Drugs in preclinical and early clinical development for the treatment of Chagas´s disease: the current status.

INTRODUCTION: Chagas disease is spreading faster than expected in different countries, and little progress has been reported in the discovery of new drugs to combat Trypanosoma cruzi infection in humans. Recent clinical trials have ended with small hope. The pathophysiology of this neglected disease and the genetic diversity of parasites are exceptionally complex. The only two drugs available to treat patients are far from being safe, and their efficacy in the chronic phase is still unsatisfactory. AREAS COVERED: This review offers a comprehensive examination and critical review of data reported in the last 10 years, and it is focused on findings of clinical trials and data acquired in vivo in preclinical studies. EXPERT OPINION: The in vivo investigations classically in mice and dog models are also challenging and time-consuming to attest cure for infection. Poorly standardized protocols, availability of diagnosis methods and disease progression markers, the use of different T. cruzi strains with variable benznidazole sensitivities, and animals in different acute and chronic phases of infection contribute to it. More synchronized efforts between research groups in this field are required to put in evidence new promising substances, drug combinations, repurposing strategies, and new pharmaceutical formulations to impact the therapy.

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 antigen detection in blood to assess treatment efficacy and cure in mice models of Chagas disease.

Introduction: Chagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi. Although endemic mainly in Latin America, CD has become a global public health problem due to migration of infected individuals to non-endemic regions. Despite progress made in drug development, preclinical assays for drug discovery are required to accelerate the development of new drugs with reduced side effects, which are much needed for human treatment. Methods: We used a cure model of infected mice treated with Fexinidazole (FZ) to further validate a novel Enzyme Linked Aptamer (ELA) assay that detects parasite biomarkers circulating in the blood of infected animals. Results: The ELA assay showed cure by FZ in ~71% and ~77% of mice infected with the VL-10 and Colombiana strains of T. cruzi, respectively. The ELA assay also revealed superior treatment efficacy of FZ compared to Benznidazole prior to immunosuppression treatment. Discussion: Our study supports the use of ELA assay as an alternative to traditional serology or blood PCR to assess the efficacy of antichagasic drugs during their preclinical phase of development. Further, the combination of high sensitivity and ease of use make this parasite antigen detection assay an attractive new tool to facilitate the development of much needed new therapies for CD.

The entrance route: Oral, mucous, cutaneous, or systemic has a marked influence on the outcome of Trypanosoma cruzi experimental infection.

In 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.

The Impact of the CTHRSSVVC Peptide Upon Experimental Models of Trypanosoma cruzi Infection.

Chagas disease (CD), caused by the hemoflagellate protozoan Trypanosoma cruzi, affects more than six million people worldwide and presents an unsatisfactory therapy, based on two nitroderivatives, introduced in clinical medicine for decades. The synthetic peptide, with CTHRSSVVC sequence (PepA), mimics the CD163 and TNF-α tripeptide "RSS" motif and binds to atheromatous plaques in carotid biopsies of human patients, spleen tissues, and a low-density lipoprotein receptor knockout (LDLr-/-) mouse model of atherosclerosis. CD163 receptor is present on monocytes, macrophages, and neutrophils, acting as a regulator of acute-phase processes and modulating aspects of the inflammatory response and the establishment of infections. Due to the potential theranostic role of PepA, our aim was to investigate its effect upon T. cruzi infection in vitro and in vivo. PepA and two other peptides with shuffled sequences were assayed upon different binomials of host cell/parasite, including professional [as peritoneal mouse macrophages (PMM)] and non-professional phagocytes [primary cultures of cardiac cells (CM)], under different protocols. Also, their impact was further addressed in vivo using a mouse model of acute experimental Chagas disease. Our in-vitro findings demonstrate that PepA and PepB (the peptide with random sequence retaining the "RS" sequence) reduced the intracellular parasitism of the PMM but were inactive during the infection of cardiac cells. Another set of in-vitro and in-vivo studies showed that they do not display a trypanocidal effect on bloodstream trypomastigotes nor exhibit in-vivo efficacy when administered after the parasite inoculation. Our data report the in-vitro activity of PepA and PepB upon the infection of PMM by T. cruzi, possibly triggering the microbicidal arsenal of the host professional phagocytes, capable of controlling parasitic invasion and proliferation.

N6-modification of 7-Deazapurine nucleoside analogues as Anti-Trypanosoma cruzi and anti-Leishmania agents: Structure-activity relationship exploration and In vivo evaluation.

Chagas disease and leishmaniasis are two poverty-related neglected tropical diseases that cause high mortality and morbidity. Current treatments suffer from severe limitations and novel, safer and more effective drugs are urgently needed. Both Trypanosoma cruzi and Leishmania are auxotrophic for purines and absolutely depend on uptake and assimilation of host purines. This led us to successfully explore purine nucleoside analogues as chemotherapeutic agents against these and other kinetoplastid infections. This study extensively explored the modification of the 6-amino group of tubercidin, a natural product with trypanocidal activity but unacceptable toxicity for clinical use. We found that mono-substitution of the amine with short alkyls elicits potent and selective antitrypanosomal and antileishmanial activity. The methyl analogue 15 displayed the best in vitro activity against both T. cruzi and L. infantum and high selectivity versus host cells. Oral administration for five consecutive days in an acute Chagas disease mouse model resulted in significantly reduced peak parasitemia levels (75, 89 and 96% with 12.5, 25 and 50 mg/kg/day, respectively). as well as increased animal survival rates with the lower doses (83 and 67% for 12.5 and 25 mg/kg/day, respectively).

