WO2018068114A1 - Antimalarial composition, method for treating malaria and use of an antimalarial composition - Google Patents

Abstract

The present invention relates to a combination of the drugs curcumin (Cur), which is a natural polyphenol, and sodium diethyldithiocarbamate (DETC), or its precursor disulfiram (DS), which exhibits synergistically increased antimalarial activity.

Description

 "ANTIMALARIC COMPOSITION, METHOD FOR TREATING MALARIA, AND USE OF AN ANTIMALARIC COMPOSITION"

Field of the Invention

 The present invention is a combination of curcumin.

(Cure), which is a natural polyphenol, and sodium diethyl dithiocarbamate (DETC), or its precursor, disulfiram (DS) (Ι, Γ, Γ ', Γ "- [disulfanedilbis (carbonothioylnitrile)] tetraethane) where such a combination of drugs presents increased antimalarial activity.

Background of the Invention

 Malaria is an infectious disease caused by parasitic protozoa of the genus Plasmodium. There are different species that affect man, they are: Plasmodium falciparum, the most common on the planet and which causes the highest number of deaths; Plasmodium vivax, responsible for 77% of infections in the Americas; Plasmodium malariae and Plasmodium ovale (World Health Organization - WHO, 2013). It is also known the natural infection of humans by P. knowlesi (Jongwutiwes et al., 2004; Antinori et al., 2013), a species initially described as a monkey parasite, indicating the zoonotic character of this infection. Recently, the existence of P. ovale subspecies has been described (Calderaro et al., 2013).

Plasmodium falciparum and, to a lesser extent, P. vivax are the main etiologies of morbidity and mortality among the human malaria-causing plasmids. Malaria is the product of parasite-host interaction, geographic and social factors, and its evolution is directly associated with factors linked to both.

Chemotherapy is not only the main therapeutic strategy for malaria, but also one of the main ways of controlling disease progression. The three most widely used and effective drug groups are quinine-derived quinoline drugs obtained from the Cinchona officinalis plant and its analogues. Another widely used group are the dihydrofolate reductase inhibitors, always used in combination, the best known being proguanil, sulfas, pyrimethamine and trimethoprim. Drugs derived from the Artemisia annua plant are also very effective in treating malaria, including artesunate and arthemeter (Farooq & Mahajan, 2004).

 Importantly, among the major obstacles to reducing the incidence of malaria in the world is the spread of cases refractory to therapy (Farooq & Mahajan, 2004; WHO, 2013), particularly related to chloroquine resistance (QC). ) and the sulfadoxine / pyrimethamine (SP) combination.

 [006] The therapeutic regimen is very important to ensure the clinical cure of patients in the case of malaria. The rapid improvement of the condition is fundamental for the prevention of disease transmission. The use of sub-therapeutic doses increases the risk of treatment failure and furthermore constitutes a selective pressure for resistant phenotypes (Barnes et al., 2008a); and in malaria, resistant parasites are associated with increased latent infections, as well as increased gametocytemia, which leads to a higher likelihood of disease transmission, which explains the rapid spread of resistance (Barnes et al., 2008b).

 [007] Malaria chemotherapy, including the action of chloroquine, is primarily directed towards the elimination of asexual forms of protozoan, which actively degrade hemoglobin (Peters, 1970).

There are reports indicating that chloroquine interferes with the process of hemoglobin degradation and consequent formation of hemozoin (Hz - malarial pigment). The primary mechanism of action of chloroquine (QC) is associated with its ability to enter the parasite's digestive vacuole, considered an important cellular target in the mechanism of action of anti-malarial compounds (Wunderlich et al., 2012), to protonate in the acidic compartment. , as it is a weak base. Once in protonated form, the charged compound no longer crosses the parasite's digestive vacuole membrane, where it accumulates, leading to pH change and thus preventing the process of forming the crystal of Hz and leading to increased oxidative stress by the pro-oxidant effect of heme. free (Fitch, 1998; Sullivan, 2002). However, it should be noted that QC can exert oxygen-reactive species (ROS) -dependent annulus-Plasmodium effects associated with the induction of programmed cell death triggered by the permeabilization of the digestive vacuole membrane (Ch'ng et al., 2011).

[009] Since the publication of the first edition of the guidelines of the

World Health Organization In 2006, most countries where P. falciparum is endemic developed up-to-date treatment policies, replacing treatment with QC and SP for artemisinin-based combination therapies, which is currently considered the best treatment. for uncomplicated falciparum malaria. However, the high cost of treatment with artemisin derivatives (WHO, 2013) has made it difficult to combat this disease.

 The major molecules used as antimalarials in use and in development today are: quinine, pachycin, chloroquine, mepacrine, proguanil, amodiaquine, primaquine, pyrimethamine, sulfadoxine, artemisinin, mefloquine, halofantrine, pyromaridine, piperaquine, artemeter, , dihydroartesimine, autovaquone, lumefantrine, tafenoquine and ablaquine (Kaur et al., 2010).

The main pharmacological strategy against malaria proposed by WHO involves artemisinin-based combination therapy (ACT), in which one or more drugs are used in conjunction with artemisinin (or its derivatives). ) in order to achieve maximum effect and minimum resistance. WHO currently suggests the use of artemeter and lumefantrine (AL), artesunate and amodiaquine (AS + AQ), artesunate and mefloquine (AS + MQ) and artesunate and sulfadoxine pyrimethamine (AS + SP). Many of these combinations do not yet have an established mechanism of action, as they may act synergistically or not (WHO, 2013).

 However, resistance to drugs used to treat malaria is the biggest challenge in reducing mortality caused by Plasmodium. There is widespread concern about the emerging resistance of parasites to artemisinin combination therapies, which generates tremendous interest in discovering a new generation of drugs for these purposes (WHO, 2013).

 Although several known antimalarials are used to good effect in treating malaria, these compounds also have unwanted side effects, and there is a continuing need to improve the effectiveness of the drugs.

 Thus, there is a great need to provide a new stable combination due to the fact that infectious agents become resistant.

 US patent 7,858,659 relates to a treatment for tumor cells and malignant cells by altering the redox state or environment of the cell. This treatment aims to alter the balance of glutathione (GSH) to oxidized glutathione (GSSG). This patent mentions a synergistic combination of a GSH oxidizing agent, preferably DS. The synergistic combination includes Cure. and enzyme antagonists.

Clark et al. (1983) provide evidence that malaria-causing parasites are susceptible to ROS. In this study, DETC was used which was shown to reverse the antimalarial effect of alloxan.

[0017] Suckow and Suckow (2006) investigated the possibility that diets high in antioxidants promote the extension of life span. It is mentioned that turmeric (Curcuma longa) is a spice, also known as turmeric, which has been consumed as a condiment and used for medicinal purposes for many centuries in Asia. Turmeric contains the powerful antioxidant, Cure. Numerous tests were conducted employing Cure. in several experimental models. It was observed that Cure. significantly increases the longevity of Caenorhabditis elegans (Liao et al., 2011), Drosophila melanogaster (Suckow & Suckow, 2006) and tribolium castaneum (Bingsohn et al., 2016) and murines (Shen et al., 2013), but in humans the findings do not look very promising (Sadowska-Bartosz & Bartosz et al., 2014; Soare et al., 2014). This process depends, at least in part, on autophagy modulation (Petrovski & Das, 2010).

For the reasons given above, there is still a pressing demand for research and development (R&D) focusing on safe, effective and cost-effective treatment for malaria. This is a definite necessity not only in more than 100 endemic countries, but also for more than half of the world's population at risk of malaria. It is noteworthy that the transmission of this parasitosis has already been confirmed in aircraft in Europe and in homes near airports, proving that mosquitoes Anopheles sp. They are transported between nations and continents. Moreover, with global warming, their distribution, among other vectors, is being expanded, which may increase the incidence of parasitosis, as well as its endemicity.

 Therefore, the present invention aims to increase the efficacy and safety of a pharmaceutical preparation for malaria chemotherapy by employing Cure. and DS or its derivative, DETC.

Since the combined compounds may promote antiparasitic actions but mutually counterbalance the redox and / or toxicity effects, they may exert cytoprotective effects and thus the pharmaceutical preparations of the present invention employing low dosages may further extend the effect of the antigen. reach or therapeutic application of pharmaceutical preparations and in particular also to extend it for the treatment of groups that require special care, such as children, pregnant women, and immunocompromised individuals.

Summary of the Invention

 The present invention deals with a combination of the drugs

Cure, which is a natural polyphenol, and DETC, or its precursor DS, which has synergistically enhanced antimalarial activity.

[0022] The present invention features the combination of two drugs, already approved for long time use in humans, both of which are very well tolerated (ie showing low toxicity) that showed promising effect on both in vitro and in vivo malaria chemotherapy. DS is the active ingredient in Antabuse ^® or Anti-ethanol ^® in Brazil and is successfully used for the treatment of alcoholism and other forms of addiction, as well as the treatment of AIDS, cancer and heavy metal poisoning. less toxic than aspirin (reviewed in Gessner & Gessner, 1992).

Being the Cure. generally considered an antioxidant and DS and its derivative, DETC, characterized as pro-oxidant drugs, by the ability to inhibit superoxide (0 ₂ ^~ ) dismutase (SOD) activity, it would be expected that these compounds exert mutually antagonistic effects. In fact, DS reverses the aforementioned increase in longevity caused by Cure. in D. melanogaster (Suckow & Suckow, 2006), a fact that can be explained, at least in part, by Cure's call. to thiol groups (Aggarwal et al., 2007b). Although very well tolerated, DS has reports of neurotoxic effect (Torre et al., 2010; Stone et al., 2014), however some of these studies are based on patients with several years of alcoholism and eventually narcotics and report eventual, one single (Stone), two (Filosto et al., 2008; Tran et al., 2016) or three (Torre et al., 2010) cases. However, it should be noted that the occurrence of seizures during withdrawal syndrome may have a genetic etiology (Shirley et al., 2004). Besides that, It is worth mentioning the well-known neuroprotective activity of Cure. (eg Cole et al., 2007) in lesions produced by arsenic (Srivastava et al., 2014), fluoride (Sharma et al., 2014) ischemia (Tu et al., 2014), hemorrhage (Yang et al., 2014 ). Conflicting data may reflect the complexity of modes of action as well as the demand for determining precise conditions of use. The cure. may be anticlastogenic (Alaikov et al., 2007) as well as induce chromosomal damage (Araújo et al., 1999).

