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Antimicrobial Agents and Chemotherapy, July 1998, p. 1771-1777, Vol. 42, No. 7
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Antiproliferative Effects and Mechanism of Action of SCH
56592 against Trypanosoma (Schizotrypanum)
cruzi: In Vitro and In Vivo Studies
Julio A.
Urbina,1,*
Gilberto
Payares,2
Lellys M.
Contreras,1
Andreína
Liendo,1
Cristina
Sanoja,2
Judith
Molina,2
Marta
Piras,3
Romano
Piras,3
Norma
Perez,1,4
Patrick
Wincker,4 and
David
Loebenberg5
Laboratorio de Química Biológica, Centro de
Biofísica y Bioquímica, Instituto Venezolano de
Investigaciones Científicas, Caracas
1020A,1
Departmento de
Parasitología, Instituto de Zoología Tropical,
Universidad Central de Venezuela, Caracas
1041,2 and
Unidad de Investigacion,
Centro Medico Docente "La Trinidad," Caracas
1060,3 Venezuela;
Laboratoire
`Genome des Parasites,' Faculté de Medecine, Université
de Montpellier, Montpellier, France4; and
Schering-Plough Research Institute, Kenilworth, New Jersey
07033-05395
Received 23 February 1998/Returned for modification 1 April
1998/Accepted 4 May 1998
 |
ABSTRACT |
We have investigated the antiproliferative effects of SCH 56592, a
new experimental triazole, against Trypanosoma
(Schizotrypanum) cruzi, the etiological agent
of Chagas' disease in Latin America. SCH 56592 blocked the
proliferation of the epimastigote form of the parasite in vitro at 30 nM, a concentration 30- to 100-fold lower than that required with the
reference compounds ketoconazole and itraconazole. At that
concentration all the parasite's endogenous sterols (ergosterol,
24-ethyl-cholesta-5,7,22-trien-3
-ol, and its 22-dihydro analogs),
were replaced by methylated sterols (lanosterol and
24-methylene-dihydrolanosterol), as revealed by high-resolution gas
chromatography coupled with mass spectrometry. This indicated that the
primary mechanism of action of the drug was inhibition of the
parasite's sterol C-14
demethylase. Against the clinically relevant
intracellular amastigote form, grown in cultured Vero cells at 37°C,
the MIC of SCH 56592 was 0.3 nM, again 33- to 100-fold lower than that
of ketoconazole or itraconazole. In a murine model of acute Chagas'
disease, SCH 56592 given at 
10 mg/kg of body weight/day for a
total of 43 doses allowed 85 to 100% survival and 90 to 100% cure of
the surviving animals, as verified by parasitological, serological, and
PCR-based tests, while ketoconazole given at 30 mg/kg day allowed 60%
survival but only 20% cure. In a murine model of chronic Chagas'
disease, SCH 56592 was again more effective than ketoconazole,
providing 75 to 85% protection from death, with 60 to 75%
parasitological cures of the surviving animals, while no
parasitological cures were observed with ketoconazole. The results
indicate that SCH 56592 is the most powerful sterol biosynthesis
inhibitor ever tested against T. cruzi and may be useful in
the treatment of human Chagas' disease.
| |
INTRODUCTION |
Chagas' disease (American
trypanosomiasis) is a parasitic disease caused by the kinetoplastid
protozoon Trypanosoma (Schizotrypanum) cruzi, which afflicts 16 to 18 million people in Latin
America and causes an estimated loss of 2.7 disability-adjusted years annually. It accounts for the largest parasitic disease burden in the
region and the third largest worldwide, following malaria and
schistosomiasis (42). Thirty to 40% of patients who survive the initial acute phase (ca. 90%) develop irreversible heart and gastrointestinal lesions over years or decades, and these frequently lead to death (30). Chemotherapy of this disease is still
very unsatisfactory, since it is based on nitrofurans (nifurtimox; Bayer) and nitroimidazoles (benznidazole; Roche), which act through the induction of oxidative or reductive damage of the parasite but
which can also produce serious toxic effects in the host (6, 7,
29, 30). These compounds have been used in the acute phase, but
their efficacy varies according to the geographical area, due to
differences in drug susceptibility among different T. cruzi
strains (10). However, there is no available treatment for
the prevalent chronic form of the disease (6, 7, 29, 30).
