DICHLOROACETATE COMPOUNDS FOR USE IN TREATING A DISEASE CAUSED BY A GLYCOLYTIC PARASITE

STATE OF THE ART

LEISHMANIASES are diseases caused by species of the kinetoplastid parasite Leishmania spp. and transmitted by hematophagous sandflies. Leishmaniases represent a major, although grossly underestimated, health problem: over 350 million people are at risk, Leishmaniases are endemic in 88 countries, the worldwide prevalence is > 12 million cases/year and the incidence is > 2.5 million cases/year. The estimated disease burden is 2.4 million disability-adjusted life years (DALYs). The clinical presentation is dependent upon both the parasite species and the host's immune response. Visceral Leishmaniasis is the most severe form of the disease with deadly epidemics that periodically flare up but go mostly unnoticed. Visceral Leishmaniasis has an estimated annual incidence of 500000, a 90 % mortality rate if left untreated, and accounts for around 70000 deaths per year, thus ranking second only to malaria for mortality and fourth for morbidity amongst tropical parasitic diseases^, 2). Among the various species, Leishmania donovani causes Anthroponotic Visceral Leishmaniasis, whereas L. infantum (syn. L. chagasi) is the causative agent of the zoonotic form, with the domestic dog as its main reservoir. The dog itself also suffers the lethal Canine Leishmaniasis. Leishmaniases control remains a difficult issue and eradication of the disease is even more difficult. It depends on personal protection from sandfly bites and on reservoir or vector control. The current Leishmaniases control programs have largely failed mainly because of the insufficient regional health delivery systems and due to the limited local resources. Research is being conducted on antileishmanial vaccines(3, 4) but, at present, there are only 3 vaccines marketed for use in the dog and phase IV studies are lacking. As it is the case for most parasitic diseases, the control of Leishmanises is almost confined to far from effective chemotherapy. Thus, the World Health Organization has acknowledged leishmaniases as a category 1 disease ("emerging or uncontrolled diseases") that has been severely neglected, and urging intensified research programs to improve vector control, diagnostics and therapeutic arsenal to contain further incidence and morbidity(5). In addition, Visceral Leishmaniasis and Human Immunodeficiency Virus co-infection is on the rise in all ecozones and poses a new and difficult challenge to containment efforts.

There are limited numbers of drugs available for treating leishmaniases. They require long periods of administration, induce serious side effects, prone to resistance development, and not affordable for the poor(6). Furthermore, most of these drugs are largely dependent on a non-damaged immune system, which is not usually the case.

Pentavalent antimonials are still the first line drugs according to World Health Organization. Resistances have increased due to improper use and to Human Immunodeficiency Virus co-infection(7). Initial treatments respond well, but in India there is a large proportion of primary failures(8, 9) and there is fear for spreading to other ecozones. They can cause severe adverse reactions: cardiac arrhythmias, acute pancreatitis, nephrotoxicity and death( iO). It is also the first line drug for Canine Leishmaniasis. Amphotericin B replaces pentavalent antimonials in ecozones with therapeutic failures above 60% There are few and contradictory studies on resistance in

Leishmaniasis ( 12), thus the emergence of resistances should be monitored in extended use ( 13). Adverse reactions are frequent: fever and chills, and other potentially fatal as hypokalemia, nephrotoxicity or anaphylaxis^). Liposomal amphotericin B preparations exhibit fewer adverse reactions, but this feature comes at the cost of increased cost. Currently, it is often the first choice drug in developed countries, but it is still too expensive for underdeveloped endemic countries. The results have been contradictory in Canine Leishmaniasis, with high neurotoxicity^^ and little evidence for use in this species^).

Miltefosine, developed as antineoplastic, is the first effective oral drug against Visceral Leishmaniasis^ / ⁷ ) . In India in phase IV trials it reached effectiveness ~ 90% ( 18), and in Ethiopia was safe and as effective as pentavalent antimonials in the absence of Human Immunodeficiency Virus co-infection, but less effective in patients co- infected (19). It is teratogenic and causes gastrointestinal problems, sometimes serious. The sensitivity is variable with fully resistant strains(20) but it is easy to induce in vitro resistance^?). Data from India suggests that after treatment the rate of relapse doubles(22). Its use in Canine Leishmaniasis may aggravate resistances(23).

