In Vitro Activity of a New Isothiazoloquinolone, ACH-702, against Mycobacterium tuberculosis and Other Mycobacteria

Abstract

In this work, we describe the activity of ACH-702 against clinical isolates of Mycobacterium tuberculosis and six different nontuberculous mycobacteria. The MIC₅₀ and MIC₉₀ of both susceptible and drug-resistant M. tuberculosis strains tested were 0.0625 and 0.125 μg/ml, respectively. The MIC₅₀ and MIC₉₀ values for Mycobacterium fortuitum isolates were 0.0625 μg/ml in both cases; Mycobacterium avium complex isolates showed MIC₅₀ and MIC₉₀ values of 0.25 and 4 μg/ml, respectively.

Tuberculosis (TB) remains a public health problem, with 1.3 million deaths among HIV-negative incident cases of TB and an additional 456,000 deaths among HIV-positive incident TB cases in 2007 (33). The infection can be treated properly if an early diagnosis is made and the appropriate therapy is administered. However, due to multiple factors, multidrug-resistant strains of M. tuberculosis are becoming more numerous, increasing the urgency for alternative therapeutic options.

Recently, some compound analogs related to the quinolone class, the isothiazoloquinolones (ITQs), have been identified, and their chemical synthesis and preliminary biological profiling have been reported (13, 29). These ITQs target bacterial replication and are potent inhibitors of both DNA gyrase and topoisomerase IV (5). A lead ITQ compound, ACH-702, has demonstrated potent antibacterial activity against a number of medically relevant bacteria, including drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) (21, 28, 31). In the present work, we studied the antibacterial activity of ACH-702 against clinical isolates of Mycobacterium tuberculosis, including drug-resistant isolates, and additional isolates of nontuberculous mycobacteria (NTM).

Sixty clinical isolates of M. tuberculosis were tested, including nine isolates resistant to isoniazid and 20 clinical isolates resistant to both isoniazid and rifampin, with susceptibility assays to these drugs performed by the proportion method. The isolates were obtained from the Clínica de Tuberculosis (CIPTIR) from the Hospital Universitario José E. González in Monterrey, México. Also, a total of 30 NTM clinical isolates corresponding to Mycobacterium fortuitum (n = 11), Mycobacterium chelonae (n = 2), Mycobacterium abscessus (n = 2), Mycobacterium intracellulare (n = 7), Mycobacterium avium complex (n = 3), Mycobacterium kansasii (n = 3), and Mycobacterium gordonae (n = 2) were tested. All the atypical mycobacteria were identified by phenotypic characteristics and by using PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene as previously described (25).

ACH-702 was obtained from Achillion Pharmaceuticals, Inc., New Haven, CT. A stock solution (1 mg/ml) of ACH-702 was prepared in 100% dimethyl sulfoxide and diluted in 7H9GC broth (4.7 g of Middlebrook 7H9 broth base [Difco, Detroit, MI], 20 ml of 10% [vol/vol] glycerol, 1 g of Bacto Casitone [Difco], 880 ml of distilled water, 100 ml of oleic acid-albumin-dextrose-catalase [Becton Dickinson, MD]) (9). The final drug concentration range was 0.03 to 8 μg/ml. In order to determine the susceptibility of M. tuberculosis to ACH-702, the broth microdilution method with Alamar Blue was utilized (9). M. tuberculosis H37Rv was run as a susceptible-strain control; moxifloxacin was also tested for activity comparison.

For NTM, MIC was determined as recommended by the Clinical and Laboratory Standards Institute (CLSI, formerly the National Committee for Clinical and Laboratory Standards) by a broth microdilution method in cation-adjusted Mueller-Hinton broth (CA-MHB) for rapidly growing mycobacteria (RGM) and in CA-MHB with the addition of an oleic acid-albumin-dextrose-catalase supplement (Becton Dickinson, Sparks, MD) at a final concentration of 10% for slowly growing mycobacteria (20). The final drug concentration range was 0.06 to 16 μg/ml. The MIC was determined for the RGM after 72 h of incubation at 30°C and for the slowly growing mycobacteria after 7 days of incubation at 35°C. For external controls, we utilized Staphylococcus aureus ATCC 29213.

In Table 1, we show the susceptibility of clinical isolates of Mycobacterium tuberculosis to ACH-702. All M. tuberculosis isolates were inhibited with ≤0.125 μg/ml, including the drug-resistant isolates. Moxifloxacin showed MIC₅₀ and MIC₉₀ values of 0.125 and 0.25 μg/ml, respectively (data not shown). ACH-702 displayed antibacterial activity equal or superior to that of other currently marketed quinolones against quinolone-susceptible M. tuberculosis clinical isolates and superior antibacterial activity against quinolone-resistant clinical isolates, including XDR strains as well as laboratory-selected quinolone-resistant mutants (M. Pucci, M. Ackerman, J. Thanassi, C. Schoen, and M. Cynamon, submitted for publication).

