WO2011042565A2

WO2011042565A2 - Antiprotozoal activity

Info

Publication number: WO2011042565A2
Application number: PCT/EP2010/065189
Authority: WO
Inventors: Vincent Fernand Paul Delespaux
Original assignee: Instituut Voor Tropische Geneeskunde
Priority date: 2009-10-09
Filing date: 2010-10-11
Publication date: 2011-04-14
Also published as: WO2011042565A3

Abstract

The invention generally relates to methods and compositions for use in the treatment of parasitic protozoal infections. The methods and compositions of the present invention are particularly useful in the treatment of parasitic protozoal infections; in particular in the treatment of parasitic protozoal infections wherein the parasite causing the infection is resistant to said anti-protozoal drug.

Description

ANTI PROTOZOAL ACTIVITY Field of the Invention

The invention generally relates to methods and compositions for use in the treatment of parasitic protozoal infections. The methods and compositions of the present invention are particularly useful in the treatment of parasitic protozoal infections; more in particular in the treatment of parasitic protozoal infections wherein the parasite causing the infection is resistant to said anti-protozoal drug.

Background to the Invention

Human parasitic diseases are endemic in many parts of the world and are often caused by flagellate protozoa from the class of Kinetoplastidae including the following orders Oikomonadidae, Amphimonadidae, Monodidae, Bodonidae, Cryptobiidae and Trypanosomatidae . Amongst the order of Trypanosomatidae, Trypanosoma and Leishmania, are the most important genuses, causing parasitic diseases in humans as well as in animals.

Leishmaniasis for example (a parasitic disease caused by an obligate intracellular protozoan transmitted by the bite of some species of sand flies) is found in approximately 90 tropical and subtropical countries around the world and in southern Europe. More than 90% of the world's cases of cutaneous leishmaniasis are in Afghanistan, Algeria, Brazil, Iran, Iraq, Peru, Saudi Arabia, and Syria. However, approximately 75% of the cases that are evaluated in the United States were acquired in Latin America, where leishmaniasis occurs from northern Mexico (occasionally in rural southern Texas) to northern Argentina. More than 90% of the world's cases of visceral leishmaniasis occur in Bangladesh, Brazil, India, Nepal, and Sudan. Similarly, the geographical distribution of Chagas ' Disease (caused by a flagellate protozoan parasite, Trypanosoma cruzi, transmitted to humans by triatomine insects) extends from Mexico to the south of Argentina. The disease affects 16 - 18 million people and some 100 million, i.e. about 25% of the population of Latin America, is at risk of acquiring Chagas' disease. Even people staying for a short time in parasite-endemic areas can become infected and parasitic diseases are becoming a problem in the developed world as a result from the increase in global travel.

Not only for humans, also for the animal feed stock in tropical and sub-tropical regions around the globe, parasitic infections, in particular trypanosomiasis, are the major pathological threat for cattle breeders in those regions. Trypanosomiasis is one of the main pathological constraints of breeding in western and central Africa, where they are considered the diseases most frequently transmitted to cattle by a vector. The trypanosomiasis is caused by protozoan parasites of the genus Trypanosoma which include Trypanosoma vivax, Trypanosoma congolense, Trypanosoma brucei, Trypanosoma simiae, Trypanosoma equiperdum and Trypanosoma evansi.

In Africa, these parasites are usually transmitted by the bite of a fly of the genus glossina or "tsetse fly" (including 31 species and subspecies have been identified on the African continent) previously contaminated by the blood of an animal already infected by trypanosomes . These have serious health effects on humans as the "sleeping sickness", but also on livestock in which they induce weight loss, a fall of lactation, spontaneous abortions, failure to effort, may cause rapid death of animals that are infected, but are most often present in a chronic form.

The losses that result, are estimated at 1.5 billion U.S. dollars per year, slowing the development of mixed farming but also the supply of protein (meat, milk) to the population. Treatment typically involves the use of Trypanocidal drugs such as; isometamidium chloride (ISM), diminazene aceturate (DA) and homidium. All these molecules are on the market for veterinary drugs for over five decades and as a consequence, with on the one hand, liberalization of the practice of veterinary medicine and secondly the low purchasing power of most farmers, frequent and long-term use and improper dosage, has led to the phenomena of resistance in many countries in Africa.

