Activity of a new class of isonicotinoylhydrazones used alone and in combination with isoniazid, rifampicin, ethambutol, para-aminosalicylic acid and clofazimine against Mycobacterium tuberculosis

Alessandro De Logua,*, Valentina Onnisb, Barbara Saddic, Cenzo Congiub, Maria Laura Schivoa and Maria Teresa Coccob

a Sezione di Microbiologia e Virologia, Dipartimento di Scienze Chirurgiche e Trapianti d'Organo, Università di Cagliari, Viale Frà Ignazio 38, 09123 Cagliari; b Dipartimento di Tossicologia, Università di Cagliari, Cagliari; c Laboratorio di Analisi Ospedale SS. Trinità ASL 8, Cagliari, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The activities of six derivatives of a new class of isonicotinoylhydrazones were investigated in vitro against Mycobacterium tuberculosis H37Rv ATCC 27294, isoniazid-resistant M. tuberculosis ATCC 35822, rifampicin-resistant ATCC 35838, pyrazinamide-resistant ATCC 35828, streptomycin-resistant ATCC 35820 and 16 clinical isolates of M. tuberculosis. Several compounds showed interesting antimycobacterial activity against both ATCC strains and clinical isolates, but were less active against isoniazid-resistant M. tuberculosis. Combinations of five isonicotinoylhydrazone derivatives and rifampicin, ethambutol, para-aminosalicylic acid, isoniazid and clofazimine were also investigated against M. tuberculosis H37Rv ATCC 27294 and against ATCC drug-resistant strains. Addition of sub-MICs of some isonicotinoylhydrazone derivatives resulted in a four- to 16-fold reduction in MICs of ethambutol, para-aminosalicylic acid and rifampicin with fractional inhibitory concentrations (FICs) ranging between 0.17 and 0.37, suggesting a synergic interaction against M. tuberculosis H37Rv. Increased activity was also observed with other combinations (FICs 0.53–0.75), including isoniazid, and a synergic interaction between one of the isonicotinoylhydrazone derivatives and isoniazid (FIC 0.26) was shown against isoniazid-resistant M. tuberculosis ATCC 35822, whereas no effects were observed on combining the isonicotinoylhydrazones with clofazimine. The ability of isonicotinoylhydrazones to inhibit specifically the growth of M. tuberculosis, the high selectivity index and their ability to enhance the activity of standard antituberculous drugs in vitro indicate that they may serve as promising lead compounds for future drug development for the treatment of M. tuberculosis infections.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Tuberculosis is still a leading cause of death among those infections with a single aetiology and one-third of the world population is latently infected with Mycobacterium tuberculosis.1 First-line drugs, with superior efficacy and acceptable toxicity, used for treating infections caused by M. tuberculosis include isoniazid, rifampicin, ethambutol, streptomycin and pyrazinamide. Second-line drugs are usually characterized by lower efficacy or greater toxicity.2,3

However, the increase in AIDS-associated infections and recent outbreaks of infections sustained by multidrug-resistant (MDR) M. tuberculosis indicate the need for new effective anti-tuberculosis drugs4–7 and for alternative therapy regimens.8,9 Several tuberculosis control programmes and multidrug regimens have been proposed to prevent the spread of tuberculosis and MDR M. tuberculosis infections.10–12 Furthermore, new formulations employing drug-loaded microspheres13 and several drug combinations have been studied in order to increase the therapeutic efficacy and to reduce the toxicity of anti-mycobacterial agents.14–16

Isonicotinoylhydrazones are compounds structurally related to isoniazid and have antibacterial and antimycobacterial activities.17–20 We have synthesized a new class of isonicotinoylhydrazones, and a few members showed specific activity against M. tuberculosis NCTC 8337 ATCC 25584 with MIC of 6.25 mg/L but were ineffective in inhibiting the growth of Mycobacterium avium and Gram-positive and Gram-negative bacteria.21 Here, we describe in vitro studies of particular isonicotinoylhydrazones against 16 strains of M. tuberculosis isolated from clinical specimens and five reference strains, including four drug-resistant strains, in which we used quantitative assessments of MICs for the evaluation of the antimycobacterial activities of the compounds used alone, and in combination with isoniazid, para-aminosalicylic acid, rifampicin, clofazimine and ethambutol.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Compounds