Although great progress has been made in recent years in the control of
the vectorial and transfusional transmission of the disease,
particularly through the Southern Cone Initiative, involving Brazil,
Argentina, Paraguay, and Uruguay, progress is uneven throughout
the continent, and the problem of treating persons already infected
remains unresolved.
Despite the fact that T. cruzi requires specific endogenous
sterols and is extremely sensitive to sterol biosynthesis inhibitors in
vitro (17, 35, 37, 39, 40), currently available sterol biosynthesis inhibitors which are highly successful in the
treatment of fungal diseases are not powerful enough to eradicate
T. cruzi from experimentally infected animals or human
patients (1, 18, 23). We have recently demonstrated that
D0870 (Zeneca Pharmaceuticals), a bistriazole derivative which is the
R(+) enantiomer of ICI 195,739 (15, 17, 31,
36), is capable of inducing parasitological cures of both
acute and chronic experimental Chagas' disease (34,
38), the first compound ever to display such activity.
Unfortunately, the development of this compound has recently been
discontinued. SCH 56592 {(
)-4-[4-[4-[4-[[(2R- cis)-5-(2,4-difluorophenyl)-tetrahydro-5-(1H-1,2,4-triazol-1-yl- methyl)furan-3-yl]methoxy]phenyl]-2,4-dihydro-2-[(S)-1-ethyl- 2(S)-hydroxypropyl]-3H-1,2,4-triazol-3-one;
Schering-Plough} (Fig. 1) is an
experimental triazole derivative which has remarkable and
broad-spectrum antifungal activity and is particularly effective in the
treatment of experimental systemic mycoses (11, 26-28, 33). In this article we describe the results of a study on the in
vitro and in vivo anti-T. cruzi activity of SCH 56592, which indicate that this compound has antiparasitic activity
comparable or superior to that of D0870.
| |
MATERIALS AND METHODS |
Parasite.
For the in vitro studies, the EP (8)
and Y strains of T. cruzi were used with similar results;
for the in vivo studies, the Y and Bertoldo (34, 38) strains
were used. Live T. cruzi was handled according to
established guidelines (12).
In vitro studies.
The epimastigote form of the parasite was
cultivated in a modification of liver infusion-tryptose (LIT) medium
(8) supplemented with 10% newborn calf serum (Gibco) at
28°C with strong agitation (120 rpm); the cultures were initiated
with a cell density of 2 × 106 epimastigotes per ml,
and the drugs were added when cell density reached 1 × 107 epimastigotes per ml. Cell densities were measured with
an electronic particle counter (model ZBI; Coulter Electronics, Inc.,
Hialeah, Fla.) and by direct counting with a hemocytometer. Cell
viability was followed by trypan blue exclusion using light microscopy. Amastigotes were cultured in Vero cells maintained in minimal essential
medium supplemented with 2% fetal calf serum in a humidified 95%
air-5% CO2 atmosphere at 37°C as previously described
(16, 35-37, 39). Cells were infected with 10 tissue
culture-derived trypomastigotes per cell for 2 h and then were
washed three times with phosphate-buffered saline to remove nonadherent
parasites. Fresh medium with or without drugs was added, and the cells
were incubated for 96 h, with a change of medium at 48 h.
Quantification of the number of infected cells and of the number of
parasites per cell by use of light microscopy and statistical analysis
of the results were carried out as described elsewhere (16,
35-37, 39).
Studies of lipid composition.
