A chloroquinoline derivate presents effective in vitro and in vivo antileishmanial activity against Leishmania species that cause tegumentary and visceral leishmaniasis

Article abstract of DOI:10.1016/j.parint.2019.101966

The identification of new therapeutics to treat leishmaniasis is desirable, since available drugs are toxic and present high cost and/or poor availability. Therefore, the discovery of safer, more effective and selective pharmaceutical options is of utmost

Full text of DOI:10.1016/j.parint.2019.101966

ParasitologyInternational73(2019)101966
Contents lists available at ScienceDirect
Parasitology International
journal homepage: www.elsevier.com/locate/parint
A chloroquinoline derivate presents effective in vitro and in vivo
antileishmanial activity against Leishmania species that cause tegumentary
and visceral leishmaniasis
Jessica K.T. Sousaa,1, Luciana M.R. Antinarellia,1, Débora V.C. Mendonçaa,1, Daniela P. Lagea,1
Grasiele S.V. Tavaresa,1, Daniel S. Diasa, Patrícia A.F. Ribeiroa, Fernanda Ludolfa,
Vinicio T.S. Coelhoa, João A. Oliveira-da-Silvaa, Luísa Perina, Bianka A. Oliveirab,
,
Denis F. Alvarengab, Miguel A. Chávez-Fumagallia, Geraldo C. Brandãoc, Vandack Nobrea,
⁎
Guilherme R. Pereirab, Elaine S. Coimbrad, Eduardo A.F. Coelhoa,e,
a Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, 30130-100 Belo
Horizonte, Minas Gerais, Brazil
b Pontifícia Universidade Católica de Minas Gerais, Departamento de Física e Química, Instituto de Ciências Exatas e Informática, 30535-901 Belo Horizonte, Minas
Gerais, Brazil
c Escola de Farmácia, Universidade Federal de Ouro Preto, 35400-000 Ouro Preto, Minas Gerais, Brazil
d Departamento de Parasitologia, Microbiologia e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, 36036-900 Juiz de Fora, Minas
Gerais, Brazil
e Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Minas Gerais, Brazil
A R T I C L E I N F O
Keywords:
Abstract: The identification of new therapeutics to treat leishmaniasis is desirable, since available drugs are toxic
and present high cost and/or poor availability. Therefore, the discovery of safer, more effective and selective
pharmaceutical options is of utmost importance. Efforts towards the development of new candidates based on
molecule analogs with known biological functions have been an interesting and cost-effective strategy. In this
context, quinoline derivatives have proven to be effective biological activities against distinct diseases. In the
present study, a new chloroquinoline derivate, AM1009, was in vitro tested against two Leishmania species that
cause leishmaniasis. The present study analyzed the necessary inhibitory concentration to preclude 50% of the
Leishmania promastigotes and axenic amastigotes (EC50 value), as well as the inhibitory concentrations to pre-
clude 50% of the murine macrophages and human red blood cells (CC50 and RBC50 values, respectively). In
addition, the treatment of infected macrophages and the inhibition of infection using pre-treated parasites were
also investigated, as was the mechanism of action of the molecule in L. amazonensis. To investigate the in vivo
therapeutic effect, BALB/c mice were infected with L. amazonensis and later treated with AM1009.
Parasitological and immunological parameters were also evaluated. Clioquinol, a known antileishmanial qui-
noline derivate, and amphotericin B (AmpB), were used as molecule and drug controls, respectively. Results in
both in vitro and in vivo experiments showed a better and more selective action of AM1009 to kill the in vitro
parasites, as well as in treating infected mice, when compared to results obtained using clioquinol or AmpB.
AM1009-treated animals presented significantly lower average lesion diameter and parasite burden in the in-
fected tissue and organs evaluated in this study, as well as a more polarized antileishmanial Th1 immune re-
sponse and low renal and hepatic toxicity. This result suggests that AM1009 should be considered a possible
therapeutic target to be evaluated in future studies for treatment against leishmaniasis.
Antileishmanial activity
Chloroquinoline derivate
Treatment
Leishmaniasis
Toxicity
Mammalian hosts
⁎ Corresponding author at: Laboratório de Pesquisa do, Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de
Medicina, Universidade Federal de Minas Gerais, Avenida Prof. Alfredo Balena, 190, 30.130-100, Belo Horizonte, Minas Gerais, Brazil.
E-mail address: eduardoferrazcoelho@yahoo.com.br (E.A.F. Coelho).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.parint.2019.101966
Received 17 December 2018; Received in revised form 27 June 2019; Accepted 26 July 2019
Availableonline27July2019
1383-5769/©2019ElsevierB.V.Allrightsreserved.

J.K.T. Sousa, et al.
ParasitologyInternational73(2019)101966
1. Introduction
purpose is to identify new candidates to be tested in future studies for
treatment against leishmaniasis, which can present higher activity and
selectivity when compared to previously described antileishmanial
agents.
Leishmaniases are diseases caused by protozoan parasites of the
Leishmania genus, which present an annual estimated incidence of 1.5
to 2.0 million new cases, which range between 1.0 and 1.5 million cases
of tegumentary leishmaniasis (TL), coupled with approximately 0.5
million new cases of visceral leishmaniasis (VL). Epidemiological data
have proven that this disease complex is endemic in 98 countries,
mainly in subtropical and tropical regions in the world [1,2]. TL can
lead to self-healing cutaneous lesions, even causing mutilation and
morbidity in patients. The disease is usually caused by Leishmania
braziliensis, L. amazonensis, L. guyanensis, L. mexicana, and L. lainsoni
species in the Americas, whereas VL, which is considered a fatal disease
if acute and left untreated, can be caused by L. donovani and L. infantum
species [3,4].