For example, on the Adamaoua plateau in Cameroon up to 100% of the tested isolates were found resistant to ISM and to diminazene aceturate (DA) leaving farmers helpless. As no new drug is expected to be available in a near future and as the spreading of this phenomenon seems very rapid, a five fold increase in DA resistance within a seven years interval was observed in Eastern Province of Zambia (Delespaux et al . , 2008a), alternatives are urgently needed to circumvent Trypanocidal drug resistance (TDR) . Given the shortcomings of the currently available treatments of parasitic protozoal infections, it is an object of the present invention to provide a solution in the treatment of parasitic protozoal infections wherein the parasite causing the infection gained resistance to the typical Trypanocidal drugs. To achieve said objective, it has now surprisingly been found that antibiotics like tetracycline potentiate the trypanocidal activity of trypanocidal drugs in resistant strains of parasitic protozoal infections.

This and other aspects of the present invention will be explained in more detail hereinafter.

Brief Description of the Drawings

Figure 1 Survival analysis of mice infected with the resistant IL3343 Trypanosoma strain over a period of 140 days. Dots: untreated mice; squares: mice treated with tetracycline alone; triangles: mice treated with ISM alone; crosses: mice treated with ISM in combination with tetracycline.

Figure 2 Survival analysis of mice infected with the highly resistant TRT57 Trypanosoma strain over a period of 140 days. Dots: untreated mice; squares: mice treated with tetracycline alone; triangles: mice treated with ISM alone; crosses: mice treated with ISM in combination with tetracycline.

Figure 3 Parasitaemic analysis of cattle infected with the resistant IL3343 Trypanosoma congolense strain over a period of 95 days. Hairline: cattle treated with ISM alone; Line: cattle treated with ISM in combination with OTC; Bold line: cattle treated with ISM in combination with FQE .

Detailed Description of the Invention

In a first embodiment, the present invention provides the use of an antibiotic to potentiate the anti-protozoal activity of anti-protozoal drugs. As demonstrated in the examples hereinafter, it has been found that the complementation of a typical anti-protozoal treatment with an antibiotic like tetracyclines, significantly improves the survival of an animal challenged with resistant parasitic protozoal infections. Even upon challenge with highly resistant strains, complementation with an antibiotic like tetracyclines has a curative effect.

This enhanced activity against resistant parasitic protozoal infections is unprecedented over earlier publications on particular treatments of malaria and trypanosmiasis .

At the time of filing of the present application it was generally accepted (see for example Jennings, Veterinary Science, Vol.43, 1987, pages 173-176 & Yunmbam and Roberts, Comparitive Biochemistry and Physiology, Part C: Pharmacology, Toxicology & endocrinology, Vol. 105C(3), 1993, pages 521-524) that drug combination therapies of anti- protozoal drugs with antibiotics had no additive or synergistic effects. It was accordingly not to be expected that one would be able to potentiate the anti-protozoal activity of anti-protozoal drugs against resistant parasitic protozoal infections through a drug combination therapy of anti- protozoal drugs with antibiotics

This general understanding that the mere combination of anti- protozoal drugs with antibiotics has no additive or synergistic effects, is for example confirmed in the European patent application by Bourdichon A.J. (EP1220668) .

In said application it is shown that the presence of a sodium channel inhibitor like procaine or lidocaine is a requisite to potentiate the anti-protozoal effect of diminazene di aceturate, pentamidine or isometamidium chloride. In said reference, there is no indication that the same effect and in particular in the treatment of resistant parasitic protozoal infections, can be achieved through the use of a drug combination therapy of anti^¬ protozoal drugs with an efflux-inhibitor, i.e. antibiotics.

As explained in more detail hereinafter, the efflux inhibitors of the present invention interfere with the extrusion of the anti-protozoal drug from the parasite and would not include ion-channel inhibitors like procaine or lidocaine .

