Activity of pentamidine and pentamidine analogs against Toxoplasma gondii in cell cultures


Lindsay, D.S.; Blagburn, B.L.; Hall, J.E.; Tidwell, R.R.

Antimicrobial Agents and ChemoTherapy 35(9): 1914-1916

1991


The capabilities of pentamidine and nine pentamidine analogs to inhibit the development of Toxoplasma gondii were examined in vitro. Treatment of infected cultures with pentamidine and five of its analogs caused a significant (P<0.05) reduction in the numbers of tachyzoites produced. Analogs of pentamidine may be useful agents in the treatment of toxoplasmosis.

ANTIMICROBIAL
AGENTS
AND
CHEMOTHERAPY,
Sept.
1991,
p.
1914-1916
0066-4804/91/091914-03$02.00/0
Copyright
1991,
American
Society
for
Microbiology
NOTES
Vol.
35,
No.
9
Activity
of
Pentamidine
and
Pentamidine
Analogs
against
Toxoplasma
gondii
in
Cell
Culturest
DAVID
S.
LINDSAY,
1
*
BYRON
L.
BLAGBURN,
1
JAMES
EDWIN
HALL,
2
AND
RICHARD
R.
TIDWELL
3
Department
of
Pathobiology,
College
of
Veterinary
Medicine,
Auburn
University,
Auburn,
Alabama
36849,
1
and
Department
of
Epidemiology,
School
of
Public
Health,'
and
Department
of
Pathology,
School
of
Medicitie,
3
University
of
North
Carolina
at
Chapel
Hill,
Chapel
Hill,
North
Carolina
27599
Received
12
April
1991/Accepted
14
June
1991
The
capabilities
of
pentamidine
and
nine
pentamidine
analogs
to
inhibit
the
development
of
Toxoplasma
gondii
were
examined
in
vitro.
Treatment
of
infected
cultures
with
pentamidine
and
fi
ve
of
its
analogs
caused
a
significant
(P
<
0.05)
reduction
in
the
numbers
of
tachyzoites
produced.
Analogs
of
pentamidine
may
be
useful
agents
in
the
treatment
of
toxoplasmosis.
Toxoplasma
gondii
is
an
ubiquitous
protozoan
parasite
that
can
be
an
important
disease
-producing
agent
in
humans
and
warm-blooded
animals
(6).
In
humans,
T.
gondii
infec-
tions
in
immunocompetent
patients
are
usually
asympto-
matic,
whereas
infections
in
immunocompromised
patients
or
transplacentally
infected
ants
can
be
life
-threatening.
Encephalitis
caused
by
T.
gt;
dii
has
become
recognized
as
a
serious,
often
fatal,
manifestation
of
the
infection
seen
in
many
patients
with
AIDS
(9,
15,
21).
Toxoplasmic
enceph-
alitis
(TE)
is
probably
caused
by
reactivation
of
latent
tissue
cyst
stages
(7).
Pyrimethamind
alone
or
in
combination
with
sulfadiazine
or
trisulfapyrimidines
have
been
the
standard
treatments
for
acute
toxoplasmosis
and
TE,
although
ad-
verse
reactions
are
common
(9,
15,
17).
Relapse
of
TE
is
common
once
treatment
has
been
stopped.
Additional
com-
pounds
that
do
not
cause
adverse
side
effects
and
that
have
activity
against
T.
gondii
must
be
discovered
and
evaluated
for
the
treatment
of
acute
toxoplasmosis
and
TE.
Pentamidine
and
its
analogs
have
been
shown
to
have
a
broad
spectrum
of
activity
against
intracellular
and
extracel-
lular
protozoan
parasites
as
well
as
Pneumocystis
carinii
(2,
11, 12,
18-20).
Because
of
this
demonstrated
effectiveness
against
parasitic
protozoa,
we
examined
the
capabilities
of
pentamidine
and
nine
of
its
analogs
to
inhibit
replication
of
the
highly
pathogenic
RH
isolate
of
T.
gondii
in
cell
cultures.
Pentamidine
and
the
pentamidine
analogs
(Table
1)
used
in
this
study
were
synthesized
in
the
laboratories
of
Richard
R.
Tidwell,
Department
of
Pathology,
School
of
Medicine,
University
of
North
Carolina
at
Chapel
Hill.
The
methods
of
their
production
and
examinations
for
purity
have
been
described
previously
(19).
Pentamidine
and
its
analogs
were
examined
at
100
µg/ml;
compounds
found
to
be
toxic
at
this
concentration
were
subjected
to
further
study
at
lower
concentrations
(Tables
2
and
3).