Treatment against leishmaniasis has been based on the use of pen-
tavalent antimonials, although other drugs, such as free and liposomal
amphotericin B (AmpB), miltefosine, paramomycin, and pentamidine
have been also used [5]. However, problems related to their toxicity,
high cost, and/or development of resistant strains have limited the ef-
ficacy of these therapeutics [6,7]. The prolonged administration of
these compounds can cause adverse effects, such as renal, hepatic, and
cardiac toxicity. These can also show variable efficacy according to the
parasite strain, immune status of the hosts, and the emergence of drug
resistance, all of which have been observed in the subcontinent of India
and other regions [8,9]. Consequently, since the search to identify new
antileishmanial targets is hampered due to the high investment neces-
sary to develop new products, as well as by the lack of a profitable drug
market [10], alternative means through which to identify new antil-
eishmanial agents, such as plant derivates or synthetic products, could
help to solve this relevant problem and make it possible to identify new
therapeutics to be applied against leishmaniasis [11,12].
2. Materials and methods
2.1. Synthesis of AM1009
To perform the synthesis of AM1009, 220 mg (0.668 mmol) N-(3-
bromopropyl)-7-chloroquinolin-4-amine was added to a round bottom
flask containing 464 mg (4.68 mmol) of cyclohexanamine, 462 mg
(3.34 mmol) of potassium carbonate, and 1 mL of dimethylformamide
PA. The flask was shaken for 20 h at 120 °C, and the reaction was
completed by thin layer chromatography. After, the compound was
mixed using distilled water and dichloromethane PA, and AM1009 was
purified through a filtration using silica, which was eluted with 50%
ethyl acetate and 50% methanol. The molecule was dried on the rotary
evaporator, and characterized by 1H 13C NMR and mass spectrometry,
presenting an estimated yield of 76.0%.
2.2. Mice and parasites
This study was approved by the Committee for the Ethical Handling
of Research Animals (CEUA) from the Federal University of Minas
Gerais (UFMG), logged under protocol number 085/2017. BALB/c mice
(female, 8 weeks old) were purchased from the UFMG Institute of
Biological Sciences, and were kept in pathogen-free conditions.
Leishmania amazonensis (IFLA/BR/1967/PH-8) and L. infantum
(MHOM/BR/1970/BH46) strains were used. Stationary promastigotes
were grown in complete Schneider's medium (Sigma-Aldrich, USA),
which consisted of a medium plus 20% heat-inactivated fetal bovine
serum (FBS, Sigma-Aldrich, USA) and 20 mM L-glutamine pH 7.4, at
24 °C. The soluble L. amazonensis antigenic extract (SLA) was prepared
from stationary promastigote cultures as described [33]. To obtain the
axenic amastigotes, 109 stationary promastigotes were washed three
times in sterile phosphate buffer saline (PBS 1x) and incubated for 48 or
72 h (L. amazonensis and L. infantum, respectively) in 5 mL FBS at 37 °C.
The parasites were then washed three times in cold PBS 1×, and their
morphology was evaluated after staining by the Giemsa method in an
optical microscope as described [19].
The interest in natural and/or synthetic molecules has increased in
recent decades, and new agents have been evaluated in experimental
trials [13–16], although most have been tested in in vitro studies, and
few in vivo treatment experiments have been developed using mam-
malian models [17–19]. Distinct molecule classes, such as flavonoids,
terpenoids, quinolines, among others, have shown variable degrees of
antileishmanial activity [20–23]. In this aspect, quinoline derivatives
have been applied as an important drug class, showing potent anti-
tumoral, antiprotozoal, antimicrobial, and anti-inflammatory activity,
among others, and did not cause significant toxicity in the hosts
[24–27].
2.3. In vitro antileishmanial activity
Research groups have demonstrated the selective antileishmanial
activity of the quinoline-based compounds against Leishmania models
[28–30]. One of these molecules, clioquinol (5-chloro-7-iodoquinolin-8-
ol), demonstrated high efficacy against L. amazonensis and L. infantum
promastigotes and amastigotes, without causing toxicity in murine
macrophages. It was also effective in the treatment of infected macro-
phages and in the inhibition of the infection of these cells using pre-
treated parasites [31]. In another study, clioquinol was incorporated to
a Poloxamer 407-based delivery system, and the formulation proved to
be effective in the treatment of L. amazonensis-infected BALB/c mice
[32].
The in vitro inhibition of Leishmania growth was evaluated by in-
cubating L. infantum or L. amazonensis stationary promastigotes and
axenic amastigotes (106 cells; each) in the presence of varied con-
centrations of AM1009 (0, 0.06, 0.12, 0.25, 0.49, 0.98, 1.97, 3.93, 7.87,
15.73, and 31.46 μM) or clioquinol (0, 0.07, 0.13, 0.26, 0.51, 1.03,
2.05, 4.09, 8.19, 16.37, and 32.73 μM), in 96-well culture plates (Nunc,
Nunclon, Roskilde, Denmark) for 48 h at 24 °C. AmpB (0, 0.04, 0.07,
0.14, 0.27, 0.54, and 1.08 μM; Sigma-Aldrich, USA) was used as a drug
control. Cell viability was assessed by the 3-(4.5-dimethylthiazol-2-yl)-
2.5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) method. The
In this context, in the present study, a new chloroquinoline derivate,
optical density (OD) values were read in a microplate spectro-
AM1009
[N1-(7-chloroquinolin-4-yl)-N3-cyclohexylpropane-1,3-dia-
photometer (Molecular Devices, Spectra Max Plus, San Jose, CA, USA)
at 570 nm. The data were entered into Microsoft Excel (version 10.0)
mine], was in vitro tested against two Leishmania species that cause VL
and TL around the world. The 50% Leishmania inhibitory concentration
(EC50) and the 50% macrophage inhibitory concentration (CC50) in
murine macrophages and in human red blood cells (RBC50) were in-
vestigated. Additionally, the treatment of infected macrophages and the
inhibition of infection using pre-treated parasites were also evaluated,
as were the mechanism of action in L. amazonensis and the in vivo
therapeutic efficacy in treating Leishmania-chronically infected BALB/c
mice. As a molecule control, clioquinol was used in the in vitro and in
vivo experiments, as was AmpB, which was used as a drug control. Our
spreadsheets, and the 50% Leishmania inhibitory concentration (EC50
)
evaluated for promastigotes and axenic amastigotes was calculated by
applying a sigmoidal regression of the dose-response curve [34].
