In vitro and in vivo antiparasitic activity of Physalis angulata L. concentrated ethanolic extract against Trypanosoma cruzi

BackgroundThe current treatment of Chagas disease, endemic in Latin America and emerging in several countries, is limited by the frequent side effects and variable efficacy of benznidazole. Natural products are an important source for the search for new drugs.Aim/hypothesisConsidering the great potential of natural products as antiparasitic agents, we investigated the anti-Trypanosoma cruzi activity of a concentrated ethanolic extract of Physalis angulata (EEPA).MethodsCytotoxicity to mammalian cells was determined using mouse peritoneal macrophages. The antiparasitic activity was evaluated against axenic epimastigote and bloodstream trypomastigote forms of T. cruzi, and against amastigote forms using T. cruzi-infected macrophages. Cell death mechanism was determined in trypomastigotes by flow cytometry analysis after annexin V and propidium iodide staining. The efficacy of EEPA was examined in vivo in an acute model of infection by monitoring blood parasitaemia and survival rate 30 days after treatment. The effect against trypomastigotes of EEPA and benznidazole acting in combination was evaluated.ResultsEEPA effectively inhibits the epimastigote growth (IC₅₀ 2.9 ± 0.1 µM) and reduces bloodstream trypomastigote viability (EC₅₀ 1.7 ± 0.5 µM). It causes parasite cell death by necrosis. EEPA impairs parasite infectivity as well as amastigote development in concentrations noncytotoxic to mammalian cells. In mice acutely-infected with T. cruzi, EEPA reduced the blood parasitaemia in 72.7%. When combined with benznidazole, EEPA showed a synergistic anti-T. cruzi activity, displaying CI values of 0.8 ± 0.07 at EC₅₀ and 0.83 ± 0.1 at EC₉₀.ConclusionEEPA has antiparasitic activity against T. cruzi, causing cell death by necrosis and showing synergistic activity with benznidazole. These findings were reinforced by the observed efficacy of EEPA in reducing parasite load in T. cruzi-mice. Therefore, this represents an important source of antiparasitic natural products.     

Adenylate cyclase activation by GTP analogs

Benznidazole (a nitroimidazole derivative used for the treatment of Chagas' disease) is reduced by rat liver microsomes to the nitro anion radical, as indicated by ESR spectroscopy. Addition of benznidazole to rat liver microsomes produced an increase of electron flow from NADPH to molecular oxygen, and generation of both superoxide anion and hydrogen peroxide. The benznidazole-stimulated O₂ consumption and O₂^? formation was greatly inhibited by NADP⁺ and p-chloromercuribenzoate but not by SKF-525-A and metyrapone. The former inhibitions indicated the involvement of NADPH-cytochrome P-450 (c) reductase, while the lack of inhibition by SKF-525-A and metyrapone ruled out any major role for cytochrome P-450 in benznidazole reduction. In contrast to nifurtimox, a nitrofuran derivative (R. Docampo and A. O. M. Stoppani, 1979, Arch. Biochem. Biophys.197, 317–321), benznidazole was not reduced to the nitro anion radical, nor did it stimulate oxygen consumption, O₂^? production, and H₂O₂ generation by Trypanosoma cruzi cells or microsomal fractions. A different mechanism of benznidazole toxicity in T. cruzi and the mammalian host is postulated.     

New potent 5-nitroindazole derivatives as inhibitors of Trypanosoma cruzi growth: Synthesis,biological evaluation,and mechanism of action studies

New 5-nitroindazole derivatives were developed and their antichagasic properties studied. Eight compounds (14–18, 20, 26 and 28) displayed remarkable in vitro activities against Trypanosoma cruzi (T. cruzi). Its unspecific cytotoxicity against macrophages was evaluated being not toxic at a concentration at least twice that of T. cruzi IC₅₀, for some derivatives. The electrochemical studies, parasite respiration studies and ESR experiment showed that 5-nitroindazole derivatives not be able to yield a redox cycling with molecular oxygen such as occurs with nifurtimox (Nfx). The study on the mechanism of action proves to be related to the production of reduced species of the nitro moiety similar to that observed with benznidazole.     

In Vitro and In Vivo High-Throughput Assays for the Testing of Anti-Trypanosoma cruzi Compounds

Background

The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.

Methods

In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC₅₀ lower than that of BZ.

Findings

This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.

Conclusion

These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.     

