GB2175502A

GB2175502A - Inhibiting the development of protozoa

Info

Publication number: GB2175502A
Application number: GB08608021A
Authority: GB
Inventors: Pierre Regis Carle; Alain Richard
Original assignee: Roussel Uclaf SA
Current assignee: Sanofi Aventis France
Priority date: 1985-04-03
Filing date: 1986-04-02
Publication date: 1986-12-03
Also published as: GB8608021D0; AP8600031A0; PH24218A; FR2579867A1; BR8601509A; FR2579867B1; AP15A; GB2175502B; OA08274A

Abstract

A process for combatting the endemics transmitted to living beings by protozoa is characterized in that a sufficient quantity of a compound of pyrethrinoid type is used in the place where the protozoa are found in order to prevent the development of the protozoa. The pyrethrimoid compounds may be administered to vertebrates, e.g. in food or water, or applied to rooms etc., or may be applied to the habitat of vectors (particularly insect vectors) of the protozoa. Preferably the amount of pyrethrimoid used is non-lethal to such vectors.

Description

SPECIFICATION Method of inhibiting the development of protozoa The present invention is concerned with a process for combatting the protozoa responsible for illness of vertebrates and in particular human and animal endemics, and compositions intended for use in this process.

The subject of the invention is a process for combatting the endemics transmitted to living beings by protozoa, characterized in that a sufficient quantity of a compound of pyrethrinoid type is used in the place where the protozoa are found in order to prevent the development of the protozoå.

By living being, there is intended man, mammals, fish and birds.

As protozoa at which the present invention aims, there can be cited spirochetes, treponema, ameba, trypanosomes, hematozoa, particularly the hematozoa of malaria or plasmodium and the leishmania.

The invention is therefore concerned with compositions intended for combatting very diverse illnesses, such as the spirochetoses, illnesses caused by treponemes, amebiases, malaria, trypanosomiases or leishmanoses.

The pyrethrinoid compounds aimed at by the invention are compounds known in a general way; they answer to the general formula CO2B in which: - -- either represents a radical:

in which -eitherZ1 and Z2 each represent a methyl radical, - or Z1 represents a hydrogen atom and - eitherZ2 represents a radical

in which Z3 represents a hydrogen or halogen atom and either T1 and T2 identical or different, represent a hydrogen atom, a halogen atom, an alkoxyl radical, an alkyl radical containing from 1 to 8 carbon atoms, a CF3 or CN radical or a phenyl nucleus possibly substituted by a halogen, or together form a cycloalkyl radical containing from 3 to 6 carbon atoms or a radical:

in which X represents an oxygen or sulphur atom:: - orZ2 represents a radical:

in which a, b, c and d, identical or different, each represent a halogen atom, - orZ2 represents a radical:

in which D represents a hydrogen or halogen atom, or an alkoxyl radical, G represents an oxygen or sulphur atom and J represents either a linear, branched or cyclic alkyl radical, saturated or unsaturated, containing from 1 to 8 carbon atoms, possibly substituted by one or more functional groups, identical or different, - or an aryl group containing from 6 to 14 carbon atoms, possibly substituted by one or more functional groups, identical or different, -ora heterocyclic radical possibly substituted by one or more functional groups, identical or different, - orA represents a radical::

In which U, in any position on the benzene nucleus, represents a halogen atom, an alkyl radical containing from 1 to 8 carbon atoms, or an alkoxyl radical containing from 1 to 8 carbon atoms, m represents the numeral 0, 1 or 2.

11 - and B represents a group:

in which R1 represents a hydrogen atom, a methyl ethynyl or cyano radical and R2 and R3, identical or different, represent a hydrogen, fluorine or bromine atom.

The subject of the invention is quite particularly the compositions intended to destroy the pathogenic protozoa of vertebrates and invertebrates, or to limit their multiplaction.

As vector vertebrates, there can be cited man as well as mammals, notably cattle, sheep, pigs, goats and horses, birds like fowls, batrachians, reptiles, fish and the various aquatic species.

As invertebrate vectors, there can be cited the terrestriai and marine arthropods, notably the acarina, the insects, the molluscs, notably the gasteropods and the worms.

As insect vectors, there can be cited mosquitoes, flies, bugs, fleas and lice.

The combat process of the invention, in the case where the protozoa combatted are carried by arthropods and notably by insects, is particularly interesting from an economic and ecologic view-point; it is possible to use doses of pyrethrinoids lower than the lethal doses for the arthropod vectors. The treatments can be applied in the development areas of these animals, differing from those of their vertebral hosts (e.g., water-mosquito); these treatments are easier and cost less than those aimed at suppressing the insect vectors. Furthermore, by not suppressing the arthropod totally, the equilibrium of the natural surroundings is better respected.