Pyrimethamine
(lot
no.
3f0991)
and
piritrexim
(lot
no.
87/5089-036;
Burroughs
Well-
come
Co.,
Research
Triangle
Park,
N.C.)
were
used
at
10
p.g/m1
of
medium
in
two
experiments
to
ensure
that
our
assay
would
identify
effective
compounds.
*
Corresponding
author.
t
This
is
publication
no.
2223
of
the
College
of
Veterinary
Medicine,
Auburn
University.
African
Green
monkey
kidney
cells
(Vero)
(ATTC
CCL
81;
American
Type
Culture
Collection,
Rockville,
Md.)
were
grown
to
monolayers
in
25-cm
2
plastic
tissue
culture
fl
asks
in
RPMI
1640
medium
containing
10%
(vol/vol)
fetal
bovine
serum,
100
U
of
penicillin
G
per
ml,
100
lig
of
dihydrostrep-
tomycin(GIBCO,
Grand
Island,
N.Y.)
per
ml,
and
5
x
10
-2
mM
2-mercaptoethanol
(Sigma
Chemical
Co.,
St.
Louis,
Mo.).
Cell
cultures
were
maintained
in
an
identical
medium;
but
the
concentration
of
fetal
boyine
serum
was
lowered
to
2%
(vol/vol).
Cell
cultures
were
incubated
at
37°C
in
a
95%
air
-5%
CO
2
atmosphere.
Tachyzoites
of
the
RH
isolate
of
T.
gondii
are
maintained
in
the
laboratories
of
David
S.
Lindsay
and
Byron
L.
Blagburn,
Department
of
Pathobiology,
Auburn
University,
by
serial
passage
in
infected
Vero
cell
cultures.
Tachyzoites
were
collected
for
inoculation
of
cell
cultures
by
fi
rst
remov-
ing
the
cell
culture
medium
and
replacing
it
with
fresh
maintenance
medium.
The
Vero
cells
were
then
scraped
off
the
plastic
growth
surface
into
the
medium,
and
the
suspen-
sion
was
forced
through
a
syringe
equipped
with
a
27
-gauge
needle
to
rupture
most
infected
Vero
cells.
The
free
tachy-
zoites
were
separated
from
intact
cells
and
most
cellular
debris
by
fi
ltration
through
a
sterile
3
-p.m
-pore
-size
polycar-
bonate
fi
lter
(Nuclepore
Corp.,
Pleasanton,
Calif.).
The
numbers
of
tachyzoites
present
were
determined
by
count-
ing
them
in
a
hemacytometer,
and
the
volume
of
inoculum
was
adjusted
so
that
1
ml
contained
2
x
10
5
tachyzoites.
For
each
treatment,
four
25-cm
2
fl
asks
were
each
inoculated
with
2
x
10
5
tachyzoites.
Two
hours
postinoculation
(p.i.),
the
tachyzoite
inoculum
was
removed
and
replaced
with
test
compounds
in
maintenance
medium.
Pentamidine,
pentami-
dine
analogs,
and
piritrexim
were
solubilized
in
deionized
water
and
diluted
in
maintenance
medium,
whereas
py-
rimethamine
was
solubilized
in
100%
ethanol.
Controls
received
maintenance
medium
to
which
an
equal
amount
of
deionized
water
was
added.
Media
were
removed
from
the
infected
cells
4
to
4.5
days
p.i.,
the
volume
was
recorded,
and
the
numbers
of
tachyzoites
present
were
determined
by
counting
them
in
a
hemacytometer.
The
total
number
of
tachyzoites
present
in
each
fl
ask
was
determined
by
multi-
plying
the
volume
of
the
medium
by
the
numbers
of
tachy-
zoites
present
(mean
of
six
counts
per
fl
ask)
per
milliliter
of
1914
VOL.
35,
1991
NOTES
1915
TABLE
1.
Structures
and
names
of
pentamidine
analogs
5
5
Y
o—(cH2).-
4
Compound
no.
