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JAC Advance Access originally published online on November 16, 2006
Journal of Antimicrobial Chemotherapy 2007 59(2):212-218; doi:10.1093/jac/dkl463
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© 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: journals.permissions@oxfordjournals.org

Efficacy of nitazoxanide, tizoxanide and tizoxanide/albendazole sulphoxide combination against Taenia crassiceps cysts

Francisca Palomares-Alonso1, Juan Carlos Piliado1, Guadalupe Palencia2, Alma Ortiz-Plata3 and Helgi Jung-Cook1,4,*

1 Laboratorio de Neuropsicofarmacología, Instituto Nacional de Neurología y Neurocirugía 14269 México D.F., México 2 Laboratorio de Neuroinmunología Instituto Nacional de Neurología y Neurocirugía 14269, México D.F., México 3 Laboratorio de Neuropatología Instituto Nacional de Neurología y Neurocirugía 14269, México D.F., México 4 Facultad de Química, UNAM México, D.F., México

*Correspondence address. Instituto Nacional de Neurología y Neurocirugía, Insurgentes Sur 3877, Col. La Fama, Tlalpan 14269, México City, México. Tel/Fax: +52-54-24-08-08; E-mail: helgi{at}servidor.unam.mx

Received 12 April 2006; returned 19 September 2006; revised 22 September 2006; accepted 17 October 2006


   Abstract
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Objectives: Neurocysticercosis is a common parasitic disease in the CNS in humans caused by the metacestode Taenia solium, with high incidence in developing countries. Albendazole is the drug of choice. However, a wide interindividual variability in the response has been reported. In order to evaluate alternative treatment options, the in vitro efficacy of nitazoxanide, its main metabolite tizoxanide as well as the tizoxanide and albendazole sulphoxide combination was tested against Taenia crassiceps cysts.

Methods: T. crassiceps cysts were incubated in culture medium containing different concentrations of nitazoxanide, tizoxanide and albendazole sulphoxide (0.037–0.42 µg/mL). The effect of the tizoxanide and albendazole sulphoxide combination was evaluated in a fixed-concentration ratio (1:1). Isobolographic analyses were used to define the kind of interaction between drugs. Morphological and ultrastructural alterations over the parasite tissue were observed by light and transmission electron microscopy.

Results: Nitazoxanide and tizoxanide exhibited cestocidal activity which was time–concentration-dependent. The EC50 values were 0.15, 0.12 and 0.080 µg/mL for nitazoxanide, tizoxanide and albendazole sulphoxide, respectively. No statistical differences between EC50 values were found, indicating that nitazoxanide and tizoxanide are equally potent as albendazole sulphoxide. The effect of the tizoxanide and albendazole sulphoxide combination was faster than that observed with each drug alone. Isobolographic analysis showed that the effect of the combination was additive. Nitazoxanide and tizoxanide had an effect on the germinal layer, where lipid droplets were found. Nitazoxanide and tizoxanide produced less damage than albendazole sulphoxide on the germinal layer. After the tizoxanide and albendazole sulphoxide combination, a high accumulation of lipid droplets within the germinal layer of the parasite was found.

Conclusions: Our results suggest that nitazoxanide in combination with albendazole could be useful for treatment of cysticercosis infections. Additional in vivo studies are required to confirm this hypothesis.

Keywords: antiparasitics , in vitro activity , drug interactions , metacestodes ultrastructure


   Introduction
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Neurocysticercosis is a common parasitic disease in the CNS in humans caused by the metacestode Taenia solium, with high incidence in developing countries of Latin America, Africa and Asia.1 Albendazole is the drug of choice for neurocysticercosis treatment; however, a wide interindividual variability in the response has been reported. While some patients require one course of albendazole, others require repeated courses and in some cases failure of the treatment has been reported;2 therefore new alternatives are needed.

