Amlodipine Increases the Therapeutic Potential of Ravuconazole upon Trypanosoma cruzi Infection.

Mining existing agents that enhance the therapeutic potential of ergosterol biosynthesis inhibitors (EBI) is a promising approach to improve Chagas disease chemotherapy. In this study, we evaluated the effect of ravuconazole, an EBI, combined with amlodipine, a calcium channel blocker, upon Trypanosoma cruzi experimental infection. In vitro assays confirmed the trypanocidal activity of both compounds in monotherapy and demonstrated an additive effect (sum of the fractional inhibitory concentration [ΣFIC] > 0.5) of the combined treatment without additional toxicity to host cells. In vivo experiments, using a murine model of the T. cruzi Y strain in a short-term protocol, demonstrated that amlodipine, although lacking trypanocidal activity, dramatically increased the antiparasitic activity of underdosing ravuconazole regimens. Additional analysis using long-term treatment (20 days) showed that parasitemia relapse until 60 days after treatment was significatively lower in mice treated with the combination (4 out of 14 mice) than ravuconazole monotherapy (10 out of 14 mice), even in the presence of immunosuppressant pressure. Furthermore, the combined therapy was well tolerated and protected the mice from mortality. The treatments also impacted on the cellular and humoral immune response of infected animals, inducing a reduction of serum cytokine levels in all ravuconazole-treated mice. Our findings demonstrate that amlodipine is efficacious in enhancing the antiparasitic activity of ravuconazole in an experimental model of T. cruzi infection and indicates a potential strategy to be explored in Chagas disease treatment.

Parasitaemia and parasitic load are limited targets of the aetiological treatment to control the progression of cardiac fibrosis and chronic cardiomyopathy in Trypanosoma cruzi-infected dogs.

It is still unclear whether the progression of acute to chronic Chagas cardiomyopathy is predominantly associated with the limited efficacy of aetiological chemotherapy, or with the pharmacological resistance profiles and pathogenicity of specific Trypanosoma cruzi strains. Thus, we tested the hypothesis that parasitic load could be a limited target of aetiological chemotherapy to prevent chronic cardiomyopathy in dogs infected by different T. cruzi strains. Animals were infected with benznidazole-susceptible (Berenice-78) and -resistant (VL-10 and AAS) strains of T. cruzi. A quantitative real-time PCR strategy was developed to comparatively quantify the parasite load of the three different strains using a single standard curve. For dogs infected with the VL-10 strain, benznidazole treatment reduced cardiac parasitism during the acute phase of infection. However, similar parasite load and collagen deposition were detected in the myocardium of treated and untreated animals in the chronic phase of the infection. In animals infected with the AAS strain, benznidazole reduced parasite load, myocarditis and type III collagen deposition in the acute phase. However, increased type III collagen deposition was verified in the chronic phase. Dogs infected with the Berenice-78 strain showed a parasitological cure and no evidence of myocardial fibrosis. Parasitic load and cardiac fibrosis presented no correlation in acute or chronic phases of T. cruzi infection. Our findings in a canine model of Chagas disease suggest that parasite burden is a limited predictor for disease progression after treatment and show that benznidazole, although not inducing parasitological cure, is able to prevent total fibrosis in the early stages of infection, as well as complete prevention of cardiac damage when it eliminates parasites at the onset of infection.

Outcome of E1224-Benznidazole Combination Treatment for Infection with a Multidrug-Resistant Trypanosoma cruzi Strain in Mice.

Combination therapy has been proposed as an alternative therapeutic approach for the treatment of Chagas disease. In this study, we evaluated the effect of treatment with benznidazole combined with E1224 (ravuconazole prodrug) in an experimental murine model of acute infection. The first set of experiments assessed the range of E1224 doses required to induce parasitological cure using Trypanosoma cruzi strains with different susceptibilities to benznidazole (Y and Colombian). All E1224 doses were effective in suppressing the parasitemia and preventing death; however, parasitological cure was observed only in mice infected with Y strain. Considering these results, we evaluated the effect of combined treatment against Colombian, a multidrug-resistant T. cruzi strain. After exclusion of antagonistic effects using in vitro assays, infected mice were treated with E1224 and benznidazole in monotherapy or in combination at day 4 or 10 postinoculation. All treatments were well tolerated and effective in suppressing parasitemia; however, parasitological and PCR assays indicated no cure among mice treated with monotherapies. Intriguingly, the outcome of combination therapy was dependent on treatment onset. Early treatment using optimal doses of E1224-benznidazole induced a 100% cure rate, but this association could not eliminate a well-established infection. The beneficial effect of combination therapy was evidenced by further reductions of the patent parasitemia period in the group receiving combined therapy compared with monotherapies. Our results demonstrated a positive interaction between E1224 and benznidazole against murine T. cruzi infection using a multidrug-resistant strain and highlighted the importance of a stringent experimental model in the evaluation of new therapies.

