Efficacy of nitazoxanide, tizoxanide and tizoxanide glucuronide against Cryptosporidium parvum development in sporozoite-infected HCT-8 enterocytic cells

Gilles Gargalaa,*, Agnès Delaunaya, Xunde Lib, Philippe Brasseurb, Loic Favennecb and Jean Jacques Balleta

a Laboratoire d'Immunologie et Immunopathologie, UPRES-EA 2128, CHU, 14033 Caen; b Laboratoire de Parasitologie Expérimentale, UPRES-JE 2008, CHU, 76000 Rouen, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effects of nitazoxanide and its metabolites, tizoxanide and tizoxanide glucuronide, on the development of the asexual and sexual stages of Cryptosporidium parvum in differentiated human enterocytic HCT-8 cells were evaluated in a quantitative alkaline phosphatase immunoassay. Nitazoxanide, tizoxanide and tizoxanide glucuronide were inhibitory for up to 46 h when added after sporozoite invasion (MIC50 1.2, 22.6 and 2.2 mg/L, respectively). Tizoxanide had only limited activity, but nitazoxanide and tizoxanide glucuronide strongly inhibited asexual and sexual stages, respectively.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cryptosporidium parvum is a common cause of diarrhoea in immunocompetent individuals. In immunodeficient subjects, cryptosporidiosis may lead to life-threatening chronic diarrhoea and, because of the incidence of AIDS, it is a public health concern in developing countries where AIDS is endemic.1,2 Of the many chemotherapeutic and immunotherapeutic agents tested in recent years, few have shown anti-cryptosporidial activity.3,4 Infectious relapses are often observed after anti-cryptosporidial therapy, both in animal models and in clinical studies, primarily because parasites persist in the biliary tract, which is not reached by active agents, such as paromomycin, that are not absorbed from the digestive tract.5 This underlines the potential interest in agents such as nitazoxanide, which has already been shown to be active in vitro and to be readily absorbed from the gut. In humans, nitazoxanide is metabolized in vivo into tizoxanide and tizoxanide glucuronide, and these are the only metabolites detected in blood samples.6

The aim of this study was to investigate in vitro the efficacy of nitazoxanide, tizoxanide and tizoxanide glucuronide on sporozoite invasion and asexual and sexual development of C. parvum in the enterocytic cell line HCT-8. Asexual and sexual stages of parasite development were assessed by both enzyme immunoassay and immunofluorescence.7,8


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nitazoxanide, tizoxanide and tizoxanide glucuronide were supplied by Romark Laboratories, Tampa, FL, USA. Stock solutions were prepared in co-culture medium containing 1% (v/v) dimethylsulphoxide (DMSO) at 1 g/L for nitazoxanide and tizoxanide, and 2 g/L for tizoxanide glucuronide, and frozen at –20°C until used. Final dilutions in culture wells ranged from 1 to 50 mg/L for nitazoxanide and tizoxanide, and from 1 to 100 mg/L for tizoxanide glucuronide.

HCT-8 cells (CCL-244) were obtained from the American Type Culture Collection, Rockville, MD, USA. They were maintained in culture in BHK 21 (Eagle's modified Dulbecco's) medium supplemented with 1% non-essential amino acids and 20% fetal calf serum (Gibco BRL, Gaithersburg, MD, USA) in a 5% CO2 humidified atmosphere. HCT-8 cells were cultured in 96-well Nunclon microplates (Nunc, Naperville, IL, USA) at 1.5 x 105 cells/well. Cultures were simultaneously performed in 16-well tissue culture chambers (LabTek Chamber Slides; Nunc).

