Benznidazole Research Articles

Abstract 11768: Regression of Inflammation by Cardiac Magnetic Resonance in Chronic Chagasic Patients Treated With Benznidazole

Introduction: Chagas disease (CD) is a neglected disease caused by Trypanosoma cruzi ( T. cruzi ), and benznidazole (BZN) is the most prescribed trypanocidal drug in Brazil in function of its best tolerability profile. While treatment with this drug is effective in acute disease, there is no consensus on its effectiveness in adults with chronic infection. Although it is now accepted that treatment directed against T. cruzi is necessary to avoid the long-term sequelae of the disease, the lack of reliable markers for assessment of treatment efficacy is a major obstacle to evaluating new drugs. Hypothesis: the objective of this study is to determine if BZN treatment reduces cardiac inflammation/fibrosis as assessed by magnetic resonance imaging (MRI), using parametric mapping techniques. Methods: Prospective, multi-center study of chronic CD patients underwent MRI T1 and T2 mapping and extracellular volume measurement (ECV) using the modified Look-Locker inversion recovery (MOLLI) sequence, undergoing treatment with BZN, at a dose of 300 mg/kg daily divided into two doses and taken for 60 days. Absolute change T1 and T2 mapping, ECV, left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV) and left ventricle ejection fraction (LVEF) were analyzed from baseline to 6-month follow-up. Results: 53 patients (females 83%, 53.9 ± 11.9 years old) were studied. LVEDV (149 ± 27ml vs 147 ± 29ml, p = 0.52), LVESV (66 ± 29 vs 64 ± 15, p = 0.65) and LVEF (55 ± 11% vs 57 ± 8%, p = 0,28) did not change between baseline and post treatment. Native T1 time showed reduced values among anterior (1030 ± 41ms vs 1011 ± 52ms, p = 0.011), septal (1052 ± 53ms vs 1006 ± 38ms, p = 0.000) and lateral (1042 ± 55ms vs 1019 ± 44ms, p = 0.003) LV walls. T2 mapping showed reduction in LV septal wall (53 ± 4ms vs 52 ± 3ms, p = 0.005). ECV showed significant reduction from baseline to 6-month follow-up (29.9 ± 3,3% vs 28.2 ± 3.5%, p = 0.0001). Conclusions: For the first time, we observe favorable changes in CMR ECV, T1 and T2 mapping induced by benznidazole therapy in chronic Chagas disease patients. These findings suggest that there may be an opportunity to decrease fibrosis/inflammation in chronic CD patients with the use of this therapeutic approach.

Anti-Trypanosoma cruzi Activity, Mutagenicity, Hepatocytotoxicity and Nitroreductase Enzyme Evaluation of 3-Nitrotriazole, 2-Nitroimidazole and Triazole Derivatives.

Chagas disease (CD), which is caused by Trypanosoma cruzi and was discovered more than 100 years ago, remains the leading cause of death from parasitic diseases in the Americas. As a curative treatment is only available for the acute phase of CD, the search for new therapeutic options is urgent. In this study, nitroazole and azole compounds were synthesized and underwent molecular modeling, anti-T. cruzi evaluations and nitroreductase enzymatic assays. The compounds were designed as possible inhibitors of ergosterol biosynthesis and/or as substrates of nitroreductase enzymes. The in vitro evaluation against T. cruzi clearly showed that nitrotriazole compounds are significantly more potent than nitroimidazoles and triazoles. When their carbonyls were reduced to hydroxyl groups, the compounds showed a significant increase in activity. In addition, these substances showed potential for action via nitroreductase activation, as the substances were metabolized at higher rates than benznidazole (BZN), a reference drug against CD. Among the compounds, 1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanol (8) is the most potent and selective of the series, with an IC50 of 0.39 µM and selectivity index of 3077; compared to BZN, 8 is 4-fold more potent and 2-fold more selective. Moreover, this compound was not mutagenic at any of the concentrations evaluated, exhibited a favorable in silico ADMET profile and showed a low potential for hepatotoxicity, as evidenced by the high values of CC50 in HepG2 cells. Furthermore, compared to BZN, derivative 8 showed a higher rate of conversion by nitroreductase and was metabolized three times more quickly when both compounds were tested at a concentration of 50 µM. The results obtained by the enzymatic evaluation and molecular docking studies suggest that, as planned, nitroazole derivatives may utilize the nitroreductase metabolism pathway as their main mechanism of action against Trypanosoma cruzi. In summary, we have successfully identified and characterized new nitrotriazole analogs, demonstrating their potential as promising candidates for the development of Chagas disease drug candidates that function via nitroreductase activation, are considerably selective and show no mutagenic potential.

