INTRODUCTION

The present invention relates to compositions

comprising neonicotinoids (as active ingredient ( s )) , and specifically their systemic use in humans against free- living ectoparasites. Importantly, although such compounds have been used in and on companion animals against

temporary and permanent ectoparasites in the veterinary and plant-breeding sectors, the present invention relates to the surprising findings of their hitherto unexplored application in humans for the control of free-living ectoparasites and the diseases they transmit.

Free-living ectoparasites visit their blood hosts for the duration of blood feeding only and live elsewhere in the environment outside this activity (e.g. mosquitoes) . Temporary ectoparasites stay significantly longer on their hosts, (i.e. several) days (e.g. fleas and ticks), whereas permanent ectoparasites complete their entire life-cycle on the host, never leaving it (e.g. head lice) . Beyond the novel application in humans against free- living ectoparasites the present invention is also the first to demonstrate that single neonicotinoids are effective killing agents for free-living ectoparasites when present in the bloodstream of humans. The invention

therefore creates multiple opportunities for systemic use in humans to interfere with the transmission, by free- living ectoparasites, of disease-causing pathogens. Given the huge public health burden caused by diseases like malaria (300 million cases per annum, with 800.000 deaths) and dengue (50-100 million cases per annum), as reported by the World Health Organization, such novel application has high significance for alleviating human suffering,

particularly in developing countries.

BACKGROUND

Neonicotinoids are an important class of synthetic insecticides (Tomizawa and Casida, 2003), with a common mode of action that affects the central nervous system of arthropods, initially causing paralysis followed by death. Compounds of this class of insecticides act on the central nervous system of insects by binding at a specific site, the postsynaptic nicotinic acetylcholine receptor channels (nAChRs) .

In blocking this specific neural pathway, which is more common in arthropods than in warm-blooded animals, toxicity is far more pronounced in the former than the latter group of organisms, rendering WHO/EPA classification of these compounds in toxicity class II or III.

Advantageously neonicotionids may, through their unique mode of action that differs from that of classical insecticides used in arthropod pest control, like

organochlorines , organophosphates , carbamates, and

synthetic pyrethroids, be used to manage pests that have developed (physiological) resistance to these classes of insecticides. Neonicotinoids have a fast mode of action. Moreover, their low mammalian toxicity (see below) offers unique prospects for systemic use beyond plants or animals, i.e. in humans, which forms part of the current invention. Uses of neonicotinoids are reported from both the veterinary and plant breeding sectors, whereby use of the compounds in the environment (e.g. through spraying or soaking) as well as systemic uptake (in plant nutrient substrates) or topical and oral administration (in

companion animals, notably cats and dogs) in different combinations is practiced. For instance, oral application of a single dose (at recommended levels of 1 mg/kg

bodyweight) of nitenpyram provides 100% kill of flea infestations on cats for up to 48 hours (Rust et al . ,

2003); see also WO 00/21371. Oral use in tablet form of 11.4 mg can be used in animals of 2 pounds bodyweight, essentially extending the dose range from 1 - 10 mg/kg bodyweight (Novartis, 2011) .

Additional uses are described in:

US5750548 : 1- [N- (halo-3-pyridylmethyl ) ] -N-methy1amino- 1- alklamino-2-nitroethylene derivatives for controlling fleas in domestic animals ;

WO2008062821 : Composition for controlling plant disease and insect damage and method for preventing plant disease and insect damage ; For example nitenpyram (N- ( ( 6-chloro-3- pyridinyl) methyl) -N ethyl-N' -methyl-2-nitro-l , 1- ethenediamine ) , is a neonicotinoid that acts as an agonist of nAChRs and is used as a systemic flea control drug for companion animals. Nitenpyram does not inhibit

acetylcholinesterase. Nitenpyram, when administered orally to cats/dogs, acts within 30 min, and kills 90% of fleas within 4-6 hours. Oral (ORL) and dermal (SKN) toxicity of nitenpyram is low, ORL-Rat (male) LD ₅₀ 1680 mg kg ^-1 , ORL-Rat (female) LD ₅₀ 1575 mg kg ^-1 , SKN-Rat LD ₅₀ > 2000 mg kg ^-1 (MSDS, 2006) . A summary of safety and dose-effectiveness trials with nitenpyram is available (FDA) .

