Cristiane França da Silva

In Vitro and In Vivo Studies of the Trypanocidal Activity of a Diarylthiophene Diamidine against Trypanosoma cruzi

ABSTRACT Aromatic diamidines are DNA minor groove-binding ligands that display excellent antimicrobial activity against fungi, bacteria, and protozoa. Due to the currently unsatisfactory chemotherapy for Chagas’ disease and in view of our previous reports regarding the effect of diamidines and analogues against both in vitro and in vivo Trypanosoma cruzi infection, this study evaluated the effects of a diarylthiophene diamidine (DB1362) against both amastigotes and bloodstream trypomastigotes of T. cruzi, the etiological agent of Chagas’ disease. The data show the potent in vitro activity of DB1362 against both parasite forms that are relevant for mammalian infection at doses which do not exhibit cytotoxicity. Ultrastructural analysis and flow cytometry studies show striking alterations in the nuclei and mitochondria of the bloodstream parasites. In vivo studies were performed at two different drug concentrations (25 and 50 mg/kg/day) using a 2-day or a 10-day regimen. The best results were obtained when acutely infected mice were treated with two doses at the lower concentration, resulting in 100% survival, compared to the infected and untreated mice. Although it did not display higher efficacy than benznidazole, DB1362 reduced both cardiac parasitism and inflammation, and in addition, it protected against the cardiac alterations (determined by measurements) common in T. cruzi infection. These results support further investigation of diamidines and related compounds as potential agents against Chagas’ disease.

In Vitro and In Vivo Studies of the Antiparasitic Activity of Sterol 14α-Demethylase (CYP51) Inhibitor VNI against Drug-Resistant Strains of Trypanosoma cruzi

ABSTRACT Chagas disease affects more than 10 million people worldwide, and yet, as it has historically been known as a disease of the poor, it remains highly neglected. Two currently available drugs exhibit severe toxicity and low effectiveness, especially in the chronic phase, while new drug discovery has been halted for years as a result of a lack of interest from pharmaceutical companies. Although attempts to repurpose the antifungal drugs posaconazole and ravuconazole (inhibitors of fungal sterol 14α-demethylase [CYP51]) are finally in progress, development of cheaper and more efficient, preferably Trypanosoma cruzi-specific, chemotherapies would be highly advantageous. We have recently reported that the experimental T. cruzi CYP51 inhibitor VNI cures with 100% survival and 100% parasitological clearance both acute and chronic murine infections with the Tulahuen strain of T. cruzi. In this work, we further explored the potential of VNI by assaying nitro-derivative-resistant T. cruzi strains, Y and Colombiana, in highly stringent protocols of acute infection. The data show high antiparasitic efficacy of VNI and its derivative (VNI/VNF) against both forms of T. cruzi that are relevant for mammalian host infection (bloodstream and amastigotes), with the in vivo potency, at 25 mg/kg twice a day (b.i.d.), similar to that of benznidazole (100 mg/kg/day). Transmission electron microscopy and reverse mutation tests were performed to explore cellular ultrastructural and mutagenic aspects of VNI, respectively. No mutagenic potential could be seen by the Ames test at up to 3.5 μM, and the main ultrastructural damage induced by VNI in T. cruzi was related to Golgi apparatus and endoplasmic reticulum organization, with membrane blebs presenting an autophagic phenotype. Thus, these preliminary studies confirm VNI as a very promising trypanocidal drug candidate for Chagas disease therapy.

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.

Trypanosoma cruzi: Activity of heterocyclic cationic molecules in vitro

Chagas disease remains a serious public health problem in several Latin American countries. New chemotherapy is urgently needed since current drugs are limited in efficacy and exhibit undesirable side effects. Aromatic diamidines and analogs are well known anti-parasitic agents and in this study, we have evaluated the in vitro trypanocidal effect of several different heterocyclic cationic compounds, including diamidines (DB1195, DB1196 and DB1345), a monoamidine (DB824), an arylimidamide (DB613A) and a guanylhydrazone (DB1080) against amastigotes and bloodstream trypomastigotes of Trypanosoma cruzi, the etiological agent of Chagas disease. Our present findings showed that all compounds exerted, at low-micromolar doses, a trypanocidal effect upon both intracellular parasites and bloodstream trypomastigotes of T. cruzi. The activity of DB1195, DB1345, DB824 and DB1080 against bloodstream forms was reduced when these compounds were assayed in the presence of mouse blood possibly due to their association with plasma constituents and/or due to metabolic instability of the compounds. However, trypanocidal effects of DB613A and DB1196 were not affected by plasma constituents, suggesting their potential application in the prophylaxis of banked blood. In addition, potency and selectivity of DB613A, towards intracellular parasites, corroborate previous results that demonstrated the highly promising activity of arylimidamides against this parasite, which justify further studies in experimental models of T. cruzi infection.

