1179
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(4): 1179-1188, October-December 2021 (Published Nov. 17, 2021)
Anti-leishmanial activity of 29 daily sessions of intralesional-pentavalent
antimony administration on Leishmania (Viannia)-infected BALB/c mice
Angélica María Vera1; http://orcid.org/0000-0002-3352-2978
Julio Cesar Mantilla2; http://orcid.org/0000-0002-9834-5028
Patricia Escobar1*; http://orcid.org/0000-0002-7527-2364
1. Centro de Investigación en Enfermedades Tropicales (CINTROP-UIS), Departamento de Ciencias Básicas,
Universidad Industrial de Santander, Bucaramanga, Colombia; angelica_vera01@hotmail.com, pescobar@uis.edu.co
(*Correspondence)
2. Departamento de Patología, Universidad Industrial de Santander (PAT-UIS), Bucaramanga, Colombia;
jumantil@uis.edu.co
Received 19-IX-2021. Corrected 10-X-2021. Accepted 02-XI-2021.
ABSTRACT
Introduction: Intralesional-pentavalent antimonials (IL-SbV) are recommended for simple cutaneous leishmani-
asis (CL). Few treatment sessions (1-5) and drug volumes (1-5 ml each), relative to lesion size (LS), are recom-
mended. There is not a validated IL-SbV protocol using doses calculated as mg/kg body weight and administered
over a large number of IL-sessions, with small injection volumes.
Objective: The study aim was to determine the efficacy of different concentrations of IL-SbV administered in
29 daily sessions of 100 μL each, on CL infected mice.
Methods: Leishmania (Viannia) panamensis and L. (V.) braziliensis-infected mice (N = 6) were treated with
150, 50, and 16.6 mgSbV/kg/day x 29 days. Percentage of lesion area reduction, aesthetic and final (no lesions,
no parasites) efficacy and effective dose (ED)50 were determined. In vitro-SbV activity against parasites was
evaluated for both species.
Results: The ED50 values were 72.2 and 66.3 (at the end of treatment), 54.3 and 37.7 (15-days pt.), and 145.3
and 148.6 (60-days pt.) for each species, respectively. Differences were observed between Leishmania species at
15-days pt., but not later. At 60-day pt., IL-SbV-150 mg showed final cure rates of 66.6 % for L. (V.) panamensis
and 33.3 % for L. (V.) braziliensis-infected mice. After 15 days pt., lesion reactivation was observed in some
“aesthetically cured” mice. Glucantime was not active in in vitro assays.
Conclusions: The IL-SbV use with a dose calculated as mg/kg body weight and administered over a large num-
ber of IL-sessions, with small injection volumes each day could be effective against L. (V.) panamensis and L.
(V.) braziliensis-CL infection. An appropriate SbV-dose (higher than 150 mg/kg/day x less than 29 days) must
be evaluated.
Key words: Glucantime; New World cutaneous leishmaniasis; intralesional drugs; Leishmania panamensis;
Leishmania braziliensis.
Vera, A. M., Mantilla, J. C., & Escobar, P. (2021). Anti-
leishmanial activity of 29 daily sessions of intralesional-
pentavalent antimony administration on Leishmania
(Viannia)-infected BALB/c mice. Revista de Biología
Tropical, 69(4), 1179-1188. https://doi.org/10.15517/rbt.
v69i4.47368
https://doi.org/10.15517/rbt.v69i4.47368
New World cutaneous leishmaniasis (NW-
CL) is the most common form of leishmani-
asis in Latin America with an annual average
of 56 000 cases of cutaneous and mucosal
leishmaniasis. It is produced by an intracel-
lular protozoan parasite belonging to subgenus
Viannia or Leishmania, transmitted to humans
and reservoirs by infected female sand-flies
BIOMEDICINE
1180
Revista de Biología Tropical, ISSN: 2215-2075 Vol. 69(4): 1179-1188, October-December 2021 (Published Nov. 17, 2021)
of the genus Lutzomyia (Pan American Health
Organization, 2018). Symptomatic NW-CL is
characterized by skin lesions, such as papules,
nodules, or ulcerative volcano-like lesions,
with varied outcomes, in terms of lesion num-
ber and size, scar type, disability, spontaneous
healing, and destructive mucosal inflammation
(Bailey et al., 2017).
Pentavalent antimonials (SbV, Glucantime,
and Pentostam) remain the standard treatment
for all forms of leishmaniasis in some coun-
tries (Burza, et al.,2018). They are adminis-
tered parentally in 10-30 doses of 20 mg/kg/
day, depending on the clinical manifestations.
