Field of the Invention

The present invention broadly relates to emulsion compositions comprising allylisothiocyanate and the use thereof.

Background of the Invention

Allylisothiocyanate is a naturally occurring compound known to have antimicrobial properties. However, the use of allylisothiocyanate in antimicrobial applications is severely limited by its instability in aqueous compositions.

Against this background the present inventors have developed aqueous emulsion compositions comprising allylisothiocyanate which possess improved stability and potent fungicidal activity.

Summary of the Invention

In a first aspect the present invention provides a stable emulsion composition comprising:

(i) water;

(ii) allylisothiocyanate (AITC);

(iii) one or more surfactants; and

(iv) an oil other than AITC.

The emulsion composition may be a liquid emulsion.

The emulsion composition may be an oil-in-water emulsion.

The emulsion composition may be a nanoemulsion.

The one or more surfactants may have a HLB between about 8 and 16. In one embodiment the one or more surfactants have a HLB of about 12.

The one or more surfactants may be non-ionic surfactants.

The emulsion composition may comprise between about 0.1% and about 5% by weight of AITC.

The emulsion composition may comprise between about 55% and about 85% by weight of water.

The emulsion composition may comprise between about 1 % and about 15% by weight of the one or more surfactants.

The oil other than AITC may be a plant-based oil. The emulsion composition may comprise between about 10% and about 30% by weight of the plant-based oil.

The plant-based oil may be obtained from a plant of the genus Angophora, Eucalyptus, Corymbia, Melaleuca, Pinus or Citrus. In one embodiment the plant-based oil is obtained from Corymbia citriodora, Eucalyptus globulus, Melaleuca alternifolia, Pinus sylvestris or Citrus limon.

The plant-based oil may be an essential oil obtained from Corymbia citriodora, Eucalyptus globulus, Melaleuca alternifolia, Pinus sylvestris or Citrus limon. In one embodiment the plant-based oil is an essential oil obtained from Corymbia citriodora.

In certain examples, the composition comprises more than one plant-based oil other than AITC. The more than one plant-based oil may comprise an essential oil obtained from a species of Jasminum, Gardenia or Cymbopogon.

The emulsion composition may be free, or substantially free, of mineral oil.

In a second aspect the present invention provides a pharmaceutical composition comprising the emulsion composition of the first aspect and one or more pharmaceutically acceptable excipients.

The composition may be adapted for topical administration.

In a third aspect the present invention provides a method for treating a fungal infection in a subject in need thereof, the method comprising administration to the subject of a therapeutically effective amount of the emulsion composition of the first aspect, or the pharmaceutical composition of the second aspect.

In a fourth aspect the present invention provides use of the emulsion composition of the first aspect in the manufacture of a medicament for treating a fungal infection.

The fungal infection may be caused by a fungus from the genus Trichophyton.

The fungal infection may be athlete’s foot.

The fungal infection may be onychomycosis.

The administration may be topical administration, for example at the site of the fungal infection.

In a fifth aspect, the present invention provides a method of repelling an insect or a pest from a subject, the method comprising administering to the subject a therapeutically effective amount of the emulsion composition of the first aspect, or the pharmaceutical composition of the second aspect.

In certain examples, the insect is a mosquito or a fly. In certain examples, the pest is a tick or a leech.

In a sixth aspect, the present invention provides a use of the emulsion composition of the first aspect, or the pharmaceutical composition of the second aspect in the manufacture of a medicament for repelling an insect or a pest from a subject.

In a seventh aspect, the present invention provides a method of removing mold from a surface the method comprising contacting the mold with the emulsion composition of the first aspect.

Definitions

The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely".

In the context of this specification the term "oil-in-water emulsion" refers to a colloidal dispersion system in which liquid oil is dispersed in small droplets in an aqueous medium.

In the context of this specification the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

In the context of this specification the terms "a" and "an" are used herein to refer to one or to more than one (i.e to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

In the context of the present specification, the term "substantially free" is understood to mean less than about 0.01 %, or less than about 0.005%, or less than about 0.001 %, or less than about 0.0001% of the recited component by weight in the composition.

