Download Evaluation of the efficacy of albendazole sulphoxide and

Journal of Antimicrobial Chemotherapy (2006) 57, 482–488
doi:10.1093/jac/dki484
Advance Access publication 12 January 2006
Evaluation of the efficacy of albendazole sulphoxide and praziquantel
in combination on Taenia crassiceps cysts: in vitro studies
Francisca Palomares1, Guadalupe Palencia2, Javier R. Ambrosio3,
Alma Ortiz2 and Helgi Jung-Cook1,4*
1
Laboratorio de Neuropsicofarmacologı́a, Instituto Nacional de Neurologı́a y Neurocirugı́a 14269, México D.F.,
México; 2Laboratorio de Neuroinmunologı́a, Instituto Nacional de Neurologı́a y Neurocirugı́a 14269, México D.F.,
México; 3Facultad de Medicina, UNAM, Departamento de Microbiologı́a y Parasitologı́a, México D.F., México;
4
Facultad de Quı́mica, UNAM, México D.F., México
Received 22 October 2004; returned 23 June 2005; revised 28 November 2005; accepted 2 December 2005
Objectives: Praziquantel and albendazole are currently used for chemotherapeutic treatment of
neurocysticercosis. Albendazole has been found to be more effective than praziquantel; however, it is
well known that not all patients will show a complete resolution of cysts. Searching for more
effective treatments, this study was designed to evaluate the effect of the combination of praziquantel
and albendazole sulphoxide in a Taenia crassiceps in vitro model as well as the kind of interaction between
both drugs.
Methods: In order to determine the concentration that produced 50% effect (EC50), T. crassiceps cysts
were incubated in culture medium containing praziquantel (0.005–0.04 mg/mL), albendazole sulphoxide
(0.021–0.16 mg/mL) or the combination of praziquantel and albendazole sulphoxide in a fixed-dose ratio
(1 : 1). The experimental concentration (EC50Exp) of the combination was determined from the concentration-response curve constructed from the combined drug treatment. Isobolographic analyses were used
to define the nature of the interaction between praziquantel and albendazole sulphoxide. Morphological
and ultrastuctural alterations after different treatments over the parasite tissue were observed by light
and transmission electron microscopy.
Results: The changes in ultrastructure were more marked with the praziquantel and albendazole
sulphoxide combination. Also the cysticidal effect of the combination was observed earlier than that of
each drug alone. When isobolographic analysis was employed, we found that the experimental EC50
value (0.042 mg/mL) was not significantly different from the theoretical EC50 value (0.035 mg/mL), which
indicates an additive interaction between praziquantel and albendazole sulphoxide.
Conclusions: Our study suggests that the combination of praziquantel and albendazole sulphoxide could
potentially improve the current neurocysticercosis treatment.
Keywords: neurocysticercosis, T. crassiceps, drug–drug interactions
Introduction
Neurocysticercosis is a common cause of neurological disease in
developing countries. Among the diverse clinical manifestations of
human neurocysticercosis, seizures are the most common, but other
clinical problems may occur, depending upon the localization and
viability of the parasite (Taenia solium). Although praziquantel and
albendazole are used in the treatment of this disease, albendazole
has been found to be more effective.1–3 The action of albendazole
has been shown to be due to its principal metabolite, albendazole
sulphoxide. Although both drugs are used for the treatment of
neurocysticercosis it is well known that not all patients will
show a complete resolution of cysts.2,3 Searching for more effective treatments, we considered that the combination of praziquantel
and albendazole sulphoxide could be an alternative to consider.
Although a few studies have evaluated the efficacy of the
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*Corresponding author. Instituto Nacional de Neurologı́a y Neurocirugı́a, Insurgentes Sur 3877, Col. La Fama, Tlalpan 14269, México City,
México. Tel/Fax: (52) 54-24 08 08; E-mail: [email protected]
.............................................................................................................................................................................................................................................................................................................................................................................................................................
