PROADIFEN AND TRYPANOSOMA CRUZI
TRYPANOCIDAL EFFECT OF SKF525A, PROADIFEN, ON DIFFERENT
DEVELOPMENTAL FORMS OF TRYPANOSOMA CRUZI.*
BERTA M. FRANKE DE
CAZZULO1, ADRIANA BERNACCHI2, MONICA I. ESTEVA3, ANDRES M. RUIZ3, JOSE
A. CASTRO2, JUAN J. CAZZULO1
1 Instituto de
Investigaciones Biotecnológicas, Universidad Nacional de General San
Martín, San Martín, e Instituto de Invstigaciones Bioquímicas
Fundación Campomar; 2 Centro de Investigaciones Toxicológicas
CITEFA/CONICET, Villa Martelli; 3 Instituto Nacional de Parasitología
Dr. Mario Fatala Chabén, Ministerio de Salud y Acción Social, Buenos
*Presented at XII Annual Meeting of the Brazilian Society of
Protozoology y XXIII Annual Meeting of Basic Research in Chagas’
Disease, Caxambu, Brasil, 1996.
Key words: Trypanosoma cruzi, SKF525A, growth inhibition,
blood bank sterilization
an inhibitor and inducer of cytochrome P450, was tested on different
developmental stages of Trypanosoma cruzi. Growth, motility and
structure of epimastigotes, motility and infectivity of
trypomastigotes, and infectivity of trypomastigotes to Vero cells in
culture were abolished by the drug at 10-100 µM concentrations. When
blood from infected mice was treated with the drug, and used to infect
8 day-old mice, no parasites were observed at 0.6-1 mM, and all
animals survived. Blood cell morphology was well preserved, and the
sleeping time of pentobarbital-treated mice inoculated with the same
amount of drug was not increased. The present results suggest that
SKF525A or other related inhibitors of cytochrome P450 coned be tested
as an additive for blood sterilization in blood banks.
tripanocida de SKF 525A, proadifen, sobre diferentes estadios del
desarrollo del Try- panosoma cruzi. El crecimiento, la motilidad y la
estructura de epimastigotes, la motilidad y la infectividad de
tripomastigotes y la infectividad de tripomastigotes sobre células
Vero en cultivo fueron suprimidas totalmente con concentraciones de la
droga entre 10 y 100 µM. Cuando sangre de ratón infectado se trató
con la droga, y luego se la utilizó para infectar ratones de 8 días
de edad, no se observaron parásitos entre 0.6 y 1 mM, y todos los
animales sobrevivieron. La morfología de las células sanguíneas se
preservó y el tiempo de sueño de los ratones tratados con
pentobarbital e inoculados con la misma cantidad de droga no se vio
aumentado. Los presentes resultados sugieren que el SKF 525A u otras
drogas relacionadas inhibidores del P450 podrían probarse como
aditivos en la esterilización en bancos de sangre.
Postal address: Dr. Juan J Cazzulo, Instituto de
Investigaciones Biotecnológicas, Universidad Nacional de General San
Martín, Casilla de Correo 30, 1650 San Martín, Prov. de Buenos
Aires, Argentina. Fax: 54-1-752-9639; E-mail: email@example.com
Received: 31-III-1998 Accepted: 22-VI-1998
A large number of drugs have been tested against Trypanosoma cruzi,
the parasitic flagellate which causes the American Trypanosomiasis,
Chagas disease. Among these, there are antibiotics, amphiphilic drugs,
azole derivatives, nitroheterocyclics, purine derivatives,
naphthoquinones, metallic complexes, antioxidants, cysteine proteinase
and tripanothione reductase inhibitors1-4. New drugs are necessary,
not only for the treatment of chagasic patients, but for the treatment
of blood to prevent transfusional transmission of the disease. The
drugs available (Nifurtimox and Benznida-zole for treatment, Gentian
Violet for transfusion) present a number of undesirable properties5,
6. It is necessary, therefore, to search for new drugs, more effective
and less toxic than those already available.
The aim of this study is to determine the potential trypanocidal
effects of b-diethylaminoethyl-diphenyl-propyl acetate hydrochloride
(SKF525A or PROADIFEN). This drug interacts with the parasite’s
cytochrome P450 (as azole derivatives, such as ketoconazole and
fluoconazole)7-9 and shares other properties with drugs proven to be
active against the parasite3, since it has an amphiphilic structure4,
10 and presents antioxidant properties11.
Materials and Methods
Obtention of parasites. Epimastigotes (Tul2 or RA strains) were
grown and harvested as previously described12. Cell-culture
trypomastigotes (RA strain) were obtained from infected Vero cells13.