Mild to severe side effects, including arthral-
gia, myalgia, cardiotoxicity, nephropathy, and
pain during intramuscular administration, are
observed (Burza et al., 2018; Goto & Lindoso,
2010). In some regions endemic for visceral
leishmaniasis, such as Bihar, in Eastern India,
therapeutic failure (TF) can be observed in up
to 65 % of cases, while estimates of antimo-
nial TF rates on NW-CL range from 15-32 %
(Castro, et al.,2017; Sundar et al., 2000). In
cases with simple CL (single or a few lesions,
up to 900 mm2), pregnant women, and patients
with kidney, heart disease, or other concomi-
tant diseases, local/topical treatments, such as
cryotherapy, localized heat, intralesional drugs,
or topical application of semisolid formulations
directly onto the lesion are alternative therapies
(Burza et al., 2018).
Intralesional (IL)-SbV treatment is widely
used in treatment of Old World-CL, and is
applied alone, or in combination with cryo-
therapy. Further, intralesional (IL)-SbV therapy
is recommended for simple NW-CL cases,
where lesions do not involve the face or joints
(PAHO, 2018). Intralesional-SbV injection is
easily administered by trained staff, does not
require hospitalization, can be given at a low
dose, and results in less systemic drug absorp-
tion, side effects, and treatment costs; 1-5 ses-
sions of intradermal IL-SbV injections, with
1-5 ml per session, at the border or base of the
CL-lesion every 3-7 days are recommended
(PAHO, 2018). The overall clinical effica-
cy of IL-SbV using an injectable Glucantime
solution (the same one used for intramuscular
route treatment) is reported as 75 to 77 % (de
Oliveira Duque et al., 2019; Oliveira-Neto et
al., 1997). In Bolivian patients with CL, three
or five sessions of 2-3 ml each (650 μg SbV per
mm2), every other day resulted in cure rates
from 57 to 73 % at 6 months (Soto et al., 2016).
Overall TF was 30 % (like parenteral SbV) and
no recurrences were recorded in cured patients
for ≥ 6 months (de Oliveira Duque et al., 2019;
Oliveira-Neto et al., 1997). There is not a vali-
dated IL-SbV protocol using doses calculated
as mg/kg body weight and administered over a
large number of IL-sessions, with small injec-
tion volumes in humans. In addition, there is
not an IL-SbV protocol available as a positive
control for NW-CL intralesional drug discov-
ery able to demonstrate both efficacy and some
SbV-IL risks such as lesion reactivation and TF
on experimental models.
The present study aim was to determine
the efficacy of different concentrations of IL-
SbV (Glucantime) administered in 29 daily
sessions of 100 μL each, on mice infected with
two common NW-CL species, L. (V.) panamen-
sis and L. (V.) braziliensis.
MATERIALS AND METHODS
Drugs and reagents: Meglumine antimo-
niate, Glucantime® (Sanofi-Aventis, Brazil; lot
357345) was kindly donated by the Secretary
of Health of Santander, Colombia. One bottle
of Glucantime contained 1.5 g of meglumine
antimoniate (405 mg of SbV). Schneider’s
insect medium, RPMI medium, and heat inac-
tivated FBS (hiFBS) were from Gibco (Grand
Island, NY, USA).
Parasites and cells: Promastigotes of L.
(V.) panamensis (MHOM/PA/71/LS94) and L.
(V.) braziliensis (MHOM/BR/75/M2903) were
maintained in Schneider’s medium plus 10
% hiFBS at 28 °C. Human leukaemia THP-1
cells (ATCC, USA) were cultured in RPMI
1640 medium plus 10 % hiFBS at 37 °C in a
5 % CO2 /95 % air mixture. THP-1 cells were
transformed to adherent cells using 40 ng/ml
1181
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(4): 1179-1188, October-December 2021 (Published Nov. 17, 2021)
phorbol 12-myristate 13-acetate for 48 h, and
these macrophages infected with stationary-
phase promastigotes at a cell: parasite ratio of
1:5 for 24 h at 32 °C. The percentage of infec-
tion was determined microscopically by Giem-
sa staining. Axenic amastigotes were obtained
from late-phase promastigotes after 8 days of
transformation by progressive changes of tem-
perature (from 27 to 32 °C) and pH (from pH
7.0 to 5.5). The Leishmania species used were
susceptible to miltefosine, pentamidine and
ketoconazole (Neira, et al., 2019a).
Animals, ethics: Female and male
BALB/c mice (10-12 weeks of age) were sup-
plied by the National Health Institute (Bogotá,
Colombia). Mice were housed with a 12 h
light/dark cycle, at 23 °C, 55 % ± 5 % relative
humidity, with access to water and food pellets
ad libitum. Studies were approved by the UIS-
Ethics Committee (CIENCI, Code 17-2017).
Animals were anesthetized by intraperitoneal
injection of a ketamine/xylazine cocktail and
euthanized by cervical dislocation.