In the context of this specification, the term "therapeutically effective amount" includes a non-toxic but sufficient amount of the composition to which it refers to provide the desired therapeutic effect. Those skilled in the art will appreciate that the exact amount of a composition required will vary based on a number of factors and thus it is not possible to specify an exact "therapeutically effective amount". However, for any given case an appropriate "therapeutically effective amount" may be determined by one of ordinary skill in the art by routine trial and experimentation.

In the context of this specification, the terms "treating" and "treatment" refer to any and all uses which remedy, hinder, retard or reverse the progression of a fungal infection or symptoms thereof. Thus, the terms "treating" and "treatment" are to be considered in their broadest context. For example, treatment does not necessarily imply that a subject is treated until total recovery.

In the context of this specification, the term "subject" includes human and also non human animals. Preferably, the subject is a human.

Detailed Description of the Invention

The present inventors have successfully developed aqueous emulsion compositions comprising allylisothiocyanate which possess improved stability and potent fungicidal activity. In one aspect the present invention relates to a stable emulsion composition comprising:

(i) water;

(ii) allylisothiocyanate (AITC);

(iii) one or more surfactants; and

(iv) an oil other than AITC.

Typically, the emulsion compositions are liquid emulsions.

The composition may be stable for at least three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months or twenty-four months. In alternative embodiments the composition may be stable for a period beyond three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months or twenty-four months. In further embodiments the composition may be stable for a period of up to three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months or twenty-four months.

The composition may be stable for the above noted periods when stored at a temperature of about 5 °C.

Emulsion compositions in accordance with the present invention may be microemulsions or nanoemulsions. Microemulsions generally comprise droplets having a diameter in the range of about 1000 nm to about 500 mGh. Nanoemulsions comprise smaller droplets with a diameter of less than about 1000 nm. In some embodiments the nanoemulsions of the present invention may comprise droplets having an average diameter of about 1000 nm or less, about 950 nm or less, about 900 nm or less, about 850 nm or less, about 800 nm or less, about 750 nm or less, about 700 nm or less, about 650 nm or less, about 600 nm or less, about 550 nm or less, about 500 nm or less, about 450 nm or less, about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 200 nm or less, about 150 nm or less, about 100 nm or less, or about 50 nm or less.

Surfactants suitable for use in the compositions include anionic, cationic, amphoteric and non-ionic surfactants. Surfactants are well known to those skilled in the art. Non limiting examples of anionic surfactants include sulfate esters, sulfonate esters, phosphate esters and alkyl carboxylates. Non-limiting examples of cationic surfactants include quaternary ammonium compounds, such as cetyl trimethylammonium bromide, cetylpyridinium chloride, benzethonium chloride and dioctadecyldimethylammonium bromide. Non-limiting examples of amphoteric surfactants include betaines, imino acetates and imino propionates. Non-limiting examples of non-ionic surfactants include fatty alcohols, glucosides, maltosides, polyethoxylated tallow amines, cocoamides, sorbitan alkyl esters, block copolymers of polyethylene glycol and polypropylene glycol, ethoxylated amines and ethoxylated alcohols. In one embodiment the surfactant is a polysorbate (i.e., an ethoxylated sorbitan esterified with one or more fatty acids). In another embodiment the surfactant is a mixture of polysorbates, such as for example, a mixture of polysorbate 20 and polysorbate 80. Polysorbates are commercially available under the trade name Tween®.

The surfactant, or surfactant mixture, may have a hydrophilic-lipophilic balance (HLB) between about 10 and about 16, or between about 1 1 and about 15, or between about 11 and about 14, or between about 11 and about 13, or about 12.

The surfactant, or surfactant mixture may be present in the composition in an amount between about 1 % and about 15% by weight, or in an amount between about 1 % and about 10% by weight, or in an amount between about 1 % and about 8% by weight, or in an amount between about 3% and about 8% by weight, or in an amount of about 6% by weight.

AITC for use in the compositions of the invention may be obtained from the seeds of Brassica nigra or Brassica juncea by, for example, steam distillation. Alternatively, AITC may be synthesised as shown below: AITC may also be purchased commercially from Sigma-Aldrich (St Louis, MO, USA).

