Experimental Parasitology 164 (2016) 20e30

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Experimental Parasitology
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Full length article

In vitro and in vivo identification of tetradentated polyamine

complexes as highly efficient metallodrugs against Trypanosoma cruzi
Francisco Olmo a, Olaf Cusso
b, Clotilde Marín a, Maria Jose

Rosales a, Kristína Urbanova

a,
R. Luise Krauth-Siegel c, Miquel Costas b, Xavi Ribas b, Manuel Sa
nchez-Moreno a, *
a
n Biosanitaria (ibs.GRANADA), Hospitales Universitarios De Granada/Universidad de Granada,
Departamento de Parasitología, Instituto de Investigacio
Granada, Spain
b
QBIS Research Group, Institut de Quimica Computacional i Cata lisi (IQCC), and Departament de Química, Universitat de Girona, Campus de Montilivi, E-
17071, Girona, Spain
c
Biochemie-Zentrum der Universita €t Heidelberg, Im Neuenheimer Feld 328, 69120, Heidelberg, Germany

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Low toxicity alternative treatment

against Trypanosoma cruzi in mice.

Tetraamine 2 in vitro was 108 times
better than Bz.

Parasitemia reactivation of only 10.1%
in mice treated with Tetraamine 2.

50% of cure in the mice treated with
Tetraamine 3 after chronic phase.

a r t i c l e i n f o a b s t r a c t

Article history: In order to identify new compounds to treat Chagas disease during the acute phase with higher activity
Received 24 July 2015 and lower toxicity than the reference drug benznidazole (Bz), a series of tetraamine-based compounds
Received in revised form was prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light micro-
4 February 2016
scopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays
Accepted 5 February 2016
Available online 10 February 2016
against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were
quantified by fresh blood examination; the assignment of a cure was determined by PCR and reactivation
of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at
Keywords:
Trypanosomiasis
metabolic and ultra-structural levels by 1H NMR and TEM studies. Finally, as tetraamines are potentially
Chemotherapy capable of casuing oxidative damage in the parasites, the study was completed by assessing their activity
Polyamines as potential iron superoxide dismutase (Fe-SOD) and trypanothione reductase (TR) inhibitors. High-
Murine model selectivity indexes observed in vitro were the basis of promoting three of the tested compounds to
Chagas in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better
parasitemia inhibition values than those found for Bz. Tetraamines 2 and 3 induced a remarkable
decrease in the reactivation of parasitemia after immunosuppression and curative rates of 33 and 50%,
respectively. Tetraamine 3 turned out to be a great inhibitor of Fe-SOD and TR. The high anti-parasitic
activity and low toxicity render these tetraamines appropriate molecules for the development of an
affordable anti-Chagas agent.
© 2016 Elsevier Inc. All rights reserved.

* Corresponding author.

nchez-Moreno).
E-mail address: [email protected] (M. Sa

http://dx.doi.org/10.1016/j.exppara.2016.02.004
0014-4894/© 2016 Elsevier Inc. All rights reserved.
 F. Olmo et al. / Experimental Parasitology 164 (2016) 20e30 21

