Ivermectin presents effective and selective antileishmanial activity
- Category: Ivermectin
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Ivermectin presents effective and selective anti-leishmanial activity in vitro and in vivo against Leishmania infantum and is therapeutic against visceral leishmaniasis
Treatment for visceral leishmaniasis (VL) is hindered mainly by the toxicity and/or high cost of therapeutic drugs. In addition, parasite resistance has been registered. Thus, there is an urgent need for the identification of novel, effective and low-cost antileishmanial agents. Since drug discovery is a long and expensive process, drug repositioning for treatment of leishmaniasis should be considered. In the present study, Ivermectin (IVE), a broad-spectrum drug used for treatment of parasitic diseases, was evaluated in vitro and in vivo against Leishmania infantum species.
Results in vitro showed that IVE presented 50% Leishmania and macrophage inhibitory concentrations (IC50 and CC50, respectively) of 3.64 ± 0.48 μM and 427.50 ± 17.60 μM, respectively, with a selectivity index (SI) of 117.45; whereas Amphotericin B (AmpB), which was used as control, showed IC50 and CC50 values of 0.12 ± 0.05 μM and 1.06 ± 0.23 μM, respectively, with a corresponding SI of 8.90. Treatment with IVE effectively reduced the infection percentage and parasite burden in infected and treated macrophages and displayed a prophylactic activity by inhibiting macrophage infection with pre-treated parasites. Furthermore, preliminary studies suggested that IVE targets the parasite's mitochondria. Activity of IVE in its free format or incorporated into Pluronic® F127-based polymeric micelles (IVE/Mic) was also evaluated in vivo as a treating drug for L. infantum-infected BALB/c mice. Miltefosine was used as a control. Results showed that Miltefosine, IVE and IVE/Mic-treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as well as development of an antileishmanial Th1-type immune response one and 15 days after treatment. Notably, IVE/Mic showed a better parasitological and immunological response in comparison to other alternative treatments. In conclusion, results suggest that IVE/Mic could be considered in future studies as a therapeutic alternative to treat VL.
Leishmaniases are tropical neglected diseases endemic in 98 countries in the world, with about 380 million people exposed to the risk of infection by Leishmania parasite (WHO, 2018). This disease complex includes tegumentary leishmaniasis (TL), which is characterized by cutaneous, mucosal and cutaneous-diffuse forms of disease, and visceral leishmaniasis (VL) (Grimaldi and Tesh, 1993). TL is caused by distinct parasite species, such as Leishmania major, L. tropica, L. braziliensis, L. aethiopica, L. mexicana, among others; while VL is caused by L. donovani and L. infantum species (Alvar et al., 2012). Acute VL can cause anemia, fever, wasting, hepatosplenomegaly, immune suppression and it can be fatal if left untreated (Sundar and Singh, 2016).
Previous studies have shown that the protective response against infection relies on the induction of Th1-type immunity, characterized by the production of pro-inflammatory cytokines such as IFN-γ, IL-12, GM-CSF, among others, which stimulates infected host cells to kill parasites (Kedzierski and Evans 2014). On the contrary, a Th2-type immune response based on the production of cytokines such as IL-4, IL-5 and IL-10, contributes to disease progression by deactivation of parasitized macrophages (Dayakar et al., 2019).
VL treatment has long relied on the use of pentavalent antimonials. Despite antimonials have been reported to be effective against parasites, they are also toxic to the patients who can develop myalgia, arthralgia, anorexia and fever, as well as renal, cardiac and hepatic toxicity (Jain and Jain, 2013; Sundar and Singh, 2018). Amphotericin B (AmpB) has been used as a second line drug. It was originally developed as an antifungal agent but its antileishmanial activity was also demonstrated against distinct Leishmania species (Zauli-Nascimento et al., 2010; Corral et al., 2014; Mesquita et al., 2014). As with antimonials, however, treatment with AmpB is mainly related to renal and cardiac alterations, hemolysis and liver damage (Ortega et al., 2017). AmpB-based liposomal formulations are instead less toxic but their high costs limit their broad use as prophylactic drugs against VL (Sundar et al., 2019).