Paromomycin is used parenterally or applied topically with few adverse reactions(24). Its effectiveness depends on the species(25) and the ecozone(26). Because it is an aminoglycoside, a rapid rise in resistances is expected, especially in monotherapy(27). Use in Canine Leishmaniasis is limited by significant adverse reactions and frequent relapses(28).

MALARIA is caused by glycolytic parasites of the Plasmodium genus, threatening 3.2 billion people in 97 countries every year and infecting and developing disease in 200 million people from which 627 000 will die, 90 % of which were in the African Region, followed by Southeast Asia (7 %) and the Eastern Mediterranean (3 %). About 482 000 malaria deaths are estimated to occur in children under 5 years of age, constituting 77 % of the global total. Most of these deaths due to Plasmodium falciparum. However, P. vivax is now increasingly recognized as a cause of severe malaria and death (30). For decades, drug resistance has been one of the greatest obstacles in fighting malaria. Drug resistance has been reported in three of the five Plasmodium species that is, P. falciparum, P. vivax and in P. malariae which are the causative agents for human malaria^i). Malaria is nowadays considered the most important parasitic diseases all around.

Chloroquine has been the agent of choice for many decades because of its safety, efficacy and affordability, but resistances were observed for first time back in 1957 in Thailand, that later spread to South America and to Africa. At present, chloroquine remains effective only in some parts of Central America.

Amodiaquine was found to be more effective than chloroquine in areas of persistent chloroquine resistance and combination with artesunate was adopted as the first-line treatment by several countries. But again highly resistant strains to amodiaquine have been reported in East Africa.

Sulfadoxine-pyrimethamine, has been used to treat chloroquine-resistant malaria in several countries of South America and Central and Middle East Asia, with a treatment failure rate low, as compared to the failure rate in eastern Africa (52.8 %) for this combination.

Resistance to mefloquine is a concern in South East Asia, where artesunate- mefloquine is still used as first-line treatment. In order to maximize the effectiveness of artemisinin and its derivatives and to protect them from the development of resistance, WHO recommended combining them with other drugs that have different mechanisms of action and longer half-lives. However, remarkable failure rates of these combinations have been observed in several African countries.

The next challenge for Malaria is to identify new classes of drugs that will attack novel molecular targets, with sufficient therapeutic lifespans that will not be compromised by the rapid development of resistance, and to develop novel technologies, that will effectively clear Plasmodium with maximum precision, thus minimising the risk of drug resistance(32).

The organisms of the clades formerly grouped in the kingdom PROTISTA diverged early in evolution, and present variations on what were considered paradigms of the domain Eukarya that can be used as therapeutic targets. One of them is to partially degrade glucose to CO2 even aerobically (aerobic glycolysis), excreting to the medium reduced products (lactate ...). This "aerobic fermentation" is accompanied by absence of the "Pasteur effect". The Kinetoplastida also have the first seven enzymes of glycolysis in a unique organelle called glycosome, which provides a high glycolytic flux with high bioenergetic performance, and protects against toxic intermediates produced in this autocatalytic way that lacks the allosteric control present in other organisms. The glycosome and their enzymes are also involved in other metabolic pathways depending on the phase of the cycle (gluconeogenesis ...). In these organisms the production of ATP and metabolic intermediates to generate biomass is mainly based on aerobic fermentation.

The present inventors surprisingly found that one can inhibit the growth of glycolytic parasites, including Leishmania spp. and Plasmodium spp., by blocking the glycolytic pathway. A pharmaceutically acceptable dichloroacetate compound is provided for use in a method for treating the diseases caused by such parasites in mammals. The compound inhibits parasite growth while growth of host cells is not inhibited or less inhibited.