Accordingly, the present invention provides the combination of an antibiotic and an anti-protozoal drug for use in the treatment of parasitic protozoal infections; in particular in the treatment of resistant parasitic protozoal infections. Resistant parasitic protozoal infections as used herein, refers to parasitic protozoal infections wherein the parasite causing the infection gained resistance to said anti-protozoal drug. As used, the individual components in the aforementioned combination, i.e. the antibiotic (component a) and the anti-protozoal drug (component b) , may be present in the same or separate compositions and are accordingly suitable for sequential, separate and/or simultaneous use in the treatment of parasitic protozoal infections; in particular in the treatment of resistant parasitic protozoal infections, i.e. infections wherein the parasite causing the infection gained resistance to anti-protozoal drugs.

Thus, in a further objective, the present invention also provides a kit of parts comprising an antibiotic and an anti-protozoal drug for use in the treatment of parasitic protozoal infections; in particular in the treatment of resistant parasitic protozoal infections, i.e. infections wherein the parasite causing the infection gained resistance to said anti-protozoal drug. In a particular embodiment, the individual components in said kit are present in separate unit dosage forms, together with instructions for simultaneous or sequential use. It thus provides a commercial package comprising as active ingredients the combination according to the present invention (in the form of two or three or more separate units of the components (a) or (b) ) , together with instructions for its simultaneous, separate or sequential use, or any combination thereof, in the treatment of parasitic protozoal infections; in particular in the treatment of resistant parasitic protozoal infections, i.e. infections wherein the parasite causing the infection gained resistance to said anti-protozoal drug.

Alternatively, and as already mentioned herein before, in a further embodiment of the present invention, the individual components in the aforementioned combination, i.e. the antibiotic (component a) and the anti-protozoal drug (component b) , may be present in the same compositions. It accordingly provides a pharmaceutical composition comprising an antibiotic and an anti-protozoal drug for use in the treatment of parasitic protozoal infections; in particular in the treatment of resistant parasitic protozoal infections, i.e. infections wherein the parasite causing the infection gained resistance to said anti-protozoal drug.

The pharmaceutical compositions of the present invention can be prepared by any known or otherwise effective method for formulating or manufacturing the selected product form. Methods for preparing the pharmaceutical compositions according to the present invention can be found in "Remington's Pharmaceutical Sciences", Mid . Publishing Co., Easton, Pa., USA.

For example, the compounds can be formulated along with common excipients, diluents, or carriers, and formed into oral tablets, capsules, sprays, mouth washes, lozenges, treated substrates (e. g. oral or topical swabs, pads, or disposable, non-digestible substrate treated with the compositions of the present invention) ; oral liquids (e. g. suspensions, solutions, emulsions), powders, or any other suitable dosage form.

Non-limiting examples of suitable excipients, diluents, and carriers can be found in "Handbook of Pharmaceutical Excipients", Second edition, American Pharmaceutical Association, 1994 and include: fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as acetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite ; carriers such as propylene glycol and ethyl alcohol or ligation to nanobodies, and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.

As used herein, the anti-protozoal drugs are in particular diamidines including diminazene aceturate (DA) (e.g. Berenil®, Veriben®, Dophanil®) and pentamidine (e.g. Lomidine®, Pentam®, Nebupent®) ; melaminophenyl arsenicals including Melarsoprol (e.g. Arsobal®, MelB®) and Melarsomine (e.g. Cymelarsan®) and phenanthridines including isometamidium chloride (ISM) (e.g. Samorin®, Trypamidium®, Veridium®) and ethidium bromide (e.g. Homidium®, Novidium®) . For said second component, the effective amount which is required to achieve a therapeutical effect will, of course, vary with the particular composition, the route of administration, the age and condition of the recipient, and the particular disorder or disease being treated. For example anti-trypanosomal treatments such as for example melaminophenyl arsenicals, diamidines and phenanthridines, in particular selected from the group consisting of melarsoprol, cymelarsan, isometamidium chloride (ISM), pentamidine, diminazene aceturate (DA) and homidium; and anti Leishmania treatments such as for example the diamidine pentamidine. Generally, said amount of anti-protozoal drug will be determined on a case by case by an attending physician . Generally, a suitable dose of an anti-protozoal drug is one that results in a concentration of the anti-protozoal drug at-the treatment site in the range of 0.1 mM to 50 mM, and more usually 1 mM to 10 mM. To obtain these treatment concentrations, an animal in need of treatment likely will be administered an amount of about 0.01 to 5 mg/kg; in particular 0.1 to 3 mg/kg; in a further embodiment from 1.5 to 5 mg/kg. As noted hereinbefore, the above amounts may vary on a case-by-case basis. In these methods of treatment the compounds according to the invention are preferably formulated prior to admission. As described herein before, suitable pharmaceutical formulations are prepared by known procedures using well-known and readily available ingredients .

