Patent Application
20110008471
The presently-disclosed subject matter relates to methods for treating parasitic infections and compositions useful for treating parasitic infections. It also relates to screening systems and methods for developing agents and compositions useful for treating parasitic infections
Parasitic infections of plants, humans, and other animals pose a worldwide problem. For example, more than 650 million people are at risk for gastrointestinal parasitic infection, and about 200 million are actually infected. Various conditions contribute to the development and spread of parasitic infections, including poor sanitary conditions; low host resistance; population expansion; and inadequate control of vectors and infection reservoirs.
Such parasitic infections present an abundance of medical and social problems. For example, parasitic infection can undermine child development, educational achievement, reproductive health, and social and economic development. Indeed, some parasitic infections can cause morbidity and mortality. Notwithstanding the severe impact that parasitic infections can have, relatively few treatment options are available.
Available treatments are limited, and treatments for some parasitic infections are non-existent. In the 1960s, niclosamide (also known as yomesan) was identified for use in treating certain helminthic parasitic infections; however, niclosamide has certain drawbacks. For example, in many cases a single dose of niclosamide does not provide a curative effect, rather, a relapse ensues because the compound has difficulty accessing cysticercoids buried deeply within the mucosal villi. As such, satisfactory results require an extended treatment with niclosamide for approximately 7 days. See Davis, Drug treatment of intestinal helminthiasis, World Health Organization (WHO), Geneva, 1973.
Another drug that has been used to treat helminthic parasitic infections is Praziquantel (2-(cyclohexylcarbonyl)-1,2,3,6,7,11b-hexahydro-4H-pyrazino(2,1-a)isoquinolin-4-one; also known as Biltracide). See Pearson and Gurrant, Praziquantel: a major advance in anthelminthic therapy. Annals of Internal Medicine, 99:195-198, 1983. Praziquantel can be administered in a single dose; however, treatment strategies making use of Praziquantel are at risk because of the possibility of the development of resistance to Praziquantel. Accordingly, there remains a need in the art for non-harmful compositions that are effective for treating parasitic infections.
The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of the information provided in this document.
This Summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Disclosure of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.
The presently-disclosed subject matter includes compositions and methods for treating parasitic infections, and methods of screening for and selecting compositions useful for treating a parasitic infection.
In some embodiments, the parasitic infections are caused by parasites classified as endoparasites, ectoparasites, human parasites, animal parasites, or agricultural parasites.
In some embodiments, the composition for treating a parasitic infection in a subject includes two or more compounds selected from: trans-anethole, para-cymene, linalool, α-pinene, and thymol.
In some embodiments, the composition includes two more compounds selected from: para-cymene, linalool, α-pinene, and thymol. In some embodiments, the composition includes three or more compounds selected from: para-cymene, linalool, α-pinene, and thymol. In some embodiments, the composition includes para-cymene, linalool, α-pinene, and thymol. In some embodiments, the composition further includes soy bean oil.
In some embodiments, the composition includes 25-35% by weight para-cymene, 1-10% by weight linalool, 1-10% by weight α-pinene, 35-45% by weight thymol, and 20-30% by weight soy bean oil. In some embodiments, the composition includes 28.39% by weight para-cymene, 6.6‰ by weight linalool, 3.8% by weight α-pinene, 37.2% by weight thymol, and 24% by weight soy bean oil.
In some embodiments, the composition includes 25-35% by volume para-cymene, 1-10% by volume linalool, 1-10% by volume α-pinene, 35-45% by volume thymol and 20-30% by volume soy bean oil. In some embodiments, the composition includes 30% by volume para-cymene, 7% by volume linalool, 4%>by volume α-pinene, 35% by volume thymol, and 24% by volume soy bean oil.
In some embodiments, the composition includes three or more compounds selected from: trans-anethole, para-cymene, linalool, α-pinene, and thymol. In some embodiments, the composition includes four or more compounds selected from: trans-anethole, para-cymene, linalool, α-pinene, and thymol. In some embodiments, the composition includes trans-anethole, para-cymene, linalool, α-pinene, and thymol.
In some embodiments, the composition includes 15-25% by weight trans-anethole, 30-40% by weight para-cymene, 1-10% by weight linalool, 1-10% by weight α-pinene, and 35-45% by weight thymol. In some embodiments, the composition includes 18.2% by weight trans-anethole, 34.4% by weight para-cymene, 4.7% by weight linalool, 1.9% by weight α-pinene, and 40.8% by weight thymol.
In some embodiments, the composition includes 10-20% by volume trans-anethole, 30-40% by volume para-cymene, 1-10% by volume linalool, 1-10% by volume α-pinene, and 35-45% by volume thymol. In some embodiments, the composition includes 17% by volume trans-anethole, 37% by volume para-cymene, 5% by volume linalool, 2% by volume α-pinene, and 39% by volume thymol.
In some embodiments, the composition includes 15-25% by weight trans-anethole, 1-10% by weight para-cymene, 35-45% by weight linalool, 1-10% by weight α-pinene, and 30-40% by weight thymol. In some embodiments, the composition includes 18.2% by weight trans-anethole, 1.9% by weight para-cymene, 40.8% by weight linalool, 4.7% by weight α-pinene, and 34.4% by weight thymol.
In some embodiments, the composition includes 15-25% by volume trans-anethole, 1-10% by volume para-cymene, 35-45% by volume linalool, 1-10% by volume α-pinene, and 30-40% by volume thymol. In some embodiments, the composition includes 17% by volume trans-anethole, 2% by volume para-cymene, 39% by volume linalool, 5% by volume α-pinene, and 37% by volume thymol.
In some embodiments, the compounds of the composition together demonstrate a synergistic anti-parasitic effect. In some embodiments, the actual percent effect of the composition is greater than the expected percent effect of the composition. In some embodiments the coefficient of synergy relative to a component of the composition is greater than 5, 10, 25, 50, 75, or 100.
In some embodiments, the parasitic infection is by a protozoan parasite. In some embodiments, the parasite is selected from intestinal protozoa, tissue protozoa, and blood protozoa. In some embodiments, the parasite is selected from: Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Cryptosporidium parvum, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis.
In some embodiments, the parasitic infection is by a helminthic parasite. In some embodiments, the parasite is selected from nematodes. In some embodiments, the parasite is selected from Adenophorea. In some embodiments, the parasite is selected from Secementea. In some embodiments, the parasite is selected from: Trichuris trichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, Dracunculus medinensis. In some embodiments, the parasite is selected from trematodes. In some embodiments, the parasite is selected from: blood flukes, liver flukes, intestinal flukes, and lung flukes. In some embodiments, the parasite is selected from: Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica, Heterophyes heterophyes, Paragonimus westermani, and Opishorchis sinensis.
In some embodiments, the parasite is selected from cestodes. In some embodiments, the parasite is selected from Taenia solium, Taenia saginata, Hymenolepis nana, Echinococcus granulosus, and Diplyidium caninum.
In some embodiments, the composition is provided in a formulation. The formulation can include the composition and a carrier, such as a food product. In some embodiments the formulation includes the composition encapsulated or microencapsulated with an outer shell material.
The presently-disclosed subject matter includes a method of treating a parasitic infection in a subject. In some embodiments, the method includes administering to the subject an effective amount of a composition as described herein.
The presently-disclosed subject matter includes a method for selecting a composition for use in treating a parasitic infection. In some embodiments, the method includes: providing a cell expressing a tyramine receptor; contacting test compounds to the cell; measuring the receptor binding affinity of the compounds; measuring at least one parameter selected from, (i) intracellular cAMP level, and (ii) intracellular Ca2+ level; identifying a first compound for the composition that is capable of altering at least one of said parameters, and which has a high receptor binding affinity for the tyramine receptor; identifying a second compound for the composition that is capable of altering at least one of said parameters, and which has a low receptor binding affinity for the tyramine receptor; and selecting a composition including the first and second compounds. In some embodiments, the selected composition demonstrates an anti-parasitic effect that exceeds the anti-parasitic effect of any of the compounds when used alone.
An embodiment of the present disclosure provides an antiparasitic composition, comprising a synergistic combination of two or more compounds from a blend listed in Table E.
An embodiment of the present disclosure provides an antiparasitic composition, comprising a synergistic combination of three or more compounds from a blend listed in Table E.
An embodiment of the present disclosure provides an antiparasitic composition, comprising a synergistic combination of four or more compounds from a blend listed in Table E.
An embodiment of the present disclosure provides an antiparasitic composition, comprising a synergistic combination of all compounds from a blend listed in Table E.
An embodiment of the present disclosure provides an antiparasitic composition wherein the amount of each compound is within a range obtained by multiplying the amount in Table E by Factor 1.
An embodiment of the present disclosure provides an antiparasitic composition, wherein the amount of each compound is within a range obtained by multiplying the amount in Table E by Factor 2.
An embodiment of the present disclosure provides an antiparasitic composition, wherein the amount of each compound is within a range obtained by multiplying the amount in Table E by Factor 3.
An embodiment of the present disclosure provides an antiparasitic composition, wherein the amount of each compound is within a range obtained by multiplying the amount in Table E by Factor 4.
An embodiment of the present disclosure provides an antiparasitic composition, wherein each compound is present in the amount stated in Table E.
An embodiment of the present disclosure provides an antiparasitic composition, wherein a coefficient of synergy relative to a component of the composition is greater than 5, 10, 25, 50, 75, or 100.
An embodiment of the present disclosure provides an antiparasitic composition, wherein the composition exhibits synergistic effects on a parasite selected from the group consisting of: a protozoan parasite, a helminthic parasite, a pest of the subclass Acari, a louse, a flea, or a fly.
An embodiment of the present disclosure provides an antiparasitic composition, wherein the composition exhibits synergistic effects on a parasite having a host selected from the group consisting of: canola, cat, dog, goat, horse, man, maize, mouse, ox, pig, poultry, rabbit, rice, sheep, soybean, tobacco, and wheat.
An embodiment of the present disclosure provides any of the above antiparasitic compositions, additionally comprising an ingredient selected from the group consisting of a surfactant and a fixed oil.
An embodiment of the present disclosure provides an antiparasitic composition, comprising a synergistic combination of two or more compounds listed in any of Tables B, B1, C, D, or E.
An embodiment of the present disclosure provides a formulation comprising the composition of any of the above antiparasitic compositions and a carrier.
An embodiment of the present disclosure provides the above formulation, wherein the carrier is a food product.
An embodiment of the present disclosure provides any of the above antiparasitic compositions as a medicament for the treatment or prevention of parasitic disease or infestation.
An embodiment of the present disclosure relates to the any of the above antiparasitic compositions as an antiparasitic agent for the treatment or prevention of parasitic disease or infestation.
An embodiment of the present disclosure relates to a method of treating a parasitic infection in a subject, comprising administering an effective amount of any of the above antiparasitic compositions to the subject.
An embodiment of the present disclosure relates to the above metho, where the parasitic infection is caused by a parasite in a classification selected from the group consisting of endoparasites, ectoparasites, human parasites, animal parasites, or agricultural parasites.
An embodiment of the present disclosure relates to a method of selecting a composition for use in treating a parasitic infection, comprising: providing a cell expressing a receptor selected from the group consisting of a tyramine receptor and a receptor of the olfactory cascade; contacting test compounds to the cell; measuring the receptor binding affinity of the compounds; measuring at least one parameter selected from (i) intracellular cAMP level; and (ii) intracellular Ca2+ level; identifying a first compound for the composition that is capable of altering at least one of said parameters, and which has a high receptor binding affinity for the receptor; and identifying a second compound for the composition that is capable of altering at least one of said parameters, and which has a low receptor binding affinity for the receptor; and selecting a composition including the first and second compounds.
An embodiment of the present disclosure relates to a method of selecting a composition for use in treating a parasitic infection, comprising: providing a cell expressing a receptor selected from the group consisting of the receptors listed in Table F; contacting test compounds to the cell; measuring the receptor binding affinity of the compounds; measuring at least one parameter selected from (i) intracellular cAMP level; and (ii) intracellular Ca2+ level; identifying a first compound for the composition that is capable of altering at least one of said parameters, and which has a high receptor binding affinity for the receptor; and identifying a second compound for the composition that is capable of altering at least one of said parameters, and which has a low receptor binding affinity for the receptor; and selecting a composition including the first and second compounds.
An embodiment of the present disclosure relates to a method of selecting a composition for use in treating a parasitic infection, comprising: providing a cell comprising a molecular target selected from the group consisting of the molecular targets listed in Table G; contacting test compounds to the cell; measuring the binding affinity of the compounds for the molecular target; measuring at least one parameter selected from (i) intracellular cAMP level; and (ii) intracellular Ca2+ level; identifying a first compound for the composition that is capable of altering at least one of said parameters, and which has a high binding affinity for the molecular target; and identifying a second compound for the composition that is capable of altering at least one of said parameters, and which has a low binding affinity for the molecular target; and selecting a composition including the first and second compounds.
The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.
The presently-disclosed subject matter includes compositions and methods for treating parasitic infections, and methods of screening for and selecting compositions useful for treating a parasitic infection.
As used herein, the term “parasitic infection” refers to the infection of a plant or animal host by a parasite, such as a successful invasion of a host by an endoparasite, including for example a protozoan parasite or a helminthic parasite.
As used herein, the term “parasite” includes parasites, such as but not limited to, protozoa, including intestinal protozoa, tissue protozoa, and blood protozoa. Examples of intestinal protozoa include, but are not limited to: Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, and Cryptosporidium parvum. Examples of tissue protozoa include, but are not limited to: Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, and Trichomonas vaginalis. Examples of blood protozoa include, but are not limited to Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium falciparum. Histomonas meleagridis is yet another example of a protozoan parasite.
As used herein, the term “parasite” further includes, but is not limited to: helminthes or parasitic worms, including nematodes (round worms) and platyhelminthes (flat worms). Examples of nematodes include, but are not limited to: animal and plant nematodes of the adenophorea class, such as the intestinal nematode Trichuris trichiura (whipworm) and the plant nematode Trichodorus obtusus (stubby-root nematode); intestinal nematodes of the secementea class, such as Ascaris lumbricoides, Enterobius vermicularis (pinworm), Ancylostoma duodenale (hookworm), Necator americanus (hookworm), and Strongyloides stercoralis; and tissue nematodes of the secementea class, such as Wuchereria bancrofti (Filaria bancrofti) and Dracunculus medinensis (Guinea worm). Examples of plathyeminthes include, but are not limited to: Trematodes (flukes), including blood flukes, such as Schistosoma mansoni (intestinal Schistosomiasis), Schistosoma haematobium, and Schistosoma japonicum; liver flukes, such as Fasciola hepatica, and Fasciola gigantica; intestinal flukes, such as Heterophyes heterophyes; and lung flukes such as Paragonimus westermani. Examples of platheminthes further include, but are not limited to: Cestodes (tapeworms), including Taenia solium, Taenia saginata, Hymenolepis nana, and Echinococcus granulosus.
Furthermore, the term “parasite” further includes, but is not limited to those organisms and classes of organisms listed in the following Table A:
Entamoeba
coli
dispar
histolytica
gingivalis
Balantidium
coli
Giardia
intenstinalis
lamblia
Trichomonas
vaginalis
Histomonas
meleagridis
Trypanosoma
avium
brucei
cruzi
equiperdum
evansi
vivax
Eimeria
acervulina
brunetti
jemezi
maxima
nextrix
tenella
stiedae
meleagridis
Isospora
belli
felis
canis
Cyclospora
cayetanensis
Cryptosporidium
parvum
hominis
canis
felis
hominis
meleagridis
muris
Sarcocystis
cruzi
hominis
muris
Toxoplasma
gondii
Neospora
caninum
Babesia
major
microti
divergens
duncani
gibsoni
Plasmodium
falciparum
vivax
ovale
malariae
knowlesi
gigliolii
Leishmania
aethiopica
donovani
major
mexicana
tropica
braziliensis
Fasciola
hepatica
magna
gigantica
jacksoni
Dicrocoelium
dendriticum
Schistosoma
mansoni
japonicum
mekongi
intercalatum
haematobium
Taenia
crassiceps
pisiformis
saginata
solium
Dipylidium
caninum
Echinococcus
granulosus
multilocularis
shiquicus
Aphelenchoides
fragariae
ritzemabosi
besseyi.
Heterodera
Globodera
solanacearum
virginiae
tabacum
Nacobbus
dorsalis
Pratylenchus
brachurus
penetrans
Ditylenchus
dipsaci
Xiphinema
americanum
Longidorus
sylphus
Paratrichodorus
minor
Dioctophyma
renale
Meloidogyne
hapla
incognita
javanica
Trichostrongylus
tenius
Ostertagia
Nematodirus
Haemonchus
Cooperia
Trichuris
Ascaris
Parascaris
Oxyuris
Toxascaris
Toxocara
T. catti, refs back to non-migratory Toxascaris
Trichinella
Oesophagostomum
Chabertia
Cyathostomes
Strongylus
vulgaris
Bunostomum
Uncinaria
Ancylostoma
Dictyocaulus
Metastrongylus
Parafilaria
Dirofialria
Cercospora
zeae-maydis
Ustilago
maydis
Magnaporthe
grisea
Bipolaris
oryzae
Psoroptes ovis,
Chorioptes
Sarcoptes,
Knemidocoptes
Demodex,
Trombicula,
Cheyletiella
Dermanyssus,
Ornithonyssus
Ixodes ricinus
Linognathus and
Haematopinus sp.
Trichodectes and
Felicola
Lipeurus,
Cuclotogaster,
Menopon
Ctenocephalides felis
Ceratophyllus and
Echidnophaga
Haematobia and
Stomoxys
Melophagus ovinus
Phlebotomus sand
Lucilia cuprina
Hypoderma bovis
Gasterophilus and
Oestrus bots
Alternaria black spot =
Alternaria brassicae
Alternaria brassicicola
Alternaria japonica =
Alternaria raphani
Colletotrichum gloeosporioides
Glomerella cingulata [teleomorph]
Colletotrichum higginsianum
Leptosphaeria maculans
Phoma lingam [anamorph]
Rhizopus stolonifer
Aphanomyces raphani
Rhizoctonia solani
Thanatephorus cucumeris [teleomorph]
Cercospora brassicicola
Plasmodiophora brassicae
Peronospora parasitica
Fusarium oxysporum fsp. conglutinins
Botrytis cinerea
Botryotinia fuckeliana [teleomorph]
Rhizoctonia solani
Thanatephorus cucumeris [teleomorph]
Alternaria alternata
Ascochyta spp.
