There is no effective treatment for the prevalent chronic form of Chagas' disease in Latin America. Its causative agent, the protozoan parasite Trypanosoma cruzi, has an essential requirement for ergosterol, and ergosterol biosynthesis inhibitors, such as the antifungal drug posaconazole, have potent trypanocidal activity. The antiarrhythmic compound amiodarone, frequently prescribed for the symptomatic treatment of Chagas' disease patients, has also recently been shown to have antifungal activity. We now show here for the first time that amiodarone has direct activity against T. cruzi, both in vitro and in vivo, and that it acts synergistically with posaconazole. We found that amiodarone, in addition to disrupting the parasites' Ca(2+) homeostasis, also blocks ergosterol biosynthesis, and that posaconazole also affects Ca(2+) homeostasis. These results provide logical explanations for the synergistic activity of amiodarone with azoles against T. cruzi and open up the possibility of novel, combination therapy approaches to the treatment of Chagas' disease using currently approved drugs.
Chagas' disease, a protozoan infection by the kinetoplastid Trypanosoma cruzi, constitutes a major public health problem in Latin America. With the use of mouse models of both short- and long-term forms of the disease, the efficacy of D0870, a bis-triazole derivative, was tested. D0870 was able to prevent death and induced parasitological cure in 70 to 90 percent of animals, in both the short- and long-term disease. In contrast, currently used drugs such as nifurtimox or ketoconazole prolonged survival but did not induce significant curing effects. D0870 may be useful in the treatment of human long-term Chagas' disease, a condition that is currently incurable.
High field31 P nuclear magnetic resonance spectroscopy showed that inorganic pyrophosphate (P 2 O 7 4؊ ) is more abundant than ATP in Trypanosoma cruzi, the causative agents of Chagas' disease. These results were confirmed by specific analytical assays, which showed that in epimastigotes, the concentrations of inorganic pyrophosphate and ATP were 194.7 ؎ 25.9 and 37.6 ؎ 5.5 nmol/mg of protein, respectively, and for the amastigote form, the corresponding concentrations were 358.0 ؎ 17.0 and 36.0 ؎ 1.9 nmol/mg of protein. High performance liquid chromatographic analysis of perchloric acid extracts of epimastigotes labeled for 3 h with 32 P-orthophosphate showed a significant incorporation of the precursor into inorganic pyrophosphate. Inorganic pyrophosphate was not uniformly distributed in T. cruzi but was shown by 31 P-NMR and chemical analysis to be particularly associated with acidocalcisomes, organelles shown previously to contain large amounts of phosphorus and various elements. Electron microscopy analysis of pyrophosphatase-treated permeabilized epimastigotes showed disappearance of the electron density of the acidocalcisomes. Nonmetabolizable analogs of pyrophosphate, currently used for the treatment of bone resorption disorders, selectively inhibited the proliferation of intracellular T. cruzi amastigotes and produced a profound suppression in the number of circulating trypomastigotes in mice with an acute infection of T. cruzi, offering a potentially new route to chemotherapy.Infections caused by Trypanosoma cruzi are among the most widespread parasitic diseases in Latin America and are responsible for heavy socioeconomic losses. There is therefore considerable interest in developing novel chemotherapeutic approaches, based on unique aspects of the structure and metabolism of this parasite. T. cruzi develops intracellularly in its vertebrate hosts and is confronted during its life cycle with drastic changes in its microenvironment. Survival through such complex environmental changes requires appropriate reserves of carbon and energy sources, as well as signaling species, such as Ca 2ϩ . Recent work has led to the identification of an acidic calcium pool (acidocalcisome) in this organism (1-4) that contains most of its cellular Ca 2ϩ , together with large amounts of magnesium, sodium, zinc, and phosphorus (3). However, the precise chemical nature of the phosphorus compound(s) in these vacuoles was not determined.In this work, we used 31 P nuclear magnetic resonance (NMR) spectroscopy to investigate the nature of the acidocalcisomal phosphorus.31 P NMR has been extensively used in the past to study the energy metabolism of several microorganisms, as well as that of living vertebrate tissues (5, 6). It has been particularly useful in the identification of phosphorus-based storage compounds, such as polyphosphates (7,8), and sugar phosphates and diphosphates (9, 10). Our results indicate that pyrophosphate is more abundant than ATP in the replicating forms of the parasite and is partly located in acidocalci...