Nitazoxanide is a broad-spectrum parasiticidal agent with activity against bacteria, protozoa, nematodes and trematodes.3,4 It has been available for several years in developing countries where tapeworm and liver fluke infestations are common.5,6 Once orally administered, nitazoxanide is rapidly metabolized to tizoxanide,7 which also shows parasiticidal activity.8 In 1984, Rossignol and Maisonneuve,9 in a clinical trial in humans showed the activity of nitazoxanide against the cestode of Taenia saginata. Considering that to date there is no information about the efficacy of nitazoxanide against T. solium, the aim of this study was to investigate the in vitro efficacy of this drug and its main metabolite, tizoxanide, as well as the combined treatment of tizoxanide plus albendazole sulphoxide (active metabolite of albendazole) against Taenia crassiceps cysts.


   Materials and methods
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Drugs and reagents

Nitazoxanide was donated by Laboratorios Liomont (México), and tizoxanide and albendazole sulphoxide were synthesized and donated by Dr Rafael Castillo (Facultad de Química, UNAM, México). Dimethyl sulphoxide (DMSO; Merck, Shuchardt, Germany; assay 99%) was analytical reagent grade. The culture medium used was Dulbecco's modified minimal essential medium (Sigma-Aldrich Co., USA), 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).

Parasites

Studies were performed on T. crassiceps cysts (strain ORF), which have been used previously as a model for the evaluation of cestocidal drugs, due to its similarity with T. solium cysts.10,11 Male BALB/c mice (2 months old) were experimentally infected with T. crassiceps cysts. After 3 months of infection, mice were killed by cervical dislocation and the cysts were removed from the peritoneal cavity and washed several times with sterile 0.9% saline solution. Only those cysts exhibiting intact bladder surface were used for the experiments. The study was approved by the local ethics committee.

In vitro studies

Two studies were performed: a single drug exposure study to evaluate the efficacy of nitazoxanide and tizoxanide; and a drug combination exposure study to evaluate the interaction between tizoxanide and albendazole sulphoxide.

Single drug exposure study

Stock solutions of nitazoxanide, tizoxanide and albendazole sulphoxide were prepared in DMSO. Working solutions of each drug were prepared in culture medium to obtain concentrations from 0.037 to 0.42 µg/mL. A solution of 0.05% DMSO was prepared as a control. Cell-culture flasks were carefully filled with 5 mL of culture medium containing each drug or DMSO, and 25 cysts were deposited into each flask and were incubated at 37°C in a 5% CO2 atmosphere and with 98% relative humidity for 11 days. The medium was changed every day. Each experiment was performed in triplicate. During incubation, the cysts were observed every day 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 mortality was confirmed on day 11 using the Trypan Blue exclusion test.

The results were analysed using non-linear regression to obtain the corresponding EC50 and the confidence limits of each drug. The analysis was done using SPSS software (version 9).

Drug combination exposure study

Considering that nitazoxanide and albendazole are extensively metabolized to their active metabolites, we evaluated the effect of tizoxanide and albendazole sulphoxide in combination. For the evaluation of the interaction between tizoxanide and albendazole sulphoxide the isobolographic method was selected.12 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)


Formula
where n is 2, 4, 8, 16, 32 ... and CTZD and CABZSO are the concentrations of tizoxanide and albendazole sulphoxide, respectively.

Drug fraction

In order to calculate CTZD and CABZSO in the combination, the EC50 of each drug was transformed to fractions (XTZD and XABZSO) using the following equations:

Formula

Formula
Therefore the concentration of each drug in the combination was calculated as follows:

Formula

Formula
Thus the final concentrations of tizoxanide and albendazole sulphoxide in the culture medium were, respectively: 0.0078 + 0.0052, 0.0156 + 0.0103, 0.0311 + 0.0206 and 0.0622 + 0.0413 µg/mL. Cysts incubated in culture medium containing 0.05% DMSO served as a control. 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 was 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.13 To visualize the type of interaction, an isobologram was constructed using the EC50 value of tizoxanide (on the x-axis), the EC50 value of albendazole sulphoxide (on the y-axis) and the EC50Exp and EC50Add values of the combination were included in the isobologram.

