AR-12 Exhibits Direct and Host-Targeted Antibacterial Activity toward Mycobacterium abscessus

Therapeutic options for Mycobacterium abscessus infections are extremely limited. New or repurposed drugs are needed. The anti-M. abscessus activity of AR-12 (OSU-03012), reported to express broad-spectrum antimicrobial effects, was investigated in vitro and in vivo. Antimicrobial susceptibility testing was performed on 194 clinical isolates. Minimum bactericidal concentration and time-kill kinetics assays were conducted to distinguish the bactericidal versus bacteriostatic activity of AR-12.

A detailed evaluation of the anti-M. abscessus activity of AR-12 was undertaken. AR-12 was active against M. abscessus growing in vitro and in vivo and proved especially effective in inhibiting the growth of intracellular organisms. Moreover, AR-12 did not act antagonistically toward the most frequently used anti-M. abscessus drugs. To our knowledge, this is the first report of the anti-M. abscessus activity of AR-12. As such, this work may provide a novel approach to treating M. abscessus infections.

RESULTS
AR-12 is active against M. abscessus. A total of 194 clinical M. abscessus isolates (148 subsp. abscessus and 46 subsp. massiliense) were collected. AR-12 displayed similar antimicrobial activity against both subspecies. The MICs ranged from 2 to 16 mg/liter, with an MIC 50 and an MIC 90 of 4 and 8 mg/liter (8.7 and 17.4 M), respectively, emphasizing the broad sensitivity of M. abscessus to AR-12 (Table 1; see also Table S1 in the supplemental material).
AR-12 exhibits bactericidal activity. The MBC/MIC ratio of AR-12 for all isolates tested ranged from 2 to Ͼ4 (Table 2). Most isolates (81.8%, 9/11) exhibited an MBC/MIC ratio of Յ4, which is indicative of bactericidal activity. AR-12 exhibited essentially the same bactericidal activity toward both subspecies. Indeed, no obvious trait (e.g., increased sensitivity to other antibiotics such as amikacin) differentiated the isolates that were and were not subject to the bactericidal activity of AR-12.
AR-12 is comparable to amikacin in bacterial killing kinetics assay. To determine the potency of the killing kinetics of AR-12 against M. abscessus, AR-12 was tested at 1ϫ and 8ϫ MIC for 7 days; amikacin served as a positive control. At 1ϫ MIC, there was no reduction in log 10 CFU of either ATCC 19977 or G137 compared to the abscessus reference strain (ATCC 19977) (unpublished data). Thus, it is unlikely that the regrowth shown here is due to increased antibiotic resistance. Rather, regrowth is more likely due to the loss of antibiotic potency with time in culture.  (Table 3; Tables S2 and S3). Only the interaction of AR-12 with imipenem in a subsp. abscessus strain showed synergy (FICI ϭ 0.5). No antagonism between drugs was observed.

AR-12 is indifferent in its interaction
AR-12 inhibits the growth of intracellular M. abscessus. Experiments were undertaken to access and compare the effects of AR-12 and amikacin on the intracellular survival of M. abscessus subsp. abscessus (reference strain ATCC 19977) and subsp. massiliense (clinical isolate G137 chosen at random) in primary peritoneal macrophages. Both AR-12 and amikacin at 0.5ϫ and 1.0ϫ MIC significantly inhibited the intracellular growth of subsp. abscessus relative to the untreated control; AR-12 demonstrated greater inhibition than amikacin ( Fig. 2A). AR-12 exerted a far more pronounced effect on subsp. massiliense, which exhibited an ϳ0.5 log 10 reduction in intracellular organisms during 48 h incubation with 1ϫ MIC (Fig. 2B). Treatment with 1ϫ MIC amikacin, on the other hand, only slowed the intracellular growth M. abscessus subsp. massiliense.