Pyrenopeziza brassicae
Cylindrosporium concentricum [anamorph]
Alternaria alternats
Cladosporium spp.
Erysiphe polygoni
Erysiphe cruciferarum
Mycosphaerella brassicicola
Asteromella brassicae [anamorph]
Alternaria alternata
Fusarium spp.
Macrophomina phaseolina
Phymatotrichopsis omnivora
Phytophthora megasperma
Pythium debaryanum
Pythium irregulare
Rhizoctonia solani
Thanatephorus cucumeris [teleomorph]
Sclerotium rolfsii
Athelia rolfsii [teleomorph]
Sclerotinia stem rot
Sclerotinia sclerotiorum
Alternaria spp.
Fusarium spp.
Gliocladium roseum
Nectria ochroleuca [teleomorph]
Pythium spp.
Rhizoctonia solani
Thanatephorus cucumeris [teleomorph]
Rhizopus stolonifer
Sclerotium rolfsii
Urocystis brassicae
Sclerotium rolfsii
Verticillium wilt
Verticillium longisporum
Rhizoctonia solani
Thanatephorus cucumeris [teleomorph]
Pseudocercosporella capsellae =
Cercosporella brassicae
Mycosphaerella capsellae [teleomorph]
Albugo candida =
Albugo cruciferarum
Fusarium oxysporum
Besnoitia sp. (oocysts)
Isospora felis
Isospora rivolta
Sarcocystis gigantea (sporocysts)
Sarcocystis hirsuta (sporocysts)
Sarcocystis medusijormis (sporocysts)
Sarcocystis muris (sporocysts)
Sarcocystis sp. (sporocysts)
Toxoplasma gondii (cysts)
Toxoplasma gondii (oocysts)
Giardia intestinalis
Hammondia heydorni (oocysts)
Isospora canis
Isospora ohioensis
Neospora caninum
Sarcocystis arieticanis (sporocysts)
Sarcocystis capracanis (sporocysts)
Sarcocystis cruzi (sporocysts)
Sarcocystis tenella (sporocysts)
Sarcocystis sp. (sporocysts)
Toxoplasma gondii (cysts)
Giardia intestinalis
Cryptosporidium sp.
Eimeria alijevi
Eimeria apsheronica
Eimeria arloingi
Eimeria capralis
Eimeria caprina
Eimeria caprovina
Eimeria charlestoni
Eimeria christenseni
Eimeria hirci
Eimeria jolchejevi
Eimeria masseyensis
Eimeria ninakohlyakimovae
Eimeria punctata
Eimeria tunisiensis
Sarcocystis capracanis (cysts)
Toxoplasma gondii (cysts)
Giardia sp.
Eimeria leuckarti
Klossiella equi
Sarcocystis sp. (cysts)
Ciyptosporidium sp.
Isospora hominis*
Plasmodium sp.*
Toxoplasma gondii (cysts)
Chilomastix mesnili
Dientamoeba fragilis
Endolimax nana
Entamoeba coli
Entamoeba hartmanni
Entamoeba histolytica
Giardia intestinalis
Iodamoeba buetschlii
Leishmania donovani*
Trichomonas hominis
Trichomonas vaginalis
Colletotrichum graminicola
Glomerella graminicola
Glomerella tucumanensis
Glomerella falcatum
Aspergillus ear and kernel rot
Aspergillus flavus
Rhizoctonia solani = Rhizoctonia microsclerotia
Thanatephorus cucumeris
Acremonium strictum = Cephalosporium
acremonium
Lasiodiplodia theobromae = Botryodiplodia
theobromae
Marasmiellus sp.
Physoderma maydis
Cephalosporium kernel rot
Acremonium strictum = Cephalosporium
acremonium
Macrophomina phaseolina
Corticium ear rot
Thanatephorus cucumeris = Corticium sasakii
Curvularia leaf spot
Curvularia clavata
C. eragrostidis = C. maculans
Cochliobolus eragrostidis
Curvularia inaequalis
C. intermedia
Cochliobolus intermedius
Curvularia lunata
Cochliobolus lunatus
Curvularia pallescens Cochliobolus pallescens
Curvularia senegalensis
C. tuberculate
Cochliobolus tuberculatus
Didymella leaf spot
Didymella exitalis
Diplodia ear rot and stalk rot
Diplodia frumenti
Botryosphaeria festucae
Diplodia ear rot
Diplodia maydis
Diplodia leaf spot or leaf streak
Stenocarpella macrospora = Diplodia
macrospora
Sclerophthora rayssiae
Sclerophthora macrospora = Sclerospora
macrospora
Sclerospora graminicola
Graminicola downy mildew
Peronosclerospora maydis = Sclerospora
maydis
Peronosclerospora philippinensis = Sclerospora
philippinensis
Sorghum downy mildew
Peronosclerospora sorghi = Sclerospora sorghi
Spontaneum downy mildew
Peronosclerospora spontanea = Sclerospora
spontanea
Peronosclerospora sacchari = Sclerospora
sacchari
Nigrospora oryzae
Khuskia oryzae
Alternaria alternata = A. tenuis
Aspergillus glaucus
A. niger
Aspergillus spp.
Botrytis cinerea
Botryotinia fuckeliana
Cunninghamella sp.
Curvularia pallescens
Doratomyces stemonitis = Cephalotrichum
stemonitis
Fusarium culmorum
Gonatobotrys simplex
Pithomyces maydicus
Rhizopus microsporus
R. stolonifer = R. nigricans
Scopulariopsis brumptii
Claviceps gigantea
Sphacelia sp.
Aureobasidium zeae = Kabatiella zeae
Fusarium ear and stalk rot
Fusarium subglutinans = F. moniliforme
Fusarium kernel, root and stalk rot, seed rot and
Fusarium moniliforme
Gibberella fujikuroi
Fusarium stalk rot
Fusarium avenaceum
Gibberella avenacea
Gibberella ear and stalk rot
Gibberella zeae
Fusarium graminearum
Botryosphaeria zeae = Physalospora zeae
Macrophoma zeae
Cercospora sorghi = C. sorghi
Cercospora leaf spot
C. zeae-maydis
Helminthosporium root rot
Exserohilum pedicellatum = Helminthosporium
pedicellatum
Setosphaeria pedicellata
Hormodendrum ear rot
Cladosporium cladosporioides =
Cladosporium rot
Hormodendrum cladosporioides
C. herbarum
Mycosphaerella tassiana
Hyalothyridium leaf spot
Hyalothyridium maydis
Cephalosporium maydis
Alternaria alternata
A. tritici
A. zeicola
Bipolaris victoriae = Helminthosporium
victoriae
Cochliobolus victoriae
C. sativus
Bipolaris sorokiniana = H. sorokinianum = H. sativum
Epicoccum nigrum
Exserohilum prolatum = Drechslera prolata
Setosphaeria prolata
Graphium penicillioides
Leptosphaeria maydis
Leptothyrium zeae
Ophiosphaerella herpotricha
Scolecosporiella sp.
Paraphaeosphaeria michotii
Phoma sp.
Septoria zeae
S. zeicola
S. zeina
Setosphaeria turcica
Exserohilum turcicum = Helminthosporium
turcicum
Cochliobolus carbonum
Helminthosporium ear rot (race 1)
Bipolaris zeicola = Helminthosporium
carbonum
Penicillium ear rot
Penicillium spp.
P. chrysogenum
P. expansum
P. oxalicum
Phaeocytostroma stalk rot and root rot
Phaeocytostroma ambiguum =
Phaeocytosporella zeae
Phaeosphaeria leaf spot
Phaeosphaeria maydis = Sphaerulina maydis
Physalospora ear rot
Botryosphaeria festucae = Physalospora zeicola
Botryosphaeria ear rot
Diplodia frumenti
Hemiparasitic bacteria and fungi
Pyrenochaeta stalk rot and root rot
Phoma terrestris = Pyrenochaeta terrestris
Pythium root rot
Pythium spp.
P. arrhenomanes
P. graminicola
Pythium stalk rot
Pythium aphanidermatum = P. butleri
Epicoccum nigrum
Rhizoctonia ear rot
Rhizoctonia zeae
Sclerotial rot
Waitea circinata
Rhizoctonia root rot and stalk rot
Rhizoctonia solani
R. zeae
Alternaria alternata
Cercospora sorghi
Dictochaeta fertilis
Fusarium acuminatum Gibberella acuminata
F. equiseti
G. intricans
F. oxysporum
F. pallidoroseum
F. poae
F. roseum
G. cyanogena
F. sulphureum
Microdochium bolleyi
Mucor sp.
Periconia circinata
Phytophthora cactorum
P. drechsleri
P. nicotianae
Rhizopus arrhizus
Rostratum leaf spot
Setosphaeria rostrata = Helminthosporium
Helminthosporium leaf disease, ear and stalk
rostratum
Puccinia sorghi
Puccinia polysora
Physopella pallescens
P. zeae = Angiopsora zeae
Sclerotium ear rot
Sclerotium rolfsii
Athelia rolfsii
Bipolaris sorokiniana
B. zeicola = Helminthosporium carbonum
Diplodia maydis
Exserohilum pedicillatum
Exserohilum turcicum = Helminthosporium
turcicum
Fusarium avenaceum
F. culmorum
F. moniliforme
Gibberella zeae
F. graminearum
Macrophomina phaseolina
Penicillium spp.
Phomopsis spp.
Pythium spp.
Rhizoctonia solani
Sclerotium rolfsii
Spicaria spp.
Selenophoma leaf spot
Selenophoma sp.
Gaeumannomyces graminis
Myrothecium gramineum
Monascus purpureus
M. ruber
Ustilaso zeae = U. maydis
Ustilasinoidea virens
Sphacelotheca reiliana = Sporisorium holci-
sorghi
Cochliobolus heterostrophus
Bipolaris maydis = Helminthosporium maydis
Stenocarpella macrospora = Diplodia
macrospora
Cercospora sorghi
Fusarium episphaeria
F. merismoides
F. oxysporum
F. poae
F. roseum
F. solani
Nectria haematococca
F. tricinctum
Mariannaea elegans
Mucor spp.
Rhopographus zeae
Spicaria spp.
Aspergillus spp.
Penicillium spp. and other fungi
Phyllachora maydis
Trichoderma ear rot and root rot
Trichoderma viride = T. lignorum
Hypocrea sp.
Stenocarpella maydis = Diplodia zeae
Ascochyta ischaemi
Phyllosticta maydis
Mycosphaerella zeae-maydis
Gloeocercospora sorghi
Dolichodorus spp.
D. heterocephalus
Ditylenchus dipsaci
Radopholus similis
Heterodera avenae
H. zeae
Punctodera chalcoensis
Xiphinema spp.
X. Americanum X. mediterraneum
Nacobbus dorsalis
Hoplolaimus Columbus
Hoplolaimus spp.
H. galeatus
Pratylenchus spp.
P. brachyurus
P. crenatus
P. hexincisus
P. neglectus
P. penetrans
P. scribneri
P. thornei
P. zeae
Longidorus spp.
L. breviannulatus
Criconemella spp.
C. ornata
Meloidogyne spp.
M. chitwoodi
M. incognita
M. javanica
Helicotylenchus spp.
Belonolaimus spp.
B. longicaudatus
Paratrichodorus spp.
P. christiei
P. minor
Quinisulcius acutus
Trichodorus spp.
Tylenchorhynchus dubius
Hepatozoon musculi
Sarcocystis muris (cysts)
Giardia intestinalis
Giardia muris
Cryptosporidium sp.
Eimeria alabamensis
Eimeria auburnensis
Eimeria bovis
Eimeria brasiliensis
Eimeria bukidnonensis
Eimeria canadensis
Eimeria cylindrica
Eimeria ellipsoidalis
Eimeria subspherica
Eimeria wyomingensis
Eimeria zurnii
Isospora sp.
Neospora caninum
Sarcocystis cruzi (cysts)
Sarcocystis hirsuta (cysts)
Theileria orientalis
Tritrichomonas foetus
Balantidium coli
Cryptosporidium sp.
Eimeria cerdonis
Eimeria debliecki
Eimeria neodebliecki
Eimeria porci
Eimeria scabra
Eimeria suis
Isospora suis
Sarcocystis sp. (cysts)
Toxoplasma gondii (cysts)
Balantidium coli
Histomonas meleagridis
Hexamita meleagridis
Eimeria spp.
Ascaridia galli
Ascaridia dissimilis
Ascardidia columbae
Capillaria contorta
Capillaria obsingata
Capillaria caudinflata
Heterakis gallinarum
Heterakis isolonche
Syngamus trachea
Cnemidocoptes mutans
Cnemidocoptes gallinae
Dermanyssus gallinae
Lamiosioptes cysticola
Ornithonyssus slyvarium
Ceratophyllus gallinae
Echindnophaga gallinacea
Menacanthus stramineus
Eimeria flavescens
Eimeria irresidua
Eimeria media
Eimeria petforans
Eimeria pyriformis
Eimeria stiedae
Hepatozoon cuniculi
Sarcocystis sp. (cysts)
Toxoplasma gondii (cysts)
Ceratobasidium oryzae-sativae
Rhizoctonia oryzae-sativae
Curvularia lunata
Cochliobolus lunatus
Pyricularia grisea =
Pyricularia oryzae
Magnaporthe grisea
Cochliobolus miyabeanus
Bipolaris oryzae
Gaeumannomyces graminis
Sclerophthora macrospora
Drechslera gigantea
Ustilaginoidea vixens
Tilletia barclayana =
Neovossia horrida
Entyloma oryzae
Microdochium oyvzae =
Rhynchosporium oryzae
Cercospora janseana =
Cercospora oryzae
Sphaerulina oryzina
Cochliobolus miyabeanus
Curvularia spp.
Fusarium spp.
Microdochium oryzae
Sarocladium oryzae and other fungi.
Fusarium spp.
Pythium spp.
Pythium dissotocum
Pythium spinosum
Cochliobolus miyabeanus
Curvularia spp.
Fusarium spp.
Rhizoctonia solani
Sclerotium rolfsii
Athelia rolfsii
Thanatephorus cucumeris
Rhizoctonia solani
Sarocladium oryzae =
Acrocylindrium oryzae
Rhizoctonia oryzae
Alternaria padwickii
Magnaporthe salvinii
Sclerotium oryzae
Achlya conspicua
Achlya klebsiana
Fusarium spp.
Pythium spp.
Pythium dissotocum
Pythium spinosum
Aphelenchoides besseyi
Meloidogyne spp.
Hirschmanniella oryzae
Ditylenchus angustus
Ctyptosporidium sp.
Eimeria ahsata
Eimeria crandallis
Eimeria faurei
Eimeria granulosa
Eimeria intricata
Eimeria ovinoidalis
Eimeria ovis
Eimeria pallida
Eimeria pama
Eimeria punctata
Eimeria weybridgensis
Sarcocystis arieticanis (cysts)
Sarcocystis gigantea (cysts)
Sarcocystis medusiformis (cysts)
Sarcocystis tenella (cysts)
Toxoplasma gondii (cysts)
Alternaria leaf spot
Alternaria spp.
Anthracnose
Collelotrichum truncatum
Collelotrichum demalium f. truncatum
Glomerella glycines
Colletotrichum destructivum
Arkoola nigra
Thielaviopsis basicola
Chalara elegans [synanamorph]
Septoria glycines
Mycosphaerella usoenskajae
Phialophora gregata =
Cephalosporium gregatum
Macrophomina phaseolina
Choanephora leaf blight
Choanephora infundibulifera
Choanephora trispora
Rhizoctonia solani
Thanatephorus cucumeris
Pythium aphanidermatum
Pythium debaryanum
Pythium irregulare
Pythium myriotylum
Pythium ultimum
Peronospora manshurica
Drechslera blight
Drechslera glycines
Cercospora sojina
Fusarium root rot
Fusarium spp.
Leptosphaerulina leaf spot
Leptosphaerulina trifolii
Mycoleptodiscus root rot
Mycoleptodiscus terrestris
Neocosmospora stem rot
Neocosmospora vasinfecta
Acremonium spp.
Phomopsis seed decay
Phomopsis spp.
Phytophthora root and stem rot
Phytophthora sojae
Phyllosticta leaf spot
Phyllosticta sojaecola
Phymatotrichum root rot = cotton root rot
Phymatotrichopsis omnivora =
Phymatotrichum omnivorum
Diaporthe phaseolorum
Phomopsis sojae
Microsphaera diffusa
Cercospora kikuchii
Pyrenochaeta leaf spot
Pyrenochaeta glycines
Pythium rot
Pythium aphanidermatum
Pythium debaryanum
Pythium irregulare
Pythium myriotylum
Pythium ultimum
Cylindrocladium crotalariae
Calonectria crotalariae
Dactuliochaeta glycines =
Pyrenochaeta glycines
Dactuliophora glycines [synanamorph]
Rhizoctonia aerial blight
Rhizoctonia solani
Thanatephorus cucumeris
Rhizoctonia root and stem rot
Rhizoctonia solani
Phakopsora pachyrhizi
Spaceloma glycines
Sclerotinia stem rot
Sclerotinia sclerotiorum
Sclerotium rolfsii
Athelia rolfsii
Diaporthe phaseolorum
Diaporthe phaseolorum var. caulivora
Phomopsis phaseoli
Stemphylium leaf blight
Stemphylium botryosum
Pleospora tarda
Fusarium solani f.sp. glycines
Corynespora cassiicola
Nematospora coryli
Hoplolaimus columbus
Hoplolaimus galeatus
Hoplolaimus magnistylus
Pratylenchus spp.
Paratylenchus projectus
Paratylenchus tenuicaudatus
Rotylenchulus reniformis
Criconemella ornata
Meloidogyne arenaria
Meloidogyne hapla
Meloidogyne incognita
Meloidogyne javanica
Hemicycliophora spp.
Heterodera glycines
Helicotylenchus spp.
Belonolainus gracilis Belonolainus
longicaudatus
Paratrichodorus minor
Quinisulcius acutus
Tylenchorhynchus spp.
Collelotrichum destructivum
Glomerella glycines
Cercospora nicotianae
Thielaviopsis basicola
Phytophthora nicotianae
Peronospora tabacina =
Peronospora hyoscyami f. sp. tabacina
Alternaria alternata
Macrophomina phaseolina
Sclerotinia sclerotiorum
Pythium spp.
