Anissa Daliry

Experimental chemotherapy for Chagas disease: 15 years of research contributions from in vivo and in vitro studies

Chagas disease, which is caused by the intracellular parasite Trypanosoma cruzi, is a neglected illness with 12-14 million reported cases in endemic geographic regions of Latin America. While the disease still represents an important public health problem in these affected areas, the available therapy, which was introduced more than four decades ago, is far from ideal due to its substantial toxicity, its limited effects on different parasite stocks, and its poor activity during the chronic phase of the disease. For the past 15 years, our group, in collaboration with research groups focused on medicinal chemistry, has been working on experimental chemotherapies for Chagas disease, investigating the biological activity, toxicity, selectivity and cellular targets of different classes of compounds on T. cruzi. In this report, we present an overview of these in vitro and in vivo studies, focusing on the most promising classes of compounds with the aim of contributing to the current knowledge of the treatment of Chagas disease and aiding in the development of a new arsenal of candidates with anti-T. cruzi efficacy.

In vitro and in vivo investigation of the efficacy of arylimidamide DB1831 and its mesylated salt form--DB1965--against Trypanosoma cruzi infection.

Chagas disease is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi. At present, nifurtimox and benznidazole, both compounds developed empirically over four decades ago, represent the chemotherapeutic arsenal for treating this highly neglected disease. However, both drugs present variable efficacy depending on the geographical area and the occurrence of natural resistance, and are poorly effective against the later chronic stage. As a part of a search for new therapeutic opportunities to treat chagasic patients, pre-clinical studies were performed to characterize the activity of a novel arylimidamide (AIA - DB1831 (hydrochloride salt) and DB1965 (mesylate salt)) against T.cruzi. These AIAs displayed a high trypanocidal effect in vitro against both relevant forms in mammalian hosts, exhibiting a high selectivity index and a very high efficacy (IC50 value/48 h of 5–40 nM) against intracellular parasites. DB1965 shows high activity in vivo in acute experimental models (mouse) of T.cruzi, showing a similar effect to benznidazole (Bz) when compared under a scheme of 10 daily consecutive doses with 12.5 mg/kg. Although no parasitological cure was observed after treating with 20 daily consecutive doses, a combined dosage of DB1965 (5 mg/kg) with Bz (50 mg/kg) resulted in parasitaemia clearance and 100% animal survival. In summary, our present data confirmed that aryimidamides represent promising new chemical entities against T.cruzi in therapeutic schemes using the AIA alone or in combination with other drugs, like benznidazole.

In vitro analyses of the effect of aromatic diamidines upon Trypanosoma cruzi

OBJECTIVES Aromatic diamidines (ADs) have been recognized as promising antiparasitic agents. Therefore, in the present work, the in vitro trypanocidal effect of 11 ADs upon the relevant clinical forms of Trypanosoma cruzi was evaluated, as well as determining their toxicity to mammalian cells and their subcellular localization. METHODS The trypanocidal effect upon trypomastigotes and amastigotes was evaluated by light microscopy through the determination of the IC(50) values. The cytotoxicity was determined by the MTT colorimetric assay against mouse cardiomyocytes. For the subcellular localization, transmission electron microscopy and fluorescence approaches were used. The fluorescence intensity within the kinetoplast DNA (kDNA) and nuclear DNA (nDNA) of treated parasites was determined using the Image J program. RESULTS Compounds 2, 5 and 7 showed the lowest IC(50) values (micromolar range) against intracellular amastigotes and trypomastigotes. In the presence of blood, all the tested ADs exhibited a reduction of their activity. The compounds did not exhibit toxicity to cardiac cells and the highest selectivity index (SI) was achieved by compound 5 with an SI of >137 for trypomastigotes and compound 7 with an SI of >107 for intracellular parasites. The subcellular effects upon bloodstream forms treated with compounds 5 and 7 were mainly on kDNA, leading to its disorganization. The higher accumulation in the kDNA observed for all tested ADs was not directly related to their efficacy. CONCLUSIONS Our results show the high activity of this new series of ADs against both trypomastigote and amastigote forms, with excellent SIs, especially compound 7, which merits further in vivo evaluation.

