PROTECTION AGAINST ARTHROPOD PARASITES WITH PURINERGIC RECEPTOR (OPR) ANTAGONISTS

Abstract

This invention concerns a product comprising one or more purinergic receptor (PR) antagonist for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite by exposing the arthropod parasite to one or more PR antagonist, or by contacting the arthropod parasite with one or more PR antagonist. The invention also concerns a pharmaceutical composition comprising the product for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite, and screening methods to identify suitable PR antagonist.

Claims

1. A product comprising one or more purinergic receptor (PR) antagonist for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite by exposing the arthropod parasite to one or more PR antagonist, or by contacting the arthropod parasite with one or more PR antagonist.

2. Product comprising one or more purinergic receptor (PR) antagonist for use in prevention and/or treatment of parasitic infestation of a human and/or animal host according to claim 1, wherein the one or more PR antagonist inhibits the arthropod parasite from feeding on host tissue, including blood.

3. Product for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite according to claim 1, wherein the product comprises at least one of (i) a P1 PR antagonist, or (ii) a P2X PR antagonist selected from a group consisting of P2X.sub.2, P2X.sub.3, or a P2X.sub.2/3 PR antagonist, (iii) or a combination thereof.

4. Product for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite according to claim 3, wherein the one or more P1 PR antagonist is a xanthine, such as 1,3-dimethylxanthine or 8-cyclopentyl-1,3-dimethylxanthine.

5. Product for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite according to claim 3, wherein the one or more P2X.sub.2, P2X.sub.3, or P2X.sub.2/3 PR antagonist is selected from a group consisting of a polysulfonated naphthylamine such as suramin, pyridoxal phosphate-6-azophenyl-2,4-disulfonate (PPADS), an anthraquinone-sulfonic acid compound such as 1-amino-4-[[4-[[4-chloro-6-[[3 (or 4)-sulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl]amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid (Reactive Blue-2), a tricarboxylate such as 5-[(3-phenoxyphenyl)methyl-[(1S)-1,2,3,4-tetrahydronaphthalen-1-yl]carbamoyl]benzene-1,2,4-tricarboxylic acid (A-317491), a pyrazine alkaloid such as 2,3,5,6-tetramethylpyrazine, and a heptapeptide such as spinorphin.

6. Product for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite according to claim 3, comprising suramin and 8-cyclopentyl-1,3-dimethylxanthine, or suramin and 2,3,5,6-tetramethylpyrazine, or suramin and 1,3-dimethylxanthine, or suramin and a tricarboxylate such as 5-[(3-phenoxyphenyl)methyl-[(1S)-1,2,3,4-tetrahydronaphthalen-1-yl]carbamoyl]benzene-1,2,4-tricarboxylic acid (A-317491), or suramin and spinorphin, or comprising suramin and 2,3,5,6-tetramethylpyrazine and 8-cyclopentyl-1,3-dimethylxanthine, or suramin and 2,3,5,6-tetramethylpyrazine and 1,3-dimethylxanthine, or suramin and spinorphin and 8-cyclopentyl-1,3-dimethylxanthine, or suramin and spinorphin and 1,3-dimethylxanthine, or suramin and 2,3,5,6-tetramethylpyrazine and A-317491, or comprising 2,3,5,6-tetramethylpyrazine and 8-cyclopentyl-1,3-dimethylxanthine, or 2,3,5,6-tetramethylpyrazine and 1,3-dimethylxanthine, or 2,3,5,6-tetramethylpyrazine and A-317491, or comprising pyridoxal phosphate-6-azophenyl-2,4-disulfonate and 8-cyclopentyl-1,3-dimethylxanthine, or pyridoxal phosphate-6-azophenyl-2,4-disulfonate and 1,3-dimethylxanthine, or comprising Reactive Blue-2 and 8-cyclopentyl-1,3-dimethylxanthine, or Reactive Blue-2 and 1,3-dimethylxanthine, or comprising A317491 and 8-cyclopentyl-1,3-dimethylxanthine, or A317491 and 1,3-dimethylxanthine, or comprising spinorphin and 8-cyclopentyl-1,3-dimethylxanthine, or spinorphin and 1,3-dimethylxanthine.

7. Product for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite according to claim 1, further comprising at least one PR metal modulator.

8. Product for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite according to claim 1, wherein the product is directed against blood-feeding arthropod ectoparasites, for example against biting insects such as mosquitoes, sandflies, tsetse flies, black flies, tabanid horse and deer flies, stable flies, horn flies, midges, lice, bed bugs, triatomine bugs, fleas, and/or against mites or ticks, such as species of the group of the Ixodidae or the Argasidae, or against crustacean ectoparasites.

9. Product for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite according to claim 1, wherein the product is directed against arthropod endoparasites, such as an endoparasitic mite, a crustacean endoparasite, endoparasitic larvae of the botfly families Cuterebridae and Hypodermatidae, endoparasitic gadflies, endoparasitic flies of the genus Hypoderma, or endoparasitic flies of the families Calliphoridae, Muscidae and Sacrophagidae.

10-12. (canceled)

13. Product for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite according to claim 1, wherein the product is suitable for topical application onto humans and/or host animals, for application onto any solid surface, for spraying and/or for soaking of a fabric, such as clothing, a bed net, or a cover for a host animal.

14. A pharmaceutical composition comprising the product for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite according to claim 1.

15. The pharmaceutical composition according to claim 14 being suitable for topical application.

16. The pharmaceutical composition according to claim 14 being suitable for systemic delivery, for example by means of subcutaneous, intravenous or transdermal administration, or through ingestion.

17. The pharmaceutical composition according to claim 11, wherein the pharmaceutical formulation further comprises an insecticide, an insecticide synergist, an acaricide, a parasiticide, an endectocide, a chemosterilant, a repellent, and/or a surfactant.

18. A method for preventing an arthropod parasite from feeding, comprising the step of applying the product according to claim 1 topically onto the host, or applying said product to clothing or covers for hosts, or applying the product to bed nets or fly screens or to any means of restraining or housing a host animal, or applying the product topically onto the arthropod parasite, or dispersing the product in facilities for a host, or dispersing the product in a habitat occupied by the arthropod parasite.

19. A screening method for selecting an antagonist for a PR of an arthropod parasite comprising: Arranging at least one test animal such as to expose at least one animal cell expressing one or more PRs or at least one portion of said test animal comprising one or more purine-sensitive cells for electrophysiological measurement, Stimulating the one or more PRs carried on the animal cell, or on the animal portion by presenting at least one PR agonist, Determining the response of the animal cell or of the animal portion comprising the one or more PR over a defined period during stimulation, for example by determining the action potential frequency, Presenting a test compound in combination with the at least one PR agonist to the one or more PR carried on the animal cell or on the animal portion, Determining the response of the one or more PR carried on the animal cell or on the animal portion over a defined period during presentation of the test compound together with the at least one PR agonist, for example by determining the action potential frequency, Comparing the response of the animal cell or of the at least one animal portion comprising the one or more PR in absence of the test compound to the response of the animal cell, or of the animal portion when presented with the test compound, Determining the suitability of the test compound for use as a PR antagonist based on the result of this comparison.

20-21. (canceled)

22. A screening method for determining the ability of a PR antagonist to prevent infestation of a human and/or animal host by an arthropod parasite comprising: Contacting at least one the cell expressing one or more PRs of at least one test animal, or at least one portion comprising one or more purine-sensitive cells of at least one test animal with a warm substrate or warm body, Determining the biting rate of said at least one test animal over a defined period on said warm substrate or said warm body, Presenting at least one test compound on said warm substrate or said warm body contacting the at least one cell or the at least one portion of the at least one test animal, Determining the biting rate of said at least one test animal over a defined period in presence of the at least one test compound on said warm substrate or said warm body, Comparing the biting rates of said at least one test animal in the presence and in the absence of the at least one test compound; Determining the suitability of the at least one test compound for use as a feeding inhibitor based on the result of this comparison.

23. A screening method for selecting an antagonist for a PR of an arthropod parasite comprising: Arranging a test animal or at least one portion of said test animal such as to expose at least one animal cell expressing one or more PRs or at least one portion of said test animal comprising one or more purine-sensitive cells, or arranging a cell, a tissue or an organoid of vertebrate or invertebrate origin expressing on or more PR in or onto a medium containing a fixed dose of at least one PR agonist, Determining the cellular activity of said animal cell, of said at least one portion of test animal, or of said cell, tissue or organoid of vertebrate of invertebrate origin, and/or determining the growth rate of said animal cell, of said at least one portion of test animal, or of said cell, tissue or organoid of vertebrate or invertebrate origin over a defined period in presence of the at least one PR agonist in the medium, Presenting at least one test compound in combination with the at least one PR agonist to said animal cell, to said portion of test animal, or to said cell, tissue or organoid of vertebrate or invertebrate origin, Determining the cellular activity of said animal cell, of said at least one portion of test animal, or of said cell, tissue or organoid of vertebrate or invertebrate origin, or determining the growth rate of said animal cell, of said portion of test animal, or of said cell, tissue or organoid of vertebrate or invertebrate origin over a defined period in presence of the at least one PR agonist and the at least one test compound in the medium, Comparing the cellular activity of said animal cell, of said at least one portion of test animal, or of said cell, tissue or organoid of vertebrate or invertebrate origin expressing the one or more PR, or determining the growth rate of said of said animal cell, of said at least one portion of test animal, or of said cell, tissue or organoid of vertebrate or invertebrate origin expressing the one or more PR in the presence and in the absence of the at least one test compound, Determining the suitability of the at least one test compound for use as a PR antagonist based on the result of this comparison.

24. The screening method according to claim 23, being performed using a transgenic animal comprising an altered allele for a gene that naturally encodes and expresses at least one functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite, for example a blood-feeding arthropod parasite, or using a transgenic animal comprising an altered allele for a reporter gene, or using a transgenic animal comprising an altered allele for a gene that naturally encodes and expresses at least one functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of a non-parasitic animal where said PRs respond to an agonist of at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of the arthropod parasite, for the purpose of selecting antagonists for said PRs.

25. The screening method according to claim 23, being performed using a heterologous system, comprising a host cell of vertebrate or invertebrate origin, containing an allele for the gene that naturally encodes and expresses at least one functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite, such as a blood-feeding arthropod parasite, for the purpose of selecting antagonists for said PRs.

26. A pharmaceutical composition comprising the product for use in prevention and/or treatment of parasitic infestation of a human and/or animal host by an arthropod parasite according to claim 7.

27. A method for preventing an arthropod parasite from feeding, comprising the step of applying the product according to claim 7 topically onto the host, or applying said product to clothing or covers for hosts, or applying the product to bed nets or fly screens or to any means of restraining or housing a host animal, or applying the product topically onto the arthropod parasite, or dispersing the product in facilities for a host, or dispersing the product in a habitat occupied by the arthropod parasite.

28. The screening method according to claim 23, being performed using a heterologous system, comprising cells expressing a functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR or any combination of said PRs belonging to any organism within the Protozoa or belonging to any invertebrate or vertebrate organism where said PR or PR combination responds to an agonist of at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite, such as a blood-feeding arthropod parasite, for the purpose of selecting antagonists for said PRs.

Description

SHORT DESCRIPTION OF THE DRAWINGS

[0076] Exemplar embodiments of the invention are disclosed in the description and illustrated by the drawings in which:

[0077] FIGS. 1A and B: Mean spike frequencies/2 s (s.e.m.) generated in response to 200 pM doses of nucleotides, nucleosides, uracil and adenine presented to Anopheles gambiae (A. gambiae) labellum sensilla (FIG. 1A) and representative 2 s recordings (FIG. 2B). Responses, which are significantly different to the water negative control are indicated by h (P<0.05) and spike frequencies significantly lower than those recorded for ATP are indicated by a (P<0.05). Treatment repetitions (n) are reported in Materials and Methods.

[0078] FIG. 2A to 2D: Number of spikes (means.e.m.) per 2s recorded from A. gambiae labellum sensilla in response to increasing doses of ATP and AMP-CPP (FIG. 2A), to adenosine and AMP (FIG. 2B), to 50 pM of adenosine, 50 pM of ATP, and to an equimolar mixture of 50 pM ATP and adenosine with water as negative control (0 spikes) (FIG. 2C), and to increasing equimolar doses of ATP admixed with adenosine (continuous line) compared to responses to increasing doses of ATP and adenosine (respectively, dashed and dotted lines, data as in A and B) (FIG. 2D). Treatment repetitions (n) are reported in Materials and Methods.

[0079] FIG. 3A: Two second recordings from a female A. gambiae labellum sensillum presented with ATP, adenosine and an equimolar 200 pM mixture of ATP plus adenosine. Discrimination for action potentials of similar amplitudes and durations (on right) indicate that treatments elicited responses characterized by one spike class.

[0080] FIG. 3B: Typical 2 second recordings from the sensillum of FIG. 3A presented with 200 pM ATP, 1 mM sucrose and 1 mM sucrose admixed to 200 pM ATP. Spike amplitudes elicited by sucrose are 1.7 times higher than those elicited by ATP or adenosine, permitting discrimination of two spike classes each distinguished by similar amplitudes and durations (on right) elicited by sucrose (open arrowhead at bottom) and by ATP (closed arrowhead at bottom). A spike doublet (d) resulting from near simultaneous firing by the sucrose-sensitive and ATP-sensitive cells is indicated. Treatment repetitions (n) are reported in Materials and Methods.

[0081] FIGS. 4A to 4D: The effect of CPT (FIG. 4A) and suramin (FIG. 4B) on mean (s.e.m.) action potential frequencies recorded from A. gambiae labellum sensilla in response to adenosine and ATP each at 200 pM. The test sequence in FIGS. 4A and 4B was agonist, agonist admixed with antagonist, followed by agonist, presented to the sensillum at approximately 2 min intervals; doses of CPT and suramin are indicated. Antagonism lasted for a least 2 min (final response to an agonist compared to the initial response). Typical 2s spike trains from such recording sequences where 10 nM CPT (FIG. 4C) and 1 nM suramin (FIG. 4D) were admixed with adenosine. *P<0.05, **P<0.001 indicate significant reductions in spike frequency. Treatment repetitions (n) are reported Materials and in Methods.

[0082] FIG. 5A: Number of spikes (means.e.m.) per 2s recorded from A. gambiae labellum sensilla in response to increasing doses of ATP (solid line; data as in FIG. 2A), and to increasing doses of TMP (black dots) and A-317491 (triangles) admixed with 200 pM ATP (positive control, 0 on abscissa). Doses up to 100 nM TMP are presented for purposes of clarity, for full dose range of TMP tested see FIG. 6E.

[0083] FIGS. 5B and 5C: Two second recordings obtained with 10 nM doses of TMP and A-317491 admixed with 200 pM ATP following the sequence ATP (top trace), ATP+antagonist and ATP, presented at approximately 2 min intervals to the sensillum. Antagonism lasted at least 2 min (spike count for the final response to ATP compared to the initial response). Treatment repetitions (n) are reported in Materials and Methods.

[0084] FIGS. 6A to 6F: Number of spikes (means.e.m.) per 2s recorded from A. gambiae labellum sensilla presented with 200 pM ATP (positive control, 0 on abscissa) and to increasing doses of the P2 PR antagonists suramin (FIG. 6A) and PPADS (FIG. 6B), the P2X.sub.2 PR antagonist RB-2 (FIG. 6C), the P2X.sub.3/P2X.sub.2/3 PR antagonist A-317491 (FIG. 6D), and the P2X.sub.3 PR antagonists TMP (FIG. 6E) and spinorphin (FIG. 6F), each dose admixed with 200 pM ATP. Treatment repetitions (n) are reported in Materials and Methods.