TABLE 1.

In vitro susceptibility of 60 clinical isolates of M. tuberculosis to ACH-702

M. tuberculosis isolates with phenotype (n):	MIC (μg/ml)a
	Range	50%	90%
Susceptible to both isoniazid and rifampin (31)	≤0.03-0.12	0.06	0.12
Resistant to isoniazid or rifampin (9)	≤0.03-0.12	0.12	0.12
Resistant to both isoniazid and rifampin (20)	0.06-0.25	0.06	0.12

^a50% and 90%, MICs at which 50% and 90% of the M. tuberculosis strains are inhibited, respectively.

The activity of ACH-702 against M. avium and M. fortuitum is presented in Table 2. The MIC value for two isolates of M. chelonae was 8 μg/ml; the MIC for two isolates of M. abscessus was 16 μg/ml. The range of MICs for M. kansasii (n = 3) was 0.12 to 16 μg/ml, and the MIC values for two M. gordonae isolates were 0.12 and 4 μg/ml. The MIC for quality control strains was determined with each lot of microtiter plates prepared, and it was ≤0.06 μg/ml for S. aureus ATCC 29213.

TABLE 2.

In vitro susceptibility of clinical isolates of M. avium complex and M. fortuitum to ACH-702

Species	MIC (μg/ml)a
	Range	50%	90%
M. avium complex (n = 10)	0.12-8	0.25	4
M. fortuitum (n = 11)	≤0.06-0.12	≤0.06	≤0.06

^a50% and 90%, MICs at which 50% and 90% of the Mycobacterium sp. strains are inhibited, respectively.

Fluoroquinolones are utilized as second-line anti-TB drugs (4, 19, 32). Moxifloxacin and gatifloxacin are being investigated in clinical trials, and they are candidates for shortening TB treatment since they have the lowest MICs (3, 10, 11) and greatest bactericidal activity (14, 16, 24). Quinolones have a long history of chemical modifications giving rise to new compounds with enhanced antibacterial activity. One example is the recently reported ITQ class (30, 31), an underexplored chemotype related to quinolones first described by Chu and coworkers at Abbott in the late 1980s (7, 8). ITQs have a substitution in the typical 3-carboxyl group by an isothiazolone ring and have demonstrated good antibacterial activity (28, 31). ACH-702 is an ITQ lead compound that has been studied against a number of bacterial pathogens, including MRSA, VRE, and Nocardia brasiliensis, with promising results (21, 27, 28, 31).

In this work, we observed that both susceptible and drug-resistant M. tuberculosis isolates were inhibited with MICs of ≤0.25 μg/ml ACH-702 for all 60 isolates tested. Comparing these data with previously published in vitro data for quinolones like ciprofloxacin, gatifloxacin, ofloxacin, garenoxacin, and levofloxacin, it appears that ACH-702 may have antibacterial activity superior to that of these representative quinolones (1, 22, 10). In our work, we also tested moxifloxacin as a quinolone control and obtained MIC values in agreement with previously reported results.

In addition, the incidence of diseases caused by NTM appears to be increasing worldwide, particularly due to its association with HIV AIDS (12). Management of NTM infections is difficult in most cases due to intrinsic resistance to many antimycobacterial agents. The fluoroquinolones have been shown to be active in vitro against many mycobacterial species, such as M. fortuitum, and some strains of M. kansasii and the M. avium-intracellulare (MAI) complex (2, 15, 18, 23, 26). Ciprofloxacin, ofloxacin, gatifloxacin, and sparfloxacin are the best studied of these agents to date and are among the most active of this group against NTM (2, 6, 17, 18, 26). Some of them are recommended in the prophylaxis and treatment against NTM disease (12), but many strains of MAI are resistant to fluoroquinolones. Although for ACH-702 is not yet possible to establish breakpoints, all the NTM species tested were inhibited with ≤16 μg/ml, obtaining the best results for M. fortuitum isolates with both MIC₅₀ and MIC₉₀ values of 0.0625 μg/ml. Depending on the pharmacokinetics in humans, these MIC values, particularly those of M. fortuitum, raise the possibility that at least some of these NTM may be susceptible in vivo as well.

In conclusion, based on in vitro data, the ITQ ACH-702 appears to be a promising drug against mycobacterial disease, although it will be important to test it in animal models in order to determine its possible usefulness in the treatment of human infections. We believe that this improved antibacterial activity is due to a more potent inhibition of the mycobacterial gyrase enzyme than that with quinolones, and this hypothesis is supported by recent biochemical assay data (M. Pucci et al., submitted for publication).