Evaluation of the in vitro and in vivo antileishmanial activity of a chloroquinolin derivative against Leishmania species capable of causing tegumentary and visceral leishmaniasis

Highlights

• A new chloroquinoline derivative, GF1059, was evaluated against Leishmania.

• The molecule was highly effective in in vitro experiments.

• It showed a very low toxicity in mammalian cells.

• The mechanism of action was based on apoptosis/necrosis of the parasites.

• Additional studies concerning treatment against disease should be performed.

Abstract

The treatment against leishmaniasis presents problems, since the currently used drugs are toxic and/or have high costs. In addition, parasite resistance has increased. As a consequence, in this study, a chloroquinolin derivative, namely 7-chloro-N,N-dimethylquinolin-4-amine or GF1059, was in vitro and in vivo tested against Leishmania parasites. Experiments were performed to evaluate in vitro antileishmanial activity and cytotoxicity, as well as the treatment of infected macrophages and the inhibition of infection using pre-treated parasites. This study also investigated the GF1059 mechanism of action in L. amazonensis. Results showed that the compound was highly effective against L. infantum and L. amazonensis, presenting a selectivity index of 154.6 and 86.4, respectively, against promastigotes and of 137.6 and 74.3, respectively, against amastigotes. GF1059 was also effective in the treatment of infected macrophages and inhibited the infection of these cells when parasites were pre-incubated with it. The molecule also induced changes in the parasites’ mitochondrial membrane potential and cell integrity, and caused an increase in the reactive oxygen species production in L. amazonensis. Experiments performed in BALB/c mice, which had been previously infected with L. amazonensis promastigotes, and thus treated with GF1059, showed that these animals presented significant reductions in the parasite load when the infected tissue, spleen, liver, and draining lymph node were evaluated. GF1059-treated mice presented both lower parasitism and low levels of enzymatic markers, as compared to those receiving amphotericin B, which was used as control. In conclusion, data suggested that GF1059 can be considered a possible therapeutic target to be tested against leishmaniasis.

Introduction

Leishmaniasis is a disease complex that is considered to be endemic in 98 countries around the world, with approximately 350 million people at risk of contracting infection by the Leishmania parasite, coupled with an annual incidence of 58,000 cases of visceral leishmaniasis (VL) and 220,000 cases of tegumentary leishmaniasis (TL) (Alvar et al., 2013, Araújo et al., 2017, World Health Organization, 2018). Different parasite species can cause TL, such as Leishmania major, L. tropica, L. aethiopica, L. panamensis, L. peruviana, L. braziliensis, and L. amazonensis. VL is caused mainly by L. donovani and L. infantum species (Ullah et al., 2016). The main clinical manifestations of TL include cutaneous leishmaniasis, the most frequent form, which can be cured spontaneously, and mucosal leishmaniasis, which is characterized by debilitating lesions due to the uncontrolled parasite replication leading to patient mutilation and morbidity. VL can cause symptoms, such as prolonged fever, splenomegaly, hypergammaglobulinemia, and pancytopenia, and the disease can be fatal if acute or left untreated (Kevric et al., 2015).

Treatment against leishmaniasis has been based on the use of pentavalent antimonials, pentamidine, amphotericin B (AmpB) and its liposomal formulation, paramomycin, miltefosine, among others. However, in spite of the success against the parasites, these compounds are toxic and/or present high costs. In addition, parasite resistance has increased (Ghorbani and Farhoudi, 2017, Sundar and Chakravarty, 2015, Uliana et al., 2018). However, the research for new drugs is not considered attractive, since it is an expensive process, requiring many years to occur and an investment of more than $1.0 billion to identify, characterize, and develop new antileishmanial agents (Hughes et al., 2011, Renslo and McKerrow, 2006). In this aspect, there is an urgent need to search for new compounds, such as those identified in plants, which could be promising leads for the development of novel and non-toxic therapeutics.

AmpB is a known antileishmanial drug; however, the side effects, such as renal, hepatic, and cardiac toxicity have limited its application in large scale. AmpB-containing lipid-based formulations have presented higher efficacy and lower toxicity, as compared to the use of free drugs; however, their high cost is a limiting factor (Copeland and Aronson, 2015, Cunha et al., 2015, Frézard and Demicheli, 2010). On the other hand, natural compounds have been considered a valuable source of new products with effective biological action (Cheuka et al., 2016, Pinto et al., 2014). The interest in plant-derived compounds has increased in recent years, and new antileishmanial agents have been identified (Duarte et al., 2016, Lage et al., 2015, Mendonça et al., 2018). However, although such candidates have presented a satisfactory and selective antileishmanial action, to date, few compounds have reached the clinical development in the field of leishmaniasis.

Host-directed immunotherapeutics have the major advantage of reducing the potential emergence of drug resistance, as well as interfering in the mechanisms of immune evasion which Leishmania parasites can perform, aiming to promote their survival within the phagolysosome of infected macrophages. One such evasion mechanism is the parasite's ability to reduce macrophage activation by manipulating their cell membranes (Masihi, 2000). As a consequence, the use of compounds able to display protective effects, when explored for its prophylactic potential, is desirable. Mechanistically, the previous contact of macrophages or parasites with these candidates could result in the inhibition of infection in these cells, either due to the increased production of hydrogen peroxide and phagosome maturation of the macrophages or by reducing the infectivity of pre-treated parasites (Parihar et al., 2016).

The high potential of quinoline-based natural products and their derivatives in medicinal chemistry has led to the identification of novel targets exhibiting antibacterial, anti-HIV, and anticancer activities (Kallander et al., 2005, Silva et al., 2009, Wang et al., 2010). These compounds are among the most biologically active class of heterocyclic products, presenting a wide range of pharmacological use, such as against Leishmania (Duarte et al., 2016, Lage et al., 2016, Mendonça et al., 2018, Tavares et al., 2018). In this context, in the present study, the in vitro and in vivo antileishmanial activity of a chloroquinolin derivative, namely 7-chloro-N,N-dimethylquinolin-4-amine, or GF1059, was evaluated against L. infantum and L. amazonensis. The cytotoxicity in murine macrophages and the hemolytic potential in human red blood cells, as well as the efficacy of GF1059 for the treatment of infected macrophages and in the inhibition of the infection using pre-treated parasites, were investigated. The mechanism of action of GF1059 and the in vivo therapeutic efficacy in L. amazonensis-infected BALB/c mice were also analyzed.