Pythium aphanidermatum
Pythium ultimum
Cercospora nicotianae
Fusarium wilt
Fusarium oxysporum
Botrytis cinerea
Botryotinia fuckeliana
Mycosphaerella leaf spot
Mycosphaerella nicotianae
Olpidium seedling blight
Olpidium brassicae
Phyllosticta leaf spot
Phyllosticta nicotiana
Erysiphe cichoracearum
Phoma exigua var. exigua =
Ascochyta phaseolorum
Hymenula affinis =
Fusarium affine
Rhizoctonia solani
Thanatephorus cucumeris
Sclerotium rolfsii
Athelia rolfsii
Pythium spp.
Rhizoctonia solani
Verticillium wilt
Verticillium albo-atrum
Verticillium dahliae
Ditylenchus dipsaci
Globodera solanacearum =
Globodera virginiae
Globodera tabacum
Xiphinema americanum
Aphelenchoides ritzemabosi
Pratylenchus brachyurus
Pratylenchus penetrans
Pratylenchus spp.
Rotylenchulus reniformis
Meloidogvne arenaria
Meloidogvne hapla
Meloidogvne incognita
Meloidogvne javanica
Helicotylenchus spp.
Paratrichodorus spp.
Trichodorus spp.
Merlinius spp.
Tylenchorhynchus spp.
Alternaria leaf blight
Alternaria triticina
Colletotrichum graminicola
Glomerella graminicola
Ascochyta leaf spot
Ascochyta tritici
Aureobasidium decay
Microdochium bolleyi =
Aureobasidium bolleyi
Alternaria spp.
Cladosporium spp.
Epicoccum spp.
Sporobolomyces spp.
Stemphylium spp. and other genera
Cephalosporium stripe
Hymenula cerealis =
Cephalosporium gramineum
Tilletia tritici =
Tilletia caries
Tilletia laevis =
Tilletia foetida
Cochliobolus sativus
Bipolaris sorokiniana =
Helminthosporium sativum
Coprinus psychromorbidus
Fusarium spp.
Fusarium pseudograminearum
Gibberella zeae
Fusarium graminearum Group II
Gibberella avenacea
Fusarium avenaceum
Fusarium culmorum
Dilophospora leaf spot = twist
Dilophospora alopecuri
Sclerophthora macrospora
Tilletia controversa
Claviceps purpurea
Sphacelia segetum
Tapesia vallundae
Ramulispora herpotrichoides =
Pseudocercosporella herpotrichoides W-
T. acuformis
Ramulispora acuformis =
Pseudocercosporella herpotrichoides var.
acuformis R-pathotype
Gibellina cerealis
Urocystis agropyri
Fusarium spp.
Pseudoseptoria donacis =
Selenophoma donacis
Tilletia indica =
Neovossia indica
Puccinia triticina
Puccinia recondita f. sp. tritici
Puccinia tritici-duri
Leptosphaeria leaf spot
Phaeosphaeria herpotrichoides =
Leptosphaeria herpotrichoides
Stagonospora sp.
Ustilaso tritici =
Ustilaso segetum var. tritici
Ustilaso segetum var. nuda
Ustilaso segetum var. avenae
Microscopica leaf spot
Phaeosphaeria microscopica =
Leptosphaeria microscopica
Phoma spot
Phoma spp.
Phoma glomerata
Phoma sorghina =
Phoma insidiosa
Microdochium nivale =
Fusarium nivale
Monographella nivalis
Platyspora leaf spot
Clathrospora pentamera =
Platyspora pentamera
Erysiphe graminis f. sp. tritici
Blumeria graminis =
Erysiphe graminis
Oidium monilioides
Pythium root rot
Pythium aphanidermalum
Pythium arrhenomanes
Pythium graminicola
Pythium myriotylum
Pythium volutum
Rhizoctonia root rot
Rhizoctonia solani
Thanalephorus cucumeris
Pyrenophora seminiperda =
Drechslera campanulata
Drechslera wirreganensis
Fusarium spp.
Gibberella zeae
Fusarium graminearum Group II
Gibberella avenacea
Fusarium avenaceum
Fusarium culmorum
Microdochium nivale =
Fusarium nivale
Monographella nivalis
Sclerotinia snow mold = snow
Myriosclerotinia borealis =
Sclerotinia borealis
Sclerotium wilt (see Southern
Sclerotium rolfsii
Athelia rolfsii
Septoria blotch
Septoria tritici
Mycosphaerella graminicola
Rhizoctonia cerealis
Ceratobasidium cereale
Pythium spp.
Pythium aristosporum
Pythium iwayamae
Pythium okanoganense
Sclerotium rolfsii
Athelia rolfsii
Typhula idahoensis
Typhula incarnata
Typhula ishikariensis
Typhula ishikariensis var. canadensis
Cochliobolus sativus
Bipolaris sorokiniana =
Helminthosporium sativum
Stagonospora blotch
Phaeosphaeria avenaria f. sp. triticae
Stasonospora avenae f. sp. triticae =
Septoria avenae f. sp. triticea
Phaeosphaeria nodorum
Stagonospora nodorum =
Septoria nodorum
Puccinia graminis =
Puccinia graminis f. sp. tritici
Aspersillus spp.
Penicillium spp.
Puccinia striiformis
Uredo glumarum
Gaeumannomyces graminis var. tritici
Gaeumannomyces graminis var. avenae
Pyrenophora tritici-repentis
Drechslera tritici-repentis
Phyllachora graminis
Linochora graminis
Masnaporthe grisea
Lagena radicicola
Ligniera pilorum
Olpidium brassicae
Rhizophydium graminis
Compositions of the invention can be used to treat parasitic infections. In some embodiments, the compositions can include compounds that are generally regarded as safe (GRAS compounds). In some embodiments, the compositions can include compounds of a plant origin, such as plant essential oils or monoterpenoids of plant essential oils. In some embodiments, the compositions include two or more compounds. In some embodiments, the compositions can include any of the following oils, or mixtures thereof:
In other embodiments, methods can be used to assess or screen the anti-parasitic effect of a particular small molecule other than the essential oils described above. These small molecules can include, for example, any of the following small molecules, or the like, or any other small molecules that include these groups, or different groups of the like. In the following table, the bolded designations indicate generic terms for small molecules sharing particular characteristics, while non-bolded terms following the bolded generic terms indicate individual small molecules within the genus described by the bolded term.
Cumulenes:
Polycyclic heteroarenes:
Allenes:
Pseudohalogens:
Functional Classes:
Monocyclic heterarnes:
Heteroaryl groups:
Arynes:
Polycyclic Arenes:
Organic Hetro
Monocyclic Compounds
Heteroaryl Groups:
Organic
Heterobicyclic
Compounds:
Monocyclic Heteroarenes:
Alkanes:
Alicyclic Compounds:
Hydrocarbylidene
Groups:
Crown Compounds:
Arylene groups
Carbonyl Group
Organyl Groups
Alkenylidene Groups
Organoheteryl
Groups:
Elemental Carbon:
Hydrocarbylidyne Group:
In some embodiments, compositions include two or more compounds selected from the following compounds:
In some embodiments of the compositions that include lilac flower oil, one or more of the following compounds can be substituted for the lilac flower oil: tetrahydrolinalool; ethyl linalool; heliotropine; hedion; hercolyn D; and triethyl citrate.
In some embodiments of the compositions that include black seed oil, one or more of the following compounds can be substituted for the black seed oil: α-thujene, α-pinene, β-pinene, p-cymene, limonene, and tert-butyl-p-benzoquinone.
In some embodiments of the compositions that include thyme oil, one or more of the following compounds can be substituted for the thyme oil: thymol, α-thujone; α-pinene, camphene, β-pinene, p-cymene, α-terpinene, linalool, borneol, and β-caryophyllene. In some embodiments of the compositions that include thymol, thyme oil can be substituted. In some embodiments of the compositions that include thyme oil, it can be desirable to include a specific type of thyme oil. In this regard, thyme oil (white) is preferred to thyme oil (red) because the latter has been found to cause negative side effects for the subject or host.
Compounds used to prepare embodiments of the compositions can be obtained, for example, from the following sources: Millennium Chemicals, Inc. (Jacksonville, Fla.), Ungerer Company (Lincoln Park, N.J.), SAFC (Milwaukee, Wis.), IFF Inc. (Hazlet, N.J.); Sigma Chemical Co. (St. Louis, Mo.); and The Lebermuth Company, Inc. (Southbend, Ind.).
In some embodiments of the compositions, it can be desirable to include a naturally-occurring version or a synthetic version of a compound. For example, in certain embodiments it can be desirable to include Lime Oil 410, a synthetic lime oil that can be obtained, for example, from Millennium Chemicals, Inc. In certain exemplary compositions, it can be desirable to include a compound that is designated as meeting Food Chemical Codex (FCC), for example, geraniol Fine FCC or Tetrahydrolinalool FCC, which compounds can be obtained, for example, from Millennium Chemicals, Inc.
In some embodiments of the compositions, it can be desirable to include a compound having a specific purity. In some embodiments of the compositions, it can be desirable to include compounds each having a purity of at least about 80%, 81%, 82%, 83%), 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. For example, in some embodiments of the compositions including α-pinene, an α-pinene that is at least about 98% pure can be selected. For another example, in embodiments of the compositions including linalool, a linalool that is at least about 97-99% pure (e.g., linalool coeur) can be selected.
In some embodiments of the compositions, it can be desirable to include compounds each having a purity of about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. For example, in some embodiments of the compositions that include geraniol, it can be desirable to include a geraniol that is at least about 60%, 85% or 95%) pure. In some embodiments, it can be desirable to include a specific type of geraniol. For example, in some embodiments, the compositions can include: geraniol 60, geraniol 85, or geraniol 95. When geraniol is obtained as geraniol 60, geraniol 85, or geraniol 95, then forty percent, fifteen percent, or five percent of the oil can be Nerol. Nerol is a monoterpene (C10H18O), which can be extracted from attar of roses, oil of orange blossoms, and oil of lavender.
In some embodiments, compositions include two or more compounds selected from the following compounds: linalool, thymol, α-pinene, para-cymene, and trans-anethole. In some embodiments, compositions include three or more compounds selected from the following compounds: linalool, thymol, α-pinene, para-cymene, and Trans-Anethole. In some embodiments, compositions include four or more compounds selected from the following compounds: linalool, thymol, α-pinene, para-cymene, and Trans-Anethole. In some embodiments, compositions include: linalool, thymol, α-pinene, para-cymene, and Trans-Anethole. In some embodiments, it is preferred that an α-pinene that is at least about 98% pure is used. In some embodiments, it is preferred that a linalool that is a linalool coeur is used. In some embodiments, the composition can further include soy bean oil.
In some embodiments, compositions include two or more compounds selected from the following compounds: linalool, thymol, α-pinene, and para-cymene. In some embodiments, compositions include three or more compounds selected from the following compounds: linalool, thymol, α-pinene, and para-cymene. In some embodiments, compositions include: linalool, thymol, α-pinene, and para-cymene. In some embodiments, it is preferred that an α-pinene that is at least about 98% pure is used. In some embodiments, it is preferred that a linalool that is a linalool coeur is used. In some embodiments, the composition can further include soy bean oil.
In some embodiments, each compound can make up between about 1% to about 99‰, by weight (wt/wt %) or by volume (vol/vol %), of the composition. For example, composition can comprises about 1% α-pinene and about 99% thymol. As used herein, % amounts, by weight or by volume, of compounds are to be understood as referring to relative amounts of the compounds. As such, for example, a composition including 7% linalool, 35% thymol, 4% α-pinene, 30% para-cymene, and 24‰ soy bean oil (vol/vol %) can be said to include a ratio of 7 to 35 to 4 to 30 to 24 linalool, thymol, α-pinene, para-cymene, and soy bean oil, respectively (by volume). As such, if one compound is removed from the composition, or additional compounds or other ingredients are added to the composition, it is contemplated that the remaining compounds can be provided in the same relative amounts. For example, if soy bean oil was removed from the exemplary composition, the resulting composition would include 7 to 35 to 4 to 40 linalool, thymol, α-pinene, and para-cymene, respectively (by volume). This resulting composition would include 9.21% linalool, 46.05% thymol, 5.26% α-pinene, and 39.48% para-cymene (vol/vol %). For another example, if safflower oil was added to the original composition to yield a final composition containing 40% (vol/vol) safflower oil, then the resulting composition would include 4.2% linalool, 21% thymol, 2.4% α-pinene, 18% para-cymene, 14.4% soy bean oil, and 40% safflower oil (vol/vol %).
In some embodiments, the composition includes about 1-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40-45%, about 45-50%, about 50-60%, about 60-75%, or about 75-99% linalool, as measured by volume (vol/vol %). In some embodiments, the composition includes about 4.5-5.5% linalool, as measured by volume. In some embodiments, the composition includes about 5‰ linalool, as measured by volume. In some embodiments, the composition includes about 6.5-7.5% linalool, as measured by volume. In some embodiments, the composition includes about 7% linalool, as measured by volume. In some embodiments, the composition includes about 38-40% linalool, as measured by volume. In some embodiments, the composition includes about 39‰ linalool, as measured by volume.
In some embodiments, the composition includes about 1-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40-45%, about 45-50%, about 50-60%, about 60-75%, or about 75-99% linalool, as measured by weight (wt/wt %). In some embodiments, the composition includes about 4.2-5.2% linalool, as measured by weight. In some embodiments, the composition includes about 4.7% linalool, as measured by weight. In some embodiments, the composition includes about 6.1-7.1% linalool, as measured by weight. In some embodiments, the composition includes about 6.6% linalool, as measured by weight. In some embodiments, the composition includes about 40.3-41.3% linalool, as measured by weight. In some embodiments, the composition includes about 40.8% linalool, as measured by weight.
In some embodiments, the composition includes about 1-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40-45%, about 45-50%, about 50-60%, about 60-75%, or about 75-99% thymol, as measured by volume (vol/vol %). In some embodiments, the composition includes about 38-40% thymol, as measured by volume. In some embodiments, the composition includes about 39% thymol, as measured by volume. In some embodiments, the composition includes about 36-38% thymol, as measured by volume. In some embodiments, the composition includes about 37% thymol, as measured by volume. In some embodiments, the composition includes about 34-36% thymol, as measured by volume. In some embodiments, the composition includes about 35% thymol, as measured by volume.
In some embodiments, the composition includes about 1-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40-45%, about 45-50%, about 50-60%, about 60-75%, or about 75-99% thymol, as measured by weight (wt/wt %). In some embodiments, the composition includes about 40.3-41.3% thymol, as measured by weight. In some embodiments, the composition includes about 40.8% thymol, as measured by weight. In some embodiments, the composition includes about 33.9-34.9% thymol, as measured by weight. In some embodiments, the composition includes about 34.4% thymol, as measured by weight. In some embodiments, the composition includes about 36.7-37.7% thymol, as measured by weight. In some embodiments, the composition includes about 37.2% thymol, as measured by weight.
In some embodiments, the composition includes about 1-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40-45%, about 45-50%, about 50-60%, about 60-75%, or about 75-99% α-pinene, as measured by volume (vol/vol %). In some embodiments, the composition includes about 1.5-2.5% α-pinene, as measured by volume. In some embodiments, the composition includes about 2% α-pinene, as measured by volume. In some embodiments, the composition includes about 4.5-5.5% α-pinene, as measured by volume. In some embodiments, the composition includes about 5% α-pinene, as measured by volume. In some embodiments, the composition includes about 3.5-4.5% α-pinene, as measured by volume. In some embodiments, the composition includes about 4% α-pinene, as measured by volume.
In some embodiments, the composition includes about 1-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40-45%, about 45-50%, about 50-60%, about 60-75%, or about 75-99% α-pinene, as measured by weight (wt/wt %). In some embodiments, the composition includes about 1.4-2.4% α-pinene, as measured by weight. In some embodiments, the composition includes about 1.9% α-pinene, as measured by weight. In some embodiments, the composition includes about 4.2-5.2% α-pinene, as measured by weight. In some embodiments, the composition includes about 4.7% α-pinene, as measured by weight. In some embodiments, the composition includes about 3.3-4.3% α-pinene, as measured by weight. In some embodiments, the composition includes about 3.8% α-pinene, as measured by weight.
In some embodiments, the composition includes about 1-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40-45‰, about 45-50%, about 50-60%, about 60-75%, or about 75-99% para-cymene, as measured by volume (vol/vol %). In some embodiments, the composition includes about 36.5-37.5% para-cymene, as measured by volume. In some embodiments, the composition includes about 37% para-cymene, as measured by volume. In some embodiments, the composition includes about 29.5-30.5% para-cymene, as measured by volume. In some embodiments, the composition includes about 30% para-cymene, as measured by volume. In some embodiments, the composition includes about 1.5-2.5% para-cymene, as measured by volume. In some embodiments, the composition includes about 2% para-cymene, as measured by volume.
In some embodiments, the composition includes about 1-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40-45%, about 45-50%, about 50-60%, about 60-75%, or about 75-99% para-cymene, as measured by weight (wt/wt %). In some embodiments, the composition includes about 33.9-34.9‰ para-cymene, as measured by weight. In some embodiments, the composition includes about 34.4% para-cymene, as measured by weight. In some embodiments, the composition includes about 1.4-2.4% para-cymene, as measured by weight. In some embodiments, the composition includes about 1.9% para-cymene, as measured by weight. In some embodiments, the composition includes about 27.9-28.9% para-cymene, as measured by weight. In some embodiments, the composition includes about 28.4% para-cymene, as measured by weight.
In some embodiments, the composition includes about 1-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40-45%, about 45-50%, about 50-60%, about 60-75%, or about 75-99% trans-anethole, as measured by volume (vol/vol %). In some embodiments, the composition includes about 16.5-17.5‰ trans-anethole, as measured by volume. In some embodiments, the composition includes about 17% trans-anethole, as measured by volume.
In some embodiments, the composition includes about 1-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 30-35%, about 35-40%, about 40-45%, about 45-50%, about 50-60%, about 60-75%, or about 75-99% trans-anethole, as measured by weight (wt/wt %). In some embodiments, the composition includes about 17.7-18.7% trans-anethole, as measured by weight. In some embodiments, the composition includes about 18.2% trans-anethole, as measured by weight.
In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as wt/wt: 15-25% trans-anethole, 30-40% para-cymene, 1-10% linalool, 1-10% α-pinene, and 35-45% thymol. In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as % wt/wt: 18.2% trans-anethole, 34.4% para-cymene, 4.7% linalool, 1.9% α-pinene, and 40.8% thymol.