For the analysis of the
effects of drugs on the sterol composition of the epimastigotes, total
lipids from control and drug-treated cells were extracted and
fractionated by silicic acid column chromatography and gas-liquid
chromatography (16, 38-40). The neutral lipid fractions
were preliminarily analyzed by thin-layer chromatography (on Merck 5721 silica gel plates with heptane-isopropyl ether-glacial acetic acid
[60:40:4] as the developing solvent) and conventional gas-liquid
chromatography (isothermic separation in a 4-m glass column packed with
3% OV-1 on Chromosorb 100/200 mesh, with nitrogen as the carrier gas
at 24 ml/min and flame ionization detection in a Varian 3700 gas
chromatograph). For quantitative analysis and structural assignments,
the neutral lipids were separated in a capillary high-resolution column
(25 m by 0.20 mm [inner diameter] Ultra-2 column, 5%
phenyl-methyl-siloxane, 0.33-µm film thickness) in a Hewlett-Packard
5890 series II gas chromatograph equipped with an HP5971A mass
sensitive detector. Lipids were injected in ethyl acetate, and the
column was kept at 50°C for 1 min; the temperature was then increased
to 270°C at a rate of 25°C min
1 and finally to
300°C at a rate of 1°C min1. The carrier gas (He)
flow was kept constant at 1.0 ml · min1. The
injector temperature was 250°C, and the detector temperature was kept
at 280°C.
In vivo studies.
Studies were carried out as described
previously (17, 37, 39) by following the initial studies of
McCabe et al. (19-21) for the acute model and the
procedures described by Urbina and coworkers (34, 38) for
the chronic model. For the acute model, groups of 8 to 11 outbred NMRI
albino female mice weighing 25 to 30 g were inoculated
intraperitoneally with 105 blood trypomastigotes of the Y
strain, and treatment was initiated 24 h later. For the chronic
model, animals were inoculated with 104 blood
trypomastigotes of the Bertoldo strain, and treatment was initiated in
surviving animals when no circulating parasites could be observed,
usually 45 to 60 days postinfection (p.i.). The drugs were suspended in
aqueous 2% methylcellulose plus 0.5% Tween 80 and given by gavage;
controls (untreated animals) received the vehicle as a placebo, which
had no detectable toxic effects. Treatment was given once daily for 28 consecutive days, followed by a 7-day rest and another 15 days of
treatment. Surviving animals were monitored for up to 100 to 120 days
p.i. Parasitemia was measured in a hemocytometer by using tail blood.
The Kaplan-Meier nonparametric method was used to estimate the survival
functions of the different experimental groups, and rank tests were
used to compare them; these analyses were done by using the Survival
Tools for StatView 4.5 run in a Power Macintosh 7100/66 computer.
Hemocultures were carried out by inoculating 2 ml of liver infusion
medium with 0.4 ml of blood obtained from experimental animals by
cardiac puncture; the cultures were microscopically examined for the
presence of proliferative epimastigote forms weekly for 4 weeks.
Surviving animals were sacrificed, and organs (spleen, heart, and
liver) were minced individually in 1 ml of sterile phosphate-buffered saline with 10 mM D-glucose. Portions (0.4 ml) of these
suspensions were inoculated in juvenile (15- to 20-g) animals.
Hemoinoculation (50 µl of blood diluted to 100 µl with sterile
phosphate-buffered saline) was done subcutaneously in 10- to 12-day-old
mice. Xenodiagnosis was done with 10 2nd-stage Rodnius
prolixus nymphs per mouse. After 2 weeks the feces were analyzed
for T. cruzi metacyclic forms, and the examination was
repeated weekly thereafter for 1 month. The presence of circulating
anti-T. cruzi antibodies was detected by immunoprecipitation
of 125I-labeled total-surface epimastigote antigens with
experimental sera in the presence of protein A, followed by analysis of
the precipitate by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. DNA extraction and PCR of blood samples were carried
out as described previously (2, 3, 41), by using the
T. cruzi-specific primers
5'-AAATAATGTACGGG(T/G)GAGATGCATGA-3' and
5'-GGTTCGATTGGGGTTGGTGTAATATA-3'.
