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Item Amlodipine increases the therapeutic potential of ravuconazole upon Trypanosoma cruzi infection.(2020) Machado, Yara Almeida; Bahia, Maria Terezinha; Caldas, Ivo Santana; Mazzeti, Ana Lia; Novaes, Rômulo Dias; Vilas Boas, Breno Raimundo; Santos, Lorena Júnia de Souza; Martins Filho, Olindo Assis; Marques, Marcos José; Diniz, Lívia de FigueiredoMining 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 [FIC] 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.Item Antitrypanosomal activity of fexinidazole metabolites, potential new drug candidates for Chagas disease.(2014) Bahia, Maria Terezinha; Nascimento, Álvaro Fernando da Silva do; Mazzeti, Ana Lia; Marques, Luiz F.; Gonçalves, Karolina Ribeiro; Mota, Ludmilla Walter Reis; Diniz, Lívia de Figueiredo; Caldas, Ivo Santana; Silva, André Talvani Pedrosa da; Shackleford, David M.; Koltun, Maria; Saunders, Jessica; White, Karen L.; Scandale, Ivan; Charman, Susan A.; Chatelain, EricThis study was designed to verify the in vivo efficacy of sulfoxide and sulfone fexinidazole metabolites following oral administration in a murine model of Chagas disease. Female Swiss mice infected with the Y strain of Trypanosoma cruzi were treated orally once per day with each metabolite at doses of 10 to 100 mg/kg of body weight for a period of 20 days. Parasitemia was monitored throughout, and cures were detected by parasitological and PCR assays. The results were compared with those achieved with benznidazole treatment at the same doses. Fexinidazole metabolites were effective in reducing the numbers of circulating parasites and protecting mice against death, compared with untreated mice, but without providing cures at daily doses of 10 and 25 mg/kg. Both metabolites were effective in curing mice at 50 mg/kg/day (30% to 40%) and 100 mg/kg/day (100%). In the benznidazole- treated group, parasitological cure was detected only in animals treated with the higher dose of 100 mg/kg/day (80%). Single-dose pharmacokinetic parameters for each metabolite were obtained from a parallel group of uninfected mice and were used to estimate the profiles following repeated doses. Pharmacokinetic data suggested that biological efficacy most likely resides with the sulfone metabolite (or subsequent reactive metabolites formed following reduction of the nitro group) following administration of either the sulfoxide or the sulfone and that prolonged plasma exposure over the 24-h dosing window is required to achieve high cure rates. Fexinidazole metabolites were effective in treating T. cruzi in a mouse model of acute infection, with cure rates superior to those achieved with either fexinidazole itself or benznidazole.Item Benznidazol em combinação com alopurinol : aumento da atividade anti-Trypanosoma cruzi in vitro e in vivo.(2014) Mazzeti, Ana Lia; Bahia, Maria Terezinha; Diniz, Lívia de FigueiredoO tratamento da doença de Chagas apresenta limitações relacionadas à sua eficácia variável, particularmente na fase crônica, e aos efeitos adversos frequentes, que podem levar à interrupção do tratamento. A terapia de combinação de fármaco parece ser uma estratégia ideal, já que pode contribuir para o aumento da eficácia do tratamento, diminuição da toxicidade e da probabilidade de desenvolvimento de resistência. No presente estudo foi avaliada a atividade do alopurinol (Al) em combinação com o benznidazol (Bz) na infecção experimental por T. cruzi. Inicialmente, foi verificada a atividade da combinação Bz/Al sobre a infecção de células H9c2 infectadas pela cepa Y de T. cruzi, incubando-as com diferentes concentrações de cada composto isoladamente ou em combinação. Foi utilizado, in vitro, o método de diluição “tabuleiro de dama” (checkerboard) e os resultados foram analisados utilizando índice de combinação (CI) calculado pelo software Calcusyn. A avaliação global dos resultados demonstrou que os CI variaram no intervalo de 0,62 a 1,76, o que sugeriu um efeito aditivo resultante da associação in vitro dos compostos avaliados. Posteriormente, foi realizada a avaliação in vivo da combinação Bz/Al. Para isso, camundongos Swiss fêmeas foram inoculados com 5000 formas tripomastigotas sanguíneos da cepa Y de T. cruzi. O tratamento foi administrado por via oral durante 20 dias consecutivos, iniciado quatro dias após a inoculação. Os animais foram tratados com Bz (100, 