Quite specially, the subject of the invention is a combat process characterized in that the protozoa are destroyed which are carried by the insects.

Therefore the invention has as its subject a combat process as defined above, characterized in that a compound of pyrethrinoid type is used at a dose less than the lethal dose of the insect vectors.

Quite specially, the subject of the invention is a combat process characterized in that the insect vectors are anopheles.

As anopheles more specially aimed at by the invention, there can be cited: -Anopheles gambiae, Anopheles moucheti, Anopheles funestus, Anopheles hancocki, Anopheles paludis, Anopheles nili, Anopheles littoralis, Anopheles aconitus, Anopheles flavirostris, Anopheles mangyanus, Anopheles stephensi, etc.

More particularly, the subject of the invention is a process of combat characterized in that the protozoa combatted are the plasmodia, such for example, as Plasmodium falci pa rum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale or Plasmodium yoelii.

The fight against malaria is of very great interest; even in our days malaria is one of the great scourges of the modern world, which makes ravages in very numerous countries, notably in Africa, for example in Nigeria, in Upper Volta, in the Cameroons, in the Congo, in Kenya or in Ethiopia, in Latin America, particularly in Venezuela and in Guatemala, or in South-East Asia; there are at present reckoned to be about 1 million children killed in the world by malaria each year.

The invention has more particularly as its subject a process characterized in that the pyrethrinoid utilized is deltamethrin; it also has as its subject a process characterized in that the pyrethrinoid is chosen from the group of photostable or photolabile pyrethrins constituted by tralomethrin, cypermethrin, permethrin, alphamethrin, cyfluthrin, cyhalothrin, fenfluthrin, fencythrin, and fenvalerate.

To combat protozoa in an aqueous environment, according to the invention process, compositions containing the pyrethrinoid are spread in the zone where the arthropod vectors of the protozoa are found on and in marshes, lakes, pools of water, canals, rivers, in forests and savannas. To combat the protozoa in the habitation premises of human beings or animals, the compositions according to the invention are spread by treatment of surfaces, walls, roofs, mosquito nets, etc.

The compositions according to the invention are spread by aeroplane, by soil treatment (liquid or powdering treatment), on the vertebrates to be protected and in the premises frequented by man and the vertebrates to be protected, in residual or atmospheric treatment on the various supports of habitations.

The invention has also as its subject a combatting process characterized in that a sufficient quantity of compound of pyreth rinoid type is administered directly to the living beings in order to prevent the development of the parasitic protozoa.

In this particular combatting process, the compounds of the pyrethrinoid type can advantageously be incorporated in the drinks or foodstuffs to be consumed.

When it is a matter of combatting the parasitic protozoa of vertebrates, the compositions of the invention can be incorporated in the drinking water or in the food compositions combined with a nutritive mixture adapted for animal feeding. The nutritive mixture can vary according to the animal species, it can contain cereals, sugars and seeds, soya, ground-nut and sunflower cakes, meals of animal origin, for example fish-meals, synthetic amino acids, mineral salts, vitamins and anti-oxidants.

Quite naturally, the subject of the invention is compositions intended for putting the above process into operation; these compositions are characterized in that they contain a sufficient quantity of pyrethrinoid to prevent the development of the protozoa.

Because the protozoa are notably carried by insects, the invention has particularly as its subject compositions characterized in that they contain a compound of pyrethrinoid type at a dose less than the lethal dose of the vector insects.

Quite specially, the subject of the invention is compositions characterized in that the pyrethrinoid utilized is deltamethrin; it also has as its subject compositions characterized in that the pyrethrinoid utilized is chosen from the following group of compounds: tralomethrin, cypermethrin, permethrin, alphamethrin, cyfluthrin, cyhalothrin, fenfluthrin, fencythrin and fenvalerate.

The invention compositions intended for the treatment of premises preferably contain from 0.05 mg/l.

to 1 girl. of pyrethrinoid; among the preferred compositions, there can quite specially be cited compositions containing from 0.25 to 5 mg/litre of deltamethrin.

These compositions are prepared according to the usual processes of the agrochemical or agroalimentary industry. They can also have added to them one or more other pesticidal agents. These compositions can be presented in the form of powders, granules, suspensions, emulsions, solutions, solutions for aerosols, combustible strips, and baits.

In addition to the active principle, these compositions in general contain a vehicle and/or a non-ionic surface-active agent which furthermore ensures a uniform dispersion of the substances of which the mixture is made up. The vehicle utilized can be a liquid such as water, alcohol, a hydrocarbon or other organic solvent, a mineral, animal or vegetable oil, a powder such as talc, clays, silicates, kieselguhr or a combustible solid.

The following examples illustrate the invention, without nevertheless limiting it.

EXAMPLE 1: Composition intended for treatment of marshes.

