Methods and compositions for the treatment of infections and arthropod infestation

Abstract

The invention relates to biting arthropod repellent compositions comprising nootkatone. The application also relates to a method of repelling said biting arthropods by application of the nootkatone composition to a hard or soft surface, to a method of treating an arthropod infestation and to a method of reducing the transmission of a parasite or disease pathogen spread by a biting arthropod using the nootkatone composition.

Claims

1. A method of repelling a biting arthropod, comprising: applying an effective amount of a nootkatone composition to a surface, wherein the composition comprises nootkatone ex valencene, wherein the nootkatone ex valencene is limonene-free and bergapten-free, and wherein the nootkatone composition has a reduced risk for causing skin irritation when applied topically compared to a nootkatone composition comprising plant-derived nootkatone.

2. The method of claim 1, wherein the nootkatone composition is applied to one or more of fur, hair, skin, hide, or scalp of an animal.

3. The method of claim 1, wherein the nootkatone composition is applied to one or more of untreated lumber, treated lumber, a wood beam, a wood board, cardboard, particle board, joist, a stud, a baseboard, wood trim, a hardwood floor, a window sill, a screen, a porch floor, a deck, a door, a wall, a ceiling, interior furniture, or exterior furniture.

4. The method of claim 1, wherein the nootkatone composition is applied to one or more of a carpet, a curtain, a rug, padded furniture, a cushion, a mattress, a box spring, a mattress cover, a bedbug repellent mattress pad, a bed sheet, a blanket, a pillow, a doll, a stuffed animal, an insect trap, or a net.

5. The method of claim 1, wherein the nootkatone composition is applied to a bracelet, a necklace, a bandage, a hat, a glove, pants, a shirt, underwear, a coat, a bandana, a scarf, a sock, a shoe, a shoelace, a footwear liner, a footwear insert, gaiters, hunting clothes, or military clothes.

6. A method of treating an arthropod infestation, comprising: applying an effective amount of a nootkatone composition comprising a carrier and at least about 0.5% nootkatone to a surface, wherein the nootkatone composition comprises nootkatone ex valencene, wherein the nootkatone ex valencene is limonene-free and bergapten-free, and wherein the nootkatone composition has a reduced risk for causing skin irritation when applied topically compared to a nootkatone composition comprising plant-derived nootkatone.

7. The method of claim 6, wherein the carrier is one or more of an aqueous liquid carrier, water, a gel, a powder, a zeolite, a cellulosic material, a microcapsule, an alcohol, a hydrocarbon, a polymer, a wax, a fat, or an oil.

8. The method of claim 7, wherein the nootkatone composition is formulated as a spray, a powder, a paint, a stain, a wax, a gel, or a paste.

9. The method of claim 6 further comprising: allowing the nootkatone composition to remain on the surface for at least about 1 hour; and removing the nootkatone composition from the surface.

10. The method of claim 9, wherein the nootkatone composition is removed by one or more of vacuuming, rinsing, steaming, washing, drying, or scrubbing.

11. The method of claim 9 further comprising reapplying the nootkatone composition to the surface.

12. The method of claim 9 further comprising agitating the surface to which the nootkatone composition is being applied to clean the surface at the same time as the composition is being applied and/or facilitate penetration of the composition into the surface.

13. The method of claim 12, wherein the agitating is accomplished by sweeping, vacuuming, or scrubbing.

14. The method of claim 1, wherein the biting arthropod is one or more of a cat flea, a bed bug, a termite, a fire ant, a centipede, a chigger, a drain fly, a biting fly, a black fly, a deer fly, a sand fly, a horse fly, a stable fly, a kissing bug, an earwig, a biting gnat, a louse, a millipede, a dust mite, a scabies mite, or a mosquito.

15. A method of reducing the transmission of a parasite or disease pathogen spread by a biting arthropod, by application of a nootkatone composition comprising an effective amount of nootkatone ex valencene to an animal capable of acting as a host or being infected by the parasite or disease pathogen, wherein the nootkatone ex valencene is limonene-free and bergapten-free, and wherein the nootkatone composition has a reduced risk for causing skin irritation when applied topically compared to a nootkatone composition comprising plant-derived nootkatone.

16. The method of claim 15, wherein the nootkatone ex valencene is applied to one or more of fur, hair, skin, hide, or scalp of an animal capable of acting as a host or being infected by the parasite or disease pathogen.

17. The method of claim 15, wherein the disease pathogen is an arbovirus that causes or is associated with dengue fever, chikungunya, yellow fever, or Zika virus infection.

18. The method of claim 1, wherein the nootkatone composition is limonene-free and bergapten-free.

19. The method of claim 6, wherein the nootkatone composition is limonene-free and bergapten-free.

20. The method of claim 15, wherein the nootkatone composition is limonene-free and bergapten-free.

21. The method of claim 20, wherein the biting arthropod is a mosquito.

22. The method of claim 1, wherein the biting arthropod is a mosquito.

23. The method of claim 1, wherein the nootkatone composition is applied to the skin of a human.

24. The method of claim 23, wherein the biting arthropod is a mosquito.

25. The method of claim 6, wherein the biting arthropod is a cat flea, a bed bug, a termite, or a deer tick.

26. The method of claim 25, wherein the biting arthropod is a termite.

27. The method of claim 15, wherein the biting arthropod is a mosquito.

28. The method of claim 18, wherein the biting arthropod is one or more of a cat flea, a bed bug, a termite, a fire ant, a centipede, a chigger, a drain fly, a biting fly, a black fly, a deer fly, a sand fly, a horse fly, a stable fly, a kissing bug, an earwig, a biting gnat, a louse, a millipede, a dust mite, a scabies mite, or a mosquito.

29. The method of claim 28, wherein the biting arthropod is a mosquito.

30. The method of claim 18, wherein the nootkatone composition is applied to the skin of a human.

31. The method of claim 30, wherein the biting arthropod is a mosquito.

32. The method of claim 19, wherein the biting arthropod is a cat flea, a bed bug, a termite, or a deer tick.

33. The method of claim 32, wherein the biting arthropod is a termite.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings:

(2) FIG. 1 illustrates a biosynthetic pathway for producing nootkatone.

(3) FIG. 2 shows the average repellency of 1% nootkatone compositions against Ae. aegypti mosquitoes compared to untreated, ethanol only, and 20% DEET treated mosquitoes. The 1% nootkatone compositions (both ethanol and acetone based) demonstrated repellency similar to that of 20% DEET.

(4) FIG. 3 shows the average percent of Ae. aegypti mosquitoes affected upon continuous exposure to 1% nootkatone compositions for 30 min, 1, 4, and 24 hr compared to untreated mosquitoes. By 4 hours, over 90% of the mosquitoes were affected. All mosquitoes were affected after 24 hr.

(5) FIG. 4 shows the average percent of Ae. aegypti mosquitoes killed upon continuous exposure to 1% nootkatone compositions for 30 min, 1, 4, and 24 hr compared to untreated mosquitoes. Mosquito killing began by 4 hr and was complete by 24 hr.

(6) FIG. 5 shows the average percent of Ae. aegypti mosquitoes knocked down upon continuous exposure to 1% nootkatone compositions for 30 min, 1, 4, and 24 hr compared to untreated mosquitoes. Mosquito knockdown began by 30 min and was complete before 24 hr.

(7) FIG. 6 is GC-FID chromatogram overlay of Frutarom® nootkatone (i.e., citrus-derived nootkatone) and the nootkatone (N×V) used for the arthropod studies described herein (see Examples below).

(8) FIG. 7 is a GC-MS NIST library match of an unknown peak in Frutarom® nootkatone. The peak was identified as limonene. No limonene nor bergapten was found in the nootkatone used in the present application.

DETAILED DESCRIPTION OF THE INVENTION

(9) All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

(10) Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to a “nucleic acid” means one or more nucleic acids.

(11) It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

(12) For the purposes of describing and defining the present invention, it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

(13) As used herein, the term “vector” refers to an arthropod carrying an arbovirus.

(14) As used herein, the terms “or” and “and/or” is utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.”.

(15) As used herein, the term “about” refers to ±10% of a given value.

(16) As used herein, the term “nootkatone” refers to a compound seen in FIG. 1 that may be synthesized, isolated, and purified from of a mixture of products produced in a host modified to express enzymes of the nootkatone biosynthetic pathway or that can be produced from naturally occurring sources, such as citrus plants. “Nootkatone” also refers to a mixture of chemical compounds containing or enriched for the nootkatone compound and derived from a modified host, such as a microorganism, or isolated or derived from plant extracts. “Nootkatone” further refers to derivatives and analogs thereof. For example, the nootkatone compound contemplated for use herein may be produced in vivo through expression of one or more enzymes involved in the nootkatone biosynthetic pathway in a recombinant yeast or in vitro using isolated, purified enzymes involved in the nootkatone biosynthetic pathway, such as those described in U.S. Patent Application Publication Nos. 2015/0007368 and 2012/0246767. Therefore, nootkatone as defined herein can differ chemically from other sources of nootkatone, such as extracts from plants and derivatives thereof, or may include such plant extracts and derivatives thereof.