The antibiotics as used in the present invention are selected from the group consisting of tetracyclines, quinolones, aminoglycosides and fluoroquinolones. In a particular embodiment the antibiotic is a tetracycline, such as for example tetracycline hydrochloride, or oxytetracycline ; or a fluoroquinolone, such as for example enrofloxacine, difloxacine, ciprofloxacine .

Tetracyclines are a group of broad-spectrum antibiotics whose are defined as "a subclass of polyketides having an octahydrotetracene-2-carboxamide skeleton". They are collectively known as "derivatives of polycyclic naphthacene carboxamide" and act through the inhibition of protein synthesis by inhibiting the binding of aminoacyl-tRNA to the mRNA-ribosome complex. They do so mainly by binding to the 30S ribosomal subunit in the mRNA translation complex. Examples of tetracyclines include the naturally-occuring tetracyclines such as Tetracycline, Chlortetracycline, Oxytetracycline, and Demeclocycline ; and the semi-synthetic tetracyclines, such as Doxycycline, Lymecycline, Meclocycline, Methacycline, Minocycline, and

Rolitetracycline.

The quinolones and fluoroquinolones are a family of synthetic broad-spectrum antibiotics. The term quinolone(s) refers to potent synthetic chemotherapeutic antibacterials, the first generation of which was derived from an attempt to create a synthetic form of chloroquine, which was used to treat malaria during World War II. The parent of the quinolone class is nalidixic acid. The majority of quinolones in clinical use belong to the subset of fluoroquinolones, which have a fluorine atom attached to the central ring system, typically at the 6-position or C-7 position. Quinolones inhibit the bacterial DNA gyrase or the topoisomerase IV enzyme, thereby inhibiting DNA replication and transcription. Quinolones can enter cells easily via porins and therefore are often used to treat intracellular pathogens such as Legionella pneumophila and Mycoplasma pneumoniae. For many gram-negative bacteria DNA gyrase is the target, whereas topoisomerase IV is the target for many gram-positive bacteria.

Examples of quinolones that have been widely used in agriculture, include danofloxacin (Advocin, Advocid) , difloxacin (Dicural, Vetequinon) , enrofloxacin (Baytril) , ibafloxacin (Ibaflin), marbofloxacin (Marbocyl, Zenequin) , orbifloxacin (Orbax, Victas) , and sarafloxacin (Floxasol, Saraflox, Sarafin) .

In a particular embodiment of the present invention, the antibiotic used is a tetracycline selected from the group consisting of tetracycline hydrochloride, and oxytetracycline . Alternatively, the antibiotic used is a fluoroquinolone, such as for example the enrofloxacine .

Generally, the antibiotic drug will be administered an amount of about 0.01 to 500 mg/kg; in particular from about 0.1 to 300 mg/kg; in a further embodiment from about 1 to 100 mg/kg. As hereinbefore, the above amounts may vary on a case-by-case basis. In these methods of treatment the compounds according to the invention are preferably formulated prior to admission. As described herein before, suitable pharmaceutical formulations are prepared by known procedures using well-known and readily available ingredients .

In a particular embodiment of the combinations of present invention the antibiotic component is tetracycline hydrochloride, and the anti-protozoal component is ISM.

All of the aforementioned embodiments are particularly useful when the parasitic protozoal infection is caused by trypanosomatidae in particular Leishmania or Trypanosoma, more in particular Leishmania selected from the group consisting of Leishmania major, Leishmania infantum, Leishmania donovani, Leishmania Mexicana, Leishmania Braziliensis and Leishmania Tropica; or Trypanosoma selected from the group consisting of Trypanosoma vivax, Trypanosoma congolense, Trypanosoma brucei, Trypanosoma simiae, Trypanosoma equiperdum and Trypanosoma evansi.