Epidemiology and pathogenesis of maternal-fetal transmission of Trypanosoma cruzi and a case for vaccine development against congenital Chagas disease

Trypanos o ma cruzi (T. cruzi or Tc) is the causative agent of Chagas disease (CD). It is common for patients to suffer from non-specific symptoms or be clinically asymptomatic with acute and chronic conditions acquired through various routes of transmission. The expecting women and their fetuses are vulnerable to congenital transmission of Tc. Pregnant women face formidable health challenges because the frontline antiparasitic drugs, benznidazole and nifurtimox, are contraindicated during pregnancy. However, it is worthwhile to highlight that newborns can be cured if they are diagnosed and given treatment in a timely manner. In this review, we discuss the pathogenesis of maternal-fetal transmission of Tc and provide a justification for the investment in the development of vaccines against congenital CD.     

Trypanosoma cruzi: Immunological predictors of benznidazole efficacy during experimental infection

C3H/HeN male mice were infected with a lethal population of Trypanosoma cruzi and treated with benznidazole (Bz). Parasitemia, body weight and survival rate were registered during the therapy with significant improvement for T. cruzi-infected Bz-treated animals. Besides, flow cytometry resulted a useful method to discriminate between cured animals from those not cured by monitoring IgG₁ bound to live trypomastigotes levels. At the end of Bz therapy, the LT splenocyte compartment was studied for activation/memory cell surface markers ( and ). Cytofluorometric analysis showed that T. cruzi-infected untreated mice increased their activated LT numbers and this effect was completely abolished only in cured mice at the end of Bz administration. The same behavior was observed for the memory LT subpopulation correlating to an effector memory () displayed by T. cruzi infection. Bz treatment was able to modulate the immunological response by reducing the deleterious effect of the acute phase in all T. cruzi-infected mice.     

The Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection against Oxidative Stress but Is Not a Determinant of Virulence

Background

The neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in Trypanosoma cruzi, the causative agent. T. cruzi expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy.

Methodology/Principal Findings

To assess the importance of TcAPx in protecting T. cruzi from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle in vitro, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on T. cruzi replication, dissemination or tissue tropism in vivo.

Conclusions/Significance

TcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design.     

Trypanosoma cruzi: Antiproliferative effect of indole phytoalexins on intracellular amastigotes in vitro

American trypanosomiasis (Chagas disease) continues to be a significant public health problem, and the therapeutic potential of current antichagasic agents (nifurtimox and benznidazole) is rather limited. Here we report on the antitrypanosomal effect of 1-methoxyspirobrassinol and other indole phytoalexins—secondary metabolites produced by Cruciferous plants. These compounds, that previously demonstrated antimicrobial and anticancer properties, displayed significant antiproliferative effects on intracellular amastigotes of Trypanosoma cruzi and may be prospective candidates for antichagasic drug design and development.     

Synthesis of benzologues of Nitazoxanide and Tizoxanide: a comparative study of their in vitro broad-spectrum antiprotozoal activity

We have synthesized two new benzologues of Nitazoxanide (NIT) and Tizoxanide (TIZ), using a short synthetic route. Both compounds were tested in vitro against six protozoa (Giardia intestinalis, Trichomonas vaginalis, Entamoeba histolytica, Plasmodium berghei, Leishmania mexicana and Trypanosoma cruzi). Compound 1 (benzologue of NIT) showed broad antiprotozoal effect against all parasites tested, showing IC₅₀’s <5μM. This compound was five-times more active than NIT, and 18-times more potent than metronidazole against G. intestinalis. It was 10-times more active than pentamidine against L. mexicana, and it was sevenfold more potent than benznidazole versus T. cruzi. This compound could be considered as a new broad spectrum antiprotozoal agent.     

Preventive effect of Ligularia fischeri on inhibition of nitric oxide in lipopolysaccharide-stimulated RAW 264.7 macrophages depending on cooking method

Background

Ligularia fischeri (common name Gomchwi) is known for its pharmaceutical properties and used in the treatment of jaundice, scarlet-fever, rheumatoidal arthritis, and hepatic diseases; however, little is known about its anti-inflammatory effect. In this study the influence of blanching and pan-frying on the anti-inflammatory activity of Ligularia fischeri (LF) was evaluated.

Results

Fresh LF and cooked LF showed no significant effect on the viability of macrophages after 24 h incubation. Fresh LF was found to be the most potent inhibitor of nitric oxide (NO) production at 100 μg/ml, while pan-fried LF showed little inhibitory effect on lipoloysaccharide (LPS) stimulated murine machrophage RAW264.7 cells. In contrast with its effect on NO production, pan-fried LF showed significant attenuation of the expression of inducible nitiric oxide synthase (iNOS) compared with fresh LF. In the cooking method of LF, PGE₂ production was not affected in the LPS-induced RAW 264.7 cells. In LPS-induced RAW 264.7 cells, pretreatment by fresh and cooked LF increased COX2 mRNA expression. The 3-O-caffeoylquinic acid content of blanching and pan-frying LF increased by 4.92 and 9.7 fold with blanching and pan-frying respectively in comparison with uncooked LF.

