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

ABSTRACT: 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 [?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.

SUBMITTER: Machado YA

PROVIDER: S-EPMC7526806 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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

Machado Yara Almeida YA Bahia Maria Terezinha MT Caldas Ivo Santana IS Mazzeti Ana Lia AL Novaes Rômulo Dias RD Vilas Boas Breno Raimundo BR Santos Lorena Júnia de Souza LJS Martins-Filho Olindo Assis OA Marques Marcos José MJ Diniz Lívia de Figueiredo LF

Antimicrobial agents and chemotherapy 20200722 8

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 <i>Trypanosoma cruzi</i> experimental infection. <i>In vitro</i> assays confirmed the trypanocidal activity of both compounds in monotherapy and demonstrated an additive effect (sum of the fractional inhibito ...[more]

PMID: 32423960

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Project description:Chagas disease (CD) is a neglected chronic infection caused by the protozoan parasite Trypanosoma cruzi (T. cruzi). A significant portion of infected people develops cardiac or digestive alterations over a lifetime. Since several chronic infections associated with antigen persistence and inflammation have been shown to lead to T cell exhaustion, new therapies targeting co-inhibitory receptors to regain T cell activity are under consideration. This study explored immune therapeutic approaches targeting the inhibitory PD-1/PD-L pathway in an experimental model for CD. Infected PD-L1 knockout mice (PD-L1 KO) showed increased systemic parasitemia in blood although no significant differences in parasite load were observed in different organs. Furthermore, we found no significant differences in the frequency of activated T cells or proinflammatory cytokine production when compared to WT counterparts. PD-L1 deficiency led to the production of IL-10 by CD8+ T cells and an upregulation of Tim-3 and CD244 (2B4). Unexpectedly, the lack of PD-L1 did not contribute to a significantly improved T cell response to infection. Single blockade and combined blockade of PD-1 and Tim-3 using monoclonal antibodies confirmed the results observed in infected. PD-L1 KO mice. Our results describe for the first time that the interruption of the PD-1/PD-L1 axis during acute T. cruzi infection does not necessarily enhance the immune response against this parasite. Its interruption favors increased levels of parasitemia and sustained upregulation of other co-inhibitory receptors as well as the production of regulatory cytokines. These results suggest that the clinical application of immune therapeutic approaches targeting the PD-1/PD-L1 axis in CD might be risky and associated with adverse events. It highlights that more research is urgently needed to better understand the immune regulation of T cells in CD before designing immune therapeutic approaches for a clinical context.

Project description:Metacyclic trypomastigote (MT) forms of Trypanosoma cruzi have been shown to release into medium gp82 and gp90, the stage-specific surface molecules that regulate host cell invasion, either in vesicles or in soluble form. Here, we found that during interaction of poorly invasive G strain with the host cell, gp82 and gp90 were released in vesicle-like forms, whereas no such release by highly invasive CL strain was observed. Shedding of vesicles of varying sizes by CL and G strains was visualized by scanning electron microscopy, and the protein profile of conditioned medium (CM) of the two strains was similar, but the content of gp82 and gp90 differed, with both molecules being detected in G strain as bands of high intensity in Western blotting, whereas in CL strain, they were barely detectable. Confocal images revealed a distinct distribution of gp82 and gp90 on MT surface of CL and G strains. In cell invasion assays, addition of G strain CM resulted in decreased CL strain internalization. Depletion of gp82 in G strain CM, by treatment with specific mAb-coupled magnetic beads, increased its inhibitory effect on CL strain invasion, in contrast to CM depleted in gp90. The effect of cholesterol-depleting drug methyl-β-cyclodextrin (MβCD) on gp82 and gp90 release by MTs was also examined. G strain MTs, untreated or treated with MβCD, were incubated in serum-containing medium or in nutrient-depleted PBS++, and the CM generated under these conditions was analyzed by Western blotting. In PBS++, gp82 and gp90 were released at lower levels by untreated MTs, as compared with MβCD-treated parasites. CM from untreated and MβCD-treated G strain, generated in PBS++, inhibited CL strain internalization. Treatment of CL strain MTs with MβCD resulted in increased gp82 and gp90 shedding and in decreased host cell invasion. The involvement of phospholipase C (PLC) on gp82 and gp90 shedding was also investigated. The CM from G strain MTs pretreated with specific PLC inhibitor contained lower levels of gp82 and gp90, as compared with untreated parasites. Our results contribute to shed light on the mechanism by which T. cruzi releases surface molecules implicated in host cell invasion.

Dataset Information

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

Publications

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

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