Activity of nitazoxanide alone and in combination with azithromycin and rifabutin against Cryptosporidium parvum in cell culture

Andrea Giacometti*, Oscar Cirioni, Francesco Barchiesi, Fausto Ancarani and Giorgio Scalise

Institute of Infectious Diseases and Public Health, University of Ancona, Ancona, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro activity of nitazoxanide alone and in combination with azithromycin and rifabutin was investigated against four clinical isolates of Cryptosporidium parvum. The susceptibility tests were performed by inoculation of the isolates on toe cell monolayers and determination of the parasite count after 48 h incubation at 37°C. The culture medium was supplemented with Dulbecco's modified Eagle's medium containing serial dilutions of each agent. Antibiotic-free plates were used as controls. Experiments were performed in triplicate. Nitazoxanide showed moderate anticryptosporidial activity: it suppressed the growth of parasites by >50% at 8 mg/L. A parasite reduction of 79.8–83.9% was observed when nitazoxanide 8 mg/L was combined with azithromycin 8 mg/L and rifabutin 8 mg/L. The study suggests that nitazoxanide may be active in inhibiting C. parvum growth in vitro upon combination with azithromycin or rifabutin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In humans and livestock, especially calves, lambs and kids, cryptosporidiosis is a serious enteric disease caused by the coccidial zoonotic protozoan parasite Cryptosporidium parvum. In immunocompetent individuals it causes an influenza-like illness that is usually resolved in 2–3 weeks. In immunodeficient individuals, especially those with AIDS, the disease is often protracted and severe and may become life threatening.1,2 Despite considerable efforts to identify and develop new anticryptosporidial agents, no effective therapy for immunodeficient individuals with cryptosporidiosis exists and no compound has as yet received regulatory approval for treatment or prevention of this infection.35

Nitazoxanide is a nitrothiazole benzamide compound [20(acetolyloxy)-N-(5-nitro-2-thiazolyl) benzamide] that has a wide range of antimicrobial activity against parasites and bacterial pathogens. The broad spectrum of in vivo activity is related to its desacetyl derivative, tizoxanide, and includes intracellular and extracellular protozoa (microsporidia, C. parvum, Giardia intestinalis, Entamoeba and Trichomonas spp.), helminths (Taenia and Hymenolepis spp.) and aerobic and anaerobic bacteria.69 Its mechanism of action is not known. Previous reports have demonstrated that nitazoxanide has a dose-dependent effect on DNA synthesis and that the nitro group of the compound must be reduced in the target organism.10 Pharmacokinetic studies in healthy volunteers showed that its active derivative, tizoxanide, was the only measurable compound in plasma, where it reached a maximum concentration of 1.9 mg/L (range 11–2.5) 2–6 h after ingestion of a single dose of 500 mg of nitazoxanide.11

Azithromycin is a macrolide antibiotic recently introduced as a potential anticryptosporidial drug. In vitro studies have shown azithromycin to be active in inhibiting C. parvum growth at concentrations close to those achievable in vivo.12 It has also been reported to be effective as suppressive therapy for cryptosporidiosis in steroid-immunosuppressed rodents, and a prompt clinical improvement has been described in two children with cancer and severe cryptosporidiosis-associated diarrhoea after administration of this drug.1316 Azithromycin is currently undergoing clinical trials as an anticryptosporidial agent.17

Rifabutin is a spiro-piperidyl-rifamycin derived from rifamycin-S. It is structurally related to and shares many properties of rifampicin.18 Although there are few or no published studies of rifabutin in treating cryptosporidiosis, a recent study demonstrated a robust and statistically significant protective effect of rifabutin, used for Mycobacterium avium complex chemoprophylaxis or treatment in immunosuppressed HIV-positive individuals, in preventing cryptosporidiosis.19

To study new drug combinations for the treatment of cryptosporidiosis the in vitro activity of nitazoxanide alone and combined with azithromycin and rifabutin is here investigated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Organisms

Four strains of C. parvum, isolated from stools of four patients with AIDS, were used throughout the study.

Drugs

Nitazoxanide (Unimed Pharmaceuticals, Inc., Buffalo Grove, IL, USA), azithromycin (Pfizer/Roerig, Rome, Italy) and rifabutin (Pharmacia & Upjohn, Milan, Italy) were dissolved in 50% methanol/50% acetone at a concentration of 1 mg/mL. Solutions of drugs were made fresh on the day of assay or stored at –80°C in the dark for short periods.