1. Introduction dichloromethane (3 mL) and the solution filtered off through cel-
ite©. Slow diethyl ether diffusion over the resultant solution
The World Health Organisation (WHO) lists Chagas disease as afforded, in a few days, 81 mg of white crystals (0.10 mmol, yield
one of the most neglected tropical diseases. Chagas disease is 65%). Anal. Calcd for C28H40F6MnN4O8S2: C, 42.37; H, 5.08; N, 7.06%.
endemic in Latin America; it is estimated that 10 million people are Found: C, 42.13; H, 4.89; N, 7.13%. FT-IR (ATR) n, cm1: 2934e2872
infected worldwide and more than 25 million are living at risk of (CeH)sp3, 1599, 1479, 1306, 1233, 1211, 1158, 1081, 1026, 879, 633,
infection (World Health Organization, 2012). The main drugs used 511. ESI-HRMS calcd. for C27H40F3MnN4O5S [M-OTf]þ: 644.2047,
for the treatment of Chagas disease are benznidazole (Bz, LAFEPE) found: 644.2061.
and nifurtimox (Lampit®, Bayer), which present significant side
effects and cure <20% of patients with chronic Chagas disease 2.3. Parasite strain culture
(Mckerrow et al., 2009). Thus, many researchers have combined
efforts to discover drugs against new targets, looking for lower T. cruzi SN3 strain of IRHOD/CO/2008/SN3 was isolated from
toxicity and a greater tolerance in patients, aiming not only for domestic Rhodnius prolixus; biological origin is Guajira (Colombia)
efficiency in the acute phase, but also in the chronic phase.
llez-Meneses et al., 2008). Epimastigote forms were grown in
(Te
One of the self-defence strategies of Trypanosoma cruzi (the axenic Grace's insect medium (Gibco) supplemented with 10%
etiological agent of Chagas disease) is its highly active and exclusive inactivated fetal bovine serum (FBS) at 28  C in tissue-culture
iron superoxide dismutase (Fe-SOD), which differs from CueZn- flasks, Roux flasks (Corning, USA) with a surface area of 75 cm2,
SOD or Mn-SOD operating in mammals. Fe-SOD is an extremely as described by us (Gonz
alez et al., 2005).
efficient enzyme for preventing any oxidative damage from the
host in combination with peroxidases. The other self-defence 2.4. Transformation of epimastigotes to metacyclic forms
strategy is trypanothione, a parasitic molecule involved in proto-
zoan protection against oxidative stress, which is now considered a Metacyclogenesis was induced by culturing a 5-day-old culture
virulence factor of Chagas disease (Piacenza et al., 2013) Thus, the of epimastigote forms of T. cruzi that was harvested by centrifu-
use of molecules that interfere with the enzyme trypanothione gation at 7000 g for 10 min at 10  C according to Cardoso and
reductase (TR), which keep the trypanothione molecule in its Soares, 2010.
reduced status, may lead to parasite death. In this sense, we focused
on complexes capable affecting or interfering with these exclusive 2.5. Cell culture and cytotoxicity tests
enzymes of T. cruzi.
In previous work, we designed iron and manganese coordina- Vero cells (Flow) were grown in RPMI and MEM (Gibco), sup-
tion complexes containing polyamine ligands, which are capable of plemented with 10% iFBS and the procedure followed was as in
generating highly oxidising species (Cusso
et al., 2013b; García-
lez et al., 2005.
Gonza
Bosch et al., 2012; Company et al., 2011) Highly reactive Mn(IV)]
O and Fe(IV)]O species embedded in these ligand scaffolds have 2.6. In vitro activity assays, extracellular forms
been prepared and their oxidative reactivity studied. Furthermore,
these polyamine ligands bind very strongly to iron and manganese 2.6.1. Epimastigotes assay
ions, forming coordination complexes that are stable under highly T. cruzi epimastigotes were collected in the exponential growth
acidic and oxidative conditions. phase and distributed in culture trays (with 24 wells) at a final
Herein, we report on the in vitro and in vivo anti-trypanosomal concentration of 5  104 parasites/well. The effects on the parasite
properties of Mn-based polyamines and polyamine complexes growth were tested according to Olmo et al., 2013.
(Fig. 1), which represent a class of compounds that have, so far, only
rarely been explored for Chagas disease chemotherapy. Finally, we 2.6.2. Blood trypomastigote forms assay
analyse the possible mechanism of action over the parasite struc- Compounds 4e5 were also evaluated in blood trypomastigote
ture and function as well as the protective enzymes mentioned forms of T. cruzi. BALB/c female mice infected with T. cruzi were
above. used 7 days after infection. Blood was obtained by cardiac puncture
using 3.8% sodium citrate as an anticoagulant in a 7:3 blood:-
2. Materials and methods anticoagulant ratio. The parasitaemia in the infected mice was
about 1  105 parasites/mL. The compounds were diluted in
2.1. Chemistry phosphate-buffered saline solution (PBS) to give a final concen-
tration 10, 25, and 50 mM for each product. Aliquots (20 mL) of each
Most of our compounds (1, 2, 4 and 5) have been previously solution were mixed in culture trays (96 wells) with 55 mL of
synthesized for organic synthesis purposes (Cusso
et al., 2013a; infected blood containing the parasites at a concentration of
Costas and Que, 2002) On the other hand, the compound 3 has approximately 1  106 parasites/mL. Infected blood with PBS, at the
been designed as a structural variant of compound 2 and synthe- same concentrations as the products, was used as control. The
sized for the first time in this work. The structures are shown in plates were shaken for 10 min at room temperature and kept at 4  C
Fig. 1. for 24 h. Each solution was examined microscopically (Olympus
CX41) for parasite counting using the Neubauer haemocytometric
2.2. Compound 3 synthesis chamber (a dilution of 1:100 in PBS was necessary to get into the
range of counting). The activity (percent of parasites reduction) was
A suspension of Mn(OTf)2 (69.7 mg, 0.16 mmol) in anhydrous compared with that of the control.
dichloromethane (1 mL) was added dropwise to a vigorously stirred
solution of (R,R)-dMMBPMCN (Cusso
et al., 2013a) (55.8 mg, 2.7. In vitro activity assays, intracellular forms: amastigotes assay
0.16 mmol) in dichloromethane (1 mL). After a few seconds the
solution became cloudy and a white precipitate appeared. After Vero cells were cultured in RPMI medium supplemented with
stirring for 1 h the solution was filtered off and the resultant white 10% iFBS, in a humidified 95% air and 5% CO2 atmosphere at 37  C.
solid dried under vacuum. This solid was solved in Then the cells were infected and treated as in Olmo et al., 2013.
22 F. Olmo et al. / Experimental Parasitology 164 (2016) 20e30

Fig. 1. Structure of the tetraamine-based compounds screened in this study.

