WO2003075656A2 - Acide gras nematicide et composes apparentes a des esters d'acide gras - Google Patents

Acide gras nematicide et composes apparentes a des esters d'acide gras Download PDF

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Publication number
WO2003075656A2
WO2003075656A2 PCT/US2003/006525 US0306525W WO03075656A2 WO 2003075656 A2 WO2003075656 A2 WO 2003075656A2 US 0306525 W US0306525 W US 0306525W WO 03075656 A2 WO03075656 A2 WO 03075656A2
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Prior art keywords
substituted
group
carbon chain
unsubstituted
hydroxy
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PCT/US2003/006525
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English (en)
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WO2003075656A3 (fr
Inventor
Deryck J. Williams
Andrew P. Kloek
Michelle Coutu Hresko
Barry J. Shortt
Jennifer A. Davila-Aponte
John D. Bradley
James P. Mccarter
Merry B. Mclaird
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Divergence, Inc.
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Priority claimed from US10/090,527 external-priority patent/US6887900B2/en
Application filed by Divergence, Inc. filed Critical Divergence, Inc.
Priority to AU2003225651A priority Critical patent/AU2003225651A1/en
Priority to EP03744163A priority patent/EP1480633A2/fr
Priority to CA002478589A priority patent/CA2478589A1/fr
Priority to US10/655,165 priority patent/US20040157803A1/en
Publication of WO2003075656A2 publication Critical patent/WO2003075656A2/fr
Publication of WO2003075656A3 publication Critical patent/WO2003075656A3/fr
Priority to PCT/US2004/029015 priority patent/WO2005023989A2/fr
Priority to US12/105,591 priority patent/US20080249160A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/06—Unsaturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing within the same carbon skeleton a carboxylic group or a thio analogue, or a derivative thereof, and a carbon atom having only two bonds to hetero atoms with at the most one bond to halogen, e.g. keto-carboxylic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/20—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom three- or four-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin

Definitions

  • Nematodes (derived from the Greek word for thread) are active, flexible, elongate, organisms that live on moist surfaces or in liquid environments, including films of water within soil and moist tissues within other organisms. While only 20,000 species of nematode have been identified, it is estimated that 40,000 to 10 million actually exist. Some species of nematodes have evolved to be very successful parasites of both plants and animals and are responsible for significant economic losses in agriculture and livestock and for morbidity and mortality in humans (Whitehead (1998) Plant Nematode Control. CAB International, New York).
  • Nematode parasites of plants can inhabit all parts of plants, including roots, developing flower buds, leaves, and stems. Plant parasites are classified on the basis of their feeding habits into the broad categories: migratory ectoparasites, migratory endoparasites, and sedentary endoparasites. Sedentary endoparasites, which include the root knot nematodes (Meloidogyne) and cyst nematodes (Globodera and Heterodera) induce feeding sites and establish long-term infections within roots that are often very damaging to crops (Whitehead, supra).
  • Sedentary endoparasites which include the root knot nematodes (Meloidogyne) and cyst nematodes (Globodera and Heterodera) induce feeding sites and establish long-term infections within roots that are often very damaging to crops (Whitehead, supra).
  • the macrocyclic lactones e.g., avermectins and milbemycins
  • delta-toxins from Bacillus thuringiensis Bt
  • Bt Bacillus thuringiensis
  • Bt delta toxins must be ingested to affect their target organ, the brush border of midgut epithelial cells (Marroquin et al. (2000) Genetics. 155(4):1693-1699). Consequently they are not anticipated to be effective against the dispersal, non-feeding, juvenile stages of plant parasitic nematodes in the field. Because juvenile stages only commence feeding when a susceptible host has been infected, nematicides may need to penetrate the plant cuticle to be effective. Transcuticular uptake of a 65-130 kDa protein - the size of typical Bt delta ends toxins - is unlikely. Furthermore, soil mobility is expected to be relatively poor.
  • Fatty acids are a class of natural compounds that have been investigated as alternatives to the toxic, non-specific organophosphate, carbamate and fumigant pesticides (Stadler et al. (1994) Planta Medica 60(2): 128-132; US Pat. Nos. 5,192,546; 5,346,698; 5,674,897; 5,698,592; 6,124,359). It has been suggested that fatty acids derive their pesticidal effects by adversely interfering with the nematode cuticle or hypodermis via a detergent (solubilization) effect, or through direct interaction of the fatty acids and the lipophilic regions of target plasma membranes (Davis et al.
  • fatty acids are used in a variety of pesticidal applications including as herbicides (e.g., SCYTHE by Dow Agrosciences is the C9 saturated fatty acid pelargonic acid), bactericides and fungicides (US Pat. Nos. 4,771,571; 5,246,716) and insecticides (e.g., SAFER INSECTICIDAL SOAP by Safer, Inc.).
  • herbicides e.g., SCYTHE by Dow Agrosciences is the C9 saturated fatty acid pelargonic acid
  • bactericides and fungicides US Pat. Nos. 4,771,571; 5,246,716
  • insecticides e.g., SAFER INSECTICIDAL SOAP by Safer, Inc.
  • Ricinoleic acid the major component of castor oil, has been shown to have an inhibitory effect on water and electrolyte abso ⁇ tion using everted hamster jejunal and ileal segments (Gaginella et al. (1975) J Pharmacol Exp Ther 195(2):355-61) and to be cytotoxic to isolated intestinal epithelial cells (Gaginella et al. (1977) J Pharmacol Exp Ther 201(l):259-66). These features are likely the source of the laxative properties of castor oil which is given as a purgative in humans and livestock (e.g., castor oil is a component of some de-worming protocols because of its laxative properties). In contrast, the methyl ester of ricinoleic acid is ineffective at suppressing water absorption in the hamster model (Gaginella et al. (1915) J Pharmacol Exp Ther 195(2):355-61).
  • short- and medium-chain fatty acids and salts e.g., C6 to C12
  • the commercial fungicidal and moss killing product De-Moss comprises mainly fatty acids and salts in this size range.
  • the phytotoxicity of these shorter fatty acids also makes them suitable as broad-spectrum herbicides when used at higher concentrations as is exemplified by the commercial herbicide SCYTHE which comprises the C9 fatty acid pelargonic (nonanoic) acid.
  • SCYTHE which comprises the C9 fatty acid pelargonic (nonanoic) acid.
  • C16 to C20 fatty acids and salts such as oleic acid (C18:l) are suitable insecticidal fatty acids.
  • Insecticidal fatty acid products such as M-PEDE and SAFER Insecticidal Concentrate whose active ingredients comprise longer chain fatty acids rich in C16 and C18 components represent real world applications of this scientific information.
  • the prior art provides little guidance for the selection of suitable broad-spectrum nematicidal fatty acids and what information exists is often contradictory.
  • Stadler and colleagues (Stadler et al. (1994) Planta Medica 60(2): 128-132) tested a series of fatty acids against L4 and adult C. elegans stages and found that a number of common longer chain fatty acids such as linoleic (C18:2), myristic (C14:0), palmitoleic (C16:l) and oleic (C18:l) acids had significant nematicidal activity. C. elegans was not very sensitive to C6 to CIO (medium chain) fatty acids. Stadler et al.
  • Castor beans are plowed under as a green manure before a seed crop is set.
  • a significant drawback of the castor plant is that the seed contains toxic compounds (such as ricin) that can kill humans, pets, and livestock and is also highly allergenic.
  • toxic compounds such as ricin
  • the active principle(s) for plant nematicidal activity has not been discovered and it remains difficult to derive commercially successful nematicidal products from these resistant plants or to transfer the resistance to agronomically important crops such as soybeans and cotton.
  • nematodes Genetic resistance to certain nematodes is available in some commercial cultivars (e.g., soybeans), but these are restricted in number and the availability of cultivars with both desirable agronomic features and resistance is limited.
  • the production of nematode resistant commercial varieties by conventional plant breeding based on genetic recombination through sexual crosses is a slow process and is often further hampered by a lack of appropriate germplasm.
  • Nematode parasites of vertebrates include gut roundworms, hookworms, pinworms, whipworms, and filarial worms. They can be transmitted in a variety of ways, including by water contamination, skin penetration, biting insects, or by ingestion of contaminated food.
  • nematode control or "de- worming" is essential to the economic viability of livestock producers and is a necessary part of veterinary care of companion animals.
  • Parasitic nematodes cause mortality in animals (e.g., heartworm in dogs and cats) and morbidity as a result of the parasites' inhibiting the ability of the infected animal to absorb nutrients.
  • the parasite-induced nutrient deficiency leads to disease and stunted growth in livestock and companion animals. For instance, in cattle and dairy herds, a single untreated infection with the brown stomach worm can permanently restrict an animal's ability to convert feed into muscle mass or milk.
  • hookworms examples include hookworms, filarial worms, and pinworms.
