Rodrigo López-Muñoz

Chagas disease: Present status of pathogenic mechanisms and chemotherapy

There are approximately 7.8 million people in Latin America, including Chile, who suffer from Chagas disease and another 28 million who are at risk of contracting it. Chagas is caused by the flagellate protozoan Trypanosoma cruzi. It is a chronic disease, where 20%-30% of infected individuals develop severe cardiopathy, with heart failure and potentially fatal arrhythmias. Currently, Chagas disease treatment is more effective in the acute phase, but does not always produce complete parasite eradication during indeterminate and chronic phases. At present, only nifurtimox or benznidazole have been proven to be superior to new drugs being tested. Therefore, it is necessary to find alternative approaches to treatment of chronic Chagas. The current treatment may be rendered more effective by increasing the activity of anti-Chagasic drugs or by modifying the hosts immune response. We have previously shown that glutathione synthesis inhibition increases nifurtimox and benznidazole activity. In addition, there is increasing evidence that cyclooxygenase inhibitors present an important effect on T. cruzi infection. Therefore, we found that aspirin reduced the intracellular infection in RAW 264.7 cells and, decreased myocarditis extension and mortality rates in mice. However, the long-term benefit of prostaglandin inhibition for Chagasic patients is still unknown.

Buthionine Sulfoximine Has Anti-Trypanosoma cruzi Activity in a Murine Model of Acute Chagas' Disease and Enhances the Efficacy of Nifurtimox

ABSTRACT l-Buthionine (S,R)-sulfoximine (BSO) at a dose of 220 mg/kg of body weight/day showed an anti-Trypanosoma cruzi effect in infected mice, increasing their survival rate and decreasing the parasitemias and parasite burden in the hearts. Treatment with BSO plus nifurtimox caused an increase in the survival rate in comparison to the rates with treatment with each drug alone.

Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole.

Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an antiparasitic response, which the parasite evades through an increase of tumor growth factor beta and PGE(2) activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect. Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolographic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively. The observed effect of aspirin upon T. cruzi infection was not related with the PGE(2) synthesis inhibition. Nevertheless, NO() levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macrophage antiparasitic activity improvement. Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to increase antiparasitic activity of macrophages.

Benznidazole prevents endothelial damage in an experimental model of Chagas disease.

OBJECTIVES To evaluate the effect of benznidazole on endothelial activation in a murine model of Chagas disease. METHODS A low (30mg/kg/day) and a high (100mg/kg/day) dose of benznidazole were administered to mice infected with Trypanosoma cruzi during the early phases of the infection. The effects of the treatments were assessed at 24 and 90 days postinfection by evaluating the parasitaemia, mortality, histopathological changes and expression of ICAM in the cardiac tissue. The blood levels of thromboxane A2, soluble ICAM and E-selectin were also measured. T. cruzi clearance was assessed by the detection of parasite DNA in the heart tissue of infected mice. RESULTS Benznidazole decreased the cardiac damage induced by the parasite, and amastigote nests disappeared at 90 days postinfection. Both doses cleared the parasite from the cardiac tissue at 24 and 90 days postinfection. In addition, benznidazole decreased the thromboxane levels and normalized the plasma sICAM and sE-selectin levels by 90 days postinfection. CONCLUSIONS Early administration of benznidazole at a dose as low as 30mg/kg eradicates T. cruzi from cardiac tissue. Additionally, benznidazole prevents cardiac damage and modulates endothelial activation as part of its antichagasic activity.

2-Phenylaminonaphthoquinones and related compounds: Synthesis, trypanocidal and cytotoxic activities

A series of new 2-aminonaphthoquinones and related compounds were synthesized and evaluated in vitro as trypanocidal and cytotoxic agents. Some tested compounds inhibited epimastigote growth and trypomastigote viability. Several compounds showed similar or higher activity and selectivity as compared with current trypanocidal drug, nifurtimox. Compound 4l exhibit higher selectivity than nifurtimox against Trypanosoma cruzi in comparison with Vero cells. Some of the synthesized quinones were tested against cancer cells and normal fibroblasts, showing that certain chemical modifications on the naphthoquinone moiety induce and excellent increase the selectivity index of the cytotoxicity (4g and 10). The results presented here show that the anti-T. cruzi activity of 2-aminonaphthoquinones derivatives can be improved by the replacement of the benzene ring by a pyridine moiety. Interestingly, the presence of a chlorine atom at C-3 and a highly lipophilic alkyl group or aromatic ring are newly observed elements that should lead to the discovery of more selective cytotoxic and trypanocidal compounds.

Toxic and therapeutic effects of Nifurtimox and Benznidazol on Trypanosoma cruzi ex vivo infection of human placental chorionic villi explants

Nifurtimox (Nfx) and Benznidazole (Bnz) are the only available drugs in use for the treatment of Chagas disease. These drugs are recommended but not fully validated in evidence-based medicine and reports about the differential toxicity of both drugs are controversial. Here, we evaluated the toxic and therapeutic effects of Nfx and Bnz on human placental chorionic villi explants (HPCVE) during ex vivo infection of Trypanosoma cruzi, performing histopathological, histochemical, immunohistochemical as well as immunofluorescence analysis of the tissue. Additionally, we determined the effect of both drugs on parasite load by real time PCR. Bnz prevents the parasite induced tissue damage in ex vivo infected HPCVE compared to Nfx, which is toxic per se. The presence of T. cruzi antigens and DNA in infected explants suggests that these drugs do not impair parasite invasion into the HPCVE. Additionally, our results confirm reports suggesting that Bnz is less toxic than Nfx and support the need for the development of more effective and better-tolerated drugs.

