The most potent compounds from our previous studies were subjected to the following set of tests: determination of MIC against different SDR strains of M. tuberculosis, MBC, oral bioavailability, MTD and in vivo efficacy in mice.
Table shows the MIC values obtained against SDR strains of M. tuberculosis, including those resistant to isoniazid, rifampicin, thiacetazone, ethambutol, ciprofloxacin, kanamycin, ethionamide and p-aminosalicylic acid. MIC was also retested against a susceptible strain. In general, all the compounds showed good MIC values against resistant strains. Results showed that the most moderate activity was observed against the ciprofloxacin-resistant strain, and MICs ranged between 6.25 and 12.5 mg/L, although compound 9 revealed the poorest activity against an isoniazid-resistant strain, with an MIC value of 100 mg/L. The susceptibilities of rifampicin, thiacetazone, ethambutol and p-aminosalicylic acid-resistant strains can be considered comparable to those of H37Rv, as was indicated by the ratios of MICs against resistant and non-resistant strains (Table ), which were generally ~1. This indicates that there is a little, if any, cross-resistance with the current anti-TB drugs thereby supporting a novel mechanism of action. These results are promising for the development of new effective compounds against the growing number of drug-resistant strains. Only compound 9 showed resistance with the isoniazid-resistant strain, with a ratio >31.9. The reason for this finding is unknown.
Determination of MIC against strains of SDR M. tuberculosis
Ratios of MICs against resistant and non-resistant strains
was also active against MDR-TB strains, including strains with resistance to additional TB drugs and quinolones (Table ). The activity on the drug-susceptible strain and the four MDR-TB strains varied only 2-fold, which is within the variation of MIC determinations. One of the strains tested was resistant to isoniazid, rifampicin, streptomycin, ethambutol and pyrazinamide, indicating that quinoxaline 1,4-di-N
-oxides will maintain activity on MDR and poly drug-resistant strains. Such activity is particularly important in light of the recent reports of XDR strains of TB.3–5,7–9,26
Overall, compound 5
was tested and found to be active on M
strain Erdman, M
strain BCG Montreal (also a human pathogen), four independent clinical isolates of M
from China with multiple resistance phenotypes and eight SDR strains of M
Activity of compound 5 and moxifloxacin control (mg/L) on drug-resistant clinical isolates of M. tuberculosis
is likely activated via bioreduction in bacteria, similar to the reduction observed for other substituted N
Since PA-824, a nitroimidazole in clinical trials for treating TB,28
is bioreduced to an active intermediate,29–31
we tested the activity of compound 5
against an isogenic set of M
strains with defined resistance to PA-824 (Table ). PA-824 is bioreduced to an active form by a pathway involving the deazaflavin F420
cofactor-dependent glucose dehydrogenase (Fdg1)29
and other cellular factors including molybdopterin, a cofactor for many oxido-reductase enzymes. Thus, loss of function of Fdg1 or loss of the ability to synthesize the F420
cofactor leads to resistance to PA-824 (Table ). Compound 5
was active on all PA-824-resistant M
strains tested, thus showing the lack of cross-resistance and supporting a different pathway of drug activation.
Activity of compound 5 (µM) in the LORA and on M. bovis strains with defined resistance to PA-824, and M. tuberculosis strain H37Rv with and without a reporter gene
The antitubercular activity of compound 5 was concurrently tested against M. tuberculosis strain H37Rv using MABA and strain Rv containing the luciferase reporter, both under aerobic conditions; activity was comparable in both cases (Table ). Niclosamide was included as another control compound that is structurally different from PA-824 and activated by a different pathway. Table also shows that compound 5 is equally active on growing bacteria and non-replicating persistent (NRP) bacteria adapted to low oxygen in the LORA test. In this test, activity was 1.65 and 1.21 µM (0.42 and 0.31 mg/L) against growing and NRP bacteria, respectively. This is another unique and important property of quinoxaline 1,4-di-N-oxides that may translate to a faster sterilization of infected tissues. The long continuation phase for the treatment of TB is believed to be in part due to the presence of non-replicating organisms that persist even in the presence of antitubercular drugs. PA-824 is an experimental nitroimidazole that is in Phase I clinical trials. PA-824 is bioreduced by M. tuberculosis to an active component but unlike compound 5, PA-824 is only about 1/10 as active against NRP bacteria compared with aerobically growing cells.
