In Vitro Effects of Albendazole Sulfoxide and Praziquantel against Taenia
solium and Taenia crassiceps Cysts
Francisca Palomares, Guadalupe Palencia, Rodolfo Pérez, Dinora González-Esquivel, Nelly Castro, and Helgi Jung Cook, 作者单位？Laboratorio de
Neuropsicofarmacología, 1 Laboratorio de Neuroinmunología, Instituto Nacional de Neurología y Neurocirugía 1426 2 Facultad de Química, UNAM, México,
Several studies ( 3, 8 ) have evaluated the effects of praziquantel (PZQ) and albendazole sulfoxide (ABZSO) against different species
of metacestodes, but this study is the first to evaluate the minimum exposure times required for activity and the effective concentrations
of PZQ and ABZSO against Taenia solium and Taenia crassiceps cysts.
T. crassiceps cysts (ORF strain) were obtained from experimentally infected male BALB/c mice (age, 2 months). The mice were killed
by cervical dislocation, and the metacestodes were removed from the peritoneal cavity. T. solium cysts were obtained from the skeletal
muscle of naturally infected pigs. The parasites were washed with sterile 0.9% saline solution, and only those which exhibited an intact
bladder surface were used for the experiments.
Stock solutions of ABZSO and PZQ were prepared in dimethyl sulfoxide (DMSO) and ethanol, respectively. Working solutions of the
drugs were prepared with culture medium to obtain PZQ concentrations from 0.001 to 0.3 µg/ml and ABZSO concentrations from 0.0125 to 2.5 µg/ml. Culture media containing 0.25% DMSO or 0.05% ethanol were prepared as controls. Each cell culture flask contained 25 cysts in 5 ml of culture medium and was incubated at 37?C with 5% CO 2. The parasites were observed with an inverted
light microscope (Reichert 569). The criteria used to assess cyst mortality in vitro were total paralysis of the membrane and cyst
collapse. In order to determine the exposure time required for activity for PZQ, the cysts were observed each 15 min for 6 h; after this
time, observations were made each 24 h for 11 days. For ABZSO, the parasites were observed each 24 h for 11 days. The culture medium was changed every day. Each experiment was performed in quadruplicate.
The effective concentrations that killed 50 and 99% of the parasites (EC 50 s and EC 99 s, respectively) were determined at 4 and 6 h
and 11 days for PZQ and at 11 days for ABZSO. Each experiment was performed in triplicate. Data were analyzed by logistic regression by using SPSS software (version 9.0).
When the cysts were exposed to the drugs, an immediate contraction of the membrane was found; extensive vacuolization of the teguments and evagination were also observed.
Figure 1A shows the loss of the cystic fluid of T. solium cysts after treatment with PZQ, and Fig. 1B shows the loss of the integrity of the
membrane when the T. crassiceps cysts were exposed to the drugs.
FIG. 1. (A) T. solium cysts: control cysts (left) and dead cysts after treatment with PZQ (right). Note the loss of cystic fluid and that only
the scolex is visible after the drug treatment. (B) T. crassiceps cysts: control live cysts (left) and cysts after treatment with the drugs
(right). Note the size reduction and the loss of cystic fluid in the parasites.
The maximum effects on T. crassiceps and T. solium cysts were observed after 4 and 6 h, respectively, when the concentrations of
PZQ were in the range of 0.012 to 0.1 µg/ml. When the effects of PZQ against T. crassiceps cysts were evaluated at lower
concentrations (0.001 to 0.04 µg/ml), we found that the maximum effect was obtained after 9 days of exposure. These results show that
the effects of PZQ on T. solium and T. crassiceps cysts are time and concentration dependent. These results are in accordance with
those presented in previous reports of studies with other metacestodes ( 2, 3, 4, 8 ).
Data on the mortality of T. solium cysts were plotted against data on the mortality of T. crasssiceps cysts after PZQ treatment. A linear
relationship with a slope of 1.36 was found. These results indicate that T. crassiceps could be used as a reliable model to evaluate the
in vitro effects of cestocidal drugs.
When the effect of ABZSO against T. solium cysts was evaluated, we found that after 5 days of incubation, 50% of the cysts evaginated
and transformed to young Taenia, which survived for only 24 h. These changes were also observed in control cultures; therefore, we
could perform the study only with the T. crassiceps model. With this model the effect of ABZSO was slower than that of PZQ, and the
maximum effect was attained at 11 days of exposure. The lag time in the effect could be due to the mechanism of action of ABZSO,
since the efficacies of benzimidazole carbamates have been demonstrated to be slow ( 5, 8 ).
The results of the present study show that the effect of ABZSO, like that of PZQ, is time and concentration dependent; however, longer
incubation periods in vitro are required with ABZSO to attain the maximum effect. Our observations agree with those found for other
metacestodes species ( 1, 6, 7 ).
In order to calculate the EC 50 s and EC 99 s of PZQ and ABZSO, the maximal rate of mortality of Taenia cysts was plotted against
different concentrations of each drug (Fig. 2 ). The results are given in Table 1. We found that the EC 50 s of PZQ for T. crassiceps
decreased from 0.023 to 0.011 µg/ml when the exposure time was increased from 4 h to 11 days. The EC 50 of PZQ was slightly higher
for T. solium cysts (0.034 µg/ml).
FIG. 2. In vitro efficacies of PZQ and ABZSO. Each point represents the mean ? standard deviation percentage of dead cysts from
three different experiments., PZQ against T. solium cysts at 6 h;, PZQ against T. crassiceps cysts at 4 h;, PZQ against T. crassiceps
cysts at 11 days;, ABZSO against T. crassiceps at 11 days.
TABLE 1. In vitro effective concentrations of PZQ and ABZSO against T. crassiceps and T. solium cysts
The mean EC 99 s of PZQ for T. crassiceps cysts at 4 h and 11 days were similar (0.113 and 0.105 µg/ml, respectively), which
indicates that at concentrations higher than 0.1 µg/ml the effect of PZQ could be independent of the incubation time. The mean EC 99
of PZQ for T. solium cysts was 0.537 µg/ml, which is five times higher than that obtained for T. crassiceps cysts (0.113 µg/ml). The EC 50 s and EC 99 s of ABZSO for T. crassiceps cysts at 11 days of exposure were 0.068 and 0.556 µg/ml, respectively, showing that
PZQ is more potent than ABZSO.
The results of the present study indicate that the time of exposure and the effective concentrations are important for destruction of the parasite and could provide the basis for the establishment of an effective therapy for the treatment of neurocysticercosis. Also, in vivo studies are required in order to relate our in vitro data to the concentrations of the drugs effective clinically.
We thank Alma Rosa Cortés for statistical assistance.
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