Parasitemia and antibody response to benznidazole treatment in a cohort of patients with chronic Chagas disease

BackgroundEvaluating the effectiveness of Chagas disease treatment poses challenges due to the lack of biomarkers for disease progression and therapeutic response. In this study, we aimed to assess the clearance of Trypanosoma cruzi (T. cruzi) parasites in a group of benznidazole (BNZ)-treated chronic Chagas disease patients using high-sensitivity quantitative PCR (qPCR) and track T. cruzi antibody levels through a semiquantitative chemiluminescent assay.MethodsA total of 102 T. cruzi seropositive patients with previous PCR-positive results were enrolled in the study. We collected samples 30 days before treatment (T-30d), on the day before initiating BNZ treatment (T0d), and at follow-up visits 60 days (T60d), 6 months (T6M), 12 months (T12M), and 36 months (T36M) after treatment initiation. Treatment efficacy was assessed by testing of serial samples using a target-capture qPCR assay specific to satellite T. cruzi DNA and the ORTHO T. cruzi ELISA Test System for antibody quantitation.ResultsOf the enrolled individuals, 87 completed at least 50% of the treatment course, and 86 had PCR results at follow-up visits T6M, T12M, and T36M. PCR results exhibited fluctuations before and after treatment, but levels were significantly lower post-treatment. Only 15 cases consistently tested PCR-negative across all post-treatment visits. Notably, nearly all participants demonstrated a declining antibody trajectory, with patients who tested PCR-negative at T36M exhibiting an earlier and more pronounced decline compared to PCR-positive cases at the same visit.ConclusionOur study suggests that serial PCR results pose challenges in interpretation. In contrast, serial antibody levels may serve as an ancillary, or even a more reliable indicator of parasite decline following BNZ treatment. Monitoring antibody levels can provide valuable insights into the efficacy of treatment and the persistence of parasites in Chagas disease patients.

Identifying inhibitors of Trypanosoma cruzi nucleoside diphosphate kinase 1 as potential repurposed drugs for Chagas’ disease

Trypanosoma cruzi is the causative agent of Chagas’ disease, an endemic and neglected disease. The treatment is limited to only two drugs, benznidazole (BZL) and nifurtimox (NFX), introduced more than fifty years ago and no new advances have been made since then. Nucleoside diphosphate kinases (NDPK) are key metabolic enzymes which have gained interest as drug targets of pathogen organisms. Taking advantage of the computer-assisted drug repurposing approaches, in the present work we initiate a search of potential T. cruzi nucleoside diphosphate kinase 1 (TcNDPK1) inhibitors over an ∼ 12,000 compound structures database to find drugs targeted to this enzyme with trypanocidal activity. Four medicines were selected and evaluated in vitro, ketorolac (KET, an anti-inflamatory), dutasteride (DUT, used to treat benign prostatic hyperplasia), nebivolol and telmisartan (NEB and TEL, used to treat high blood pressure). The four compounds were weak inhibitors and presented different trypanocidal effect on epimastigotes, trypomastigotes and intracellular stages. NEB and TEL were the most active drugs with increased effect on intracellular stages, (IC50 = 2.25 µM and 13.21 µM respectively), and selectivity indexes of 13.01 and 8.59 respectively, showing comparable effect to BZL, the first line drug for Chagas’ disease treatment. In addition, both presented positive interactions when combined with BZL. Finally, transgenic epimastigotes with increased expression of TcNDPK1 were more resistant to TEL and NEB, suggesting that TcNDPK1 is at least one of the molecular targets. In view of the results, NEB and TEL could be repurposed medicines for Chagas’ disease therapy.

The Therapeutic Potential of Angeli's Salt in Mitigating Acute Trypanosoma cruzi Infection in Mice.