Anti-leishmanial activity in vivo: Mice
were infected by subcutaneous injection in the
shaven rump with 5 × 105 stationary-phase par-
asites. When lesion size (LS) was in the range
40-42 mm2 (8 weeks after infection), mice were
randomly allocated into four groups (N = 6 per
group); different concentrations of SbV (150,
50, and 16.6 mg SbV/kg body weight /day) and
vehicle (0.9 % saline solution) (100 µL) were
injected intralesionally (IL) for 29 days. Doses
of SbV were calculated for mice using mean
body weight (27 g), containing 4.1, 1.4, and 0.4
mg SbV per mouse and a total of 117.5, 39.2,
and 13.9 mg SbV/mouse over 29 days. Lesion
size was measured weekly using a digital cal-
liper and lesion area (mm2) calculated (Neira et
al., 2019a). Follow-up period was determined
in a preliminary experiment (Appendix 1).
Briefly, Leishmania infected mice (N = 1 per
species) were treated with IL-SbV-150 mg/kg/
day × 29 days and sacrificed 15, 22, 29, and
42-days post-treatment. The chosen follow-up
time was 60 days’ post treatment (pt.).
At the end of the experiment, animals
were sacrificed, and smears (imprints) pre-
pared from lesions fixed in methanol and
stained with Giemsa for detection and calcula-
tion of parasites by microscopy. Additionally,
lesion samples were collected and processed
for histopathological examination. Amastigote
number per microscopic field (400×) was semi-
quantitatively scored [No parasites = 0, scarce
= 1-5 (+), moderate = 6-10 (++), abundant > 11
(+++) parasites)] and percentage LS reduction
(LSred) calculated (Neira, et al.,2019b). Aes-
thetic efficacy (eE) was calculated as (N mice
with > 75 % of LSred /6 mice) × 100, and final
efficacy (fE) as (N mice with both 100 % of
LSred and no parasites/6) × 100. Reactivation
was defined as the appearance of a new lesion
in a previously aesthetically 100 % healed site.
Mean effective dose (ED)50 (dose of SbV able
to reduce LS by 50 %) with 95 % confidence
interval (95 % CI) was calculated by sigmoidal
regression using MsxlfitTM software.
Adverse effects: Mice weight was mea-
sured weekly using a digital balance. Skin irri-
tation was registered by visual inspection and
signs of irritation at application sites classified
from 0 = no irritation to 4 = severe irritation.
Histopathological analysis: Samples were
fixed with 10 % neutral formalin, embedded in
paraffin, and sectioned into 5 µm thick sec-
tions using a microtome. Dewaxed slices were
stained with haematoxylin-eosin and examined
by microscopy. Histopathological parameters
and parasites were semi-quantified and scored
as follows: -, absent; +, mild; ++, moderate;
and +++, severe (Neira et al., 2019b).
In vitro assay: Promastigotes and axenic
amastigotes were incubated with serial 1:3
dilutions of Glucantime (33.3 to 900 µg/ml)
for 72 h at 27 and 32 °C, respectively (Gupta
et al., 2001). Control cells were incubated in
culture medium. Drug activities were assessed
using a resazurin colorimetric test. Absorbance
values were measured using a Synergy H1
microplate reader at 570 and 600 nm. For
1182
Revista de Biología Tropical, ISSN: 2215-2075 Vol. 69(4): 1179-1188, October-December 2021 (Published Nov. 17, 2021)
intracellular parasites, infected THP-1 cells
were treated with Glucantime for 5 days, at
32 °C (Neira et al., 2019a). Parasite growth
inhibition was determined by counting infected
and non-infected cells on Giemsa-stained slides
by microscopy. IC50 values were calculated as
described for ED50.
Statistical analysis: Differences were
analyzed using the Student’s t-test. P values ≤
0.05 were considered statistically significant.
Area under the curve values were calculated
for comparisons of SbV dose-response effects
using GraphPad Prism software, version 6.0
for Windows and two-way ANOVA and Sidak
post-hoc method.
RESULTS
Efficacy of treatment with IL-SbV-150
mg/kg × 29: For L. (V.) panamensis infection,
eE of 83.3 % (5/6 mice; M1–M4, M6) and LSred
values of 87.0-100 % were observed both at the
end of treatment and 15-days pt. At 60-days pt.,
LS reactivation (Re) was observed in mouse
M6 (Table 1, Fig. 1). Therapeutic failure was
also observed in mouse M5failure. At the end of
treatment and until 43-day pt., M5failure showed
LSred of almost 50 %; however, a subsequent
increase of LS by almost 8 times was observed
(Table 1). In conclusion, 29 doses of IL-
SbV-150 mg induced a fE value of 66.6 % in L.
(V.) panamensis-infected mice.