It is also an object of the present invention to provide the mechanism of action related to the drug resistance observed in the aforementioned parasitic protozoal infections. Where it is known that anti-protozoal drugs like ethidium bromide have affinity for both the primary-active and the secondary- active transporters, it has now surprisingly been found that the antibiotic drugs (supra) interfere (reduce or inhibit) with the extrusion of the anti-protozoal drug from the parasite, allowing a prolonged anti-protozoal action and accordingly enhancing the anti-protozoal activity of anti^¬ protozoal drugs. Hence, in a further aspect, the present invention provides a method of treatment of a parasitic protozoal infection, said method comprising administering to a subject in need thereof a therapeutic amount of an anti^¬ protozoal drug in combination with a compound that interferes with, i.e. prevents or reduces, the extrusion of the anti-protozoal drug from the parasite, and accordingly acts as a chemosensitizing agent of the parasite for the anti-protozoal drug. As used herein, the compound that interferes with the extrusion of the anti-protozoal drug (hereinafter also referred to as an efflux inhibitor) is typically an inhibitor of either a primary-active transporter; i.e. a transmembrane transport protein that is dependent on ATP hydrolysis for the translocation of various substrates across the membrane, in particular ATP-binding cassette transporters (ABC-transporters) ; or of a secondary- active transporter, i.e. transmembrane transport proteins that are driven by electrochemical gradients that exist across the plasma membrane for the translocation of various substrates, in particular transporters belonging to the Major Facilitator Superfamily of transporters (MFS transporters) .

As is evident from the examples hereinafter, in a particular embodiment said efflux inhibitors consist of the antibiotic drugs as provided herein, but as will be evident to the skilled artisan should not be limited thereto. Any compound known to inhibit the aforementioned transporters can be used in combination with anti-protozoal drugs in the treatment of parasitic protozoal infections, and in particular in the treatment of resistant parasitic protozoal infections as provided herein. Other efflux inhibitors are for example described in J. Antimicrob. Chemother. (2007) 59, 1247-1260, and include alkaloids, such as reserpine; oligosaccharides, such as orizabin; diterpenes, such as carnosol; ethoxylated flavones, such as chrysosplenol-D; and isoflavones, such as chrysoplenetin .

This invention will be better understood by reference to the Experimental Details that follow, but those skilled in the art will readily appreciate that these are only illustrative of the invention as described more fully in the claims that follow thereafter. Additionally, throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains .

EXAMPLES

The following examples illustrate the invention. Other embodiments will occur to the person skilled in the art in light of these examples.

EXAMPLE 1 : Evaluation of the effect of tetracycline hydrochloride on the anti-protozoal activity of isometamidium chloride

Material and methods

Trypanosome strains

A drug-resistant T. congolense savannah type stock TRT57_Cio was isolated from cattle in 1996 in Eastern Zambia, cloned and conserved as stabilate in liquid nitrogen. It was found highly resistant to Isometamidium Chloride (ISM) and showed a CD50 > 10 mg/kg as tested according to the protocol described by Eisler et al . (2001). CD50 is determined as the curative dose that gives complete cure in 50% of the animals .

The second T. congolense savannah type cloned strain IL3343 was found resistant to ISM when tested in mice and has a CD50 in mouse of 1.7 mg/kg (Peregrine et al . , 1997) .

Group inoculation and treatment

The stabilates of the cloned T congolense isolates were reactivated in mice and blood was collected under terminal anaesthesia by heart puncture, when the parasitaemia reached 8 on Herbert and Lumsden' s scale. Subsequently 128 adult OF1 (Oncins France 1) mice weighing on average 30g were divided in 8 groups of 16 mice each and inoculated with one of the two trypanosome clones, as indicated in table 1. Each mouse was inoculated with 5xl0⁵ trypanosomes through intraperitoneal injection.