AR-12 inhibits the replication of M. abscessus in a mouse model of lung infection.
AR-12 exerted a potent inhibitory effect on the intra-and extracellular growth of M. abscessus in vitro. Additional experiments were conducted to evaluate and compare the effects of AR-12 and amikacin in vivo. A study was conducted using a murine respiratory infection model under immunocompromised conditions that mimicked the clinical presentations of NTM lung disease. AR-12 (50 mg/kg) and amikacin (50 mg/kg) caused significant reductions of ϳ3.7 log 10 CFU and ϳ3.0 log 10 CFU, respectively, in lungs accessed at 14 days postinfection (Fig. 3A). Immunostaining also revealed a reduction in the inflammatory pathology and bacterial counts in the lungs of AR-12-treated mice compared to untreated mice (Fig. 3B). Overall, AR-12 exhibited bactericidal activity in vivo, which was consistent its effects on the intracellular survival of M. abscessus in vitro.

DISCUSSION
Treatment of M. abscessus infections is particularly difficult, a high rate of failure (45.1 to 46.0%) has been reported (6,7). Developing new antimicrobial strategies to control M. abscessus is urgently needed. AR-12, used as a host-directed therapeutic or a direct antibacterial agent, is a prospective drug for treatment of multiple pathogens. It offers of variety of advantages such as oral delivery, a broad anti-pathogen spectrum, and effective in treating MDR bacteria . The efficacy of AR-12 in the treatment of M. abscessus infections remains to be determined.
In the present study, 194 clinical M. abscessus isolates were collected. AR-12 exhibited significant antimicrobial activity against these isolates in vitro; the MIC 50 and MIC 90  were 4 and 8 mg/liter (8.7 and 17.4 M), respectively. Pharmacokinetic studies in mice indicated that the peak serum concentration of oral AR-12 administered at 200 mg/kg exceeded 20 M. At the end of a 28-day period, the mice exhibited no visible signs of toxicity. All the animals maintained stable body weights and lacked gross pathological abnormalities upon necropsy (C.-S. Chen, unpublished data) (18). Similarly, an AR-12 dose of Յ200 mg/kg proved to be safe in mouse models of viral and fungal infections (22,24).
Data obtained in a phase I clinical trial, exploring the effectivity of AR-12 as a targeted anticancer drug, showed that an 8 M or 3.7 mg/liter serum concentration was safely attained and well tolerated by patients administered 800 mg twice daily (19). This serum concentration is similar to the MIC 50 of AR-12 for M. abscessus in vitro determined here, indicating that a safe and effective dose can be achieved to treat clinical M. abscessus infections. The best dose and formulation of AR-12 required to optimize therapeutic outcomes, however, remain to be determined.
It has been suggested that AR-12 is a host-directed therapeutic rather than an antibiotic. Reportedly, it enhances host-dependent eradication of numerous intracellular infectious agents via multiple mechanisms that culminate in an antiproliferative effect (10)(11)(12)(13)(14)(15)(16). AR-12, for example, has no direct effect on Francisella strains but inhibits the intracellular survival of Francisella indirectly by inducing host cell autophagy (13). In a second study, however, AR-12 inhibited ampicillin-and kanamycin-resistant Escherichia coli and MDR N. gonorrhoeae directly by reducing DnaK expression (31). The latter study indicates that AR-12 can target certain bacteria and is a valuable, bioavailable small molecule that should be evaluated for the breadth of its antimicrobial activity. In the present study, AR-12 killed M. abscessus by a direct, concentration-dependent mechanism and was comparable to amikacin in activity expressed in vitro.