75, 50 e 25 mg/kg) ou Al (90, 60 e 30 mg/kg) isoladamente ou em combinação (Bz/Al: 75/90 75/60 75/30, 50/90 , 50/60, 50/30, 25/90 , 25/60 e 25/30 mg/kg ). Os seguintes parâmetros foram utilizados para determinação da cura parasitológica: exame de sangue a fresco durante e até 60 dias após o tratamento e ensaio de PCR em tempo real de amostras de sangue (30 e 180 dias após o tratamento). A análise dos níveis de parasitemia após o tratamento com Bz mostrou um efeito tripanocida dose-dependente. Entretanto, mesmo havendo uma redução significativa do nível de parasitemia após o tratamento, a cura parasitológica foi identificada apenas nos animais tratados com 75 e 100mg de Bz por kg de peso corporal (20 e 70 % de cura, respectivamente). De forma diferente, não foi verificada efeito dose-dependente resultante do tratamento com Al. Além disso, não foi observada redução da parasitemia nem cura entre os animais que receberam diferentes doses desse fármaco. Posteriormente, foi avaliado o efeito das combinações entre Bz/Al, in vivo. O efeito benéfico das combinações dos fármacos foi demonstrado pela redução significativa da parasitemia detectada em todos os animais que receberam a terapia de combinação. Além disso, foi observado um aumento no índice de cura entre os animais tratados com os fármacos em combinação, uma vez que a administração de Bz-75 mg/kg + Al-90 mg/kg ou Bz-75 mg/kg + Al-60 mg/kg induziu 100 % de cura, e a combinação de Bz-75 mg/kg + Al-30 mg/kg ou Bz-50 mg/kg + Al-60 mg/kg induziu, respectivamente, 80 e 20 % de cura. Em conclusão, a combinação de Bz e Al apresentou um efeito benéfico, uma vez que foi identificada cura parasitológica em uma proporção maior de animais tratados com os medicamentos em associação do que naqueles que receberam as mesmas doses de cada fármaco em monoterapia. Nossos resultados reforçam a importância da identificação de combinações de compostos já comercializados que apresentem uma interação positiva no tratamento da infecção por T. cruzi. Esta abordagem pode contribuir para otimizar os custos e tempo investidas na pesquisa relacionada à toxicidade e a biodisponibilidade de novas drogas para o consumo humano.Item Benznidazole and Posaconazole in experimental Chagas disease: positive interaction in concomitant and sequential treatments.(2013) Diniz, Lívia de Figueiredo; Urbina, Julio Alberto; Andrade, Isabel Mayer de; Mazzeti, Ana Lia; Martins, Tassiane Assíria Fontes; Caldas, Ivo Santana; Silva, André Talvani Pedrosa da; Ribeiro, Isabela; Bahia, Maria TerezinhaBackground: 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.Item Benznidazole self-emulsifying delivery system : a novel alternative dosage form for Chagas disease treatment.(2020) Mazzeti, Ana Lia; Oliveira, Liliam Teixeira; Gonçalves, Karolina Ribeiro; Schaun, Géssica C.; Mosqueira, Vanessa Carla Furtado; Bahia, Maria TerezinhaBenznidazole (BZ) tablets are a unique form of treatment available for treating Chagas disease. Development of a liquid formulation containing BZ easy to administer orally for the treatment of paediatric patients, particularly for newborns is urgently required, with the same efficacy, safety and suitable biopharmaceutical properties as BZ tablets. Self-emulsifying drug delivery systems (SEDDS) may improve bioavailability of drugs such as BZ, which have poor water solubility and low permeability. In this context, the aim of this work was to develop a liquid BZ-SEDDS formulation as an alternative to tablets and to evaluate its cytotoxicity in different host cell lines and its efficacy in experimental Trypanosoma cruzi infection in mice. The optimized SEDDS formulation (25 mg/ml of BZ) induced no cytotoxicity in H9c2, HepG2 and Caco2 cells in vitro at 25 μM level. BZ-SEDDS and free-BZ showed similar in vitro trypanocidal activity in H9c2 cells infected by T. cruzi Y strain, with IC50 values of 2.10 ± 0.41 μM and 1.29 ± 0.01 μM for BZ and BZ-SEDDS, respectively. A follow up of efficacy in an acute model of infected mice resulted in the same percentage of cure (57%) for both free-BZ and BZ-SEDDS- groups according to established parameters. Furthermore, no additional in vivo toxicity was observed in animals treated with BZ-SEDDS. Taken together, in vitro and in vivo data of BZ-SEDDS showed that the incorporation of BZ into SEDDS does not alter its potency, efficacy and safety. Thus, BZ-SEDDS can be a more practical and personalized orally administered liquid dosage form compared to suspension of crushed BZ-tablets to treat newborn and young children by emulsifying SEDDS in different aqueous liquids with advantage of dosing flexibility.Item Benznidazole/Itraconazole combination treatment enhances anti-Trypanosoma cruzi activity in experimental Chagas disease.