 [0024] There is no doubt that Cure. has numerous promising pharmaceutical applications (Aggarwal et al., 2007b; Goel et al., 2008) However, the use of Cure. must be viewed carefully. Unlike a panacea based on innocuous seasoning, the Cure. may pose a risk (Marathe et al., 2009; 2011) by reducing the microbicidal activity of ciprofloxacin against Salmonella typhimurium and S. typhi (Marathe et al., 2013), promoting intracellular proliferation of Salmonella (Marathe et al., 2012b). . The anti-inflammatory effect of Cure. can modulate cellular immune response (THi) by aggravating the condition in visceral leishmaniasis model (Adapala & Chan, 2008), as well as increasing pathogenicity of murine Salmonella (Marathe et al., 2010). This bimodal action of Cure. It may be linked to its anti-inflammatory effect as well as its ability to act as an antioxidant or pro-oxidant, depending on conditions such as concentration. This property goes back to the definition of Galen's drug, correlating drug and poison. The search for synergistic and / or additive combinations can be a great advantage as it allows for lower dosages, and can prevent the production of side effects. Being a compound widely employed in the nutraceutical industry, it is undoubtedly well tolerated by the human organism. However, the aforementioned side effects need to be avoided, which can be achieved by dosing.

Although DETC elevates parasitemia in infection with Plasmodium berghei (Nair et al., 1982), its immediate precursor, DS has antimalarial effect on Plasmodium falciparum (Scheibel et al., 1979). This drug has a very relevant property for this infection as it is able to reverse the resistance phenotype, inhibiting the activity of P-glycoproteins (Sauna et al., 2005), responsible for drug extrusion in the MDR (multiple drug resistance) phenotype. ). Infected cells and protozoan per se present this enzyme, which is a virulence factor and marker of severe malaria.

 The other compound employed is the natural product Cure. ((E, E) -1,7-bis (4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione) already tested in different nosological entities (Aggarwal et al., 2007b), in dosages ranging from 1 to 8 g / day (per os), being widely consumed in the form of the turmeric spice obtained from Curcuma longa turmeric (with Cure being a major component) in several countries by millions / billions of people. This polyphenol has activity in various pathologies, acting mainly as anti-inflammatory (Goel et al., 2008), but also having antiparasitic action, including on Plasmodium sp., And having demonstrated considerable antimalarial activity (Haddad et al., 2011). .

Cure. and DETC demonstrated in vitro antimalarial action with IC ₅₀ of 3.4 and 8.0 μg / mL, respectively. The combinations of these drugs showed highly synergistic antimalarial effect, as demonstrated by the Fractional Inhibitory Concentration (FIC) value of 0.000175, as well as the isobologram plotting. It is noteworthy that the P. falciparum strain employed is chloroquine resistant.

 We also observe that Cure. inhibits hemozoma (Hz) formation in the same concentration range as chloroquine, and induces the formation of reactive oxygen species in intracellular parasites detectable by fluorescence microscopy

The ultrastructural analysis showed alterations of compartments of the endocytic pathway and formation of Hz, corroborating the biochemical dosages. The presence of Hz crystals in the P. falciparum cytoplasm unambiguously indicates the rupture of the membranes of digestive vacuoles and, therefore, cellular autolysis. It is noteworthy that this effect was not associated with hemolysis.

Cytotoxicity measurements as measured by incorporation of [ ³ H] Thymidine by murine splenocytes revealed the IC ₅₀ of DETC in the 14.22 μg / mL combination, whereas in P. falciparum proliferation in combination with Cure . DETC IC ₅₀ was 0.0014 μg / mL, demonstrating that the combination is about 10,000 times more toxic to parasites than to mammalian cells, with no haemolytic activity. The selectivity index (IS) calculated for the combination was 10,157.14, while for artemisinin, the gold standard drug for malaria therapy, the IS obtained was 27.57. We observed low systemic toxicity by measuring liver transaminases in mouse plasma (Mus musculus).

 In vivo experiments employing Plasmodium berghei mouse infection demonstrated that both drugs had lower antimalarial activity than the positive control with chloroquine, but the Curc.-DETC combination had increased activity (more than 40% promoting survival), approaching the activity exerted by chloroquine.

 Description of the Figures

 Figure 1 shows inhibition of proliferation / survival of

Plasmodium falciparum in vitro, measured by incorporation of [ ³ H] hypoxanthine by erythrocytes infected and incubated with Cure.

Figure 2 shows inhibition of proliferation / survival of

In vitro P. falciparum, as measured by [ ³ H] hypoxanthine incorporation, by infected erythrocytes and incubated with DETC. Figure 3 shows inhibition of proliferation / survival of

P. falciparum in vitro, measured by incorporation of [ ³ H] hypoxanthine by erythrocytes infected and incubated with DS.

 Figure 4 shows the effects of the Cure combination. like

DETC on P. falciparum proliferation in vitro for 24h.

 [0036] Figure 5 shows the effects of combining DETC with

Heal P. falciparum proliferation, measured by the incorporation of

[ ³ H] hypoxanthine by in vitro infected erythrocytes for 24h.

 [637] Figure 6 shows a graph with the combination of Cure. with DS in P. falciparum proliferation, measured by the incorporation of

[ ³ H] hypoxanthine by in vitro infected erythrocytes for 24h.

 [0038] Figure 7 shows the effects of combining DS with

Heal P. falciparum proliferation, measured by the incorporation of

[ ³ H] hypoxanthine by in vitro infected erythrocytes for 24h.

 Figure 8 shows the isobologram of antiparasitic activity of different combinations between Cure. and the DETC on P. falciparum in vitro.

 Figure 9 shows the isobologram of antiparasitic activity of different combinations between Cure. and the DS on P. falciparum in vitro.

Figure 10 shows the cytotoxic effect of Cure. on in vitro murine splenocyte proliferation for 24h.

Figure 11 shows the cytotoxic effect of DETC on murine splenocytes in vitro for 24h.

 Figure 12 shows the cytotoxic effect of DS on murine splenocyte proliferation in vitro for 24h.

 Figure 13 shows the effect of the Cure combination. with different concentrations of DETC on murine splenocyte proliferation for 24h.

Figure 14 shows the effect of the Cure combination. with different concentrations of DS on murine splenocyte proliferation for 24h.

 Figure 15 shows the effect of combining DETC with different Cure concentrations. on murine splenocyte proliferation for 24h.

 Figure 16 shows the effect of combining DS with different Cure concentrations. on murine splenocyte proliferation for 24h.

 [0048] Figure 17 shows the evaluation of the reversibility of the effect of

Heal for 48h on P. falciparum infected red blood cells in the presence and absence of the urate antioxidant.

 Figure 18 shows the evaluation of the reversibility of the effect of

DETC for 48h on P. falciparum-infected red blood cells in the presence and absence of urate.

 Figure 19 shows the evaluation of the reversibility of the effect of

DS for 48h on P. falciparum infected red blood cells in the presence and absence of urate.

 Figure 20 shows the evaluation of the ability of 24h urate preincubation to reverse the effect of DS on P. falciparum proliferation in vitro.

 Figure 21 shows the assessment of the ability of 24h urate preincubation to reverse the effect of DETC on

P. falciparum proliferation in vitro.

 Figure 22 shows the assessment of the ability of 24h urate preincubation to reverse the effect of Cure. on P. falciparum proliferation in vitro.

 [0054] Figure 23 shows the assessment of hemolytic capacity of

Heal for 1h.

[0055] Figure 24 shows the assessment of hemolytic capacity of the DETC for 1h.

 Figure 25 shows the assessment of hemolytic capacity of the

DS for 1h.

 Figure 26 shows the assessment of hemolytic capacity of the

Heal in combination with DETC for 1h.

 [0058] Figure 27 shows the assessment of hemolytic capacity of

Heal in combination with the DS for lh.

 [0059] Figure 28 shows the effect of Cure. for 24h on in vitro hemozoin formation in the presence of 40 mM SDS and 200 μΜ hemine.

[0060] Figure 29 shows the effect of 24h DETC on in vitro hemozoin formation in the presence of 40 mM SDS and 200 μΜ hemine.

 [0061] Figure 30 shows the effect of DS for 24h on in vitro hemozoin formation in the presence of 40 mM SDS and 200 μΜ hemine.

Figure 31 shows hemozoin dosage in the presence of P. falciparum extracts treated with drug IC ₅₀ .

Figure 32 shows the measurement of lipid peroxidation by the measurement of thiobarbituric acid reactive substances (TBARS) per μg of P. falciparum protein cultures incubated with compound IC50 for 24h.

 Figure 33 shows flow cytometry using the DHE probe from P. falciparum infected red blood cells. A- untreated control; B- cells treated with 10 μg / mL DETC for 2h; C-Cure treated cells. 10 μg / mL for 2h; D- cells treated with DETC 10 μg / mL with Cure. 10 μg / mL for 2h; E- Fluorescence microscopy of P. falciparum infected red blood cells and incubated with Cure. 10 μg / mL, whose labeling was partially reversed by N-acetyl-L-cysteine.

Figure 34 shows flow cytometry of P. falciparum-infected red blood cells whether or not treated with the isolated compounds and combined for 2h, using the DHE probe, showing the median of all populations.

 Figure 35 shows flow cytometry of P. falciparum-infected red blood cells from the M3 population whether or not treated with the isolated compounds and combined for 2h, labeled with the DHE probe.

Figure 36 shows flow cytometry of P. falciparum-infected red blood cells from the M4 population whether or not treated with compounds alone and combined for 2h, labeled with the DHE probe.

Figure 37 shows flow cytometry of P. falciparum infected red blood cells from the M5 population whether or not treated with the isolated compounds and combined for 2h, labeled with the DHE probe.

Figure 38 shows flow cytometry of untreated P. falciparum infected red blood cells labeled with the MitoSox probe.

Figure 39 shows flow cytometry of P. falciparum-infected red cells, treated with 10 μg / mL DETC for 2h and labeled with the MitoSox probe.

 Figure 40 shows flow cytometry of P. falciparum infected red blood cells treated with 10 μg / mL DETC and Cure. 10 μg / mL for 2h and labeled with the MitoSox probe.