Miltefosine was originally described as an anti-tumor drug and later as an antileishmanial agent against parasite species causing VL (Mbui et al., 2019; Pijpers et al., 2019). Miltefosine presents a selective activity against parasites (Sane et al., 2010; Neira et al., 2019) by inhibiting the biosynthesis of the glycosylphosphatidylinositol receptor, which is a key molecule for Leishmania intracellular survival (Zhang et al., 2018). However, it causes teratogenicity and parasite resistance has been reported (Sundar and Singh, 2018). Therefore, the search to identify novel, effective and low-cost antileishmanial agents continues till date.
Previous studies have highlighted the relevance of drug repositioning for treatment against leishmaniasis. Rub et al. (2019) evaluated glyburide, an effective sugar lowering drug used for treatment of diabetes, regarding its antileishmanial activity against L. donovani. Results showed that glyburide inhibited parasite viability, suggesting its potential use as a drug to treat VL. In a similar fashion, hydroxyurea, an anti-tumor agent which is used for treatment of myeloid leukemia and melanoma, was also evaluated as an antiparasitic agent against L. mexicana species. In that study, the authors demonstrated that the drug caused alterations in the parasite cell cycle and effectively reduced infection of macrophages after treatment by its antileishmanial effect against intracellular amastigotes (Martinez-Rojano et al., 2008). Cisplatin, another anti-tumor agent, was evaluated against L. donovani and was proved to cause an increase in the levels of thiols and reactive oxygen species in the parasite, ultimately leading to Leishmania death (Kaur et al., 2010).
In the present study, Ivermectin (IVE) was tested in vitro and in vivo against L. infantum. IVE is a broad-spectrum antiparasitic agent commonly used for treatment of lice infestation, onchocerciasis, strongyloidiasis, scabies and cutaneous larva migrans (Omondi et al., 2018; Repetto et al., 2018). Here, IVE was evaluated in its free format or incorporated into a Poloxamer 407 (Pluronic® F127)-based polymeric micelle system (IVE/Mic) to treat L. infantum-infected BALB/c mice. Parasitological analyses by estimation of parasite load in spleen, liver, bone marrow (BM) and draining lymph nodes (dLN), as well as immunological assays, based on the evaluations of cellular and humoral responses, were performed after euthanasia of infected mice, one and 15 days post-therapy.
Miltefosine (C21H46NO4P), IVE (C25H43NO18), AmpB (C47H73NO17) and Poloxamer 407 (Pluronic® F127) were purchased from Sigma-Aldrich (catalog numbers: 58,066-85-6, 70,288-86-7, 1397-89-3, and 16,758, respectively; St. Louis, USA).
Experimental animals and parasites
BALB/c mice (female, 8 weeks old) were purchased from the Institute of Biological Sciences from Federal University of Minas Gerais (UFMG; Belo Horizonte, Minas Gerais, Brazil) and were kept under specific pathogen-free conditions. The study was approved by the Committee
Biological activity in vitro
Antileishmanial activity of IVE was evaluated in vitro against L. infantum and its cytotoxicity, against murine macrophages and human red blood cells. Results showed IC50 and CC50 values of 3.64 ± 0.48 μM and 427.50 ± 17.60 μM, respectively, with corresponding SI of 117.45 (Table 1). AmpB showed IC50 and CC50 values of 0.12 ± 0.05 μM and 1.06 ± 0.23 μM, respectively, and SI of 8.83. RBC50 values were 482.60 ± 29.00 μM and 11.10 ± 1.40 μM for IVE and AmpB, respectively. L. infantum-infected
Searching for new antileishmanial agents is a resource-intensive, expensive and time-consuming task. Thus, drug repositioning should be considered as an alternative strategy. In the present study, IVE, a well-described antiparasitic drug, was evaluated in vitro and in vivo against L. infantum species. Results clearly showed that IVE was highly effective against both L. infantum promastigotes and amastigotes, while displaying low toxicity to murine and human cells. Treatment of infected
Declaration of competing interest
The authors confirm that they have no conflicts of interest in relation to this work.
This work was supported by grant MR/R005850/1 from Medical Research Council (VAccine deveLopment for complex Intracellular neglecteD pAThogEns - VALIDATE), UK, and grant APQ-408675/2018–7 from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. The authors also thank the Brazilian agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), CNPq and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the student scholarships.
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