Dichloroacetate has been previously used in the treatment of diseases producing metabolic disorders related to lactic acidosis, including diabetes mellitus, MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) or malaria, among others. The use in malaria is based on its ability to reduce blood lactate levels in the metabolic acidosis of severe complicated malaria. Dichloroacetate, acts there as a cofactor that stimulates the activity of pyruvate dehydrogenase, accelerates the oxidative removal of lactate, and the consumption of glucose. Dichloroacetate has resulted in improvements in lactic acidosis— as it is the case for the above mentioned diseases— in children with severe malaria(35), thus it has been proposed as targeted intervention. However it has never been described and it has never been used as a treatment for malaria itself in order to kill the parasite, just as an adjuvant short term treatment for lactic acidosis associated to cerebral malaria.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutically acceptable dichloroacetate compound selected from a salt of dichloroacetic acid, an ester of dichloroacetic acid with a C-I-C6 alkanol, C1-C6 acyloxy-methanol or C1-C6 alkoxycarbonyloxy-methanol and mixtures thereof for use in a method for treating a disease caused by a glycolytic parasite in a mammal.

In an embodiment, the inventions relates to a pharmaceutically acceptable dichloroacetate compound selected from a salt of dichloroacetic acid, an ester of dichloroacetic acid with a C1-C6 alkanol, C1-C6 acyloxy-methanol or C1-C6 alkoxycarbonyloxy-methanol and mixtures thereof for use as an anti-"glycolytic parasite" agent. In a preferred embodiment, the pharmaceutically acceptable dichloroacetate compound is a sodium salt of dichloroacetic acid.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a pharmaceutically acceptable dichloroacetate compound selected from a salt of dichloroacetic acid, an ester of dichloroacetic acid with a C1-C6 alkanol, C1-C6 acyloxy-methanol or C1-C6 alkoxycarbonyloxy-methanol and mixtures thereof for use in a method for treating a disease caused by a glycolytic parasite in a mammal.

The term "treating" means preventing, alleviating or curing the disease disease caused by the glycolytic parasite in the mammal.

The term "glycolytic parasite" as used herein refers to a parasite that is more sensitive to inhibition of pyruvate dehydrogenase kinase activity than its host cell. In a preferred embodiment, the parasite is a glycolytic protozoan parasite, in particular a glycolytic protozoan parasite selected from Leishmania spp., Trypanosoma spp., Plasmodium spp., and Toxoplasma spp, such as Leishmania major, Leishmania infantum, Leishmania braziliensis, Leishmania donovani, Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi, and Toxoplasma gondii. In a particularly preferred embodiment, the parasite is selected from Leishmania spp. such as Leishmania major, Leishmania infantum, Leishmania braziliensis, and Leishmania donovani. A "mammal" as used herein is a mammal that has or is suspected of having a disease caused by the glycolytic parasite, i. e. Leishmaniasis spp. Preferably said mammal is a human, a horse, a cat or a dog. In particularly preferred embodiments, the mammal is a human. The pharmaceutically acceptable dichloroacetate compound is preferably a salt of dichloroacetic acid. Any salt of dichloroacetate with minimum toxicity for the mammal is preferred. A salt of dichloroacetic acid that comprises a cation selected from alkali metal ions, alkaline earth metal ions, ammonium ions, substituted ammonium ions, and positively charged amino acids is particularly preferred. Exemplary substituted ammonium ions are substituted with 1 to 4 Ci-C4-alkyl residues, wherein the Ci-C4-alkyl residues may be the same or different. Other exemplary substituted ammonium ions are substituted with 1 to 4 hbN-alkylene-, H3N ⁺ -alkylene- and/or HO-alkylene- residues, i. e. ethylene diammonium. The salt may for example be selected from the sodium, potassium, calcium, magnesium, ethyl ammonium, diethyl ammonium, diisopropyl ammonium, dicyclohexyl ammonium, triethyl ammonium, butyl ammonium, ethylenediamine, hydroxyethyl ammonium, di(hydroxyethyl) ammonium, tri(hydroxyethyl) ammonium, piperazine, benzyl ammonium, phenylbenzyl ammonium, choline, meglumine, tromethamine, lysine, arginine, and histidine salt. Highly preferred pharmaceutically acceptable dichloroacetates are sodium dichloroacetate, potassium dichloroacetate, and diisoproyl ammonium dichloroacetate. The most preferred pharmaceutically acceptable dichloroacetate compound is sodium dichloroacetate.

