Synergistic effect of Ir-(COT)-pentamidine alizarin red and pentamidine, amphotericin B, and paromomycin on Leishmania donovani


Mbongo, N.; Loiseau, P.M.; Craciunescu, D.G.; Robert Gero, M.

Acta Tropica 70(2): 239-245

1998


The treatment of various human infections caused by parasitic organisms of the genus Leishmania is still based on pentavalent antimonials, an empirical treatment developed more than 50 years ago. Pentamidine is a molecule of high interest for curing early stages of African trypanosomiasis and both visceral and mucocutaneous leishmaniasis, refractory to pentavalent antimonials which is increasing in all areas of endemicity. The combination between a metallic structure and an organic moiety is one of the strategies in finding new antileishmanial drugs since it was demonstrated that organometallic compounds were less toxic and usually more active.

ACTA
TROPICA
ELSEVIER
Acta
Tropica
70
(1998)
239-245
Short
communication
Synergistic
effect
of
Ir-(COT)-pentamidine
alizarin
red
and
pentamidine,
amphotericin
B,
and
paromomycin
on
Leishmania
donovani
N.
Mbongo
a
'
11
,
P.M.
Loiseau
b,
*,
D.G.
Craciunescu
C,
M.
Robert-Gero
a
a
Centre
National
de
la
Recherche
Scientifique,
Institut
de
Chimie
des
Substances
Naturelles,
91190-Gif
sur
Yvette,
France
b
Biologie
et
Contr
Ole
des
Organismes
Parasites,
Faculte
de
Pharmacie,
92296-Chatenay-Malabry,
France
Quimica
Inorganica
y
Bioinorganica,
Universidad
Complutense,
Madrid,
Spain
Received
13
June
1997;
received
in
revised
form
15
October
1997;
accepted
16
January
1998
Keywords:
Synergistic
effect;
Ir-(COT)-pentamidine;
Amphotericin
B
The
treatment
of
various
human
infections caused
by
parasitic
organisms
of
the
genus
Leishmania
is
still
based
on
pentavalent
antimonials,
an
empirical
treatment
developed
more
than
50
years
ago
(Croft,
1988,
Herwaldt
and
Berman,
1992,
011iaro
and
Bryceson,
1993).
Pentamidine
is
a
molecule
of
high
interest
for
curing
early
stages
of
African
trypanosomiasis
and
both
visceral
and
mucocutaneous
leishmaniasis,
refractory
to
pentavalent
antimonials
which
is
increasing
in
all
areas
of
endemicity
(Sands
et
al.,
1985,
011iaro
and
Bryceson,
1993).
The
combination
between
a
metallic
structure
and
an
organic
moiety
is
one
of
the
strategies
in
fi
nding
new
antileishmanial
drugs
since
it
was
demonstrated
that
organometallic
compounds
were
less
toxic
and
usually
more
active
(Farrell
et
al.,
1984,
Gayral
et
*
Corresponding
author.
Tel.:
+
33
146
835554;
fax:
+
33
146
835557.
0001-706X/98/$19.00
©
1998
Elsevier
Science
B.V.
All
rights
reserved.
PH
S0001
-706X(98)00018-7
240
N.
Mbongo
et
al.
/
Acta
Tropica
70
(1998)
239-245
al.,
1992,
Loiseau
et
al.,
1992).
In
a
previous
study,
we
investigated
the
in
vitro
antileishmanial
activity
of
15
organometallic
complexes
of
pentamidine
on
Leish-
mania
donovani
DD8
promastigotes
(Mbongo
et
al.,
1997).
The
most
promising
compound
selected
from
this
primary
screening
was
Ir-(COT)-pentamidine
alizarin
red.
IC
50
and
IC
90
values
of
Ir-(COT)-pentamidine
alizarin
red
on
L.
donovani
DD8
promastigotes
were
found
at
3.1
and
11.2
itM,
respectively,
whereas
the
corre-
sponding
values
for
pentamidine
isethionate
were
7.7
and
>
50
itM,
respectively
(Mbongo
et
al.,
1997).