[0085] FIG. 7A to 7I: Number of spikes (means.e.m.) per 2s recorded from A. gambiae labellum sensilla presented in sequence with 200 pM ATP, the indicated nM dose of a PR antagonist admixed with 200 pM ATP, and to 200 pM ATP: suramin (FIG. 7A), PPADS (FIG. 7B), MRS 2159 (FIG. 7C), RB-2 (FIG. 7D), A-317491 (FIG. 7E), TMP (FIG. 7F), spinorphin (FIG. 7G), paroxetine (FIG. 7H) and A-438079 (FIG. 7I). Treatments were presented to sensilla at approximately 2 min intervals. The dose of antagonist used approximates its estimated ID.sub.95 value, whereas the dose of a substance failing to inhibit is the maximum dose tested (see Table 4). *P<0.05, **P<0.01 compared to the initial response to ATP, NS not significant. Treatment repetitions (n) are reported in Materials and Methods.

[0086] FIG. 8A Mean percentage (s.e.m.) A. gambiae feeding on increasing doses of ATP in the feeding solution.

[0087] FIG. 8B: Percentage A. gambiae feeding on the feeding solution containing 20 pM ATP (positive control, 0 on abscissa) with increasing doses of TMP (black dots) and suramin (triangles) added. Treatment repetitions (n) are reported in Materials and Methods.

[0088] FIG. 9A to 9E: Mean proportion (s.e.m.) of A. gambiae feeding on a solution containing 20 pM ATP (positive control, 0 on abscissae) and on the same solution containing 20 pM ATP with increasing doses added of the P2 PR antagonists suramin (FIG. 9A) and PPADS (FIG. 9B), the P2X.sub.2 PR antagonist RB-2 (FIG. 9C), the P2X.sub.3, P2X.sub.2/3 PR antagonist A-317491 (FIG. 9D), and the P2X.sub.3 PR antagonist TMP (FIG. 9E). Treatment repetitions (n) are reported in Materials and Methods.

[0089] FIG. 10: Percentage A. gambiae feeding on the feeding solution (buffered NaCl/dextrose), on 20 pM ATP+200 pM adenosine as feeding stimulant in the feeding solution, on the P1A.sub.1 PR antagonist CPT at 20 M+the P2X.sub.3 PR antagonist TMP at 7.5 M+the feeding stimulant in the feeding solution, and on TMP alone at 7.5 M+the feeding stimulant in the feeding solution. Bars accompanied by same lower-case letter indicate a significant difference. Treatment repetitions (n) and exact P values are reported on Table 6.

DETAILED DESCRIPTION AND EMBODIMENTS OF THE PRESENT INVENTION

Basic Embodiments

[0090] The present invention comprises a means to inhibit feeding by an arthropod parasite using purinergic-receptor antagonists identified for at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of said organism using the means described in the invention. The present invention also comprises methods for selecting compounds that antagonize P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite for the purpose of using such antagonists to inhibit feeding by an arthropod parasite on or in a host animal.

[0091] A suitable PR antagonist is for example Suramin. Suramin is a symmetric molecule of formula 1:

##STR00001##

wherein R1 is -methyl in Suramin, and wherein the methyl group may be replaced in suramin derivatives which may be used as PR antagonists according to this invention, by moieties selected from a group consisting of H, an ethyl group, an isopropyl group, a phenyl group, F, Cl, a methoxy group, or CH.sub.2OCH.sub.3.

[0092] Derivatives of suramin, which may be used as PR antagonists according to this invention, include compounds of formula 1 wherein the carbamide moiety forming R2 is replaced by methylene, carbonyl, or carbonate.

[0093] Derivatives of suramin, which may be used as PR antagonists according to this invention also include compounds of formula 1 wherein benzene ring with amide linkers forming R3 is replaced by benzyl, benzofuran, and indole.

[0094] Derivatives of suramin, which may be used as PR antagonists according to this invention further include compounds of formula 1 wherein the amidotoluene forming R4 is replaced by thiazolidinone, thiazinanone, or thiobenzamide.

[0095] Derivatives of suramin, which may be used as PR antagonists according to this invention further include compounds of formula 1 wherein the naphthalene rings forming R5 are replaced by benzene rings, cyclohexane rings, or 1,3,4-triazine rings.

[0096] Derivatives of suramin, which may be used as PR antagonists according to this invention further include compounds of formula 1 wherein sulfonate group as in R6 is replaced by a methyl group, a methoxy group, or an amino group.

[0097] Suramin-derived P2X2, P2X3 and P2X2/3 PR antagonist also comprise the following compounds:

##STR00002## ##STR00003##

PR antagonists may also be small molecule inhibitors. P2PX2 PR antagonists may be selected from compounds comprising an anthraquinone moiety of the formula:

##STR00004##

such as compound RB-2 having the formula:

##STR00005##

P2PX2 PR antagonists may also be selected from compounds comprising a pyridoxal phosphate moiety of the formula:

##STR00006##

such as PPADS having the following formula:

##STR00007##

[0098] P2PX.sub.3 and P2X.sub.2/3 PR antagonists may also be selected from the following compounds:

##STR00008##

[0099] The present invention also concerns assay methods for selecting compounds capable of modulating at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite, in particular a blood-feeding arthropod parasite. Such assay methods can be physiological and/or biochemical assays and/or behavioural assays. Examples of behavioural assays are described herein in Material and Methods and in Examples, but suitable assays are not limited to the examples provided in Material and Methods and in Examples herein. Assay methods can be used to select compounds for their ability to modulate a P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite from available libraries containing hundreds of thousands of molecules or substances.

[0100] In one embodiment of this invention a product comprising at least one PR antagonist is directed against arthropod parasites of a vertebrate host, preferably warm-blooded vertebrate host.

[0101] In another embodiment, the product is directed against parasites of an invertebrate host, such as an insect, for example a bee.

[0102] In an embodiment the product is directed against arthropod parasites. The product may be directed against arthropod endoparasites such as mange mites, botfly larvae, myisasis fly larvae, and/or against arthropod ectoparasites such as blood-feeding micro-predators, mites and ticks.

[0103] In an embodiment the product is directed against arthropod biofouling, such as against barnacles.

[0104] In an embodiment at least one P1 PR of an arthropod parasite, preferably a blood-feeding arthropod parasite, is modulated by a P1 PR antagonist or by a combination of P1 PR antagonists;

[0105] In an embodiment at least one P1 A.sub.1 PR of an arthropod parasite, preferably a blood-feeding arthropod parasite, is modulated by a P1 A.sub.1 PR antagonist or by a combination of P1 A.sub.1 PR antagonists;

[0106] In an embodiment at least one P2X.sub.2 PR of an arthropod parasite, preferably a blood-feeding arthropod parasite, is modulated by a P2X.sub.2 PR antagonist or by a combination of P2X.sub.2 PR antagonists.

[0107] In an embodiment at least one P2X.sub.3 PR of an arthropod parasite, preferably a blood-feeding arthropod parasite, is modulated by a P2X.sub.3 PR antagonist or by a combination of P2X.sub.3 PR antagonists;

[0108] In an embodiment at least one P2X.sub.2/3 PR of an arthropod parasite, preferably a blood-feeding arthropod parasite, is modulated by a P2X.sub.2/3 PR antagonist or by a combination of P2X.sub.2/3 PR antagonists;

[0109] In an embodiment at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite, preferably a blood-feeding arthropod parasite, is modulated by a combination of P1, P2X.sub.2, P2X3.sub.3, P2X.sub.2/3 PR antagonists;

[0110] In an embodiment the invention comprises modulation of any combination of P1, P2X.sub.2, P2X.sub.3 and P2X.sub.2/3 PRs of an arthropod parasite, preferably a blood-feeding arthropod parasite, by a combination of P1, P2X.sub.2, P2X.sub.3 and P2X.sub.2/3 PR antagonists;

Methods for Selecting PR Antagonists

[0111] The present invention comprises a method for selecting an antagonist for a P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite by making, for example, electrophysiological recordings from a cell expressing at last one of said PRs. This is achieved when a test compound is presented in combination with an agonist such as adenosine or ATP and brought into contact in solution with the PR. A PR antagonist causes a decrease, for example, in action potential frequency recorded from the cell compared to the action potential frequency recorded from the same cell when an agonist such as adenosine or ATP is presented alone as positive control.

[0112] In an embodiment, the invention comprises an electrophysiological method for selecting an antagonist for a purine-sensitive cell of an arthropod parasite expressing at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR. In a preferred embodiment the test compound is presented in solution to contact the cell at increasing doses over the 1 pM to 100 nM range in combination with a fixed dose of an agonist such as adenosine or ATP. Alternatively, 1 pM to 100 M dose range for the test compound can be chosen for testing. Inhibition induced by an antagonist occurs within seconds as measured by the reduction in the number of action potentials compared to the number of action potentials recorded for adenosine or ATP presented alone as positive control. This is shown herein in Examples on FIGS. 4 A and C for the P1 antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT), and on FIG. 5 for the P2X.sub.3 PR antagonist 2,3,5,6-tetramethylpyrazine (TMP) and for the P2X.sub.3/P2X.sub.2/3 PR antagonist A-317491. This permits calculation of a dose-response function and ID.sub.50 value for a PR antagonist which can be in the 300 pM to 15 nM range as shown herein in Examples on FIGS. 5 and 6, and on Table 4 for the P2 PR antagonists suramin and PPADS, for the P2X.sub.2 PR antagonist RB-2, for the P2X.sub.3 PR antagonists TMP and spinorphin, and for the P2X.sub.3/P2X.sub.2/3 PR antagonist A-317491. Nevertheless, an ID.sub.50 value of an antagonist for a P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite is not limited to a particular molar range.

[0113] The present invention comprises a method for selecting an inverse agonist for a P1, P2X.sub.2, P2X P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite by making, for example, electrophysiological recordings from a cell expressing at last one of said PRs. This is achieved when a test compound is brought into contact in solution with the PR. An inverse agonist causes, for example, a decrease in action potential frequency recorded from the cell compared to the action potential frequency recorded from the same cell when the inverse agonist is absent from the solution.

Use of Transgenic Animals

[0114] For the selection of suitable PR antagonists it may be advantageous to use transgenic animals.

[0115] In an embodiment the present invention provides for a method for selecting an antagonist for a P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite by making, for example, recording from a cell, organ or tissue of a transgenic arthropod parasite containing an altered allele for a reporter gene that encodes and expresses for example a fluorescent protein in addition to at least one functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR. Presence of the encoded fluorescent protein permits recording of Ca.sup.++ transients in the cell, organ or tissue. Selection of a PR antagonist can be achieved when a test compound is presented in combination with an agonist such as adenosine or ATP and brought into contact in solution with the cell, organ or tissue of the transgenic arthropod parasite. A PR antagonist causes, for example, a decrease in cellular activity as measured by Ca.sup.++ transients recorded from said cell, organ or tissue compared to Ca.sup.++ transients recorded from the same cell, organ or tissue when adenosine or ATP is presented alone as positive control. In this case, modulation by the PR antagonist comprises inhibition of functional PR activity in the transgenic arthropod parasite's cell, organ or tissue.

[0116] In an embodiment the present invention provides for a method for selecting an antagonist for a P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite by making, for example, electrophysiological recording from a cell of a non-parasitic transgenic animal containing an altered allele for a gene that naturally encodes and expresses at least one functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite. This is achieved when a test compound is presented in combination with an agonist such as adenosine or ATP and brought into contact in solution with the PR. A PR antagonist causes, for example, a decrease in action potential frequency recorded from said cell compared to the action potential frequency recorded from the same cell when adenosine or ATP is presented alone as positive control. In the present case, modulation comprises inhibition of the functional PR of the non-parasitic transgenic animal.

[0117] In an embodiment the present invention provides for a method for selecting an antagonist for a P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite by making, for example, electrophysiological recordings from a cell of a non-parasitic transgenic animal containing an altered allele for a gene that naturally encodes and expresses at least one functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of a non-parasitic organism. It is understood that the functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of the non-parasitic organism responds to an agonist of at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of the arthropod parasite. Selection of an antagonist is achieved when a test compound is presented in combination with an agonist such as adenosine or ATP and brought into contact in solution with the PR. A PR antagonist causes, for example, a decrease in action potential frequency recorded from said cell compared to the action potential frequency recorded from the same cell when adenosine or ATP is presented alone as positive control. In the present case, modulation comprises inhibition of the functional PR of the transgenic animal.

[0118] In one embodiment the selection method is performed using a transgenic animal containing an altered allele for the gene that naturally encodes and expresses a functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of a non-parasitic organism such as humans or a rat;

[0119] In an embodiment the selection method is performed using a transgenic animal containing an altered allele for the gene that naturally encodes and expresses a functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite such as a biting insect or a tick;

[0120] In an embodiment the selection method is performed using a transgenic animal possessing an altered allele for a functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR with a phenotype that shows enhanced sensitivity to adenosine and/or ATP as a feeding stimulant compared to the wild-type animal. It is understood that such as transgenic animal is a non-parasitic one;

[0121] In an embodiment the selection method is performed using a transgenic animal possessing an altered allele for a non-functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR that renders it less sensitive to adenosine or ATP as a feeding stimulant. Such a transgenic animal is a knockout (KO) transgenic animal. Further, such a KO transgenic animal can be either an arthropod parasite or a non-parasitic animal.

Use of Heterologous System to Select PR Antagonists

[0122] It may furthermore be useful to base a selection method for a PR antagonist on a heterologous system.

[0123] In an embodiment the selection method is performed using a heterologous cell system, using host cells of vertebrate or invertebrate origin, containing an allele for the gene that naturally encodes and expresses at least one functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite for the purpose of selecting antagonists for said PRs. The host cell in such a system expresses a nucleic acid sequence or sequences that is not part of its own genome. Host cells can have a variety of origins, for example being human in origin such as human HEK cells, or in another example being amphibian in origin such as the Xenopus oocyte. In such a system a functional PR is constituted when polypeptide subunits synthesized by the host cell combine in the cell membrane to form a functional PR. The PR is activated by a nucleoside such as adenosine or a nucleotide such as ATP in solution through ion flux, and/or ion channel activation and/or induction of secondary messengers. Such activation by an agonist can be used to select antagonists for P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite through changes brought about by the antagonist in solution such as changes in ion flux when the antagonist contacts the functional PR. Such changes in ion flux can be quantified by a variety of techniques well known in the art as indicative of cellular activity, for example by measurement of changes of cell membrane potential, measurement of cell membrane currents, using radiolabelled ion flux assays, using fluorescence assays for voltage sensitive dyes, and by patch clamp electrophysiological techniques. In the case of P1 PRs such measurements that are indicative of cellular activity can also include quantification of secondary messengers such as adenylate cyclase and inositol 1,4,5-triphosphate (IP.sub.3). Automation of such measurements permits high-throughput screening of compound libraries using said heterologous systems to select antagonists of said PRs. Alternatively, the ability of cells expressing said functional PRs of an arthropod parasite to grow in a feeding medium in presence of a test compound can be used to select antagonists for P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite.

[0124] In an embodiment the selection method is performed using a heterologous cell system for the purpose of selecting antagonists for P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite where host cells express a functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR or any combination of said PRs belonging to any organism within the Protozoa or belonging to any invertebrate or vertebrate organism. It is understood that the functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR or said PR combination expressed in the heterologous cell system responds to an agonist of at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of the arthropod parasite. Human astrocytoma cells expressing human and rat P2X.sub.3 and P2X.sub.2/3 PRs permitted identification of A-317491 as a non-nucleotide antagonist of Ca.sup.++ flux by ATP-gated P2X.sub.3 and P2X.sub.2/3 PRs of humans and the rat as documented by Jarvis et al., 2002 in A-317491, a novel potent and selective non-nucleotide antagonist of P2X.sub.3 and P2X.sub.2/3 PRs, reduces chronic inflammatory and neuropathic pain in the rat in PNAS 99(26), doi:10.1073/pnas.252537299.

[0125] As described herein on FIGS. 5, 6 and 7 and Table 4 of Examples, A-317491 inhibits the malaria mosquito PR at an ID.sub.50 of 320 pM in the electrophysiological assay and, as described herein on FIG. 9 and Table 7 of Examples, A-317491 inhibits feeding by the malaria mosquito at an ID.sub.50 of 240 pM. Furthermore, as described herein on Tables 8, 9 and 10 of Examples, A-317491 inhibits feeding by the Asian mosquito vector of malaria, by biting flies and by ticks.