C. parvum sporozoites were obtained from the faeces of experimentally infected calves (kindly provided by Dr Naciri, Laboratoire de Pathologie Aviaire, INRA, Nouzilly, France) as previously described.9 Briefly, C. parvum oocysts were concentrated on a sucrose gradient and then excysted using a 1.5% taurocholate solution. Culture medium was removed from 24 h HCT-8 cultures and 100 µL per well of BHK 21 medium containing 1.5–2 x 105 freshly isolated sporozoites was added to cell monolayers for 2 h at 37°C. Two hundred microlitres of BHK 21 medium supplemented with paraminobenzoic acid 4 mg/L (Sigma, St Louis, MO, USA), ascorbic acid 35 mg/L (Sigma), glucose 25 mmol/L (Merck, Darmstadt, Germany), insulin 100 IU/L (Novo Nordisk Pharmaceutique, BoulogneBillancourt, France), HEPES 15 mmol/L (Sigma), streptomycin 500 mg/L (Gibco BRL), penicillin 105 IU/L (Gibco BRL) and fetal calf serum 20% (v/v) was added to each well, and cells were cultured for an additional 46 h.10,11

Enzyme immunoassay (EIA) for the detection of C. parvum in HCT-8 cultures was performed as previously described8 using rat polyclonal antibodies against Cryptosporidium and biotin–SP-conjugated anti-rat IgG and IgM (heavy and light chain) goat F(ab')2 fragments 2 mg/L (Jackson Immunoresearch, Westgrove, PE, USA) as secondary antibody revealed using alkaline phosphatase-coupled avidin (ABC Reagent; Vector Laboratories, Burlingame, CA, USA).

Inhibitory activities of agents are expressed as percentages, the per cent inhibition was defined as

(A405 in infected cells with agent)

1 – – (A405 in uninfected cells with agent) x 100

(A405 in infected cells without agent)

– (A405 in uninfected cells without agent)

The MIC50 was defined as the concentration (in mg/L of culture) of agent that resulted in a 50% inhibition of C. parvum development.

The cytotoxicities of nitazoxanide, tizoxanide and tizoxanide glucuronide were determined using trypan blue exclusion and nitroblue tetrazolium chloride monohydrate reduction assays (CellTiter 96 AQueous non-radioactive cell proliferation assay; Promega, Madison, WI, USA). Controls included infected and uninfected cells in culture medium. Results were expressed as the decrease in absorbance at 450 nm (A450) expressed as percentages of the A450 values in control cultures. From preliminary studies we verified that HCT-8 cell cytotoxicity was minimal (decrease of A450 from 0 to 14%) for all controls and at all concentrations of agents, except nitazoxanide at 25 and 50 mg/L (A450 decrease of 36% and 39%, respectively).

A405 values in control cultures in which heat-inactivated sporozoites were added instead of viable sporozoites, were not significantly different from A405 values in the absence of heat-inactivated sporozoites. For uninfected wells, one s.d. accounted for <10% and 15% of the mean A405 for each microplate and for the pooled data of five microplates, respectively. For infected wells, one s.d. accounted for <10% and 21% of the mean A405 for each microplate and for the pooled data of five microplates, respectively.

In the EIA, background A405, resulting from the intense colour of the solutions, was higher in uninfected wells exposed to drugs than in control wells without agent, and for this reason they were used as controls for infected wells containing the same agent concentration. In addition, disruption of infected monolayers and/or peeling off of HCT-8 cells occurred during EIA in wells containing tizoxanide or tizoxanide glucuronide at 10–50 mg/L. For tizoxanide and tizoxanide glucuronide, high background A405 was observed at concentrations of >50 mg/L, preventing any conclusions from being drawn.

The effects of nitazoxanide, tizoxanide and tizoxanide glucuronide on the various stages of the C. parvum life cycle were studied by adding the tested compounds, at concentrations ranging from 1 to 50 mg/L, at the start of the culture (sporozoite stage), 2 h after adding sporozoites (trophozoite stage) and 18 h after adding sporozoites (sexual stages).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effects of nitazoxanide, tizoxanide and tizoxanide glucuronide on the asexual and sexual stages of parasite development were investigated by exposure to the agents for 46 h starting 2 h after adding sporozoites (Figure 1Go and TableGo). To test the effects of agents on penetration of sporozoites into HCT-8 cells, the agents were added at the same time as sporozoites, and culture supernatants were removed and replaced by agent-free medium 2 h later; EIA detection of parasite development was performed after a further 46 h incubation. Nitazoxanide and tizoxanide were active during the sporozoite penetration stage, while the effect of tizoxanide glucuronide appeared limited (Figure 2Go and TableGo). The effects on asexual or sexual development were investigated by adding agents for 4 h, 2 h after adding sporozoites (for asexual development) or 18 h after adding sporozoites (for sexual development). As shown in Figure 3Go and the TableGo, nitazoxanide inhibited asexual development more than tizoxanide glucuronide, while tizoxanide had no significant effect. Figure 4Go and the TableGo depict the marked inhibitory effect of tizoxanide glucuronide on sexual stages, and the lesser effects of nitazoxanide and tizoxanide.