In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as vol/vol: 10-20% trans-anethole, 30-40% para-cymene, 1-10% linalool, 1-10% α-pinene, and 35-45% thymol. In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as vol/vol: 17% trans-anethole, 37% para-cymene, 5% linalool, 2% α-pinene, and 39% thymol.
In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as wt/wt: 15-25% trans-anethole, 1-10% para-cymene, 35-45% linalool, 1-10% α-pinene, and 30-40% thymol. In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as % wt/wt: 18.2% trans-anethole, 1.9% para-cymene, 40.8% linalool, 4.7% α-pinene, and 34.4% thymol.
In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as % vol/vol: 15-25% trans-anethole, 1-10% para-cymene, 35-45% linalool, 1-10% α-pinene, and 30-40% thymol. In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as % vol/vol: 17% trans-anethole, 2% para-cymene, 39% linalool, 5% α-pinene, and 37% thymol.
In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as wt/wt: 25-35% para-cymene, 1-10% linalool, 1-10% α-pinene, 20-30% soy bean oil, and 35-45% thymol. In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as % wt/wt: 28.39% para-cymene, 6.6% linalool, 3.8% α-pinene, 24% soy bean oil, and 37.2% thymol.
In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as vol/vol: 25-35% para-cymene, 1-10% linalool, 1-10% α-pinene, 20-30% soy bean oil, and 35-45% thymol. In some embodiments, the composition includes the following compounds in the following relative amounts, where the relative amounts of the compounds are expressed as vol/vol: 30% para-cymene, 7% linalool, 4% α-pinene, 24% soy bean oil, and 35% thymol.
In some embodiments the composition can include, for example, any of the following compounds from Table D, or active components of any of the compositions listed as “blends” in Table E, or the like:
Citronella Oil
Citronella Oil
Furthermore, in addition to the specific amounts of ingredients listed for each blend inn Table E above, ranges of amounts are also contemplated that may be derived by multiplying each specific amount by the following four factors: Factor 1 (±200%); Factor 2 (±100%); Factor 3 (±40%); and Factor 4 (±10%). The resulting ranges will not, of course, containing any values less than 0% or greater than 100%.
In some embodiments, compositions are specifically contemplated that comprise a synergistic combination of at least two compounds listed in any of Tables B, B1, C, D, or E above.
Surprisingly, by blending certain compounds in certain relative amounts, the resulting composition demonstrates an anti-parasitic effect that exceeds the anti-parasitic effect of any component of the composition. As used herein, “component of a composition” refers to a compound, or a subset of compounds included in a composition, e.g., the complete composition minus at least one compound. As used herein, an “anti-parasitic effect” refers to any measurable parameter related to the efficacy of a composition for treating a parasitic infection. The effect can be a parameter related to viability, killing, prophylaxis, or another useful and quantifiable parameter for a set time point, or it can be time to achieve a defined result, e.g., time to achieve 100% killing with a set dose. In this regard, when a first effect and a second effect are compared, the first effect can indicate a greater efficacy for treating a parasitic infection if it exceeds the second effect. For example, when the effect being measured is a time to achieve 100% killing, a shorter time is an anti-parasitic effect that exceeds a longer time. For another example, when the effect being measured is a % killing of target parasites, a greater % killing is an anti-parasitic effect that exceeds a lesser % killing. Effects that can be measured include, but are not limited to: time to kill a given percentage of a target parasite in vivo or in vitro; percent viability or percent killing of a target parasite in vivo or in vitro; percent viability of eggs of a target parasite; percent of a host population that is cured of an infestation by a target parasite; percent of a host population that is protected against infection by a target parasite (prophylactic effect); perturbation of a cell message or cell signal in a target parasite, such as, e.g., calcium, cyclic-AMP, and the like; and diminution of activity or downstream effects of a molecular target in a target parasite.
An exemplary in vivo method for assessing the anti-parasitic effect of a particular composition, or component of the composition, can be conducted using host animals. The host animals are infected with a target parasite. The composition or component of interest is administered to the host animal. Administration of the composition or component of interest can be initiated at various times before and/or after infection of the host animal, depending on the target parasite being tested. The eggs generated by the parasite in the host animal are quantified. For example, the eggs in a stool sample collected from the animal can be quantified. The quantification of eggs generated by the parasite in the host animal receiving the composition or component of interest can be compared the quantification of eggs generated by the parasite in another host animal, such as a host animal receiving another composition or component of interest, or a host animal serving as a control, e.g., uninfected control, or untreated control.
An exemplary in vitro method for assessing the anti-parasitic effect of a particular composition or component can be conducted using target parasites provided in test plates. The composition or component of interest is contacted with the target parasites, and the effect is observed, e.g., the effect of the composition or component of interest on the vitality of the target parasites. The effect of the treatment on the target parasites can be compared to the effect of another treatment on target parasites, such as target parasites treated with another composition or component of interest, or target parasites serving as a control, e.g., uninfected control, or untreated control.
Other methods can be used to assess the anti-parasitic effect of a particular composition or component, which methods will be evident to one of ordinary skill in the art, or can be can be determined for use in a particular case by one of ordinary skill in the art using only routine experimentation. Additional information related to assessing anti-parasitic effect can be found in the Examples set forth in this document.
In some embodiments, a synergistic anti-parasitic effect is achieved when certain compounds are blended, and the synergistic effect can be enhanced when certain compounds are blended in certain relative amounts or ratios. In other words, the compositions including certain combinations of the compounds can have an enhanced ability to treat parasitic infections, as compared to each of the compounds taken alone.
As used herein, “synergy” and “synergistic effect” can refer to any substantial enhancement, in a composition of at least two compounds, of a measurable effect, e.g., an anti-parasitic effect, when compared with the effect of a component of the composition, e.g., one active compound alone, or the complete blend of compounds minus at least one compound. Synergy is a specific feature of a blend of compounds, and is above any background level of enhancement that would be due solely to, e.g., additive effects of any random combination of ingredients.
In some embodiments, a substantial enhancement of a measurable effect can be expressed as a coefficient of synergy. A coefficient of synergy is an expression of a comparison between measured effects of a composition and measured effects of a comparison composition. The comparison composition can be a component of the composition. In some embodiments, the synergy coefficient can be adjusted for differences in concentration of the complete blend and the comparison composition.
Synergy coefficients can be calculated as follows. An activity ratio (R) can be calculated by dividing the % effect of the composition (AB) by the % effect of the comparison composition (Xn), as follows:
R=A
B
/X
n Formula 1
A concentration adjustment factor (F) can be calculated based on the concentration (Cn), i.e., % (wt/wt) or % (vol/vol), of the comparison composition in the composition, as follows:
F=100/Cn Formula 2
The synergy coefficient (S) can then be calculated by multiplying the activity ratio (R) and the concentration adjustment factor (F), as follows:
S=(R)(F) Formula 3
As such, the synergy coefficient (S) can also by calculated, as follows:
S=[(AB/Xn)(100)]/Cn Formula 4
In Formula 4, AB is expressed as % effect of the blend, Xn is expressed as % effect of the comparison composition (Xn), and Cn is expressed as % (wt/wt) or % (vol/vol) concentration of the comparison composition in the blend.
In some embodiments, a coefficient of synergy of about 1.1, 1.2, 1.3, 1.4, or 1.5 can be substantial and commercially desirable. In other embodiments, the coefficient of synergy can be from about 1.6 to about 5, including but not limited to about 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5. In other embodiments, the coefficient of synergy can be from about 5 to 50, including but not limited to about 10, 15, 20, 25, 30, 35, 40, and 45. In other embodiments, the coefficient of synergy can be from about 50 to about 500, or more, including but not limited to about 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, and 450. Any coefficient of synergy above 500 is also contemplated within embodiments of the compositions.
Given that a broad range of synergies can be found in various embodiments describe herein, it is expressly noted that a coefficient of synergy can be described as being “greater than” a given number and therefore not necessarily limited to being within the bounds of a range having a lower and an upper numerical limit. Likewise, in some embodiments described herein, certain low synergy coefficients, or lower ends of ranges, are expressly excluded. Accordingly, in some embodiments, synergy can be expressed as being “greater than” a given number that constitutes a lower limit of synergy for such an embodiment. For example, in some embodiments, the synergy coefficient is equal to or greater than 25; in such an embodiment, all synergy coefficients below 25, even though substantial, are expressly excluded.
In some embodiments, synergy or synergistic effect associated with a composition can be determined using calculations similar to those described in Colby, S. R., “Calculating synergistic and antagonistic responses of herbicide combinations,” Weeds (1967) 15:1, pp. 20-22, which is incorporated herein by this reference. In this regard, the following formula can be used to express an expected % effect (E) of a composition including two compounds, Compound X and Compound Y:
E=X+Y−(X*Y/100) Formula 5
In Formula 5, X is the measured actual % effect of Compound X in the composition, and Y is the measured actual % effect of Compound Y of the composition. The expected % effect (E) of the composition is then compared to a measured actual % effect (A) of the composition. If the actual % effect (A) that is measured differs from the expected % effect (E) as calculated by the formula, then the difference is due to an interaction of the compounds. Thus, the composition has synergy (a positive interaction of the compounds) when A>E. Further, there is a negative interaction (antagonism) when A<E.
Formula 5 can be extended to account for any number of compounds in a composition; however it becomes more complex as it is expanded, as is illustrated by the following formula for a composition including three compounds, Compound X, Compound Y, and Compound Z:
E=X+Y+Z−((XY+XZ+YZ)/100)+(X*Y*Z/10000) Formula 6
An easy-to-use formula that accommodates compositions with any number of compounds can be provided by modifying Formulas 5 and 6. Such a modification of the formula will now be described. When using Formulas 5 and 6, an untreated control value (untreated with composition or compound) is set at 100%, e.g., if the effect being measured is the amount of target parasites killed, the control value would be set at 100% survival of target parasite. In this regard, if treatment with Compound A results in 80% killing of a target parasite, then the treatment with Compound A can be said to result in a 20% survival, or 20%>of the control value. The relationship between values expressed as a percent effect and values expressed as a percent-of-control are set forth in the following formulas, where E′ is the expected % of control of the composition, Xn is the measured actual % effect of an individual compound (Compound Xn) of the composition, Xn′ is the % of control of an individual compound of the composition, and A′ is the actual measured % of control of the of the composition.
E=100−E′ Formula 7
Xn=100=Xn′ Formula 8
A=100−A′ Formula 9
By substituting the percent-of-control values for the percent effect values of Formulas 5 and 6, and making modifications to accommodate any number (n) of compounds, the following formula is provided for calculating the expected % of control (E′) of the composition:
According to Formula 10, the expected % of control (E′) for the composition is calculated by dividing the product of the measured actual % of control values (Xn′) for each compound of the composition by 100n
Compositions containing two or more compounds in certain ratios or relative amounts can be tested for a synergistic effect by comparing the anti-parasitic effect of a particular composition of compounds to the anti-parasitic effect of a component the composition. Additional information related to making a synergy determination can be found in the Examples set forth in this document.
It is contemplated that the compositions of the presently-disclosed subject matter can be formulated for and delivered by carriers, including food products. For example, additives are added to baked goods, such as cookies, breads, cakes, etc., to enhance or modify flavor or color, increase shelf life, enhance their nutritional value, and generally produce a desired effect. Similarly, compositions of the presently-disclosed subject matter can be formulated with food products as carriers and delivered by ingestion to produce their desired effect. Of course, numerous types of foods can be used to deliver the compositions, including but not limited to: beverages, breakfast cereals, and powdered drink mixes.
Further, the compositions disclosed herein can take such forms as suspensions, solutions or emulsions in oily or aqueous carriers, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. For oral administration, the compositions can take the form of, for example, tablets or capsules prepared by a conventional technique with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods known in the art. For example, a composition disclosed herein can be formulated having an enteric or delayed release coating which protects the composition until it reaches the colon.
Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions. Such liquid preparations can be prepared by conventional techniques with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Liquid preparations for oral administration can also be formulated for delayed release, such as for example in “gel caps”.
In certain embodiments, the compositions can be provided in an encapsulated or microencapsulated form. Microencapsulation is a process where small particles of the composition are coated or encapsulated with an outer shell material for controlling the release of the composition or for protecting the composition. Exemplary outer shell material includes proteins, polysaccharides, starches, waxes, fats, natural and synthetic polymers, and resins. Microencapsulation can be done either chemically or physically. For example, physical methods of encapsulating the compositions can include: spray drying, spray chilling, pan coating, or coextrusion. Chemical methods of encapsulation can include coacervation, phase separation, solvent extraction, or solvent evaporation.
As one example, for coextrusion of a liquid core, liquid core and shell materials are pumped through concentric orifices, with the core material flowing in the central orifice, and the shell material flowing through the outer annulus. An enclosed compound drop is formed when a droplet of core fluid is encased by a layer of shell fluid. The shell is then hardened by appropriate means; for example, by chemical cross-linking in the case of polymers, cooling in the case of fats or waxes, or solvent evaporation. Additional information about methods and systems for providing compositions formulated for and delivered via food products can be found in U.S. Pat. Nos. 5,418,010, 5,407,609, 4,211,668, 3,971,852, and 3,943,063, each of which is incorporated herein by this reference.
The compositions of the presently-disclosed subject matter can be used for treating parasitic infections. The presently-disclosed subject matter includes methods for treating a parasitic infection in a subject, including administering an effective amount of a composition described herein.
As used herein, the terms “host” and “subject” are used interchangeably and refer to a plant or an animal capable of being infected by a parasite. The animal can be a vertebrate. The vertebrate can be warm-blooded. The warm-blooded vertebrate can be a mammal. The mammal can be a human. The human can be an adult or a child. As used herein, the terms “host” and “subject” include human and animal hosts and subjects. Thus, veterinary therapeutic uses are provided in accordance with the presently-disclosed subject matter. As such, the presently-disclosed subject matter provides for the treatment of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers or snow leopards; of economic importance, such as animals raised on farms for consumption by humans; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered and/or kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), poultry, and the like.
As used herein, the terms “treat,” “treating,” and “treatment” refer to: conferring protection against infection; preventing infection; alleviating infection; reducing the severity of symptoms and/or sequelae of infection; eliminating infection; and/or preventing relapse of infection. As used herein, the terms “treat,” “treating,” and “treatment” also refer to conferring protection against, preventing, alleviating, reducing the severity of, eliminating, and/or preventing relapse associated with a disease or symptoms caused by a parasitic infection.
As used herein, the term “effective amount” refers to a dosage sufficient to provide treatment for a parasitic infection. The exact amount that is required can vary, for example, depending on the target parasite, the treatment being affected, age and general condition of the subject, the particular formulation being used, the mode of administration, and the like. As such, the effective amount will vary based on the particular circumstances, and an appropriate effective amount can be determined in a particular case by one of ordinary skill in the art using only routine experimentation.
The presently-disclosed subject matter includes methods of screening for compositions useful for treating a parasitic infection. In some embodiments, the screening method is useful for narrowing the scope of possible compounds that are identified as components for a composition for treating a parasitic infection.
In some embodiments, a method of selecting a composition for use in treating a parasitic infection includes the following. A cell expressing a tyramine receptor is provided and is contacted with test compounds. The receptor binding affinity of the compounds is measured. At least one parameter selected from the following parameters is measured: intracellular cAMP level, and intracellular Ca2+ level. A first compound for the composition is identified, which is capable of altering at least one of the parameters, and which has a high receptor binding affinity for the tyramine receptor; and a second compound for the composition is identified, which is capable of altering at least one of the parameters, and which has a low receptor binding affinity for the tyramine receptor. A composition is selected that includes the first and second compounds. In some embodiments, a composition is selected that includes the first and second compounds and demonstrates an anti-parasitic effect that exceeds the anti-parasitic effect of any of the compounds when used alone.
The cell used for the method can be any cell capable of being transfected with and express a Tyramine Receptor (TyrR). Examples of cells include, but are not limited to: insect cells, such as Drosophila Schneider cells, Drosophila Schneider 2 cells (S2 cells), and Spodoptera frugiperda cells (e.g., Sf9 or Sf21); or mammalian cells, such as Human Embryonic Kidney cells (HEK-293 cells), African green monkey kidney fibroblast cells (COS-7 cells), HeLa Cells, and Human Keratinocyte cells (HaCaT cells). Additional information about preparing cells expressing receptors can be found in U.S. patent application Ser. Nos. 10/832,022; 11/086,615; and 11/365,426, which are incorporated herein in their entirety by this reference.
The tyramine receptor (TyrR) can be a full-length TyrR, a functional fragment of a TyrR, or a functional variant of a TyrR. A functional fragment of a TyrR is a TyrR in which amino acid residues are deleted as compared to the reference polypeptide, i.e., full-length TyrR, but where the remaining amino acid sequence retains the binding affinity of the reference polypeptide for tyramine. A functional variant of a TyrR is a TyrR with amino acid insertions, amino acid deletions, or conservative amino acid substitutions, which retains the binding affinity of the reference polypeptide for tyramine. A “conservative amino acid substitution” is a substitution of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. A conservative amino acid substitution also includes replacing a residue with a chemically derivatized residue, provided that the resulting retains the binding affinity of the reference polypeptide for tyramine. Examples of TyrR5 include, but are not limited to: TyrR5, such as, Drosophila melanogaster TyrR (GENBANK® accession number (GAN) CAA38565), Locusta migratoria TyrR (GAN: Q25321), TyrR5 of other invertebrates, and TyrR5 of nematodes, including Ascaris.
In some embodiments, other receptors, such as G-protein coupled receptors (GPCRs), whether having native affinity for tyramine or other ligands, can be employed in methods of screening for compositions useful for treating a parasitic infection. Examples of receptors that can be used include, but are not limited to: Anopheles gambiae (GAN: EAA07468), Heliothis virescens (GAN: Q25188), Mamestra brassicae (GAN: AAK14402), Tribolium castaneum (GAN: XP—970290), Aedes aegypti (GAN: EAT41524), Boophilus microplus (GAN: CAA09335); Schistosoma mansoni (GAN: AAF73286); and Schistosoma mansoni (GAN: AAW21822).