  • Hookworms (1.3 billion infections) are the major cause of anemia in millions of children, resulting in growth retardation and impaired cognitive development.
  • Filarial worm species invade the lymphatics, resulting in permanently swollen and deformed limbs (elephantiasis), and the eyes, causing African river blindness.
  • the large gut roundworm Ascaris lumbricoides infects more than one billion people worldwide and causes malnutrition and obstructive bowel disease.
  • pinworms are common and often transmitted through children in daycare.
  • nematodes can still deprive the host of valuable nutrients and increase the ability of other organisms to establish secondary infections. In some cases, infections can cause debilitating illnesses and can result in anemia, diarrhea, dehydration, loss of appetite, or death.
  • C. elegans is a small free-living bacteriovorous nematode that for many years has served as an important model system for multicellular animals (Burglin (1998) Int. J. Parasitol. 28(3):395-411). The genome of C. elegans has been completely sequenced and the nematode shares many general developmental and basic cellular processes with vertebrates (Ruvkin et al. (1998) Science 282:2033-41). This, together with its short generation time and ease of culturing, has made it a model system of choice for higher eukaryotes (Aboobaker et al. (2000) Ann. Med. 32:23-30). Although C.
  • elegans serves as a good model system for vertebrates, it is an even better model for study of parasitic nematodes, as C. elegans and other nematodes share unique biological processes not found in vertebrates.
  • nematodes produce and use chitin, have gap junctions comprised of innexin rather than connexin and contain glutamate-gated chloride channels rather than glycine-gated chloride channels (Bargmann (1998) Science 282:2028-33).
  • the latter property is of particular relevance given that the avermectin class of drugs is thought to act at glutamate-gated chloride receptors and is highly selective for invertebrates (Martin (1997) Fet. J 154:11-34).
  • a subset of the genes involved in nematode-specific processes will be conserved in nematodes and absent or significantly diverged from homologues in other phyla. In other words, it is expected that at least some of the genes associated with functions unique to nematodes will have restricted phylogenetic distributions.
  • the completion of the C. elegans genome project and the growing database of expressed sequence tags (ESTs) from numerous nematodes facilitate identification of these "nematode-specific" genes.
  • conserved genes involved in nematode- specific processes are expected to retain the same or very similar functions in different nematodes. This functional equivalence has been demonstrated in some cases by transforming C.
  • RNA interference a technique that provides a powerful experimental tool for the study of gene function in nematodes (Fire et al. (1998) Nature 391(6669):806-811; Montgomery et al. (1998) Proc. Natl. Acad Sci USA 95(26): 15502-15507).
  • Treatment of a nematode with double-stranded RNA of a selected gene can destroy expressed sequences corresponding to the selected gene thus reducing expression of the corresponding protein.
  • RNA interference a process that provides a powerful experimental tool for the study of gene function in nematodes.
  • Treatment of a nematode with double-stranded RNA of a selected gene can destroy expressed sequences corresponding to the selected gene thus reducing expression of the corresponding protein.
  • By preventing the translation of specific proteins their functional significance and essentiality to the nematode can be assessed. Determination of essential genes and their corresponding proteins using C. elegans as a model system will assist in the rational design of anti
  • the present invention describes compositions which shows surprising nematicidal activity in part due to selective inhibition of metabolic processes demonstrated to be essential to nematodes and either absent or non-essential in vertebrates and plants. This invention therefore provides urgently needed compounds and methods for the environmentally safe control of parasitic nematodes.
  • the invention concerns compositions and processes for controlling nematodes.
  • the subject invention comprises the use of certain fatty acids, fatty acid esters and related compounds to control nematodes that infest plants or the situs of plants. Nematodes that parasitize animals can also be controlled using the methods and compounds of this invention.
  • Certain of the useful nematicidal fatty acids (free fatty acids and salts) and analogs (e.g., esters) are predicted inhibitors of nematode delta- 12 fatty acid desaturases (also referred to herein as a nematode delta-12 desaturases).
  • fatty acids and analogs can be, for example, an epoxide, a cyclopropane, a cyclopropene, an oxo, methylated, or hydroxylated fatty acid or analog.
  • the compounds can also contain sulfur in place of carbon at certain positions.
  • the fatty acids and analogs are delta-12 desaturase inhibiting fatty acids and esters.
  • the term fatty acid analog is also used to denote free fatty acids, fatty acid salts and fatty acid esters of unusual fatty acids (i.e., those containing for example, epoxide, cyclopropane, cyclopropene, oxo, hydroxy, conjugated double bonds, triple bonds or other unusual groups).
  • Preferred fatty acids and esters also include thia fatty acids and thia fatty acid esters with sulfur in place of carbon at positions 12, 13 or 12 and 13. Most preferred compounds are C16 to C18 in length.
  • Examples include, ricinoleic (12- hydroxy-cis-9-octadecenoic) acid and ricinoleic acid methyl ester, ricinelaidic (12- hydroxy-trans-9-octadecenoic) acid and ricinelaidic acid methyl ester, vernolic ((12,13)-epoxy-cis-9-octadecenoic) acid and vernolic acid methyl ester, (12,13)-epoxy- trans-9-octadecenoic acid and (12,13)-epoxy-trans-9-octadecenoic acid methyl ester, 12-oxo-9(Z)-octadecenoic acid and 12-oxo-9(Z)-octadecenoic acid methyl ester, 12- oxo-9(E)-octadecenoic acid and 12-oxo-9(E)-octadecenoic acid methyl ester.
  • delta-12 desaturases e.g., cis-9- octadecenoate (oleate), cis-9-hexadecenoate (palmitoleate), isomers of the substrate such as trans-9-octadecenoate (elaidate) and the normal products of delta-12 desaturases (e.g., cis-9,12-octadecadienoate (linoleate), cis-9,12-hexadecadienoate).
  • delta-12 desaturases e.g., cis-9- octadecenoate (oleate), cis-9-hexadecadienoate.
  • Fatty acid compounds where the only modifications are a single cis or trans double bond at the delta-9 position (i.e., a cis or trans double bond between C9 and CIO), or double bonds at both the delta-9 (cis or trans double bond between C9 and CIO) and delta-12 positions (i.e., a cis or trans double bond between C12 and C13) as well as certain naturally occurring esters such as triglycerides, diacylglycerides and phospholipids are generally less preferred.
  • preferred sulfur containing fatty acid analogs include methyl 12-thia-oleate and methyl 13-thia-oleate.
  • Fatty acids analogs e.g., free fatty acids, salts, esters
  • a specific inhibitor of delta-12 desaturase inhibit the activity of a nematode delta-12 desaturase to a lesser extent in the presence of the product of a delta- 12 fatty acid desaturase (e.g., linoleate) than in the presence of the substrate of the enzyme (e.g., oleate).
  • the substrate e.g., oleate
  • the product e.g., linoleate
  • delta-12 fatty acid desaturase also referred to herein as a delta-12 desaturase
  • the inhibitor, the substrate and product of the delta-12 desaturase are present in equal concentrations.
  • the invention also features compounds that inhibit the expression of a delta-12 desaturase at the level of transcription or translation. Also within the invention are compounds that that that impair the modification of a delta-12 desaturase resulting in change in the activity or localization of the desaturase.
  • the invention also features compounds that are relatively selective inhibitors of one or more nematode delta-12 desaturase polypeptides relative to one or more plant or animal fatty acid desaturase-like polypeptides.
  • the compounds can have a K; for a nematode fatty acid desaturase that is 10-fold, 100-fold, 1,000-fold or more lower than for a plant or animal fatty acid desaturase-like polypeptides, e.g., a host plant or host animal of the nematode.
  • the invention further features relatively non-selective inhibitors as well as completely non-selective inhibitors.
  • the invention features a method of treating a disorder (e.g., an infection) caused by a nematode, (e.g., M. incognita, H. glycines, H. contortus, A. suum) in a subject, e.g., a host plant or host animal.
  • a disorder e.g., an infection
  • a nematode e.g., M. incognita, H. glycines, H. contortus, A. suum
  • the method includes administering to the subject an effective amount of a fatty acid (free fatty acids or salts) or fatty acid analog (esters) of the invention, e.g., a compound that is an inhibitor of a delta-12 desaturase polypeptide activity or an inhibitor of expression of a delta-12 desaturase polypeptide or an inhibitor that impairs the modification of a delta-12 desaturase resulting in change in the activity or localization of the desaturase.
  • the inhibitor may be delivered by several means including as a feed additive, as a pill or by injection.
  • methods of inhibiting a nematode e.g., M. incognita, H. glycines, H. contorius, A. suum
  • delta-12 desaturase(s) are provided.
  • Such methods can include the steps of: (a) providing a nematode, e.g., a nematode that contains a delta-12 fatty acid desaturase-like gene; (b) contacting the nematode with a fatty acid (free fatty acids or salts) or fatty acid analog (esters) of the invention.