2.4. Cytotoxicity in murine macrophages and human red cells
The cytotoxicity was evaluated in murine macrophages (5 × 105
cells), which were incubated with varied concentrations of AM1009 (0,
0.06, 0.12, 0.25, 0.49, 0.98, 1.97, 3.93, 7.87, 15.73, and 31.46 μM) or
2

J.K.T. Sousa, et al.
ParasitologyInternational73(2019)101966
clioquinol (0, 0.07, 0.13, 0.26, 0.51, 1.03, 2.05, 4.09, 8.19, 16.37, and
32.73 μM) in RPMI 1640 medium and in 96-well plates (Nunc) for 48 h
at 37 °C and 5% CO2. AmpB (0, 0.08, 0.17, 0.34, 0.68, 1.35, 2.70, 5.40,
and 10.80 μM) was used as a control. The macrophage viability was also
assessed by the MTT method. The data were entered into Microsoft
Excel (version 10.0) spreadsheets, and the 50% macrophage inhibitory
concentration (CC50) was calculated by applying a sigmoidal regression
of the dose-response curve [34]. The cytotoxicity was also evaluated in
human cells, when the 50% red blood cell inhibitory concentration
(RBC50) was determined by incubation of AM1009, clioquinol, or
AmpB, in the same concentrations as described above, with a 5% red
blood cell (human O+ type) suspension, for 1 h at 37 °C. The material
was centrifuged by 1000 ×g for 10 min, when the cell lyses was de-
termined spectrophotometrically at 570 nm. Negative (saline) and po-
sitive (distilled water) controls were used, and the values were calcu-
lated by also applying a sigmoidal regression of the dose-response
curve.
control [35].
2.6.3. Cell cycle analysis and autophagy vacuole formation
Initially, to evaluate the cell cycle, promastigotes (107 cells) were
cultured in the absence or presence of AM1009 (2.41 and 4.84 μM) for
24 h at 25 °C. Parasites were then fixed with 70% ethanol for 1 h at 4 °C
and incubated with ribonuclease A (200 μg/mL; Sigma-Aldrich, USA)
for 1 h at 37 °C. Cells were stained with propidium iodide (7.0 μg/mL;
Sigma-Aldrich, USA) for 20 min in the dark at room temperature. For
each sample, 10,000 events were acquired in a FACsCanto II flow cyt-
ometer (Becton Dickinson, Rutherford, NJ, USA), which was equipped
with DIVA software (Joseph Trotter, Scripps Research Institute, La
Jolla, CA, USA), as described [36]. To investigate the autophagy va-
cuole formation, treated parasites were incubated with mono-
dansylcadaverin reagent (100 μM; Sigma-Aldrich, USA) for 1 h in the
dark at 25 °C. Cells were washed twice with PBS 1×, and 200 μL of
suspension were added into a black 96-well plate, at which time they
were analyzed in a spectrofluorometer, by using 335 nm and 460 nm for
excitation and emission, respectively [37].
2.5. Treatment of infected macrophages and inhibition of the infection
To evaluate the efficacy of compounds in treatment-infected mac-
rophages, cells (5 × 105) were plated on round glass coverslips within
24-well plates in RPMI 1640 medium, which was supplemented with
20% FBS and 20 mM L-glutamine, pH 7.4, and incubated for 24 h at
37 °C in 5% CO2. The stationary promastigotes were the added to the
wells, and cultures (10 parasites per macrophage) were incubated for
48 h at 37 °C in 5% CO2. Free parasites were removed by extensive
washing with RPMI 1640 medium, and infected macrophages were
treated with AM1009 or clioquinol (0, 0.5, 1.0, 2.5, and 5.0 μM; each)
for 48 h at 24 °C in 5% CO2. AmpB (0, 0.11, 0.54, and 1.08 μM) was
used as a control. To evaluate the inhibition of infection using pre-
treated parasites, promastigotes (5 × 106 cells) were first incubated
with AM1009, clioquinol, or AmpB, in the same concentrations as de-
scribed above, for 4 h at 24 °C. The cells were then washed three times
in RPMI 1640 medium, quantified, and added to infect murine mac-
rophages (10 parasites per one macrophage), for 24 h at 37 °C in 5%
CO2. After fixation with 4% paraformaldehyde, cells were washed,
stained with Giemsa and the evaluation of the infection, as well as the
number of recovered amastigotes per infected cell, were determined by
counting 200 cells in triplicate by using an optical microscope.
2.7. In vivo antileishmanial activity
2.7.1. Infection and treatment schedule
To evaluate the in vivo therapeutic efficacy of AM1009, clioquinol,
and AmpB in the treatment of BALB/c mice, animals (n = 8 per group)
were subcutaneously infected in the base of the tail with L. amazonensis
stationary promastigotes (106 cells). Fifty to 60 days after infection,
mice were separated into groups, aiming to ensure a similar average
lesion diameter between them. They then received subcutaneous in-
jections near the site of infection, once a day and during 7 days, using
saline (PBS 1× pH 7.4), AmpB (1 mg/kg body weight), clioquinol
(5 mg/kg body weight), or AM1009 (5 mg/kg body weight). After the
treatment, the lesion average diameter was measured using an elec-
tronic caliper (799–6/150 model, Starrett®, Brazil); 15 days later, they
were euthanized, when parasitological and immunological evaluations
were performed.
2.7.2. Parasite load in treated and infected animals
The infected tissue, spleen, liver, and draining lymph nodes (dLN) of
treated and infected animals were collected, and the parasite load was
evaluated by limiting dilution technique. For this, infected tissue and
organs were macerated in a glass tissue grinder using sterile PBS 1×,
tissue debris were removed by centrifugation at 150 ×g, and cells were
concentrated by centrifugation at 2000 xg. The pellets were re-
suspended in 1 mL of complete Schneider's medium. Log-fold serial
dilutions were performed in Schneider's medium with a 10−1 to 10−12
dilution, and each sample was plated in triplicate and read 7 days after
the beginning of the culture at 24 °C. Results were expressed as the
negative log of the titer (i.e., the dilution corresponding to the last
positive well) adjusted per milligram of tissue or organ.