In some embodiments, receptors of the nuclear hormone receptor superfamily can be employed in methods of screening for compositions useful for treating a parasitic infection. Examples of receptors that can be used include, but are not limited to receptors from parasites or invertebrates that are analogous to the DAF family of nuclear receptors such as DAF-2 and DAF-12. In other embodiments, nuclear receptor proteins from Drosophila or other invertebrate can be employed, such as: nuclear receptors of subfamily 1 such as E78, E75, DHR3, EcR, and DHR96; nuclear receptors of subfamily 2 such as USP, DHR78, HNF4, SVP, TLL, DSF, DHR51, or DHR83; nuclear receptors of subfamily 3 such as ERR, nuclear receptors of subfamily 4 such as DHR38; nuclear receptors of subfamily 5 such as FTZ-F1 or DHR39; or nuclear receptors of subfamily 6 such as DHR4. In other embodiments, invertebrate or parasite nuclear receptor proteins analogous to certain human nuclear receptors can be employed, such as: nuclear receptors of subfamily 1 such as PPAR, RAR, TR, REV-ERB, ROR, FXR, LXR, VDR, SXR, or CAR; nuclear receptors of subfamily 2 such as RXR, TR2/TR4, HNF4, COUP-TF, TLX, or PNR; nuclear receptors of subfamily 3 such as ERR, ER, or MR/PR/AR/GR; nuclear receptors of subfamily 4 such as NURRI/NGFIB; nuclear receptors of subfamily 5 such as LRH/SF1; or nuclear receptors of subfamily 6 such as GCNF. In other embodiments, invertebrate or parasite nuclear receptor proteins having as their native ligand naturally occurring hormones such as 1a, 25(OH)2-vitamin D3, 17p-oestradiol, testosterone, progesterone, cortisol, aldosterone, all-trans retinoic acid, 3,5,3′-L-triiodothyronine, cc-ecdysone, or brassinolide, among others, can be employed.
In other embodiments, invertebrate or parasite nuclear receptor proteins analogous to certain human nuclear receptors can be employed, such as the receptors listed in Table F below. In the Table, a, b and g correspond to the Greek letters α, β and gamma, respectively.
When such nuclear receptors are employed in a screening platform, known downstream effects of the receptors can be used as indicative of an effect of an agent or blend of agents on the receptor. For example, levels of RNA transcribed from known targets of activated receptors can be assessed, or downstream effects of known regulatory cascades can be assessed.
Other molecular targets of interest include those listed in Table G:
Entamoeba
histolytica
Entamoeba histolytica depends on activity of
Giardia lamblia
Cryptosporidium
muris
Cryptosporidium
Cryptosporidium parvum encodes 11
parvum
hominis sporozoites. The p30 gene is
parva, Toxoplasma gondii and Eimeria
tenella) have been identified. The architecture
Trypanosomatidae
cruzi
cruzi, parvulin TcPINl is a homolog of the
Leishmania brucei
Leishmania spp.
Toxoplasma gondii
Plasmodium spp.
Babesia
Trichomonas
vaginalis
Schistosoma spp.
Taenia spp.
ovis 45 W gene family. These domains are
Eimeria spp.
Fasciola spp.
Cladosporium spp.
Colletotrichum
Saccharomyces cerevisiae SSD1.
Neospora spp.
Sarcocystis spp.
Ustilago maydis
Magnaporthe
grisea
Fusarium spp.
Aspergillus spp.
In response to ligand binding, GPCRs can trigger intracellular responses such as changes in levels of Ca2+ or cAMP. G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs) leads to GPCR desensitization. GRK-mediated receptor phosphorylation promotes the binding of β-arrestins, which in addition to uncoupling receptors from heterotrimeric G proteins also target many GPCRs for internalization in clathrin-coated vesicles. B-arrestin proteins play a dual role in regulating GPCR responsiveness by contributing to both receptor desensitization and internalization.
Following desensitization, GPCRs can be resensitized. GPCR sequestration to endosomes is thought to be the mechanism by which GRK-phosphorylated receptors are dephosphorylated and resensitized. The identification of β-arrestins as GPCR trafficking molecules suggested that β-arrestins can be determinants for GPCR resensitization. However, other cellular components also play pivotal roles in the de- and resensitization (D/R) process, including, for example, GRK, N-ethylmaleimide-sensitive factor (NSF), clathrin adaptor protein (AP-2 protein), protein phosphatases, clathrin, dynamin, and the like. In addition to these molecules, other moieties such as, for example, endosomes, lysosomes, and the like, also influence the D/R process. These various components of the D/R cycle provide opportunities to disrupt or alter GPCR “availability” to extracellular stimuli, and thus attenuate or intensify the effect of those extracellular stimuli upon target organisms. Attenuation, achieved, for example, by inhibition of the resensitization process, or the like, can limit the effects of extracellular stimuli (such as, for example, UV exposure, toxins, or the like) on the GPCR signaling process. Intensifying a signal cascade, achieved, for example, by inhibition of the desensitization process, or the like, can increase the effects of extracellular stimuli (such as, for example, pharmaceuticals, insecticides, or the like) on the GPCR signaling process.
Embodiments in accordance with the present disclosure can include a method to disrupt or alter parasite GPCR D/R by altering or disrupting the various signal cascades triggered through GPCR action. Certain embodiments can disrupt or alter parasite GPCR D/R in various ways, including, for example, the application of small molecules, including, for example, essential oils, and the like. These small molecules can include, for example, any of the following, or the like:
Alternatively, the small molecules can include members of any of the non-essential oil small molecule classes described above.
Embodiments in accordance with the present disclosure can include a method for screening a composition for indirect parasite GPCR desensitization inhibitory activity. In certain embodiments in accordance with the present disclosure, an indication that the test composition has indirect parasite GPCR desensitization inhibitory activity can be apparent when a test composition has parasite GPCR desensitization inhibitory activity with respect to different GPCRs. In certain embodiments, an indication that the test composition has indirect parasite GPCR desensitization inhibitory activity can be apparent when parasite GPCR cycling is inhibited without the composition binding the receptor itself. In certain embodiments in accordance with the present disclosure, indications of desensitization can include a reduced response to extracellular stimuli, such as, for example, a reduction in GPCR recycling from the plasma membrane to the cell's interior and back to the plasma membrane, or the like. Such a reduced response can result in lowered receptor dephosphorylation and recycling, thus leading to the presence of fewer sensitized receptor molecules on the cell surface. Another indication can be an altered period for the GPRC regulated activation of the Ca2+ cascade or the cAMP levels in the organism.
Embodiments in accordance with the present disclosure can include a method for screening a composition for indirect parasite GPCR resensitization inhibitory activity. In certain embodiments in accordance with the present disclosure, an indication that the test composition has indirect parasite GPCR resensitization inhibitory activity can be apparent when a test composition has parasite GPCR resensitization inhibitory activity with respect to different GPCRs. In certain embodiments, an indication that the test composition has indirect parasite GPCR resensitization inhibitory activity can be apparent when parasite GPCR cycling is inhibited without the composition binding the receptor itself. In certain embodiments in accordance with the present disclosure, indications of resensitization can include a reduced response to extracellular stimuli, such as, for example, a reduction in GPCR recycling from the plasma membrane to the cell's interior and back to the plasma membrane, or the like. Where the receptor does not require segregation to endosomal compartments to undergo dephosphorylation, such a reduction in GPCR cycling can result in the presence of more sensitized receptor molecules on the cell surface. Another indication can be a recovery to normal or static level of Ca2+ or cAMP.
Embodiments in accordance with the present disclosure can include a method for screening a composition for non-specific parasite GPCR desensitization inhibitory activity. The method can include screening a test composition for parasite GPCR desensitization inhibitory activity against two or more different parasite GPCRs. In certain embodiments in accordance with the present disclosure, an indication that the test composition has non-receptor-specific parasite GPCR desensitization inhibitory activity can be apparent when a test composition has parasite GPCR desensitization inhibitory activity with respect to each of the two or more different GPCRs. In certain embodiments in accordance with the present disclosure, indications of desensitization inhibitory activity can include a reduced response to extracellular stimuli, such as, for example, a reduction in GPCR recycling from the plasma membrane to the cell's interior and back to the plasma membrane, or the like. Another indication can be an altered period for the GPRC regulated activation of the Ca2+ cascade or the cAMP levels in the organism.
Embodiments in accordance with the present disclosure can include a method for screening a composition for non-specific parasite GPCR resensitization inhibitory activity. The method can include screening a test composition for parasite GPCR resensitization inhibitory activity against two or more different parasite GPCRs. In certain embodiments in accordance with the present disclosure, an indication that the test composition has non-receptor-specific parasite GPCR resensitization inhibitory activity can be apparent when a test composition has parasite GPCR resensitization inhibitory activity with respect to each of the two or more different parasite GPCRs. In certain embodiments in accordance with the present disclosure, indications of resensitization inhibition can include a reduced response to extracellular stimuli, such as, for example, a reduction in GPCR recycling from the plasma membrane to the cell's interior and back to the plasma membrane, or the like. Another indication can be an altered period for the GPRC regulated activation of the Ca2+ cascade or the cAMP levels in the organism.
In an embodiment in accordance with the present disclosure, one cell can be used to screen a test composition for indirect parasite GPCR desensitization inhibitory activity. In such an embodiment, the cell can express two or more parasite GPCRs that are different from each other such that a detection method can be used for determining whether there is an indication that a test composition has parasite GPCR desensitization inhibitory activity with respect to each of the different parasite GPCRs.
In some embodiments in accordance with the present disclosure, a multi-well format can be used to screen a test composition for indirect parasite GPCR desensitization inhibitory activity. In some embodiments, each well of the plate can contain at least one cell that includes a parasite GPCR, and the assay can include adding a compound in an amount known to activate that parasite GPCR, and thus affect intracellular Ca2+ levels, to each well. In some embodiments, at least one test compound can also be added to each well. In some embodiments, Ca2+ level can be tested at various time points after adding the at least one test compound. In certain embodiments, time points used for testing intracellular Ca2+ level can extend beyond the time points where an increase in Ca2+ level can be seen without the presence of the at least one test compound. In some embodiments, methods in accordance with the present disclosure can identify compounds that prolong agonist effect on GPCRs. In some embodiments in accordance with the present disclosure, cAMP levels can be evaluated to gauge the effect of the at least one test compound on GPCR response.
In some embodiments in accordance with the present disclosure, a multi-well format can be used to screen a test composition for indirect GPCR desensitization inhibitory activity. In some embodiments, each well of the plate can contain at least one cell that includes a GPCR, and the assay can include adding a compound in an amount less than that required to activate that GPCR, and thus affect intracellular Ca2+ levels, to each well. In some embodiments, at least one test compound can also be added to each well. In some embodiments, Ca2+ level can be tested at various time points after adding the at least one test compound. In certain embodiments, time points used for testing intracellular Ca2+ level can extend beyond the time points where an increase in Ca level can not be seen without the presence of the at least one test compound. In certain embodiments, time points used for testing intracellular Ca level can extend beyond the time points where an increase in Ca2+ level can be seen with the presence of an GPCR-activating dose of the agonist compound. In some embodiments, methods in accordance with the present disclosure can identify compounds that enhance agonist effect on GPCRs. In some embodiments in accordance with the present disclosure, cAMP levels can be evaluated to gauge the effect of the at least one test compound on GPCR response.
Some of the receptors disclosed herein are cross-referenced to GENBANK accession numbers. The sequences cross-referenced in the GENBANK® database are expressly incorporated by reference as are equivalent and related sequences present in GENBANK® or other public databases. Also expressly incorporated herein by reference are all annotations present in the GENBANK® database associated with the sequences disclosed herein.
As used herein, the term “receptor binding affinity” refers to an interaction between a composition or component, e.g., compound, and a receptor binding site. The interaction between a composition or component, and the receptor binding site, can be identified as specific or non-specific. In some embodiments, the specificity of an interaction between a composition or component, and a TyrR binding site, can be determined in the following manner. A wild type fly (Drosophila melanogaster) and a mutant fly are provided, where the mutant fly lacks a TyrR. The wild type and mutant flies are exposed to a composition or component of interest. If the exposure negatively affects the wild type fly, (e.g., knock down, death), but does not negatively affect the mutant fly, then the treatment with the composition or component of interest can be said to be specific for the TyrR. If the exposure negatively affects the wild type fly and the mutant fly, then the treatment with the composition or component of interest can be said to be non-specific for the TyrR.
A “high receptor binding affinity” can be a specific interaction between a composition or component, and the receptor binding site. In some embodiments, a high receptor binding affinity is found when the equilibrium dissociation constant (Kd) is less than about 100 nM, 75 nM, 50 nM, 25 nM, 20 nM, 10 nM, 5 nM, or 2 nM. In some embodiments, a high receptor binding affinity is found when the equilibrium inhibitor dissociation constant (Ki) is less than about is less than about 100 μM, 75 μM, 50 μM, 25 μM, 20 μM, 10 μM, 5 μM, or 2 μM, when competing with tyramine. In some embodiments, a high receptor binding affinity is found when the effective concentration at which tyramine binding is inhibited by 50% (EC50) is less than about 500 μM, 400 μM, 300 μM, 100 μM, 50 μM, 25 μM, or 10 μM.
A “low receptor binding affinity” can be a non-specific interaction between a composition or component, and the receptor binding site. In some embodiments, a low receptor binding affinity is found when the equilibrium dissociation constant (Kd) is greater than about 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, or 250 nM. In some embodiments, a low receptor binding affinity is found when the equilibrium inhibitor dissociation constant (Ki) is greater than about 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 225 μM, or 250 μM, when competing with tyramine. In some embodiments, a low receptor binding affinity is found when the effective concentration at which tyramine binding is inhibited by 50% (EC50) is greater than about 500 μM, 625 μM, 750 μM, 875 μM, 1000 μM, 1125 μM, or 1250 μM.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently-disclosed subject matter belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods and materials are now described.
Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter. As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration, or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples can include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the presently-disclosed subject matter. The following examples include prophetic examples.
An example of a parasite that commonly infects humans is Hymenolepsis nana, which is an intestinal parasite. H. nana is a difficult worm to eliminate from the human intestine. See John Rim, Treatment of Hymenolepis nana infection. Post-Graduate Doctor Journal. Middle East Edition, 5:330-334, 1985. H. nana is found worldwide and infection can occur in humans of any age; however, due to the increased likelihood of exposure to human feces, small children have the highest risk of contracting hymenolepiasis, the disease associated with H. nana infection.
H. nana has a characteristic life cycle of about 7 days. When a host has been infected, the H. nana eggs pass into the ileum of the small intestine and hatch into oncospheres, motile larvae of H. nana, which penetrate the lamina propria of the villus of the small intestine. Within about 3 to 4 days, the larvae mature into pre-adult cysticercoids, which then enter the gut lumen, attaching to the mucosa of the villus of the small intestine. Many infections are asymptomatic, keeping some infected individuals from seeking medical treatment and being cured. Symptomatic forms of the infection are characterized by irritability, diarrhea, abdominal pain, restless sleep, anal pruritus, nasal pruritus, behavior disturbance, and seizures.
In the present Examples, H. nana is selected as an exemplary parasite used to study the efficacy in vitro and in vivo of compositions disclosed herein for treating parasitic infections. Laboratory-raised Swiss albino mice are used as host animals. Uninfected males and females are used. Pregnant females are isolated from other mice. The newly born litters are maintained to avoid infection thereof. The mother mice are checked twice weekly by direct saline fecal smear and the negative sample is re-examined by zinc sulphate centrifugation floatation and saline sedimentation techniques to exclude those parasitologically infected. See Melvin and Brooke, Laboratory procedures for the diagnosis of intestinal parasites. DHEW Publications No. (CDC) 76-828, Public Health Services, 1975, incorporated herein by reference in its entirety.
After weaning the litters, the mice are checked twice weekly and uninfected litters are used for the Examples. Mice are kept under scrupulous hygienic conditions and fed one day milk and the other day wheat. Diet and water are available ad libitum.
Eggs of H. nana, free of debris, teased from gravid segments are used for infection. See Ito, In vitro oncospheral agglutination given by immune sera from mice infected and rabbits injected with eggs of Hymenolepis nana. Parasito, 71: 465, 1975, incorporated herein by reference in its entirety. Prior to inoculation, the egg shells are removed and every mouse is inoculated with a known number of eggs to maintain the infection cycle. See Bernetzen and Voge, In vitro hatching of oncosphere of Hymenolepidid cestodes. J. Parasitol., 5:235, 1965, incorporated herein by reference in its entirety.
Maximum tolerated dose (MTD) of each test agent is determined before starting the in vivo study. Worm-free 5 weeks old mice (25-30 grams) are used in the experiment. Each mouse is inoculated with 150 eggs. Then they are subdivided into groups, each group containing 15 mice. Each of these groups is specified for testing the efficacy of one test agent as a potential therapeutic drug against adult worm of H. nana. A control group composed of 15 mice is also infected with the same number of eggs but not subjected to the test agents. Infection is monitored and a base egg count from feces is determined for each mouse (experimental and control groups).
The following compositions were each tested for anti-parasitic effects against H. nana in vivo: Rx1—Black seed cumin oil; Rx2—Lilac flower oil; Rx3—thyme oil (white); Rx4—carvacrol; Rx5—geraniol; Rx6—cineol; and Rx7—wintergreen oil; Rx8—Lilac Flower oil-V3; Rx9—trans-anethole; Rx10—p-cymene; Rx11—thymol.
Each mouse in the experimental groups was inoculated orally with 400 mg/kg body weight of the specified test compound (Rx) daily for 5 successive days beginning 24 hours following detection of eggs in feces. At the same time, each mouse of the control group was inoculated orally with 400 mg/kg body weight of the suspension material only, i.e. soybean oil, daily for 5 successive days. The egg count of every mouse (experimental and control) was determined daily during the periods of treatment and for further 2 days after the last dose treatment. On the 3rd day after the last dose treatment the cure rate was determined. The criteria for cure was assessed according to: (1) determination of egg-reduction rate; and (2) the absence of the adult worms. The mouse being assessed was killed by decapitation and the small intestine dissected for detecting the adult worms.
With reference to Table 1 and
Post treatment dissection of the positive infected mice showed the following: the worms were intact, living, and active; the scolex (head) of the worm was intact keeping its anatomical feature with moving rostellum and contracting suckers; the neck, which is considered the area of segmentation (producing new segments), was intact; and the strobila (the body of the worm) was intact, maintaining its anatomical feature with 3 groups of segments (immature segments or segments with immature reproductive organs, mature segments or segments with mature reproductive organs, and gravid segments or segments with uteri full of mature eggs). Worms were absent or dead in mice treated for 5 consecutive days with Rx2 (71%), Rx5 (67%), and Rx7 (60%).