  • Certain such compounds may inhibit the activity of a delta-12 desaturase.
  • methods of rescuing the effect of the inhibitor comprise the steps of: (a) inhibiting the enzyme and (b) providing delta-12 unsaturated fatty acids exogenously (e.g., linoleate).
  • methods of reducing the viability or fecundity or slowing the growth or development or inhibiting the infectivity of a nematode using a nematicidal fatty acid or fatty acid analog of the invention e.g., an inhibitor of a delta-12 desaturase are provided.
  • Such methods comprise the steps of (a) providing a nematode that contains a delta-12 desaturase-like gene; (b) contacting the nematode with specific fatty acid or fatty acid analog, e.g., an inhibitor of a delta-12 fatty acid desaturase; (c) reducing the viability or fecundity of the nematode.
  • methods of rescuing the effect of the fatty acid desaturase inhibitors or other inhibitors can involve contacting the nematode with delta-12 unsaturated fatty acids exogenously.
  • the invention features a method for reducing the viability, growth, or fecundity of a nematode, the method comprising exposing the nematode to a fatty acid or fatty acid analog of the invention, e.g., a compound that inhibits the activity of a fatty acid desaturase-like polypeptide (e.g., a delta-12 fatty acid desaturase) and a method for protecting a plant from a nematode infection, the method comprising applying to the plant, to the soil, or to seeds of the plant a fatty acid or fatty acid analog of the invention.
  • a fatty acid or fatty acid analog of the invention e.g., a compound that inhibits the activity of a fatty acid desaturase-like polypeptide (e.g., a delta-12 fatty acid desaturase) and a method for protecting a plant from a nematode infection, the method comprising applying to the plant, to the soil, or to seeds
  • the invention also features a method for protecting a vertebrate (e.g., a bird or a mammal) from a nematode infection, the method comprising administering to the mammal a fatty acid or fatty acid analog of the invention, e.g., an inhibitor of a nematode fatty acid desaturase-like polypeptide (e.g., a delta-12 fatty acid desaturase).
  • the inhibitor does not significantly inhibit the activity of a fatty acid desaturase-like polypeptide expressed by the vertebrate or at least does not do so to the extent that the growth of the plant is significantly impaired.
  • the bird can be a domesticated fowl (e.g., a chicken, turkey, duck, or goose).
  • the mammal can be a domesticated animal, e.g., a companion animal (e.g., a cat, dog, or rabbit) or livestock (e.g., a cow, sheep, horse, pig, goat, alpaca, or llama).
  • a companion animal e.g., a cat, dog, or rabbit
  • livestock e.g., a cow, sheep, horse, pig, goat, alpaca, or llama.
  • the invention process is particularly valuable to control nematodes attacking the roots of desired crop plants, ornamental plants, and turf grasses.
  • the desired crop plants can be, for example, soybeans, cotton, strawberries, tomatoes, banana, sugar cane, sugar beet, potatoes, or citrus.
  • the invention features a nematicidal composition comprising:
  • Ri H, a cation (e.g., Na + , K + , NH ) or a C1-C5 (i.e., a one, two, three, four or five carbon) substituted or unsubstituted carbon chain (e.g., a straight chain), wherein the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano, cyclopropane, epoxy and a substituted or unsubstituted C1-C2 (i.e., a one or two carbon) carbon chain; and
  • R 2 a C15-C19 (i.e., a 15, 16, 17, 18, or 19 carbon) substituted or unsubstituted carbon chain (e.g., a straight chain) having a cis or trans double bond between the 9 th and 10 th carbons counting from the carbonyl (carboxyl) carbon and either: (i) a triple bond between the 12 th and 13 th carbons counting from the carbonyl (carboxyl) carbon or (ii) either a single or double bond between the 12 th and 13 th carbons and at least one substituant at one or both of the 12 th and 13 th carbons, wherein the substituants are selected from the group consisting of hydroxy, oxo, halogen, amino, cyano, azido, cyclopropane, cyclopropene, epoxy and a substituted or unsubstituted C1-C2 carbon chain.
  • the C15-C19 carbon chain can be substituted at positions other than the 12 th
  • the composition can be produced in concentrated form that includes little or no water.
  • the composition can be diluted with water or some other solvent or combination of solvents prior to use to treat plants, seeds, soil or vertebrates.
  • the cation can be bivalent and complexed with two fatty acid or fatty acid ester molecules.
  • Ri is H or a cation
  • Ri is a C1-C5 substituted or unsubstituted carbon chain (i.e., a carbon chain, e.g., a linear carbon chain, having 1, 2, 3, 4 or 5 carbons), wherein the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano, cyclopropane, epoxy and a substituted or unsubstituted C1-C2 carbon chain
  • Ri is a C1-C5 substituted or unsubstituted carbon chain, wherein the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano, cyclopropane, epoxy and an unsubstituted C1-C2 carbon chain
  • the C1-C2 carbon chain of one or both of Ri and R 2 is substituted and the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano, and epoxy
  • the C1-C2 carbon chain of R 2 is substituted and the substituants are selected from the group consisting of: hydroxy, oxo, halogen, amino, cyano, azido, and epoxy; the C1-C2 carbon chain of R 2 is substituted and the substituants are selected from the group consisting of: hydroxy, oxo, halogen, azido, and amino; the C1-C2 carbon chain of R 2 is singly substituted; Ri is H; Rj is a cation; R 2 is substituted only at one or both of 12 th and 13 th carbons counting from the carbonyl (carboxyl) carbon; R 2 is substituted only at the 12 th carbon counting from the carbonyl (carboxyl) carbon; R 2 is substituted only at the 13 th carbon counting from the carbonyl (carboxyl) carbon; with R 2 the substituants are polar and are selected from the group consisting of: hydroxy, oxo, epoxy, halogen, amino,
  • the invention features a nematicidal composition
  • a fatty acid or salt or ester selected from the group consisting of: ricinoleic acid, ricinelaidic acid, 12-oxo-9(Z)-octadecenoic acid, 12-oxo-9(E)- octadecenoic acid, (12,13)-epoxy-trans-9-octadecenoic acid and vernolic acid; and (b) an aqueous surfactant.
  • the aqueous surfactant is selected from the group consisting of: ethyl lactate, Span 20, Span 40, Span 80, Span 85, Tween 20, Tween 40, Tween 80, Tween 85 and Igepal CO 630;
  • the composition further comprises a permeation enhancer (e.g., cyclodextrin);
  • the composition further comprises a co- solvent (e.g., isopropanol, acetone, 1,2-propanediol, a petroleum based-oil (e.g., aromatic 200) or a mineral oil (e.g., paraffin oil));
  • the composition further comprises a nematicide selected from the group consisting of: avermectins, ivermectin, and milbemycin;
  • the composition further comprises an inhibitor of oxidation (e.g., butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)).
  • composition comprises at least two different compounds having the formula
  • compositions comprising, consisting or consisting essentially of at least one such fatty acid or fatty acid analog and an aqueous surfactant, a co-solvent or an anti-oxidant.
  • the invention also features the use of such compositions in the control of nematodes.
  • compositions comprising, consisting or consisting essentially of at least one such fatty acid or fatty acid analog and an aqueous surfactant and an anti-oxidant.
  • Certain compositions contain two, three, four or more different fatty acids or fatty acid analogs.
  • the fatty acids and fatty acid analogs are mixtures of compounds and in other embodiments the surfactants are mixtures of compounds. In another embodiment both the fatty acids or fatty acid analogs and surfactants are mixtures of compounds.
  • a permeation enhancer is generally an agent that facilitates the active compounds of the invention, e.g., the fatty acids or esters of the invention, to pass through cellular membranes.
  • a co-solvent i.e., a latent solvent or indirect solvent
  • a latent solvent or indirect solvent is an agent that becomes an effective solvent in the presence of an active solvent and can improve the properties of the primary (active) solvent.
  • compositions can also include one more nematicides such as an avermectin, ivermectin, and milbemycin.
  • the invention also features a method for control of unwanted nematodes, the method comprising administering to a vertebrate, a plant, a seed or soil a composition comprising:
  • Ri H, a cation or a C1-C5 (i.e., a carbon chain having 1, 2, 3, 4 or 5 carbons) substituted or unsubstituted carbon chain, wherein the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano, cyclopropane, epoxy and a substituted or unsubstituted C1-C2 carbon chain; and
  • R 2 a C15-C19 substituted or unsubstituted carbon chain having a cis or trans double bond between the 9 th and 10 th carbons counting from the carbonyl (carboxyl) carbon and either: (i) a triple bond between the 12 l and 13 th carbons counting from the carbonyl (carboxyl) carbon or (ii) either a single or double bond between the 12 th and 13 th carbons and at least one substituant at one or both of the 12 th and 13 th carbons, wherein the substituants are selected from the group consisting of hydroxy, oxo, halogen, amino, cyano, azido, cyclopropane, cyclopropene, epoxy and a substituted or unsubstituted C1-C2 carbon chain.
