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was used) of the drug from a stock solution in dimethylsulfoxide
(DMSO). The final concentration of DMSO in the culture milieu
never exceeded 0.4%. Cultures with nontreated epimastigotes and
0.4% DMSO were included as negative controls, while cultures
with 8 mm of Nifurtimox were used as positive controls. The per-
centage of growth inhibition (PGI) was calculated as follows: PGI
(%)={1-[(ApÀA0p)/(AcÀA0c)]}100, where Ap =A600 of the culture
containing the drug at day 5; A0p =A600 of the culture containing
the drug just after adding the inoculum (day 0); Ac =A600 of the
culture in the absence of drug (negative control) at day 5; A0c =
A600 in the absence of the drug at day 0. In order to determine the
50% inhibitory concentration (IC50) values, parasite growth was fol-
lowed in the absence (negative control) and presence of increasing
concentrations of the corresponding drug. At day 5, the absorb-
ance of the culture was measured and related to the control. The
IC50 value was taken as the concentration of drug needed to de-
crease the absorbance ratio to 50%. All IC50 values in this work
were obtained by analysis with the program OriginLab8.5, using
sigmoidal regression (PGI vs. logarithm of the compound concen-
tration) and triplicate samples. The positive control PGI was always
around 50%.
tions of 5103 blood trypomastigotes (CL Brener). One group
(eight animals) was used as control (inoculated orally with the ve-
hicle), and two groups of animals were treated with the studied
derivatives (eight animals) or Benznidazole (seven animals), respec-
tively. The first parasitemia developed five days postinfection
(week 1), and the treatment began seven days later (when 80% of
the animals were infected). Compounds were administered orally,
using the aforementioned formulation, 0.2 mL at 50 mgkgÀ1 body
weight/day, during 15 days (daily, once a day). Parasitemia, in con-
trol and treated mice, was determined in tail-vein blood once
a week after the first administration during 60 days, and the mor-
tality rate was recorded. The number of parasites (trypomastigotes
form) in blood were counted manually in an optical microscope (at
40 magnification). The numbers of parasites in blood were aver-
aged for each group, and the number of parasites in blood vs.
time post-infection in days was graphed. The experimental proto-
cols with animals were evaluated and supervised by the local
Ethics Committee, and the research adhered to the Principles of
Laboratory Animal Care. These recommend five to eight animals
per group for a good relation between the number of parasites
and errors.
Nonspecific cytotoxicity assay.[17] J774.1 murine macrophage cells
(ATCC, USA) were grown in Dulbecco’s Modified Eagle’s Medium
(DMEM) culture milieu containing 4 mm l-glutamine and supple-
mented with 10% heat-inactivated fetal calf serum. The cells were
seeded in a 96-well plate (5104 cells in 200 mL culture medium)
and incubated at 378C in a 5% CO2 atmosphere for 48 h, to allow
cell adhesion prior to drug testing. Afterwards, cells were exposed
for 48 h to the compounds (12.5–400 mm) or vehicle for control
(0.4% DMSO), and additional controls (cells in milieu) were used in
each test. Cell viability was then assessed by measuring the mito-
chondria-dependent reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) to formazan. For this purpose,
MTT in sterile PBS (0.2% glucose) pH 7.4 was added to the macro-
phages to achieve a final concentration of 0.1 mgmLÀ1 and the
cells were incubated at 378C for 3 h. After removing the milieu,
formazan crystals were dissolved in DMSO (180 mL) and MTT buffer
(20 mL, 0.1m glycine, 0.1m NaCl, 0.5 mm EDTA, pH 10.5), and the
absorbance at 560 nm was measured. The IC50 was defined as the
drug concentration at which 50% of the cells were viable, relative
to the control (no drug added), and was determined by analysis
using OriginLab8.5 sigmoidal regression (% of viable cells vs. loga-
rithm of the compound concentration) for triplicate samples.
