ravuconazole and Chagas-Disease

This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real-time quantitative PCR (qPCR) for baseline detection and quantification of parasitic loads and posttreatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely, DNDi-CH-E1224-001 (ClinicalTrials.gov registration no. NCT01489228) and the MSF-DNDi PCR Sampling Optimization Study (NCT01678599). Patients from Cochabamba (

Topics: Adolescent; Adult; Chagas Disease; DNA, Protozoan; Humans; Middle Aged; Monitoring, Physiologic; Nitroimidazoles; Parasite Load; Placebos; Real-Time Polymerase Chain Reaction; Thiazoles; Treatment Outcome; Triazoles; Trypanocidal Agents; Trypanosoma cruzi; Young Adult

Chagas disease is a major neglected vector-borne disease. In this study, we investigated the safety and efficacy of three oral E1224 (a water-soluble ravuconazole prodrug) regimens and benznidazole versus placebo in adult chronic indeterminate Chagas disease.. In this proof-of-concept, double-blind, randomised phase 2 clinical trial, we recruited adults (18-50 years) with confirmed diagnosis of Trypanosoma cruzi infection from two outpatient units in Bolivia. Patients were randomised with a computer-generated randomisation list, which was stratified by centre and used a block size of ten. Patients were randomly assigned (1:1:1:1:1) to five oral treatment groups: high-dose E1224 (duration 8 weeks, total dose 4000 mg), low-dose E1224 (8 weeks, 2000 mg), short-dose E1224 (4 weeks + 4 weeks placebo, 2400 mg), benznidazole (60 days, 5 mg/kg per day), or placebo (8 weeks, E1224-matched tablets). Double-blinding was limited to the E1224 and placebo arms, and assessors were masked to all treatment allocations. The primary efficacy endpoint was parasitological response to E1224 at the end of treatment, assessed by PCR. The secondary efficacy endpoints were parasitological response to benznidazole at end of treatment, assessed by PCR; sustainability of parasitological response until 12 months; parasite clearance and changes in parasite load; incidence of conversion to negative response in conventional and non-conventional (antigen trypomastigote chemiluminescent ELISA [AT CL-ELISA]) serological response; changes in levels of biomarkers; and complete response. The primary analysis population consisted of all randomised patients by their assigned treatment arms. This trial is registered with ClinicalTrials.gov, number NCT01489228.. Between July 19, 2011, and July 26, 2012, we screened 560 participants with confirmed Chagas disease, of whom 231 were enrolled and assigned to high-dose E1224 (n=45), low-dose E1224 (n=48), short-dose E1224 (n=46), benznidazole (n=45), or placebo (n=47). Parasite clearance was observed with E1224 during the treatment phase, but no sustained response was seen with low-dose and short-dose regimens, whereas 13 patients (29%, 95% CI 16·4-44·3) had sustained response with the high-dose regimen compared with four (9%, 2·4-20·4) in the placebo group (p<0·0001). Benznidazole had a rapid and sustained effect on parasite clearance, with 37 patients (82%, 67·9-92·0) with sustained response at 12-month follow-up. After 1 week of treatment, mean quantitative PCR repeated measurements showed a significant reduction in parasite load in all treatment arms versus placebo. Parasite levels in the low-dose and short-dose E1224 groups gradually returned to placebo levels. Both treatments were well tolerated. Reversible, dose-dependent liver enzyme increases were seen with E1224 and benznidazole. 187 (81%) participants developed treatment-emergent adverse events and six (3%) developed treatment-emergent serious adverse events. Treatment-emergent adverse events were headaches, nausea, pruritus, peripheral neuropathy, and hypersensitivity.. E1224 is the first new chemical entity developed for Chagas disease in decades. E1224 displayed a transient, suppressive effect on parasite clearance, whereas benznidazole showed early and sustained efficacy until 12 months of follow-up. Despite PCR limitations, our results support increased diagnosis and access to benznidazole standard regimen, and provide a development roadmap for novel benznidazole regimens in monotherapy and in combinations with E1224.. Drugs for Neglected Diseases initiative.

