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Antimicrobial Agents and Chemotherapy, July 2003, p. 2299-2302, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2299-2302.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Inhibitory Effect of NO-Releasing Ciprofloxacin (NCX 976) on Mycobacterium tuberculosis Survival

R. Ciccone,1 F. Mariani,2 A. Cavone,1 T. Persichini,3 G. Venturini,3 E. Ongini,4 V. Colizzi,1 and M. Colasanti3*

Department of Biology, University of Rome "Tor Vergata,",1 Institute of Neurobiology and Molecular Medicine, National Council of Research,2 Department of Biology University of Rome "Roma Tre," Rome,3 Nicox Research Institute, Bresso, Milan, Italy4

Received 18 June 2002/ Returned for modification 29 July 2002/ Accepted 21 March 2003


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ABSTRACT
 
Here, we report the antimycobacterial activity of NCX 976, a new molecule obtained adding a NO moiety to the fluoroquinolone ciprofloxacin, on Mycobacterium tuberculosis H37Rv strain, both in a cell-free model and in infected human macrophages. Unlike unaltered ciprofloxacin, NCX976 displayed a marked activity also at low-nanomolar concentrations.


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TEXT
 
Mycobacterium tuberculosis, which causes tuberculosis (TB), is the greatest single infectious cause of mortality worldwide, killing roughly 2 million people per year. Estimates indicate that one-third of the world population is infected with latent M. tuberculosis. The synergy between TB and the AIDS epidemic, and the surge of multidrug-resistant (MDR) clinical isolates of M. tuberculosis have reaffirmed TB as a primary public health threat. Hence, the development of new drugs, as well as improvement of the existing drugs, as new potential antitubercular agents could have a profound impact on the TB therapy (16).

Nitric oxide (NO) has been found to possess microbicidal effects against a number of pathogens, including DNA and RNA virus families (1, 5) and intracellular and extracellular protozoa (4;M. Colasanti, L. Salvati, G. Venturini, P. Ascenzi, and L. Gradoni, Letter, Trends Parasitol. 17:575, 2001). The production of NO and other reactive nitrogen intermediates by innate immune cells is also considered to be an effective host-defense mechanism against bacterial pathogens, including M. tuberculosis (3, 9, 12, 14). Recently, a mycobactericidal action of exogenous NO has been unambiguously observed for both the H37Rv laboratory strain and a multidrug-resistant wild M. tuberculosis strain, thus suggesting that exogenous NO can have a potential use in the treatment of tuberculosis (10).

The synthesis of NO-releasing molecules is an innovative strategy to design novel drugs (7). These compounds slowly release NO via an enzymatic pathway, exhibiting new biological activities. In this respect, ciprofloxacin, a fluoroquinolone currently used to treat mycobacterial infections (2), has been modified by addition of a chemical group releasing NO. Here, we compared the antimycobacterial activity of NCX 976 with that of the native compound (i.e., ciprofloxacin) on M. tuberculosis H37Rv strain, both in a cell-free model and in infected human macrophages.

Ciprofloxacin 2-methoxy-4-(3-[4'-(nitrooxy)butoxy]-3-oxo-1-propenyl]phenyl ester (NCX 976; molecular weight, 671.07) (Fig. 1) was from the Nicox Research Institute (Milan, Italy). Since NO release from NCX 976 occurs enzymatically, NCX 976 was used either in the absence or presence of esterase (2 U/ml; Sigma-Aldrich, Milan, Italy). In addition, unaltered ciprofloxacin monohydrochloride (molecular weight, 366.4; UQIFA, Barcelona, Spain) was used as a control. Both drugs were used at concentrations ranging between 1 nM and 1 µM. This range is compatible with the mean maximum serum concentration (about 15 µM) achieved following 60-min intravenous infusion of 400 mg of ciprofloxacin to normal volunteers; at 12 h, the concentration goes down to 0.2 µg/ml (0.6 µM), as it occurs after a 500-mg oral dose.



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  		FIG. 1. Chemical structure of ciprofloxacin and NCX 976 (2-methoxy-4-(3-[4'-(nitrooxy)butoxy]-3-oxo-1-propenyl]phenyl ester).

To test the antimycobacterial activity of NCX 976, the virulent strain of M. tuberculosis H37Rv was employed. First, the morphology of the acid-fast bacilli (AFB) stained by the Ziehl-Neelsen protocol was observed in the presence of NCX 976 and ciprofloxacin (Fig. 2). After 2 days of treatment of M. tuberculosis H37Rv strain with 10 nM NCX 976 plus 2 U/ml esterase, the normal morphology of AFB resulted dramatically disrupted (Fig. 2A), when compared to either unaltered ciprofloxacin (10 nM) (Fig. 2B) or 10 nM NCX 976 in the absence of esterase (data not shown). The NCX976-treated mycobacteria, as shown in Fig. 2, lost their resistance to alcohol-acid destaining and their typical red rod appearance.