482
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Evaluation of albendazole sulphoxide and praziquantel combination
praziquantel plus albendazole combination and the praziquantel
plus albendazole sulphoxide combination in Echinococcus granulosus and Echinococcus multilocularis,4–6 to date there is no
information about the effect of such combinations in the Taenia
species. Considering that in a previous study we found that Taenia
crassiceps could be used as a reliable model to evaluate the in vitro
effect of cestocidal drugs,7 the main objectives of this study were
to evaluate the efficacy of the praziquantel and albendazole
sulphoxide combination in the in vitro model of T. crassiceps,
to examine the ultrastructural changes in the parasitic tissue and
to evaluate the kind of interaction between praziquantel and
albendazole sulphoxide.
concentration-response curves fitted using logistic regression; we
used the SPSS software (version 9).
Drug combination study. For the evaluation of the interaction between
drugs the isobolographic method was selected.8,9 This is a convenient
tool that assumes that the combination of drugs is made from equipotent doses of the individual drugs (EC50). Therefore the concentrations
of the combination were calculated based on the EC50 values of each
individual agent (in a constant concentration ratio, 1 : 1) using the
following equations:
Total concentration of the combination (CT).
CT ¼
ðPZQ EC50 þ ABZSO EC50 Þ
¼ CPZQ þ CABZSO
n
where n is 2, 4, 8, 16, 32, etc. and CPZQ and CABZSO are
the concentrations of praziquantel and albendazole sulphoxide,
respectively.
Materials and methods
Drugs and chemicals
Praziquantel was obtained from Sigma Chemical Co. (St Louis, Mo,
USA) (assay 99%) and albendazole sulphoxide was synthesized and
kindly donated by Dr Rafael Castillo (Facultad de Quı́mica, UNAM,
México). The solvents used, dimethyl sulphoxide (DMSO; Merck,
Shuchardt, Germany; assay 99%) and ethanol (J.T. Baker, Mexico;
assay 99.7%), were of analytical reagent grade. The culture medium
used was Dulbecco’s modified minimal essential medium (SigmaAldrich Co.), supplemented with 10% fetal calf serum, 2 mM
L-glutamine, 8 mg/dL gentamicin sulphate and 200 000 IU/dL penicillin G sodium (Gibco, USA). All the assays were carried out in cell
culture flasks (Corning, USA).
Drug fraction
In order to calculate CPZQ and CABZSO in the combination, the EC50 of
each drug was transformed to fractions (XPZQ and XABZSO) using the
following equations:
XPZQ ¼
PZQ EC50
ðPZQ EC50 þ ABZSO EC50 Þ
XABZSO ¼
ðABZSO EC50 Þ
ðPZQ EC50 þ ABZSO EC50 Þ
Therefore the concentration of each drug in the combination was
calculated.
Parasites
Male BALB/c mice (2 months old) were experimentally infected with
T. crassiceps cysts (ORF strain). After 3 months of infection, mice
were killed by cervical dislocation and metacestodes were removed
from the peritoneal cavity and washed several times with sterile 0.9%
saline solution. Only those parasites exhibiting an intact bladder surface were used for the experiments. The study was approved by the
local ethics committee.
In vitro evaluations
In order to evaluate the drug interaction, two in vitro studies were
performed: a single drug exposure study and a drug combination
exposure study.
Single drug exposure study. Stock solutions of albendazole sulphoxide
and praziquantel were prepared in DMSO and ethanol, respectively. To
determine the concentration that produced 50% effect (EC50), working
solutions of praziquantel and albendazole sulphoxide were prepared in
culture medium to obtain concentrations from 0.005 to 0.04 mg/mL and
from 0.021 to 0.16 mg/mL, respectively. Cysts incubated in the culture
medium containing 0.0125% DMSO or 0.01% ethanol served as controls. Cell culture flasks were carefully filled with 5 mL of the culture
medium containing each drug, DMSO or ethanol; 25 cysts were
deposited into each flask and were incubated at 37 C with a 5%
CO2 atmosphere and 98% relative humidity. The culture medium
was changed every day. Each experiment was run in triplicate. During
incubation, the cysts were observed every day for 11 days with an
inverted light microscope (Reichert, 569) and the mortality of the cysts
was registered. The criteria to assess parasite mortality were loss of
vesicular fluid, paralysis of membrane and collapse of parasites.