Parasite preparation

Oocysts were pretreated by suspending samples of stock oocysts in a bleach solution containing 9 parts sterile deionized water and 1 part 5.25% sodium hypochlorite for 10 min. The oocysts were then washed twice in sterile water, centrifuged again and resuspended in Dulbecco's modified Eagle's medium (DMEM, BioWhittaker, Inc., Walkersville, MD, USA) warmed to 37°C. Excystation of sporozoites was achieved by incubating oocysts in phosphate-buffered saline (PBS; pH 7.2) containing 0.25% trypsin and 0.75% sodium taurocholate for 60 min at 37°C. Free sporozoites were pelleted by centrifugation (200g for 20 min), resuspended in DMEM, counted in a haemocytometer and aliquoted for culture.

Cell cultures

A-549 cells (BioWhittaker) were maintained in 25 cm2 tissue culture flasks. The medium consisted of DMEM with 10% fetal calf serum (BioWhittaker), 1% L-glutamine (BioWhittaker), 20 mM N-2-hydroxyethylpiperazine-N-ethanesulphonic acid (HEPES; Sigma-Aldrich S.r.l., Milan, Italy), penicillin G (100 U/mL), streptomycin (100 mg/L) and amphotericin B (0.5 mg/L). Cells were lifted from the surface of flasks using a solution of 0.25% trypsin and 0.53 mM EDTA in PBS; then they were counted using a haemocytometer. Forty-eight hours before parasite inoculation, A549 cells were plated on to 35 mm diameter tissue culture plates at a concentration of 105 viable cells in a total volume of 5 mL. Viability was assessed by trypan blue exclusion. The infection of the cell monolayer was initiated by adding 104 sporozoites in 0.2 mL of medium. After incubation for 4 h at 37°C in 5% CO2 to allow attachment and penetration of sporozoites, the monolayers were washed with DMEM to remove non-invasive sporozoites, residual oocysts and non-adherent epithelial cells, and 5 mL of new growth medium with or without antimicrobial agents was added. Infected cell cultures were kept at 37°C in 5% CO2 throughout the study.12,20

Susceptibility test

Nitazoxanide, azithromycin and rifabutin were examined singly at concentrations of 0.5, 2 and 8 mg/L. In experiments to test drug interactions, nitazoxanide was combined with azithromycin and rifabutin. Antibiotic-free plates were used as controls. Experiments were performed in triplicate. C. parvum sporozoites were added at a concentration of 104 sporozoites per plate. The monolayers were incubated for 48 h at 37°C in 5% CO2. Following four washes in PBS to remove free oocysts and non-adherent epithelial cells, 5 mL of new growth medium was added and the monolayers were observed under Nomarski interference contrast optics at 1000x.21,22 Parasite growth was assessed 48 h after infection in 50 random oil fields. Only meronts and gamonts were enumerated to avoid counting nonviable, but adherent sporozoites or merozoites.2123

Cytotoxicity assay

The cytotoxicities of nitazoxanide and its combinations were determined by CellTiter 96 AQ cell proliferation assay (Promega Corp., Lyon, France). Controls for each cytotoxicity assay included (i) uninfected cells incubated in DMEM, (ii) infected cells incubated in DMEM and (iii) cells exposed to a freeze–thaw lysate containing 104 oocyst equivalents in DMEM.

Analysis of results

The activities of each agent alone and in combination were evaluated by counting parasites from plates with antimicrobial-supplemented medium compared with control plates without drugs. The percentage reduction in parasite counts (PC) was calculated as [(mean PC of controls – mean PC of treated cultures)/mean PC of controls] x 100. The percentage cytotoxicity was calculated from the optical density (OD) as [(mean OD of uninfected cells – mean OD of infected cells)/mean OD of uninfected cells] x 100.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In control plates without drugs the average number of parasites in 50 random oil fields was 41.1 (range 29.4–52.8). A high preponderance of meronts over microgamonts was observed; macrogamonts were not seen 48 h after infection.