AITC may be present in the compositions in an amount between about 0.01% and about 10% by weight, or in an amount between about 0.1 % and about 10% by weight, or in an amount between about 0.5% and about 5% by weight, or in an amount between about 0.5% and about 3% by weight, or in an amount between about 0.5% and about 2% by weight, or in an amount of about 1% by weight.

The oil other than AITC may be a minteral oil such as paraffin oil. In certain examples, the oil other than AITC is a plant-based oil.

In some embodiments the plant-based oil is obtained from a plant of the genus Angophora, Eucalyptus, Corymbia, Melaleuca, Pinus or Citrus.

Angophora are trees and shrubs native to Australia, most of which have rough bark. Exemplary species include: Angophora bakeri, Angophora costata, Angophora euryphylla, Angophora crassifolia, Angophor exul, Angophora floribunda, Angophora hispida, Angophora inopina, Angophora leiocarpa, Angophora melanoxylon, Angophora paludosa, Angophora robur, Angophora subvelutina and Angophora woodsiana.

Eucalyptus is a genus of flowering trees and shrubs. There are more than 700 species of Eucalyptus and most are native to Australia. Exemplary Eucalyptus species include: Eucalyptus absita, Eucalyptus acies, Eucalyptus aenea, Eucalyptus ammophila, Eucalyptus ancophila, Eucalyptus apiculata, Eucalyptus coolabah, Eucalyptus crenulata, Eucalyptus cupularis, Eucalyptus cyanoclada, Eucalyptus darwinensis, Eucalyptus globulus Eucalyptus halophila, Eucalyptus incerata, Eucalyptus odorata, Eucalyptus oxymitra, Eucalyptus seeana, Eucalyptus sparsa and Eucalyptus staeri.

Corymbia is a genus comprising about 113 tree species that were classified as Eucalyptus species until the mid 1990s. Exemplary Corymbia species include: Corymbia aparrerinja, Corymbia aspera, Corymbia calophylla, Corymbia citriodora, Corymbia dichromophloia, Corymbia exemia, Corymbia ficifolia, Corymbia gummifera, Corymbia henryi, Corymbia maculate, Corymbia opaca, Corymbia ptychocarpa, Corymbia variegata and Corymbia Watsoniana.

The Angophora, Eucalyptus and Corymbia genera are closely related and are often referred to as "eucalypts".

Melaleuca is a genus comprising about 300 species of plants ranging from shrubs to tall trees. Most species of Melaleuca are endemic to Australia. Exemplary Melaleuca species include: Melaleuca acacioides, Melaleuca acuminata, Melaleuca alsophila, Melaleuca alternifolia, Melaleuca armillaris, Melaleuca bracteata, Melaleuca micromera, Melaleuca nanophylla, Melaleuca pearsonii, Melaleuca parviceps, Melaleuca orophila, Melaleuca pulchella and Melaleuca radula.

Pinus is a genus of approximately 120 extant tree and shrub species. Exemplary Pinus species include: Pinus brutia, Pinus pinaster, Pinus pinea, Pinus cubensis, Pinus glabra, Pinus jaliscana, Pinus Lawsonii, Pinus roxburghii, Pinus Pringiei, Pinus radiate, Pinus teocote, Pinus syivestris, Pinus cooperi, Pinus coulteri, Pinus strobus and Pinus cembra.

Citrus is a genus of flowering trees and shrubs that produce citrus fruits. Exemplary Citrus species include: Citrus maxima, Citrus Japonica, Citrus medica, Citrus micrantha, Citrus reticulate, Citrus x limon and Citrus x paradise and Citrus x sinensis.

The plant-based oil may be obtained from any part of the plant, for example bark, leaves, seeds, roots, stems, fruit or flowers.

In some embodiments the oil is an essential oil obtained from a plant of the genus Angophora, Eucalyptus, Corymbia, Melaleuca, Pinus or Citrus. Essential oils are concentrated hydrophobic liquids having the characteristic odour of the plant or other source from which they are extracted.