The structures of the compounds used in this study are presented in Table 1Go. The isonicotinoylhydrazones were obtained by addition of isonicotinoylhydrazine to aryloxyacetonitriles in the presence of catalytic amounts of sodium ethoxide in anhydrous ethanolic solution as described previously.21


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Table 1. MICs (mg/L) of isonicotinoylhydrazones, isoniazid, rifampicin, ethambutol, para-aminosalicylic acid and clofazimine against five ATCC strains and 16 clinical isolates of M. tuberculosis
 
Ethambutol, clofazimine, rifampicin, para-aminosalicylic acid and isoniazid were purchased from Sigma Chemical Company (St Louis, MO, USA). Initial stock solutions of these drugs and isonicotinoylhydrazones were made in dimethyl sulphoxide (DMSO) at 10 or 20 g/L from which further dilutions were made in 7H9 broth (Difco Laboratories, Detroit, MI, USA). To avoid interference by the solvent, the highest DMSO concentration was 0.5%.22

Mycobacterial strains

The M. tuberculosis strains used in this study consisted of five ATCC strains, including H37Rv ATCC 27294, pyrazinamide-resistant ATCC 35828, rifampicin-resistant ATCC 35838, streptomycin-resistant ATCC 35820, isoniazid-resistant ATCC 35822 and 16 M. tuberculosis isolates recovered in our laboratory from clinical specimens and identified by standard biochemical tests. Both the clinical isolates and standard strains were maintained on Löwenstein–Jensen (bioMérieux, Marcy l'Étoile, France) agar slants until needed.

Antimicrobial susceptibility testing

MICs of the isonicotinoylhydrazone derivatives and isoniazid, rifampicin, clofazimine, ethambutol and para-aminosalicylic acid, employed as reference drugs, were determined by a standard two-fold agar dilution method.23 Briefly, 1 mL of 7H11 agar (Difco Laboratories, Detroit, MI, USA) supplemented with 10% oleic acid–albumin– dextrose–catalase (OADC) enrichment (Difco Laboratories) containing the drugs or isonicotinoylhydrazones in 24-multiwell plates was inoculated with 10 µL of a suspension containing M. tuberculosis 1.5 x 103 cfu/mL obtained as described below and incubated at 37°C in an atmosphere of 5% CO2. After cultivation for 21 days, MICs were read as minimal concentrations of drugs completely inhibiting visible growth of mycobacteria.

Determination of in vitro synergic activity

The determination of the effects of combinations of isonicotinoylhydrazones with isoniazid, para-aminosalicylic acid, clofazimine, ethambutol or rifampicin was studied in 7H11 agar as described for the antimicrobial susceptibility testing. Ten microlitres of the appropriate dilution of compounds in 7H9 broth were dissolved in 1 mL of 7H11 agar supplemented with 10% OADC in 24-multiwell plates to obtain final concentrations of two drugs that ranged from six dilutions below the MIC to 2x MIC, using two-fold dilutions according to Krogstar & Moellering.24 Each well received 10 µL of the test bacterial suspensions containing 1.5 x 103 cfu/mL. Plates were incubated at 37°C in 5% CO2 atmosphere for 21 days.