Use of Ex Vivo and In Vivo Systems to Select PR Antagonists

[0126] It is also possible, to perform the method of selecting PR antagonists using an ex vivo or an in vivo system.

[0127] In an embodiment the method of selecting antagonists for P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite is performed employing cell lines, organoids and tissue originating from arthropod parasites or originating from any organism within the Protozoa or from any invertebrate or vertebrate organism that expresses native P1, P2X.sub.2, P2X.sub.3 and P2X.sub.2/3 PRs or expresses any combination of said PRs. Alternatively, transformed cell lines, organoids and tissue from said organisms expressing P1, P2X.sub.2, P2X.sub.3 and P2X.sub.2/3 PRs may be used to select antagonists for P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite. The ability of cells expressing said native PRs or of said transformed cells expressing PRs to grow in a feeding medium in presence of a test compound can be used to select antagonists for P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite. Notably, sensory cells of dorsal root ganglia and nodose ganglia of vertebrate animals express P1 PRs that respond to adenosine and P2X.sub.2, P2X.sub.3 and P2X.sub.2/3 PRs that respond to ATP and accordingly provide an appropriate medium in which to select antagonists for P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite.

[0128] In an embodiment the present invention provides for a method for selecting an antagonist for a P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite by making, for example, recording ex vivo from a cell, organ or tissue of a transgenic arthropod parasite containing an altered allele for a gene that encodes and expresses for example a fluorescent protein in addition to at least one functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR. Examples of an arthropod-parasite organ could be the labellum of a mosquito or the hypostome of a tick. Presence of the encoded fluorescent protein in the arthropod parasite permits recording of Ca.sup.++ transients in the cell, organ or tissue indicative of activation of said PRs in presence of an agonist such as adenosine or ATP. Selection of a PR antagonist can be achieved when a test compound is presented in combination with an agonist such as adenosine or ATP and brought into contact in solution with the cell, organ or tissue of the transgenic arthropod parasite. A PR antagonist causes, for example, a decrease in cellular activity as measured by Ca.sup.++ transients recorded from said cell, organ or tissue compared to the Ca.sup.++ transient activity recorded from the same cell, organ or tissue when adenosine or ATP is presented alone as positive control. In this case, modulation by the PR antagonist comprises inhibition of functional PR activity in the transgenic arthropod parasite's cell, organ or tissue.

[0129] In an embodiment the selection method is performed by selecting antagonists for P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite by using for example electrophysiological recordings of action potentials, recording cell membrane potentials, measurement of cell membrane currents, use of ion flux assays, use of fluorescence assays for voltage sensitive dyes and fluorescent proteins, patch clamp electrophysiological recording techniques or cell imaging techniques that measure cellular activity of any cell, tissue or organ of any organism within the Protozoa or of any invertebrate, including arthropod parasites, or of any vertebrate organism expressing a P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR or expressing any combination of said PRs that responds to an agonist of at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of the arthropod parasite.

Methods of Selecting Feeding Inhibitors

[0130] In an embodiment the present invention provides for a method of selecting antagonists of P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs that inhibit feeding by an arthropod parasite through inhibition of at least one of its P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs.

[0131] In an embodiment the present invention provides for selecting a compound that inhibits feeding by an arthropod parasite possessing at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR. This is achieved by measuring feeding by the arthropod parasite on a feeding medium to which a test compound plus a feeding stimulant such as adenosine or ATP is added in solution. A compound that inhibits feeding causes a reduction in feeding by the arthropod parasite compared to its feeding, for example, on the positive control where only the feeding stimulant is added to the feeding medium. In this case, modulation comprises inhibition by the antagonist of the PR that interferes with the ability of the arthropod parasite to sense the feeding stimulant in the feeding medium.

[0132] In an embodiment, a compound that inhibits feeding can be selected using an arthropod parasite expressing at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR. In a preferred embodiment the test compound is presented in solution to the arthropod parasite at increasing doses over the 10 nM to 10 M range as shown herein on FIG. 8 B of Examples for TMP and suramin when added to a solution containing a fixed dose of ATP. Alternatively, a 1 pM to 1 mM dose range of a test compound can be chosen for testing. Inhibition of feeding induced by a test compound is measured within a period of a few minutes to over an hour, depending on the arthropod parasite in question, and is expressed, for example, relative to the number of the arthropod parasites feeding on the positive control where only the feeding stimulant is added to the feeding medium. This permits calculation of a dose-response function and ID.sub.50 value for a feeding inhibitor which can be in the 200 pM to 800 nM range as shown herein on FIGS. 8 and 9 and on Table 7 of Examples for the P2 PR antagonists suramin and PPADS, for the P2X.sub.2 PR antagonist RB-2, for the P2X.sub.3 PR antagonists TMP and spinorphin, and for the P2X.sub.3/P2X.sub.2/3 PR antagonist A-317491. Nevertheless, the ID.sub.50 value for a compound that inhibits feeding by an arthropod parasite is not limited to a particular molar range.

[0133] An embodiment of the present invention comprises selecting a compound that inhibits feeding by an arthropod parasite possessing at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR where a test compound is presented on or anywhere around a substrate such as a membrane or surface of a reservoir held at a temperature at or near 37 C. on which the arthropod parasite has the ability to feed on a feeding medium. This is achieved by measuring feeding by the arthropod parasite on the feeding medium to which a feeding stimulant such as adenosine or ATP is added in solution and where the test compound is present on the substrate over the feeding medium. Inhibition of feeding induced by a test compound is measured within a period of a few minutes to over an hour, depending on the arthropod parasite in question. A compound that inhibits feeding causes a reduction in feeding by the arthropod parasite compared to its feeding on the positive control where, for example, an untreated membrane is placed over the feeding medium incorporating the feeding stimulant. This is shown herein on Tables 10 and 11 of Examples where the P2X3 antagonist TMP applied onto the feeding membrane inhibits feeding by ticks and biting flies. In this case, modulation comprises inhibition by the antagonist of the PR that interferes with the arthropod parasite's ability of the feed through the membrane. Feeding rates of the arthropod in the presence and in the absence of at least one test compound are compared. The comparison allows for selection of compounds which are suitable feeding inhibitors.

[0134] An embodiment of the present invention comprises selecting a compound that inhibits feeding by an arthropod parasite possessing at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR where a test compound is presented on or anywhere around a substrate or warm body held at a temperature at or near 37 C. Once an arthropod parasite, such as a mosquito, has contacted said substrate or warm body the arthropod parasite starts probing the surface with its mouthparts leading to biting attempts. Such probing and biting activity on said substrate or warm body is typical of mosquito behaviour once it has landed on a host animal. This biting behaviour induced on a warm body has been exploited to test repellents for arthropod parasites as described in Krber et al., 2010, An in vitro assay for testing mosquito repellents employing a warm body and carbon dioxide as a behavioral activator, Journal of the American Mosquito Control Association 26, 381-386; doi.org/10.2987/10-6044.1. Selection of a compound that inhibits feeding is made by measuring the biting rate by the arthropod parasite on the warm body onto which the test compound is applied. Inhibition of the biting rate induced by the test compound is measured within a period of a few minutes to over an hour, depending on the arthropod parasite in question. A compound that inhibits feeding causes a reduction in the biting rate by the arthropod parasite compared to its biting rate on the positive control where, for example, an untreated warm body is presented. In this case, modulation comprises inhibition by the antagonist of the PR that interferes with the arthropod parasite's ability of bite. Biting rates of the arthropod in the presence and in the absence of at least one test compound are compared. The comparison allows for selection of compounds which are suitable feeding inhibitors.

[0135] The present invention comprises a method for selecting an antagonist for a P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite by recording, for example, a feeding electrogram from said parasite expressing at last one of said PRs. The feeding electrogram technique is used to measure the feeding rate by the arthropod parasite on a feeding medium to which a test compound plus a feeding stimulant such as adenosine or ATP is added in solution. The feeding electrogram permits recording of changes in potential, measured in mV, across the mouthparts and action potential frequencies generated by cells of the arthropod parasite's mouthparts. A regular feeding rate is sustained, for example, by sucking bouts induced by activation of pharyngeal muscles, and salivation induced by closing of the pre-pharyngeal valve accompanied by lowering of the salivarium floor. Use of the feeding electrogram to measure the feeding rate of an arthropod parasite is described in Lsel et al., 1992, Feeding electrogram studies on the African cattle brown ear tick Rhipicephalus appendiculatus: evidence for an antifeeding effect of tick resistant serum, Physiological Entomology 17, 342-350; doi.org/10.1111/i.1365-3032.1992.tb01032x. The arthropod parasite can feed on said medium with the test compound plus the feeding stimulant added either across a membrane, or directly on said medium with the test compound and the feeding stimulant added that is held, for example, in a micro reservoir such as a glass capillary. Inhibition of feeding induced by the test compound is measured within a period of a few minutes to over an hour, depending on the arthropod parasite in question. Both the membrane-feeding and glass-capillary feeding methods are described herein in Materials and Methods. Feeding rates measured from the arthropod parasite in the presence and in the absence of at least one test compound are compared to detect, for example, a decrease in the frequency of sucking events caused by the test compound. The comparison allows for selection of compounds which are suitable feeding inhibitors. Sucking and salivation events that indicate the feeding rate of an arthropod parasite can also be measured by macro-video photography as described in Lsel et al., 1992, ibid.

[0136] An embodiment of the present invention comprises selecting a compound that inhibits feeding by an arthropod parasite through use of a non-parasitic transgenic animal containing cells with an altered allele for a gene that naturally encode and express at least one functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite. This is achieved when feeding by the transgenic animal is lower on a feeding medium to which a feeding stimulant such as adenosine or ATP plus a test compound is added in solution than feeding by the same transgenic animal on the feeding medium that serves as positive control to which only the feeding stimulant is added. In this case, modulation comprises inhibition by the feeding inhibitor of the functional PR that interferes with the ability of the transgenic animal to sense the feeding stimulant in the feeding medium.

[0137] In an embodiment the present invention provides for selecting a compound that inhibits feeding by an arthropod parasite using of a non-parasitic transgenic animal possessing an altered allele for a gene that naturally encodes and expresses at least one functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of a non-parasitic organism. It is understood that the functional P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of the non-parasitic organism responds to an agonist of at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of the arthropod parasite. Selection of an antagonist is achieved when feeding by the transgenic animal is lower on a feeding medium to which a feeding stimulant such as adenosine or ATP plus a test compound is added in solution than feeding by the same transgenic animal on the feeding medium that serves as positive control to which only the feeding stimulant is added. In this case, modulation comprises inhibition by the feeding inhibitor of the functional PR that interferes with the ability of the transgenic animal to sense the feeding stimulant in the feeding medium.

Embodiments for Feeding Inhibitors

[0138] In an embodiment the present invention comprises an antagonist of at least one P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR of an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, as a feeding inhibitor for said organism.

[0139] In an embodiment the present invention comprises an antagonist of any combination of P1, P2X.sub.2, P2X.sub.3 and P2X.sub.2/3 PRs of an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, as a feeding inhibitor for said organism.

[0140] In an embodiment the present invention comprises an antagonist of at least one P1 PR of an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, as a feeding inhibitor for said organism.

[0141] In an embodiment the present invention comprises an antagonist of at least one P1 A.sub.1 PR of an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, as a feeding inhibitor for said organism.

[0142] In an embodiment the present invention comprises an antagonist of at least one P2X.sub.2 PR of an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, as a feeding inhibitor for said organism.

[0143] In an embodiment the present invention comprises an antagonist of at least one P2X.sub.3 PR of an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, as a feeding inhibitor for said organism.

[0144] In an embodiment the present invention comprises an antagonist of at least one P2X.sub.2/3 PR of an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, as a feeding inhibitor for said organism.

[0145] In an embodiment the present invention comprises an antagonist of any combination of P1, P2X.sub.2, P2X.sub.3 and P2X.sub.2/3 PRs of an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, as a feeding inhibitor for said organism.

[0146] For the purposes of this invention it is understood that inhibition of P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs by an antagonist can be achieved in different ways, for example, by binding to the ion channel pore thus blocking opening of the ion channel by the agonist, by blocking the ion channel pore in the open state, by acting as a competitive agonist for binding by the agonist with the PR, or by binding to a non-agonist allosteric site on the PR to prevent its activation.

[0147] In an embodiment this invention comprises suramin (antrypol, a polysulfonated naphthylamine), PPADS (pyridoxal-phosphate-6-azophenyl-2,4-disulfonate), RB-2 (an anthraquinone-sulfonic acid), spinorphin (a heptapeptide), 2,3,5,6-tetramethylpyrazine (TMP) or A-317491 (a tricarboxylate) as an antagonist for P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, that respond to ATP or AMP-CCP as an agonist, as disclosed herein on FIGS. 4 B and D, 5, 6 and 7 and on Table 4 of Examples. The invention also comprises any combination of said PR antagonists and pharmacologically admissible salts of said PR antagonists for an arthropod parasite. An antagonist for P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite may also comprise compounds selected from P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PR antagonists listed herein.

[0148] In an embodiment this invention comprises suramin, PPADS, RB-2, spinorphin, TMP or A-317491 as a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, where said organism responds to ATP or AMP-CPP as a feeding stimulant, as disclosed herein on FIGS. 8 B, 9 and 10, and on Tables 7, 8, 9, and 10 of Examples. The invention also comprises any combination of said feeding inhibitors and pharmacologically admissible salts of said feeding inhibitors for an arthropod parasite. A feeding inhibitor for an arthropod parasite may also comprise compounds selected from P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PR antagonists listed herein.

[0149] In an embodiment this invention comprises 1,3-dimethyxanthine (theophylline) or 8-cyclopentyl-1,3-dimethylxanthine (8-cyclopentyltheophylline or CPT) as an antagonist of a P1 PR of an arthropod parasite that respond to adenosine or AMP as an agonist, as herein disclosed on FIGS. 4 A and C and in the corresponding text in Examples. The invention also comprises a combination of said antagonists and pharmacologically admissible salts of said antagonists. An antagonist for P1 PRs of an arthropod parasite may also comprise compounds selected from P1 PR antagonists listed herein.

[0150] In an embodiment this invention comprises 8-cyclopentyl-1,3-dimethylxanthine as a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, that responds to adenosine as a feeding stimulant, as herein disclosed on Table 6 of Examples. The invention also comprises a pharmacologically admissible salt of 8-cyclopentyl-1,3-dimethylxanthine as a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect. A feeding inhibitor for an arthropod parasite may also comprise compounds selected from P1 PR antagonists listed herein.

[0151] In an embodiment this invention comprises the P1A.sub.1 PR antagonist 8-cyclopentyl-1,3-dimethylxanthine combined with the P2X.sub.3 PR antagonist 2,3,5,6-tetramethylpyrazine as a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, that responds to adenosine with ATP as a feeding stimulant, as herein disclosed on FIG. 10 and Table 6 of Examples. A feeding inhibitor for an arthropod parasite may also comprise combinations of compounds selected from P1 PR antagonists listed herein.