2.8. Infectivity assay levels independent of the treatment. Therefore, on day 120, para-
sitaemia was shown to be undetectable by fresh blood microscopic
Vero cells were cultured in RPMI medium supplemented with examination, and the mice received 4 intraperitoneal injections of
10% iFBS as described above. Afterward, the cells were infected 200 mg/kg of body mass of cyclophosphamide monohydrate (CP)
in vitro with metacyclic trypomastigote forms of T. cruzi at a ratio of (ISOPAC®) on alternate days, as previously described (Cencig et al.,
10:1. The assay was performed as in Olmo et al., 2013. 2011). Within 1 week of the last CP injection, parasitaemia was
evaluated according to the procedure described for acute phase to
2.9. In vivo trypanosomicidal activity assay quantify the presence of blood trypomastigote forms as reac-
tivation rate. Finally, mice were bled out, under gaseous anaes-
2.9.1. Mice infection and treatment thesia (CO2), via heart puncture and blood was collected. Blood was
This experiment was performed using the rules and principles of incubated for 2 h at 37  C and then over night at 4  C in order to
the International Guide for Biomedical Research in Experimental allow clotting and then the serum was obtained from samples after
Animals and with the approval of the ethical committee of the centrifuging the supernatant twice at 1000 and 2700 g, consecu-
University of Granada, Spain. Groups of six BALB/c albino female tively. The serum was aliquoted and used for ELISA and biochemical
mice (6e8 weeks old, 25e30 g weight), maintained under a 12-h analysis, as explained below. Hearts were harvested and immedi-
dark/light cycle (lights on at 07:30 h) at a temperature of ately flushed free of blood by gentle infusion of 10 ml of pre-
22 ± 3  C and provided with water and standard chow ad libitum, warmed PBS through the left ventricle (Ye et al., 2008) in order to
were inoculated via the intraperitoneal route with 5  105 blood avoid contamination of the collected tissue with blood parasites.
trypomastigotes of T. cruzi obtained from previously infected mice After this, samples were frozen at 80  C and stored until used for
blood. The animals were divided as follows: I, positive control DNA extraction.
group (mice infected but not treated); II, study group (mice infected
and treated with the compounds under study). The administration 2.9.3. ELISA tests
of the testing compounds was begun on the seventh day of infes- Fe-SOD excreted from the parasite, cultured and processed as
tation once the infection was confirmed by intraperitoneal route, described in Lopez-Cespedes et al., 2012, was used as the antigen
and doses of 5 mg/kg body mass per day were used for 5 consec- fraction. The ELISA test to measure the antibodies against T. cruzi
utive days (7e12 days post-infection). Peripheral blood was ob- used was performed as in Olmo et al., 2014b.
tained from the mandibular vein of each mouse (5 mL samples) and
dissolved in 495 mL of a PBS solution at a dilution of 1:100. The 2.9.4. DNA extraction and PCR
circulating parasite numbers were quantified with a Neubauer's Hearts were defrosted and then ground using a Potter-Elvehjem
chamber for counting blood cells. This counting was performed to follow the purification procedure of the Wizard® Genomic DNA
every 3 days during a 40 day period (acute phase). The number of Purification Kit (Promega). PCR was performed using two primers
bloodstream forms was expressed as parasites/mL. designed in our laboratory (Olmo et al., 2014a), based on the pub-
lished sequence of the enzyme superoxide dismutase T. cruzi CL
2.9.2. Cyclophosphamide-induced immune suppression and Brenner (GenBank accession No. XM_808937), which amplifies a
assessment of cure fragment of approximately 300bp belonging to the superoxide
After day 60, the animals entered the chronic phase of the dismutase gene b of T. cruzi. The PCR was run in a total volume of
experiment where parasitaemia showed progressively decreasing 20 ml. Next, the amplification products were subjected to
 F. Olmo et al. / Experimental Parasitology 164 (2016) 20e30 23

electrophoresis on 1.6% agarose gel containing 1:10,000 GelRed™ excluded because the signal to noise ratio in this region was poorer
Nucleic Acid Gel Stain, for 90 min at 90 V. compared to that of the aliphatic region. The peak (2.6 ppm) cor-
responding to DMSO was removed before binning. The resulting
2.9.5. Toxicity tests by clinical chemistry measurements integrals were normalised to the working region (1.0e3.4) ppm of
A fraction of the serum obtained as it was shown above was the spectrum to correct for inter-sample differences in dilution. The
send to the Biochemical service in the University of Granada where binning and normalisations were achieved using Mestrenova 9.0
a series of parameters were measured according to their commer- software. The matrix obtained in Mestrenova was imported to
cial kits acquired from Cromakit® by BS-200 Chemistry Analyser Microsoft Excel for further data analyses.
Shenzhen Mindray (Bio-medical Electronics Co., LTD). With the
levels obtained for different populations of sera (n ¼ 15, n ¼ 6) we 2.10.3. Superoxide dismutase inhibition assay
calculated the mean value and standard deviation. Finally, we also The parasites cultured as described above were centrifuged. The
calculated the confidence interval for the mean normal populations pellet was suspended in 3 mL of STE buffer (0.25 M sucrose, 25 mM
based on a confidence level of 95% (100  (1a) ¼ 100 TriseHCl, 1 M EDTA, pH 7.8) and disrupted by three cycles of sonic
(10.05)%). The ranges obtained are shown in Table 2, which allows disintegration, 30 s each at 60 W. The sonicated homogenate was
comparison and analysis of the sera studied in this work. centrifuged at 1500 g for 5 min at 4  C, and the pellet was washed
three times in ice-cold STE buffer. This fraction was centrifuged
2.10. Assays to figure out the mechanism of action (2500 g for 10 min at 4  C) and the supernatant was collected. The
protein concentrations were determined using the Bradford
2.10.1. Ultrastructural alterations method (Bradford, 1972). Iron and copperezinc superoxide dis-
The parasites were cultured at a density of 5  105 cells/mL in mutases (Fe-SOD and CueZn-SOD) activities were determined us-
each corresponding medium containing the compounds tested at ing the method described by Beyer and Fridovich (Beyer and
the concentration of IC25. After 96 h, these cultures were centri- Fridovich, 1987).
fuged at 400 g for 10 min and the pellets produced were washed in
PBS before being mixed with 2% (v/v) paraformaldehyde/glutaral- 2.10.4. TR inhibition studies
dehyde in 0.05 M cacodylate buffer (pH 7.4) for 24 h at 4  C. Recombinant TR from T. cruzi (TcTR) was prepared following a
Following this, the pellets were prepared for transmission electron published procedure (Sullivan and Walsh, 1991). TS2 was generated
microscopy study using a technique described by our group enzymatically as described previously (Comini et al., 2009). The TR
(Ferna
ndez-Becerra et al., 1997).
activity was measured in a total volume of 1 mL of 40 mM Hepes
and 1 mM EDTA at pH 7.5 in the presence of 100 mM NADPH by
2.10.2. Metabolite excretion varying the concentrations of TS2 (20, 40, 80, 100 and 200 mM) and/
Cultures of T. cruzi epimastigote forms (initial concentration of or the inhibitor (0, 28 and 100 mM). The absorption decrease, owing
5  105 cells/mL) received IC25 of the compounds (except for the to NADPH oxidation, was followed at 340 nm and 25  C. Stock so-
control cultures). After incubation for 96 h at 28  C, the cells were lutions of the inhibitors were prepared in water according to their
centrifuged at 400 g for 10 min. The supernatants were collected in solubility.
order to determine the excreted metabolites through 1H NMR, and The Ki and Ki0 values were determined using the following
the chemical shifts were expressed in parts per million (ppm), us- equations (Dixon and Webb, 1986):
ing dimethyl sulphoxide (DMSO) as the reference signal. One-
dimensional 1H NMR spectra were acquired on VARIAN DIRECT ½I
Ki ¼

