiminished even at a 1 : 00 dilution.
  • SS showed less effect at a 1 :100 dilution inhibiting influenza A infection by 55%.
  • These two lipid preparations at 1 : 1000 dilution displayed only weak inhibitory effect on vims infectivity at the range of 22-29% (FIG. 23B). Since BCTP and SS both showed strong inhibitory effect on vims infectivity, PRA was used to verify data obtained from cellular ELISA.
  • BCTP reduced the number of plaques from an average of 50.88 to 0 at a 1 :10 dilution (Table 21). At dilution 1 :100, BCTP maintained vimcidal effectiveness. At dilution 1 :100 SS reduced the number of plaques only approximately 7% as compared with untreated virus. Table 21
  • Virus was incubate with SLPs for 30 minutes. b Number of plaques.
  • Anti-influenza A efficacy of BCTP Since TRITON X-100 detergent has anti-viral activity (Maha and Igarashi, 1997; Portocala et al, 1976), it was investigated whether TRITON X-100 alone or combined with individual BCTP components inhibits influenza A infectivity to the same extent as BCTP. Influenza A virus was treated with: 1) BCTP,-2).(he combination of tri(n-butyl)phosphate, TRITON X-100, and soybean oil (TTO), 3) TRITON X-100 and soybean oil (TO), or 4) TRITON X-100 (T) alone.
  • BCTP was significantly more effective against influenza A vims at 1 : 10 and 1 : 100 dilutions (TRITON X-1 00 dilution of 1 :500, and 1 :5000) than TRITON X-1 00 alone or mixed with the other components tested (FIG. 23).
  • BCTP TRITON X-100 dilution of 1:50,000
  • BCTP does not affect infectivity of non-enveloped virus: To investigate whether BCTP may affect the infectivity of non-enveloped vims, genetically engineered adenovims containing LacZ gene was used, encoding ⁇ -galactosidase. This adenovims constmct was deficient in the transfomiing gene and therefore can replicate and transform only permissive cells containing the transfomiing gene of adenovims 5. The 293 cells, which constitutively express transfomiing gene, were employed to promote adenovims replication and production of ⁇ -galactosidase enzyme. As shown in FIG.
  • BCTP treatment did not affect the ability of adenovirus to replicate and express ⁇ -galactosidase activity in 293 cells. Both BCTP treated and untreated adenovims produced approximately 0.11 units of ⁇ -galactosidase enzyme.
  • Action of BCTP on enveloped virus Since BCTP only altered the infectivity of enveloped vimses, the action of this nanoemulsion on enveloped virus integrity was further investigated using electron microscopy. As shown in FIG. 25D, after a 60 min incubation with 1 : 100 dilution of BCTP, the structure of adenovirus is unchanged. A few recognizable influenza A virions were located after 15 min incubation with BCTP (FIG.
  • Figures 31 and 32 show the treatment of Salmonellae with different emulsions of the present invention with the addition of 0.1% EDTA,
  • the EDTA improved the bactericidal activity of the emulsion at both 40°C ( Figure 32) .and 50°C ( Figure 33).
  • the emulsions were tested at 10.0%, 1,0%, and 0.1% > dilutions.
  • the emulsion X8PC is composed of about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1% of CPC, about 64 vol. % of soybean oil, and about 19 vol. % of DiH 2 0 and the emulsion W 2 o5EC is composed of from about 5 vol. % of TWEEN 20, from about 8 vol. % of ethanol, from about 1 vol, % of CPC, about 64 vol. % of oil (e.g., soybean oil), and about 22 vol. % of DiH 2 0.
  • W 2 o5EC were tested for their ability to reduce the growth of a number of microorganisms under various conditions.
  • Figure 35 shows the log reduction of ' Mycobacteria fortuitum by X8PC at 10%, 1% and 0.1% dilutions at room temperature and 37°C.
  • a 2% emulsion of W 20 5EC (with and without 1%, 2%, and 3% Natrosol) each showed an approximately 2 log reduction in E. coli for both dry and wet bacteria after a 15 minute incubation at room temperature.
  • a 2% emulsion of W 20 5EC (with and without 1%, 2%, and 3% Natrosol) each showed an approximately 4 log reduction in S.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Agronomy & Crop Science (AREA)
  • Environmental Sciences (AREA)
  • Pest Control & Pesticides (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicinal Preparation (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention concerne des compositions et des méthodes destinées à réduire l'infectiosité, la morbidité et le taux de mortalité liés à divers virus et organismes pathogènes. L'invention concerne également des méthodes et des compositions de décontamination de zones colonisées ou infectées par des virus et organismes pathogènes. L'invention concerne encore des méthodes et des compositions destinées à réduire l'infectiosité d'organismes pathogènes dans des produits alimentaires.
PCT/US2004/031346 2003-09-24 2004-09-24 Compositions antimicrobiennes en nanoemulsion et methodes associees WO2005030172A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US66986503A 2003-09-24 2003-09-24
US10/669,865 2003-09-24