2.6. Mechanism of action in Leishmania amazonensi
2.6.1. Evaluation of mitochondrial membrane potential
Stationary promastigotes (107 cells) were cultured in the absence or
presence of AM1009 (2.41 and 4.84 μM, corresponding to one and two
times the EC50 values, respectively), for 24 h at 25 °C. The cells were
then washed and incubated with JC-1 reagent (10 μg/mL; Sigma-
Aldrich, USA) for 30 min and in the dark. After washing twice with
HBSS, cells were added to a black 96-well plate, and the mitochondrial
membrane potential (Δψm) was measured in a spectrofluorometer
(FLx800, BioTek Instruments, Inc., Winooski, VT, USA) at 528 and
600 nm, using 485 nm as the excitation wavelength. FCCP (1.0 μM)-
incubated promastigotes were used as a positive control. Δψm values
were calculated by the ratio between the reading at 600 nm and
528 nm, as described [35].
2.7.3. Investigating the immunological profile
Cell response was evaluated in spleen cells of treated and infected
animals. For this, splenocytes (5 × 106 cells) were plated in duplicate in
24-well plates (Nunc) and incubated in complete DMEM (medium),
which was composed by medium plus 20% FBS and 20 mM L-glutamine
pH 7.4, or stimulated with L. amazonensis SLA (25.0 μg/mL), for 48 h at
37 °C, 5% CO2. IFN-γ, IL-4, IL-10, IL-12p70, and GM-CSF levels were
measured in the cell supernatant by capture ELISA (BD Pharmingen®,
San Diego, CA, USA), according to the manufacturer's instructions. The
nitrite production was also evaluated in the supernatant by the Griess
method [37]. The humoral response was investigated by determining
anti-parasite IgG1 and IgG2a antibody levels. For this, SLA was added
in the ELISA plates (1.0 μg per well), sera samples were diluted at 1:100
in PBS-T (PBS 1× plus 0.05% Tween 20), and anti-mouse IgG1 and
IgG2a horseradish-peroxidase conjugated antibodies (Sigma-Aldrich)
2.6.2. Reactive oxygen species production
Stationary promastigotes (107 cells) were cultured in the absence or
presence of AM1009 (2.41 and 4.84 μM) for 24 h at 25 °C. Parasites
were then incubated with H2DCFDA reagent (20 μM; Sigma-Aldrich,
USA) for 30 min in the dark at room temperature. To evaluate the re-
active oxygen species (ROS) production, the fluorescence intensity was
measured in a spectrofluorometer (Varioskan® Flash, Thermo Scientific,
USA) at 485 and 528 nm for excitation and emission, respectively.
Miltefosine (22.1 μM)-treated promastigotes were used as a positive
3

J.K.T. Sousa, et al.
ParasitologyInternational73(2019)101966
were employed both in a 1:10,000 dilution in PBS-T. Reactions were
developed using H2O2, ortho-phenylenediamine and citrate-phosphate
buffer, pH 5.0, for 30 min and in the dark, and were stopped by adding
H2SO4 2 N. The OD values were measured in an ELISA microplate
spectrophotometer (Molecular Devices, Spectra Max Plus, Canada) at
492 nm.
such data, the SI values were calculated, and results were 61.80, 77.63,
and 6.54 against L. amazonensis promastigotes, and of 144.6, 270.1, and
2.5 against the amastigotes, respectively. For the L. infantum species,
the SI values were 392.0, 137.8, and 9.4 against the promastigotes,
respectively, and of 152.0, 168.0, and 4.7 against the axenic amasti-
gotes, respectively (Table 2).
3.2. Treatment of infected macrophages and inhibition of infection
2.7.4. Evaluating the organic toxicity
To verify if the compounds were toxic for treated and infected an-
imals, commercial kits (Labtest Diagnostica®, Belo Horizonte) were
tested using sera samples of these mice, at which time the hepatic (AST
and ALT) and renal (urea and creatinine) functions were evaluated.
Samples from non-treated and non-infected animals (n = 4) were em-
ployed as a control.
The treatment of infected macrophages was evaluated, and results
showed that all therapeutics were able to reduce the percentage of in-
fected macrophages and the number of recovered amastigotes after
treatment. With the obtained values, reductions in the infectiveness
degree and in the number of amastigotes by macrophage were calcu-
lated, and data showed that both AM1009 and clioquinol showed most
significant reductions in the parasitism in the treated cells, when
compared to the treatment using AmpB (Table 3). The inhibition of
infection using pre-treated parasites also demonstrated that AM1009
and clioquinol significantly reduced the infection and parasitism in the
mammalian cells, presenting similar values when both the percentage
of infection and the number of recovered amastigotes were evaluated
(Table 4).
2.8. Statistical analysis
The EC50, CC50, and RBC50 values were entered into Microsoft Excel
(version 10.0) spreadsheets and calculated by dose-response curves,
which were plotted in GraphPad Prism 5.03. Results were analyzed by
the one-way analysis of variance (ANOVA), followed by the Dunnett's
post-test to comparison between the groups. Results were expressed as
mean
standard deviation. Three independent experiments, which
3.3. Mechanism of action in L. amazonensis
presented similar results, were performed. Differences were considered
significant with P < .05.
The mechanism of action of AM1009 in Leishmania was evaluated
using L. amazonensis species. The evaluation of ΔΨm illustrated that
treatment with this molecule induced significant parasite membrane
depolarization, in the order of 24.6%, when 2.41 μM of AM1009 was
used. The positive control showed a ΔΨm reduction of 35.9% (Fig. 1).