 Figure 41 shows flow cytometry of P. falciparum-infected red blood cells, treated with DETC 10 μg / mL and Tocopherol 10 μg / mL for 2h and labeled with the MitoSox probe.

 Figure 42 shows flow cytometry of P. falciparum infected red blood cells treated with DETC 10 μg / mL, Cure. 10 μg / mL and Tocopherol 10 μg / mL for 2h and labeled with the MitoSox probe.

Figure 43 shows flow cytometry of P. falciparum-infected red blood cells whether or not treated with compounds isolated and combined for 2h, labeled with the MitoSox probe.

Figure 44 shows the evaluation of the effect of DS 5 and 25 mg / kg cumulative mortality of P. berghei-infected mice.

Figure 45 shows the evaluation of the effect of DS 50 and 100 mg / kg on the cumulative mortality of P. berghei infected mice.

 Figure 46 shows the survival assessment of P. berghei-infected mice treated with different Cure concentrations.

 Figure 47 shows the survival assessment of P. berghei infected mice treated with different concentrations of DETC, DMSO (negative control) and QC (positive control).

Figure 48 shows the survival assessment of P. berghei-infected mice treated with the combination of DETC and Cure.

 Figure 49 shows the evaluation of hepatic transaminases.

A: Alanine aminotransferase (ALT) and B: Aspartate aminotransferase (AST) in plasma of DETC and Cure treated mice isolated and combined for 5 consecutive days.

 [0081] Figure 50 shows the evaluation of activity (A) creatine kinase (CK) and plasma urea levels (B) in plasma from DETC and Cure treated mice isolated and combined for 5 consecutive days.

 Detailed Description of the Invention

 According to the World Health Organization (WHO), in

In 2015, 95 countries reported at least 214 million cases of malaria, resulting in 438,000 deaths (WHO, 2015). It is alarming to think that more than three billion people are at risk of infection, particularly in the face of climate change, which favors the distribution of transmitting insects.

[0083] Infection begins with hematophagy of female anopheline during which inoculation of sporozoites in the insect's saliva occurs in the host's bloodstream. In approximately 60 minutes these sporozoites reach the hepatocytes, initiating the exoerythrocytic phase.

In 2002, Mota et al. Reported that sporozoite needs activation to successfully infect the hepatocyte, and this process occurs during migration through host cells that allows the parasite to contact specific molecules in the cytoplasm, activating a Ca ^{2+ -} dependent signaling cascade, which is fundamental for inducing sporozoite exocytosis, a process necessary for the formation of the parasite vacuole in the hepatocyte. Thus, migration through host cells is critical to the establishment of infection.

 [0085] The process of hepatocyte invasion by sporozoite occurs through the binding of the circumsporozoite protein (CSP) to hepatocyte heparan sulfated proteoglycan receptors (HSPGs) (Frevert et al., 1993). Liver heparan sulfate has a higher degree of sulfation when compared to other tissues. This may explain the recognition of liver heparan sulfate by the CSP protein, and why the initial replication site of malaria parasites in the mammalian host is the liver (Ying et al, 1997).

 In infections caused by P. vivax and P. ovale there is the possibility of a second schizogony, as some parasites remain quiescent in hepatocytes. This stage is called hypnozoite (Krotoski et al., 1982), which may be involved in relapses of infection.

Sporozoites begin the process of multiplication into exoerythrocytic merozoites. About 10 to 12 days, the hepatocytes break down releasing thousands of merozoites that will then infect the erythrocytes and begin the erythrocytic cycle of the disease, and it is at this stage. most symptomatology occurs (Haldar et ah, 2007). The process of invasion by the parasite is similar between the different species of Plasmodium spp. It is a complex process that is mediated by multifactorial molecular interactions.

 [0088] The process of red blood cell invasion by plasmodia occurs through the interaction of the parasite with red cell membrane receptors. In the case of P. vivax and P. knowlesi the interaction occurs with the Duffy receptor; P. falciparum, on the other hand, has the ability to interact with various receptors, including glycophorins A, B and C, and with Band 3 protein (Gaur et al., 2004).

 The parasite interacts with receptors on the erythrocyte membrane and reorients adhesion to the host cell so that it is mediated by the apical complex region. The parasite induces the formation of a vacuole derived from the red cell plasma membrane and with parasitic components and then penetrates the vacuole through mobile junctions. Three organelles of the parasite are mainly involved in the invasion process: roptrias, micrones and dense granules. The receptors that mediate the process of invasion into the parasite are located in micrones, cell surface and roptrias, and their localization within organelles protects the parasite from the antibody-mediated neutralization process; as their release from the apex organelles is subsequent to contact with the red blood cells, this ultimately limits exposure to antibodies.

Already in the erythrocyte, the parasite begins the process of asexual cell division. The young ring-shaped trophozoite is the most immature stage of the parasite. Each mature trophozoite in P. vivax and P. falciparum infections is capable of giving rise to 20 merozoites, with each merozoite infecting a red blood cell (Miller, 2002). Trophozoite growth is accompanied by a significant increase in metabolic rate, which includes glycolysis, ingestion of host cell cytoplasm, and proteolysis of the hemoglobin in amino acids.

 Plasmodium does not degrade free tetrapyrrolic or heme rings, and these and the iron present therein are toxic to the microorganism by its oxidizing effect (Har-El et al., 1993). However, the effects of heme on parasites incubated with QC may be independent of ROS or reactive nitrogen species (Ch'ng JH et al., 2011). Thus, during the process of hemoglobin degradation, most of the released heme is polymerized as hemozoin or malarial pigment (Stiebler et al., 2011). Compounds that inhibit this process of biocrystallization have antimalarial activity (Ziegler et al., 2001; Hempelmann, 2007). In addition, there is biochemical evidence of hemozoin depolymerization by quinoline compounds such as chloroquine (Pandey & Tekwani, 1997).

The biogenesis of hemozoin-containing digestive vacuoles plays a central role in the pathophysiology of malaria (Dasari & Bhakdi, 2012), activating complement and coagulation cascades, processes observed in severe malaria cases (Dasari et al., 2012), modulating the immune response (Schumann, 2007) and inhibiting effector cell function (Perkins et al., 2011) as macrophages (Arese & Schwarzer, 1997), monocytes (Schwarzer et al., 2008), neutrophils (Perkins et al. , 2011) and dendritic cells (Urban & Todryk, 2006; Millington et al., 2006). Naturally, for the vacuolar content to exert such effects, hemolysis is required. The pathophysiology of malaria is largely triggered by erythrocyte lysis, a process that places parasite components in the bloodstream, including hemozoin, and the red blood cell itself that will trigger an intense host response associated with paroxysm (Garcia, 2010). Treatment of malaria with compounds such as primaquine, particularly in patients deficient in glucose-6-phosphate dehydrogenase (G6PD) can lead to severe hemolytic conditions (Ramos Júnior et al., 2010), but many patients with normal levels of G6PD have so-called "black water fever" (loc. cit. White & Ho, 1992). Even combinations involving artesunate (Raffray et al., 2014; Boillat et al., 2015; Chavada et al., 2015; Rolling et al., 2015) and artemeter (De Nardo et al., 2013) can cause hemolysis, both in (Anaba et al., 2012) as in humans (De Nardo et al., 2013). It is noteworthy that, in the invention presented herein, the combinations did not cause in vitro detectable hemolysis.

[0093] Hemozoin production may be related to resistance to antimalarial drugs such as artemisinin (Meunier & Robert, 2010; Witkowski et al., 2012), which act on the digestive vacuole of intracellular parasites (dei Pilar Crespo et al., 2008 ). Thus, the endocytic pathway of this parasite is not only an important experimental model for understanding the disease, but also a promising target for antimalarial chemotherapy. Thus, the formation of hemozoin crystals from heme is an important parasitic strategy to escape oxidative stress (see below). It is noteworthy that blockade of hemozoin biogenesis can trigger programmed cell death in P. falciparum by permeabilizing the membrane of the digestive vacuole (Ch'ng et al., 2011).

When gametocytes are ingested by the vector (which is the definitive host), gametes mature within the mosquito's intestines. Microgametes (male gametes) undergo nuclear division, followed by a process called exflagellation, as gametes are flagellate. They become mobile, break red blood cells and penetrate macrogametes (female gametes) thus forming the fertilized, zygote stage. The zygote lengthens and becomes mobile and is then called the kinetide, which crosses the wall of the insect's digestive tract and attaches to its face facing the general cavity of the body. At this stage it becomes an oocyst, within which hundreds of sporozoites are formed. The oocysts rupture releasing sporozoites that migrate to the salivary glands, from where they are introduced into the vertebrate hosts along with saliva during blood meal, restarting the cycle.

 These intraerythrocytic forms circulate in the bloodstream in all species of plasmodium that infect humans. However, in falciparum malaria, infected cells tend to adhere to the endothelium of different organs in the body, so that only ring and gametocyte forms usually appear in peripheral blood.

 Homeostasis requires the maintenance of an appropriate redox balance medium, minimizing the generation of ROS such as superoxide anions, hydrogen peroxide and hydroxyl radicals that cause damage to nucleic acids, proteins, membrane lipids (Imlay, 2003). In an attempt to preserve this reducing environment, aerobic and anaerobic organisms use antioxidant systems and oxide-reduction reactions (Jakob & Reichmann, 2013). The redox state of the cell is mediated by oxidized and reduced purine nucleotide rates and thiols such as glutathione / glutathione disulfide and thioredoxin / thioredoxin disulfide (Ghezzi et al., 2005), important system in P. falciparum (Kanzok et al., 2002).

Oxidative stress in malaria is relevant both at the level of the infected cell (Muller, 2004) and that of the patient (Pabón et al., 2003). Since effector cells of the immune system generate oxidizing species with microbicidal activity, the antioxidant mechanisms of parasites are usually important escape strategies and therefore virulence factors.

[0098] It has been shown in the literature that malaria-causing parasites are particularly vulnerable to oxidative stress during their erythrocyte life stage (Becker et al., 2004). This is understandable given that the parasite inhabits a pro-oxidant environment that presents oxygen and iron, the main prerequisites for the formation of ROS, such as hydroxyl anions (OH ^" ) and hydroxyl radicals (· ΟΗ) via reaction of Fenton (Liochev & Fridovich, 1999).