For L. (V.) braziliensis infected mice, an
eE value of 83.3 % (5/6 mice, M1, M2, M4,
M5, M6) and LSred from 80.9 to 100 % were
observed both at the end of treatment and at
15-days pt. At 50-day pt., lesion reactivation
was detected in mice M4, M5, and M6. Addi-
tionally, mouse M2, an aesthetically cured
animal, tested positive for parasites in biopsy
samples (Table 2, Fig. 2). Mouse M3 showed
a slight decrease in LS at 15 days pt. and a
complete aesthetic response, without observ-
able parasites, at the end of the experiment.
Consequently, fE was 33.3 %.
All non-cured mice showed abundant
(+++) intra and extracellular amastigotes in
CL-lesion samples.
None of the mice treated with 29 doses of
IL-SbV showed body weight loss, skin irrita-
tion, or any signs of pain or loss of well-being.
Efficacy of lower doses (IL-SbV-50 mg/
kg x 29 days and IL-SbV-16.6 mg/kg x
29 days): At 15-day pt., L. (V.) panamen-
sis infected mice M4, M5, and M6 treated
with IL-SbV-50 mg, displayed considerable
TABLE 1
Efficacy of 29-doses of IL-SbV at 150 mg/kg/day in mice infected with L. (V.) panamensis
Mice Before
mm2
29-doses-IL-SbV-150 mg on L. (V.) panamensis infected mice
end treatment 15-day pt. 60-day pt.
mm2LSred
(%) eE
(%) mm2LSred
(%) eE
(%) mm2LSred
(%) eE
(%)
Parasites fE
imprints biopsies
M1 36.6 3.5 90.5
83.3
0 100
83.3
0 100
66.6
- -
66.6
M2 35.4 0 100 0 100 0 100 - -
M3 32.5 0 100 0 100 0 100 - -
M4 45.9 6.2 86.6 0 100 0 100 - -
M5 50.3 35.2 29.9 19.6 61.0 153.2 0 +++ +++
M6 46.7 3.1 93.3 0 100 31.6 Reb+++ +++
SS 13.8 28.7 0 NDc30.1 0 ND 46.5 0 ND +++ +++ ND
The table shows details of individual lesion size (LS) before and at the end of treatment and at 15 and 60 days post treatment
(pt.) (N = 6). Parasite loads were scored at the end of treatment. LSred: lesion size reduction; eE: aesthetic efficacy; %eE:
percentage of mice with LSred between 75-100 %); fE, final efficacy: with a complete aesthetic and parasite response; SS:
saline solution (a representative control mouse); Re: reactivation; ND: not determined.
1183
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(4): 1179-1188, October-December 2021 (Published Nov. 17, 2021)
Fig. 1. Response of mice infected with L. (V.) panamensis to IL-SbV-150 mg/kg/day x 29. Photographs of CL lesions in mice
M1-M6 at the beginning, 15 days and at the end of treatment (29 days); and at 15 and 60 days pt.
improvement (LSred 89.5-100 %) (Appendix
2, Appendix 3) and stable effects were also
observed in mice M1, M2, and M3 (LSred
43.7-58.4 %); however, at 60-day pt., reactiva-
tion or increase of LS was observed. In mice
treated with IL-SbV-16.6 mg, LSred values from
0 to 51.6 % were observed both at the end of
treatment and 15 days pt.; however, at the end
of the experiment, fE was zero and abundant
(+++) parasites were observed by microscopy
in imprint smears and biopsies (Appendix 2,
Appendix 4).
For L. (V.) braziliensis infected mice, IL-
SbV-50mg treatment induced LSred. of 80-100
% in mice M2 and M4-M6 at 15 days pt.; how-
ever, reactivation occurred in two of these mice
(M2 and M6) and there was an increase of LS
in another (M4) with parasites present in all of
1184
Revista de Biología Tropical, ISSN: 2215-2075 Vol. 69(4): 1179-1188, October-December 2021 (Published Nov. 17, 2021)
them, including M5 (Appendix 5, Appendix 6).
Treatment with IL-SbV-16.6mg induced a LSred
of 87.2 % in one mouse (M6) at 15 days pt.;
however, TF occurred at 60-day pt. Parasites
were observed in lesions from all treated mice
by microscopy (Appendix 5, Appendix 7).
Cutaneous leishmaniasis lesions after
relapse (Re): Mouse M6Re treated with IL-
SbV-150 mg and mice M4Re and M6Re treated
with IL-SbV-50 mg after infection with L. (V.)
panamensis had new lesions of LS of 31.6,
11.6, and 3.5 mm2, respectively, at the end
of the experiment. Mice M4Re–M6Re, treated
with IL-SbV-50 mg and M2Re and M6Re, with
IL-SbV-16.6 mg, following L. (V.) braziliensis
infection, relapsed with new lesions of LS 30.6,
11.9, 8.6, 7.8, and 20.0 mm2, respectively, at
the end of the experiment. Lesions reappeared
at the inoculation site and began as papules,
Fig. 2. L. (V.) braziliensis -infected mice response to IL-SbV-150 mg/kg/day x 29. Photographs of CL lesions in mice M1-M6
at the beginning, 15 days and at the end of treatment (29 days); and at 15 and 60 days pt.