Myocarditis in different experimental models infected by Trypanosoma cruzi is correlated with the production of IgG1 isotype.

This study was designed to verify the relationship between IgG antibodies isotypes and myocarditis in Trypanosoma cruzi infection using mice and dogs infected with different T. cruzi strains. The animals were infected with benznidazole-susceptible Berenice-78 and benznidazole-resistant AAS and VL-10 strains. The IgG subtypes were measured in serum samples from dogs (IgG, IgG1, and IgG2) and mice (IgG, IgG1, IgG2a, and IgG2b). The infection of dogs with VL-10 strain induced the highest levels of heart inflammation while intermediate and lower levels were detected with Berenice-78 and AAS strains, respectively. Similar results were found in mice infected with VL-10, but not in those infected with AAS or Berenice-78 strains. The AAS strain induced higher levels of heart inflammation in mice, while Berenice-78 strain was not able to induce it. Correlation analysis between myocarditis and antibody reactivity index revealed very interesting results, mainly for IgG and IgG1, the latter being the most exciting. High IgG1 showed a significant correlation with myocarditis in both experimental models, being more significant in dogs (r=0.94, p<0.0001) than in mice (r=0.58, p=0.047). Overall, our data suggest that IgG1 could be a good marker to demonstrate myocarditis intensity in Chagas disease.

Mir-190b negatively contributes to the Trypanosoma cruzi-infected cell survival by repressing PTEN protein expression.

Chagas disease, which is caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.

Mir-190b negatively contributes to the Trypanosoma cruzi- infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoanTrypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.

Therapeutic responses to different anti-Trypanosoma cruzi drugs in experimental infection by benznidazole-resistant parasite stock.

SUMMARY This study describes the role of parasite clearance time induced by benznidazole, fexinidazole and posaconazole treatments upon mice infection with a benznidazole-resistant Trypanosoma cruzi strain in the pathological outcomes. Trypanosoma cruzi-infected mice were treated with different drugs and parasite clearance time was detected by blood and tissue qPCR, to determine the dynamic relationship between the efficacy of the treatments and the intensity of heart lesion/serum inflammatory mediators. Our results indicate that anti-T. cruzi treatments were able to reduce parasite replication and consequently induce immunomodulatory effects, where the degree of the immunopathology prevention was related to the time of parasite clearance induced by different treatments. Nevertheless, in benznidazole and posaconazole treatments, parasite rebounding was detected with parasitism reaching levels similar to infected and non-treated mice; the time for parasitic rebound being earlier among benznidazole-treated mice. In parallel, an increase of cardiac lesions and plasma chemokine levels was also detected and was more accentuated in benznidazole-treated animals. Interestingly, in the presence of parasitological cure (fexinidazole treatment), basal levels of these inflammatory mediators were evidenced as well as an absence of cardiac inflammation or fibrosis. Overall, our data indicate that all treatments have positive effects on the clinical evolution of T. cruzi infection, with success in preventing cardiac alterations being drug-dependent.

Therapeutical approaches under investigation for treatment of Chagas disease.

INTRODUCTION: A century after its discovery, American trypanosomiasis or Chagas disease remains a serious health problem in Latin America, where it affects around 7 - 8 million people. The prevalence of Chagas disease in the poorer parts of the world has meant that it has largely been neglected with limited progress that made in identifying new drugs for the treatment. The nitroheterocyclic drugs nifurtimox and benznidazole are first-line drugs available for Chagas disease with limitations that include variable efficacy, long treatment courses and toxicity. AREAS COVERED: This review focuses on different therapeutic strategies that have been used for the discovery of new treatments for Chagas disease. These include combination chemotherapy, drug repositioning, re-dosing regimens for current drugs and the identification of new drugs with specified target profiles. EXPERT OPINION: There are currently several reasons for a more optimistic view about chemotherapy with Chagas disease. However, despite some progress being made in preclinical studies, there is yet to be an ideal drug or formulation for human treatment. One major drawback in the evaluation of potential Chagas disease therapeutics is the lack of tools available to perform the said evaluation. Indeed, there is a great need to discover a better biomarker that could determine the efficacy of potential chemotherapeutics in treated patients.