AR-12 was effective in inhibiting the growth of intracellular M. abscessus in vitro at concentrations effective in inhibiting organisms growing extracellularly. Similarly, AR-12 inhibited M. abscessus growth in a mouse model of lung infection. Interestingly, AR-12 exhibited a slight advantage over amikacin in these latter two cases, which was not evident when accessing the effects of AR-12 on extracellular growth in vitro. This finding was most likely due to the combined effects of the direct bactericidal activity of AR-12 and its host-directed therapeutic function. This is a significant advantage of AR-12 over other drugs used in treating bacteria, such as M. abscessus, growing intracellularly. It exhibits single-use bactericidal activity and simultaneously enhances bacterial clearance, acting as a small molecule adjuvant.
Hydrophobicity is a major concern associated with AR-12, making it difficult to achieve therapeutic doses. Modification and enhanced delivery systems may be needed to generate higher drug concentrations in vivo (11). In this regard, AR-12 encapsulated in acetalated dextran microparticles maintained pharmaceutical activity while exhibiting reduced cytotoxicity (27)(28)(29).
M. abscessus infections generally require treatment with multidrug combinations. AR-12 is an attractive option for treating M. abscessus infections, although its optimal effectiveness may necessitate its incorporation into multidrug therapy. In the present study, no antagonism was observed between AR-12 and five antibiotics (clarithromycin, amikacin, imipenem, cefoxitin, and tigecycline) found to be clinically important in treating M. abscessus infections according to guidelines (4,5). Although there are currently no reports of AR-12 antagonism, one study showed that AR-12 can potentiate the antibacterial activity of polymyxins purportedly by modifying lipopolysaccharide, altering the bacterial outer membrane, and promoting polymyxin uptake (30). Koselny et al. reported that AR-12 could enhance the activity of fluconazole in a mouse model of cryptococcosis (25). AR-12 also acts as a potentiator in colistin (polymyxin E)-sensitive strains (32). These results indicate that AR-12 does not antagonize other drugs and suggest the ease with which AR-12 could be integrated into current anti-M. abscessus drug combinations.
This study is limited by the fact that all isolates used were obtained from a single hospital, Shanghai Pulmonary Hospital, a designated treatment center for Chinese tuberculosis and NTM infections, which attracts NTM cases from across the country. Acquisition of M. abscessus from other sources is difficult. Consequently, many of the isolates may be related genetically and/or epidemiologically. In conclusion, AR-12 is active against M. abscessus both in vitro and in vivo and does not antagonize other antibiotics most frequently used to treat M. abscessus infections. As such, AR-12 is a potential candidate to include in novel therapeutic anti-M. abscessus regimens.