The biological in vitro effect and selectivity of aromatic dicationic compounds on Trypanosoma cruzi

Trypanosoma cruzi is a parasite that causes Chagas disease, which affects millions of individuals in endemic areas of Latin America. One hundred years after the discovery of Chagas disease, it is still considered a neglected illness because the available drugs are unsatisfactory. Aromatic compounds represent an important class of DNA minor groove-binding ligands that exhibit potent antimicrobial activity. This study focused on the in vitro activity of 10 aromatic dicationic compounds against bloodstream trypomastigotes and intracellular forms of T. cruzi. Our data demonstrated that these compounds display trypanocidal effects against both forms of the parasite and that seven out of the 10 compounds presented higher anti-parasitic activity against intracellular parasites compared with the bloodstream forms. Additional assays to determine the potential toxicity to mammalian cells showed that the majority of the dicationic compounds did not considerably decrease cellular viability. Fluorescent microscopy analysis demonstrated that although all compounds were localised to a greater extent within the kinetoplast than the nucleus, no correlation could be found between compound activity and kDNA accumulation. The present results stimulate further investigations of this class of compounds for the rational design of new chemotherapeutic agents for Chagas disease.

Delta Opioid Receptors: The Link between Exercise and Cardioprotection

This study investigated the role of opioid receptor (OR) subtypes as a mechanism by which endurance exercise promotes cardioprotection against myocardial ischemia-reperfusion (IR) injury. Wistar rats were randomly divided into one of seven experimental groups: 1) control; 2) exercise-trained; 3) exercise-trained plus a non-selective OR antagonist; 4) control sham; 5) exercise-trained plus a kappa OR antagonist; 6) exercise-trained plus a delta OR antagonist; and 7) exercise-trained plus a mu OR antagonist. The exercised animals underwent 4 consecutive days of treadmill training (60 min/day at ∼70% of maximal oxygen consumption). All groups except the sham group were exposed to an in vivo myocardial IR insult, and the myocardial infarct size (IS) was determined histologically. Myocardial capillary density, OR subtype expression, heat shock protein 72 (HSP72) expression, and antioxidant enzyme activity were measured in the hearts of both the exercised and control groups. Exercise training significantly reduced the myocardial IS by approximately 34%. Pharmacological blockade of the kappa or mu OR subtypes did not blunt exercise-induced cardioprotection against IR-mediated infarction, whereas treatment of animals with a non-selective OR antagonist or a delta OR antagonist abolished exercise-induced cardioprotection. Exercise training enhanced the activities of myocardial superoxide dismutase (SOD) and catalase but did not increase the left ventricular capillary density or the mRNA levels of HSP72, SOD, and catalase. In addition, exercise significantly reduced the protein expression of kappa and delta ORs in the heart by 44% and 37%, respectively. Together, these results indicate that ORs contribute to the cardioprotection conferred by endurance exercise, with the delta OR subtype playing a key role in this response.

Active Transport of Glutamate in Leishmania (Leishmania) amazonensis

ABSTRACT. Leishmania spp. are the causative agents of leishmaniasis, a complex of diseases with a broad spectrum of clinical manifestations. Leishmania (Leishmania) amazonensis is a main etiological agent of diffuse cutaneous leishmaniasis. Leishmania spp., as other trypanosomatids, possess a metabolism based significantly on the consumption of amino acids. However, the transport of amino acids in these organisms remains poorly understood with few exceptions. Glutamate transport is an important biological process in many organisms. In the present work, the transport of glutamate is characterized. This process is performed by a single kinetic system (Km=0.59±0.04 mM, Vmax=0.123±0.003 nmol/min per 20 × 106 cells) showing an energy of activation of 52.38±4.7 kJ/mol and was shown to be partially inhibited by analogues, such as glutamine, aspartate, α‐ketoglutarate and oxaloacetate, methionine, and alanine. The transport activity was sensitive to the extracellular concentration of H+ but not to Na+ or K+. However, unlike other amino acid transporters presently characterized, the treatment with specific ionophores confirmed the participation of a K+, and not H+ membrane gradient in the transport process.

Anti-adrenergic and muscarinic receptor autoantibodies in a canine model of Chagas disease and their modulation by benznidazole

Chronic chagasic cardiomyopathy (CCC) is a public health problem still without a defined physiopathology. It results from a chronic infection by Trypanosoma cruzi and is characterized by irreversible lesions to the heart. The contribution of autoimmune processes to cardiac dysfunctions observed in CCC has been hypothesized by several authors [1–3]. Those studies demonstrated the presence of IgG components in sera of patients with CCC and dilated cardiomyopathy interacting with cardiac β1-adrenergic (anti-β1-AR) and muscarinic membrane receptors (anti-M2-CR) [3].