Chagas disease (CD), caused by Trypanosoma cruzi, is a neglected tropical disease prevalent in Latin America. Infected patients are treated to eliminate the parasite, reduce the cardiomyopathy risk, and interrupt the disease transmission cycle. The World Health Organization recognizes benznidazole (BZ) and nifurtimox as effective drugs for CD treatment. In the chronic phase, both drugs have low cure rates and serious side effects. T. cruzi infection causes intense tissue inflammation that controls parasite proliferation and CD evolution. Compounds that liberate nitric oxide (NO) (NO donors) have been used as anti-T. cruzi therapeutics. Currently, there is no evidence that nitroxyl (HNO) affects T. cruzi infection outcomes. This study investigated the effects of the HNO donor Angeli's salt (AS) on C57BL/6 mice infected with T. cruzi (Y strain, 5 × 103 trypomastigotes, intraperitoneally). AS reduced the number of parasites in the bloodstream and heart nests and increased the protective antioxidant capacity of erythrocytes in infected animals, reducing disease severity. Furthermore, in vitro experiments showed that AS treatment reduced parasite uptake and trypomastigote release by macrophages. Taken together, these findings from the murine model and in vitro testing suggest that AS could be a promising therapy for CD.

Drug-excipient compatibility studies in formulation development: A case study with benznidazole and monoglycerides

Monoglycerides (MGs) such as glycerol monolaurate (GML) and glycerol monostearate (GMS) have been used as excipients in oral formulations because of their emulsifying effect as well as their ability to inhibit the precipitation and intestinal efflux of drugs. Excipient-drug compatibility studies, however, have been underexplored. In this study, benznidazole (BNZ) was selected as a drug model due to the difficulty in improving its solubility and because of the potential impact on public health (it is the only drug currently used to treat Chagas disease). The effect of different processing conditions (maceration, ball milling, and melting) on the physical-chemistry properties of BNZ/MGs mixtures was investigated to guide the rational development of new solid formulations. GML was more effective in improving the solubility of BNZ, which could be due to its more malleable structure, less hydrophobic nature, and greater interaction with BNZ. The formation of hydrogen bonds between the imidazole group of BNZ and the polar region of GML was confirmed by spectroscopy analyses (IR, 1H NMR). The higher the monoglyceride content in the mixture, the higher the BNZ solubility. Regardless of the method of processing the mixture, the drug was found to be crystalline. Polarized light microscopy analysis showed the presence of spherulites. Overall, these findings suggest that preparation methods of BNZ:MGs formulations that involve thermal or/and mechanical treatment have a low impact on the solid properties of the material, and this allows for the production of formulations with reproducible performance.

Thermal energy and tableting effects in benznidazole product: the impacts of industrial processing

Objective The investigation of benznidazole (BZN), excipients, and tablets aims to evaluate their thermal energy and tableting effects. They aim to understand better the molecular and pharmaceutical processing techniques of the formulation. Significance The Product Quality Review, part of Good Manufacturing Practices, is essential to highlight trends and identify product and process improvements. Methods A set of technique approaches, infrared spectroscopy, X-ray diffraction, and thermal analysis with isoconversional kinetic study, were applied in the protocol. Results X-ray experiments suggest talc and α-lactose monohydrate dehydration and conversion of β-lactose to stable α-lactose upon tableting. The signal crystallization at 167 °C in the DSC curve confirmed this observation. A calorimetric study showed a decrease in the thermal stability of BZN tablets. Therefore, the temperature is a critical process parameter. The specific heat capacity (Cp) of BZN, measured by DSC, was 10.04 J/g at 25 °C and 9.06 J/g at 160 °C. Thermal decomposition required 78 kJ mol−1. Compared with the tablet (about 200 kJ mol−1), the necessary energy is two-fold lower, as observed in the kinetic study by non-isothermal TG experiment at 5; 7.5; 10; and 15 °C min−1. Conclusions These results indicate the necessity of considering the thermal energy and tableting effects of BZN manufacturing, which contributes significantly to the molecular mechanistic understanding of this drug delivery system.

Population pharmacokinetics of benznidazole in neonates, infants and children using a new pediatric formulation.

