In Vitro and In Vivo Biological Effects of Novel Arylimidamide Derivatives against Trypanosoma cruzi

Abstract

bChagas disease (CD), a neglected tropical disease caused by Trypanosoma cruzi, remains a serious public health problem in several Latin American countries. The available chemotherapies for CD have limited efficacy and exhibit undesirable side effects. Aromatic diamidines and arylimidamides (AIAs) have shown broad-spectrum activity against intracellular parasites, including T. cruzi. Therefore, our aim was to evaluate the biological activity of eight novel AIAs (16DAP002, 16SAB079, 18SAB075, 23SMB022, 23SMB026, 23SMB054, 26SMB070, and 27SMB009) against experimental models of T. cruzi infection in vitro and in vivo. Our data show that none of the compounds induced a loss of cellular viability up to 32 M. Two AIAs, 18SAB075 and 16DAP002, exhibited good in vitro activity against different parasite strains (Y and Tulahuen) and against the two relevant forms of the parasite for mammalian hosts. Due to the excellent selective indexes of 18SAB075, this AIA was moved to in vivo tests for acute toxicity and parasite efficacy; nontoxic doses (no-observed-adverse-effect level [NOAEL], 50 mg/kg) were employed in the tests for parasite efficacy. In experimental models of acute T. cruzi infection, 18SAB075 reduced parasitemia levels only up to 50% and led to 40% protection against mortality (at 5 mg/kg of body weight), being less effective than the reference drug, benznidazole. Chagas disease (CD) is a neglected tropical disease caused by the intracellular flagellate protozoan Trypanosoma cruzi, which presents a complex life cycle with distinct morphological stages in its obligatory passage through vertebrate and invertebrate hosts (1). Currently, there are approximately 10 million infected individuals in areas of Latin America where CD is endemic, and many reports also point to the occurrence of CD in geographical areas where it is not endemic, such as the United States and Europe, mainly attributed to migration of infected people (2-6). CD can be transmitted by Triatominae insect feces, blood transfusion, organ transplantation, and laboratory accidents and through oral and congenital routes (7,8). This pathology has two successive phases, a short, acute phase characterized by a patent parasitemia, followed by a chronic phase in which most of the infected individuals remain asymptomatic (indeterminate form), but about onethird may later manifest cardiac and/or digestive complications, developed progressively for years or decades after infection (9, 10). Benznidazole (Bz) and nifurtimox, introduced into clinical therapy about 40 years ago, are the only available drugs. Both have several shortcomings related to their required long periods of treatment, high toxicity, variable results, and low efficacy during the chronic phase, justifying the identification of novel therapies (11-13). Aromatic diamidines and analogues exhibit broad-spectrum activity against pathogenic microorganisms, including T. cruzi (14). Among the different tested derivatives of amidine compounds, the most effective against T. cruzi have been the bisarylimi...

In Vitro and In Vivo Trypanosomicidal Action of Novel Arylimidamides against Trypanosoma cruzi

“…Immediately after the purification step, the parasites were resuspended in RPMI 1640 medium (pH 7.2 to 7.4) without phenol red (Gibco BRL) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine, as reported previously Reagents and conditions were as follows: a, sodium bis(trimethylsilyl)amide; tetrahydrofuran; and 25°C. (18). The effect against the intracellular forms was investigated through the use of L929 cell lineages infected with tissue culture-derived trypomastigotes (the Tulahuen strain expressing the Escherichia coli ␤-galactosidase gene), using a 10:1 parasite/host cell ratio.…”

“…Primary cultures of embryonic cardiac cells (CCs) were obtained from Swiss Webster mice as previously reported (20). After purification, the CCs were seeded into 24-and 96-well microplates containing gelatin-coated coverslips at densities of 0.2 ϫ 10 6 and 0.05 ϫ 10 6 cells/well, respectively, as reported previously (18). The cardiac cell cultures were then sustained at 37°C in Dulbecco's modified Eagle medium (DMEM; without phenol red; Sigma-Aldrich) supplemented with 10% horse serum, 5% fetal bovine serum, 2.5 mM CaCl 2 , 1 mM L-glutamine, and 2% chicken embryo extract.…”

Anti-parasitic effect of novel amidines against Trypanosoma cruzi: phenotypic and in silico absorption, distribution, metabolism, excretion and toxicity analysis

“…L929 cell lineages incubated for 24 and 96 h at 37°C, with different concentrations of each compound diluted in RPMI and their cellular viability determined by the AlamarBlue test as reported (Timm et al 2014). The maximum concentration of each compound was 96 µM due to molecule precipitation.…”

“…The cardiac cells were incubated for 24 h at 37°C with different concentrations of each compound diluted in RPMI and then, the morphology, cell density and spontaneous contractibility evaluated by light microscopy and their cellular viability determined by the Presto Blue test as reported (Timm et al 2014). L929 cell lineages incubated for 24 and 96 h at 37°C, with different concentrations of each compound diluted in RPMI and their cellular viability determined by the AlamarBlue test as reported (Timm et al 2014).…”

“…After compound incubation, the death parasite rates were determined by light microscopy through the direct quantification of the number of live parasites using a Neubauer chamber, and the EC 50 concentration (the compound concentration that reduces in 50% the number of parasites) was calculated (Timm et al 2014). For the assay on intracellular forms, culture-derived trypomastigotes of T. cruzi (Tulahuen strain expressing β-galactosidase; DTU VI) (Zingales et al 2009) used to infect L929 infected-cells cultures using a ratio of 10:1 (parasite: host cell).…”