The EC50 values and 95% confidence limits of praziquantel
and albendazole sulphoxide were calculated by the log
CPZQ ¼ CT * XPZQ
CABZSO ¼ CT * XABZSO
Thus the final concentrations of praziquantel and albendazole
sulphoxide in the culture medium were, respectively, 0.0004 +
0.0018, 0.0007 + 0.0036, 0.0014 + 0.0073, 0.0028 + 0.0145 and
0.0056 + 0.029 mg/mL.
Cysts incubated in the culture medium containing 0.0125% DMSO
and 0.01% ethanol served as controls. The incubation procedure and
criteria to assess parasite mortality were the same as those used in the
single drug study.
The experimental concentration (EC50Exp) of the combination and
its respective 95% confidence limits were determined from the concentration-response curve constructed from the combined drug treatment. Also, the theoretical additive concentration (EC50Add) and its
confidence limits were calculated according to Tallarida et al.9 To
visualize the type of interaction, the isobologram was constructed
using the EC50 value of praziquantel (on the x-axis) and the EC50
value of albendazole sulphoxide (on the y-axis), and the EC50Exp
and EC50Add values of the combination were included in this
isobologram.
To evaluate the difference between the experimental value
(EC50Exp) and the theoretical value, we performed a Student’s t-test
(P < 0.05). Additivity is found when the theoretical and experimental
EC50Exp values do not differ.
Evaluation by microscopy
In order to observe the effect of praziquantel 0.0056 mg/mL, albendazole sulphoxide 0.029 mg/mL, and the praziquantel 0.0056 mg/mL and
albendazole sulphoxide 0.029 mg/mL combination (which represent
the in vitro EC25 of each drug and the EC50 for the drug combination)
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Palomares et al.
over the parasite tissue, living cysts were removed on Day 11 and
were observed by microscopy. The cysts were washed with 0.9% saline
solution, fixed with 2.5% glutaraldehyde (1 h), washed in 0.1 M
phosphate buffer solution (PBS, pH 7.4), post-fixed in 0.5% osmium
tetroxide (Merck, Germany), dehydrated in series of graded ethanol
and embedded in EPON 812 resin (Merck, Germany). Afterwards,
polymerization of the resin was carried out at 60 C overnight and
then sections were cut using an ultramicrotome (Reicher, Mod.
570 0.7 TO 4.2X).
Light microscopy. Sections of 1 mm thickness were cut and recovered
over glass slides and stained with 1% Toluidine Blue and examined by
light microscopy (LM). Micrographs were taken and captured by computerized digital image (Leica DMLS, software IM 1000).
(a)
Transmission electron microscopy. Ultra-thin sections of 60 nm were
cut and recovered over covered formvar copper grids, stained with 3%
uranyl acetate/0.3% lead citrate and were examined using a transmission electron microscope (TEM) (JEOL JEM-1200 EXII) at
70 kV. Electron microscopy photographs were taken and scanned
(Scanner HP, 3300). Addition of scale bars, lettering and arrows
was performed using Adobe Photoshop version 6.0.
(b)
(c)
(e)
(f)
(g)
(h)
(d)
Results
Morphological and ultrastructural alterations
The morphological alterations on the parasites of T. crassiceps after
the different drug treatments were observed by light microscopy.
