Current drug therapy and pharmaceutical challenges for Chagas disease

Abstract

One of the most significant health problems in the American continent in terms of human health, and socioeconomic impact is Chagas disease, caused by the protozoan parasite Trypanosoma cruzi. Infection was originally transmitted by reduviid insects, congenitally from mother to fetus, and by oral ingestion in sylvatic/rural environments, but blood transfusions, organ transplants, laboratory accidents, and sharing of contaminated syringes also contribute to modern day transmission. Likewise, Chagas disease used to be endemic from Northern Mexico to Argentina, but migrations have earned it global. The parasite has a complex life cycle, infecting different species, and invading a variety of cells -including muscle and nerve cells of the heart and gastrointestinal tract- in the mammalian host. Human infection outcome is a potentially fatal cardiomyopathy, and gastrointestinal tract lesions. In absence of a vaccine, vector control and treatment of patients are the only tools to control the disease. Unfortunately, the only drugs now available for Chagas' disease, Nifurtimox and Benznidazole, are relatively toxic for adult patients, and require prolonged administration. Benznidazole is the first choice for Chagas disease treatment due to its lower side effects than Nifurtimox. However, different strategies are being sought to overcome Benznidazole's toxicity including shorter or intermittent administration schedules -either alone or in combination with other drugs. In addition, a long list of compounds has shown trypanocidal activity, ranging from natural products to specially designed molecules, re-purposing drugs commercialized to treat other maladies, and homeopathy. In the present review, we will briefly summarize the upturns of current treatment of Chagas disease, discuss the increment on research and scientific publications about this topic, and give an overview of the state-of-the-art research aiming to produce an alternative medication to treat T. cruzi infection.

Full text

Acta
Tropica
156
(2016)
1–16
Contents
lists
available
at
ScienceDirect
Acta
Tropica
jo
ur
nal
home
p
age:
www.elsevier.com/locate/actatropica
Current
drug
therapy
and
pharmaceutical
challenges
for
Chagas
disease
José
Bermudeza,1,
Carolina
Daviesb,1,
Analía
Simonazzia,
Juan
Pablo
Realc,
Santiago
Palmac,∗
aINIQUI-CONICET,
Universidad
Nacional
de
Salta,
Salta,
Argentina
bIPE-CONICET,
Universidad
Nacional
de
Salta,
Salta,
Argentina
cUNITEFA-CONICET,
Universidad
Nacional
de
Córdoba,
Córdoba,
Argentina
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
17
October
2015
Received
in
revised
form
23
December
2015
Accepted
25
December
2015
Available
online
30
December
2015
Keywords:
Chagas
disease
Trypanosoma
cruzi
Trypanocidal
treatment
Benznidazol
Nifurtimox
New
medication
a
b
s
t
r
a
c
t
One
of
the
most
significant
health
problems
in
the
American
continent
in
terms
of
human
health,
and
socioeconomic
impact
is
Chagas
disease,
caused
by
the
protozoan
parasite
Trypanosoma
cruzi.
Infection
was
originally
transmitted
by
reduviid
insects,
congenitally
from
mother
to
fetus,
and
by
oral
ingestion
in
sylvatic/rural
environments,
but
blood
transfusions,
organ
transplants,
laboratory
accidents,
and
sharing
of
contaminated
syringes
also
contribute
to
modern
day
transmission.
Likewise,
Chagas
disease
used
to
be
endemic
from
Northern
Mexico
to
Argentina,
but
migrations
have
earned
it
global.
The
parasite
has
a
complex
life
cycle,
infecting
different
species,
and
invading
a
variety
of
cells
–
including
muscle
and
nerve
cells
of
the
heart
and
gastrointestinal
tract
–
in
the
mammalian
host.
Human
infection
outcome
is
a
potentially
fatal
cardiomyopathy,
and
gastrointestinal
tract
lesions.
In
absence
of
a
vaccine,
vector
control
and
treatment
of
patients
are
the
only
tools
to
control
the
disease.
Unfortunately,
the
only
drugs
now
available
for
Chagas’
disease,
Nifurtimox
and
Benznidazole,
are
relatively
toxic
for
adult
patients,
and
require
prolonged
administration.
Benznidazole
is
the
first
choice
for
Chagas
disease
treatment
due
to
its
lower
side
effects
than
Nifurtimox.
However,
different
strategies
are
being
sought
to
overcome
Benznidazole’s
toxicity
including
shorter
or
intermittent
administration
schedules—either
alone
or
in
combination
with
other
drugs.
In
addition,
a
long
list
of
compounds
has
shown
trypanocidal
activity,
ranging
from
natural
products
to
specially
designed
molecules,
re-purposing
drugs
commercialized
to
treat
other
maladies,
and
homeopathy.
In
the
present
review,
we
will
briefly
summarize
the
upturns
of
current
treatment
of
Chagas
disease,
discuss
the
increment
on
research
and
scientific
publications
about
this
topic,
and
give
an
overview
of
the
state-of-the-art
research
aiming
to
produce
an
alternative
medication
to
treat
T.
cruzi
infection. ©
2015
Elsevier
B.V.
All
rights
reserved.
Contents
1.
Introduction
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2
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Treatment
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3
2.1.
Benznidazole.
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2.2.
Nifurtimox
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4
3.
Public
health
information
and
specific
research
on
chagas
disease
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4
4.
Challenges
for
new
therapies
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5
4.1.
Improvements
to
current
treatment
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5
4.2.
Molecular
targets
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4.2.1.
Nitroreductase
type
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8
∗Corresponding
author.
E-mail
address:
sdpalma@fcq.unc.edu.ar
(S.
Palma).
1These
authors
contributed
equally
to
this
paper.
http://dx.doi.org/10.1016/j.actatropica.2015.12.017
0001-706X/©
2015
Elsevier
B.V.
All
rights
reserved.

2
J.
Bermudez
et
al.
/
Acta
Tropica
156
(2016)
1–16
4.2.2.
Ergosterol
synthesis
inhibitors
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4.2.3.
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4.2.4.
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4.2.5.
Trans-sialidase
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4.3.
Discovering
new
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10
5.
Concluding
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.12
Acknowledgements.
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.12
References
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12
1.
Introduction
Neglected
Tropical
Diseases
are
a
group
of
17
parasitic
infections
that
affect
people
living
with
low
income
mainly
in
developing
countries,
causing
large
physical,
economic
and
health
problems
in
patients
and
their
communities.
Accord-
ing
to
the
World
Health
Organization
(WHO),
these
infections
include
dengue,
rabies,
trachoma,
human
African
trypanosomi-
asis
(sleeping
sickness),
leishmaniasis,
leprosy,
Chagas
disease,
Buruli
ulcer,
echinococcosis,
lymphatic
filariasis,
onchocerciasis,
schistosomiasis,
dracunculiasis
(Guinea
worm
disease),
foodborne
trematodiases,
taeniasis/cysticercosis,
soil-transmitted
helminth
infection,
and
yaws
(WHO,
2013).
American
trypanosomiasis,
also
called
Chagas
disease
after
the
Brazilian
physician
Carlos
Chagas
who
described
the
infection
in
1909,
is
a
vector-borne
infection
caused
by
the
protozoan
parasite
Trypanosoma
cruzi
(Chagas,
1909).
It
was
originally
found
throughout
South
and
Central
America,
but
owing
to
migrations
it
has
been
recorded
in
every
continent.
The
endemicity
of
the
disease
is
a
complex
phenomenon
that
roots
in
history,
when
sedentary
and
grain
accumulation
developed
in
ancient
human
populations
facilitating
their
interaction
with
vec-
tors
(Brenière
et
al.,
1998;
Aufderheide
et
al.,
2004;
Coura,
2007)
and
in
the
modern
economic
and
social
scenery
of
Latin
America
(Dias
et
al.,
2014;
WHO,
2002).
Chagas
disease
represents
one
of
the
most
significant
health
problems
in
the
American
continent
in
terms
of
human
health
(i.e.,
number
of
people
infected
with
and
dying
from
it),
socioeconomic
impact,
and
geographic
distribution.
Even
though
the
incidence
of
new
infections
decreased
in
Brazil
and
other
countries
due
to
urbanization
and
improved
living
con-
ditions,
an
estimated
number
of
8
million
people
remains
infected
(WHO,
2013).
In
its
natural
life
cycle,
T.
cruzi
is
transmitted
by
reduviid
insects
to
vertebrates
while
they
sleep.
While
feeding,
infected
reduvi-
ids
(Order
Hemiptera,
Family
Reduviidae,
Sub-Family
Triatominae)
defecate
on
the
sleeping
vertebrate
and
parasites
present
in
the
feces
(metacyclic
trypomastigotes)
enter
the
host
through
skin
abrasions
provoked
by
scratching
the
bite.
Due
to
this
complex
life
cycle
that
can
be
sustained
in
both
sylvatic
and
urban
environ-
ments,
T.
cruzi
oral
transmission
has
been
reported
in
rural
areas
in
close
association
with
the
sylvatic
cycle.
One
of
the
main
syl-
vatic
reservoirs
is
Didelphis
spp.,
marsupials
that
have
anal
glands
where
all
T.
cruzi
stages
can
be
found
(epi,
trypo
and
amastigote
forms).
With
these
glands’
secretions,
oppossums
can
contaminate
with
parasites
fruits
and
utensils
used
to
prepare
juice
in
syl-
vatic/rural
environments
in
the
Amazon
region
(Shikanai-Yasuda
et
al.,
1991;
Roque
et
al.,
2008;
Coura,
2015;
Barbosa
et
al.,
2015).
Crushing
infected
bugs
among
the
fruits
and
ingestion
of
raw
or
undercooked
meat
has
also
been
reported
as
a
source
of
oral
out-
breaks
(Pereira
et
al.,
2010;
Cardoso
et
al.,
2006).
Oral
transmission
is
usually
manifested
as
acute
Chagas,
and
it
has
been
demon-
strated
that
metacyclic
trypomastigotes
invade
the
gastric
mucosal
epithelium
(Camandaroba
et
al.,
2002;
Pinto
et
al.,
2008;
Pinto
Dias,
2006;
Yoshida,
2009).
A
comparison
between
oral
and
gastric
infections
in
mice
showed
that
oral
infections
induced
higher
lev-
els
of
parasitemia,
mortality,
liver
lessions,
and
pro-inflammatory
cytokines
(INF-␥
and
TNF-␣)
than
gastric
infections
(Barreto-de-
Albuquerque
et
al.,
2015).
Similarly,
intragastric
infections
showed
slower
development
of
parasitemia,
with
lower
peaks,
as
well
as
lower
mortality
than
intraperitoneally
infected
mice
(Castellanos-
Domínguez
et
al.,
2015),
demonstrating
that
the
initial
site
of
parasite
entrance
strongly
affects
the
host
immune
response
and
disease
outcome.
Human
infection
results
in
a
myriad
of
clinical
symptoms
arising
from
the
initial
deposition
of
infective
trypomastigotes,
occasion-
ally
originating
swelling
or
“chagoma”
at
the
site
of
infection
(WHO,
2002).
The
development
of
Chagas’
disease
varies
considerably
and
there
are
marked
differences
between
individuals
and
geographic
localities.
This
suggests
that
genetic
differences
on
both
the
par-
asite
and
the
host
(Trischmann
and
Bloom,
1982;
Anon.,
1992;
Silva
et
al.,
1992;
Williams-Blangero
et
al.,
2012)
are
important
for
disease
outcome,
which
is
characterized
by
three
phases.
In
the
early,
acute
phase
of
infection
trypomastigotes
circulate
in
blood
(parasitemia),
and
infect
cells
where
they
transform
into
the
asexually-multiplying
amastigotes.
When
the
cell
containing
amastigotes
is
broken,
parasites
are
released
to
the
blood
and
infect
other
cells
in
a
cycle
lasting
a
few
weeks.
During
this
period
there
are
unspecific
symptoms
(fever,
allergic
reactions,
and
more
rarely
acute
heart
failure
or
meningoencephalitis).
Acute
Chagas
disease
can
be
life
threatening
if
acute
myocarditis
develops,
but
it
can
also
be
a
non-specific
febrile
illness
that
in
some
cases
resolves
spon-
taneously
without
diagnosis
or
therapy
(WHO,
2002).
The
acute
phase
might
be
fatal
in
children,
but
most
patients
survive
to
enter
a
prolonged,
asymptomatic
indeterminate
phase
where
parasites
reach
and
establish
in
their
target
organs,
forming
amastigote
nests
(Estani
et
al.,
1998).
Chronic
Chagas
disease
progresses
at
a
rel-
atively
slow
pace
and
70%
of
chronic
patients
have
no
further
evidence
of
disease.
Only
30%
develop
chronic
Chagasic
cardiomy-
opathy
or
mega-organs—esophagus,
liver
or
intestines-
decades
later
(WHO,
2002).
Chronic
Chagasic
cardiomyopathy
(CCC)
is
char-
acterized
by
heart
hypertrophy
and
dilatation,
which
cause
severe
arrhythmias
and
progressive
systolic
dysfunction
(Pearson
et
al.,
2003).
Severe
inflammation
of
the
myocardium
(myocarditis)
was
found
to
be
positively
associated
with
parasite
persistence
and
interstitial
fibrosis
(Benvenuti
et
al.,
2008).
Destroyed
myocardial
cells
are
also
found,
with
lymphocyte,
plasma
cell,
and
macrophage
infiltration
often
forming
“microabscesses”
that
later
heal
by
fibro-
sis.
Mega-organ
disease
is
associated
with
destruction
of
the
myenteric
plexus
in
the
gastrointestinal
tract
(Pearson
et
al.,
2003).
Inflammatory
infiltrate
cells
and
their
cytokine
and
chemokine
expression
in
CCC
heart
lesions
are
well
characterized.
Moreover,
given
the
fact
that
only
20–40%
of
patients
develop
CCC
(Rassi
et
al.,
2009;
Bern,
2015;
Organization
WH,
2015),
and
the
importance
of
inflammatory
mechanisms
in
its
development,
it
is
expected
to
find
genetic
polymorphisms
and
susceptibility
markers
to
CCC
in
genome-wide
association
studies
(Cunha-Neto
and
Chevillard,
2014).
A
consequence
of
research
about
Chagas
disease
has
been
the
description
and
study
of
its
causative
agent,
T.
cruzi
(Domain:
Eukarya,
Phylum:
Euglenozoa,
Class:
Kinetoplastea,
Order:
Tri-
panosomatida,
Family:
Tripanosomatidae,
Genus:
Trypanosoma,