There is a major need for information on pharmacokinetics (PK) of benznidazole (BNZ) in children with Chagas disease (CD). We conducted a multicentre population PK, safety and efficacy study in children, infants and neonates with CD treated with BNZ (formulated in 100 mg tablets or 12.5 mg dispersible tablets, developed by the pharmaceutical company LAFEPE, in a collaboration with DNDi). 81 children 0-12 years old were enrolled at 5 pediatric centers in Argentina. Diagnosis of T. cruzi infection was confirmed by direct microscopic examination, or at least two positive conventional serological tests. Subject enrolment was stratified by age: newborns to 2 years (minimum of 10 newborns) and >2-12 years. BNZ 7.5 mg/kg/d was administered in two daily doses for 60 days. Five blood samples per child were obtained at random times within pre-defined time windows at Day 0 at 2-5 h post-dose; during steady state, one sample at Day 7 and at Day 30; and two samples at 12-24 h after final BNZ dose at Day 60. The primary efficacy endpoint was parasitological clearance by qualitative PCR at the end of treatment. Forty-one (51%) patients were under 2 years of age (including 14 newborns <1 month of age). Median age at enrolment was 22 months (mean: 43.2; interquartile range (IQR) 7-72 months). The median measured BNZ Cmax was 8.32 mg/L (IQR 5.95-11.8; range 1.79-19.38). Median observed BNZ Cmin (trough) concentration was 2 mg/L (IQR 1.25-3.77; range 0.14-7.08). Overall median simulated Css was 6.3 mg/L (IQR 4.7-8.5 mg/L). CL/F increased quickly during the first month of postnatal life and reached adult levels after approximately 10 years of age. Negative qPCR was observed at the end of treatment in all 76 patients who completed the treatment. Five patients discontinued treatment (3 due to AEs and 2 due to lack of compliance). We observed lower BNZ plasma concentrations in infants and children than those previously reported in adults treated with comparable mg/kg doses. Despite these lower concentrations, pediatric treatment was well tolerated and universally effective, with a high response rate and infrequent, mild AEs. Registered in clinicaltrials.gov #NCT01549236.

Transcriptomic analysis of benznidazole-resistant and susceptible Trypanosoma cruzi populations

BackgroundChagas disease (CD), caused by the parasite Trypanosoma cruzi, is a serious public health concern in Latin America. Nifurtimox and benznidazole (BZ), the only two drugs currently approved for the treatment of CD, have very low efficacies in the chronic phase of the disease and several toxic side effects. Trypanosoma cruzi strains that are naturally resistant to both drugs have been reported. We performed a comparative transcriptomic analysis of wild-type and BZ-resistant T. cruzi populations using high-throughput RNA sequencing to elucidate the metabolic pathways related to clinical drug resistance and identify promising molecular targets for the development of new drugs for treating CD.MethodsAll complementary DNA (cDNA) libraries were constructed from the epimastigote forms of each line, sequenced and analysed using the Prinseq and Trimmomatic tools for the quality analysis, STAR as the aligner for mapping the reads against the reference genome (T. cruzi Dm28c—2018), the Bioconductor package EdgeR for statistical analysis of differential expression and the Python-based library GOATools for the functional enrichment analysis.ResultsThe analytical pipeline with an adjusted P-value of < 0.05 and fold-change > 1.5 identified 1819 transcripts that were differentially expressed (DE) between wild-type and BZ-resistant T. cruzi populations. Of these, 1522 (83.7%) presented functional annotations and 297 (16.2%) were assigned as hypothetical proteins. In total, 1067 transcripts were upregulated and 752 were downregulated in the BZ-resistant T. cruzi population. Functional enrichment analysis of the DE transcripts identified 10 and 111 functional categories enriched for the up- and downregulated transcripts, respectively. Through functional analysis we identified several biological processes potentially associated with the BZ-resistant phenotype: cellular amino acid metabolic processes, translation, proteolysis, protein phosphorylation, RNA modification, DNA repair, generation of precursor metabolites and energy, oxidation–reduction processes, protein folding, purine nucleotide metabolic processes and lipid biosynthetic processes.ConclusionsThe transcriptomic profile of T. cruzi revealed a robust set of genes from different metabolic pathways associated with the BZ-resistant phenotype, proving that T. cruzi resistance mechanisms are multifactorial and complex. Biological processes associated with parasite drug resistance include antioxidant defenses and RNA processing. The identified transcripts, such as ascorbate peroxidase (APX) and iron superoxide dismutase (Fe-SOD), provide important information on the resistant phenotype. These DE transcripts can be further evaluated as molecular targets for new drugs against CD.Graphical

1,3-Thiazole derivatives as privileged structures for anti-Trypanosoma cruzi activity: Rational design, synthesis, in silico and in vitro studies