To evaluate the difference between the experimental (EC50Exp) and the theoretical value, a Student's t-test was performed (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 0.13 µg/mL nitazoxanide, 0.06 µg/mL tizoxanide, 0.04 µg/mL albendazole sulphoxide, and 0.06 µg/mL tizoxanide plus 0.04 µg/mL albendazole sulphoxide (which represent the in vitro EC25 of each drug and the EC50 for the drug combination) 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 with 0.1 M phosphate-buffered saline (PBS; pH 7.4), post-fixed in 0.5% osmium tetroxide (Merck, Germany), dehydrated in a 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 a ultramicrotome (Reicher, Mod. 570 0.7 TO 4.2X).

Light microscopy

Sections of 1 µm thickness were cut and recovered over glass slides and stained with 1% Toluidine Blue and examined by light microscopy. Micrographs were taken and captured by computerized digital image (Leica DMLS, software IM 1000).

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.


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Morphological and ultrastructural alterations

Figure 1 shows the morphological alterations by light microscopy on cysts after the different treatments. It can be seen that after 11 days of incubation, the control parasites did not exhibit changes in their morphology. The ruggedness and distribution of microtriches were unaltered (Figure 1a and b). Parasites treated with nitazoxanide and tizoxanide exhibited partial loss of the characteristic ruggedness of the tegument (Figure 1c and d). In the germinal layer a high formation of large lipid droplets was found (Figure 1e). In the case of the albendazole sulphoxide treatment, a high level of damage to the parasite tissue was observed. In many areas the germinal layer was largely disintegrated, the connective tissue was damaged and only necrotic tissue was observed (Figure 1f). Parasites treated with tizoxanide plus albendazole sulphoxide showed changes in morphology. The tegumental layer lost its characteristic structure and the germinal layer showed an increased number of lipoidal inclusions (Figure 1g and h).


Figure 1
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  		Figure 1. Light microscopy of T. crassiceps cysts after 11 days of in vitro treatment. A representative section of the parasite tissue after the different treatments is presented in each panel. Control (DMSO 0.05%) (a and b), nitazoxanide (NTZ, 0.13 µg/mL) (c and d), tizoxanide (TZD, 0.06 µg/mL) (e), albendazole sulphoxide (ABZSO, 0.04 µg/mL) (f) and the combination of TZD (0.06 µg/mL) plus ABZSO (0.04 µg/mL) (g and h). t, Tegument; gl, germinal layer; mt, microtriches; ld, lipid droplets. (a, c, e, f and g) Bars = 5 µm. (b and d) Bars = 400 µm. (h) Bar = 1 µm.

 
Observations by transmission electron microscopy (Figure 2) revealed that all parasite tissue, including the microtriches, remained unaltered; also tegumental cells were normal in the controls (Figure 2a). After nitazoxanide and tizoxanide exposure, the high accumulation of lipid droplets within the germinal layer was confirmed at the ultrastructural level (Figure 2c). The microtriches in the tegument and muscular cells and germinal layer were not affected (Figure 2b and d) and the tegumental cells remained joined through cytoplasmic bridges (Figure 2e). Our data show that nitazoxanide and its metabolite exhibited the same effect on the parasite tissue.


Figure 2
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  		Figure 2. Transmission electron microscopy of T. crassiceps cysticerci tissue after 11 days of in vitro treatment. Cyst control (a). Cysts treated with nitazoxanide (NTZ; 0.13 µg/mL) (b and c), tizoxanide (TZD; 0.06 µg/mL) (d and e), albendazole sulphoxide (ABZSO, 0.04 µg/mL) (f) and the combination 0.06 µg/mL TZD plus 0.04 µg/mL ABZSO (g, h and i). t, Tegument; gl, germinal layer; mt, microtriches; ld, lipid droplets; mc, muscular cell; tc, tegumental cell; fm, muscular fibre. (a, b and d) Bars = 40 µm. (c, e, g and h) Bars = 1 µm. (f) Bar = 2 µm. (i) Bar = 3 µm.