(17) As used herein, the term “valencene derivative” includes nootkatone and other valencene derived compounds, such as valencene-13-ol.

(18) In one embodiment, nootkatone used herein does not contain bergapten (or bergaptine).

(19) As used herein, the term “nootkatone ex valencene” refers to nootkatone derived from oxidation of valencene that was produced by fermentation, such as by microorganisms harboring one or more valencene synthases and/or other molecules that catalyze formation of valencene. Further, nootkatone ex valencene refers to a combination of chemical compounds derived from oxidation of a valencene-containing fermentation product produced by culturing microorganisms harboring one or more valencene synthases and/or other molecules that catalyze formation of valencene. Nootkatone ex valencene can be purified to maximize the percent of nootkatone relative to other chemical compounds. For example, nootkatone ex valencene can be less than about 50%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 98% nootkatone.

(20) As used herein, the term “active ingredient” refers to a chemical compound or mixture of chemical compounds that kills and/or repels an insect or a pest, such as a tick and related species.

(21) As used herein, the term “individual” refers to a human. Further, the term “subject” refers to an animal, such as a mammal, bird, or reptile.

(22) As used herein, the term “effective amount” refers to an amount of an arthropod-repellent composition that achieves at least 50% repellency for a given arthropod.

(23) As used herein, the terms “therapeutic amount” or “therapeutically effective amount” can be used interchangeably and refer to an amount of a compound that becomes available through the appropriate route of administration to treat or prevent an arthropod from landing on or biting an individual.

(24) As used herein, “biting” refers to contact from an arthropod, such as by mouth parts, that breaks or pierces the skin of an animal and which can cause irritation, itching, pain, and/or swelling at the site of the bite wound. In some instances, biting serves to provide an arthropod with a blood meal, which can serve as sustenance for the arthropod and/or a prerequisite for reproduction, such as egg development. In other instances, biting can be defensive. Non-limiting examples of biting arthropods include cat fleas, bed bugs, termites, fire ants, centipedes, chiggers, drain flies, biting flies, black flies, deer flies, sand flies, horse flies, stable flies, kissing bugs, earwigs, biting gnats, lice, millipedes, mites, and mosquitoes, among others.

(25) Disclosed herein are compositions and methods for preventing vector-borne viral infections and include compositions capable of repelling and killing virus-carrying air-borne pests, such as mosquitoes.

(26) The compositions for killing or repelling virus-carrying air-borne pests and repelling arthropods provided herein can include a carrier and at least about 0.1%, or at least about 0.5%, or at least about 1%, or at least about 2%, or at least about 5%, or at least about 7.5%, or at least about 10%, or greater than about 10%, or greater than about 15%, or greater than about 20%, or greater than about 25%, or greater than about 50% by weight nootkatone or other valencene derivative. In some applications, a nootkatone or other valencene derivative can be present in an amount that is greater than about 60%, about 70%, about 80%, about 90%, about 95% or about 99% by weight of the composition. In one example, the provided compositions contain nootkatone or other valencene derivative in an amount at or about 0.01% to at or about 75% by weight of the composition. In another example, a composition may contain nootkatone or other valencene derivative in an amount of from at or about 1% to at or about 50% by weight of the composition. In another example, a composition may contain nootkatone or other valencene derivative in an amount of from at or about 5% to at or about 40% by weight of the composition. In another example, a composition may contain nootkatone or other valencene derivative in an amount of from at or about 10% to at or about 30% by weight of the composition. In another example, a composition may contain nootkatone or other valencene derivative in an amount of from at or about 15% to at or about 25% by weight of the composition. In another example, a composition may contain nootkatone or other valencene derivative in an amount of from at or about 1% to at or about 90% by weight of the composition. In another example, a composition may contain nootkatone or other valencene derivative in an amount of about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 50% by weight of the composition. In another example, a composition may contain nootkatone or other valencene derivative or a combination thereof in an amount of up to 99% by weight of the composition.

(27) In another embodiment, a contemplated formulation can be seen in Table No. 1.

(28) TABLE-US-00001 TABLE NO. 1 Contemplated formulation Ingredient Approximate Wt. % nootkatone or other valencene 0.01-75   derivative Additional active Ingredients 0-30 Carriers  25-99.9 Additives 0-50

(29) In certain embodiments, compositions contemplated herein may include nootkatone and one or more additional active ingredients, such as DEET, a pyrethroid, or any other synthetic or natural insecticide or pesticide or repellent. Further examples of additional active ingredients include, for example, those disclosed in U.S. Pat. Nos. 6,897,244, 7,129,271, 7,629,387, and 7,939,091. An additional active ingredient may also be added to a composition in an amount of about 1% to about 30%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 50% by weight of the composition.

(30) In another embodiment, compositions contemplated herein may include nootkatone or other valencene derivative in combination with one or more additives, such as a fragrance, a preservative, a propellant, a pH buffering agent, a colorant, a surfactant, an emulsifier, a solvent, a salt, an antibiotic, an analgesic, and the like. An additive may be added to a composition in an amount of greater than about 1% to about 50%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 50% by weight of the composition.

(31) In another embodiment, compositions contemplated herein may include nootkatone in combination with one or more synergists that increase one or more of repellency, knockdown, lethality, and/or longevity of effectiveness (residual activity). Examples of synergists include piperonyl butoxide, DMSO, and vanillin.

(32) In another embodiment, compositions may include a carrier, such as an aqueous liquid carrier, water, a gel, a powder, a zeolite, a cellulosic material, a microcapsule, an alcohol such as ethanol, a hydrocarbon, a polymer, a wax, a fat, and/or an oil, and the like. A carrier may be added to a composition in an amount of about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 50% by weight of the composition. In some applications, a carrier can be present in an amount that is at or greater than about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% by weight of the composition.

(33) In certain embodiments, a composition may be formulated for application topically on an exterior surface of an individual, for example, to the lips, skin, scalp or hair. For example, the composition may be provided as an aerosol, a solution, an emulsion, an oil, a lotion, a soap, a shampoo, a conditioner, a spray, a gel, a cosmetic, a perfume, or a cologne.

(34) In further embodiments, a composition may be formulated for application onto an exterior surface of an animal, such the fur, hair, skin, hide, and/or scalp of a human, a domesticated animal, livestock, or a pet.

(35) In other embodiments, a composition may be formulated for ingestion by an animal to convey insect or pest repellency through secretion of an active ingredient onto the skin, hair, fur, and the like. For example, the composition may be formulated as animal feed, such as an extruded animal food, a grain, a canned food, an animal treat, a dog bone, and the like. In still further embodiments, a composition may be formulated for ingestion such as may be added to a water reservoir to control mosquito larvae and oral/systemic formulations for small animals that serve as disease reservoirs or companion animals.

(36) Compositions containing nootkatone to treat or prevent arthropod infestations in humans (especially children), farm animals (such as sheep, pigs, horses, cows, camels), or companion animals (such as dogs and cats) can be formulated for ingestion as a food supplement, a drinking water supplement, a paste, gel, or syrup that is eaten, or a liquid drench to be given orally. Contemplated compositions can be made in many different forms including a granular form to be added to food, pill form, chew tablets, and liquid suspensions. In one example, the composition may be formulated as animal feed, such as an extruded animal food, a grain, a canned food, an animal treat, a dog bone, an additive to fish food, a coating on animal forage, and the like.

(37) Compositions containing nootkatone to treat or prevent arthropod infestations can be placed into the form of pharmaceutical dosage forms, such as solid oral forms such as tablets, capsules granules, pellets, or sachet, for example, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration, or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms thereof can comprise conventional ingredients in conventional proportions, with or without additional active compounds, and such unit dosage forms can contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.

(38) Compositions containing nootkatone to treat or prevent arthropod infestations can also contain one or more inactive pharmaceutical excipients such as diluents, lubricants, solubilizers, alcohols, binders, controlled release polymers, enteric polymers, disintegrants, colorants, flavorants, sweeteners, antioxidants, preservatives, pigments, additives, fillers, suspension agents, surfactants (for example, anionic, cationic, amphoteric and nonionic), and the like. Various FDA-approved inactive ingredients are found at the FDA's “The Inactive Ingredients Database” that contains inactive ingredients specifically intended as such by the manufacturer, whereby inactive ingredients can also be considered active ingredients under certain circumstances, according to the definition of an active ingredient given in 21 CFR 210.3(b)(7).