Chagas disease is a deadly and centenary neglected disease that is recently surging as a potential global threat. Approximately 30% of infected individuals develop chronic Chagas cardiomyopathy and current treatment with the reference benznidazole (BZN) is ineffective for this stage. We presently report the structural planning, synthesis, characterization, molecular docking prediction, cytotoxicity, in vitro bioactivity and mechanistic studies on the anti-T. cruzi activity of a series of 16 novel 1,3-thiazoles (2–17) derived from thiosemicarbazones (1a, 1b) in a two-step and reproducible Hantzsch-based synthesis approach. The anti-T. cruzi activity was evaluated in vitro against the epimastigote, amastigote and trypomastigote forms of the parasite. In the bioactivity assays, all thiazoles were more potent than BZN against epimastigotes. We found that the compounds presented an overall increased anti-tripomastigote selectivity (Cpd 8 was 24-fold more selective) than BZN, and they mostly presented anti-amastigote activity at very low doses (from 3.65 μM, cpd 15). Mechanistic studies on cell death suggested that the series of 1,3-thiazole compounds herein reported cause parasite cell death through apoptosis, but without compromising the mitochondrial membrane potential. In silico prediction of physicochemical properties and pharmacokinetic parameters showed promising drug-like results, being all the reported compounds in compliance with Lipinski and Veber rules. In summary, our work contributes towards a more rational design of potent and selective antitripanosomal drugs, using affordable methodology to yield industrially viable drug candidates.

Oxidative stress, cardiomyocytes premature senescence and contractile dysfunction in in vitro and in vivo experimental models of Chagas disease

AimsThe relationship between redox imbalance and cardiovascular senescence in infectious myocarditis is unknown. Thus, the aim of this study was to investigate whether cardiomyocytes parasitism, oxidative stress and contractile dysfunction can be correlated to senescence-associated β-galactosidase (SA-β-Gal) activity in Trypanosoma cruzi-infection in vitro and in vivo. MethodsUninfected, T. cruzi-infected untreated and benznidazole (BZN)-treated H9c2 cardiomyocytes and rats were investigated. Parasitological, prooxidant, antioxidant, microstructural, and senescence-associated markers were quantified in vitro and in vivo. ResultsT. cruzi infection triggered intense cardiomyocytes parasitism in vitro and in vivo, which was accompanied by reactive oxygen species (ROS) upregulation, lipids, proteins and DNA oxidation in cardiomyocytes and cardiac tissue. Oxidative stress was parallel to microstructural cell damage (e.g., increased cardiac toponin I levels) and contractile dysfunction in cardiomyocytes in vitro and in vivo, whose severity accompanied a premature cellular senescence-like phenotype revealed by increased senescence-associated β-galactosidase (SA-β-Gal) activity and DNA oxidation (8-OHdG). Cellular parasitism (e.g., infection rate and parasite load), myocarditis and T. cruzi-induced prooxidant responses were attenuated by early BZN administration to interrupt the progression of T. cruzi infection, protecting against SA-β-gal-based premature cellular senescence, microstructural damage and contractile deterioration in cardiomyocytes from T. cruzi-infected animals. ConclusionOur findings indicated that cell parasitism, redox imbalance and contractile dysfunction were correlated to SA-β-Gal-based cardiomyocytes premature senescence in acute T. cruzi infection. Therefore, in addition to controlling parasitism, inflammation and oxidative stress; inhibiting cardiomyocytes premature senescence should be further investigated as an additional target of specific Chagas disease therapeutics.

Effectiveness of Nitazoxanide and Electrolyzed Oxiding Water in Treating Chagas Disease in a Canine Model.

Chagas disease (CD) is caused by the protozoan Trypanosoma cruzi, and affects seven million people in Latin America. Side effects and the limited efficacy of current treatment have led to new drug research. The objective of this work was to evaluate the effectiveness of nitazoxanide (NTZ) and electrolyzed oxidizing water (EOW) in a canine model of experimental CD. Náhuatl dogs were infected with the T. cruzi H8 strain and NTZ- or EOW-treated orally for 10 days. Seronegativity was shown at 12 months post-infection (mpi) in the NTZ-, EOW-, and benznidazole (BNZ)-treated groups. The NTZ and BNZ groups had high levels of IFN-γ, TNF-α, IL-6, IL-12B, and IL-1β at 1.5 mpi and low levels of IL-10. Electrocardiographic studies showed alterations from 3 mpi and worsening at 12 mpi; NTZ treatment produced fewer cardiac pathomorphological changes compared to EOW, similar to BNZ treatment. There was no cardiomegaly in any group. In conclusion, although NTZ and EOW did not prevent changes in cardiac conductivity, they were able to avoid the severity of heart damage in the chronic phase of CD. NTZ induced a favorable proinflammatory immune response after infection, being a better option than EOW as a possible treatment for CD after BNZ.