The MBCs of several compounds against H37
Rv were determined (Table ). A compound is generally considered to be bactericidal if the ratio of MIC to MBC is ≤4;32
so, compounds 1
could be considered to be bactericidal due to the low ratios obtained. On the other hand, compounds 3
, which showed higher MBC/MIC ratios for H37
Rv, may be less bactericidal.
MBCs against H37Rv and SDR strainsa
was chosen for evaluation in in vivo
assays. The MTD of compound 5
was determined by using an escalating dose of drug given to mice by oral gavage. No adverse effects or reactions were observed at a dose of 500 mg/kg in this test of acute, single dose toxicity. Compound 5
was orally bioavailable as assessed in the bioassay method22
with an estimated blood level of ~5 mg/L at 30 min post-oral dosing of mice with 200 mg/kg.
Preliminary in vivo
evaluation of compound 5
was made at a dose of 300 mg/kg in infected GKO C57BL/6 mice.24
This compound afforded significant reductions of 2.7 and 2.82 log10
cfu in the lung and spleen tissues, respectively, versus the untreated controls. Compound 5
was bactericidal in vivo
because the cfu present in the lung and spleen at the start of therapy (day 15) are lowered by greater than 2 logs (i.e. 99% killing) following 9 days of treatment. Visual inspection showed little lung pathology with a few small granulomas. Spleens appeared visibly normal and mice appeared normal and active. The efficacy of the compound was statistically better than controls (P
< 0.05) and equivalent to the efficacy of isoniazid (P
< 0.05). The protection shown by compound 5
is similar to clinically available compounds.33
In this same in vivo
run, isoniazid at 25 mg/kg/day gave a reduction in cfu of 3.1 and 4.37 log10
in the lungs and spleen, respectively, and was bactericidal.
In a second experiment, the dose–response of compound 5 in vivo was determined using the GKO mouse model at doses of 25, 100 and 300 mg/kg (Table ). Compound 5 was active in the lung and spleen at 100 and 300 mg/kg (P < 0.001), respectively. At 25 mg/kg, it was active in the spleen (P < 0.05) but not statistically active in the lung. Activity at 300 mg/kg dosing was striking in that it lowered the cfu by 4.04 and 5.19 log cfu, respectively, in the lung and spleen. Bactericidal activity was detected at the higher doses of 100 and 300 mg/kg (Table ). No clear toxicity was apparent in the first in vivo experiment, while in the dose–response experiment, some toxicity was observed at the highest dose; two mice in the 300 mg/kg group died during treatment, which was truncated to 7 days instead of the usual 9 days. No cfu was recovered from the lungs of two of the surviving mice or from the spleen of one of the surviving mice dosed at 300 mg/kg, indicating that the organs may have been sterilized. Preliminary studies indicate that both in vitro (cytotoxicity) and in vivo toxicity can be separated from the antitubercular activity.
Activity of compound 5 (TAACF 118845) in the mouse low-dose aerosol model
In conclusion, an extended evaluation of the in vitro and in vivo antimycobacterial activities of quinoxaline 1,4-di-N-oxide derivatives was performed. All of them displayed good inhibitory activity against resistant strains and only compound 9 showed a significant resistance in an isoniazid-resistant strain. Compounds 1, 5 and 8 can be considered to be bactericidal due to the low MBC/MIC ratios. Furthermore, compound 5 showed strong in vivo activity comparable to clinically used TB drugs, although a relatively high dose of compounds was required to obtain equivalent reductions in lung cfu. Overall, these data also suggest the importance of the chlorine group in position 7 of the benzene moiety. The activity of compound 5 is unique in that it is active on: (i) SDR strains; (ii) poly drug-resistant clinical isolates, including MDR-TB; and (iii) NRP mycobacteria. This latter activity may prove important for attaining cures in a shorter amount of time, since the presence of NRP bacteria is believed to be a major factor responsible for the prolonged nature of antitubercular therapy. Additional studies are planned to further assess the in vivo efficacy of compound 5 alone and in combination with other clinically used and antitubercular drugs in the standard mouse model of TB.