(2015) Martins, Tassiane Assíria Fontes; Diniz, Lívia de Figueiredo; Mazzeti, Ana Lia; Nascimento, Álvaro Fernando da Silva do; Caldas, Sérgio; Caldas, Ivo Santana; Andrade, Isabel Mayer de; Ribeiro, Isabela; Bahia, Maria TerezinhaThe nitroheterocyclic drugs nifurtimox and benznidazole are first-line drugs available to treat Chagas disease; however, they have limitations, including long treatment courses and toxicity. Strategies to overcome these limitations include the identification of new drugs with specific target profiles, re-dosing regimens for the current drugs, drug repositioning and combination therapy. In this work, we evaluated combination therapy as an approach for optimization of the current therapeutic regimen for Chagas disease. The curative action of benznidazole/itraconazole combinations was explored in an established infection of the mice model with the T. cruzi Y strain. The activities of the benznidazole/itraconazole combinations were compared with the results from those receiving the same dosage of each individual drug. The administration of benznidazole/itraconazole in combination eliminated parasites from the blood more efficiently than each drug alone. Here, there was a significant reduction of the number of treatment days (number of doses) necessary to induce parasitemia suppression with the benznidazole/itraconazole combination, as compared to each compound administered alone. These results clearly indicate the enhanced effects of these drugs in combination, particularly at the dose of 75 mg/kg, as the effects observed with the drug combinations were four times more effective than those of each drug used alone. Moreover, benznidazole/itraconazole treatment was shown to prevent or decrease the typical lesions associated with chronic experimental Chagas disease, as illustrated by similar levels of inflammatory cells and fibrosis in the cardiac muscle tissue of healthy and treated mice. These results emphasize the importance of exploring the potential of combination treatments with currently available compounds to specifically treat Chagas disease.Item Chagas cardiomyopathy : the potential effect of benznidazole treatment on diastolic dysfunction and cardiac damage in dogs chronically infected with Trypanosoma cruzi.(2016) Santos, Fabiane Matos dos; Mazzeti, Ana Lia; Caldas, Sérgio; Gonçalves, Karolina Ribeiro; Lima, Wanderson Geraldo de; Torres, Rosália Morais; Bahia, Maria TerezinhaCardiac involvement represents the main cause of mortality among patients with Chagas disease, and therelevance of trypanocidal treatment to improving diastolic dysfunction is still doubtful. In the presentstudy, we used a canine model infected with the benznidazole-sensitive Berenice-78 Trypanosoma cruzistrain to verify the efficacy of an etiologic treatment in reducing the parasite load and ameliorating cardiacmuscle tissue damage and left ventricular diastolic dysfunction in the chronic phase of the infection. Theeffect of the treatment on reducing the parasite load was monitored by blood PCR and blood cultureassays, and the effect of the treatment on the outcome of heart tissue damage and on diastolic function wasevaluated by histopathology and echo Doppler cardiogram. The benefit of the benznidazole-treatmentin reducing the parasite burden was demonstrated by a marked decrease in positive blood culture andPCR assay results until 30 days post-treatment. At this time, the PCR and blood culture assays yieldednegative results for 82% of the treated animals, compared with only 36% of the untreated dogs. However,a progressive increase in the parasite load could be detected in the peripheral blood for one year post-treatment, as evidenced by a progressive increase in positive results for both the PCR and the bloodculture assays at follow-up. The parasite load reduction induced by treatment was compatible with thelower degree of tissue damage among animals euthanized in the first month after treatment and withthe increased cardiac damage after this period, reaching levels similar