We found that the control cysts remained unaltered during the
experiment, showing the classical movements and normal appearance. The first effects of the treatment with praziquantel over the
cysts, such as size increase and spasmodic movements, were
observed after 2 days of incubation. Albendazole sulphoxide produced a slower effect than praziquantel. After 3 days the cysts
remained unaltered. With the praziquantel and albendazole sulphoxide combination the effects were seen earlier, after 1 day of
treatment; 10% of the cysts showed a size increase and after 6
days of incubation the mortality was higher than with each drug
alone. Figure 1 shows the results obtained by LM after the different
treatments. In Figure 1 (a and e) a control cyst from culture medium
after 11 days of incubation with its characteristic ruggedness and
the distribution of the microtriches in the surface of the tegument
can be seen. After the treatment with praziquantel, an increase in
the parasite size and stretching of outer and parenchymal tissues
was observed (Figure 1b). Praziquantel also induced high vacuolization of the tegument of the parasite near to the basal membrane
and the germinal layer presented alterations in its cellular components (Figure 1f). Albendazole sulphoxide treatment produced
parasite collapse, its form was altered and the content of vesicular
fluid was reduced (Figure 1c). This treatment produced different
alterations in the parasite tissue. Components such as microtriches
disappeared in some zones and the germinal layer was visibly
affected (Figure 1g). When the praziquantel and albendazole sulphoxide combination was evaluated, we found that this treatment
produced the most destructive effect in the parasite tissue
(Figure 1d). A dramatic reduction in the length of microtriches
in the tegument thickness as well as a total cellular degeneration
was observed (Figure 1h). In order to evaluate the effect of the drug
combination treatment, the length of microtriches, thickness of
tegumental tissue and vesicular tissue of cysticerci were
measured. Table 1 shows the results obtained. It can be seen
that after the combined treatment the length of microtriches
Figure 1. Light microscopy of different sections of T. crassiceps cysticerci after
11 days of in vitro treatment. A representative cysticercus after the different
treatments is presented in each panel: control (a), praziquantel (PZQ; 0.0056 mg/
mL) (b), albendazole sulphoxide (ABZSO; 0.029 mg/mL) (c), and the PZQ +
ABZSO combination (0.0056 + 0.029 mg/mL) (d). As whole parasites, a representative section of them is presented: control (e), PZQ alone (f), ABZSO alone
(g), and the drug combination (h). Differences in size and form of whole parasites,
changes in thickness and surface of the vesicular bladder wall, and components
of tegumental and germinal layer were seen. MT, microtriches; T, tegument;
GL, germinal layer. Arrows demonstrate vacuolization and damage of tissues
(f–h). Bars: 400 mm (a–d) and 5 mm (e–h).
decreased significantly with respect to the treatments with each
drug alone. The thickness of tegumental tissue was more affected
with the treatment with praziquantel, and the vesicular tissue was
affected significantly with the praziquantel and albendazole
sulphoxide combination.
TEM
Observations by TEM revealed that, after 11 days of incubation in
the control medium, the microtriches in the germinal layer and the
flame cells in the cyst were unaltered (Figure 2a and e). With
praziquantel, albendazole sulphoxide and the drug combination
we found several ultrastructural changes, such as alterations in
the protonephridial system at flame cell level. These alterations
were clearly produced in inner microtubules of cilia at the apical
portion. The cilia of central microtubules disappeared and in some
flame cells a complete disintegration of external and internal
tubules that surround the apical portions of the cells was found
(Figure 2f, g and h).
Praziquantel treatment induced an apparent fusion of the tegument and the germinal layer (Figure 2b) which was related to the
high vacuolization of tegumental tissue, previously seen by light
484
Evaluation of albendazole sulphoxide and praziquantel combination
Table 1. Effect of praziquantel (PZQ), albendazole sulphoxide (ABZSO) and the combined drug treatment in relation to the length of
microtriches and thickness of tegumental tissue and vesicular tissue of Taenia crassiceps cysticerci
Treatment
Length of microtriches (mm)
Thickness of tegumental tissue (mm)
Vesicular tissue (mm)
Control
PZQ
(0.0056 mg/mL)
ABZSO
(0.029 mg/mL)
Combination
(PZQ 0.0056 mg/mL +
ABZSO 0.029 mg/mL)
2.3 – 0.5
1.7 – 0.5
116 – 22
2.4 – 0.4
3.3 – 0.5*
73 – 9*
2.3 – 0.4
2.1 – 0.3
27 – 8*
0.7 – 0.1*
1.2 – 0.1
1.2 – 0.1**
Data are the means – SD of 10 measurements in different sections of tissue.
*Statistical difference in relation to control, P < 0.05.