J.
Bermudez
et
al.
/
Acta
Tropica
156
(2016)
1–16
3
Species:
cruzi).
Classification
of
unicellular
eukaryotes
is
under
constant
review,
but
the
most
recent
studies
state
that
Phylum
Euglenozoa
comprises
single-cell,
flagellated
organisms
with
dif-
ferent
nutrition
forms,
including
parasitism.
Among
these,
Class
Kinetoplastea
groups
obligatory
parasites
characterized
by
the
“kinetoplast”,
a
mitochondrion-like
organelle
with
extensive
DNA
content
(kDNA).
Family
Tripanosomatidae
groups
many
organisms
of
human
medical
importance
baring
only
one
flagellum,
such
as
Trypanosoma
brucei,
T.
cruzi
and
Leishmania
spp.
(Lukes,
2009).
In
particular,
T.
cruzi
not
only
has
unique
enzymes
that
are
absent
in
other
organisms,
such
as
trans-sialidase
and
cruzi
pain,
but
also
unique
metabolic
pathways
(RNA
trans-splicing),
or
proteins
shared
with
others
but
with
distinctive
characteristics.
Since
T.
cruzi
reproduces
asexually
in
both
its
vertebrate
and
invertebrate
hosts
it
is
postulated
to
have
a
clonal
population
struc-
ture,
with
high
diversity
reflected
by
its
isoenzymes
(Morel
et
al.,
1980)
and
their
codifying
genes
(Tibayrenc
et
al.,
1986;
Tibayrenc
and
Breniere,
1988;
Tibayrenc
et
al.,
1993).
However,
some
authors
suggested
that
genetic
hybridization
between
parasites
may
also
take
place
(Sturm
and
Campbell,
2010).
It
is
assumed
that
parasite
strains
or
isolates
obtained
from
mammals
or
vectors
are
multi-
clonal
entities,
and
their
filogenetic
relationships
have
impact
on
the
parasites’
diversity
and
biological
characteristics
(Oliveira
et
al.,
1998).
Current
classification
of
T.
cruzi
variability
is
based
on
Dis-
crete
Typing
Units
(DTUs)
that
describes
“a
set
of
stocks
that
are
genetically
more
similar
to
each
other
than
to
any
other
stock,
and
are
identifiable
by
common
genetic,
molecular,
or
immunological
markers,
constituting
relevant
units
for
molecular
epidemiology
and
experimental
evolution”
(Tibayrenc,
2003).
There
are
six
main
DTUs,
each
one
having
distinct
biological
properties
such
as
infec-
tivity,
tissue
tropism,
and
drug
susceptibility
(Zingales
et
al.,
2009).
These
characteristics
must
be
taken
into
account
when
perform-
ing
experiments
on
new
drugs,
for
example,
because
different
DTUs
show
variations
in
expression
and
activity
of
some
metabolic
enzymes
(Zingales
et
al.,
2014).
Besides
humans,
T.
cruzi
infects
a
wide
range
of
domestic
and
wild
mammals
including
dogs,
cats,
bats,
rats
and
armadil-
los.
Therefore,
control
of
Chagas
disease
is
difficult
because
there
are
many
potential
parasite
reservoirs
in
different
vertebrate
species
and
environmental
settings
(PAHO,
2008).
In
addition,
some
triatomine
populations
have
developed
insecticide
resistance
(Vassena
and
Picollo,
2003;
Cardozo
et
al.,
2010).
Other
trans-
mission
paths
of
T.
cruzi
infection
are
blood
transfusions,
organ
donation,
congenitally
from
mother
to
fetus,
and
laboratory
acci-
dents.
Although
numerous
studies
have
been
conducted
aiming
to
produce
a
vaccine
against
Chagas’
disease,
success
has
been
impeded
by
two
main
difficulties:
finding
protective
antigens,
and
generating
attenuated
parasites
that
will
not
trigger
pathology
in
the
long
term
(Sánchez-Valdéz
et
al.,
2014;
Vázquez-Chagoyán
et
al.,
2011).
Therefore,
in
absence
of
vaccines,
control
measures
of
Chagas
disease
are
limited
to
case
detection
and
treatment,
vec-
tor
control
by
insecticide
applications
within
domestic
buildings,
screening
of
blood
banks
and
organ
donors,
detection
of
infected
pregnant
women
and
congenital
cases
(WHO,
2002).
Based
on
these
facts,
we
will
discuss
the
treatment
of
human
Chagas
disease
with
the
drugs
currently
in
use,
as
well
as
the
poten-
tial
new
drugs
under
trial
and
innovative
strategies
for
treating
Chagas
disease.
2.
Treatment
Since
Neglected
Tropical
Diseases
occur
mainly
in
developing
countries,
some
of
the
drugs
needed
to
treat
them
are
not
autho-
rized
to
use
in
industrialized
countries,
and
therefore
have
not
been
approved
by
a
regulatory
drug
agency.
The
guarantee
of
product
quality
is
evaluated
at
the
time
of
its
registration
in
every
country.
National
regulatory
drug
agencies
of
Neglected
Tropical
Diseases
in
many
endemic
countries
have
limited
ability
to
assess
whether
drug
producers
comply
with
guidelines
established
by
WHO
in
its
Good
Manufacturing
Practices.
The
risk
associated
with
these
prod-
ucts
is
further
complicated
because
some
drugs
are
outdated,
and
guidance
on
the
product’s
specifications
is
not
available
in
interna-
tional
pharmacopoeia
monographs.
In
the
particular
case
of
T.
cruzi
infection,
Benznidazole
(BZL)
and
Nifurtimox
(NFX),
launched
in
the
early
1970s,
are
the
only
drugs
approved
for
human
treatment.
Both
compounds
share
some
char-
acteristics:
better
tolerance
by
children,
more
effectiveness
during
the
acute
phase
of
T.
cruzi
infection,
higher
toxicity
in
adults,
and
different
susceptibility
among
T.
cruzi
DTUs.
There
is
documented
evidence
that
some
DTUs
are
naturally
resistant
to
nitrohetero-
cyclic
compounds
(Filardi
and
Brener,
1987;
Andrade
et
al.,
1985;
Toledo
et
al.,
2004;
Martins
et
al.,
1998),
but
drug-induced
resis-
tant
parasites
can
also
be
obtained
in
response
to
the
selective
pressure
of
adding
drugs
to
the
culture
medium
(Buckner
et
al.,
1998;
Wilkinson
et
al.,
2008;
Campos
et
al.,
2014).
In
addition,
P-
glycoprotein
(Pgp)
efflux
and
Pgp
ATPase
activity
were
described
in
T.
cruzi,
and
Pgp
inhibitors
cyclosporine
A
and
verapamil
were
able
to
revert
BZL
resistance
in
treated
parasites,
thus
implicating
Pgp
efflux
pumps
in
drug
resistance
(Campos
et
al.,
2013).
How-
ever,
overexpression
of
Pgp
genes
did
not
seem
to
play
a
role
in
T.
cruzi
drug
resistance,
rather
than
qualitative
differences
in
Pgp
function
(Murta
et
al.,
2001).
The
controversy
about
the
relationship
between
drug
resistance
and
parasite
virulence
is
also
a
matter
to
be
settled;
some
authors
claim
that
resistant
strains
are
more
viru-
lent
(Andrade
et
al.,
1985)
while
others
did
not
find
this
association
(Filardi
and
Brener,
1987).
According
to
WHO,
there
is
enough
evidence
supporting
that
parasitic
persistence
is
the
main
cause
of
progression
towards
car-
diomyopathy,
and
therefore
treatment
with
BZL
or
NFX
during
the
chronic
phase
can
soften
its
development.
For
that
reason,
there
has
been
a
change
of
paradigm
in
the
guidelines
of
WHO
that
now
recommends
treatment
to
all
patients.
However,
treatment
must
be
handled
with
care
in
advanced
chronic
patients
since
the
exist-
ing
pathology
may
not
be
reversed,
but
those
with
gastrointestinal
manifestations
could
have
lower
risk
of
developing
cardiomyopa-
thy
after
treatment
(WHO,
2002;
Viotti
et
al.,
2014).
2.1.
Benznidazole
Benznidazole
(BZL)
or
N-benzyl-2-(2-nitro-1H-imidazol-1-yl)
acetamide,
is
a
2-nitroimidazole
derivative
used
as
the
first
line
treatment
of
Chagas
disease
(Fig.
1A).
According
to
Maximiano
et
al.
(2010),
based
on
its
low
solubility
in
water
BZL
can
be
assigned
to
class
4
in
the
Biopharmaceutical
Classification
Sys-
tem
(Leonardi
et
al.,
2009).
It
is
recommended
that
BZL
treatment
should
be
given
orally
for
60
days
on
a
daily
basis
at
5–7
mg/kg
for
adults,
and
10
mg/kg
for
children
(WHO,
2002).
The
low
solu-
bility
of
BZL,
combined
to
a
high-dose
treatment
for
a
long
period
of
time
trigger
adverse
reactions
(WHO,
2013;
Lima
et
al.,
2011)
that
include
hypersensitivity
-ranging
from
light-sensitive
rashes
to
exfoliative
dermatitis,
bone
marrow
suppression
(thrombocy-
topenia,
neutroenia,
agranulocytosis)
and
peripheral
neuropathy
(Weiss
Louis
et
al.,
2011).
The
low
solubility
of
BZL
also
affects
its
bioavailability,
decreasing
its
effectiveness
during
the
chronic
phase
of
infection—when
parasites
are
localized
mainly
in
skeletal
and
heart-muscle
cells.
Pinazo
et
al.
(2013)
tested
whether
adverse
reactions
were
related
to
BZL
concentration
in
serum,
and
there-
fore
analyzed
54
patients
diagnosed
with
T.
cruzi
infection
that
were
given
BZL
regardless
the
phase
of
disease.
BZL
concentra-
tion
in
serum
was
not
statistically
different
in
either
patients
that
stopped
treatment
due
to
adverse
effects
or
those
who
finished