Structure
component
Position
Y
Compound
name
n
1
3
—OCH
3
Am
4
1,3-Di(4-amindino-2-methoxyphenoxy)propane
2
3
—H
Im
4
1,3-Di(4-imidazolinophenoxy)propane
3
4
—OCH
3
Im
4
1,4-Di(4-imidazolino-2-methoxyphenoxy)butane
4 4
—H
Am
4
1,4-Di(4-amidinophenoxy)butane
5
3
—OCH
3
Im
4
1,3-Di(4-imidazolino-2-methoxyphenoxy)propane
[DIMP]
6
3
—OCH
3
Im
5
1,3-Di(5-imidazolino-2-methoxyphenoxy)propane
7
5
—OCH
3
Im
4
1,5-Di(4-imidazolino-2-methoxyphenoxy)pentane
8
4
—OCH
3
Am
4
1,4-Di(4-amidino-2-methoxyphenoxy)butane
[dimethoxybutane]
9
6
—H
Am
4
1,6-Di(4-amidinophenoxy)hexane
[hexamidine]
10
5
—H
Am
4
1,5-Di(4-amidinophenoxy)pentane
[pentamidine]
H
Am
=
N
H
Itn
=
medium.
Mean
total
tachyzoite
production
was
analyzed
by
a
nonparametric
one
-tailed
Mann
-Whitney
test.
Designation
of
significant
differences
in
all
experiments
was
based
on
a
cutoff
of
P
<
0.05.
The
percent
reduction
in
tachyzoites
was
calculated
by
subtracting
the
mean
treated
values
from
the
mean
control
value,
dividing
this
numerator
by
the
mean
control
value,
and
multiplying
the
product
by
100.
Eight
experiments
were
conducted
(Tables
2
and
3).
Pentamidine
and
compounds
3,
6,
7,
8,
and
9
reduced
the
numbers
of
T.
gondii
RH
tachyzoites
in
cell
cultures
(Tables
TABLE
2.
Activities
of
pentamidine
analogs
against
T.
gondii
in
Vero
cell
cultures"
Expt
no.
Compound"
Total
no.
of
tachyzoites/
fl
ask
(mean
±
SD
[101)
%
Reduction'
1
Control
597
±
166
NA
1
(100)
1,088
±
408
0
2
(100)
950
287
0
3
(100)
45
±
11
92.5*
2
Control
672
±
173
NA
5
(100)
1,325
±
633
0
3
Control
1,367
±
141
NA
4
(50)
1,065
±
233
22.1
6
(100)
649
±
304
52.5*
4
Control
479
±
120
NA
7
(100)
28
±
13
94.2*
8
(100)
102
±
51
78.7*
Pyrimethamine
(10)
3
±
3
99.4*
"
Pentamidine
and
compound
9
were
toxic
at
100
and
50
1.4m1,
and
compound
4
was
toxic
at
100
1.1.g/ml.
"
Control
cultures
received
no
drug
treatment
and
treated
cultures
received
treatment
at
the
concentration
(in
micrograms
per
milliliter)
given
in
paren-
theses.
NA,
not
applicable;
negative
percent
reductions
are
expressed
as
0;
*,
significant
reduction
in
the
number
of
tachyzoites
per
fl
ask
compared
with
that
in
control
fl
asks
(P
<
0.05).
2
and
3).
Because
compounds
3,
7,
and
8
appeared
to
be
highly
effective
at
100
ILg/ml,
additional
doses
were
exam-
ined
(Table
3).
Compounds
1,
2,
4,
and
5
had
no
significant
effects
on
tachyzoite
production.
Pentamidine
and
com-
pound
9
were
toxic
at
100
and
50
µg/ml,
and
compound
4
was
toxic
at
100
µg/ml
for
Vero
cell
cultures.
These
compounds
were
considered
toxic
because
many
of
the
Vero
cells
became
rounded
and
detached
from
the
plastic
growth
surface
by
1
to
3
days
p.i.
in
these
fl
asks,
whereas
Vero
cells
in
fl
asks
infected
with
T.
gondii
and
not
treated
did
not
exhibit
these
cytopathic
changes.
When
pentamidine
was
evaluated
at
25
and
10
µg/ml
(Table
3),
significant
effects
on
TABLE
3.
Dosage
evaluations
of
pentamidine
and
compounds
3,
7,
8,
and
9
against
T.
gondii
in
Vero
cell
cultures
Expt
no.
(compound)
Dose
(14,/mIr
Total
no.
of
tachyzoites/flask
(mean
±
SD
[10
4
])
%
Reduction"
5
(compound
3)
Control
1,093
±
108
NA
10
859
268
21.4
50
176
±
97
83.9*
6
(pentamidine)
Control
579
±
116
NA
10
230
±
56
60.3*
25
182
±
95
68.6*
7
Control
316
±
157
NA
Compound
7
10
423
±
85
0
50
62
±
23
80.4*
Compound
8
10
809
±
234
0
50
132
±
66
58.2
8
Control
506
±
125
NA
Compound
9
10
92
±
62
81.8*
Piritrexim
10
1
99.8*
"
Control
cultures
received
no
drug
treatment.
