J. Med. Chem. 2009, 52, 5789–5792 5789 DOI: 10.1021/jm9010719
*To whom correspondence should be addressed. Phone: 212-7466505. Fax: 212-746-8587. E-mail:
[email protected]. a Abbreviations: Mtb, Mycobacterium tuberculosis; CFU, colony forming unit; FOR, frequency of resistance; NTZ, nitazoxanide; FDA, U.S. Food and Drug Administration; TIZ, tizoxanide; PFOR, pyruvate ferredoxin oxidoreductase; MIC, minimal inhibitory concentration; BCG, bacillus Calmette-Guerin; DMSO, dimethyl sulfoxide; BSA, bovine serum albumin; HIV, human immunodeficiency virus.
tizaxonide (TIZ), is believed to enter cells and inhibit pyruvate-ferredoxin oxidoreductase (PFOR)17 and perhaps also nitroreductases and peptide disulfide isomerases.18-20 The presumed essentiality of PFOR in anaerobes and microaerophils is thought to account for the preferential activity of NTZ against such microbes. In contrast, NTZ is nearly inactive against aerobically cultured Staphylococcus, Enterococcus, Pseudomonas, and Enterobacteriaceae.21 Besides its wide spectrum of activity, NTZ is remarkable for its apparent lack of toxicity and the absence of reports of microbial resistance during its clinical use.22 We describe here the first studies of the potential of NTZ against mycobacteria. NTZ’s MIC values were the same (16 μg/mL) for Mtb and M. bovis var. BCG, in liquid and solid media. TIZ’s MIC for Mtb in liquid medium was also 16 μg/ mL. Because of this similarity, subsequent tests were performed with the parent compound NTZ. MICs can only be determined under conditions that support bacterial replication. We assayed bactericidal activity under replicating and nonreplicating conditions (Figure 1). Instead of standard inocula of ∼106 CFU/mL, inocula of ∼108 CFU/mL were used here to mimic the concentrations of Mtb reported to be present in cavitary lesions3 and to demonstrate NTZ’s ability to afford multilog killing. In the absence of the drug, neither growth nor killing was observed under the nonreplicating conditions. Under replicating conditions in the absence of NTZ, Mtb grew even at this high density, as shown by the increase in CFU over the initial inoculum. With replicating Mtb, 4 days of exposure to NTZ at 125 μg/mL reduced the CFU by 2 log10 below that of DMSOtreated controls. When replication of Mtb was halted by nitrite at a mildly acidic pH, the same concentration of NTZ produced 6-8 log10 reduction in CFU. Within the range tested (105-108/mL), the starting inoculum had little impact on the mycobactericidal activity of NTZ under growth-promoting conditions, as illustrated in the Figure 2 inset. In contrast, under growth-inhibitory conditions, there was an inverse correlation between the inoculum and the extent of killing (Figure 2 inset). Extending the exposure time led to markedly greater killing of Mtb in response to a single addition of NTZ (Figure 3), indicative of time-dependent killing. Under replicating conditions, NTZ at 62 μg/mL reduced the number of viable Mtb by 1 log10 at 4 days and by ∼3.5 log10 at 10 days. Under nonreplicating conditions, CFU was reduced by g5 log10 (below the limit of detection) in 6 days. TIZ binds extensively to serum proteins.19 Consistent with that, a sharp decrease in the MIC for NTZ was observed as the BSA concentration was lowered from 5 to 0 mg/mL (Figure 4). A limiting value of 1 μg/mL was obtained in the absence of BSA, suggesting that the intrinsic mycobactericidal potency of NTZ is high and that its target(s) is (are) essential under the conditions tested. Using a standard method for evaluation of FOR of antimycobacterial agents,4,23-25 we were unable to observe any colonies at any time up to 5 weeks following exposure of up to 1.78 1012 CFU of Mtb to NTZ at 60 and 100 μg/mL (∼4 and 6 MIC, respectively). This experiment was repeated twice at 4 MIC and 3 times at 6 MIC. Under similar conditions,
r 2009 American Chemical Society
Published on Web 09/08/2009
Nitazoxanide Kills Replicating and Nonreplicating Mycobacterium tuberculosis and Evades Resistance Luiz Pedro S. de Carvalho, Gang Lin, Xiuju Jiang, and Carl Nathan* Department of Microbiology and Immunology, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065 Received July 20, 2009 Abstract: We report here that nitazoxanide (NTZ) and its active metabolite kill replicating and nonreplicating M. tuberculosis at low μg/mL levels. NTZ appears to evade resistance, as we were unable to recover resistant colonies, using up to 1012 colony forming units. Therefore, NTZ is a novel lead compound that kills replicating and nonreplicating M. tuberculosis by a novel mechanism of action, which appears to bypass the development of resistance.