(39) In other embodiments, a composition may be formulated for application to a hard surface, such as a structural surface, including but not limited to untreated lumber, treated lumber, a wood beam, a wood board, cardboard, particle board, joist, stud and the like, a baseboard, wood trim, a hardwood floor, a window sill, a screen, a porch floor, a deck, a door, a wall, a ceiling, interior furniture, exterior furniture, and the like. Similarly, a composition may be formulated for application to a soft surface, such as a carpet, a curtain, a rug, padded furniture, a cushion, a mattress, a box spring, a mattress cover, a bedbug repellent mattress pad, a bed sheet, a blanket, a pillow, a doll, a stuffed animal, a net, and the like.

(40) In a further example, the composition may be formulated for reconstitution, such that it may be dispensed in a dry state, such as in a powder, grain, or mixture with other materials, such as animal forage, seed, or fertilizer. Once dispensed, the material may be reconstituted when combined with water.

(41) In further embodiments, a composition contemplated herein can be formulated to be sprayed, sprinkled, poured, or brushed onto a surface. In accordance, the composition may be formulated as a spray, a powder, a paint, a stain, a wax, a gel, a paste, or in any other form.

(42) In another embodiment, a composition may be formulated for application to an outdoor area, such as a lawn, a flowerbed, a forest, a field, and the like. For example, the composition can be placed in a bug bomb, or a pressurized canister adapted to dispense the composition a distance of up to about 1 meter, or up to about 5 meters, or up to about 10 meters. In another example, the composition may be formulated for inclusion in a sprayer device to be connected to a water source and thereby dispensed over a large area.

(43) In further embodiments, a composition contemplated herein can be impregnated within a passive insect repellent dispenser and/or charged within a reservoir of an active insect repellent dispenser either of which can be wearable by an individual or placeable in an interior or exterior volume. For example, the composition may be formulated as or applied to a surface of a bracelet, a necklace, a bandage, or an article of clothing to be worn by an individual or an animal, such as a collar, harness, and the like. Examples of clothing contemplated include hats, gloves, pants, shirts, underwear, coats, bandanas, scarves, socks, shoes, shoelaces, footwear liners and inserts, gaiters, hunting clothes, military clothes, and the like. Further, the composition may be formulated as a blanket, a netting, an insect trap, such as or similar to fly paper, a glue trap, a colored trap, and the like.

(44) Further, the composition may be formulated as part of a bandage, which further can include an antibiotic. The bandage may be configured with an adherent portion and a non-stick composition-carrying absorptive portion. Alternatively, the bandage may consist of a porous carrier portion that is adherent.

(45) In another embodiment, a device including an attractant may be used to carry a contemplated composition. For example, the device may include an insect or pest food and/or a pheromone and/or a scent and/or a lure and/or may emit light and/or sound including subsonic emissions, and the like.

(46) In one embodiment, compositions contemplated herein may be applied to one or more surfaces using an applicator having a reservoir for carrying the composition in a wet form or a dry form. Examples of applicators that may be used include an aerosol container with a spray nozzle with or without a spray straw to focus delivery of the composition, a spray gun, an impregnated sheet, film, and/or matrix where the composition is released onto the surface by a releasing agent, such as water or other carrier. Additional examples include a roll-on dispenser, such as is used for liquid deodorant application, a telescopic gel/wax dispenser, similar to a gel deodorant applicator, lip balm dispenser, or a glue stick, a squeeze tube, a pen, such as is used for a bleach pen or an anti-itch composition applicator, a felt-tip marker, a temporary tattoo, a stamp such as a self-inking stamp, a pump sprayer, a trigger sprayer, a pressurized spraying device, a sponge, a squeegee, an airbrush, a brush, a pet brush, a broom, a mop, a roller, a powder dispenser, such as for dispensing foot powder, a hair brush that dispenses composition into the bristles when the brush used on hair, a comb that dispenses composition onto the teeth when the comb is used on hair.

(47) Methods of treating arthropod infestations, as contemplated herein, can include application of a nootkatone-containing composition to a surface. Application can be by any applicator or dispenser disclosed herein and others. Additional methods of treating arthropod infestations can include application of a nootkatone-containing composition to a surface, allowing the composition to remain on the surface for a period of time, and then removing the composition from the surface. The composition can be removed after application by waiting for at least about 1, 5, 10, or 30 seconds, at least about 1, 2, 5, 10, 15, or 30 minutes, at least about 1, 2, 4, 8, 12, or 24 hours, at least about 1, 2, 4, or 6 days, or at least about 1, 2, or 4 weeks. The composition can be removed by vacuuming, rinsing, steaming, washing, drying, scrubbing, and combinations thereof. After the composition has been removed, another application of the nootkatone-containing composition can be reapplied to the surface and either left in place or similarly removed. In some embodiments, the process of application can also include brushing or agitating the surface to which the composition is being applied to clean the surface at the same time as the composition is being applied and/or to facilitate penetration of the composition into the surface. The brushing or agitating can be accomplished by sweeping, vacuuming, or scrubbing. Similarly, in the context of a soft surface, such as a carpet, normal walking on the surface can also facilitate penetration of the composition into the soft surface.

EXAMPLES

(48) The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.

Example No. 1. Nootkatone Repels Aedes aegypti Mosquitoes

(49) In the following example, the effectiveness of 1% nootkatone compositions in repelling mosquitoes was determined in comparison with DEET in a choice design test.

(50) Methods

(51) Compositions of 1% nootkatone were formulated in either ethanol or acetone and coated onto collagen membrane-covered wells filled with warm blood.

(52) Two hundred and fifty female mosquitoes aged 4-8 days were sucrose deprived for 18 hr and then introduced to the collagen membrane-covered wells. Mosquito probing of the treated collagen membranes was recorded every 2 min during a 20 min period.

(53) Five replicates were performed. A 20% DEET composition was used as a positive control and an ethanol only solution served as a negative control.

(54) Results

(55) As seen in FIG. 2, 1% nootkatone exhibits comparable repellency against Ae. aegypti as a 20% DEET formulation.

Example No. 2. Nootkatone Repels Aedes aegypti, Culex quinquefasciatus, and Anopheles quadrimaculatus Mosquitoes

(56) A laboratory trial was required to evaluate the repellency of 1% Nootkatone against female Aedes aegypti, Culex quinquefasciatus, and Anopheles quadrimaculatus mosquitoes using an in vitro method.

(57) Methodology

(58) Test System

(59) Female adult laboratory reared Aedes aegypti, Culex quinquefasciatus, and Anopheles quadrimaculatus mosquitoes (susceptible) were obtained from Benzon Research in Carlisle, Pa. 250 mosquitoes were used per replicate (5 replicates per test, per species). Adult mosquitoes were 3-8 days old.

(60) Test Treatments and Application

(61) Nootkatone was diluted in ethanol and acetone (note that Nootkatone diluted in acetone was not tested against An. quadrimaculatus). A positive control consisting of 20% DEET in ethanol was prepared using stock solutions. Negative controls consisted of no treatment, and ethanol-only treatment.

(62) The treatments were applied directly to prepared collagen membranes by pipetting 25 μL of the appropriate substance onto a membrane and spreading it evenly with the tip of the pipette.

(63) Experimental Design

(64) Exposure container.

(65) A 30.5 cm×30.5 cm×30.5 cm rigid plastic frame supported by four, 4 cm high legs, with a sleeved entry on one side and a sliding door on the bottom.

(66) Membrane feeder.

(67) Five wells (3 cm in diameter×8 mm in depth) in line on a hollow plastic block (6 cm wide×22 cm long×3 cm deep), which fits through the sliding door in the bottom of the exposure container. Hoses attached to each side of the block circulate heated water that is pumped from a water bath. The sliding door in the bottom of the exposure container covers and uncovers the wells in the membrane feeder, allowing mosquitoes to access the wells.

(68) Test set-up.

(69) The membrane feeder was connected to a heated water bath, and warm water passed through the feeder via a circulating pump so that the wells were warmed to 89-95° F. Seventy-two (72) mg of ATP (disodium salt) were added to 26 mL of warmed citrated bovine blood, which was poured into the wells until they were completely full.

(70) The collagen membranes were placed on vacuum greased wax paper for treatment. They were then treated as described above and left for five minutes to dry. After they dried, the membranes were placed over each of the wells (using vacuum grease as an adhesive), completely covering the blood.

(71) Exposure to mosquitoes.

(72) The mosquitoes were released into the exposure container just prior to exposure to the membranes. The exposure container was then placed on the membrane feeder and the sliding door opened, allowing the mosquitoes to access the wells. The number of mosquitoes probing each membrane was recorded every two minutes for twenty minutes.

(73) The above procedures were repeated until five replicates were completed. A new batch of 250 female mosquitoes and fresh blood were used for each replicate, and the wells were cleaned in between replicates. The position of the treatments was rotated for each replicate, so that each treatment was tested on each of the five wells.

(74) Statistical Analyses

(75) Percent repellency was calculated for each replicate using the total number of probes with the following formula:

(76) $\mathrm{Repellency}=\frac{C-T}{C}\times 100$ where C=the total number of probes on the untreated well and T=the total number of probes on the treated well

(77) Average percent repellency and standard error were then calculated across all five replicates. Shapiro-Wilks tests were conducted to determine the normality of the data, based on total number of probes per replicate. Depending on normality, student t-tests (with concurrent F-tests for variance) or Mann-Whitney tests were then used to determine significance between the treatments.