A dichloroacetate compound can also be an ester of dichloroacetic acid with a C1-C6 alkanol, C1-C6 acyloxy-methanol or C1-C6 alkoxycarbonyloxy-methanol. Preferred esters are methyl dichloroacetate and ethyl dichloroacetate. In humans, the disease that is preferably treated with the pharmaceutically acceptable dichloroacetate compound of the present invention is selected from Leishmaniasis, Malaria, Trypanosomiasis, and Toxoplasmosis. Particular Leishmaniases that are preferably treated with the pharmaceutically acceptable dichloroacetate compound of the present invention are Visceral Leishmaniasis, Cutaneous Leishmaniasis, Diffuse cutaneous Leishmaniasis, Mucocutaneous Leishmaniasis. Particular Trypanosomiases that are preferably treated with the pharmaceutically acceptable dichloroacetate compound of the present invention are African Trypanosomiasis and American Trypanosomiasis. The disease that is most preferably treated with the pharmaceutically acceptable dichloroacetate compound of the present invention is Visceral Leishmaniasis.

Examples of pentavalent antimonials are, among others, meglumine antimoniate or sodium stibogluconate.

Further examples of glycolytic inhibitors besides dichloroacetate are, among others, the following: Bromopyruvic acid; 2-Deoxy-Dglucose; Lonidamine; Chetomin; Cryptotanshinone; (3E,5E)-3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone; [2-oxo-2- (p-tolyl)ethyl] 3-[(2,4-dinitrobenzoyl)amino]benzoate; 2-[(1 -methylpropyl)dithio]-1 H- imidazole; Deferoxamine mesylate salt; Dimethyloxalylglycine; L-Mimosine; Ethyl 3,4-dihydroxybenzoate; Acadesine; 1 ,1 -Dimethylbiguanide hydrochloride; Phenformin hydrochloride; 6-[4-(2-Piperidin-1 -ylethoxy)phenyl]-3-pyridin- 4-ylpyrazolo[1 ,5-a]pyrimidine; (±)-2,2'-bis(8-Formyl-1 ,6,7-trihydroxy-5-isopropyl-3- methylnaphthalene); Aminooxoacetic acid sodium salt; 1 ,4a-Dimethyl-7-isopropyl- 1 ,2,3,4,4a,9,10,10a-octahydro-1 -phenanthrenemethylamine.

The pharmaceutically acceptable dichloroacetate compound can be administered through known routes of pharmaceutical administration. Preferably the route of administration is selected from at least one or more of: oral administration, injection (such as direct injection), topically, parenteral administration, mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, or transdermal administration. The pharmaceutically acceptable dichloroacetate compound may be administered in the form of a pharmaceutical formulation. The term "pharmaceutical formulation" refers to a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects.

In one embodiment, the pharmaceutically acceptable dichloroacetate compound is administered in the form of a pharmaceutical formulation which may comprise a pharmaceutically acceptable carrier, excipient or nanoparticles. Depending on the route of administration, the pharmaceutical formulation may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. For parenteral application, particularly suitable are injectable sterile solutions, preferably oil or aqueous solutions, as well as suspensions, emulsions or implants, including suppositories. Ampules are convenient unit dosages. The pharmaceutical formulation can be sterilized and, if desired, mixed with stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers or other substances that do not react deleteriously with the specific dichloroacetate, i. e. salt or ester, being present. Specific pharmaceutical formulations include (i) tablets optionally containing excipients, i. e. starch or lactose; (ii) capsules or ovules either alone or in admixture with excipients; (iii) elixirs; (iv) solutions; (v) suspensions optionally containing flavouring or colouring agents; (vi) injections, i. e. intravenous, intramuscular or subcutaneous injections; (vii) suppositories or pessaries; and (viii) lotions, solutions, creams, ointments, dusting powders, skin patches for topical application. In one embodiment, the pharmaceutical formulation may be designed to be administered by a number of routes. In one embodiment, the pharmaceutical formulation is a form suitable for oral administration. Injections may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. The pharmaceutical formulation may comprise substances that are customarily used in pharmaceuticals.

Suitable carriers include starch, lactose, glucose, sucrose, dextrin, cellulose, paraffin, aliphatic glyceride, water, alcohol, acacia.

The excipients may comprise binding agents, wetting agents, and/or vehicles, i. e. water and alcohols. See, for example reference 29. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical formulation. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. The pharmaceutical formulation may also contain an auxiliary substance, stabilizer, emulsifier, lubricant, binder, pH-adjuster, isotonic agent and other conventional additives, as necessary.