to those in untreated animals atthe one-year follow-up. The two infected groups also presented similar, significantly smaller values forearly tissue septal velocity (E’ SIV) than the non-infected dogs did at this later time. Moreover, in thetreated animals, an increase in the E/E’ septal tissue filling pressure ratio was observed when comparedwith basal values as well as with values in non-infected dogs. These findings strongly suggest that thetemporary reduction in the parasite load that was induced by benznidazole treatment was not able toprevent myocardial lesions and diastolic dysfunction for long after treatment.Item Chagas cardiomyopathy : the potential effect of benznidazole treatment on diastolic dysfunction and cardiac damage in dogschronically infected with Trypanosoma cruzi.(2016) Santos, Fabiane Matos dos; Mazzeti, Ana Lia; Caldas, Sérgio; Gonçalves, Karolina Ribeiro; Lima, Wanderson Geraldo de; Torres, Rosália Morais; Bahia, Maria TerezinhaCardiac involvement represents the main cause of mortality among patients with Chagas disease, and therelevance of trypanocidal treatment to improving diastolic dysfunction is still doubtful. In the presentstudy, we used a canine model infected with the benznidazole-sensitive Berenice-78 Trypanosoma cruzistrain to verify the efficacy of an etiologic treatment in reducing the parasite load and ameliorating cardiacmuscle tissue damage and left ventricular diastolic dysfunction in the chronic phase of the infection. Theeffect of the treatment on reducing the parasite load was monitored by blood PCR and blood cultureassays, and the effect of the treatment on the outcome of heart tissue damage and on diastolic function wasevaluated by histopathology and echo Doppler cardiogram. The benefit of the benznidazole-treatmentin reducing the parasite burden was demonstrated by a marked decrease in positive blood culture andPCR assay results until 30 days post-treatment. At this time, the PCR and blood culture assays yieldednegative results for 82% of the treated animals, compared with only 36% of the untreated dogs. However,a progressive increase in the parasite load could be detected in the peripheral blood for one year post-treatment, as evidenced by a progressive increase in positive results for both the PCR and the bloodculture assays at follow-up. The parasite load reduction induced by treatment was compatible with thelower degree of tissue damage among animals euthanized in the first month after treatment and withthe increased cardiac damage after this period, reaching levels similar to those in untreated animals atthe one-year follow-up. The two infected groups also presented similar, significantly smaller values forearly tissue septal velocity (E’ SIV) than the non-infected dogs did at this later time. Moreover, in thetreated animals, an increase in the E/E’ septal tissue filling pressure ratio was observed when comparedwith basal values as well as with values in non-infected dogs. These findings strongly suggest that thetemporary reduction in the parasite load that was induced by benznidazole treatment was not able toprevent myocardial lesions and diastolic dysfunction for long after treatment.Item Combination therapy using nitro compounds improves the efficacy of experimental Chagas disease treatment.(2021) Mazzeti, Ana Lia; Gonçalves, Karolina Ribeiro; Mota, Suianne Letícia Antunes; Pereira, Dario Elias; Diniz, Lívia de Figueiredo; Bahia, Maria TerezinhaDrug combinations have been evaluated for Chagas disease in an attempt to improve efficacy and safety. In this line, the objective of this work is to assess the effects of treatment with nitro drugs combinations using benznidazole (BZ) or nifurtimox (NFX) plus the sulfone metabolite of fexinidazole (fex-SFN) in vitro and in vivo on Trypanosoma cruzi infection. The in vitro interaction of fex-SFN and BZ or NFX against infected H9c2 cells by the Y strain was classi- fied as an additive (0.5⩾ΣFIC<4), suggesting the possibility of a dose reduction in the in vivo T. cruzi infection. Next, the effect of combining suboptimal doses was assessed in an acute model of murine T. cruzi infection. Drug combinations led to a faster suppression of parasit- emia than monotherapies. Also, the associations led to higher cure levels than those in the reference treatment BZ 100 mg day−1 (57.1%) (i.e. 83.3% with BZ/fex-SFN and 75% with NFX/fex-SFN). Importantly, toxic effects resulting from the associations were not observed, according to weight gain and hepatic enzyme levels in the serum of experimental animals. Taken together, this study is a starting point to explore the potential effects of nitro drugs combinations in preclinical models of kinetoplastid-related infections.Item Higher oral efficacy of ravuconazole in self-nanoemulsifying systems in shorter treatment in experimental chagas disease.