Essential oils can be obtained by methods known to those skilled in the art, for example distillation (e.g. steam distillation), pressing and solvent extraction. Essential oils may also be obtained from commercial sources.

The plant-based oil other than AITC may be present in the compositions in an amount between about 10% and about 40% by weight, or in an amount between about 10% and about 35% by weight, or in an amount between about 10% and about 30% by weight, or in an amount between about 15% and about 30% by weight, or in an amount between about 15% and about 25% by weight, or in an amount of about 19% by weight.

In some embodiments, the oil other than AITC comprises a mineral and a plant-based oil.

In some embodiments, the composition comprises more than one plant-based oil other than AITC. The more than one plant-based oil may include a plant-based oil that provides a desirable aroma to the composition or comprises an adjuvant. The more than one plant-based oil may comprise, for example, an essential oil obtained from Jasmine ( Jasminum spp ), Gardenia ( Gardenia spp.), lemon grass ( Cymbopogon spp.), tea tree (eg, Camellia spp., Melaleuca spp., Leptospermum spp., Kunzea spp., Taxandria spp. or Lycium spp.), citronella (eg, Citronella spp., Pelargonium spp. or Cymbopogon spp.) and/or any one or more of the plant-based oils described elsewhere herein.

Water may be present in the compositions in an amount between about 50% and about 90% by weight, or in an amount between about 55% and about 85% by weight, or in an amount between about 60% and about 80% by weight, or in an amount between about 65% and about 80% by weight, or in an amount between about 70% and about 80% by weight, or in an amount of about 74% by weight.

In one embodiment the emulsion composition comprises:

(i) water in an amount between about 60% and 80% by weight;

(ii) AITC in an amount between about 0.1 % and 5% by weight;

(iii) one or more surfactants in an amount between about 3% and 10% by weight; and

(iv) an oil obtained from a plant of the genus Angophora, Eucalyptus, Corymbia, Melaleuca, Pinus or Citrus in an amount between about 10% and 30% by weight.

In another embodiment the emulsion composition comprises:

(i) water in an amount between about 70% and 80% by weight;

(ii) AITC in an amount between about 0.5% and 2% by weight;

(iii) one or more surfactants in an amount between about 3% and 8% by weight; and

(iv) an oil obtained from a plant of the genus Angophora, Eucalyptus, Corymbia,

Melaleuca, Pinus or Citrus in an amount between about 15% and 25% by weight.

In a further embodiment the emulsion composition comprises:

(i) water in an amount between about 60% and 80% by weight;

(ii) AITC in an amount between about 0.1 % and 5% by weight;

(iii) one or more surfactants in an amount between about 3% and 10% by weight; and

(iv) an oil obtained from a plant of the genus Corymbia in an amount between about 10% and 30% by weight.

In another embodiment the emulsion composition comprises:

(i) water in an amount between about 70% and 80% by weight;

(ii) AITC in an amount between about 0.5% and 2% by weight;

(iii) one or more surfactants in an amount between about 3% and 8% by weight; and (iv) an oil obtained from a plant of the genus Corymbia in an amount between about 15% and 25% by weight.

In another embodiment the emulsion composition comprises;

(i) water in an amount between about 60% and 80% by weight;

(ii) AITC in an amount between about 0.1% and 5% by weight;

(iii) one or more surfactants in an amount between about 3% and 10% by weight;

(iv) an oil obtained from a plant of the genus Angophora, Eucalyptus, Corymbia, Melaleuca, Pinus or Citrus in an amount between about 10% and 30% by weight; and

(v) an oil obtained from a plant of the genus Jasminum, Gardenia or Cymbopogon in an amount between about 0.1 % and 5% by weight.

In another embodiment the emulsion composition comprises:

(i) water in an amount between about 70% and 80% by weight;

(ii) AITC in an amount between about 0.5% and 2% by weight;

(iii) one or more surfactants in an amount between about 3% and 8% by weight;

(iv) an oil obtained from a plant of the genus Corymbia in an amount between about 10% and 25% by weight; and

(v) an oil obtained from a plant of the genus Jasminum, Gardenia or Cymbopogon in an amount between about 0.1 % and 1% by weight.