Topics: Administration, Oral; Adolescent; Adult; Bolivia; Chagas Disease; Double-Blind Method; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Incidence; Male; Middle Aged; Nitroimidazoles; Parasite Load; Placebos; Polymerase Chain Reaction; Prospective Studies; Thiazoles; Treatment Outcome; Triazoles; Trypanocidal Agents; Trypanosoma cruzi; Young Adult

The discovery and development of new drugs for the treatment of Chagas disease is urgent due to the high toxicity and low cure efficacy, mainly during the chronic phase of this disease. Other chemotherapeutic approaches for Chagas disease treatment are being researched and require screening assays suitable for evaluating the effectivity of new biologically active compounds. This study aims to evaluate a functional assay using the internalization of epimastigotes forms of Trypanosoma cruzi by human peripheral blood leukocytes from healthy volunteers and analyses by flow cytometry of cytotoxicity, anti-T. cruzi activity, and immunomodulatory effect of benznidazole, ravuconazole, and posaconazole. The culture supernatant was used to measure cytokines (IL-1-β, IL-6, INF-γ, TNF and IL-10) and chemokines (MCP-1/CCL2, CCL5/RANTES and CXCL8/IL-8). The data showed a reduction in the internalization of T. cruzi epimastigote forms treated with ravuconazole, demonstrating its potential anti-T. cruzi activity. In addition, an increased amount of IL-10 and TNF cytokines was observed in the supernatant of cultures upon the addition of the drug, mainly IL-10 in the presence of benznidazole, ravuconazole and posaconazole, and TNF in the presence of ravuconazole and posaconazole. Moreover, the results revealed a decrease in the MCP-1/CCL2 index in cultures in the presence of benznidazole, ravuconazole, and posaconazole. A decrease in the CCL5/RANTES and CXCL8/IL-8 index in cultures with BZ, when compared to the culture without drugs, was also observed. In conclusion, the innovative functional test proposed in this study may be a valuable tool as a confirmatory test for selecting promising compounds identified in prospecting programs for new drugs for Chagas disease treatment.

Topics: Chagas Disease; Cytokines; Flow Cytometry; Humans; Interleukin-10; Interleukin-8; Nitroimidazoles; Trypanocidal Agents; Trypanosoma cruzi

Neglected tropical diseases (NTDs) are communicable diseases that are uncommon in developed countries but epidemic in developing countries in tropical and subtropical regions of the world. One of the important contributions expected of pharmaceutical companies is the development and provision of drugs effective against NTDs. Eisai's efforts toward improving global health have resulted in a rich portfolio of assets addressing six infectious diseases: malaria, tuberculosis, Chagas disease, lymphatic filariasis, leishmaniasis, and mycetoma. As the most advanced project, Eisai has developed E1224 (fosravuconazole l-lysine ethanolate), which is available in both intravenous and oral formulations, and provides ravuconazole, an active form of fosravuconazole, with a long plasma half-life. The first clinical trials of E1224, for Chagas disease, have already been completed, led by the Drugs for Neglected Diseases initiative (DNDi). As a result, parasite clearance was observed with E1224 during the treatment phase, but parasite regrowth was observed after the end of drug administration, suggesting that the mechanism of action of E1224 on Trypanosoma cruzi is static rather than parasiticidal. On the other hand, a clinical trial for eumycetoma in collaboration with DNDi is ongoing supported by the Global Health Innovative Technology Fund, and is examining the efficacy of weekly treatment with E1224 versus the current standard of care, daily treatment with itraconazole. In this manner, Eisai will continue its drug-discovery research projects in collaboration with various PDPs and academia supported by funding agencies.

Topics: Chagas Disease; Global Health; Neglected Diseases; Thiazoles; Triazoles; Tropical Medicine; Trypanocidal Agents; Trypanosoma cruzi

Topics: Animals; Chagas Disease; Emulsions; Female; Hep G2 Cells; Humans; Inhibitory Concentration 50; Mice; Myocytes, Cardiac; Nanostructures; Rats; Thiazoles; Triazoles; Trypanosoma cruzi

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

Topics: Amlodipine; Animals; Chagas Disease; Mice; Nitroimidazoles; Parasitemia; Thiazoles; Triazoles; Trypanocidal Agents; Trypanosoma cruzi

Ergosterol biosynthesis inhibitors, such as posaconazole and ravuconazole, have been proposed as drug candidates for Chagas disease, a neglected infectious tropical disease caused by the protozoan parasite Trypanosoma cruzi. To understand better the mechanism of action and resistance to these inhibitors, a clone of the T. cruzi Y strain was cultured under intermittent and increasing concentrations of ravuconazole until phenotypic stability was achieved. The ravuconazole-selected clone exhibited loss in fitness in vitro when compared to the wild-type parental clone, as observed in reduced invasion capacity and slowed population growth in both mammalian and insect stages of the parasite. In drug activity assays, the resistant clone was above 300-fold more tolerant to ravuconazole than the sensitive parental clone, when the half-maximum effective concentration (EC