Interpretation of the data was achieved by calculating the fractional inhibitory concentration (FIC) as follows: FIC = (MICa combination/MICa alone) + (MICb combination/ MIC b alone).24 The FIC was interpreted as follows: FIC < 1, synergic activity; FIC = 1, indifference; FIC > 1, antagonistic activity. Employing the chequerboard technique, the lowest concentration of each agent that inhibited the organisms was plotted as an isobologram and the effect of a drug combination was considered synergic when the MIC for each drug was reduced to one-quarter of the original MIC in order to have the sum of the FICs equal to or less than 0.5.25

Inoculum preparation

Suspensions of M. tuberculosis to be used for antimicrobial susceptibility testing and for the determination of synergic activity were prepared by inoculating the organisms grown on Löwenstein–Jensen slants in tubes containing 7H9 broth supplemented with 10% albumin–dextrose–catalase (ADC) enrichment (Difco Laboratories) and Tween 80 0.05% (v/v). Suspensions were incubated aerobically for 14 days. Cells were then washed, suspended in 7H9 broth, shaken and sonicated in an ultrasonicator until visible clumps were disrupted (usually 15–30 s). Suspensions were diluted in 7H9 broth to a turbidity of no. 1 McFarland and then diluted in the same medium to 1.5 x 105 cfu/mL.

Cytotoxicity assay

Cell cytotoxicity of isonicotinoylhydrazones was tested in vitro by a cell viability assay as reported previously.26,27 Monolayers of Vero cells in 96-multiwell plates were incubated with the test compounds at concentrations of 1000–62.5 mg/L in Roswell Park Memorial Institute (RPMI) 1640 (Gibco, Rockville, MD, USA) with 5% fetal calf serum (FCS; Gibco) for 48 h and the medium replaced with 50 µL of a 1 g/L solution of 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) in RPMI without phenol red (Sigma). Cells were incubated at 37°C for 3 h, the untransformed MTT removed and 50 µL of 0.04 M HCl isopropanolic solution were added to each well. After a few minutes at room temperature to ensure that all crystals were dissolved, the plates were read using an automatic plate reader with a 650 nm test wavelength and a 690 nm reference wavelength. In the Vero cell toxicity test, sodium lauryl sulphate and isoniazid were included as positive and negative controls, respectively.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Vero cell toxicity

The cytotoxicity of isonicotinoylhydrazones as determined by the colorimetric method proposed by Denizot & Mosmann26,27 in Vero cells is reported in Table 1Go and is expressed as maximal non-toxic dose (MNTD). The results obtained indicate that the MNTDs of the test compounds ranged between 500 and 1000 mg/L and were comparable to that of isoniazid (1000 mg/L).

Determination of MICs of isonicotinoylhydrazones

The structures of compounds described in this study are shown in Table 1Go. MICs of isonicotinoylhydrazones were determined according to a standard two-fold agar dilution method under the defined conditions described above. The MIC values obtained for the ATCC strains and clinical isolates of M. tuberculosis in comparison with those of isoniazid, rifampicin, para-aminosalicylic acid and ethambutol are reported in Table 1Go. For M. tuberculosis H37Rv ATCC 27294, isonicotinoylhydrazones showed MIC values ranging between 3.12 mg/L (2c, R = 3-CH3; 2j, R = 4-Cl; and 2i, R = 3-Cl) and 12.5 mg/L, and are consistent with our results described previously for M. tuberculosis NCTC 8337 ATCC 25584.21 The compounds studied were also active in inhibiting the growth of rifampicin-resistant M. tuberculosis ATCC 35838, pyrazinamide-resistant ATCC 35828 and streptomycin-resistant ATCC 35820. In particular, 2c showed MIC values ranging between 1.56 and 12.5 mg/L. Isoniazid-resistant M. tuberculosis ATCC 35822 was inhibited by higher concentrations of isonicotinoylhydrazones with MICs ranging between 12.5 (2i) and 100 mg/L.

