The Synthesis, Characterization, and Study of
Leishmanicidal Activity of
Meglumine Antimoniate (Glucantime)
In Promastigote Forms of Leishmania major
2
1
4
3
U. Barrie , Prof. M. Meneghetti , R. Omena , G. Melo
1 University at Albany, 2 Federal University of Alagoas,
3 State University of New York at Oswego
Introduction To Leishmania
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.
Introduction To Glucantime
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.
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.
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]
• 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 .004 mol of N-MethylD-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
Synthesis of
Meglumine
Antimoniate
• After cooling, precipitation was
introduced by addition of 75 ml
of Acetone
• Precipitation is filtered and
dried
Characterization
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
• Dissolve .01 mol of N-Methyl-DGlucamine 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
Antileishmanial Assay Against Leishmania major
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 1x105 cells 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).
Results
• Place the mixture in the freezer
until the next day
• Filter and dry the precipitation
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 p value 0.01 were considered significant in relation of medium group
and p value 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