(78) Results

(79) Results are summarized in Table No. 2 and FIG. 2. Nootkatone diluted in both ethanol and acetone provided significantly higher protection (α=0.05) against Ae. aegypti and Cx. quinquefasciatus than untreated or ethanol alone, and was not significantly different from 20% DEET in its protection level. Against An. quadrimaculatus, nootkatone diluted in ethanol provided an average of 90.3% protection; however, this was not found to be significantly different from ethanol alone, which provided an average of 44.9% protection.

(80) TABLE-US-00002 TABLE NO. 2 Percent repellency (average ± SE, n = 5) and comparison p-values (α = 0.05) between formulations for female mosquitoes exposed over twenty minutes in an in vitro assay. Comparison p-values Percent 20% Nootkatone Nootkatone Treatment repellency Ethanol DEET (acetone) (ethanol) Aedes aegypti Untreated — 0.66 0.01 0.0002 <0.0001 Ethanol 12.6 ± 5.5 0.01 0.0005 0.0004 20% DEET 99.4 ± 0.6 0.16 0.056 Nootkatone 91.5 ± 2.1 0.72 (acetone) Nootkatone 92.0 ± 3.4 (ethanol) Culex quinquefasciatus Untreated — 0.333 0.01 0.005 0.011 Ethanol  29.2 ± 10.0 0.01 0.02 0.025 20% DEET 95.9 ± 4.1 0.119 0.105 Nootkatone  80.1 ± 10.0 0.68 (acetone) Nootkatone 83.8 ± 5.5 (ethanol) Anopheles quadrimaculatus Untreated — 0.174 0.007 — 0.009 Ethanol  44.9 ± 18.8 0.007 — 0.084 20% DEET 100.0 ± 0.0  — 0.007 Nootkatone 90.3 ± 5.3 (ethanol)

Example No. 3. Nootkatone Kills Aedes aegypti Mosquitoes

(81) In the following example, the effectiveness of a 1% nootkatone composition in killing Ae. aegypti mosquitoes was determined.

(82) Methods

(83) A composition of 1% nootkatone formulated in ethanol was added to filter paper, which was then introduced into a Fluon®-coated container. Ten Ae. aegypti mosquitoes were added to the container and observed at 30 min, 1, 4, and 24 hr. Five replicates were performed. Knockdown (KD) occurred when an insect could right itself when placed on its back or initiate directional movement and/or could not fly, but exhibits still movement in the limbs and antennae with or without tactile stimulation. Mortality occurred when the insect exhibited no movement, even with tactile stimulation. An untreated population was observed as a negative control.

(84) Results

(85) Compared to the negative control, 1% nootkatone in ethanol begins affecting Ae. aegypti mosquitoes after 30 minutes and after 4 hr has affected nearly 100% of the Ae. aegypti treatment population (see FIGS. 3-5). Maximal mortality required a 24 hr exposure, however, mortality was first observed after 4 hr of exposure suggesting a rapid increase in the rate of mortality after 4 hr of exposure. Knockdown rate was observed after 30 min exposure and more than doubled after 1 hr and was greater than 90% after 4 hr. Therefore, these results indicate that an environmental dispersed 1% nootkatone formulation begins controlling Ae. aegypti mosquitos shortly after exposure. These results suggest that an environmentally dispersed nootkatone formulation would be an effective tool against Ae. aegypti mosquito populations.

Example No. 4. Nootkatone Kills a Broad Spectrum of Arthropods

(86) Overview

(87) In the following example, the effectiveness of a 1% nootkatone composition in killing Ae. aegypti and other mosquitoes as well as other insects was determined.

(88) A laboratory trial was conducted to assess the efficacy of nootkatone applied to filter paper against aphids (Schizaphis gramium), dust mites (Dermatophagoides sp.), termites (Reticulitermes flavipes), deer ticks (Ixodes scapularis), biting midges (Culicoides sp.), fire ants (Solenopsis invicta), and mosquitoes (Aedes aegypti and Culex), in terms of knockdown (KD) and mortality. One concentration (1% in ethanol) of nootkatone was assessed for most species, but three concentrations (0.25%, 0.5%, and 1% in ethanol) were assessed for mosquitoes. A negative control group, consisting of filter paper treated with ethanol, was also assessed for comparison purposes for each species.

(89) Test systems were introduced to treated filter paper, allowed to dry, and then introduced into treatment chamber, where treated arthropods were monitored at intervals up to 72 hours. If 100% mortality was obtained prior to 72 hours, the individual study was halted.

(90) Nootkatone at 1% concentration produced 94-100% mortality in mosquitoes, 90% mortality in termites, and 100% mortality in dust mites, deer ticks, fire ants, aphids, and biting midges (respectively) during the experimental duration. The time required for greater than 90% killing varied among species tested, ranging from 30 minutes for aphids, to 24 hours for mosquitoes, fire ants, and biting midges, and 72 hours for termites, deer ticks, and dust mites. At 0.5% concentration, nootkatone in ethanol produced 82-95% mortality in both mosquito species by 72 hours post treatment.

(91) As shown below, it can be concluded that nootkatone has significant efficacy as a pesticide against biting arthropods including mosquitoes, dust mites, termites, deer ticks, fire ants, and biting midges especially at or over the 1% concentration.

(92) Aim

(93) A laboratory trial was required to assess the efficacy of a compound applied to filter paper against dust mites (Dermatophagoides sp.), termites (Reticulitermes flavipes), deer ticks (Ixodes scapularis), biting midges (Culicoides sp.), fire ants (Solenopsis invicta), and mosquitoes (Aedes aegypti and Culex), in terms of knockdown (KD) and mortality.

(94) Methods

(95) Fire ants—5 replicates of 10 insects were tested per treatment. A Petri dish lid 3.54 inches (9 cm) in diameter was inverted and serve as the substrate for a 2.75 inch (7 cm) treated filter paper circle. The small Petri dish bottom was inverted over the paper and had its sides treated with Fluon® to prevent climbing and force exposure with the filter paper. The entire Petri dish was enclosed with two rubber bands.

(96) Termites—3 replicates of 10 termites were tested per treatment. One long glass tube with screened ends (to provide ventilation) was used for each test container. Test containers were placed into a larger container to prevent escapes, and were kept in darkness and at high humidity for the duration of the experiment. Filter papers were cut into strips and fitted inside the glass tubes. Two paper strips were pushed end to end to form one continuous strip, leaving a 2 mm gap running the entire length of the tube for viewing termite activity.

(97) Deer Ticks and Dust mites—5 replicates of 10 ticks or 35 dust mites were tested per treatment. Envelopes made from treated 2.5 inch (6.35 cm) filter paper squares served as the containers. After the ticks or dust mites were placed inside the envelopes, and the openings were sealed using small binder clips.

(98) Midges and mosquitoes—5 replicates of 10 insects per treatment were tested. Inverted 10 ounce (295 ml) clear plastic cups with a 0.5 inch (1.27 cm) hole burned into the center of the bottom, and with the sides coated with Fluon® was used at the treatment containers. The bottoms of the containers consisted of Petri dish lids 3.54 inches (9.0 cm) in diameter lined with a treated filter paper circle the same diameter. The cup was placed over the filter paper in the lid and both were secured in place using 2 rubber bands. The insects were introduced through the hole in the cup, and a plastic disc was placed over the opening to prevent escapes.

(99) Arthropods were observed at +30 minutes, and +1, +4, +24, +48, and +72 hours post introduction to the test containers. At each observation period, arthropods were scored according to the following criteria: Knockdown (KD)—cannot right itself when placed on its back or otherwise initiate directional movement and/or cannot fly (if applicable), but still exhibits movement in the limbs and antennae with or without tactile stimulation; and Dead (mortality)—exhibits no movement, even with tactile stimulation. Readings were discontinued if treated mortality reaches 1100%, or control mortality exceeds 20%.

(100) Results

(101) Mosquitoes:

(102) Assessment results are summarized in Table Nos. 3-4 below.

(103) Nootkatone at 0.5, and 1% concentration produced 92%, mortality in, Aedine mosquitoes, and the same concentrations produced 82% and 100% mortality in Culicine mosquitoes during the experimental duration. At 1% concentration, the formulation affected (knockdown+mortality) at least 80% of mosquitoes in both species by 8 hours post treatment. Nootkatone produced less than 80% mortality in mosquitoes at 0.25%, the lowest tested concentration.

(104) Nootkatone has efficacy as a pesticide against yellow fever mosquitoes and southern house mosquitoes, especially at or over the 0.5% concentration.