Blood samples containing bloodstream trypomas-tigotes (Tulahuén
strain, Tul 2 stock) were obtained by cardiac puncture from infected
BALB/c mice. Blood samples from inbred CF-1 male mice were used to
simulate blood bank conditions by adding cell-culture trypomastigotes,
and for control determinations of the effect of SKF525A on
pentobarbital sleeping time.
Assay of inhibition of motility. Epimastigotes and culture
trypomastigotes (in both cases at a concentration of 5 x 106
parasites/ml), were suspended in fresh culture medium containing the
drug concentrations stated. The suspensions were observed under the
microscope, and the time for complete immobilization was recorded.
Assay of inhibition of epimastigote growth. Parasites were grown at
28° C, in the absence or in the presence of the drug concentrations
stated in Fig. 1, and growth was followed by daily counting using a
Assay of the effect of SKF525A on the parasite cycle in Vero cells.
Vero cells (4.4 x 104/ml) were cultured at 37°C in Modified Eagle’s
Medium (MEM) containing 5% (v/v) fetal calf serum, in 24-well plate
dishes containing glass coverslips. After 48 hr the cultures were
inoculated with RA strain cell-culture trypo-mastigotes (3.5 x
105/ml), with or without preincubation (4 hr at 37°C without drug or
with 10 µM SKF525A). After 24 hr the medium, containing the
non-internalized parasites, was removed; fresh medium, with or without
drug, was added, and the infected cells were incubated for 72 hr, and
stained with May-Grünwald-Giemsa. The percentage of infected cells
and the number of intracellular parasites were estimated by observing
500 cells in a Zeiss Photomicroscope II. The results are expressed as
the endocytic index (product of the % of cells infected and the number
Assay of the effect of SKF525A on RA strain trypomas-tigotes suspended
in mouse blood. Vero cell-derived trypo-mastigotes suspended in MEM
containing 5% fetal calf serum were added to mouse blood, at a final
concentration of 5 x 105 trypomastigotes/ml. Aliquots (100 µl) were
sampled in microwell plates and added SKF525A at the final
concentrations stated under Results. After 24 hr at 4°C, the
parasites in 5 µl aliquots were counted under the microscope.
Aliquots were diluted with the same medium to a final concentration of
9.0 x 103 trypo-mastigotes/ml, and used to infect Vero cell cultures.
Culture medium and non-internalized parasites were removed after 24
hr, fresh medium was added, and the flasks were incubated at 37°C for
up to 20 days, with periodic changes of medium. The number of released
parasites was counted daily until the end of the experiment.
Assay of the effect of SKF525A on Tul 2 strain bloodstream
trypomastigotes in mouse blood. The incubation of the parasites was
performed as in the previous experiment, but the samples, after
counting the trypomastigotes under the microscope, were inoculated
into 8-days old mice. Groups of 5 male BALB/c mice from the same
litter were used for each drug concentration. Parasitemia (determined
in blood taken from the tail) and survival were followed up to 30
days. The animals surviving after 30 days were sacrificed, and
homogenates of heart or liver, and blood, were inoculated into 8
Assay of the pentobarbital sleeping time in CF-I mice treated with
SKF525A suspended in blood. Sodium pentobarbital (40 mg/kg in 0.9%
NaCI) was injected i.p. When the animals (5 male mice per group) got
asleep, blood treated with or without 0.5 mM SKF525A for 24 hr at 4°C
(0.1 ml/20 g body weight) was given i.v., and the sleeping time was
Electron microscopy. Epimastigotes were fixed by suspen-sion in 2.5%
glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 60 min. at room
temperature. Afterwards samples were treated as described14, and thin
section were observed in a Philips EM 300 electron microscope.
Chemicals. SKF525A was obtained from Smith, Kline and French Labs,
Philadelphia, PA, U.S.A. All other chemicals used were analytical
reagents of the highest purity available.
Results and discussion
Incubation of epimastigotes (Tul 2 or RA strains) or cell-culture
trypomastigotes (RA strain) with 0.1 mM SKF525A led to their complete
immobilization in 360, 160 or 18 min., respectively. The presence of
the drug during axenic culture of Tul 2 epimastigotes led to complete
inhibition of growth and progressive parasite lysis even at a
concentration as low as 50 mM SKF525A (Fig. 1). Incubation of the
epimastigotes with the drug resulted in progressive damage to cellular
structure (Fig. 2). After 6 hr the cells looked almost like empty
bags, with damage to the plasma membrane. The kinetoplast and the
subpellicular microtubules were relatively little affected. This
suggests that, at variance with other amphiphilic drugs, like
trifluoperazine14, the mitochondrion is not the primary target of
When the drug was tested on the parasite cycle in cultured Vero cells
(Table 1) the presence of SKF525A during infection and subsequent
culture caused a marked decrease in the endocytic index in a
concentration dependent manner. Considerable decrease was also
observed when the drug was present only during infection, or when the
trypomastigotes were preincubated with 10 µM SKF525A. On the other
hand, when the drug was added 24 hr after infection, while changing
the culture medium, there was little effect on the endocytic index.
