A quantitative colorimetric assay using the oxidation-reduction indicator resazurin was developed to measure cytotoxicity of compounds against the protozoan parasite Trypanosoma cruzi. This method is based on the detection of colorimetric changes caused by the oxidation (blue) and reduction (pink) capabilities of resazurin dye, an indicator for metabolic cell function. To validate the assay, the experimental conditions were adjusted, such as number of parasites, dye concentration, and time of incubation, with respect to linearity and lower limit of detection. We found that absorbances increased linearly, with the plating density of parasites as low as 5-100 x 10(4)/well (r=0.99; p<0.001) when they were incubated for 5 h at 28 degrees C in the presence of 10% resazurin solution (3 mM). When the cytotoxicity of the reference drugs nifurtimox and benznidazole was measured with this assay and compared to the microscopic counting method, the same range was obtained, demonstrating that the resazurin microtiter assay is valid for the screening of new trypanocidal compounds. This test is very simple, fast, sensitive, and cheap.

The method most commonly used in screening of drugs for the treatment of Chagas' disease, microscopic counting of viable trypanosomes, is time-consuming, labor-intensive, and dependent on the observer. Although the tetrazolium dye [MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is comparatively quick and accurate, it requires careful attention in design as well as in interpretation of the results. Therefore, we examined under various conditions the sensitivity and specificity of the MTT assay versus microscopic counting for determination of the viability of Trypanosoma cruzi for drug-screening purposes. We tested different concentrations of MTT in phenazine methosulfate (PMS) against T. cruzi epimastigotes of the Y strain in different stages of logarithmic growth. In our model, in tests of benznidazole and nifurtimox the optimal concentration of MTT was 2.5 mg/ml of PMS and the optimal incubation period was 75 min. This method detected parasite concentrations of approx. 500,000 epimastigotes/ml (P<0.01), and the linear correlation between absorbance values and numbers of epimastigotes per milliliter was very strong (approx. R = 0.99). The present MTT assay results in faster determination of the activity of compounds, is more objective, and enables testing of several drugs simultaneously.

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In the search for new therapeutic tools against Chagas disease (American trypanosomiasis) palladium and platinum complexes of the bioactive ligand pyridine-2-thiol N-oxide were exhaustively characterized and evaluated in vitro. Both complexes showed high in vitro growth inhibition activity (IC(50) values in the nanomolar range) against Trypanosoma cruzi, the causative agent of the disease. They were 39-115 times more active than the antitrypanosomal drug Nifurtimox. The palladium complex showed an approximately threefold enhancement of the activity compared with the parent compound. In addition, owing to their low unspecific cytotoxicity on mammalian cells, the complexes showed a highly selective antiparasite activity. To get an insight into the mechanism of action of these compounds, DNA, redox metabolism (intraparasite free-radical production) and two parasite-specific enzymes absent in the host, namely, trypanothione reductase and NADH-fumarate reductase, were evaluated as potential parasite targets. Additionally, the effect of metal coordination on the free radical scavenger capacity previously reported for the free ligand was studied. All the data strongly suggest that trypanocidal action of the complexes could mainly rely on the inhibition of the parasite-specific enzyme NADH-fumarate reductase.

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The trypanocidal effect of six lignan lactones, (-)-cubebin (1), (-)-O-methyl cubebin (2), (-)-O-benzyl cubebin (3), (-)-6,6'-dinitrohinokinin (4), (-)-hinokinin (5) and dimethoxymorelensin (6), previously synthesized by our research group, was evaluated in vitro and in vivo. The compounds with higher anti-epimastigote activity were screened against intracellular amastigote of Trypanosoma cruzi. Among these, compound 5 was selected to be assayed in vivo. It was observed that compounds 5, 6 and 2 showed higher trypanocidal activity against epimastigote forms of T. cruzi, displaying inhibitory concentration (IC(50)) values of 0.67, 3.89 and 31.35 muM, respectively. These compounds were also evaluated against intracellular amastigote forms of T. cruzi, with five displaying similar activity to benznidazole. In vivo assays showed significant reduction of parasitaemia after administration of five in mice infected.

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