Drugs.
SCH 56592 was provided by Schering-Plough Research
Institute through David Loebenberg, while ketoconazole and itraconazole were provided by Janssen Pharmaceutica, Caracas, Venezuela. The drugs
were added to cultures as dimethyl sulfoxide solutions; the final
dimethyl sulfoxide concentration in the culture medium never exceeded
1% (vol/vol), and dimethyl sulfoxide had no effect by itself on the
proliferation of the parasites or Vero cells.
| |
RESULTS AND DISCUSSION |
In vitro studies.
Figure 2
presents the effects of SCH 56592 on the epimastigote form of T. cruzi, a form equivalent to that present in its reduviid vectors,
grown in modified LIT medium at 28°C (8). It can be seen
that with all the concentrations tested there were no immediate effects
on epimastigote proliferation, but at 30 nM, growth arrest and
subsequent rounding up and lysis of the cells (verified by light
microscopy and trypan blue exclusion) ensued. This delayed lytic effect
is a characteristic effect of sterol biosynthesis inhibitors,
associated with the time required for the complete depletion of the
parasite's endogenous sterols (16, 36-40). This was
directly confirmed by analysis of the parasite's sterols with
high-resolution gas-liquid chromatography coupled to mass spectrometry.
Table 1 shows that in epimastigotes
incubated for 120 h with concentrations of the SCH 56592 equal to
or greater than the MIC (30 nM), all the endogenous 4,14-desmethyl
sterols (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3-ol, and
precursors) were replaced by 4,14-dimethyl and -trimethyl sterols such
as lanosterol, 24-methylene-dihydrolanosterol, and
4,14-dimethyl-ergosta-8,24(24')-3-ol (obtusifoliol). This confirmed
that the primary target of the drug was the parasite's sterol
C-14-demethylase, as was seen in fungi (24, 32, 43).
Together with the accumulation of the C-14-methyl sterols, significant
amounts of the apolar precursor squalene were detected, reaching ca.
40% of the total weight of sterols and precursors in the cells at 3 µM SCH 56592 (100 times the MIC). This accumulation is most probably
due to mass-action effects and indicated a very effective blockade of
the metabolic flow at the level of sterol C-14-demethylase, which in
turn suggested tight binding of this drug to its target enzyme. In
fact, the concentration of SCH 56592 required for complete endogenous
sterol depletion and growth arrest, associated with subsequent cell
lysis of the epimastigotes (Fig. 2), was 33- to 100-fold lower that that required for ketoconazole (5, 14, 35, 37), D0870 (16, 38), or itraconazole (data not shown). These results are in line with those of Sanglard et al. (32), who have
recently shown that azole-resistant Candida albicans
isolates which had mutations in their sterol 14-demethylase which
sharply reduced their affinity for fluconazole or itraconazole
nevertheless had normal affinity for SCH 56592.
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FIG. 2.
Effects of SCH 56592 on the proliferation of T. cruzi epimastigotes. Epimastigotes were cultured in modified LIT
medium at 28°C as described in Materials and Methods. The arrow
indicates the time of addition of the drug, at the indicated
concentrations. Each experimental point corresponds to the mean of
three independent cultures; the standard deviations of the measurements
were equal to or less than 10% of the means.
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TABLE 1.
Free sterols and precursors present in T. cruzi epimastigotes (EP stock) grown in the absence or presence of
SCH 56592 for 120 ha
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|
Against the clinically relevant intracellular amastigote form,
proliferating in cultured Vero cells at 37°C, SCH 56592 was
again
remarkably active. Figure
3A shows that
the minimal concentration
of SCH 56592 required to eradicate the
parasite from the host
cells was just 0.3 nM, again 33 to 100 times
lower than that required
with ketoconazole (Fig.