In one embodiment the emulsion composition comprises:

(i) water in an amount between about 60% and 80% by weight;

(ii) AITC in an amount between about 0.1% and 5% by weight;

(iii) one or more surfactants in an amount between about 3% and 10% by weight; and

(iv) an oil other than AITC in an amount between about 10% and 30% by weight.

In one embodiment the emulsion composition comprises:

(i) water in an amount between about 60% and 80% by weight;

(ii) AITC in an amount between about 0.1 % and 5% by weight;

(iii) one or more surfactants in an amount between about 3% and 10% by weight; and

(iv) a mineral oil in an amount between about 10% and 30% by weight. In one embodiment the emulsion composition comprises:

(i) water in an amount between about 60% and 80% by weight;

(ii) AITC in an amount between about 0.1 % and 5% by weight;

(iii) one or more surfactants in an amount between about 3% and 10% by weight; and

(iv) paraffin oil in an amount between about 10% and 30% by weight.

In the above embodiments the one or more surfactants may have a HLB between about 10 and about 16, or between about 10 and about 14, or about 12. In the above embodiments the emulsions may be oil-in-water emulsions. In the above embodiments the emulsions may be nanoemulsions. In the above embodiments the oil may be an essential oil.

A variety of different methods may be used to prepare the emulsion compositions of the present invention. For example, high energy methods such as ultrasonication and high pressure homogenisation may be used to generate micro- or nano-sized droplets for the purposes of producing a microemulsion or a nanoemulsion. Lower energy methods such as phase inversion temperature, solvent displacement and emulsion inversion point (EIP) may also be employed. An example of the EIP method involves the gradual addition of an aqueous phase to a water/oil (or oil/water) microemulsion, thereby causing the microemulsion to disintegrate into smaller, nano-sized droplets. Other suitable approaches for producing microemulsions and nanoemulsions in accordance with the present invention include the bubble bursting (Feng et at. Nat. Phys., 2014, 10, 606-612) and evaporative ripening (Fryd and Mason, J. Phys. Chem. Lett., 2010, 1 , 3349-3353) methods. Those skilled in the art will be aware of other methods that may be used to produce emulsions in accordance with the present invention.

In another aspect the present invention relates to pharmaceutical compositions comprising the emulsion composition of the first aspect and one or more pharmaceutically acceptable additives. The pharmaceutical composition may be formulated in a variety of pharmaceutically acceptable forms known by those skilled in the art, such as for example, a cream, an ointment, a gel, a spray, a paste, an aerosol, a lotion, a powder, a wet wipe, a slow-release vaporiser or a foam.

