Abstract
Current therapy for pulmonary tuberculosis involves 6 months of treatment with isoniazid, pyrazinamide, rifampin, and ethambutol or streptomycin for reliable treatment efficacy. The long treatment period increases the probability of noncompliance, leading to the generation of multidrug-resistant isolates of Mycobacterium tuberculosis. A treatment option that significantly shortened the course of therapy, or a new class of antibacterial effective against drug-resistant M. tuberculosis would be of value. ABT-255 is a novel 2-pyridone antibacterial agent which demonstrates in vitro potency and in vivo efficacy against drug-susceptible and drug-resistant M. tuberculosis strains. By the Alamar blue reduction technique, the MIC of ABT-255 against susceptible strains of M. tuberculosis ranged from 0.016 to 0.031 μg/ml. The MIC of ABT-255 against rifampin- or ethambutol-resistant M. tuberculosis isolates was 0.031 μg/ml. In a murine model of pulmonary tuberculosis, 4 weeks of oral ABT-255 therapy produced a 2- to 5-log10 reduction in viable drug-susceptible M. tuberculosis counts from lung tissue. Against drug-resistant strains of M. tuberculosis, ABT-255 produced a 2- to 3-log10 reduction in viable bacterial counts from lung tissue. ABT-255 is a promising new antibacterial agent with activity against M. tuberculosis.
Length of therapy and patient noncompliance with treatment regimens remain clinical problems in the treatment of Mycobacterium tuberculosis infections. Current therapies reduce the pulmonary bacterial burden, but treatment periods of 6 months for nonimmunosuppressed individuals and at least 9 months for immunosuppressed patients are required for reliable treatment efficacy. The long treatment period increases the probability of noncompliance, leading to the generation of drug-resistant strains of M. tuberculosis (11). A recent survey determined that drug-resistant M. tuberculosis usually arises as a recrudescence of an existing infection, rather than as a primary infection (19). Also, the population in need of therapy often does not comply with the lengthy treatment regimen, causing further potential for treatment failure or an increase in drug resistance. In New York City, only 11% of the patients under care for M. tuberculosis infection reported back to an outpatient clinic to continue therapy (3). Protocols utilizing directly observed therapy have increased compliance, but additional public health resources are required (4, 14, 18).
In the United States, M. tuberculosis is most prevalent in immunosuppressed individuals and AIDS patients. Single and combination therapies are used following M. tuberculosis exposure or infection. Current recommended therapy of active infection with drug-susceptible M. tuberculosis is a 6-month regimen of isoniazid, pyrazinamide, rifampin, and ethambutol or streptomycin (5). For treatment of drug-resistant M. tuberculosis, a 24-month regimen of at least three drugs is recommended (7). A treatment option that allowed significant shortening of the course of therapy or represented a new drug class for M. tuberculosis therapy would be a useful advance.
The 2-pyridones are a promising class of antibacterial agents that possess broad-spectrum in vitro potency and in vivo efficacy. Like the fluoroquinolones, the 2-pyridones are inhibitors of bacterial DNA gyrase (10). ABT-719, a representative 2-pyridone, was approximately 10-fold more potent in vitro than ciprofloxacin against gram-positive bacterial strains. Against gram-negative bacterial strains, the 2-pyridones were approximately equal in potency to ciprofloxacin (8, 9). In mouse studies, oral efficacy was obtained at dosages that were 6- to 20-fold lower than those of ciprofloxacin against gram-positive bacterial infections and approximately equivalent to those of ciprofloxacin against gram-negative bacterial infections (2). M. tuberculosis isolates are generally susceptible to fluoroquinolones, although resistant isolates have been found (16). However, cross-resistance to fluoroquinolones and isoniazid and rifampin has not been found in M. tuberculosis (1). ABT-719 and structurally similar 2-pyridones produced MICs of ≤0.4 μg/ml against drug-susceptible and drug-resistant M. tuberculosis strains (data not shown). ABT-255 is an analog of ABT-719 with improvements in the therapeutic margin against common gram-positive and -negative bacterial infections (2). The efficacy of ABT-255 was compared to that of the antituberculosis agents isoniazid and rifampin against drug-sensitive and -resistant strains of M. tuberculosis in a murine model of pulmonary tuberculosis.