The ROS production was also evaluated as an indicator of oxidative
stress, and the results showed that AM1009-treated parasites presented
higher production of this molecule, when compared to the control
(Fig. 2). AM1009-treated parasites also showed significant alterations
in their cell cycle, due to the occurrence of cells in the sub-G0/G1 phase
(Fig. 3), as well as by the formation of autophagic vacuoles, thus in-
dicating cell apoptosis (Fig. 4).
3. Results
3.1. Antileishmanial activity, cytotoxicity, and selectivity index
The in vitro antileishmanial activity of AM1009 was evaluated
against L. amazonensis and L. infantum promastigotes and axenic
amastigotes. As molecule and drug control, clioquinol and AmpB were
used, respectively. The EC50 values for AM1009, clioquinol, and AmpB
against L. amazonensis promastigotes were 2.41
and 0.13
and 0.34
Regarding the L. infantum species, the EC50 values against the pro-
mastigotes were of 0.38
respectively, and of 0.98
0.28, 7.90
0.22, 2.27
0.65,
0.44,
0.04 μM, respectively, and of 1.03
0.11 μM against the axenic amastigotes, respectively.
3.4. In vivo therapeutic effect against L. amazonensis infection
0.13, 4.45
0.15, 3.65
0.98, and 0.09
0.25, and 0.18
0.02 μM,
0.05 μM
To evaluate the in vivo therapeutic effect of compounds against
Leishmania infection in a mammalian model, BALB/c mice were in-
fected with L. amazonensis promastigotes. At 50 to 60 days post-infec-
tion, the mice were separated into groups, and received saline or were
treated with AM1009, clioquinol, or AmpB. The therapeutic efficacy
was then evaluated by measuring the average lesion diameter and
parasite load in the animals' infected tissue, liver, spleen, and dLN.
Results showed significant reductions in the average lesion diameter
and in the parasite load in the treated mice, when compared to saline
group (Fig. 5). Comparing the results between them, animals receiving
AM1009 presented lower lesion diameter (Fig. 5A) and parasite load
(Fig. 5B) in the infection site and evaluated organs, when compared to
clioquinol and AmpB groups. In an attempt to investigate the im-
munological profile generated in the animals, Th1 and Th2-type cyto-
kines were measured in the cell supernatant of stimulated splenocytes.
Results showed that clioquinol or AM1009-treated mice presented a
more polarized Th1 response, with specific production of IFN-γ, IL-12,
and GM-CSF, which were associated with lower antileishmanial IL-4
and IL-10 production (Fig. 6). Treatment using AmpB also induced the
production of IFN-γ, IL-12, and GM-CSF but only in lower levels when
compared to values obtained in the other groups (Fig. 6A). In addition,
AM1009-treated mice, as compared to the clioquinol group, showed
higher IFN-γ and lower IL-4 and IL-10 production, although no sig-
nificant difference has been found between them. In an attempt to
evaluate the parasite-specific activation of macrophages in the treated
groups, the nitrite presence in culture supernatants was assayed as an
against the amastigote forms (Table 1). The cytotoxicity in murine
macrophages
613.27 20.53, and 0.85
AmpB, respectively; while the RBC50 values were 1739.69
showed
CC50
0.16 μM for AM1009, clioquinol, and
79.69,
64.35, and 13.93 1.95 μM, respectively (Table 2). With
values
of
148.94
10.12,
1409.85
Table 1
Antileishmanial activity against Leishmania promastigotes and axenic amasti-
gotes. L. amazonensis and L. infantum stationary promastigotes and axenic
amastigotes (106 cells; each) were incubated with AM1009 (0, 0.06, 0.12, 0.25,
0.49, 0.98, 1.97, 3.93, 7.87, 15.73, and 31.46 μM), clioquinol (0, 0.07, 0.13,
0.26, 0.51, 1.03, 2.05, 4.09, 8.19, 16.37, and 32.73 μM), or AmpB (0, 0.04,
0.07, 0.14, 0.27, 0.54, and 1.08 μM) for 48 h at 24 °C. The cell viability was
analyzed by the MTT method. The 50% Leishmania inhibitory concentrations
(EC50) evaluated for each parasite stage were calculated by applying a sig-
moidal regression of the dose-response curve. Results were expressed as
mean
standard deviation.
Compounds
Antileishmanial activity (EC50; in μM)
Leishmania amazonensis
Leishmania infantum
Promastigotes Axenic
Promastigots Axenic
amastigotes
amastigotes
AM1009
Clioquinol
Amphotericin B 0.13
2.41
7.90
0.28
0.65
0.04
1.03 0.22
0.38
4.45
0.09
0.13 0.98
0.98 3.65
0.02 0.18
0.15
0.25
0.05
2.27
0.34
0.44
0.11
4

J.K.T. Sousa, et al.
ParasitologyInternational73(2019)101966
Table 2
Evaluation of the cytotoxicity and selectivity index. Murine macrophages (5 × 105 cells) were incubated with AM1009 (0, 0.06, 0.12, 0.25, 0.49, 0.98, 1.97, 3.93,
7.87, 15.73, and 31.46 μM), clioquinol (0, 0.07, 0.13, 0.26, 0.51, 1.03, 2.05, 4.09, 8.19, 16.37, and 32.73 μM) or AmpB (0, 0.04, 0.07, 0.14, 0.27, 0.54, and 1.08 μM)
for 48 h at 37 °C. The cell viability was analyzed by the MTT method. The cytotoxicity was also evaluated in human cells, at which time the 50% red blood cell
inhibitory concentration was determined through the incubation of AM1009, clioquinol, or AmpB, in the same concentrations as described above, with a 5% red
blood cell (human O+ type) suspension, for 1 h at 37 °C. In both cases, the CC50 and RBC50 values were calculated by applying a sigmoidal regression of the dose-
response curve. Results were expressed as a mean
standard deviation.