DRIVE 400 MHz Bruker spectrometer with AutoX probe using D2O. ½I
KmðobsÞ 1þK 0
The assignments of metabolites were based on 1D NMR spectrum. i

Km 1
The chemical shifts used to identify the respective metabolites
were consistent with those described previously by our group
(Ferna
ndez-Becerra et al., 1997). In addition, the human metab- ½I
Ki0 ¼ Vmax
olome database (http://www.hmdb.ca/) was also used for this
VmaxðobsÞ 1
purpose. The spectral region of 1.0e5.5 ppm was bucketed into a
frequency window of 0.1 ppm. The region corresponding to water The ability of the compounds to induce the oxidase activity of TR
(4.5e5.5 ppm) was excluded during binning to avoid artefacts due was measured in the presence of 100 mM NADPH and about 2 U/mL
to pre-saturation of water, and the region corresponding to glucose of TR, with 40 or 100 mM of the specified compound in a total
(3.4e3.8 ppm) was also excluded. The aromatic region was volume of 1 mL. Under these conditions, spontaneous NADPH

Table 1
In vitro activity, toxicity and selectivity index for the polyamine compounds and complexes on different forms of Trypanosoma cruzi.

Compound IC50 (mM)a Toxicity IC50 Vero cell (mM) SIb

Epimastigote forms Amastigote forms Trypomastigote forms Epimastigote forms Amastigote forms Trypomastigote forms

Bz 15.8 ± 1.1 23.3 ± 4.6 22.4 ± 1.9 13.6 ± 0.9 0.8 0.6 0.6
Comp 1 41.5 ± 3.0 22.7 ± 1.4 23.5 ± 1.8 31.0 ± 2.3 0.7 (1) 1.4 (2) 1.3 (2)
Comp 2 8.2 ± 0.6 1.2 ± 0.2 1.4 ± 0.2 78.0 ± 11,0 9.5 (12) 65 (108) 55.7 (93)
Comp 3 13.6 ± 0.9 9.1 ± 0.3 4.4 ± 0.2 88.1 ± 12.0 6.5 (8) 9.7 (16) 20 (33)
Comp 4 33.6 ± 0.7 14.5 ± 0.8 9.5 ± 0.5 108.0 ± 10.0 3.2 (4) 7.5 (12) 11.4 (19)
Comp 5 10.4 ± 0.3 14.4 ± 0.5 13.6 ± 0.7 248.3 ± 8.6 23.9 (30) 17.2 (29) 18.3 (30)

The values are the mean plus standard deviation derived from four separate determinations.
a
IC50 ¼ the concentration that inhibits cell proliferation by 50%. In each case, the IC50 was estimated using the regression function with higher goodness of fit (R2), at six
different concentrations (0.1e100 mM).
b
Selectivity Index ¼ IC50 Vero cells/IC50 T. cruzi. The numbers in parenthesis give the ratio of SI values for the compound compared to the reference drug benznidazole (Bz).
24 F. Olmo et al. / Experimental Parasitology 164 (2016) 20e30

Table 2
Summarizes the Biochemical clinical parameters tested in different groups of Balb/c Mice infected with Trypanosoma cruzi at different experimental situations.