4
J.
Bermudez
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/
Acta
Tropica
156
(2016)
1–16
Fig.
1.
Structures
of
benznidazole
(A)
and
nifurtimox
(B),
drugs
currently
used
against
Chagas
disease.
it
without
complications.
Even
though
the
authors
did
not
find
any
association
between
BZL
concentration
in
serum
and
adverse
effects,
they
established
that
BZL
given
at
5
mg/kg/day
resulted
in
mean
serum
concentrations
of
3–6
␮g/mL-assumed
to
be
the
high-
est
trypanocidal
concentration,
thus
indicating
that
5
mg/kg/day
is
appropriated
to
obtain
therapeutic
drug
concentrations
in
serum
(Pinazo
et
al.,
2013).
In
agreement
with
these
results,
empirical
data
from
patients
treated
with
5
mg/kg/day
of
BZL
during
the
chronic
phase
have
shown
lower
clinical
progression
to
cardiomyopathy
than
untreated
patients
(Viotti
et
al.,
1994;
Viotti
et
al.,
2011).
2.2.
Nifurtimox
Nifurtimox,
or
(RS)-3-methyl-N-[(1E)-(5-nitro-2-furyl)
methy-
lene]
thiomorpholin-4-amine
1,1-dioxide
(Fig.
1B)
is
produced
by
Bayer,
and
has
been
the
mainstay
of
therapy
for
Chagas’
dis-
ease
in
the
United
States.
For
more
than
a
decade
Bayer
has
been
providing
the
WHO
with
1
million
tablets
a
year
free
of
charge,
along
with
financial
assistance
for
its
distribution
mainly
in
Honduras
and
El
Salvador
(AGB,
2015).
Doses
recommended
by
WHO
are
8–10
mg/kg
daily
in
three
divided
doses
for
adults,
and
15–20
mg/kg
daily
in
four
divided
doses
for
children,
during
60–90
days
(WHO,
2002).
Gastrointestinal
maladies
(nausea,
vom-
iting,
abdominal
pain)
are
predominant
adverse
effects
observed
with
nifurtimox,
but
30%
of
patients
can
also
experience
central
nervous
system
perturbations
such
as
polyneuritis,
confusion
or
focal
or
generalized
seizures,
and
even
psychosis
that
resume
when
treatment
is
stopped
(Weiss
Louis
et
al.,
2011).
Some
patients
may
also
develop
skin
rashes,
and
individuals
with
glucose-6-phosphate
dehydrogenase
deficiency
can
experience
drug-induced
hemolytic
anemia.
An
increase
in
chromosomal
aberrations
has
been
seen
in
children
given
nifurtimox
(Gorla
et
al.,
1989).
NFX
has
shown
higher
toxicity
and
adverse
effects
than
BZL,
including
increased
oxidative
stress
in
rat
pancreas
(de
Mecca
et
al.,
2007)
and
heart
(Mecca
et
al.,
2008).
In
the
context
of
heart
damage
caused
by
the
parasite
dur-
ing
a
chronic
infection,
treatment
with
NFX
may
represent
a
higher
risk
of
heart
failure
than
BZL
treatment.
For
these
reasons,
NFX
is
not
the
treatment
of
choice
for
Chagas
disease
in
most
endemic
countries.
It
is
accepted
that
toxicity
of
nitro-compounds
arises
from
their
metabolic
conversion
after
enzymatic
reduction
of
the
nitro
group,
which
results
in
molecules
considered
free
radicals
(Castro
and
Diaz,
1988).
This
statement
stands
true
for
both
BZL
and
NFX,
although
there
are
some
differences
in
the
nature
of
their
reac-
tive
metabolites.
Several
enzymes
are
proposed
to
be
involved
in
BZL
and
NFX
metabolism,
including
trypanothione
reductase
(Henderson
et
al.,
1988)
and
nitroreductases
(Wilkinson
et
al.,
2008;
Kubata
et
al.,
2002).
The
general
mechanism
proposed
cyclic
reduction
of
the
nitro
group
that
yields
a
nitro-radical,
which
in
turn
undergoes
auto-oxidation
and
forms
superoxide,
regenerat-
ing
the
parent
compound.
Wilkinson
et
al.
(2008)
proposed
that
type
I
nitroreductase
(NTR)
is
the
main
enzyme
involved
in
the
bioactivation
of
nitroheterocyclic
drugs
in
T.
cruzi
and
T.
brucei,
and
that
impairment
of
its
activity
confers
resistance
to
BZL
and
NFX.
In
further
experiments,
Hall
and
Wilkinson
(2012)
demonstrated
that
reduction
of
BZL
generates
a
hydroxylamine
derivative
that
ultimately
converts
to
glyoxal,
a
highly
cytotoxic
and
mutagenic
compound
(Hall
and
Wilkinson,
2012).
However,
in
a
recent
work,
Trochine
et
al.
(2014)
failed
to
detect
glyoxal
in
a
metabolomics
study
carried
out
on
T.
cruzi
treated
with
BZL.
Instead,
they
found
that
BZL
binds
to
low
molecular
weight
thiols,
and
to
protein
thi-
ols
thus
inactivating
enzymes
whose
active
site
involves
cysteine
residues,
such
as
the
tryparedoxin
peroxidase-tryparedoxin
sys-
tem.
They
also
found
BZL
covalently
bound
to
pyroglutamic
acid
and
valine
residues.
Hence
the
authors
proposed
a
new
mechanism
of
action
BZL,
but
its
exact
pathway
and
enzymes
involved
will
be
discovered
in
future
experiments
(Trochine
et
al.,
2014).
On
the
other
side,
reduction
of
NFX
by
NTR
brakes
the
furan
ring
yielding
an
unsaturated
open
chain
nitrile
that
is
as
cytotoxic
as
the
par-
ent
compound,
evidencing
that
NFX
typanocidal
activity
does
not
necessarily
involve
oxidative
stress
(Hall
et
al.,
2011).
3.
Public
health
information
and
specific
research
on
chagas
disease
Disease
profiles
show
variations
among
developing
and
fully
developed
countries.
These
profiles
are
also
reflected
in
the
infor-
mation
about
health
needs
of
different
countries.
Taking
the
number
of
scientific
publications
as
a
general
measurement
of
health
information,
it
is
possible
to
observe
that
the
vast
majority
of
global
knowledge
on
health
topics
-including
clinical
trials-
is
pro-
duced
by
developed
countries
in
response
to
their
own
local
needs.
This
situation
also
raises
concern
about
information
on
research
infrastructure
in
lower
income
populations.
However,
the
num-
ber
of
research
articles
remains
an
imperfect
approach
of
accurate
knowledge
about
biomedical
development.
Since
some
diseases
are
harder
to
understand,
prevent,
diagnose,
and
treat
than
oth-
ers,
many
publications
contain
a
high
amount
of
data
aiming
to
tackle
these
difficulties,
often
with
limited
success.
In
the
present
section
we
will
describe
how
scientific
publications
about
Chagas
disease
have
shifted
during
the
past
100
years,
in
terms
of
num-
ber
of
publications,
topics,
and
countries
that
make
contributions
to
the
subject.
It
has
been
mentioned
before
that
Chagas
disease
was
first
described
in
1909,
and
some
articles
of
that
time
have
been
indexed
by
Latin-American
search
engines
such
as
LILACS
and
Scielo.
The
first
references
in
search
engines
such
as
Scopus
and
Pubmed
were
registered
in
1933
(Villela
and
Villela
Von,
1933)
and
1938
(Wood,
1938),
when
presence
of
non-infectious
forms
of
T.
cruzi
were
described
in
USA.
Nevertheless,
the
increasing
inter-
est
of
scientific
research
on
Chagas
disease
has
been
boosted
by
the
combination
of
different
factors.
One
of
them,
and
perhaps
the
main,
is
a
consequence
of
the
economic
support
derived
from
policies
implemented
following
the
“Special
Program
for
Research
and
Training
in
Tropical
Diseases”
(TDR)
by
WHO
(Rowe,
1977).
Other
factors
include
the
general
improvement
in
global
com-