[0152] In an embodiment this invention comprises combinations of PR antagonists. A combination of said PR antagonists may comprise suramin with 8-cyclopentyl-1,3-dimethylxanthine. A combination of said PR antagonists may comprise suramin with 1,3-dimethylxanthine. A combination of said PR antagonists may comprise suramin with 2,3,5,6-tetramethylpyrazine. A combination of said PR antagonists may comprise suramin with a tricarboxylate such as A-317491. A combination of said PR antagonists may comprise suramin with spinorphin. A combination of said PR antagonists may comprise PPADS with 8-cyclopentyl-1,3-dimethylxanthine. A combination of said PR antagonists may comprise PPADS with 1,3-dimethylxanthine. A combination of said PR antagonists may comprise RB-2 with 8-cyclopentyl-1,3-dimethylxanthine. A combination of said PR antagonists may comprise RB-2 with 1,3-dimethylxanthine. A combination of said PR antagonists may comprise 2,3,5,6-tetramethylpyrazine with 1,3-dimethylxanthine. A combination of said PR antagonists may comprise A317491 with 8-cyclopentyl-1,3-dimethylxanthine. A combination of said PR antagonists may comprise A317491 with 1,3-dimethylxanthine. A combination of said PR antagonists may comprise spinorphin with 8-cyclopentyl-1,3-dimethylxanthine. A combination of said PR antagonists may comprise spinorphin with 1,3-dimethylxanthine. A combination of said PR antagonists may comprise RB-2 with 2,3,5,6-tetramethylpyrazine. A combination of said PR antagonists may comprise s RB-2 with A317491. A combination of said PR antagonists may comprise RB-2 with spinorphin. A combination of said PR antagonists may comprise PPADS with 2,3,5,6-tetramethylpyrazine. A combination of said PR antagonists may comprise PPADS with A317491. A combination of said PR antagonists may comprise PPADS with spinorphin. A combination of said PR antagonists may comprise s 2,3,5,6-tetramethylpyrazine with spinorphin. A combination of said PR antagonists may comprise suramin with 2,3,5,6-tetramethylpyrazine and A-317491. A combination of said PR antagonists may comprise suramin with 8-cyclopentyl-1,3-dimethylxanthine and 2,3,5,6-tetramethylpyrazine. A combination of said PR antagonists may comprise suramin with 8-cyclopentyl-1,3-dimethylxanthine and A317491. A combination of said PR antagonists may comprise suramin with 1,3-dimethylxanthine and A317491. A combination of said PR antagonists may comprise suramin with 1,3-dimethylxanthine and 2,3,5,6-tetramethylpyrazine. A combination of said PR antagonists may comprise RB-2 with 2,3,5,6-tetramethylpyrazine and A-317491. A combination of said PR antagonists may comprise RB-2 with 8-cyclopentyl-1,3-dimethylxanthine and 2,3,5,6-tetramethylpyrazine. A combination of said PR antagonists may comprise RB-2 with 1,3-dimethylxanthine and 2,3,5,6-tetramethylpyrazine. A combination of said PR antagonists may comprise RB2 with 8-cyclopentyl-1,3-dimethylxanthine and A317491. A combination of said PR antagonists may comprise 2,3,5,6-tetramethylpyrazine with A-317491.

[0153] In an embodiment of the present invention a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, comprises an antagonist of P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PRs or an antagonist for any combination of said PRs belonging to any invertebrate or vertebrate organism.

[0154] In an embodiment of the present invention a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, comprises antagonists of P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PRs of said arthropod parasites as disclosed herein, combined with a compound or compounds selected from P1 PR antagonists listed herein, such as DPCPX, and/or combined with a compound or compounds selected from P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR antagonists listed herein, such as AF-353.

[0155] In an embodiment of this invention a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, comprises one or more compounds selected from a group consisting of: pyridoxalphosphate-6-azophenyl-2,5-disulfonic acid (iso-PPADS); 2-[[2-hydroxy-5-(hydroxymethyl)-2-oxo-4H-[1,3,2]dioxaphosphinino[4,5-c]pyridin-8-yl]diazenyl]benzene-1,4-disulfonic acid (MRS 2220); MRS 2160; [3-[[4-formyl-5-hydroxy-6-methyl-3-(phosphonooxymethyl)pyridin-2-yl]diazenyl]-5-[[hydroxy(oxido)phosphoryl]methyl]phenyl]methyl-hydroxyphosphinate (MRS 2257); P.sup.1P.sup.5-di-(inosine-5)pentaphosphate (Ip.sub.5I); aurintricarboxylic acid; 2,3-O-benzyldiene-ATP; 3-deoxy-3-(3,5-dimethoxybenzamido)-ATP (DT-0111); 4-[(2-amino-3,5-dibromophenyl)methylamino]cyclohexan-1-ol (ambroxol); 5-O-[2-[benzyl(methyl)amino]ethyl]3-O-methyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (nicardipine).

[0156] In an embodiment of this invention a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, comprises one or more compounds selected from a group consisting of: polysulfonated aromatic compound; sulfonamide tethered (hetero)aryl ethylidene compound; diaminopyrimidine compound; biaryl benzamide compound; teatrahydropyrido-pymiridine compound; pyrrolopyrimidin-7-one; pyrrolinone; minodronic acid; benzamide compound containing piperazine, tetrazole, thiazole, imidazole, triazole or pyrazole. The invention also comprises a pharmacologically admissible salt of said compounds.

[0157] In an embodiment of this invention a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, comprises one or more compounds selected from a group consisting of: imidazopyridine; piperidino-aminopyrimidyl; benzamide; pyrrolidino-aminopyrimidyl; benzoimidazole; phenoxy-diaminopyrimidine; benzyl-diaminopyrimidine; carboxamide; benzoxazole; pyrrol; oxo-triazine; pyrimidinone. The invention also comprises a pharmacologically admissible salt of said compounds.

[0158] In an embodiment of this invention a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, comprises one or more compounds selected from a group consisting of: diaminopyrimidine; arylamide; pyrazole; oxazole; pyridine; pyrimidine; pyrazolo-pyrimidine; imidazo-pyridine. The invention also comprises a pharmacologically admissible salt of said compounds.

[0159] In an embodiment of this invention a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, comprises one or more compounds selected from a group consisting of: 2(3)-O-(2,4,6-trinitrophenyl)-adenosine 5-triphosphate (TNP-ATP); 2,3-O-(2,4,6-trinitrophenyl)-ADP (TNP-ADP); 2,3-O-(2,4,6-trinitrophenyl)-AMP (TNP-AMP); 2,3-O-(2,4,6-trinitrophenyl)-GTP (TNP-GTP); 5-[[4,5-dirnethoxy-2-(1-methylethyl)phenyl]methyl]-2,4-pyrimidinediamine (RO-3); 2,3-O-cyclohexylideneATP; 2,3-O-cyclohexylidene-,-methylene ATP; 5-(5-iodo-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (AF-353 or RO-4); tetrasodium 4-amino-6-[[4-[4-[(8-amino-1-hydroxy-5,7-disulfonatonaphthalen-2-yl)diazenyl]-3-methylphenyl]-2-methylphenyl]diazenyl]-5-hydroxynaphthalene-1,3-disulfonate (Evans Blue); sodium 1-amino-9,10-dihydro-9,10-dioxo-4-(phenylamino)-2-anthracenesulfonate (Acid Blue 25); 5-(2,4-diamminopyrimidin-5-yloxy)-4-isopropyl-2-methoxybenzenesulfonamide (AF-219, MK-7264 or gefapixant); 9-(5-iodo-2-isopropyl-4-methoxybenzyl)-N.sup.6-methyl-9H-purine-2,6-diamine; 2-(R)-(4-fluoro-2-methyl-phenyl)-4-(5)-((8aS)-6-oxo-hexahydro-pyrrolo[1,2-a]-pyrazin-2-yl)-piperidine-1-carboxylic acid [1-(R)-(3,5-bis-trifluoromethyl-phenyl)-ethyl]-methylamide (orvepitant); 5-(4-methoxy-5-methylsulfonyl-2-propan-2-ylphenoxy)pyrimidine-2,4-diamine (AF-130); 5-(5-isobutyltetrazol-1-yl)-4-methylbiphenyl-3-carboxylic acid ((S)-2-hydroxy-1-methylethyl)amide; N-[1(R)-(5-fluoropyridin2-yl)ethyl]-3-(5-methylpyridin-2-yl)-5-[5(S)-(2-pyridyl)-4,5-dihydroisoxazol-3-yl]benzamide (MK-3901); 3-(5-methylthiazol-2-yl)-5-(((R)-tetrahydrofuran-3-yl)oxy)-N((R)-1-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)benzamide (eliapixant or BAY-1817080); methyl (S)-3-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-7-methylimidazo [1,2-a]pyridin-3-yl)methyl)piperidine-1-carboxylate (BLU-5937); (2S)-3-[(4E)-3-[(4-chlorophenyl)methyl]-2,6-dioxo-4-({4-[(pyridin-2-yl)oxy]phenyl}imino)-1,3,5-triazinan-1-yl]-2-methylpropanoic acid (sivopixant or S-600918); 5-cyclohexyl-3-hydroxy-1-(4-(isoxazol-4-yl)phenyl)-4-(4-methoxybenzoyl)-1,5-dihydro-2H-pyrrol-2-one; biphenyl and phenyl-pyridine amides; oxazolone and pyrrolidinone-substituted arylamides; amino quinazoline; fused heterocyclics; triazole-substituted arylamide; oxalamide; indole arylamide; indazole arylamide; benzimidazole arylamide. The invention also comprises a pharmacologically admissible salt of said compounds.

[0160] In an embodiment of this invention a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect, comprises one or more compounds selected from a group consisting of: 8-[[3-[[3-[(4,6,8-trisulfonaphthalen-1-yl)carbamoyl]phenyl]carbamoylamino]benzoyl]amino]naphthalene-1,3,5-trisulfonic acid (NF023); tetrasodium 4-[[3-[[3,5-bis[(4-sulfonatophenyl)carbamoyl]phenyl]carbamoylamino]-5-[(4-sulfonatophenyl)carbamoyl]benzoyl]amino]benzenesulfonate (NF110); hexasodium 8--[[4-[[4-[[4-[[4-[(4,6,8-trisulfonatonaphthalen-1-yl)carbamoyl]phenyl]carbamoyl]phenyl]carbamoylamino]benzoyl]amino]benzoyl]amin o]naphthalene-1,3,5-trisulfonate (NF279); 5-methoxy-3-[[3-[[3-[[3-[[5-[(8-methoxy-3,6-disulfonaphthalen-2-yl)carbamoyl]-2-methylphenyl]carbamoyl]phenyl]carbamoylamino]benzoyl]amino]-4-methylbenzoyl]amino]naphthalene-2,7-disulfonic acid (NF770); 2-[[4-Methyl-5-[[3-[[3-[[2-methyl-5-[(1,3,7-trisulfonaphthalen-2-yl)carbamoyl]phenyl]carbamoyl]phenyl]carbamoylamino]cyclohexa-2,5-diene-1-carbonyl]amino]cyclohexa-2,4-diene-1-carbonyl]amino]naphthalene-1,3,7-trisulfonic acid (NF778); 1-amino-4-[3-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]anilino]-9,10-dioxoanthracene-2-sulfonic acid (PSB-10211); 1-amino-4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)-4-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (PSB-1011); 2-[[4-amino-5-(5-iodo-4-methoxy-2-propan-2-ylphenoxy)pyrimidin-2-yl]amino]propane-1,3-diol (AF-906 or RO-51); N-[(2R)-1-(4-acetylpiperazin-1-yl)propan-2-yl]-1-methyl-3-phenylthieno[2,3-c]pyrazole-5-carboxamide (RO-85); 5-[5-Methyl-2-(1-methylethyl)phenoxy]-2,4-pyrimidinediamine (TC-P 262); 8-cyclopentyl-1,3-dipropylxanthine (DPCPX): 1,3-dipropyl-8-[2-(5,6-epoxy)norbornyl]xanthine (ENX); (S-1,3-dipropyl-8[2-(5,6-epoxynorbornyl)]xanthine (CVT124); (5R)-5-[[9-methyl-8-[methyl(propan-2-yl)amino]purin-6-yl]amino]bicyclo[2.2.1]heptan-2-ol (WRC-0571); [2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol (mefloquine); trans-4-[(2-Phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexanol (SLV 320); 8-(Hexahydro-2,5-methanopentalen-3a(1H)-yl)-3,7-dihydro-1,3-dipropyl-1H-purine-2,6-dione (KW 3902); N-(2-Aminoethyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-acetamide dihydrochloride (XAC); 3-[[5-(2,3-dichlorophenyl)tetrazol-1-yl]methyl]pyridine (A-438079). The invention also comprises a pharmacologically admissible salt of said compounds.

[0161] In an embodiment this invention comprises a peptide, a polypeptide or a protein, and a pharmacologically admissible salts thereof, as a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect. Purotoxin-1, a 35 amino acid peptide of the wolf spider, antagonizes rat and human P2X.sub.3 PRs at nanomolar doses. Spinorphin is an endogenous antinociceptive heptapeptide and a known antagonist of human P2X.sub.3 PRs.

[0162] As herein disclosed in FIGS. 6 and 7 and in Table 4 of Examples, spinorphin serves as an antagonist of a purine-sensitive PR of the malaria mosquito at sub-nanomolar doses, and spinorphin inhibits feeding by the malaria mosquito at 100 nM as disclosed herein on Table 7 of Examples. A feeding inhibitor for an arthropod parasite may also comprise compounds structurally related to spinorphin, such as heptapeptides AVVYPWT, LAVYPWT, LVAYPWT, LVVAPWT and the cyclic peptide LWVYPWT that serve as selective P2X.sub.3 PR antagonists.

[0163] In an embodiment this invention comprises a natural product and pharmacologically admissible salts thereof as a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect. The isoflavone puerarin and polyphenols emodin and resveratrol are reported as P2X.sub.3 PR antagonists. Theophylline, a constituent of tea, coffee and chocolate, is a non-selective P1 PR antagonist and as disclosed herein in Examples, theophylline antagonizes the response of the malaria mosquito P1 PR to adenosine. 2,3,5,6-Tetramethylpyrazine (TMP), synonym to ligustrazine, from Ligusticum wallichii, employed as an analgesic in Chinese medicine, is a P2X.sub.3 PR antagonist. As disclosed herein on FIGS. 5, 6 and 7 and on Table 4 of Examples, TMP antagonizes a PR of the malaria mosquito at an ID.sub.50 of 12 nM. Furthermore, and as disclosed herein on FIGS. 8 and 9 and on Table 7 of Examples, TMP inhibits feeding by the malaria mosquito at an ID.sub.50 of 15 nM. In addition, TMP inhibits feeding by the Asian mosquito vector of malaria and by the horn fly as disclosed herein on Tables 8 and 10 of Examples. As disclosed herein on Tables 9 and 10 of Examples, TMP inhibits feeding when applied onto a membrane through which a biting fly and tick species feeds on a feeding medium with ATP added as feeding stimulant.

[0164] In an embodiment this invention comprises an antibody as a feeding inhibitor for an arthropod parasite, for example a blood-feeding arthropod parasite, such as a biting insect. A means of using vascular endothelial growth factor (VEGF) antibody to inhibit the expression of the P2X.sub.2 and P2X.sub.3 PRs has been described, and application of monoclonal antibodies produced against human P2X.sub.3 PRs induce significant inhibition of human P2X.sub.3 and P2X.sub.2/3 PRs.

Embodiments for Metal Modulators of P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs

[0165] In an embodiment the product used for the protection against arthropod parasites comprises metal ions as modulators of P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs of an arthropod parasite further comprising PR antagonist compounds. Metals such as copper, magnesium, iron, nickel and zinc modulate P2X.sub.2 and P2X.sub.3 PR function. As disclosed herein in Table 5 of Examples, Zn.sup.++ significantly increases the response to ATP by the malaria mosquito purine-sensitive cell at 100 pM but not at double that dose, and 200 pM Mg.sup.++ inhibits the response of the malaria mosquito purine-sensitive cell to ATP.

[0166] In an embodiment the present invention comprises a product for inhibiting feeding by an arthropod parasite using at least one metal ion modulator of its P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs

[0167] In an embodiment the present invention comprises a product for inhibiting feeding by an arthropod parasite using at least one metal ion modulator of its P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs further comprising PR antagonist compounds.

Embodiments for Inhibiting Feeding by an Arthropod Parasite, Preferably a Blood-Feeding Arthropod Parasite, Using an Antagonist for at Least One of its PRs as a Feeding Inhibitor

[0168] In an embodiment the present invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, comprising an antagonist of at least one of its PRs.

[0169] In an embodiment the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, comprising an antagonist for at least one of its P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PRs or for any combination of said PRs where said antagonist is selected by means of assay methods described in Materials and Methods and in Examples herein, or selected using other suitable assay methods described herein. Compounds structurally related to the selected antagonist, isomers thereof, esters thereof, prodrugs thereof and pharmaceutically admissible salts thereof are herein included.