  • the C15-C19 carbon chain can be substituted at positions other than the 12 th and/or 13 th carbons.
  • the composition further comprises an aqueous surfactant, Ri is H or a cation; Ri is a C1-C5 substituted or unsubstituted carbon chain, wherein the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano, cyclopropane, epoxy and a substituted or unsubstituted C1-C2 carbon chain; R !
  • Ri is a substituted CI methyl
  • Ri is a C1-C2 substituted or unsubstituted carbon chain
  • R 2 is a C15-C19 substituted or unsubstituted carbon chain having a cis or trans double bond between the 9 th and 10 th carbons counting from the carbonyl (carboxyl) carbon and either:
  • the C1-C2 carbon chain of R 2 is substituted and the substituants aie selected from the group consisting of: hydroxy, oxo, halogen, amino, cyano, azido, and epoxy; the C1-C2 carbon chain of R 2 is substituted and the substituants are selected from the group consisting of: hydroxy, oxo, halogen, azido, and amino; the C1-C2 carbon chain of R 2 is singly substituted; Ri is H; Ri is a cation; R 2 is substituted only at one or both of 12 th and 13 th carbons counting from the carbonyl (carboxyl) carbon; R 2 is substituted only at the 12 th carbon counting from the carbonyl (carboxyl) carbon; R is substituted only at the 13 th carbon counting from the carbonyl (carboxyl) carbon; with R 2 the substituants are polar and are selected from the group consisting of: hydroxy, oxo, epoxy, halogen, amino,
  • the invention features a method comprising administering to a vertebrate, a plant, a seed or soil a composition comprising (a) a fatty acid or salt or ester selected from the group consisting of: ricinoleic acid, ricinelaidic acid, 12-oxo-9(Z)-octadecenoic acid, 12-oxo-9(E)-octadecenoic acid, (12,13)-epoxy- trans-9-octadecenoic acid and vernolic acid; and (b) an aqueous surfactant.
  • a fatty acid or salt or ester selected from the group consisting of: ricinoleic acid, ricinelaidic acid, 12-oxo-9(Z)-octadecenoic acid, 12-oxo-9(E)-octadecenoic acid, (12,13)-epoxy- trans-9-octadecenoic acid and vernolic acid
  • the aqueous surfactant is selected from the group consisting of: ethyl lactate, Span 20, Span 40, Span 80, Span 85, Tween 20, Tween 40, Tween 80, Tween 85 and Igepal CO 630;
  • the composition further comprises a permeation enhancer (e.g., cyclodextrin);
  • the composition further comprises a co- solvent (e.g., isopropanol, acetone, 1,2-propanediol, a petroleum based-oil (e.g., aromatic 200) or a mineral oil (e.g., paraffin oil));
  • the composition further comprises a nematicide selected from the group consisting of: avermectins, ivermectin, and milbemycin;
  • the composition further comprises an inhibitor of oxidation (e.g., butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)).
  • Ri H, a cation or a C1-C5 substituted or unsubstituted carbon chain, wherein the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano, cyclopropane, epoxy and a substituted or unsubstituted C1-C2 carbon chain; and
  • R 2 a C15-C19 substituted or unsubstituted carbon chain having a cis or trans double bond between the 9 th and 10 th carbons counting from the carbonyl (carboxyl) carbon and either: (i) a triple bond between the 12 th and 13 th carbons counting from the carbonyl (carboxyl) carbon or (ii) either a single or double bond between the 12 th and 13 th carbons and at least one substituant at one or both of the 12 th and 13 th carbons, wherein the substituants are selected from the group consisting of hydroxy, oxo, halogen, amino, cyano, azido, cyclopropane, cyclopropene, epoxy and a substituted or unsubstituted C1-C2 carbon chain.
  • the nematode infects plants and the composition is applied to the soil or to plants, the composition is applied to soil before planting, the composition is applied to soil after planting, the composition is applied to soil using a drip system, the composition is applied to soil using a drench system, the composition is applied to plant roots; the composition is applied to seeds; the nematode infects a vertebrate (e.g., a mammal, a bird, a non-human mammal, a human); the composition is formulated as a drench to be administered to a non-human vertebrate; and the composition is formulated as an orally administered drug; the composition is formulated as an injectable drug.
  • a vertebrate e.g., a mammal, a bird, a non-human mammal, a human
  • the composition is formulated as a drench to be administered to a non-human vertebrate
  • the composition is formulated as an orally administered drug
  • the composition is
  • the invention also features a feed for a non-human vertebrate comprising:
  • Ri H, a cation or a C1-C5 substituted or unsubstituted carbon chain, wherein the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano, cyclopropane, epoxy and a substituted or unsubstituted C1-C2 carbon chain; and
  • R 2 a C15-C19 substituted or unsubstituted carbon chain having a cis or trans double bond between the 9 th and 10 th carbons counting from the carbonyl (carboxyl) carbon and either: (i) a triple bond between the 12 th and 13 th carbons counting from the carbonyl (carboxyl) carbon or (ii) either a single or double bond between the 12 th and 13 th carbons and at least one substituant at one or both of the 12 th and 13 th carbons, wherein the substituants are selected from the group consisting of hydroxy, oxo, halogen, amino, cyano, azido, cyclopropane, cyclopropene, epoxy and a substituted or unsubstituted C1-C2 carbon chain.
  • the feed further comprises (b) an aqueous surfactant; the feed has been treated to reduce linoleic acid content, linolenic acid content or both; the feed has been treated to reduce both the gamma linolenic acid content and the alpha linolenic acid content; and the feed is selected from the group consisting of: soy, wheat, corn, sorghum, millet, alfalfa, clover, and rye.
  • the invention also features a nematicidal composition comprising: (a) an effective amount of a compound having the formula
  • Ri a C1-C5 substituted or unsubstituted carbon chain, wherein the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano, cyclopropane, epoxy and a substituted or unsubstituted C1-C2 carbon chain;
  • R 3 a CI 1 substituted or unsubstituted carbon chain having a cis double bond between the 9 th and 10 th carbons counting from the carbonyl (carboxyl) carbon, wherein the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano, cyclopropane, cyclopropene, epoxy and a substituted or unsubstituted C1-C2 carbon chain;
  • R 4 a C2-C6 (i.e., a 2, 3, 4, 5 or 6 carbon) substituted or unsubstituted carbon chain wherein the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano,
  • X and Y are independently a substituted or unsubstitued methyl or S, provided at least one or X and Y is S and wherein the substituants on the methyl selected from the group consisting of: halogen, hydrogen, amino, and hydroxy.
  • composition further comprises an aqueous surfactant; and one of X and Y is CH 2 .
  • the invention also features a nematicidal composition
  • a nematicidal composition comprising; (a) an effective amount of a compound having the formula
  • Ri a C1-C5 substituted or unsubstituted carbon chain, wherein the substituants are selected from the group consisting of: hydroxy, halogen, amino, cyano, cyclopropane, epoxy and a substituted or unsubstituted C1-C2 carbon chain; and
  • R 2 a C15-C19 substituted or unsubstituted carbon chain having a single bond between the 9 th and 10 th carbons counting from the carbonyl (carboxyl) carbon and either: (i) a triple bond between the 12 th and 13 th carbons counting from the carbonyl (carboxyl) carbon or (ii) either a single or double bond between the 12 th and 13 th carbons counting from the carbonyl (carboxyl) carbon and at least one substituant at one or both of the 12 th and 13 th carbons, wherein the substituants are selected from the group consisting of hydroxy, halogen, amino, cyano, cyclopropane, cyclopropene, epoxy and a substituted or unsubstituted C1-C2 carbon chain.
  • the composition further comprises an aqueous surfactant
  • R 2 is a C15-C19 substituted or unsubstituted carbon chain having a single bond between the 9 th and 10 th carbons and a single bond between the 12 th and 13 th carbons counting from the carbonyl (carboxyl) carbon and at least one substituant at one or both of the 12 th and 13 th carbons counting from the carbonyl (carboxyl) carbon, wherein the substituants are selected from the group consisting of hydroxy, halogen, amino, cyano, cyclopropane, cyclopropene, epoxy and a substituted or unsubstituted C1-C2 carbon chain; the 12 th and 13 th carbons are substituted with an epoxy group; and 12 th carbon is substituted with a hydroxy group.
  • compositions comprising, consisting or consisting essentially of at least one such nematicidal compound and an aqueous surfactant or a co-solvent or a permeation enhancer or an anti-oxidant and their use in control nematodes. Also within the invention are compositions comprising, consisting or consisting essentially of at least one such nematicidal compound and an aqueous surfactant and an anti-oxidant. Certain compositions contain two, three, four or more different such nematicidal compounds.