There are seven neonicotinoid insecticides currently on the market: three cyclic compounds, that is,

neonicotinoids with five-membered ring systems such as imidacloprid and thiacloprid (Bayer CropScience) and the six-membered neonicotinoid thiamethoxam (Syngenta) , and four noncyclic compounds, that is, nitenpyram

(Sumitomo Chemical Takeda Agro Co.), acetamiprid (Nippon Soda) , clothianidin (Sumitomo Chemical Takeda Agro Co. /Bayer CropScience), and dinotefuran (Mitsui Chemicals) .

Clothianidin (i) is known for the publication EP 0376279 A2 :

Thiacloprid (ii) is known for the publication EP 0235725 A2.

Dinotefuran (iii) is known for the publication EP 0649845 Al .

Acetamiprid (iv) is known for the publication WO 91/0465 Al .

Nitenpyram (v) is known for the publication EP 0302389 A2.

Imidacloprid (vi)is known for the publication EP 0192060 Al .

And Thiamethoxam (vii)is known for the publication EP 0580553 A2.

There are several ways to subdivide the seven commercially available neonicotinoids into different groups. They can be classified by their pharmacophore moieties [-N— C(E)dX—Y] (see Fig.l: part ii), as N-nitroguanidines

( imidacloprid, thiamethoxam, clothianidin, and

dinotefuran) , nitromethylenes (nitenpyram) , and N- cyanoamidines (acetamiprid and thiacloprid) .

Another way to subdivide them is via their different nicotyl groups. This method subdivides them into

chloronicotinyl (imidacloprid, nitenpyram, acetamiprid and thiacloprid) , thianicotinyl (thiamethoxam and

clothianidin) and furanicotinyl (dinotefuran) molecules.

A more general way to subdivide the neonicotinoids is division into ring systems (imidacloprid, thiacloprid and thiamethoxam) and non-cyclic compounds (Nitenpyram,

Acetamiprid, Clothianidin and Dinotefuran) . The overall chemical structure of both rings systems and non-cyclic compounds consists of three segments (see Fig.l) : i. for five and six-membered ring systems the bridging fragment -Z- between R ¹ and R ² including the two substituents and

for noncyclic neonicotinoids the separate substituents (Rl, R2) . i. the hetarylmethyl or heterocyclylmethyl group R i . the functional group [ = X-Y] as part of the dif- ferent pharmacophore types [-N-C(E) = X-Y] .

X-Y

Figure 1 : The general chemical structure for

neonicotinoids . The different substituents are shown in Table 1.

Table 1: Different known chemical structures belonging to the neonicotinoids

Noncyclic neonicotinoids

Commercial

R R ¹ X-Y Ξ R ² names

6-chloro-pyrid-3-

H CHN0 ₂ NH ₂ \ ylmethyl (CPM)

6-chloro-pyrid-3-

H CHN0 ₂ NH N(CH ₃ ) ₂ ylmethyl (CPM)

6-chloro-pyrid-3- CH ₃

CHN0 ₂ NH CH ₃ ylmethyl (CPM) (=Me)

6-chloro-pyrid-3-

CH ₃ CHN0 ₂ NH CH ₂ CH ₃ ylmethyl (CPM)

6-chloro-pyrid-3- CH ₂ CH ₃

CHN0 ₂ NH CH ₃

Nitenpyram ylmethyl (CPM) (=Et)

6-chloro-pyrid-3-

CH ₂ CH ₃ CHN0 ₂ NH CH ₂ CH ₃ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ CH ₃ CHN0 ₂ NCH ₂ CH ₃ CH ₂ CH ₃ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ CH ₃ CHN0 ₂ NH cyclopropyl ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ CH ₃ CHN0 ₂ N(CH ₂ ) ₄ \ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ CH ₂ F CHN0 ₂ NH ₂ \ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ CH ₂ CH ₃ CHN0 ₂ NH ₂ \ ylmethyl (CPM)

6-chloro-pyrid-3-

CH(CH ₃ ) ₂ CHN0 ₂ NH CH ₃ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₃ NCN CH ₃ \

Acetamiprid ylmethyl (CPM)

2-chloro-l, 3-thiazol-

H NN0 ₂ NH CH ₃

Clothianidin 5-ylmethyl (CTM)

