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Antimicrobial Agents and Chemotherapy, September 2000, p. 2498-2502, Vol. 44, No. 9
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
In Vitro Antiproliferative Effects and Mechanism of
Action of the New Triazole Derivative UR-9825 against the Protozoan
Parasite Trypanosoma (Schizotrypanum)
cruzi
Julio A.
Urbina,1,*
Renee
Lira,1
Gonzalo
Visbal,1 and
Javier
Bartrolí2
Laboratorio de Química
Biológica, Centro de Biofísica y Bioquímica,
Instituto Venezolano de Investigaciones Científicas, Caracas
1020A, Venezuela,1 and Research Center,
Uriach & Cia, 08026 Barcelona, Spain2
Received 27 January 2000/Returned for modification 4 May
2000/Accepted 20 June 2000
 |
ABSTRACT |
We describe the in vitro antiproliferative effects of the new
triazole derivative UR-9825 against the protozoan parasite
Trypanosoma (Schizotrypanum) cruzi,
the causative agent of Chagas' disease in Latin America. The compound
was found to be extremely active against the cultured (epimastigote)
form of the parasite, equivalent to that present in the reduviid
vector, with a MIC of 30 nM, a concentration 33-fold lower than that
required with the reference compound ketoconazole. At that MIC, growth
arrest coincided with depletion of the parasite's 4,14-desmethyl
endogenous sterols (ergosterol, 24-ethylcholesta-5,7,22-trien-3b-ol,
and precursors) and their replacement by methylated sterols
(lanosterol, 24-methylenedihydrolanosterol, and obtusifoliol), as
revealed by high-resolution gas chromatography coupled with mass
spectrometry. This indicated that the primary mechanism of action of
UR-9825 was inhibition of the parasite's sterol C14
demethylase, as
seen with other azole derivatives. The phospholipid composition of
growth-arrested epimastigotes was also altered, when compared to
controls, with a significant increase in the content of
phosphatidylethanolamine and phosphatidylserine and a concomitant
reduction of the content of phosphatidylcholine. The clinically
relevant intracellular amastigote form, grown in cultured Vero cells at
37°C, was even more sensitive to UR-9825, with a MIC of 10 nM,
comparable to that for ketoconazole. The results showed that UR-9825 is
among the most potent azole derivatives tested against this parasite
and support in vivo studies with this compound.
| |
INTRODUCTION |
The largest parasitic disease burden
on the American continent is Chagas' disease, caused by the
kinetoplastid protozoon Trypanosoma (Schizotrypanum) cruzi, with 16 to 18 million
people infected and an estimated annual loss of 2.7 disability-adjusted
years (41). The parasite develops intracellularly in its
mammalian hosts, leading to severe tissue damage, particularly in the
heart and gastrointestinal tract, which, coupled with intense
inflammatory reactions, underlies the pathogenesis of the disease
(1, 5). Although >90% of those infected survive the
initial acute phase, 30 to 40% of individuals with chronic T. cruzi infection will develop, over years or decades, irreversible
heart and gastrointestinal lesions which are frequently lethal (1,
6, 23, 25). Current chemotherapeutic approaches for the treatment
of this condition, based on nitrofurans and nitroimidazoles, are
unsatisfactory due to limited efficacy, particularly for the prevalent
chronic form of the disease, and frequent toxic side effects (8,
10, 23, 25). Recently, great advances have been made in the
control of both vectorial and transfusional transmission of the
disease, particularly through the Southern Cone and Andean Initiatives (41). However, transmission continues in other parts of the continent and the problem of those already infected, most of them in
the undetermined chronic phase, remains a challenge (8, 28,
29).
Sterol biosynthesis inhibitors (SBI) are currently the most advanced
and widely used drugs in the treatment of fungal diseases (26, 38,
39). Although T. cruzi requires specific endogenous sterols for cell viability and proliferation and thus is extremely sensitive to SBI in vitro (reviewed in references 27,
28, and 30), currently available SBI are
not powerful enough to eradicate T. cruzi from
experimentally infected animals or human patients (4, 12, 20,
22). However, we have recently shown that new triazole
derivatives such as D08070 (Zeneca Pharmaceuticals) and SCH 56592 (Schering-Plough), both selective inhibitors of the parasite's sterol
C14 demethylase with high intrinsic anti-T. cruzi
activity and special pharmacokinetic properties (long terminal half-lives and large volumes of distribution), are capable of inducing
radical parasitological cure of both acute and chronic experimental
Chagas' disease (21, 28, 30, 34, 35). UR-9825 [(1R,2R)-7-chloro-3-[2-(2,4-difluoropenyl) - 2 - hydroxyl - 1 - methyl - 3 - (1H - 1,2,4 - triazol - 1 - yl)propyl]quinazolin-4(3H)-one] (Uriach & Cia) (Fig. 1) is a new triazole
derivative with potent and broad-spectrum antifungal activity and a
long terminal life in dogs and cynomolgous monkeys (3,
24; J. Bartolí, E. Turmo, M. Algueró, E. Boncompte, M. L. Vericat, L. Conte, J. Ramis, J. García-Rafanell, and J. Forn, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-67, 1997). In this article we
describe the results of a study on the in vitro anti-T.