  • a “purified polypeptide”, as used herein, refers to a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated.
  • the polypeptide can constitute at least 10, 20, 50, 70, 80 or 95% by dry weight of the purified preparation.
  • isolated nucleic acid is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid, or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes.
  • the term therefore covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e.,
  • nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones in a DNA library such as a cDNA or genomic DNA library.
  • Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” refers to the sequence of the nucleotides in the nucleic acid molecule, the two phrases can be used interchangeably.
  • substantially pure polypeptide as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules.
  • the substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • the "percent identity" of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
  • B12seq performs a comparison between the subject sequence and a target sequence using either the BLASTN (used to compare nucleic acid sequences) or BLASTP (used to compare amino acid sequences) algorithm.
  • BLASTN used to compare nucleic acid sequences
  • BLASTP used to compare amino acid sequences
  • the default parameters of a BLOSUM62 scoring matrix, gap existence cost of 11 and extension cost of 1, a word size of 3, an expect value of 10, a per residue cost of 1 and a lambda ratio of 0.85 are used when performing amino acid sequence alignments.
  • the output file contains aligned regions of homology between the target sequence and the subject sequence. Once aligned, a length is determined by counting the number of consecutive nucleotides or amino acid residues (i.e., excluding gaps) from the target sequence that align with sequence from the subject sequence starting with any matched position and ending with any other matched position. A matched position is any position where an identical nucleotide or amino acid residue is present in both the target and subject sequence. Gaps of one or more residues can be inserted into a target or subject sequence to maximize sequence alignments between structurally conserved domains (e.g., ⁇ -helices, ⁇ -sheets, and loops).
  • structurally conserved domains e.g., ⁇ -helices, ⁇ -sheets, and loops.
  • nucleic acid or amino acid target sequence that aligns with a subject sequence can result in many different lengths with each length having its own percent identity.
  • percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2.
  • length value will always be an integer.
  • conserved regions in a template, or subject, polypeptide can facilitate homologous polypeptide sequence analysis.
  • conserved regions can be identified by locating a region within the primary amino acid sequence of a template polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains at http://www.sanger.ac.uk Pfam/ and http://genome.wustl.edu/Pfam/. A description of the information included at the Pfam database is described in Sonnhammer et al. (1998) Nucl. Acids Res. 26: 320-322;
  • transgene means a nucleic acid sequence (encoding, e.g., one or more subject polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic plant, animal, or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic plant, animal, or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the plant's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can include one or more transcriptional regulatory sequences and other nucleic acid sequences, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
  • transgenic cell refers to a cell containing a transgene.
  • a transgenic plant is any plant in which one or more, or all, of the cells of the plant includes a transgene.
  • the transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by T-DNA mediated transfer, electroporation, or protoplast transformation.
  • the transgene may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as a leaf, root, seed, or stem.
  • tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as a leaf, root, seed, or stem.
  • hybridizes under high stringency conditions refers to conditions for hybridization in 6X sodium chloride/sodium citrate (SSC) buffer at about 45°C, followed by two washes in 0.2 X SSC buffer, 0.1% SDS at 60°C or 65°C.
  • hybridizes under low stringency conditions refers to conditions for hybridization in 6X SSC buffer at about 45°C, followed by two washes in 6X SSC buffer, 0.1 % (w/v) SDS at 50°C.
  • an agent with “anthelmintic activity” is an agent, which when tested, has measurable nematode-killing activity or results in reduced fertility or sterility in the nematodes such that fewer viable or no offspring result, or compromises the ability of the nematode to infect or reproduce in its host, or interferes with the 5 growth or development of a nematode.
  • the agent may also display nematode repellant properties.
  • the agent is combined with nematodes, e.g., in a well of microtiter dish, in liquid or solid media or in the soil containing the agent. Staged adult nematodes are placed on the media. The time of survival, viability of offspring, and/or the movement of the nematodes are measured.
  • An agent with "anthelmintic activity" o can, for example, reduce the survival time of adult nematodes relative to unexposed similarly staged adults, e.g., by about 20%, 40%, 60%, 80%, or more.
  • an agent with "anthelmintic activity” may also cause the nematodes to cease replicating, regenerating, and/or producing viable progeny, e.g., by about 20%, 40%, 60%, 80%, or more. The effect may be apparent immediately or in successive 5 generations.
  • binding refers to the ability of a first compound and a second compound that are not covalently linked to physically interact.
  • the apparent dissociation constant for a binding event can be 1 mM or less, for example, 10 nM, 1 nM, and 0.1 nM or less.
  • the term "binds specifically” refers to the ability of an antibody to discriminate between a target ligand and a non-target ligand such that the antibody binds to the target ligand and not to the non-target ligand when simultaneously exposed to both the given ligand and non-target ligand, and when the target ligand and the non- target ligand are both present in molar excess over the antibody.
  • altering an activity refers to a change in level, either an increase or a decrease in the activity, (e.g., an increase or decrease in the ability of the polypeptide to bind or regulate other polypeptides or molecules) particularly a fatty acid desaturase-like or fatty acid desaturase activity (e.g., the ability to introduce a double bond at the delta-12 position of a fatty acid).
  • the change can be detected in a 0 qualitative or quantitative observation.
  • a "substituted" carbon, carbon chain, or methyl, alkyl can have one or more hydrogens replaced by another group, e.g., a halogen or a hydroxyl group.
  • a carbon or carbon chain can be singly or multiply substituted.
  • the nematicidal fatty acids and fatty acid analogs described herein provide an effective, environmentally safe means of inhibiting nematode metabolism, growth, viability, fecundity, development, infectivity and/or the nematode life-cycle.
  • the compounds may be used alone or in combination with other nematicidal agents.
  • the greater activity reduced phytotoxicity at active concentrations (i.e., greater therapeutic window) of many of the compounds of the invention compared to prior art non-specific nematicidal fatty acid compounds allows for application post-planting and reduced handling costs providing economic incentives in addition to the environmental benefits.
  • Fig. 1 is a photograph of C. elegans grown on oleic acid methyl ester.
  • Fig. 2 is a photograph of C. elegans grown on linoleic acid methyl ester.
  • Fig. 3 is a photograph of C. elegans grown on ricinoleic acid methyl ester.
  • Fig. 4 is a photograph of C. elegans grown on vernolic acid methyl ester.
  • Fig. 5 is a set of drawings depicting the structures of ricinoleic acid, ricinelaidic acid, 12-oxo-9(Z)-octadecenoic acid, 12-oxo-9(E)-octadecenoic acid, (12,13)-epoxy- trans-9-octadecenoic acid and vernolic acid. The numbering of the carbons is indicated with the carbonyl (carboxyl) carbon being carbon 1.
  • Fig. 6 depicts the result of soil drench phytotoxicity experiments at 500 parts per million (ppm) and 100 ppm for ricinoleic acid (Ra), ricinelaidic acid (Rea) and perlargonic acid methyl ester (P).
  • Fig 7 depicts the result of experiment testing the effect of several formulations of methyl ricinelaidate on a root knot nematode (Meloidogyne incognita)app ⁇ ied to tomato plants grown in pots.
  • nematicidal fatty acids and analogs including nematicidal fatty acids and esters that have activity consistent with that of an inhibitor of a delta-12 fatty acid desaturase.
  • Unsaturated fatty acids are essential to the proper functioning of biological membranes. At physiological temperatures, polar glycerolipids that contain only saturated fatty acids cannot form the liquid-crystalline bilayer that is the fundamental structure of biological membranes.
  • NAD(P)H NAD(P)H
  • cytochrome b5 reductase cytochrome b5 reductase
  • cytochrome b5 reductase cytochrome b5 reductase
  • cytochrome b5 reductase cytochrome b5
  • C double bond
  • Many eukaryotic desaturases are endoplasmic reticulum (ER) bound non-heme diiron-oxo proteins which contain three conserved histidine-rich motifs and two long stretches of hydrophobic residues. These hydrophobic alpha helical domains are thought to position the protein with its bulk exposed to the cytosolic face of
  • linoleate (18:2 ⁇ 9,12) and linolenate (18:3 ⁇ 9,12,15) must be obtained from the diet and, thus, are termed essential fatty acids. These dietary fatty acids come predominately from plant sources, since flowering plants readily desaturate the ⁇ 12 and the ⁇ 15 positions. Certain invertebrate animals, including some insects and nematodes, can synthesize de novo all their component fatty acids including linoleate and linolenate.
  • the nematode C. elegans can synthesize de novo a broad range of polyunsaturated fatty acids including arachidonic acid and eicosapentaenoic acids, a feature not shared by either mammals or flowering plants (Spychalla et al. (1997) Proc. Natl. Acad. Sci USA 94(4): 1142-7).