 
With albendazole sulphoxide treatment, the microtriches appeared truncated or even absent. Undifferentiated cells disappeared, and cellular residues were present in large parts of the germinal layer (Figure 2f). After tizoxanide plus albendazole sulphoxide treatment, a partial loss of microtriches on the tegument was observed. Also the cell–cell contacts and the muscular fibres appeared disorganized in some areas (Figure 2g). The formation of large lipid droplets was extended into the cytoplasm (Figure 2h and i).

Pharmacological effect

Figure 3 (a and b) shows the time–mortality curve of T. crassiceps cysts at different concentrations of nitazoxanide and tizoxanide. It can be seen that nitazoxanide and tizoxanide exhibited a cestocidal effect and the maximum effect was attained at 11 days of exposure. These results indicate that, like the benzimidazoles, the effect of nitazoxanide and tizoxanide was time–concentration-dependent. Figure 3(c) shows the mortality of T. crassiceps cysts after exposure to the tizoxanide plus albendazole sulphoxide combination as well as to tizoxanide and albendazole sulphoxide alone at the same concentrations used in the combination. It can be seen that the effect of each drug alone was slower than that observed with the combination. Thus after tizoxanide and albendazole sulphoxide treatments, the mortality became evident after 4 days of exposure, while after the combination the mortality was clearly evident after 2 days. Figure 4 shows the concentration–response curves of nitazoxanide, tizoxanide and albendazole sulphoxide. The EC50 values obtained for nitazoxanide, tizoxanide, albendazole sulphoxide and the tizoxanide and albendazole sulphoxide combination were 0.15, 0.12, 0.08 and 0.098 µg/mL, respectively (Table 1). A t-test between nitazoxanide, tizoxanide and albendazole sulphoxide showed that there were no significant differences between EC50 values, which indicates that in vitro nitazoxanide and tizoxanide are equally potent as albendazole sulphoxide. The isobolographic analysis of the combination revealed that there were no significant differences between the experimental EC50Exp value (0.098 µg/mL) and the theoretical EC50Add value (0.103 µg/mL) (P > 0.05), which indicates an additive interaction between both drugs.


Figure 3
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  		Figure 3. Time–mortality curves of T. crassiceps cysts after in vitro exposure to nitazoxanide (NTZ), tizoxanide (TZD) and the tizoxanide and albendazole sulphoxide (TZD + ABZSO) combination. (a) NTZ: 0.062 (filled circles), 0.092 (filled triangles), 0.135 (open upside-down triangles), 0.197 (filled diamonds), 0.29 (open circles) and 0.42 µg/mL (open squares). (b) TZD: 0.037 (filled circles), 0.054 (filled triangles), 0.08 (open upside-down triangles), 0.116 (filled diamonds), 0.17 (open circles) and 0.25 µg/mL (open squares). (c) Combination of 0.06 µg/mL TZD + 0.04 µg/mL ABZSO (filled diamonds), 0.06 µg/mL TZD (filled triangles) and 0.04 µg/mL ABZSO (open circles). (a–c) Control (DMSO 0.05%) (filled squares). Each point represents the mean percentage of mortality of cysts resulting from three different experiments.

 


Figure 4
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  		Figure 4. Concentration–response curves of nitazoxanide (filled triangles), tizoxanide (open squares), and albendazole sulphoxide (filled circles). Each point represents the mean ± SD of three experiments.

 


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  		Table 1. EC50 values of nitazoxanide (NTZ), tizoxanide (TZD), albendazole sulphoxide (ABZSO) and the combined drug treatment (TZD + ABZSO) for T. crassiceps cysts

 

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Although there are several studies in which the pharmacological activity of nitazoxanide against different parasites, including T. saginata,14,15 has been evaluated, this is the first study that reports the effect of nitazoxanide and its main active metabolite, tizoxanide, against T. crassiceps cysts as well as the effect of the tizoxanide and albendazole sulphoxide combination.