**Statistical difference in relation to control, P < 0.01.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 2. Transmission electron microscopy of T. crassiceps cysticerci tissue after 11 days of in vitro treatment. Cyst control (a and e). Cysts treated with praziquantel
(PZQ; 0.0056 mg/mL) (b and f), albendazole sulphoxide (ABZSO; 0.029 mg/mL) (c and g) or the PZQ 0.0056 mg/mL + ABZSO 0.029 mg/mL combination (d and h).
Note the partial loss of the microtriches (MT), the alteration of tegumental (T) and germinal layer (GL) components of the parasite treated with the PZQ + ABZSO
combination (d) and disruptions in the organization of microtubules of cilia in flame cells (h) (arrows). Arrows in (e), (g) and (h) indicate cilia and asterisks indicate
absence of central protofilaments. Bars: 40 mm (a–g) and 50 mm (h).
microscopy (Figure 1f). With albendazole sulphoxide treatment,
the microtriches disappeared in some regions and, in addition, the
vacuolization of the tegument was observed (Figure 2c). With the
praziquantel and albendazole sulphoxide combination the effect
was more pronounced than with praziquantel or albendazole sulphoxide alone. In addition, important alterations of the inner germinal
layer, including disappearance of cellular integrity, destruction of
the longitudinal and circular muscle systems with disruption of the
muscular cells at their myofibril portion, as well as release of
myofilaments, were observed. Also the striated fibres that support
the anchorage to the cytoplasm and their nucleus were lost
(Figure 2d).
Pharmacological effects
Figure 3 shows the mortality of T. crassiceps cysts after exposure to
the praziquantel and albendazole sulphoxide combination as well
as to praziquantel and albendazole sulphoxide alone at the same
concentrations used in the combination. Figure 4 shows the
concentration-response curves for praziquantel, albendazole sulphoxide and the praziquantel and albendazole sulphoxide combination. The EC50 values obtained for praziquantel, albendazole
sulphoxide and the combination were 0.0112, 0.058 and 0.043
mg/mL, respectively. The isobologram at EC50 shown in Figure 5
revealed there were no significant differences between the EC50Exp
485
Palomares et al.
0.08
Albendazole sulphoxide (µg/mL)
50
Mortality (%)
40
30
20
10
0
0
2
4
6
Time (days)
8
10
0.06
0.04
0.02
0.00
0.00
12
0.004
0.008
0.012
0.016
Praziquantel (µg/mL)
Figure 3. Mortality of T. crassiceps cysts after in vitro exposure to praziquantel
(PZQ, 0.0056 mg/mL) (open squares), albendazole sulphoxide (ABZSO,
0.029 mg/mL) (filled diamonds) and the combination of PZQ (0.0056 mg/mL)
plus ABZSO (0.029 mg/mL) (filled triangles). Each point represents the mean
percentage of live parasites from three different experiments – SD.
Figure 5. Isobologram for the interaction between praziquantel and albendazole
sulphoxide against T. crassiceps cysts. The straight line that connects the experimental EC50 points for the single drug treatments (filled squares) is the theoretical
additive line, with the theoretical additive EC50Add (filled circle) and the experimental EC50Exp (open circle) for the combination. Each point on the graph
represents the mean – SEM of three experiments.
100
Mortality (%)
80
60
40
20
0
–3.0
–2.5
–1.5
–2.0
LogConc (µg/mL)
–1.0
–0.5
Figure 4. Concentration-response curves of praziquantel (PZQ, filled squares),
albendazole sulphoxide (ABZSO, filled diamonds) and the PZQ + ABZSO
combination (open triangles). Each point represents the mean – SD of three
experiments.
value (0.043 mg/mL) and the theoretical EC50Add value
(0.035 mg/mL) (P < 0.05), which indicates that the effect of the
combination is additive.
Discussion
Although the effectiveness of praziquantel plus albendazole and
praziquantel plus albendazole sulphoxide combinations has been
demonstrated in E. granulosus and E. multilocularis metacestodes,5,6,10,11 this is the first study describing the in vitro efficacy
of the praziquantel and albendazole sulphoxide combination in
Taenia metacestodes. Also, this is the first report that evaluates
the kind of interaction between both drugs in vitro.