Benznidazole and posaconazole in experimental Chagas disease: positive interaction in concomitant and sequential treatments.

BACKGROUND: Current chemotherapy for Chagas disease is unsatisfactory due to its limited efficacy, particularly in the chronic phase, with frequent side effects that can lead to treatment discontinuation. Combined therapy is envisioned as an ideal approach since it may improve treatment efficacy whilst decreasing toxicity and the likelihood of resistance development. We evaluated the efficacy of posaconazole in combination with benznidazole on Trypanosoma cruzi infection in vivo. METHODS AND FINDINGS: Benznidazole and posaconazole were administered individually or in combination in an experimental acute murine infection model. Using a rapid treatment protocol for 7 days, the combined treatments were more efficacious in reducing parasitemia levels than the drugs given alone, with the effects most evident in combinations of sub-optimal doses of the drugs. Subsequently, the curative action of these drug combinations was investigated, using the same infection model and 25, 50, 75 or 100 mg/kg/day (mpk) of benznidazole in combination with 5, 10 or 20 mpk of posaconazole, given alone or concomitantly for 20 days. The effects of the combination treatments on parasitological cures were higher than the sum of such effects when the drugs were administered separately at the same doses, indicating synergistic activity. Finally, sequential therapy experiments were carried out with benznidazole or posaconazole over a short interval (10 days), followed by the second drug administered for the same period of time. It was found that the sequence of benznidazole (100 mpk) followed by posaconazole (20 mpk) provided cure rates comparable to those obtained with the full (20 days) treatments with either drug alone, and no cure was observed for the short treatments with drugs given alone. CONCLUSIONS: Our data demonstrate the importance of investigating the potential beneficial effects of combination treatments with marketed compounds, and showed that combinations of benznidazole with posaconazole have a positive interaction in murine models of Chagas disease.

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.

Real-time PCR strategy for parasite quantification in blood and tissue samples of experimental Trypanosoma cruzi infection.

The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (p<0.05) reduction of parasitism after 60 days of infection. Later, this methodology was used to monitor the evolution of infection in animals treated with itraconazole (Itz). Itz-treatment induced a reduction of parasite load in both blood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our data show that real-time PCR strategy used was suitable for studies of murine T. cruzi infection and may prove useful in investigations involving experimental chemotherapy of the disease and the benefits of treatment in relation to parasitism and inflammatory response.

Effects of ravuconazole treatment on parasite load and immune response in dogs experimentally infected with Trypanosoma cruzi.

In this work, we investigated the in vivo activity of ravuconazole against the Y and Berenice-78 Trypanosoma cruzi strains using acutely infected dogs as hosts. Ravuconazole was well tolerated, as no significant side effects were observed during the treatment using 6.0 mg/kg twice a day (12 mg/kg/day) for up to 90 days. In all treated animals, parasitemia was permanently suppressed by the first day of treatment, independently of the parasite strain. Cultures of blood obtained posttreatment were negative for 90% of the animals, confirming that the drug induced a marked reduction in the parasite load. The results of PCR tests for T. cruzi in blood performed 1 month posttreatment were consistently negative for three of five and two of five animals infected with the Y and Berenice-78 strains, respectively. All ravuconazole-treated dogs consistently had negative serological test results during and until 30 days after treatment, regardless of the therapeutic scheme used. However, after the end of treatment, an increase in specific antibody levels was observed in all treated animals, although the antibody levels were always significantly lower than those of the nontreated control dogs. Despite being unable to induce a parasitological cure, ravuconazole treatment led to significant reductions in the levels of gamma interferon expression and lesions in cardiac tissues in animals infected with the Y strain, while the level of interleukin-10 mRNA expression increased. We conclude that ravuconazole has potent suppressive but not curative activity in the canine model of acute Chagas' disease, probably due to its unfavorable pharmacokinetic properties (half-life, 8.8 h). The longer half-life of ravuconazole in humans (4 to 8 days) makes it a promising drug for assessment for use as chemotherapy in human Chagas' disease.