The quest for new drug candidates targeting neglected parasitic diseases has become increasingly urgent over the past decades. Advancements in formulating and optimizing drug delivery systems begin with basic research, including direct assays to evaluate the activity of molecules against parasitic stages maintained in laboratories; i.e., promastigotes. In the context of leishmaniasis, an endemic disease worldwide, the cultivation of Leishmania parasites can vary significantly across different laboratories. Factors such as culture media composition, pH, supplementation, and temperature can lead to varied drug responses in in vitro activity assays. This study aims to compile the parameters used in Leishmania spp. promastigotes cultivation protocols described in scientific articles published in indexed journals over the past ten years. The data reveal a lack of uniformity among Leishmania culture protocols, suggesting a potential bottleneck in comparing the leishmanicidal potential of in vitro drug candidates reported by different research groups. This condition is crucial to consider, because viability/inhibition assays should begin with fully-grown, healthy promastigote cultures capable of homogeneous division, thereby producing more reproducible results.

Leishmaniasis is a worldwide disease caused by more than 20 species of Leishmania parasites. Leishmania amazonensis and L. braziliensis are among the main causative agents of cutaneous leishmaniasis, presenting a broad spectrum of clinical forms. As these pathologies lead to unsatisfactory treatment outcomes, the discovery of alternative chemotherapeutic options is urgently required. In this investigation, a leishmanicidal bioassay-guided fractionation of the growth media extract produced by Aspergillus terreus P63 led to the isolation of the cyclic depsipeptide beauvericin (1). The viability of L. amazonensis, L. braziliensis and mammalian cells (macrophages and L929 fibroblasts) was assessed in 1 incubated cultures. Leishmania promastigotes were sensitive to 1, with EC₅₀ values ranging from 0.7 to 1.3 μM. Microscopy analysis indicated that Leishmania spp. parasites showed morphological abnormalities in a dose-dependent manner in the presence of 1. L. amazonensis intracellular amastigotes were more sensitive to 1 than promastigotes (EC₅₀ = 0.8 ± 0.1 μM), with a good selectivity index (22-30). 1 reduced the infectivity index at very low concentrations, maintaining the integrity of the primary murine host cell for up to the highest concentration tested for 1. In vivo assays of 1 conducted using BALB/c mice infected with stationary-phase promastigotes of L. amazonensis in the tail base presented a significant reduction in the lesion parasite load. A second round of in vivo assays was performed to assess the efficacy of the topical use of 1. The results demonstrated a significant decrease in the total ulcerated area of mice treated with 1 when compared with untreated animals. Our results present promising in vitro and in vivo leishmanicidal effects of beauvericin, emphasizing that systemic inoculation of 1 led to a decrease in the parasite load at the lesion site, whereas topical administration of 1 delayed the progression of leishmaniasis ulcers, a cure criterion established for cutaneous leishmaniasis management.