Expression and purification of TIMs. TcTIM, TbTIM, and HsTIM
were expressed in Escherichia coli and purified as described in the
literature.[7–11] After purification, the enzymes were dissolved in
100 mm triethanolamine, 10 mm EDTA ,and 1 mm dithiothreitol
(DTT, pH 8) and were precipitated with (NH4)2SO4 (75% saturation)
for storage at 48C. Before use, extensive dialysis against 100 mm
triethanolamine, 10 mm EDTA (pH 7.4) was performed. The purity
of the protein was analyzed by SDS-PAGE electrophoresis (TIM mo-
nomer is 27 kDa). Protein concentration was determined by ab-
sorbance readings at 280 nm. The e (mÀ1 cmÀ1) were 36440 for
TcTIM, 33460 for TbTIM, and for HsTIM concentration was deter-
mined by Bradford’s method, using the Bio-Rad protein assay, with
bovine serum albumin as standard.
TIM enzymatic activity and inhibition assays.[14,21,22] Enzymatic ac-
tivity was determined following the conversion of glyceraldehyde
3-phosphate (GAP) into dihydroxyacetone phosphate. The de-
crease in absorbance at 340 nm due to oxidation of NADH in a cou-
pled enzyme assay was followed in a multicell Hewlett–Packard
spectrophotometer at 258C. The reaction mixture (1 mL, pH 7.4)
contained 100 mm triethanolamine, 10 mm EDTA, 0.2 mm NADH,
1 mm GAP, and 0.9 units of a-glycerol phosphate dehydrogenase.
The reaction was initiated by addition of 1.0 nm of the correspond-
ing TIM or the corresponding TIM preincubated with the studied
compounds (from the mixture as described below). In these cases
of the inhibition assays, the enzymes at 1.0 mm were preincubated
for 2 h at 378C with the studied compounds (at different concen-
trations) in 10% DMSO. The average specific activity of TcTIM with
1 mm GAP as substrate was 3400 mmol(minmg)À1 as 100% of ac-
tivity. The IC50 was defined as the drug concentration at which
there is only 50% of the initial velocity, relative to the control (no
drug added), and was determined by analysis using OriginLab8.5
sigmoidal regression (% of enzymatic activity vs. logarithm of the
compound concentration). All assays were done in triplicate, and
the average error for each measurement did not exceed 10%.
Formulation for in vivo assays.[14,17,30] The lipid-based drug deliv-
ery system was prepared using 1.0 g of surfactant (460 mg polyox-
yl-40 hydrogenated castor oil, 360 mg of sodium oleate, and
180 mg of soya phosphatidylcholine), 1.0 g of cholesterol, and
phosphate buffer enough to make 10 mL of the vehicle. Prepara-
tion: each compound was pulverized in a porcelain mortar and
mixed with cholesterol, phosphatidylcholine, and polyoxyl-40 hy-
drogenated castor oil. The mixture was dissolved in CHCl3, and this
was evaporated under vacuum to dryness. To ensure complete re-
moval of the CHCl3, a stream of N2 was passed through the vial for
5 min. In parallel, sodium oleate was dissolved in phosphate buffer
and shaken for 12 h at rt in an orbital shaker. This solution was
then added to the mix containing the compounds, and the mix-
ture was homogenized and immersed in an ultrasonic bath at full
power for 30–60 min until the desired homogeneity and consisten-
cy were reached.
Cruzipain enzymatic activity and inhibition assays..[23,32] Cruzipain
was purified according to the work of Cazzulo et al.[33] Cruzipain
(2.5 mm e=58285mÀ1 cmÀ1) was incubated in 50 mm acetate buffer
pH 5.5 with 50 mm DTT, and 100 mm inhibitor was added, and the
solution was shaken for 15 min at 278C. The derivatives were
added diluted in DMSO, and the controls contained the same sol-
In vivo anti-T. cruzi activity (acute model).[14,17,31] BALB/c male
mice (30 days old, 25–30 g) were infected by intraperitoneal injec-
ChemMedChem 2016, 11, 1328 – 1338
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