Light microscopy observations showed that the main effect of nitazoxanide and tizoxanide was on the germinal layer, where lipid droplets were found, reflecting the metabolic stress that occurred after the treatment. This effect has been observed in other species of metacestodes like Echinococcus granulosus.16 On the other hand, structures like microtriches and tegumental cells remained unaltered. These observations did not agree with previous reports in other species of parasites, where nitazoxanide produced large amounts of cellular detritus and the complete absence of microtriches in the tegument.8,16 This fact could be due to the differences in the concentrations used to evaluate morphological alterations. Thus in our study concentrations of nitazoxanide and tizoxanide were 0.13 and 0.06 µg/mL, respectively, whereas Stettler et al.8 used concentrations of 5 and 10 µg/mL of nitazoxanide and tizoxanide and Walker et al.16 used 10 µg/mL. Another contributory factor could be the differences in susceptibility between species to the drugs. The alterations after albendazole sulphoxide treatment found in T. crassiceps cysts are in agreement with those found in other species of metacestodes like Echinococcus multilocularis and E. granulosus.17,18 When morphological alterations were compared, we found that nitazoxanide and tizoxanide produced less damage than albendazole sulphoxide. These differences could be related to the mode of action of these drugs. The benzimidazole derivatives bind to ß-tubulin, and prevent the uptake of glucose by disrupting cellular microtubular structures,19 whereas it has been postulated that in helminths nitazoxanide interferes with the pyruvate ferredoxin oxidoreductase enzyme-dependent anaerobic metabolism.8,16

In the case of the tizoxanide and albendazole sulphoxide combination, a high accumulation of lipid droplets within the germinal layer of the parasite was found, which could be related to the effect of tizoxanide; however, the damage in the microtriches and tegumental cells was less than that observed with albendazole sulphoxide. To date there are no reports about the ultrastructural effect of the combined treatment of tizoxanide and albendazole sulphoxide in any metacestode species and clearly more detailed studies will be required.

Our pharmacological results showed that as in other species of metacestodes, nitazoxanide and tizoxanide exhibited a cestocidal effect against T. crassiceps,8,16 which was time–concentration-dependent. To date there are no reports about the potency of nitazoxanide and tizoxanide. The results of this study show that against T. crassiceps cysts these drugs were equally potent as albendazole sulphoxide.

In order to evaluate the interaction between tizoxanide and albendazole sulphoxide we used the isobolographic method. This analysis is very useful when the mechanism of action of the drugs in the combination is different.13 The cestocidal effect of the tizoxanide and albendazole sulphoxide combination was additive, which indicates that the total effect is the sum of the individual effects of each drug and each one contributes to the total effect according to its own potency.20 Also the additive action indicates that both drugs are active and neither drug influences the action of the other.20 The mechanism underlying the additive cestocidal activity between tizoxanide and albendazole sulphoxide, against T. crassiceps cysts, is not clear; therefore, further and more detailed investigations are required to explain the results.

Our results suggest that nitazoxanide in combination with albendazole could be useful for in vivo treatment of cysticercosis infections. Additional studies are required to confirm this hypothesis.


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None to declare.


   Acknowledgements
 
We thank Biol. Olivia Reynoso Ducoing and Javier Ambrosio PhD (Facultad de Medicina de la UNAM, México) for providing the Taenia crassiceps cysts strain ORF. There was no external source of funding for this project.


   References
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1 Schantz P. (2002) Taenia solium cysticercosis: an overview of global distribution and transmission. In Singh G and Sudesh P (Eds.). Taenia solium Cysticercosis: From Basic to Clinical Science(CABI Publishing, London, UK) pp. 63–73.