Topics: 14-alpha Demethylase Inhibitors; Animals; Cell Line; Chagas Disease; Drug Resistance, Multiple; Genes, Protozoan; Mutation; Nitroimidazoles; Sterol 14-Demethylase; Thiazoles; Triazoles; Trypanocidal Agents; Trypanosoma cruzi

The Pharmaceutical Industry is expected to play a proactive global role in combatting neglected tropical diseases (NTDs) and other tropical diseases affecting low-income countries. Such a role would include novel medicine R&D, manufacturing and distribution. In order to succeed in this role, several challenges need to be overcome: a) the economic challenge or cost benefit balance for the development of these medicines, and b) sparse in-house experience with these diseases within the Industry. During the last decade, the Product Development Partnership (PDP) model has become an effective strategy to address such challenges. Organizations such as the Medicines for Malaria Venture (MMV), Drugs for Neglected Diseases initiative (DNDi), TB alliance, PATH (formerly the Program for Appropriate Technology in Health), and others have linked pharmaceutical companies, funding organizations, academic researchers and others, and have thus been able to successfully populate treatment pipelines directed at NTDs, Malaria, tuberculosis (TB), and human immunodeficiency virus (HIV)/AIDS. In this paper, our experience working with one of these organizations, DNDi, is described. We have been collaborating with DNDi in evaluating the actions of Eisai's antifungal compound, E1224, in a clinical study for treating Chagas Disease. In addition, other Eisai initiatives directed at NTDs and improving patients' access to medicines are introduced.

Topics: Animals; Antifungal Agents; Chagas Disease; Cost-Benefit Analysis; Drug Discovery; Drug Industry; Global Health; Humans; Intersectoral Collaboration; Mice; Neglected Diseases; Prodrugs; Thiazoles; Triazoles; Tropical Medicine

Chagas disease affects more than 10 million people worldwide, and yet, as it has historically been known as a disease of the poor, it remains highly neglected. Two currently available drugs exhibit severe toxicity and low effectiveness, especially in the chronic phase, while new drug discovery has been halted for years as a result of a lack of interest from pharmaceutical companies. Although attempts to repurpose the antifungal drugs posaconazole and ravuconazole (inhibitors of fungal sterol 14α-demethylase [CYP51]) are finally in progress, development of cheaper and more efficient, preferably Trypanosoma cruzi-specific, chemotherapies would be highly advantageous. We have recently reported that the experimental T. cruzi CYP51 inhibitor VNI cures with 100% survival and 100% parasitological clearance both acute and chronic murine infections with the Tulahuen strain of T. cruzi. In this work, we further explored the potential of VNI by assaying nitro-derivative-resistant T. cruzi strains, Y and Colombiana, in highly stringent protocols of acute infection. The data show high antiparasitic efficacy of VNI and its derivative (VNI/VNF) against both forms of T. cruzi that are relevant for mammalian host infection (bloodstream and amastigotes), with the in vivo potency, at 25 mg/kg twice a day (b.i.d.), similar to that of benznidazole (100 mg/kg/day). Transmission electron microscopy and reverse mutation tests were performed to explore cellular ultrastructural and mutagenic aspects of VNI, respectively. No mutagenic potential could be seen by the Ames test at up to 3.5 μM, and the main ultrastructural damage induced by VNI in T. cruzi was related to Golgi apparatus and endoplasmic reticulum organization, with membrane blebs presenting an autophagic phenotype. Thus, these preliminary studies confirm VNI as a very promising trypanocidal drug candidate for Chagas disease therapy.