Determination of in vitro synergic activity

The MICs obtained by the combination of some isonicotinoylhydrazones with ethambutol, rifampicin, para-aminosalicylic acid and isoniazid against M. tuberculosis H37Rv ATCC 27294 are listed in Table 2Go. Sub-inhibitory concentrations of isonicotinoylhydrazones enhanced the antimycobacterial activity of ethambutol, rifampicin and para-aminosalicylic acid. However, synergic activity as determined by a four-fold decrease in the MIC of each agent in the combination, and thus determined by a combined FIC of <=0.5 or less according to Stratton & Coosey,25 was observed only with some combinations. In particular, the combinations 2d (R = 4-CH3)–para-aminosalicylic acid and 2i–para-aminosalicylic acid were found to be synergic, as demonstrated by the combined FICs of 0.17 and 0.36, respectively, and by the individual FICs plotted as isobolograms in the Figure (a)Go. The MIC of para-aminosalicylic acid (individual MIC 0.39 mg/L) was lowered to one-eighth (0.045 mg/L) when it was used in combination with concentrations of 2d equal to 1/16x MIC (0.39 mg/L) (individual MIC 6.25 mg/L, combined FIC 0.17) and to 0.09 mg/L in the presence of concentrations of 2d equal to 1/64x MIC (combined FIC 0.27). Compound 2i (individual MIC 3.12 mg/L) 0.78 mg/L and 0.39 mg/L lowered the MIC of para-aminosalicylic acid to 0.045 mg/L (combined FIC 0.37) and 0.19 mg/L (combined FIC 0.62), respectively.


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Table 2. Combination testing with isonicotinoylhydrazones plus antituberculous drugs against M. tuberculosis H37Rv ATCC 27294
 


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Figure. Synergic activity of isonicotinoylhydrazones: (a) 2d ({blacksquare}) or 2i (•) and para-aminosalicylic acid (PAS), (b) 2c ({blacksquare}) or 2i (•) and ethambutol (EMB), and (c) 2d and rifampicin (RMP) against M. tuberculosis H37Rv ATCC 27294 as determined by the chequerboard method. The broken lines show the theoretical plot for an additive effect.

 
The synergy observed on combining 2c or 2i with ethambutol against M. tuberculosis H37Rv ATCC 27294 is shown in the isobolograms in the Figure (b)Go. The MIC of ethambutol (individual MIC 6.25 mg/L) was lowered to 1.56 mg/L in the presence of 2c 0.39 mg/L (FICethambutol 0.25, combined FIC 0.37) or to 0.78 mg/L in the presence of 2i 0.78 mg/L (FICethambutol 0.12, combined FIC 0.37). Synergic antimycobacterial activity was also observed between 2d and rifampicin as shown in Table 2Go and in the isobologram in the Figure (c)Go, while a non-synergic increase in the activity of rifampicin against M. tuberculosis H37Rv was observed with 2c, 2j, 2i and 2k (R = 4-NO2) (combined FIC 0.75). The MIC of rifampicin for M. tuberculosis H37Rv ATCC 27294 (individual MIC 0.19 mg/L) was lowered to 0.04 mg/L in the presence of 2d 0.78 mg/L (FICrifampicin 0.25, combined FIC 0.37). Interestingly, 2j (R = 4-Cl) and 2k (R = 4-NO2) enhanced the effects of ethambutol, para-aminosalicylic acid and rifampicin against M. tuberculosis H37Rv, even though a synergic effect was not observed according to Stratton & Coosey25 (combined FICs 0.56–0.75). The inhibition of growth of M. tuberculosis H37Rv by isoniazid was enhanced by 2d (combined FIC 0.53) and 2c (combined FIC 0.75), whereas an additive effect was detected combining isoniazid with 2j, 2i or 2k (combined FIC 1). However, the combination 2d–isoniazid was found to be synergic when tested against isoniazid-resistant M. tuberculosis ATCC 35822 and the MIC of isoniazid was lowered from 200 mg/L as determined when it was tested alone to 50 mg/L when tested in the presence of concentrations of 2d equal to 1/64x MIC (combined FIC 0.26, data not shown). No influence of the isonicotinoylhydrazone derivatives was detected on the antimycobacterial activity of clofazimine.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The resurgence of tuberculosis and the recent increase in cases caused by MDR strains of M. tuberculosis have renewed interest in the development of effective anti-tuberculous drugs and new treatment regimens. In this study we investigated the ability of isonicotinoylhydrazone derivatives to inhibit the growth of M. tuberculosis in vitro. We observed that the MIC values of isonicotinoylhydrazones obtained by a standard two-fold agar dilution method were comparable to those of ethambutol and clofazimine, but were higher than the MICs of isoniazid, rifampicin and para-aminosalicylic acid against the isoniazid-susceptible strains. However, isonicotinoylhydrazone derivatives were more active than several newly synthesized and promising compounds affecting mycolic acid synthesis, such as N-octanesulphanylacetamide.28 The compounds tested were less effective in inhibiting the growth of isoniazid-resistant M. tuberculosis ATCC 35822, showing MICs ranging between 12.5 and 100 mg/L, probably as a consequence of their structure, which is chemically related to isoniazid. However, they were significantly more active than isoniazid against isoniazid-resistant M. tuberculosis ATCC 35822 (MIC 200 mg/L). Even if some reports demonstrated that higher lipophilicity was important for anti-mycobacterial activity,29 since the mycobacterial cell wall contains unique lipophilic substances such as mycolic acid and its distinctive formation may play a role in hindering drug penetration, the lipophilicity of isonicotinoylhydrazones was not the critical factor affecting their potency.