To survive this oxidative stress, parasites and hosts had to develop a plethora of antioxidant defenses. Some of these are functional only in parasites or act differently from human cells, thus constituting promising selective therapeutic targets. In this sense, one of the first enzymes in antioxidant defenses is superoxide dismutase (SOD), which is responsible for detoxification of superoxide (O ₂ ^{* "} ). SODs can be characterized as second cofactors necessary for their catalytic activity, which are metals such as copper. , zinc, manganese In mammalian cells, including red blood cells, cytoplasm has Cu-Zn-SOD, while mitochondrial matrix has Mn-SOD Some bacteria and parasites have iron as a cofactor of SOD activity Plasmodium falciparum also has Fe-SOD activity (Bécuwe et al., 1996), which may be a highly selective target for antimalarial chemotherapy (Soulère et al., 2003) It is noteworthy that SOD activity has been reported to be reduced in P. vivax-infected patients (Bilgin et al. , 2012) and in severe cases of malaria (Narsaria et al., 2012) However, plasma SOD-1 activity may represent a marker of severity in vivax malaria (Andrade et al., 20 10), being increased in the most severe cases and reduced after treatment, but in contrast, a reduction in this activity has been reported both after infection and its therapy (Farombi et al., 2003). These apparently conflicting data indicate that oxidative stress and SOD are involved in the pathogenesis of malaria in a complex way.

 Another important factor in the study of malaria is the occurrence of cases refractory to available treatments. The extensive and prolonged use of antimalarial drugs has led to the emergence of resistant strains (Barnes et al., 2008).

P-glycoprotein (Pgp) is an efflux-dependent efflux pump. ATP, located on the cell membrane. This protein extrudes drugs and, consequently, confers resistance from parasites and mammalian cells. Drug susceptibility is often inversely proportional to Pgp expression and activity (Sauna et al., 2005).

Pgp activity has been described in Plasmodium sp. and is related to chloroquine and mefloquine resistance (Borst & Ouellette, 1995). Chloroquine resistance may be mediated by Pgp-dependent mechanisms that effect chloroquine efflux on resistant parasites (Ullman, 1995), in which treatment with verapamil, a classic PgP inhibitor, reverses chloroquine resistance (Watt et al. , nineteen ninety). However, in addition to inhibiting this ATPase, verapamil acts as a calcium blocker, acting on both the electrical conduction of the sinoatrial beam and the endothelium, causing vasodilation, thus constituting the calcium blocker with the most lethal cases reported (reviewed by DeWitt & Waksman , 2004).

 Loo et al. in 2004, showed that DS metabolites exert inhibitory effects on Pgp. They demonstrated that these effects are not related to inhibition of protein synthesis, but to the interaction of DS with two cisterns located at the protein ATP ligand site. When ATP sites are accessible, DS interacts with cisterns; thus inhibiting ATP hydrolysis and inactivating the protein.

The present invention utilizes a synergistic combination containing Cure. and DETC or its precursor, DS, and these drugs have the ability to inhibit PgP, like Cure, and may presumably act to reverse parasite resistance to drugs. Heal

Cure. [(E, E) -1,7-bis (4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5 dione] is a natural polyphenol found in the Curcuma longa rhizome that has the following molecular structure ( formula I):

 Formula I

Cure. presents a series of potential therapeutic properties such as: anti-inflammatory, antioxidant, antineoplastic, among others. Thus, it is possible to realize that Cure. It has a number of molecular and cellular targets. This possibly explains the pleiotropic activities of the substance for various diseases, including cancer.

Similarly to DETC, Cure. It is capable of inhibiting ABC cassette pumps by reversing the MDR phenotype in tumor cells (reviewed in Aggarwal et al., 2007b) and Candida albicans, and may be employed in combinations (Sharma et al., 2009; Garcia-Gomes et al., 2012 ).

Natural products, including Cure, are known to be important sources of antiparasitic agents. Different formulations and regimens of Cure. have had antiparasitic activities (Haddad et al., 2011) against Trypanosoma cruzi, Leishmania spp., Schistosoma mansoni, Giardia lamblia, Cryptosporidium parvum, Eimeria tenella, Opisthorchis viverrini, and antimalarial activity (Reddy et al., 2005). However, Cure. has not been tested in combination with thiocarbamates in the treatment of any parasitic infection or other nosological entity. DS and DETC

 Diethylditiocarbamate is the first metabolite of DS, a drug used in the treatment of alcoholism in the last 50 years. In mammalian cells, this compound acts as a selective agent for protein carbamylation in sulfhydryl groups (Nagendra et al., 1997).

Dithiocarbamates demonstrate antioxidant and metal chelating activity (reviewed in Hogarth, 2012). They are recognized by its ability to inactivate the enzyme superoxide dismutase (SOD-Cu / Zn) in eukaryotic cells by acting as a metal chelator at the enzyme's active site (Halliwell & Gutteridge, 1990).

 [00111] The chemical structures of DS (Ι, Γ, Γ ', Γ "-

[disulfanedylbis (carbonothioylnitrile)] tetraethane) and DETC are given below as formulas II and III, respectively.

 Formula II Formula III

[00112] The DETC may carry out various activities. In addition to chelating metal ions such as iron, zinc and copper and acting as a free radical suppressant, this compound inhibits SOD, promoting superoxide-mediated damage, and binds to thiol and glutathione-rich proteins, forming disulfide bridges, inhibiting, thus possible antioxidant mechanisms (Halliwell & Gutteridge, 1990).

 [00113] The search for new therapies leads us to address the possibility of synergism between drugs, improving their effectiveness even at lower concentrations, thus decreasing the side effects of both. In this sense, oxidative stress regulating systems appear as a possible target.

In addition to possible effects on SOD, both DETC (Arnelle et al., 1997; Ningaraj et al., 2001; Rahden-Staron et al., 2012) and its precursor, DS (Nagendra et al., 1994; van Gorp et al., 1997; Burkitt et al., 1998; Balakirev & Zimmer, 2001; Kwolek-Mirek et al., 2012), may negatively regulate reduced glutathione (GSH) levels and may thus promote antimalarial action of quinoline drugs (Deharo et al., 2003). DETC has been used in humans as an immunomodulator (imuthiol) in patients with HIV / AIDS (Reisinger et al., 1990) and in cutaneous leishmaniasis (Khouri et al., 2010), but had not yet been tested in malaria models or combinations.

 Aware of the state of the art, the inventors have analyzed the action of thiocarbamate combinations with the natural product Cure. malaria and found the relevant efficacy of the Cure combination. and DETC in the treatment of malaria.

 To achieve their objectives, the inventors conducted several experiments to evaluate the synergistic combination of the present invention.

 The following were specifically evaluated:

 (1) The effect of Cure. isolated and in combination with DETC and DS on the survival of Plasmodium falciparum in vitro;

 (2) the cytotoxic potential of single and combined substances;

 (3) the possible synergistic effects between the combined drugs;

 (4) the possible mechanisms of action of drugs isolated and combined with antagonists of antioxidant systems through ultrastructural analysis and biochemical / cytochemical techniques of Plasmodium falciparum and P. berghei;

(5) the in vivo effect of the above-mentioned drugs and combinations on the survival of Plasmodium berghei-infected Swiss webster mice. It should be mentioned that both DS and Cure. has DNA radioprotective activity in Saccharomyces cerevisiae (Nemavarkar et al., 2004) and rodent (Srinivasan et al., 2007; 2008;) and human cells in vitro (Srinivasan et al., 2006; Sebastià et al., 2014; Shafaghati et al., 2014), as well as in in vivo experimental models (Ozgen et al., 2012; Cho et al., 2013). In these models the Cure. reverses the oxidative stress produced by radiation (Srinivasan et al., 2007). The effects of Cure have already been tested. in radiotherapy patients and the antioxidant capacity of the plasma was increased, but there was no difference in clinical outcome in the adopted regimen (Hejazi et al., 2016).

 [00118] The present invention will now be defined with reference to the following examples which are not to be construed as limiting the scope of the invention.

Methodology

 [00119] Human Blood Collection The procedures for collection in the peripheral veins were performed according to the technique recommended by the Ministry of Health (Brazil). Inclusion criteria were: healthy individuals of both sexes who had type O blood and positive HR factor (0+), aged over 21 years and weighing over 50 kg. The selected individuals donated 10 mL of blood, which was used only for in vitro Plasmodium culture. It is noteworthy that no form of diagnosis or characterization of the collected samples was made. In vivo assays were performed on a murine experimental model only. Cultivation of Plasmodium falciparum in erythrocytic phase

The experiments were performed using P. falciparum clone W2 (CQ-resistant), cultured in human red blood cells as described by Trager and Jensen, 1976. The parasites were kept in 50 to 150 ml plastic culture bottles at 37 ° C in RPMI 1640 (GibcoBRL) medium supplemented with 25mM Hepes (Sigma-Aldrich), 1mM glucose (Sigma-Aldrich), µg / ml gentamicin (Sigma-Aldrich), 5% sodium bicarbonate. filtered sodium (Sigma-Aldrich), hypoxanthine (Sigma-Aldrich) and 10% (v / v) 0 ⁺ (inactivated) human plasma collected at the Bahia Blood Center (HEMOBA). Cultures were maintained with 5% hematocrit in 90% N ₂ gasogenic mixture; 5% 02; 5% CO ₂ . Changes of culture medium and gasogenic mixture were performed daily. Parasitemia was monitored by Panotic-stained and fixed blood smears. Separation of P. falciparum from red blood cells: Red blood cell lysis was performed according to Gallo et al., 2009. Parasites were isolated from red blood cells using 20 times the pellet volume of a buffer containing 7 mM K ₂ HP0. _4, 1 mM NaH ₂ P0 _4, 11 mM NaHC0 _3, 58 mM KC1, 56 mM NaCl, 1 mM MgCl _2, 14 mM glucose and 0.02% saponin. The pellets were washed 2 times at 1500 g for 5 minutes in this buffer.

Synchronization of Cultures: Synchronization to obtain cultures with predominantly "ring" or young trophozoite parasites used for in vitro chemotherapeutic tests was performed according to Lambros and Vanderberg, 1979. Briefly, 15 mL of the 90 µM culture % of parasites in evolutionary "ring" form were centrifuged for 5 minutes at 2039 g. The cells were then resuspended in 10 mL of 5% sorbitol (VETEC) and incubated for 5 minutes at 37 ° C. The cells were then centrifuged at 2039 g for 6 minutes and maintained in culture as described.