[0170] In an embodiment, the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, comprising an antagonist for at least one of its P1 PRs.

[0171] In an embodiment, the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, comprising an antagonist for at least one its P1A.sub.1 PRs.

[0172] In an embodiment, the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, comprising an antagonist for at least one of its P2X.sub.2 PRs.

[0173] In an embodiment, the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, comprising an antagonist for at least one of its P2X.sub.3 PRs.

[0174] In an embodiment, the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, comprising an antagonist for at least one its P2X.sub.2/3 PRs.

[0175] In an embodiment, the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, comprising an antagonist for any combination of its P1, P2X.sub.2, P2X.sub.3 and P2X.sub.2/3 PRs.

[0176] In an embodiment the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, through inhibition of at least one of its P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PRs by at least one P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PR antagonist.

[0177] In an embodiment the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, comprising an antagonist for at least one of its P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs accompanied by an antagonist for another PR of the arthropod parasite. For example, feeding inhibition can be achieved employing an antagonist for at least one P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PR combined with an antagonist for at least one P1 PR of the arthropod parasite as disclosed herein on FIG. 10 and Table 6 of Examples where the P1 PR antagonist 8-cyclopentyl-1,3-dimethylxanthine combined with the P2X.sub.3 PR antagonist 2,3,5,6-tetramethylpyrazine serves to inhibit feeding by the malaria mosquito.

[0178] In an embodiment the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, using at least one metal modulator of P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PRs further comprising at least one P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PR antagonist.

[0179] In an embodiment the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, comprising an antagonist for at least one of its P1, P2X.sub.2, P2X.sub.3, P2X.sub.2/3 PRs and further comprising at least one metal modulator of P2X.sub.2, P2X.sub.3 or PX.sub.2/3 PRs.

[0180] In an embodiment the invention comprises a product for inhibiting feeding by an arthropod parasite, preferably an arthropod parasite, comprising an antagonist for at least one of its P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PRs where said antagonist can be modified through derivatization, conjugation, or esterification to facilitate, among others, stability, miscibility, resistance to light exposure and for target delivery to the arthropod parasite.

[0181] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, using a feeding inhibitor comprising an antagonist for at least one of its P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PRs where said inhibitor is applied onto a host animal for the prevention and/or treatment of an infestation by the arthropod parasite, preferably a blood-feeding arthropod parasite. This control method aims to inhibit the arthropod parasite, preferably a blood-feeding arthropod parasite, from feeding on the host animal. For example, in the case of a flying biting insect such as a mosquito or a biting fly, this can lead to the organism flying away in a starved state from a host animal.

[0182] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite using a product comprising a PR antagonist where said product affects an endosymbiont of the arthropod parasite, thereby adversely affecting development, reproduction and survival of the arthropod parasite.

[0183] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite using a PR antagonist where said antagonist is applied onto a host animal for the prevention and/or treatment of an infestation by the arthropod parasite. Said control method aims to inhibit host recognition, biting, entering the host animal's body, establishing a feeding site, feeding on host animal blood or on body fluids or on body tissues by the arthropod parasite, and/or inhibit the arthropod parasite from absorbing nutrients from the host animal through its integument or plasma membrane so as to prevent the arthropod parasite's survival, growth and reproduction. Examination of an adequate number of host animals for presence of the arthropod parasite made at regular intervals following application of the antagonist, such as is made by following a test protocol, allows assessment of the PR antagonist as a feeding inhibitor to control the arthropod parasite infestation, and permits establishment of an effective dose of the feeding inhibitor by a person skilled in the art.

[0184] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite, preferably a blood-feeding arthropod parasite, using a PR antagonist where said antagonist is applied at an effective dose for the prevention and/or treatment of an infestation by the arthropod parasite. An effective dose is the amount of a feeding inhibitor that achieves the desired effect of inhibiting feeding by the arthropod parasite, preferably a blood-feeding arthropod parasite.

[0185] Establishing an effective dose of a PR antagonist that achieves the desired effect of inhibiting feeding by an arthropod parasite on a host animal considers several variables comprising, in case of arthropod parasites for example, the life stage and species of the arthropod parasite in question, the population density of the arthropod parasite, the type of compound, formulation of the compound, the method of application of said formulation and the required duration of the effect. Accordingly, the effective dose of a feeding inhibitor in a formulation or in any use form of the feeding inhibitor may vary over a wide range from 10.sup.9 wt % to 50 wt %, preferably from 10.sup.7 wt % to 5 wt % of the feeding inhibitor as a proportion of the formulation or of any use form of the feeding inhibitor. Details of suitable formulation methods for active ingredients used in the control of parasites are known in the art. A person skilled in the art of controlling an arthropod parasite infestation can therefore determine the amount of a compound of this invention that is required as a feeding inhibitor for the prevention and/or treatment of the infestation.

[0186] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite on a host animal using a product comprising a PR antagonist where said product is applied topically onto a host animal's pelage or skin, and/or onto a neck collar, a saddle, a cover for a host animal such as a horse cover or fly rug, and/or onto a fly screen or any other protective screen or netting for a host animal, and/or onto a fly mask, cap or blinkers for a host animal, and/or onto a harness, bridle, or any means of restraining a host animal.

[0187] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite on a host animal using a product comprising a PR antagonist where said product is applied in the form of a spray onto a host animal's pelage or skin, and/or onto a neck collar, a saddle, a cover for a host animal, such as a horse cover or fly rug, and/or onto a fly screen or any other protective screen or netting for a host animal, and/or onto a fly mask, cap or blinkers for a host animal, and/or onto a harness, bridle, or any means of restraining a host animal.

[0188] In an embodiment the invention comprises inhibiting feeding by an arthropod ectoparasite for the purpose of preventing and/or treating an infestation by said parasite on a host animal using a product comprising a PR antagonist where said product is applied topically in the form of a solution or in the form of a spray onto a predilected biting or feeding site or onto a preferred egg deposition site of the arthropod parasite on a host animal.

[0189] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite on a host animal using a product containing PR antagonist where said product that may optionally contain a surfactant is applied, for example, in the form of a solution, emulsion, shampoo, cream, gel, lotion, ointment, by microencapsulation, as a nanoparticle formulation such as a nanoemulsion, as a sustained-release formulation, or as an ear tag preparation, or by applying any other suitable pour-on or pharmaceutical preparation, or by applying a solution or by applying a device onto the host animal's pelage or skin, and/or onto a neck collar, a saddle, a cover for a host animal, such as a horse cover or fly rug, and/or onto a fly screen or any other protective screen or netting for a host animal, and/or onto a fly mask, cap or blinkers for a host animal, and/or onto a harness, bridle, or any means of restraining a host animal.

[0190] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite using a product comprising a PR antagonist where said product is applied at an effective dose onto clothing of a person by use of a spray or by immersing a person's clothing in an appropriate solution containing said feeding inhibitor.

[0191] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite using a product comprising a PR antagonist where said product is applied at an effective dose onto a bed net by use of a spray or by immersing the bed net in an appropriate solution containing said PR antagonist.

[0192] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite using a product comprising a PR antagonist where said product is applied topically in the form of a solution onto the arthropod parasite.

[0193] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite using a product comprising a PR antagonist where said product is applied in the form of the spray onto the arthropod parasite.

[0194] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite using a product comprising a PR antagonist where said product is applied topically in the form of a solution or in the form of a spray onto any life stage of the arthropod parasite.

[0195] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite using a product comprising a PR antagonist where said product is incorporated at an appropriate dose into an attractive bait, for example a sugar bait, fed on by the arthropod ectoparasite.

[0196] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite using a product comprising a PR antagonist where said product that may optionally contain a surfactant is applied in the form of a spray or solution, or applied using a slow-release device, or applied by microencapsulation, or applied in a sustained-release long-lasting formulation onto and/or into, for example, any facility for a host animal comprising a refuge, and/or a tent, and/or a dwelling, and/or a barn, and/or a stable, and/or a perch, and/or a cage, and/or an animal rearing or housing facility or any part thereof, and/or a breeding site of an arthropod parasite, and/or a swarm occupied by an arthropod parasite, and/or a terrestrial zone, and/or an aquatic zone, and/or a biotope, and/or a natural or man-made habitat occupied by the arthropod parasite or by any life stage of said parasite. Details of suitable preparation methods for P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 antagonist application are found in Knowles D. A. 1998, Chemistry and Technology of Agrochemical Formulations, Springer Dordrecht.

[0197] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite using a product comprising a PR antagonist where said product optionally comprises an insecticide, insecticide synergist, acaricide, parasiticide, repellent or a chemosterilant, each at its effective dose, in the form of a solution or a spray, in the form of a shampoo, or from a slow-release device, or by microencapsulation, or in a sustained-release long-lasting formulation for topical application onto a host animal's pelage or skin, onto any life stage of the arthropod parasite, and/or for application onto a neck collar, a saddle, a cover for a host animal such as a horse cover or fly rug, and/or onto a fly screen or any other protective screen or netting for a host animal, and/or onto a fly mask, cap or blinkers for a host animal, and/or onto a harness or bridle, or any means of restraining a host animal.

[0198] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite using a product comprising a PR antagonist where said product optionally comprises an insecticide, insecticide synergist, acaricide, parasiticide, repellent or a chemosterilant, each at its effective dose, in the form of a solution or a spray, or from a slow-release device, or by microencapsulation, or in a sustained-release long-lasting formulation for application onto or into any facility for a host animal comprising a refuge, a tent, a dwelling, a barn, a stable, a perch, a cage, an animal rearing or housing facility or any part thereof, a breeding site of the arthropod parasite, a swarm occupied by the arthropod parasite, a terrestrial zone, an aquatic zone, a biotope, or a natural or man-made habitat occupied by the arthropod parasite or occupied by any life stage of the arthropod parasite. An effective dose of an insecticide, insecticide synergist, acaricide, parasiticide or a chemosterilant is one which permits controlling the arthropod parasite infestation.

[0199] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite for the purpose of preventing and/or treating an infestation by said parasite using a product comprising a PR antagonist where said product optionally comprises an insecticide, insecticide synergist, acaricide, repellent or parasiticide or a chemosterilant, each at its effective dose, for application onto a person's clothing and/or onto a bed net in the form of a spray or by immersion of the clothing or bed net in an appropriate solution containing the combination of said product and the insecticide, insecticide synergist, acaricide, parasiticide or chemosterilant.

Embodiments for Inhibiting Feeding by an Arthropod Parasite with a Therapeutically Effective does of a PR Antagonist

[0200] In an embodiment the invention comprises inhibiting feeding by an arthropod endoparasite using a product comprising a PR antagonist where said product is administered as a pharmaceutical formulation onto or internally to a host animal for the prevention and/or treatment of an arthropod endoparasite infestation. This control method aims to inhibit the arthropod endoparasite from recognizing the host, entering the host animal's body, establishing a feeding site, inhibit the arthropod endoparasite from feeding on host animal blood or on body fluids or on body tissues, and/or inhibit the arthropod endoparasite from absorbing or adsorbing nutrients from the host animal through its integument or plasma membrane so as to prevent the arthropod endoparasite's survival, growth and reproduction. Examination of an adequate number of host animals for presence of the arthropod endoparasite made at regular intervals following administration of the PR antagonist, such as is made by following a test protocol, allows assessment of a compound of this invention as a feeding inhibitor and permits a person skilled in the art to determine a therapeutically effective dose of the PR antagonist comprised in said product for the prevention and/or treatment of the arthropod endoparasite infestation.

[0201] In an embodiment the invention comprises inhibiting feeding by an arthropod endoparasite or an arthropod ectoparasite for the prevention and/or treatment of an infestation by said parasite using a product comprising a PR antagonist where said product is administered at a therapeutically effective dose onto or internally to a host animal. The product, that may optionally contain a surfactant, can be administered as a pharmaceutically acceptable formulation suitable for parenteral, oral, buccal, enteral, rectal, vaginal, nasal, intramuscular, intravenous, intraarterial, intrathecal, subcutaneous, and transdermal delivery, or by any suitable means for systemic delivery of said pharmaceutical formulation to the host animal. Establishing a therapeutically effective dose for the PR antagonist that achieves the desired effect of inhibiting feeding by the arthropod endoparasite or an arthropod ectoparasite considers several variables, comprising the type of compound, the method of administration of the formulated compound onto and/or internally to the host animal, the pharmacokinetics of the compound, the pharmacodynamics of the compound, the species of endoparasite being targeted, the severity of a endoparasite's burthen on the host animal, the age and health of the host animal and the duration of protection against the endoparasite that is desired.

[0202] The therapeutically effective dose of a PR antagonist that achieves the desired effect of inhibiting feeding by an arthropod endoparasite or an arthropod ectoparasite to be used in a pharmaceutically acceptable formulation, or in a sustained-release formulation or in any use form of the feeding inhibitor may vary over a wide range from 0.000000001 wt % to 50 wt %, preferably between 0.0000001 wt % to 5 wt % by weight of the PR antagonist as a proportion of the pharmaceutical formulation or of any use form of the feeding inhibitor. A person skilled in the art of controlling endoparasitic and ectoparasitic arthropods can determine the amount of a compound or compounds of this invention that is required to inhibit feeding by the endoparasite for the prevention and/or treatment of the infestation by said parasite.

[0203] In an embodiment the invention comprises inhibiting feeding by an arthropod parasite using a product comprising a PR antagonist where said product is purposely synthesized to inhibit P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PRs of both humans and animals, formulation of said compound as a pharmaceutical preparation and administration of said compound at a therapeutically effective dose for the prevention and/or treatment of an infestation by an arthropod parasite. Details of pharmaceutically suitable preparation methods for P1, P2X.sub.2, P2X.sub.3 or P2X.sub.2/3 PR antagonist administration onto or internally to a host animal are provided in Felton L. 2013, in Remington: Essentials of Pharmaceutics, Pharmaceutical Press London.

[0204] In an embodiment the invention comprises inhibiting feeding by an arthropod endoparasite or an arthropod ectoparasite for the prevention and/or treatment of an infestation by said parasite using a product comprising a PR antagonist where said product optionally comprises an insecticide, insecticide synergist, acaricide, parasiticide, endectocide or a chemosterilant, each at its therapeutically effective dose, for administration onto or internally to a host animal as a pharmaceutical formulation, or as a sustained-release formulation or in any use form suitable for parenteral, oral, buccal, enteral, rectal, vaginal, nasal, intramuscular, intravenous, intraarterial, intrathecal, subcutaneous, and transdermal delivery, or by any suitable means for systemic delivery said pharmaceutical formulation to the host animal. A therapeutically effective dose of an insecticide, insecticide synergist, acaricide, parasiticide or a chemosterilant is one which permits controlling the arthropod endoparasite.

Materials and Methods

Mosquitoes, Ticks and Biting Flies:

[0205] Anopheles gambiae Giles (Diptera, Culicidae) 16cSS-strain colony derived from wild-caught adults in Lagos, Nigeria in 1974. The Anopheles stephensi mysorensis Liston (Diptera, Culicidae) colony originated from the Swiss Tropical Institute, Basel. Colonies were maintained under standardized conditions. A 10% sucrose solution and water were supplied ad libitum on cotton for adults. Females were deprived of sucrose the night before electrophysiological recordings.