Although tuberculosis is curable, treatment is more prolonged than for almost any other infectious disease. Nonoptimal or discontinuous antibiotic treatment selects for strains of Mycobacterium tuberculosis (Mtba) resistant to multiple drugs. Such strains now cause an estimated 450 000 cases of tuberculosis a year.1,2 Mtb’s success in becoming drug-resistant is due in large part to its burden in the host with advanced disease, estimated to reach 107-109 colony forming units (CFU) per cavity,3 coupled with a frequency of resistance (FOR) to existing anti-infectives high enough to make it likely that resistant clones of Mtb pre-exist in a population of that size. FOR has been measured in vitro for all currently approved and experimental drugs in clinical trials for tuberculosis and ranges from 10-6 (e.g., isoniazid, pyrazinamide) to 10-7-10-8 (e.g., rifampicin, nitroimidazoles, diarylquinoline) to 10-8-10-9 (linezolid).4-6 Selection for resistant clones can be eliminated by combination chemotherapy but not if treatment is discontinuous or prematurely terminated. Combination therapy requires 6 months or more; this is thought to reflect that most of the agents used in current regimens, except for rifamycins and pyrazinamide, are relatively inactive against a nonreplicating subpopulation of Mtb. Thus, there is great interest in identifying new antimycobacterial chemophores with activity against nonreplicating Mtb7-9 that have a low FOR. Nitazoxanide (NTZ) was approved by the U.S. Food and Drug Administration (FDA) in 2002 for the treatment of infections caused by the protozoans Giardia and Cryptosporidium. Currently, NTZ is in clinical trials for infections caused by the bacteria Helicobacter and Clostridium and the viruses hepatitis B and C.10-16 NTZ is a prodrug (Scheme 1) that undergoes deacetylation in the stomach. Its active metabolite,
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Scheme 1. Schematic Representation of NTZ’s Activation Mechanism to TIZ and Possible Targets
Figure 1. Killing of replicating and nonreplicating Mtb by NTZ. Mtb (∼108 CFU/mL) was exposed to various concentrations of NTZ under growth promoting conditions, 7H9 medium at pH 6.6 (circles), and under growth inhibitory conditions, pH 5.5 with 0.5 mM NaNO2 (squares). Open or closed symbols represent data from two independent experiments. After 4 days, serial dilutions were plated in media free of drug and CFU determined 3 weeks later. Dashed line represents the limit of detection. Asterisk indicates samples in which no colony was detected. Points are averages of triplicates from one experiment. Error bars show standard errors. Inocula were 3.4 108 and 1.6 108 CFU/mL for growth promoting conditions and 1.5 108 and 3.1 108 CFU/mL for growth inhibitory conditions.
Figure 3. Time dependence of NTZ killing of Mtb under growth sustaining and growth inhibitory conditions. Mtb in 7H9 medium at pH 6.6 (closed circles) and in 7H9 medium at pH 5.5 with 0.5 mM NaNO2 (open circles) was exposed to NTZ at 62 μg/mL for 4, 6, 8, and 10 days, at which times serial dilutions were plated for CFU. Dashed line indicates the limit of detection. Asterisks indicate samples in which no colony was detected. This result is representative of two experiments. Points are averages of triplicates from one experiment, and error bars show standard errors.
Figure 4. Effect of BSA on the MIC for NTZ. Closed and open bars represent two independent experiments performed in triplicate. Standard errors were zero. 7H9 medium was prepared as described in Supporting Information. Mtb was exposed to a range of NTZ concentrations for 8 days at 37 °C.