  • the C. elegans desaturase gene fat-2 has been expressed in S. cerevisiae and shown to be a delta-12 fatty acid desaturase (Peyou-Ndi et al. (2000) Arch. Biochem. Biophys. 376(2):399-408).
  • This enzyme introduces a double bond between the 12th and the 13th carbons (from the carboxylate end) and can convert the mono-unsaturated oleate (18:1 ⁇ 9) and palmitoleate (16:1 ⁇ 9) to the di-unsaturated linoleate (18:2 ⁇ 9,12) and 16:2 ⁇ 9,12 fatty acids, respectively.
  • the nematode delta-12 enzymes are potentially good targets for anti-nematode compounds for several reasons. Firstly, as mentioned above, mammals are thought not to have delta-12 fatty acid desaturases. Thus, inhibitors of the enzyme are likely to be non-toxic to mammals. In addition, the enzymes appear to be phylogenetically diverged from their homologs in plants, having less than 40% pairwise sequence identity at the amino acid level and phylogenetic analyses demonstrate clustering of nematode delta-12 and ⁇ -3 desaturases away from homologs in plants.
  • Sterculic acid a cyclopropenoid fatty acid analog of oleic acid, is a potent inhibitor of delta-9 fatty acid desaturases (Schrnid & Patterson (1998) Lipids 23(3):248-52; Waltermann & Steinbuchel (2000) FEMS Microbiol Lett.l90(l):45-50).
  • cyclopropenoid analogs of linoleic acid may similarly inhibit delta-12 fatty acid desaturases (Dulayymi et al. (1997) Tetrahedron 53(3): 1099-1110). It is worth noting however that malvalate, a delta-8 cyclopropene fatty acid, seems to be equally inhibitory to delta-9 desaturases in some systems, as the delta-9 cyclopropene fatty acid sterculate (Schrnid & Patterson (1998) Lipids 23(3):248-52), demonstrating how difficult it is to predict inhibitory profiles for some fatty acid analogs. Thia fatty acid analogs are also potential inhibitors of fatty acid desaturases (Skrede et al.
  • Certain analogs of linoleic acid that may be specific delta-12 inhibitors include the epoxy fatty acid (vernolic acid), the acetylenic fatty acid (crepenynic acid), 12-oxo- 9(Z)-octadecenoic acid methyl ester or the hydroxy fatty acids (ricinoleic and ricinelaidic acid).
  • fatty acid analogs such as crepenynate, ricinoleate and vernolate acids were not thought to be specific inhibitors of the endogenous delta-12 desaturase desaturase (Broun & Somerville (1997) Plant. Physiol.
  • fatty acids and methyl esters of certain fatty acid analogs are nematicidal and have activity consistent with that of specific inhibitors of nematode delta-12 desaturases.
  • the fatty acids and methyl esters show significantly enhanced activity over other eighteen carbon free fatty acids and esters such as oleate, elaidate and linoleate.
  • fatty acid analogs that are predicted delta-12 desaturase inhibitors show dramatically reduced phytoxicity and can therefore be used effectively while minimizing undesirable damage to non-target organisms.
  • Fatty acid analogs or other types of inhibitors may be supplied to plants exogenously, through sprays for example.
  • the fatty acid analogs may also be applied as a seed coat.
  • the host organism or organism on which the nematode feeds may or may not be engineered to produce lower amounts of linoleate.
  • a host cell that does not naturally produce an inhibitor of a nematode fatty acid desaturase-like polypeptide can be transformed with genes encoding enzymes capable of making inhibitory analogs and provided with appropriate precursor chemicals exogenously if necessary.
  • the active inhibitors and precursors can be made endogenously by the expression of the appropriate enzymes.
  • yeast or other organisms can be modified to produce inhibitors. Nematodes that feed on such organisms would then be exposed to the inhibitors.
  • transgenic cells and/or organisms could be generated that produce enzymes active on fatty acids (e.g., desaturating, hydroxylating, and/or epoxygenating enzymes). Such enzymes may be expressed, for example, in plants, vertebrates, and/or nematodes. These enzymes may produce fatty acids, analogs, or other inhibitors that can then act as specific inhibitors for other enzymes such as a fatty acid desaturase (e.g., a delta-12 epoxygenase from Crepis palaestina produces vernolic acid in transgenic Arabidopsis) (Singh et. al. (2000) Biochem. Society Trans. 28:940- 942; Lee et al.
  • fatty acids e.g., desaturating, hydroxylating, and/or epoxygenating enzymes
  • Such enzymes may be expressed, for example, in plants, vertebrates, and/or nematodes. These enzymes may produce fatty acids, analogs, or other inhibitors that can then
  • the fatty acid analogs used in the invention can be applied to animals, plants or the environment of plants needing nematode control or to the food of animals needing nematode control.
  • the compositions may be applied by, for example drench or drip techniques. With drip applications fatty acid analogs can be applied directly to the base 5 of the plants or the soil immediately adjacent to the plants.
  • the composition may be applied through existing drip irrigation systems. This procedure is particularly applicable for cotton, strawberries, tomatoes, potatoes, vegetables and ornamental plants.
  • a drench application can be used where a sufficient quantity of nematicidal composition is applied such that it drains to the root area of the plants.
  • the o drench technique can be used for a variety of crops and turf grasses.
  • the drench technique can also be used for animals.
  • the nematicidal compositions would be administered orally to promote activity against internal parasitic nematodes. Nematicidal compositions may also be administered in some cases by injection of the host animal. 5
  • a compound of the invention will be applied as an aqueous micro-emulsion.
  • the concentration of the nematicidal composition should be sufficient to control the nematode without causing phytotoxicity to the desired plant or undue toxicity to the animal host.
  • An important aspect of the invention is the surprising discovery that certain fatty acid analogs (e.g., 0 ricinoleic acid, methyl ricinoleate, ricinelaidic acid, methyl ricinelaidate, methyl vernolate) that are predicted to be specific inhibitors of nematode delta-12 desaturases are nematicidal and show significantly enhanced activity over non-specific pesticidal fatty acid esters such as oleate, elaidate and linoleate. Moreover, the compounds show reduced phytotoxicity compared to non-specific short chain pesticidal fatty acid esters 5 such as pelargonate (pelargonic acid or methyl pelargonate). Thus, the compositions of this invention show excellent nematicidal activity at concentrations that are not phytotoxic.
  • fatty acid analogs e.g., 0 ricinoleic acid, methyl ricinoleate, ricinelaidic acid, methyl ricinelaidate
  • nematicidal fatty acid analogs of the invention can be applied in conjunction with another nematicidal agent.
  • the second agent may, for example, be 0 applied simultaneously or sequentially.
  • Such nematicidal agents can include for example, avermectins for animal applications.
  • a nematicidal fatty acid analog may also be coupled to an agent such as glyphosate to improve phloem mobility to the roots of plants.
  • the aforementioned nematicidal fatty acid ester compositions can be used to treat diseases or infestations caused by nematodes of the following non-limiting, exemplary genera: Anguina, Ditylenchus, Tylenchorhynchus, Pratylenchus, Radopholus, Hirschmanniella, Nacobbus, Hoplolaimus, Scutellonema, Rotylenchus, Helicotylenchus, Rotylenchulus, Belonolaimus, Heterodera, other cyst nematodes, Meloidogyne, Criconemoides, Hemicycliophora, Paratylenchus, Tylenchulus, Aphelenchoides, Bursaphelenchus, Rhadinaphelenchus, Longidorus, Xiphi
  • Dioctophyme Dioctophyme, Dipetalonema, Dracunculus, Enterobius, Filaroides, Haemonchus, Lagochilascaris, Loa, Manseonella, Muellerius, Necator, Nematodirus, Oesophagostomum, Ostertagia, Parafilaria, Parascaris, Physaloptera, Protostrongylus, Setaria, Spirocerca, Stephana gilaria, Strongyloides, Strongylus, Thelazia, Toxascaris, Toxocara, Trichinella, Tricho strongylus, Trichuris, Uncinaria, and Wuchereria.
  • nematodes including Dirofilaria, Onchocerca, Brugia, Acanthocheilonema, Dipetalonema, Loa, Mansonella, Parafilaria, Setaria, Stephanofilaria, and Wucheria, Pratylenchus, Heterodera, Meloidogyne, Paratylenchus.
  • Species that are particularly preferred are: Ancylostoma caninum, Haemonchus contorius, Trichinella spiralis, Trichurs muris, Dirofilaria immitis, Dirofilaria tenuis, Dirofilaria repens, Dirofilari ursi, Ascaris suum, Toxocara canis, Toxocara cati, Strongyloides ratti, Parastrongyloides trichosuri, Heterodera glycines, Globodera pallida, Meloidogyne javanica, Meloidogyne incognita, and Meloidogyne arenaria, Radopholus similis, Longidorus elongatus, Meloidogyne hapla, and Pratylenchus penetrans.