Design, synthesis and antitrypanosomatid activity of 2-nitroimidazole-3,5-disubstituted isoxazole compounds based on benznidazole

Chagas disease and leishmaniasis are neglected diseases of high priority as a public health problem. Pharmacotherapy is based on the administration of a few drugs, which exhibit hazardous adverse effects and toxicity to the patients. Thus, the search for new antitrypanosomatid drugs is imperative to overcome the limitations of the treatments. In this work, 46 2-nitroimidazole 3,5-disubstituted isoxazole compounds were synthesized in good yields by [3 + 2] cycloaddition reaction between terminal acetylene (propargyl-2-nitroimidazole) and chloro-oximes. The compounds were non-toxic to LLC-MK2 cells. Compounds 30, 35, and 44 showed in vitro antichagasic activity, 15-fold, 12-fold, and 10-fold, respectively, more active than benznidazole (BZN). Compounds 30, 35, 44, 45, 53, and 61 acted as substrates for the TcNTR enzyme, indicating that this might be one of the mechanisms of action involved in their antiparasitic activity. Piperazine series and 4-monosubstituted compounds were potent against T. cruzi parasites. Besides the in vitro activity observed in compound 45, the in vivo assay showed that the compound only reduced the parasitemia levels by the seventh-day post-infection (77%, p > 0.001) compared to the control group. However, 45 significantly reduced the parasite load in cardiac tissue (p < 0.01) 11 days post-infection. Compounds 49, 52, and 54 showed antileishmanial activity against intracellular amastigotes of Leishmania (L.) amazonensis at the same range as amphotericin B. These findings highlight the antitrypanosomatid properties of 2-nitroimidazole 3,5-disubstituted isoxazole compounds and the possibility in using them as antitrypanosomatid agents in further studies.

Frequency Variation and Dose Modification of Benznidazole Administration for the Treatment of Trypanosoma cruzi Infection in Mice, Dogs, and Nonhuman Primates.

Trypanosoma cruzi naturally infects a broad range of mammalian species and frequently results in the pathology that has been most extensively characterized in human Chagas disease. Currently employed treatment regimens fail to achieve parasitological cure of T. cruzi infection in the majority of cases. In this study, we have extended our previous investigations of more effective, higher dose, intermittent administration protocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice and in naturally infected dogs and nonhuman primates (NHP). Collectively, these studies demonstrate that twice-weekly administration of BNZ for more than 4 months at doses that are ~2.5-fold that of previously used daily dosing protocols, provided the best chance to obtain parasitological cure. Dosing less frequently or for shorter time periods was less dependable in all species. Prior treatment using an ineffective dosing regimen in NHPs did not prevent the attainment of parasitological cure with an intensified BNZ dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed nearly identical susceptibility to BNZ as those obtained prior to treatment, confirming the low risk of induction of drug resistance with BNZ and the ability to adjust the treatment protocol when an initial regimen fails. These results provide guidance for the use of BNZ as an effective treatment for T. cruzi infection and encourage its wider use, minimally in high value dogs and at-risk NHP, but also potentially in humans, until better options are available.

Poly-ε-Caprolactone Implants for Benznidazole Prolonged Release: An Alternative to Chagas Disease Oral Treatment.

Benznidazole (BZ) tablets are the currently prescribed treatment for Chagas disease. However, BZ presents limited efficacy and a prolonged treatment regimen with dose-dependent side effects. The design and development of new BZ subcutaneous (SC) implants based on the biodegradable poly-ɛ-caprolactone (PCL) is proposed in this study for a controlled release of BZ and to improve patient compliance. The BZ-PCL implants were characterized by X-ray diffraction, differential scanning calorimetry, and scanning electron microscopy, which indicated that BZ remains in its crystalline state dispersed in the polymer matrix with no polymorphic transitions. BZ-PCL implants, even at the highest doses, induce no alteration of the levels of hepatic enzymes in treated animals. BZ release from implants to blood was monitored in plasma during and after treatment in healthy and infected animals. Implants at equivalent oral doses increase the body's exposure to BZ in the first days compared with oral therapy, exhibiting a safe profile and allowing sustained BZ concentrations in plasma to induce a cure of all mice in the experimental model of acute infection by the Y strain of T. cruzi. BZ-PCL implants have the same efficacy as 40 daily oral doses of BZ. Biodegradable BZ implants are a promising option to reduce failures related to poor adherence to treatment, with more comfort for patients, and with sustained BZ plasma concentration in the blood. These results are relevant for optimizing human Chagas disease treatment regimens.