Compouns
CC50 (μM)
RBC50 (μM)
Selectivity index
Leishmania amazonensis
Promastigotes
Leishmania infantum
Axenic amastigotes
Promastigotes
Axenic amastigotes
AM1009
Clioquinol
Amphotericin B
148.94
613.27
0.85
10.12
20.53
0.16
1739.69
1409.85
13.93
79.69
64.35
1.95
61.80
77.63
6.54
144.60
270.16
2.50
391.95
137.81
9.44
152.0
168.0
4.7
Table 3
Treatment of infected macrophages. Murine macrophages were infected with L. amazonensis promastigotes (10 parasites per one macrophage) and later treated with
AM1009 or clioquinol (0, 0.5, 1.0, 2.5, and 5.0 μM; each) for 48 h at 24 °C in 5% CO2. AmpB (0, 0.11, 0.54, and 1.08 μM) was used as the control. The reduction of
infectivity and the percentage of amastigotes in treated and infected macrophages were determined after counting 200 cells in triplicate. Results were expressed as
mean
standard deviation.
Compounds
Concentration (μM)
Infectiveness reduction (%)
L. amazonensis L. infantum
Number of recovered amastigotes in treated macrophages
L. amazonensis L. infantum
AM1009
5.0
2.5
1.0
0.5
0
5.0
2.5
1.0
0.5
0
91.7
72.9
53.4
28.9
(−)
80.9
53.2
28.7
18.8
(−)
89.1
79.2
65.2
(−)
5.5
4.6
3.8
2.9
89.9
70.3
52.4
24.5
(−)
67.7
40.8
18.9
10.4
(−)
85.8
71.8
51.7
(−)
6.7
5.2
4.9
3.3
0.3
0.7
1.8
2.9
6.8
2.0
2.8
3.5
4.4
6.8
1.7
2.2
3.7
6.8
0.1
0.2
0.3
0.7
1.6
0.6
0.9
1.7
1.1
1.6
0.5
0.6
1.2
1.6
0.2
0.4
1.4
2.5
4.7
1.5
1.9
2.6
3.5
4.7
1.3
2.0
3.1
4.7
0
0.2
0.4
1.0
0.5
0.6
0.7
1.0
0.8
0.5
0.4
0.7
1.2
0.5
Clioquinol
5.1
4.7
3.3
2.5
4.0
2.7
3.4
1.6
Amphotericin B
1.08
0.54
0.11
0
3.1
4.2
5.4
2.5
3.0
2.7
Table 4
Inhibition of infection of macrophages using pre-treated parasites. The inhibition of infection of macrophages using pre-treated parasites was evaluated by incubating
cells (5 × 106) with AM1009 or clioquinol (0, 0.5, 1.0, 2.5, and 5.0 μM; each) for 1 h at 24 °C. AmpB (0, 0.11, 0.54, and 1.08 μM) was used as a control. Parasites were
then washed, quantified, and incubated with macrophages (10 parasites per one macrophage) for 24 h at 37 °C in 5% CO2. The percentage of infection using pre-
treated parasites and the number of recovered amastigotes per infected macrophage were determined after counting 200 cells in triplicate. Results were expressed as
mean
standard deviation.
Compounds
Concentration (μM)
Percentage of infection using pre-treated parasites
L. amazonensis L. infantum
Number of recovered amastigotes per infected macrophage
L. amazonensis L. infantum
AM1009
5.0
2.5
1.0
0.5
0
5.0
2.5
1.0
0.5
0
10.2
21.2
48.5
66.5
85.5
23.6
51.5
64.3
73.5
85.5
15.5
29.8
45.5
85.5
1.7
3.5
4.3
5.7
3.4
3.3
3.8
4.4
5.3
3.4
1.6
3.2
4.2
3.4
11.3
24.3
45.5
58.8
63.3
26.2
49.7
52.6
58.6
63.3
17.7
26.7
41.2
63.3
2.0
2.5
5.0
4.4
3.2
2.7
6.0
4.9
3.4
3.2
2.1
1.8
3.2
3.2
0.5
1.0
2.3
3.5
6.5
1.5
2.5
3.6
4.1
6.5
0.5
1.5
2.8
6.5
0.2
0.4
0.9
0.9
1.4
0.8
0.7
0.7
1.3
1.4
0.1
0.3
0.5
1.4
0.4
0.9
2.0
3.1
4.5
1.3
2.0
2.7
3.6
4.5
0.5
1.3
2.3
4.5
0.2
0.3
0.6
1.1
0.3
0.3
0.6
1.0
0.7
0.3
0.2
0.2
0.6
0.3
Clioquinol
Amphotericin B
1.08
0.54
0.11
0
indicator of NO production. Results showed that the nitrite production
was higher in the AM1009-treated and infected animals, when com-
pared to the other groups (Fig. 6B). To characterize the humoral re-
sponse triggered by the treatments, we have analyzed the profile of
parasite-specific IgG1 and IgG2a isotypes. Results showed that treated
animals produced higher IgG2a isotype levels, as compared to the IgG1
production, consequently presenting a higher IgG2a/IgG1 ratio when
compared to the values obtained in the saline group (Fig. 6C). AM1009-
treated mice were those presenting higher antibody isotype ratios when
compared to the other groups. The organic toxicity showed that AmpB-
5

J.K.T. Sousa, et al.
ParasitologyInternational73(2019)101966
Fig. 1. Evaluation of the mitochondrial membrane potential in AM1009-treated
parasites. L. amazonensis promastigotes (107 cells) were treated with 2.41 and
4.84 μM of AM1009 (corresponding to one and two times the EC50 values, re-
spectively) for 24 h, when they were probed with JC-1 reagent. FCCP (1 μM)-
treated promastigotes were used as a positive control. The analysis of labeling
was performed in a fluorometer microplate reader. Data were expressed as
Fig. 4. Formation of autophagy vacuoles in AM1009-treated parasites. The
formation of autophagy vacuoles in AM1009-treated L. amazonensis promasti-
gotes was evaluated by fluorimetry, after incubation with AM1009 (2.41 and
4.84 μM, corresponding to one and two times the EC50 values, respectively) for
24 h. Non-treated promastigotes were used as a positive control. Results were
mean
standard deviation of the groups. (⁎⁎⁎) indicates statistically sig-
nificant difference in relation to the control (P < .001).
expressed as mean
standard deviation of the groups. (⁎⁎) and (⁎⁎⁎) indicate
statistically significant difference in relation to the control (P < .01 and
P < .001, respectively).