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Acta
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156
(2016)
1–16
5
Fig.
2.
Number
of
publications
by
year/database.
munications
that
triggered
systematic
organization
of
specialized
meetings,
the
existence
of
resources
exclusively
dedicated
to
cer-
tain
topics,
along
with
discussion
places
for
researchers
and
public
exposure
of
research
papers.
The
increasing
scientific
interest
on
Chagas
disease
can
be
clearly
seen
in
Fig.
2,
which
shows
the
rising
number
of
pub-
lished
articles
using
the
keywords
“Chagas”;
“Chagas
disease”;
“Trypanosoma
cruzi”;
“Chagas
cardiomyopathy”
and
“Drug
Ther-
apy”
in
different
databases.
While
WHO
and
PAHO
policies
triggered
an
increase
in
publica-
tion
numbers,
there
was
a
weak
articulation
between
knowledge
production
and
specific
intervention
opportunities
on
the
disease.
More
than
70%
of
the
total
amount
of
papers
published
on
the
sub-
ject
refers
to
basic/experimental
research,
and
the
most
explored
topics
are
related
to
the
insect
vector,
including
descriptions
of
its
biochemical
functions,
its
feeding,
reproductive
habits,
geographi-
cal
distribution,
and
insecticide
resistance.
Less
than
10%
of
the
total
amount
of
publications
is
related
to
clinical
trials.
Leaving
aside
research
about
the
biology
of
T.
cruzi
and
the
different
cellular
and
animal
models
employed
for
its
study,
case
reports
are
by
far
the
largest
type
of
study
on
Chagas
disease
(Fig.
3).
Private-funded
laboratories
lack
interest
on
research
about
Cha-
gas
disease,
while
equipment
and
technological
restrictions
in
public
laboratories
jeopardize
the
development
of
new
drugs
and
treatment
strategies,
leading
to
the
actual
situation
where
cur-
rently
available
drugs
are
the
same
as
in
1970.
Considering
that
research
about
this
topic
are
mainly
produced
by
the
academy,
we
embraced
a
“classic”
scientific
production
evaluation
criterion
(quantity
and
quality
of
publications)
to
assess
the
development
of
interest
on
Chagas
disease.
In
that
regard,
the
number
of
publica-
tions
related
to
other
diseases
such
Alzheimer,
hypertension,
HIV,
tuberculosis
is
at
least
triple
than
the
number
of
publications
on
Chagas
disease
(Fig.
4).
Academically,
the
number
of
publications
should
be
analyzed
using
a
criterion
that
allows
assessing
their
quality
as
well.
The
impact
factor
can
be
used
as
a
(rather
subjective)
measure
of
qual-
ity.
Since
it
is
obtained
by
calculating
the
average
of
the
times
an
article
published
in
a
certain
journal
is
referenced,
it
is
therefore
assumed
that
the
higher
the
impact
factor
is,
the
higher
the
vis-
ibility
of
the
journal,
and
the
greater
the
number
of
researchers
who
will
intend
to
publish
in
that
journal.
Resulting
from
that
com-
petition,
there
is
a
correlation
between
the
quality
and
impact
of
the
publications
in
a
journal
with
its
impact
factor.
There
are
two
indicators
for
scientific
journals
(journal
metrics):
Scimago
Journal
&
Country
Rank
(SJR),
and
Scopus
Source
Normalized
Impact
per
Paper
(SNIP).
The
journal
metrics
show
that
articles
about
Chagas
disease
are
more
frequently
published
in
Latin-American
journals
with
low
impact
factor
(Fig.
5).
It
is
not
surprising
that
the
highest
number
of
research
articles
are
published
in
-and
proceed
from-
American
countries
(Fig.
6),
since
Chagas
is
endemic
in
21
countries
of
the
region.
Moreover,
since
1991
there
are
explicit
PAHO
initiatives
committed
to
elimi-
nate
or
at
least
reduce
neglected
diseases,
implementing
policies
to
promote
active
research
and
scientific
development
supported
by
the
Ministries
of
Health
of
Argentina,
Bolivia,
Brazil,
Chile,
Paraguay
and
Uruguay.
Increased
interest
on
research
about
Chagas
disease
from
US
and
European
countries
arose
from
higher
migrations
rates
in
recent
decades,
causing
a
new
health
problem
in
non-endemic
countries.
Therefore
the
affected
countries
acknowledged
the
new
situation,
taking
measures
to
prevent
congenital
transmission,
carrying
out
controls
at
blood
banks,
and
last
but
not
least,
developing
new
lines
of
research
for
treatment
of
this
old
disease.
4.
Challenges
for
new
therapies
Taking
a
deeper
insight
on
the
information
presented
above,
one
of
the
fields
studied
with
equal
interest
in
higher
and
lower
income
countries
is
the
improvement
of
existing
therapies,
and
the
development
of
new
ones
to
treat
American
trypanosomiasis.
Given
the
complexity
of
Chagas
disease,
the
characteristics
of
the
parasite,
and
the
evidence
gathered
in
clinical
practice,
it
is
assumed
that
any
new
drug
would
be
administered
for
at
least
30
days,
and
in
order
to
achieve
patient
compliance
in
low
income
settings
it
needs
to
be
given
orally
(Buckner,
2011).
In
this
section
we
will
briefly
summarize
the
most
recent
upturns
in
treatment
with
BZL,
and
the
new
drug
candidates
that
may
struggle
to
reach
the
market
in
the
future.
4.1.
Improvements
to
current
treatment
According
to
new
WHO
recommendations
(2002)
of
giving
treatment
to
all
Chagasic
patients
regardless
the
phase
of
the
disease
they
are
suffering,
there
have
been
many
initiatives
to

6
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/
Acta
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156
(2016)
1–16
Fig.
3.
Main
types
of
studies
on
human
Chagas
disease.
evaluate
BZL
treatment
in
the
chronically
infected
population.
One
of
these
initiatives
was
the
trial
“Benznidazole
Evaluation
for
Interrupting
Trypanosomiasis
(BENEFIT)”,
designed
to
clarify
the
role
of
treatment
with
BZL
in
patients
with
CCC,
and
its
effect
on
disease
progression
in
endemic
countries
(Argentina,
Bolivia,
Brazil,
Colombia,
and
El
Salvador)
(Morillo
et
al.,
2015).
It
was
a
multicenter,
international,
randomized,
double-blind,
placebo-
controlled
trial
in
which
2854
patients
received
treatment
during
40–80
days
with
either
BZL
(5
mg/kg/day,
later
adjusted
by
body
weight
to
300
mg/day)
or
placebo,
and
were
followed
up
for
at
least
5
years.
Patients
were
evaluated
twice
intra-treatment,
at
the
end
of
treatment,
at
6
months,
and
then
annually
until
the
end
of
the
study
for
side-effects,
liver-function,
ECG,
and
parasitic
burden
in
blood.
Parasite
load
was
determined
by
conventional
PCR
in
50%
of
the
population,
and
serum
samples
were
stored
for
future
studies.
Adverse
effects,
which
include
cutaneous
rash,
gastrointestinal
symptoms
and
nervous
system
disorders,
lead
to
significantly
higher
interruption
of
treatment
in
BZL
(23.9%)
than
in
placebo
groups
(9.5%).
In
conclusion,
the
study
demonstrated
that
treatment
with
BZL
significantly
diminished
parasite
load
circulat-
ing
in
blood,
but
this
reduction
did
not
correlate
with
ameliorating
cardiac
deterioration.
An
alternative
often
proposed
is
the
combination
of
BZL
with
other
compounds
to
increase
effectiveness.
Allopurinol
and
allop-
urinol
riboside
have
been
under
investigation
for
a
long
time
as
possible
treatments
of
American
trypanosomiasis
(Gallerano
et
al.,
1990),
and
it
has
been
demonstrated
that
allopurinol
and
itracona-
zole,
alone
or
in
combination
have
produced
beneficial
responses
in
patients
with
chronic
disease
(Apt
et
al.,
1998;
Apt
et
al.,
2003).
Recently,
Perez-Mazliah
et
al.
(2013)
have
shown
that
a
combi-
nation
of
BZL
and
allopurinol
is
well
tolerated,
and
effective
in
reducing
infection
parameters
(parasite
burden
and
changes
in
B-
and
T-cell
response)
in
chronically
infected
humans.
Nowadays,
BZL
is
only
available
as
100
mg
tablets,
and
they
must
be
fractioned
for
administration
in
suspension
to
newborns
and
children,
leading
to
inadequate
doses
and
possible
adverse
reac-
tions.
Since
a
large
part
of
the
population
affected
by
Chagas
disease
are
children,
one
line
of
research
is
focused
on
the
discovery
of
new
pediatric
formulations.
Tarragona
et
al.
(2013)
developed
BZL
sug-
ary
gels
and
chewable
tablets
for
pediatric
administration.
These
were
innovative
formulations
that
masked
the
bad
taste
of
the
drug
and
increased
its
dissolution
profile
compared
to
the
commercially
available
tablet.
Moreover,
Manarin
et
al.
(2013)
formulated
novel
BZL-water-polyethylene
glycol
400
solutions
at
pH
2.5,
which
were
useful
vehicles
that
highly
improved
BZL
solubility.
The
advantage
Fig.
4.
Number
of
publications
related
to
Chagas
disease
and
other
diseases
until
present
day.

J.
Bermudez
et
al.
/
Acta
Tropica
156
(2016)
1–16
7
Fig.
5.
Impact
factor
of
the
top
10
journals
arranged
by
number
of
publications
about
Chagas,
according
Scopus
(left
to
right).
of
liquid
formulations
is
to
avoid
manual
disruption
of
BZL
tablets
needed
to
prepare
suspensions
suitable
for
small
children.
Regarding
the
children
population,
recent
advances
were
reported
by
Alonso-Vega
et
al.
(2013)
on
Bolivia’s
Congenital
Chagas
Disease
Program.
All
children
diagnosed
with
congenital
Chagas
were
treated
with
BZL
at
10
mg/kg/day
for
30
days,
in
two
daily
doses,
excepting
the
first
week
when
it
was
7
mg/kg/day
in
two
doses.
Children
were
monitored
to
control
compliance
with
treatment,
side
effects
and
dose-adjustment
according
to
weight
variations.
There
were
no
reports
of
side
effects
in
more
than
90%
treated
children,
and
those
who
presented
adverse
effects
were
treated
accordingly.
Side
effects
were
not
a
cause
of
withdrawal,
rather
than
migration
to
other
places
in
Bolivia.
Treatment
out-
come
was
evaluated
by
serological
tests
(HAI
and
ELISA)
6
months
after
treatment;
if
the
result
was
positive
the
test
was
repeated
6
months
later,
and
if
that
was
still
positive
it
was
considered
treat-
ment
failure,
and
the
child
received
a
second
round
of
treatment.
Serological
negativization
(treatment
success)
was
found
in
98%
of
children,
demonstrating
that
BZL
is
effective
as
an
anti-Chagasic
medication
in
the
children
population
of
several
Bolivian
depart-
ments
(Alonso-Vega
et
al.,
2013).
Results
about
the
effectiveness
of
an
alternative
drug
to
BZL
were
published
by
Molina
et
al.
(2014)
that
conducted
CHAGA-
ZOL,
a
randomized,
open-labeled
clinical
trial
carried
out
in
Catalunya,
Spain.
The
aim
was
to
evaluate
the
efficacy
of
posacona-
zole
vs.
BZL
in
chronic
Chagasic
patients
in
a
1:1:1
ratio,
who
randomly
recived
oral
treatments
twice
daily
for
60
days
(BZL
group:
150
mg,
posaconazole
(low-dose):
100
mg,
posaconazole
(high-dose):
400
mg).
They
evaluated
treatment
outcome
only
by
real
time
PCR
(qPCR)
determination
of
T.
cruzi
DNA
circulating
in
blood
at
8,
16,
24,
and
40
weeks
after
the
end
of
the
treatment.
They
found
treatment
failure
(measured
as
positive
qPCR)
in
90%
of
patients
treated
with
high
dose
posaconazole,
in
80%
of
low
dose
posaconazole,
and
in
5.9%
of
BZL-treated
patients.
Positive
results
occurred
at
an
earlier
time
in
posaconazole-treated
patients
than
in
those
receiving
BZL.
Side
effects
were
not
reported
in
either
of
the
Fig.
6.
Distribution
of
publications
about
Chagas
disease
by
regions.
posaconazole
groups,
but
5
patients
withdraw
from
BZL
treatment
due
to
adverse
side
effects.
Even
though
the
authors
did
not
evalu-
ate
changes
in
the
serologic
profile
after
treatment,
they
postulate
that
posaconazole
was
not
as
effective
as
BZL
in
chronic
Chaga-
sic
patients
(Molina
et
al.,
2014).
However,
as
reviewed
by
Urbina
(2015);
this
failure
of
posaconazole
treatment
in
human
patients
compared
to
better
results
in
mice
models
is
related
to
a
suboptimal
dosage
and
administration
schedules.
Posaconazole
concentration
measured
in
human
plasma
is
10–20%
of
that
measured
in
mice,
which
could
be
improved
by
new
formulations
of
the
drug
with
better
plasma
exposure
(currently
under
development).
Likewise,
increasing
time
of
administration
may
improve
parasitological
cure
rates
(Wood,
1938).
In
2012,
Alvarez
et
al.
(2012)
suggested
that
BZL
dosing
may
be
reduced
and
still
be
trypanocidal
in
human
subjects.
Since