In
this
study,
the
compound
is
evaluated
in
vitro
on
macrophages
infected
by
L.
donovani
LV9
and
a
research
of
synergy
with
glucantime,
pentamidine,
ampho-
tericin
B
and
paromomycin
was
carried
out
on
promastigote
and
amastigote
forms.
Furthermore,
Ir-(COT)-pentamidine
alizarin
red
was
compared
to
pentamidine
isethionate
on
the
L.
donovani
LV9/BALB/C
mouse
model.
Pentamidine
isethionate
was
kindly
supplied
by
Roger
Bellon,
(Neuilly
sur
Seine,
France);
amphotericin
B,
paromomycin
and
alizarin
red
were
purchased
from
Sigma
(Saint
Quentin
Fallavier,
France).
The
organometallic
compound,
also
named
P2368
had
the
following
general
formula:
[Mgt(
Y),(L)J
2
2X
or
X'
where
M
is
Iridium,
I
is
coordination
index
indicating
that
the
compound
is
a
square
planner
complex,
Y
is
1,3-1,5-cyclooctatetraene
(COT),
L
is
pentamidine,
and
X
is
alizarin
red.
The
formula
of
the
compound
is
presented
in
Fig.
1.The
ability
of
the
compound
to
reach
the
intracellular
forms
was
assessed
on
mouse
peritoneal
macrophages
infected
by
L.
donovani
LV9
(MHOM/ET/67/L82)
amastigotes.
This
strain
was
provided
by
Dr
S.L.
Croft
(London
School
of
Hygiene
and
Tropical
Medicine).
Uninfected
mouse
macrophages
were
used
for
cytotoxicity
assays
over
a
5
-day
period
according
to
the
Mossman
method
(Mossman,
1983).
2+
H,
I
I
Ir
H
2
0
--(cHz15-
0
\ /
H
\
H
Ir''
I
I
H
0
S0
3
-
OH
Fig.
1.
Chemical
structure
of
Ir-(COT)-pentamidine
alizarin
red
(compound
P2368).
N.
Mbongo
et
al.
/
Acta
Tropica
70
(1998)
239-245
241
Leishmanicidal
activities
against
amastigotes
were
performed
on
infected
-mouse
macrophages
following
the
method
of
Neal
and
Croft
(1984).
The
toxicity
of
the
compound
to
uninfected
macrophages
was
verified
in
order
to
determine
the
range
of
drug
concentrations
to
be
used.
Ir-(COT)-pentamidine
alizarin
red
had
similar
toxicity
to
pentamidine
isethionate
toward
the
uninfected
macrophages
(IC
50
at
35
and
30
itM,
respectively).
The
organometallic
compound
was
at
least
twice
as
active
as
pentamidine
isethionate
on
the
L.
donovani
LV9
amastigotes
in
macrophages
with
IC
50
,
respectively,
at
6
and
15
it
M.
The
maximum
tolerated
concentration
by
infected
macrophages
were
25
itM
for
the
organometal-
lic
compound
and
20
itM
for
pentamidine
isethionate.
The
organometallic
compound
was
more
active
than
Platinum
complexes
since
Pt(II)-pentamidine
displayed
only
31%
of
amastigotes
inhibition
at
30
itM
on
L.
donovani
LV9
(Croft
et
al.,
1992).
Compound
P2368
is
therefore
more
active
in
vitro
than
pentamidine
isethionate
against
L.
donovani
intracellular
amastigotes
and
its
toxicity
on
macrophages
is
identical
to
that
of
pentamidine.
The
synergistic
effect
with
other
drugs
was
determined
both
on
promastigote
forms
of
L.
donovani
(MHOM/IN/80/DD8)
originated
from
the
WHO
strain
collection
in
London
School
of
Hygiene
and
Tropical
Medicine
and
amastigote
forms
of
L.
donovani
LV9
in
mouse
peritoneal
macrophages.
Promastigotes
were
cultivated
in
Hepes
(25
mM)
buffered
RPMI
1640
medium
enriched
with
fetal
calf
serum
10%
and
1000
IU
penicillin/ml
at
27°C.