(2021) Spósito, Pollyanna Álvaro; Mazzeti, Ana Lia; Castro, Kelly Christyne Miranda Pereira de; Mendes, Priscila Fagundes; Urbina, Julio Alberto; Bahia, Maria Terezinha; Mosqueira, Vanessa Carla FurtadoWe investigated the in vitro activity and selectivity, and in vivo efficacy of ravuconazole (RAV) in self- nanoemulsifying delivery system (SNEDDS) against Trypanosoma cruzi. Novel formulations of this poorly solu- ble C14-α-demethylase inhibitor may improve its efficacy in the experimental treatment. In vitro activity was determined in infected cardiomyocytes and efficacy in vivo evaluated in terms of parasitological cure induced in Y and Colombian strains of T. cruzi-infected mice. In vitro RAV-SNEDDS exhibited significantly higher potency of 1.9-fold at the IC50 level and 2-fold at IC90 level than free-RAV. No difference in activity with Colombian strain was observed in vitro. Oral treatment with a daily dose of 20 mg/kg for 30 days resulted in 70% of cure for RAV- SNEDDS versus 40% for free-RAV and 50% for 100 mg/kg benznidazole in acute infection (T. cruzi Y strain). Long-term treatment efficacy (40 days) was able to cure 100% of Y strain-infected animals with both RAV preparations. Longer treatment time was also efficient to increase the cure rate with benznidazole (Y and Colombian strains). RAV-SNEDDS shows greater efficacy in a shorter time treatment regimen, it is safe and could be a promising formulation to be evaluated in other pre-clinical models to treat T. cruzi and fungi infections.Item Neuronal parasitism, early myenteric neurons depopulation and continuous axonal networking damage as underlying mechanisms of the experimental intestinal Chagas' disease.(2020) Ricci, Mayra Fernanda; Béla, Samantha Ribeiro; Moraes, Michele Macedo; Bahia, Maria Terezinha; Mazzeti, Ana Lia; Oliveira, Anny Carolline Silva; Oliveira, Luciana Souza de; Radi, Rafael; Piacenza, Lucía; Arantes, Rosa Maria EstevesThere is a growing consensus that the balance between the persistence of infection and the host immune response is crucial for chronification of Chagas heart disease. Extrapolation for chagasic megacolon is hampered because research in humans and animal models that reproduce intestinal pathology is lacking. The parasite-host relationship and its consequence to the disease are not well-known. Our model describes the temporal changes in the mice intestine wall throughout the infection, parasitism, and the development of megacolon. It also presents the consequence of the infection of primary myenteric neurons in culture with Trypanosoma cruzi (T. cruzi). Oxidative neuronal damage, involving reactive nitrogen species induced by parasite infection and cytokine production, results in the denervation of the myenteric ganglia in the acute phase. The long-term inflammation induced by the parasite’s DNA causes intramuscular axonal damage, smooth muscle hypertrophy, and inconsistent innervation, affecting contractility. Acute phase neuronal loss may be irreversible. However, the dynamics of the damages revealed herein indicate that neuroprotection interventions in acute and chronic phases may help to eradicate the parasite and control the inflammatory-induced increase of the intestinal wall thickness and axonal loss. Our model is a powerful approach to integrate the acute and chronic events triggered by T. cruzi, leading to megacolon.Item Nitrotriazole-based compounds as antichagasic agents in a long-treatmenti In vivo assay.