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Figure 1. Effects of nitazoxanide, tizoxanide and tizoxanide glucuronide on the development of the asexual and sexual stages of C. parvum in HCT-8 cells. Agents were added to cultures 2 h after the addition of 1.5–2 x 105 sporozoites/well and incubated for 46 h. Results are expressed as mean (± s.d.) inhibition percentages. Pooled data from 20 independent experiments. ——, Nitazoxanide; ...., tizoxanide; - - - -, tizoxanide glucuronide.

 

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Table. MIC50 (mg/L) of nitazoxanide, tizoxanide and tizoxanide glucuronide on complete development (asexual and sexual stages), sporozoite, asexual and sexual stages of Cryptosporidium parvum in HCT-8 cells
 


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Figure 2. Effects of nitazoxanide, tizoxanide and tizoxanide glucuronide on the penetration of C. parvum sporozoite into HCT-8 cells. Agents were added to cultures at the same time as 1.5–2 x 105 sporozoites/well, and incubated for 2 h. Results of EIA detection, performed after a further 46 h incubation, are expressed as mean (± s.d.) inhibition percentages. Pooled data from five independent experiments. Key as in Figure 1Go.

 


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Figure 3. Effects of nitazoxanide, tizoxanide and tizoxanide glucuronide on the asexual development of C. parvum in HCT-8 cells. Agents were added to cultures 2 h after the addition of 1.5–2 x 105 sporozoites/well and incubated for 4 h. Results are expressed as mean (± s.d.) inhibition percentages. Pooled data from three independent experiments. Key as in Figure 1Go.

 


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Figure 4. Effects of nitazoxanide, tizoxanide and tizoxanide glucuronide on the sexual development of C. parvum in HCT-8 cells. Agents were added to cultures 18 h after the addition of 1.5–2 x 105 sporozoites/well and left in culture for 4 h. Results are expressed as mean (± s.d.) inhibition percentages. Pooled data from seven independent experiments. Key as in Figure 1Go.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, nitazoxanide was found to be highly effective in vitro against the development of the asexual and sexual stages of C. parvum. This is in agreement with a previous study which found that nitazoxanide inhibits C. parvum development.12 Our findings, which indicate that tizoxanide glucuronide inhibits parasite development, suggest that nitazoxanide activity is at least in part due to this metabolite. Interestingly, nitazoxanide and tizoxanide appear to be more active against the extracellular sporozoite stage, whereas tizoxanide glucuronide acts primarily on intracellular development. This was confirmed using simultaneous immunofluorescent assays (data not shown). Whether this is because tizoxanide glucuronide is better at penetrating the cell membrane requires additional investigation.

Previous clinical studies have shown that nitazoxanide is clinically active against cryptosporidiosis.13,14 This compound has a wide range of antimicrobial activities, particularly against intestinal protozoa and helminths.15 Present data underline the potential interest in nitazoxanide metabolites as candidates for in vivo studies. Interestingly, studies in an immunosuppressed rat model suggest that relapses are less frequent after treatment with nitazoxanide than with the non-absorbable paromomycin and sinefungin (Li, X., unpublished results). Taken together, these data indicate the need for additional studies on the biliary clearance of nitazoxanide metabolites and their potential activity on biliary tract cryptosporidiosis.


    Notes
 
* Correspondence address. Service d'Immunologie et Immunopathologie, CHU Clemenceau, 14033 Caen Cedex, France. Tel: +33-2-31272551; Fax: +33-2-31272550; E-mail: ballet-jj{at}chu-caen.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Griffiths, J. K. (1998). Human cryptosporidiosis: epidemiology, transmission, clinical disease, treatment, and diagnosis. Advances in Parasitology 40, 37–85.[ISI][Medline]

2 . Kelly, P., Davies, S. E., Mandanda, B., Veitch, A., McPhail, G., Zulu, I. et al. (1997). Enteropathy in Zambians with HIV related diarrhoea: regression modelling of potential determinants of mucosal damage. Gut 41, 811–6.[Abstract/Free Full Text]

3 . Blagburn, B. B. L. & Soave, R. (1997). Prophylaxis and chemotherapy: human and animal. In Cryptosporidium and Cryptosporidiosis, (Fayer, R., Ed.), pp. 111–27. CRC Press, Boca Raton, FL.