1185
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(4): 1179-1188, October-December 2021 (Published Nov. 17, 2021)
that gradually evolved into nodules with raised
edges. In some cases, they presented as typical
clean, pink ulcers, with granular tissue, and
rounded with regular and raised edges.
Dose response activities of SbV: The
mean effective dose (ED)50 values for L. (V.)
panamensis and L. (V.) braziliensis infected
mice were 33.99 (CI, 18.89-61.14) mg and
36.96 (CI, 17.87-76.45) mg at the end of treat-
ment; 28.62 (CI, 16.53-49.55) mg and 12.40
(CI, 4.41-34.91) mg at 15 days pt.; and 142.6
(CI, 57.56-353.3) mg and 157.8 (CI, 58.99-
421.9) mg at 60 pt., respectively. At 43 days
pt., IL-SbV was more active in mice infected
with L. (V.) braziliensis than L. (V.) panamen-
sis (P < 0.001); however, no difference was
observed at the end of the experiment (60 days
pt.). No significant differences were detected in
the response to IL-SbV between mice infected
with the two species of Leishmania based on
analysis of LS or area under the curve values
for lesion changes after treatment with at 150,
50, or 16.6 mg/kg/day IL-SbV × 29.
Histopathological characteristics:
Responders (cured) mice (M1–M4 for L. (V.)
panamensis; M1 and M3 for L. (V.) braziliensis)
showed none to mild changes in the epidermis
and dermis (Appendix 8, Appendix 9, Appen-
dix 10). In contrast, M5failed or M6Re for L. (V.)
panamensis, M4Re–M6Re for L. (V.) brazilien-
sis, and negative control mice presented with
severe inflammatory infiltrates, comprising
lymphocytes, neutrophils, macrophages, and
giant cells. Some mice presented with severe
acanthosis and moderate spongiosis and necro-
sis (Appendix 8).
In vitro drug activity: Based on IC50
values, SbV was inactive against all live forms
of parasites evaluated, since IC50 values were
> 900 µgSbV/ml for both Leishmania strains.
DISCUSSION
In this work, we evaluated the responses
of CL-infected mice to Glucantime, admin-
istered over a large number of IL-sessions,
with small injection volumes each day (e.g.,
not until lesions blanched) and SbV regimens
calculated by body weight (kg), instead of LS
(mm2). Administration of the highest dose of
IL-SbV (equivalent to 98.78 µg SbV/mm2 × 29
daily, and 2 864.6 µg SbV/mm2 in total) was
effective in 4/6 and 2/6 mice infected with
TABLE 2
Efficacy of 29 doses of IL-SbV at 150 mg/kg in mice infected with L. (V.) braziliensis
Mice Before
(mm2)
29-doses-IL-SbV-150 mg on L. (V.) braziliensis infected mice
end of treatment 15-day pt. 60-day pt.
mm2LSred
(%) eE
(%) mm2LSred
(%) eE
(%) mm2LSred
(%) eE
(%)
Parasites fE
Im
prints biopsies
M1 55.9 0 100
83.3
0 100
83.3
0 100
50
- -
33.3
M2 34.9 0 100 0 100 0 100 - ++
M3 14.5 23.2 0 10.4 28.6 0 100 - -
M4 32.8 6.28 80.9 0 100 30.6 Reb+++ +++
M5 35.7 0 100 0 100 11.9 Re +++ +++
M6 43.1 0 100 0 100 8.7 Re +++ +++
SSa13.65 49.64 0 NDc57.8 0 ND 113.5 0 ND +++ +++ ND
The table shows details of individual lesion size (LS) before and at the end of treatment and at 15 and 60 days’ post treatment
(pt.) (N = 6). Parasite loads were scored at the end of treatment. LSred: lesion size reduction; eE: aesthetic efficacy; %eE:
percentage of mice with LSred between 75-100 %); fE, final efficacy: with a complete aesthetic and parasite response; SS:
saline solution (a representative control mouse); Re: reactivation; ND: not determined.
1186
Revista de Biología Tropical, ISSN: 2215-2075 Vol. 69(4): 1179-1188, October-December 2021 (Published Nov. 17, 2021)
L. (V.) panamensis and L. (V.) braziliensis at
60 days pt.