Combined therapy with metformin and insulin attenuates systemic and hepatic alterations in a model of high-fat diet-/streptozotocin-induced diabetes

In this study we have explored the pathogenesis of the hepatic alterations which occur in diabetes and the modulation of these complications by the combination of metformin adjunct treatment and insulin monotherapy. For this purpose, diabetic rats were treated with insulin (DM + Ins) or metformin plus insulin (DM + Met + Ins). Biochemical and cardiometabolic parameters were analysed by spectrophotometry. Intravital microscopy was used to study the hepatic microcirculation. In the liver tissue, real‐time PCR was used to analyse oxidative stress enzymes, inflammatory markers and receptors for advanced glycation end products (AGE) (RAGE) gene expression. Lipid peroxidation was assessed by thiobarbituric acid reactive species (TBARs) analyses. AGE deposition and RAGE protein expression were studied by fluorescence spectrophotometry and Western blot respectively. Body weight, %HbA1c, urea, total proteins and oxidative stress parameters were found to be similarly improved by insulin or Met + Ins treatments. On the other hand, Met + Ins treatment showed a more pronounced effect on fasting blood glucose level than insulin monotherapy. Fructosamine, uric acid, creatinine, albumin and amylase levels and daily insulin dose requirements were found to be only improved by the combined Met + Ins treatment. Liver, renal and pancreatic dysfunction markers were found to be more positively affected by metformin adjunct therapy when compared to insulin treatment. Liver microcirculation damage was found to be completely protected by Met + Ins treatment, while insulin monotherapy showed no effect. Our results suggest that oxidative stress, microcirculatory damage and glycated proteins could be involved in the aetiology of liver disease due to diabetes. Additionally, metformin adjunct treatment improved systemic and liver injury in induced diabetes and showed a more pronounced effect than insulin monotherapy.

The impact of early aerobic exercise on brain microvascular alterations induced by cerebral hypoperfusion

The therapeutic potential of early exercise training following cerebral hypoperfusion was investigated on brain perfusion and inflammation in rats with permanent bilateral occlusion of the common carotid arteries (2VO). Wistar rats were subjected to 2VO or sham surgery and each group was then subdivided randomly into sedentary or exercise groups. Early exercise training was initiated after three days of 2VO or sham surgery and consisted of seven days of treadmill training (30min/day at ∼60% of maximal exercise test), composing four groups: 1) Sham sedentary (Sham-Sed), 2) Sham exercised (Sham-Ex), 3) 2VO sedentary (2VO-Sed) and 4) 2VO exercised (2VO-Ex). Microvascular cerebral blood flow (MCBF) and NADPH oxidase and eNOS gene expression were evaluated by laser speckle contrast imaging and RT-PCR, respectively, and brain functional capillary density and endothelial-leukocyte interactions were evaluated by fluorescence intravital video-microscopy. The 2VO-Sed group presented a decrease in MCBF (Sham-Sed: 230.9±12.2 vs. 2VO-Sed: 183.6±10.6 arbitrary perfusion units, P<0.05) and in functional capillary density (Sham-Sed: 336.4±25.3 vs. 2VO-Sed: 225.5±28.1capillaries/mm2, P<0.05). Early intervention with physical exercise was able to prevent the cerebral microvascular inflammation by decreasing endothelial-leukocyte interactions (2VO-Ex: 0.9±0.3 vs. 2VO-Sed: 5±0.6cells/min/100μm, P<0.0001) and reducing brain NADPH oxidase gene expression (2VO-Ex: 1.7±0.1 arbitrary units, P<0.05). Cerebral microcirculatory and inflammatory alterations appear to be triggered during the first days after 2VO surgery, and early intervention with physical exercise may represent a means of preventing the microvascular alterations induced by chronic cerebral hypoperfusion.

The efficacy of novel arylimidamides against Trypanosoma cruzi in vitro

The present study aimed to determine the in vitro biological efficacy and selectivity of 7 novel AIAs upon bloodstream trypomastigotes and intracellular amastigotes of Trypanosoma cruzi. The biological activity of these aromatic compounds was assayed for 48 and 24 h against intracellular parasites and bloodstream forms of T. cruzi (Y strain), respectively. Additional assays were also performed to determine their potential use in blood banks by treating the bloodstream parasites with the compounds diluted in mouse blood for 24 h at 4°C. Toxicity against mammalian cells was evaluated using primary cultures of cardiac cells incubated for 24 and 48 h with the AIAs and then cellular death rates were determined by MTT colorimetric assays. Our data demonstrated the outstanding trypanocidal effect of AIAs against T. cruzi, especially DB1853, DB1862, DB1867 and DB1868, giving IC50 values ranging between 16 and 70 nanomolar against both parasite forms. All AIAs presented superior efficacy to benznidazole and some, such as DB1868, also demonstrated promising activity as a candidate agent for blood prophylaxis. The excellent anti-trypanosomal efficacy of these novel AIAs against T. cruzi stimulates further in vivo studies and justifies the screening of new analogues with the goal of establishing a useful alternative therapy for Chagas disease.