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  		FIG. 2. Morphology of the AFB-stained by the Ziehl-Neelsen protocol. A virulent strain of M. tuberculosis (H37Rv) was treated with 10 nM NCX 976 (A) and 10 nM ciprofloxacin (B) for 2 days. The NO-releasing drug disrupted the normal morphology and red staining of the AFB. No effect was observed in the presence of unaltered ciprofloxacin.

The significant changes observed in the mycobacterial shape most likely led the bacilli to death. To address this question, the effect of NCX 976 on growth of M. tuberculosis H37Rv was determined in a cell-free model. Mycobacteria were grown in Sauton's medium, in which the bacilli grow as a layer on the medium surface (11), in the presence of the NO-releasing drug and the native compound. The mycobacterial clumps present in the Sauton's culture were dispersed by means of sonication in a water bath sonicator (50W, 20 kHz; UST), regulated at a maximal power of 50 W, in sterile glass tubes. The tubes were then kept in ice prior to putting the bacteria in culture plates, to prevent mycobacteria to clump again. Furthermore, in order to exclude any influence of the culture media on the bacilli sensitivity to the drug, we performed the same series of treatments with M. tuberculosis H37Rv cultivated in 7H9 Middlebrook medium, in which mycobacteria grow in a homogeneous resuspension (8). The results obtained with this second batch of mycobacteria are absolutely comparable with the ones performed with the Sauton's medium cultivated mycobacteria.

After a 24-h treatment with NCX 976 and ciprofloxacin at different concentrations (in a total range between 1 nM and 1 µM) and in the absence and presence of esterase (2 U/ml), the number of CFU was evaluated by culturing the different samples on 7H10 plates. Then, mycobacteria were washed twice with sterile phosphate-buffered saline (PBS), and incubated 30 min in 0.5 ml of 0.1% saponin-PBS. The dilutions 10-1, 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7 of each sample (in 0.01% Tween 80-PBS) were plated as 50-µl droplets on 7H10 Middlebrook (Becton Dickinson, Franklin Lakes, N.J.) medium. CFU were checked after about 21 days of culture in a 5% CO2 incubator (11). For each drug concentration and treatment studied, at least two separate experiments were performed. The colony counts from all experiments with each drug concentration and treatment were then averaged. These data were analyzed by two-way analysis of variance (ANOVA) with the F statistic to test for independent effects of drug concentration and treatment. When compared to ciprofloxacin, treatment with NCX 976 showed a significant antimycobacterial activity (F, 214.18 [P < 0.0001]). Notably, the efficacy of NCX 976 in killing the growth of cell free M. tuberculosis H37Rv was significantly high also at low drug concentrations (1 to 10 nM), when ciprofloxacin was ineffective (Fig. 3). Differences in CFU between drug concentrations were significant by two-way ANOVA (F, 38.43 [P < 0.0001]). In order to demonstrate that the antimicrobial effect of the NO-releasing drug was attributable to NO release, NCX 976 was used in the absence of esterase. Under these experimental conditions, NCX 976 (1000 nM) was unable to kill M. tuberculosis. Furthermore, esterase (2 U/ml) alone or in the presence of unaltered ciprofloxacin did not affect M. tuberculosis growth (data not shown). Note that the effect of NCX 976 was both mycobactericidal and mycobacteriostatic. In fact, when the culture plates for the CFU enumeration were kept in culture up to 45 days, the CFU number did not change, suggesting a bactericidal effect. Moreover, the samples where NCX 976 showed the higher mycobactericidal effects, were the same in which the M. tuberculosis colonies grew more slowly, thus showing that the NO-releasing drug possesses a parallel inhibitory effect.



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  		FIG. 3. Antimycobacterial activity of NCX 976 and ciprofloxacin against M. tuberculosis grown in Sauton's medium. Drugs were added to the indicated concentrations, and incubation was continued for 24 h. Then, cultures were diluted and plated for counting of CFU. Each bar represents the mean ± standard deviation (error bar) (n = 3). (F, 38.43 [P < 0.0001]). For further details see the text.