Some positive results were obtained with
IL-SbV in a hamster CL model (100 % lesion
reduction and no parasites in organs) (Travi,
et al., 1993; Yépez, et al.,1999); however, no
complete cure of CL-infected mice has been
documented. Per example, treatment of mice
infected with L. (L.) amazonensis, with IL-SbV
28 mg/kg/day × 5, every 4-5 days resulted
in partial parasite burden reduction with no
increase in footpad thickness, but not in cure
(Cos et al., 2018; Fournet et al., 1996). Dif-
ferences in numbers of SbV-IL sessions, dose,
site of infection, parasite species, and follow-
up time could explain the different results
obtained in this study. In addition, type of CL
lesion could be also involved; ulcerated lesions
(Fig. 1, Fig. 2) without parasite dissemination
or spontaneous cure are characteristic of our
CL-mouse model (Neira, et al., 2019b). The
final SbV doses/per mouse (at a body weight,
27 g) also differed. At 150, 50, and 16.6 mg/
kg/day × 29, the final doses were 117.5, 39.2
and 13.9 mg SbV, while at 28 mg/kg × 5 the
dose was 3.78 mg 224 SbV (almost three times
less than the minimum non-effective dose used
in our study).
IL-SbV treatment induced a dose-response
effect; drug potency was higher soon after
treatment than at the end of experiment, as
ED50 values were lower at 15 than 60 days pt.
Hence, the maximal SbV concentration used
may have been unable to kill 100 % of parasites
on all treated mice, with remaining parasites
able to reactivate lesions, as was observed in
some mice at the end of the experiment. In
a previous study we reported the histopatho-
logical characteristics of CL lesions before
treatment (Neira et al., 2019b). They were
characterized moderate hyperkeratosis and
acanthosis, mild spongiosis in the epidermis,
with a severe diffuse inflammatory infiltrate
predominantly comprised of lymphocytes and
plasma cells, and abundant amastigote-infected
macrophages in the dermis. It is possible that a
higher mgSbV/kg dose will be necessary to kill
all parasites.
Next, we decided to evaluate SbV activity
in vitro on promastigotes, axenic amastigotes,
and intracellular amastigotes of both Leish-
mania strains infecting THP-1 cells, using the
same batch as that used for in vivo experiments.
Unfortunately, we were unable to calculate IC50
values (drug potency) at the maximal doses
evaluated in any of the live parasite forms of
either parasite species. The antileishmanial
activities of SbV have been demonstrated in
various in vitro models, showing some pecu-
liarities compared with other drugs such as
miltefosine or pentamidine isethionate (Fernán-
dez et al., 2014). Most notably, SbV is unable to
kill promastigotes and, to a lesser extent, axen-
ic amastigotes (Sereno et al., 1998); however,
activities have been demonstrated against intra-
cellular amastigotes infecting different types of
macrophage and reference strains or isolates
collected worldwide (IC50 2.9-146 µgSbV/ml)
(Fernández et al., 2014; Pérez-Franco et al.,
2016). In general, drug susceptibility (in vitro)
is supposed as a method to predict treatment
outcome. However, this was not our case as
SbV was active on some CL infected mice
but not on free or intracellular parasites life
forms. Both reference strains used are not clas-
sified as Sb-resistant strains. In contrast, SbV
activity has been demonstrated against L. (V.)
braziliensis /M2903 intracellular amastigotes
infecting U937 differentiated cells (IC50 26-50
µgSbV/ml) (Pérez-Franco et al., 2016). Before
our experiments, the parasites had never been
exposed to antimonials in neither in vitro or in
vivo assays; however, they have been cultivated
in vitro for a relatively extended period, by
quarterly passage in BALB/mice. Leishmania
resistance mechanisms or other factors related
to drug (drug quality, intrinsic drug properties,
drug physicochemical characteristics), parasite
(lower intrinsic susceptibility to the drug, para-
site infection by RNA viruses, parasite adap-
tations, manipulation skills of the parasites,
higher parasite fitness) or host (immunological
factors, pharmacokinetics, genetics) could be
involved with SbV treatment efficacy or failure
(Vanaerschot et al. 2014).
1187
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(4): 1179-1188, October-December 2021 (Published Nov. 17, 2021)
Independent of the lack of in vitro SbV
response, the use of intralesional SbV with a
dose calculated as mg/kg body weight and
administered over a large number of IL-ses-
sions, with small injection volumes each day
could be effective against L. (V.) panamensis
and L. (V.) braziliensis-CL infection. An appro-
priate SbV-dose (higher than 150 mg/kg/day x
20) must be evaluated.
Ethical statement: the authors declare
that they all agree with this publication and
made significant contributions; that there is no
conflict of interest of any kind; and that we fol-
lowed all pertinent ethical and legal procedures
and requirements. All financial sources are
fully and clearly stated in the acknowledge-
ments section. A signed document has been
filed in the journal archives.