Conclusions

Regardless of the cooking method, Ligularia fischeri exhibited potent inhibition of NO production through expression of iNOS in LPS-induced RAW264.7 cells.     

Synthesis,in vitro antitrypanosomal and antibacterial activity of phenoxy,phenylthio or benzyloxy substituted quinolones

Chagas’ disease, caused by Trypanosoma cruzi (T. cruzi), is one of the most serious parasitic diseases in Latin America. The currently available chemotherapy, based on nifurtimox or benznidazole, is unsatisfactory due to the limited efficacy in the prevalent chronic stage of the disease and toxic side effects. In order to address these deficiencies, a series of quinolones based novel molecules have been synthesized and evaluated as potential antitrypanosomal agents. The most active analogue 10 inhibited T. cruzi with an IC₅₀ of 1.3 μg/mL. The results of this study have implications in the development of novel quinolone’s antitrypanosomal agents.     

Evaluation of thiosemicarbazones and semicarbazones as potential agents anti-Trypanosoma cruzi

Synthetic thiosemicarbazones and semicarbazones were evaluated for their Trypanosoma cruzi trypomastigotes obtained from LLC-MK2 cell cultures. In general, thiosemicarbazone derivatives were most effective and among them the 4-N-(2′-methoxy styryl)-thiosemicarbazone was chosen, to compare the in vitro effect against amastigotes of T. cruzi lodged in mouse peritoneal and human macrophages. A potent trypanocidal effect was observed that was more pronounced against parasites internalized in human macrophages. A potential target for this compound was also evaluated by measuring the nitric oxide synthase activity through NADPH consumption. A significant decrease in enzyme activity was observed. In contrast to the cytotoxic effect observed with benznidazole, no macrophage toxicity was observed for any of the compounds, indicating that their activity was specific for the parasite forms investigated.     

Fexinidazole: A Potential New Drug Candidate for Chagas Disease

Background

New safe and effective treatments for Chagas disease (CD) are urgently needed. Current chemotherapy options for CD have significant limitations, including failure to uniformly achieve parasitological cure or prevent the chronic phase of CD, and safety and tolerability concerns. Fexinidazole, a 2-subsituted 5-nitroimidazole drug candidate rediscovered following extensive compound mining by the Drugs for Neglected Diseases initiative and currently in Phase I clinical study for the treatment of human African trypanosomiasis, was evaluated in experimental models of acute and chronic CD caused by different strains of Trypanosoma cruzi.

Methods and Findings

We investigated the in vivo activity of fexinidazole against T. cruzi, using mice as hosts. The T. cruzi strains used in the study were previously characterized in murine models as susceptible (CL strain), partially resistant (Y strain), and resistant (Colombian and VL-10 strains) to the drugs currently in clinical use, benznidazole and nifurtimox. Our results demonstrated that fexinidazole was effective in suppressing parasitemia and preventing death in infected animals for all strains tested. In addition, assessment of definitive parasite clearance (cure) through parasitological, PCR, and serological methods showed cure rates of 80.0% against CL and Y strains, 88.9% against VL-10 strain, and 77.8% against Colombian strain among animals treated during acute phase, and 70% (VL-10 strain) in those treated in chronic phase. Benznidazole had a similar effect against susceptible and partially resistant T. cruzi strains. Fexinidazole treatment was also shown to reduce myocarditis in all animals infected with VL-10 or Colombian resistant T. cruzi strains, although parasite eradication was not achieved in all treated animals at the tested doses.

Conclusions

Fexinidazole is an effective oral treatment of acute and chronic experimental CD caused by benznidazole-susceptible, partially resistant, and resistant T. cruzi. These findings illustrate the potential of fexinidazole as a drug candidate for the treatment of human CD.     

Inhibition of Trypanosoma cruzi α-Hydroxyacid Dehydrogenase-isozyme II by N-Isopropyl Oxamate and its Effect on Intact Epimastigotes

The effect of N-isopropyl oxamate on the activity of α-hydroxyacid dehydrogenase-isozyme II (HADH-isozyme II) from Trypanosoma cruzi was investigated. The kinetic studies showed that this substance was a competitive inhibitor of this isozyme. The attachment of the nonpolar isopropylic branched chain to the nitrogen of oxamate increased 12-fold the affinity of N-isopropyl oxamate for the active site of T. cruzi HADH-isozyme II. N-isopropyl oxamate was a selective inhibitor of HADH-isozyme II, since other T. cruzi dehydrogenases were not inhibited by this substance. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with inhibitors of this isozyme. However, although it was not possible to detect any trypanocidal activity with N-isopropyl oxamate when the ethyl ester was tested as a possible trypanocidal prodrug, the expected trypanocidal effect was obtained, comparable to that obtained with nifurtimox and benznidazole.     