The differences in the mode of action of praziquantel and
albendazole sulphoxide on T. crassiceps cysts at the ultrastructural
level are shown in Figure 2. The effect of praziquantel was detected
in the inner region of the germinal layer where large vacuoles
appeared in the distal cytoplasm and in some areas a loss of
the tissue integrity was observed. This effect could be considered
as an indirect effect of the general metabolic disruption of the
cysts.12,13 Praziquantel treatment also produced alterations in
the contractility of the parasites. These changes were associated
with damage of the muscular fibres. Our results are in accordance
with a previous in vitro study on T. solium cysts.12 Praziquantel did
not show any effect on microtriches, which remained unaltered.
These observations are in accordance with those found in E. granulosus.13 On the other hand, albendazole sulphoxide induced several
changes in the morphology of the parasite as well as a partial loss of
the intracellular tissue, which is associated with the mechanism of
action of benzimidazoles, since the benzimidazole carbamates destroy the microtubules and interfere with cyst nutrition.14–17 The
activity of flame cells can be considered a good parameter for
evaluation of vitality.18 After the treatment with albendazole sulphoxide, we observed that the flame cells presented severe morphological damage. The disruption of the flame cells reflects the
impaired excretory functions due to the damage in the microtubule
structures, localized at cilia.16–18 It is well known that the primary
mode of action of benzimidazoles is via inhibition of b-tubulin.16 In
our study it was possible to observe that albendazole sulphoxide
produced an effect over the central protofilaments (in the flame
cells), which disappeared completely. This effect reflected the
damage to the tubulin components. Lacey18 demonstrated that
albendazole is effective at destroying the specific organization
of protofilaments and it is possible that the action of albendazole
sulphoxide is also directed towards a specific isoform of tubulin.
Some studies14,15,19 have evaluated the effect of albendazole sulphoxide on the microtriches, showing a partial loss in some sites of the
distal cytoplasm. In our study we found that the number of microtriches decreased considerably; however, the length of the remaining microtriches was not affected.
The microscopic observations after treatment with the drug
combination showed clear changes in parasite tissue, therefore
we decided to quantify these changes by performing measurements
of the length of microtriches, thickness of the tegumental tissue and
486
Evaluation of albendazole sulphoxide and praziquantel combination
the vesicular bladder (Table 1). A dramatic reduction in the length
of microtriches (threefold with respect to the control) was found
only after the drug combination treatment. Currently there is no
explanation for these findings; however, it is possible that the
parasite reacted against the combined treatment by reducing
their absorbing and desorbing surface. Other studies have to be
performed in order to explain this effect. The increase in the thickness of the tegumental tissue could be due to the vacuolation of the
tegument, which is related to the specific action of each drug. The
primary effect of praziquantel is to induce changes in intracellular
calcium with subsequent vacuolization of the tegument and
increased tegumental tissue thickness.5,12 Meanwhile, albendazole
sulphoxide interacts with b-tubulin affecting the cytoskeletal structure required for maintaining the dynamics of vesicular traffic, and
this action produces a vacuolation in the tegumental layer.14 In the
case of the praziquantel and albendazole sulphoxide combination
treatment, the thickness of the tegumental tissue was similar to that
obtained in the control group; however, a marked increase in damage at the ultrastructural level was observed. Figure 2(d) shows
clear damage of the tegumental tissue; the germinal layer appeared
fused to a disrupted tegumental basal membrane as seen under
unravelling myofibrils in apparently open pockets. This tissue
destruction, at the level of tegumental and germinal layers, is
extended to the more profound tissue layer where flame cells
are localized as seen in Figure 2(h). Owing to this damage no
vacuoles and lipid droplets were present, which could explain
the fact that the thickness of the tegumental tissue remained
unaltered.