There may be different composition/formulation requirements dependent on the delivery system and way of administration. By way of example, the pharmaceutical formulation may be formulated to be administered using a mini-pump or by a mucosal route, for example, as an ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.

The pharmaceutical formulation described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.

In the present invention, the dosage and type of pharmaceutically acceptable dichloroacetate compound to be administered will depend on a variety of factors, which may be readily monitored in patients. These factors include the type and severity of the disease.

The pharmaceutically acceptable dichloroacetate compound can be administered in combination with other pharmaceutically active agents. Accordingly, in one embodiment, the compound for use may additionally comprise at least one other agent for use in the method for treating the disease caused by the glycolytic parasite in the mammal. The other agent can be administered at the same time or at different times, but in a combination treatment regimen, such as combination regimens know to those skilled in the art. The pharmaceutically acceptable dichloroacetate compound and the other agent that is effective for treating the disease can be formulated into the same pharmaceutical compositions. Alternatively, the pharmaceutically acceptable dichloroacetate compound and the other agent that is effective for treating the disease can be formulated into different pharmaceutical compositions. The other agent is preferably selected from pentavalent antimonials, Amphotericin B, Miltefosine, Paromomycin or a glycolytic inhibitor.

A "therapeutically effective amount" is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. The dosage regime may be adjusted to provide the optimum therapeutic response in a particular patient.

In another aspect, the present invention relates to a method for inhibiting proliferation of a glycolytic parasite in mammalian host cells in vitro, which comprises treating host cells infected with the parasite with a pharmaceutically acceptable dichloroacetate compound of the invention. The host cells may for example be in the form of a cell culture.

The invention will now be described by the following non-limiting examples. EXAMPLES

The invention will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention in any way.

Toxicity of dichloroacetate to human macrophages

Cell line U397 (European Collection of Cell Cultures) was cultured at a density of 5 ^χ 10 ⁴ per well in RU937 (RPMI-1640, 10 % Bovine Fetal Calf Serum, 1 % de Penicillin/ Streptomycin) at 37 °C and 5 % CO2. Differentiation into adherent macrophages was induced by exposure of the cells to phorbol-12-myristate-13-acetate (PMA) at 25 nM. After 48 h incubation, cells were washed with phosphate buffered saline (PBS) and resuspended in RU937 and increasing concentrations of dichloroacetate were added. The assessment was carried out in triplicate. After 48 h of incubation, Thiazolyl Blue Tetrazolium Blue (MTT) was added to a final concentration of 0.5 mg/mL. After 4 h at 37 °C and 5 % C0 ₂ , 100 μΙ_ of MTT Solubilization Solution (HCI 0.1 N in anhydrous isopropanol + 10 % Triton X-100 plus) was added to each well and was shaken for 5 min. Absorbance at 570 nm was recorded using 690 nm as reference. Results are expressed as the rate of survival in relation to non-treated cells (Figure 1 ). Concentrations below 25 mM of DCA do not significantly affect survival of human macrophages. Efficacy of dichloroacetate on intracellular amastigotes of L. infantum

Cell line U397 (European Collection of Cell Cultures) was cultured in 25 mm ³ flasks and differentiated as explained above. After elimination of PMA macrophages were incubated during 24 h at 37 °C and 5 % CO2 with L. infantum promastigotes at a parasite/cell ratio of 10/1 . Non-internalized promastigotes were then removed by washing adherent cells with PBS. Infected macrophages were resuspended in RU937 and incubated at a density of 5 ^χ 10 ⁴ per well with increasing concentrations of dichloroacetate. Meglumine antimoniate at a concentration of 100 μ-JmL was used as positive control. Giemsa stain was used to estimate the rate of infected macrophages and the parasite load (Figure 2). Concentrations of DCA in the range of 2 mM-25 mM achieve growth inhibition of intracellular amastigotes of L. infantum between 50 %-90 %, higher than those obtained with the first choice drug meglumine antimoniate at recommended doses. Efficacy of dichloroacetate in the in vivo hamster model of experimental leishmaniasis