The compositions of the invention may possess potent fungicidal activity and therefore find application in methods for the treatment of fungal infections. Fungal infections that may be treated include, but are not limited to, tinea and other infections of the skin or nail. Genera of fungi or yeast that may be associated with a skin or nail infection include Microsporum, Epidermophyton, Trichophyton, Candida, Aspergillus, Fusarium, Phoma, Scopulariopsis, Scytalidium, Alternaria, Epicoccum, Curvularia, Acremonium, Chaetomium and Paeciiomyces. Examples of species within the Microsporum genus include Microsporum canis, Microsporum audouinii, Microsporum gallinae, Microsporum ferrugineum, Microsporum distortum, Microsporum nanum, Microsporum cookie, Microsporum vanbreuseghemii and Microsporum gypseum. Examples of species within the Trichophyton genus include Trichophyton rubrum, Trichophyton ajelioi, Trichophyton concentricum, Trichophyton equinum, Trichophyton erinacei, Trichophyton fiavescens, Trichophyton gioriae, Trichophyton interdigitale, Trichophyton megini, Trichophyton mentagrophytes, Trichophyton phaseoiiforme, Trichophyton schoenleini, Trichophyton simii, Trichophyton Soudanese, Trichophyton terrestre, Trichophyton tonsurans, Trichophyton vanbreuseghemii, Trichophyton verrucosum, Trichophyton violaceum, Trichophyton yaoundei, Trichophyton tonsurans and Trichophyton mentagrophytes. Examples of species within the Epidermophyton genus include Epidermophyton floccosum and Epidermophyton stockdaleae. Yeasts of the Candida genus are also responsible for a variety of skin and nail infections, which are occasionally referred to as candidiasis. Examples of species within the Candida genus include Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Candida lusitaniae, Candida kefyr, Candida guiliiermondii, Candida dubliniensis and Candida krusei. Examples of species within the Aspergillus genus include Aspergillus terreus, Aspergillus sydowii, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus oryzae, Aspergillus ustus and Aspergillus versicolor. Examples of species within the Fusarium genus include Fusarium solani, Fusarium oxysporum and Fusarium semitectum. Examples of species within the Paeciiomyces genus include Paeciiomyces variotii and Paeciiomyces lilacinus. Examples of species within the Chaetomium genus include Chaetomium atrobrunneum, Chaetomium globosum, Chaetomium funicola and Chaetomium strumarium. Examples of species within the Scopulariopsis genus include Scopulariopsis Candida, Scopulariopsis koningii, Scopulariopsis brevicaulis, Scopulariopsis acremonium, Scopulariopsis fiava, Scopulariopsis cinerea, Scopulariopsis trigonospora, Scopulariopsis brumptii, Scopulariopsis chartarum, Scopulariopsis fusca and Scopulariopsis asperula. Examples of species within the Scytalidium genus include Scytalidium hyalinum, Scytalidium dimidiatum, Scytalidium infestans, Scytalidium japonicum and Scytalidium lignicola. Examples of species within the Alternaria genus include Alternaria teunissima, Alternaria stemphyloides, Alternaria infectoria, Alternaria geophilia, Alternaria dianthicola, Alternaria chartarum and Alternaria alternate. Examples of species within the Curvularia genus include Curvularia brachyspora, Curvularia clavata, Curvularia geniculata, Curvularia lunata, Curvularia pallescens, Curvularia senegalensis and Curvularia verruculosa.

Compositions of the invention may be used to treat fungal infections caused by, or otherwise associated with, one or more of the fungi recited above. In embodiments of the invention the fungal infection may be caused by a fungus from the genus Trichophyton. In other embodiments, the composition of the present invention is used to remove, or slow the growth of, mold such house or indoor mold (eg, Stachybotrys spp., Aspergillus spp. or Penicillium spp.).

In certain embodiments, the composition of the present invention is used to treat or prevent a bacterial infection or bacterial contamination. The bacterial infection or bacterial contamination may be caused by Staphylococcus aureus or Listeria monocytogenes.

In some embodiments, the composition of the present invention is used as a deodorizer. For example, the composition may be used to deodorize a shoe or a refrigerator.

In some embodiments, the composition of the present invention is used to repel an insect. Insects that may be repelled by the composition of the present disclosure include mosquitos (eg, Aedes aegypti), flies such as vinegar flies (eg, Drosophila melanogaster), garden pests such as aphids, moths (eg, codling moths) and citrus leaf miners, silverfish, hair lice (eg, Pediculus humanus capitis), weevils such as flour weevils and moths such as dried fruit moth and clothes moth. The composition of the present invention may be used to repel pests such as ticks (eg, Ixodes holocyclus) and leeches (eg, Hirudinea spp.). In certain embodiments, the composition of the present invention is used to repel an animal such as a cat, a dog or a possum.

Examples

The invention will now be described in more detail, by way of illustration only, with respect to the following examples. The examples are intended to serve to illustrate this invention and should not be construed as limiting the generality of the disclosure of the description throughout this specification.

Example 1 - Preparation of a nanoemulsion composition

An exemplary nanoemulsion was prepared according to the following procedure:

1. A surfactant combination having a weight of 6.0 g (comprising 42.4% w/w polysorbate 80 and 57.6% polysorbate 20) was prepared having a HLB of 12.0. 2. The mixture from step 1 was added to a mixture of 1.0 g of AITC and 19.0 g of essential oil obtained from Corymbia citriodora.