(2017) Papadopoulou, Maria V.; Bloomer, William D.; Rosenzweig, Howard S.; Mazzeti, Ana Lia; Gonçalves, Karolina Ribeiro; Mendes, Priscila Fagundes; Bahia, Maria Terezinha3-Nitrotriazole-based compounds belonging to various chemical subclasses were found to be very effective against Chagas disease both in vitro and in vivo after a short administration schedule. In this study, five compounds with specific characteristics were selected to be administered for longer periods of time to mice infected with the virulent Trypanosoma cruzi Y strain to further evaluate their effectiveness as antichagasic agents and whether or not potential adverse effects occur. Benznidazole was included for comparison purposes. Complete parasitemia depletion, weight gain, 100% survival, and a lack of myocardial inflammation were observed with four of the compounds and benznidazole administered intraperitoneally at 15 or 20 mg/kg of body weight/day for 40 days. There was a significant reduction in the number of treatment days (number of doses) necessary to induce parasitemia suppression with all four compounds compared to that required with benznidazole. Partial cures were obtained with only one compound tested at 15 mg/kg/day and on the schedule mentioned above but not with benznidazole. Taken together, our data suggest that these compounds demonstrate potent trypanocidal activity comparable to or better than that of the reference drug, benznidazole, when they are administered at the same dose and on the same schedule.Item Outcome of E1224-Benznidazole combination treatment for infection with a multidrug-resistant Trypanosoma cruzi strain in mice.(2018) Diniz, Lívia de Figueiredo; Mazzeti, Ana Lia; Caldas, Ivo Santana; Ribeiro, Isabela; Bahia, Maria TerezinhaCombination 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.Item A potential role of cholinergic dysfunction on Impaired Colon Motility in experimental intestinal Chagas disease.(2022) Ricci, Mayra Fernanda; Béla, Samantha Ribeiro; Barbosa, Joana Lobato; Moraes, Michele Macedo; Mazzeti, Ana Lia; Bahia, Maria Terezinha; Horta, Laila Sampaio; Santiago, Helton da Costa; Cruz, Jader dos Santos; Capettini, Luciano dos Santos Aggum; Arantes, Rosa Maria EstevesBackground/Aims Chagasic megacolon is caused by Trypanosoma cruzi, which promotes in several cases, irreversible segmental colonic dilation. This alteration is the major anatomic-clinical disorder, characterized by the enteric nervous system and muscle wall structural damage. Herein, we investigate how T. cruzi-induced progressive colonic structural changes modulate the colonic contractile pattern activity. Methods We developed a murine model of T. cruzi-infection that reproduced long-term modifications of the enlarged colon. We evaluated colonic and total intestinal transit time in animals. The patterns of motor response at several time intervals between the acute and chronic phases were evaluated using the organ bath assays. Enteric motor neurons were stimulated by electric field stimulation. The responses were analyzed in the presence of the nicotinic and muscarinic acetylcholine receptor antagonists. Western blot was performed to evaluate the expression of nicotinic and muscarinic receptors. The neurotransmitter expression was analyzed by real-time polymerase chain reaction. Results In the chronic phase of infection, there was decreased intestinal motility associated with decreased amplitude and rhythmicity of intestinal contractility. Pharmacological tests suggested a defective response mediated by acetylcholine receptors. The contractile response induced by acetylcholine was decreased by atropine in the acute phase while the lack of its action in the chronic phase was associated with tissue damage, and decreased expression of choline acetyltransferase, nicotinic subunits of acetylcholine receptors, and neurotransmitters. Conclusions T. cruzi-induced damage of smooth muscles was accompanied by motility disorders such as decreased intestinal peristalsis and cholinergic system response impairment. This study allows integration of the natural history of Chagasic megacolon motility disorders and opens new perspectives for the design of effective therapeutic.Item Ravuconazole self-emulsifying delivery system : in vitro activity against Trypanosoma cruzi amastigotes and in vivo toxicity.