INTRODUCTION
The immune system consists of a complex network of organs, cells, and molecules, to maintain the body's homeostasis by defending against various threats. However, species of the Leishmania genus disrupt the immune system's homeostasis and often lead to pathological conditions during the establishment of defense mechanisms (Cruvinel et al., 2010).
Visceral leishmaniasis (VL) is a zoonotic infectious disease that affects humans and animals, with a worldwide distribution in Asia, Europe, Africa, and the Americas. Reports of the disease date back to colonial times in the Americas, where it primarily affected children in the Mediterranean region. The differences in the causative organism of leishmaniasis between regions led to recognizing a distinct species, Leishmania infantum in 1908 (Rath et al., 2003).
VL is a vector-borne disease transmitted by sandflies of the genus Phlebotomus (in Africa, Asia, and Europe) and Lutzomyia in the Americas. Unlike most species causing pathology in humans, which reside in macrophages of epithelial and lymphatic vessels, L. infantum systematically spreads in the internal organs, primarily in the liver, spleen, bone marrow, and lymphatic vessels (Kumar; Nylén, 2012).
In Brazil, VL was primarily considered a rural anthroponosis until recently. However, since the 1980s, it has expanded into periurban regions of major cities. Several factors, including the emergence of the AIDS (Acquired Immune Deficiency Syndrome) epidemic and urbanization of vectors due to disorganized urban expansion and deforestation in rural areas, have contributed to the renewed medical significance of VL (Aguiar; Rodrigues, 2017).
In Brazil, VL is significant due to its high incidence and broad distribution. It can also assume severe and lethal forms when associated with malnutrition and concurrent infections. With the disease's expanding range and a significant increase in the number of cases, VL has been recognized by the World Health Organization (WHO) as a priority among tropical diseases (Gontijo; Melo, 2004).
Most cases of VL are treatable, and the most commonly used drugs are pentavalent antimonials (SbV), Miltefosine, and Amphotericin B, all of which are expensive and associated with toxicity. In India, resistance to SbV is already occurring, and there are reports of resistance to other drugs, including Amphotericin B, which justifies studies in search of alternatives. Currently, there is no anti-Leishmania vaccine, highlighting the need to develop new therapeutic strategies (Kumar; Nylén, 2012). Thus, there is a clear need for studies to identify substances with potential for new therapies, providing lower side effects and higher efficacy in patients.
Isatin and its analogs are versatile substrates, which can be used to synthesize numerous heterocyclic compounds. Isatin and its derivatives are used in organic synthesis, and in evaluating new products that possess different biological activities, including leishmanicidal activity. A variety of biological activities are associated with isatin including CNS activities as potentiation of pentobarbitone-induced necrosis, analgesic, anticonvulsant, antidepressant, antiinflammatory, antimicrobial, and effects on the central nervous system (Bhrigu, 2010).
Therefore, it’s expected that the results of this project will contribute to understanding the leishmanicidal properties of isatins compounds and their role in the immune response. Furthermore, it will assist in developing new studies for treating leishmaniasis in infected individuals caused by Leishmania infantum.
AIMS
Main Aim
To study the anti-Leishmania activity in L. infantum of fractions rich in isatins compounds.
Specific Aim:
To evaluate the anti-Leishmania activity of isatins on promastigote forms of L. infantum to determine the minimum inhibitory concentration values, representing the inhibition of 50% of promastigote growth (IC50).
METHODS
Culturing and Maintenance of Parasites in vitro
Promastigote forms of L. infantum were cultured in vitro in Schneider medium at pH 7, supplemented with 20% fetal bovine serum, 1% antibiotics, and 1% human male urine, referred to in this study as supplemented Schneider medium. The cultures were maintained at a temperature of 26 ± 1°C in a biological oxygen demand (BOD) incubator and subcultured weekly, not exceeding twenty passages, to sustain the cultures.
For cryopreservation to maintain the promastigote cultures, at the beginning of the stationary phase, they were cryopreserved in 12% sterile glycerol with constant agitation for 15 minutes and then stored in a freezer at -80°C. Additionally, promastigote forms were also cryopreserved in 7.5% dimethyl sulfoxide (DMSO), supplemented with 20% fetal bovine serum and 70% RPMI-1640 culture medium (Cultilab, São Paulo, Brazil), and stored in a freezer at -80°C for the same purpose.
Anti-Leishmania Activity of isatins on L. infantum Promastigotes
The anti-Leishmania activity of isatins on L. infantum was assessed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In a 96-well plate, 50?L of supplemented Schneider medium was added, followed by the addition of isatins in duplicate, previously diluted in supplemented Schneider medium to a final volume of 100 ?L for each well, at concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, and 3.12 ?M. Finally, approximately 1 x 10^6 promastigotes per well were added, a concentration achieved through adjustment of the control culture after counting in a Neubauer chamber, with the addition of supplemented Schneider medium to reach the required cell concentration.