3. The mixture obtained from step 2 was placed in a conical flask on a heated magnetic stirrer held at 30 °C.

4. Deionised water (74.0 g) was then added to the conical flask at a rate of 0.5 mL/minute with constant stirring.

5. The resultant nanoemulsion was transferred to an aluminium canister which was sealed and subsequently stored at 5 °C until use.

Example 2 - Antifungal testing

Objective

The objective of this study was to determine the antifungal activity of two compositions against clinical dermatophyte strains by minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Terbinafine was used as a comparator.

Materials and methods

Five recent clinical strains each of Trichophyton rubrum and T. mentagrophytes (the major cause of onychomycosis) were tested, along with the QC strains T. rubrum ATCC MYA-4438 and T. mentagrophytes ATCC MYA-4439 as controls.

Composition B is the nanoemulsion composition prepared in Example 1 above. Composition A is identical to composition B, except that in composition A the essential oil obtained from Corymbia citriodora is replaced by pharmaceutical grade mineral oil.

Minimum Inhibitory Concentration (MIC): MIC testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 methodology ¹ . Briefly, RPMI 1640 was the test medium, the incubation temperature and time were 35 °C and 4 days, respectively, and the inoculum size was 1 to 3x10 ³ conidia/ml. The MIC inhibition endpoint was 80% as compared to the growth control.

Minimum Fungicidal Concentration (MFC): MFC testing was performed to determine whether the composition is fungicidal. Briefly, MFC determinations were performed according to the method previously described by Ghannoum and Isham ² . Specifically, the total contents of each clear well from a microdilution plate where organisms were exposed to different concentrations of drug was subcultured on potato dextrose agar. To avoid antifungal carryover the aliquots were allowed to soak into the agar and then streaked for isolation once dry, thus removing the cells from the drug source. Agar plates were incubated at 35 °C for 48 hours and the number of colony forming units (CFUs) determined. Cidal activity was defined as a >99.9% reduction in the number of CFUs/ml from the starting inoculum count. Static activity was defined as < 99.9% reduction.

Results

Initial MIC testing of these compounds resulted in inconsistent fungal growth in the microtiter plate wells. It was determined that this was due to fumes emanating from the compositions effecting fungal growth in adjacent wells, including growth control wells. The experiment was modified using an adhesive covering on the microtiter plate to prevent fumes from affecting growth in adjacent wells. This modification allowed determination of the efficacy of the test compositions against dermatophytes.

Table 1 below shows the MIC and MFC endpoints for the compositions tested. The endpoints for compositions A and B are given as a percentage of the original composition, while the endpoints for the comparator (terbinafine) are given as pg/ml. The MIC range for Composition A against the Trichophyton isolates tested was 0.4% to 1.6% and the MFC range was 3.1 % to >3.1 %. The MFCs for composition A were more than 4 wells greater than the MIC, which indicates a fungistatic effect.

The MIC range for Composition B against the Trichophyton isolates tested was 0.1 % to 0.4% and the MFC range was 0.2 to 0.4%. Interestingly, the MFCs for Composition B were less than 4 wells different than the MIC, which indicates that the composition possesses fungicidal activity. As expected, the MIC range for terbinafine was 0.002 to 0.008 pg/ml and the MFC range was 0.008 - 0.031 pg/ml.

Table 1 - MIC and MFC values for the test compounds. Results are given as a percent of the original formulation

^* Due to complications during initial testing these strains could not be evaluated.

Conclusion

The data shows that test composition B possesses potent fungicidal activity against the T. rubrum and T. mentagrophytes isolates tested. References

1. CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard- Second Edition. CLSI document M38-A2 [ISBN 1 -56238-668-9] CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898 USA, 2008.

2. Ghannoum M.A., Isham N. 2007. Voriconazole and Caspofungin Cidality Against Non -albicans Candida Species. Infectious Diseases in Clinical Practice. 15(4):250-253.

Example 3 - Antileech testing

An adult leech was placed onto the abdomen of a male subject, allowed to penetrate the subject's skin and to commence feeding. The leech was then sprayed with an emulsion composition comprising the following components:

(i) water in an amount of 74% by weight;

(ii) AITC in an amount of 0.5% by weight;

(iii) surfactants in an amount of 6% by weight (Tween 80 [HLB 16.7] and Span 80 [HLB 4.3] mixed to achieve a HLB of 12); and

(iv) paraffin oil in an amount of 19.5% by weight.