More interestingly, sub-inhibitory concentrations of isonicotinoylhydrazones induced a significant increase in the antimycobacerial activities of rifampicin, ethambutol and para-aminosalicylic acid against M. tuberculosis H37Rv, whereas no effects were observed when the isonicotinoylhydrazone derivatives were used in association with clofazimine. In particular, the associations 2c–ethambutol (combined FIC 0.37), 2i–ethambutol (combined FIC 0.37), 2d–para-aminosalicylic acid (combined FIC 0.17), 2i–para-aminosalicylic acid (combined FIC 0.36) and 2d–rifampicin (combined FIC 0.37) were found to be synergic, as demonstrated by low FIC values. The synergic effects between the isonicotinoylhydrazone derivatives and ethambutol or para-aminosalicylic acid were also observed against isoniazid-resistant, rifampicin-resistant, pyrazinamide-resistant and streptomycin-resistant strains, but were not observed between 2d and rifampicin against rifampicin-resistant M. tuberculosis ATCC 35838.

Ethambutol is a first-line drug used in many regimens suitable for directly observed treatment programmes because its activity against both extracellular and intracellular bacilli inhibits the development of resistant M. tuberculosis.30 Furthermore, ethambutol can enhance the activity of clarithromycin,14 co-amoxiclav,31 cefepime16 and many other antimycobacterial drugs such as aminoglycosides, rifamycins, quinolones and macrolides against mycobacteria, including susceptible and MDR M. tuberculosis. The synergy with ethambutol may be explained by an effect on the integrity of the mycobacterial cell wall,32 and is particularly important in MDR tuberculosis patients since clinical resistance to ethambutol is uncommon. However, ethambutol is toxic to retinal ganglion cells in vitro and in vivo,33 and ocular toxicity has been reported.34,35

Para-aminosalicylic acid, although less frequently employed, is an important drug as it may prevent the emergence of resistance to streptomycin and leads to a substantial enhancement of the efficacy of monotherapy with isoniazid.36 However, dose-related side effects such as nausea, vomiting, severe diarrhoea, hepatotoxicity, rash and fever have been described.30 Therefore, a reduction in the therapeutic doses of both ethambutol and para-aminosalicylic acid, achievable by the co-administration of isonicotinoylhydrazones, might be particularly helpful in reducing their side effects.