[0206] N3 or N4 nymphs of the eyeless tampan, Ornithodorus moubata Murray (Ixodida, Argasidae), were from an in-house culture of individuals collected in Ulanga District, Tanzania and originally maintained at the Swiss Tropical Institute, Basel. Ixodes ricinus Linnaeus (Ixodida, Ixodidae) and Amblyomma hebraeum Koch (Ixodida, Ixodidae) were from colonies at Novartis, Centre de Recherche Sant Animale SA, St Aubin, Switzerland. Glossina pallidipes Austen (Diptera, Glossinidae) pupae were obtained from the International Atomic Energy Agency (IAEA) Siebersdorf Laboratories, Austria. Adult tsetse were sexed at emergence and maintained separately in cotton-netting cages (1-mm mesh; 251515 cm, tg tubular bandage, Lohmann & Rauscher, St Gallen, Switzerland) in an environmental cabinet at 24 C., 80% relative humidity (RH) under 10 h photophase:14 h scotophase with 1 hour light ramps in the first and last hour of the photophase. Pupae of the horn fly, Haematobia irritans Linnaeus (Diptera, Muscidae), obtained from a rearing at BAYER Animal Health GmbH, Monheim, Germany were maintained in cages as tsetse using 0.5-mm mesh netting and provided with water-soaked cotton on the cage roof from emergence.

Chemicals:

[0207] Compounds used for the characterisation of purine-sensitive sensory cells in A. gambiae are listed in Tables 1 and 2. Compounds were dissolved in nanopure water and serially diluted. Solutions were kept at 20 C. for less than a week and defrosted before experiments. D-(+)-sucrose (purity>99.5%) was from Sigma-Aldrich, Switzerland.

Electrophysiology:

[0208] The tip recording method was used to record action potentials (spikes) from sensory cells in sensilla on A. gambiae labellum lobes. Contrary to other labellum sensilla, the six most distally placed trichoid type 1 (T1) 8v sensilla do not house water-sensitive neurones in A. gambiae, allowing water to be used as a negative control in recordings.

[0209] Sensilla were exposed to test compounds and PR antagonists for ca. 2-4 s and spikes were counted in the first 2 s of recordings. Up to 4 test compounds were tested on a sensillum to evaluate potential agonists, each presented at 200 pM. To study responses as a function of dose for agonists ATP, adenosine, AMP, AMP-CPP and ATP+adenosine, up to 7 recordings were made at increasing doses of agonist from the same sensillum. Unless otherwise stated, recordings were made throughout from 4 to 16 sensilla from individual 4-6-day-old females per dose of agonist, antagonist and metal ion modulator. For example, a response curve established over 6 doses of an antagonist using 8 sensilla per dose involved recording from a sensillum of 48 separate mosquitoes. A given dose of a PR antagonist or metal ion modulator was only presented once to a sensillum. Two-second recordings were imported as ASCII files into DataView software for analysis of spike amplitude. Differences in spike amplitude generated by stimulation of one 8v sensillum/female with 1 mM sucrose (n=294 spikes from 6 females) 200 pM adenosine (n=207 spikes from 7 females) and 200 pM ATP (n=192 spikes from 7 females) were compared by measuring the spike amplitude in mV between the highest point of depolarisation and the lowest point of hyperpolarisation.

Feeding Assays:

[0210] Experiments with A. gambiae and A. stephensi were made in a climate chamber at 28 C. and 80% RH during the last 6 hours of scotophase. As a sugar meal can interfere with the blood meal by reducing the number of females that feed, the sucrose solution supplied in rearing cages to mosquitoes employed for feeding experiments was reduced to 5%. This did not affect mosquito survival but induced higher appetence. The 5% sucrose solution was presented to mosquitoes from emergence on four 1 cm diam. cotton dental rolls (Hartmann A G, Neuhausen, Switzerland) inserted in 10 ml upturned glass vials placed in holes at each corner of the rearing cage ceiling. Cotton rolls were turned daily in vials and renewed every two days. Mosquitoes were not fed blood and were deprived of sucrose 17 to 20 hours before experiments. Thirty min before a feeding experiment, between 10-25, 6-day-old females that responded to activation by human breath were removed with a mouth aspirator from the rearing cage wall and placed in polystyrol cylinders with the floor cut out (10 cm dm15 cm high; Semadeni, Ostermundigen, Switzerland) that served as feeding cages. Each cage was covered with mosquito netting at one end and a soft polyurethane lid at the other.

[0211] Mosquitoes were fed for 30 min using a feeding system (Hemotek, Hemotek Ltd, Blackburn, UK) consisting of 3.5 ml stainless steel reservoirs 37 mm in diameter covered with a 60-90 m thick silicone-impregnated membrane (Krber and Guerin, 2007 in Feeding assays for hard ticks, Trends in parasitology 23, 445-449; doi 10.1016/j.pt.2007.07.010). Feeding reservoirs reached a temperature of 37 C. within 10 min and were placed on the netting of feeding cages. Mosquitoes were activated using a 200 ms pulse of pure CO.sub.2 (volume 6.25 ml) from a pressurised cylinder (Carbagas, Switzerland) using a solenoid valve that caused the CO.sub.2 level in the feeding cage to rise to at least 1,500 ppm. Between 3 and 4 sets of 10 feeding cages held in a custom-built Ertacetal POM frame were tested per day, depending of the number of female mosquitoes available from the colony.

[0212] All compounds were tested in a mosquito feeding solution (FS) consisting of 25 mM dextrose, 25 mM NaCl buffered with 8.93 mM NaHCO.sub.3 adjusted to pH 7.4. Feeding responses of A. gambiae and A. stephensi to ATP as a function of dose were tested with between 7 to 10 feeding cages per dose. Each feeding experiment with an antagonist systematically included a treatment containing the FS as negative control along with a treatment containing the FS with 20 pM ATP added as positive control for A. gambiae and with 160 pM ATP added as positive control for A. stephensi. These positive and negative controls permitted to monitor any effect of experimental day on feeding responses as a function of dose for antagonists that were recorded over several days. Each dose of PR antagonist was tested in the FS with 20 pM ATP for A. gambiae and with 160 pM ATP for A. stephensi. Four to 10 cages each with 10 to 29 mosquitoes were tested per antagonist dose. The experimental procedure for testing antagonists as feeding inhibitors was set up using suramin as test compound for A. gambiae, hence 43 positive controls for this compound. The number of feeding cages used to test other compounds and their mixtures as feeding stimulants or as feeding inhibitors is listed on Table 6. The percentage of fed mosquitoes was estimated by visual inspection of abdominal distention that is indicative of engorgement.

TABLE-US-00001 TABLE 1 Substances tested for characterization of A. gambiae PRs Synonym/ CAS Substance abbreviation Number Purity 6-Aminopurine 1 Adenine 73-24-5 >98% 9--D-Ribofuranosyladenine 2 Adenosine 58-61-7 >99% Adenosine 5-monophosphate 3 AMP 61-19-8 99% Adenosine 5-diphosphate 4 ADP 58-64-0 97% Adenosine 5-O-(2-thiodiphosphate) 5 ADP--S 73536-95-5 >80% Adenosine 5-triphosphate 6 ATP 34369-07-8 99% ,-Methyleneadenosine-5-triphosphate AMP-CPP 1343364-54-4 >98% 7* 2-Deoxyadenosine 5-triphosphate 8 dATP 1927-31-7 >99% 2,3-Dideoxyadenosine-5-triphosphate 9 ddATP 178451-61-1 98% 9--D-Ribofuranosyl-guanine 10 Guanosine 118-00-3 >99% Guanosine 5-monophosphate 11 GMP 5550-12-9479 99% Guanosine 5-diphosphate 12 GDP 43139-22-6 96% Guanosine 5-triphosphate 13 GTP 36051-31-7 95% 2,4(1H,3H)-Pyrimidinedione 14 Uracil 66-22-8 >99% 1--D-Ribofuranosyluracil 15 Uridine 58-96-8 >99% Uridine-5-monophosphate 16 UMP 3387-36-8 98% Uridine-5-diphosphate 17 UDP 27821-45-0 >96% Uridine 5-triphosphate 18 UTP 116295-90-0 >80% Substances were purchased from Sigma-Aldrich, Schnelldorf, Germany and *Tocris Bioscience, Bristol, UK.

[0213] I. ricinus adults were prefed for 3-4 days and A. hebraeum adults for 10 days on a membrane over bovine blood (Krber T. and Guerin P. M., 2007, supra). Partly fed I. ricinus and A. hebraeum, freshly detached from the membrane, were tightly fixed by the scutum and alloscutum to double-sided adhesive tape (Tesafix 4964, Beiersdorf, Hamburg, Germany) on a glass slide and secured with plasticine. Test compounds were fed to these partly-fed female ticks using borosilicate capillary tubes (Clark GC120-10, 0.69 mm i.d., 1.2 mm o.d., 50 mm long; Clark Electromedical Instruments, Pangbourne, Reading, UK) with drawn out tips adapted to fit around the mouthparts of individual ticks. Feeding capillaries were filled with a solution of 100 mM NaCl, 8.9 mM NaHCO.sub.3 with 1000 M ATP added, hereafter termed tick feeding solution (TFS) that served as positive control in each feeding experiment. PR antagonists were added to the TFS and pH of the solution was adjusted to 7.2-7.8. Capillary-fed ticks were kept in a plastic box on a warm plate at 34 C. and 80% RH using wet paper. After 1 h, the amount of solution imbibed by a tick was indicated by the drop of the meniscus in the capillary and measured using Vernier callipers and converted to mg. Numbers of ticks (n) tested per treatment are provided in Table 9.

[0214] O. moubata nymphs were fed for 25 min through a membrane (50-75 m thick) as for hard ticks in glass feeding unit on TFS containing 1000 M ATP as positive control in each feeding experiment. PR antagonists were added to the TFS as for hard ticks. One compound, TMP was applied onto the membrane in aqueous solution (8 g TMP in 70 l) in small drops using a micropipette. Individual nymphs were marked on the dorsum with nail varnish in distinguishable colours and weighed before and after the experiment. Numbers of ticks (n) tested per treatment are provided in Table 9.

[0215] Flies of both species were transferred 30 min before feeding experiments to feeding cages as used for mosquitoes at 24 C., 80% RH in an environmental cabinet. Teneral tsetse were fed at 2 to 4 days old during the photophase with between 7 and 13 flies per feeding cage, covered with a black cloth. Horn flies were tested at 1 and 2 days old in the photophase with between 6 and 21 flies per cage. Both fly species were fed using small Hemotek feeding reservoirs (29 mm diameter, 1 ml) equipped with a membrane as for mosquitoes. Horn flies were fed for 15 min with feeding reservoirs placed on the cage. For tsetse, cages were closed with black mosquito netting at the bottom to allow flies to feed through the membrane placed under the cage. Feeding time (1.5 to 6 min) for tsetse was adjusted as a function of age to achieve at least 30% feeding on the positive control included in each feeding experiment. The positive control for both fly species contained 150 mM NaCl, 8.9 mM NaHCO.sub.3 with 100 M ATP added, hereafter termed fly feeding solution (FFS). The pH of the FFS was adjusted as for ticks. The effect of TMP on feeding by G. pallidipes was also tested at 16 g TMP/cm.sup.2 dissolved in 140 l hexane and applied homogeneously to the membrane surface using a micropipette. Hexane was used to treat the membrane with TMP to avoid the test compound being dispersed by fly wing-fanning. Flies of both species were activated just prior to feeding on a treatment by exhalation into the cage. Tsetse that had fed were identified with swollen abdomens and counted at the end of a feeding experiment. Since it was difficult to ascertain by eye whether a horn fly had fed on a treatment, 10 l/ml of blue food colorant (No. 4866908, Trawosa AG, St. Gallen, Switzerland) was added. This facilitated the detection of ingested liquid when the abdomen of flies, killed by freezing, was squashed onto filter paper. Only experiments in which the feeding rate by flies was >30% on the positive control were included in analyses. Numbers of flies tested per treatment (n) are provided in Table 10.

Data Analysis

[0216] Significance level for all tests was set at P=0.05. Asterisks on bar graphs represent significance levels at *P<0.05, **P<0.01, and ***P<0.001; NS not significant.

TABLE-US-00002 TABLE 2 P1 PR and P2 PR antagonists tested for characterization of A. gambiae PRs CAS Purinergic Purin-ergic number/ receptor Name/ receptor other antagonist abbreviation subtype identifier Purity 8-[[4-methyl-3-[3-[[3-[[2- Suramin P2 145-63-1 99% methyl-5-[(4,6,8- trisulfonaphthalen-1-yl) carbamoyl]phenyl]carbamoyl]phenyl] carbamoylamino]benzoyl] amino]benzoyl] amino]naphthalene-1,3,5- trisulfonic acid 19* pyridoxal phosphate-6- PPADS P2 149017-66-3 98% azophenyl-2,4-disulfonic acid 20 [2-[(4- MRS 2159 P2X.sub.1, (P2X.sub.7) PubChem >98% carboxyphenyl)diazenyl]- CID 4-formyl-6-methyl-5- 135484644 oxidopyridin-3-yl]methyl phosphate 21 1-amino-4-[[4-[[4-chloro- Reactive P2X.sub.2, 12236-82-7 94% 6-[[3 (or 4)- Blue-2, sulfophenyl]amino]-1,3,5- RB-2 triazin-2-yl]amino]-3- sulfophenyl]amino]-9,10- dihydro-9,10-dioxo-2- anthracenesulfonic acid 22** 5-[(3- A-317491 P2X.sub.3, P2X.sub.2/3 475205-49-3 >98% phenoxyphenyl)methyl- [(1S)-1,2,3,4- tetrahydronaphthalen-1- yl]carbamoyl]benzene- 1,2,4-tricarboxylic acid 23 2,3,5,6- TMP P2X.sub.3 1124-11-4 >98% tetramethylpyrazine L-leucyl-L-valyl-L-valyl-L- Spinorphin P2X.sub.3 137201-62-8 98% tyrosly-L-prolyl-L- tryptophyl-L-threonine 24* (3S,4R)-3-(1,3- Paroxetine P2X.sub.4 61869-08-7 >98% benzodioxol-5- yloxymethyl)-4-(4- fluorophenyl)piperidine 25 3-[[5-(2,3- A-438079 P2X.sub.7 899507-36-9 >98% dichlorophenyl)tetrazol-1- yl]methyl]pyridine 26 1,3-dimethylxanthine 27 Theophylline P1 58-55-9 >99% 8-cyclopentyl-1,3- 8- P1A.sub.1 35873-49-5 >98% dimethylxanthine 28 Cyclopentylth eophylline/ CPT Substances were purchased from Sigma-Aldrich Schnelldorf, Germany; *Tocris Bioscience, Bristol, UK; and **Chemical Point (Deisenhofen, Germany)

[0217] Spike counts elicited by 200 pM doses of nucleotides, nucleosides, purines, pyrimidines and water were log(x+1.25)-transformed (optimal constant numerically established using a maximum likelihood procedure) and analysed using a linear mixed effects model (function lme in nlme R package) with mosquito number as random effect. To model inhomogeneous variance in residuals (near normal distribution, P=0.045, Shapiro-Wilk test, W=0.98604), the variance function varldent in the nlme package that allows for different variance per treatment was used. Statistical differences were identified between treatments using post-hoc-Tukey test with contrasts adjusted by Holm's family wise error compensation (FWEC). Spike counts elicited by ATP, AMP, adenosine and their binary mixtures were analysed using a generalised linear model (LM) of log (x+10) transformed data and differences were identified using post-hoc-Tukey test with Holm's FWEC.

[0218] Log transformed spike amplitudes generated in response to ATP, adenosine and sucrose were compared using linear mixed effects models using the lme function of the nlme R package, with spike amplitude as the dependent variable, treatment as the fixed factor and mosquito number within treatment as random effect. Residuals were normally distributed (Shapiro-Wilk test P>0.0563) and their variances homogeneous (Bartlett test P>0.9799); P-values were corrected using Holm's FWEC.

[0219] Pairwise comparisons of A. gambiae electrophysiological (EP) responses to ATP and adenosine alone and combined with a given dose of PR antagonist, EP responses to ATP alone and combined with a given dose of a metal modulator, and EP responses to ATP prior to and after exposure of a sensillum to a given dose of a PR antagonist were made with a one-tailed Wilcoxon signed-rank test for data showing a non-normal distribution, with a paired Student's t-test for normally distributed data showing homogeneous variance and with a Welch's t-test for normally distributed data showing non-homogeneous variance. Effects on feeding of adding ATP, AMP-CPP, adenosine, ATP+adenosine, AMP and ADP-S, CPT with ATP or adenosine, TMP with ATP+adenosine, TMP+CPT with ATP+adenosine to the FS for A. gambiae, and of adding PR antagonists with ATP to the FS for A. stephensi were compared using one-tailed Mann-Whitney U-test or Welch's t-test, depending on data distribution; Holm's FWEC was applied where treatment comparisons involved the same positive control. Percent feeding inhibition induced by PR antagonists in A. stephensi was calculated using Abbott's formula.