Percoll cell concentration: To increase the rate of parasitemia, it was necessary to use the concentration of the parasitized cells by using the percoll gradient according to Kutner et al., 1985. Synchronized and hematocrit cultures of 5 % in the mature trophozoite stage were centrifuged at 550 g and 27 ° C for 5 minutes. The pellets were washed in phosphate buffered saline - PBS (137 mM NaCl - 2.7 mM KCl - 1.24 mM Na ₂ HPO ₄ - 0.14 mM KH ₂ P0 ₄ pH 7.2). The infected red blood cells were then resuspended in 2 mL of 6% sorbitol PBS (VETEC). Red blood cell suspensions in PBS-sorbitol were added over percoll gradients (GE Healthcare) (90% - 80% - 70% - 60%). After this procedure the tubes were centrifuged at 1500 g for 20 minutes at room temperature in a fixed angle rotor (Beckman) centrifuge.

Proliferation Tests: Proliferation tests have been Desjardins et al., 1979. The parasites were previously cultured in hypoxanthine-free medium for at least 72 hours and subsequently synchronized. Parasites were incubated with 5 (five) varying concentrations of compounds using the 1/3 ratio (10 (æg / mL to 1.23 μg / mL) for 24 hours in 96-well plates, after which time they were added to the medium). 25 μΕ / well [ ³ H] -hypoxanthine (0.5 μθ / ροςο), and incubated for a further 24 hours at 37 ° C. The plates were then frozen at - 20 ° C for 6-18 hours for lysis of the After this time, they were thawed and collected in glass capillaries on filter papers (Perkin-Elmer), in which 4 mL of scintillation fluid was added, and then placed in pouches and emerged in a flow scintillation. β-Matrix 9600 radioactive emission (Perkin-Elmer) The concentration of tritiated hypoxanthine incorporated into the parasites was assessed by reading the incorporated radioactivity The measurement of [ ³ H] -hypoxanthine incorporation was performed by counting per minute, being proportional viability of the parasite. To determine the IC ₅₀ of the drugs, five (5) drug concentrations were used. Ring stages (200 μΐ per well, with 5% hematocrit and 1-2% parasitemia) were exposed to drugs. All tests were performed in triplicate. Parasitemia and parasite morphology were analyzed on Giemsa stained smears and visualized under optical microscopy. IC ₅₀ values were calculated by the GRAPHPAD.PRISM5.0 program.

Evaluation of splenocyte cytotoxicity: 1 x 10 ⁶ splenocyte inocula from BALB / c mice were incubated in complete RPMI medium (GibcoBRL) supplemented with 10% (v / v) fetal bovine serum and 10 μΐ . [ ³ H] -thymidine / well to give a concentration of 1 μθ / ροςο in the presence or absence of drugs. To stimulate splenocyte proliferation, 10 μg / mL concanavalin A (Sigma-Aldrich) was used. After 24 hours, the cells were collected for radioactivity counting incorporated using the Matrix 9600 (Perkin-Elmer) counter. The inhibition percentage will be calculated relative to the control and the CC5 ₀ determined.

Evaluation of macrophage cytotoxicity: Macrophage collection was performed by peritoneal lavage using PBS at 4 ° C, pH 7.4. The collected peritoneum cells were transferred to sterile tubes, kept in an ice bath, centrifuged three times at 363 g for 5 minutes at 4 ° C in phosphate buffer for washing. The pelleted cells were resuspended in complete RPMI - 1640 culture medium at a concentration of 5x10 ⁶ cells / mL. From this suspension, 100 μΐ, added to each well of the plate (96 wells). Then the compounds, diluted in complete RPMI culture medium, were added in triplicate 100 μ of each. Incubations were performed for 24 hours (37 ° C, 7.5% CO ₂ ). After this period, 100 μΐ of a 3- (4,5-dimethylthiazol-2-yl) -2-5-diphenyltetrazolium (MTT) solution diluted in RPMI medium in a 1: 1 ratio were incubated in each well for 3 hours (37 ° C, 7.5% CO ₂ ). After the period, 100 μΐ were removed from the supernatant and DMSO was added in a 1: 1 portion. The cells were homogenized and centrifuged for 10 minutes at 1400 g. Cytotoxicity was measured by analyzing mitochondrial cell activity through the MTT reduction method. The supernatant was read on a VersaMax spectrophotometer at 570 nm.

In vitro hemolysis rates: To assess the ability of the compounds to cause hemolysis, the technique proposed by Wang and colleagues 2010 was used. Heparin tube blood was collected, centrifuged at 796 g for 10 minutes at 10 ° C. 4 ° C. The supernatant was removed and the red blood cells washed 4 times with PBS. Red blood cells (1% hematocrit) were incubated with varying concentrations of the substances (100 μg / mL - 50 μg / mL - 25 μg / mL - 12.5 μg / mL and IC ₅₀ of the substance) at 37 ° C in a greenhouse. 5% CO ₂ for 1 hour. A 1% (w / v) saponin solution was used as a positive control, generating 100% hemolysis. After incubation, the plates were centrifuged at 286 g for 10 minutes and 100 μΐ. of the supernatant were transferred to another microplate. The reading was taken at 540 nm in a spectrophotometer. The hemolysis rate of the samples was calculated according to the formula below:

% Hemolysis = sample absorbance - blank x 100 / saponin control absorbance

Selectivity Index: After determining the IC ₅₀ and CC ₅₀ of the isolated compounds and the combination, the selectivity indices (IS) were determined with the following formula: selectivity index = ^{L "}

For a compound to be considered safe, the IS must be equal to or greater than 10 (Bézivin et al., 2003).

Determination of Fractional Inhibitory Concentrations (ICF): For the determination of ICF and further characterization of the type of association between drugs, cell proliferation assays were performed. The assays were performed according to the previously mentioned methodology. However, for the determination of FIC, it was necessary to combine varying concentrations of one compound, keeping the concentration of the other fixed (and vice versa). Subsequently, the IC ₅₀ of the compound that had its concentration varied in the combination between the drugs was determined and later these results were plotted in the following formula aiming at the determination of the ICF:

From then on the IC ₅₀ of the drug combinations was calculated and subsequently these results were applied in the following formula: FIC = (IC ₅₀ of the drug combination in AB / _IC50 of drug A) + _(IC50 combination of drugs BA / _IC50 of drug B)

FIC results below 0.5 characterize synergistic effects (Hallander et al., 1982).

 [00133] Isobologram: The isobologram plots have been

built using the CompuSyn program (ComboSyn, Paragon, NJ, USA). For this purpose, FICs were determined in varying proportions (25%, 50%, 75%, 100%).

 Lipid peroxidation: Infected red blood cells were incubated in the presence and absence of drugs and, after treatment, the cells were centrifuged and then the red blood cells lysed according to the protocol described. After lysis, cells were resuspended in 200 μΐ PBS. 200 μΐ of 1% TBA (thiobarbituric acid) in distilled water and 1: 1 glacial acetic acid were added for further incubation at 100 ° C for a period of 3 hours. TBA-reactive substances (TBARS) were measured in a spectrophotometer at 532 nm and the results were expressed as μg of protein.

Transmission Electron Microscopy: Cells were concentrated by percoll gradient and samples were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2, post-fixed in osmium tethoxide 1% in 0.1M sodium cacodylate buffer, 0.8% potassium ferricyanide and 5mM calcium chloride protected from light for 40 minutes at room temperature. The cells were then washed in the same buffer and dehydrated in increasing acetone concentrations (30 - 100%) for 10 minutes each. Samples were infiltrated and embedded in Polybed epoxy resin (Polysciences). After polymerization, ultrafine sections were obtained on ultramicrotome and collected in copper grids. 400 mesh were then counterstained in 5% aqueous acetate and uranyl solutions for 20 min. and 3% lead citrate for 5 min. and observed under the transmission electron microscope Zeiss EM 109 at 80kV.

Fluorescence microscopy: Culture flasks containing Cure-treated synchronized Plasmodium falciparum infected red blood cells. and 10 µg / ml DETC for 2 hours were washed with PBS and centrifuged at 1,500 rpm (639 g) for 5 minutes. They were subsequently incubated with 10 μΜ of Dihydroetide (DHE) or 5 μΜ of MitoSox in Hank's balanced salt solution at 37 ° C. After this time, the cells were observed under the inverted fluorescence microscope.

Mitochondrial Superoxide Detection: For the detection of mitochondrial superoxide radicals, the MitoSox ^® (Molecular Probes ^® ) fluorescent probe was used. Plasmodium falciparum infected red blood cells were incubated in the presence of 5 μΜ MitoSox in HBSS for 10 minutes at 37 ° C in the dark. The cells had been previously treated with the test drugs at a concentration of 10 μg / mL for a period of 3 hours. After incubation, cells were washed in HBSS and evaluated by flow cytometry at 510/580 nm (excitation / emission).

Detection of reactive oxygen species: To detect reactive oxygen species, the DHE fluorescent probe was used. Plasmodium falciparum-infected red blood cells were treated for 3 hours with the test drugs, and were subsequently incubated with 10 μΜ DHE for 30 minutes at 37 ° C in PBS. After incubation, cells were washed and evaluated by flow cytometry and fluorescence microscopy.

[00139] Formation of β-Hematin / w Vitro: The synthesis of β-hematin in vitro allows us to investigate the influence of drugs on the formation of Hz, based on the principle presented by Egan et al. (2001) and modifications to the methods described by Nhien et al. In 2011. Were incubated in ΙΟΟμΜ hemin microtubes in 0.1M NaOH with dodecil solution 40 mM sodium sulfate (SDS). The final volume of 1 mL was completed with 0.5 M sodium acetate buffer (pH 4.8). After sixteen hours in an oven at 28 ° C, the tubes were centrifuged at 7,000 g for 10 min. The precipitates were then resuspended in 0.1 M 2.5% SDS sodium bicarbonate buffer (pH 9.1). . This procedure was repeated three more times, and then the precipitate was diluted in 0.1 M NaOH, quantifying free hemine at the end of the process by absorbing the samples at 400 nm on the VERSAmax microplate spectrophotometer.