Generation of superoxide anion and hydrogen peroxide induced by nifurtimox in Trypanosoma cruzi.

Addition of nifurtimox (a nitrofuran derivative) to Trypanosoma cruzi culture (epimastigote) forms induced an increase in the respiratory rate and the release of H₂O₂ from the whole cells to the suspending medium. Growth-inhibiting concentrations of nifurtimox were able to stimulate O_2? production by the T. cruzi mitochondrial fraction supplemented with NADH (or succinate), and also to enhance the generation of O_2? by the microsomal fraction with NADPH as reductant.     

A Quinoxaline Derivative as a Potent Chemotherapeutic Agent,Alone or in Combination with Benznidazole,against Trypanosoma cruzi

Background

Chagas’ disease is a condition caused by the protozoan Trypanosoma cruzi that affects millions of people, mainly in Latin America where it is considered endemic. The chemotherapy for Chagas disease remains a problem; the standard treatment currently relies on a single drug, benznidazole, which unfortunately induces several side effects and it is not successful in the cure of most of the chronic patients. In order to improve the drug armamentarium against Chagas’ disease, in the present study we describe the synthesis of the compound 3-chloro-7-methoxy-2-(methylsulfonyl) quinoxaline (quinoxaline 4) and its activity, alone or in combination with benznidazole, against Trypanosoma cruzi in vitro.

Methodology/Principal Findings

Quinoxaline 4 was found to be strongly active against Trypanosoma cruzi Y strain and more effective against the proliferative forms. The cytotoxicity against LLCMK₂ cells provided selective indices above one for all of the parasite forms. The drug induced very low hemolysis, but its anti-protozoan activity was partially inhibited when mouse blood was added in the experiment against trypomastigotes, an effect that was specifically related to blood cells. A synergistic effect between quinoxaline 4 and benznidazole was observed against epimastigotes and trypomastigotes, accompanied by an antagonistic interaction against LLCMK₂ cells. Quinoxaline 4 induced several ultrastructural alterations, including formations of vesicular bodies, profiles of reticulum endoplasmic surrounding organelles and disorganization of Golgi complex. These alterations were also companied by cell volume reduction and maintenance of cell membrane integrity of treated-parasites.

Conclusion/Significance

Our results demonstrated that quinoxaline 4, alone or in combination with benznidazole, has promising effects against all the main forms of T. cruzi. The compound at low concentrations induced several ultrastructural alterations and led the parasite to an autophagic-like cell death. Taken together these results may support the further development of a combination therapy as an alternative more effective in Chagas’ disease treatment.     

Trypanosoma cruzi: Biological characterization of a isolate from an endemic area and its susceptibility to conventional drugs

We describe some biological and molecular characteristics of a Trypanosoma cruzi isolate derived from a Triatomine captured in Nicaragua. PCR based typification showed that this isolate, named Nicaragua, belonged to the lineage Tc I. Nicaragua infected culture cells were treated with allopurinol, showing different behavior according to the cellular compartment, being cardiomyocyte primary cultures more resistant to this drug. The course of the infection in a mice experimental model and its susceptibility to benznidazole and allopurinol was analyzed. In benznidazole treatment, mice reverted the high lethal effect of parasites during the acute infection, however, a few parasites were detected in the heart of 88% of mice 1 year post-infection. Since T. cruzi is a heterogeneous species population it is important to study and characterize different parasites actually circulating in humans in endemic areas. In this work we show that T. cruzi Nicaragua isolate, is sensitive to early benznidazole treatment.     

Synthesis and biological evaluation of 2-methyl-1H-benzimidazole-5-carbohydrazides derivatives as modifiers of redox homeostasis of Trypanosoma cruzi

Twelve novel benzimidazole derivatives were synthesized and their in vitro activities against epimastigotes of Trypanosoma cruzi were evaluated. Two derivatives (6 and 7), which have 4-hydroxy-3-methoxyphenyl moiety in their structures, proved to be the most active in inhibiting the parasite growth. Compound 6 showed a trypanocidal activity higher than benznidazole (IC₅₀ = 5 µM and 7.5 µM, respectively) and less than nifurtimox (IC₅₀ = 3.6 µM). In addition, the ability of 6 and 7 to modify the redox homeostasis in T cruzi epimastigote was studied; cysteine and glutathione increased in parasites exposed to both compounds, whereas trypanothione only increased with 7 treatment. These results suggest that the decrease in viability of T. cruzi may be attributed to the change in cellular redox balance caused by compound 6 or 7. Furthermore, compounds 6 and 7 showed CC₅₀ values of 160.64 and 160.66 µM when tested in mouse macrophage cell line J774 and selectivity indexes (macrophage/parasite) of 32 and 20.1, respectively.