  • a double stranded RNA (dsRNA) molecule can be used to inactivate a delta-12 fatty acid desaturase (delta-12 fat-2) gene in a cell by a process known as RNA mediated-interference (Fire et al. (1998) Nature 391:806-811, and G ⁇ nczy et al. (2000) Nature 408:331-336).
  • the dsRNA molecule can have the nucleotide sequence of a delta-12 fat-2 nucleic acid (preferably exonic) or a fragment thereof.
  • the dsRNA molecule can be delivered to nematodes via direct injection, or by soaking nematodes in aqueous solution containing concentrated dsRNA, or by raising bacteriovorous nematodes on E. coli genetically engineered to produce the dsRNA molecule.
  • RNAi by injection To examine the effect of inhibiting delta-12 fat-2 activity, a dsRNA corresponding to the C. elegans delta-12 fat-2 gene was injected into the nematode, basically as described in Mello et al. (1991) EMBO J. 10:3959-3970. Briefly, a plasmid was constructed that contains a portion of the C. elegans delta-12 fat- 2 sequence, specifically a fragment 651 nucleotides long, containing the entire first exon and terminating just before the conserved intron splice junction between the first exon and first intron. This construct encodes approximately the first 217 amino acids of the C. elegans delta-12 fat-2 gene.
  • RNAs Single-stranded RNAs were transcribed from these fragments using T7 RNA polymerase and SP6 RNA polymerase (the RNAs correspond to the sense and antisense RNA strands). RNA was precipitated and resuspended in RNAse free water. For annealing of ssRNAs to form dsRNAs, ssRNAs were combined, heated to 95° for two minutes then allowed to cool from 70° to room temperature over 1.5-2.5 hours.
  • DsRNA was injected into the body cavity of 15-20 young adult C. elegans hermaphrodites. Worms were immobilized on an agarose pad and typically injected at a concentration of 1 mg/ml. injections were performed with visual observation using a Zeiss Axiovert compound microscope equipped with 10X and 40X DIG objectives, for example. Needles for microinjection were prepared using a Narishige needle puller, stage micromanipulator (Leitz) and a N2-powered injector (Narishige) set at 10-20 p.s.i.
  • GFP Green Fluorescent Protein
  • C. elegans delta-12 FAT RNAi injection phenotype presented as a strongly reduced FI hatch-rate, with the few surviving individuals arrested in an early larval stage.
  • RNAi by feeding C. elegans can be grown on lawns of E. coli genetically engineered to produce double stranded RNA (dsRNA) designed to inhibit delta-12 fat-2 expression.
  • dsRNA double stranded RNA
  • E. coli were transformed with a genomic fragment of a portion of the C. elegans fat-2 gene sequence, specifically a fragment 651 nucleotides long, containing the entire first exon and terminating just before the conserved intron splice junction between the first exon and first intron. This construct encodes approximately the first 217 amino acids of the C. elegans delta-12 FAT gene.
  • the 651 nucleotide genomic fragment was cloned into an E. coli expression vector between opposing T7 polymerase promoters.
  • RNAi Green Fluorescent Protein
  • the sequence of the fat-2 gene is of sufficiently high complexity (i.e., unique) such that the RNAi is not likely to represent cross reactivity with other genes.
  • coli expressing dsRNA and those injected with dsRNAfrom the delta-12 FAT gene were strongly impaired indicating that the fatty acid desaturase-like gene provides an essential function in nematodes and that dsRNA from the fatty acid desaturase-like gene is lethal when ingested by or injected into C. elegans.
  • the C. elegans delta-12 fatty acid desaturase (FAT-2 protein) converts the mono-unsaturated oleic acid to the di-unsaturated fatty acid linoleic acid.
  • the delta-12 FAT RNAi prevents expression of the delta-12 fatty acid desaturase, which is predicted to cause a decrease in levels of linoleic acid in the nematode, leading to arrested development and death.
  • Addition of 3 mM linoleic acid methyl ester to the NGM media used for the RNAi experiment brings about a partial rescue of the delta-12 FAT RNAi feeding phenotype. Addition of 3 mM oleic acid methyl ester does not rescue the delta-12 FAT RNAi feeding phenotype (see Table 1 below).
  • Table 1 C. elegans delta-12 fat-2 RNAi feeding phenotypes (starting with C. elegans L4 larvae as the P0 animal)
  • fatty acid and Igepal CO 630 After careful mixing of fatty acid and Igepal CO 630, 850 ⁇ l of ddH 2 0 was added and mixed by gentle pipetting until a homogeneous solution was obtained. Finally, 120 ⁇ l of pure isopropanol was added and mixed by gentle pipetting. 1% stock emulsions were also prepared for the potassium salt of ricinoleic acid, the sodium salt of ricinelaidic acid, and ricinelaidic free acid. For the potassium salt of ricinoleic acid, 0.01 grams were dissolved in 100 ⁇ l of ddH 2 0, and combined with 20 ⁇ l of the surfactant Igepal CO 630 in a 1.5 ml eppendorf tube.
  • acetone control emulsion was prepared by combining 100 ⁇ l of acetone, 20 ⁇ l of the surfactant Igepal CO 630, 760 ⁇ l of ddH 2 0, and 120 ⁇ l of pure isopropanol in a 1.5 ml eppendorf tube and mixing to homogeneity.
  • fatty acid emulsions or control emulsions were added and rapidly mixed by swirling. Nematode viability was scored by visual observation and motility assays at various time points 24 hours following addition of emulsions or controls.
  • the fatty acid emulsions tested were methyl esters of nonanoic (pelargonic) acid, ricinoleic acid, vernolic acid, linoleic acid, oleic acid, and control emulsions lacking fatty acids.
  • the structures of ricinoleic acid, ricinelaidic acid and vernolic acid are depicted in Fig. 5.
  • Nonanoic (pelargonic) and ricinoleic acid methyl ester emulsions are strongly nematicidal at a concentration of 0.1%.
  • Nonanoic methyl ester emulsions cause an almost immediate cessation of nematode movement and subsequent death whereas ricinoleic methyl ester emulsions require up to several hours before strong killing effects are apparent.
  • nonanoic acid methyl ester emulsions temporarily "stunned" C. elegans, initially giving the appearance of a 100% death phenotype. Several hours post inoculation, many nematodes recover and start moving again. This "stun" effect was not observed with the other fatty acid emulsions. Oleic acid methyl ester was not nematicidal.
  • ricinoleic and ricinelaidic acid, salt and methyl ester and (12,13)-epoxy-trans-9-octadecenoic acid methyl ester show significant nematicidal activity against both normally fed C. elegans ⁇ 2 worms and C. elegans dauer larvae.
  • the dauer pathway is an alternate life-cycle entered in response to crowding and starvation.
  • Nematodes and emulsions were incubated with shaking at room temperature for 48 hours. The contents of each well were transferred to a small spot on individual NGM plates lacking bacteria. About 24 hours after the transfer to plates, worms on and off the inoculation spot were counted as not viable or viable, respectively. Worms were considered viable if they had crawled away from the inoculation spot, or if they were moving. Worms were considered non- viable if they remained at the inoculation spot.
  • Nonanoic, vernolic and ricinoleic acid methyl ester emulsions have significant nematicidal activity against root knot nematodes (Meloidogyne spp.) at a concentration of 0.1%.
  • Sterilized tomato seeds were germinated in magenta jars containing Gamborg's agar media. After two weeks of growth, seedlings were treated with 250 ⁇ l of 1% fatty acid methyl ester emulsion (nonanoic acid, ricinoleic acid, ricinelaidic acid, oleic acid, or a control emulsion lacking any fatty acid), applied directly to the stem-media interface. Tomato seedlings were scored at various times after application of emulsions. Of the fatty acids tested, only 1% nonanoic acid methyl ester emulsion showed obvious effects on the tomatoes.
  • nonanoic acid emulsion application Within 18 hours of nonanoic acid emulsion application, those tomatoes showed a distinct loss of turgor pressure (wilting phenotype) and had become noticeably less green in appearance. Within 24 hours, nonanoic acid treated tomatoes were almost entirely bleached to a pale white color and had nearly totally collapsed with most leaves lying directly on the agar media surface. Importantly, none of the tomatoes treated with the other fatty acid methyl ester emulsions showed visible effects. Therefore, ricinoleic and ricinelaidic (see examples 9-12) acid methyl esters show excellent potential as anthelmintic chemicals based on their combination of high nematicidal properties and with favorable low phytotoxicity.