Kidney markers profile Heart markers profile Liver markers profile

Urea Uric acid CK-MB (U/ LDH (U/L) AST/GOT ALT/GPT Total Alkalyne
(mg/dL) (mg/dL) L) (U/L) (U/L) Bilirubin(mg/ phosphatase (U/L)
dL)

UNINFECTED MICE (n ¼ 15) 39 [36 5 [4.3e5.5] 453 [215 3086 [2108 126 [103 46 [37 0.23 [0.17 133 [104e161]
e43] e690] e4064] e148] e54] e0.28]
INFECTED MICE-ACUTE PHASE (n ¼ 15) 49 [39 4.5 [3.7 681 [400 2910 [1589 129 [100 48 [38 0.15 [0.12 231 [161e300]
e60] e5.5] e950] e4232] e157] e58] e0.18]
120 days POST INFECTION MICE (n ¼ 6) 40 4.56 742 3104 133 52.34 0.209 164.5
120 days POST INFECTION and Compound 2 15 mg/kg$w ¼ - ¼ – – ¼ ¼ ¼
TREATED MICE (n ¼ 6)
120 days POST INFECTION and Compound 3 15 mg/kg$w þþþ þ þþþ – – ¼ ¼ ¼
TREATED MICE (n ¼ 6)

Key: ¼, variation no larger than 10%; þ, up to 10% of increasing over the range; þþ, up to 30% of increasing over the range; þþþ, up to 40% of increasing over the range; þþþþ,
more than 50% of increasing over de range; -, up to 10% of decreasing over the range; - -, up to 30% of decreasing over the range; - - -, up to 40% of decreasing over the range; - -
- -, more than 50% of decreasing over de range.

oxidation results in a minimal absorption decrease. This activity cell (Fig. 2a), the results were consistent with those mentioned
was not accelerated in the presence of the compounds, ruling out above for infection rates. After treatment, compounds 2, 3 and 5
the possibility that they act as subversive substrates (data not significantly reduced the number of amastigote forms per cell by
shown). 81, 59 and 56%, respectively, whereas Bz showed reduction of only a
39%. The average number of amastigote forms increased to a
3. Results and discussion maximum on the sixth day of culture, but decreased thereafter
(Fig. 2b); the rupture of Vero cells released trypomastigote forms. In
3.1. Chemistry terms of the number of trypomastigote forms found per millilitre of
culture medium (Fig. 2c), a maximum was reached on day 10
Manganese-based coordination complexes (1e3) were selected (9.5  103 trypomastigote forms/mL) for the control, whereas this
for their ability to generate high-oxidation-state compounds, value was substantially reduced by the reference drug (41%), but
which are potentially capable of causing oxidative damage in the even more so by compounds 2, 3 and 5 (84, 76 and 72%, respec-
parasite. Additionally, water-soluble compounds (4, 5) were stud- tively). In summary, the results of the parasite spreading in Vero
ied to evaluate potential Fe-chelation in causing Fe-SOD dysfunc- cells are in agreement with those of the trypanocidal activity re-
tion and inducing parasite death. ported in Table 1 for intracellular and extracellular forms of T. cruzi,
with compound 2 being much better than any of the other drug
candidates tested.
3.2. In vitro trypanosomicidal evaluation

All five polyamines showed better selectivity indexes (SI ¼ IC50 3.3. In vivo activity of tetradentate polyamines
Vero cells/IC50 extracellular and intracellular forms of T. cruzi)
compared to the reference drug Bz. As summarised in Table 1, The promising in vitro results prompted us to study the in vivo
compound 2, with SI values 108 and 93 times higher than those of activity of these polyamines in female BALB/c mice. As the effec-
Bz, was the best compound, independent of the parasite stage. This tiveness of the drugs currently in use against Chagas disease varies
activity meets the criteria given by Nwaka and co-workers (Nwaka widely between the acute and chronic phase, we decided to eval-
et al., 2011) and Romanha and co-workers (Romanha et al., 2010) uate the impact of our compounds on both phases. For the acute
and is, therefore, considered as a strong candidate to undergo phase experiments, we considered the first 40 days after infection
in vivo studies. On the other hand, compounds 3 and 5 also showed (dpi), whereas the effect on the chronic phase was studied between
quite good trypanocidal activity, thus resulting SI values that were days 40 and 120 after infection. The intraperitoneal (i.p.) doping
even better than those of Bz. route was preferred over the intravenous procedure, because it is
To obtain more accurate information about the most active well known that i.p. treatment substantially reduces animal mor-
polyamines (2, 3, and 5), the development of the parasites in Vero tality (Da Silva et al., 2008). In fact, no mouse died in any of our
cells was studied by measuring the rates of infection and the experiments with either the control or with the compounds
average number of amastigote and trypomastigote forms present assayed at the concentrations used (15 mg/kg of body mass).
during a ten-day treatment period. Vero cells were infected with However, the survival percentage for the mice treated with Bz was
metacyclic forms of T. cruzi and the gradual differentiation into only about 80%. The female mice were inoculated with trypomas-
amastigote forms was followed. During the observation time, in the tigote forms, as described in the Experimental section, and i.p.
control group, the rate of infection of the host cells gradually treatment with the compounds was started five days post-infection
increased, reaching 99% invasion at the end of the experiment and maintained for five consecutive days. A group of mice (control
(Fig. 2a). The test was repeated in the presence of the reference group) was treated in the same manner, but using only the vehicle.
drug and in the presence of compounds 2, 3 or 5 at their IC25 During the study of acute-phase activity, the level of parasitemia
concentrations. It was found that the rate of infection decreased in was determined every two days. Fig. 3 shows the number of try-
all cases, with respect to the control, and compounds 2, 3 and 5 pomastigote forms found during the acute phase (1e40 dpi). On the
showed reductions in the infection rate of 84, 66 and 55%, day of maximum parasite burden (20e22 days after infection), all of
respectively, thus with much a higher efficiency than Bz (19% the tested compounds greatly reduced the number of blood try-
reduction). pomastigote forms compared with the control group. Furthermore,
In terms of the average number of amastigote forms per Vero compounds 2, 3 and 5 lowered the level of parasitemia on day 40 by
 F. Olmo et al. / Experimental Parasitology 164 (2016) 20e30 25