MATERIALS AND METHODS
Antimicrobial agents.
Isoniazid was obtained from Barr Laboratories, Pomona, N.Y.; rifampin was from Merrell Dow Pharmaceutical, Kansas City, Mo., and ethambutol was from Lederle Laboratories, Pearl River, N.Y. Ciprofloxacin was obtained from Miles Laboratories (Spokane, Wash.). ABT-255 (Fig. 1) was synthesized at Abbott Laboratories (12). For in vitro studies, stock solutions of isoniazid, rifampin, and ethambutol were prepared in 95% ethanol. For in vivo studies, compounds were prepared in distilled water.
FIG. 1.
ABT-255.
Bacterial strains.
All M. tuberculosis bacterial strains were obtained from the American Type Culture Collection, Bethesda, Md. Cultures were grown in Middlebrook 7H10 broth with 10% OADC enrichment (Gibco) at 37°C in 5% CO2 to obtain visible turbidity. Staphylococcus aureus 10649, Streptococcus pneumoniae 6303, and Escherichia coli JUHL were from the Abbott culture collection.
In vitro tests.
MICs against M. tuberculosis were determined colorimetrically by utilizing an Alamar blue assay (20). In brief, suspensions of the M. tuberculosis strains were grown in 7H10 broth to visible turbidity (greater than or equal to a McFarland no. 1 standard). Dilutions of different concentrations of drugs ranging from 0.008 to 100 μg/ml in a volume of 1.0 ml of 7H10 broth were combined with a 20-μl aliquot of a 1:5-diluted culture. This suspension was incubated for 7 days at 37°C in 5% CO2. On day 7, 20 μl of a 10× stock of Alamar blue solution and 50 μl of a 5% Tween 80 solution were added to each tube, and the tubes were incubated at 50°C for 2 h. A change in color from blue to pink indicated the growth of M. tuberculosis. The MIC was recorded as the lowest drug concentration that prevented growth. MICs against S. aureus, S. pneumoniae, and E. coli were determined by agar dilution testing as described by the National Committee for Clinical Laboratory Standards (15).
In vivo tests.
The basic efficacy of ABT-255 was determined in mouse models of acute bacterial infection as described previously (6). In brief, CF1 female mice (Charles River, Wilmington, Mass.) weighing 20 to 25 g were inoculated intraperitoneally with overnight cultures of S. aureus NTCC 10649, S. pneumoniae 6303, or E. coli JUHL. The cultures were adjusted to yield approximately 100 times the 50% lethal dose (LD50). Concurrently with each trial, the challenge LD50 was validated by inoculation of untreated mice with log10 dilutions of the bacterial inoculum. A 5-log dilution range of the bacterial challenges was inoculated into five groups of 10 mice each. A mortality rate of 100% was produced in all groups of untreated mice with the challenge inoculum of 100 times the LD50. ABT-255 and ciprofloxacin were formulated in sterile injectable water (Abbott Laboratories) and were administered subcutaneously or orally 1 and 5 h postchallenge. Mortality was recorded for 7 days, and the mean effective dose necessary to protect 50% of the mice (ED50) was calculated from cumulative mortality by using trimmed-logit analysis (10).
For the murine model of pulmonary tuberculosis, 4- to 6-week-old female outbred CF-1 mice (Charles River) were infected intravenously through a caudal tail vein. Each mouse received approximately 107 viable M. tuberculosis cells suspended in 0.2 ml of modified 7H10 broth. There were 10 mice per group. Treatment was started 7 days postinfection and administered once daily for 28 days (days 7 to 34 days postinfection). All drugs were administered by gavage in a volume of 0.5 ml. Animals were sacrificed on day 35 approximately 24 h after administration of the final drug dose. Lungs were aseptically removed and ground in a contained tissue homogenizer (Tekmar, Cincinnati, Ohio). The number of viable organisms was determined by dilution plating on 7H11 agar plates.
Statistics.
Mean log10 values were calculated from bacterial burden counts. Student’s t test was used to compare means between test and control groups. A P value of ≤0.05 was considered significant.
RESULTS
In vitro tests of the potency of 2-pyridones against drug-susceptible and -resistant M. tuberculosis.
Table 1 shows the MICs of ABT-255 against two drug-susceptible M. tuberculosis isolates, ATCC 35801 and ATCC 25618, and two single-drug-resistant M. tuberculosis isolates, ATCC 35837 (ethambutol resistant) and ATCC 35838 (rifampin resistant). Two experiments were conducted under the same conditions. By the Alamar blue reduction technique, the MICs of ABT-255 against drug-susceptible isolates of M. tuberculosis ranged from 0.016 to 0.031 μg/ml. The MIC of ABT-255 against both ethambutol- and rifampin-resistant isolates of M. tuberculosis was 0.031 μg/ml. The MICs of isoniazid against both drug-susceptible and drug-resistant isolates of M. tuberculosis ranged from 0.5 to 0.78 μg/ml. The MICs of rifampin against drug-susceptible and ethambutol-resistant isolates of M. tuberculosis ranged from 0.016 to 0.5 μg/ml. The MIC of rifampin against the rifampin-resistant isolate of M. tuberculosis was 12.5 μg/ml. The MICs of ethambutol against the drug-susceptible and rifampin-resistant isolates of M. tuberculosis ranged from 0.78 to 1.56 μg/ml. The ethambutol MIC against the ethambutol-resistant isolate was >25 μg/ml.
TABLE 1.
MICs of ABT-255 and other antituberculosis drugs against M. tuberculosis strains
| Drug | MIC (μg/ml) for strain:
|
|||
|---|---|---|---|---|
| 35801a | 25618a | 35837b | 35838c | |
| ABT-255 | 0.016 | 0.031 | 0.031 | 0.031 |
| Isoniazid | 0.78 | 0.5 | 0.5 | 0.5 |
| Rifampin | 0.016 | 0.5 | 0.5 | 12.5 |
| Ethambutol | 1.56 | 0.78 | >25.0 | 0.78 |
Drug susceptible.
Ethambutol resistant.
Rifampin resistant.
In vivo tests. (i) Efficacy of ABT-255 against common bacterial pathogens.
ABT-255 demonstrated both in vitro potency and in vivo efficacy superior to those of ciprofloxacin against S. aureus NTCC 10649 and S. pneumoniae 6303. Efficacy and potency were equal to those of ciprofloxacin against E. coli JUHL. Efficacy was demonstrated by both the oral and subcutaneous routes (Table 2).
TABLE 2.
MICs and ED50s of ABT-255 and ciprofloxacin versus systemic bacterial infections
| Drug |
S. aureus
|
S. pneumoniae
|
E. coli
|
||||||
|---|---|---|---|---|---|---|---|---|---|
| MICa | ED50b
|
MIC | ED50
|
MIC | ED50
|
||||
| PO | SC | PO | SC | PO | SC | ||||
| ABT-255 | 0.015 | 2.5 | 0.5 | 0.03 | 3.6 | 2.2 | 0.015 | 1.0 | 0.2 |
| Ciprofloxacin | 0.25 | 28.2 | 4.1 | 2.0 | >100 | 19.1 | 0.015 | 1.0 | 0.1 |
In micrograms per milliliter.
ED50s are in milligrams per kilogram. PO, peroral administration; SC, subcutaneous administration.
(ii) Efficacy of ABT-255 against drug-susceptible M. tuberculosis.
ABT-255 produced a dose-responsive 0- to 5.5-log reduction in pulmonary M. tuberculosis bacterial counts at daily dosages of 3.13 to 25 mg/kg. The efficacy of ABT-255 at 25 mg/kg per day was comparable to that of isoniazid against M. tuberculosis ATCC 35801 (Erdman strain). ABT-255 at 12.5 and 25 mg/kg per day and isoniazid at 3.12 to 25 mg/kg per day showed significant reductions in viable organism cell counts from lung tissue (P ≤ 0.05) compared to untreated infected mice (Table 3).
TABLE 3.
In vivo activity of ABT-255 against M. tuberculosis ATCC 35801 (strain Erdman)a in CF-1 mice
| Drug and doseb (mg/kg day−1) or group | Mean log10 no. of CFU/lung ± SE | Mean log10 reductionc |
|---|---|---|
| ABT-255 | ||
| 25 | 1.43 ± 0.49 | 5.46 |
| 12.5 | 3.58 ± 0.64 | 3.31 |
| 6.25 | 5.55 ± 1.34 | 1.34 |
| 3.13 | 7.00 ± 0.23 | 0 |
| Isoniazid | ||
| 25 | 1.69 ± 0.57 | 5.20 |
| 12.5 | 2.00 ± 0.56 | 4.89 |
| 6.25 | 1.96 ± 0.44 | 4.93 |
| 3.13 | 2.37 ± 0.59 | 4.52 |
| Infected controls | 6.89 ± 0.23 |
Inoculum of log10 7.00 mycobacteria.
Mice were dosed orally daily, from days 7 to 34 postinfection.
Difference in the mean log number of CFU from that of the day 35 late control group.
(iii) Efficacy of ABT-255 against single-drug-resistant M. tuberculosis.
Against ethambutol-resistant M. tuberculosis, ABT-255 produced a dose-related 1- to 3-log reduction in viable bacterial counts compared to infected control mice (P ≤ 0.05). Isoniazid at 25 mg/kg per day showed a significant 5-log reduction in viable organism cell counts, while ethambutol at a dose of 150 mg/kg per day produced no reduction in the M. tuberculosis burden (Table 4). Against rifampin-resistant M. tuberculosis, ABT-255 produced a dose-responsive 1- to 2.5-log reduction in bacterial colony counts (P ≤ 0.05). Isoniazid produced a greater-than-3-log reduction in bacterial colony counts compared to infected untreated mice (P ≤ 0.05). As expected, rifampin was not effective against rifampin-resistant M. tuberculosis (Table 5).
TABLE 4.
In vivo activity of ABT-255 against ethambutol-resistanta M. tuberculosis ATCC 35837 in CF-1 mice
| Drug and doseb (mg/kg day−1) or group | Mean log10 no. of CFU/lung ± SE | Mean log10 reductionc |
|---|---|---|
| ABT-255 | ||
| 25 | 4.13 ± 0.15 | 3.10 |
| 12.5 | 5.62 ± 0.21 | 1.61 |
| 6.25 | 6.52 ± 0.21 | 0.71 |
| Isoniazid, 25 | 2.08 ± 0.48 | 5.15 |
| Ethambutol, 150 | 7.28 ± 0.17 | 0 |
| Infected controls | 7.23 ± 0.14 |
Inoculum of log10 7.60 mycobacteria.
Mice were dosed orally daily, from 7 to 34 days postinfection.
Difference in mean log number of CFU from that of the day 35 late control group.
TABLE 5.
In vivo activity of ABT-255 against rifampin-resistanta M. tuberculosis ATCC 35838 in CF-1 mice
| Drug and doseb (mg/kg day−1) or group | Mean log10 no. of CFU/lung ± SE | Mean log10 reductionc |
|---|---|---|
| ABT-255 | ||
| 25 | 5.24 ± 0.33 | 2.66 |
| 12.5 | 6.58 ± 0.31 | 1.32 |
| 6.25 | 7.46 ± 0.10 | 0.44 |
| Isoniazid, 25 | 4.48 ± 0.25 | 3.42 |
| Rifampin, 50 | 7.37 ± 0.35 | 0.53 |
| Infected controls | 7.90 ± 0.09 |
Inoculum of log10 8.00 mycobacteria.
Mice were dosed orally daily, from 7 to 34 days postinfection.
Difference in mean log number of CFU from that of the day 35 late control group.
DISCUSSION
There is a need for alternative therapies for M. tuberculosis infections. Current therapy for pulmonary tuberculosis involves at least 6 months of treatment with any of the first-line medications (11). Patient noncompliance has created a growing number of persons with drug-resistant tuberculosis. Treatment of multidrug-resistant tuberculosis with multiple-drug regimens produced a response rate of only 50% with a mortality rate of 22% (7). While there is no adequate therapy for multidrug-resistant tuberculosis, clinical data suggests that the quinolone ofloxacin shows promise (13). In addition, the new quinolone levofloxacin has demonstrated greater in vitro potency than ofloxacin against M. tuberculosis (17).
The 2-pyridones are a new class of antibacterial agent similar in structure to fluoroquinolones but different by one nitrogen atom at the ring juncture. ABT-255 is a novel 2-pyridone antibacterial agent which demonstrated in vitro potency and in vivo efficacy against drug-susceptible and drug-resistant M. tuberculosis strains. By the Alamar blue technique, ABT-255 yielded in vitro potency against drug-susceptible and rifampin- or ethambutol-resistant strains of M. tuberculosis. Caution is needed in explaining differences in the potency of ABT-255 versus those of rifampin and ethambutol without additional pharmacokinetic studies. ABT-255 was more effective against the drug-susceptible strains, with an up to 5-log reduction of viable M. tuberculosis produced, compared to a 3-log reduction versus drug-resistant isolates, where ethambutol and rifampin therapy failed. ABT-255 did not sterilize mouse lung tissue, suggesting that it could best be used to supplement, rather than replace, existing therapies.
ABT-255 is a promising compound for development against M. tuberculosis due to the efficacy demonstrated against both drug-sensitive and -resistant strains of M. tuberculosis. The apparent lack of cross-resistance between quinolones and established M. tuberculosis drugs is encouraging. Combination therapies with drugs utilizing different mechanisms of action produce better efficacy with less probability of drug resistance. Greater preclinical efficacy could also translate into a shorter course of treatment. ABT-255 could be a useful addition to therapeutic treatments against M. tuberculosis infection.
ACKNOWLEDGMENTS
We are grateful to Al Dutkiewicz, Lori Gaede, Leann Mitchell, and Joanne Rover (Animal Husbandry).
REFERENCES
- 1.Alangaden G J, Manavathu E K, Vakulenko S B, Zvonok N M, Lerner S A. Characterization of fluoroquinolone-resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients. Antimicrob Agents Chemother. 1995;39:1700–1703. doi: 10.1128/aac.39.8.1700. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Alder J, Clement J, Meulbroek J, Shipkowitz N, Mitten M, Jarvis K, Oleksijew A, Hutch T, Paige L, Flamm R, Chu D, Tanaka K. Efficacies of ABT-719 and related 2-pyridones, members of a new class of antibacterial agents, against experimental bacterial infections. Antimicrob Agents Chemother. 1995;39:971–975. doi: 10.1128/aac.39.4.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Brudney K, Dobkin J. Resurgent TB in New York City. Human immunodeficiency virus, homelessness and the decline of TB programs. Am Rev Respir Dis. 1991;144:745. doi: 10.1164/ajrccm/144.4.745. [DOI] [PubMed] [Google Scholar]
- 4.Centers for Disease Control and Prevention. Improving adherence to antituberculosis therapy—South Carolina and New York. Morbid Mortal Weekly Rep. 1993;42(4):74. [PubMed] [Google Scholar]
- 5.Centers for Disease Control and Prevention. Initial therapy for tuberculosis in the era of multidrug resistance. Recommendations of the Advisory Council for the Elimination of Tuberculosis. Morbid Mortal Weekly Rep. 1993;42(RR-7):4. [PubMed] [Google Scholar]
- 6.Clement J, Tanaka S, Alder J, Vojtko C, Beyer J, Hensey D, Ramer N, McDaniel D, Chu D. In vitro and in vivo evaluations of A-80556, a new fluoroquinolone. Antimicrob Agents Chemother. 1994;38:1071–1078. doi: 10.1128/aac.38.5.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Cohn, D. L. 1995. Treatment of multidrug resistant tuberculosis. J. Hosp. Infect. 30(Suppl.):322–328. [DOI] [PubMed]
- 8.Cooper M, Andrews J, Ashby J, Matthews R, Wise R. In vitro activity of sparfloxacin, a new quinolone antimicrobial agent. J Antimicrob Chemother. 1990;26:667–676. doi: 10.1093/jac/26.5.667. [DOI] [PubMed] [Google Scholar]
- 9.Flamm R, Vojtko C, Chu D, Li Q, Beyer J, Hensey D, Ramer N, Clement J, Tanaka S. In vitro evaluation of ABT-719, a novel DNA gyrase inhibitor. Antimicrob Agents Chemother. 1995;39:964–970. doi: 10.1128/aac.39.4.964. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Hardy D, Swanson R, Hensey D, Ramer N, Bower R, Hansen C, Chu D, Fernandez P. Comparative antibacterial activities of temafloxacin hydrochloride (A-62254) and two reference fluoroquinolones. Antimicrob Agents Chemother. 1987;31:1768–1774. doi: 10.1128/aac.31.11.1768. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Hopewell, P., M. Cynamon, J. Starke, M. Iseman, and R. O’Brien. 1992. Evaluation of new-infective drugs for the treatment and prevention of tuberculosis. Clin. Infect. Dis. 15(Suppl. 1):S282–S295. [DOI] [PubMed]
- 12.Li Q, et al. Synthesis and structure-activity relationships of 2-pyridones: a novel series of potent DNA gyrase inhibitors as antibacterial agents. J Med Chem. 1996;39:3070–3088. doi: 10.1021/jm960207w. [DOI] [PubMed] [Google Scholar]
- 13.Maranetra, K. N. 1996. Treatment of multidrug resistant tuberculosis in Thailand. Chemotherapy 42(Suppl. 3):10–15. [DOI] [PubMed]
- 14.McDonald R J, Memon A M, Reichman L B. Successful supervised ambulatory management of TB treatment failures. Ann Intern Med. 1982;96:297. doi: 10.7326/0003-4819-96-3-297. [DOI] [PubMed] [Google Scholar]
- 15.National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically. Approved standard M7-A2. Villanova, Pa: National Committee for Clinical Laboratory Standards; 1990. [Google Scholar]
- 16.Perlman D C, et al. Susceptibility to levofloxacin of Mycobacterium tuberculosis isolates from patients with HIV-related tuberculosis and characterization of a strain with levofloxacin monoresistance. Community Programs for Clinical Research on AIDS 019 and the AIDS Clinical Trials Group 222 Protocol Team. AIDS. 1997;11(12):1473–1478. doi: 10.1097/00002030-199712000-00011. [DOI] [PubMed] [Google Scholar]
- 17.Saito H, Sato K, Tomioka H, Dekio S. In vitro antimycobacterial activity of a new quinolone, levofloxacin (DR-3355) Tuberc Lung Dis. 1995;76(5):377–380. doi: 10.1016/0962-8479(95)90001-2. [DOI] [PubMed] [Google Scholar]
- 18.Sbarbaro J A, Johnson S. TB chemotherapy for recalcitrant outpatients administered twice-weekly. Am Rev Respir Dis. 1968;97:895. doi: 10.1164/arrd.1968.97.5.895. [DOI] [PubMed] [Google Scholar]
- 19.Sepkowitz K A, Raffalli J, Riley L, Kiehn T E, Armstrong D. Tuberculosis in the AIDS era. Clin Microbiol Rev. 1995;8:180–199. doi: 10.1128/cmr.8.2.180. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Yajko M D, Madej J J, Lancaster M V, Sanders C A, Cawthon V L, Gee B, Babst A, Hadley W K. Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis. J Clin Microbiol. 1995;33:2324–2327. doi: 10.1128/jcm.33.9.2324-2327.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]