  • Nematicidal Activity of Single Fattv Acid Methyl Ester Emulsions Against a Spectrum of Free-Living, Animal Parasitic, and Plant Parasitic Nematodes Briefly, the indicated fatty acid emulsions were added and rapidly mixed by swirling. Nematode viability was scored by visual observation and motility assays 24 hours following addition of emulsions (48 hours for plant parasitic nematodes Meloidogyne and Heterodera species). The fatty acid emulsions tested were methyl
  • the Caenorhabiditis elegans were mixed stage populations. Similar effects seen on several other free-living nematode species.
  • the Parastrongyloides trichosuri (parasite of Australian bushtail possum) were dauer-like infective 3 rd stage larva. Similar effects are also seen against free-living stages.
  • the Meloidogyne incognita and Meloidogyne javanica were 2 nd stage juveniles (dauer-like infective stage).
  • the Heterodera glycines sub-bean cyst nematode
  • the Pratylenchus scribneri (corn lesion nematode) were mixed stage populations.
  • Example 9 Some nematodes assayed as described in Example 9 lacked tolerance to emulsions made with the surfactant Igepal CO 630. For this reason some assays were repeated with Tween-20-based emulsions.
  • fatty acid methyl ester emulsions made with Tween-20 replacing Igepal CO 630 exhibited comparable nematicidal activity to Igepal-based emulsions.
  • isopropanol was replaced with one of two cyclodextrins (Methyl- ⁇ - Cyclodextrin or 2-Hydroxypropyl- ⁇ -Cyclodextrin).
  • Table 8 Nematicidal activity of various fatty acid methyl ester emulsions with cyclodextrins against C. elegans
  • fatty acid emulsion mixtures or control emulsions were added and rapidly mixed by swirling. Nematode viability was scored by visual observation and motility assays at various time points 24 hours following addition of emulsions or controls.
  • the fatty acid emulsion mixtures tested were methyl esters of ricinelaidic acid, ricinoleic acid, oleic acid and control emulsions lacking fatty acids.
  • N.B. Blank is the stock emulsion with no fatty acid added. Since there is no fatty acid in the blank, Ricinelaidic/Ricinoleic, Ricinelaidic/Oleic, and Ricinelaidic/blank mixture comparisons have equal amounts of emulsion components and equal amounts of Re when compared at the same percentages. However, the Re/blank experiment has less total fatty acid so the 0.1 and 0.01% assays have to be multiplied by the % Re to get the total fatty acid added.
  • fatty acid emulsion mixtures or control emulsions were added and rapidly mixed by swirling. Nematode viability was scored by visual observation and motility assays at various time points 24 hours following addition of emulsions or controls.
  • the fatty acid emulsion mixtures tested were ricinelaidic acid, ricinoleic acid, oleic acid and control emulsions lacking fatty acids.

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Abstract

Cette invention concerne certains acides gras et composés connexes permettant de combattre efficacement les nématodes qui infestent les plantes ou leur site. Ces méthodes et composés agissent également contre les nématodes qui parasitent les animaux.
PCT/US2003/006525 2002-03-04 2003-03-04 Acide gras nematicide et composes apparentes a des esters d'acide gras WO2003075656A2 (fr)

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CA002478589A CA2478589A1 (fr) 2002-03-04 2003-03-04 Acide gras nematicide et composes apparentes a des esters d'acide gras
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WO2004093886A1 (fr) * 2003-04-24 2004-11-04 Galderma S.A. Formulation a usage topique d'ivermectine pour le traitement de conditions dermatologiques
EP1773110A2 (fr) * 2004-08-04 2007-04-18 Divergence, Inc. Acides nucléiques codant des agents anthelminthiques et plantes fabriquées à partir de ceux-ci
FR2899061A1 (fr) * 2006-03-31 2007-10-05 Cid Lines Nv Composition d'hygiene et/ou comestique et/ou desinfectante contenant au moins un agent anti-parasitaire repulsif et/ou insectifuge.
WO2007096833A3 (fr) * 2006-02-27 2008-01-31 Univ Northwest Préparation de support de plantes, véhicule pour l'administration et la translocation de substances bénéfiques sur le plan phytologique et compositions les contenant
US7939713B2 (en) 2003-02-05 2011-05-10 Divergence, Inc. Method for screening transgenic plants for anthelmintic activity
WO2014100874A1 (fr) * 2012-12-26 2014-07-03 Empresa Brasileira De Pesquisa Agropecuária-Embrapa Composition nématotoxique à effet synergique, et utilisation d'une composition nématotoxique à effet synergique
US8815816B2 (en) 2003-04-24 2014-08-26 Galderma S.A. Topical application of ivermectin for the treatment of dermatological conditions/afflictions
US9089587B2 (en) 2013-07-08 2015-07-28 Galderma S.A. Treatment of papulopustular rosacea with ivermectin
US9782425B2 (en) 2013-07-08 2017-10-10 Galderma S.A. Treatment of papulopustular rosacea with ivermectin
WO2018161131A1 (fr) * 2017-03-10 2018-09-13 Leite Miguel Produit bactéricide/germicide/fongicide/sporicide extrait des acides gras à administrer par voie orale à des animaux d'élevage monogastriques et/ou des ruminants, et procédés d'obtention
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US11033565B2 (en) 2003-04-24 2021-06-15 Galderma Holding SA Topical application of ivermectin for the treatment of dermatological conditions/afflictions
US8815816B2 (en) 2003-04-24 2014-08-26 Galderma S.A. Topical application of ivermectin for the treatment of dermatological conditions/afflictions
US8598129B2 (en) 2003-04-24 2013-12-03 Galderma S.A. Topical application of ivermectin for the treatment of dermatological conditions/afflictions
US8470788B2 (en) 2003-04-24 2013-06-25 Galderma S.A. Topical application of ivermectin for the treatment of dermatological conditions/afflictions
US8415311B2 (en) 2003-04-24 2013-04-09 Galderma S.A. Topical application of ivermectin for the treatment of dermatological conditions/afflictions
US7550440B2 (en) 2003-04-24 2009-06-23 Galderma S.A. Topical application of ivermectin for the treatment of dermatological conditions/afflictions
WO2004093886A1 (fr) * 2003-04-24 2004-11-04 Galderma S.A. Formulation a usage topique d'ivermectine pour le traitement de conditions dermatologiques
US8093219B2 (en) 2003-04-24 2012-01-10 Galderma S.A. Topical application of ivermectin for the treatment of dermatological conditions/afflictions
US8080530B2 (en) 2003-04-24 2011-12-20 Galderma S.A. Topical application of ivermectin for the treatment of dermatological conditions/afflictions
US7667095B2 (en) 2004-02-04 2010-02-23 Divergence, Inc. Transgenic plants having anthelmintic activity and methods of producing them
US7932435B2 (en) 2004-02-04 2011-04-26 Divergence, Inc. Method of screening transgenic plants for anthelmintic activity
US7919680B2 (en) 2004-02-04 2011-04-05 Divergence, Inc. Nucleic acids encoding anthelmintic agents and plants made therefrom
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EP1773110A4 (fr) * 2004-08-04 2008-09-17 Divergence Inc Acides nucléiques codant des agents anthelminthiques et plantes fabriquées à partir de ceux-ci
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WO2007096833A3 (fr) * 2006-02-27 2008-01-31 Univ Northwest Préparation de support de plantes, véhicule pour l'administration et la translocation de substances bénéfiques sur le plan phytologique et compositions les contenant
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FR2899061A1 (fr) * 2006-03-31 2007-10-05 Cid Lines Nv Composition d'hygiene et/ou comestique et/ou desinfectante contenant au moins un agent anti-parasitaire repulsif et/ou insectifuge.
EP1842425A1 (fr) 2006-03-31 2007-10-10 Cid Lines N.V. Composition d'hygiène et/ou cosmétique et/ou désinfectante contentant au moins un agent anti-parasitaire répulsif et/ou insectifuge
WO2014100874A1 (fr) * 2012-12-26 2014-07-03 Empresa Brasileira De Pesquisa Agropecuária-Embrapa Composition nématotoxique à effet synergique, et utilisation d'une composition nématotoxique à effet synergique
US9089587B2 (en) 2013-07-08 2015-07-28 Galderma S.A. Treatment of papulopustular rosacea with ivermectin
US9233117B2 (en) 2013-07-08 2016-01-12 Galderma S. A. Treatment of inflammatory lesions of rosacea with ivermectin
US9233118B2 (en) 2013-07-08 2016-01-12 Galderma S.A. Treatment of papulopustular rosacea with ivermectin
US9782425B2 (en) 2013-07-08 2017-10-10 Galderma S.A. Treatment of papulopustular rosacea with ivermectin
US10206939B2 (en) 2013-07-08 2019-02-19 Galderma S.A. Treatment of papulopustular rosacea with ivermectin
US11122801B2 (en) 2015-07-09 2021-09-21 Apreslabs Ltd. Inhibitors of enzymes that confer pesticide resistance
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