* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Evaluation of the efficacy of albendazole sulphoxide and
Survey
Document related concepts
Neuropsychopharmacology wikipedia , lookup
Drug design wikipedia , lookup
Drug discovery wikipedia , lookup
Psychopharmacology wikipedia , lookup
Prescription drug prices in the United States wikipedia , lookup
Polysubstance dependence wikipedia , lookup
Pharmacogenomics wikipedia , lookup
Neuropharmacology wikipedia , lookup
Prescription costs wikipedia , lookup
Pharmaceutical industry wikipedia , lookup
Pharmacokinetics wikipedia , lookup
Transcript
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 ............................................................................................................................................................................................................................................................................................................................................................................................................................. *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 The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected] 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) 483 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. Transparency declarations No declarations were made by the authors of this paper. References 1. Del Bruto O, Sotelo J, Román G. Neurocysticercosis: A Clinical Handbook. The Netherlands Swets & Zeitlinger: Lisse, 1998. 487 Palomares et al. 2. Garcı́a H, Evans C, Nash T et al. Current consensus guidelines for treatment of neurocysticercosis. Clin Microbiol Rev 2002; 15: 747–56. 3. Sotelo J, Del Bruto O. Review of Neurocysticercosis. Neurosurg Focus 2002; 12: 1–7. 4. Casado N, Urrea-Parı́s M, Moreno M et al. Combined and albendazole chemoprophylaxis in experimental hydatidosis. Parasitol Res 2001; 87: 787–9. 5. Moreno M, Urrea-Parı́s M, Casado N et al. Praziquantel and albendazole in the combined treatment of experimental hydatid disease. Parasitol Res 2001; 87: 235–8. 6. Taylor D, Morris D. In vitro culture of Echinococcus multilocularis: protoscolicidal action of praziquantel and albendazole sulphoxide. Trans R Soc Trop Med Hyg 1988; 82: 265–7. 7. Palomares F, Palencia G, Pérez R et al. In vitro effects albendazole sulfoxide and praziquantel against Taenia solium and Taenia crassiceps cysts. Antimicrob Agents Chemother 2004; 48: 2302–4. 8. Tallarida R, Porreca F, Cowan A. Statistical analysis of drug-drug and site-site interactions with isobolograms. Life Sci 1989; 45: 947–61. 9. Tallarida R. Drug Synergism and Dose-Effect Data Analysis. Boca Ratón, FL: Chapman Hall/CRC Press, 2000. 10. Taylor D, Morris D, Richards K. Combination chemotherapy of Echinococcus granulosus—in vitro studies. Trans R Soc Trop Med Hyg 1988; 82: 263–4. 11. Urrea-Parı́s M, Moreno M, Casado N et al. In vitro effect of praziquantel and albendazole combination therapy on the larval stage of Echinococcus granulosus. Parasitol Res 2000; 86: 957–64. 12. Garcı́a-Domı́nguez C, Correa D, Rabiela M et al. Praziquantel treatment of muscle Taenia solium cysticercosis. Reversible in vitro effect. Parasitol Res 1991; 77: 691–6. 13. Morris D, Taylor D, Daniels D et al. Determination of minimum effective concentration of praziquantel in in vitro cultures of protoscoleces of Echinococcus granulosus. Trans R Soc Trop Med Hyg 1987; 81: 494–7. 14. Casado N, Pérez-Serrano J, Denegri G et al. Development of a Chemotherapy Model for the in vitro screening of drugs against Echinococcus granulosus cysts: the effects of an albendazolealbendazole sulphoxide combination. Int J Parasitol 1996; 26:59–65. 15. Ingold K, Bigler P, Wolfgang T et al. Efficacies of albendazole sulphoxide and albendazole sulfone against in vitro-cultivated Echinococcus multilocularis metacestodes. Antimicrob Agents Chemother 1999; 43: 1052–61. 16. Lacey E. Mode of action of benzimidazoles. Parasitol Today 1990; 6: 112–15. 17. Lubega G, Prichard R. Interaction of benzimidazole anthelmintics with Haemonchus contortus tubulin: binding affinity and anthelmintic efficacy. Exp Parasitol 1991; 73: 203–13. 18. Lacey E. Role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles. Int J Parasitol 1988; 18: 885–936. 19. Pérez-Serrano J, Denegri G, Casado N et al. In vivo effect of oral albendazole and albendazole sulphoxide on development of secondary Echinococcosis in mice. Int J Parasitol 1997; 27: 1341–5. 20. Tallarida R. Drug synergism: its detection and applications. J Pharmacol Exp Ther 2001; 289: 865–72. 488