Fig. 2. Effect of compounds 2, 3, 5 and Bz on the infectivity of T. cruzi in Vero cells. (A) Rate of infection, (B) mean number of amastigote forms per infected Vero cell and (C) number
of trypomastigote forms in the culture medium after treatment with the IC25 of the control (filled circles), Bz (filled squares), compound 2 (filled triangles), compound 3 (open
triangles) and compound 5 (open squares). The results are the mean values of three separate experiments and the error bars represent the mean ± standard deviation.

59, 35 and 27%, respectively. This effect was significantly greater reactivation of parasitemia after immunosuppression. It can be
than that found for the reference drug (11%). The decreasing order appreciated that the control group recovers half of its initial peak
of in vivo activity on trypomastigote forms in the acute phase was of parasitemia, with a reactivation of 55.5%. In contrast, the treated
found to be 2 >> 3 > 5 > Bz. groups show a parasitemia load of only 10.1% with respect to their
The chemical structure of these coordination complexes (2, 3 burden peak when the mice have been administered with com-
and 5) is fundamentally different from that of Bz. Therefore, the pound 2, as well as a low reactivation (12.7%) for those treated
mechanisms underlying their anti-trypanosomal activity most with compound 3; less significant was the decrease in reactivation
likely differ, and simple structureeactivity correlations should be for the group treated with compound 5, which was closer to that
regarded with caution. of the untreated group (40.2%). ELISA assays offer an alternative
Therefore, these group of mice, treated with polyamines 2 and method for verifying the effectiveness of these three compounds
3, where kept until 120 dpi was achieved under the same condi- in the chronic phase after being challenged with an immuno-
tions. The experiment was concluded with an immunosuppression suppression cycle. Fig. 4b shows the total Ig-G levels of anti-T. cruzi
test on the treated mice 120 days after p.i. (late chronic phase, in and, according to previous reports, the titer of Ig-G in Balb/c be-
which there are no parasites remaining in the bloodstream that comes stable during chronic phase of the disease (el Bouhdidi
are quantifiable by fresh blood analysis). Fig. 4a shows the et al., 1994); this is confirmed in Fig. 4b. The control group
26 F. Olmo et al. / Experimental Parasitology 164 (2016) 20e30

showed an almost triplication in the Ig-G levels in response to the experiments and modify the schedule of treatment for a better
presence of the parasite in the bloodstream after immunosup- exposure of the compound in the bloodstream.
pression. However, in the treated group, the differences between Biochemical clinical parameters were tested in different groups
immunosuppressed (IS) and non-immunosuppressed (non-IS) of BALB/c mice infected with T. cruzi in different experimental sit-
groups were smaller and not significant in the case of compounds uations (Table 2), showing that an increase in the dosage does not
2 and 3; however, treatment with compound 5 showed a doubled cause a toxicity risk. All biochemical parameters tested, regarding
Ig-G value compared to the non-IS group. This means that the the kidney, heart and liver profiles, were not altered after com-
parasite-specific Ig-G levels were not higher than the remaining pound administration, with the exception of compound 3, for
amount of total unspecific Ig-G caused by the hyper- which the uric acid, urea and CK-MB values increased significantly.
gammaglobulinaemia characteristic of the infection by T. cruzi The latter could be related to its excretion, which could cause some
(Bryan et al., 2010). abnormalities in kidney function. Regarding compound 2, this
Finally, Fig. 4c shows the polymerase chain reaction (PCR) re- analysis could allow us to increase the dosage for a better efficacy,
sults after necropsy. The hearts were grinded and underwent a total considering that no toxicity was observed. It also remarkable how
DNA extraction and amplification of a fragment within the parasite the LDH parameter, which is a marker of heart damage associated
SOD gene; the hearts of the control animals showed the ubiquitous
presence of the parasite. In contrast, the hearts from mice treated
with compound 2 were relatively clean from parasites (33%) and
50% were clean when treated with compound 3, thus confirming
the partial curative effect of these compounds at this dosage. No
mice were clean of parasites when they were treated with com-
pound 5.
The in vivo study clearly showed that compounds 2 and 3 were
the two most effective compounds; therefore, they were selected
for a deeper study to gain insight into their mechanism of action. In
order to improve the effectiveness of compounds 2 and 3, we must
take into consideration an increase in the dosage for future