2 García HH, Evans CA, Nash TE, et al. (2002) Current consensus guidelines for treatment of neurocysticercosis. Clin Microbiol Rev 15:747–56.[Abstract/Free Full Text]

3 Fonseca-Salamanca F, Martínez-Grueiro MM, Martínez-Fernández AR. (2003) Nematocidal activity of nitazoxanide in laboratory models. Parasitol Res 91:321–4.[CrossRef][ISI][Medline]

4 White A. (2003) Nitazoxanide: an important advance in anti-parasitic therapy. Am J Trop Med Hyg 68:382–3.[Free Full Text]

5 Rodríguez-García R, Rodríguez-Guzmán LM, Cruz-Del Castillo AH. (1999) Effectiveness and safety of mebendazole compared to nitazoxanide in the treatment of Giardia lamblia in children. Rev Gastroenterol Mex 64:122–6.[Medline]

6 Romero C, Robert G, Muñoz G, et al. (1997) Nitazoxanide for the treatment of intestinal protozoan and helminthic infections in México. Trans R Soc Trop Med Hyg 91:701–3.[CrossRef][ISI][Medline]

7 Broekhuysen J, Stokis A, Lins R, et al. (2000) Nitazoxanide: pharmacokinetics and metabolism in man. Int J Clin Pharmacol Ther 38:387–94.[ISI][Medline]

8 Stettler M, Fink R, Walker M, et al. (2003) In vitro parasiticidal effect of nitazoxanide against Echinococcus multilocularis metacestodes activity. Antimicrob Agents Chemother 47:467–74.[Abstract/Free Full Text]

9 Rossignol JF and Maisonneuve H. (1984) Nitazoxanide in the treatment of Taenia saginata and Hymenolepis nana infections. Am J Trop Med Hyg 33:511–2.[Abstract/Free Full Text]

10 Rossi N, Rivas I, Hernández M, et al. (2000) Immunodiagnosis of neurocysticercosis: comparative study of antigenic extracts from Cysticercus cellulosae and Taenia crassiceps. Rev Cubana Med Trop 52:157–64.[Medline]

11 Palomares F, Palencia G, Pérez R, et al. (2004) In vitro effects albendazole sulfoxide and praziquantel against Taenia solium and Taenia crassiceps cysts. Antimicrob Agents Chemother 48:2302–4.[Abstract/Free Full Text]

12 Tallarida R, Porreca F, Cowan A. (1989) Statistical analysis of drug–drug and site–site interactions with isobolograms. Life Sci 45:947–61.[CrossRef][ISI][Medline]

13 Tallarida R. (2000) Combinations of chemicals. Drug Synergism and Dose-effect Data Analysis(Chapman & Hall/CRC, New York) pp. 1–72.

14 Belkind-Valdovinos U, Belkind-Gerson J, Sánchez-Francia D, et al. (2004) Nitazoxanide vs albendazole against intestinal parasites in a single dose for three days. Salud Publica Mex 46:333–40.[ISI][Medline]

15 Gilles HM and Hoffman PS. (2002) Treatment of intestinal parasitic infections: a review of nitazoxanide. Trends Parasitol 18:95–7.[CrossRef][ISI][Medline]

16 Walker M, Rossignol JF, Torgerson P, et al. (2004) In vitro effects of nitazoxanide on Echinococcus granulosus protoscoleces and metacestodes. J Antimicrob Chemother 54:609–16.[Abstract/Free Full Text]

17 Ingold K, Bigler P, Thormann W, et al. (1999) Efficacies of albendazole sulfoxide and albendazole sulfone against in vitro-cultivated Echinococcus multilocularis metacestodes. Antimicrob Agents Chemother 43:1052–61.[Abstract/Free Full Text]

18 Urrea-París MA, Moreno MJ, Casado N, et al. (2000) In vitro effect of praziquantel and albendazole combination therapy on the larval stage of Echinococcus granulosus. Parasitol Res 86:957–64.[CrossRef][ISI][Medline]

19 Morgan UM, Reynoldson JA, Thompson RC, et al. (1993) Activities of several benzimidazoles and tubulin inhibitors against Giardia spp. in vitro. Antimicrob Agents Chemother 37:328–31.[Abstract/Free Full Text]

20 Tallarida R. (2001) Drug synergism: its detection and applications. J Pharmacol Exp Ther 289:865–72.


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