8
J.
Bermudez
et
al.
/
Acta
Tropica
156
(2016)
1–16
side
effects
are
the
main
cause
of
BZL-treatment
withdrawal,
the
authors
analyzed
a
cohort
of
81
patients
that
were
treated
for
only
10
days
and
found
that
20%
meet
the
criteria
of
cure.
These
data
suggested
that
BZL
dosing
regimen
could
be
adjusted
to
avoid
side
effects
and
achieve
cure
(Alvarez
et
al.,
2012).
Different
authors
have
found
evidence
that
supports
Alvarez
and
cols.
data,
and
demonstrated
that
reducing
BZL
dosing
to
diminish
adverse
effects
while
maintaining
its
trypanocidal
effect
is
possible
in
mice
mod-
els.
In
addition,
co-administration
of
BZL
with
azoles
can
improve
BZL
effectiveness
and
contribute
to
reduce
its
dosing.
Evaluation
of
the
interaction
between
BZL
and
itraconazole
(ITC)
by
Moreira
da
Silva
et
al.
(2012)
demonstrated
that
co-administration
of
both
drugs
in
mice
diminished
maximum
BZL
plasma
concentration,
improving
its
half-life
and
volume
of
distribution.
Since
BZL
and
ITC
are
metabolized
by
the
same
cytochrome
P450
enzymes,
the
prolonged
half-life
of
BZL
could
be
due
to
the
preference
of
ITC
for
the
metabolizing
enzymes,
but
this
hypothesis
needs
confirmation
in
animals
with
larger
volumes
of
blood
than
mice
(Moreira
da
Silva
et
al.,
2012).
In
a
recent
work,
the
interaction
between
BZL
and
ITC
was
shown
again
in
an
experiment
of
combined
therapy
at
subop-
timal
doses
of
both
compounds.
In
a
mice
model
of
acute
T.
cruzi
infection,
treatment
with
suboptimal
doses
(50
and
75
mg/kg/day)
or
optimal
doses
(100
mg/kg/day)
of
BZL/ITC
in
combination
ther-
apy
had
equivalent
or
superior
efficacies
than
these
compounds
given
at
their
optimal
doses
separately
(Assíria
Fontes
Martins
et
al.,
2015).
This
is
a
promising
scenario
for
a
combined
therapy,
since
the
good
pharmacokinetic
properties
of
ITC
can
be
used
to
reduce
the
dosage
and
time
of
administration
of
BZL,
concomitantly
low-
ering
the
emergence
of
side-effects.
Similarly,
a
synergistic
effect
using
a
combined
treatment
of
BZL
and
the
azole
posaconazole
was
observed
by
other
authors.
Cencig
et
al.
(2012)
demonstrated
that
BZL
and
NFX,
alone
or
in
combination
with
posaconazole
or
ampho-
tericin
B
(Ambisome)
administered
during
shorter
periods
of
time
were
able
to
cure
T.
cruzi
infection
in
mice.
The
best
combination
was
BZL
+
posaconazole
in
both
susceptible
and
partially
resistant
strains;
whereas,
BZL
+
Ambisomedid
not
cure
the
infection
(Cencig
et
al.,
2012).
The
synergistic
effect
of
BZL-posaconazole
at
sub-
optimal
doses
or
shorter
treatment
schedules
during
the
acute
phase
of
T.
cruzi
infection,
even
with
the
BZL-resistant
strain
VL-
10,
has
also
been
shown
(Diniz
Lde
et
al.,
2013).
The
authors
used
1/2
(50
mg/kg/day)
and
1/4
(25
mg/kg/day)
of
the
optimal
doses
of
BZL
and
posaconazole
for
the
combined
therapies.
Similarly,
a
sequential
treatment
at
optimal
dosage
starting
with
posacona-
zole
followed
by
BZL
significantly
reduced
parasite
burden.
The
authors
suggest
that
BZL
would
rapidly
reduce
parasite
biomass
for
subsequent
action
of
posaconazole,
facilitated
by
its
large
vol-
ume
of
distribution
and
long
half-life.
These
results
indicate
that
it
is
possible
to
reduce
dosage
and/or
treatment
duration
to
dimin-
ish
BZL
toxicity
and
the
azole
cost.
This
is
a
promising
scenario
for
life-threatening
acute
Chagas
cases,
such
as
reactivation
after
an
immunosuppression.
However,
these
combinations
remain
to
be
tested
in
chronic
phase
models
of
the
infection
to
assess
their
efficacy
in
reducing
the
progression
of
CCC.
Likewise,
using
a
short,
intermittent
dosage
schedule,
Bustamante
et
al.
(2014)
also
found
promising
results
in
mice
infected
with
susceptible
and
resistant
T.
cruzi
strains
treated
with
BZL,
NFX,
allopurinol,
posaconazole,
an
oxaborale,
and
a
nitrotriazole,
alone
and
in
combination
(Bustamante
et
al.,
2014).
Mice
infected
with
susceptible
strains
were
cured
under
regimens
of
intermintent
administration
of
BZL
or
NFX
over
a
reduced
period
of
time
(13
doses
given
at
5
days
intervals).
A
short-term,
intermittent
regime
of
a
combination
of
BZL
+
posaconazole
ren-
dered
the
same
result
as
a
40
consecutive
days
of
BZL
or
NFX
alone.
In
contrast,
the
oxaborale
was
effective
only
in
a
40-day
administration
schedule.
Therefore
the
authors
suggested
that
BZL
may
have
a
maximum-dose
mechanism
of
action,
which
displays
an
extended
post-antibiotic
effect
even
after
the
compound
has
been
completely
removed
(Craig,
1998).
This
finding
is
opposite
to
the
previous
assumption
that
BZL
should
be
maintained
in
a
concentration
above
the
minimum
inhibitory
concentration,
con-
sidering
its
half-life
of
1–2
h
in
mice
and
12
in
humans.
In
addition,
Planer
et
al.
(2014)
found
some
new
combinations
of
amlodipine
(a
calcium-channel
blocker)
and
posaconazole,
as
well
as
the
combination
of
clemastine
(anti-histamine)
and
posaconazole,
to
be
effective
in
reducing
parasitemia
in
mice
(Planer
et
al.,
2014).
4.2.
Molecular
targets
Since
T.
cruzi
diverged
from
other
eukaryotes
a
long
time
ago,
there
are
many
metabolic
pathways
and
enzymes
unique
to
this
parasite
that
represent
excellent
molecular
targets
for
drug
development.
However,
despite
the
specificity
of
new
compounds
targeting
parasite
molecules,
the
effect
of
these
drugs
on
mam-
malian
metabolism
must
be
carefully
addressed
before
they
reach
the
market.
A
few
of
the
most
studied
compounds
targeting
specific
T.
cruzi
enzymes
will
be
reviewed
in
this
section.
4.2.1.
Nitroreductase
type
I
This
enzyme
is
involved
in
activation
of
nitroheterocycles,
as
mentioned
earlier
(Wilkinson
et
al.,
2008),
and
therefore
represents
a
molecular
target
for
BZL/NFX-susceptible
strains.
New
com-
pounds
that
inhibit
NTR
have
shown
anti-T.
cruzi
activity,
including
aziridinyl
nitrobenzamide
derivatives
(Bot
et
al.,
2010;
Wilkinson
et
al.,
2011),
and
3-nitrotriazoles,
the
latter
against
amastigotes
and
infected
mice
(Papadopoulou
et
al.,
2013;
Papadopoulou
et
al.,
2015).
Moreover,
nitroheterocyclic
compounds
dependent
on
NTR
activation
demonstrated
to
be
more
effective
than
ergosterol
biosynthesis
inhibitors
when
tested
on
the
same
clones.
Experi-
ments
conducted
on
amastigotes
of
the
same
clones
and
strains
representative
of
each
DTU
showed
that
nitroheterocyclic
com-
pounds
were
rapidly
active
towards
a
broader
range
of
clones
and
strains;
whereas,
ergosterol
synthesis
inhibitors
showed
variable
activity
and
were
unable
to
eradicate
intracellular
infection
after
7
days
of
continuous
compound
exposure
(Moraes
et
al.,
2014).
In
turn,
these
findings
could
explain
results
obtained
in
mice
treated
with
itraconazole
(Toledo
et
al.,
2004),
and
in
patients
treated
with
posaconazole
(Molina
et
al.,
2014).
4.2.2.
Ergosterol
synthesis
inhibitors
The
existence
of
T.
cruzi
populations
naturally
resistant
to
ben-
znidazole
and
nifurtimox
(Murta
et
al.,
1998;
Camandaroba
et
al.,
2003;
Mejía-Jaramillo
et
al.,
2012)
led
to
search
for
compounds
with
a
different
mechanism
of
action,
such
as
ergosterol
biosynthesis
inhibitors.
Ergosterol
is
a
precursor
in
the
synthesis
of
choles-
terol,
and
therefore
essential
for
membrane
structure
in
animal
cells.
One
step
in
its
synthesis
reaction
is
catalized
by
a
member
of
the
cytochrome
P450
family,
the
sterol
C14-demethylase
(CYP51),
which
in
T.
cruzi
is
highly
specific
and
different
from
organisms
closely
related,
like
T.
brucei
(Lepesheva
et
al.,
2006).
This
finding
led
to
test
T.
cruzi
susceptibility
to
several
azole
compounds
that
inhibit
C14-demethylase
synthesis,
which
were
already
approved
to
treat
fungal
infections
(Lepesheva
et
al.,
2007).
Azole
molecules
with
trypanocidal
activity
include
the
compound
TAK-187
that
showed
promising
results
in
vitro
(Corrales
et
al.,
2005)
but
whose
production
was
discontinued,
and
some
commercially
available
antifungals
such
as
fluconazole,
itraconazole,
ravuconazole,
and
posaconazole.
Fluconazole
resistance
was
in
vitro-induced
in
T.
cruzi,
shown
to
be
stable
in
absence
of
the
drug,
and
even
main-
tained
in
animal
models
thus
hampering
its
further
use
as
an
alternative
treatment
(Buckner
et
al.,
1998).
In
addition,
strains
nat-
urally
resistant
to
itraconazole
(ITC)
were
reported.
ITC-resistance
was
apparently
linked
to
phylogenetic
relationships
among
popu-