 Dosage of hemozoin formation in parasite extracts: Ring-shaped infected red blood cells were incubated in the presence and absence of drugs after a period of 18 hours, whereas as mature trophozoites of Plasmodium falciparum, red blood cells were found and parasites were resuspended in 1 ml PBS. Subsequently, they were centrifuged at 14,000 g for 5 minutes at room temperature and the supernatant was discarded. The pellets were resuspended in 1.0 mL of 0.1 M sodium bicarbonate buffer pH 9.1 with 2.5% SDS. The samples were stirred for 15 minutes and centrifuged again at 14000 g in 10 minutes at room temperature (this procedure was repeated 3 times). After this process, the samples were resuspended in 1 mL of distilled water and vortexed for 1 minute. Again they were centrifuged at 14000 g for 10 minutes at room temperature, and the supernatant was discarded (this procedure was repeated 2 times). The samples were then resuspended in 1.0 mL of 0.1 M NaOH, stirred for 30 minutes and total heme concentrations were quantified by spectrophotometer at 400 nm.

In vivo experiments: They were performed on Swiss Webster mice weighing approximately 20 + 5 g from the CPqGM FIOCRUZ / BA vivarium. Mice were inoculated intraperitoneally with P. berghei infected erythrocytes (1 x 10 ⁵ ) as described by Peters W., 1965. Animals were treated orally daily for 5 days with the test substances. Two control groups (n = 5) were used for each experiment, one group treated with curing doses of chloroquine (25 mg / kg) and another group treated with test substance diluent (saline or dimethyl sulfoxide - DMSO). Survival of treated groups was calculated relative to untreated control groups. All substances were tested in 3 independent experiments.

In vivo toxicity assessment: For in vivo toxicity assessment, Swiss webster mice were orally treated for five consecutive days with Cure. and DETC isolated and combined. After 36 hours of the last treatment, blood and plasma samples were collected from these animals to evaluate the parameters indicative of liver damage: AST (Aspartate aminotransferase), ALT (Alanine aminotransferase), CK (Creatine kinase) and Urea. Samples were evaluated using Reflotron ^® Plus, ROCHE (Una Health Ltd).

Drugs Used

 (1) Chloroquine (6-chloro-4- (4-diethyl-amino-1-methylbutylamino) -quinoline)

 (2) DETC

 (3) Cure. ([(E, E) -1,7-bis (4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5 dione])

 (4) DS (Ι, Ι ', Γ, Γ'- [disulfanedylbis (carbonothioylnitrile)] tetraethane)

 Chloroquine and DS were obtained from Farmanguinhos / FIOCRUZ, and Cure. and DETC were purchased from Sigma-Aldrich. These drugs were tested only in vitro and in mice.

Statistical analysis: The data obtained were represented as the mean ± standard deviation and statistically analyzed by the tests. YEAR VA and Tukey post-test with significance level of p <0.05. All experiments were performed with at least three independent replicates in triplicate. The graphs presented in this patent application are representative of the results, confirming the synergistic effect of the combination proposed in the present invention.

[00145] Ethical Considerations: The animals were kept and handled within the norms recommended by FIOCRUZ-BA's Ethics Committee on Animal Use (CEUA), according to Law no. 1,153, Sérgio Arouca, 1995. The project was approved and approved by the aforementioned committee (license No. ² 020/2015).

 EXAMPLE 1: Inhibition of P. falciparum Growth in vitro

To evaluate protozoan susceptibility to different drugs tested, P. falciparum-infected human erythrocytes were incubated in vitro with Cure, DS and DETC for 24 hours, and cell proliferation was evaluated by tritiated hypoxanthine incorporation technique. The drugs were analyzed against the control with DMSO, solvent used to dilute Cure. The graphs are representative of the experiments and were repeated at least three times. Approximate IC ₅₀ values were obtained in at least four independent dose-response experiments employing five concentrations of single or combined drugs.

Figures 1, 2 and 3 show inhibition of P. falciparum proliferation / survival in vitro as measured by incorporation of [ ³ H] hypoxanthine incubated with Cure. (Fig 1), with DETC (Fig. 2) and with DS (Fig. 3). The IC ₅₀ values obtained with Cure, DETC and DS were 3.4 μg / mL, 8.0 μg / mL and 5.9 μg / mL, respectively.

EXAMPLE 2: In vitro growth inhibition of P. falciparum by drug combination

[00148] To evaluate the effect of drug combination on proliferation of the parasites, they were incubated with the combinations for a period of 24 hours, and FIC (fractional inhibitory concentration) values were calculated to determine whether combinations of different drugs have antagonistic, additive or synergistic effects.

The results are shown in Figures 4, 5, 6 and 7. The

Figure 4, which is the combination of Cure. with the DETC, shows the IC ₅₀ value of the DETC in the 0.0014 μg / mL combination, which determined the FIC value of 0.000175, thus characterizing a very synergistic effect for the combination at these concentrations, given that FIC values below 1 determine drug synergism in combinations.

Figure 5, which shows the combination of DETC and Cure, shows Cure IC ₅₀ . in the combination of 0.36 μg / mL, which determined the FIC value of 0.1, thus characterizing a synergistic effect.

Figure 6, which represents the combination of Cure. with DS, it shows the IC ₅₀ of the DS in the combination of 0.0017 μg / mL, which determined the FIC value of 0.00028, thus characterizing a highly synergistic effect.

Figure 7, which represents the combination of DS and Cure, shows Cure IC ₅₀ . in the combination of 0.44 μg / mL, which determined the FIC value of 0.129, thus characterizing a very synergistic effect. EXAMPLE 3: Isobolograms of Drug Combinations

To evaluate whether the combination used had a synergistic effect for different fractional combinations, as initially indicated by the FIC, the isobolograms were plotted.

Example 3A - Cure. + DETC

The isobologram result of different combinations between the Cure. and the DETC is shown in Figure 8 and demonstrates that of the eight combinations evaluated, 7 showed a synergistic effect, with values of combinations below 1. This result demonstrates that the combination of the two substances on P. falciparum proliferation is effective.

Example 3B - Cure. + DS

 The isobologram result of different combinations between Cure. and the DS is shown in Figure 9 and demonstrates that of the eight combinations evaluated, 6 had a synergistic effect, with combination values below 1. This result demonstrates that the combination of the two substances on P. falciparum proliferation is effective, same as with results inferior to the combination with sodium diethyldithiocrabamate (Example 3A).

 EXAMPLE 4 - Mammalian Cell Cytotoxicity

 To evaluate the possible in vitro selectivity of the tested substances, the cytotoxicity of the drugs was evaluated by the tritiated thymidine incorporation technique showing the proliferation of murine splenocytes.

The concentration-response relationship shows the effect of Cure. (Figure 10), DETC (Figure 11) and DS (Figure 12) on splenocyte proliferation in vitro, revealing IC ₅₀ (ie CC ₅₀ ) values of 11.2, 3.4 and 4.5 μg / mL, respectively.

 EXAMPLE 5 - Cytotoxicity of Splenocyte Combinations

 Example 5 was performed to evaluate the possible cytotoxic effects of the combined substances tested on splenocyte survival to assess the possible selective effects of the combined substances.

[00159] Figure 13 shows the effect of the Cure combination. with the DETC on splenocyte proliferation. The IC ₅₀ (ie CC ₅₀ ) of the DETC in the combination was 14.22 μg / mL, and when compared to its isolated CC ₅₀ (3.4 μg / mL), it can be concluded that the his combination with Cure. It is able to decrease its toxicity. [00160] Figure 14 shows the effect of the Cure combination. with the DS on splenocyte proliferation. It can be observed that the IC ₅₀ (ie CC ₅₀ ) of DS in the combination was 1.75 μg / mL.

[00161] Figure 15 shows the effect of combining DETC with Cure. on splenocyte proliferation. The IC ₅₀ (ie CC ₅₀ ) of the Cure combination is observed. was 7.76 μg / mL.

[16162] Figure 16 shows the effect of combining DS with Cure. on splenocyte proliferation. Since the toxicity of the combined compounds was very low, it was not possible to calculate the IC ₅₀ (ie CC ₅₀ ).

 [00163] Table 1 below shows the selectivity indices (IS) of the compounds in combination.

 Table 1: Selectivity of combination activity over Plasmodium

Selectivity indices were calculated by the ratio of IC ₅₀ found for the splenocyte combination (ie CC ₅₀ ) to the IC ₅₀ of the combination in P. falciparum cultures. Data were based on the average determined by the experiments performed for each evaluation.

Table 1 shows a much lower splenocyte cytotoxicity than the toxicity of combinations on P. falciparum cultures, which is extremely interesting for a chemotherapy combination. The IS of the DETC + Cure combination. was greater than 10,000, ie well above the gold standard drug ie artemisinin, whose IS obtained was 27.57 (more than 368 times more selectivity). Could not calculate Cure selectivity index. in combination with DS, as it is impossible to determine the value of cytotoxicity in splenocytes of this combination. The IS demonstrates a high security, as these are higher than 3-10 and are considered highly selective.

(Bézivin et al. 2003; Prayong et al. 2008).

 EXAMPLE 6 - Reversibility of Antiparasitic Effect

 Example 6 was performed to evaluate the cytotoxic or cytostatic effects of drugs on the proliferation of Plasmodium falciparum. The pretreatment with the urate antioxidant was intended to assess whether the effect on cells had any relation to oxidative stress. Results are shown in Figures 17, 18 and 19.

 [00167] Figure 17 shows the evaluation of the effect of Cure. on red blood cells infected with Plasmodium falciparum in the presence and absence of urate. After 48 hours of incubation, the drug was withdrawn and the cells cultured. Even after the drug was removed from the medium, the cells did not proliferate again, demonstrating a cytotoxic or irreversible effect of the drug under these conditions. It is noteworthy that preincubation with urate did not reverse or prevent the effect of the drug.

[00168] Figure 18 shows the evaluation of the effect of DETC on Plasmodium falciparum infected red blood cells in the presence and absence of urate. After 48 hours of incubation, the drug was withdrawn and the cells cultured. It can be observed that after drug withdrawal from the medium, cell proliferation was restarted, which demonstrates a possible reversible cytostatic effect of the drug under these conditions. It is noteworthy that preincubation with urate inhibited the effect of the drug during the first 48 hours, demonstrating that the effect of the drug can be reversed through the use of antioxidant.