(2017) Spósito, Pollyanna Álvaro; Mazzeti, Ana Lia; Faria, Caroline de Oliveira; Urbina, Julio Alberto; Lana, Gwenaelle Elza Nathalie Pound; Bahia, Maria Terezinha; Mosqueira, Vanessa Carla FurtadoSelf-emulsifying drug delivery systems (SEDDSs) are lipid-based anhydrous formulations composed of an isotropic mixture of oil, surfactant, and cosurfactants usually presented in gelatin capsules. Ravuconazole (Biopharmaceutics Classification System [BCS] Class II) is a poorly water-soluble drug, and a SEDDS type IIIA was designed to deliver it in a predissolved state, improving dissolution in gastrointestinal fluids. After emulsification, the droplets had mean hydrodynamic diameters ,250 nm, zeta potential values in the range of -45 mV to -57 mV, and showed no signs of ravuconazole precipitation. Asymmetric flow field-flow fractionation with dynamic and multiangle laser light scattering was used to characterize these formulations in terms of size distribution and homogeneity. The fractograms obtained at 37°C showed a polydisperse profile for all blank and ravuconazole–SEDDS formulations but no large aggregates. SEDDS increased ravuconazole in vitro dissolution extent and rate (20%) compared to free drug (3%) in 6 h. The in vivo toxicity of blank SEDDS comprising Labrasol® surfactant in different concentrations and preliminary safety tests in repeated-dose oral administration (20 days) showed a dose-dependent Labrasol toxicity in healthy mice. Ravuconazole–SEDDS at low surfactant content (10%, v/v) in Trypanosoma cruzi-infected mice was safe during the 20-day treatment. The anti-T. cruzi activity of free ravuconazole, ravuconazole–SEDDS and each excipient were evaluated in vitro at equivalent ravuconazole concentrations needed to inhibit 50% or 90% (IC50 and IC90), respectively of the intracellular amastigote form of the parasite in a cardiomyocyte cell line. The results showed a clear improvement of the ravuconazole anti-T. cruzi activity when associated with SEDDS. Based on our results, the repurposing of ravuconazole in SEDDS dosage form is a strategy that deserves further in vivo investigation in preclinical studies for the treatment of human T. cruzi infections.Item Review on experimental treatment strategies against Trypanosoma cruzi.(2021) Mazzeti, Ana Lia; Oliveira, Patricia Capelari de; Bahia, Maria Terezinha; Mosqueira, Vanessa Carla FurtadoChagas disease is a neglected tropical disease caused by the protozoan Trypanosoma cruzi. Currently, only nitroheterocyclic nifurtimox (NFX) and benznidazole (BNZ) are available for the treatment of Chagas disease, with limitations such as variable efficacy, long treatment regimens and toxicity. Different strategies have been used to dis cover new active molecules for the treatment of Chagas disease. Target-based and phenotypic screening led to thousands of compounds with anti-T. cruzi activity, notably the nitroheter ocyclic compounds, fexinidazole and its metabolites. In addition, drug repurposing, drug combinations, re-dosing regimens and the development of new formulations have been evaluated. The CYP51 antifungal azoles, as posaconazole, ravuconazole and its prodrug fosravuconazole presented promising results in experimental Chagas disease. Drug combina tions of nitroheterocyclic and azoles were able to induce cure in murine infection. New treatment schemes using BNZ showed efficacy in the experimental chronic stage, including against dormant forms of T. cruzi. And finally, sesquiterpene lactone formulated in nanocar riers displayed outstanding efficacy against different strains of T. cruzi, susceptible or resistant to BNZ, the reference drug. These pre-clinical results are encouraging and provide interesting evidence to improve the treatment of patients with Chagas disease.Item Synergic effect of allopurinol in combination with nitro-heterocyclic compounds against Trypanosoma cruzi.(2019) Mazzeti, Ana Lia; Diniz, Lívia de Figueiredo; Gonçalves, Karolina Ribeiro; WonDollinger, Ruan Schott; Martins, Tassiane Assíria Fontes; Ribeiro, Isabela; Bahia, Maria TerezinhaCombination therapy has gained attention as a possible strategy for overcoming the limitations of the present therapeutic arsenal for Chagas disease. The aim of this study was to evaluate the effect of allopurinol in association with nitroheterocyclic compounds on infection with the Y strain of Trypanosoma cruzi. The in vitro effect of allopurinol plus benznidazole or nifurtimox on intracellular amastigotes in infected H9c2 cells was assessed in a 72-h assay. The interactions were classified as synergic for both allopurinol-nifurtimox (sums of fractional inhibitory concentrations [FICs] 0.49 0.08) and allopurinol-benznidazole (FICs 0.48 0.09). In the next step, infected Swiss mice were treated with allopurinol at 30, 60, and 90 mg/kg of body weight and with benznidazole at 25, 50, and 75 mg/kg in monotherapy and in combination at the same