J.
Bermudez
et
al.
/
Acta
Tropica
156
(2016)
1–16
9
lations,
since
all
the
studied
ITC-resistant
populations
belonged
to
DTU
I
and
susceptible
ones
were
DTU
II,
showing
an
even
clearer
pattern
than
BZL-resistance
(Toledo
et
al.,
2004;
Toledo
et
al.,
2003).
However,
promising
results
were
obtained
in
Chile,
where
after
a
20-year
follow
up
chronic
patients
treated
with
ITC
showed
normal
ECG
but
remained
serologically
positive
(Apt
et
al.,
2013).
It
is
worth
noting
that
DTU
II,
ITC-susceptible
populations
described
by
Toledo
in
2003
were
isolated
from
Chile
(Toledo
et
al.,
2003).
Ravucona-
zole
showed
potent
trypanocidal
activity
in
vitro,
but
its
activity
in
a
mice
model
was
limited
(Urbina
et
al.,
2003).
Tested
in
dogs,
ravuconazole
showed
suppressive
but
not
curative
activity
against
T.
cruzi,
probably
due
to
its
unfavorable
pharmacokinetic
properties
(a
half-life
of
8.8
h).
However,
its
half-life
in
humans
is
4–8
days,
thus
ravuconazole
might
still
be
a
good
candidate
for
a
combined
therapy
(Diniz
et
al.,
2010).
Posaconazole
has
also
been
tested
as
trypanocidal
agent,
showing
promising
results
as
described
ear-
lier
(Molina
et
al.,
2014).
Other
CYP51
inhibitors
were
also
tested
against
T.
cruzi.
Buckner
et
al.
(2012)
reported
that
the
oncologic
drug
tipifarnib,
as
well
as
some
of
its
analogs,
were
effective
against
amastigotes
in
vitro,
but
less
effective
in
mice
infection.
Their
results
suggest
that
chemical
modification
of
the
lead
molecule
may
increase
its
trypanocidal
activity
(Buckner
et
al.,
2012).
Soeiro
et
al.
(2013)
described
the
activity
of
VNI/VNF
-a
carboxamide-
containing
␤-phenyl
imidazole
designed
to
fill
the
majority
of
the
CYP51-binding
cavity-
in
vivo
against
parasite
strains
with
different
drug
susceptibility.
Even
though
sterile
cure
was
not
achieved
in
the
drug-resistant
Colombiana
strain,
ultrastructural
alterations
were
observed,
suggesting
that
further
modifications
in
the
molecule
and
different
dosage
schemes
may
improve
its
efficacy
(Soeiro
et
al.,
2013).
SQ19,
a
compound
with
activity
against
Mycobacterium
tuberculosis
has
also
shown
trypanocidal
properties
and
a
complex
mechanism
of
action
that
includes
NADPH-dependent
reductase
inhibition,
collapsing
the
mitocondrial
membrane
potential,
releas-
ing
H+from
intracellular
acidic
compartments,
and
partial
CYP51
inhibition,
in
addition
to
synergy
with
posaconazole
in
amastigotes
(Veiga-Santos
et
al.,
2015).
4.2.3.
Topoisomerase
inhibitors
DNA
topoisomerases
are
a
family
of
enzymes
that
control
DNA
supercoiling
and
entanglement
in
all
living
organisms
from
bacte-
ria
to
multicellular
eukaryotes.
Their
importance
arises
from
the
double
helical
structure
of
DNA,
which
also
determines
topoiso-
merase
subfamilies:
ATP-independent,
type
I
subfamily
passes
one
strand
of
the
DNA
through
a
break
in
the
opposing
strand;
and
ATP-dependent,
type
II
subfamily
passes
a
duplex
strand
from
the
same
or
a
different
molecule
through
a
double-brake
gap
in
the
DNA
(Champoux,
2001;
Pommier,
2013).
Type
II
topoisomerase
was
found
in
the
nucleus
of
T.
cruzi,
downregulated
in
trypomastigotes,
and
upregulated
in
the
replicative
epimastigotes
and
amastig-
otes
(Fragoso
et
al.,
1998).
Consistently,
topoisomerase
inhibitors
like
tarivin,
oxoflacin,
novobiocin
and
nalixidic
acid
were
shown
to
be
effective
against
epimastigotes
and
amastigotes
(Gonzales-
Perdomo
et
al.,
1990).
Type
I
topoisomerase
was
found
to
be
localized
in
both
nucleus
and
kinetoplast,
and
parasites
were
sus-
ceptible
to
the
inhibitor
camptothecin
(Bodley
and
Shapiro,
1995).
Due
to
their
use
as
antitumoral
agents,
there
has
been
renewed
interest
in
testing
new
topoisomerase
inhibitors
on
T.
cruzi.
Camp-
tothecin
caused
ultrastructural
alterations
in
the
kinetoplast
and
heterochromatin
unpacking
in
the
nucleous;
enoxacin
and
mitox-
antrone,
topoisomerase
II
inhibitors,
were
also
effective
to
inhibit
T.
cruzi
proliferation
(Zuma
et
al.,
2015).
However,
camptothecin
derivatives
topotecan
and
irinotecan
were
not
as
effective
as
the
parent
compound
in
imparing
T.
cruzi
growth
(Lacombe
et
al.,
2014).
Recent
work
has
revealed
that
camptothecin
treatment
trig-
gers
T.
cruzi
apoptosis,
but
some
parasites
do
not
progress
to
late
apoptosis
signaling
and
remain
in
a
“senescence-like”
state
(Zuma
et
al.,
2014).
Taken
together,
these
results
show
that
topoisomerase
inhibitors
are
compounds
that
will
still
be
studied
as
promising
trypanocidal
compounds.
4.2.4.
Cruzipain
The
main
cysteine
proteinase
of
T.
cruzi,
encoded
by
tandemly
arranged
genes
located
on
2–4
chromosomes
in
different
isolates,
constitutes
a
family
of
proteins
with
30–70%
sequence
identity
with
their
homologues
in
T.
brucei
(Campetella
et
al.,
1992).
Being
the
main
T.
cruzi
lysosomal
proteinase,
cruzipain
is
involved
in
parasite
nutrition,
but
also
in
penetration
of
trypomastigotes
into
the
host
cell,
in
the
defense
of
parasites
against
the
mammalian
immune
system,
and
in
the
differentiation
processes
from
one
stage
to
another.
Its
catalytic
domain
has
homology
with
cathepsines
B
and
L,
but
the
specificity
of
its
C-terminal
domain
renders
cruzi-
pain
as
one
of
the
most
specific
families
of
T.
cruzi
proteins,
and
therefore
it
has
been
extensively
studied
as
a
drug
target
candi-
date
(Cazzulo,
2002).
Even
though
most
cruzipain
inhibitors
were
also
able
to
inhibit
mammalian
cathepsins,
mammalian
cells
are
not
adversely
affected
at
concentrations
which
effectively
kill
the
parasite.
This
selective
effect
may
be
due
to
the
redundancy
of
pro-
teolytic
activities
in
higher
eukaryotic
cells
compared
to
parasitic
protozoa.
In
T.
cruzi,
cruzipain
inhibitor-resistant
parasites
were
not
cross-resistant
to
nifurtimox
and
benznidazole,
indicating
different
mechanisms
of
action.
Among
the
compounds
tested
against
this
enzyme,
the
most
promising
has
been
K777,
a
vinyl
sulfone
deriva-
tive
described
by
McKerrow
et
al.
(2009).
Compound
K777
was
active
against
a
wide
range
of
susceptible
and
resistant
strains
rep-
resentative
of
the
main
circulating
DTUs.
It
was
able
to
cure
T.
cruzi
acute
and
non-acute
infection
in
mice,
showing
synergistic
activ-
ity
with
BZL,
low
hepatotoxicity,
and
ameliorated
cardiac
damage
in
treated
dogs
(McKerrow
et
al.,
2009).
While
K777
is
now
under
pre-clinical
trials
(Zingales
et
al.,
2014),
some
of
its
analogs
have
been
documented
to
display
higher
trypanocidal
potency,
unex-
pectedly
due
to
inhibition
of
C14-␣-demetilase
(TcCYP51)
rather
than
inhibition
of
cruzipain
(Choy
et
al.,
2013).
4.2.5.
Trans-sialidase
(TS)
This
enzyme
has
been
extensively
studied
since
the
first
reports
of
its
activity
in
the
early
1990s
(Schenkman
et
al.,
1991).
It
has
been
demonstrated
that
TS
is
composed
by
3
domains:
the
N-terminal,
trans-sialidase
catalylic
domain;
the
globular
lectin-
like
domain
involved
in
binding
to
nerve
growth
factor
receptor,
and
the
C-terminal,
antigenic
part,
which
ends
in
a
glycophos-
phatidyl
inositol
membrane
anchor
that
provides
the
hydrolyzing
point
to
cleavage
the
molecule
for
shedding
into
the
extracel-
lular
milieu
(Rubin
and
Schenkman,
2012).
Briefly,
TS
is
related
to
viral
and
bacterial
hydrolases,
and
catalizes
transfer
of
sialic
acid
from
sialoglycoconjugates
in
the
external
milieu
of
mam-
malian
cells
to
glycoconjugates
(mucins)
in
the
cell
membrane
of
T.
cruzi
(Buschiazzo
et
al.,
2002).
Sialation
in
T.
cruzi
membranes
plays
important
roles
in
T.
cruzi
biology,
but
probably
the
most
remarkable
are
evading
the
early
complement-mediated
immune
response,
and
host-cell
invasion
(Schenkman
and
Eichinger,
1993;
Rubin-de-Celis
et
al.,
2006).
For
these
important
roles
and
its
absence
in
mammalian
organisms,
TS
has
been
proposed
as
a
possible
drug
target.
The
binding
of
sialic
acid
to
TS
triggers
con-
formational
changes
that
create
a
sugar-acceptor
binding
site
for
␤-galactose,
and
a
Tyr-residue
switch
that
allows
the
covalent
bond
with
sialic
acid
during
the
intermediary
state
of
the
reaction
(Watts
et
al.,
2003).
Agustí
et
al.
(2004)
tested
lactose
derivatives
modified
in
the
glucose
constituent,
demonstrating
that
lactitol
inhibited
trans-sialidase
reaction
in
vitro
by
being
a
preferencial
sialyl
residue
acceptor
during
the
transfer
reaction,
and
effec-
tively
interfered
with
parasite
infection
in
cultured
cells
(Agustí
et
al.,
2004).
Due
to
the
short
half-life
of
lactitol
in
blood
(Mucci