cruzi activity of UR-9825, which suggest that it is a good
candidate for in vivo chemotherapeutic studies in appropriate animal
models of Chagas' disease.
| |
MATERIALS AND METHODS |
Parasite.
The EP (11) and Y stocks of T. cruzi were used in this study with indistinguishable results.
Handling of live T. cruzi was done according to established
guidelines (13).
In vitro studies.
The epimastigote form of the parasite was
cultivated in liver infusion tryptose (LIT; see reference
11) medium, 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 at a cell density of 0.5 × 107 to 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 1% fetal calf
serum in a humidified 95% air-5% CO2 atmosphere at
37°C as previously described (18, 31-33, 36). Briefly,
the cells were infected with 10 tissue culture-derived trypomastigotes
per cell for 2 h and then washed three times with phosphate-buffered saline to remove nonadherent parasites; fresh medium
with and without drugs was added and the cells were incubated for
96 h with a medium change 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 previously (18, 31-34, 36).
Studies of lipid composition.
For the analysis of the
effects of drugs on the lipid composition of the epimastigotes, total
lipids from control and drug-treated cells were extracted and
fractionated into neutral and polar lipid fractions by silicic acid
column chromatography and gas-liquid chromatography (18,
34-37). The neutral lipid fractions were first 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
[inside 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. The
lipids were injected in ethyl acetate; the column was kept at 50°C
for 1 min and then the temperature was 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 was kept at 280°C. The
polar lipid fraction (containing mostly phospholipids) was analyzed as
described before (7); briefly, the lipid fractions eluted
from the silicic acid column with chloroform-methanol at 1:1 (vol/vol)
were pooled and further fractionated by thin-layer chromatography on
Merck 5721 silica gel plates using chloroform-methanol-32.5% ammonia
(wt/vol) (17:7:1 by volume) as the developing solvent (9).
The phospholipid spots were visualized using iodine and scraped, and
the total organic phosphorous was measured using the method of Ames and
Dubin (2).
Drugs.
UR-9825 was provided by the Research Center, Uriach & Cia, Barcelona, Spain, while ketoconazole was provided by Janssen
Pharmaceutica, Caracas, Venezuela. The drugs were added as dimethyl
sulfoxide solutions; the final dimethyl sulfoxide concentration in the
culture media never exceeded 1% (vol/vol) and had no effect by itself on the proliferation of the parasites or Vero cells.
| |
RESULTS AND DISCUSSION |
Effects on epimastigotes.
The data presented in Fig.
2 show the effects of UR-9825 on the
proliferation of the epimastigote form of T. cruzi,
equivalent to that present in its reduviid vectors, grown in LIT medium
at 28°C (11). The response observed is characteristic of
all SBI tested against this organism, with no significant effects on
the growth rate for at least one generation after the drug addition, followed, at effective doses, by complete growth arrest and cell lysis
after three to four generations (verified by light microscopy and
trypan blue exclusion). Just before lysis, epimastigotes became rounded
and large cell aggregates were observed, as seen before with other SBI
(16, 17, 40). The minimal concentration of UR-9825 required
to induce this "delayed lytic effect" after 144 h (MIC) was 30 nM, which is 33 times lower than that of ketoconazole (7, 16, 31,
33), D0870 (18, 35), or itraconazole (results not
shown) and only comparable to that of SCH 56592, the most potent SBI
known against this parasite (34).
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FIG. 2.
