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.

Quantitative PCR and unconventional serological methods to evaluate clomipramine treatment effectiveness in experimental Trypanosoma cruzi infection.

Clomipramine (CLO), a tricyclic antidepressant drug, has been used for the treatment of mice infected with Trypanosoma cruzi. In this work we evaluated the effectiveness of CLO treatment upon T. cruzi-infected mice in the chronic phase of the experimental infection using Quantitative polymerase chain reaction (qPCR) and recombinant ELISA. Sixty Swiss albino mice were inoculated with 50 trypomastigote forms of T. cruzi (Tulahuen strain). CLO treatment consisted of 5mg/kg/day during 60days by intraperitoneal injection, beginning on day 90 post infection (p.i) when the mice presented electrocardiographic (ECG) alterations compatible with the chronic phase of the disease. The evolution of experimental infection and the treatment efficacy were studied through survival, electrocardiography, serology using a mixture and individual (1, 2, 13, 30, 36 and SAPA) recombinant proteins from epimastigotes and trypomastigotes of T. cruzi; and qPCR on days 180 and 270 p.i. CLO treatment in the chronic phase decreased the parasite load, reduced the levels of antibodies against antigen 13 throughout 270days p.i and reversed the ECG abnormalities in the treated animals, from 100% of the mice with alterations at the beginning of the treatment to only 20% of the mice with alterations by day 270 p.i. This study shows that qPCR and the use of recombinant antigens are more sensitive to evaluate the effectiveness of the treatment and proves that clomipramine may be considered as a new chemotherapy for the chronic phase of the disease.

Chronic indeterminate phase of Chagas' disease: mitochondrial involvement in infection with two strains.

Chagasic cardiopathy has become one of the most frequent causes of heart failure and sudden death, as well as one of the most common causes of cardio-embolic stroke in Latin America. The myocyte response to oxidative stress involves the progression of cellular changes, primarily targeting the mitochondria and modifying therefore the energy supply. In this paper we analysed the effect of the infection of mice with 2 different strains of Trypanosoma cruzi (Tulahuen and SGO Z12) in the chronic indeterminate stage (75 days post-infection), upon the structure and function of cardiac mitochondria. The structural results showed that 83% of the mitochondria from the Tulahuen-infected mice presented an increase in their matrix and 91% of the mitochondria from the SGO Z12-infected group showed a reduction in their diameter (P < 0.05). When the Krebs cycle and mitochondrial respiratory chain functionality was analysed through the measurement of the citrate synthase and complexes I to IV activity, it showed that their activity was altered in all cases in a similar manner in both infected groups. In this paper we have demonstrated that the chronic indeterminate phase is not 'silent' and that cardiac mitochondria are clearly involved in the genesis and progression to the chronic chagasic cardiopathy when different factors alter the host-parasite equilibrium.

Chemotherapy of chronic indeterminate Chagas disease: a novel approach to treatment.

Treatment of Chagas disease is a controversial issue because the available drugs are highly toxic. Clomipramine is a tricyclic antidepressant drug that inhibits Trypanosoma cruzi's trypanothione reductase, provoking the death of the parasite and preventing the cardiac damage when used for the treatment of acutely infected mice. Here, we studied the effectiveness of clomipramine (5 mg/kg/day for one month) as chemotherapy for T. cruzi-infected mice in the chronic indeterminate stage of the infection. The animals were analyzed in the cardiac chronic phase. Survival of treated animals was 84% while for the untreated ones was 40%; most of the animals presented electrocardiographic abnormalities. Affinity and density of cardiac beta receptors from infected and treated mice were similar to those in the indeterminate phase, showing that clomipramine treatment stopped the increment of functional alterations provoked by the infection, while untreated mice presented affinity and density significantly diminished. Hearts from infected and untreated mice in the chronic stage presented mononuclear cells, necrosis and fiber dissolution while hearts from treated animals showed only isolated inflammatory infiltrates. Present results demonstrate that clomipramine used in the chronic indeterminate phase of the T. cruzi infection modified the natural evolution of the chagasic cardiopathy.