doses; as a reference treatment, another group of animals received benznidazole at 100 mg/kg. Allopurinol in monotherapy led to a smaller or nil effect in the reduction of parasite load and mortality rate. Treatment with benznidazole at suboptimal doses induced a transient suppression of parasitaemia with subsequent relapse in all animals treated with 25 and 50 mg/kg and in 80% of those that received 75 mg/kg. Administration of the drugs in combination significantly increased the cure rate to 60 to 100% among mice treated with benznidazole at 75 mg/kg plus 30, 60, or 90 mg/kg of allopurinol. These results show a positive interaction between allopurinol and benznidazole, and since both drugs are commercially available, their use in combination may be considered for the assessment in the treatment of Chagas disease patients.Item The entrance route : oral, mucous, cutaneous, or systemic has a marked influence on the outcome of Trypanosoma cruzi experimental infection.(2022) Gonçalves, Karolina Ribeiro; Mazzeti, Ana Lia; Nascimento, Alvaro Fernando da Silva do; Lacerda, Jéssica Mara Castro; Paiva, Nívia Carolina Nogueira de; Mathias, Fernando Augusto Siqueira; Reis, Alexandre Barbosa; Caldas, Sérgio; Bahia, Maria TerezinhaIn recent decades, the oral infection of Trypanosoma cruzi has gathered increased attention due to frequent outbreaks that can lead to more severe clinical signs than those usually found in the areas of vector transmission. This study addresses the main routes of infection using metacyclic trypomastigotes (MT) and blood trypomastigotes (BT). Herein, BALB/c mice were infected with the Colombian (TcI) strain via intraperitoneal (IP), oral, intragastric (IG), ocular (OC) and cutaneous (CT) routes with 106 culture-derived MT or BT. Parasitemia was intermittent and low in animals inoculated with MT, in contrast, high parasitemia levels were found in BT-mice. A tropism for the muscles was observed in oral or IG infection with BT. Differently, the parasite was widely distributed in the tissues of mice infected with MT. However, the intensity of the inflammation infiltrating the tissues was higher in oral or IG infection with BT. Animals inoculated with BT via the IG route had similar serum levels of IFN-γ and smaller IL-10 compared to those infected with MT via the IG route. TNF-α levels were higher in the serum from BT-animals, which could explain the higher intensity of heart inflammation in these animals. Our results suggest that the infective form and the route of infection differentially modulated the outcome of Trypanosoma cruzi mice infection.Item Time and dose-dependence evaluation of nitroheterocyclic drugs for improving efficacy following Trypanosoma cruzi infection : a pre-clinical study.(2018) Mazzeti, Ana Lia; Diniz, Lívia de Figueiredo; Gonçalves, Karolina Ribeiro; Nascimento, Álvaro Fernando da Silva do; Spósito, Pollyanna Álvaro; Mosqueira, Vanessa Carla Furtado; Coelho, George Luiz Lins Machado; Ribeiro, Isabela; Bahia, Maria TerezinhaBenznidazole and nifurtimox-treatments regimens currently used in human are supported by very limited experimental data. This study was designed to evaluate the time and dose dependence for efficacy of the most important nitroheterocyclic drugs in use for Chagas disease. In order to evaluate time dependence, Y strain-infected mice received benznidazole for a total of 1, 3, 7, 10, 20, and 40 days. Treatment courses of 3–10-day were effective in clearing parasitaemia and suppressing mortality, but parasitological cure was not achieved. Extending the treatments to 20 or 40 days clearly improved benznidazole efficacy. The 20-day treatment induced cure in 57.1% of Y strain infections (partially drug resistant) but failed to cure Colombian strain infections (full drug resistant), while the 40-day treatment resulted in cure of 100% of Y and 50% of Colombian strain infected mice. The increased cure rates in T. cruzi infected animals that received nifurtimox for 40 days confirm the relationship between the length of treatment and efficacy. An improvement in efficacy was observed with increasing benznidazole doses; cure was verified in 28.6% (75 mg/kg), 57.1% (100 mg/kg) and 80% (300 mg/kg). Overall, these nonclinical study data provide evidence that the efficacy of benznidazole is dose and time dependent. These findings may be relevant for optimizing treatment of human Chagas disease.