Figure 2. Effect of inoculum on the activity of NTZ against Mtb under replicating and nonreplicating conditions. Mtb was exposed to 50 μg/mL of NTZ in 7H9 medium at pH 6.6 (closed circles) and in 7H9 medium at pH 5.5 with 0.5 mM NaNO2 (open circles). The concentration of NTZ used was chosen to produce only 1-3 log10 killing after 4 days (Figure 2). Data are from two independent experiments, each performed in triplicate. Cultures were serially diluted at 4 days and plated for determination of CFU. Vertical error bars show standard errors of viable Mtb counts, and horizontal error bars show standard errors of the inoculum counts. Lines represent the linear regression of the data. Inset illustrates the reduction in CFU from the input, as a function of inoculum.
rifampicin’s FOR was estimated to be 1.1 10-8 (at 1 μg/mL rifampicin), in accordance with previous determinations.4,26 Moreover, passaging Mtb for 5 weeks in liquid medium containing a sub-MIC concentration of NTZ (10 μg/mL) did not lead to appearance of any resistant colonies after plating in 60 μg/mL NTZ. The lack of resistant colonies under all conditions tested, including very high inocula and low concentrations of drug (4 MIC), precluded the determination of the true rate of resistance or the characterization of resistant mutants. NTZ, an FDA-approved antiparasitic drug with activity against some anaerobic and microaerophilic bacteria, has
Letter
been well tolerated clinically for treatment of gastrointestinal infections caused by Giardia lamblia and Cryptosporidium parvum, with no reported incidence of resistance. PFOR, the enzyme that is considered the primary target of NTZ, has not been annotated or identified in Mtb. Thus, we were surprised to discover that NTZ was active against Mtb, particularly given that Mtb was cultured in air. It will be of interest in further studies to determine if NTZ is bacteridical to Mtb cultured under hypoxic conditions. Hypoxia is a pathologically relevant condition that Mtb is believed to face in the host.27,28 Other such conditions include mild acidity and nitrosative stress, which markedly enhanced the mycobactericidal effect of NTZ in our experiments. The mycobactericidal effect of NTZ was also markedly enhanced by the time of exposure. The time-dependent effect may indicate that killing is limited by slow penetration of the compound or by a subsequent activation step that may generate the final active species, as seen with isoniazid, PA-824, and other antimycobacterial agents.6,29,30 It was extraordinary that not a single resistant mutant could be detected in bacterial populations that are thought to be comparable in size to those in patients with advanced disease, even at relatively low concentrations of drug (4 MIC). This ultralow FOR suggests that NTZ has multiple targets in Mtb. If so, this would be an example of the “polypharmacology” that is considered highly desirable in new anti-infectives.31 All three unexpected results (killing of mycobacteria putatively lacking the presumptive target, activity against nonreplicating mycobacteria, and ability to evade resistance) underscore the importance of identifying how NTZ is metabolized in Mtb and what is/are its target(s). The MIC values for NTZ (16 and 1 μg/mL in the presence and absence of BSA, respectively) and the concentrations reducing viable, nonreplicating Mtb by 2 log10 in 4 days (and by greater amounts over longer periods) are in the range of the maximal plasma concentration of the deacetylated form achieved in adults treated with a single oral dose of 500 mg (TIZ Cmax = 10.6 ( 2.0 μg/mL).22 Mycobactericidal activity of NTZ in a host may require different dosing and/or formulation for tuberculosis than for cryptosporidiosis and giardiasis, although the IC50 or MIC for Giardia and Cryptosporidium are about 2.5 and 10 μg/mL, respectively.10,16 Controlled release tablets of NTZ are currently in use in clinical trials for viral hepatitis and are reported to improve NTZ’s blood levels.15 It could prove rewarding to screen analogues of NTZ or TIZ for improved uptake by and potency against Mtb and for lower serum protein binding and better bioavailability. Combination chemotherapy, now common in the treatment of cancer and HIV infection, was introduced to modern medicine in the treatment of tuberculosis.32 While it has become customary to abjure monotherapy in tuberculosis as a sure route to dissemination of drug resistant mutants, our inability to detect any NTZ-resistant mutants among >1012 CFU of Mtb raises the possibility that certain antimycobacterial compounds might be suitable for monotherapy. If active in experimental infection, NTZ might offer a therapeutic option for patients infected with extensively drug-resistant tuberculosis. Acknowledgment. The authors are grateful to Dr. Hui Tao for the preparation of TIZ, Caitlyn Dickinson, Yan Ling, and Julia Roberts for technical assistance, Drs. Kyu Rhee, Sabine Ehrt, Joeli Marrero, and Omar Vandal for stimulating
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discussions, and the Milstein Program in Chemical Biology of Infectious Diseases for support. The Department of Microbiology and Immunology is supported by the William Randolph Hearst Foundation. Supporting Information Available: Detailed experimental procedures employed. This material is available free of charge via the Internet at http://pubs.acs.org.
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