Studies trying to dissect VL disease are hampered by the lack of bio-models that accurately reflect the human disease, being the best the golden hamster when experimental infection is used, and the dog for natural infections(33). The golden hamster model of L. infantum infection is thus known to be the best mimicking the outcome of this disease, characterized by parasite visceralization, splenomegaly, cachexia, and progressive hypergammaglobulinemia. Twelve golden hamsters were randomly assigned to two equally sized experimental groups. Both groups were infected with an intraperitoneal injection of 1 ^χ 10 ⁷ stationary promastigotes of L. infantum. Eleven weeks after infection, one group was treated with dichloroacetate at a dose of 100 mg/kg via intraperitoneal every 24 h for 15 consecutive days, the other group was no treated. Twenty-four hours after the last dose all animals were euthanized and samples collected. An effective response against leishmaniasis would be predominantly mediated by a Th1 like immune response. In our experiments, we have observed that in the dichloroacetate treated group there is a trend on decreasing Th2 type cytokines (IL4) and Th1 -downregulating cytokines such as IL10. Expression of proinflammatory cytokines IFN-γ and TNF-a was also significantly lower than in the control group (Figure 3). Reduced immune activation associated with the use of DCA, exemplified by lower levels of both types of cytokines— proinflammatory and antiinflammatory— is also associated with a better response to the infection, lower parasite loads, and less symptomatology in other models. Animals treated with dichloroacetate also shown a significant decreased in arginase activity when compared to the control group (Figure 4). This enzyme catalyzes the L- arginine transformation into ornithine and urea and so do this to the polyamine synthesis, promoting cell proliferation. This enzyme competes with the Nitric oxide synthase (iNOS) for the same substrate (L-arginine), blocking the nitric oxide production (which is necessary for the removal of intracellular parasites) and promoting the polyamine synthesis, which leads to an increase of the parasite multiplication rate. Parasite load in spleen and liver of dichloroacetate treated group was lower than in the control group. It has been shown that during leishmaniasis induction of arginase-1 - macrophages has a detrimental role by limiting classically activated macrophages dependent parasite clearance and promoting parasite proliferation. Arginase-1 T cell suppression is also known to contribute to failure to eliminate the pathogen. Furthermore, an increased activity of arginase— one of the hallmarks of alternatively activated macrophages and a marker of disease in leishmaniasis— is responsible for the uncontrolled growth of Leishmania parasites in vivo. Finally, it has been reported that high levels of arginase in leishmaniasis human patients decline considerably once they are successfully treated. Thus, the decreased arginase activity in the dichloroacetate treated group is related to the reduction of the parasite load and to a better outcome(34).

Malaria: growth inhibition inhibition of Plasmodium parasites assay in the human blood stage.

Four % of hematocrit with a 0.8% of parasitized human erythrocites with immature trophozoites (ring stage) of P. falciparum 3D7 were cultured in 96 microwell plates and exposed to growing concentrations of DCA prepared in media culture. Chloroquine (80 nM) was used as reference drug. Chloroquine was used at a concentration able to inhibit parasite multiplication completely and results were normalized accordingly. Each concentration was analyzed in triplicate and kept at 37 °C in a 5 % CO2 and 2 % O2 atmosphere for 48 h. Parasite viability was determined by cytometry. Results are given as the inhibition rate of parasite growth (Figure 5). Concentrations of 5.9 mM inhibit 50 % growth, while 12.5 inhibit 90 % growth.

CONCLUSIONS Evidence of the relative lack of toxicity of dichloroacetate on macrophages is provided. This confers a wide therapeutic window to the glycolytic parasite treatment according to the invention. Increasing concentrations of dichloroacetate result in both increasing inhibition of intracellular Leishmania infantum amastigotes and of infected macrophages, and in increasing inhibition of rate growth of P. falciparum. The inhibition is stronger than that obtained using first choice treatment drugs: meglumine antimoniate and chloroquine. Finally, we provide evidence for the in vivo efficacy of dichloroacetate in experimental leishmaniasis.

The glycolytic phenotype occurs almost universally in the organisms of the clades formerly grouped in the kingdom Protista. Dichloroacetate is an inhibitor of pyruvate dehydrogenase kinase (PDK) that phosphorylates and inactivates pyruvate dehydrogenase (PDH), and is able to reverse the glycolytic phenotype. It is shown that glycolytic parasites, for example Leishmania spp., are sensitive to dichloroacetate, with growth inhibition being observed. The sensitivity of parasite cells ranged from around 30 % to 90 % inhibition, far greater than that obtained using first choice treatment meglumine antimoniate. Therefore, blocking the glycolytic phenotype of glycolytic parasites becomes a valid strategy for fighting the diseases caused by these parasites.