[0220] Dose-response models fitted to mosquito data were generated using the drm command in the drc R package (version 2.5-12). Best fitting models (goodness of fit) and effects of dose of a compound were assessed using, respectively, modelFit and noEffect functions in the same R package. A 3-parameter logistic model (L3) was fitted to spike-counts recorded to increasing doses of adenosine, AMP, ATP and AMP-CPP. Spike numbers recorded in response to 200 pM ATP with increasing doses of PR antagonists added were analysed using a 4-parameter log-logistic model (LL.4) for A-317491 and spinorphin, using a 4-parameter Weibull model (W1.4) for PPADS, RB-2 and TMP, and using a 3-parameter log-logistic model (LL.3) for suramin. An L3 model was fitted to proportions of females feeding on increasing doses of ATP. Feeding rates on 20 M ATP with increasing doses of a PR antagonist added were analysed using a 4-parameter log-logistic model (LL.4) with the median of the positive control (20 M ATP) fixed as the upper asymptote. Response dynamics in 3-parameter models are related to the slope b, the upper asymptote, and inflexion point of the model e (the ED.sub.50 value), whereas 4 parameter models also include the lower asymptote parameter. The upper asymptote d in EP responses represents the maximum response, which is insurmountable, i.e., the response in spike count does not rise regardless of how much the dose of agonist increases.

[0221] Effective ED.sub.50 and ED.sub.95 doses of agonists and inhibitory ID.sub.50 and ID.sub.95 doses of antagonists were estimated using the ED function of the drc package. An ED.sub.x value is the effective dose of a compound at x percent of the total response encompassed between the upper and lower asymptotes estimated by the drc model. Statistical differences between ED.sub.50 values of compounds were compared using the EDcomp and comParm functions in the drc package. In EP experiments an ID.sub.x value is the inhibitory dose of an antagonist inducing x percent of its maximum effect on the response to 200 pM ATP. Reduction in spike count in the response to 200 pM ATP induced by a PR antagonist at a dose approximating its ID.sub.95 value was compared with the response to the corresponding positive control (200 pM ATP) presented before and after the antagonist treatment using one-tailed Wilcoxon rank-sum test, Students t-test or Welch's t-test. In feeding experiments an ID.sub.x value is the dose of an antagonist reducing the feeding rate by x percent on a solution of a phagostimulant relative to the upper and lower limits of the drc estimated for the phagostimulant presented alone. The effect of a PR antagonist at a dose approximating its ID.sub.50 value on feeding by A. gambiae on 20 M ATP was compared to the positive control using the Mann Witney U-test or Student t-test, depending on the data distribution; P-values were corrected using Holm's FWEC for paroxetine and A-438079 that shared the same positive control.

[0222] Uptake of TFS containing PR antagonists, converted to mg/h for hard ticks and weight gains in mg/25 min for soft ticks, was compared to TFS uptake in the corresponding positive controls. For soft ticks, 3.5 mg was added to all measurements to compensate for negative values when ticks did not feed and lost weight through evaporative water loss. For tsetse and horn flies the percentage flies that fed per antagonist treatment served as the criterion for inhibition of feeding. As horn fly sexes emerged within 24 h, both sexes were assumed to show the same degree of appetence. In tsetse, males emerged first so a gender-related appetence could not be excluded. No sex-related feeding differences in feeding were found for testse (GLM, P>0.05) so data for the sexes were pooled. Comparisons of percentage fed flies were made using a Bayesian GLMthe most appropriate model considering the zero-inflated distributions shown by the data sets. As measurements of reduced feeding on TFS containing PR antagonists by hard ticks and weight gain by soft ticks did not fit to a particular distribution type, values were compared with corresponding positive controls using the Kruskal-Wallis test for soft tick data, the one-tailed Wilcoxon test for A. hebraeum and the one-tailed Mann-Whitney U tests for I. ricinus. P-values were corrected using Holm's FWEC in multiple comparisons of data for each tick and fly species.

Examples

Evidence for P1 and P2X PRs on Malaria Mosquito Mouthparts

[0223] The labellum, used for probing substrates on which the malaria mosquito feeds, is located at the tip of the mouthparts, and bears long (30-35 m) terminal-pore trichoid sensilla housing four chemosensory cells. One of these cells responds to sugars for nectar feeding. The inventors set out to establish whether other cells respond to stimuli and identified a cell which is particularly sensitive to ATP (detection threshold<100 pM). ATP is present in blood. The inventors therefore investigated if the capacity to sense the presence of ATP is associated with blood-feeding behaviour in the mosquito using a series of electrophysiological (EP) assays.

[0224] EP responses to a set of pertinent nucleotides, nucleosides, purines and pyrimidines (1-18, Table 1) each at 200 pM indicate how labellum sensory cells show strongest responses to adenine-containing ATP, AMP-CPP, adenosine and AMP, all at 23 to 26 spikes/2s (FIGS. 1A and B). ADP, a P2Y PR agonist, showed no activity, suggesting that biological activity of nucleotides is limited to one or three phosphates. Loss of OH groups on the ribose moiety, as in dATP and ddATP, strongly reduced activity. This led to prioritising P2X.sub.2, P2X.sub.3 and P2X.sub.2/3 PR agonists ATP and AMP-CPP, and P1A.sub.1 PR agonists adenosine and AMP as best stimulants.

[0225] Action potential frequencies generated as a function of dose for adenosine, AMP and ATP indicate an increase in spike frequency from 100 pM reaching an unsurmountable asymptote, typical of PRs, at 250 pM where approximately 30 spikes/2 s were recorded for all three compounds (FIGS. 2A and B). No difference was found between dose-response models (drc) generated for adenosine, AMP and ATP (all P>0.2) with similar ED.sub.50 (120 pM) and ED.sub.95 values (200 pM, P>0.05 in all cases). AMP-CPP (,-methylene ATP), a stronger P2X PR agonist than ATP (Coddou et al., 2011 in Activation and Regulation of Purinergic P2X Receptor Channels, Pharmacological Reviews 63, 641-683; doi 10.1124/pr.110.003129), proved the most potent agonist. This nonhydrolysable ATP phosphonic analogue, with a methylene group substituting for oxygen between the - and -phosphates, shifted the EP response curve to lower doses with a steep increase in spike frequency from 10 pM to an asymptote at 80 pM (ED.sub.95 of 50 pM) and an ED.sub.50 at 20 pM, significantly lower than for adenosine, AMP and ATP (all P<0.001; FIGS. 2A and B). The drc-slope computed for AMP-CPP was steeper compared to AMP and adenosine (P<2E-16), but no different from that estimated for ATP (P=0.1563), suggesting different kinetics in the binding of AMP and adenosine to a P1 PR than by AMP-CPP and ATP to a P2 PR. Superior activity of AMP-CPP and failure by the P2Y PR agonists ADP, ADP-S and uracil nucleotides to elicit responses (FIG. 1A) suggested presence of a P2X PR activated by ATP and AMP-CPP accompanied by a P1A.sub.1 PR responding to adenosine and AMP on A. gambiae mouthparts.

[0226] Whereas ATP and adenosine alone at 50 pM hardly elicited a response, respectively 2.50.7/2s and 1.250.62/2s, an equimolar mixture containing 50 pM of each agonist elicited a spike frequency of 16.284.68/2s, higher than to 100 pM of either compound presented alone (FIGS. 2C and D). Equimolar amounts of the two agonists were necessary since adding either 20 pM adenosine to 200 pM ATP or 20 pM ATP to 200 pM adenosine did not significantly enhance the response. Presenting adenosine admixed with ATP at increasing equimolar doses to the sensillum shifted the ED.sub.50 value to 86.0910.2 pM, significantly lower than for either adenosine or ATP alone (FIG. 2D). Despite this, the drc models and ED.sub.95 values estimated for ATP, adenosine and their combination are not different. The insurmountable asymptote reached for adenosine, AMP, ATP and AMP-CPP show no difference, suggesting presence of PRs that saturate in the same dose range. Yet adding either 200 pM adenosine or 200 pM AMP to 200 pM ATP (approximately asymptote doses of each) elicited spike frequencies higher than either agonist presented alone, whereas adenosine combined with AMP had not such effect (FIGS. 2C and D, and Table 3). Taken together, these data indicate a P1A.sub.1 PR activated by adenosine and AMP, and a P2X PR activated by ATP and AMP-CPP on A. gambiae mouthparts.

One Sensory Cell with Two PR Types on Malaria Mosquito Mouthparts

[0227] The inventors next addressed the question of where the PRs reside in labellum sensilla of A. gambiae females may be located. Action potentials recorded in response to purinergic compounds and sucrose were typically biphasic (FIGS. 3A and B). Spike amplitudes elicited by ATP (2.49 mV0.030, means.e.m.) and adenosine (2.57 mV0.031) were the same whereas sucrose systematically elicited higher amplitude spikes (4.44 mV0.047; FIGS. 3A and B). Firing of sensory cells with distinct receptor types in response to stimulation by two agonists generate doublets from near simultaneous firing by the cells. No doublets were found in responses recorded for ATP combined with adenosine whereas responses to ATP combined with sucrose did (FIGS. 3A and B). This suggests that ATP and adenosine affect PRs on the same sensory cell within mouthpart sensilla of the malaria mosquito.

TABLE-US-00003 TABLE 3 Comparisons of mean spike frequencies (s.e.m.) recorded from A. gambiae T1 labellum sensilla in response to stimulation with adenosine, AMP and ATP alone and combined. Adenosine + Adenosine + AMP + Agonist(s) * AMP ATP AMP ATP ATP Mean 28.58 33.04 24.55 53.8 44.6 spike (4.63) (3.18) (2.9) (6.59) (5.02) frequency per 2 s (SEM) Adenosine 27.14 1 0.9087 1 0.001 0.0316 (2.23) AMP 28.58 1 1 0.0033 0.0559 (4.63) ATP 33.04 0.8033 0.0254 0.3804 (3.18) Adenosine + 24.55 0.0025 0.0372 AMP (2.9) Adenosine + 53.8 1 ATP (6.59) * Each compound was tested at 200 pM alone and in binary mixtures. Treatments with spike frequencies that are significantly different have P - values in bold. Recording were made from between 9 and 25 sensilla per compound and binary mixtures of compounds.

Malaria Mosquito PR Responses are Inhibited by P1 PR and P2 PR Antagonists

[0228] Xanthines selectively block adenosine-sensitive PRs. One M of the non-selective P1 PR antagonist theophylline (27, Table 2) reduced the EP response elicited by 200 pM adenosine by almost half from 30.233.02 spikes/2s to 17.634.48 for the adenosine theophylline combination (paired t-test, P=0.0259). As ATP complexes with theophylline in solution the inventors used the high affinity and selective P1A.sub.1 PR antagonist and theophylline-related compound 8-cyclopentyl-1,3-dimethylxanthine (CPT, 28, Table 2) as an alternative. CPT inhibited the EP response to 200 pM adenosine but not that to ATP (FIGS. 4A and C). Conversely, 1 nM suramin (19, Table 2), a non-selective P2 PR antagonist, inhibited the response to ATP but not to adenosine (P>0.05; FIGS. 4B and D). Inhibition persisted for at least 2 min in both cases (FIGS. 4 A and B, and FIG. 7 A). Use of these PR inhibitors confirmed presence of P1A.sub.1 and P2X PRs on A. gambiae mouthparts.

[0229] The P2X PR expressed by the A. gambiae mouthpart sensory cell was further characterised using non-selective P2 PR and selective P2X PR antagonists (19-26, Table 2), some tested over several doses (FIGS. 5 and 6). PPADS (P2 PR antagonist) inhibited the response to ATP at an ID.sub.50 of 1.42 nM, close to that of suramin (0.55 nM; Table 4). EP responses to ATP were inhibited by the P2X.sub.2 PR antagonist RB-2, the P2X.sub.3 PR antagonists TMP and spinorphin, and by the P2X.sub.3/P2X.sub.2/3 PR antagonist A-317491 but not by antagonists for other P2X PRs (FIGS. 5, 6 and 7, Table 4). The most effective antagonists were spinorphin and A-317491 (matching ID.sub.95 values at ca. 0.4 nM; Table 1), suggesting a P2X.sub.3/P2X.sub.23 PR on A. gambiae mouthpart sensilla. P2, P2X.sub.3 and P2X.sub.23 PR antagonists, but not the P2X.sub.2 PR antagonist RB-2, induced persistent antagonism to ATP presented to the sensillum approximately 2 min later (FIGS. 5 and 7). Response curves for these antagonists, except that for suramin, indicate resistance to inhibition, characteristic of PRs, when spike counts reach ca. 10/2 s (FIGS. 5 and 6).

TABLE-US-00004 TABLE 4 Neuropharmacological effects of P2 PR antagonists on responses of A. gambiae mouthpart ATP-sensitive receptor. Mean no. of spikes per 2 s ID.sub.50 in ID.sub.95 in Antagonist (s.e.m.) PR nM.sup.1 nM.sup.1 dose ATP ATP + p- antagonist (s.e.m.) (s.e.m.) [nM] [200 pM] antagonist.sup.2 values Suramin (P2) 0.55 1.15 0.6 20.60 4.20 0.0011 0.06 0.46 2.73 1.32 PPADS (P2) 1.42 12.66 50 21.63 8.13 0.0253 1.2 24.05 3.09 2.69 MRS 2159 No block (1 M) 1000 32.71 27.14 0.5339 (P2X.sub.1) 6.54 8.95 RB-2 (P2X.sub.2) 1.04 113.02 50 27.67 12.83 0.0038 1.11 247.77 2.63 3.35 A-317491 0.32 0.41 0.5 34.17 7.5 0.0033 (P2X.sub.3, 0.33 0.51 4.95 2.29 P2X.sub.2/3) TMP(P2X.sub.3) 12.17 27.50 10 31.40 11.2 0.0203 6.61 46.49 1.17 4.86 Spinorphin 0.34 0.43 1 29. 9.8 0.0426 (P2X.sub.3) 0.47 0.36 5.26 4.43 Paroxetine No block (2 M) 2000 27 27.6 1.0 (P2X.sub.4) 6.01 3.23 A-438079 No block (1 M) 1000 27.57 22.57 0.843 (P2X.sub.7) 4.16 6.77 .sup.1ID.sub.50 and ID.sub.95 values in nM (unless otherwise stated) for PR antagonists were estimated by testing them at increasing doses admixed to 200 pM ATP that served as positive control (see FIGS. 5 and 6). The maximum dose tested is provided when an antagonist failed to inhibit. .sup.2Mean spike frequencies provided for 200 pM ATP + antagonist is for an antagonist dose near its estimated ID.sub.95 value (column 4); P - values indicating significant reductions in spike frequency compared to 200 pM ATP are indicated in bold. Treatment repetitions (n) are reported in Materials and Methods.

Malaria Mosquito P2X PR Responses are Modulated by Metal Ions

[0230] Essential trace metals like Zn.sup.2+ and macrometals like Mg.sup.2+ modulate vertebrate P2X PRs via extracellular binding sites (Coddou et al., supra). The crystal structure and EP responses of AmP2XR from the Gulf Coast tick Amblyomma maculatum indicate it functions as an ATP-gated cation channel with two extracellular metal-binding sites, M1 and M2 (Kasuya et al., 2016 in Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels, Cell Reports 14, 932-44; doi 10.1016/j.celrep.2015.12.087). The M1 site located at the trimer interface permits allosteric potentiation by Zn.sup.2+ of AmP2XR responses to ATP. LsP2X of the pond snail Lymnaea stagnalis with a similar M1 site is likewise potentiated by Zn.sup.2+. The inventors established through EP recordings from A. gambiae labellum sensilla that 200 pM Zn.sup.2+ admixed with 100 pM ATP induced a 5-fold increase in spike frequency but 200 pM Zn.sup.2+ had no such effect at the asymptote dose of 200 pM ATP (Table 5). The M2 site on AmP2XR is associated with the ATP binding site and subject to modulation by Mg.sup.2+, reducing ATP receptor affinity at higher Mg.sup.2+ levels (Kasuya et al., supra). The inventors found that adding 200 pM Mg.sup.2+ to 200 pM ATP reduced spike frequency recorded from A. gambiae labellum sensilla by half compared to 200 pM ATP alone (Table 5). These findings suggest the P2X PR of A. gambiae shares characteristics with vertebrate, tick, and snail P2X PRs.