(105) TABLE-US-00003 TABLE NO. 3 Yellow Fever mosquito (Aedes aegypti) Time post Means Standard Error introduction KD Dead Affected KD Dead Affected Untreated 30 minutes 0.0 0.0 0.0 0.0 0.0 0.0  1 hour 0.0 0.0 0.0 0.0 0.0 0.0  4 hours 0.0 0.0 0.0 0.0 0.0 0.0  8 hours 0.0 0.0 0.0 0.0 0.0 0.0 24 hours 0.0 0.0 0.0 0.0 0.0 0.0 48 hours 2.0 0.0 2.0 2.0 0.0 2.0 72 hours 0.0 4.0 4.0 0.0 2.4 2.4 Nootkatone 0.25% 30 minutes 0.0 17.8 17.8 0.0 17.8 17.8  1 hour 0.0 20.0 20.0 0.0 20.0 20.0  4 hours 0.0 20.0 20.0 0.0 20.0 20.0  8 hours 2.2 15.6 17.8 2.2 15.6 17.8 24 hours 4.4 13.3 17.8 4.4 13.3 17.8 48 hours 2.0 15.6 17.6 2.0 15.6 15.2 72 hours 8.4 17.6 26.0 4.1 15.2 18.6 Nootkatone 0.5% 30 minutes 0.0 0.0 0.0 0.0 0.0 0.0  1 hour 0.0 0.0 0.0 0.0 0.0 0.0  4 hours 5.3 0.0 5.3 3.6 0.0 3.6  8 hours 29.9 0.0 29.9 11.9 0.0 11.9 24 hours 60.6 22.5 83.1 9.7 13.4 8.8 48 hours 15.1 72.0 87.1 6.9 7.8 6.8 72 hours 7.5 92.5 100.0 3.4 3.4 3.2 Nootkatone 1% 30 minutes 0.0 0.0 0.0 0.0 0.0 0.0  1 hour 1.8 0.0 1.8 1.8 0.0 1.8  4 hours 46.9 0.0 46.9 17.6 0.0 17.6  8 hours 80.4 0.0 80.4 15.5 0.0 15.5 24 hours 18.5 65.5 84.0 6.9 15.3 16.0 48 hours 9.6 80.4 90.0 6.2 15.5 10.0 72 hours 2.0 92.0 94.0 2.0 8.0 6.0

(106) TABLE-US-00004 TABLE NO. 4 Southern house mosquito (Culex) Time post Means Standard Error introduction KD Dead Affected KD Dead Affected Untreated 30 minutes 0.0 0.0 0.0 0.0 0.0 0.0  1 hour 0.0 0.0 0.0 0.0 0.0 0.0  4 hours 0.0 0.0 0.0 0.0 0.0 0.0  8 hours 0.0 0.0 0.0 0.0 0.0 0.0 24 hours 4.0 0.0 4.0 2.4 0.0 2.4 48 hours 34.7 0.0 34.7 5.0 0.0 5.0 72 hours 4.2 36.7 40.9 2.6 4.9 5.5 Nootkatone 0.25% 30 minutes 24.0 0.0 24.0 16.9 0.0 16.9  1 hour 22.0 0.0 22.0 17.4 0.0 17.4  4 hours 32.2 0.0 32.2 15.5 0.0 15.5  8 hours 18.0 0.0 18.0 13.6 0.0 13.6 24 hours 14.0 12.2 26.2 9.8 3.7 12.0 48 hours 14.2 61.3 75.6 2.4 9.6 9.2 72 hours 2.0 74.0 76.0 2.0 12.1 10.8 Nootkatone 0.5% 30 minutes 0.0 0.0 0.0 0.0 0.0 0.0  1 hour 2.5 0.0 2.5 2.5 0.0 2.5  4 hours 4.0 0.0 4.0 2.4 0.0 2.4  8 hours 4.0 0.0 4.0 2.4 0.0 2.4 24 hours 18.5 10.5 29.0 6.5 3.2 5.6 48 hours 23.0 49.0 72.0 8.6 15.0 9.7 72 hours 0.0 82.0 82.0 0.0 6.6 6.6 Nootkatone 1% 30 minutes 2.2 0.0 2.2 2.2 0.0 2.2  1 hour 52.2 0.0 52.2 5.9 0.0 5.9  4 hours 83.7 0.0 83.7 2.8 0.0 2.8  8 hours 78.9 13.1 92.0 8.6 8.4 2.0 24 hours 2.2 97.8 100.0 2.2 2.2 0.0 48 hours 0.0 100.0 100.0 0.0 0.0 0.0 72 hours 0.0 100.0 100.0 0.0 0.0 0.0

(107) Other Arthropods:

(108) Assessment results are summarized in Table Nos. 5-10 below.

(109) Nootkatone at 1% concentration produced 100%, mortality in dust mites, 90% mortality in termites, and 98% mortality in deer ticks after 72 hours. The same 1% concentration of nootkatone produced 100% mortality in fire ants and biting midges after only 24 hours.

(110) It can be concluded that nootkatone has significant efficacy as a pesticide against dust mites, termites, deer ticks, fire ants, and biting midges.

(111) TABLE-US-00005 TABLE NO. 5 Efficacy of Nootkatone against Dust mites Dust mite Time post Means Standard Error introduction KD Dead Affected KD Dead Affected Untreated 72 hours 0.0 0.0 0.0 0.0 0.0 0.0 Nootkatone 72 hours 0.0 100.0 100.0 0.0 0.0 0.0

(112) TABLE-US-00006 TABLE NO. 6 Efficacy of Nootkatone against Termites Termite Time post Means Standard Error introduction KD Dead Affected KD Dead Affected Untreated 30 minutes 0.0 0.0 0.0 0.0 0.0 0.0  1 hour 0.0 0.0 0.0 0.0 0.0 0.0  4 hours 0.0 0.0 0.0 0.0 0.0 0.0 24 hours 0.0 0.0 0.0 0.0 0.0 0.0 48 hours 3.3 0.0 3.3 3.3 0.0 3.3 72 hours 0.0 0.0 0.0 0.0 0.0 0.0 Nootkatone 30 minutes 0.0 0.0 0.0 0.0 0.0 0.0  1 hour 0.0 0.0 0.0 0.0 0.0 0.0  4 hours 0.0 0.0 0.0 0.0 0.0 0.0 24 hours 83.3 0.0 83.3 16.7 0.0 16.7 48 hours 0.0 86.7 86.7 0.0 13.3 13.3 72 hours 0.0 90.0 90.0 0.0 10.0 10.0

(113) TABLE-US-00007 TABLE NO. 7 Efficacy of Nootkatone against Fire Ants Fire ant Time post Means Standard Error introduction KD Dead Affected KD Dead Affected Untreated 30 minutes 0.0 0.0 0.0 0.0 0.0 0.0  1 hour 0.0 0.0 0.0 0.0 0.0 0.0  4 hours 0.0 0.0 0.0 0.0 0.0 0.0 24 hours 0.0 10.0 10.0 0.0 10.0 10.0 Nootkatone 30 minutes 0.0 0.0 0.0 0.0 0.0 0.0  1 hour 0.0 0.0 0.0 0.0 0.0 0.0  4 hours 22.0 0.0 22.0 19.6 0.0 19.6 24 hours 0.0 100.0 100.0 0.0 0.0 0.0

(114) TABLE-US-00008 TABLE NO. 8 Efficacy of Nootkatone against Deer Ticks Deer tick Time post Means Standard Error introduction KD Dead Affected KD Dead Affected Untreated 24 hours 0.0 2.0 2.0 0.0 2.0 2.0 48 hours 0.0 4.0 4.0 0.0 2.4 2.4 72 hours 0.0 6.0 6.0 0.0 4.0 4.0 Nootkatone 24 hours 62.0 36.0 100.0 18.5 18.1 0.0 48 hours 16.0 82.0 100.0 5.1 5.8 0.0 72 hours 0.0 98.0 100.0 0.0 2.0 0.0

(115) TABLE-US-00009 TABLE NO. 9 Efficacy of Nootkatone against Biting Midges Biting midge Time post Means Standard Error introduction KD Dead Affected KD Dead Affected Untreated 30 minutes 0.0 2.5 2.5 0.0 2.5 2.5  1 hour 2.2 2.5 4.7 2.2 2.5 2.9  4 hours 0.0 4.7 4.7 0.0 2.9 2.9 24 hours 2.2 4.7 6.9 2.2 2.9 4.5 Nootkatone 30 minutes 100.0 0.0 100.0 0.0 0.0 0.0  1 hour 98.2 1.8 100.0 1.8 1.8 0.0  4 hours 21.3 78.7 100.0 1.9 1.9 0.0 24 hours 0.0 100.0 100.0 0.0 0.0 0.0

(116) TABLE-US-00010 TABLE NO. 10 Efficacy of Nootkatone against Aphids Aphid Time post Means Standard Error introduction KD Dead Affected KD Dead Affected Untreated 30 minutes 1.8 3.5 5.3 1.8 2.1 3.6 Nootkatone 30 minutes 6.0 94.0 100.0 6.0 6.0 0.0

Example No. 5: Comparison of Fermentation-Derived Nootkatone with Citrus-Derived Nootkatone

(117) Overview

(118) Nootkatone, as defined herein, has a particular chemical profile indicative of its constituent chemical species. Other sources of nootkatone can have different chemical profiles and therefore actually represent different chemical compositions. GC-FID analyses of the nootkatone used in the studies described above (obtained from oxidation of fermentation-derived valencene, also known as, nootkatone ex valencene (N×V)) and a citrus fruit-derived nootkatone (also known as nootkatone ex citrus, which is derived from citrus fruit and available from Frutarom®, Corona, Calif.) are shown in FIG. 6. The nootkatone used in the studies described herein lacked valencene and demonstrated a lower amount of 11,12-epoxide than the Frutarom® nootkatone. Moreover, further analysis of an unknown peak from the Frutarom® nootkatone sample revealed that the Frutarom® sample contained limonene (see FIG. 7), whereas the nootkatone used in the present studies was limonene-free. These results underscore the different chemical profile of the nootkatone used herein (N×V) compared to commercially-available nootkatone derived from citrus, such as that provided by Frutarom®.