"
NA,
not
applicable;
negative
percent
reductions
are
expressed
as
0;
*,
significant
reduction
in
the
number
of
tachyzoites
per
fl
ask
compared
with
that
in
control
fl
asks
(P
<
0.05).
1916
NOTES
ANTIMICROB.
AGENTS
CHEMOTHER.
tachyzoite
replication
were
observed.
Compound
9
was
effective
when
it
was
examined
at
10
µg/ml
(Table
3).
Pyrimethamine
and
piritrexim
both
significantly
inhibited
tachyzoite
replication
in
our
assay
at
a
concentration
of
10
p.g/ml
(Tables
2
and
3).
Many
antimicrobial
agents
have
been
evaluated
for
their
effectiveness
against
T.
gondii;
these
include
dihydrofolate
reductase
inhibitors,
macrolide
antibiotics,
polyether
iono-
phorous
antibiotics,
sulfonamides,
1,2,4-trioxanes,
hydroxy-
naphthoquinones,
and
purine
analogs
(1,
3-5,
8,
10, 13,
14,
16,
17).
However,
the
present
study
appears
to
be
the
fi
rst
to
evaluate
the
effectiveness
of
pentamidine
and
some
of
its
analogs
against
T.
gondii.
The
present
study
demonstrated
that
pentamidine
and
fi
ve
of
its
analogs
are
effective
in
inhibiting
replication
of
the
RH
isolate
of
T.
gondii
in
cell
cultures
and
suggests
that
other
analogs
of
pentamidine
may
also
be
effective
against
the
parasite.
Future
studies
should
focus
on
evaluation
of
these
additional
analogs.
Additionally,
studies
must
be
conducted
to
determine
whether
synergistic
effects
are
observed
when
these
compounds
are
administered
with
other
effective
com-
pounds.
We
thank
R.
A.
Cole
and
M.
E.
Vertuca,
Department
of
Patho-
biology,
Auburn
University,
for
technical
assistance.
This
study
was
supported
in
part
by
Public
Health
Service
contract
no.
1-A1-72648
from
the
National
Institute
of
Allergy
and
Infectious
Diseases
(to
R.R.T.)
and
U.S.
Department
of
Agriculture
Formula
Funds
(to
B.L.B.
and
D.S.L.).
REFERENCES
1.
Araujo,
F.
G.,
J.
Huskinson,
and
J.
S.
Remington.
1991.
Re-
markable
in
vitro
and
in
vivo
activities
of
the
hydroxynaphtho-
quinone
566C80
against
tachyzoites
and
tissue
cysts
of
Toxo-
plasma
gondii.
Antimicrob.
Agents
Chemother.
35:293-299.
2.
Bell,
C.
A.,
J.
E.
Hall, D.
E.
Kyle,
M.
Grogl,
K.
A.
Ohemeng,
M.
A.
Allen,
and
R.
R.
Tidwell.
1990.
Structure
-activity
rela-
tionships
of
analogs
of
pentamidine
against
Plasmodium
falci-
parum
and
Leishmania
mexicana
amazonensis.
Antimicrob.
Agents
Chemother.
34:1381-1386.
3.
Chang,
H.
R.,
C.
W.
Jefford,
and
J.
Pechere.
1989.
In
vitro
effects
of
three
new
1,2,4-trioxanes
(pentatroxane,
thia-
hexatroxane,
and
hexatroxanone)
on
Toxoplasma
gondii.
Anti-
microb.
Agents
Chemother.
33:1748-1752.
4.
Dannemann,
B.
R.,
D.
M.
Israelski,
and
J.
S.
Remington.
1988.
Treatment
of
toxoplasmic
encephalitis
with
intravenous
clinda-
mycin.
Arch.
Intern.
Med.
148:2477-2482.
5.
Derouin,
F.,
and
C.
Chastang.
1989.
In
vitro
effects
of
folate
inhibitors
on
Toxoplasma
gondii.
Antimicrob.
Agents
Chemo-
ther.
33:1753-11759.
6.
Dubey,
J.
P.,
and
C.
P.
Beattie.
1988.
Toxoplasmosis
of
man
and
animals.
CRC
Press,
Inc.,
Boca
Raton,
Fla.
7.
Frenkel,
J.
K.,
and
A.
Esrajadillo.
1987.
Cyst
rupture
as
a
pathogenic
mechanism
of
toxoplasmic
encephalitis.
Am.
J.
Trop.