See Digital Appendix at: revistas.ucr.ac.cr
ACKNOWLEDGMENTS
This work was supported by the Autono-
mous Patrimony of the National Financing
Fund for Science, Technology, and Innovation
Francisco José de Caldas, Contract RC-687-
2014. AMV was sponsored by the program
High-Level of Human Capital Training for the
Regions-Santander Department, announcement
771 of 2016, Minciencias and the Government
of Santander (Colombia).
RESUMEN
Actividad antileishmania de 29 sesiones diarias de
antimonio pentavalente intralesional en ratones
BALB/c infectados con Leishmania (Viannia)
Introducción: Los antimoniales pentavalentes aplicados
intralesionalmente (IL-SbV) se recomiendan para el trata-
miento de la leishmaniasis cutánea (LC) simple. Se reco-
miendan pocas sesiones (1-5) y volúmenes (1-5 ml cada
uno) en relación con el tamaño de la lesión (LS). No existe
un protocolo de IL-SbV validado que utilice dosis calcu-
ladas según el peso corporal (en mg/kg) y administradas
durante varias sesiones en pocos volúmenes de inyección.
Objetivo: El objetivo del estudio fue determinar la eficacia
de diferentes concentraciones de IL-SbV administradas en
29 sesiones diarias de 100 μL cada una, en ratones con LC.
Métodos: Ratones infectados con L. (V.) panamensis y L.
(V.) braziliensis (N = 6) fueron tratados intralesionalmente
con 150, 50 y 16,6 mg SbV/kg/día x 29 días. Se determinó
el porcentaje de reducción del área de la lesión, la efica-
cia estética y final (sin lesiones, sin parásitos) y la dosis
efectiva (DE)50. Adicionalmente de evaluó la actividad in
vitro del SbV.
Resultados: Los valores de DE50 fueron 72.2 y 66.3 (al
final del tratamiento), 54.3 y 37.7 (15 días pt) y 145.3 y
148.6 (60 días pt) para cada especie. Se encontraron dife-
rencias entre las especies sólo a los 15 días pt. La eficacia
del tratamiento IL-SbV-150 mg, 60 días pt., fue de 66.6 y
33.3 % en ratones infectados con L. (V.) panamensis L.
(V.) braziliensis respectivamente. Después de 15 días pt.,
se observó reactivación de la lesión en algunos ratones
“estéticamente curados”. Glucantime no fue activo in vitro.
Conclusiones: El uso intralesional de SbV con una dosis
calculada en mg/kg de peso corporal y administrada duran-
te varias sesiones, con pequeños volúmenes de inyección
cada día, podría ser eficaz en LC por L. (V.) panamensis y
L. (V.) braziliensis. Dosis adecuadas de SbV (superiores a
150 mg/kg/día x 20) deben evaluarse.
Palabras clave: Glucantime; leishmaniasis cutánea del
Nuevo Mundo; medicamento intralesional; Leishmania
panamensis; Leishmania braziliensis.
REFERENCES
Bailey, F., Mondragon-Shem, K., Hotez, P., Ruiz-Postigo,
J. A., Al-Salem, W., Acosta-Serrano, Á., & Moly-
neux, D. H. (2017). A new perspective on cutaneous
leishmaniasis-Implications for global prevalence and
burden of disease estimates. PloS Neglected Tropical
Diseases, 11(8), e0005739.
Burza, S., Croft, S. L., & Boelaert, M. (2018). Leishmania-
sis. The Lancet, 392(10151), 951–970.
Castro, M., Cossio, A., Velasco, C., & Osorio, L. (2017).
Risk factors for therapeutic failure to meglumine anti-
moniate and miltefosine in adults and children with
cutaneous leishmaniasis in Colombia: A cohort study.
PloS Neglected Tropical Diseases, 11(4), e0005515.
Cos, P., Janssens, J., Piñón, A., Cuesta-Rubio, O., Yglesias-
Rivera, A., Díaz-García, A., Vilegas, W., & Monzote,
L. (2018). Efficacy of four Solanum spp. extracts in
an animal model of cutaneous leishmaniasis. Medi-
cines, 5(2), 49.
de Oliveira Duque, M. C., Quintão Silva, J. J., Soares, P.,
Magalhães, R. S., Horta, A., Paes, L., Rosandiski
Lyra, M., Pimentel, M., de Fátima Antonio, L., de
Camargo Ferreira, E., Vasconcellos, É., Saheki, M.
N., de Almeida Marzochi, M. C., Valete-Rosalino, C.
1188
Revista de Biología Tropical, ISSN: 2215-2075 Vol. 69(4): 1179-1188, October-December 2021 (Published Nov. 17, 2021)
M., & de Oliveira Schubach, A. (2019). Comparison
between systemic and intralesional meglumine anti-
moniate therapy in a primary health care unit. Acta
Tropica, 193, 176–182.