-Deltamethrin 0.1 g - Piperonyl butoxide 4 g - Tween 80 0.25 g -TopanolA 0.1 g -Xylene q.s. for 1 litre.

EXAMPLE 2: Composition intended for treatment of premises.

-Deltamethrin 5 mg -VercorylS 899 g -TopanolA 16 g -Emcol 35 g EXAMPLE 3: Experimentation "in vitro" on cultures of Plasmodium falciparium.

The culture is carried out in polystyrene cupules for tissie culture piaced in an oven at 37" with CO2 (6%), utilising a modified RPMI 1640 medium.

Each cupule contains 0.7 ml. of parasited human blood suspension (starting level of parasitemia: 0.3%). The incubation is continued for 48 hours without renewing the medium. The anti-plasmodium products to be tested are added before incubation in the survival medium.

The reading of the results is carried out on smears after specific colouring with MGG and counting the number of parasited red blood corpuscles. The counts are referred to about 3,000 red blood corpuscles per cupule. The products to be tested are introduced into 3 cupules per concentration studied.

The results obtained are summarized in table 1. The parasited red blood corpuscles contain either trophozoites (juvenile form), or schizontes juvenile or old, or merozoites (in rosettes), all forms of the plasmodium.

The conclusion that can be drawn is that a marked activity on the development oftheplasmodium is produced with a dose of 0.75 ug/ml.

TABLE 1

Product Concentra- No. of No. of Level of Level of para tion corpuscles corpuscles corpuscles sitemia in (in uglml) counted parasited parasited comparison (in %) withthe control (%) delmethrin in aqueous formulation 0.750 9,400 118 126 42 (flowable) at 25 g/l.

EXAMPLE 4: Effect of the pyrethrinoids on the sporogonic cycle in anopheles.

The model chosen was the couple A. stephensi- P.y.yoelii of which the cycle can be easily maintained on white mice.

Plasmodium yoelii yoelii.

Plasmodium of rodents, P. y. yoelli originates from central Africa.

-Maintenance of the cycle ofP.y.yoelli.

The maintenance of the strain on white mice OF1 was carried out by intravenous inoculation of sporozoites obtained according to the following record: -Afterverification on a sample that 7580% of the anophelesfemales in a cage were carriers of sporozoites in their salivary glands, 120 to 150 females were killed, then coarsely dissected so as to keep only the head and the thorax.

These latter were put in a "Thomas pot" containing 4 ml. of RMP1 1640 maintained at 40C, then finely ground.

- The result obtained was centrifuged first at 1500 rpm, (800 9) for 5 minutes.

- 2 ml. of the supernatant was recovered with syringes, the remainder was centrifuged again for 5 minutes at 1500 rpm, and 1 ml. was recovered from the supernatant.

-After verification of the presence of sporozoites, 0.5 ml. of the supernatantwas inoculated intravenously per mouse, i.e., a total of 4 to 6 mice according to the results of the second centrifuging.

From one donor mouse, on which it had previousiy been verified that the parasitemia extended to at least 40% of the red blood corpuscles, 1 ml. of blood was removed by puncture of the retro-orbital sinus by means of a heparinated Pasteur pipette. This blood was mixed in a watch glass with 1 ml. of RPMI 640 to which a drop of heparin was added. Then, by intra-peritoneal route, 0.2 to 0.5 ml of this mixture was inoculated per mouse.

This method of infecting mice has the disadvantage of increasing the virulence of the plasmodial strain on each new passage, and above all, of making it progressively lose its capacity to produce infectious gametocytes. The donor mice are uniquely the mice infected from the start with sporozoites.

The infectivity of the mice was verified on blood smears from the 3rd day after the inoculation. The criteria considered are: presence of immature gametocytes and of microgametes of stage 0 and 1, absence of degenerate gametocytes of which the cytoplasm is sprinkled with little round vacuoles.

The mice recognized as infectious were imprisoned in a flexible netting and put into a cage containing about 150 female anopheles aged at least one week.

- Evaluation of the action of deltamethrin put into the breeding water of mosquito larvae on the development of the Plasmodium sporozoites in adult mosquitoes.

The impregnation with deltamethrin at DL50 or at lower doses was carried out on mosquito larvae not yet infectious while being reared in water. The surviving adults were then put on to mice infected with plasmodium to feed on blood.

Because the complete evolution of the sporogonic cycle of the plasmodium cannot be judged on the presence of oocysts in the stomach of the adult, the experimentation is continued by evaluating the effect of deltamethrin on the percentage of mosquitoes carrying sporozoites in the salivary glands. The dissections were carried out 14 days after feeding on blood.

All of the results are shown in the following table: Series a: Deltamethrin dose used: 2.10-4 mg/l. (DL50 of the mosquito larvae).

