The Synthesis, Characterization, and Study of Leishmanicidal Activity of

1. The Synthesis, Characterization, and Study of Leishmanicidal Activity of Meglumine Antimoniate (Glucantime) In Promastigote Forms of Leishmania major 4 2 3 1 U. Barrie , Prof. M. Meneghetti , R. Omena , G. Melo University at Albany, Federal University of Alagoas, State University of New York at Oswego 2 1 3 Introduction To Leishmania Introduction To Glucantime Leishmaniasis, is a complex of diseases caused by 17 types of the protozoan Leishmania, belonging to the family Trypanosomatidae of the genus Leishmania, transmitted by the phlebotomine sand fly vector. This parasite leads a digenetic life cycle. Human Leishmaniasis is distributed worldwide, with important emphases of infection in South and Central America, Southern Europe, North and East Africa, the Middle East, and the Indian subcontinent. It has prevalence of 12 million cases and 400,000 new cases reported annually, causing diseases ranging from skin lesions in Cutaneous Leishmaniasis (CL) to a progressive and frequently fatal hepatosplenomegaly in Visceral Leishmaniasis (VL). Leishmaniasis causes significant morbidity and mortality worldwide. • Symptoms of Leishmaniasis: • Begins as a papule that soon becomes itchy • Develops into one or more skin ulcers on exposed body parts, mostly the face, arms and legs • Sores can also be covered by a scab Meglumine Antimoniate (Glucantime®), a first choice drug for the treatment of leishmaniasis, is produced by the reaction of pentavalent antimony with N-methyl-D-glucamine. In the absence of effective vaccines and vector-control measures, the main line of defense against this disease is chemotherapy. Organic pentavalent antimonials [Sb(V) & Sb (III)] have been the first line drugs for the treatment of VL and CL for the last 60 years. Figure 1: Leishmania Life Cycle Figure 2: Symptoms of Leishmaniasis. Stanford University. Cutaneous Leishmaniasis Objectives Figure 3: Molecular Structure of Glucantime • The objectives of the project include: • Synthesis and characterization of Meglumine Antimony (Glucantime) • - Assay the cytotoxicity effect of Meglumine Antimoniate against promastigote forms of Leishmania major Figure 4: 3D Molecular Structure of Glucantime Results Using Potassium Hexahydroxoantimonate [KSb(OH)6] Using Antimony (III) Tetraoxide [Sb2O3] Antileishmanial Assay Against Leishmania major • Dissolve .004 mol of N-Methyl-D-Glucamine in 25ml of water under stirring at a constant 55 degrees C • .004 mol of KSb(OH)6 was then added to the solution and the mixture is kept at a Ph 7 with NaOH and HCl until the mixture remained clear • After cooling, precipitation was introduced by addition of 75 ml of Acetone • Precipitation is filtered and dried • Dissolve .002 mol of Sb2O3 in 15 ml of HCl 5 mol/L and add NaOH 3 mol/L at the Ph at 9. Keep the mixture at 65 degrees C and Ph 9. • Dissolve .01 mol of N-Methyl-D-Glucamine in 10 ml of H2O and add HCl 2 mol/L to keep the Ph at 7 • Leave the reaction for 2 hours and, then precipitate the reaction with 100 ml of acetic acid • Place the mixture in the freezer until the next day • Filter and dry the precipitation The cytotoxicity effect of Meglumine Antimoniate against promastigotes forms were determined. The stationary phase L. major promastigotes were plated in 96-well vessels (Nunc) at 1x105cells per well, in Schneider’s medium, supplemented with 10% FBS and 2% human urine. The Meglumine Antimoniate solution was added at serial concentrations (100, 30, 10, 3, 1 and 0.3 μM). The cell also cultured with medium free from compounds or vehicle (basal growth control) or in media with DMSO 0.1% (vehicle control). After 48h, extracellular load of L. major promastigotes was estimated by count of promastigotes in Schneider’s medium in the CELM automatic cell counter (model CC530). Synthesis of Meglumine Antimoniate Results Characterization Discussion Pentavalent antimonial drugs were used worldwide for the treatment of VL and CL for over six decades. The characterization results following the IR analysis of Glucantime using KSb(OH)6, confirmed the formation of Meglumine Antimoniate. The cytotoxicity assay of L. major followed data obtained from analysis of 1H NMR and infrared corroborate those demonstrated by Demicheli et al (2003) confirming the formation of Meglumine Antimoniate. The Meglumine Antimoniate failed to inhibit the growth of forms of L. major promastigotes in any of the concentrations tested, so this result is consistent with the demonstrated by Ashutosh, Sundar and Goyal (2007). The synthesis methodology proposed by Demicheli et al (2003) was quite satisfactory, as well as being simple and fast characterization demonstrated the formation of Meglumine Antimoniate. (Glucantime) Future experiments should focus on synthesis, characterization and in vitro cytotoxicity assay of using Sb(V) Meglumine Antimoniate instead of Sb(III). We can also compare and contrast commercial to university laboratory Glucantime. Other follow up experiments could direct to amastigotes. In vivo experiments are also necessary to analyze Glucantime inside a living organism. Figure 6: Effect of Meglumine Antimoniate against promastigote forms of L. major. The experiments were performed in triplicate. Data are reported as means ± S.E.M. Differences with a pvalue 0.01 were considered significant in relation of medium group and pvalue 0.05 were considered significant in relation of vehicle group (DMSO). Statistical differences between the treated and the control groups were evaluated by ANOVA and Dunnett hoc tests. Acknowledgements Figure 5: Comparing obtained IR of N-Methyl-D-Glucamine and Meglumine Antimony with known IR of the compounds. References • Cynthia Demicheli, Rosemary Ochoa, Ivana Silva Lula, Fabio C. Gozzo, Marcos N. Eberlin and Frederic Frezard. Pentavalent organoantimonial derivatives: two simple and efficient synthetic methods for Meglumine Antimoniate. Minas Gerais, Brazil • Ashutosh, Shyam Sundar and Neena Goyal1. Molecular mechanisms of antimony resistance in Leishmania. Varanasi, India. • Grupo de Catálise e Reatividade Química (GCaR) – Instituto de Química e Biotecnologia (IQB – UFAL) • Laboratório de Farmacologia e Imunologia (LaFI) – Instituto de Ciências Biológicas e da Sáude (ICBS – UFAL) • Maria Jania Teixeira, Clarissa Romero Teixeira, Bruno Bezerril Andrade, Manoel Barral-Netto and Aldina Barral. Chemokines in host–parasite interactions in leishmaniasis. Salvador, Bahia, Brazil.C • Center of Disease Control. Leishmaniasis. Life Cycle. Image • Stanford University. Cutaneous Leishmania. Symptoms. Image.2006