Ravuconazole self-emulsifying delivery system : in vitro activity against Trypanosoma cruzi amastigotes and in vivo toxicity.

Self-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

Benznidazol em combinação com alopurinol : aumento da atividade anti-Trypanosoma cruzi in vitro e in vivo.

Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto.O 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.Current chemotherapy for Chagas disease is unsatisfactory due to its limited and variable efficacy, particularly in chronic phase, and frequent side effects that can lead to treatment discontinuation. Combined therapy is envisioned as an ideal approach since it may improve treatment efficacy and decrease toxicity and resistance likelihood. In this work, we evaluated the activity of the alopurinol (Al) in combination with the benznidazole (Bz) upon T. cruzi infection. First, we investigated the activity of the Bz/Al combination on intracellular parasites of Y stain by incubating T. cruzi-infected H9c2 cells with different concentrations of each compound alone or in combination. In vitro drug interactions were assessed using a “checkerboard method” and the results were analyzed by calculating of the combination index (CI) using Calcusyn software.The overall assessment of the results demonstrated CI (0,62 a 1,76) suggesting an in vitro additive effect between the evaluated drug combination. Subsequently, the in vivo evaluation of the Bz/Al combination was performed in mice infected with 5000 blood tripomastigotes of Y stain. Treatment was administered orally for 20 consecutive days, beginning at 4 days after inoculation. Animals were treated with Bz (100, 75, 50 e 25 mg/kg) or All (90, 60 e 30 mg/kg) alone or in combination (Bz/Al: 75/90, 75/60, 75/30, 50/90, 50/60, 50/30, 25/90, 25/60 e 25/30 mg/kg). The parameters chosen to determine parasitological cure were parasitaemia during and up to 60 days after treatment and blood real time PCR assay (30 and 180 days post treatment). First, the effects of several doses of each drug alone on the evolution of the infection in mice were evaluated. The analysis of parasitemia levels after Bz-treatment showed a dosedependent trypanocidal effect. Although there was significant reduction in the level of parasitemia after treatment, parasitological cure was documented only in animals treated with 75 and100 mg of Bz per kg of body weight (20 and 70 % of cure, respectively). Differently, a dose-dependent effect was not observed among treated animal and the parasitemia reduction or cure was not verified among animals that received different doses of Al. In a second series of data analyses, the activities of the combinations of the Bz/Al were compared. Here the beneficial effect of the drug combinations was demonstrated by a significant reduction of parasitemia detected in all animals that received the combination therapy. Besides increase in rate of cure among animals treated with combination therapy was observed, since the combination of the Bz-75 mg/kg + Al-90 mg/kg or Bz-75 mg/kg + Al-60 mg/kg induced 100% of cure, and the combination of 75 mg/kg + Al-30 mg/kg or Bz-50 mg/kg + Al-60 mg/kg induced, respectively, 80 and 20% of cure. In conclusion, the combination of Bz and Al suggest a beneficial effect, as parasitological cure was observed in a higher proportion of animals treated with the drugs in association than in animals receiving the same dose of each of the drugs in monotherapy. Our results reinforce the importance of identifying alreadymarketed compounds with synergistic effects. This approach may help avoid expensive and time-consuming research on the toxicity and biological availability of drugs for human consumption

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

Benznidazole self-emulsifying delivery system : a novel alternative dosage form for Chagas disease treatment.

Benznidazole (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 ?M 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 ?M and 1.29 ? 0.01 ?M 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

Nitrotriazole-based compounds as antichagasic agents in a long-treatmenti In vivo?assay.

3-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

Chagas cardiomyopathy : the potential effect of benznidazole treatment on diastolic dysfunction and cardiac damage in dogschronically infected with Trypanosoma cruzi.

Cardiac 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

Synergic effect of allopurinol in combination with nitro-heterocyclic compounds against Trypanosoma cruzi.

Combination 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

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

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