Topics: 14-alpha Demethylase Inhibitors; Animals; Chagas Disease; Drug Resistance; Endoplasmic Reticulum; Golgi Apparatus; Imidazoles; Male; Mice; Microscopy, Electron, Transmission; Nitroimidazoles; Oxadiazoles; Protozoan Proteins; Sterol 14-Demethylase; Thiazoles; Triazoles; Trypanocidal Agents; Trypanosoma cruzi

Chagas disease, caused by the eukaryotic (protozoan) parasite Trypanosoma cruzi, is an alarming emerging global health problem with no clinical drugs available to treat the chronic stage. Azole inhibitors of sterol 14α-demethylase (CYP51) were proven effective against Chagas, and antifungal drugs posaconazole and ravuconazole have entered clinical trials in Spain, Bolivia, and Argentina. Here we present the x-ray structures of T. cruzi CYP51 in complexes with two alternative drug candidates, pyridine derivatives (S)-(4-chlorophenyl)-1-(4-(4-(trifluoromethyl)phenyl)-piperazin-1-yl)-2-(pyridin-3-yl)ethanone (UDO; Protein Data Bank code 3ZG2) and N-[4-(trifluoromethyl)phenyl]-N-[1-[5-(trifluoromethyl)-2-pyridyl]-4-piperi-dyl]pyridin-3-amine (UDD; Protein Data Bank code 3ZG3). These compounds have been developed by the Drugs for Neglected Diseases initiative (DNDi) and are highly promising antichagasic agents in both cellular and in vivo experiments. The binding parameters and inhibitory effects on sterol 14α-demethylase activity in reconstituted enzyme reactions confirmed UDO and UDD as potent and selective T. cruzi CYP51 inhibitors. Comparative analysis of the pyridine- and azole-bound CYP51 structures uncovered the features that make UDO and UDD T. cruzi CYP51-specific. The structures suggest that although a precise fit between the shape of the inhibitor molecules and T. cruzi CYP51 active site topology underlies their high inhibitory potency, a longer coordination bond between the catalytic heme iron and the pyridine nitrogen implies a weaker influence of pyridines on the iron reduction potential, which may be the basis for the observed selectivity of these compounds toward the target enzyme versus other cytochrome P450s, including human drug-metabolizing P450s. These findings may pave the way for the development of novel CYP51-targeted drugs with optimized metabolic properties that are very much needed for the treatment of human infections caused by eukaryotic microbial pathogens.

Topics: 14-alpha Demethylase Inhibitors; Antiprotozoal Agents; Chagas Disease; Crystallography, X-Ray; Humans; Protozoan Proteins; Sterol 14-Demethylase; Thiazoles; Triazoles; Trypanosoma cruzi

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

Topics: Animals; Chagas Disease; Dogs; Enzyme-Linked Immunosorbent Assay; Immunoglobulin G; Interferon-gamma; Interleukin-10; Myocardium; Polymerase Chain Reaction; Thiazoles; Triazoles; Trypanocidal Agents; Trypanosoma cruzi

Topics: Chagas Disease; Clinical Trials, Phase I as Topic; Drug Industry; Global Health; Humans; Latin America; Prodrugs; Research Support as Topic; Thiazoles; Triazoles

Ravuconazole is an experimental triazole derivative with potent and broad-spectrum antifungal activity and a remarkably long half-life in humans. In this work, we investigated the in vitro and in vivo activities of this compound against Trypanosoma cruzi. Ravuconazole showed very potent in vitro anti-T. cruzi activity with minimal inhibitory concentrations (MIC) of 300 and 1 nM against the extracellular epimastigote and intracellular amastigote forms, respectively. As with other azole derivatives, ravuconazole at the MIC led to an essentially complete depletion of the epimastigotes' endogenous C4,14-desmethyl sterols and their replacement by di- and tri-methylated sterols. In murine acute models of acute Chagas disease, it was found that ravuconazole treatment led to high levels of parasitological cures, but only when given twice a day (b.i.d.), consistent with its short terminal half-life in mice (4 h). Furthermore, it was found that this curative activity was restricted towards nitrofuran/nitroimidazole-susceptible (CL) and partially drug-resistant (Y) strains of T. cruzi, with no curative activity in animals infected with the fully drug-resistant Colombiana strain. No curative activity occurred in a chronic model of the disease. No toxic side effects were observed resulting from treatment with the triazole. Ravuconazole is a very potent and specific anti-T. cruzi agent in vitro but its in vivo activity in mice is limited, probably due to its unfavourable pharmacokinetic properties in this animal model. However, these results do not necessarily rule out the potential utility of ravuconazole in the treatment of human T. cruzi infections.

Topics: Animals; Chagas Disease; Dose-Response Relationship, Drug; Drug Resistance; Female; Mice; Thiazoles; Triazoles; Trypanocidal Agents; Trypanosoma cruzi