Fig. 4. Post-mortem analyses on the final day after necropsy. (A) Percentage of
reactivation of blood parasitemia after immunosuppression, (B) total Ig-G levels of
anti-T. cruzi, where the error bars represent the mean ± standard deviation and (C)
Fig. 3. Parasitemia in the murine model of acute Chagas disease (A and B). Control PCR of mouse heart tissue. M, marker; , PCR negative control; þ, PCR positive
(filled triangles) and doses of 15 mg/kg body mass of Bz (filled rhombus), compound 2 control; lanes 1e3 represent the non-IS mice in the final days of the experiment;
(open triangles), compound 3 (filled squares) and compound 5 (filled circles). The grey lanes 4e6 represent the IS mice; x, infection control group; ¶, infected and treated
shading represents the days of treatment. Values are the means of six separate mice with compound 2; ♯, infected and treated with compound 3; D, infected and treated
and error bars represent the mean ± standard deviation. with compound 5.
 F. Olmo et al. / Experimental Parasitology 164 (2016) 20e30 27

with the presence of parasites, decreased between a 30 and 40% in Similar, but less intense, effects were produced by compound 3
both treatments compared to the untreated group. (Fig. 6e, f), in which the mitochondria were much more swollen and
disorganised, as in the previous item, and were also present in a
3.4. Metabolite excretion study lighter shade than usual. The most degenerated structure was that
of K, which was almost unrecognisable, showing an enormous size
As trypanosomatids are unable to completely degrade glucose and being completely disorganised. To a lesser extent, the mito-
to CO2, they excrete a considerable portion of their hexose skeleton chondria were swollen and altered, and a dilatation of the FP could
as partially oxidised fragments in the form of fermented metabo- be appreciated. Cytoplasm of the epimastigote forms was filled
lites, whose nature and percentage depend on the pathway used for with vacuoles of different types, some were empty and some
glucose metabolism (Bringaud et al., 2006; Ginger, 2005). The contained debris, electron-granules or concentric myelin bands and
catabolism products in T. cruzi were acetate and succinate, with many lipid vacuoles. The plasma membrane was also altered, with
smaller percentages of L-alanine and D-lactate, in agreement with small blisters.
the data in previously published reports (Turrens, 1999). Detection
of large amounts of succinate as a major end product is an usual
feature, because it relies on glycosomal redox balance, enabling re-
3.6. Inhibitory effect on T. cruzi Fe-SOD
oxidation of the NADH produced in the glycolytic pathway. Succinic
fermentation requires only half of the phosphoenolpyruvate pro-
The effect of compounds 2 and 3 on T. cruzi Fe-SOD was assayed
duced to maintain the NADþ/NADH balance, and the remaining
at concentrations ranging from 0.5 to 100 mM. We used epi-
pyruvate is converted inside the mitochondria and the cytosol is
mastigote forms of T. cruzi, which excrete Fe-SOD when cultured in
converted into acetate, D-lactate, L-alanine or ethanol, according to
a medium lacking inactivated foetal calf serum (FCS) (Villagran
the degradation pathway followed by each species (Michels et al.,
et al., 2005). The results are summarised in Fig. 7 together with
2006). In order to obtain some information concerning the effect
the corresponding IC50 values. Fig. 7b shows the respective data for
of the tested compounds on glucose metabolism in the parasites,
CueZn-SOD from human erythrocytes. A comparison of Fig. 7a and
we obtained 1H NMR spectra of T. cruzi epimastigote forms after
b reveals that, in the case of compound 3, Fe-SOD was clearly
treatment with compounds 2 and 3, from which the final excretion
inhibited, whereas the effect on the human CueZn-SOD was sub-
products were identified qualitatively and quantitatively (spectra
stantially lower. Compound 3 caused 100% inhibition of Fe-SOD,
not shown). The results were compared with those obtained from
even at a concentration of 25 mM, and thus showed a lower IC50
parasites maintained in a cell-free medium (control) for four days
value compared to human SOD. Compound 2 did not significantly
after inoculation with the parasite. The characteristic presence of
interfere with any of the SOD species.
acetate, succinate, D-lactate and L-alanine was confirmed in the
Thus, in addition to their effect in glucose catabolism, the
control experiments. As expected, succinate and acetate were the
compounds interfere with the parasite Fe-SOD. As the mitochon-
most abundant end products identified. However, after treatment
dria are an essential part of the antioxidant protective response
of the parasites with compounds 2 and 3, the excretion of metab-
(Piacenza et al., 2007) the inhibition of Fe-SOD suggests that
olites was slightly altered at the dosages employed. Fig. 5 displays
compound 3 could be a good candidate for the target mentioned.
these modifications with respect to the control at the height of the
Future modelling studies will be carried out to further investigate
spectral peaks, corresponding to the most representative final
this point.
excretion products. Marked differences in the catabolic pathways
appeared, which seemed to be connected with the trypanocidal
activity mentioned above. The most actives compounds 2 and 3
decreased the amount of succinate by 11 and 10%, respectively; in 3.7. Inhibition of TR
addition, an increase in the amount of acetate, D-lactate and L-
alanine by 22, 21 and 12%, respectively, was found upon treatment The two polyamines 2 and 3 with the highest anti-parasitic
with compound 2, whereas the same metabolites increased by 25, activity were also studied as inhibitors of T. cruzi TR. Neither of
14 and 15% upon treatment with compound 3. So, in conclusion, the compounds showed spontaneous oxidation of NADPH by
even though the disturbances were quantifiable, we could not themselves. At fixed concentrations of 40 and 100 mM of both in-
attribute the metabolism of the involved enzymes to the direct hibitor and the substrate trypanothione disulfide (TS2), the degree
targets of the compounds. of inhibition varied between 23 and 72% (Fig. 8). Compound 3
showed a higher inhibitory activity, reaching 72% at 100 mM of the
3.5. Ultra-structural alterations compound and 40 or 100 mM TS2, suggesting an inhibition mech-
anism that is independent of the substrate concentration. Com-
The ultra-structural alterations produced by compounds 2 and 3 pound 2 was less effective, yielding 23% inhibition when 100 mM of
were studied against the epimastigote forms of T. cruzi. As shown in the compound and 40 mM TS2 were applied. To obtain a deeper
Fig. 6, comparing to the control (Fig. 6a), all parasites showed some insight into the mode of inhibition, compound 3 was subjected to a
common disturbances, but we also found some specific alterations. detailed kinetic analysis.
Compound 2 is more active, causing excessive expansion and Indeed, LineweavereBurk analysis revealed that compound 3
swelling at the flagellar pocket (FP) and the Golgi apparatus (GA), as behaves as a mixed-type inhibitor of TR (Fig. 9). The calculation of
shown in Fig. 6b. The mitochondria appeared extremely swollen the inhibition constants, as described in the Materials and Methods
and, in some cases, they were empty with a few crypts (Fig. 6c,d). section (Dixon and Webb, 1986), yielded average Ki and Ki’ values of
The number of lipid vacuoles was higher than usual and, in some 7 and 49 mM, respectively (Ki ¼ 40; Ki’ ¼ 6 for 28 mM inhibitor and
parasites, there were vesicles in the plasma membrane. This Ki ¼ 58; Ki’ ¼ 8 for 100 mM inhibitor), so the values for the different
disturbance in the plasma membrane was exclusive at this level concentrations of inhibitor do not differ from the average by more
and the microtubules were properly engaged. A higher number in than 12 or 16% for Ki or Ki’, respectively. The lines in the double-
ribosomes with a strong density were also noticed. The kinetoplast reciprocal plot cut above the x-axis, and thus Ki < Ki0 , which in-
(K) lost its helical structure and appeared as a dark stain within the dicates that compound 3 has a higher affinity for the free enzyme
mitochondria. compared to the TResubstrate complex.
28 F. Olmo et al. / Experimental Parasitology 164 (2016) 20e30