Our results also showed that the drug treatment induced a dramatic reduction in the vesicular tissue in contrast to the effect of
each drug alone (Table 1). The marked damage in the vesicular
tissue (Figure 1h and Figure 2d) could be due to the association of
the effect of the drugs. It has been established that both drugs could
produce, depending on the concentration and the exposure time,
common effects over the cysts as alterations in their energy metabolism related to glucose absorption, glycogen depletion and morphological changes as vacuolization of the tegument, impairment
of the microtriches and, finally, destruction of the parasitic tissues.12,15 These alterations, plus the specific action of each drug
(praziquantel changes metabolism and intracellular calcium with
the main effect of inhibition of muscular movements12 and albendazole sulphoxide affects the dynamics of vesicular traffic16), could
explain why the combination treatment induced a more serious and
irreversible damage of the parasite tissue.
When the efficacy results were evaluated, we found that the
combination was more effective than when the drugs were used
separately (Figure 3). The time factor may sometimes be important
in order to reduce the duration of the treatment. In our study, the
effect of the combination was observed in a shorter time than with
albendazole sulphoxide treatment. In this way the praziquantel and
albendazole sulphoxide combination (0.0056 + 0.029 mg/mL)
required 1 day to produce 10% mortality, while albendazole sulphoxide (0.029 mg/mL) and praziquantel (0.0056 mg/mL) required 6
days to show the same mortality. Similar results were found with
the other concentrations. Our results agree with other studies where
the praziquantel and albendazole combination showed a higher
efficacy in a shorter time than with each drug alone.6,10,11 This
rapid effect could be due to the presence of praziquantel in the
combination, since the efficacy of benzimidazole carbamates has
been demonstrated to be slow.6,7,13 These results show that the
effect of the combination is time-dependent. Figure 4 shows the
concentration-response curve obtained after the praziquantel and
albendazole sulphoxide combination treatment. It can be seen that
the effect of the combination is also concentration-dependent. In
a previous study in E. granulosus, Urrea-Parı́s et al.11 found that the
praziquantel and albendazole combination was time-dependent,
but not concentration-dependent. The differences between the
findings could be due to the methodology used; we performed a
dose–response curve and Urrea-Parı́s et al. used only two concentrations to evaluate the praziquantel and albendazole combination
(0.1 mg/mL praziquantel + 10 mg/mL albendazole and 1 mg/mL
praziquantel + 10 mg/mL albendazole).
Although it has been shown that the efficacy of the praziquantel
and albendazole combination or the praziquantel and albendazole
sulphoxide combination was greater than that obtained with each
drug alone,6,10,11 to date there is no information available about the
kind of interaction between praziquantel and albendazole sulphoxide. The present study was also focused to evaluate the nature of
the interaction between both drugs. Conceptually, an additive
effect refers to the interaction between two drugs such that,
when they are co-administered, the resultant effect approaches
the maximum effect of the sum of the effects of the two drugs
administered individually.8,9,20 Synergy describes the interaction
between two drugs such that, when given concurrently, the resultant efficacy or potency supports a greater-than-additive or multiplicative interaction compared with each drug administered
individually.9 In the present study we used the isobolographic
method to evaluate the interaction. This analysis provides more
complete information, which is very useful when the mechanism of
action of the drugs in the combination is different.20 By using a
fixed-ratio strategy, isobolographic analysis showed an additive
interaction between praziquantel and albendazole sulphoxide.
The mechanism underlying the additive effect between praziquantel and albendazole sulphoxide is not clear.
Treatment of neurocysticercosis remains a difficult clinical
problem. Our data suggest that the combination of praziquantel
and albendazole sulphoxide could be an alternative in the treatment
of neurocysticercosis. Considering that the toxicity of benzimidazole carbamates may be dose limiting, the increased efficacy
of the combination may allow a reduction in the dose and time of
treatment. Taking into account that this study was performed
in vitro, other studies should be performed in order to establish
the in vivo response of the combination.
Acknowledgements
We thank Dr Lilia Roberts (Facultad de Medicina de la UNAM,
México) for her excellent technical assistance in the electron
microscopy and Hector I. Rocha-González (Departamento de
Farmacologı́a, CINVESTAV-IPN, México) for his statistical
assistance. Dr Javier R. Ambrosio thanks the support of
DGAPA-UNAM IN201003.
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