7-Aryl-7-deazapurine 3'-deoxyribonucleoside derivative as a novel lead for Chagas' disease therapy: in vitro and in vivo pharmacology.

BACKGROUND: The protozoan Trypanosoma cruzi is auxotrophic for purines and causes Chagas' disease (CD), a neglected illness affecting >6 million people. Combining the 3-deoxyribofuranose part of cordycepin with the modified purine ring of a nucleoside 'hit' led to the discovery of 4-amino-5-(4-chlorophenyl)-N7-(3'-deoxy-ß-d-ribofuranosyl)-pyrrolo[2,3-d]pyrimidine (Cpd1), revealing promising anti-T. cruzi activity. OBJECTIVES: To further evaluate Cpd1 in vitro and in vivo to fully assess its therapeutic potential against CD, covering cell culture sterilization through washout assays, drug combination with benznidazole and long-term administration in T. cruzi-infected mice. RESULTS: Although less susceptible to Cpd1 than amastigotes, trypomastigotes present an impaired capacity to successfully establish intracellular infection of cardiac cultures. Combination of benznidazole with Cpd1 indicated no interaction (additive effect) (FIC index = 0.72) while administration to mice at one-tenth of the optimal dose (2.5 mg/kg and 10 mg/kg for Cpd1 and benznidazole, respectively) suppressed parasitaemia but failed to avoid mortality. Long-term treatment (60 days) gave a rapid drop of the parasitaemia (>98% decline) and 100% mice survival but only 16% cure. In vitro washout experiments demonstrated that although parasite release into the supernatant of infected cardiac cultures was reduced by >94%, parasite recrudescence did occur after treatment. CONCLUSIONS: Parasite recrudescence did occur after treatment corroborating the hypothesis of therapeutic failure due to subpopulations of dormant forms and/or genetic factors in persister parasites involved in natural drug resistance.

Higher oral efficacy of ravuconazole in self-nanoemulsifying systems in shorter treatment in experimental chagas disease.

We 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 soluble 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.

Combination therapy using nitro compounds improves the efficacy of experimental Chagas disease treatment.

Drug 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 classified 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 parasitemia 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.

Review on Experimental Treatment Strategies Against Trypanosoma cruzi.

Chagas 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 discover 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 nitroheterocyclic 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 combinations 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 nanocarriers 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.

Neuronal Parasitism, Early Myenteric Neurons Depopulation and Continuous Axonal Networking Damage as Underlying Mechanisms of the Experimental Intestinal Chagas' Disease.

There 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.

Impact of diminazene aceturate on renin-angiotensin system, infectious myocarditis and skeletal myositis in mice: An in vitro and in vivo study.

Although renin-angiotensin system (RAS) imbalance is manifested in cardiomyopathies with different etiologies, the impact of RAS effectors on Chagas cardiomyopathy and skeletal myositis is poorly understood. Given that diminazene aceturate (DMZ) shares trypanocidal, angiotensin-converting enzyme 2 (ACE2) and angiotensin-(1-7) stimulatory effects, we investigated the impact of DMZ on cardiomyocytes infection in vitro, renin-angiotensin system, Chagas cardiomyopathy and skeletal myositis in vivo. Cardiomyocytes and T. cruzi were used to evaluate DMZ toxicity in vitro. The impact of 20-days DMZ treatment (1 mg/kg) was also investigated in uninfected and T. cruzi-infected mice as follows: control uninfected and untreated, uninfected treated with DMZ, infected untreated and infected treated with DMZ. DMZ had low toxicity on cardiomyocytes, induced dose-dependent antiparasitic activity on T. cruzi trypomastigotes, and reduced parasite load but not infection rates in cardiomyocytes. DMZ increased ACE2 activity and angiotensin-(1-7) plasma levels but exerted no interference on angiotensin-converting enzyme (ACE) activity, ACE, ACE2 and angiotensin II levels in uninfected and infected mice. DMZ treatment also reduced IFN-γ and IL-2 circulating levels but was ineffective in attenuating parasitemia, MCP-1, IL-10, anti-T. cruzi IgG, nitrite/nitrate and malondialdehyde production, myocarditis and skeletal myositis compared to infected untreated animals. As the antiparasitic effect of DMZ in vitro did not manifest in vivo, this drug exhibited limited relevance to the treatment of Chagas disease. Although DMZ is effective in upregulating angiotensin-(1-7) levels, this molecule does not act as a potent modulator of T. cruzi infection, which can establish heart and skeletal muscle parasitism, lipid oxidation and inflammatory damage, even in the presence of high concentrations of this RAS effector.

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.

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 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.

Synergic Effect of Allopurinol in Combination with Nitroheterocyclic 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.

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.

Time and dose-dependence evaluation of nitroheterocyclic drugs for improving efficacy following Trypanosoma cruzi infection: A pre-clinical study.

Benznidazole 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.

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.

Nitrotriazole-Based Compounds as Antichagasic Agents in a Long-Treatment 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.

Benznidazole/Itraconazole Combination Treatment Enhances Anti-Trypanosoma cruzi Activity in Experimental Chagas Disease.

The 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.