3B), D0870 (
16,
38), or itraconazole
(data not shown). It can also be seen from
Fig.
3 that SCH 56592
had no effects on the proliferation of the host
cells at 100 nM
(>300 times the MIC); the same was also observed even
at 1 µM
(data not shown), indicating a very specific antiparasitic
activity.
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FIG. 3.
Concentration dependence of the effects of SCH 56592 (A)
and ketoconazole (B) on the proliferation of T. cruzi
amastigotes and Vero cells at 37°C. Shown are the percentage of
infected cells ( ), the number of amastigotes per cell ( ), and the
number of Vero cells per field ( ) after 96 h as a function of
the drug concentration. Vero cells were infected with T. cruzi as described in Materials and Methods. Each bar represents 1 standard deviation.
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Taken together, these results indicated that SCH 56592 is, in vitro,
the most potent sterol biosynthesis inhibitor and antiproliferative
agent ever tested against both proliferative stages of
T. cruzi.
In vivo studies.
In a murine model of acute Chagas' disease
previously described (17, 37, 39), a fulminant infection is
produced by inoculating 105 bloodstream trypomastigotes of
the virulent Y stock per mouse, leading to the death of all untreated
animals in 5 weeks (Fig. 4). Oral
treatment was started 24 h p.i. and given daily for 28 consecutive
days, followed by a 7-day rest and another 15 days of treatment. As
reported previously (17, 34, 37-39), ketoconazole given at
30 mg/kg/day was very effective in suppressing the proliferation of the
parasite while the drug pressure was present, but the parasite was not
eradicated, as seen by the subsequent increase in parasitemia (Fig. 4B)
and the concomitant death of the animals receiving this treatment 70 to
80 days p.i. (Fig. 4A). At low (5- to 10-mg/kg/day) dosages of SCH
56592, some animals displayed delayed parasitemia and subsequently
died, as did those receiving ketoconazole, but at higher dosages, no
evidence of circulating parasites was found during the observation
period (100 to 120 days p.i.), suggesting sterilization of the treated
animals. At those higher dosages (15 to 25 mg/kg/day), survival levels
varied from 75 to 100%. Parasitological cures (Table
2) were assessed by a combination of
xenodiagnosis, hemoculture, hemoinoculation in newborn mice, subinoculation of organs in naive mice, and a recently developed PCR-based test (2, 3, 41); animals which tested negative were also found to lack anti-T. cruzi antibodies, detected
by precipitation of 125I-labeled total-surface epimastigote
antigens, indicating that they had reverted to preimmune status. Cures
ranged from 90 to 100% of the surviving animals which received 15 to
25 mg of SCH 56592/kg/day, while only 20% of those treated with
ketoconazole were cured. The only precedents for these results are our
recent studies with the bistriazole D0870 (34, 38), carried
out under the same experimental conditions; this drug was capable of
inducing parasitological cures in 60 to 70% of treated animals, while
drugs currently used in clinical settings, such as Nifurtimox and
ketoconazole, did not produce significant levels of parasitological
cures.
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FIG. 4.
Effects of SCH 56592 and ketoconazole on survival (A)
and parasitemia (B) in a murine model of acute Chagas' disease.
Statistical analysis using both the log rank (Mantel-Cox) and
Peto-Peto-Wilcoxon tests indicated a very significant
(P < 0.0001) difference between the control
(untreated) animals and all those that received the drug
treatments. For details, see Materials and Methods.
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TABLE 2.
Effects of SCH 56592 and ketoconazole on survival and
parasitological cure in a murine model of acute
Chagas' diseasea
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|
A model of the chronic form of the disease previously described
(
34,
38) was also investigated. Animals were infected
with
10
4 trypomastigotes of the cardiotropic Bertoldo strain,
which produced
a slowly developing parasitemia that peaked ca. 25 days
p.i. but
was effectively controlled by most infected animals. Animals
that
survived this initial phase (ca. 70%) developed a condition in
which their general physical condition deteriorated slowly but
they
survived for several months, although sudden death was also
observed.