These experiments can also be conducted to study the treatment efficacy of the presently-disclosed compositions against Trichuris trichiura in vivo.
The compounds are combined to produce the compositions having anti-parasitic properties disclosed herein. The compositions tested are set forth in Table 2. An “X” in a cell of the table indicates that a particular compound is included in a particular test composition. For example, in the column labeled “S1,” there is an X in the row setting forth thymol. As such, composition “S1” includes Thymol. Composition S1 further includes carvacrol, trans-anethole, and p-cymene.
Each mouse in the experimental groups is inoculated orally with 400 mg/kg body weight of the specified test composition daily for 5 successive days. At the same time, each mouse of the control group is inoculated orally with 400 mg/kg body weight daily for 5 successive days of the suspension material only, i.e. soybean oil. The egg count of every mouse (experimental and control) is determined daily during the periods of treatment and for a further 2 days after the last dose treatment. On the 3rd day after the last dose treatment the cure rate is determined. The criteria for cure are assessed according to: (1) determination of egg-reduction rate; and (2) the absence of adult worms. The mouse being assessed is killed by decapitation and the small intestine is dissected for detecting the adult worms.
The cure rate is between about 25% and 80% following treatment with compositions S1 through S16. An infected animal is determined to be cured when it is completely free of worms and eggs at the time of assessment. Worms are absent or dead in mice treated for multiple consecutive days with the compositions having cure rates of about 60% or higher.
These experiments can also be conducted to study the treatment efficacy of the presently-disclosed compositions against Trichuris trichiura in vivo.
The following compounds and blend compositions were each tested for anti-parasitic effects against H. nana in vivo: (1) p-cymene; (2) thymol; (3) α-pinene; (4) linalool; (5) soybean oil (control); and (6) blend of 30% p-cymene, 35% thymol, 4% α-pinene, 7% linalool, and 24% soybean oil, where percentages are by weight.
Each mouse in the groups was inoculated orally with 100 mg/kg body weight of the specified compound or blend composition daily for 5 successive days. The egg count of each mouse (experimental and control) was determined daily during the periods of treatment and for 2 more days after the last dose treatment. Following the 3rd day of the last dose treatment the cure rate was determined. The criteria for cure was assessed according to: (1) determination of egg-reduction rate; and (2) the absence of the adult worms. The mouse being assessed was killed by decapitation and the small intestine dissected for detecting the adult worms.
With reference to Table 3, the cure rate ranged from 0%, for the soybean oil (control), to 100%, for the blend composition containing 30% p-cymene, 35% thymol, 4% α-pinene, 7% linalool, and 24% soybean oil. Cure rate represents the number of infected animals that demonstrate no eggs in their stool and no worms found in their intestine following treatment with the tested compounds.
As indicated by the data above, the blend composition has a synergistic effect, as compared to the individual compounds that are components of the blend. A coefficient of synergy can be calculated for the blend, relative to each individual compound, i.e., comparison composition. Such synergy coefficients are set forth in Table 4.
For example, the activity ratio for p-cymene is 7.52 because the effect of the blend is a cure rate of 100%, while the effect of p-cymene alone is 13.3% [(1.00)/(0.133)=7.52]. The concentration adjustment factor for p-cymene is 3.33 because the blend contains 30% p-cymene, as compared to the 100% p-cymene tested alone [(1.00)/(0.300)=3.33]. The synergy coefficient of the blend, relative to p-cymene (Sp-Cymene) is therefore 25.1 [((1.00)/(0.133))/(0.300)=25.1]. With further reference to Table 4, the synergy coefficients for the blend are as follows: Sp-cymene=25.1; Sthymol=8.57; Sα-.pinene=100; and Slinalool=61.3.
D. caninum, also called the cucumber tapeworm or the double-pore tapeworm, is a cyclophyllid cestode that infects organisms afflicted with fleas, including canids, felids, and pet-owners, especially children. Adult worms are about 18 inches long. Eggs (or “egg clusters” or “egg balls”) are passed in the host's feces and ingested by fleas, which are in turn ingested by another mammal after the tapeworm larvae partially develop. Examples of fleas that can spread D. caninum include Ctenocephalides canis and Ctenocephalides felis.
In the present Examples, D. caninum is selected as an exemplary parasite used to study the efficacy in vitro and in vivo of compositions disclosed herein for treating parasitic infections. Laboratory-raised Swiss albino mice are used as host animals. Uninfected males and females are used. Pregnant females are isolated from other mice. The newly born litters are maintained to avoid infection thereof. The mother mice are checked twice weekly by direct saline fecal smear and the negative sample is re-examined by zinc sulphate centrifugation floatation and saline sedimentation techniques to exclude those parasitologically infected.
After weaning the litters, the mice are checked twice weekly and uninfected litters are used for the Examples. Mice are kept under scrupulous hygienic conditions and fed one day milk and the other day wheat. Diet and water are available ad libitum.
Eggs of D. caninum, free of debris, teased from gravid segments are used for infection. Prior to inoculation, the egg shells are removed and every mouse is inoculated with a known number of eggs to maintain the infection cycle.
Maximum tolerated dose (MTD) of each test agent is determined before starting the in vivo study. Worm-free 5 weeks old mice (25-30 grams) are used in the experiment. Each mouse is inoculated with 150 eggs. Then they are subdivided into groups, each group containing 15 mice. Each of these groups is specified for testing the efficacy of one test agent as a potential therapeutic drug against adult worm of D. caninum. A control group composed of 15 mice is also infected with the same number of eggs but not subjected to the test agents. Infection is monitored and a base egg count from feces is determined for each mouse (experimental and control groups).
The following compositions are each tested for anti-parasitic effects against D. caninum in vivo: Rx1—Black seed cumin oil; Rx2—Lilac flower oil; Rx3—thyme oil (white); Rx4—carvacrol; Rx5—geraniol; Rx6—cineol; and Rx7—wintergreen oil; Rx8—Lilac Flower oil-V3; Rx9—trans-anethole; Rx10—p-cymene; Rx11—thymol.
Each mouse in the experimental groups is inoculated orally with 400 mg/kg body weight of the specified test compound (Rx) daily for 5 successive days beginning 24 hours following detection of eggs in feces. At the same time, each mouse of the control group is inoculated orally with 400 mg/kg body weight of the suspension material only, i.e. soybean oil, daily for 5 successive days. The egg count of every mouse (experimental and control) is determined daily during the periods of treatment and for further 2 days after the last dose treatment. On the 3rd day after the last dose treatment the cure rate is determined. The criteria for cure is assessed according to: (1) determination of egg-reduction rate; and (2) the absence of the adult worms. The mouse being assessed is killed by decapitation and the small intestine is dissected for detecting the adult worms.
An infected animal is determined to be cured when it is completely free of worms and eggs at the time of assessment.
Post treatment dissection of the positive infected mice show the following: the worms are intact, living, and active; the scolex (head) of the worm is intact keeping its anatomical feature with moving rostellum and contracting suckers; the neck, which is considered the area of segmentation (producing new segments), is intact; and the strobila (the body of the worm) is intact, maintaining its anatomical feature with 3 groups of segments (immature segments or segments with immature reproductive organs, mature segments or segments with mature reproductive organs, and gravid segments or segments with uteri full of mature eggs).
The compounds are combined to produce the compositions having anti-parasitic properties disclosed herein. The compositions tested are set forth in Table 5. An “X” in a cell of the table indicates that a particular compound is included in a particular test composition. For example, in the column labeled “S1,” there is an X in the row setting forth thymol. As such, composition “S1” includes Thymol. Composition S1 further includes carvacrol, trans-anethole, and p-cymene.
Each mouse in the experimental groups is inoculated orally with 400 mg/kg body weight of the specified test composition daily for 5 successive days. At the same time, each mouse of the control group is inoculated orally with 400 mg/kg body weight daily for 5 successive days of the suspension material only, i.e. soybean oil. The egg count of every mouse (experimental and control) is determined daily during the periods of treatment and for a further 2 days after the last dose treatment. On the 3rd day after the last dose treatment the cure rate is determined. The criteria for cure are assessed according to: (1) determination of egg-reduction rate; and (2) the absence of adult worms. The mouse being assessed is killed by decapitation and the small intestine is dissected for detecting the adult worms.
The cure rate is between about 25% and 80% following treatment with compositions S1 through S16. An infected animal is determined to be cured when it is completely free of worms and eggs at the time of assessment. Worms are absent or dead in mice treated for multiple consecutive days with the compositions having cure rates of about 60% or higher.
The following compounds and blend compositions are each tested for anti-parasitic effects against D. caninum in vivo: (1) p-cymene; (2) thymol; (3) α-pinene; (4) linalool; (5) soybean oil (control); and (6) blend of 30% p-cymene, 35% thymol, 4% α-pinene, 7% linalool, and 24% soybean oil, where percentages are by weight.
Each mouse in the groups is inoculated orally with 100 mg/kg body weight of the specified compound or blend composition daily for 5 successive days. The egg count of each mouse (experimental and control) is determined daily during the periods of treatment and for 2 more days after the last dose treatment. Following the 3rd day of the last dose treatment the cure rate is determined. The criteria for cure is assessed according to: (1) determination of egg-reduction rate; and (2) the absence of the adult worms. The mouse being assessed is killed by decapitation and the small intestine is dissected for detecting the adult worms.
In the present Example, Schistosoma mansoni is selected as an exemplary parasite used to study the efficacy in vivo of compositions disclosed herein for treating parasitic infections, such as compositions Rx1-Rx11 and S1-S16 described above. Assessment of the efficacy of the tested compositions against S. mansoni infection is with regard to worm load, sex ratio of worms, distribution of worms, fecundity of female worms, and egg deposition in liver and intestine.
Female Swiss Albino mice, 8 weeks in age, from 18-22 gm in weight, which can be obtained from Theodore Bilharz Research Institute, Cairo, are infected percutaneously by S. mansoni cercariae (100 cercariae/mouse). Each group consists of 15 mice.
For each test composition, three concentrations are tested. For each concentration nine groups of mice are studied. One group of S. mansoni-infected mice receives Praziquantel (PZQ), which is the present standard antischistosomal drug. Three groups of uninfected mice receive the test compound in the same schedule and concentration as the test drug groups. One group of uninfected and untreated mice and one group of S. mansoni infected mice that do not receive any treatment are maintained as controls.
Three different concentrations from each of the test compositions are determined after estimation of the LD50. The schedule for drug administration is as follows: (1) four days post-infection (PI); (2) one week PI; and seven weeks PI. Praziquantel (Distocide), 600 mg/Kg body weight, is administered seven weeks PI. All drugs are administered orally using a stomach tube.
For the parasitological studies, fecal egg counts are done for all infected groups twice weekly starting from the 5th week PI.
Mice are sacrificed 9 weeks PI. Perfusion of the portal system is done for the recovery of the schistosome worms. The total number, sex, maturation and distribution of the worms are determined. Four portions, two from the jejunum and two from the ileum, are taken from each mouse, washed with PBS, opened and compressed between two slides and examined microscopically for detection of the stage of maturation. 0.3 gram of the liver and of the intestine are digested in 4% potassium hydroxide overnight, and S. mansoni ova counted.
In the present Example, Opisthorchis sinensis is selected as an exemplary parasite used to study the efficacy in vivo of compositions disclosed herein for treating parasitic infections, such as compositions Rx1-Rx11 and S1-S16 described above. Assessment of the efficacy of the tested compositions against O. sinensis infection is with regard to worm load, sex ratio of worms, distribution of worms, fecundity of female worms, and egg deposition in liver and intestine.
Female Swiss Albino mice, 8 weeks in age, from 18-22 gm in weight, which can be obtained from Theodore Bilharz Research Institute, Cairo, are infected percutaneously by S. mansoni cercariae (100 cercariae/mouse). Each group consists of 15 mice.
For each test composition, three concentrations are tested. For each concentration nine groups of mice are studied. One group of O. sinensis-infected mice receives the present standard treatment drug. Three groups of uninfected mice receive the test compound in the same schedule and concentration as the test drug groups. One group of uninfected and untreated mice and one group of O. sinensis infected mice that do not receive any treatment are maintained as controls.
Three different concentrations from each of the test compositions are determined after estimation of the LD50. The schedule for drug administration is as follows: (1) four days post-infection (PI); (2) one week PI; and seven weeks PI. Praziquantel (Distocide), 600 mg/Kg body weight, is administered seven weeks PI. All drugs are administered orally using a stomach tube.
For the parasitological studies, fecal egg counts are done for all infected groups twice weekly starting from the 5th week PI.
Mice are sacrificed 9 weeks PI. Perfusion of the portal system is done for the recovery of the worms. The total number, sex, maturation and distribution of the worms are determined. Four portions, two from the jejunum and two from the ileum, are taken from each mouse, washed with PBS, opened and compressed between two slides and examined microscopically for detection of the stage of maturation. 0.3 gram of the liver and of the intestine are digested in 4% potassium hydroxide overnight, and O. sinensis ova counted.
Three groups of mice are treated with each test compound or composition blend of compounds. For Groups 1 and 2, treatment starts 4 and 7 days after infection, respectively. For Group 3, treatment starts 7 weeks after infection. For the control group, the mice are injected 7 weeks after infection with Praziquantel at 600 mg/kg. Efficacy of test agents is determined based on: worm load; sex ratio; distribution of worms; fecundity of female worms; and egg deposition in liver and intestine.
Adult male and female S. mansoni were collected from infected mice and transferred into 100 ml saline treated with test compositions Rx1-Rx10 (as disclosed in Example 1) or Praziquantel at varying concentrations and incubated at 37° C. in 5% CO2. In many cases adult male and females were collected as couples. Viability of worms was examined under a binuclear microscope. Controls were treated in parallel. The experiment was terminated either when all worms are dead in the treated samples or when the first death among controls is found.
Each of the compounds were tested individually at differing concentrations and the data from these experiments are presented in
The present Example provides an in vitro study testing treatment of Histomonas meleagridis, a protozoan parasite causing blackhead disease of chickens and turkeys, using the presently-disclosed compounds and blend compositions of the compounds.
H. meleagridis is cultured in vitro and prepared for use in screw-capped glass vials containing 1 ml of Dwyer's medium and inoculated with 20,000 cells. The test compounds and/or compositions are diluted to appropriate concentrations, so that the desired dose is administered to the tubes in 0.1 ml. Each treatment is replicated in duplicate cultures. The cultures are incubated for 2 days.
The number of H. meleagridis cells/ml can be counted using a standard hemocytometer (Neubauer) and the actual number of cells/ml is reported.
Each compound and/or composition is tested at 1, 0.1, 0.01, 0.001 and 0.0001%. Controls are included as untreated and with solvent (ethanol). Data from the experiments are presented in
The present Example provides an in vitro study testing treatment of Cryptosporidium parvum using the presently-disclosed compounds and blend compositions of the compounds, such as compositions Rx1-Rx11 and S1-S16 described above. Cryptosporidiosis is a parasitic infection of human and animal importance. The organism can affect the epithelial cells of the human gastrointestinal, bile duct and respiratory tracts. Over 45 different species of animals including poultry, fish, reptiles, small mammals (rodents, dogs, and cats) and large mammals (including cattle and sheep) can become infected with C. parvum. The reservoir for this organism includes people, cattle, deer and many other species of animal.
Transmission is fecal-oral, which includes contaminated food and water, animal-to-person and person-to-person. The parasite infects intestinal epithelial cells and multiplies. Oocysts are shed in the feces and can survive under very adverse environmental conditions. The oocysts are very resistant to disinfectants. People can re-infect themselves one or more times.
C. parvum is cultured in vitro and prepared for use in screw-capped glass vials containing 1 ml of Dwyer's medium and inoculated with 20,000 cells. The test compounds and/or compositions are diluted to appropriate concentrations, so that the desired dose is administered to the tubes in 0.1 ml. Each treatment is replicated in duplicate cultures. The cultures are incubated for 2 days.
The number of C. parvum cells/ml can be counted using a standard hemocytometer (Neubauer) and the actual number of cells/ml is reported. Each compound and/or composition is tested at 1, 0.1, 0.01, 0.001 and 0.0001%. Controls are included as untreated and with solvent.
Trichinellosis (previously referred to as ‘trichinosis’) is a zoonosis caused by parasitic nematodes of the genus Trichinella. The most common species is Trichinella spiralis, but other species such as Trichinella trichuris are also infective. It is a serious food born parasitic zoonosis with worldwide distribution whenever pork including domestic and wild pig is an important component of the diet (Frierson, 1989). The infection has a worldwide occurrence specifically, it has been estimated that 10 million people worldwide are infected (Jean Dupouy, 2000) and in the past 10 years an increase in the occurrence of infection has been reported among domestic pigs and wildlife, with a consequent increase among humans (Murrell & Pozio, 2000).
Transmission occurs when pork containing infective, encysted larvae is eaten. Also, inadvertent or deliberate mixing of pork with other meat products as grinding beef and pork in the same grinder or mixing pork in the same grinder or mixing pork with beef in sausages can result in infection (Kejenie and Bero, 1992). The larvae burrow beneath the mucosa of the small intestine where they mature into adult worms. Within 7 days, female worms release another generation of larvae which migrate to striated skeletal muscle and become encysted. Larvae often reach to the myocardium but do not become encysted there. The larvae produce intense allergic and inflammatory reactions which are expressed clinically as fever, muscle pains, periorbital oedema and eosinophilia. The initial intestinal infection often induces nausea, diarrhea and abdominal cramps, but these are rarely serious. However, subsequent complications such as myocarditis, pneumonia and meningoencephalitis can be fatal.
Death from trichinellosis is rare. For example, of the >6500 infections reported in the European Union in the past 25 years, only five deaths have been recorded, all of which were due to thromboembolic disease and recorded in people aged >65 years as reported by Ancelle et al. 1988. Twenty fatalities out of 10,030 cases were reported in a worldwide survey performed by the International Commission on Trichinellosis (January 1995-June 1997) as reported by Jean Dupouy, 2000.
Each case of confirmed or even suspected infection must be treated in order to prevent the continued production of larvae. The medical treatment includes anthelmintics (mebendazole or albendazole) and glucocorticosteroids. Mebendazole is usually administered at a daily dose of 5 mg/kg but higher doses (up to 20-25 mg/kg/day) are recommended in some countries. Albendazole is used at 800 mg/day (15 mg/kg/day) administered in two doses. These drugs are taken for 10-15 days. The use of mebendazole or albendazole is contraindicated during pregnancy and not recommended in children aged <2 years. The most commonly used steroid is prednisolone, which can alleviate the general symptoms of the disease. It is administered at a dose of 30-60 mg/day for 10-15 days (Jean Dupouy et., al, 2002).