The
screening
was
performed
in
fl
at-bottomed
24
-well
tissue
culture
plastic
trays
with
a
well
size
of
17.8
(diameter)
x
16
mm
(depth),
Falcon
No
3047,
maintained
at
27°C
in
an
atmosphere
of
air
95%-CO
2
5%.
Promastigote
forms
from
a
logarithmic
phase
culture
were
suspended
to
10
6
cells/ml.
Each
well
was
fi
lled
with
1
ml
of
the
parasite
suspension,
and
plates
were
incubated
at
27°C
for
1
h
before
drug
addition.
The
compounds
to
be
tested
alone
or
in
combination
were
dissolved
in
dimethyl-
sulphoxide
or
sterile
water
and
diluted
in
medium
before
to
be
added
in
each
well
under
a
10
ul
volume.
Dimethylsulphoxide
had
no
effect
on
the
parasite
growth
up
to
2%
v/v.
Serial
dilutions
(2
-fold)
of
the
tested
drugs
were
performed
and
each
concentration
or
combination
was
screened
in
triplicate.
The
checkerboard
broth
dilution
technique
was
used
to
analyze
the
effect
of
the
combination
of
Ir-(COT)-
pentamidine
alizarin
red
and
pentamidine,
amphotericin
B
and
paromomycin
on
promastigote
growth.
The
combination
with
glucantime
was
not
carried
out
since
IC
50
of
glucantime
on
promastigotes
was
superior
to
100
itM.
The
viability
of
promastigotes
was
checked
using
the
MTT
colorimetric
method
(Mossman,
1983).
The
inhibition
of
growth
is
given
in
inhibitory
concentration
50%
(IC
50
)
after
a
3
-day
incubation
period.
IC
50
of
pentamidine
isethionate
was
7.5
itM
(Table
1).
Alizarin
red
was
inactive
at
50
it
M.
Fig.
2,
Fig.
3
and
Fig.
4
show
a
synergistic
effect
3
days
after
exposure
to
P2368
combined
with
pentamidine,
amphotericin
B
and
paromomycin.
It
is
of
interest
that
P2368
and
pentamidine
were
synergistic
suggesting
that
these
compounds
have
two
distinct
mechanisms
of
action.
This
result
corroborates
a
previous
study
carried
out
on
a
similar
compound,
Ir-(COD)-pentamidine
tetraphenylborate,
that
showed
that
242
N.
Mbongo
et
al.
/
Acta
Tropica
70
(1998)
239-245
Table
1
Effect
of
Ir-(COT)-pentamidine
alizarin
red
on
the
L.
donovani
LV9/BALB/c
mouse
model
compared
reference
drugs
Compound
Dose
(i.p.)a
Stauber
count
x
10
8
S.E.M.)
Percent
of
parasites
suppression
(mean)
mg/kg
per
day
iimokkg
per
day
Ir-(COT)
230
x
2
b
150x
2
b
-pentamidine
75.5
x
5
37.5
x
5
88.5
±
17.3
48
alizarin
red
37.7
x
5
18.7
x
5
162.3
+
37.8
5
Pentacarinat
®
89
x
l
b
150
x
l
b
22
x
5
b
37.5
x
5
b
11
x
5
18.7
x
5
151.6
+
45.1
11
Glucantime
®
200
x
5
546
x
5
3.4
+
0.7
98
55
x
5
150
x
5
152.6
+
31.2
10
Controls'
Excipient
only
170.1
±
47.1
0
230
mg/kg
of
Ir-(COT)-pentamidine
alizarin
red
corresponds
to
50
mg
pentamidine
equivalent/kg.
a
n
=
10
Mice
per
group.
b
Toxic
dose
which
provokes
death
of
mice.
Treatment
with
0.2
ml
excipient
per
day
for
5
days.
pentamidine
was
not
released
from
the
complex,
which
seems
to
act
itself
by
its
own
mechanism
(Mbongo
et
al.,
in
press).
