Elsevier

Tuberculosis

Volume 98, May 2016, Pages 92-96
Tuberculosis

Drug discovery and resistance
Activity of nitazoxanide and tizoxanide against Mycobacterium tuberculosis in vitro and in whole blood culture

https://doi.org/10.1016/j.tube.2016.03.002Get rights and content

Summary

Nitazoxanide (NTZ) and its metabolite tizoxanide (TIZ) were studied as antimycobacterial agents in vitro (in mycobacterial growth indicator tube [MGIT] cultures) and in a whole blood bactericidal assay. Both NTZ and TIZ show high protein binding. In MGIT cultures (albumin concentration = 78 μM), inhibition of Mycobacterium tuberculosis growth occurred at total drug concentrations of ≥16 μg/ml, whereas in whole blood cultures (albumin concentration = 350 μM), ≥128 μg/ml was required. Free drug fractions at these two conditions were estimated to be 69% and 2%, respectively. Co-incubation of NTZ and TIZ in human plasma for 72 h nearly completely eliminated their ability to inhibit mycobacterial growth in MGIT. Interactions with plasma proteins may limit the potential of NTZ and TIZ as drugs for human tuberculosis.

Introduction

The high prevalence and mortality of tuberculosis is largely due to a lack of easy to deliver, effective treatment. Better medications are needed. Nitazoxanide (NTZ) is a synthetic nitrothiazolyl-salicylamide derivative approved for the treatment of intestinal protozoal infections. It is inexpensive, orally available, well tolerated, and safe [1]. The mechanisms of action of NTZ against Mycobacterium tuberculosis (MTB), disruption of membrane potential and pH homeostasis, differ from those against intestinal pathogens [2]. In addition to targeting MTB directly, NTZ may enhance the host's immune response by stimulating macrophage autophagy, and MTB appears to have a high threshold for developing resistance to NTZ [3], [4], [5].

NTZ is rapidly and completely hydrolyzed (half-life of 6 min) by plasma esterases into its active metabolite tizoxanide (TIZ), the only form found in the circulation [6]. This conversion to TIZ is unchanged even when plasma is diluted 10 fold [6], [7]. NTZ also hydrolyzes spontaneously to TIZ in aqueous media alone, with a half-life of 3 h under the conditions in this study [6]. TIZ is 99% protein bound at concentrations reached in plasma after oral dosing [8]. However, the impact of protein binding on the bioactivity of TIZ has not been previously reported.

Although further assessment of NTZ's anti-TB properties is warranted, studies in mice have not proven useful, as NTZ is glucuronidated to an inactive form in that species [9]. An alternative method for evaluating the intracellular activity of NTZ and TIZ against MTB is the whole blood bactericidal assay (WBA). In this assay, a small number of mycobacteria are inoculated into whole blood diluted with an equal volume of tissue culture medium. Bacilli are phagocytosed by leukocytes and allowed to grow for a 72 h incubation period [10]. This assay requires a sensitive mycobacterial growth detection method, such as the mycobacterial growth indicator tube system (MGIT) [11]. After 72 h, phagocytes are lysed and remaining mycobacteria are pelleted, and then cultured in MGIT cultures. Differences in time to positivity (TTP) of the original inoculum and experimental results reflect a change in the number of viable mycobacteria [4], [10], [11], [12], [13]. If no mycobacterial growth or killing occurred, TTP of mycobacteria at the end of the WBA equals that of the original inoculum. Increased TTP correlates with lower residual mycobacteria and indicates intracellular killing during the incubation in whole blood due to drug effects and/or host defenses [10]. NTZ has not previously been evaluated in the WBA, although it has anti-MTB activity in macrophage studies [2], [3], [9], [14]. This study evaluated the activity of NTZ and TIZ against MTB in vitro and by WBA.

Section snippets

Preparation of NTZ and TIZ

NTZ and TIZ were received from the Infectious Disease Research Institute on behalf of the Lilly MDR-TB Partnership Drug Discovery Initiative as lyophilized powder. Drugs were reconstituted in 100% DMSO to a concentration of 32 mg/ml, aliquoted and stored at −70 °C.

Preparation of MTB, Mycobacterium bovis BCG and use of MGIT system

M. tuberculosis-H37Rv (NR-13648) (MTB) was obtained from BEI Resources (Manassas, VA), grown in MGIT growth medium, aliquoted, and stored at −70 °C. MGIT growth medium consisted of Middlebrook 7H9 broth with OADC (oleic acid, bovine

Direct effect of NTZ and TIZ on MTB growth in MGIT

NTZ and TIZ directly inoculated into MGIT tubes began to inhibit MTB growth, as shown by a significantly greater TTP than “no drug”, at 16 μg/ml (p = 0.002 and 0.006, for NTZ and TIZ, respectively), with almost complete inhibition of growth at 64 μg/ml (Figure 1A). TTP was also significantly longer at 32 μg/ml when compared to 16 μg/ml and at 64 μg/ml when compared to 32 μg/ml (p = 0.002 and 0.001 for NTZ and p = 0.004 and <0.0005 for TIZ, respectively). The concentration of bovine albumin in

Discussion

Peak plasma TIZ concentrations in healthy human volunteers after 7 days of treatment with 500 mg of NTZ every 12 h (the recommended dose) reached 10 μg/ml; dosing at 1 g every 12 h produced 22 μg/ml [15]. Single doses of 4 g yielded Cmax values of 17.5 μg/ml, with an upper range of 26.5 μg/ml [6]. Our study demonstrates that NTZ and TIZ begin to have activity against MTB when directly inoculated into MGIT tubes at 16 μg/ml, although the magnitude of the response appears relatively small until

Acknowledgments

We would like to thank Roxana Rojas and Qing Li for their help and sharing of THP-1 growth results.

This research was supported by National Institute of Allergy and Infectious Diseases, NIH, contract HHSN266200700022C/NO1-AI-70022. EPH was supported by T32-AI07024. The sponsors had no role in the study or in the writing and publication of the manuscript.

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Present address: Novartis Institute for Biomedical Research, Cambridge, MA 02139, USA.

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