Trichuris trichiura is a common nematode infection worldwide. The highest prevalence occurs in tropic climates with poor sanitation practices, as it has fecal/oral transmission. T. trichiura does not migrate through the tissues, and it does not cause eosinophilia. It can survive 6 yrs. in host (average 3 years), living in the large intestine with its head imbedded in intestinal mucosa, but there is virtually no cellular response. Diagnosis of T. trichiura is made through finding the eggs in feces. Infection with T. trichiura is frequently asymptomatic. However, in heavy infection in undernourished children, T. trichiura can cause rectal prolapse following chronic bloody diarrhea.
Compounds and blended compositions of the compounds, such as compositions Rx1-Rx11 and S1-S16 described above, as disclosed herein, are tested for in vitro anti-parasitic activity using the protocols following. Ten groups (8 different concentrations of compositions and 2 controls) can be tested. Tests are performed in sterile six well plates with 1-4 worms per well. Each well contains 3 mL RPMI 1640 containing a 10× antibiotic/antimycotic (penicillin/streptomycin/amphotercin B) solution to prevent overgrowth of contaminating organisms. Worm motility is observed at all initial time points, as well as 24 hour post treatment, i.e. following wash and placement in media without test compounds.
As indicated, eight concentrations and two controls are tested. The controls indicated for these tests will be a surfactant control and a media control. The protocol utilizes 5-10× of the final concentrations of test compounds to be added to the media at the time of testing.
Once the test is initiated, motility is checked at 15, 30, 60, 120, 240, and 360 minutes post-treatment. Following the last time point, the worms are removed from the treated media, rinsed and placed into untreated media. A last motility check is performed at 24 hours post-treatment. Worms not observed to be motile are prodded with a sterile (autoclaved) wooden applicator stick to confirm lack of responsiveness. An effective concentration of the compounds and blended compositions of the compounds is determined.
The human pinworm Enterobius vermicularis is a ubiquitous parasite of man, it being estimated that over 200 million people are infected annually. It is more common in the temperate regions of Western Europe and North America and is particularly in common in children. Samples of Caucasian children in the U.S.A. and Canada have shown incidences of infection of between 30% to 80%, with similar levels in Europe, and although these regions are the parasites strongholds, it can be found throughout the world. For example in parts of South America, the incidence in children can be as high as 60%. Interestingly non-Caucasians appear to be relatively resistant to infection with this nematode. As a species, E. vermicularis is entirely restricted to man, other animals harboring related but distinct species that are non-infective to humans, although their fur can be contaminated by eggs from the human species.
The adult parasites live predominantly in the caecum. The male and females mate, and the uteri of the females become filled with eggs. Eventually the female die, their bodies disintegrating to release any remaining eggs. These eggs, which are clear and measure −55 by 30 μm, then mature to the infectious stage (containing an LI larvae) over 4 to 6. Infection of the host typically follows ingestion of these eggs, the eggs hatching in the duodenum.
Compounds and blended compositions of the compounds, such as compositions Rx1-Rx11 and S1-S16 described above, as disclosed herein, can be tested for in vitro anti-parasitic activity against E. vermicularis using the protocols following. Ten groups (8 different concentrations of compositions and 2 controls) can be tested. Tests are performed in sterile six well plates with 1-4 worms per well. Each well contains 3 mL RPMI 1640 containing a 10× antibiotic/antimycotic (penicillin/streptomycin/amphotercin B) solution to prevent overgrowth of contaminating organisms. Worm motility is observed at all initial time points, as well as 24 hour post treatment, i.e. following wash and placement in media without test compounds.
As indicated, eight concentrations and two controls are tested. The controls indicated for these tests are a surfactant control and a media control. The protocol utilizes 5-10× of the final concentrations of test compounds to be added to the media at the time of testing.
Once the test is initiated, motility is checked at 15, 30, 60, 120, 240, and 360 minutes post-treatment. Following the last time point, the worms are removed from the treated media, rinsed and placed into untreated media. A last motility check is performed at 24 hours post-treatment. Worms not observed to be motile are prodded with a sterile (autoclaved) wooden applicator stick to confirm lack of responsiveness. An effective concentration of the compounds and blended compositions of the compounds is determined.
Compounds and blended compositions of the compounds, such as compositions Rx1-Rx11 and S1-S16 described above, as disclosed herein, are tested for in vitro anti-parasitic activity using the protocols following. Ten groups (8 different concentrations of compositions and 2 controls) can be tested. Tests are performed in sterile 150 cm3 flasks with 1-2 worms per flask. Each flask contains 200 mL RPMI 1640 containing a 10× antibiotic/antimycotic (penicillin/streptomycin/amphotercin B) solution to prevent overgrowth of contaminating organisms. Worm motility is observed at all initial time points, as well as 24 hour post treatment, i.e. following wash and placement in media without test compounds.
As indicated, eight concentrations and two controls are tested. The controls indicated for these tests will be a surfactant control and a media control. The protocol utilizes 5-10× of the final concentrations of test compounds to be added to the media at the time of testing.
Once the test is initiated, motility is checked at 15, 30, 60, 120, 240, and 360 minutes post-treatment. Following the last time point, the worms are removed from the treated media, rinsed and placed into untreated media. A last motility check is performed at 24 hours post-treatment. Worms not observed to be motile are prodded with a sterile (autoclaved) wooden applicator stick to confirm lack of responsiveness. An effective concentration of the compounds and blended compositions of the compounds is determined.
Testing was conducted to determine the dose-response of test agents against larvae of Trichinella spiralis under in vitro conditions.
Two test agents were used in this Example, designated Agents A and B. Agent A comprised 7% linalool coeur, 35% thymol, 4% α-pinene, 30% p-cymene, and 24% soybean oil. Agent B comprised Agent A with the addition of 1.2% of a surfactant, the commercially available Sugar Ester OWA-1570. The stock solution (A or B) was diluted by normal sterile saline solution into five concentrates: 100 ppm, 50 ppm, 25 ppm, 10 ppm and 1 ppm. Each concentrate was agitated by vortex for 15 minutes before use.
Infective larvae were obtained from muscle samples mainly from the diaphragm taken from freshly slaughtered pigs. These were compressed by the compressorium (consisting of two glass slides of 6 mm thickness, each one measuring 20×5 cm with one hole on each side, each hole being provided with a screwed nail, and the upper surface of the lower slide being marked with a diamond pencil into 28 divisions having serial numbers to enable the examiner to examine 28 specimens at one setting) into a thin layer suitable for microscopic examination and examined for Trichinellosis by Trichinoscope in the slaughter house. The infected carcasses were obligatorily condemned. Infected muscle samples were taken, kept in ice box and transferred to the laboratory. The muscle samples were cut into small pieces (oat grains) parallel to the muscle fibers. Randomly selected muscle specimens were taken placed between two slides, pressed until obtaining a thin layer to be examined under the low power objective of the microscope (×10) to detect the encysted larvae of Trichinella spiralis in order to reconfirm the infection before doing the digestion technique (see
In this Example, the infective larvae were obtained by this method from freshly slaughtered infected pigs to test the efficacy of the tested drug agents upon the larvae, so as to simulate the natural mode of human infection. However, larvae obtained from infected laboratory animals in a lab can also be employed.
Five free active infective larvae were placed in a Petri dish (50×9 mm) and the tested agents (A or B) with different concentrations: 100 ppm, 50 ppm, 25 ppm, 10 ppm and 1 ppm were added to the infective larvae in sufficient quantity (to cover the larvae) to be examined carefully for their activities and vitality (viability testing) according to Ismail, 1979. This method reported that when adding the test material to the living larvae and their movement ceased, the larvae were stimulated with a needle to observe any further movement. When no movement occurred the larvae were transferred to another Petri dish containing hot water (38-40 C). The occurrence of a sudden movement indicates that the tested drug agent has relaxant effects on the larvae. When no signs of recovery occurred this indicates a sign for killing effect of the tested drug agent. The time duration, from adding the tested agent to the larvae till there was no movement of the all larvae in the Petri dish (5 larvae) was calculated.
The experiment for each concentration was repeated for 5 replicates, each one with 5 larvae (i.e. a total of 25 larvae for each concentration).
The following was observed for both groups of tested agents (A or B) with their different concentrations (100 ppm, 50 ppm, 25 ppm, 10 ppm & 1 ppm): once the tested agent became in contact with the larvae, the larvae showed vigorous contractions of their whole bodies, mainly the anterior and posterior ends, followed by relaxation as shown in the photos of
This observation demonstrated the killing effect of both compositions (A&B), regardless of concentration, on Trichinella spiralis larvae under in vitro conditions according to Ismail (1979), but they differ from each other according to the mean time to death of the tested larvae.
The following table, and the graph shown in
The table shows that the overall mean time to death with test agent A is significantly longer (144.76+41.35 minutes) than with test agent B (132.42+53.55 minutes). As regards concentration, the mean time to death significantly decreased with increasing concentration, with no significant difference between test agents in each concentration except for concentration 50 ppm which showed a mean of 162.52+10.58 minutes with test agent A compared to 81.79+12.78 minutes with test agent B.
Next, the infectivity of the treated larvae were tested with test agent B at a concentration of 50 ppm. This agent and concentration were chosen because the test agent B at a concentration 50 ppm exhibited a significant decrease in the mean time to death for T. spiralis larvae of about 50% (81.79+12.78 minutes) in comparison with test agent A at the same concentration (162.52+10.58 minutes).
Fifteen laboratory raised Swiss albino mice aged 6 weeks were used to execute the study. They were kept under scrupulous hygienic conditions and feed one day milk and other day wheat. Diet and water were available ad libitum. All animals were acclimatized to these conditions for 1 week prior to the experiment
Proven infected muscle samples (mainly from the diaphragm) containing encysted larvae of Trichinella spiralis were taken from freshly slaughtered pigs at slaughter house in Alexandria and immediately transferred to the laboratory. The infective free larvae were obtained by digestion technique according to Schad et al., 1987. The larvae were treated with the test agent B at a concentration of 50 ppm till no signs of recovery were obtained.
A dose of 150 treated larvae were inoculated orally per mouse (15 mice) at day 0 (Infection day). At day 7 post infection (adult stage), 5 mice were decapitated, their small intestines were washed by normal saline, opened and scraped. The content was filtered through 2 layers of gauze and centrifuged. The supernatant was poured off and the sediment was examined for the adult worms of T. spiralis.
At day 45 post infection (encysted larvae stage in muscle), the remaining 10 mice were decapitated, muscle samples were taken from the diaphragm and other skeletal muscle and examined under the low power objective of the microscope (×10) to detect the encysted larvae of T. spiralis in order to reconfirm the infection.
At day 7 post infection, no adult worms were detected. In addition no encysted larvae of T. spiralis were detected in the muscle on day 45 post infection. This demonstrates the killing effect of the test agent B at a concentration of 50 ppm. The test agent B at a concentration of 50 ppm thus had a lethal effect on T. spiralis larvae, making them non viable and non infective.
In summary, both agents A & B exhibited a killing effect on Trichinella spiralis larvae under in vitro conditions regardless of concentration, but they differed from each other according to the mean time to death of the tested larvae. The overall mean time to death with test agent A was significantly longer than with test agent B. As regards concentration, the mean time to death decreased significantly with increasing concentration, with no significant difference in the rate of decrease between test agents at each concentration, except that test agent B at 50 ppm decreased the mean time to death for T. spiralis larvae to about 50% of that of test agent A of the same concentration. The test agent B at 50 ppm was thus proved to have a lethal effect on T. spiralis larvae, makes them non viable and non infective under in vivo testing.
It is estimated that more than 1.4 billion people are infected with Ascaris lumbricoides, a nematode of the secementea class. This infected population represents 25 percent of the world population (Seltzer, 1999). Although ascariasis occurs at all ages, it is most common in children 2 to 10 years old, and prevalence decreases over the age of 15 years. Infections tend to cluster in families, and worm burden correlates with the number of people living in a home (Haswell et. al., 1989). The prevalence is also greatest in areas where suboptimal sanitation practices lead to increased contamination of soil and water. The majority of people with ascariasis live in Asia (73 percent), Africa (12 percent) and South America (8 percent), where some populations have infection rates as high as 95 percent (Sarinas and Chitkara, 1997). In the United States the prevalence of infection decreased dramatically after the introduction of modern sanitation and waste treatment in the early 1900s as reported by Jones, 1983.
Children are particularly vulnerable since they are at risk of ingesting Ascaris eggs while playing in soil contaminated with human faeces. Dust and contaminated fruits and vegetables pose a hazard to all members of the community. Once ingested, the eggs hatch in the small intestine and motile larvae penetrate the mucosal blood vessels. They are carried first to the liver and then to lungs where they ascend the bronchial tree before being swallowed. Eventually they re-enter the small intestine where they mature, over the period of two months into adult worms. Adult worm can live from 1-2 years.
This larval migration sometimes induces transient hypersensitivity and inflammatory reactions resulting in pneumonitis, bronchial asthma and urticaria. Subsequently, colonization of the gastrointestinal tract by adult worms, which survive for about one year, can cause anorexia, abdominal pain and discomfort and other gastrointestinal symptoms. From time to time all or part of the worms can be vomited or passed in the stools. Obstruction of the small intestine by worms or less frequently their migration, often subsequent to inadequate treatment into biliary tract, the appendix, the pancreatic ducts or even the upper respiratory tract can create a life-threatening emergency requiring surgical interference.
Ascaris suum (Goeze, 1782) or pig Ascaris is morphologically identical to A. lumbricoides with slight differences. The copulatory spicules are thinner and sharper on the tip in A. suum than in A. lumbricoides. The prepatent period in A. suum is shorter than in A. lumbricoides (Galvin, 1968).
Ascaris suum is commonly called the large roundworm of pigs and its predilection site is the small intestine. It is the largest and most common nematode of pigs on a worldwide basis. Boes et al., 1998 reported that the prevalence and intensity as well as the distribution observed for A. suum infection in pigs were comparable to those reported for A. lumbricoides in endemic areas, and there was an evidence for predisposition to A. suum in pigs, with an estimated correlation coefficient similar to that found in humans. They concluded that A. suum infections in pigs are a suitable model to study the population dynamics of A. lumbricoides in human populations.
The life cycle of the parasite, A. suum is similar to the one in A. lumbricoides. The adult worms are large worms (males 15-25 cm; females 20-40 cm) that occur in the small intestine. They feed upon the intestinal contents, competing with the host for food. Eggs are environmentally resistant. Female worms are very prolific producing 0.5 to 1 million eggs per day and these will survive outside the pig for many years (up to 20 years). They are resistant to drying and freezing but sunlight kills them in a few weeks. Eggs become infective after 18 to 22 days. When ingested eggs are hatched in the stomach and upper intestine, and larvae migrate to the liver and then to the lungs. After about 10 days, larvae migrate to esophagus and will be swallowed and return to the intestine, where after two molts develop to the adult worms between 15 and 18 cm long. Infection with A. suum affects pigs, principally the young. Signs include poor growth, poor coat and diarrhea due to enteritis (see photo in
The infection does not restricted to pigs only but also can infect cattle as reported by Borgsteede et al., 1992. The infection causing a sudden decrease in milk yield, increased respiratory rate and occasional coughing were observed in dairy cows on farms where pigs were also kept on these farms, and pastures grazed by the cattle had been fertilized with pig slurry. Laboratory investigations of some of the cattle showed eosinophilia and high ELISA titres of antibodies against Ascaris suum. The clinical symptoms disappeared after the animals had been treated.
Human infection occurs also as the result of exposure to the pig farms, or the use of pig manure in the vegetable gardens. An outbreak of infection with swine Ascaris lumbricoides suum with marked eosinophilia was reported from southern part of Kyushu District, Japan (Maruyama et al, 1997).
The clinical symptoms of infection with A. suum in man are similar to A. lumbricoides and high burden will cause sever diseases. As described by Phills et al (1972), four male students in Montreal, Canada who unknowingly swallowed eggs of A. suum became hospitalized with severe pneumonitis, high eosinophilia and asthma. The infection can also produce failure to thrive, stunting, pot belly and diarrhea (Merle and Nicole (2000).
Chemotherapy is the cornerstone of the strategy of control of morbidity and reduction of transmission. Individual human infections are eradicated by a single dose of pyrantel or levamisole. piperazine is also effective but it less well tolerated. The most commonly used drugs are broad-spectrum anthelminthics as benzimidazole, mebendazole, albendazole and flubendazole are each effective.
In this Example, the dose-response of two test agents against adult worms of Ascaris lumbricoides suum was determined under in vitro conditions.
Two test agents were used in this Example, designated Agents A and B. Agent A comprised 7% linalool coeur, 35% thymol, 4% α-pinene, 30% p-cymene, and 24% soybean oil. Agent B comprised Agent A with the addition of 1.2% of a surfactant, the commercially available Sugar Ester OWA-1570. The stock solution (A or B) was diluted by sterile normal saline solution into five concentrates: 100 ppm, 50 ppm, 25 ppm, 10 ppm and 1 ppm. Each concentrate was agitated by vortex for 15 minutes before use.
The adult worms of A. suum were obtained from intestines of slaughtered pigs condemned in the slaughter houses, as unfit for human consumption or use. The pig's intestines were taken and opened; their content was examined for the presence of adult worms of A. suum (see the photo in
Five living adult worms of both sexes of A. suum were placed in a suitable dish and the tested agents (A or B) with different concentrations: 1 ppm, 10 ppm, 25 ppm, 50 ppm and 100 ppm were added to the living adult worms in sufficient quantity (to cover the adult worms) to be examined carefully for their activities and vitality (viability testing) according to Is mail, 1979. This method reported that when adding the test material to the living worms and their movement ceased, the worms were stimulated with needle to observe any further movement. When no movement occurred the worms were transferred to another dish containing hot water (38-400 C). The occurrence of a sudden movement indicates that the tested agent has relaxant effects on the worms. When no signs of recovery occurred this indicates a sign for killing effect of the tested agent. The time duration, from adding the tested agent to the worms till there was no movement of the all worms in the dish (5 worms) was calculated.