Fernández, O. L., Diaz-Toro, Y., Ovalle, C., Valderrama,
L., Muvdi, S., Rodríguez, I., Gomez, M. A., &
Saravia, N. G. (2014). Miltefosine and antimonial
drug susceptibility of Leishmania Viannia species
and populations in regions of high transmission in
Colombia. PloS Neglected Tropical Diseases, 8(5),
e2871.
Fournet, A., Ferreira, M. E., Rojas De Arias, A., Torres
De Ortiz, S., Fuentes, S., Nakayama, H., Schinini,
A., & Hocquemiller, R. (1996). In vivo efficacy of
oral and intralesional administration of 2-substituted
quinolines in experimental treatment of new world
cutaneous leishmaniasis caused by Leishmania ama-
zonensis. Antimicrobial Agents and Chemotherapy,
40(11), 2447–2451.
Goto, H., & Lindoso, J. A. (2010). Current diagnosis and
treatment of cutaneous and mucocutaneous leishma-
niasis. Expert Review of Anti-Infective Therapy, 8(4),
419–433.
Gupta, N., Goyal, N., & Rastogi, A. (2001). In vitro culti-
vation and characterization of axenic amastigotes of
Leishmania. Trends in Parasitology, 17(3), 150–153.
Neira, L. F., Mantilla, J. C., & Escobar, P. (2019a). Anti-
leishmanial activity of a topical miltefosine gel in
experimental models of New World cutaneous leis-
hmaniasis. Journal of Antimicrobial Chemotherapy,
74(6), 1634–1641.
Neira, L. F., Peña, D. P., Vera, A. M., Mantilla, J. C., &
Escobar, P. (2019b). Leishmaniasis cutánea induci-
da por especies de Leishmania Viannia en ratones
BALB/c y eficacia de un tratamiento tópico. Revista
Universidad Industrial de Santander, 51, 33–42.
Oliveira-Neto, M. P., Schubach, A., Mattos, M., da Costa,
S. C., & Pirmez, C. (1997). Intralesional therapy of
American cutaneous leishmaniasis with pentavalent
antimony in Rio de Janeiro, Brazil- an area of Leis-
hmania (V.) braziliensis transmission. International
Journal of Dermatology, 36(6), 463–468.
Pan American Health Organization (PAHO). (2018). Leis-
hmaniasis in the Americas: Treatment recommenda-
tions. https://iris.paho.org/handle/10665.2/7704
Pérez-Franco, J. E., Cruz-Barrera, M. L., Robayo, M. L.,
López, M. C., Daza, C. D., Bedoya, A., Mariño,
M. L., Saavedra, C. H., & Echeverry, M. C. (2016).
Clinical and parasitological features of patients with
American cutaneous leishmaniasis that did not res-
pond to treatment with meglumine antimoniate. PloS
Neglected Tropical Diseases, 10(5), e0004739.
Sereno, D., Cavaleyra, M., Zemzoumi, K., Maquaire, S.,
Ouaissi, A., & Lemesre, J. L. (1998). Axenically
grown amastigotes of Leishmania infantum used
as an in vitro model to investigate the pentavalent
antimony mode of action. Antimicrobial Agents and
Chemotherapy, 42(12), 3097–3102.
Soto, J., Paz, D., Rivero, D., Soto, P., Quispe, J., Toledo,
J., & Berman, J. (2016). Intralesional pentamidine: a
novel therapy for single lesions of Bolivian cutaneous
leishmaniasis. The American Journal of Tropical
Medicine and Hygiene, 94(4), 852–856.
Sundar, S., More, D. K., Singh, M. K., Singh, V. P., Shar-
ma, S., Makharia, A., Kumar, P. C., & Murray, H. W.
(2000). Failure of pentavalent antimony in visceral
leishmaniasis in India: report from the center of the
Indian epidemic. Clinical Infectious Diseases, 31(4),
1104–1107.
Travi, B. L., Martinez, J. E., & Zea, A. (1993). Antimonial
treatment of hamsters infected with Leishmania
(Viannia) panamensis: assessment of parasitological
cure with different therapeutic schedules. Transac-
tions of the Royal Society of Tropical Medicine and
Hygiene, 87(5), 567–569.
Vanaerschot, M., Dumetz, F., Roy, S., Ponte-Sucre, A.,
Arevalo, J., & Dujardin, J. C. (2014). Treatment failu-
re in leishmaniasis: drug-resistance or another (epi-)
phenotype? Expert Review of Anti-Infective Therapy,
12(8), 937–946.
Yépez, J., Cazorla, D., Añez, N., & Yarbuh, A. L. (1999).
Effect of intralesional treatment with lidocaine and
glucantime in hamsters infected with Leishmania
(Viannia) braziliensis. Boletín de la Dirección de
Malariol Saneamiento Ambiental, 39, 10–20.
