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
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Abstract |
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Introduction |
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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 drugs47 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.1012 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.1416
Isonicotinoylhydrazones are compounds structurally related to isoniazid and have antibacterial and antimycobacterial activities.1720 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.
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Materials and methods |
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The structures of the compounds used in this study are presented in Table 1. 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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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 LöwensteinJensen (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 acidalbumin dextrosecatalase (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 LöwensteinJensen slants in tubes containing 7H9 broth supplemented with 10% albumindextrosecatalase (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 1530 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 100062.5 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.
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Results |
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The cytotoxicity of isonicotinoylhydrazones as determined by the colorimetric method proposed by Denizot & Mosmann26,27 in Vero cells is reported in Table 1 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 1. 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 1
. 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 2. 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 2ipara-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)
. 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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Discussion |
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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 2cethambutol (combined FIC 0.37), 2iethambutol (combined FIC 0.37), 2dpara-aminosalicylic acid (combined FIC 0.17), 2ipara-aminosalicylic acid (combined FIC 0.36) and 2drifampicin (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.
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Acknowledgements |
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Notes |
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References |
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Received 25 June 2001; returned 12 October 2001; revised 19 November 2001; accepted 26 November 2001