Synergic effect of allopurinol in combination with nitro-heterocyclic compounds against Trypanosoma cruzi.

Combination therapy has gained attention as a possible strategy for overcoming the limitations of the present therapeutic arsenal for Chagas disease. The aim of this study was to evaluate the effect of allopurinol in association with nitroheterocyclic compounds on infection with the Y strain of Trypanosoma cruzi. The in vitro effect of allopurinol plus benznidazole or nifurtimox on intracellular amastigotes in infected H9c2 cells was assessed in a 72-h assay. The interactions were classified as synergic for both allopurinol-nifurtimox (sums of fractional inhibitory concentrations [FICs] 0.49 0.08) and allopurinol-benznidazole (FICs 0.48 0.09). In the next step, infected Swiss mice were treated with allopurinol at 30, 60, and 90 mg/kg of body weight and with benznidazole at 25, 50, and 75 mg/kg in monotherapy and in combination at the same doses; as a reference treatment, another group of animals received benznidazole at 100 mg/kg. Allopurinol in monotherapy led to a smaller or nil effect in the reduction of parasite load and mortality rate. Treatment with benznidazole at suboptimal doses induced a transient suppression of parasitaemia with subsequent relapse in all animals treated with 25 and 50 mg/kg and in 80% of those that received 75 mg/kg. Administration of the drugs in combination significantly increased the cure rate to 60 to 100% among mice treated with benznidazole at 75 mg/kg plus 30, 60, or 90 mg/kg of allopurinol. These results show a positive interaction between allopurinol and benznidazole, and since both drugs are commercially available, their use in combination may be considered for the assessment in the treatment of Chagas disease patients

Parasitaemia and parasitic load are limited targets of the aetiological treatment to control the progression of cardiac fibrosis and chronic cardiomyopathy in Trypanosoma cruzi-infected dogs.

It is still unclear whether the progression of acute to chronic Chagas cardiomyopathy is predominantly associated with the limited efficacy of aetiological chemotherapy, or with the pharmacological resistance profiles and pathogenicity of specific Trypanosoma cruzi strains. Thus, we tested the hypothesis that parasitic load could be a limited target of aetiological chemotherapy to prevent chronic cardiomyopathy in dogs infected by different T. cruzi strains. Animals were infected with benznidazole-susceptible (Berenice-78) and -resistant (VL-10 and AAS) strains of T. cruzi. A quantitative real-time PCR strategy was developed to comparatively quantify the parasite load of the three different strains using a single standard curve. For dogs infected with the VL-10 strain, benznidazole treatment reduced cardiac parasitism during the acute phase of infection. However, similar parasite load and collagen deposition were detected in the myocardium of treated and untreated animals in the chronic phase of the infection. In animals infected with the AAS strain, benznidazole reduced parasite load, myocarditis and type III collagen deposition in the acute phase. However, increased type III collagen deposition was verified in the chronic phase. Dogs infected with the Berenice-78 strain showed a parasitological cure and no evidence of myocardial fibrosis. Parasitic load and cardiac fibrosis presented no correlation in acute or chronic phases of T. cruzi infection. Our findings in a canine model of Chagas disease suggest that parasite burden is a limited predictor for disease progression after treatment and show that benznidazole, although not inducing parasitological cure, is able to prevent total fibrosis in the early stages of infection, as well as complete prevention of cardiac damage when it eliminates parasites at the onset of infection

Myocarditis in different experimental models infected by Trypanosoma cruzi is correlated with the production of IgG1 isotype.

This study was designed to verify the relationship between IgG antibodies isotypes and myocarditis inTrypanosoma cruzi infection using mice and dogs infected with different T. cruzi strains. The animals wereinfected with benznidazole-susceptible Berenice-78 and benznidazole-resistant AAS and VL-10 strains.The IgG subtypes were measured in serum samples from dogs (IgG, IgG1, and IgG2) and mice (IgG, IgG1,IgG2a, and IgG2b). The infection of dogs with VL-10 strain induced the highest levels of heart inflammationwhile intermediate and lower levels were detected with Berenice-78 and AAS strains, respectively. Similarresults were found in mice infected with VL-10, but not in those infected with AAS or Berenice-78 strains.The AAS strain induced higher levels of heart inflammation in mice, while Berenice-78 strain was notable to induce it. Correlation analysis between myocarditis and antibody reactivity index revealed veryinteresting results, mainly for IgG and IgG1, the latter being the most exciting. High IgG1 showed asignificant correlation with myocarditis in both experimental models, being more significant in dogs(r = 0.94, p < 0.0001) than in mice (r = 0.58, p = 0.047). Overall, our data suggest that IgG1 could be a goodmarker to demonstrate myocarditis intensity in Chagas disease.