These results suggest that the effect of SKF525A is primarily on the
infecting trypomastigotes, and that the drug is not able to affect the
intracellular parasite growth.
Aiming at a possible application of SKF525A or related drugs for the
sterilization of banked blood samples, two different experiments were
performed. First, cell-culture trypomastigotes (RA strain) were
suspended in mouse blood, incubated for 24 hr at 4°C with variable
concentrations of drug, the surviving parasites were counted in a
Neubauer chamber, and the blood was used to infect Vero cell cultures,
as described under Methods. The control without drug contained 1.68 x
105 parasites/ml, and the Vero cells inoculated with this sample
released trypomastigotes into the medium after 6 days in culture.
Parasite numbers were reduced to 0.5 x 10.55 parasites/ml in the
presence of 0.1 mM SKF525A, and to 0 at concentrations of 0.2 mM or
higher. At 0.2 mM, very few trypomastigotes were released after 12
days; at 0.3 mM only after 20 days, and at drug concentrations of 0.4
mM or higher no parasites were observed in the cell cultures.
Table 2 shows the results of a typical experiment in which blood from
mice infected with the Tul 2 strain was incubated for 24 hr at 4°C
with increasing concentrations of the drug, and then inoculated into
8-days old BALB/c mice, five per drug concentration. Control mice
showed the parasitemia peak at 10 days p.i., and all died at day 12
p.i. Concentrations of SKF525A from 0.3 to 0.5 mM increased in a
dose-dependent manner the time required for the parasitemia peak and
100% mortality, and finally 0.6-1.0 mM SKF525A completely prevented
the development of patent parasitemia, all the animals surviving for
up to 30 days. The survivors were sacrificed, and homogenates of heart
or liver, and blood, were inoculated into 8 days-old mice, which
showed no parasitemia up to 60 days. SKF525A concentrations up to 1 mM
did not damage the red blood cells (the cells looked indeed better
than the controls), and caused no apparent damage to mice injected
with non-infected blood containing the same drug concentrations. Since
the drug is a well-known inhibitor of mammalian cytochrome P45015, it
was necessary to test the possibility that SKF525A concentrations
carried over with the injected blood might have deleterious effects,
for instance increasing the effects of anesthetics. However, the
pentobarbital sleeping time recorded for the control animals was 36 ±
19 min., and that for the animals inoculated with 0.1 ml/20 g of body
weight of 0.5 mM SKF525A in mouse blood was 35 ± 28 min., without
significant difference (P = 0.91).
The results presented herein suggest that some inhibitors of the
cytochrome P450 might be tested as potentially useful additives for
their use in blood banks.
Acknowledgements. AB, AMR, JAC and JJC are members of the
Research Career and BMFC of the Technical Career of CONICET (Consejo
Nacional de Investigaciones Científicas y Técnicas). This work was
aided by grants from CONICET, Ministerio de Salud y Acción Social
(Argentina) and SAREC (Sweden).
1. Schirmer RH, Muller JG, Krauth-Siegel RL. Disulfide reductase
inhibitors as chemotherapeutic agents: The design of drugs for
Trypanosomiasis and Malaria. Angew Chem Int Ed Engl 1995; 34: 141-54.
2. Docampo R, Moreno SNJ. Biochemical toxicology and antiparasitic
compounds used in the chemoprophylaxis of American Trypanosomiasis
(Chagas Disease). Rev Biochem Toxicol 1985; 7: 159-204.
3. De Castro SL. The challenge of Chagas disease chemo-therapy: An
update of drugs assayed against Trypanosoma cruzi. Acta Tropica 1993;
4. Croft SL, Walker JJ, Gutteridge WE. Screening of drugs for rapid
activity against Trypanosoma cruzi trypo-mastigotes in vivo. Trop Med
Parasitol 1988; 39: 145-8.
5. Docampo R, Moreno SNJ. The metabolism and mode of action of Gentian
Violet. Drug Met Rev 1990; 22: 161-78.
6. Castro JA, Toranzo EGD de. Toxic effects of Nifurtimox and
Benznidazole. Two drugs used against American Trypanosomiasis. Biomed
Environ Sci 1988; 1: 119-33.