Publications (1)

Publication Number Publication Date
WO2005030172A1 true WO2005030172A1 (fr) 2005-04-07

Family

ID=34393434

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/031346 WO2005030172A1 (fr) 2003-09-24 2004-09-24 Compositions antimicrobiennes en nanoemulsion et methodes associees

Country Status (1)

Country Link
WO (1) WO2005030172A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008137747A1 (fr) 2007-05-02 2008-11-13 The Regents Of The University Of Michigan Compositions thérapeutiques à base de nanoémulsion et leurs procédés d'utilisation
WO2010036938A2 (fr) * 2008-09-26 2010-04-01 Nanobio Corporation Compositions thérapeutiques de nanoémulsion et procédés d'utilisation de celles-ci
WO2010087964A2 (fr) 2009-01-28 2010-08-05 Nanobio Corporation Composition pour le traitement et la prévention de l'acné, procédés de fabrication des compositions, et procédés d'utilisation associés
US8226965B2 (en) 2008-04-25 2012-07-24 Nanobio Corporation Methods of treating fungal, yeast and mold infections
EP3881901A1 (fr) * 2020-03-18 2021-09-22 Corticalis AS Composition liquide améliorée pour le nettoyage, l'aseptisation et/ou la désinfection
WO2021186000A1 (fr) * 2020-03-18 2021-09-23 Corticalis As Composition liquide améliorée pour le nettoyage, l'assainissement et/ou la désinfection
WO2021202823A1 (fr) * 2020-04-02 2021-10-07 Mollick Peter J Traitement thérapeutique pour la maladie de coronavirus covid-19
WO2022032113A1 (fr) * 2020-08-07 2022-02-10 The University Of Chicago Produits pharmaceutiques pour le traitement ou la prévention de nidovirus et de picornavirus
US20220061315A1 (en) * 2020-08-26 2022-03-03 Simply Pure, LLC Persistent sanitizer

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5576016A (en) * 1993-05-18 1996-11-19 Pharmos Corporation Solid fat nanoemulsions as drug delivery vehicles
US6162448A (en) * 1997-05-28 2000-12-19 L'oreal Combination of a retinoid with a polyamine polymer
US6274150B1 (en) * 1998-12-23 2001-08-14 L'oreal Nanoemulsion based on phosphoric acid fatty acid esters and its uses in the cosmetics, dermatological, pharmaceutical, and/or ophthalmological fields

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5576016A (en) * 1993-05-18 1996-11-19 Pharmos Corporation Solid fat nanoemulsions as drug delivery vehicles
US6162448A (en) * 1997-05-28 2000-12-19 L'oreal Combination of a retinoid with a polyamine polymer
US6274150B1 (en) * 1998-12-23 2001-08-14 L'oreal Nanoemulsion based on phosphoric acid fatty acid esters and its uses in the cosmetics, dermatological, pharmaceutical, and/or ophthalmological fields