We have investigated the antiproliferative effects of SCH 56592, a new experimental triazole, against Trypanosoma (Schizotrypanum) cruzi, the etiological agent of Chagas' disease in Latin America. SCH 56592 blocked the proliferation of the epimastigote form of the parasite in vitro at 30 nM, a concentration 30-to 100-fold lower than that required with the reference compounds ketoconazole and itraconazole. At that concentration all the parasite's endogenous sterols (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3-ol, and its 22-dihydro analogs), were replaced by methylated sterols (lanosterol and 24-methylene-dihydrolanosterol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of the drug was inhibition of the parasite's sterol C-14␣ demethylase. Against the clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37°C, the MIC of SCH 56592 was 0.3 nM, again 33-to 100-fold lower than that of ketoconazole or itraconazole. In a murine model of acute Chagas' disease, SCH 56592 given at > 10 mg/kg of body weight/day for a total of 43 doses allowed 85 to 100% survival and 90 to 100% cure of the surviving animals, as verified by parasitological, serological, and PCRbased tests, while ketoconazole given at 30 mg/kg day allowed 60% survival but only 20% cure. In a murine model of chronic Chagas' disease, SCH 56592 was again more effective than ketoconazole, providing 75 to 85% protection from death, with 60 to 75% parasitological cures of the surviving animals, while no parasitological cures were observed with ketoconazole. The results indicate that SCH 56592 is the most powerful sterol biosynthesis inhibitor ever tested against T. cruzi and may be useful in the treatment of human Chagas' disease.
Chagas' disease is a serious public health problem in Latin America, and no treatment is available for the prevalent chronic stage. Its causative agent, Trypanosoma cruzi, requires specific endogenous sterols for survival, and we have recently demonstrated that squalene synthase (SQS) is a promising target for antiparasitic chemotherapy. E5700 and ER-119884 are quinuclidine-based inhibitors of mammalian SQS that are currently in development as cholesterol-and triglyceride-lowering agents in humans. These compounds were found to be potent noncompetitive or mixed-type inhibitors of T. cruzi SQS with K i values in the low nanomolar to subnanomolar range in the absence or presence of 20 M inorganic pyrophosphate. The antiproliferative 50% inhibitory concentrations of the compounds against extracellular epimastigotes and intracellular amastigotes were ca. 10 nM and 0.4 to 1.6 nM, respectively, with no effects on host cells. When treated with these compounds at the MIC, all of the parasite's sterols disappeared from the parasite cells. In vivo studies indicated that E5700 was able to provide full protection against death and completely arrested the development of parasitemia when given at a concentration of 50 mg/kg of body weight/day for 30 days, while ER-119884 provided only partial protection. This is the first report of an orally active SQS inhibitor that is capable of providing complete protection against fulminant, acute Chagas' disease.Chagas' disease, a parasitic disease caused by the kinetoplastid protozoan Trypanosoma (Schizotrypanum) cruzi, is still one of the most serious public health problems in Latin America, despite recent advances in the control of the vectorial and transfusional transmission of the parasite (36). There are ca. 18 million people currently infected with T. cruzi, most of them in the chronic phase, which can lead to severe heart and gastrointestinal lesions (36). Current chemotherapy, based on the nitrofuran nifurtimox (Bayer) and the nitroimidazole benznidazole (Roche), is unsatisfactory as these compounds are effective only for recent infections and have frequent toxic side effects (21,24,28). Studies have shown that T. cruzi has a strict requirement for specific endogenous sterols (ergosterol and analogs) for survival and growth and cannot use the abundant supply of cholesterol present in its mammalian host (21)(22)(23)25). Ergosterol biosynthesis inhibitors with potent in vitro activity and special pharmacokinetic properties in mammals (large volumes of distribution and long half-lives) can induce the radical cure of parasitic infection in experimental animal models of both acute and chronic 25). Squalene synthase (SQS; EC 2.5.1.21) catalyzes a head-to-head reductive dimerization of two molecules of farnesyl pyrophosphate (FPP) in a two-step reaction to form squalene (Fig.
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects several million people in Latin America. Myocarditis, observed during both the acute and chronic phases of the disease, is characterized by an inflammatory mononuclear cell infiltrate that includes CD4+ T cells. It is known that Th1 cytokines help to control infection. The role that Treg and Th17 cells may play in disease outcome, however, has not been completely elucidated. We performed a comparative study of the dynamics of CD4+ T cell subsets after infection with the T. cruzi Y strain during both the acute and chronic phases of the disease using susceptible BALB/c and non-susceptible C57BL/6 mice infected with high or low parasite inocula. During the acute phase, infected C57BL/6 mice showed high levels of CD4+ T cell infiltration and expression of Th1 cytokines in the heart associated with the presence of Treg cells. In contrast, infected BALB/c mice had a high heart parasite burden, low heart CD4+ T cell infiltration and low levels of Th1 and inflammatory cytokines, but with an increased presence of Th17 cells. Moreover, an increase in the expression of IL-6 in susceptible mice was associated with lethality upon infection with a high parasite load. Chronically infected BALB/c mice continued to present higher parasite burdens than C57BL/6 mice and also higher levels of IFN-γ, TNF, IL-10 and TGF-β. Thus, the regulation of the Th1 response by Treg cells in the acute phase may play a protective role in non-susceptible mice irrespective of parasite numbers. On the other hand, Th17 cells may protect susceptible mice at low levels of infection, but could, in association with IL-6, be pathogenic at high parasite loads.
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