Curzerene antileishmania activity: Effects on Leishmania amazonensis and possible action mechanisms

Highlights

• Curzerene showed antileishmanial activity in vitro to L. amazonensis;

• Curzerene exhibited selectivity indexes (SI) greater than reference drugs;

• Curzerene induced apoptosis in promastigotes forms;

• Curzerene reduced the infection index of macrophages by L. amazonensis;

• Curzerene increased TNF-α, IL-12, NO and lysosomal volume and reduced IL-10 and IL-6 levels.

Abstract

Leishmaniasis is a set of infectious diseases with high rates of morbidity and mortality, it affects millions of people around the world. Treatment, mainly with pentavalent antimonials, presents significant toxicity and many cases of resistance. In previous works we have demonstrated the effective and selective antileishmanial activity of Eugenia uniflora L. essential oil, being constituted (47.3%) by the sesquiterpene curzerene. Considering the high rate of parasite inhibition demonstrated for E. uniflora essential oil, and the significant presence of curzerene in the oil, this study aimed to evaluate its antileishmania activity and possible mechanisms of action. Curzerene was effective in inhibiting the growth of promastigotes (IC₅₀ 3.09 ± 0.14 µM) and axenic amastigotes (EC₅₀ 2.56 ± 0.12 µM), with low cytotoxicity to RAW 264.7 macrophages (CC₅₀ 83.87 ± 4.63 µM). It was observed that curzerene has direct effects on the parasite, inducing cell death by apoptosis with secondary necrotic effects (producing pores in the plasma membrane). Curzerene proved to be even more effective against intra-macrophage amastigote forms, with an EC₅₀ of 0.46 ± 0.02 µM. The selectivity index demonstrated by curzerene on these parasite forms was 182.32, being respectively 44.15 and 8.47 times more selective than meglumine antimoniate and amphotericin B. The antiamastigote activity of curzerene was associated with immunomodulatory activity, as it increased TNF-α, IL-12, and NO levels, and lysosomal activity, and decreased IL-10 and IL-6 cytokine levels detected in macrophages infected and treated. In conclusion, our results demonstrate that curzerene is an effective and selective antileishmanial agent, a candidate for in vivo investigation in models of antileishmanial activity.

Introduction

Leishmaniasis is a spectrum of infectious and parasitic diseases caused by protozoa of the genus Leishmania. It is transmitted through the bite of female insect vectors, mainly from the genus Phlebotomus (in the Old World), and Lutzomyia (in the Americas) [1]. The World Health Organization categorizes Leishmaniasis as an emerging -uncontrolled disease, with about 12 million people infected, and more than 350 million people at risk [2]. Given the groups of people it affects (usually low income) and the lack of investment in chemotherapy treatment research, Leishmaniasis is considered a Neglected Tropical Disease (NTD) [3].

The clinical manifestations of leishmaniasis vary according to the species and the host's immune response. Divided into two main groups, tegumentary leishmaniasis (TL) has two different clinical presentations: ulcerative skin lesions-cutaneous leishmaniasis (CL), or a destructive mucosal inflammation-mucocutaneous leishmaniasis (MCL) that affects oral-nasal-pharyngeal cavities. In visceral leishmaniasis, the most serious and fatal form of the disease, internal organs such as the spleen and liver and medulla are affected [4].

Although leishmaniasis has an important epidemiological profile, the reference drugs for treatment are far from adequate. This is due to a diversity of serious adverse effects and an increase in the number of parasitic resistance cases, making their use limited [5]. As the first choice, pentavalent antimonials have been used in the treatment of leishmaniasis since the 1940 s. They are toxic and have numerous adverse effects such as cardiotoxicity, hepatotoxicity, and pancreatitis. Second-line drugs, such as amphotericin B, miltefosine, paramomycin, and pentamidine are used in cases of antimonial resistance, but are even more toxic [6]. Thus, we see a global need to develop new leishmaniasis treatments.

In previous works, our research group studied antileishmanial activity with the essential oil of Eugenia uniflora L. (Myrtaceae), popularly known as pitangueira in Brazil, and found promising results with respective IC_50s of 1.75 µg/mL and 1.92 µg/mL on the promastigote and amastigote forms of L. amazonensis [7]. Through chemical analysis of the essential oil using gas chromatography coupled with mass spectrometry (GC–MS), the oxygenated sesquiterpene curzerene (at 47.3%) was revealed as the major constituent. Considering the high rate of parasite inhibition demonstrated for E. uniflora essential oil, and the significant presence of curzerene in the oil, the current work aimed to investigate the antileishmanial activity of curzerene, as well as to determine both its cytotoxicity in mammalian cells, and its mechanisms of action.