Table 1. Inoculation and treatment scheme of the mice

Twenty four hours after inoculation, groups 1 and 5, as indicated in table 1, were left untreated and served as control, groups 2 and 6 were treated for 30 days with lOOmg/kg/day tetracycline per os, groups 3 and 7 were treated with lmg/kg ISM injected intraperitoneally, and groups 4 and 8 were treated with lmg/kg ISM injected intraperitoneally associated to a 30 day treatment with lOOmg/kg/day tetracycline per os. The treatment per os was done by mixing 250mg tetracycline hydrochloride (Sigma- Aldrich) in 200ml drinking water proposed ad libitum. Mean water consumption of the mice was observed before and during the experiment and was on average 3ml/day/mouse at 18°C. This water intake was not affected by the presence of tetracycline in the drinking water. All mice were monitored three times a week for survival and presence of trypanosomes for a period of 140 days. Mice were euthanized when their health status as determined by clinical examination was deteriorating rapidly (prostration, lateral decubitus, hyperventilation, unconsciousness and/or PCV ≤ 20).

End of the experiment

All surviving mice at day 140 were euthanized and the blood collected with anticoagulant. The DNA of the whole blood sample was then extracted using a routine phenol-chloroform- isoamyl alcohol method (Sambrook et al . , 1989) . To confirm the presence or absence of parasites, the PCR technique on the small subunit of the ribosomal DNA (Ssu-rDNA) was used (Geysen et al . , 2003; Delespaux et al . , 2003).

Statistical analysis

The first appearance of trypanosomes in mice blood and the survival of the mice in the 8 groups was analysed in two separate survival models in Stata 10 using groups as an explanatory variable.

Results

Resistant IL3343 Trypanosoma strain

Survival of the mice, inoculated with the resistant Trypanosoma strain IL3343, was determined over a period of 140 days.

As shown in figure 1, all untreated mice (group 5, dots) as well as all the mice treated with tetracycline hydrochloride alone (group 6, squares) died within 20 days after inoculation. Treatment with ISM alone (group 7, triangles), and more importantly treatment with ISM in combination with tetracycline hydrochloride (group 8, crosses) gave a substantially higher survival rate as shown in figure 1, i.e 81% and 94% of the mice in groups 7 and 8 respectively survived until the end of the experiment (140 days after inoculation) .

Highly resistant TRT57_Cio Trypanosoma strain

Survival of the mice, inoculated with the highly resistant Trypanosoma strain TRT57_Cio , was determined over a period of 140 days.

As shown in figure 2, all untreated mice (group 1, dots) as well as all the mice treated with tetracycline hydrochloride alone (group 2, squares) and with ISM alone (group 3) died within 20 days after inoculation. Interestingly, mice treated with a combination of tetracycline hydrochloride and ISM (group 4, crosses) showed a significantly higher survival rate compared to all other groups; i.e. p>0.05 compared to group 1, and p = 0.01 compared to group 3. Furthermore, a single mouse even survived the infection for 140 days, which was never observed before in our laboratory. Furthermore, at day 140, the PCR was negative indicating that the mouse actively managed to completely clear the parasite .

EXAMPLE 2 : Evaluation of the combination therapy in cattle Material and methods

Trypanosome strains

The T. congolense savannah type cloned strain IL

3343 was identified as resistant to isometamidium chloride (ISM) when tested in mice (CD50 = 1.7 mg/kg) (Peregrine et al . , 1997) with the CD50 defined as the curative dose that gives complete cure in 50% of the animals. Cattle inoculation and treatment

The stabilates of the cloned isolate IL3343 were reactivated in six mice. When the parasitaemia reached 8 on Herbert and Lumsden' s scale, blood was collected under terminal anaesthesia by heart puncture. Three treated groups, hereinafter referred to as groups A, B, and C, each of 6 adult crossbred zebus weighing on average 158kg each

(extremes 140 and 201kg) were inoculated with 5x105 trypanosomes each by intra-j ugular injection 30 days after blanking with DA (7mg/kg) and deworming. One non treated control group of 2 cattle was inoculated in the same way. The 20 cattle were housed in fly-proof facilities. From day 7 after the inoculation, all animals were monitored 2 times a week during 95 days for Packed Cell Volume of Hematocrit

(PCV's) and for the presence of parasites by microscopic examination of the buffy coats and PCR (22) performed from buffy coats placed on filter paper Whatman 4. The DNA was obtained using a routine chelex-based extraction method

(21) .