TABLE-US-00005 TABLE 5 Effects of metals ions on electrophysiological responses of A. gambiae labellum ATP-sensitive receptor. Mean no. of spikes Treatment per 2 s (s.e.m.) * P-value 100 pM ATP 3 1.67 P = 0.008 compared 200 pM Zn.sup.2+ + 15.4 3.72 to 100 pM ATP 100 pM ATP 200 pM ATP 24.8 4.84 P = 0.89 compared 200 pM Zn.sup.2+ + 24.0 3.41 to 200 pM ATP 200 pM ATP 200 pM ATP 28.12 3.5 P = 0.0049 compared Mg.sup.2+ + 200 pM ATP 12 4.13 to 200 pM ATP * Treatment repetitions (n) are reported in Materials and Methods.

Feeding by Biting Insects and Ticks is Inhibited by P1 PR and P2 PR Antagonists

[0231] Considering the sensitivity shown by A. gambiae peripheral neurones for adenosine, adenine nucleotides and PR antagonists, the inventors set out to test the effects of these compounds on feeding by mosquitoes, biting flies and ticks. Adding ATP to the feeding solution induced dose dependent feeding from 1 M ATP with an ED.sub.50 at 6.54 M and an asymptote at 45 M where 70.25% mosquitoes fed (the ED.sub.95 at 22.5 M for 68.15% feeding was rounded to 20 M for feeding assays; FIG. 8A and Table 6). AMP-CPP at 20 M proved a better feeding stimulant, eliciting 84.35% feeding though not significantly different to ATP (Table 6). AMP and adenosine each presented at 20 M elicited increased feeding albeit to a significantly lower extent than 20 M ATP, whereas 1 mM adenosine induced an equivalent level of feeding as on 20 M ATP. No effect on feeding higher than on 20 M ATP was recorded when 20 M ATP was admixed with 200 M adenosine (Table 6). The increase in feeding induced by the P2Y PR agonist ADP-(3S was not significantly different to that of the feeding solution (Table 6).

TABLE-US-00006 TABLE 6 Proportion of A. gambiae feeding on the feeding solution, on nucleotides and adenosine in the feeding solution, and on the P1A1 PR antagonist CPT and the P2X.sub.3 PR antagonist TMP admixed with ATP and adenosine in the feeding solution. Different experimental series are separated by a blank line. % P-value Engorged compared to Treatment mosquitoes n* FS ** Compared to Feeding solution 24.1 1.6 71 (FS **) 20 M ATP 67.5 2.1 39 4.08E16 20 M AMP-CPP 84.3 6.8 4 4.33E12 ATP 20 M 0.0956 20 M Adenosine 39.1 5.7 10 0.0089 ATP 20 M 6.11E04 1 mM Adenosine 64.4 3.2 17 1.70E17 ATP 20 M 1 20 M ATP + 60.1 4.5 4 7.53E06 ATP 20 M 0.4831 200 M Adenosine 20 M AMP 38.3 5.1 10 0.0110 ATP 20 M 3.40E04 20 M ADP-S 37.8 4.1 4 0.0956 ATP 20 M 0.0191 20 M ATP + 65.4 10.2 5 1.08E07 ATP 20 M 1 10 M CPT 1 mM Adenosine + 48.7 7.0 6 7.02E05 Adenosine 1 mM 0.0146 10 M CPT Feeding solution 14.1 2.6 8 (FS **) 20 M ATP + 200 M 46.2 3.4 18 1.20E05 Adenosine 20 M ATP + 200 M 15.3 2.8 16 0.3961 20 M ATP + 200 M 2.32E07 Adenosine + 7.5 M Adenosine TMP + 20 M CPT 20 M ATP + 200 M 36.7 6.0 11 0.0117 20 M ATP + 200 M 0.1499 Adenosine + 7.5 M TMP Adenosine 20 M ATP + 200 M 0.0116 Adenosine + 7.5 M TMP + 20 M CPT *n =number of cages for each experimental situation; ** FS =feeding solution (buffered NaCl/dextrose).

[0232] PR antagonists identified by EP recordings could act on PRs to inhibit feeding. 10 M CPT, the selective P1A.sub.1 PR antagonist, significantly inhibited A. gambiae feeding on 1 mM adenosine but not on 20 M ATP added to the feeding solution (Table 6). Testing P2 PR antagonists over several doses shows how suramin, PPADS, RB-2, TMP, spinorphin and A-317491 inhibited feeding on a solution containing the feeding stimulant ATP (FIGS. 8B and 9, Table 7). A. gambiae feeding proved most sensitive to TMP (ID.sub.50 15 nM) and A-317491 (ID.sub.50 0.24 nM; FIGS. 8B and 9, Table 7). Except for the P2X.sub.7 PR antagonist A-438079, antagonists for other P2X PR subtypes were less effective (Table 7). Table 7. Effect of P2 PR antagonists on A. gambiae feeding on a feeding solution containing ATP.

TABLE-US-00007 TABLE 7 Effect of P2 PR antagonists on A. gambiae feeding on a feeding solution containing ATP. % fed mosquitoes (mean s.e.m.) ID.sub.50 in ID.sub.95 in Antagonist nM M ATP dose ATP + PR antagonist (s.e.m) * (s.e.m.) * [20 M] tested antagonist P-value Suramin (P2) 735 1.36 67.6 1 M 34.86 0.00001 56 0.30 1.92 4.49 PPADS (P2) 42.84 # 66.12 100 M 33.39 0.0001 111.58 3.67 3.49 M MRS 2159 No feeding inhibition 65.96 20 M 79.19 0.0633 (P2X.sub.1) (20 M) 3.1 6.43 RB-2 (P2X.sub.2) 100.55 # 64.49 0.1 M 32.88 0.0004 92.48 3.56 4.53 A-317491 0.24 # 69.12 1 nM 43.71 0.0005 (P2X.sub.3/P2X.sub.2/3) 1.36 1.59 6.5 TMP(P2X.sub.3) 15.47 1.28 69.68 10 nM 50.47 0.0084 10.58 3.00 2.52 6.80 Spinorphin (100 nM, 27.31%) 64.2 100 nM 46.62 0.0469 (P2X.sub.3) 3.74 6.79 Paroxetine No feeding inhibition 70.2 20 M 62.72 0.1624 (P2X.sub.4) (20 M) 5.7 6.63 A-438079 (20 M, 30.74%) 70.2 20 M 48.65 0.0005 (P2X.sub.7) 5.7 2.7 * ID.sub.50 values in nM and ID.sub.95 values M (unless otherwise stated) for antagonists were estimated by testing their effects at increasing doses on A. gambiae feeding on 20 M ATP (positive control) in the feeding solution (cf. FIGS. 8 and 9). # The log logistic LL.4 model did not permit a reliable estimate of the ID.sub.95 for PPDAS, RB-2 and A-317491. Data for the maximum dose tested is provided when an antagonist failed to inhibit feeding. For spinorphin and A438079 (not tested over several doses) the % reduction in feeding is provided for the dose indicated. The four columns on the right present, respectively, the proportion of mosquitoes feeding on 20 M ATP (cf. Table 6), the dose of antagonist tested nearest the ID.sub.50 value, the proportion of mosquitoes feeding on that dose of antagonist admixed with 20 M ATP, and the probability level for the comparison of the two proportions (significant values are in bold). Treatment repetitions (n) are reported in Materials and Methods.

[0233] 20 M CPT with 7.5 M TMP added to the feeding solution inhibited the feeding stimulant effect of the 20 M ATP plus 200 M adenosine combination to a level no different to that of the feeding solution, and this combination of P1A.sub.1 PR and P2X.sub.3 PR antagonists proved a better feeding inhibitor for the 20 M ATP plus 200 M adenosine combination in the feeding solution than 7.5 M TMP alone (FIG. 10, Table 6). It is noteworthy how even the highest doses of PR antagonists tested only reduced feeding to levels corresponding to that on the feeding solution tested as negative control, allowing the inventors to conclude that the action of PR antagonists is on purine-sensitive cells.

[0234] Another anopheline mosquito, A. stephensi, also responded as a function of dose to ATP as a feeding stimulant with an ED.sub.95 value at 160 M ATP (Table 8). The P2 PR antagonists suramin and PPADS, the P2X.sub.3 PR antagonist TMP and the P2X.sub.3/P2X.sub.2/3 PR antagonist A-317491 all inhibited feeding by A. stephensi on the feeding solution containing ATP in a pattern like that recorded for A. gambiae where the P2X.sub.3/P2X.sub.2/3 PR antagonists TMP and A-317491 again induced the strongest inhibition (Table 8).

TABLE-US-00008 TABLE 8 Effect of P2 PR antagonists on A. stephensi feeding on a feeding solution containing ATP. Suramin PPADS A-317491 TMP Treatment ATP (P2) (P2) (P2X.sub.3/P2X.sub.2/3) (P2X.sub.3) Dose 160 M 80 M 800 M 0.08 M 0.8 M % Fed females 72.96 5.9 30.04 7.75 37.29 3.75 29.68 7.14 26.67 2.98 (mean s.e.m.) % Inhibition 58.83 48.89 59.32 63.45 P-value * 0.0060 0.0066 0.0060 0.0027 * Treatment effects were compared to the positive control (feeding solution with 160 HM ATP added). Treatment repetitions (n) are reported in Materials and Methods.

[0235] In feeding experiments, suramin inhibited feeding on a solution containing ATP by two hard tick spp. Ixodes ricinus and Amblyomma hebraeum and by the soft tick Ornithodorus moubata, and A-317491 inhibited feeding by I. ricinus and O. moubata (Table 9). Suramin and A-317401 likewise inhibited feeding by the tsetse fly Glossina pallidipes and horn fly Haematobia irritans, and TMP inhibited feeding by the horn fly on solutions containing ATP (Table 10). TMP in solution failed to inhibit feeding by the soft tick and tsetse, yet when applied onto the feeding membrane it significantly inhibited feeding by both blood feeders (Tables 9 and 10), suggesting that antagonism of peripheral PRs is sufficient to inhibit feeding.

TABLE-US-00009 TABLE 9 Effect of P2 PR antagonists on feeding by three tick species on a feeding solution containing ATP. Fluid intake or Solution weight gain n containing Life Feeding Median (number P- ATP stage method Treatments (mg) .sup.+ Range of ticks) value .sup.# Ixodes females glass TFS * 2.6 1-8 16 ricinus capillary TFS + 1.9 1-8 14 0.0213 500 M Suramin TFS + 1 1.5 1-3 12 0.0122 M A- 317491 Amblyomma females glass TFS 11.5 3-28 10 hebraeum capillary TFS + 4.5 2-14 10 0.0225 500 M Suramin TFS 15 1-46 11 TFS + 1 6.7 1-99 10 0.1363 M A- 317491 Ornithodorus N3, N4 Silicone TFS 0.3 1-63 231 moubata membrane TFS + 0 3-6 40 0 500 M Suramin TFS + 1 0.1 4-52 21 0.7436 M A- 317491 TFS + 0 4-22 20 0.0183 100 M A- 317491 TFS + 0.1 4-29 77 0.1702 100 M TMP TTFS + 8 0 4-10 19 0.0024 g/cm.sup.2 TMP on membrane * TFS: tick feeding solution containing 100 mM NaCl + 8.9 mM NaHCO3 + 1000 M ATP as positive control; .sup.+ Feeding solution intake in mg/h for hard ticks and weight gain in mg/25 min for soft ticks; .sup.# Within species, treatments effects significantly different to the TFS are in bold.

TABLE-US-00010 TABLE 10 Effect of P2 PR antagonists on feeding by two biting fly species on a feeding solution containing ATP. n % Fed (number Fly species Treatment Median Range of flies) p-value .sup.# Glossina FFS * 63.6 30-100 41 pallidipes FFS + 50 M Suramin 45.5 0-91 17 0.0009 FFS + 1 M A-317491 22.5 0-90 20 0.0015 FFS + 150 M TMP 66.7 40-92 13 0.8551 FFS + 16 g/cm.sup.2 TMP on 40 8-70 9 0.0261 membrane Haematobia FFS 66.7 38-100 31 irritans FFS + 50 M Suramin 61.5 31-100 17 0.1888 FFS + 100 M Suramin 46.2 9-67 15 0.0020 FFS + 10 M A-317491 58.3 25-86 16 0.1790 FFS + 100 M A-317491 41.5 27-78 7 0.0193 FFS + 50 M TMP 50 0-80 11 0.0114 FFS + 100 M TMP 37.5 18-38 3 0.0162 FFS + 500 M TMP 0 0 8 0.0024 * FFS: fly feeding solution containing 150 mM NaCl + 8.9 mM NaHCO3 + 100 M ATP as positive control; .sup.# Within species, treatment effects significantly different to the FFS are in bold.

[0236] Evidence is presented herein for P1A.sub.1 and P2X.sub.3/P2X.sub.2/3 PRs on mouthparts of blood-feeding arthropods. The P2X PR is particularly sensitive to selective P2X.sub.3/P2X.sub.2/3 PR antagonists A-319471, spinorphin and TMP that inhibit feeding by all blood-feeding insects and ticks tested herein. The characteristics of the P2X.sub.3/P2X.sub.2/3 PRs described by the inventors show similarities to vertebrate homomeric P2X.sub.3 PRs and heteromeric P2X.sub.2/3 PRs found on unmyelinated C-fibres and on thinly myelinated A-sensory fibres that transmit pain. Such nociceptive fibres and their first relay neurons in dorsal root ganglia (DRG) show high expression levels of P2X.sub.3 PRs (Burnstock and Verkhratsky, 2012 in Purinergic signalling and the nervous system, Springer-Verlag Berlin Heidelberg; doi 10.1007/978-3-642-28863-0). In contrast to P2X.sub.3 PRs, slowly desensitising P2X.sub.2/3 PRs maintain currents in response to repeated stimulation (Coddou et al., 2011, supra). The inventors recorded little adaptation in response to repeated presentation of ATP to A. gambiae mouthpart sensilla at intervals varying from tens of seconds to 6 minutes. Vertebrate P2X.sub.3 and P2X.sub.2/3 PRs show high affinity for AMP-CPP and ATP, at <1 nM for the latter, and are readily antagonised by A-317491 (K.sub.d 0.9 nM; Jarvis et al., 2002, supra). The antinociceptive spinorphin, an endogenous heptapeptide inhibitor of encephalin-degrading enzyme, antagonises human P2X.sub.3 PR at an ID.sub.50 of 8.3 pM.

[0237] During blood feeding, ATP and ADP are hydrolysed by A. gambiae salivary apyrase to prevent platelet aggregation for wound healing. This results in adenosine being presented to mouthpart PRs. The inventors report how equimolar combinations of ATP and adenosine elicit spike frequencies significantly higher than either agonist presented singly. This shifts the threshold to lower doses of the agonist mix and the response surpasses the unsurmountable asymptote reached at higher doses of either agonist alone, leading to a wider dose range over which the PRs respond. Mouthpart PRs of A. gambiae are eminently equipped to detect physiologically operating levels of both ATP and adenosine, and as disclosed herein, can be readily inhibited by PR antagonists.

[0238] Even though the inventors tested and confirmed the usefulness of PR antagonists in the protection against arthropod parasites using only a number of arthropode parasite species, this invention is not limited to the arthropod species tested herein.