Interestingly, the association of isonicotinoylhydrazone derivative 2d with isoniazid was found to be synergic when tested against isoniazid-resistant M. tuberculosis ATCC 35822. It has been demonstrated that isoniazid provides clinically useful activity for treatment of patients with low-level isoniazid-resistant tuberculosis when it is used in multiple drug combination regimens, in particular with rifampicin and/or pyrazinamide. Since low-level isoniazid resistance accounts for c. 50% of isoniazid-resistant organisms in some areas, and since it has been demonstrated that higher doses of isoniazid are unlikely to be more efficacious than the standard dose,37 increased therapeutic activity of isoniazid against isoniazid-resistant M. tuberculosis could be helpful.

The uncertainties of translating susceptibility in vitro to clinical efficacy are well known. However, this study indicates that isonicotinoylhydrazone derivatives might be useful in increasing the effectiveness of standard drugs in the therapy of M. tuberculosis infections and may serve as promising compounds for future antimycobacterial drug development.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Mrs Luisa Cappai for technical assistance. This work was supported partially by MURST (Ministero dell'Università e della Ricerca Scientifica e Tecnologica) funds.


    Notes
 
* Corresponding author. Tel/Fax: +39-07-066-8001; E-mail: adelogu{at}unica.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Snider, D. E. & La Montagne, J. R. (1994). The neglected global tuberculosis problem: a report of the 1992 World Congress on Tuberculosis. Journal of Infectious Diseases 169, 1189–96.[ISI][Medline]

2 . Stead, W. W. & Dutt, A. K. (1982). Chemotherapy for tuberculosis today. American Review of Respiratory Disease 125, 94–101.[Medline]

3 . Snider, D. E., Cohn, D. L., Davidson, P. T., Hershfield, E. S., Smith, M. H. & Sutton, F. D. (1985). Standard therapy for tuberculosis. Chest 87, S117–24.[Medline]

4 . Reddy, V. M., O'Sullivan, J. F. & Gangadharam, P. R. J. (1999). Antimycobacterial activities of riminophenazines. Journal of Antimicrobial Chemotherapy 43, 615–23.[Abstract/Free Full Text]

5 . Tomioka, H., Sato, K., Akaki, T., Kajitani, H., Kawahara, S. & Sakatani, M. (1999). Comparative in vitro antimicrobial activities of the newly synthesized quinolone HSR-903, sitafloxacin (DU-6859a), gatifloxacin (AM-1155), and levofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex. Antimicrobial Agents and Chemotherapy 43, 3001–4.[Abstract/Free Full Text]

6 . Luna-Herrera, J., Reddy, M. V. & Gangadharam, P. R. J. (1995). In vitro activity of the benzoxazinorifamycin KRM-1648 against drug-susceptible and multidrug-resistant tubercle bacilli. Antimicrobial Agents and Chemotherapy 39, 440–4.[Abstract]

7 . Hirata, T., Saito, H., Tomioka, H., Sato, K., Jidoi, J., Hosoe, K. et al. (1995). In vitro and in vivo activities of the benzoxazinorifamycin KRM-1648 against Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 39, 2295–303.[Abstract]

8 . Jagannath, C., Emanuele, M. R. & Hunter, R. L. (2000). Activity of polaxamer CRL-1072 against drug-sensitive and resistant strains of Mycobacterium tuberculosis in macrophages and in mice. International Journal of Antimicrobial Agents 15, 55–63.[ISI][Medline]

9 . Kristiansen, J. E. & Amaral, L. (1997). The potential management of resistant infections with non-antibiotics. Journal of Antimicrobial Chemotherapy 40, 319–27.[Abstract]

10 . Mitchison, D. A. (1998). Basic concepts in the chemotherapy of tuberculosis. In Mycobacteria. II. Chemotherapy, (Gangadharam, P. R. J. & Jenkins, P. A., Eds), pp. 15–50. Chapman & Hall, New York.