The plates were then incubated for 72 hours in a biological oxygen demand (BOD) incubator at a temperature of 26°C. At the end of the incubation, 10 ?L of MTT diluted in phosphate-buffered saline to a final concentration of 5 mg/mL was added. They were further incubated for an additional 4 hours in a BOD incubator at 26°C. Subsequently, 50?L of 10% sodium dodecyl sulfate (SDS) were added. The plate was left overnight to dissolve the formazan crystals, and finally, the absorbance measured at 540 nm using a plate reader (Biotek model ELx800).
The negative control was maintained without substances, containing only supplemented Schneider medium. The positive control was conducted in the presence of amphotericin B as a reference drug. In contrast, the negative control consisted of promastigotes in supplemented Schneider medium without the test compounds. The percentage of growth inhibition of the evaluated substances was calculated relative to the control culture.
Statistical analysis
Subsequently, all data from the MTT assays were exported and analyzed using GraphPad Prism 5.0 software (San Diego, CA). The half-maximal inhibitory concentration (IC50) was calculated using dose-response curves, and 95% confidence intervals were included.
RESULTS
The tested isatins (IBM009, IDC003, IMC004, IBA005, IMA007, and IFA006) demonstrated leishmanicidal activity, with 13,20; 9,8; 11,15; 13,25; 25,83; 23,88 ?M IC50 values, respectively.
DISCUSSION
The IC50 values, representing the concentration at which half of the parasites were inhibited, ranged from 6.25 to 25.83 ?M for these isatins. This concentration range is significant because it encompasses relatively low doses compared to conventional treatments. IBM009 (6.25 ?M), IDC003 (9.8 ?M) and IMC004 (11.15 ?M) exhibited particularly promising activity, indicating that these isatins have a strong leishmanicidal potential and may be candidates for future research. Although IBA005 (13.20 ?M), IMA007 (23,88 ?M) and IFA006 (25.83 ?M) had slightly higher IC50 values, they still demonstrated leishmanicidal activity. The IC50 values of these molecules are comparable to other promising isatins with leishmanicidal activity (Sabt, 2023) and even with reference drugs as miltefosine (IC50 = 7.8976 ?M). These findings suggest that these isatins may be relevant even at slightly higher concentrations and warrant further investigation.
CONCLUSION
The study demonstrates promising results regarding the leishmanicidal activity of isatins against L. infantum promastigotes. Therefore, future experiments should take place to validate these findings and assess cytotoxicity to determine the potential of isatins for further use in L. infantum research.
ACKNOWLEDGMENTS
We express our sincere gratitude to CAPES, CNPQ, and FAPESQ-PB for their financial support that allowed the experiments outlined in this study. We are also deeply appreciative of the Federal University of Paraíba and the Laboratory of Immunology of Infectious Diseases for their generous assistance, which was instrumental in successfully concluding this research project. Their funding, provision of resources, and collaborative efforts have significantly contributed to the advancement of our scientific knowledge.
REFERENCES
CRUVINEL, Wilson de Melo et al. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória. Rev Bras Reumatol, [S.l.], p. 434-461, 18 maio 2010.
RATH, S. et al.. Antimoniais empregados no tratamento da leishmaniose: estado da arte. Química Nova, v. 26, n. 4, p. 550–555, jul. 2003.
KUMAR, Rajiv; NYLÉN, Susanne. Imunobiologia da leishmaniose visceral. Frontiers in Immunology, [S. l.], p. 1-10, 14 ago. 2012.
AGUIAR, Paulo Fernando; RODRIGUES, Raissa Katherine. Leishmaniose visceral no Brasil: artigo de revisão. Unimontes Científica, [S. l.], p. 191-204, 27 jun. 2017.
GONTIJO, C. M. F.; MELO, M. N.. Leishmaniose visceral no Brasil: quadro atual, desafios e perspectivas. Revista Brasileira de Epidemiologia, v. 7, n. 3, p. 338–349, set. 2004.
BHRIGU, B. et al. Search for biological active isatins: a short review. Int. J. Pharm. Sci. Drug Res, v. 2, n. 4, p. 229-235, 2010.
SABT, Ahmed. et al. New antileishmanial quinoline linked isatin derivatives targeting DHFR-TS and PTR1: Design, synthesis, and molecular modeling studies. European Journal of Medicinal Chemistry, p. 1-11, nov, 2022.
Comissão Organizadora
Francisco Mendonça Junior
Pascal Marchand
Teresinha Gonçalves da Silva
Isabelle Orliac-Garnier
Gerd Bruno da Rocha
Comissão Científica
Ricardo Olimpio de Moura
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