(119) These results are also in accord with the observation (not shown) that nootkatone obtained from fermentation-derived valencene does not contain bergapten (or bergaptine). Bergapten (5-methoxypsoralen or 5-MOP) is a compound found in bergamot and citrus essential oils that causes phototoxicity in humans. (see Gionfriddo et al. “Elimination of Furocoumarins in Bergamot Peel Oil,” Perfumer & Flavorist., 2004; 29:48-52; Ferreira Maia et al. “Plant-based insect repellents: a review of their efficacy, development and testing,” Malaria Journal, 2011; 10:Suppl1-11; and Kejlová et al. “Phototoxicity of bergamot oil assessed by in vitro techniques in combination with human patch tests.” Toxicol In Vitro. 2007; 21:1298-1303). In addition, GHS health warning statements for bergapten indicate that it can cause allergic skin reactions, allergy or asthma symptoms, or breathing difficulties if inhaled, and can cause genetic defects or cancer in animals. For such reasons, a Cosmetic Ingredient Review expert panel in assessing the safety of 14 citrus-derived peel oil ingredients concluded no more than 0.0015% (15 ppm) bergapten should be included in cosmetic products (see “Safety Assessment of Citrus-Derived Peel Oils as Used in Cosmetics,” Cosmetic Ingredient Review Expert Panel Final Report, Sep. 30, 2014: 1-31).

(120) Bergapten is present in naturally derived valencene (from citrus) and carries over through the chemical oxidation that forms nootkatone. Bergapten can be photo-activated to become a skin irritant, which can be worse around an open bite wound. Bergapten-associated adverse skin reactions around arthropod bites could mask or give false positives for the preliminary signs of an infection. Therefore, nootkatone ex valencene, which is bergapten-free, has particular safety advantages over plant-derived nootkatone and is preferable for topical application.

Example No. 6: Evaluation of the Repellency of a Compound Against Fleas, Ticks, Bed Bugs, and Termites

(121) Aim

(122) An in vitro laboratory trial was required to assess the repelling action of nootkatone applied to filter paper against cat fleas (Ctenocephalides felis), deer ticks (Ixodes scapularis), bed bugs (Cimex lectularius), and termites (Reticulitermes flavipes) in a choice test design.

(123) Methodology

(124) Test System

(125) The following test systems were obtained from the listed sources for this trial:

(126) TABLE-US-00011 Life stage/ Species Common name sex Source Ctenocephalides Cat flea Adult/mixed El Labs, Soquel, felis sex CA Ixodes Deer tick Adult/mixed OSU Tick Lab, scapularis sex Stillwater, OK Cimex Bed bug Adult/mixed Schal Lab, lectularius sex NCSU, Raleigh, NC Reticulitermes Eastern Adult workers/ Field sites in flavipes subterranean mixed sex Maryland termite

(127) Test Treatments and Application

(128) The sponsor provided a sample of nootkatone (98%), which was diluted in ethanol to a 0.5% concentration. The treatment was applied to filter paper at a rate of 1 mL for a 9 cm filter paper disc, and a rate of 1.9 mL for a 15 cm filter paper disc. A negative control group, consisting of untreated filter paper, was also assessed for comparison purposes.

(129) Experimental Design

(130) The filter paper was cut to fit the design of a suitable test container for each species as follows:

(131) Bed bugs:

(132) Petri dishes (9 cm) were modified by cutting a circular hole in the bottom of a clean petri plate. Fine mesh nylon cloth was fastened to the bottom using adhesive, covering the hole. The petri plate was then inverted, with the screened bottom serving as the top. Treated and untreated filter papers were cut in half and fitted into the new bottom of the test containers. Treated replicates received one half treated and one half untreated filter paper; negative control replicates received only untreated filter paper.

(133) Cat fleas:

(134) Treated and untreated filter papers were cut in half and fitted into the bottom of a 15 cm clear plastic cylinder. Treated replicates received one half treated and one half untreated filter paper; negative control replicates received only untreated filter paper.

(135) Deer ticks:

(136) Treated and untreated filter papers were cut in half and fitted into the bottom of a 9 cm petri dish. Treated replicates received one half treated and one half untreated filter paper; negative control replicates received untreated filter paper only. The petri dish arenas were then placed on their sides, with the control halves positioned at the lower half and the treated half at the upper half.

(137) Termites:

(138) One long glass tube with screened ends (to provide ventilation) was used for each test container. Test containers were placed into a larger container to prevent escapes, and were kept in darkness and at high humidity for the duration of the experiment. Treated and untreated filter papers were cut into strips and fitted inside the glass tubes. Treated replicates received one treated and one untreated paper; negative control replicates received two untreated strips. The two paper strips were pushed end to end to form one continuous strip, leaving a 2 mm gap running the entire length of the tube for viewing termite activity.

(139) Arthropod Introduction and Assessments

(140) Bed bugs:

(141) Bed bugs were placed in the center of each test container and covered with the fine-mesh lid. The containers were kept in darkness at ambient laboratory conditions for 24 hours. At the end of the exposure period, the distribution (on or under each filter paper) of bed bugs was recorded.

(142) Cat fleas:

(143) Cat fleas were introduced to the untreated side of each arena, and their distribution (on or under each paper) was recorded at +1, +4, and +24 hours post-introduction.

(144) Deer ticks:

(145) Ticks were introduced, one at a time, to the bottom half of the control (untreated) arena, and monitored to see how long it took them to get to the upper “treated” side of the arena. Ticks were allowed up to three minutes to cross into the upper area of each arena. Any ticks not crossing into the designated treated area (and therefore exhibiting appropriate questing behavior) were discarded. Once a tick had crossed into the upper side of the control arena, it was gently picked up with forceps and placed in the bottom half of the treated arena, and the procedure repeated. Ticks were considered “not repelled” if they crossed onto the treated side of the treated arena within three minutes.

(146) Termites:

(147) Termites were placed in the center of each test container and left to distribute undisturbed for 24 hours. At the end of the exposure period, the distribution (on or under each filter paper) of termites was recorded.

(148) Data Analysis

(149) Fleas, Bed Bugs, and Termites

(150) The number of insects in each location was converted to a percentage, for which the average and standard error were calculated across all five replicates.

(151) Percent repellency was calculated with the following formula:
% repellency=(1−((t/T)/(c/C)))*100 Where t=number of insects on treated side of treated arena T=total number of insects in treated arena c=average number of insects on right side of untreated arenas C=average total number of insects in untreated arenas

(152) Ticks

(153) The number of repelled ticks for each replicate was added together, and the average and standard error were calculated across all five replicates. The numbers were divided by the total number of ticks per replicate to obtain the percent repellency.

(154) Results

(155) Assessment results are summarized in Table No. 11 below. See Appendix I for full raw data.

(156) TABLE-US-00012 TABLE NO. 11 Calculated percent repellency (average ± SE) for several species exposed to nootkatone. Cat flea  61.9 ± 15.0 Bed bug 93.9 ± 6.1 Termite 100.0 ± 0.0  Deer tick 96.0 ± 2.4

(157) It can be concluded that in this laboratory trial, 0.5% nootkatone in ethanol was highly repellent to bed bugs, termites, and deer ticks, and showed some potential repellency against cat fleas.

(158) TABLE-US-00013 APPENDIX I Raw Data Tables Species Cat fleas (Ctenocephalides felis) Treatment: Untreated control Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Total: 13 Total: 10 Total: 11 Total: 9 Total: 10 Time post Control Control Control Control Control Control Control Control Control Control introduction Left Right Left Right Left Right Left Right Left Right  1 Hour 5 8 4 6 7 4 2 7 4 6  4 Hours 6 7 4 6 6 5 2 7 5 5 24 Hours 7 6 7 3 6 5 4 5 6 4 Treatment: Nootkatone Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Time post Total: 10 Total: 10 Total: 10 Total: 7 Total: 8 introduction Untreated Treated Untreated Treated Untreated Treated Untreated Treated Untreated Treated  1 Hour 2 8 4 6 7 3 3 4 4 3  4 Hours 9 1 7 3 6 4 4 3 6 2 24 Hours 8 2 6 4 9 1 6 1 8 0 Species Bed bugs (Cimex lectularius) Treatment: Untreated control Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Total: 13 Total: 10 Total: 11 Total: 9 Total: 10 Time post Control Control Control Control Control Control Control Control Control Control introduction Left Right Left Right Left Right Left Right Left Right 24 Hours 9 1 9 1 8 2 0 10 7 3 Treatment: Nootkatone Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Time post Total: 10 Total: 10 Total: 10 Total: 7 Total: introduction Untreated Treated Untreated Treated Untreated Treated Untreated Treated Untreated Treated 24 Hours 10 0 10 0 9 1 10 0 10 0 Species: Eastern subterranean termite (Reticulitermes flavipes) Treatment: Untreated control Replicate 1 Replicate 2 Replicate 3 Total: 10 Total: 10 Total: 10 Time post Control Control Control Control Control Control introduction Left Right Left Right Left Right 24 Hours 10 0 9 1 4 6 Treatment: Nootkatone Replicate 1 Replicate 2 Replicate 3 Time post Total: 10 Total: 10 Total: 10 introduction Untreated Treated Untreated Treated Untreated Treated 24 Hours 10 0 10 0 10 0 Raw Data Deer tick (Ixodes scapularis) Total: 10 Total: 10 Total: 10 Total: 10 Total: 10 Not Not Not Not Not Repelled repelled Repelled repelled Repelled repelled Repelled repelled Repelled repelled Treatment: Untreated 0 10 0 10 0 10  0 10 0 10 Treatment: Nootkatone 9 1 10 0 9 1 10 0 10  0

Example No 7 Production of Nootkatone Ex Valencene

(159) Nootkatone ex valencene may be produced in vivo through expression of one or more enzymes involved in the nootkatone biosynthetic pathway in a recombinant yeast or in vitro using isolated, purified enzymes involved in the nootkatone biosynthetic pathway, such as those described in U.S. Patent Application Publication Nos. 2015/0007368 and 2012/0246767. The final conversion of valencene to nootkatone may be done enzymatically in vivo or in vitro, or may be performed by chemical oxidation (typically inorganic) in vitro.

(160) Briefly, the valencene synthase gene (CVS) from Citrus sinensis cv. Valencia (Valencia orange) was cloned from RNA isolated from the juice vesicles of freshly harvested Valencia orange using the procedure previously described in Example 1 of U.S. Pat. No. 7,442,785.

(161) First, Yep-GW-URA (Takahashi et al., (2007) Biotechnol Bioeng. 97(1):170-181) was generated by inserting a gateway cloning cassette (RfB) with the form attR1-Cm.sup.R-ccdB gene-attR2 (Hartley et al., (2000) Genome Res. 10:1788-1795) into the SmaI restriction site of YEp352-URA (Bio-Technical Resources), which contains an URA3 selectable marker, an ADH1 promoter and an ADH1 terminator flanking, two BamHI sites (one 5′ to the ADH1 promoter and the other 3′ to the ADH terminator), a 2-micron or, an ampicillin resistance gene and a colE1 origin of replication. The resulting vector was designated YEp-CVS-URA.

(162) The CVS gene (set forth in SEQ ID NO: 1, and encoding amino acid sequence is set forth in SEQ ID NO: 2) was then amplified from RNA isolated from the juice vesicles of freshly harvested Valencia orange to contain restriction sites for subcloning into the yeast shuttle expression vector Yep-GW-URA. Following digestion of Yep-GW-URA with EcoRI and XbaI, the amplified product was cloned into the yeast shuttle expression vector YEp-GW-URA.

(163) The YEp-CVS-ura vector was maintained in S. cerevisiae by selecting on SD minimal medium lacking uracil at 28° C. The vector also was maintained in Escherichia coli by selecting for resistance to ampicillin on LB medium containing 100 μg/mL ampicillin.

(164) To screen for production of valencene, the Saccharomyces cerevisiae yeast cell strains CALI5-1 (ura3, leu2, his3, trp1, Δerg9::HIS3, HMG2cat/TRP1::rDNA, dpp1, sue), ALX7-95 (ura3, his3, trp1, Δerg9::HIS3, HMG2cat/TRP1::rDNA, dpp1, sue) or ALX11-30 (ura3, trp1, erg9def25, HMG2cat/TRP1::rDNA, dpp1, sue) were used.

(165) The CALI5-1 strain (see U.S. published Appl. No. US20040249219; U.S. Pat. Nos. 6,531,303 and 6,689,593) has a Δleu2 deletion, which required the introduction of leucine into its media. ALX7-95 was derived from CALI5-1 by correcting the Δleu2 deficiency of CALI5-1 with a functional LEU2 gene (see U.S. published Appl. No. US2010/0151519).

(166) ALX11-30 was constructed from CALI51 in several steps from ALX7-175.1 as described in US2010/0151519. Briefly, ALX7-95 HPS was obtained by transforming a plasmid containing the Hyoscyamus muticus premnaspirodiene synthase (HPS) into ALX7-95 strain. The YEp-HPS plasmid was obtained by cloning the gene for HPS into Yep-GW-URA to give YEp-HPS-ura (YEp-HPS). Then, an error prone PCR reaction of the ERG9 gene was performed, and the resulting DNA was transformed into ALX7-95 harboring YEpHPS. Transformants were plated on YP medium lacking ergosterol and screened for premnaspirodiene production. Those that produced high levels of premnaspirodiene were saved. One strain, ALX7-168.25 [ura3, trp1, his3, erg9.sup.def25, HMG2cat/TRP1::rDNA, dpp1, sue, YEpHPS] was transformed with a PCR fragment of the complete HIS3 gene to create a functional HIS3 gene. Transformants were isolated that were able to grow in the absence of histidine in the medium. From this transformation, ALX7-175.1 was isolated [ura3, trp1, erg9def25. HMG2cat/TRP1::rDNA, dpp1, sue YEpHPS]. Finally, the plasmid YEpHPS was removed by growing ALX7-175.1 several generations in YPD (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) and plating cells on YPD plates. Colonies were identified that were unable to grow on SD medium without uracil (0.67 Bacto yeast nitrogen base without amino acids, 2% glucose, 0.14% yeast synthetic drop-out medium without uracil). This strain was designated ALX11-30.

(167) For screening for production of valencene by valencene synthase or mutants, the YEp-CVS-ura plasmid, containing the CVS gene or modified versions of the CVS gene, was transformed into the above yeast strains using the lithium acetate yeast transformation kit (Sigma-Adrich). The ALX7-95 and ALX11-30 strains generally produced more valencene than the CALI5-1 strain. CALI5-1 was used for initial screening in vials (as described in Example 3) and production n fermenters. Subsequently, ALX7-95 or ALX11-30 were used for screening in vials and fermenters. Typically, ALX7-95 was used for screening in vials and ALX11-30 was used for fermenters.

(168) Transformants were selected on SDE-ura medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose, 0.14% yeast synthetic drop-out medium supplement without uracil, and 40 mg/L ergosterol as needed). Colonies were picked and screened for valencene production using the microculture assay described below.

(169) Production of valencene was performed in a 3-L fermentation tank (New Brunswick Bioflow 110). One liter of fermentation medium was prepared and autoclaved in the fermentation tank (20 g (NH.sub.4).sub.2SO.sub.4, 20 g KH.sub.2PO.sub.4, 1 g NaCl, MgSO.sub.4.7H.sub.2O, 4 g Solulys corn steep solids (Roquette)). The following components were then added: 20 ml mineral solution (0.028% FeSO.sub.4.7H.sub.2O, 0.029% ZnSO.sub.4.7H.sub.2O, 0.008% CuSO.sub.4.5H.sub.2O, 0.024% Na.sub.2MoO.sub.4.2H.sub.2O, 0.024% CoCl.sub.2.6H.sub.2O, 0.017% MnSO.sub.4.H.sub.2O, 1 mL HCl); 10 mL 50% glucose; 30 mL vitamin solution (0001% biotin; 0.012% calcium pantothenate, 0.06% inositol, 0.012% pyridoxine-HCl, 0.012% thiamine-HC); 10 mL 10% CaCl.sub.2, and 20 mL autoclaved soybean oil (purchased from local groceries). For sterol requiring strains, including CALI5-1 and ALX7-95, 50 mg/L cholesterol or 40 mg/L ergosterol was included in the medium.

(170) The seed culture for inoculating the fermentation on medium was prepared by inoculating 50 mL of SDE-ura-trp medium (see Example 3C.2.) with CALI5-1, ALX7-95 or ALX11-30 containing the YEp-CVS-ura plasmid. This culture was grown at 28° C. until early stationary phase (248 hr). One mL of this culture was inoculated into 500 mL of SDE-ura-trp medium and grown for 24 hr at 28° C. A 50-mL aliquot (5% inoculum) was used to inoculate the medium in the fermentation tank.

(171) The fermentor was maintained at 28° C. The air flow was 1 vvm and the dO.sub.2 was maintained above 30% by adjusting the agitation. The pH was maintained at 4.5 using phosphoric acid and NaOH or NH.sub.4OH.

(172) When the glucose concentration fell below 1 g/L, a feeding regimen was initiated such that the glucose in the fermentor was kept between 0 and 1 g/L. The glucose feed consisted of 60% glucose (w/v).

(173) At the end of the fermentation, generally about 132 hours after inoculation, sodium sulfate was added to 10-15% final concentration as was an additional 50 mL soybean oil and the contents of the fermentor were agitated for one hour. After allowing the fermentation vessel contents to settle, the oil was recovered by centrifugation and the valencene content n the oil was determined.

(174) To assay valencene, 3 mL of suspension was placed in a vial to which 3 mL of acetone containing 20 mg/L cedrene was added. After vortexing, the mixture was extracted with 6 mL hexane containing 10 mg/L hexadecane followed by additional vortexing. The organic phase was transferred to a second vial for analysis by gas chromatography using cedrene and hexadecane as internal standards for extraction efficiency and injection respectively. The CALI5-1, ALX7-95 or ALX11-30 S. cerevisiae containing Yep-CVS-ura, and expressing valencene synthase, was found to produce valencene.

(175) The valencene-containing soybean oil, produced by fermentation as described above, was concentrated and purified using wiped-film distillation at 100° C. and 350 mTorr to generate an oil that contained approximately 68% valencene by weight. This material was converted to nootkatone by two different methods described below.

(176) A. Oxidation of Valencene to Nootkatone Using Chromium Trioxide

(177) The valencene distillate produced as described above was oxidized to nootkatone using chromium trioxide and pyridine in dichloromethane as follows. Chromium trioxide (369 g. 3.69 mol, 22 eq) was added in portions to a solution of pyridine (584 g, 7.4 mol, 44 eq) in 5 L of dichloromethane. The mixture was stirred for 10 minutes, 50 grams of valencene distilate (68% w/w, 0.167 mol, 1 eq) was added over four minutes, and the mixture was stirred at 22′ C. for 18 hours. The liquor was drained from the vessel, and the solids were washed twice with 2 L of methyl tertbutyl ether (MTBE). The combined organic layers were further diluted with 2 L of MTBE and successively washed three times with 1.25 L of 5% sodium hydroxide, twice with 2 L of 5% hydrochloric acid, and once with 2 L of brine. The organic phase was dried over 200 grams of anhydrous sodium sulfate, filtered, and concentrated by evaporation to give 36.8 grams crude nootkatone (48% w/w, 0.081 mol, 48% yield).

(178) B. Oxidation of Valencene to Nootkatone Using Silica Phosphonate-Immobilized Chromium (III) Catalyst

(179) Silica phosphonate chromium (III) resin (48.9 g, PhosphonicS, Ltd.) was placed in a 5 L round bottom flask equipped with a condenser, thermowell, overhead stirrer, and sparge tube. Two (2) L of t-butano and valencene distillate (68%, 500 g, 1.67 moles, 1 eq) were added, the contents were heated to 45° C., and the heterogeneous suspension was allowed to stir as oxygen was sparged through the solution (ca 1.5 L/min) and nitrogen flushed over the head-space. 70% t-butyl hydroperoxide in water (TBHP, 315 g, 2.45 moles, 1.47 eq) was added to the solution over 2 hrs while the temperature of the reaction was heated and maintained at 60±5° C. The reaction was allowed to stir until >90% of the valencene allows consumed, as determined by gas chromatography. The reaction was then allowed to cool to room temperature and the silica catalyst removed by filtration. The flask and resin were washed with 500 mL isopropanol. One (1) L of deionized water was added to the combined organic solution (t-butanol and isopropanol), and the mixture was concentrated under reduced pressure by evaporation to afford an amber colored oil. The oil was dissolved in 3 L of toluene and washed with 3.125 L of 15% sulfuric acid for 15 minutes with vigorous agitation. The aqueous layer was removed and re-extracted with 1 L of toluene. The combined toluene layers were then washed three times with 2.5 L of 1 M sodium hydroxide, twice with 500 mL saturated sodium chloride, and dried over anhydrous magnesium sulfate. After filtration, the solvent was removed under reduced pressure by evaporation to afford 378 g of viscous amber oil (33% nootkatone by weight, 0.57 moles, 34% yield).

(180) TABLE-US-00014 Sequence Listing: (Citrus valencene synthase) SEQ ID NO: 1 atgtcgtctggagaaacatttcgtcctactgcagatttccatcctagt ttatggagaaaccatttcctcaaaggtgcttctgatttcaagacagtt gatcatactgcaactcaagaacgacacgaggcactgaaagaagaggta aggagaatgataacagatgctgaagataagcctgttcagaagttacgc ttgattgatgaagtacaacgcctgggggtggcttatcactttgagaaa gaaatagaagatgcaatacaaaaattatgtccaatctatattgacagt aatagagctgatctccacaccgtttcccttcattttcgattgcttagg cagcaaggaatcaagatttcatgtgatgtgtttgagaagttcaaagat gatgagggtagattcaagtcatcgttgataaacgatgttcaagggatg ttaagtttgtacgaggcagcatacatggcagttcgcggagaacatata ttagatgaagccattgctttcactaccactcacctgaagtcattggta gctcaggatcatgtaacccctaagcttgcggaacagataaatcatgct ttataccgtcctcttcgtaaaaccctaccaagattagaggcgaggtat tttatgtccatgatcaattcaacaagtgatcatttatacaataaaact ctgctgaattttgcaaagttagattttaacatattgctagagctgcac aaggaggaactcaatgaattaacaaagtggtggaaagatttagacttc actacaaaactaccttatgcaagagacagattagtggagttatatttt tgggatttagggacatacttcgagcctcaatatgcatttgggagaaag ataatgacccaattaaattacatattatccatcatagatgatacttat gatgcgtatggtacacttgaagaactcagcctctttactgaagcagtt caaagatggaatattgaggccgtagatatgcttccagaatacatgaaa ttgatttacaggacactcttagatgcttttaatgaaattgaggaagat atggccaagcaaggaagatcacactgcgtacgttatgcaaaagaggag aatcaaaaagtaattggagcatactctgttcaagccaaatggttcagt gaaggttacgttccaacaattgaggagtatatgcctattgcactaaca agttgtgcttacacattcgtcataacaaattccttccttggcatgggt gattttgcaactaaagaggtttttgaatggatctccaataaccctaag gttgtaaaagcagcatcagttatctgcagactcatggatgacatgcaa ggtcatgagtttgagcagaagagaggacatgttgcgtcagctattgaa tgttacacgaagcagcatggtgtctctaaggaagaggcaattaaaatg tttgaagaagaagttgcaaatgcatggaaagatattaacgaggagttg atgatgaagccaaccgtcgttgcccgaccactgctcgggacgattctt aatcttgctcgtgcaattgattttatttacaaagaggacgacggctat acgcattcttacctaattaaagatcaaattgcttctgtgctaggagac cacgttccattttga (Citrus valencene synthase) SEQ ID NO: 2 MSSGETFRPTADFHPSLWRNHFLKGASDFKTVDHTATQERHEALKEEV RRMITDAEDKPVQKLRLIDEVQRLGVAYHFEKEIEDAIQKLCPIYIDS NRADLHTVSLHFRLLRQQGIKISCDVFEKFKDDEGRFKSSLINDVQGM LSLYEAAYMAVRGEHILDEAIAFTTTHLKSLVAQDHVTPKLAEQINHA LYRPLRKTLPRLEARYFMSMINSTSDHLYNKTLLNFAKLDFNILLELH KEELNELTKWWKDLDFTTKLPYARDRLVELYFWDLGTYFEPQYAFGRK IMTQLNYILSIIDDTYDAYGTLEELSLFTEAVQRWNIEAVDMLPEYMK LIYRTLLDAFNEIEEDMAKQGRSHCVRYAKEENQKVIGAYSVQAKWFS EGYVPTIEEYMPIALTSCAYTFVITNSFLGMGDFATKEVFEWISNNPK VVKAASVICRLMDDMQGHEFEQKRGHVASAIECYTKQHGVSKEEAIKM FEEEVANAWKDINEELMMKPTVVARPLLGTILNLARAIDFIYKEDDGY THSYLIKDQIASVLGDHVPF

(181) Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention. Percentages disclosed herein may otherwise vary in amount by ±10, 20, or 30% from values disclosed herein.