Effects of UR-9825 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; each full bar represents two standard
deviations.
|
|
The onset of growth arrest and subsequent cell lysis induced by SBI in
T. cruzi have been associated with the complete depletion
of
the parasite's endogenous sterols (
18,
32-37). This was
confirmed
in epimastigotes treated with UR-9825 by analysis of the
neutral
lipid fraction using high-resolution gas-liquid chromatography
coupled with mass spectrometry. We present in Table
1 the free
sterol composition of
epimastigotes incubated for 120 h with concentrations
of UR-9825
or ketoconazole equal to or above the MIC (30 nM and
1 µM,
respectively), together with that of control (untreated)
organisms. It
can be seen that under those conditions essentially
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-dimethylergosta-8,24(24
1)-dien-3
-ol
(obtusifoliol). These results strongly suggest that
the primary
target of UR-9825 in
T. cruzi is the cytochrome
P-450-dependent
sterol C14
demethylase, as found for other azoles
(
18,
32-37).
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|
TABLE 1.
Free sterols present in T. cruzi epimastigotes
(EP stock) grown in the absence or presence of UR-9825
or ketoconazolea
|
|
Epimastigotes treated with UR-9825 concentrations which induced
complete growth arrest also displayed an altered phospholipid
composition when compared with control (untreated) cells. It can
be
seen in Table
2 that these cells
exhibited a large increase
in the content of phosphatidylethanolamine
(PE) and a concomitant
decrease in the content of phosphatidylcholine
(PC) (Table
2).
This effect has also been observed before with several
other SBI
and has been attributed to a sharp reduction in the activity
of
membrane-bound PC-PE-
N-methyl transferase, due to the
altered
sterol composition (
7). Thus, UR-9825 seemed to
induce all
the physiological alterations characterized before in
SBI-treated
epimastigotes, but at lower concentrations than
conventional azoles.
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|
TABLE 2.
Phospholipid composition of T. cruzi
epimastigotes (EP stock) grown in the absence or presence of
UR-9825 or ketoconazole for
120 ha
|
|
Effects on amastigotes.
The clinically relevant intracellular
amastigote forms, proliferating in cultured Vero cells at 37°C, were
more susceptible to UR-9825 than epimastigotes: it can be seen in Fig.
3 that the MIC required to reduce
infected cells by 99% and the IC50 (concentration of the
drug required to reduce infected cells by 50%) were in this case 10 nM
and 1 nM, respectively. The observation time was 96 h after
infection, as at this time the intracellular development of the
parasite is complete and nonproliferative trypomastigotes begin to be
released by host cells. It can also be seen that no effects on the
viability and proliferation of the host Vero cells were observed up to
the highest concentration tested (100 nM), indicating a very specific
antiparasitic activity. The MIC was essentially identical to that
observed with ketoconazole (results not shown; see also references
31 and 33), D0870 (18,
35), or itraconazole (not shown), but higher than that previously
reported for SCH 56592 (34).
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FIG. 3.
Concentration dependence of the effects of UR9825 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 full bar represents two standard deviations.
|
|
Conclusions.
The results presented above indicate that the in
vitro activity of UR-9825 against T. cruzi is comparable to
that of the most potent SBI tested against this organism, D0870 and SCH
56592, which have been shown to be able to induce radical
parasitological cure in murine models of both acute and chronic
Chagas' disease (34, 35). However, as discussed in detail
elsewhere (18, 34, 35), effective in vivo activity of SBI
against T. cruzi requires both high intrinsic (in vitro)
antiparasitic activity and special pharmacokinetic properties, such as
long terminal half-lives and large volumes of distribution. Thus,
although the extremely short terminal half-life of UR-9825 in mice
(3; Bartoli et al., 37th ICAAC) prevented us from
testing this compound in our previously described murine models
(19, 33-36), the long half-lives in dogs and cynomolgous
monkeys (51 and 24 h, respectively) (Bartoli et al., 37th ICAAC)
would suggest that this compound could have significant anti-T.
cruzi activity in these animal models. Work is currently in
progress to test this hypothesis in a dog model previously described by
Lana et al. (14, 15).
| |
ACKNOWLEDGMENTS |
This work received financial support from the UNDP/World
Bank/World Health Organization Programme for Research and Training in
Tropical Diseases (grant 970297) and the Instituto Venezolano de
Investigaciones Científicas.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratorio de
Química Biológica, Centro de Biofísica y
Bioquímica, Instituto Venezolano de Investigacíones
Científicas, Apartado 21827, Caracas 1020A, Venezuela. Phone:
58-2-5041479. Fax: 58-2-5041093. E-mail:
jaurbina{at}cbb.ivic.ve.
| |
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