In vitro and in vivo drug combination for the treatment of Trypanosoma cruzi infection: A multivariate approach.

Combination therapies based on the available drugs have been proposed as promising therapeutic alternatives for many diseases. Clomipramine (CLO) has been found to modify the evolution of the experimental infection. The objective of this study was to evaluate the combined effect of benznidazole (BZ) and clomipramine (CLO) against different life-stages of Trypanosoma cruzi in vitro and their efficacy in a murine model. Life-stages of T. cruzi, BZ-partially-resistant (Y) strain, were incubated with BZ and CLO and isobolograms and combination index (CI) were obtained. Swiss mice were infected with trypomastigotes and different treatment schedules were performed, each of which consisted of 30 consecutive daily doses. Treatment efficacy was evaluated by comparing parasitemia, qPCR, survival and histological analysis. These results were analyzed using multivariate analysis to determine the combined effect of the drugs in vivo. CLO + BZ showed synergistic activity in vitro against the clinically relevant life-stages of T. cruzi. The most susceptible forms were the intracellular amastigotes (CI: 0.20), followed by trypomastigotes (CI: 0.60), with no toxicity upon mammalian cells. The combination of both drugs CLO (1.25 mg/kg) and BZ (6.25 mg/kg), in vivo, significantly diminished the parasitic load in blood and the mortality rate. CLO + BZ presented a similar inflammatory response in cardiac and skeletal muscle (amount of inflammatory cells) to BZ (6.25 mg/kg). Finally, the results from the principal component analysis reaffirmed that both drugs administered in combination presented higher activity compared with the individual administration in the acute experimental model.

In vitro and in vivo experimental models for drug screening and development for Chagas disease.

Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

In vitro and in vivo experimental models for drug screening and development for Chagas disease

Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

Some components of the cardiac ß-adrenergic system are altered in the chronic indeterminate form of experimental Trypanosoma cruzi infection.

The chronic indeterminate form of Trypanosoma cruzi infection could be the key to knowing which patients will develop chagasic myocardiopathy. Infected mice present a period in which cardiac functional and structural alterations are different from those described for acute or chronic phases. We studied some components of the cardiac ß-adrenergic system in mouse hearts infected with T. cruzi Tulahuen strain or SGO-Z12 isolate during the chronic indeterminate phase of infection. We determined: (i) the primary messenger (epinephrine and norepinephrine) levels in plasma by reverse-phase-HPLC; (ii) the cardiac ß-adrenergic receptors' (ß-AR) density and affinity by binding with tritiated dihidroalprenolol and by immunofluorescence; (iii) the cardiac concentration of the second messenger (cAMP) (by ELISA) given its importance for the phosphorylation of the proteins involved in cardiac contraction; (iv) the cardiac contractility and functional studies of the ß-ARs as a response to the ligand binding to the receptor; and (v) the left ventricular ejection fraction as a measure of in vivo cardiac function. Plasma catecholamines levels remained similar to those found in uninfected controls. The ß-ARs' affinity decreased in both infected groups compared with the uninfected group (P<0.05) while the receptors' density increased only in the SGO-Z12 group (P<0.01). Cyclic AMP levels were higher in both infected groups (P<0.01) relative to controls, and were higher in SGO-Z12-infected mice compared with those infected with the Tulahuen strain. However, the basal contractile force remained unchanged and the response to catecholamines only increased in the Tulahuen group (P<0.05). The left ventricular ejection fraction, on the other hand, was diminished in SGO-Z12-infected mice. Heterogeneity between T. cruzi strains determine, in the chronic indeterminate form, alterations in the signaling pathways of the ß-adrenergic system at different levels: (i) between catecholamines and the ß(1)-receptors; (ii) between the receptors' activation and the adenylyl-cyclase activation; and/or (iii) between cAMP and the contractile response.

Changes in the cardiac beta-adrenergic system provoked by different T. cruzi strains.

BACKGROUND: It has been demonstrated that the beta-adrenergic signal transduction system is altered somewhere along its pathway in Trypanosoma cruzi infected hearts and we think that these alterations would differ according to the infection phase and the parasite strain. Their study would be important for the understanding of the disease's pathophysiology. METHODS: In the present work we studied important components of this system in mice hearts infected with T. cruzi, Tulahuen strain and with SGO-Z12 isolate, obtained from a patient of an endemic area, in the acute phase of the infection, determining: the plasma catecholamines levels, the beta-receptors density and affinity as well as their function, the cardiac concentration of cAMP and the cardiac contractility as the physiologic response to the initial stimulus. RESULTS: Plasma catecholamines levels were diminished in both infected groups when compared to the uninfected one (P < 0.01). The receptor's affinity was also diminished (P < 0.05) while their density was augmented only in the SGO-Z12 infected one (P < 0.01). The cAMP levels were higher in both infected groups (P < 0.01), the basal contractile force however increased only in the Tulahuen infected one (P < 0.01) while the response to catecholamines remained unchanged. The hearts infected with the SGO Z12 isolate presented an inferior response to epinephrine (P < 0.05) than the ventricles infected with the Tulahuen strain. CONCLUSIONS: This model represents an important approach to understand the biochemical, physiological and molecular changes in the cardiac beta-adrenergic signalling that clearly begin in the acute phase of Chagas' disease and reveal a clear differentiation in the alterations produced by different parasite strains.