11 . Bloch, A. B., Cauthen, G. M., Onorato, I. M., Dansbury, K. G., Kelly, G. D., Driver, C. R. et al. (1994). Nationwide survey of drug-resistant tuberculosis in the United States. Journal of the American Medical Association 271, 665–71.[Abstract]

12 . Bass, J. B., Farer, L. S., Hopewell, P. C., O'Brien, R., Jacobs, R. F., Ruben, F. et al. (1994). Treatment of tuberculosis and tuberculosis infection in adults and children. American Thoracic Society and the Centers for Disease Control and Prevention. American Journal of Respiratory and Critical Care Medicine 149, 1359–70.[Abstract]

13 . Quenelle, D. C., Winchester, G. A., Staas, J. K., Barrow, E. L. W. & Barrow, W. W. (2001). Treatment of tuberculosis using a combination of sustained-release rifampin-loaded microsphere and oral dosing with isoniazid. Antimicrobial Agents and Chemotherapy 45, 1637–44.[Abstract/Free Full Text]

14 . Cavalieri, S. J., Biehle, J. R. & Sanders, W. E. (1995). Synergistic activities of clarithromycin and antituberculous drugs against multidrug-resistant Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 39, 1542–5.[Abstract]

15 . Wiid, I., Hoal-Van Helden, E., Hon, D., Lombard, C. & Van Helden, P. (1999). Potentiation of isoniazid activity against Mycobacterium tuberculosis by melatonin. Antimicrobial Agents and Chemotherapy 43, 975–7.[Abstract/Free Full Text]

16 . Abate, G. & Hoffner, S. E. (1997). Synergistic antimycobacterial activity between ethambutol and the beta-lactam drug cefepime. Diagnostic Microbiology and Infectious Disease 28, 119–22.[ISI][Medline]

17 . Mazza, P., Orcesi, M., Pelizzi, C., Pelizzi, G., Predieri, G. & Zaini, F. (1992). Synthesis, structure, antimicrobial, and genotoxic activities of organotin compounds with 2,6-diacetylpyridine nicotinoyl- and isonicotinoylhydrazones. Journal of Inorganic Biochemistry 48, 251–70.[ISI][Medline]

18 . Ianelli, S., Mazza, P., Orcesi, M., Pelizzi, C., Pelizzi, G. & Zani, F. (1995). Synthesis, structure, and biological activity of organotin compounds with di-2-pyridylketone and phenyl(2-pyridyl) ketone 2-aminobenzoylhydrazones. Journal of Inorganic Biochemistry 60, 89–108.[ISI][Medline]

19 . Cesur, Z., Buyuktimkin, S., Buyuktimkin, N. & Derbentli, S. (1990). Synthesis and antimicrobial evaluation of some arylhydrazones of 4-[(2-methylimidazo[1,2-a]pyridine-3-yl)azo]benzoic acid hydrazine. Archiv der Pharmazie 323, 141–4.[ISI][Medline]

20 . Bottari, B., Maccari, R., Monforte, F., Ottana, R., Rotondo, E. & Vigorita, M. G. (2000). Isoniazid-related copper(II) and nickel(II) complexes with antimycobacterial in vitro activity. Part 9. Bioorganic and Medicinal Chemistry Letters 10, 657–60.[Medline]

21 . Cocco, M. T., Congiu, C., Onnis, V., Pusceddu, M. C., Schivo, M. L. & De Logu, A. (1999). Synthesis and antimycobacterial activity of some isonicotinohylhydrazones. European Journal of Medicinal Chemistry 34, 1071–6.[ISI]

22 . Jagannath, C., Reddy, V. M. & Gangadharam, P. R. (1995). Enhancement of drug susceptibility of multi-drug resistant strains of Mycobacterium tuberculosis by ethambutol and dimethyl sulphoxide. Journal of Antimicrobial Chemotherapy 35, 381–90.[Abstract]

23 . Hawkins, J. E., Wallace, R. J. & Brown, B. A. (1991). Antibacterial susceptibility tests: mycobacteria. In Manual of Clinical Microbiology, 5th edn, (Balows, A., Hausler, W. J., Herrmann, K. L., Isenberg, H. D. & Shadomy, H. J., Eds), pp. 1138–52. American Society for Microbiology, Washington, DC.