10
J.
Bermudez
et
al.
/
Acta
Tropica
156
(2016)
1–16
et
al.,
2006),
covalent
conjugation
of
lactitol
analogues
(lactose,
lactobionolactone,
and
benzyl-␤-d-galactopyranosyl-(1
→
6)-2-
amino-2-deoxy-␣-d-glucopyranoside)
with
polyethylene
glycol
(PEG)
have
proven
to
inhibit
TS
(Giorgi
et
al.,
2010).
Eight-
arm
PEG
with
a
star-shape
increased
the
molecular
weight
and
half-life
of
benzyl-␤-d-galactopyranosyl-(1
→
6)-2-amino-2-
deoxy-␣-d-glucopyranoside
(Giorgi
et
al.,
2012).
Buchini
et
al.
(2008)
described
compounds
that
specifically
bind
to
the
catalytic
site
of
the
enzyme
(BFNs:
9-benzoyl-3-fluoro-N-acetylneuraminic
acid,
and
its
3-fluorosialyl
fluorides-bearing
substituents
at
C9).
BFNs
formed
long-lived
intermediates
that
inactivated
mammalian
TS
150
times
more
slowly
than
T.
cruzi
TS
(Buchini
et
al.,
2008).
Similarly,
sulfonamide
chalcones
and
quinolones
were
specific
inhibitors
of
T.
cruzi
TS,
not
showing
inhibition
of
human
TS
even
at
high
concentrations
(Kim
et
al.,
2009).
There
is
a
mouse
mon-
oclonal
antibody
(mAb
13G9)
that
recognizes
T.
cruzi
TS
with
high
specificity
and
subnanomolar
affinity,
but
its
high
molecular
weight
must
be
chemically
modified
to
improve
its
bioavailabil-
ity
(Buschiazzo
et
al.,
2012).
More
recently,
a
virtual
screening
of
a
database
with
more
than
4
million
compounds
detected
2
molecules
(ZINC13359679
and
ZINC02576132)
that
are
promising
candidates
as
TS
inhibitors
(Miller
Iii
and
Roitberg,
2013).
However,
data
on
the
activity
of
all
these
synthetic
inhibitors
in
vivo
is
yet
to
be
published.
4.3.
Discovering
new
compounds
There
are
several
lines
of
research
that
may
lead
to
the
discov-
ery
of
new
compounds
with
anti-T.
cruzi
activity
while
having
low
toxicity
towards
mammalian
hosts.
In
fact,
many
promising
drug
candidates
have
been
documented,
but
the
process
of
validation
is
slow
and
rigorous
and
very
few
of
these
candidates
will
be
approved
in
the
end.
Some
of
them
will
be
briefly
described
in
this
section.
An
interesting
approach
was
performed
by
Wang
and
Sun
(2013)
that
reported
metal–drug
complexes
as
novel
alternatives
for
Chagas
disease
treatment.
Their
work
was
based
on
the
con-
cept
of
‘metal–drug
synergism’
described
by
Sanchez-Delgado
et
al.
(1993),
where
the
coordination
of
an
organic
drug
to
a
metal
center
stabilizes
the
drug
and
enhances
its
activity.
The
metal
com-
pounds
may
have
dual
or
even
multiple
mechanisms
of
action,
combining
the
pharmacological
properties
of
both
the
ligand
and
the
metal,
and
also
triggering
additive
effects.
Toxicity
of
the
metal
ion
is
reduced
by
complexation
of
the
ion
to
drug
ligands,
which
diminishes
its
interaction
with
biomolecules.
Santos
et
al.
(2012)
exhaustively
studied
Pd(II),
Pt(II),
Ru(II)
and
Ru(III)
coordi-
nation
compounds
of
bioactive
5-nitrofuryl
and
5-nitroacroleine
thiosemicarbazones.
Many
of
the
Pd
and
Pt
compounds
showed
increased
activity
against
trypanosomes
in
comparison
to
free
lig-
ands.
A
severe
disadvantage
is
that
these
metal
complexes
lead
to
deleterious
effects
on
mammalian
cells,
and
a
strategy
to
over-
come
it
is
to
use
commercially
available
drugs
that
have
low
toxicity.
In
that
regard,
recent
work
by
Martínez
et
al.
(2012)
showed
that
ruthenium-clotrimazole
did
not
induce
toxicity
in
normal
mammalian
cells
while
keeping
its
trypanocidal
activity
(Martínez
et
al.,
2012).
Similarly,
Iniguez
et
al.
(2013)
demonstrated
that
ruthenium-ketoconazole
complexes
are
non-toxic
to
murine
macrophages,
or
to
human
fibroblasts
and
osteoblasts
(Iniguez
et
al.,
2013).
The
development
of
bioactive
metal
complexes
with
com-
mercial
drugs
is
a
promising
new
approach
in
the
search
for
a
treatment
of
Chagas
disease.
In
this
sense,
Batista
et
al.
(2011)
obtained
MnCl2·4H2O,
CoCl2·4H2O
and
CuCl2(phen)
(phen
=
1,
10-phenanthroline)
complexes
with
the
quinolones
norfloxacin
and
sparfloxacin.
The
trypanocidal
effect
in
vitro
against
amastig-
otes
and
bloodstream
trypomastigotes
was
evaluated,
showing
that
quinolones
were
poorly
effective
against
T.
cruzi.
However,
[CoCl2(NOR)(H2O)2]
and
[CoCl2(SPAR)(H2O)2]
were
active
against
amastigotes,
and
[CuCl2(phen)(NOR)]
and
[CuCl2(phen)(SPAR)]
displayed
a
higher
activity
against
both
bloodstream
typomastig-
otes
and
amastigotes
(Batista
et
al.,
2011).
Several
bisphosphonates
that
are
used
for
the
treatment
of
bone
diseases
are
active
against
T.
cruzi.
The
main
target
of
these
compounds
is
the
parasitic
farnesyl
diphosphate
synthase
enzyme
which
is
involved
in
the
biosynthesis
of
polyisoprenoids
and
sterols.
This
enzyme
is
competitively
inhibited
by
commercial
bispho-
sphonate
drugs,
like
risedronate.
However,
a
significant
clinical
disadvantage
of
bisphosphonates
is
their
poor
oral
bioavailabil-
ity.
This
problem
could
potentially
be
attenuated
by
coordination
to
a
metal
ion.
In
this
sense,
Demoro
et
al.
(2010)
have
devel-
oped
metal
complexes
with
bioactive
bisphosphonates
as
ligands.
Results
demonstrated
that
the
coordination
of
risedronate
to
dif-
ferent
metal
ions
improved
its
antiproliferative
effect
against
amastigotes
(Demoro
et
al.,
2010).
Thiosemicarbazones
and
their
metal
complexes
represent
a
class
of
compounds
with
a
wide
range
of
pharmacologi-
cal
applications,
including
anti-T.
cruzi
activity.
An
interesting
approach
was
performed
by
Batista
et
al.
(2010)
that
obtained
Mn
(II)
complexes
with
N4-methyl-4-nitrobenzaldehyde
thiosemicar-
bazone
(H4NO2Fo4
M),
N4-methyl-4-nitroacetophenone
thiosemi-
carbazone
(H4NO2Ac4
M)
and
N4-methyl-4-nitrobenzophenone
thiosemicarbazone
(H4NO2Bz4
M).
The
molecules
H4NO2Fo4
M,
H4NO2Ac4
M,
and
their
Mn
(II)
complexes
displayed
poor
effect
on
bloodstream
trypomastigotes,
as
well
as
H4NO2Bz4
M,
but
Mn(H4NO2Bz4
M)2Cl2was
significantly
active
against
trypo-
mastigotes,
encouraging
further
in
vitro
and
in
vivo
studies
(Batista
et
al.,
2010).
However,
the
toxicity
of
these
compounds
is
an
impor-
tant
factor
that
must
be
taken
into
account
at
the
moment
of
designing
experiments
in
animal
models.
Plant-derived
products
are
an
immense
source
of
lead
com-
pounds
that
could
be
potentially
active
against
protozoa
(Croft
et
al.,
2005;
Salem
and
Werbovetz,
2006);
and
despite
being
exten-
sively
studied,
there
are
still
many
compounds
to
be
discovered.
Essential
oils
from
aromatic
plants
have
shown
many
biological
activities
against
various
microorganisms
(Boyraz
and
Özcan,
2006;
Tagboto
and
Townson,
2001;
Tepe
and
Sokmen,
2007)
including
T.
cruzi
(Santoro
et
al.,
2007)
and
Leishmania
spp.
(De
Medeiros
et
al.,
2011;
Oliveira
et
al.,
2009).
Just
as
a
brief
example,
extracts
from
worldwide
known
plants
like
rosemary
(Abe
et
al.,
2002)
and
green
tea
(Paveto
et
al.,
2004)
have
shown
trypanocidal
activity.
Borges
et
al.
(2012)
investigated
the
trypanocidal
activity
of
essential
oils
from
Brazilian
medicinal
plants
that
inhibited
parasite
growth
in
a
dose-dependent
way,
and
were
well
tolerated
by
mammalian
cells.
Similarly,
Bou
et
al.
(2014)
described
the
antileishmanial
and
trypanocidal
activities
of
casearins
isolated
from
Casearia
sylvestris
leaves,
a
plant
geographically
distributed
throughout
Latin
America
(Lorenzi
and
de
Abreu
Matos,
2002).
Among
the
latest
promising
candidates
from
vegetal
extracts
are
dehydroleucodine
(DhL)
and
helenalin,
sesquiterpene
lactones
found
in
many
plant
families.
It
has
been
demonstrated
that
DhL
and
helenalin
induce
programmed
cell
death
in
T.
cruzi
epimastigotes
and
trypomastigotes,
which
in
turn
may
help
to
modulate
the
host’s
immune
response
by
low-
ering
the
inflammation
triggered
by
non-programmed
cell
death
of
parasites.
In
addition,
the
combination
of
DhL
with
BZL
or
NFX
increases
its
trypanocidal
activity
(Jimenez
et
al.,
2014).
Actino-
mycetes
are
another
rich
source
of
bioactive
compounds.
It
has
recently
been
described
that
the
macrolide
actinoallolide
A
is
active
against
T.
cruzi
epimastigotes
at
a
lower
dose
than
BZL,
and
further
experiments
are
being
carried
out
to
elucidate
its
mechanism
of
action
and
in
vivo
activity
(Inahashi
et
al.,
2015).
(Annang
et
al.,
2015)
reported
a
high-throughput
platform
to
screen
5976
micro-
bial
extracts
from
MEDINA
Natural
Products
library,
and
found
that
actinomycin
D,
bafilomycin
B1,
chromomycin
A3,
echinomycin,