At the first parasitaemia, group A was treated once with 0.5mg/kg ISM, group B once with 0.5mg/kg ISM associated with 20mg/kg oxytetracycline (OTC) (Terramycin LA®) every 3 days for 30 days and group C once with 0.5mg/kg ISM associated with 5mg/kg fluoroquinolone enrofloxacine (FQE) (Baytril 100®) every 2 days for 30 days. The 35 injection sites for both drugs were alternatively selected in forehand and hindquarters, shaved and coloured with methylene blue and picric acid for OTC and FQE respectively. A minimal distance of 6 cm between injection sites was respected. Statistical analysis

The survival of the cattle in the three groups was analysed in the survival models in Stata 10© (Copyright 1996-2009 StataCorp LP) using groups as an explanatory variable. A log-normal distribution was used in a parametric model. The start of the model corresponded to the inoculation day and the experiment was short enough to ignore natural mortality of the animals.

- The animals' PCV values were analyzed using a cross- sectional linear regression, accounting for repeated measures from individual animals. Explanatory variables were the animal groups, post-treatment periods and the interactions between them. Three post-treatment periods containing each the same numbers of samplings were defined as follows: day 1-21, day 22-54 and day 55-98. The interaction term between the groups and the third period was used as indicator of the disease impact on PCV.

Results

Resistant IL3343 Trypanosoma strain in cattle

- The two untreated control animals became parasitaemic 11 days after inoculation and were treated with DA (7 mg/kg) on day 30 because their PCV s reached the critical value of 25.

- All 6 animals of group A (ISM) became positive for trypanosomes between days 24 and 46.

- When ISM was used in combination with either OTC (group B) or FQE (group C) , only half of the cattle became positive for trypanosomes and the prepatent period was significantly longer (p < 0.001; Fig. 3), i.e. between days 46 and 82. In the groups B (ISM-OTC) and C (ISM-FQE), the parasitaemia remained very low, with PCR results fluctuating with animals being detected parasitaemic every 2 or 3 weeks, indicating a parasitaemia oscillating just above and below the detection limit of the PCR test, i.e. 25 trypanosomes/ml blood.

- The impact of the disease on PCV was not very pronounced, even in group A (average PCV reduction 8 to 14 weeks after treatment: 5.9%; 95% CI: 4.5 - 7.3). However, this impact was lower in groups B (ISM-OTC) and C (ISM-FQE) compared to group A (ISM) (p<0.01) .

These observations indicate that even in the case of established ISM-resistance of the trypanosomes , farmers can still benefit from the use of the trypanocide when used in combination with an efflux-inhibitor like the aforementioned antibiotics, by a significant decrease of the effect of the disease on the global health status of the animals.

TC, FQE and some derivatives thereof are cheap drugs, registered for use in livestock, widely available on the African market. More importantly, these drugs are commonly used by African farmers and will not require elaborate new chemistry and safety tests. Hence, assuming that further trials confirm the effectiveness of the antibiotics in potentiating the activity of trypanocidal drugs in cattle under natural tsetse challenge, the new control approach can be implemented rapidly. It is likely that the combination ISM-TC/OTC can also be made more cost effective after dosage and duration adjustments.

Furthermore, several analogues of TC/OTC and FQE are available. These compounds are at the moment in a screening phase aiming at the optimization of the delivery system to increase the specificity of the treatment, to boost the intracellular concentration of the chemo-sensitizer within the trypanosome and to reduce the dose to achieve an effective competitive chemo-sensitization . Further research is on going to identify the best galenic solution, the optimal combination of chemo-sensitizer with ISM (qualitative and quantitative) and to test this combination in livestock under controlled and field conditions in areas with high tsetse challenge and high TDR.