Fig. 2. Production of reactive oxygen species in AM1009-treated L. amazo-
nensis. Promastigotes (107 cells) were untreated (negative control) or treated
with AM1009 (2.41 and 4.84 μM, corresponding to one and two times the EC50
values, respectively) for 24 h, at which time they were probed with the
H2DCFDA reagent. Miltefosine (22.1 μM)-treated promastigotes were used as a
positive control. The fluorescence intensity was evaluated in a fluorometer
microplate reader. Data were expressed as mean
standard deviation of the
groups. (⁎⁎) and (⁎⁎⁎) indicate statistically significant difference in relation to
the control (P < .01 and P < .001, respectively).
Fig. 5. In vivo therapeutic efficacy of the tested compounds. To evaluate the
therapeutic action of AM1009, clioquinol, and AmpB in a mammalian model,
BALB/c mice were infected with 107 L. amazonensis promastigotes. At 50 to
60 days post-infection, these mice were divided into groups (n = 8 per group)
and received saline or were treated with AmpB, clioquinol, or AM1009. The
lesion development was monitored weekly, and, 15 days after treatment, the
parasite load was evaluated in the infected tissue, liver, spleen, and draining
lymph nodes by applying the limiting dilution technique. Results showing the
lesion's average diameter (A) and parasite load (B) are expressed as the
Fig. 3. Action of AM1009 on the L. amazonensis cell cycle. Parasites (107 cells)
were incubated without (untreated cells) or with AM1009 (2.41 and 4.84 μM,
corresponding to one and two times the EC50 values, respectively) for 24 h, at
which time they were loaded with propidium iodide, and the DNA content was
analyzed by flow cytometry. The promastigote percentage in each phase of the
cell cycle was calculated and results are shown. Data were expressed as
mean
standard deviation of the groups. (⁎) indicates statistically significant
difference in relation to the control (P < .01).
mean
standard deviation of the groups. (⁎) indicates a statistically sig-
treated mice showed higher levels of renal and hepatic damage mar-
kers, when compared to the others (Fig. 7). Similar to that found in the
immunological evaluation, AM1009-treated mice were those presenting
lower levels of these enzymes, when compared to the other groups.
nificant difference in relation to the saline and AmpB groups (P < .0001).
4. Discussion
Advances in the biochemical research applied in in vitro and/or in
vivo parasitological and immunological studies to kill Leishmania
parasites have been developed in order to identify new and non-toxic
6

J.K.T. Sousa, et al.
ParasitologyInternational73(2019)101966
effectiveness in killing Leishmania parasites [40].
In the present study, a chloroquinoline derivative was tested for this
biological purpose, and results proved to be highly effective in in vitro
antileishmanial action against two important Leishmania species, L.
amazonensis and L. infantum, in both parasite stages. Moreover, the
molecule exhibited low toxicity in murine macrophages and in human
red blood cells, showing satisfactory selectivity index values. The effect
of AM1009 against intracellular amastigotes was observed in the
treatment of infected macrophages, since this compound significantly
diminished the infection percentage and the number of recovered
amastigotes. In addition, the chloroquinoline derivate also demon-
strated a potential to inhibit the macrophage infection, since the pre-
treatment of parasites with the molecule induced a lower degree of
infection in the murine cells, as well as a lower parasite load when the
number of recovered amastigotes was evaluated. Another chlor-
oquinoline derivative, clioquinol, was also used as a molecule control.
Antileishmanial activity also combatted both parasite species, together
with lower toxicity in murine macrophages and in human cells.
However, results obtained with AM1009 were most significant in terms
of efficacy of the treatment of infected macrophages and the inhibition
of infection using pre-treated parasites.
Quinolines are heterocyclic compounds found in several plant fa-
milies and that can be synthetized chemically. These are used in both
folk and traditional medicines with a distinct biological activity, such as
antimicrobicidal, anticancer, and antileishmanial agents [41]. In gen-
eral, structural modifications in defined molecules are a cheaper and
faster pathway to find new agents with biological activity [42]. In this
aspect, quinoline derivatives have been produced and tested as antil-
eishmanial agents, although the majority of the studies have performed
in vitro experiments, while few are in vivo tested in infected hosts
[32,40]. No less important, comparisons between the results obtained
using such molecules in mammalian models must also be performed,
especially when immunological and parasitological parameters are
evaluated, in an attempt to select the best candidates for future studies
in the treatment against disease.
Since the purpose of our study was to identify new antileishmanial
targets to be applied in a future moment in clinical practice, in vivo
experiments using a murine model were developed. For this, BALB/c
mice were experimentally infected with L. amazonensis promastigotes
and then treated with our molecule. Clioquinol and AmpB were used as
controls. The efficacy of the products was evaluated by means of
parasitological and immunological parameters, which were in-
vestigated 15 days after the treatment in the chronically-infected mice.
Results showed that AM1009 was the most effective against L. amazo-
nensis infection, since parasitological analyses demonstrated more sig-
nificant reductions in the parasite load in infected tissue, spleen, liver,
and dLN of the treated and infected animals, when compared to the
others. Although clioquinol and AmpB have also showed reductions in
the parasitism as compared to the saline group, values were higher in
relation to those obtained using AM1009 in the infected animals.
Similar results were found when some other natural or synthetic mo-
lecules, including those quinoline derivates, were tested in murine
models against Leishmania infection [14,43].
Fig. 6. Immune response developed in treated and infected animals.