[00169] Figure 19 shows the evaluation of the effect of DS on Plasmodium falciparum-infected red blood cells in the presence and absence of urate. After 48 hours of incubation, the drug was removed and the cells cultured again. It was observed that after drug withdrawal from the medium, the cells proliferated again, demonstrating a possible reversible cytostatic effect of the drug under these conditions. It is noteworthy that preincubation with urate partially reversed the drug effect during the first 48 hours, which possibly relates the drug effect to oxidative stress, but presumably not as the sole mechanism of action.

EXAMPLE 7 Reversal of Drug Effect by Antioxidant Urate

For the realization of this Example cells were preincubated in the presence of urate to assess whether the antioxidant had the ability to reverse the effect of drugs on parasite proliferation in vitro.

 [00171] Figure 20 shows the ability of the urate antioxidant to reverse the effect of DS on P. falciparum proliferation in vitro. Figure 21 shows the ability to reverse the effect of DETC by urate antioxidant action. Figure 22 shows the reversibility of the Cure effect. on the growth of P. falciparum in vitro by urate antioxidant action.

 EXAMPLE 8 - Hemolysis Test

 Given that Plasmodium falciparum is an intracellular parasite that infects red blood cells and that symptomatology (paroxysm) is linked to the hemolytic process, the ability of the drugs to cause hemolysis to verify whether the activity of the substances was on the blood was evaluated. parasite per se or by inducing red cell lysis; thus preventing the maintenance of the infection. Results are shown in Figures 23, 24, 25, 26 and 27.

 As shown in Figure 23, it was observed that at the concentrations tested, Cure. showed no ability to cause hemolysis.

DETC did not show hemolytic activity at the concentrations tested (Figure 24). It was observed that the DS was not able hemolysis at the concentrations tested (Figure 25). Figure 26 shows the combination of Cure. and DETC also did not produce hemolysis at the tested concentrations. Figure 27 shows that Cure. In combination with DS, it did not produce hemolysis at the concentrations tested. In these trials, the saponin detergent was used as a positive control.

Ultrastructural analysis of transmission-infected erythrocytes by transmission electron microscopy revealed marked dilations of endoplasmic reticulum cisterns that make up the nuclear envelope, which often appear to be juxtaposed to the membranes of the digestive vacuoles (data not shown). These findings suggest that the drugs cause imbalance in Ca ²⁺ homeostasis and vesicle traffic.

The permeabilization of the membranes of digestive vacuoles was evidenced by the presence of hemozoin crystals in the cytoplasm of treated protozoa (not shown), indicating parasite autolysis. Therefore, it is presumed that the combinations tested here exert both the pro-oxidant effect, evidenced by the use of fluorescent probes and lipoperoxidation, as well as the digestive vacuole permeabilization, demonstrated by microscopy. Intracellular parasites in cultures treated with the Cure combination. -DETC showed large vacuoles containing undigested hemoglobin, so we sought to biochemically measure hemozoin formation.

 EXAMPLE 9 - Inhibition of Hemozoin Formation in vitro

The formation of hemozoin is an essential metabolic process for the survival and maintenance of the Plasmodium falciparum cycle. In view of the aforementioned electron microscopy findings and changes in crystal structure, the possible effects of the substances on this process were biochemically evaluated by the hemozoin dosage in order to verify if the mechanism of action of the drug was related to crystal formation. In addition to the changes in the crystals, these they appeared to be low in abundance and large areas of host cell hemoglobin engulfment, presumably a compensatory homeostatic mechanism to reverse metabolic deficit, were detected by electron microscopy (not shown).

 [00178] Figure 28 shows the effect of Cure. β-hematin formation in the presence of 40mM SDS in vitro. At the tested concentrations no significant effect of the substance was observed on in vitro crystal formation.

 [00179] Figure 29 shows the effect of DETC on in vitro formation of β-hematin in the presence of 40mM SDS. At the tested concentrations there was no significant effect of the compound on in vitro crystal formation.

 [00180] Figure 30 shows the effect of DS on in vitro formation of β-hematin in the presence of 40mM SDS. At the tested concentrations, only the 50μΜ had a statistically significant effect (<0.05) compared to the control, using VA ANO and Tukey post-test. Although 100 μΜ DS caused apparent reduction in hemozoma formation, it was not statistically significant.

EXAMPLE 10 - Inhibition of hemozoin formation in parasite extracts.

 In view of the disagreement of some of the in vitro hemozoma dosing data with the electron microscopy images (not shown), mainly related to Cure, the hemozoma formation dosing technique was again used, this time making use of parasite extracts.

From the results of Figure 31, which show the dosage of Plasmo diumfalciparum hemozoma (HZ) treated with the drug IC ₅₀ , it can be observed that the Cure. inhibited crystal formation in vivo by more than 50%, with a concentration of hemozoma below 0.01 protein, ie levels similar to those observed in the presence of chloroquine, being highly statistically significant in relation to the control (<0.0001, Dunnett's Test). DETC and DS did not interfere with crystal formation at the concentrations tested. These data corroborate the ultrastructural observations regarding undigested hemoglobin in parasites incubated with the drug combination.

EXAMPLE 11 - Lipid Peroxidation

 When observing the presence of concentric membranes and myelin figures in transmission electron microscopy (not shown), mainly associated with treatment with DETC and DS, the occurrence of oxidative stress was hypothesized, thus the use of thiobarbituric acid reactive substances (TBARS) detection technique to measure lipid peroxidation, an event associated with the production of reactive oxygen species.

Figure 32 shows the measurement of TBARS in Plasmodium falciparum infected red blood cells incubated with drug IC ₅₀ for 24 hours. It can be observed that the DETC statistically significantly increases (<0.05, ANOVA, Tukey) the production of lipid peroxidation inducing substances, reaching about 0.03 protein absorbance, which was not observed. observed for Cure. DS, in turn, showed a significant decrease in lipid peroxidation levels when compared to control.

EXAMPLE 12 - DHE Fluorescent Probe Detection

Microscopic verification of the increase in reactive species generation by the tested substances was performed using probes such as DHE, which reaction was quantified by flow cytometry to generate reactive oxygen species such as superoxide in red blood cells. infected with Plasmodium falciparum.

Figure 33 shows red cell flow cytometry Plasmodium falciparum-infected humans labeled with the DHE probe. Flow cytometry of control (A) cultures and treated for 2 hours with 1 (µg / ml DETC (B) and 1 (µg / ml Cure. (C), with the combination of both (D) Detection of the DHE probe by fluorescence microscopy demonstrated positive placental staining with the presence of intracellular parasites, but not on non-parasitized red blood cells, which remained negative.This effect was partially reversed by incubation with N-acetyl-L-cysteine (E The labeling was intense for DETC, DS and Cure, unequivocally demonstrating the pro-oxidant effect caused by the drugs, even though Cure is a natural product marketed as an antioxidant.

 [00187] Figure 34 shows the comparison between medians and gate percentages in the different populations observed by flow cytometry when using the DHE probe. Through the median of the fluorescence intensity of M2 determined by the flow cytometry (referring to the total population) with their respective percentages of the population in the gate, it is possible to observe that the Cure. leads to increase in fluorescence intensity when compared to untreated cell. However, it is important to emphasize that the number of the population is reduced. When combined with DETC, the fluorescence intensity remains very similar to Cure. isolated, however, there was an increase in the population expressing fluorescence intensity.

Figure 35 shows the median fluorescence intensity of the M3 population when using the DHE probe, delimited by flow cytometry, with their respective population percentages at the gate. It is possible to observe that the Cure. leads to an increase in fluorescence intensity compared to untreated cells. However, it is important to emphasize that the number of the population is reduced. When combined with DETC, the fluorescence intensity remains quite similar to that of Heal isolated, however, there is an increase in the population expressing this fluorescence intensity.

 [00189] Figure 36 shows the median fluorescence intensity of the M4 population by flow cytometry when using the DHE probe with its respective population percentages at the gate. It is possible to observe that the Cure. leads to increased fluorescence intensity when compared to untreated cell. However, it is important to emphasize that the number of the population is reduced. When combined with DETC, the fluorescence intensity remains high, but there is an increase in the population expressing this fluorescence intensity.

 [00190] Figure 37 shows the median fluorescence intensity of the M5 population when using the flow cytometry DHE probe with its respective population percentages at the gate. It is possible to observe that the Cure. leads to an increase in fluorescence intensity when compared to untreated cells, which is also observed when cells were treated with DETC. In this specific case, the population with high fluorescence expression was also observed in all treatments when compared to the control population.

EXAMPLE 13 - Flow Cytometry with the MitoSox Probe

From the observation of the increase in intracellular ROS levels by the use of the DHE probe, which detects ROS as 0 ₂ ^~ , it was evaluated if this increase was due, at least in part, to the increase in radical levels 0 _2. mitochondrial ^~ in view of the DETC is an inhibitor of SOD.

[00192] Figure 38 shows flow cytometry erythrocytes infected with Plasmodium falciparum untreated MitoSox incubated with the probe to evaluate the profile of oxidative stress by detection of mitochondrial radical 0 ^~ ₂ in cells without incubation with drugs. It can be observed that the population M2 corresponds to 7.64% of the total, and presents a median of 80.58; thus showing the behavior of cells at their basal stage.

Figure 39 shows flow cytometry of P. falciparum-infected red blood cells treated with 10 μg / ml DETC for 2 hours and subsequently incubated with the MitoSox probe. It is possible to observe, compared to the control data, that there was a significant increase in the median fluorescence intensity of these cells, thus indicating that DETC increases the production of 0 ₂ ^~ in the mitochondrial compartment of these cells.

Figure 40 shows flow cytometry of Plasmodium falciparum-infected red blood cells treated with 10 μg / mL DETC and 10 μg / mL Cure. for 2 hours and subsequently incubated with the MitoSox probe. It can be observed that, compared to the control data, there was an increase in the median fluorescence intensity of these cells. However, when compared with DETC treatment, it was observed that there was a decline in fluorescence intensity and that, to a degree at 0 ₂ ^" , Cure. Has a protective effect, but not sufficient to reverse the level. of control.