Other oils, including plant-based oils such as mustard oil (mainly linolenic) may be used instead of paraffin oil.

Within 20 seconds of spraying, the leech ceased feeding. Within 30 seconds of spraying, the leech detached and fell from the subject's abdomen.

Example 4 Antifungal testing

The following four compositions, labelled A, B, C and D, were tested for their antifungal activity against clinical dermatophyte strains by MIC and MFC.

Composition A: 5% w/w AITC in mustard oil (5 g of allyly isothiocyanate in 95 g of refined mustard oil derived from Brassica juncea)

Composition B: 5% w/w AITC in tea tree oil 1 % (5 g of ally! isothiocyanate in 94 g of refined mustard oil derived from Brassica juncea and 1.0g of oil derived principally from Melaleuca aiternifolia by steam distillation of leaves and wood and largely comprised of terpinen-4-ol)

Composition C: Neat tea tree oil (Oil derived from Melaleuca aiternifolia by steam distillation of leaves and wood and largely comprised of terpinen-4-ol)

Composition D: 1 % AITC nanoemulsion comprising:

(i) water in an amount of 74% by weight; (ii) AITC in an amount of 1 % by weight;

(iii) surfactants in an amount of 6% by weight (Tween 80 [HLB 16.7] and Span 80 [HLB 4.3] mixed to achieve a HLB of 12); and

(iv) paraffin oil in an amount of 19% by weight.

Other oils, including plant-based oils such as mustard oil (mainly linolenic) may be used instead of paraffin oil.

The results are summarized in Table 2.

Table 2 - MIC and MFC values for compositions A, B, C and D against dermatophytes tested (%)

Example 5 Antiinsect testing

An experimental insect repellent comprising the following components was tested for its ability to deter dengue mosquito:

(i) water in an amount of 74% by weight;

(ii) AITC in an amount of 1% by weight;

(iii) surfactants in an amount of 6% by weight (Tween 80 [HLB 16.7] and Span 80 [HLB 4.3] mixed to achieve a HLB of 12); and

(iv) paraffin oil in an amount of 19% by weight.

Other oils, including plant-based oils such as mustard oil (mainly linolenic) may be used instead of paraffin oil.

Mosquito activity was measured as the number of bite attempts per second on judges' arms over the trial period. Measurements were taken at Bundaberg, Queensland and the mean number of bite attempts per second is indicated in Table 3.

Table 3 - Antimosquito results

In a further study, the experimental insect repellent was diluted with paraffin to produce four Exerimental Repellents as set forth in Table 4. The concentration of the intial repellent in each of the four Experimental Repellents is indicated as a percentage.

Table 4 - Experimental Repellents

The four compositions were tested for their ability to repel Dengue mosquito ( Aedes aegypti) and vinegar fly ( Drosophila melanogaster) as summarized in Table 5. Table 5 - Insect repellent experimental design

As shown in Tables 6 and 7, all of the compositions provided significant protection against dengue mosquitoes for at least 60 minutes after application of the composition when compared to untreated arms. All of the compositions provided a similar level of protection.

Table 6 - Mean number of mosquito bites per second on judges' arms

Means within the same column with a letter in common are not significantly different (P>0 05)

MAA = minutes after application

Table 7 - Mean mass of product applied to judges' arms

ns = not significant As shown in Tables 8 and 9, all of the compositions provided significant protection against vinegar fly for at least 60 minutes after application of the composition. All of the compositions provided a similar level of protection.

Table 8 - Vinegar fly landings on treated food sources in cages

ns = not significant

Table 9 - Vinegar fly landings on treated and untreated food sources over one hour

Means within the same column with a letter in common are not significantly different (P>0.05)

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

It will be appreciated by those skilled in the art that numerous variations and/or modifications may be made to the invention without departing from the spirit or scope of the invention as broadly described. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive.