WO2018178322A1

WO2018178322A1 - Use of nootkatone to treat infections caused by nematodes, trematodes, cestodes, or endoparasitic helminths

Info

Publication number: WO2018178322A1
Application number: PCT/EP2018/058263
Authority: WO
Inventors: Jean Davin Amick; P. M. Murali; K. Nalini; Anupam PALIWAL
Original assignee: Evolva Sa
Priority date: 2017-03-31
Filing date: 2018-03-29
Publication date: 2018-10-04

Abstract

Compositions and methods for treating and preventing nematode, trematode, cestode, and helminth infestations are disclosed herein.

Description

USE OF NOOTKATONE TO TREAT INFECTIONS CAUSED BY NEMATODES, TREMATODES,

CESTODES, OR ENDOPARASITIC HELMINTHS

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] This disclosure relates to the use of nootkatone-containing compositions to treat and kill helminths, nematodes, trematodes, and cestodes.

Description of Related Art

[0002] Helminths are parasitic worms grouped by similar life cycles and comprise four phyla: Nematoda, Platyhelminthes, Annelida, and Acanthocephala. Endoparasitic helminths include cestodes (tapeworms), trematodes (flukes), and nematodes (roundworms or nemathelminths).

[0003] Nematoda, otherwise known as roundworms or nematodes, is one of the most commonly occurring animal phyla in the world. Such a numerous and widespread phylum is inevitably diverse, but nematodes are typically described as being slender worms of 5 to 100 μηη thick and 0.1 to 2.5 mm long. The most well studied nematode is the well-known model species Caenorhabditis elegans, which was the first multicellular organism to undergo complete genome sequencing.

[0004] More than half of the 25,000 species of nematodes so far identified are parasitic, with numerous species being endoparasitic (living inside their hosts during one or more life cycle stages). Common nematodes that are human and/or animal parasites include ascarids (Ascaris), filiaris, Baylisascaris, trichina worms (Trichinella spiralis), heart worms (Dirofilaria immitis), hookworms, sheep worms (Haemonchus contortus), pinworms (Enterobius) and whipworms (Trichuris trichiura). Nematodes parasitic to humans and animals are typically transmitted either by ingesting nematode eggs in feces or by ingesting animal tissue from a nematode infested animal. Once eggs are swallowed, they hatch in the intestinal lumen, mature, mate, and produce eggs to be released in to the feces.

[0005] For example, the female pinworm Enterobius vermicularis lays eggs around the margin of the anus at night, causing itching or burning sensations that induce scratching and transfer of the eggs to fingers allowing re-infection or transmittal to a new host. In particular, school and preschool children who are still learning sanitary behavior are frequently infected with nematodes, either at communal toilets, sharing food, or through normal social play, and some reports estimate that up to 30% of U.S. children may be infected. Once a nematode infestation is detected, the entire human family unit must be treated at least twice interspersed by the maturation time of the nematode (current treatments are effective only against adult nematodes, not the eggs), and the low probability of effectively treating all family members of all of the infected children in a coordinated manner is further hindered by social taboos surrounding the open communication of nematode parasitization.

[0006] Another example is the intestinal roundworm (Ascaris lumbricoides). Adults are typically 5 mm in diameter, 20 to 350 mm in length, and females can lay 200,000 eggs per day that are individually encased in a thick shell that is dissolved in the digestive juices of a fresh host to release viable young nematodes. Over the subsequent eight weeks the young nematodes burrow through the intestinal wall and migrate via the bloodstream to the lungs, where they enter the alveoli and climb the bronchial tubes so that they may be swallowed back into the gastrointestinal tract to mature. Infections can cause pneumonia, anemia, and impaired childhood development.

[0007] However, transmission of parasitic nematodes also occurs via animal vectors, such as insects and arthropods. One example is the female mosquito that requires a blood meal prior to each cycle of egg production, resulting in re-feeding that may transmit nematodes from an existing host to a new host. Mosquito-transmitted nematodes include, but are not limited to, filarial worms (Wuchereria bancrofti and Brugia malayi) that cause elephantiasis. Similarly, the blackfly transmit Onchocerca volvulus, which causes onchocerciasis, also known as river blindness.

[0008] The Guinea worm, Dracunuculus medinensis, occurs in drinking water in Asia and Africa, and ingestion of infected copepods of the genus Cyclops releases nematode larvae that penetrate the human intestine, migrate to the skin, and grow to cause an ulcer which is capable of releasing fresh larvae when it comes into contact with water.

[0009] Some parasitic nematodes also infest and/or act as pathogens in humans, farmed animals (such as livestock), fish, birds, and companion animals. For example, trichina worms (Trichinella spiralis) causes potentially lethal trichinosis in rats, pigs, and humans, and is frequently transmitted across species by consumption of infected meat. Baylisascaris are a genus of roundworm nematodes that are known to infect more than fifty host species, and in paratenic hosts they may break out of the intestinal lumen, enter the bloodstream, and preferentially migrate to organs of the central nervous system including the eyes, spinal cord, or brain. Haemonchus contortus is present worldwide and is highly infectious, causing great economic damage as it is able to spread rapidly among sheep flocks. Dirofilaria immitis is a heartworm that infests the heart, arteries, and lungs of dogs and cats. The Toxocara genus includes T. canis (which infects dogs), T. cati, (which infects cats), T. leonina (which infects mostly cats but also dogs), and T. vitulorum (which infects ruminants including buffalo and cattle). Setaria is a genus of filarial roundworms that infects livestock and wildlife worldwide. There are several dozen species worldwide, such as Setaria bovis, Setaria digitata, and Setaria labiatopapillosa. Up to 40% of the cattle in India may be infected with several Setaria species. Some species (e.g., Setaria digitata, Setaria labiatopapillosa) can occasionally infect inadequate hosts such as sheep, goats, and horses and migrate to the nervous system causing life threatening mechanical damage or to the eyes (see parasitipedia.net).

[0010] Certain nematode species are plant parasites that are detrimental to commercial forestry, agriculture, horticulture, and leisure industries. Host plants include monocotyledonous, dicotyledonous, herbaceous, and woody plants including field crops, pasture, grassland, parkland, horticulture, ornamental, and vegetable crops. For example, the pine wood nematode Bursaphelenchus xylophilus infests pine tree bark in North America, Europe, and Asia. Nematodes also infest grass roots, which can lead to extensive damage of sporting fields, golf courses, parks, and residential lawns.

[0011] Root knot nematodes (Meloidogyne spp.) are known parasites of crop plants, collectively infecting the roots of approximately 2,000 plant species worldwide (including monocotyledonous, dicotyledonous, herbaceous, and woody plants) causing approximately 5% of global crop loss. Control of root-knot nematodes becomes extremely challenging using conventional methods once they become established in deep-rooted perennial crops. The Golden Nematode (Globdera rostochiensis) is a particularly harmful variety of nematode pest that has resulted in quarantines and crop failures worldwide. It forms cysts in the roots of Solanaceae plants such as potatoes, peppers, and tomatoes. In all cases, infestation of young plants leads to greater plant loss, whereas infestation of more mature plants results in significantly reduced yield.

[0012] One of the first attempts to address the serious agricultural challenge of plant-parasitic nematodes was the use of 1 ,2-dibromo-3-chloropropane (also DBCP or dibromochloropropane), but this persistent halocarbon causes male sterility in mammals exposed to high levels, and was progressively withdrawn between 1977 and 1985 after being linked to sterility in male workers. Moreover, residues have been identified in contaminated soil and groundwater nearly twenty years after the initial application. Since then, broad-spectrum carbamate insecticides (e.g., aldicarb) have been marketed and used as commercial plant-parasitic nematicides, but these too are no longer authorized for use in the European Union due to human health safety and environmental concerns. Also used are fumigants, such as methyl bromide or metam-sodium (Vapam®) and organophosphates, such as thionazin or fensulfothion. Yet, there are similar limitations associated with these chemical nematicides. [0013] While nematodes may represent a large group of helminths, other helminths cause considerable, widespread infections in humans and animals. For example, trematodes (from the class Trematoda), also known as flukes, infect humans worldwide. Flukes that cause infections in humans belong to the group of digenetic trematodes, which have complex life cycles where one or more life cycle stage occurs outside of the host organism. In contrast, monogenetic trematodes complete their entire life cycle within a single host. The most prevalent flukes causing human infections are Schistosoma species (blood fluke), Paragonimus westermani (lung fluke), and Clonorchis sinensis (liver fluke). Less prevalent flukes include the liver flukes Fasciola hepatica and Opisthorchis viverrini and the intestinal flukes Fasciolopsis buski, Heterophyes heterophyes, and Metagonimus yokogawai.

[0014] Trematode infections such as schistosomiasis (also known as bilharzia or "snail fever") have emerged as important tropical infections. An estimated 240 million people in the tropical belts of the world in as many as 78 countries may have schistosomal infection. This makes Schistosoma infection the second most prevalent tropical infectious disease in areas such as sub-Saharan Africa after malaria (see Parija et al., "Trematode Infection," Medscape, Article 876202, Nov. 23, 2015).

[0015] Schistosomiasis is transmitted by contact with contaminated fresh water (lakes and ponds, rivers, dams) inhabited by snails carrying the parasite. Swimming, bathing, fishing, doing laundry, and herding livestock can put people at risk of contracting the disease. Larvae emerge from snails and swim in the water until they come into contact with an individual and penetrate the skin. Once inside the body, the larvae develop into male and female worms which pair up and live together in the blood vessels for years. Female worms release thousands of eggs which are passed out of the body in the urine and feces and migrate to snails where they hatch and begin the cycle again. Schistosoma eggs can instead migrate to specific organs based on parasite type where they can inflict major damage. For example, urinary schistosomiasis causes scarring and tearing of the bladder and kidneys, and can lead to bladder cancer. Intestinal schistosomiasis develops slowly, causing abdominal bleeding; enlargement of the liver, lungs and spleen; and damage to the intestines.

[0016] Beyond human diseases, trematodes can also infect fish nurseries, grazing livestock, such as sheep and cattle, traditional (non-industrial) pig and poultry production, goats, horses, dogs, and cats. It follows that, trematode infections also impact agriculture. For example, economic losses associated with trematode infection of animals include costs of anthelmintics, labor associated with treating infected animals, losses in production due to mortality, reduction in meat, milk, and wool production, reduction in growth rate and fertility, and drench resistance. A non- limiting example of such a trematode is the parasitic flat worm of livestock ruminants, rumen-fluke (Paramphistomum sp.).

[0017] A further example of helminths is the cestodes, which are from the class Cestoda. Cestodes are a taxonomic class of parasitic flatworms in which the adult stage usually lives in the intestinal tract of vertebrates. Intermediate stages live in a wide variety of body locations in both vertebrate and invertebrate hosts. The bodies of most cestodes are ribbon-shaped and divided into short segments called proglottids, hence the name "tapeworm." It is estimated that at least 125 million humans are infected with tapeworms. Sources of human infection include ingestion of undercooked meat (e.g., pork, beef, and fish). However, tapeworms are known to infect all vertebrate species, therefore, their clinical and economic relevance is considerable. Examples include Taenia saginata (beef tapeworm), T. solium (pork tapeworm), Diphyllobothrium species, such as D. latum, D. klebanovskii, D. dendriticum, and D. nihonkaiense, which come from fish, and Hymenolepis or Echinococcus species that are found in areas where people live in or eat food prepared in conditions of poor hygiene.

[0018] In light of the discussion above, there remains a long felt need for safe and sustainable nematicidal, trematicidal, cesticidal, and/or helminth repellents and/or biocides for humans, animals, and plants. Preferred nematicidal, trematicidal, cesticidal, and/or helminth repellents and/or biocidal compositions would be broadly effective against a wide range of nematode, trematode, cestode, and helminth species and have low environmental impact due to rapid degradation in the environment. It would also be preferable to have compositions to treat and/or prevent nematode and other helminth infestation with an active agent that can be safely touched and/or ingested to allow repeated, safe treatment of humans, as well as agricultural, domesticated, and companion animals. Moreover, preferred compositions would be effective against multiple nematode, trematode, cestode, and helminth life cycle stages, rather than just the adult stage to reduce host to host and cross-species transmission. Therefore, there is a need to develop new compositions and methods that effectively control nematode, trematode, cestode, and helminth infestations in an efficient, safe, and environmentally friendly manner and that are effective against a wide range of nematode, trematode, cestode, and helminth species and life cycle stages.

SUMMARY OF THE INVENTION

[0019] It is against the above background that the present invention provides certain advantages and advancements over the prior art. In particular, as set forth herein, effective natural compositions and methods of their use to treat and prevent nematode, trematode, cestode, and helminth infestations are disclosed. [0020] In a first aspect, the invention provides a method of treating or preventing nematode, trematode, cestode, or helminth infection in a subject including applying a nootkatone-containing composition to a nematode, trematode, cestode, or helminth. The nootkatone-containing composition comprises an effective amount of nootkatone to repel or kill the nematode, trematode, cestode, or helminth.

[0021] In one embodiment of the first aspect, the nootkatone-containing composition is bergapten-free. In another embodiment of the first aspect, the nootkatone-containing composition comprises about 0.03% (v/v) nootkatone or greater. In a further embodiment of the first aspect, the nootkatone-containing composition is formulated for application topically on an exterior surface of a subject to be treated. In one embodiment of the first aspect, the composition is formulated as an aerosol, a solution, an emulsion, an oil, a lotion, a soap, a shampoo, a conditioner, a spray, a gel, a cosmetic, or a fragrance. In another embodiment of the first aspect, the method further includes applying a second effective amount of the nootkatone-containing composition after a period of 24 hours from the first application of the nootkatone-containing composition.

[0022] In another embodiment of the first aspect, the nootkatone-containing composition kills greater than 90% of the nematodes, trematodes, cestodes, or helminths within a period of 24 hours.

[0023] In a second aspect, the invention provides a method of treating or preventing nematode, trematode, cestode, or helminth infection including applying an effective amount of a nootkatone-containing composition to a nematode, trematode, cestode, or helminth vector.

[0024] In one embodiment of the second aspect, the nematode, trematode, cestode, or helminth vector is one or more of skin, hair, or other part of an animal, bird, fish or snail, a leaf, a stem, bark, a flower, a seed, a fruit, an edible portion of the plant, a root, tools, farming equipment, aquaculture equipment, food processing equipment, soil, a connected water system, a lake, a river, a stream, a sewer, a sewage treatment facility, a latrine, a toilet, a portable toilet, a child's potty seat, or a child's training potty.

[0025] In a third aspect, the invention provides a method of treating a nematode, trematode, cestode, or helminth infection including applying an effective amount of a nootkatone-containing composition to a nematode host, trematode host, cestode host, or helminth host.

[0026] In one embodiment of the third aspect, the nootkatone-containing composition is formulated for dispersing over an area to be treated. In another embodiment of the third aspect, the nootkatone-containing composition is formulated as a pellet, a grain, and/or a spray. In a further embodiment of the third aspect, the nootkatone-containing composition includes one or more of nitrogen, phosphorous, potassium, an herbicide, a plant seed, or a crop seed. In one embodiment of the third aspect, the nootkatone-containing composition is formulated for treating an intestinal infection. In another embodiment of the third aspect, the nootkatone-containing composition is formulated for ingestion as a food supplement, a paste, a gel, a syrup, or a liquid drench to be given orally, a granular form to be added to food, a pill, a chew tablet, or a liquid suspension. In a further embodiment of the third aspect, the nootkatone-containing composition is formulated as an extruded animal food, a grain, a canned food, an animal treat, a dog bone, an additive to fish food, or a coating on animal forage. In one embodiment of the third aspect, the nootkatone-containing composition is formulated as a tablet, a capsule, a granule, a pellet, a sachet, an emulsion, an elixir, a suppository for rectal administration. In another embodiment of the third aspect, the nootkatone-containing composition is formulated as a sterile injectable solution for parenteral use. In one embodiment of the third aspect, the nootkatone-containing composition comprises one or more of chitosan, juglone, thymol, wormwood, nematophagous fungi spores, or endospores of Pasteuria penetrans. In a further embodiment of the third aspect, the nootkatone-containing composition comprises one or more of mebendazole, albendazole, fenbendazole, moxidectin, levamisole, ivermectin, a plant essential oil compound, praziquantel, bithionol, triclabendazole, niclosamide, or derivatives thereof. In another embodiment of the third aspect, the nootkatone- containing composition comprises one or more of citronellal and piperazine citrate. In one embodiment according to any of the preceding aspects or embodiments, the nematode, trematode, cestode, or helminth infection is caused by one or more nematodes, trematodes, cestodes, or helminths of the genera Ascaris, Baylisascaris, Trichinella, Dirofilaria, Haemonchus, Enterobius, Trichuris, Wuchereria, Brugia, Onchocerca, Dracunuculus, Toxocara, Setaria, Bursaphelenchus, Meloidogyne, Globdera, Schistosoma, Paragonimus, Clonorchis, Fasciola, Opisthorchis, Fasciolopsis, Heterophyes, Metagonimus, Paramphistomum, Taenia, or Diphyllobothrium.

[0027] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.

DESCRIPTION OF DRAWINGS

[0028] Figure 1 illustrates a biosynthetic pathway for nootkatone.

[0029] Figure 2 shows the results of treating the nematode C. elegans with 0.03% nootkatone (v/v) in covered Petri dishes compared to an ethanol control. Results were measured after 24 hours. [0030] Figure 3 shows nematode worms {Toxocara vitulorum) from infected calf intestine.

[0031] Figure 4 shows nematicidal activity of 1 % and 5% nootkatone against cattle intestinal ascarid parasite T. vitulorum.

[0032] Figure 5 shows nootkatone's nematicidal efficacy against Toxocara vitulorum.

[0033] Figure 6 shows rumen fluke (Paramphistomum sp.) obtained from infected the rumen of infected calves.

[0034] Figure 7 illustrates an in-vitro assay for nootkatone's trematodicidal activity against rumen fluke.

[0035] Figure 8 shows the effects of different NK concentrations against rumen fluke.

[0036] Figure 9 is GC-FID chromatogram overlay of Frutarom® nootkatone (i.e., citrus-derived nootkatone) and the nootkatone (NxV) used for studies described herein (see Examples below).

[0037] Figure 10 is a GC-MS NIST library match of an unknown peak in Frutarom® nootkatone. The peak was identified as limonene. Neither limonene nor bergapten was found in the nootkatone used in the present application.

[0038] Figure 1 1 shows the experimental conditions for testing Groups 1 -9 (G1 -9) of Setaria bovis (round worms).

[0039] Figure 12A shows representative photographs of Setaria bovis 30 minutes post- treatment with nootkatone (NKT98%) (bottom row), citronellal (CT) (two middle rows), piperazine citrate (PC) (top center and top right dishes), or vehicle control (VC, top left dish).

[0040] Figure 12B is a graphical representation of the motility scoring of Setaria bovis 30 minutes post-treatment with nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), piperazine citrate (PC) (G2-G3), or vehicle control (VC) (G1 ).

[0041] Figure 12C is a graphical representation of the anthelmintic efficacy of nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), PC (G2-G3), or vehicle control (VC) (G1 ) in Setaria bovis at 30 minutes post-treatment.

[0042] Figure 13A shows representative photographs of Setaria bovis 1 hour post-treatment with nootkatone (NKT98%) (bottom row), citronellal (CT) (two middle rows), piperazine citrate (PC) (top center and top right dishes), or vehicle control (top left dish).

[0043] Figure 13B is a graphical representation of the motility scoring of Setaria bovis 1 hour post-treatment with nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), piperazine citrate (PC) (G2-G3), or vehicle control (VC) (G1 ). [0044] Figure 13C is a graphical representation of the anthelmintic efficacy of nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), PC (G2-G3), or vehicle control (VC) (G1 ) in Setaria bovis at 1 hour post-treatment.

[0045] Figure 14A shows representative photographs of Setaria bovis 3 hours post-treatment with nootkatone (NKT98%) (bottom row), citronellal (CT) (two middle rows), piperazine citrate (PC) (top center and top right dishes), or vehicle control (top left dish).

[0046] Figure 14B is a graphical representation of the motility scoring of Setaria bovis 3 hours post-treatment with nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), piperazine citrate (PC) (G2-G3), or vehicle control (VC) (G1 ).

[0047] Figure 14C is a graphical representation of the anthelmintic efficacy of nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), PC (G2-G3), or vehicle control (VC) (G1 ) in Setaria bovis at 3 hours post-treatment.

[0048] Figure 15A is a graphical representation of the motility scoring of Setaria bovis 6 hours post-treatment with nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), piperazine citrate (PC) (G2-G3), or vehicle control (VC) (G1 ).

[0049] Figure 15B is a graphical representation of the anthelmintic efficacy of nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), PC (G2-G3), or vehicle control (VC) (G1 ) in Setaria bovis at 6 hours post-treatment.

[0050] Figure 16A is a graphical representation of the motility scoring of Setaria bovis 10 hours post-treatment with nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), piperazine citrate (PC) (G2-G3), or vehicle control (VC) (G1 ).

[0051] Figure 16B is a graphical representation of the anthelmintic efficacy of nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), PC (G2-G3), or vehicle control (VC) (G1 ) in Setaria bovis at 10 hours post-treatment.

[0052] Figure 17A is a graphical representation of the motility scoring of Setaria bovis 18 hours post-treatment with nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), piperazine citrate (PC) (G2-G3), or vehicle control (VC) (G1 ).

[0053] Figure 17B is a graphical representation of the anthelmintic efficacy of nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), PC (G2-G3), or vehicle control (VC) (G1 ) in Setaria bovis at 18 hours post-treatment. [0054] Figure 18A is a graphical representation of the motility scoring of Setaria bovis 24 hours post-treatment with nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), piperazine citrate (PC) (G2-G3), or vehicle control (VC) (G1 ).

[0055] Figure 18B is a graphical representation of the anthelmintic efficacy of nootkatone (NKT98%) (G7-G9), citronellal (CT) (G4-G6), PC (G2-G3), or vehicle control (VC) (G1 ) in Setaria bovis at 24 hours post-treatment.

DETAILED DESCRIPTION

[0056] All publications, patents, and patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes.

[0057] 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 "an active ingredient" means one or more active ingredients. Moreover, as used herein, when the pluralized form of any word is used herein, unless otherwise indicated, the singular form of the word is contemplated. For example, reference to "plants" can contemplate a single "plant."

[0058] 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.

[0059] 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.

[0060] As used herein, the term "about" refers to ±10% of any particular value.

[0061] As used herein, the terms "or" and "and/or" are 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." [0062] As used herein, the term "active ingredient" refers to a chemical compound and mixtures of chemical compounds that kill, render immobile, prevent progression into another stage of the life cycle, or repel nematodes in or from a host or a treated surface during one or more life cycle stages. Active ingredients may include any natural or chemical nematicide and derivatives thereof.

[0063] As used herein, the term "derivative" refers to a molecule or compound that is derived from a similar compound by some chemical or physical process.

[0064] As used herein, the term "life cycle stage" refers to the life cycle stages of nematodes including egg, larva or juvenile stages, and adult stages, as well as the periods of transition between stages. Further, the term "life cycle stage" also refers to the life cycle stages of and helminths, such as trematodes, that include egg, miracidium, cercarial larva, and adult stages as well as the periods of transition between stages. In addition, the term "life cycle stage" refers to the life cycle stages of endoparasitic helminths, such as cestodes, that include egg, proglottid, hexacanth embryo, larva, and adult stages as well as the periods of transition between stages. Further, the term "life cycle stage" refers generally to any life cycle stage of helminths.

[0065] As used herein, the term "life cycle stage population" refers to all members of any particular life cycle stage, such as all nematodes, trematodes, cestodes, or helminth eggs or all nematode or other helminth adults.

[0066] As used herein, the term "nematode" refers to any nematode species. Examples include members of the phylum Nematoda. Further, the term "nematode" refers to nematodes in any life cycle stage: egg stage, larva or juvenile stages, or adult stage as well as the periods of transition between stages. For example, nematodes can be any parasitic nematode. Specific non- limiting examples include species of the genus Ascaris such as A. lumbricoides, or species of the genus Enterobius, such as E. vermicularis, or species of the genus Baylisascaris such as B. melis, B. transfuga, B. devosi, B. laevis, B. schroederi, and B. potosis, or species of the genus Meloidogyne, such as M. javanica, M. arenaria, M. incognita, and M. halpa, or species of the genus Trichuris, such as T. trichiura, or species of the genus Trichinella, such as T. spiralis, or species of the genus Globodera such as G. rostochiensis, or species of the genus Toxocara, such as T. canis, T. cati, T. leonina, and T. vitulorum. In addition, "nematode" refers to any nematodes that cause the diseases ascariasis, trichuriasis, hookworm, enterobiasis, strongyloidiasis, filariasis, setariasis / setariosis, trichinosis, and anisakiasis.

[0067] As used herein, the term "trematode" refers to any trematode species. Examples include members of the phylum Platyhelminthes, class Trematoda. Further, the term "trematode" refers to trematodes in any life cycle stage: egg stage, larva or juvenile stages, or adult stage as well as the periods of transition between stages. For example, trematodes can be any parasitic trematode. Specific non-limiting examples include species of the genera Schistosoma (blood fluke) and Alaria or for example, the species Paragonimus westermani (lung fluke) and Clonorchis sinensis (liver fluke). Additional examples include liver flukes (e.g., Fasciola hepatica and Opisthorchis viverrini), intestinal flukes (e.g., Fasciolopsis buski, Heterophyes heterophyes, and Metagonimus yokogawai), and rumen flukes (e.g., Paramphistomum sp.). In addition, "trematode" refers to any trematodes that cause the diseases schistosomiasis, clonorchiasis, fasciolosis, echinostomiasis, opisthorchiasis, paragonimiasis, metagonimiasis, fasciolopsiasis, metorchiasis, and dicrocoeliasis. Specific non-limiting examples of endoparasitic trematodes include species of the genus Schistosoma, such as S. mansoni, S. japonicum, and S. haematobium, species of the genus Paragonimus, such as P. westermani, species of the genus Clonorchis, such as C. sinensus, species of the genus Fasciola, such as F. hepatica, species of the genus Opisthorchis, such as O. viverrini, species of the genus Fasciolopsis, such as F. buski, species of the genus Heterophyes, such as H. heterophyes, and species of the genus Metagonimus, such as M. yokogawai.

[0068] As used herein, the term "cestode" refers to any cestode species. Examples include members of the phylum Platyhelminthes, class Cestoda. Further, the term "cestode" refers to cestodes in any life cycle stage: egg stage, larva or juvenile stages, or adult stage as well as the periods of transition between stages. For example, cestodes can be any parasitic cestode. Specific non-limiting examples include the species of Taenia saginata (beef tapeworm) and T. solium (pork tapeworm), Raillientina species, Diphyllobothrium species, such as D. latum, D. klebanovskii, D. dendriticum, D. mansonoides, and D. nihonkaiense, and Hymenolepis or Echinococcus species. In addition, "cestode" refers to any cestodes that cause the diseases diphyllobothriasis or echinococcosis. Specific non-limiting examples of endoparasitic cestodes include species of the genus Taenia, such as T saginata and T solium, species of the genus Diphyllobothrium, such as

D. latum, D. klebanovskii, D. dendriticum, and D. nihonkaiense, species of the genus Hymenolepis, such as H. diminuta and H. nana, and species of the genus Echinococcus, such as E. granulosus,

E. multilocularis, E. vogeli, and E. oligarthrus.

[0069] As used herein, the term "helminth" refers to a parasitic worm. Possible hosts for a particular helminth are typically relatively specific because of co-evolution of the host-parasite interaction, but as a class, the known hosts of helminths include but are not limited to humans, plants, animals, birds, fish, snails, large crustaceans, reptiles, and sharks. Examples of helminths include endoparasitic nematode species, as well as endoparasitic trematode and cestode species. Further, the terms "nematodes, trematodes, cestodes, or helminths" " refer to nematodes, trematodes, cestodes, or helminths in any life cycle stage as well as the periods of transition between stages. All nematodes, trematodes, cestodes, or helminths lay ova and form larvae that hatch and mature into adults.

[0070] As used herein, the term "subject to be treated" refers to a host, such as a paratenic host, an intermediate host, or definitive host, or non-host human, animal, plant, harvested crop, or part thereof in or on which at least one nematode, trematode, cestode, or helminth could survive, grow, or reproduce. Examples of plant hosts include but are not limited to monocotyledonous and dicotyledonous, herbaceous, and woody plants. Additional examples include grasses, bananas, potatoes, tomatoes, cucurbits, cassava, soybean, corn, sugar beet, citrus plants, grapes, coffee, and others. Further examples of hosts, according to the present invention include areas including infected plants, such as lawns, playing fields, recreational areas, and agricultural fields.

[0071] As used herein, the term "nematode, trematode, cestode, or helminth vector" refers to any host or surface where a nematode, trematode, cestode, or helminth, could survive, grow, reproduce, or be transferred to a host. Examples of surfaces include skin, hair, intestines, muscle, internal organs, vasculature, or other part of an animal where an nematode, trematode, cestode, or helminth can exist, a plant part such as a leaf, a stem, bark, a flower, a seed, a fruit, an edible portion of the plant, or a root, soil, tools, farming equipment, a connected water system, a water container for use in farming or aquaculture practices suitable for introduction of the host for parasite treatment, bodies of water such as lakes, rivers, streams, and surrounding areas, sewers, sewage treatment facilities, latrines, toilets, portable toilets, a child's potty seat, a child's training potty, and any other surface where nematodes, trematodes, cestodes, or helminths in any stage of their life cycle are likely be found.

[0072] As used herein, the term "treatment of nematodes," "treatment of trematodes," "treatment of cestodes," or "treatment of helminths" refers to a process by which a nematode, trematode, cestode, or helminth in any life cycle stage is at least one of contacted with a composition including an active ingredient, killed, repelled from a surface, prevented from laying eggs, prevented from transitioning from one life cycle stage to the next, or prevented from infecting a host. Treatment of a nematode, trematode, cestode or helminth can occur within a host administered an effective concentration of an active ingredient, such as in medicament, in drinking water, food, feed, or supplements or constituents thereof, or ex vivo, such as on the ground, in a cattle yard, a poultry yard, in drinking water, in a water body, in a treatment dip, in a treatment tank of a well boat, on a plant part, in feces, and other places where an ex vivo nematode, trematode, cestode, or helminth life cycle stage occurs.

[0073] As used herein, the term "effective concentration" refers to a concentration of an active ingredient (such as nootkatone) within a composition such that when the composition is applied to a nematode, trematode, cestode, or helminth or to a relevant surface, a nematode, trematode, cestode, or helminth that is contacted by the composition is repelled and/or experiences one or more of paralysis, poisoning, neuro-muscular damage, or death. An "effective concentration" is also one that prevents egg laying, transitioning from one life cycle stage to the next, or infection of a host.

[0074] As used herein, the term "effectively treat" refers to at least one of directly (e.g., by contacting nematode, trematode, cestode, or helminth, or its immediate surroundings) or indirectly (e.g., by contacting a nematode, trematode, cestode, or helminth infection site or other surface that a nematode, trematode, cestode, or helminth will be affected by) repelling, paralyzing, poisoning, damaging neuro-muscular tissue of, killing, preventing egg laying or transitioning from one life cycle stage to the next, or preventing infection of a host. Further, as used herein, the term "applying" refers to directly or indirectly contacting a substance to an intended target. For example, by applying a nootkatone-containing composition to a nematode, trematode, cestode, or helminth, the composition may be directly applied to the nematode, trematode, cestode, or helminth, or conversely, the composition may be applied to an infected individual or host and subsequently the nootkatone-containing composition contacts (indirectly) the nematode, trematode, cestode, or helminth.

[0075] As used herein, the term "nootkatone" refers to a compound seen in Figure 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.

[0076] As used herein, the term "nootkatone ex valencene" refers to nootkatone derived from oxidation of valencene that was produced by culturing (also called fermentation) recombinant microorganisms, 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 produced by recombinant microorganisms and nootkatone derived from valencene produced by recombinant microorganisms are free of bergapten. 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.

OVERVIEW

COMPOSITIONS

[0077] Nootkatone-containing compositions contemplated herein can be formulated for direct application to a host or surface to effectively treat existing nematode, trematode, cestode, or helminth life cycle stage populations or as a prophylactic to prevent the growth or spreading of nematode, trematode, cestode, or helminth to new hosts.

[0078] Generally and without limitation, compositions contemplated herein can be in the form of an aqueous liquid, an oil-based liquid, a concentrated liquid, a gel, a foam, an emulsion, a slurry, a paint, a clear coat, a wax, a block, a pellet, a puck, a dunk, a granule, a powder, a capsule, a vesicle, a tablet, an effervescent tablet, a slow release tablet, an impregnated dissolvable sheet or film, an impregnated material, and combinations thereof. Further compositions may be configured for immediate release, delayed release, intermittent release, or extended release by inclusion of excipients and/or packaging structures and/or materials that enable such release profiles.

[0079] In certain aspects, a composition may be formulated as a liquid or aerosol formulation suitable for application in a spray, an intravenous or intramuscular infusion, a roll on, a dip, a detergent, a foam, or suitable for inhalation.

[0080] In certain aspects, a composition may be formulated for application by dispensing into or onto an area of a connected water system to be distributed throughout the system. In this context, the composition can be provided as a solution, an emulsion, an oil, a spray, a gel, a powder, a foam, a block, a pellet, a dunk, a puck, a composition-filled dissolvable pouch, a granule, a vesicle, a capsule, an intravenous or intramuscular infusion, and combinations thereof.

[0081] Deworming compositions to treat intestinal nematode infestations in humans (especially children), farm animals (such as sheep, pigs, horses, cows, camels), companion animals (such as dogs and cats), or in aquaculture can be formulated for ingestion as a drinking water or food 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, wettable powders 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.

[0082] Deworming compositions 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, in aerosol form for inhalation; or in the form of sterile injectable solutions for parenteral (including subcutaneous, intravenous, or intramuscular) 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 may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.

[0083] Compositions for oral consumption to treat nematodes, trematodes, cestodes, or helminths 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).

[0084] In still further embodiments, a composition may be formulated for ingestion such as may be added to a water reservoir to control nematode or other nematode, trematode, cestode, or helminth larvae and oral/systemic formulations for animals that serve as disease reservoirs. In other embodiments, a composition may be formulated for ingestion by an animal to convey nematode, trematode, cestode, or helminth repellency or resistance through the slow release of active ingredient (such as nootkatone) or through secretion of an active ingredient onto the skin, hair, fur, and the like or passing an active ingredient in urine or feces onto a surface.

[0085] Compositions containing nootkatone to treat or prevent nematode, trematode, cestode, or helminth infestations in humans, farm animals (such as sheep, pigs, horses, cows, camels, or fish), or companion animals (such as dogs and cats) can be formulated for ingestion as a food 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.

[0086] Compositions for ingestion can be administered hourly, daily, weekly, or monthly, as required. Compositions can be administered prophylactically, for example, when an individual is anticipated to be in an area where nematode, trematode, cestode, or helminth infestation is expected or when a change of season occurs where an increase in nematode, trematode, cestode, or helminth populations or different nematode, trematode, cestode, or helminth species is expected.

[0087] In certain embodiments, a composition dosage form can provide an amount of active ingredient (e.g., nootkatone) per dose to an individual in the range of about 5 mg to about 1000 mg, or in the range of about 5 mg to about 800 mg, or in the range of about 5 mg to about 500 mg, or in the range of about 5 mg to about 200 mg, or in the range of about 5 mg to about 100 mg, or in the range of about 5 mg to about 50 mg, or in the range of about 10 mg to about 1000 mg, or in the range of about 10 mg to about 800 mg, or in the range of about 10 mg to about 500 mg, or in the range of about 10 mg to about 200 mg, or in the range of about 10 mg to about 100 mg, or in the range of about 10 mg to about 50 mg.

[0088] Compositions to treat lawns, playing fields, recreational areas, and/or agricultural fields, for example, can be formulated for broadcasting or dispersing over the area to be treated, such as in the form of pellets, grains, powders and/or sprays. In addition to an active ingredient, they can be formulated to include "turf builders" that provide nutrients to grasses and plants, such as nitrogen, phosphorous, and potassium. Forms of nitrogen that can be used include ammonium (NH₄ ⁺), nitrate (N0₃ ^"), and urea. In one embodiment, a composition for treating lawns, playing fields, and/or recreational areas can further be formulated to include herbicides to control weeds as well as grass seed or other plant seeds. In another embodiment, a composition for treating an agricultural field can further include a pesticide, an herbicide, and crop seeds (e.g., for corn, wheat, soybeans, etc.). In one aspect of this embodiment, the pesticide is effective against a vector of a nematode, trematode, cestode, or helminth. In another aspect, the active ingredient effective at treating or preventing infection by a nematode, trematode, cestode, or helminth is also effective as a pesticide against at least one vector and/or temporary host (such as a sap-sucking insect) of a nematode, trematode, cestode, or helminth.

[0089] In other embodiments of the invention, compositions contemplated herein can contain any amount of nootkatone. In another embodiment, compositions contemplated herein can contain a carrier and at least about 0.001 %, or at least about 0.005%, or at least about 0.01 %, or at least about 0.02%, or at least about 0.03%, or at least about 0.04%, or at least about 0.05%, or at least about 0.06%, or at least about 0.07%, or at least about 0.08%, or at least about 0.09%, or at least about 0.1 %, or at least about 0.2%, or at least about 0.3%, or at least about 0.4%, or at least about 0.5%, or at least about 0.6%, or at least about 0.7%, or at least about 0.8%, or at least about 0.9%, or at least about 1 %, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, 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 30%, or greater than about 35%, or greater than about 40%, or greater than about 45%, or greater than about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99% by weight nootkatone.

[0090] In one example, the provided compositions contain nootkatone in an amount at or about 0.001 % to at or about 2%, or about 0.01 % to at or about 5%, or about 0.01 % to at or about 75% by weight of the composition. In another example, a composition may contain nootkatone 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 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 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 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 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 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 in an amount of up to about 99% or more by weight of the composition.

[0091] In one particular embodiment, a contemplated nootkatone-containing composition is provided as a concentrate. For example, a nootkatone-containing composition may be provided as a 20X, or a 10X, or a 5X, or a 3X concentrate that can be diluted by an end user with an appropriate solvent or by application to a surface to be treated to achieve a 1 X (or other desired) working concentration. Alternatively, a nootkatone-containing composition may be provided to an end user at a 1X working concentration. However, any concentration is contemplated for use herein. For example, compositions provided as concentrates can be used without dilution at all or may be diluted from a highly concentrated concentrate {e.g., about 20X to about 100X, or about 30X to about 60X, or about 30X, or about 60X) to some multiple of concentration higher than 1 X, such as 2X, 2.5X, 3X, etc. or can be used at a more dilute concentration, such as 1/2X, 1/4X, 1/1 OX, etc.

[0092] In one embodiment, a final working concentration can be based on the relative surface area to be treated, such as in the context of a lawn or agricultural field. Specific final working concentration examples can be about 5 mmol/m², or about 10 mmol/m², about 15 mmol/m², about 25 mmol/m², about 50 mmol/m², about 60 mmol/m², about 70 mmol/m², about 80 mmol/m², about 90 mmol/m², about 100 mmol/m², or higher.

[0093] However, in the context of an application to an infected individual, the final working concentration can be determined by an appropriate dose-response curve.

[0094] In another embodiment, a contemplated composition may be seen in Table No. 1 , where ingredients can be measured in percent volume per volume, percent weight per volume, or percent by weight.

[0095] Table No. 1. Contemplated composition formulation.

[0096] In certain embodiments, compositions contemplated herein may include nootkatone and one or more additional active ingredients. The one or more additional active ingredients may be effective against a nematode, trematode, cestode, or helminth. In another embodiment, a contemplated composition may include one or more active ingredients against a specific life cycle stage population, such as nematode, trematode, cestode, or helminth eggs, and one or more active ingredients against a different life cycle stage population, such as adult nematodes, trematodes, cestodes, or helminths. In another embodiment, an additional active ingredient can have a different effective treatment profile than nootkatone (e.g., it may be life cycle stage population specific).

[0097] Additional active ingredients include any natural or chemical nematicide, trematicide, cestide, or helminth biocide or repellent, and derivatives thereof. Examples of current commercial nematicides include chitosan, a natural biocontrol agent able to elicit plant defense responses to destroy parasitic cyst nematodes on roots of soybean, corn, sugar beet, potato and tomato crops. Additional biocontrol agents include nematophagous fungi, such as Paecilomyces lilacinus and Verticillium chlamydosporium, bacterial parasites, such as Pasteuria penetrans, and plant derivatives, such as juglone, thymol, and wormwood, among others. Another example of an active ingredient includes nootkatone and/or a derivative thereof. In one embodiment, compositions contemplated herein may include nematophagous fungi spores and/or endospores of Pasteuria penetrans.

[0098] Additional examples of additional active ingredients include drugs such as mebendazole and albendazole that can be administered orally to humans, animals, birds and fish. Other drugs that can be used herein include fenbendazole, moxidectin, levamisole, and ivermectin. Further drugs that can be used include praziquantel, bithionol, triclabendazole, and niclosamide. Additional active ingredients include piperazine citrate.

[0099] Further examples of additional active ingredients include plant essential oil compounds or derivatives thereof. Examples include aldehyde C16 (pure), oterpineol, amyl cinnamic aldehyde, amyl salicylate, anisic aldehyde, benzyl alcohol, benzyl acetate, cinnamaldehyde, cinnamic alcohol, carvacrol, carveol, citral, citronellal, citronellol, p-cymene, diethyl phthalate, dimethyl salicylate, dipropylene glycol, eucalyptol (cineole) eugenol, is-eugenol, galaxolide, geraniol, guaiacol, ionone, menthol, methyl salicylate, methyl anthranilate, methyl ionone, methyl salicylate, opheliandrene, pennyroyal oil perillaldehyde, 1 - or 2-phenyl ethyl alcohol, 1 - or 2- phenyl ethyl propionate, piperonal, piperonyl acetate, piperonyl alcohol, D-pulegone, terpinen-4-ol, terpinyl acetate, 4-tert butylcyclohexyl acetate, thyme oil, thymol, lavender oil, rosemary oil, peppermint oil, neem oil, clove extract, metabolites of trans-anethole, vanillin, and ethyl vanillin.

[00100] In another embodiment, a contemplated composition can include a nootkatone to additional active ingredient ratio of about 1 :10, or about 1 :8, or about 1 :6, or about 1 :4, or about 1 :2, or about 1 :1 , or about 2:1 , or about 4:1 , or about 6:1 , or about 8:1 , or about 10:1 .

[00101] In other embodiments, compositions contemplated herein can include nootkatone in combination with one or more additives, such as a fragrance, a preservative, a propellant, a pH buffering agent, a UV blocker, a pigment, a dye, a surfactant, an emulsifier, a solvent, a salt, an acid, a base, an emollient, a sugar, and combinations thereof. Additional additives include disinfectants, larval-stage nematode, trematode, cestode, or helminth attractants or chemical lures and detergents. Contemplated disinfectants include quaternary ammonium compounds, phenol- based antimicrobial agents, and botanical oils with disinfectant properties. Additional additives include inactive ingredients for pharmaceutical formulations, such as diluents, lubricants, solubilizers, alcohols, binders, controlled release polymers, enteric polymers, disintegrants, colorants, flavorants, sweeteners, antioxidants, preservatives, pigments, fillers, suspension agents, surfactants (for example, anionic, cationic, amphoteric and nonionic), and the like.

[00102] In certain embodiments, a nootkatone-containing composition suitable for treating or preventing infection by nematodes, trematodes, cestodes, or helminths can be formulated for application topically on an exterior surface of a subject to be treated, for example, to the lips, hands, perianal region, or skin of a human or other animal. 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, or a fragrance.

[00103] In other embodiments, nootkatone-containing compositions can include a carrier, such as an aqueous liquid carrier, water, a saline, a gel, an inert powder, a zeolite, a cellulosic material, a microcapsule, an alcohol such as ethanol, a hydrocarbon, a polymer, a wax, a fat, an oil, a protein, a carbohydrate, and combinations thereof. Some carriers include time release materials where a nootkatone-containing composition may be released over a period of hours, or days, or weeks.

[00104] Carriers 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. In another embodiment, a carrier can be included in an amount up that when added to the amount of nootkatone included in the composition amounts to 100% by volume.

METHODS

[00105] According to some aspects of the current invention, nootkatone-containing compositions suitable for treating or preventing infection by nematodes, trematodes, cestodes, or helminths can be directly applied to nematodes, trematodes, cestodes, or helminths and vectors or temporary hosts of nematodes, trematodes, cestodes, or helminths. According to other aspects of the current invention, nootkatone-containing compositions suitable for treating or preventing infection by nematodes, trematodes, cestodes, or helminths can be applied to a surface area and/or material where nematodes can occur during transport from one host to another or inhabit during any stage of their life cycle, or surfaces and objects on which nematodes, trematodes, cestodes, or helminths at any stage of their life cycle can be detected or that commonly act as vectors for their transportation. Examples of such surfaces include, without limitation, water surfaces (e.g., of ponds, lakes, canals, creeks, ditches, irrigation channels, or marshy areas), the edges of water bodies (e.g., shorelines, pool liners and/or covers, banks, etc.), sewers, sanitation systems (including but not limited to taps, toilets, toilet roll holders, sinks, wash basins, showers, bathrooms, changing rooms), cattle yards, manure piles, lawns, playing fields, recreational areas, and/or agricultural fields, or portions thereof, animal troughs, catch basins, farm or aquaculture wash tanks or treatment tanks, or any surface of equipment, or tool used in conjunction with any of the aforementioned objects.

[00106] Treatment for nematode, trematode, cestode, or helminth infestation may be routine or prophylactic based on changing environmental conditions (such as raised humidity or temperature), seasonal changes (such as transitions from spring to summer to fall to winter to spring), flooding, observation of nematode, trematode, cestode, or helminth larvae, or in response to reports of nematode, trematode, cestode, or helminth infestations amongst peers or associates. Alternatively or in addition, contemplated methods include application of nootkatone-containing compositions suitable for treating or preventing infection by nematodes, trematodes, cestodes, or helminths to toilets, bathrooms, swimming pools, changing tables for infants, laundry, bedsheets, diapers, underwear, and pajamas.

[00107] According to some aspects of the current invention, nootkatone-containing compositions suitable for treating or preventing infection by nematodes, trematodes, cestodes, or helminths may be applied about once per day, about once every 3 days, about once per week, about twice per week, about once per two weeks, about once per month, about once per two months, or about once per three months, or about once per season.

[00108] According to some aspects of the current invention, nootkatone-containing compositions suitable for treating or preventing infection by nematodes, trematodes, cestodes, or helminths may be applied with a frequency calculated based on a particular nematode, trematode, cestode, or helminth to be treated and at particular temperatures at which they will be treated such that if a first treatment is applied to a surface or vector, a second treatment may be applied to the same surface or vector before the end of the adult stage as counted from the day before the first treatment was applied. In this manner, the first treatment is effective against adult nematodes, trematodes, cestodes, or helminths and/or any other life cycle stage present at that time, and the second treatment is effective against nematodes, trematodes, cestodes, or helminths resulting from eggs laid by mature nematodes, trematodes, cestodes, or helminths of the last generation immediately prior to the first treatment that would have been in adult form during the first treatment. If any stage of a life cycle is shorter than the adult stage, several treatments may be applied until the maximum time for adult stage has passed. For example, when a first application of nootkatone- containing composition suitable for treating or preventing infection by nematodes, trematodes, cestodes, or helminths is applied to a surface or nematode vector on d=0, a second application of nootkatone-containing compositions may be applied at d=1 or 2, 1 or 2 days later to treat any larvae newly hatched from eggs laid by nematodes, trematodes, cestodes, or helminths that were adults when the first nootkatone-containing compositions was applied. Optionally, additional treatments of nootkatone-containing composition may be applied to the surface or vector approximately every subsequent day (e.g., d=3, d=4, d=5) and so on until the maximum adult life of the nematode, trematode, cestode, or helminth that emerged from a larva the day before treatment (d=-1 ) has expired.

[00109] Various methods according to some aspects of the current invention may be employed to contact nematodes, trematodes, cestodes, or helminths, objects, surfaces, hosts, potential hosts, vectors, and environments rich in nematodes, trematodes, cestodes, or helminths with nootkatone-containing compositions.

[00110] Nootkatone can be applied, such as by directly pouring the composition into the water or placing a composition dispenser within a connected water system or nematode, trematode, cestode, or helminth vector such that the surface or environment to be treated comes into contact with the nootkatone at an effective concentration of, for example, between 100 and 2,000 ppm, preferably between 200 and 400 ppm, most preferably approximately 300 ppm. In some embodiments, the applied nootkatone-containing composition suitable for treating or preventing infection by nematodes, trematodes, cestodes, or helminths can be left without active removal to degrade naturally.

DISPENSERS/APPLICATORS

[00111] In some embodiments, dispensers or applicators for dispensing or applying a composition contemplated herein are intended to be reused. For example, upon dispensing a nootkatone-containing composition suitable for treating or preventing infection by nematodes, trematodes, cestodes, or helminths, the dispenser or applicator can be refilled. In other embodiments, a dispenser or applicator is a single-use device or substance that functions as a nootkatone composition carrier that is, itself, dispensed or degraded. For example, a dispenser or applicator can be a dissolvable vehicle such as a pouch, a puck, a pellet, a block, a granule, a vesicle, or a capsule that contains at least one additional substance (i.e., carrier) that contributes to at least one of the structure of the dispenser or application or a controlled release of the nootkatone from the dispenser or applicator. [00112] Topical compositions are also contemplated herein that may be dispensed using a dispenser or applicator including one or more of a spray bottle, a brush, a dropper, a sponge, a soft-tipped marking device with reservoir, a pressurized dispenser, an aerosol can, a roll on bottle, a wipe, a tissue, a tube, and other devices suitable for application to surfaces, objects, or environments rich in nematodes, trematodes, cestodes, or helminths.

[00113] In one embodiment, compositions contemplated herein may be applied to one or more surfaces using an applicator having a reservoir for carrying a composition in a wet form and/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 pump sprayer, a trigger sprayer, or a pressurized spraying device. The composition may alternatively be applied by spraying or dispersing over at least a portion of an area susceptible to infestation by nematodes, trematodes, cestodes, or helminths, including but not limited to spraying from a tractor, boat, irrigation spray, helicopter, crop duster or airplane or by way of a broadcast spreader.

[00114] In a further embodiment, methods for treating connected water systems may include utilization of nootkatone-containing compositions suitable for treating or preventing infection by nematodes, trematodes, cestodes, or helminths via dispensers that release a contemplated composition into a body of water (treatment locale, including but not limited to agriculture, aquaculture, or harvested product thereof treatment tank) or drinking water supply over a period of time of minutes, hours, days, or weeks. Contemplated dispensers include floating dispensers that float and dispense at the surface of a body of water. For example, contemplated floating dispensers include those that are used for chlorine dispensing in swimming pools or for dispensing water supplements in drinking troughs or for dispensing fertilizer in irrigation systems. Floating dispensers may float on the surface of a locale either freely or may be anchored. Further, when anchored, floating dispensers may float subsurface at a predetermined and adjustable depth.

[00115] Other dispensers include sink-floats that may be immersed within a treatment area and sink to or near the bottom of a treatment locale until such time as the treatment composition is completely released, at which time the dispenser floats to the surface to be recharged. In another embodiment, a weighted and buoyed dispenser may be used that includes a weighted composition dispenser connected to a floating buoy that suspends the dispenser at a predetermined depth at which depth the composition is dispensed.

[00116] In another embodiment, it is contemplated that a "use up cue" may be included in the contemplated dispensers, such as, for example, a beacon that gives off light and/or sound or changes color when a treatment composition has been nearly or completely used up. The use up cue may be based on a timer, in that, after a predetermined length of time that coincides with the time when the treatment composition is nearly or fully dispensed, the use up cue is triggered by the timer.

[00117] In the context of the sink float, the use up cue may function based on the rate of solubility of the treatment composition, such that when the treatment composition is fully dissolved, the weight loss from the sink float causes the float to rise to the surface. Alternatively, the use up cue of the sink float may be based on the rate of solubility of a companion substance within the sink float that dissolves at a rate corresponding to the rate of dispensing of the treatment composition.

[00118] In a further embodiment, a dispenser for use at the edge of a body of water may be in the form of a spike or similar device that can be driven into the bed of the body of water or at the edge of the body of water. The nootkatone composition within the device can then leach out into the body of water to effectively treat any present nematodes, trematodes, cestodes, or helminths and prevent further infestation of the connected water system.

[00119] Another aspect of the current invention includes pretreatment of surfaces, objects, environments prone to infestation with nematodes, trematodes, cestodes, or helminths in any stage of the life cycle, such as ova/eggs. This may be accomplished by coating the surfaces or objects with compositions that resist removal from the surface and contain an amount of a nootkatone, such as a paint, a clear coat, a wax, an oil, an adhesive, a resin, a cleaning solution, and combinations thereof. Another approach includes lining the surfaces, objects, environments rich in hosts or otherwise prone to infestation with nematodes with one or more nootkatone- impregnated materials, such as thermoplastic or thermoset sheets impregnated with nootkatone.

[00120] A further treatment approach is to construct surfaces or objects with nootkatone- impregnated or nootkatone-coated materials, such as plastics, wood, cloth, textiles, composites, or porous materials to prevent infestation of connected water systems and other areas where nematodes, trematodes, cestodes, or helminths may spend part of their life cycle or transit between hosts. The approaches disclosed herein can be used alone or in any combination.

EXAMPLES

[00121] The Examples that follow are illustrative of specific embodiments of the invention, and the use thereof. It is set forth for explanatory purposes only and is not taken as limiting the invention. In particular, the example demonstrates the effectiveness of nootkatone-containing compositions against nematodes, trematodes, cestodes, or helminths.

Example No. 1 : Susceptibility of nematodes to treatment with nootkatone formulations.

[00122] This example describes a laboratory bioassay in which nematodes were exposed to a nootkatone-containing composition to determine the susceptibility of nematodes to nootkatone.

[00123] The organisms used for testing were mixed life stages of the nematode C. elegans (Blaxter, Lobos and Burglin "Caenorhabditis elegans as a model for parasitic nematodes" Int. J Parasitol. 1998 Mar;18(3):395-411). C. elegans were purchased from Carolina Biologicals (Burlington, North Carolina). Worms were propagated on solid Nematode Growth Agar with E. coli as a food source until they were used in the nootkatone assay.

Media

[00124] Synthetic Dropout (SD) media powder was purchased from U.S. Biological (Salem, Massachusetts). Liquid media was prepared according to the package instructions and was autoclaved prior to use. The SD media was supplemented with the addition of sterile yeast extract (Fisher Scientific; Hampton, New Jersey) and sterile sucrose to a final concentration of 2% each (w/v). Just prior to the nootkatone assay, worms were gently washed off of Nematode Agar slabs into 25 mL of SD media plus yeast extract, and the media plus worms was transferred into 100 mm diameter Petri plates.

Treatment

[00125] Two hundred and fifty microliters of a solution of nootkatone in ethanol was applied directly to 25 milliliters of liquid media containing nematodes, using a micropipette. The media used was liquid SD media plus yeast extract. The treatment condition used 3% (v/v) nootkatone in ethanol, for a final concentration of 0.03% (v/v). One-fourth milliliter of pure ethanol was used in a separate Petri plate as a control. Two replicates of >250 nematodes were tested per treatment or control. Clean, unused 100 mm diameter Petri plates were used as test containers.

Assessments

[00126] Nematodes were observed after 24 hours post-introduction to the test containers. The nematodes were scored according to the following criteria: Morbidity (M): does not move to feed and breathe or otherwise initiate

directional movement, but still exhibits movement with or without tactile stimulation; or

Dead (mortality): exhibits no movement, even with tactile stimulation.

Results

[00127] The results for 0.03% nootkatone treatment are shown in Figure 2.

[00128] Approximately 7% of the worms in the control plates that were exposed to 1 % ethanol were affected after 24 hours. In the two replicates that were exposed to 0.03% nootkatone (final concentration, v/v), at the end of 24 hours of exposure, 92% were killed, on average.

[00129] The test results indicate very high mortality rates for nematodes exposed to low concentrations of nootkatone compared to control after 24 hours of exposure. Therefore, nematodes are highly susceptible to low concentrations of nootkatone.

Example No. 2: In vitro evaluation of nootkatone's anti-nematicidal activity against cattle round worm.

Overview

[00130] The purpose of the present study was to determine the efficacy of nootkatone for controlling Toxocara vitulorum taken from calves and adult cattle.

Introduction

[00131] Toxocara vitulorum (syn. Neoascaris vitulorum) is a parasitic ascarid of Bubalus and Bos spp. (Roberts, "The extraparasitic life cycle of Toxocara vitulorum in the village environment of Sri Lanka," Vet Res Commun. 13(5):377-88, 1989) found in tropical and subtropical climates (Starke et al., "Skin hypersensitivity tests in buffaloes parasitized with Toxocara vitulorum," Veterinary Parasitology 63, 283-90, 1996). T vitulorum is the most important parasite that is responsible for calf mortality and morbidity under 3 months of age in tropical countries. Heavy burdens of adult T vitulorum in young calves could result in a high mortality rate and economic losses. Intestinal toxocariasis is associated with diarrhea, poor performance, intestinal and biliary obstruction, and death (Roberts et al., "Toxocara vitulorum in the milk of buffalo (Bubalus bubalis) cows," Research in Veterinary Science, 49:289-91 , 1990; Kassai, Veterinary Helminothology. Butterworth-Heinemann, Boston, MS, pp. 102-103, 1999). Adult stage T vitulorum can be effectively treated with piperazine, pyrantel, febantel, and oxfendazole (Roberts et al., "Toxocara vitulorum: treatment based on the duration of the infectivity of buffalo cows (Bubalus bubalis) for their calves," Journal of Veterinary Pharmacological Therapy 12:5-13, 1989), ivermectin (Mahieu et al., "Incidence of Toxocara vitulorum in Creole Calves of Guadeloupe," Tropical Animal Health Production, 40:243-8, 2008), fenbendazole (Davila et al., "Toxocara vitulorum in beef calves in North Central Florida," Veterinary Parasitology 168:261 -3, 2010), and eprinomectin (Avcioglu et al., "Efficacy of eprinomectin against Toxocara vitulorum in calves," Trop Anim Health Prod 43:283-6, 201 1 ). However, prolonged application to calves of various treatments cited above leads to side effects associated with nervous, respiratory, and gastrointestinal systems. Hence an alternative safe, effective nematicidal natural compound is required against this species of endoparasitic helminth.

METHODOLOGY

[00132] Collection of worms: Live worms from the intestine of infected calves were collected from a local slaughter house, washed, and stored in PBS buffer with 2% glucose (pH -7.4) until testing.

[00133] Test Formulations: PBS buffer (1 x) with 2% glucose was used as media for all experiments. Nootkatone ("NK") solutions at 0.1 %, 0.5%, 1 % and 5% concentrations were obtained by dissolving 98% pure NK in 100% EtOH (250 mg/mL) and later diluted to achieve 0.1 % and 5% solutions with PBS buffer. For control, 100% ethanol 4 ml added in PBS buffer was used (appropriate volume of solution used for each test).

[00134] Selection of worms: Actively moving 1 in control and 2 in treatment (5-6 cm worms of same size) were kept in individual 6 well plates with 10 ml treatment solution either with NK or control.

[00135] Treatment conditions: The whole experiment was carried out at 39±1 °C with 5% C0₂. During treatment, worms were maintained in 5-10 mL PBS-2% glucose solution (pH -7.4).

[00136] Assessment / Observations: Round worms were under continuous observation until loss of motility or death. A standard relative motility (RM) assay protocol was followed to see treatment effect as the paralysis and/or death of the worm. Briefly, worms treated under different groups were continuously monitored for whole body movements as, very sinuously motile, sluggishly motile (intermittent jerky movements), coiling, immotile, and other movements. Worms were observed continuously for 30 min and 5 min intervals for 1 hr.

RESULTS

[00137] Slender, white nematode worms (5-7 cm) were collected from infected calf intestine and identified as T vitulorum (Figure 3). Cessation of coiling movement, followed by exhibition of a stiff paralytic body was observed within 5 min of 1 % or 5% NK exposure (Figure 4). By 30 min of exposure to 1 % NK, more than 40% of worms were dead, this increased to 100% for 5% NK. Induction of 100% mortality (100% efficacy; observed as complete cessation of body movement, even upon mechanical stimuli) occurred by 3 hr of exposure for 1 % NK exposure (Figure 5). Less than 40% efficacy was observed for worms exposed with control solution with 5% EtOH for 3 hours. These results indicate that as little as 1 % NK can kill more than 40% of exposed T. vitulorum within 30 min and confirm NK to be a potent nematicide.

Example No. 3: In vitro evaluation of nootkatone's anti-trematicidal activity against cattle rumen-fluke (stomach flat worm).

Overview

[00138] Using standard in vitro anthelmintic activity assays nootkatone (nootkatone 98%; NK) was investigated for its trematicidal activities against the parasitic flat worm of livestock ruminants, rumen-fluke (Paramphistomum sp.).

Introduction

[00139] The Paramphistomum species or rumen fluke have a worldwide distribution. They are approximately 1 cm long and resemble pink, fleshy maggots on the surface of the rumen and reticulum and are considered to be harmful parasites in a number of ruminant species. While they are prevalent particularly in tropical and subtropical areas, they have been found increasingly in British and Irish livestock over the past few years (Tilling, "Rumen fluke in cattle in the UK: a review," L/Vesfocfr 18(6):223-7, 2013).

[00140] Flukicides, such as oxyclozanide, has been reported to have activity against adult and immature rumen flukes. However, commercial flukicides containing oxyclozanide lack a specific label claim for rumen flukes. Liver flukes have been shown to be susceptible to oxyclozanide, but only adult liver flukes. Indeed, oxyclozanide is only a narrow-spectrum anthelmintic effective against a few fluke species. It is also ineffective against roundworms, tapeworms, or external parasites. It is used scarcely in ruminants (mainly in drenches) but not in other livestock, horses or pets. Therefore, there is a need for additional tools to combat flukes.

METHODOLOGY

[00141] Collection of flukes: Live parasites were collected from infected livestock rumen, from a slaughterhouse in Chennai, washed, and stored in PBS buffer with 2% glucose (saline solution, pH -7.4.) until subjected to different test conditions. Visible movements were observed to confirm vitality.

[00142] Preparation of Test Formulations

1 . Buffer: 1x PBS with 2% glucose (100 mL of 10X PBS + 900 mL of MQ water), 2 g of glucose was added

2. Nootkatone solutions: Stock solution: 250 mg + 1 mL ethanol (250,000 ppm)

[00143] For making different concentration solutions of 50 mL volume, volumes of stock solution were mixed with 50 mL PBS buffer as shown in Table No. 2.

[00144] Table No. 2. Preparation of Test Formulations.

[00145] Five percent ΝΚ = 5 g in 100 mL (250 mg of NK was first dissolved in 1 mL EtOH; therefore, 5 g of NK was dissolved in 20 mL EtOH, Buffer was added).

[00146] Positive control: A commercial anti-helminthic, albendazole (ABZ) formulation was used as the positive control (at 1 , 5, 10, 100 & 500 ppm concentrations).

[00147] Control: 20 mL ethanol diluted in PBS buffer was used as a control to confirm the effect of ethanol, if any.

[00148] Selection of flukes: Live parasite worms of different ages and sizes were picked from the collection pool, washed with buffer, and kept in individual 6 well plates with 10 mL treatment solution either with NK or control. The collected worms were identified to be Paramphistomum sp. commonly known as rumen fluke (Figure 6).

[00149] Treatment conditions: The whole experiment was carried out at 39±1 °C temperature with 5% C0₂. During treatment, flukes were maintained in 10 mL PBS-2% glucose solution (pH -7.4). Five-six flukes of different age and size were subjected to in vitro treatment (see Figure 7) with different doses of NK (10, 20, 50, 250 and 500 ppm NK and 1 and 5% NK). A commercial anthelmintic, albendazole (ABZ) formulation was used as the positive control (at 1 , 5, 10, 100 & 500 ppm dilutions). [00150] Assessment / Observations: Fluke worms were under continuous observation until loss of motility /death. A standard relative motility (RM) assay protocol was followed to observe treatment effects (paralysis and/or death of flukes).

[00151] Fluke motility/movement was closely monitored with 5 min intervals, for 1 hr and then every 30 min until death (-6 hr). Flukes treated under different groups were continuously monitored for whole body movements as very sinuously motile, sluggishly motile (intermittent jerky movements), swellings, and blebbings.

RESULTS

[00152] At the start of the 1 hr incubation, most of the worms (>4) in each group, showed active movement of some part of the body (see Figure 8). During the 1 hr incubation, a rapid loss of movement (even upon external stimuli) was noticed for NK and ABZ treated groups, at concentrations >500 ppm. Severe parasite mortality was observed, as early as within 5 min post- treatment with 1 and 5% NK treatment, wherein, swellings followed by blebbings that later ruptured, leading to 100% mortality were observed. These observations indicate that NK has promising flukicidal activity.

Example No. 4: Comparison of nootkatone with citrus-derived nootkatone

Overview

[00153] 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 (NxV)) and a citrus fruit-derived nootkatone (also known as nootkatone ex citrus, which is derived from citrus fruit and available from Frutarom®, Corona, CA) are shown in Figure 9. The nootkatone used in the studies described herein lacked valencene and demonstrated a lower amount of 1 1 ,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 Figure 10), whereas the nootkatone used in the present studies was limonene-free. These results underscore the different chemical profile of the nootkatone used herein (NxV) compared to commercially-available nootkatone derived from citrus, such as that provided by Frutarom®.

[00154] 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 photosensitivity or phototoxicity in humans (resulting in sunburn, scabs, pigment spots and possibly enhanced carcinogenesis). (Gionfriddo et al. "Elimination of Furocoumarins in Bergamot Peel OW," Perfumer & Flavorist., 2004; 29:48-52; Ferreira Maia et al. "Plant-based insect repellents: a review of their efficacy, development and testing," Malaria Journal, 201 1 ; 10:Suppl1 - 1 1 ; and Kejlova 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, September 30, 2014: 1 -31 ).

[00155] Bergapten is present in naturally derived valencene (from citrus), so protective measures should be taken to safeguard the health of workers repeatedly handling concentrated forms of citrus-derived valencene. Any bergapten that carries over through the chemical or enzymatic oxidation of citrus-derived valencene into nootkatone must be removed to acceptable levels in the final product and concentrated formulations thereof. Therefore, bergapten-free nootkatone obtained from fermentation-derived valencene has particular advantages over plant- derived nootkatone.

Example No. 5: Production of Nootkatone ex valencene

[00156] 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.

[00157] 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.

[00158] First, Yep-GW-URA (Takahashi et al., (2007) Biotech nol Bioeng. 97(1 ):170-181 ) was generated by inserting a gateway cloning cassette (RfB) with the form attR1 -Cm^R-ccdB gene-attR2 (Hartley et al., (2000) Genome Res. 10:1788-1795) into the Smal 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 ori, an ampicillin resistance gene and a colE1 origin of replication. The resulting vector was designated YEp-CVS-URA.

[00159] 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 Xbal, the amplified product was cloned into the yeast shuttle expression vector YEp-GW-URA.

[00160] 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.

[00161] To screen for production of valencene, the Saccharomyces cerevisiae yeast cell strains CALI5-1 (ura3, Ieu2, his3, trpl , Aerg9::HIS3, HMG2cat/TRP1 ::rDNA, dppl , sue), ALX7-95 (ura3, his3, trpl , Aerg9::HIS3, HMG2cat/TRP1 ::rDNA, dppl , sue) or ALX1 1 -30 (ura3, trpl , erg9def25, HMG2cat/TRP1 ::rDNA, dppl , sue) were used.

[00162] The CALI5-1 strain (see U.S. published Appl. No. US20040249219; U.S. Pat. Nos. 6,531 ,303 and 6,689,593) has a Aleu2 deletion, which required the introduction of leucine into its media. ALX7-95 was derived from CALI5-1 by correcting the Aleu2 deficiency of CALI5-1 with a functional LEU2 gene (see U.S. published Appl. No. US2010/0151519).

[00163] ALX1 1 -30 was constructed from CALI5-1 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, trpl , his3, erg9^de 25, HMG2cat/TRP1 ::rDNA, dppl , 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, trpl , erg9def25, HMG2cat/TRP1 ::rDNA, dppl , 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 ALX1 1 -30.

[00164] 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- Aldrich). The ALX7-95 and ALX1 1 -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 in fermenters. Subsequently, ALX7-95 or ALX1 1 -30 were used for screening in vials and fermenters. Typically, ALX7-95 was used for screening in vials and ALX1 1 -30 was used for fermenters.

[00165] 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.

[00166] Production of valencene was performed in a 3-L fermentation tank (New Brunswick Bioflow 1 10). One liter of fermentation medium was prepared and autoclaved in the fermentation tank (20 g (NH₄)₂S0₄, 20 g KH₂P0₄, 1 g NaCI, MgS0₄.7H₂0, 4 g Solulys corn steep solids (Roquette)). The following components were then added: 20 ml mineral solution (0.028% FeS0₄.7H₂0, 0.029% ZnS0₄.7H₂0, 0.008% CuS0₄.5H₂0, 0.024% Na₂Mo0₄.2H₂0, 0.024% CoCI₂.6H₂0, 0.017% MnS0₄.H₂0, 1 mL HCI); 10 mL 50% glucose; 30 mL vitamin solution (0.001 % biotin; 0.012% calcium pantothenate, 0.06% inositol, 0.012% pyridoxine-HCI, 0.012% thiamine- HCI); 10 mL 10% CaCI₂, 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.

[00167] The seed culture for inoculating the fermentation medium was prepared by inoculating 50 mL of SDE-ura-trp medium (see Example 3.C.2.) with CALI5-1 , ALX7-95 or ALX1 1 -30 containing the YEp-CVS-ura plasmid. This culture was grown at 28° C. until early stationary phase (24-48 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.

[00168] The fermentor was maintained at 28° C. The air flow was 1 vvm and the d0₂ was maintained above 30% by adjusting the agitation. The pH was maintained at 4.5 using phosphoric acid and NaOH or NH₄OH. [00169] 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).

[00170] 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 in the oil was determined.

[00171] 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 ALX1 1 -30 S. cerevisiae containing Yep-CVS-ura, and expressing valencene synthase, was found to produce valencene.

[00172] 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.

A. Oxidation of Valencene to Nootkatone Using Chromium Trioxide

[00173] 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 distillate (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 tert-butyl 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).

B. Oxidation of Valencene to Nootkatone Using Silica Phosphonate-lmmobilized Chromium (III) Catalyst [00174] 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-butanol 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 was 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).

Example No. 6. In vitro evaluation of nootkatone against adult Setaria bovis

Overview

[00175] The purpose of the current study was to evaluate various concentrations of nootkatone for anthelmintic efficacy on Setaria bovis (round worms). Citronellal and piperazine citrate were used as positive controls.

METHODOLOGY

[00176] Collection of Worms. Round worms measuring 3-5 cm were collected from a local slaughter house. The worms underwent a motility evaluation, and those showing good motility were selected and kept in RPMI 1640 medium until the start of the experiment.

[00177] Experimental Conditions. 98% pure nootkatone (hereinafter "NKT98%") was diluted with ethanol to final concentrations of 1 %, 0.05% (500 ppm), and 0.01 % (100 ppm). Positive controls were also diluted to final concentrations of 1 %, 0.05%, and 0.01 % for citronellal and 5 mg/mL and 10 mg/mL for piperazine citrate (see Figure 1 1 ). 1 . 25 ml. of RPMI 1640 medium comprising NKT98% (solubilized in ethanol) at final dilutions of 1 %, 100 ppm, and 500 ppm,

2. 25 mL RPMI 1640 medium comprising piperazine citrate (solubilized in ethanol) at dilutions of 5 mg/mL and 10 g/mL (positive control), and

3. 25 mL RPMI 1640 medium comprising citronella (solubilized in ethanol) at dilutions of 1 .0%, 100 ppm, and 500 ppm (positive control).

[00178] Eight worms were transferred into a 90 mm petri-dish that comprised 25 mL of NKT98%, citronellal, piperazine citrate, or ethanol carrier (vehicle control or VC, as a negative control). Plates were incubated at room temperature for 24 hours. Post treatment inhibition of motility and/or mortality of worms was observed at 0.5, 1 , 3, 6, 10, 18, 24, and 48 hours, and scores were recorded. Motility was scored as shown below:

1 . Score 3 - Moving whole body

2. Score 2 - Moving only parts of the body

3. Score 1 - Immobile but alive

4. Score 0 - Dead

[00179] Percent efficacy of each treatment was assessed using the formula below. Where (N1 ) is representative of the number of worms alive in the control group, and (N2) is representative of the number of worms alive per treatment group. Scoring was represented graphically. Post- treatment worms were photographed. Assay results (motility and mortality) were analyzed for determination of statistical significance comparing control group with treatment groups with a oneway ANOVA method using Graphpad prism software version 5.0.

Efficacy (%) = (N1 ) - (N2) x 100

(N1 )

RESULTS

[00180] At 30 minutes post-treatment, worms exposed to the ethanol vehicle control and low concentrations (0.01 %) of citronellal or NKT98% were found to be normal in appearance ("motile"). Slightly sluggish (moving only parts of the body, "immobile" but alive) movement of worms was observed in groups treated with 0.05% citronellal and 1 .0% NKT98% compared to the vehicle control. Mortality of 60%, 85%, and 60% was observed in 5 mg/mL piperazine citrate, 10 mg/mL piperazine citrate, and 1.0% NKT98%, respectively, compared to the vehicle negative control (see Table No. 3. and Figures 12A-12C). [00181] Table No. 3. Motility of Round worms after 30 minute treatment with Nootkatone (NK), Citronellal (CT), Piperazine citrate (PC), and Vehicle Control (VC).

[00182] At 1 hour post-treatment, worms in the vehicle negative control and lowest concentration (0.01 %) of citronellal and NKT98% groups were found to have normal appearance (i.e., were "motile"). Slightly sluggish (moving only parts of the body, "immobile" but alive) movement of worms was observed in groups treated with 0.05% citronellal and worms treated with 0.05% NKT98%. 100% mortality of worms was observed in groups treated with 5 mg/mL or 10 mg/mL piperazine citrate, in groups treated with 1 .0% citronellal, and in groups treated with 1 .0% NKT98% (see Table No. 4 and Figures 13A-13C).

[00183] Table No. 4. Motility of Round worms 1 hour post-treatment with Nootkatone (NK), Citronellal (CT), Piperazine citrate (PC), and Vehicle Control (VC).

Mean 3.0 0.1 0.0 2.8 0.6 0.0 2.6 0.6 0.0

SD 0.0 0.4 0.0 0.5 0.9 0.0 0.5 0.9 0.0

[00184] At 3 hours post-treatment, worms in the vehicle negative control and worms in the lowest concentration (0.01 %) of citronellal and NKT98% were found to be normal ("motile"). 80% and 100% of mortality was observed in groups treated with citronellal (0.05%) and NKT98% (0.05%) compared to vehicle negative control (see Table No. 5 and Figures 14A-14C).

[00185] Table No. 5. Motility of Round worms 3 hours post-treatment with Nootkatone (NK), Citronellal (CT), Piperazine citrate (PC), and Vehicle Control (VC).

[00186] At 6 hours post-treatment, slightly sluggish (moving only parts of the body, "immobile" but alive) movement of worms was observed in groups treated with citronellal and NKT98% (0.01 %) groups compared to vehicle negative control (see Table No. 6 and Figures 15A-15B).

[00187] Table No. 6. Motility of Round worms 6 hours post- treatment with Nootkatone (NK), Citronellal (CT), Piperazine citrate (PC), and Vehicle Control (VC).

5 3 0 0 3 0 0 0 0 0

6 2 0 0 1 0 0 3 0 0

7 3 0 0 0 0 0 1 0 0

8 1 0 0 1 0 0 3 0 0

Mean 2.6 0.0 0.0 1.8 0.0 0.0 2.3 0.0 0.0

SD 0.7 0.0 0.0 1.4 0.0 0.0 1.2 0.0 0.0

[00188] At 10 hours post-treatment, slightly sluggish (moving only parts of the body, "immobile" but alive) movement of worms was observed in groups treated with citronellal and NKT98% (0.01 %) groups compared to vehicle negative control (see Table No. 7 and Figures 16A-16B).

[00189] Table No. 7. Motility of Round worms 10 hours post-treatment with Nootkatone (NK), Citronellal (CT), Piperazine citrate (PC), and Vehicle Control (VC).

[00190] At 18 hours post-treatment, 50% and 100% mortality of worms were observed in groups treated with citronellal (0.01 %) and NKT98% (0.01 %), respectively, compared to vehicle negative control (see Table No. 8 and Figures 17A-17B).

[00191] Table No. 8. Motility of Round worms 18 hours post-treatment with Nootkatone (NK), Citronellal (CT), Piperazine citrate (PC), and Vehicle Control (VC).

3 3 0 0 2 0 0 0 0 0

4 2 0 0 0 0 0 0 0 0

5 3 0 0 1 0 0 0 0 0

6 2 0 0 0 0 0 0 0 0

7 0 0 0 0 0 0 0 0 0

8 0 0 0 0 0 0 0 0 0

Mean 2.0 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.0

SD 1.3 0.0 0.0 0.9 0.0 0.0 0.0 0.0 0.0

[00192] At 24 hours post-treatment, greater than 50% mortality of worms was observed in groups treated with citronellal (0.01 %) compared to vehicle negative control (see Table No. 9 and Figures 18A-18B).

[00193] Table No. 9. Motility of Round worms 24 hours post-treatment with Nootkatone (NK), Citronellal (CT), Piperazine citrate (PC), and Vehicle Control (VC).

CONCLUSION

[00194] Groups of round worms treated with 1 .0% NKT98% showed over 50% mortality after 30 minutes of treatment, while after 1 .0 hour of treatment they showed 100% mortality. This efficacy was comparable to the positive controls citronellal (0.01 %) and piperazine citrate (5 and 10 mg/mL). Groups of round worms having received 3.0 hours of treatment with NKT98% (0.05%) or citronellal (0.05%) showed 100% mortality. NKT98% at 0.05% and 1.0% concentrations showed anthelmintic activity against round worms within 30 minutes of treatment and showed 100% mortality of worms within 3.0 hour of treatment at these concentrations. Sequence Listing: SEQ ID NO: 1 (Citrus valencene synthase) atgtcgtctg gagaaacatt tcgtcctact gcagatttcc atcctagttt atggagaaac 60

catttcctca aaggtgcttc tgatttcaag acagttgatc atactgcaac tcaagaacga 120

cacgaggcac tgaaagaaga ggtaaggaga atgataacag atgctgaaga taagcctgtt 180

cagaagttac gcttgattga tgaagtacaa cgcctggggg tggcttatca ctttgagaaa 240

gaaatagaag atgcaataca aaaattatgt ccaatctata ttgacagtaa tagagctgat 300

ctccacaccg tttcccttca ttttcgattg cttaggcagc aaggaatcaa gatttcatgt 360

gatgtgtttg agaagttcaa agatgatgag ggtagattca agtcatcgtt gataaacgat 420

gttcaaggga tgttaagttt gtacgaggca gcatacatgg cagttcgcgg agaacatata 480

ttagatgaag ccattgcttt cactaccact cacctgaagt cattggtagc tcaggatcat 540

gtaaccccta agcttgcgga acagataaat catgctttat accgtcctct tcgtaaaacc 600

ctaccaagat tagaggcgag gtattttatg tccatgatca attcaacaag tgatcattta 660

tacaataaaa ctctgctgaa ttttgcaaag ttagatttta acatattgct agagctgcac 720

aaggaggaac tcaatgaatt aacaaagtgg tggaaagatt tagacttcac tacaaaacta 780

ccttatgcaa gagacagatt agtggagtta tatttttggg atttagggac atacttcgag 840

cctcaatatg catttgggag aaagataatg acccaattaa attacatatt atccatcata 900

gatgatactt atgatgcgta tggtacactt gaagaactca gcctctttac tgaagcagtt 960

caaagatgga atattgaggc cgtagatatg cttccagaat acatgaaatt gatttacagg 1020

acactcttag atgcttttaa tgaaattgag gaagatatgg ccaagcaagg aagatcacac 1080

tgcgtacgtt atgcaaaaga ggagaatcaa aaagtaattg gagcatactc tgttcaagcc 1140

aaatggttca gtgaaggtta cgttccaaca attgaggagt atatgcctat tgcactaaca 1200

agttgtgctt acacattcgt cataacaaat tccttccttg gcatgggtga ttttgcaact 1260

aaagaggttt ttgaatggat ctccaataac cctaaggttg taaaagcagc atcagttatc 1320

tgcagactca tggatgacat gcaaggtcat gagtttgagc agaagagagg acatgttgcg 1380

tcagctattg aatgttacac gaagcagcat ggtgtctcta aggaagaggc aattaaaatg 1440

tttgaagaag aagttgcaaa tgcatggaaa gatattaacg aggagttgat gatgaagcca 1500

accgtcgttg cccgaccact gctcgggacg attcttaatc ttgctcgtgc aattgatttt 1560

atttacaaag aggacgacgg ctatacgcat tcttacctaa ttaaagatca aattgcttct 1620

gtgctaggag accacgttcc attttga 1647

SEQ ID NO: 2 (Citrus valencene synthase)

MSSGETFRPT ADFHPSLWRN HFLKGASDFK TVDHTATQER HEALKEEVRR MITDAEDKPV 60

QKLRLIDEVQ RLGVAYHFEK EIEDAIQKLC PIYIDSNRAD LHTVSLHFRL LRQQGIKISC 120

DVFEKFKDDE GRFKSSLIND VQGMLSLYEA AYMAVRGEHI LDEAIAFTTT HLKSLVAQDH 180

VTPKLAEQIN HALYRPLRKT LPRLEARYFM SMINSTSDHL YNKTLLNFAK LDFNILLELH 240

KEELNELTKW WKDLDFTTKL PYARDRLVEL YFWDLGTYFE PQYAFGRKIM TQLNYILSII 300

DDTYDAYGTL EELSLFTEAV QRWNIEAVDM LPEYMKLIYR TLLDAFNEIE EDMAKQGRSH 360

CVRYAKEENQ KVIGAYSVQA KWFSEGYVPT IEEYMPIALT SCAYTFVITN SFLGMGDFAT 420

KEVFEWI SNN PKWKAASVI CRLMDDMQGH EFEQKRGHVA SAIECYTKQH GVSKEEAIKM 480

FEEEVANAWK DINEELMMKP TVVARPLLGT ILNLARAIDF IYKEDDGYTH SYLIKDQIAS 540

VLGDHVPF 548

[00195] Having described the invention in detail and by reference to specific aspects and/or 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 may be identified herein as particularly advantageous, it is contemplated that the present invention is not limited to these particular aspects of the invention. Percentages disclosed herein may vary in amount by ±10, 20, or 30% from values disclosed and remain within the scope of the contemplated invention.

Claims

WHAT IS CLAIMED IS:

1 . A method of treating or preventing nematode, trematode, cestode, or helminth infection in a subject, comprising:

applying a nootkatone-containing composition to a nematode, trematode, cestode, or helminth,

wherein the nootkatone-containing composition comprises an effective amount of nootkatone to repel or kill the nematode, trematode, cestode, or helminth.

2. The method of claim 1 , wherein the nootkatone-containing composition is bergapten-free.

3. The method of claim 1 , wherein the nootkatone-containing composition comprises about 0.03% (v/v) nootkatone or greater.

4. The method of claim 3, wherein the nootkatone-containing composition is formulated for application topically on an exterior surface of a subject to be treated.

5. The method of claim 4, wherein the composition is formulated as an aerosol, a solution, an emulsion, an oil, a lotion, a soap, a shampoo, a conditioner, a spray, a gel, a cosmetic, or a fragrance.

6. The method of claim 1 further comprising applying a second effective amount of the nootkatone-containing composition after a period of 24 hours from the first application of the nootkatone-containing composition.

7. The method of claim 1 , wherein the nootkatone-containing composition kills greater than 90% of the nematodes, trematodes, cestodes, or helminths within a period of 24 hours.

8. A method of treating or preventing nematode, trematode, cestode, or helminth infection, comprising:

applying an effective amount of a nootkatone-containing composition to a nematode, trematode, cestode, or helminth vector.

9. The method of claim 8, wherein the nematode, trematode, cestode, or helminth vector is one or more of skin, hair, or other part of an animal, a leaf, a stem, bark, a flower, a seed, a fruit, an edible portion of the plant, a root, tools, farming equipment, soil, a connected water system, a lake, a river, a stream, a sewer, a sewage treatment facility, a latrine, a toilet, a portable toilet, a child's potty seat, or a child's training potty.

10. A method of treating a nematode, trematode, cestode, or helminth infection, comprising: applying an effective amount of a nootkatone-containing composition to a nematode host, trematode host, cestode host, or helminth host.

1 1 . The method of claim 10, wherein the nootkatone-containing composition is formulated for dispersing over an area to be treated.

12. The method of claim 1 1 , wherein the nootkatone-containing composition is formulated as a pellet, a grain, and/or a spray.

13. The method of claim 1 1 , wherein the nootkatone-containing composition includes one or more of nitrogen, phosphorous, potassium, a herbicide, a plant seed, or a crop seed.

14. The method of claim 10, wherein the nootkatone-containing composition is formulated for treating an intestinal infection.

15. The method of claim 14, wherein the nootkatone-containing composition is formulated for ingestion as a food supplement, a paste, gel, a syrup, or a liquid drench to be given orally, a granular form to be added to food, a pill, a chew tablet, or a liquid suspension.

16. The method of claim 14, wherein the nootkatone-containing composition is formulated as an extruded animal food, a grain, a canned food, an animal treat, a dog bone, an additive to fish food, or a coating on animal forage.

17. The method of claim 14, wherein the nootkatone-containing composition is formulated as a tablet, a capsule, a granule, a pellet, a sachet, an emulsion, an elixir, or a suppository for rectal administration.

18. The method of claim 10, wherein the nootkatone-containing composition is formulated as a sterile injectable solution for parenteral use.

19. The method of claim 1 1 , wherein the nootkatone-containing composition comprises one or more of chitosan, juglone, thymol, wormwood, nematophagous fungi spores, or endospores of Pasteuria penetrans.

20. The method of claim 14, wherein the nootkatone-containing composition comprises one or more of mebendazole, albendazole, fenbendazole, moxidectin, levamisole, ivermectin, a plant essential oil compound, praziquantel, bithionol, triclabendazole, niclosamide, or derivatives thereof.

21 . The method of claim 14, wherein the nootkatone-containing composition comprises one or more of citronellal and piperazine citrate.

22. The method of any preceding claims, wherein the nematode, trematode, cestode, or helminth infection is caused by one or more nematodes, trematodes, cestodes, or helminths of the genera Ascaris, Baylisascaris, Trichinella, Dirofilaria, Haemonchus, Enterobius, Trichuris, Wuchereria, Brugia, Onchocerca, Dracunuculus, Toxocara, Setaria, Bursaphelenchus, Meloidogyne, Globdera, Schistosoma, Paragonimus, Clonorchis, Fasciola, Opisthorchis, Fasciolopsis, Heterophyes, Metagonimus, Paramphistomum, Taenia, or Diphyllobothrium.

23. A composition comprising nootkatone for use in a method of treating or preventing nematode, trematode, cestode or helminth in a subject comprising applying the composition to a nematode, trematode, cestode or helminth wherein the composition comprises an effective amount of nootkatone to repel or kill the nematode, trematode, cestode or helminth.

24. The composition of claim 23 for use according to claim 23, wherein the composition is bergapten-free.

25. The composition of claim 23 for use according to claim 23, wherein the composition comprises about 0.03% (v/v) nootkatone or greater.

26. The composition of claim 25 for use according to claim 25, wherein the composition is formulated for application topically on an exterior surface of a subject to be treated.

27. The composition of claim 26 for use according to claim 26, wherein the composition is formulated as an aerosol, a solution, an emulsion, an oil, a lotion, a soap, a shampoo, a conditioner, a spray, a gel, a cosmetic, or a fragrance.

28. The composition of claim 23 for use according to claim 23, further comprising applying a second effective amount of the composition after a period of 24 hours from the first application of the composition.

29. The composition of claim 23 for use according to claim 23, wherein the composition kills greater than 90% of the nematodes, trematodes, cestodes or helminths within a period of 24 hours.

30. A composition comprising nootkatone for use in a method of treating or preventing nematode, trematode, cestode or helminth infection comprising applying an effective amount of a the composition to a nematode, trematode, cestode or helminth vector.

31 . The composition of claim 30 for use according to claim 30, wherein the nematode vector is one or more of skin, hair, or other part of an animal, a leaf, a stem, bark, a flower, a seed, a fruit, an edible portion of the plant, a root, tools, farming equipment, soil, a connected water system, a lake, a river, a stream, a sewer, a sewage treatment facility, a latrine, a toilet, a portable toilet, a child's potty seat, or a child's training potty.

32. A composition comprising nootkatone for use in a method of treating a nematode trematode, cestode or helminth infection, comprising applying an effective amount of the composition to a nematode host, trematode host, cestode host or helminth host.

33. The composition of claim 32 for use according to claim 32, wherein the composition is formulated for dispersing over an area to be treated.

34. The composition of claim 33 for use according to claim 33, wherein the composition is formulated as a pellet, a grain, and/or a spray.

35. The composition of claim 33 for use according to claim 33, wherein the composition includes one or more of nitrogen, phosphorous, potassium, a herbicide, a plant seed, or a crop seed.

36. The composition of claim 32 for use according to claim 32, wherein the composition is formulated for treating an intestinal infection.

37. The composition of claim 36 for use according to claim 36, wherein the composition is formulated for ingestion as a food supplement, a paste, gel, a syrup, or a liquid drench to be given orally, a granular form to be added to food, a pill, a chew tablet, or a liquid suspension.

38. The composition of claims 36 for use according to claim 36, wherein the composition is formulated as an extruded animal food, a grain, a canned food, an animal treat, a dog bone, an additive to fish food, or a coating on animal forage.

39. The composition of claim 36 for use according to claim 36, wherein the composition is formulated as a tablet, a capsule, a granule, a pellet, a sachet, an emulsion, an elixir, a suppository for rectal administration.

40. The composition of claim 32 for use according to claim 32, wherein the composition is formulated as a sterile injectable solution for parenteral use.

41 . The composition of claim 33 for use according to claim 33, wherein the composition comprises one or more of chitosan, juglone, thymol, wormwood, nematophagous fungi spores, or endospores of Pasteuria penetrans.

42. The composition of claim 36 for use according to claim 36, wherein the composition comprises one or more of mebendazole, albendazole, fenbendazole, moxidectin, levamisole, ivermectin, a plant essential oil compound, praziquantel, bithionol, triclabendazole, niclosamide, or derivatives thereof.

43. The composition of claim 36 for use according to claim 36, wherein the composition comprises one or more of citronellal and piperazine citrate.

44. The composition of any preceding claim for use according to any proceeding claim, wherein the nematode, trematode, cestode, or helminth infection is caused by one or more nematodes, trematodes, cestodes, or helminths of the genera Ascaris, Baylisascaris, Trichinella, Dirofilaria, Haemonchus, Enterobius, Trichuris, Wuchereria, Brugia, Onchocerca, Dracunuculus, Toxocara, Setaria, Bursaphelenchus, Meloidogyne, Globdera, Schistosoma, Paragonimus, Clonorchis, Fasciola, Opisthorchis, Fasciolopsis, Heterophyes, Metagonimus, Paramphistomum, Taenia, or Diphyllobothrium.