Med.
Hyg.
36:517-522.
8.
Harris,
C.,
M.
P.
Salgo,
H.
B.
Tanowitz,
and
M.
Wittner.
1987.
In
vitro
assessment
of
antimicrobial
agents
against
Toxoplasma
gondii.
J.
Infect.
Dis.
157:14-22.
9.
Haverkos,
H.
W.
1987.
Assessment
of
therapy
of
toxoplasmic
encephalitis.
Am.
J.
Med.
82:907-914.
10.
Hoffiin,
J.
M.,
and
J.
S.
Remington.
1987.
Clindamycin
in
a
murine
model
of
toxoplasmic
encephalitis.
Antimicrob.
Agents
Chemother.
31:492-496.
11.
Jones,
S.
K.,
J.
E.
Hall,
M.
A.
Allen,
S.
D.
Morrison,
K.
A.
Ohemeng,
V.
V.
Reddy,
J.
D.
Geratz,
and
R.
R.
Tidwell.
1990.
Novel
pentamidine
analogs
in
the
treatment
of
experimental
Pneumocystis
carinii
pneumonia.
Antimicrob.
Agents
Chemo-
ther.
34:1026-1030.
12.
Kapusnik,
J.
E.,
and
J.
Mills.
1988.
Pentamidine,
p.
299-311.
In
P.
K.
Peterson
and
J.
Verhoef
(ed.),
The
antimicrobial
agents
annual
3.
Elsevier
Science
Publishers
BV,
Amsterdam,
The
Netherlands.
13.
Luft,
B.
J.
1986.
Potent
in
vivo
activity
of
arprinocid,
a
purine
analogue,
against
murine
toxoplasmosis.
J.
Infect.
Dis.
154:692-
694.
14.
Luft,
B.
J.
1987.
In
vivo
and
in
vitro
activity
of
roxithromycin
against
Toxoplasma
gondii
in
mice.
Eur.
J.
Clin.
Microbiol.
6:479-481.
15.
Luft,
B.
J.,
and
J.
S.
Remington.
1988.
Toxoplasmic
encephali-
tis.
J.
Infect.
Dis.
157:1-6.
16.
Melton,
M.
L.,
and
H.
G.
Sheffield.
1975.
Activity
of
the
anticoccidial
compound,
lasalocid,
against
Toxoplasma
gondii
in
cultured
cells.
J.
Parasitol.
61:713-717.
17.
Remington,
J.
S.,
and
B.
J.
Luft.
1988.
Drugs
used
in
the
treatment
of
toxoplasmosis,
p.
327-336.
In
P.
K.
Peterson
and
J.
Verhoef
(ed.),
The
antimicrobial
agents
annual
3.
Elsevier
Science
Publishers
BV,
Amsterdam,
The
Netherlands.
18.
Tidwell,
R.
R.,
S.
K.
Jones,
J.
D.
Geratz,
K.
A.
Ohemeng,
C.
A.
Bell,
B.
J.
Berger,
and
J.
E.
Hall.
1990.
Development
of
pentamidine
analogues
as
new
agents
for
the
treatment
of
Pneumocystis
carinii
pneumonia.
Ann.
N.Y.
Acad.
Sci.
661:
421-441.
19.
Tidwell,
R.
R.,
S.
G.
Kilgore,
J.
D.
Geratz,
K.
A.
Ohemeng,
M.
Cory,
and
J.
E.
Hall.
1990.
Analogues
of
1,5-bis(4
amidinophe-
noxy)pentane
(pentamidine)
in
the
treatment
of
experimental
Pneumocystis
carinii
pneumonia.
J.
Med.
Chem.
33:1252-1257.
20.
Tidwell,
R.
R.,
S.
G.
Kilgore,
K.
A.
Ohemeng,
J.
D.
Geratz,
and
J.
E.
Hall.
1989.
Treatment
of
experimental
Pneumocystis
carinii
pneumonia
with
analogues
of
pentamidine.
J.
Protozool.
36:S74
-S76.
21.
Wanke,
C.,
C.
U.
Tuazon,
A.
Kovacs,
T.
Dina,
D.
0.
Davis,
N.
Barton,
D.
Katz,
M.
Lunde,
C.
Levy,
F.
K.
Conley,
H.
C.
Lane,
A.
S.
Fauci,
and
H.
Masur.
1987.
Toxoplasma
encephalitis
in
patients
with
acquired
immune
deficiency
syndrome:
diagnosis
and
response
to
therapy.
Am.
J.
Trop.
Med.
Hyg.
36:509-516.