(+/-) -6-tetrahydrofur-

H NN0 ₂ NH CH ₃

Dinotefuran 3-ylmethyl (TFM) Cyclic neonicotinoids

Commercial

R R ¹ X-Y Ξ R ² z names

3-Pyridylmethyl CH ₂ CHN0 ₂ NH 0 CH ₂

6-chloro-pyrid-3-

CH ₂ CHN0 ₂ NH 0 CH ₂ ylmethyl (CPM)

6-Methyl-pyrid-3-

CH ₂ CHN0 ₂ NH 0 CH ₂ ylmethyl

6-chloro-pyrid-3-yl CH ₂ CHN0 ₂ NH 0 CH ₂

2- (6-chloro-3-

CH ₂ CHN0 ₂ NH 0 CH ₂ pyridyl) ethyl

2-Pyridylmethyl CH ₂ CHN0 ₂ NH 0 CH ₂

4-Pyridylmethyl CH ₂ CHN0 ₂ NH 0 CH ₂

Benzyl CH ₂ CHN0 ₂ NH 0 CH ₂

4-chloro-benzyl CH ₂ CHN0 ₂ NH 0 CH ₂

5- (2-C1-

CH ₂ CHN0 ₂ NH 0 CH ₂ thiazolyl ) methyl

3-Pyridylmethyl CH ₂ NN0 ₂ NH 0 CH ₂

6-chloro-pyrid-3-

CH ₂ NN0 ₂ NH 0 CH ₂

Imidacloprid ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ NN0 ₂ NCH ₃ 0 CH ₂ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ NCN NH 0 CH ₂ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ CHCN NH 0 CH ₂ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ CHN0 ₂ CH ₂ 0 CH ₂ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ CHN0 ₂ 0 0 CH ₂ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ CHN0 ₂ S 0 CH ₂ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ CHN0 ₂ NH CH ₂ CH ₂ ylmethyl (CPM)

6-chloro-pyrid-3-

CH ₂ NCN S 0 CH ₂

Thiacloprid ylmethyl (CPM)

(+/-) -6-tetrahydrofur-

CH ₂ NN0 ₂ NCH ₃ CH ₂ 0

Thiamethoxam 3-ylmethyl DESCRIPTION

The present invention aims at killing free-living blood feeding ectoparasites, in particular the novel use of neonicotinoids for this purpose, shortly after feeding on a human host, and in this way prevent such arthropod

ectoparasites from surviving to subsequently pass on pathogens to other humans. Compositions comprising one or more active compounds belonging to the group of

neonicotinoids can thereto be made suitable for

administration in humans, for instance oral administration.

Surprisingly, we found that systemic use of only nitenpyram without additional compounds in humans, at 1,2 mg/kg bodyweight, results in 100% kill of free-living ectoparasites within 12 hours post blood feeding at 1-24 hours post drug administration (Table 2) . Given that pathogen development in free-living ectoparasites (the so- called extrinsic incubation period) exceeds this period of survival, this novel use in humans can therefore be applied to stop transmission of pathogens. Nitenpyram is rapidly excreted from circulation (through the urinary system) and insects feeding on humans after 24 hours survive mostly unscathed .

Table 2. Effect of oral administration of nitenpyram on a free-living blood feeding arthropod (the dengue mosquito Aedes aegypti) at various times post-administration.

Now therefore, neonicotinoids , notably nitenpyram, can be used to kill free-living blood feeding ectoparasites, shortly after feeding on a human host, and in this way prevent such ectoparasites from surviving to pass on pathogens to other humans/livestock. Such free-living ectoparasites are considered to be mosquitoes {Culicidae) , tsetse flies (Glossinidae) , deer and horse flies

{Tabanidae) , midges {Ceratopogonidae) , black flies

{ Simuliidae) , stable flies (Muscidae) , kissing bugs

(Reduviidae) , and sand flies ( Psychodidae) .

The invention specifically focuses on the second medical use of neonicotinoids, notably nitenpyram, as a killing agent in human blood for free living ectoparasites. These compositions can be used for:

1. Altruistic drugs for dengue virus (DENV)

transmission control. There is, at present, no specific medication or vaccination against DENV. Mosquitoes that transmit DENV, notably the Yellow Fever mosquito Aedes aegypti L., preferably reside within the premises close to its breeding grounds. Infections with DENV therefore often lead to disease clusters, whereby those living in the immediate vicinity (i.e. within the same household) of a dengue-infected person also become infected.