The experiment for each concentration was repeated for 5 replicates, each with 5 adult worms (i.e. a total of 25 adult worms of both sexes for each concentration). The following was observed for both groups of tested agents (A or B) regardless of concentration: once the tested agent came into contact with the adult worms, the worms showed vigorous contractions of their whole bodies (see the upper photo in
It is worth noting that the damage caused by the adult worms seems largely related to their size. The large and muscular adult worms do not attach to the intestinal wall but maintain their position by constant movement. They occasionally force their way into extra intestinal sites or if present in large numbers form tangled masses that occlude the bowel as reported by Markell et al., 1999. This fact can be used to explain the importance of the relaxant effect of the tested agents A or B to expel the worms out of the intestine, if given the test agent then followed by giving a suitable purgative.
The following table and the graph shown in
The table shows that the overall mean time to show the relaxing effect on the adult worms of A. suum with test agent B is significantly longer (21.20+0.73 hours) than with test agent A (9.02+1.35 hours).
As regards concentration, the mean time to show this effect was significantly decreasing by increasing concentration with significant difference between the test agents in each concentration. Multiple comparisons among means showed that with test agent A each concentration had shorter time to bring relaxation than the preceding concentration while with test agent B no significant change was gained after 25 ppm. A significant interaction effect of test agents and concentration was revealed which indicated that increase dose of test agent A significantly decreased the time to bring the relaxing effect from 10.07 hours with 1 ppm to 6.47 hours with 100 ppm. On the other hand increase dose of test agent B showed minimal decrease of time to show the relaxing effect from 22.02 hours with 1 ppm to 20.74 hours with 100 ppm.
In sum, both tested agents (A and B), regardless of concentration, exhibited a relaxant effect on the adult worms of Ascaris lumbricoides suum under in vitro conditions, but they differ from each other according to the mean time to show this effect. The overall mean time to show the relaxing effect on the adult worms of A. suum with test agent B is significantly longer than with test agent A. A significant interaction effect of test agents and concentration was revealed which indicated that increase dose of test agent A significantly decreased the time to bring the relaxing effect from 10.07 hours with 1 ppm to 6.47 hours with 100 ppm. On the other hand, an increased dose of test agent B showed minimal decrease in the time required to produce the relaxing effect, from 22.02 hours with 1 ppm to 20.74 hours with 100 ppm. This result indicated that test agent A at 100 ppm is more potent, in that it causes a relaxing effect on the adult worms of A. suum in a short time of about 6 hours.
The results of Examples 16 and 17 indicate that the test agents A and B had different modes of action on nematode parasites. Both agents had a lethal effect on the larvae of Trichinella spiralis under in vitro conditions, with test agent B exhibiting a shorter mean time to show its effect than test agent A, and both made the larvae non-viable and non-infective under in vivo testing. Both agents had a relaxing effect on the adult worms of Ascaris lumbricoides suum under in vitro conditions, with test agent A exhibiting a shorter mean time to show its effect than test agent B.
Based on these results, the efficacy of the test agent B at different concentrations on the treatment of Trichinella spiralis is assessed in experimentally infected mice. Female Swiss Albino mice, 8 weeks in age, from 18-22 gm in weight, which can be obtained from Theodore Bilharz Research Institute, Cairo, are infected with by T. spiralis larvae (100 larvae/mouse). Each group consists of 15 mice.
For each test composition, three concentrations are tested. For each concentration nine groups of mice are studied. One group of T. spiralis-infected mice receives the present standard treatment drug. Three groups of uninfected mice receive the test compound in the same schedule and concentration as the test drug groups. One group of uninfected and untreated mice and one group of T. spiralis infected mice that do not receive any treatment are maintained as controls.
Three different concentrations from each of the test compositions are determined after estimation of the LD50. The schedule for drug administration is as follows: (1) four days post-infection (PI); (2) one week PI; and seven weeks PI. All drugs are administered orally using a stomach tube.
For the parasitological studies, fecal egg counts are done for all infected groups twice weekly starting from the 5th. week PI.
Mice are sacrificed 9 weeks PI. Perfusion of the portal system is done for the recovery of the worms. The total number, sex, maturation and distribution of the worms are determined. Four portions, two from the jejunum and two from the ileum, are taken from each mouse, washed with PBS, opened and compressed between two slides and examined microscopically for detection of the stage of maturation. 0.3 gram of the intestine are digested in 4% potassium hydroxide overnight, and T. spiralis larvae counted.
Due to the relaxant effect of the tested agents A or B on the adult worms of Ascaris lumbricoides suum, they will be useful in treating Ascaris-infected subjects so as to expel the worms out of the intestine of the infected hosts after giving a suitable purgative.
An exemplary test composition is used, which comprises: 7% (vol/vol) linalool; 35% (vol/vol) thymol; 4% (vol/vol) α-pinene; 30% (vol/vol) p-cymene; and 24% (vol/vol) soy bean oil. Test doses are: 1 mg/kg Body Weight (BW), 10 mg/kg BW, 20 mg/kg BW, and 100 mg/kg BW.
Criteria of cure used for the experiments are: (1) exposure time and efficacious dose level to produce 100% kill of H. nana in a minimum of 80% of infected mice (e.g., cure=0 viable worms in intestine and 0 viable eggs in stool). The short life cycle of H. nana can facilitate rapid prophylactic testing. H. nana has about a 14-day life cycle from egg infection until maturation and egg laying.
Several administration protocols are implemented to test the efficacy of the exemplary composition against infection. In a first protocol, an oral dose is administered to 5 groups of mice via gel capsule at 3 days prior to infection and daily until mice are sacrificed. In a second protocol, an oral dose is administered to 5 groups of mice via gel capsule at 3 weeks prior to infection and daily until mice are sacrificed. In a third protocol, an oral dose is administered to 5 groups of mice via gel capsule daily starting 3 weeks prior to infection, and treatment is discontinued after infection until mice are sacrificed. Control groups of mice in each of the protocols are dosed with soy bean oil only. Data from the three protocols using different mg/kg BW of the exemplary test composition are presented in Tables 8-12.
An exemplary test composition is used, which comprises: 7% (vol/vol) linalool; 35% (vol/vol) thymol; 4% (vol/vol) α-pinene; 30% (vol/vol) p-cymene; and 24% (vol/vol) soy bean oil.
Test groups of mice are provided for infection and treatment, each containing about 20 mice (e.g., 5 test groups×20 mice per test group=100 mice). Animals are selected and examined to ensure they are worm-free. The following test groups are designated to be infected and to received the following treatment:
Group 1: soy bean oil carrier only;
Group 2: 1 mg/kg body weight (BW) composition;
Group 3: 10 mg/kg BW composition;
Group 4: 20 mg/kg BW composition; and
Group 5: 100 mg/kg BW composition.
An additional control group that is not infected can be provided and administered the exemplary composition. Test groups of mice designated for infection are infected, for example with H. nana. About 150 viable eggs per mouse is determined to be useful for infecting mice such that test animal exposure to the parasite's infective stage is predictive of realistic environmental exposure.
An oral dose is administered via gel capsule to the test groups of mice at 2 days after egg shedding is observed. The oral dose is administered daily until mice are sacrificed. Half-life of doses of exemplary composition can be determined in mammalian blood to guide specification of prophylactic and therapeutic regiments.
Resistance studies of exemplary compositions are conducted. An exemplary test composition is used, which comprises: 7% (vol/vol) linalool; 35% (vol/vol) thymol; 4% (vol/vol) α-pinene; 30% (vol/vol) p-cymene; and 24% (vol/vol) soy bean oil.
Test groups of mice are provided, each containing about 20 mice (e.g., 5 test groups×20 mice per test group=100 mice). Animals are selected and examined to ensure they are worm-free. The following test groups are designated to be infected and to received the following treatment:
Group 1: soy bean oil carrier only;
Group 2: 1 mg/kg body weight (BW) composition;
Group 3: 10 mg/kg BW composition;
Group 4: 20 mg/kg BW composition; and
Group 5: 100 mg/kg BW composition.
An additional control group that is not infected can be provided and administered the exemplary composition.
Test groups of mice designated for infection are infected, for example with H. nana. About 150 viable eggs per mouse is determined to be useful for infecting mice such that test animal exposure to the parasite's infective stage is predictive of realistic environmental exposure. Target DNA from the eggs used for the initial infection is sequenced prior to treatment with exemplary compositions, for use as a control sequence.
An oral dose is administered via gel capsule to the test groups of mice at 2 days after egg shedding observed. The oral dose is administered daily until the mice are sacrificed. The viable eggs are counted and collected. The collected viable eggs are used to re-infect the previously uninfected animal test group, which are then treated with the exemplary composition as before. The step is repeated, for a total of three counts and collections of viable eggs. Following the third count and collection of viable eggs, the viable egg target DNA is sequenced.
The parasite is assumed to have gone through three reproductive cycles. The control unexposed DNA sequence can be compared to the target DNA sequence obtained from eggs after the third cycle, having three successive exposures to the exemplary treatment compositions. Resistance is determined by considering: no change in exposed target DNA sequence vs. control target DNA sequence results in one or more amino acid changes.
Safety studies are of exemplary compositions are conducted. Safety studies include acute toxicity tests (range finding), in vitro genetic toxicology studies, and sub-chronic rodent toxicity study (90-day) conducted under Good Laboratory Practices (GLP).
Animals are exposed to daily doses of the therapeutic compositions being tested. For example, an exemplary test composition can be used, which comprises: 7% (vol/vol) linalool; 35% (vol/vol) thymol; 4% (vol/vol) α-pinene; 30% (vol/vol) p-cymene; and 24% (vol/vol) soy bean oil. The following test groups are designated to receive the following treatment:
Group 1: soy bean oil carrier only;
Group 2: 0.07 g/kg body weight (BW) per day;
Group 3: 0.7 g/kg BW per day; and
Group 4: 7 g/kg BW per day.
All appropriate observational and clinical tests (including histopathology) are performed to assess any treatment-related effects. Safety measures (see Table 10) are made at 100× the efficacious dose using a prophylactic efficacy protocol. For example, if the efficacious dose is 10 mg/kg, the safety test dose is 1 g/kg.
Relative palatability of exemplary compositions is also tested. Synergistic combinations of compounds can be designed to favor compounds with preferred palatability.
A receptor gene encoding the Tyramine receptor (TyrR) has been isolated from the American cockroach, fruit fly, mosquito, and other organisms. The present subject matter provides methods of utilizing the TyrR protein expressed in cells to screen for compounds useful for treating parasitic infections.
In the present Example, the genes encoding TyrR were incorporated into model cells in culture that mimic receptors in insects. The screening process uses the cultured cells in combination with [Ca2+]i and [cAMP]i measuring assays to quantitatively determine effectiveness of test compound to treat parasitic infections. The screening process allows for identification of compounds that produce highly efficacious anti-parasitic compositions.
The assay steps are as follows. A cell expressing a tyramine receptor is contacted with a test compound and the receptor binding affinity of the test compound is measured. Cells which can be used include, for example, HEK293 cells, COS cells, Drosophila Schneider or S2 cells, SF9, SF21, T.ni cells, or the like. cAMP and/or Ca2+ levels within the cell are also monitored and any changes from contacting the test compound with the cell are noted for each compound tested. A test compound is identified as a potential therapeutic compound If it exhibits a high receptor binding affinity for the tyramine receptor as well as an ability to effect change in cAMP and/or Ca2+ levels within the cell. A test compound is also identified as a potential therapeutic compound If it exhibits a low receptor binding affinity for the tyramine receptor as well as an ability to effect change in cAMP and/or Ca levels within the cell. A composition for use in treating a parasitic formulation can then be selected that includes a plurality of the identified compounds. In particular, the composition can comprise at least one compound identified as having a high receptor binding affinity for the tyramine receptor as well as an ability to effect change in cAMP and/or Ca2+ levels within the cell and at least one additional compound identified as having a low receptor binding affinity for the tyramine receptor as well as an ability to effect change in cAMP and/or Ca2+ levels within the cell.
Table 14 lists compounds tested with the present screening method and the determined capacity of each compound to bind the tyramine receptor, affect intracellular Ca2+, and affect intracellular cAMP. These results can then be utilized to select a composition comprising two or more of the tested compounds with desirable characteristics. For example, p-cymene and linalool can be select to include in a composition for treating parasitic infections according to the screening method criteria since p-cymene exhibits low tyramine receptor binding affinity, linalool exhibits high tyramine receptor binding affinity, and both compounds effect change in cAMP and/or Ca2+ levels. Similarly, p-cymene and thymol can be select to include in a composition for treating parasitic infections according to the screening method criteria since p-cymene exhibits low tyramine receptor binding affinity, thymol exhibits high tyramine receptor binding affinity, and both compounds effect change in cAMP and/or Ca2+ levels. Further, compositions for treating parasitic infections can be formulated that include more than two compounds, such as for example a composition that includes α-pinene, p-cymene, linalool, thymol, and soybean oil. It can be preferable to formulate a composition that displays an anti-parasitic effect exceeding the anti-parasitic effect of any of the compounds when used alone.
HEK293 cells are transfected with the pcDNA3.1/V5-HisA vector using Lipofectamine (Invitrogen). The vector contains a full-length construct of the C. elegans tyramine receptor. 48 h after transfection cells are selected in a culture medium containing 0.5 mg/ml G418 (Invitrogen). Cells that survive from the first round of G418 selection are further subjected to limiting dilution for single clone selection. Clones are selected and then cell stocks are grown for assay purposes.
Growth media is replaced with serum free media (i.e., Eagle's minimum essential medium (EMEM) buffered with 10 mM HEPES (N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid)) 24 hours after plating of the cells.
Linalool is used as the receptor activator for the assay, and is added to each well on each plate. Sufficient linalool is added to ensure receptor activation and a resulting increase in intracellular Ca2+ levels.
Essential oil test compounds of varying concentrations are added to the wells of each of the four plates (four plates are used per replicate). The assay is conducted at room temperature.
At time points of 30 seconds, 60 seconds, 90 seconds, 120 seconds, 180 seconds, 240 seconds, 300 seconds, and 600 seconds post-addition of test compound, the assay is terminated and the cells are analyzed to determine intracellular Ca2+ levels.
HEK293 cells are transfected with the pcDNA3.1/V5-HisA vector using Lipofectamine (Invitrogen). The vector contains a full-length construct of the C. elegans tyramine receptor. 48 h after transfection cells are selected in a culture medium containing 0.5 mg/ml G418 (Invitrogen). Cells that survive from the first round of G418 selection are further subjected to limiting dilution for single clone selection. Clones are selected and then cell stocks are grown for assay purposes.
Growth media is replaced with serum free media (i.e., Eagle's minimum essential medium (EMEM) buffered with 10 mM HEPES (N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid)) 24 hours after plating of the cells.
Linalool is used as the receptor activator for the assay, and is added to each well on each plate. The amount of linalool added is less-than that required to ensure receptor activation and a resulting increase in intracellular Ca2+ levels.
Essential oil test compounds of varying concentrations are added to the wells of each of the four plates (four plates are used per replicate). The assay is conducted at room temperature.
At time points of 30 seconds, 60 seconds, 90 seconds, 120 seconds, 180 seconds, 240 seconds, 300 seconds, and 600 seconds post-addition of test compound, the assay is terminated and the cells are analyzed to determine intracellular Ca2+ levels.
HEK293 cells are transfected with the pcDNA3.1/V5-HisA vector using Lipofectamine (Invitrogen). The vector contains a full-length construct of the C. elegans tyramine receptor as well as an arrestin-GFP conjugate. For transient transfection, cells are harvested 48 h after transfection. For stable transfection, 48 h after transfection cells are selected in a culture medium containing 0.5 mg/ml G418 (Invitrogen). Cells that survive from the first round of G418 selection are further subjected to limiting dilution for single clone selection. Clones are selected and then cell stocks are grown for assay purposes.
Growth media is replaced with serum free media (i.e., Eagle's minimum essential medium (EMEM) buffered with 10 mM HEPES (N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid)) 24 hours after plating of the cells. Per replicate, two plates are incubated for 10 minutes at room temperature and atmospheric CO2 and two plates are incubated for 10 minutes at 37 C and 5% CO2.
Each test compound is solvated using 100% dimethyl sulfoxide (DMSO). Multiple solutions of each compound are prepared at varying concentrations for testing in separate wells of each plate. The solutions are sonicated to increase solubility.
Each of the solutions of varying concentrations of the fifteen compounds is added to a well on each of the four plates (four plates are used per replicate). Two plates per replicate are incubated for 30 minutes at room temperature and atmospheric CO2. The other two plates per replicate are incubated for 30 minutes at 37 C and 5% CO2.
Agonist is then added to each well. For each compound to be tested, 100 nM isoproterenol (0.4% weight/volume ascorbic acid) is added to one of the 37 C plates and one of the RT plates. 100 nM arginine vasopressin is added to one of the 37 C plates and one of the RT plates.
The assay is terminated using 1% paraformaldehyde containing 1 uM DRAQ5 DNA probe to fix the cells. The cells are analyzed using a line scanning, confocal imaging system to quantitate the localization of the arrestin-GFP conjugate for the cells in each well using the Amersham Biosciences granularity analysis GRNO algorithm. This algorithm finds the nucleus of cells and then dilates out a specified distance in which fluorescent spots of arrestin-GFP localization are identified based on size and fluorescent intensity. The average of the fluorescent intensity of the identified grains per cell in an acquired image is determined for each well on the plates.
Control wells are used on each plate to determine the basal level of fluorescent spots for the cells on the different plates as well as to determine the maximally stimulated level of fluorescent spots for the cells on the different plates. The cells in the control wells are subjected to the method described above, but no test compound or agonist is added to the wells. The cells in the “agonist” control wells are subjected to the method described above, including the addition of agonist, but no test compound is added to the wells.
Formulations in accordance with embodiments of the present disclosure are also useful as repellants against other biting anthropod vectors such as sand flies, mosquitoes and bugs that transmit deadly infections in both human and animals. Experimental hosts such as mice (for bugs) dogs (for sand flies) and human (mosquitoes) are well known in the art. Such host animals are treated with the formulations of the present disclosure and the ability of the arthropod vectors to feed on the host are evaluated. Appropriate dosages of the formulations are readily determined by methods such as those described above well known in the art.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US08/88342 | 12/24/2008 | WO | 00 | 9/29/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 61017145 | Dec 2007 | US | |
| 61017157 | Dec 2007 | US | |
| 61018879 | Jan 2008 | US |