J.
Bermudez
et
al.
/
Acta
Tropica
156
(2016)
1–16
11
hygrolidin,
and
nonactin
were
active
against
T.
cruzi
in
vitro
at
sub-
nanomolar
concentrations,
which
support
further
studies
of
these
promising
compounds.
Similarly,
(Neitz
et
al.,
2015)
performed
a
high-throughput
screening
of
160,000
compounds
from
Novar-
tis
library
and
found
promising
results
in
xanthine
and
xanthine
derivatives
that
warrant
further
in
vivo
studies.
Since
both
commercial
trypanocidal
drugs
have
a
nitroaromatic
moiety—NFX
is
a
nitrofuran
and
BZL
is
a
nitroimidazole,
the
chemi-
cal
modification
of
nitroaromatic
molecules
is
another
approach
to
search
for
new
anti-T.
cruzi
compounds.
For
example,
some
authors
have
designed
compounds
combining
a
furoxan—a
widely
studied
nitric
oxide
donor
with
cytotoxic
and
cytostatic
activity
(Cerecetto
and
Porcal,
2005),
and
N-acylhydrazone—a
molecule
with
potential
to
interact
with
different
biological
targets
(Hernández
et
al.,
2013;
Massarico
Serafim
et
al.,
2014).
In
previous
work,
quinoxaline-
N-acylhydrazones
proved
to
have
trypanocidal
activity
against
epimastigotes
by
inhibition
of
cruzipain,
the
main
T.
cruzi
cysteine
proteinase
(Romeiro
et
al.,
2009).
N-acylhydrazone
compounds
are
stable
in
both
simulated
biological
matrixes
and
plasma,
have
low
mutagenic
potential,
and
target
cruzipain
(Romeiro
et
al.,
2009;
Hernández
et
al.,
2013).
In
addition,
some
of
these
compounds
have
shown
lower
cytotoxicity
than
the
reference
drug
BZL
in
vitro
(Massarico
Serafim
et
al.,
2014).
Repositioning,
or
finding
new
therapeutic
uses
for
already
known
drugs
is
also
a
valid
approach
taken
to
discover
new
active
compounds
with
anti-T.
cruzi
activity.
Phenothiazines
are
tricyclic
drugs
employed
as
antidepressant,
anxiolytic
and
antipsychotic
in
psychiatric
treatments,
but
have
also
been
found
to
have
trypanoci-
dal
activity
by
inhibiting
trypanothione
reductase,
equivalent
to
mammalian
glutathione
reductase
(Paglini-Oliva
and
Rivarola,
2003;
Lo
Presti
et
al.,
2015).
One
example
is
clomipramine,
a
drug
prescribed
to
treat
obsessive-compulsive
disorder.
Clomipramine
has
been
used
to
treat
T.
cruzi
infected
mice
during
acute
phase
at
5
mg/kg/day
and
40
mg/kg/day
intraperitoneally,
showing
a
decrease
in
parasitemia,
structural
heart
damage
and
electrocar-
diographic
alterations
in
comparison
with
untreated
mice
(Rivarola
et
al.,
2001;
Rivarola
et
al.,
2005).
For
chronic
phase
treatment,
a
dose
of
5
mg/kg/day
induced
a
significatively
lower
amount
of
T.
cruzi
DNA
in
heart
and
skeletal
muscle,
milder
inflammatory
infiltrates,
and
reduction
in
heart
fibrosis,
as
well
as
a
decrease
in
anti-T.
cruzi
antibody
titers
and
longer
survival
(Bazán
et
al.,
2008;
Fauro
et
al.,
2013).
Similar
results
were
found
with
thiori-
dazine
treatment
(Lo
Presti
et
al.,
2015;
Lo
Presti
et
al.,
2004).
These
results
point
towards
a
decreased,
retarded
chagasic
cardiomyopa-
thy
after
treatment
with
clomipramine,
but
further
studies
will
be
needed
to
test
its
effectiveness
in
oral
doses
and
in
different
DTUs,
as
well
as
possible
side
effects
of
equivalent
doses
in
humans.
Bellera
and
co-workers
(Bellera
et
al.,
2015)
used
a
computer-aided
drug
screening,
combined
with
biochemical,
cellular
and
preclin-
ical
tests
to
study
clofazimine
(used
to
treat
leprosy),
benidipine
(for
hypertension
and
angina
pectoris
treatment)
and
saquinavir
(an
antiviral)
as
potential
trypanocidal
agents.
Only
clofazimine
and
binidipine
were
tested
on
mice
models,
because
saquinavir
maximum
steady
state
concentration
was
below
the
concentra-
tion
needed
to
kill
parasites.
Clofazimine
and
binidipine
were
not
as
effective
as
BZL
at
the
doses
tested
in
the
mice
model,
but
showed
promising
results
to
be
tested
in
further
studies.
Using
the
concept
of
latentiation
by
which
a
pro-drug
is
metabolically
converted
to
an
active
compound,
Chung
et
al.
(2003)
described
the
trypanocidal
activity
of
hydroxymethylnitrofurazone
(NFOH).
Even
though
NFOH
is
a
precursor
of
nitrofurazone
(NF)
in
the
chemical
synthesis
from
the
nitrofuran
ring,
it
can
also
be
obtained
from
NF
in
a
single
step.
Nitrofurazone
(NF)
has
proven
to
be
effective
against
Gram
positive
and
Gram
negative
bacteria,
and
to
have
trypanocidal
activity
(Henderson
et
al.,
1988)
but
is
also
a
known
carcinogen
that
affects
the
reproductive
system
in
both
male
and
female
mice
(Heindel
et
al.,
1997),
causing
single
strand
DNA
brakes
and
oxidative
damage
(Takegawa
et
al.,
2000;
Hiraku
et
al.,
2004).
For
these
reasons,
FDA
regulations
only
allow
commercialization
of
NF
as
topical
medications
formulated
for
dermatologic
applications
(FDA,
1998).
However,
the
remarkable
properties
of
NFOH
as
a
trypanocidal
agent
guaranteed
further
aca-
demic
investigation.
In
that
sense,
NFOH
was
shown
to
be
4
times
less
mutagenic
in
Ames
test
than
its
parental
compound
(Guido
et
al.,
2001),
demonstrated
higher
trypanocidal
activity
than
BZL
against
amastigotes
and
typomastigotes
in
vitro,
and
a
LD50
higher
than
2000
mg/kg
in
rats
(Melo,
2006).
Its
proposed
mechanism
of
action
involves
the
classical
nitroaromatic
activation,
interference
with
mRNA
transcription
(Barbosa
et
al.,
2007),
and
inhibition
of
cruzipain
(Trossini
et
al.,
2010).
Tested
in
vivo,
NFOH
was
as
effec-
tive
as
BZL
treatment
during
the
acute
phase
of
infection
in
mice
(Davies
et
al.,
2010).
In
addition
to
its
trypanocidal
activity,
NFOH
showed
higher
solubility
in
water,
and
lower
adverse
effects
than
BZL
or
the
parental
compound
NF.
Research
about
its
pharmaco-
logical
showed
that
50%
of
NFOH
was
converted
to
NF
in
human
plasma,
with
a
volume
of
distribution
20
times
higher
than
that
of
NF.
In
rats,
levels
of
NF
converted
from
NFOH
were
4
times
lower
than
those
of
NF
directly
administered
(Serafim
et
al.,
2013).
Toxicity
studies
conducted
in
HepG2
cells
and
in
liver
of
healthy,
non-infected
mice
showed
that
NFOH
was
less
hepatotoxic
than
BZL
(Davies
et
al.,
2014).
The
N-hydroxymethylation
at
the
primary
amide
of
NF
leading
to
NFOH
–
equivalent
to
that
occurring
in
phase
I
and
II
during
liver
metabolism
of
xenobiotics
–
resulted
in
higher
water
solubility,
lower
liver
toxicity
and
other
favorable
pharma-
cological
properties
of
NFOH
(Serafim
et
al.,
2013;
Davies
et
al.,
2014;
Nogueira
Filho
et
al.,
2013)
that
ensure
further
studies
of
this
promising
molecule.
Olmo
et
al.
(2015)
synthesized
some
abietic
acid
derivatives
(abietane
diterpenoids)
that
show
anti
T.
cruzi
activity.
Abietane
diterpenoids
have
shown
antiprotozoal
activity
against
T.
brucei
and
Plamsmodium
spp,
low
toxicity
against
Vero
cells,
and
good
efficacy
against
T.
cruzi
both
in
vitro
and
in
mice
models
of
acute
infection.
In
addition
to
their
good
trypanocidal
activity
and
low
toxicity,
being
easily
synthesized
from
non-expensive
substrates
makes
these
abietane
diterpenoids
good
candidates
to
develop
new
trypanocidal
agents.
Another
interesting
advance
was
performed
by
Cogo
et
al.
(2015).
They
synthesized
2,3-disubstituted
quinoxalinederivates
showing
effectiveness
against
T.
cruzi.
Their
activity
was
directly
related
to
the
methylsulfoxyl,
methylsulfonyl,
and
amine
groups
as
well
as
the
presence
of
chorine
or
bromine
in
the
molecules.
The
authors
assume
that
these
molecules
are
promising
candi-
dates
because
of
their
privileged
scaffold,
but
their
effectivity
in
vivo
remains
to
be
evaluated.
Finally,
homeopathic
treatment
has
also
been
considered
as
an
alternative
in
T.
cruzi
mice
models
of
infection.
A
suspension
of
107
blood
trypomastigotes/mL
from
mice
on
the
7th
day
of
infection,
diluted
1:107in
7%
ethanol
has
been
chosen
for
administration
by
gavage
to
mice
infected
with
1400
T.
cruzi,
Y
strain
trypomastigotes
(Aleixo
et
al.,
2012;
Sandri
et
al.,
2015).
This
dilution
given
to
mice
at
4
days
post-infection
delayed
the
appearance
of
parasitemia
and
increased
the
survival
of
treated
(27
days)
vs.
untreated
(15
days)
mice;
however,
mortality
was
still
high
in
the
treated
(92%)
com-
pared
to
the
untreated
group
(100%)
(Aleixo
et
al.,
2012).
The
age
of
treated
mice
was
an
important
factor
in
susceptibility
to
home-
opathic
treatment
given
at
the
5th
day
post-infection
for
20
days.
Mice
4-week
old
were
less
susceptible
than
8-week
old,
the
lat-
ter
showing
lower
inflammatory
infiltrates,
higher
apoptosis,
lower
TGF-␤,
and
lower
intracellular
parasitism
in
spleen
and
liver
cells
(Sandri
et
al.,
2015).
These
results
confirm
the
finely
tuned
immune
response
and
exquisite
relationship
between
T.
cruzi
and
its
mam-

12
J.
Bermudez
et
al.
/
Acta
Tropica
156
(2016)
1–16
malian
hosts,
pointing
towards
many
more
years
of
interesting
research.
5.
Concluding
remarks
Chagas
disease
currently
poses
one
of
the
greatest
therapeutic
challenges
in
tropical
medicine,
because
T.
cruzi
has
a
complex
life
cycle
in
sylvatic,
rural
and
urban
environments,
and
susceptibility
factors
in
both
hosts
and
parasites
have
different
influence
on
dis-
ease
outcome.
This
complexity
has
also
hampered
the
development
of
a
vaccine
against
T.
cruzi
even
using
methodologies
as
different
as
designing
specific
protective
antigens,
or
by
generating
atten-
uated
parasites.
An
additional
problem
remains
in
the
progressive
loss
of
interest
and
visibility
of
Chagas
disease
as
it
disappears
from
endemic
areas
after
implementation
of
vector
control
policies
from
Public
Health
Organizations
in
endemic
countries.
Since
climate
change
is
re-shaping
our
landscapes,
remaining
foci
of
vectors
and
insecticide-resistant
triatomines
will
constitute
permanent
reser-
voirs
of
T.
cruzi.
Prolonged
treatment
course,
side-effects
and
naturally
resistant
parasite
populations
hinder
treatment
with
BZL
or
NFX.
Recent
research
demonstrated
that
shorter
treatment
schedules
with
BZL
are
as
effective
as
the
classical
2-month
protocol
(Viotti
et
al.,
2014;
Alvarez
et
al.,
2012;
Bustamante
et
al.,
2014),
and
that
there
are
synergistic
effects
when
BZL
is
administered
with
commercial
ergosterol
synthesis
inhibitors
like
posaconazole
or
ITC.
Both
have
good
pharmacokinetic
properties—such
as
the
affinity
of
ITC
for
CYP450
enzymes
that
improves
the
half-life
of
BZL
by
substrate
competition
(Moreira
da
Silva
et
al.,
2012),
or
the
large
volume
of
distribution
of
pozaconazole.
Posaconazole
does
not
seem
to
be
effective
as
a
monotherapy
against
chronic
T.
cruzi
infection,
as
suggested
by
the
persistence
of
parasites
circulating
in
blood
during
the
CHAGAZOL
study
(Molina
et
al.,
2014).
In
contrast,
treat-
ment
with
ITC
improved
ECG
outcome
in
chronic
patients,
but
these
results
belong
to
a
limited
cohort
in
Chile
(Apt
et
al.,
2003).
Being
azole
molecules
with
the
same
molecular
target,
ITC
and
posaconazole
show
the
same
synergistic
behavior
when
admin-
istered
in
combination
with
BZL
or
in
short-alternate
schedules
during
the
acute
phase
in
mice
models
(Assíria
Fontes
Martins
et
al.,
2015;
Cencig
et
al.,
2012;
Diniz
Lde
et
al.,
2013;
Bustamante
et
al.,
2014).
The
possibility
of
using
these
combined
schemes
of
treat-
ment
aims
to
reduce
the
dose
and
time
of
exposure
of
BZL
thus
lowering
its
side-effects
while
diminishing
the
economic
cost
of
either
posaconazole
or
ITC
administration.
This
is
especially
impor-
tant
in
life-threatening
acute
cases
such
as
in
immunosuppressed
patients,
in
oral
outbreacks,
or
in
congenital
cases.
Moreover,
the
combination
of
BZL
+
posaconazole
was
effective
even
against
the
BZL-resistant
strain
VL-10,
which
is
a
promising
result
for
treat-
ment
of
infections
with
naturally
resistant
parasites
(Diniz
Lde
et
al.,
2013).
Therefore,
in
the
near
future
it
is
reasonable
to
expect
that
Chagas
disease
treatment
will
involve
combinations
of
BZL
and
other
approved
compounds
such
as
CYP51
inhibitors
(posacona-
zole
and/or
ITC),
in
short
and
intermittent
administration
schedules
to
minimize
BZL
overdosing
and
its
side
effects.
New
formulations
with
increased
water
solubility,
higher
volume
of
distribution
or
plasma
concentration
are
also
expected
for
posaconazole,
ITC,
and
BZL.
In
the
long
term,
some
molecules
from
the
list
of
promis-
ing
compounds
will
finally
hit
the
market
as
new
trypanocidal
drugs
with
high
efficacy
and
no
secondary
effects,
particularly
to
treat
chronic
cases
of
Chagas
disease.
This
list
includes
natural
products,
molecules
specifically
designed
to
inhibit
a
particular
enzyme,
chemically-modified
existing
molecules
to
increase
their
trypanocidal
activity,
and
drugs
that
were
approved
to
treat
other
maladies.
From
these,
the
cruzipain
inhibitors
that
have
passed
phase
II
trials
are
the
closest
to
be
approved
as
new
medications
for
Chagas
disease
(Zingales
et
al.,
2014;
McKerrow
et
al.,
2009),
while
metal–drug
complexes
will
take
longer
to
hit
the
market
due
to
the
metal
toxicity
towards
mammalian
cells.
Acknowledgements
The
authors
would
like
to
thank:
CONICET
(Argentina)
for
research
fellowships,
CIUNSa
(Grant
1469/0,
1471
and
1895),
CON-
ICET
(PIP
11220120100313CO),
ANPCyT
(PICT
2012-2643)
and
ANPCyT-MICINN
(Argentina–Spain)
(PICT
2011-2751).
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