Figure 41 shows flow cytometry of 10 μg / mL DETC-treated Plasmodium falciparum-infected red blood cells in the presence of 10 μg / mL tocopherol incubated with the MitoSox probe. It can be observed that tocopherol has a protective effect on cells, returning the 0 ₂ ^~ mitochondrial levels to the control level.

Figure 42 shows flow cytometry of Plasmodium falciparum-infected red blood cells treated with 10 μg / mL DETC and 10 μg / mL Cure in the presence of 10 μg / mL tocopherol and incubated with the MitoSox probe. Tocopherol can be seen to have a protective effect on cells, reversing mitochondrial 0 ₂ ^" levels below the control level, possibly a joint reaction with Cure. Figure 43 shows the comparison of fluorescence intensities of the MitoSox probe with 10 μg / mL of the various P. falciparum infected erythrocyte treatments. It can be seen that the treatment with DETC significantly increased the fluorescence intensity of the cells compared to the control. This effect was reversed by both Tocopherol and the combination with Cure.

EXAMPLE 14 In vivo Testing

 Survival of Plasmodium berghei-infected mice treated with the compounds of interest alone and in combination was evaluated.

 Figure 44 shows the evaluation of the effect of DS at concentrations of 5 and 25 mg / kg on survival of P. berghei-infected mice. It was observed that the drug at a concentration of 5 mg / kg had a survival of 80% at 25 days and 20% on the thirtieth day (end of the experiment), while in the negative control (DMSO) animals perished before the twentieth day. .

 Figure 45 shows the evaluation of the effect of DS at concentrations of 50 and 100 mg / kg on the cumulative mortality of P. berghei-infected mice. It was observed that the DS, at the tested concentrations, had a longer survival than the negative control between days 10 and 20, but later the survival rates were similar to the control, that is, around 15%. Positive control with chloroquine maintained the survival rate of 60% at the end of the experiment.

Figure 46 shows the survival assessment of P. berghei-infected mice treated with different concentrations of Cure. It is possible to observe that, among the tested concentrations, Cure. 25 mg / kg had a better effect, both in increasing the animals' survival time, almost reverting to the positive control level, and maintaining a survival rate of approximately 28%, while the negative control (DMSO) maintained a survival rate of approximately 15%.

 Figure 47 shows survival assessment of P. berghei infected mice treated with different concentrations of DETC, DMSO (negative control) and chloroquine (positive control). It was possible to observe that, among the tested concentrations, 100 mg / kg DETC was more effective, increasing the animals' life span and presenting a survival rate of approximately 45%, very close to the positive control, which presented approximately 57% survival.

 Figure 48 shows the survival assessment of P. berghei-infected mice treated with the combination of DETC 100 mg / kg and Cure. 25 mg / kg because these concentrations were the best results when evaluated alone. It can be seen that the combination of the drugs showed an increase in animal survival and a survival rate of approximately 42%.

 EXAMPLE 15

 Systemic toxicity was assessed by the measurement of hepatic transaminases in plasma from DETC and Cure treated mice, isolated and combined for 5 consecutive days.

 Figure 49 shows the evaluation of the plasma activity of Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST). It was observed that, in the animals that were administered the combinations, the activities ALT and AST remained below the controls.

Figure 50 shows in Creatine kinase (CK) dosage and B plasma urea levels in mice treated as above. It was observed that, in the animals that were administered the combinations, the dosages did not differ from the negative controls. [00207] As commented here, there is a consensus to promote the use of drugs already approved by regulatory agencies for new uses in the clinic. Among these drugs, it is worth mentioning the DS (Namazi, 2008; Chavali et al., 2012). DS and consequently DETC have antiparasitic activity against Trypanosoma brucei and T. rhodesiense; T. cruzi; Leishmania; Giardia lamblia; Trichuris muris, Plasmodium falciparum and potentiator of chloroquine antimalarial action in vivo. However, DETC reverses the antimalarial effect of alloxan and may increase parasitemia in P. berghei-infected animals (Nair et al., 1982).

Similarly, Cure. has reported antimalarial activity (Haddad et al., 2011) and has been used in combination with antimalarial drugs (Mimche et al., 2011) and, given with artemeter, can prevent the recurrence of murine Plasmodium berghei infection (Vathsala et al. 2012).

 Although Cure. considered a natural antioxidant, the compound exerts anu- Plasmodium activity associated with the generation of ROS (Cui et al., 2007). This apparent discrepancy is presumably due to the dose-dependent bimodal effect of Cure. which at low concentration (i.e. 20 μΜ) inhibits ROS production, while at 100 μΜ the drug proves to be pro-oxidant in Hep3B cells (Kang et al., 2005). Meanwhile to Cure. may be pro-oxidant even at low concentrations, induces glutathione-mediated responses (Leong et al., 2012) and is a potent inhibitor of P. falciparum chloroquine resistance enzyme glutathione transferase (Mangoyi et al., 2010 ).

[00210] The anti-malarial effect of Cure. can be explained, at least in part, by the induction of Cure-induced programmed erythrocyte cell death or erythroptosis (Föller et al., 2008). It is noteworthy that oxidative stress, including the poor functioning of antioxidant systems can induce erythroptosis (Lang et al., 2010). In addition to inhibition of SOD (Heikkila et al, 1978) DETC and hence its precursor DS have the ability to oxidize glutathione, promoting oxidative stress (Rahden-Staron et al, 2012).

A Cure. It is also capable of modulating glutathione metabolism (Heeba et al, 2012) and its depletion by ROS production can trigger the apoptosis process (Kizhakkayil et al., 2012).

The results reported here indicate that the combination of

Heal and DETC has a prooxidant effect revealed by the production of ROS. DETC-induced lipoperoxidation indicates that these combinations can cause significant oxidative stress on parasites. The extensive dilatation of the endoplasmic reticulum that delimits trophozoite nuclei may have been due to oxidative stress, as this process increases cytoplasmic calcium levels, which may be a signaling pathway for different pathways of cell death.

 Once the Cure. inhibited hemozoin formation with the same efficiency as chloroquine, it is possible that free heme rings exert a high pro-oxidant effect on parasites incubated with the combined drugs. The free heme generation by Cure. and combinations may cause discontinuity of the digestive vacuole membrane, as reported in the treatment of P. falciparum-infected red blood cells treated with chloroquine (Ch'ng et al., 2011), as evidenced by fluorescence and electron microscopy (not shown).

It is noteworthy that the increase in calcium, suggested by the marked dilatation of the endoplasmic reticulum that delimits the nuclei of the parasites, may be related to the permeabilization of the digestive vacuole, since this compartment accumulates Ca ²⁺ in the protozoan.

[00216] The present invention is based on the combination of Cure. and DETC, which acts as an antimalarial drug.

Although illustrated and described herein with reference to certain Specific representations, the present invention is not intended to be limited to the details given. Instead, various modifications may be made to the details within the scope and equivalence scope of the claims and without departing from the spirit of the invention.

 [00218] The enthusiasm regarding Cure's medicinal potential. and the tumeric motivated the publication of numerous books (eg Majeed & Badmaev 1999; McBarron, 2013; Stine, 2013; Harris, 2014; Geoffreys 2014) and articles (eg Aggarwal et al., 2007a; Goel et al., 2008) that they exalt supposedly miraculous properties (eg Mansour, 2010; Lee, 2016; Ahmed, 2014; Daniels 2014).

 [00219] In recent years, reports by many research groups on various experimental models have shown that Cure. may also have toxic or deleterious activities both in vitro (Goodpasture & Arrighi, 1976; Holy, 2002; Bielak-Zmijewska et al., 2010; Sebastià et al., 2012) as well as in vivo (Giri et al., 1990; Nair et al., 2005), but most of the deleterious effects appear to be due to methodological problems such as concentration of use and type of solvent employed (Kurien et al., 2011). Such unconditional differences may explain why some data are conflicting. The cure. may be genotoxic (Sebastià et al., 2012) or antigenotoxic (Shukla et al., 2003; Ahmad et al., 2004); carcinogenic (National Toxicology Program, 1993) and anticancer (e.g. Basnet & Skalko-Basnet, 2011; Shehzad et al., 2014; Hasima & Aggarwal, 2014; Li & Zhang, 2014; Rahmani et al., 2014). Some of these studies employed two-year treatment regimens (López-Lázaro, 2008).

The pharmaceutical compositions of the invention comprise the mixture of Cure, DETC or DS and pharmaceutically acceptable excipients and described in official compendium, providing immediate and / or controlled release medicament suitable for oral and mucosal administration (Rowe et al. , 2009). Pharmaceutical forms suitable for the compositions of The invention, without limitation, is: solution, syrup, suspension, emulsion, tablet (as example, plain, coated or special such as dispersible, sublingual and multilayer tablet), capsule, multiparticulate (such as powder, granulate and / or pellet). ) suppository, aerosol and formulations from solid dispersions and / or micro or nanoencapsulation (Aulton, 2005).

The dosages to be employed in the final formulation will certainly not be those employed in murine models, due to metabolic differences that drastically affect bioavailability. Thus, we are working on testing fixed-dose combinations in proofs of concept employing up to 250 mg / day of DS, which is well tolerated in humans (reviewed in Gessner & Gessner, 1992) and commonly employed (Antabuse ^® or Antiethanol ^®). ) at twice the concentration ie 500 mg / day - adults); associated with 1000 mg of Cure. daily, which do not cause side effects. Clinical trials have been reported in which healthy individuals are evaluated receiving up to 8 g Curc./day (Aggarwal et al., 2007b).

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Claims

 An antimalarial composition, characterized in that it comprises a therapeutically effective amount of curcumin and disulfiram or disulfiram derivatives and a pharmaceutically acceptable carrier.  Composition according to Claim 1, characterized in that therapeutically effective and well tolerated dosages of curcumin and disulfiram used in the composition in fixed-dose combinations for proof of concept tests employing up to 250 mg / day disulfiram. or its derivatives, which is well tolerated in humans combined with 1000 mg of curcumin daily, which causes no side effects.  Composition according to Claim 1, characterized in that it has a pharmaceutically acceptable carrier, comprising a pharmaceutical form suitable for administration.  A method for treating malaria, comprising administering to an individual in need of treatment an effective amount of the composition as defined in claim 1.  Use of an antimalarial composition as defined in claims 1 to 3, characterized in that it is in the preparation of an antimalarial medicament.