Pharmaceutically acceptable dichloroacetate compound are thus useful preventing, alleviating or curing a disease caused by glycolytic parasites, such as Leishmaniasis.

REFERENCES

1 . C. Bern, J. H. Maguire, J. Alvar, PLoS Negl Trop Dis 2, e313 (2008).

2. L. Kedzierski, J Glob Infect Dis 2, 177-185 (May-Aug, 2010).

3. A. Rodriguez-Cortes et al., Vaccine 25, 7962 (Nov 14, 2007).

4. F. Todoli et al., PLoS One 7, e51 181 (2012).

5. P. Desjeux, Comp Immunol Microbiol Infect Dis 27, 305 (Sep, 2004).

6. S. L. Croft, S. Sundar, A. H. Fairlamb, Clin Microbiol Rev 19, 1 1 1 (Jan, 2006).

7. J. Carrio, C. Riera, M. Gallego, E. Ribera, M. Portus, J Antimicrob Chemother 47, 120 (Jan, 2001 ).

8. S. Sundar, Trop Med Int Health 6, 849 (Nov, 2001 ).

9. S. Rijal ef a/., Trans R Soc Trop Med Hyg 97, 350 (May-Jun, 2003).

10. S. Collin et al., Clin Infect Dis 38, 612 (Mar 1 , 2004).

1 1 . S. Sundar ef a/., Clin Infect Dis 31 , 1 104 (Oct, 2000).

12. C. Di Giorgio et al., J Antimicrob Chemother44, 71 (Jul, 1999).

13. R. Durand et al., Antimicrob Agents Chemother42, 2141 (Aug, 1998).

14. S. Sundar ef a/., Clin Infect Dis 45, 556 (Sep 1 , 2007). 15. J. Lamothe, J Small Anim PractAl, 170 (Apr, 2001 ).

16. C. Noli, S. T. Auxilia, Vet Dermatol 16, 213 (Aug, 2005).

17. J. Berman, Expert Opin Pharmacother6, 1381 (Jul, 2005).

18. S. K. Bhattacharya et al., J Infect Dis 196, 591 (Aug 15, 2007).

19. K. Ritmeijer ef a/., Clin Infect Dis 43, 357 (Aug 1 , 2006).

20. P. Escobar, S. Matu, C. Marques, S. L. Croft, Acta Trap 81 , 151 (Feb, 2002).

21 . K. Seifert ef a/., I nt J Antimicrob Agents 30, 229 (Sep, 2007).

22. S. Sundar, H. W. Murray, Bull World Health Organ 83, 394 (May, 2005).

23. J. Alvar, S. Croft, P. Olliaro, Adv Parasitoie'l , 223 (2006).

24. S. Sundar, T. K. Jha, C. P. Thakur, P. K. Sinha, S. K. Bhattacharya, N Engl J Med 356, 2571 (Jun 21 , 2007).

25. R. A. Neal, S. Allen, N. McCoy, P. Olliaro, S. L. Croft, J Antimicrob Chemother

35, 577 (May, 1995).

26. C. N. Chunge, J. Owate, H. O. Pamba, L. Donno, Trans R Soc Trop Med Hyg

84, 221 (Mar-Apr, 1990).

27. S. Teklemariam et al., Trans R Soc Trop Med Hyg 88, 334 (May-Jun, 1994).

28. A. Poli, S. Sozzi, G. Guidi, P. Bandinelli, F. Mancianti, Vet Parasitol l†, 263 (Aug, 1997).

29. Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, 17th edition, 1985.

30. WHO. 2013. World Malaria Report. World Health Organization: Geneva.

31. Global report on Antimalarial Drug efficacy and Drug Resistance: World Health Organization. 2000-2010.

32. Cowman AF ef a/. FEBS Letters, 476, 84-88 (2000).

33. Duthie MS et al. Vaccine 30:134-141 (2012).

34. Osorio EY et al. PLoS Pathog 10(6): e1004165. doi:10.1371/journal.ppat.1004165.

35. S. Krishna, et al. QJM., 88 (1995), pp. 341-349