Mycobacteria are intracellular pathogens that survive and grow in host macrophages. The interaction between host cell and microbe is delicately balanced, gene-expression patterns being different in the course of intra (human macrophages)- and extracellular growth. To verify possible differences in M. tuberculosis survival in the two habitats of replication, the ability of NCX 976 to inhibit M. tuberculosis growth was also studied in infected human macrophages. Buffy coats were collected from healthy donors and mononuclear cells were separated on Ficoll gradient. After 1 h, the macrophages were separated by adherence. Infection of macrophages was performed the seventh day, at a multiplicity of infection of 10:1 bacilli: cells, keeping mycobacteria and cells in 1 ml of medium for 2 h, then washing out the bacteria three times with warm PBS, and finally putting cells in culture for other 24 h in the presence of NCX 976 and ciprofloxacin at different concentrations (in a total range between 1 nM and 1 µM). Then, the cells were washed twice with sterile PBS and incubated 30 min in 0.5 ml of lysis buffer (0.1% saponin-PBS). Each cell lysate was diluted and plated as described above and CFU checked after 21 days of culture. Experiments and statistics were performed as previously described. In agreement with the effect of NCX 976 against M. tuberculosis grown in liquid medium, treatment of infected macrophages with NCX 976 showed a significant antimycobacterial activity compared to the relative control (i.e., unaltered ciprofloxacin) (F, 95.09 [P < 0.0001]). As already observed in the experiments with extracellular M. tuberculosis, the efficacy of NCX 976 in killing the growth of intracellular M. tuberculosis H37Rv was significantly high also at low drug concentrations (1 to 10 nM) (Fig. 4). Differences in CFU between drug concentrations were significant by two-way ANOVA (F, 20.40 [P < 0.0001]). As expected, NCX 976 (1,000 nM) in the absence of esterase was unable to kill M. tuberculosis. Also, esterase (2 U/ml) alone or in the presence of unaltered ciprofloxacin did not affect M. tuberculosis growth (data not shown).



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  		FIG. 4. Antimycobacterial activity of NCX 976 and ciprofloxacin against M. tuberculosis in human macrophages. Infection of macrophages was performed at a multiplicity of infection of 10:1 (bacilli:cells). Infected cells were treated for 24 h in the presence of NCX 976 and ciprofloxacin at the indicated concentrations. Then, cells were lysed, and each cell lysate was diluted and plated for counting of CFU after 21 days of culture. Each bar represents the mean ± standard deviation (error bar) (n = 3). (F, 95.09 [P < 0.0001]). For further details see the text.

Taken together, our data indicate that the NO-releasing ciprofloxacin (NCX 976) exerted an antimicrobial activity against both extracellular and intracellular M. tuberculosis, also when ciprofloxacin was ineffective (i.e., at low doses). Consistently, exposure of extracellular M. tuberculosis to low concentrations of NO for short periods (24 h or less) results in microbial killing in vitro (10). However, the possibility that NO may enhance the sensitivity of M. tuberculosis to ciprofloxacin cannot be ruled out.

Also in vivo (e.g., murine models of TB), NO plays an essential role in the killing of M. tuberculosis by mononuclear phagocytes. For example, in the mouse strain with a genetic disruption for inducible NO synthase (NOS-II-/-), infection with M. tuberculosis is associated with a significantly higher risk of dissemination and mortality (3). Moreover, it has been established that NO is required for the control of murine tuberculosis infection caused by both laboratory and clinical strains of M. tuberculosis (14). This protective role of NO is essential for the control of the infection established by either intravenous or aerogenic challenge (14). There is a growing body of evidence, although still controversial, that also in humans, NO as produced by TB-infected macrophages and by epithelial cells, possesses antibacterial effects against M. tuberculosis (9). Interestingly, mobilization of NO-dependent antimicrobial mechanisms in human host cells appears to be critical for quinolone-mediated elimination of intracellular M. tuberculosis (17).

As a whole, this finding may be important for the screening of new quinolones with more potent anti-M. tuberculosis activities. In this respect, NO-releasing ciprofloxacin, more effective than unaltered ciprofloxacin, might both shorten the time of the chemotherapy and reduce the rise of drug-resistant strains. It should be pointed out, however, that M. tuberculosis is able to evoke a response to combat the toxic effect of NO (6, 8, 12, 13, 15, 18). Moreover, the effect of NO-releasing drugs in vivo may be limited for the presence of NO scavengers as well as reductants. Therefore, further studies are needed to evaluate the possible use of these compounds, alone or in combination with other antimycobacterial drugs, in the treatment of TB.


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ACKNOWLEDGMENTS
 
This study was supported by a grant from Nicox S.A. (Sophia Antipolis, France) to M.C.

We thank L. Mattace for editorial assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, University "ROMA TRE," Viale Marconi, 446, I-00146 Rome, Italy. Phone: 39 06 55176342. Fax: 39 06 55176383. E-mail: colasant{at}uniroma3.it. Back


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Antimicrobial Agents and Chemotherapy, July 2003, p. 2299-2302, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2299-2302.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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