Fig. 5. Variation percentages in the height of the peaks corresponding to metabolites excreted by T. cruzi epimastigote forms in the presence of tetradentate polyamines at their IC25
compared to a control sample. Values are the means of three separate experiments and error bars represent the mean ± standard deviation.

Fig. 6. Ultra-structural alterations of epimastigote forms of Trypanosoma cruzi treated with tetradentated polymanines. (A) Control of T. cruzi with typical aspect, (BeD) epi-
mastigote forms treated with compound 2, (EeF) epimastigote forms treated with compound 3. Structures codes: nucleus (N), mitochondrion (M), kinetoplast (K), ribosomes (R),
glicosomes (G), Flagellar pocket (FP), lipid vacuoles (LV) and vacuoles (V). Scale bar ¼ 1 mm.
 F. Olmo et al. / Experimental Parasitology 164 (2016) 20e30 29

Fig. 9. LineweaverBurk plot for the inhibition of TcTR by compound 3. The assays
contained 100 mM NADPH and either zero or a fixed concentration of inhibitor, as
indicated in the graph. The TS2 concentration was varied between 20, 40, 80, 100 and
200 mM.

Financial & competing interests disclosure

This work was supported by: Spanish Ministry of Economy and

Competitiveness (MINECO), CONSOLIDER-INGENIO 2010 CSD2010-
00065. European Research Foundation for Project ERC-2009-StG-
239910, MICINN for project CTQ2009-08464. INNPLANTA project
INP-2011-0059-PCT-420000-ACT1. Authors declare no conflicts of
interest. No written assistance has been required for the content
Fig. 7. (A) In vitro inhibition (%) of Fe-SOD from T. cruzi epimastigote forms by the but the English has been corrected by the company Proof Reading
compounds (activity 20.77/3.18 U/mg). (B) In vitro inhibition (%) of CueZn-SOD from Service (Devonshire Business Centre. Works Road. Letchworth
human erythrocytes for the compounds (activity 23.36/4.21 U/mg). The differences
Garden City. SG6 1GJ. United Kingdom).
between the activities of the control homogenate and those incubated with com-
pounds were obtained according to the NewmaneKeuls test. Values in the legend
represented in brackets are the IC50, which is the concentration required to give 50% Acknowledgements
inhibition and was calculated by linear-regression analysis from the Kc values at the
concentrations employed (0.5e100 mM). The values are the average of three separate
determinations and the error bars represent the mean ± standard deviation. We acknowledge Generalitat de Catalunya for an ICREA
Academia Award (X.R. and M.C.). F.O. is grateful for a FPU Grant
from the Ministry of Education of Spain (AP-2010-3562) and he also
thanks Natalie Dirdjaja and Dr. Alejandro Leroux for their support
with the kinetic analysis of TR.

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