Trypanosoma cruzi: Does the intake of nanoencapsulated benznidazole control acute infections?

Chagas Disease (CD) affects around eight million people worldwide. It is considered a neglected disease that presents few treatment options with efficacy only in the acute phase. Nanoparticles have many positive qualities for treating parasite infections and may be effectively and widely employed in clinical medicine. This research aimed to evaluate the nanoencapsulated benznidazole treatment in animals experimentally infected with Trypanosoma cruzi. To analyze the treatment efficacy, we evaluated survival during thirty days, parasitemia, genotoxicity, and heart and liver histopathology. Thirty-five female Swiss mice were organized into seven groups characterizing a dose curve: A – Negative control (uninfected animals), B – Positive control (infected animals), C – Benznidazole (BNZ) 100 mg/kg (infected animals), D – 5 mg/kg Benznidazole nanocapsules (NBNZ) (infected animals), E − 10 mg/kg Benznidazole nanocapsules (infected animals), F – 15 mg/kg Benznidazole nanocapsules (infected animals), G – 20 mg/kg Benznidazole nanocapsules (infected animals). The animals were infected with the Y strain of T. cruzi intraperitoneally. The treatment was administered for eight days by oral gavage. It was possible to observe that the treatment with the highest NBNZ dose presented efficacy similar to the standard benznidazole drug. The 20 mg/kg NBNZ dose was able to reduce parasitemia, increase survival, and drastically reduce heart and liver tissue damage compared to the 100 mg/kg BNZ dose. Moreover, it showed a lower DNA damage index than the BNZ treatment. In conclusion, the nanoencapsulation of BNZ promotes an improvement in parasite proliferation control with a five times smaller dose relative to the standard dose of free BNZ, thus demonstrating to be a potential innovative therapy for CD.

Vaccine-linked chemotherapy improves cardiac structure and function in a mouse model of chronic Chagas disease.

Chagas disease, caused by chronic infection with the protozoan parasite Trypanosoma cruzi, affects 6-7 million people worldwide. The major clinical manifestation of Chagas disease is chronic Chagasic cardiomyopathy (CCC), which encompasses a spectrum of symptoms including arrhythmias, hypertrophy, dilated cardiomyopathy, heart failure, and sudden death. Current treatment is limited to two antiparasitic drugs, benznidazole (BNZ) and nifurtimox, but both have limited efficacy to halt the progression of CCC. We developed a vaccine-linked chemotherapy strategy using our vaccine consisting of recombinant Tc24-C4 protein and a TLR-4 agonist adjuvant in a stable squalene emulsion, in combination with low dose benznidazole treatment. We previously demonstrated in acute infection models that this strategy parasite specific immune responses, and reduced parasite burdens and cardiac pathology. Here, we tested our vaccine-linked chemotherapy strategy in a mouse model of chronic T. cruzi infection to evaluate the effect on cardiac function. Female BALB/c mice infected with 500 blood form T. cruzi H1 strain trypomastigotes were treated beginning 70 days after infection with a low dose of BNZ and either low or high dose of vaccine, in both sequential and concurrent treatments streams. Control mice were untreated, or administered only one treatment. Cardiac health was monitored throughout the course of treatment by echocardiography and electrocardiograms. Approximately 8 months after infection, endpoint histopathology was performed to measure cardiac fibrosis and cellular infiltration. Vaccine-linked chemotherapy improved cardiac function as evidenced by amelioration of altered left ventricular wall thickness, left ventricular diameter, as well as ejection fraction and fractional shortening by approximately 4 months of infection, corresponding to two months after treatment was initiated. At study endpoint, vaccine-linked chemotherapy reduced cardiac cellular infiltration, and induced significantly increased antigen specific IFN-γ and IL-10 release from splenocytes, as well as a trend toward increased IL-17A. These data suggest that vaccine-linked chemotherapy ameliorates changes in cardiac structure and function induced by infection with T. cruzi. Importantly, similar to our acute model, the vaccine-linked chemotherapy strategy induced durable antigen specific immune responses, suggesting the potential for a long lasting protective effect. Future studies will evaluate additional treatments that can further improve cardiac function during chronic infection.