Treatment was started 45 to 60 days p.i., when no circulating
parasites
were found. SCH 56592 at 10 to 15 mg/kg/day, given for
a total of 43 doses as described above, provided 75 to 85% protection
from death and
60 to 75% parasitological cures of the surviving
animals, while
ketoconazole at 30 mg/kg/day had no significant
effect on the level of
parasitological cures compared with those
for untreated controls (Table
3). Autopsy of control (untreated)
animals revealed a significant increase (ca. 50%) in the size
of the
heart and spleen, with focal inflammatory lesions, characteristic
of
human and murine chagasic cardiomyopathy (
29,
30), while
the
organs from animals receiving SCH 56592 at curative doses
had normal
morphology. These results are again comparable only
to those previously
reported for D0870 (
34,
38), which was
the first report of
parasitological cure of chronic experimental
Chagas' disease. More
recently (
22a), we have found that SCH
56592 is capable of
inducing parasitological cures of experimental
T. cruzi
infections caused by strains which are moderately to
strongly resistant
to benznidazole and nifurtimox (
10); this
result also was
previously obtained only with D0870 (
22). As
we have
discussed in previous publications (
17,
37), the effective
doses of antifungal azoles are, on a weight basis, approximately
10 times smaller in humans than in mice. If this is also valid
for SCH
56592, it will suggest that the doses of this compound
required for
anti-
T. cruzi activity in humans will be well within
the
range which is already known to be well tolerated (
13).
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TABLE 3.
Effects of SCH 56592 and ketoconazole on survival and
parasitological cure in a murine model of chronic
Chagas' diseasea
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|
In the case of D0870, we have previously argued (
16,
38)
that its exceptional in vivo anti-
T. cruzi activity could be
explained only by its special pharmacokinetic properties (
4,
9), since its in vitro anti-
T. cruzi activity was
comparable
to that of ketoconazole or nifurtimox, and these drugs have
no
curative activity in our in vivo models. For SCH 56592, both its
high intrinsic anti-
T. cruzi activity, reflected in the in
vitro
results, and the long terminal half-lives and large volumes of
distribution in animals (
11,
25) and humans (
13)
should
contribute to its in vivo antiparasitic activity.
In conclusion, we have shown that SCH 56592 has outstanding and
specific in vitro activity against both proliferative stages
of
T. cruzi, particularly against the clinically relevant
intracellular
amastigote form. We have also demonstrated that this
compound
exhibits curative rather than suppressive activity in murine
models
of acute and chronic Chagas' disease, and in this respect it is
similar or superior to D0870 (
34,
38). SCH 56592 is
currently
in Phase I/II clinical trials as a systemic antifungal agent
and
should be a logical candidate for clinical trials for the treatment
of human Chagas' disease.
| |
ACKNOWLEDGMENTS |
This work received financial support from the UNDP/World
Bank/World Health Organization Programme for Research and Training in
Tropical Diseases (grant 930161), the National Research Council of
Venezuela (CONICIT; grant RP-IV-110034), and the Instituto Venezolano
de Investigaciones Científicas. J.A.U. is a John Simon Guggenheim Foundation Fellow.
We gratefully acknowledge the technical support of Gonzalo Visbal and
Reneé Lira.
| |
FOOTNOTES |
*
Corresponding author. Present address: School of
Chemical Sciences, Chemistry and Life Sciences Laboratory, Rm. A106,
University of Illinois at Urbana-Champaign, 600 S. Mathews Ave.,
Urbana, IL 61821. Phone: (217) 333-8328. Fax: (217) 244-0997. E-mail: jaurbina{at}churchill.scs.uiuc.edu.
| |
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Abstract
Introduction