The control group was infected first.

None of the 19 mosquitoes dissected in the treated group was a carrier of sporozoites, while in the control group, 14 out of 20 were carriers. On the evidence, the difference is very significant.

Series b: Deltamethrin dose used: 2.10-4 mg/l. (DL50 of the mosquito larvae).

The control group was infected first.

Here also, no mosquito was found to be a carrier of sporozoites in the treated group (0/42), while more than 50% of the mosquitoes were carriers in the control group (16/31). On the evidence, the difference is very significant.

CONCLUSION: The complete sporogonic cycle cannot take place in insects which have been in contact with deltamethrin.

TABLE Distribution of the mosquitoes carrying sporozoites in the salivary glands, according to the control groups and the groups treated with deltamethrin.

Series Dose Group Negative Positive Effective a Control 6 14 20 2.10-4 mg/l. treated 19 0 19 b Control 15 16 31 2.10-4 mgn. treated 42 0 42 EXAMPLE 5 An experiment similar to that of example 4 was carried out on Anopheles in a larval state, using a deltamethrin dose less than 2,510 mg/l.

It was again determined that the complete sporogonic cycle could not be realized in adult insects which had been put into larval contact with deltamethrin.

EXAMPLE 6 The experiment as in example 4 was carried out on Anopheies in the adult state, put into contact with a class support treated with a sub-lethal dose of deltamethrin of 2,510 mg/l. It was again determined that the complete sporogonic cycle could not be realized in insects which had been put into contact with deltamethrin.

Claims

1. Process for combatting endemics transmitted to living beings by protozoa, characterized in that a quantity of compound of pyrethrinoid type sufficient to prevent the development of the protozoa is applied where the protozoa are found.

2. Process for combatting according to Claim 1, characterized in that the pathogenic protozoa of vertebrates and invertebrates are destroyed or their multiplication is limited.

3. Process for combatting according to Claim 2, characterized in that the protozoa are carried by insects.

4. Process for combatting according to Claim 3, characterized in that a compound of pyrethrinoid type is utilized art a dose less than the lethal dose of the vector insects.

5. Process according to Claim 3 or 4 characterized in that the vector insects are anopheles.

6. Process for combatting according to any one of the Claims 1 to 5 characterized in that the protozoa combatted are plasmodia.

7. Process for combatting according to any one of the Claims 1 to 6, characterized in that the pyrethrinoid utilized is deltamethrin.

8. Process for combatting according to any one of the Claims 1 to 6, characterized in that the pyrethrinoid is chosen from the following group of compounds: tralomethrin, cypermethrin, permethrin, alphamethrin, cyfluthrin, cyhalothrin, fenfluthrin, fencythrin and fenvalerate.

9. Process for combatting according to any one of the Claims 1 to 8, characterized in that a quantity of pyrethrinoid is utilized less than that necessary to kill the vector arthropod.

10. Process for combatting according to any one of the Claims 1 to 8, characterized in that a sufficient quantity of compound of the pyrethrinoid type to prevent the development of the parasitic protozoa is administered directly to the living beings.

11. Compositions intended for putting into operation the process according to any one of the Claims 1 to 10, characterized in that they contain a sufficient quantity of pyrethrinoid to prevent the development of the protozoa.

12. Compositions according to Claim 11, characterized in that they contain a compound of pyrethrinoid type at a dose less than the lethal dose of the vector insects.

13. Compositions for use in inhibiting or preventing the development of protozoa comprising, as active ingredient, an effective amount of at least one compound of pyrethrinoid type.

14. Compositions according to Claim 11, 12 or 13, characterized in that the pyrethrinoid utilized is deltamethrin.

15. Compositions according to Claim 11, 12 or 13, characterized in that the pyrethrinoid utilized is chosen from the following group of compounds: tralomethrin, cypermethrin, permethrin, alphamethrin, cyfluthrin, cyhalothrin, fenfluthrin, fencythrin and fenvalerate.

16. A method of inhibiting the development of protozoa which comprises applying to a site infested with or susceptible to infestation by protozoa an effective amount of a compound of pyrethrinoid type.

17. A method according to claim 16 wherein the amount of pyrethrinoid applied is less than the lethal dose of the vector of the protozoa.

18. A method of improving vertebrates by inhibiting the development of protozoa which comprises administering to the vertebrate an effective amount of a compound of pyrethrinoid type.

19. Use of a compound of pyrethrinoid type in the preparation of a composition for inhibiting the development of protozoa.

20. A method or use according to any one of claims 16 to 19 wherein the pyrethrinoid is selected from deltamethrin, tralomethrin, cypermethrin, permethrin, alphamethrin, cyfluthrin, cyhalothrin, fenfluth rin, fencythrin and fenvalerate.