4 . Tzipori, S. (1998). Cryptosporidiosis: laboratory investigations and chemotherapy. Advances in Parasitology 40, 187–221.[ISI][Medline]

5 . Bissuel, F., Cotte, L., Rabodonirina, M., Rougier, P., Piens, M. A. & Trepo, C. (1994). Paromomycin: an effective treatment for cryptosporidial diarrhea in patients with AIDS. Clinical Infectious Diseases 18, 447–9.[ISI][Medline]

6 . Stockis, A., Lins, R., Deroubaix, X., Jeanbaptiste, B., Calderon, P. & Rossignol, J. F. (1996). Pharmacokinetics of nitazoxanide after single oral dose administration in 6 healthy volunteers. International Journal of Clinical Pharmacology and Therapeutics 34, 349–51.[ISI][Medline]

7 . Egraz-Bernard, M., Favennec, L., Agnamey, P., Ballet, J. J. & Brasseur, P. (1996). Inhibition of complete development of Cryptosporidium parvum in Caco-2 cells. European Journal of Clinical Microbiology and Infectious Diseases 15, 897–900.[ISI][Medline]

8 . Gargala, G., Delaunay, A., Favennec, L., Brasseur, P. & Ballet, J. J. (1999). Enzyme immunoassay detection of Cryptosporidium parvum inhibition by sinefungin in sporozoite infected HCT-8 enterocytic cells. International Journal for Parasitology 29, 703–9.[ISI][Medline]

9 . Buraud, M., Forget, E., Favennec, L., Bizet, J., Gobert, J. G. & Deluol, A. M. (1991). Sexual stage development of cryptosporidia in the Caco-2 cell line. Infection and Immunity 59, 4610–3.[ISI][Medline]

10 . Maillot, C., Favennec, L., François, A., Ducrotte, P. & Brasseur, P. (1997). Sexual and asexual development of Cryptosporidium parvum in five oocyst- or sporozoite-infected human enterocytic cell lines. Journal of Eukaryotic Microbiology 44, 582–5.[ISI][Medline]

11 . Upton, S. J., Tilley, M. & Brillhart, D. B. (1995). Effects of select medium supplements on in vitro development of Cryptosporidium parvum in HCT-8 cells. Journal of Clinical Microbiology 33, 371–5.[Abstract]

12 . Theodos, C. M., Griffiths, J. K., D'Onfro, J., Fairfield A. & Tzipori, S. (1998). Efficacy of nitazoxanide against Cryptosporidium parvum in cell culture and in animal models. Antimicrobial Agents and Chemotherapy 42, 1959–65.[Abstract/Free Full Text]

13 . Doumbo, O., Rossignol, J. F., Pichard, E., Traore, H. A., Dembele, T. M., Diakite, M. et al. (1997). Nitazoxanide in the treatment of cryptosporidial diarrhea and other intestinal parasitic infections associated with acquired immunodeficiency syndrome in tropical Africa. American Journal of Tropical Medicine and Hygiene 56, 637–9.[ISI][Medline]

14 . Rossignol, J. F., Hidalgo, H., Feregrino, M., Higuera, F., Gomez, W. H., Romero, J. L. et al. (1998). A double-blind placebo-controlled study of nitazoxanide in the treatment of cryptosporidial diarrhoea in AIDS patients in Mexico. Transactions of the Royal Society of Tropical Medicine and Hygiene 92, 663–6.[ISI][Medline]

15 . Abaza, H., El-Zayadi, A., Kabil, S. M. & Rizk, H. (1998). Nitazoxanide in the treatment of patients with intestinal protozoan and helminthic infections: a report on 546 patients in Egypt. Current Therapeutic Research 59, 116–21.[ISI]

Received 19 July 1999; returned 30 November 1999; revised 16 December 1999; accepted 6 February 2000