In vitro and in silico analysis of imatinib analogues as anti-Trypanosoma cruzi drug candidates.

AbstractChagas disease (CD) is a neglected tropical disease caused by the intracellular protozoan Trypanosoma cruzi that remains a serious public health issue affecting more than 6 million people worldwide. The available treatment includes 2 nitro derivatives, benznidazole (BZ) and nifurtimox, that lack in efficacy in the later chronic phase and when administered against the several naturally resistant parasite strains and present several side-effects, demanding new therapeutic options. One strategy is based on repurposing by testing drugs already used for other illness that may share similar targets. In this context, our previous data on imatinib (IMB) and derivatives motivated the screening of 8 new IMB analogues. Our findings showed that all except 1 were active against bloodstream trypomastigotes reaching drug concentration capable of inducing a 50% of parasite lysis (EC50) values &lt; 12 μm after 2 h while BZ was inactive. After 24 h, all derivatives were more potent than BZ, exhibiting EC50 values 1.5–5.5 times lower. Against intracellular forms, 7 out of 8 derivatives presented high activity, with EC50 values ≤ BZ. LS2/89 stood out as one of the most promising, reaching EC90 values of 1.68 and 4.9 μm on intracellular and trypomastigote forms, respectively, with the best selectivity index (&gt;60) towards the proliferative forms. Physicochemical parameters as well as the absorption, distribution, metabolism, excretion and toxicity properties were predicted to be acceptable and with good chance of a favourable oral bioavailability. The promising results motivate further studies such as in vivo and combinatory assays aiming to contribute for a novel safer and effective therapy for CD.

Benznidazole treatment decreases IL-6 levels in Trypanosoma cruzi-infected human adipocytes differentiated from adipose tissue-derived stem cells.

Trypanosoma cruzi, which causes Chagas disease (CD), is a versatile haemoparasite that uses several strategies to evade the host's immune response, including adipose tissue (AT), used as a reservoir of infection. As it is an effective barrier to parasite evasion, the effectiveness of the drug recommended for treating CD, Benznidazole (BZ), may be questionable. To this end, we evaluated the parasite load and immunomodulation caused by BZ treatment in the culture of adipocytes differentiated from human adipose tissue-derived stem cells (ADSC) infected with T. cruzi. The ADSC were subjected to adipogenic differentiation. We then carried out four cultures in which we infected the differentiated AT with trypomastigote forms of the Y strain of T. cruzi and treated them with BZ. After the incubation, the infected AT was subjected to quantitative polymerase chain reaction (qPCR) to quantify the parasite load and transmission electron microscopy (TEM) to verify the infection. The supernatant was collected to measure cytokines, chemokines, and adipokines. We found elevated secretion of IL-6, CXCL-10/IP-10, CCL2/MCP-1, CCL5/RANTES, and leptin in infected fat cells. However, treatment with BZ promoted a decrease in IL-6. Therefore, we believe that BZ has a beneficial role as it reduces inflammation in infected fat cells.

Silver and copper-benznidazole derivatives as potential antiparasitic metallodrugs: Synthesis, characterization, and biological evaluation

Currently the only drug available to treat Chagas disease in Brazil is benznidazole (BZN). Therefore, there is an urgent need to discover and develop new anti- Trypanosoma cruzi candidates. In our continuous effort to enhance clinical antiparasitic drugs using synergistic strategy, BZN was coordinated to silver and copper ions to enhance its effectiveness to treat that illness. In this work, the syntheses of four novel metal-BZN complexes, [Ag(BZN)2]NO3·H2O (1), [CuCl2(BZN)(H2O)]·1/2CH3CN (2), [Ag(PPh3)2(BZN)2]NO3·H2O (3), and [Cu(PPh3)2(BNZ)2]NO3·2H2O (4), and their characterization using multiple analytical and spectroscopic techniques such as Infrared (FTIR), Nuclear Magnetic Resonance (1H, 13C, 31P), UV–Visible (UV–Vis), Electron Paramagnetic Resonance (EPR), conductivity and elemental analysis are described. IC50 (Half-maximal inhibitory concentration) values of Ag-BZN compounds are about five to ten times lower than benznidazole itself in both proliferation stages of the parasite (epimastigotes and amastigotes). The cytotoxicity of both compounds in human cells (fibroblasts and hepatocytes) are comparable to BZN, indicating that Ag-BZN complexes can be more selective than BZN.