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9801842B2 (en) 2007-05-02 2017-10-31 The Regents Of The University Of Michigan Nanoemulsion therapeutic compositions and methods of using the same
EP2152304A1 (fr) * 2007-05-02 2010-02-17 The Regents of the University of Michigan Compositions thérapeutiques à base de nanoémulsion et leurs procédés d'utilisation
WO2008137747A1 (fr) 2007-05-02 2008-11-13 The Regents Of The University Of Michigan Compositions thérapeutiques à base de nanoémulsion et leurs procédés d'utilisation
US8747872B2 (en) * 2007-05-02 2014-06-10 The Regents Of The University Of Michigan Nanoemulsion therapeutic compositions and methods of using the same
US20140287047A1 (en) * 2007-05-02 2014-09-25 The Regents Of The University Of Michigan Nanoemulsion therapeutic compositions and methods of using the same
EP2152304A4 (fr) * 2007-05-02 2015-03-25 Univ Michigan Compositions thérapeutiques à base de nanoémulsion et leurs procédés d'utilisation
US8226965B2 (en) 2008-04-25 2012-07-24 Nanobio Corporation Methods of treating fungal, yeast and mold infections
WO2010036938A2 (fr) * 2008-09-26 2010-04-01 Nanobio Corporation Compositions thérapeutiques de nanoémulsion et procédés d'utilisation de celles-ci
WO2010036938A3 (fr) * 2008-09-26 2010-06-10 Nanobio Corporation Compositions thérapeutiques de nanoémulsion et procédés d'utilisation de celles-ci
WO2010087964A2 (fr) 2009-01-28 2010-08-05 Nanobio Corporation Composition pour le traitement et la prévention de l'acné, procédés de fabrication des compositions, et procédés d'utilisation associés
EP3881901A1 (fr) * 2020-03-18 2021-09-22 Corticalis AS Composition liquide améliorée pour le nettoyage, l'aseptisation et/ou la désinfection
WO2021186000A1 (fr) * 2020-03-18 2021-09-23 Corticalis As Composition liquide améliorée pour le nettoyage, l'assainissement et/ou la désinfection
WO2021202823A1 (fr) * 2020-04-02 2021-10-07 Mollick Peter J Traitement thérapeutique pour la maladie de coronavirus covid-19
GB2595427A (en) * 2020-04-02 2021-11-24 J Mollick Peter Therapeutic treatment for the coronavirus disease COVID-19
US11865154B2 (en) 2020-04-02 2024-01-09 Peter Joseph Mollick Therapeutic treatment for the coronavirus disease COVID-19
WO2022032113A1 (fr) * 2020-08-07 2022-02-10 The University Of Chicago Produits pharmaceutiques pour le traitement ou la prévention de nidovirus et de picornavirus
US20220061315A1 (en) * 2020-08-26 2022-03-03 Simply Pure, LLC Persistent sanitizer

Similar Documents

Publication Publication Date Title
AU2002350587B2 (en) Antimicrobial nanoemulsion compositions and methods
US6559189B2 (en) Non-toxic antimicrobial compositions and methods of use
US8771731B2 (en) Antimicrobial nanoemulsion compositions and methods
US8232320B2 (en) Antimicrobial nanoemulsion compositions and methods
EP2364709B1 (fr) Composition antimicrobielle et procédé d'utilisation
US7767216B2 (en) Antimicrobial compositions and methods of use
AU2002350587A1 (en) Antimicrobial nanoemulsion compositions and methods
AU2004273779B2 (en) Compositions for inactivating pathogenic microorganisms, methods of making the compositions, and methods of use thereof
EP1528937B1 (fr) Vaccins sous forme de nanoemulsion
US20050208083A1 (en) Compositions for inactivating pathogenic microorganisms, methods of making the compositons, and methods of use thereof
Pavoni et al. Microemulsions: An effective encapsulation tool to enhance the antimicrobial activity of selected EOs
WO2005030172A1 (fr) Compositions antimicrobiennes en nanoemulsion et methodes associees
JP2010209109A (ja) 抗微生物ナノエマルジョンの組成物および方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase