Abstract
The in vitro activities of DC-159a against seven species of Mycobacterium were compared with moxifloxacin, gatifloxacin, levofloxacin, and rifampin. DC-159a was the most active compound against quinolone-resistant multidrug-resistant M. tuberculosis (MIC90, 0.5 μg/ml) as well as drug-susceptible isolates (MIC90, 0.06 μg/ml). The anti-tubercle bacilli activity of DC-159a was 4- to 32-fold more potent than those of currently available quinolones. DC-159a also demonstrated the highest activities against clinically important nontuberculous mycobacteria.
To be effective, tuberculosis (TB) treatment with a multidrug regimen must be continued for at least 6 months. This complicated regimen makes TB control difficult because of the long treatment duration, resulting in nonadherence of treatment, the development of multidrug-resistant TB (MDR-TB), and an additional problem of TB and human immunodeficiency virus (HIV) coinfection. Consequently, new anti-TB agents are urgently needed to overcome these problems (14).
Quinolones such as ofloxacin and levofloxacin are classified and used as second-line drugs for MDR-TB cases because they inhibit the supercoiling action of DNA gyrase, which is different from the target enzymes of first-line drugs; however, their bactericidal activities against Mycobacterium tuberculosis are weak in clinical use (15). Currently, moxifloxacin and gatifloxacin, which are 8-methoxy fluoroquinolones, have proven to have more potent activities and may enable the duration of treatment to be shortened. Therefore, combination regimens consisting of new quinolones are expected to improve the treatment of TB (3, 11, 13). However, there is a problem of cross-resistance among quinolones caused by the previous usage of old quinolones in the treatment of MDR-TB and other respiratory infections. Thus, the emergence of quinolone-resistant M. tuberculosis is a concern (4).
DC-159a is a newly synthesized broad-spectrum 8-methoxy fluoroquinolone. This compound has been shown previously to have potent activities against various respiratory pathogens, including quinolone-resistant strains (2, 7). The present study was performed to compare the in vitro antimycobacterial activities of DC-159a with three currently available quinolones and also rifampin against M. tuberculosis, including quinolone-resistant MDR (QR-MDR) strains and six species of clinically important nontuberculous mycobacteria (NTM).
(This work was presented in part at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, September 2006.)
Bacterial strains were isolated from Japanese patients between 1997 and 2003. The 32 M. tuberculosis isolates included drug-susceptible isolates (n = 21) and QR-MDR isolates (n = 11; levofloxacin MICs, ≥2 μg/ml; rifampin MICs, ≥6 μg/ml; isoniazid MICs, ≥4 μg/ml). The NTM isolates comprised slowly growing mycobacteria, including M. kansasii (n = 22), M. avium (serovar 4 [n = 10] and serovar 8 [n = 23]), and M. intracellulare serovar 16 (n = 17), and rapidly growing mycobacteria, including M. fortuitum (n = 10), M. abscessus (n = 12), and M. chelonae (n = 10). The bacterial strains were isolated from clinical specimens and cultured in Middlebrook 7H9 broth medium (Difco, MD) supplemented with 10% albumin-dextrose-catalase enrichment and 0.2% glycerol to provide 107-CFU/ml stock cultures and stored at −80°C until use.
Antimicrobial agents were obtained as pure substances from their manufacturers: DC-159a and levofloxacin (LVX; both from Daiichi-Sankyo Pharmaceuticals Co., Ltd., Tokyo, Japan), moxifloxacin (MXF; Bayer Yakuhin, Ltd., Osaka, Japan), gatifloxacin (GAT; Kyorin Co., Ltd., Tokyo, Japan), rifampin (RIF; Sigma-Aldrich Co., Tokyo, Japan).
MICs were determined according to the standard agar dilution method recommended by NCCLS (10). A multipoint inoculator (Microplanter; Sakuma Seisakusho, Tokyo, Japan) was used to inoculate approximately 104 CFU per 2-μl spot onto Middlebrook 7H10 agar (Difco, MD) supplemented with 10% oleic acid-albumin-dextrose-catalase and 0.5% glycerol. The tested agar plates contained antimicrobial agents ranging from 0.015 to 128 μg/ml in 2-fold dilutions. The inoculated agar plates were incubated at 37°C for 21 days for M. tuberculosis, 14 days for M. avium and M. intracellulare, 7 days for M. kansasii, and 3 days for M. fortuitum and M. abscessus. M. chelonae was incubated at 30°C for 3 days. The MIC was defined as the lowest drug concentration yielding no visible colony formation on the agar surface.
Summaries of the in vitro activities of DC-159a against M. tuberculosis and NTM in comparison with those of the other three quinolones and RIF are shown in Tables 1 and 2, respectively. MICs of currently available quinolones were generally similar to those of previous reports (1, 5, 8). DC-159a was the most active compound against M. tuberculosis. The MIC90 of DC-159a against drug-susceptible M. tuberculosis was 0.06 μg/ml, which was 2-fold lower than that of RIF and 4- to 8-fold lower than those of other tested quinolones. The MIC90 of DC-159a against QR-MDR M. tuberculosis was 0.5 μg/ml, which was 4- to 32-fold lower than those of other tested quinolones and equal to that of LVX against drug-susceptible M. tuberculosis.
TABLE 1.
Comparison of MICs of DC-159a with other quinolones and rifampin against M. tuberculosis
| M. tuberculosis isolate type (n) | Drug | MIC (μg/ml) |
||
|---|---|---|---|---|
| Range | 50% | 90% | ||
| Drug susceptible (21) | DC-159a | 0.03-0.125 | 0.06 | 0.06 |
| MXF | 0.125-0.5 | 0.25 | 0.25 | |
| GAT | 0.06-0.25 | 0.125 | 0.25 | |
| LVX | 0.25-1 | 0.5 | 0.5 | |
| RIF | ≤0.015-0.25 | 0.06 | 0.125 | |
| QR-MDR (11) | DC-159a | 0.125-0.5 | 0.125 | 0.5 |
| MXF | 1-4 | 2 | 4 | |
| GAT | 0.5-2 | 1 | 2 | |
| LVX | 2-16 | 8 | 16 | |
| RIF | 16 to >128 | 128 | >128 | |
TABLE 2.
Comparison of MICs of DC-159a with other quinolones and rifampin against nontuberculous mycobacteria
| Mycobacterium species (no. of isolates) | Drug | MIC (μg/ml) |
||
|---|---|---|---|---|
| Range | 50% | 90% | ||
| Slowly growing mycobacteria | ||||
| M. kansasii (22) | DC-159a | 0.03-0.25 | 0.06 | 0.125 |
| MXF | 0.06-1 | 0.125 | 0.25 | |
| GAT | 0.06-1 | 0.125 | 0.5 | |
| LVX | 0.25-2 | 0.25 | 1 | |
| RIF | 0.06-128 | 0.25 | 0.25 | |
| M. avium (33): serovar 4 (10) and serovar 8 (23) | DC-159a | 0.25-8 | 1 | 2 |
| MXF | 1-16 | 2 | 4 | |
| GAT | 1-32 | 2 | 8 | |
| LVX | 2-64 | 8 | 32 | |
| RIF | 0.25 to >128 | 32 | 128 | |
| M. intracellulare serovar 16 (17) | DC-159a | 0.25-8 | 1 | 4 |
| MXF | 0.5-32 | 2 | 8 | |
| GAT | 1-32 | 4 | 16 | |
| LVX | 2-32 | 8 | 32 | |
| RIF | 0.25-128 | 2 | 32 | |
| Rapidly growing mycobacteria | ||||
| M. fortuitum (10) | DC-159a | 0.03-0.25 | 0.125 | 0.25 |
| MXF | 0.06-0.5 | 0.125 | 0.25 | |
| GAT | 0.03-0.25 | 0.125 | 0.25 | |
| LVX | 0.06-2 | 0.25 | 0.5 | |
| RIF | 1-128 | 32 | 64 | |
| M. chelonae (10) | DC-159a | 4-16 | 4 | 8 |
| MXF | 8-64 | 32 | 32 | |
| GAT | 4-64 | 16 | 32 | |
| LVX | 4-128 | 32 | 64 | |
| RIF | >128 | >128 | >128 | |
| M. abscessus (12) | DC-159a | 4-32 | 8 | 16 |
| MXF | 16-128 | 64 | 128 | |
| GAT | 8-64 | 32 | 64 | |
| LVX | 16 to >128 | 64 | 128 | |
| RIF | >128 | >128 | >128 | |
Furthermore, DC-159a inhibited the growth of slowly growing NTM and yielded the lowest MIC90 among the tested quinolones with M. kansasii (MIC90, 0.125 μg/ml), M. avium (MIC90, 2 μg/ml), and M. intracellulare (MIC90, 4 μg/ml). The MIC90 of DC-159a against the M. avium-M. intracellulare complex was 2- to 16-fold lower than those of other tested quinolones.
In the rapidly growing mycobacteria, it is well known that M. fortuitum is susceptible to quinolones, but M. chelonae and M. abscessus are naturally resistant to quinolones (6). DC-159a also showed the same pattern of activity against rapidly growing NTM as the other quinolones. The MIC90 of DC-159a against M. fortuitum (0.25 μg/ml) was comparable to those of MXF and GAT; however, the MIC90s of DC-159a against M. chelonae (8 μg/ml) and M. abscessus (16 μg/ml) were 4- to 8-fold lower than those of other tested quinolones.
In summary, the order of susceptibility of Mycobacterium species to DC-159a was M. tuberculosis > M. kansasii > M. fortuitum > M. avium > M. intracellulare > M. chelonae > M. abscessus.
In this study, we compared the in vitro activities of DC-159a with those of LVX, MXF, GAT, and RIF as reference drugs. DC-159a showed the lowest MICs and the narrowest MIC distribution against M. tuberculosis and NTM isolates for the tested drugs. A good activity of DC-159a against quinolone-resistant strains in other bacteria has been reported previously (2, 7). The results of this study also confirmed the characteristic advantage of DC-159a against QR-MDR M. tuberculosis. This finding may be attributed to the high inhibitory activities of DC-159a against the mutant GyrA enzymes in quinolone-resistant M. tuberculosis (12). The exact mechanism of DC-159a resistance in M. tuberculosis is under investigation.
According to the pharmacokinetic study in a monkey model in which an oral dose of 5 mg/kg of body weight was administered, DC-159a achieved a higher peak concentration (Cmax; 2.20 μg/ml) and area under the concentration-time curve from 0 to 24 h (AUC0-24; 16.9 μg·h/ml) than the MIC against M. tuberculosis, showed better pharmacokinetic properties than LVX (Cmax, 1.68 μg/ml; AUC0-24, 15.3 μg·h/ml), and lacked interaction with cytochrome P450 3A4 (9).
For substantiation of DC-159a as a novel agent for TB, MDR-TB, extensively drug-resistant TB, and TB/HIV coinfection cases, further intracellular, pharmacokinetic, drug-drug interaction, and in vivo evaluation studies are essential. DC-159a deserves further study as a promising candidate for a better TB cure, with a focus on the establishment of a 3- to 4-month new standard regimen in the near future.
Acknowledgments
This work was supported by Daiichi-Sankyo Pharmaceuticals Co., Ltd., Tokyo, Japan.
Footnotes
Published ahead of print on 5 April 2010.
REFERENCES
- 1.Brown-Elliott, B. A., R. J. Wallace, Jr., C. J. Crist, L. Mann, and R. W. Wilson. 2002. Comparison of in vitro activities of gatifloxacin and ciprofloxacin against four taxa of rapidly growing mycobacteria. Antimicrob. Agents Chemother. 46:3283-3285. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Clark, C., K. Smith, L. Ednie, T. Bogdanovich, B. Dewasse, P. McGhee, and P. C. Appelbaum. 2008. In vitro activity of DC-159a, a new broad-spectrum fluoroquinolone, compared with that of other agents against drug-susceptible and -resistant pneumococci. Antimicrob. Agents Chemother. 52:77-84. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Cynamon, M., M. R. Sklaney, and C. Shoen. 2007. Gatifloxacin in combination with rifampicin in a murine tuberculosis model. J. Antimicrob. Chemother. 60:429-432. [DOI] [PubMed] [Google Scholar]
- 4.Drlica, K., X. Zhao, and B. Kreiswirth. 2008. Minimising moxifloxacin resistance with tuberculosis. Lancet Infect. Dis. 8:273-275. [DOI] [PubMed] [Google Scholar]
- 5.Gillespie, S. H., and O. Billington. 1999. Activity of moxifloxacin against mycobacteria. J. Antimicrob. Chemother. 44:393-395. [DOI] [PubMed] [Google Scholar]
- 6.Guillemin, I., V. Jarlier, and E. Cambau. 1998. Correlation between quinolone susceptibility patterns and sequences in the A and B subunits of DNA gyrase in mycobacteria. Antimicrob. Agents Chemother. 42:2084-2088. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Hoshino, K., K. Inoue, Y. Murakami, Y. Kurosaka, K. Numba, Y. Kashimoto, S. Uoyama, R. Okumura, S. Higuchi, and T. Otani. 2008. In vitro and in vivo antibacterial activities of DC-159a, a new fluoroquinolone. Antimicrob. Agents Chemother. 52:65-76. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Jacobs, M. R. 2004. Fluoroquinolones as chemotherapeutics against mycobacterial infections. Curr. Pharm. Des. 10:3213-3220. [DOI] [PubMed] [Google Scholar]
- 9.Komoriya, S., H. Inagaki, Y. Sugimoto, M. Nagamochi, R. Miyauchi, J. Kuroyanagi, T. Kitamura, K. Hoshino, Y. Murakami, M. Tachibana, Y. Imamura, H. Takahashi, and M. Takemura. 2006. DC-159a, a new generation of respiratory quinolone: synthesis, design, and biological evaluations, abstr. F1-0477. Abstr. 46th Intersci. Conf. Antimicrob. Agents Chemother., American Society for Microbiology, Washington, DC.
- 10.NCCLS. 2003. Susceptibility testing of mycobacteria, nocardia, and other aerobic actinomycetes; approved standard M24-A. National Committee for Clinical Laboratory Standards, Wayne, PA. [PubMed]
- 11.Nuermberger, E. L., T. Yoshimatsu, S. Tyagi, R. J. O'Brien, A. N. Vernon, R. E. Chaisson, W. R. Bishai, and J. H. Grosset. 2004. Moxifloxacin-containing regimen greatly reduces time to culture conversion in murine tuberculosis. Am. J. Respir. Crit. Care Med. 169:421-426. [DOI] [PubMed] [Google Scholar]
- 12.Onodera, Y., T. Hirata, K. Hoshino, and T. Otani. 2007. DC-159a, a novel quinolone, showed high inhibitory activity against altered topoisomerases of Streptococcus pneumoniae and Mycobacterium tuberculosis, abstr. F1-2126. Abstr. 47th Intersci. Conf. Antimicrob. Agents Chemother., American Society for Microbiology, Washington, DC.
- 13.Rustomjee, R., C. Lienhardt, T. Kanyok, G. R. Davies, J. Levin, T. Mthiyane, C. Reddy, A. W. Strum, F. A. Sirgel, J. Allen, D. J. Coleman, B. Fourie, D. A. Mitchison, and the Gatifoxacin for TB (OFLOTUB) Study Team. 2008. A phase II study of the sterilising activities of ofloxacin, gatifloxacin and moxifloxacin in pulmonary tuberculosis. Int. J. Tuberc. Lung Dis. 12:128-138. [PubMed] [Google Scholar]
- 14.van den Boogaard, J., G. S. Kibiki, E. R. Kisanga, M. J. Boeree, and R. E. Aarnoutse. 2009. New drug against tuberculosis: problems, progress, and evaluation of agents in clinical development. Antimicrob. Agents Chemother. 53:849-862. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.World Health Organization. 2008. Guideline for the programmatic management of drug-resistant tuberculosis. World Health Organization, Geneva, Switzerland.