Splenocytes of treated and infected animals were collected 15 days after
treatment, and cells were unstimulated (medium) or stimulated with L. ama-
zonensis SLA (25.0 μg/mL) for 48 h at 37 °C in 5% CO2. IFN-γ, IL-4, IL-10, IL-
12p70, and GM-CSF levels (in A) were measured in the cell supernatant, using
commercial kits. The nitrite production was also evaluated in the supernatants
by Griess reaction (in B). In addition, sera samples were also collected of the
animals, and the parasite-specific IgG1 and IgG2a isotype levels were evaluated
by an indirect ELISA, at which time the ratio between the IgG2a/IgG1 values
The immune profile evaluated in the treated and infected animals
showed the development of polarized Th1 response in AM1009-treated
animals, which was based on high levels of IFN-γ, IL-12, GM-CSF, and
nitrite, as well as by the presence of the parasite-specific IgG2a isotype
antibody. The nitrite production by activated macrophages is known to
play a major role in the elimination of parasites, such as Leishmania
[44]. Data showed here suggest the macrophage activation in AM1009-
treated animals, based on the higher antileishmanial nitrite levels that
were found, thus relating to the lower parasitism found in infected
tissue and organs of these animals. Clioquinol or AmpB-treated and
infected mice also showed an antileishmanial Th1 response, although
their spleen cells have produced lower IFN-γ and nitrite levels, as well
as higher IL-4 and IL-10 levels, when compared to the AM1009-treated
were calculated and are shown (in C). Bars represent the mean
standard
deviation of the groups. (⁎) indicate statistically significant difference in rela-
tion to the saline and AmpB groups (P < .0001).
and cost-effective antileishmanial products to control this neglected
disease in the world [38]. However, current therapeutics present pro-
blems, such as side effects, including arthralgia, myalgia, fever, weak-
ness, besides renal, hepatic and cardiac toxicity, together with high cost
and/or poor availability. In addition, the parasite resistance against
some drugs is increasing in several countries worldwide [39]. As a
consequence, the search continues to find new antileishmanial targets,
which should present a low toxicity and a lower cost but a high
7

J.K.T. Sousa, et al.
ParasitologyInternational73(2019)101966
Fig. 7. Evaluation of in vivo toxicity in treated and infected mice. To evaluate the organic toxicity induced by the therapeutics, alanine aminotransferase (A),
aspartate aminotransferase (B), urea (C), and creatinine (D) levels were measured in the sera samples collected from treated and infected animals (n = 8 per group),
at 15 days post-treatment. Samples from naive (untreated and uninfected) animals (n = 4) were used as the control. Bars indicate the mean
standard deviation of
the groups. (⁎) indicates statistically significant difference in relation to the saline and AmpB groups (P < .0001).
group. On the other hand, saline group mice mounted a polarized Th2
response, which was characterized by high levels of IL-4, IL-10, and
anti-Leishmania IgG1 antibody, corroborating with the susceptibility
profile found when this mouse lineage is infected with L. amazonensis
[45,46]. As a limitation of the study, the therapeutic efficacy in dif-
ferent periods of time after the treatment was not evaluated, and dif-
ferent dose schedules and the incorporation to delivery systems were
not performed. Nevertheless, data presented here allow one to infer
about an in vitro and in vivo effective antileishmanial action of AM1009,
as well as by lower toxicity found in two distinct types of mammalian
cells. Thus, this molecule can be considered to be an alternative antil-
eishmanial derivate for studies against this disease.
The toxicity induced in treated and infected BALB/c mice was
evaluated, and the results demonstrated that AM1009 or clioquinol
caused no significant toxicity in the animals, since renal and hepatic
markers were near the values found for the non-treated and non-in-
fected animals. By contrast, AmpB-treated mice presented high levels of
AST, ALT, urea, and creatinine, thus reflecting the toxicity of this drug
when used in mammalians, such as that reported in other studies
[47,48]. Although AmpB is considered to be an immunomodulatory
agent, as it is able to induce the production of IL-1, TNF-α, and nitric
oxide, it is also considered to be potentially nephrotoxic, the acute
toxicity of which can cause fever, chills, nausea, vomiting, diarrhea,
and headaches in patients [49,50].
high levels, it can cause oxidative stress and cell death. This production
can also induce deleterious effects in the cell mitochondrial membrane,
leading to ΔΨm depolarization and cell death [51,52]. Alterations in
the sub-G0/G1 phase cell cycle indicate parasite stress, with the pre-
sence of a cell subpopulation presenting low DNA content, which is
compatible with cell degradation [53]. In conclusion, these findings
suggest that mitochondria were the target organelles in AM1009-
treated parasites. Other quinoline derivates have also demonstrated a
similar mechanism of action in Leishmania, such as 4-hydrazinoquino-
line [54], QuinDer1 [55], and Flau-A [56].
Efforts towards the development of new antileishmanial candidates
have been performed. This action has led to the discovery of new
products with higher therapeutic efficacy, reduced toxicity, and
broader utility. Here, a new chloroquinolin derivate presented a more
selective action against two important Leishmania species. In addition, it
was not toxic to mammalian cells derived from mice and humans and
was effective in the treatment of infection caused in a mammalian host.
Although other quinoline derivates are also under production and are
being tested as antileishmanial targets, new options containing more
effective and selective molecules are desirable, since the therapeutic
arsenal available to treat against leishmaniasis is scarce, and the
pharmaceutical industry is not interested in developing new products
against this neglected disease. As a result, this study presents AM1009
as a new candidate to be tested in future studies for treatment against
leishmaniasis in mammalian hosts.
The mechanism of action of AM1009 was evaluated in L. amazo-
nensis. Results showed that the molecule induced ΔΨm depolarization,
stimulated ROS production, and promoted alterations in the Leishmania
cell cycle by increasing the sub-G0/G1 phase cell population. The ROS
production is considered relevant for organisms, since it regulates the
signaling pathways and cell proliferation; however, when present in
Declaration of Competing Interest
The authors confirm that they have no conflicts of interest in rela-
tion to this work.
8

J.K.T. Sousa, et al.
ParasitologyInternational73(2019)101966
Acknowledgments
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