The
IC
50
obtained
on
L.
donovani
LV9
amastigotes
in
macrophages
were
3.5
itM
for
P2368,
14.7
itM
for
glucantime,
14.9
itM
for
pentamidine
isethionate,
0.08
itM
for
amphotericin
B
and
26.2
itM
for
paromomycin.
No
significant
synergistic
effect
was
observed
when
P2368
was
combined
with
these
drugs
on
amastigotes
in
macrophages
due
probably
to
low
penetration
into
macrophages
in
relation
to
drugs
interaction.
The
in
vitro
results
justified
an
in
vivo
evaluation.
Female
BALB/c
mice
were
infected
with
10'
amastigotes
of
L.
donovani
LV9
by
the
retro-orbital
sinus.
The
mice
were
randomly
divided
into
groups
of
10,
one
week
after
infection.
Ir-(COD)-pentamidine
alizarin
red
in
DMSO
and
pentami-
dine
isethionate
in
sterile
water
were
given
by
the
subcutaneous
route
or
intra-
peritoneally
at
doses
up
to
232
mg/kg
(50
mg
pentamidine
equivalent/kg)
and
87
mg/kg
respectively.
Mice
were
dosed
once
per
day
for
5
consecutive
days
and
sacrificed
3
days
after
the
completion
of
treatment.
Drug
activity
was
estimated
by
counting
the
number
of
amastigotes/500
liver
cells
in
Giemsa
stained
impression
smears
prepared
from
the
weighed
livers
of
treated
and
untreated
mice
(Neal
and
Croft,
1984).
P2368
was
partially
active
at
37.5
itmol/kg
per
day
x
5
(48%
of
parasite
suppression)
whereas
pentamidine
isethionate
was
toxic
at
this
dose.
Lower
dose
of
pentamidine
(18.7
itmol/kg
per
day
x
5)
was
poorly
active
(11%
of
parasite
N.
Mbongo
et
al.
/
Acta
Tropica
70
(1998)
239-245
243
[Pentamidine]
(JAM)
10
0
1
2
3
[P2368]
(14M)
Fig.
2.
Isobologram
demonstrating
synergistic
action
of
compound
P2368
and
pentamidine
acting
alone
and
simultaneously
on
L.
donovani
DD8
promastigotes.
The
points
show
the
IC
50
when
compound
P2368
and
pentamidine
are
used
in
combination.
suppression)
and
not
toxic.
The
in
vivo
evaluation
demonstrated
the
advantage
of
P2368
comparatively
to
pentamidine.
In
conclusion,
the
fact
that
synergism
was
observed
between
Ir-(COT)-pentamidine
alizarin
red
and
pentamidine
isethionate
suggests
two
possibilities:
the
existence
of
two
different
transport
systems
or
two
different
targets
for
these
compounds.
The
complex
has
therefore
a
specific
action
[AmphoB]
(14M)
0,6
0,4
-
0,2
-
0,0
0
1
2
3
[P2368]
(yM)
Fig.
3.
Isobologram
demonstrating
synergistic
action
of
compound
P2368
and
amphotericin
B
acting
alone
simultaneously
on
L.
donovani
DD8
promastigotes.
The
points
show
the
IC
50
when
compound
P2368
amphotericin
B
are
used
in
combination.
244
N.
Mbongo
et
al.
/
Acta
Tropica
70
(1998)
239-245
[Paromomycine]
(AA)
40
30
20
-
1
0
-
2
[P2368]
(JAM)
3
Fig.
4.
Isobologram
demonstrating
synergistic
action
of
compound
P2368
and
paromomycin
acting
alone
and
simultaneously
on
L.
donovani
DD8
promastigotes.
The
points
show
the
IC„
when
compound
P2368
paromomycin
are
used
in
combination.
which
is
different
from
that
of
pentamidine.
Further
studies
will
be
directed
to
understand
the
absence
of
synergistic
effect
on
infected
macrophages.
One
advantage
of
such
complexes
is
their
long
half-life
in
mammal
serum,
which
is
responsible
for
an
extended
antiparasitic
effect,
even
after
administration
of
a
single
dose
(Dreyfuss
et
al.,
1993).
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245
Mbongo,
N.,
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