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.

Trypanosoma cruzi Modulates the Expression of Rabs and Alters the Endocytosis in Mouse Cardiomyocytes In Vitro

Chagas disease is an incurable illness caused by the protozoan Trypanosoma cruzi. Cardiomyocytes represent important targets for the parasite infection and alterations in their physiology were reported. Because endocytosis is involved in different cellular events and guanosine triphosphatase (GTPase) Rab proteins play important roles in various aspects of the membrane traffic, our aim was to characterize the expression of Rab proteins in T. cruzi-infected cardiomyocytes, which displayed a downregulation of Rab7 and Rab11, whereas the expression of Rab5a was maintained in the infected cultures even after longer periods of parasite internalization, but early endosome antigen 1 was partially downregulated. The parasite infection also decreased the uptake of fluid phase ligands by the cardiac cultures. The regulation of GTPase proteins and effector molecules can contribute to the altered physiology of the host cells by modifying the normal incoming of nutrients as well as interfering with other important events related to the endocytic pathway.

Activities of Psilostachyin A and Cynaropicrin against Trypanosoma cruzi In Vitro and In Vivo

ABSTRACT In vitro and in vivo activities against Trypanosoma cruzi were evaluated for two sesquiterpene lactones: psilostachyin A and cynaropicrin. Cynaropicrin had previously been shown to potently inhibit African trypanosomes in vivo, and psilostachyin A had been reported to show in vivo effects against T. cruzi, albeit in another test design. In vitro data showed that cynaropicrin was more effective than psilostachyin A. Ultrastructural alterations induced by cynaropicrin included shedding events, detachment of large portions of the plasma membrane, and vesicular bodies and large vacuoles containing membranous structures, suggestive of parasite autophagy. Acute toxicity studies showed that one of two mice died at a cynaropicrin dose of 400 mg/kg of body weight given intraperitoneally (i.p.). Although no major plasma biochemical alterations could be detected, histopathology demonstrated that the liver was the most affected organ in cynaropicrin-treated animals. Although cynaropicrin was as effective as benznidazole against trypomastigotes in vitro, the treatment (once or twice a day) of T. cruzi-infected mice (up to 50 mg/kg/day cynaropicrin) did not suppress parasitemia or protect against mortality induced by the Y and Colombiana strains. Psilostachyin A (0.5 to 50 mg/kg/day given once a day) was not effective in the acute model of T. cruzi infection (Y strain), reaching 100% animal mortality. Our data demonstrate that although it is very promising against African trypanosomes, cynaropicrin does not show efficacy compared to benznidazole in acute mouse models of T. cruzi infection.

In vitro and in vivo biological effects of novel arylimidamide derivatives against Trypanosoma cruzi

ABSTRACT Chagas disease (CD), a neglected tropical disease caused by Trypanosoma cruzi, remains a serious public health problem in several Latin American countries. The available chemotherapies for CD have limited efficacy and exhibit undesirable side effects. Aromatic diamidines and arylimidamides (AIAs) have shown broad-spectrum activity against intracellular parasites, including T. cruzi. Therefore, our aim was to evaluate the biological activity of eight novel AIAs (16DAP002, 16SAB079, 18SAB075, 23SMB022, 23SMB026, 23SMB054, 26SMB070, and 27SMB009) against experimental models of T. cruzi infection in vitro and in vivo. Our data show that none of the compounds induced a loss of cellular viability up to 32 μM. Two AIAs, 18SAB075 and 16DAP002, exhibited good in vitro activity against different parasite strains (Y and Tulahuen) and against the two relevant forms of the parasite for mammalian hosts. Due to the excellent selective indexes of 18SAB075, this AIA was moved to in vivo tests for acute toxicity and parasite efficacy; nontoxic doses (no-observed-adverse-effect level [NOAEL], 50 mg/kg) were employed in the tests for parasite efficacy. In experimental models of acute T. cruzi infection, 18SAB075 reduced parasitemia levels only up to 50% and led to 40% protection against mortality (at 5 mg/kg of body weight), being less effective than the reference drug, benznidazole.

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.