No compound produced complete inhibition of parasite growth. Nitazoxanide exhibited the highest anticryptosporidial activity: growth of meronts and gamonts was suppressed by 56.1% at 8 mg/L, whereas at the same concentration azithromycin and rifabutin produced decreases in parasite counts of 25.5 and 22.9%, respectively.

The cumulative anticryptosporidial activities of nitazoxanide, azithromycin and rifabutin tested in combination reached 80% inhibition in parasite counts when the drugs were combined at the highest concentrations. Nitazoxanide 8 mg/L with azithromycin 8 mg/L showed the highest activity against C. parvum, effecting a reduction of 83.9%. The combination of nitazoxanide 8 mg/L and rifabutin 8 mg/L was less potent, producing a decrease in parasite counts of 79.8%. The results are summarized in the TableGo.


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Table. Inhibitory effects of nitazoxanide in combination with azithromycin and rifabutin on C. parvum in A-549 cells: parasite count and percentage reduction versus control plates without antimicrobials
 
The cytotoxic effect was practically absent at all concentrations of drugs tested alone (cytotoxicity –8.9 to 11.2%) and for all nitazoxanide combinations (cytotoxicity –6.5 to 8.4%).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although several papers have been devoted to studying development of C. parvum in cell culture, there continues to be a lack of a reproducible model system for studying host cell–parasite interactions in vitro.24 There is some controversy about the procedures used to test for in vitro susceptibility. These can vary in the method of infection with oocysts or sporozoites, the timing of the start of drug treatment, the duration of incubation, the method of quantitation and the analysis of results.25

In the present study, nitazoxanide was evaluated in an in vitro system in combination with azithromycin and rifabutin. Parasite growth was assessed by quantifying meronts and gamonts after 48 h incubation at 37°C in an atmosphere of CO2.

Nitazoxanide was combined with two agents that can play a role in multiple opportunistic pathogen prophylaxis (MOPP). In immunocompromised individuals MOPP is premised on the advantages of using agents with activity against a variety of opportunistic pathogens. Rifabutin has a potential role to play in a MOPP regimen because it is active against mycobacteria, Gram-positive cocci, Haemophilus influenzae, Legionella pneumophila, Helicobacter pylori, Campylobacter jejuni, Chlamydia trachomatis and Toxoplasma gondii.5,18,26 On the other hand, azithromycin is active against several Gram-positive and Gram-negative bacteria, mycobacteria and T. gondii. Studies of the efficacy of azithromycin or combination regimens are in progress.5

Our data demonstrate the good in vitro activity of the new drug combinations and partially confirm the activity of nitazoxanide tested singly. Although our results for nitazoxanide tested alone are not in agreement with the high efficacies observed in other animal models and in cell cultures, combinations with this drug markedly decreased parasite counts in the plates with the highest concentrations.25,27 Moreover, the cytotoxicity assay suggests that these combinations are safe.

The mechanism of the additive anticryptosporidial effect of nitazoxanide combined with azithromycin and rifabutin appears to be complex. As mentioned above, nitazoxanide has a dose-dependent effect on DNA synthesis, while the action of azithromycin and rifabutin results in inhibition of protein synthesis. The antimicrobial activity of azithromycin is known to result from its ability to inhibit protein synthesis by binding to the transpeptidation site of the larger ribosomal subunit,12 whereas that of rifabutin is produced by inhibition of DNA-dependent RNA polymerase and subsequent initiation of transcription.18 We presume that the additive effect observed between nitazoxanide and the other two agents may be a consequence of the cumulative inhibitory effect on different and essential metabolic pathways. Our results showed that the combinations tested were active in inhibiting C. parvum at concentrations that appeared not to be toxic to the human cell monolayer.

Despite advances in anticryptosporidial chemoprophylaxis studies, no comprehensive strategy for prevention and treatment of cryptosporidiosis has yet been developed. We believe it would be useful to explore the feasibility of combining the most widely used anticryptosporidial drugs in order to find active combinations against cryptosporidiosis.


    Notes
 
* Corresponding address. Clinica Malattie Infettive, c/o Azienda Ospedaliera Umberto I, Piazza Cappelli 1, 60121 Ancona, Italy. Tel: +39-71-596-3467; Fax: +39-71-596-3468; E-mail: cmalinf{at}popcsi.unian.it Back


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 2 June 1999; returned 19 August 1999; revised 12 October 1999; accepted 10 November 1999