The period of infectiousness of DENV for mosquitoes (i.e. the period during which humans can pass the virus to mosquitoes) coincides with the period of intense febrile illness in humans (WHO, 2009) , when normally medical care will be sought. This infective period typically lasts for 4-8 days. Prescription of neonicotinoids , notably

nitenpyram, by health providers during the infective period, with daily administration of 1 mg/kg bodyweight, will result in the killing of all biting potential mosquito vectors within 24 hours, thus completely blocking virus transmission. Although the patient remains ill, he/she will avoid further dissemination of the virus in the

environment, hence the term Altruistic drug' .

Alternatively, slow-release formulations can be

administered so that a single or few doses will suffice to kill all mosquitoes during the host's infectious period.

Alternatively, if the viremic patient is not capable of taking such drugs, those living in the patient's

vicinity may do so to halt the spread of DENV. The option described here does also include use by both the patient and those living nearby.

2. Mass drug administration, in elimination or

eradication campaigns of ectoparasite-borne infectious diseases, for example malaria, and particularly for

diseases of which the ectoparasites that transmit them have a strong innate tendency to consume human blood (i.e.

anthropophilic arthropods), such as the malaria mosquitoes Anopheles gambiae sensu stricto Giles, An. funestus Giles, and the arbovirus vectors Aedes albopictus Skuse and Aedes aegypti . Such mass drug administrations can for instance be integrated with conventional mosquito control strategies, in such manner as to wipe out remaining pockets of

infestation .

3. A means to interrupt transmission of diseases like malaria, through prescriptive treatment following recovery from infection with asexual parasites. Such recovery is normally followed by a period in which human hosts are infectious to mosquito vectors. Carriage of infective parasites (so-called gametocytes) often occurs after the patient is sick, and leads to further transmission of the disease to mosquitoes, which can thus be overcome. Most drugs to cure malaria do not affect the infectious stage of the disease (with the exception of primaquine) , and

nitenpyram thus provides a novel means to stop transmission of malaria.

4. Mass drug administration if an epidemic disease outbreak occurs. Epidemic outbreaks of many ectoparasite- borne diseases are often restricted to so-called hotspots (Bousema et al . , 2010) . Targeted interventions based on mass drug administration in such hotspots can be used to prevent the epidemic from spreading further. For instance, models have shown that 80% of malaria transmission

originates from 20% of the population, thus targeted interventions may have a huge impact on disease prevalence and incidence (Bousema et al . , 2010) . Similar uses are foreseen for outbreaks of leishmaniasis, (remaining) foci of river blindness (onchocerciasis), and foci of human sleeping sickness (trypanosomiasis) . This latter use may include treatment of livestock as a means to eliminate their role as a parasite reservoir from which human

infections may culminate.

5. A means to control intra-domiciliary ectoparasitic arthropods transmitting pathogens, particularly kissing bugs (e.g. Triatoma infestans) that spend the vast majority of their lifecycle indoors and transmit the causative parasite of Chagas disease, Trypanosoma cruzii.

What is set out in the above for nitenpyram also holds for other members of the group of active neonicotinoid compounds including imidacloprid, thiacloprid, acetamiprid, thiamethoxam, and/or its subgroups e.g. nitroguanidines such as clothianidine .

In the above mentioned compositions the one or more compounds belonging to the group of neonicotinoids may be mixed with pesticides, fungicides and/or insecticides, as long as the thus practically administered composition is not too toxic if administered to humans.

References :

Bousema T, Drakeley C, Gesase S, Hashim R, Magesa S, Mosha F, Otieno S, Carneiro I, Cox J, Msuya E, Kleinschmidt

I, Maxwell C, Greenwood B, Riley E, Sauerwein

R, Chandramohan D, and Gosling R. (2010) . Identification of hot spots of malaria transmission for targeted malaria control. J Infect Dis, 201, 1764-1774. Food and Drug Administration. NADA 141-175. See:

http : //www. fda . gov/downloads/AnimalVeterinary/Products/Appr ovedAnimalDrugProducts/FOIADrugSummaries/ucml 17258. pdf

MSDS (2006) . Material Safety Data Sheet for Nitenpyram. See: http://msds.chem.ox.ac.uk/NI/nitenpyram.html

Tomizawa, M. and Casida, J.E. (2003) . Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors. Annu Rev Entomol., 38, 339- 364.

World Health Organization (2009) . Dengue: Guidelines for diagnosis, treatment, prevention and control. TDR/WHO publication WHO/HTM/NTD/DEN/2009.1.