Time and dose-dependence evaluation of nitroheterocyclic drugs for improving efficacy following Trypanosoma cruzi infection : a pre-clinical study.

Benznidazole and nifurtimox-treatments regimens currently used in human are supported by very limited experimental data. This study was designed to evaluate the time and dose dependence for efficacy of the most important nitroheterocyclic drugs in use for Chagas disease. In order to evaluate time dependence, Y strain-infected mice received benznidazole for a total of 1, 3, 7, 10, 20, and 40 days. Treatment courses of 3?10-day were effective in clearing parasitaemia and suppressing mortality, but parasitological cure was not achieved. Extending the treatments to 20 or 40 days clearly improved benznidazole efficacy. The 20-day treatment induced cure in 57.1% of Y strain infections (partially drug resistant) but failed to cure Colombian strain infections (full drug resistant), while the 40-day treatment resulted in cure of 100% of Y and 50% of Colombian strain infected mice. The increased cure rates in T. cruzi infected animals that received nifurtimox for 40 days confirm the relationship between the length of treatment and efficacy. An improvement in efficacy was observed with increasing benznidazole doses; cure was verified in 28.6% (75 mg/kg), 57.1% (100 mg/kg) and 80% (300 mg/kg). Overall, these nonclinical study data provide evidence that the efficacy of benznidazole is dose and time dependent. These findings may be relevant for optimizing treatment of human Chagas disease

Increased type 1 chemokine expression in experimental Chagas disease correlates with cardiac pathology in Beagle dogs.

Chemokines and chemokine receptors interaction have presented important role in leukocyte migration to specific immune reaction sites. Recently, it has been reported that chemokine receptors CXC (CXCR3) and CC (CCR5) were preferentially expressed on Th1 cells while CCR3 and CCR4 were preferentially expressed on Th2 cells. This study evaluated the mRNA expression of type 1 and type 2 chemokine and chemokine receptors in the cardiac tissue of Beagle dogs infected with distinct genetic groups of Trypanosoma cruzi (Y, Berenice- 78 and ABC strains) during acute and chronic phases. To analyze the correlation between chemokine and chemokine receptors expression and the development of heart pathology, the chronic infected animals were divided into groups, according to the parasite strain and based on the degree of heart damage: cardiac and indeterminate form of Chagas disease. Our results indicated that cardiac type1/2 chemokines and their receptors were partially dependent on the genetic diversity of parasites as well as the polarization of clinical forms. Also, dogs presenting cardiac form showed lower heart tissue mRNA expression of CCL24 (type 2) and higher expression of CCL5, CCL4 and CXCR3 (type 1) when compared with those with indeterminate form of disease. Together, these data reinforce a close-relation between T. cruzi genetic population and the host specific type 1 immune response and, for the first time, we show the distribution of type 1/2 chemokines associated with the development of cardiac pathology using dogs, a well similar model to study human Chagas disease

Antitrypanosomal activity of fexinidazole metabolites, potential new drug candidates for Chagas disease.

This study was designed to verify the in vivo efficacy of sulfoxide and sulfone fexinidazole metabolites following oral administration in a murine model of Chagas disease. Female Swiss mice infected with the Y strain of Trypanosoma cruzi were treated orally once per day with each metabolite at doses of 10 to 100 mg/kg of body weight for a period of 20 days. Parasitemia was monitored throughout, and cures were detected by parasitological and PCR assays. The results were compared with those achieved with benznidazole treatment at the same doses. Fexinidazole metabolites were effective in reducing the numbers of circulating parasites and protecting mice against death, compared with untreated mice, but without providing cures at daily doses of 10 and 25 mg/kg. Both metabolites were effective in curing mice at 50 mg/kg/day (30% to 40%) and 100 mg/kg/day (100%). In the benznidazole- treated group, parasitological cure was detected only in animals treated with the higher dose of 100 mg/kg/day (80%). Single-dose pharmacokinetic parameters for each metabolite were obtained from a parallel group of uninfected mice and were used to estimate the profiles following repeated doses. Pharmacokinetic data suggested that biological efficacy most likely resides with the sulfone metabolite (or subsequent reactive metabolites formed following reduction of the nitro group) following administration of either the sulfoxide or the sulfone and that prolonged plasma exposure over the 24-h dosing window is required to achieve high cure rates. Fexinidazole metabolites were effective in treating T. cruzi in a mouse model of acute infection, with cure rates superior to those achieved with either fexinidazole itself or benznidazole