ESR, electrochemical, molecular modeling and biological evaluation of 4-substituted and 1,4-disubstituted 7-nitroquinoxalin-2-ones as potential anti-Trypanosoma cruzi agents

Electrochemical and ESR studies were carried out in this work with the aim of characterizing the reduction mechanisms of 4-substituted and 1,4-disubstituted 7-nitroquinoxalin-2-ones by means of cyclic voltammetry in DMSO as aprotic solvent. Two reduction mechanisms were found for these compounds: the first, for compounds bearing a labile hydrogen by following a self-protonation mechanism (ECE steps), and the second, for compounds without labile hydrogen, based on a purely electrochemical reduction mechanism (typical of nitroheterocycles). The electrochemical results were corroborated using ESR spectroscopy allowing us to propose the hyperfine splitting pattern of the nitro-radical, which was later corroborated by the ESR simulation spectra. All these compounds were assayed as growth inhibitors against Trypanosoma cruzi: first, on the non-proliferative (and infective) form of the parasite (trypomastigote stage), and then, the ones that displayed activity, were assayed on the non-infective form (epimastigote stage). Thus, we found four new compounds highly active against T. cruzi. Finally, molecular modeling studies suggest the inhibition of the trypanothione reductase like one of the possible mechanisms involved in the trypanocidal action.

Biological and chemical study of fused tri- and tetracyclic indazoles and analogues with important antiparasitic activity

A series of fused tri- and tetracyclic indazoles and analogues compounds (NID) with potential antiparasitic effects were studied using voltamperometric and spectroscopic techniques. Nitroanion radicals generated by cyclic voltammetry were characterized by electron spin resonance spectroscopy (ESR) and their spectral lines were explained and analyzed using simulated spectra. In addition, we examined the interaction between radical species generated from nitroindazole derivatives and glutathione (GSH). Biological assays such as activity against Trypanosoma cruzi and cytotoxicity against macrophages were carried out. Finally, spin trapping and molecular modeling studies were also done in order to elucidate the potentials action mechanisms involved in the trypanocidal activity.

Simvastatin and Benznidazole-Mediated Prevention of Trypanosoma cruzi-Induced Endothelial Activation: Role of 15-epi-lipoxin A4 in the Action of Simvastatin.

Trypanosoma cruzi is the causal agent of Chagas Disease that is endemic in Latin American, afflicting more than ten million people approximately. This disease has two phases, acute and chronic. The acute phase is often asymptomatic, but with time it progresses to the chronic phase, affecting the heart and gastrointestinal tract and can be lethal. Chronic Chagas cardiomyopathy involves an inflammatory vasculopathy. Endothelial activation during Chagas disease entails the expression of cell adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) through a mechanism involving NF-κB activation. Currently, specific trypanocidal therapy remains on benznidazole, although new triazole derivatives are promising. A novel strategy is proposed that aims at some pathophysiological processes to facilitate current antiparasitic therapy, decreasing treatment length or doses and slowing disease progress. Simvastatin has anti-inflammatory actions, including improvement of endothelial function, by inducing a novel pro-resolving lipid, the 5-lypoxygenase derivative 15-epi-lipoxin A4 (15-epi-LXA4), which belongs to aspirin-triggered lipoxins. Herein, we propose modifying endothelial activation with simvastatin or benznidazole and evaluate the pathways involved, including induction of 15-epi-LXA4. The effect of 5 μM simvastatin or 20 μM benznidazole upon endothelial activation was assessed in EA.hy926 or HUVEC cells, by E-selectin, ICAM-1 and VCAM-1 expression. 15-epi-LXA4 production and the relationship of both drugs with the NFκB pathway, as measured by IKK-IKB phosphorylation and nuclear migration of p65 protein was also assayed. Both drugs were administered to cell cultures 16 hours before the infection with T. cruzi parasites. Indeed, 5 μM simvastatin as well as 20 μM benznidazole prevented the increase in E-selectin, ICAM-1 and VCAM-1 expression in T. cruzi-infected endothelial cells by decreasing the NF-κB pathway. In conclusion, Simvastatin and benznidazole prevent endothelial activation induced by T. cruzi infection, and the effect of simvastatin is mediated by the inhibition of the NFκB pathway by inducing 15-epi-LXA4 production.

Dehydroepiandrosterone effect on Plasmodium falciparum and its interaction with antimalarial drugs

Dehydroepiandrosterone (DHEA) inhibits glucose 6-phosphate dehydrogenase (G6PDH) of different species and may potentially decrease intracellular glutathione. Therefore, it can have and enhance anti-parasitic action against Plasmodium spp. We evaluated the antiplasmodial activity and the interaction of DHEA with several antimalarial drugs. The inhibitory effect of DHEA on erythrocytic and G6PDH activity and changes in the content of total and reduced gluthatione Plasmodium falciparum content were also evaluated. DHEA showed antiplasmodial activity in vitro, but the potency was low (IC(50) 118.5 μM). DHEA inhibits G6PDH from healthy erythrocyte and decreases GSH content in both erythrocytes and P. falciparum. DHEA did not synergize or antagonize the antiplasmodial effect of several antimalarial drugs. The most important actions of DHEA were the inhibition of G6PDH activity, and the decrease in both P. falciparum and erythrocyte GSH. Since most of the GSH in Plasmodium spp. infected erythrocytes comes from the parasite itself, it is possible that DHEA analogs could act with higher selectivity, better potency, and might interact synergistically with antimalarials drugs.