An effective combination of ISM and chemo-sensitizer (s) , i.e. efflux inhibitor, should result in (i) a decrease in the proportion of circulating strains resistant to ISM (ii) a decrease in the proportion of circulating strains resistant to diminazene aceturate (DA) since the association ISM-chemo-sensitizer will eliminate both strains resistant to DA and strains with DA/ISM multi-resistance and (iii) a decreased impact of the disease on the health status of the cattle. Strategic use of this approach may result in an increased efficacy of currently available trypanocidal drugs in extensive areas of sub-Saharan Africa where their use was severely curtailed as a result of the development of resistance in trypanosomes .

Ref erences

Eisler MC, Brandt J, Bauer B, Clausen PH, Delespaux V, Holmes PH, et al . Standardised tests in mice and cattle for the detection of drug resistance in tsetse-transmitted trypanosomes of African domestic cattle. Vet Parasitol 2001 Jun 12; 97 (3) : 171-82.

Peregrine AS, Gray MA, Moloo SK. Cross-resistance associated with development of resistance to isometamidium in a clone of Trypanosoma congolense. Antimicrob Agents Chemother 1997 Jul; 41 (7) :1604-6.

Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Arbor; 1989.

Geysen D, Delespaux V, Geerts S. PCR-RFLP using Ssu-rDNA amplification as an easy method for species-specific diagnosis of Trypanosoma species in cattle. Vet Parasitol 2003 Jan 2 ; 110 (3-4) : 171-80.

Delespaux V, Ayral F, Geysen D, Geerts S. PCR-RFLP using Ssu-rDNA amplification: applicability for the diagnosis of mixed infections with different trypanosome species in cattle. Vet Parasitol 2003 Nov 14; 117 (3) : 185-93.

Claims

1. Use of an efflux inhibitor to potentiate the anti^¬ protozoal activity of anti-protozoal drugs.

2. A combination of an efflux inhibitor and an antiprotozoal drug for use in the treatment of parasitic protozoal infections; in particular for use in the treatment of resistant parasitic protozoal infections.

3. A kit of parts comprising an efflux inhibitor and an anti-protozoal drug for use in the treatment as defined in claim 2.

4. A pharmaceutical composition comprising an efflux inhibitor and an anti-protozoal drug for use in the treatment as defined in claim 2.

5. The use according to claim 1; the combination according to claim 2; the kit of parts according to claim 3; and the pharmaceutical composition according to claim 4 ; wherein the efflux inhibitor is an antibiotic.

6. The use according to claim 1; the combination according to claim 2; the kit of parts according to claim 3; and the pharmaceutical composition according to claim 4 ; wherein the anti-protozoal drugs are selected from the group consisting of melaminophenyl arsenicals, diamidines and phenanthridines , in particular melarsoprol, cymelarsan, isometamidium chloride (ISM) , pentamidine, diminazene aceturate (DA) and homidium.

7. The use according to claim 1; the combination according to claim 2; the kit of parts according to claim 3; and the pharmaceutical composition according to claim 4; wherein the efflux inhibitor is an antibiotic selected from the group consisting of tetracyclines, quinolones and aminoglycosides.

8. The use; the combination; the kit of parts; and the pharmaceutical composition according to claim 7; wherein the antibiotic is tetracycline hydrochloride, and the anti-protozoal drug is ISM.

9. The combination according to claim 2; the kit of parts according to claim 3; and the pharmaceutical composition according to claim 4; wherein the parasitic protozoal infection is caused by a Kinetoplastidae, in particular a Trypanosoma or a Leishmania.

10. The combination according to claim 2; the kit of parts according to claim 3; and the pharmaceutical composition according to claim 4; wherein the parasitic protozoal infection is caused by a Trypanosoma, in particular a Trypanosoma selected from the group consisting of Trypanosoma vivax, Trypanosoma congolense, Trypanosoma brucei, Trypanosoma simiae, Trypanosoma equiperdum and Trypanosoma evansi; more in particular Trypanosoma congolense.

11. The combination according to claim 2; the kit of parts according to claim 3; and the pharmaceutical composition according to claim 4; wherein the parasitic protozoal infection is caused by a Leishmania, in particular a Leishmania selected from the group consisting of Leishmania major, Leishmania infantum, Leishmania donovani, Leishmania Mexicana, Leishmania braziliensis , Leishmania tropica.