24 . Krogstar, D. J. & Moellering, R. C. (1986). Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 2nd edn, (Lorian, V., Ed.), pp. 537–95. Williams & Wilkins, Baltimore, MD.

25 . Stratton, C. W. & Cooksey, R. C. (1991). Susceptibility tests: special tests. In Manual of Clinical Microbiology, 5th edn, (Balows, A., Hausler, W. J., Herrmann, K. L., Isenberg, H. D. & Shadomy, H. J., Eds), pp. 1153–65. American Society for Microbiology, Washington, DC.

26 . Denizot, F. & Lange, R. (1986). Rapid colorimetric assay for cell growth and survival. Modification to the tetrazolium dye procedure giving improved sensitivity and reliability. Journal of Immunological Methods 89, 271–7.[ISI][Medline]

27 . Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. Journal of Immunological Methods 65, 271–7.

28 . Parrish, N. M., Houston, T., Bones, P. B., Townsend, C. & Dick, J. D. (2001). In vitro activity of a novel antimycobacterial compound, N-octanesulfonylacetamide, and its effects on lipid and mycolic acid synthesis. Antimicrobial Agents and Chemotherapy 45, 1143–50.[Abstract/Free Full Text]

29 . Haemers, A., Leysen, D. C., Bollaert, W., Zhang, M. & Pattyn, S. R. (1990). Influence of N substitution on antimycobacterial activity of ciprofloxacin. Antimicrobial Agents and Chemotherapy 34, 496–7.[ISI][Medline]

30 . Dutt, A. K. & Metha, J. B. (1998). Chemotherapy of tuberculosis in developed countries. In Mycobacteria. II. Chemotherapy, (Gangadharam, P. R. J. & Jenkins, P. A., Eds), pp. 131–60. Chapman & Hall, New York, NY.

31 . Abate, G. & Miörner, H. (1998). Susceptibility of multidrug-resistant strains of Mycobacterium tuberculosis to amoxycillin in combination with clavulanic acid and ethambutol. Journal of Antimicrobial Chemotherapy 42, 753–40.

32 . Young, D. B. (1994). Strategies for new drug development. In Tuberculosis: Pathogenesis, Protection and Control, (Bloom, B. R., Ed.), pp. 559–67. American Society for Microbiology, Washington, DC.

33 . Heng, J. E., Vorwerk, C. K., Lessell, E., Zuralowski, D., Levin, L. A. & Dreyer, E. B. (1999). Ethambutol is toxic to retinal ganglion cells via an excitotoxic pathway. Investigative Ophthalmology and Visual Science 40, 190–6.[Abstract]

34 . Joubert, P. H., Strobele, J. G., Ogle, C. W. & Van der Merwe, C. A. (1986). Subclinical impairment of colour vision in patients receiving ethambutol. British Journal of Clinical Pharmacology 21, 213–6.[ISI][Medline]

35 . Yoon, Y. H., Jung, K. H., Sadun, A. A., Shin, H. C. & Koh, J. Y. (2000). Ethambutol-induced vacuolar changes and neuronal loss in rat retinal cell culture: mediation by endogenous zinc. Toxicology and Applied Pharmacology 162, 107–14.[ISI][Medline]

36 . Radhakrishna, S. (1998). Controlled clinical trials in tuberculosis: lessons to be drawn. In Mycobacteria. II. Chemotherapy, (Gangadharam, P. R. J. & Jenkins, P. A., Eds), pp. 98–130. Chapman & Hall, New York, NY.

37 . Cynamon, M. H., Zhang, Y., Harpster, T., Cheng, S. & Destefano, M. S. (1999). High-dose isoniazid therapy for isoniazid-resistant murine Mycobacterium tuberculosis infection. Antimicrobial Agents and Chemotherapy 43, 2922–4.[Abstract/Free Full Text]

Received 25 June 2001; returned 12 October 2001; revised 19 November 2001; accepted 26 November 2001