7. Agosin M, Náquira C, Capdevila J, Paulin J. Hemoproteins in
Trypanosoma cruzi with emphasis on microsomal pigments. Int J Biochem
1976; 7: 585-91.
8. Berger BJ, Fairlamb AH. Cytochrome P450 in trypanoso-matids.
Biochem Pharmacol 1993; 46. 149-57.
9. Agosin M, Náquira C, Paulin J, Capdevila J. Cytochrome P450 and
drug metabolism in Trypanosoma cruzi: Effects of phenobarbital.
Science 1976; 194: 195-7.
10. Lee IP, Yamamura HI, Dixon RL. The effects of
b-diethy-laminoethyl-diphenylpropyl acetate (SKF525A) on biolo-gical
membranes. Biochem Pharmacol 1968; 17: 1671-81.
11. Castro JA, Cignoli EV. Effect of inhibitors of drug meta-bolizing
enzymes on carbon tetrachloride hepatotoxicity. Toxicol Appl Pharmacol
1971; 18: 625-37.
12. Cazzulo JJ, Franke de Cazzulo BM, Engel JC, Cannata JJB. End
products and enzyme levels of aerobic glucose fermentation in
trypanosomatids. Mol Biochem Parasitol 1985; 16: 329-43.
13. Franke de Cazzulo BM, Martínez J, North MJ, Coombs G, Cazzulo JJ.
Effects of proteinase inhibitors on the growth and differentiation of
Trypanosoma cruzi. FEMS Microbiol Lett 1994; 124: 81-6.
14. Lacuara JL, Barioglio SR, Oliva PP, Bernacchi AS, Castro JA,
Franke de Cazzulo BM, et al. Disruption of mitochondrial function as
the basis of the trypanocidal effect of trifluoroperazine on
Trypanosoma cruzi. Expe-rientia 1991; 47: 612-6.
15. Castro JA, Sasame HA, Sussman H, Gillette JT. Diverse effects of
SKF525A and antioxidants on carbon tetra-chloride-induced changes in
liver microsomal P450 content and ethylmorphine metabolism. Life Sci
1968; 7: 129-36.
TABLE 1.– Effect of SKF525A on the parasite cycle in Vero cells.
The experiment was performed as described under Materials and Methods.
a) Drug present during infection and subsequent culture; b) Drug
present only during infection; c) Drug added 24 hr after infection; d)
Trypomastigotes pre-incubated for 4 hr with 10 µM SKF525A before
infection, which was performed at the drug concentrations stated in
Treatment Infected cells Amastigotes/cell Endocytic index
Control 11.9 ± 1.3 16.4 ± 14.3 196
5 µM SKF525A
a 5.6 ± 1.1 8.8 ± 10.4 49
b 6.3 ± 0.02 10.1 ± 9.2 64
c 13.8 ± 1.9 11.1 ± 10.6 153
d 6.3 ± 0.09 8.8 ± 9.9 55
10 µM SKF525A
a 2.6 ± 0.2 5.3 ± 4.9 14
b 5.1 ± 0.1 10.7 ± 9.0 54
c 13.6 ± 1.6 10.8 ± 9.6 147
d 1.1 ± 0.36 5.1 ± 3.6 6
TABLE 2.– Effect of SKF525A on Tul 2 strain bloodstream
trypomastigotes in mouse blood. The experiment was performed as
described under Materials and Methods. The results are given as
average of 5 determinations ± SE
Concentration of Parasitemia (tryps/ml x 104) at day Mortality (at
SKF525A (mM) p.i. p.i.)
6 10 13 17 20 25
0 7.67 ± 0.17 258 ± 10.5 - - - - 100% (12)
0.3 0 26.3 ± 9.9 147.5 ± 57.9 - - - 100% (16)
0.4 0 6 ± 5.9 76.2 ± 45.5 215 - - 100% (18)
0.5 0 3.2 ± 1.8 23 ± 10 198 ± 69.6 - - 100% (19)
0.6 0 0 0 0 0 0 0%
0.8 0 0 0 0 0 0 0%
1.0 0 0 0 0 0 0 0%
Fig. 1.– Effect of SKF525A on growth of epimastigotes, Tul2
stock, in axenic culture. The drug concentrations used were 0 (l), 25
(¡), 50 (D), 75 (s) and 100 (n) µM.
Fig. 2.– Effect of SKF525A on the ultrastructure of Trypanosoma
cruzi epimastigotes. The epimastigotes were incubated at 28°C in the
absence (1) or in the presence of 0.1 mM SKF525A for 164 min, (2) or
360 min, (3) Samples were processed for electron microscopy as
described under Materials and Methods 1, x 14.000; 2, x 36.500; 3, x