MATERIALS AND METHODS
Bacterial strains. A total of 194 clinical M. abscessus isolates were acquired retrospectively from sputum and bronchoalveolar lavage fluid specimens that were obtained between January 2014 and December 2017 and stored at Shanghai Pulmonary Hospital. Shanghai Pulmonary Hospital is one of the designated treatment centers for Chinese tuberculosis and NTM infections, attracting NTM cases from across the country. Both MGIT960 medium culture and p-nitrobenzoic acid testing were used as preliminary screening methods for NTM. The positive isolates were divided into subsp. abscessus and subsp. massiliense based upon multilocus sequencing of the rpoB and erm(41) genes. Staphylococcus aureus ATCC 29213, Mycobacterium peregrinum (ATCC 700686), and M. abscessus ATCC 19977 reference strains were purchased from the American Type Culture Collection (ATCC; Manassas, VA). M. abscessus was grown at 37°C on Middlebrook 7H10 (M7H10) agar plates supplemented with 10% oleic acidalbumin-dextrose-catalase (OADC) and 0.2% glycerol or with continuous shaking in Middlebrook 7H9 (M7H9) broth supplemented with 10% OADC and 0.05% Tween 80 (7H9sB). Suspensions of all isolates were stored at -80°C and cultured fresh before each assay. Middlebrook 7H9 broth, M7H10 agar, cation-adjusted Mueller-Hinton II broth (CAMHB), and OADC supplement were purchased from Becton, Dickinson, and Company (BD; Franklin Lakes, NJ).
Antimicrobial susceptibility testing. Antimicrobial susceptibility testing was conducted in accordance with CLSI document M24-A2. In brief, individual M. abscessus colonies were picked from M7H10 agar plates, grown to logarithmic phase in M7H9 broth, diluted to McFarland 0.5 with sterile saline, and adjusted to 1 ϫ 10 5 to 5 ϫ 10 5 CFU/ml using CAMHB. AR-12 was serially diluted 1:2 in a 96-well microtiter plate (final concentrations ranged from 16 to 0.008 g/ml), and 100 l of bacteria in suspended CAMH was added to each well. The plates were incubated at 37°C under aerobic conditions for 3 to 4 days until the control wells without AR-12 exhibited visible growth. Mycobacterium peregrinum (ATCC 700686) and Staphylococcus aureus (ATCC 29213) served as control reference strains. The MIC was defined as the lowest drug concentration at which no bacterial growth was observed by visual examination. All assays were repeated twice.
Minimum bactericidal concentration. The minimum bactericidal concentration (MBC) was determined in order to distinguish between the bacteriostatic and bactericidal activities of AR-12. M. abscessus subsp. abscessus and subsp. massiliense isolates were selected randomly for determination. After the antimicrobial susceptibility test at day 4 of AR-12 incubation, wells in which the drug concentrations were higher than the MIC were evenly resuspended. Portions (100 l) of the contents from wells were serially diluted (10-fold) and plated to count the remaining CFU. The CFU were determined after 5 days of incubation at 37°C. The MBC values were defined as the minimum drug concentration with no colony growth. An antibiotic is considered bactericidal when the MBC/MIC ratio is Յ4; otherwise, it is considered bacteriostatic.
Time-kill kinetics assay. We used the subsp. abscessus ATCC 19977 and a clinical subsp. massiliense isolate (G137) to evaluate the potency of the killing kinetics of AR-12 against M. abscessus. Tubes (5 ml) of 7H9sB containing AR-12 or amikacin at 1ϫ or 8ϫ MIC were inoculated with 5 ϫ 10 5 CFU of bacteria growing exponentially; the tubes were incubated for 7 days at 37°C with shaking (100 rpm). The bacteria were enumerated on days 0, 1, 2, 3, 5, and 7 by plating serial dilutions on lysogeny broth agar plates; CFU were enumerated after an additional 5 days incubation at 37°C. All assays were performed in duplicate.
Synergy with frequently used antimycobacterial drugs. Synergy between AR-12 and clarithromycin, amikacin, imipenem, cefoxitin, and tigecycline was assessed in vitro using the broth microdilution chequerboard titration technique. Briefly, serial 2-fold dilutions of AR-12 were added to the columns of wells, and serial dilutions of the five antibiotics were added to the rows of wells in a 96-well microtiter plate, yielding a two-dimensional matrix composed of AR-12 in combination with various antibiotic concentrations. The 2-fold dilutions of AR-12 or each antibiotic alone and the medium without AR-12 or antibiotics were used as controls. Randomly selected M. abscessus subsp. abscessus and subsp. massiliense isolates (ϳ10 6 CFU/ml) were added to each well, and the plates were incubated for 3 to 4 days at 37°C. The final MIC of each drug combination was determined as described for antimicrobial susceptibility testing above. The results of synergy testing were interpreted based on the FICI determined according to the following formula: FICI ϭ (MIC of antibiotic A in combination/MIC of antibiotic A alone) ϩ (MIC of AR-12 in the combination/MIC of AR-12 alone). Drug interactions exhibit three patterns: synergy (FICI Յ 0.5), indifference (0.5 Ͻ FICI Յ 4.0), and antagonism (FICI Ͼ 4.0). Intracellular killing. Mouse peritoneal macrophages, frequently used in studies undertaken to examine the factors that affect intracellular replication of Mycobacterium species, were obtained by previously described methods (33)(34)(35). The cells were infected at a multiplicity of infection of 5 with M. abscessus ATCC 19977 suspended in RPMI 1640 with 10% fetal bovine serum and no antibiotics. After 4 h of incubation at 37°C in 5% CO 2 , the cells were washed three times with warm phosphate-buffered saline (PBS) to remove the extracellular organisms. Fresh RPMI 1640 supplemented with 10% human serum and then AR-12 or amikacin at 0.5ϫ to 1ϫ MIC was added; infected control cells were treated with culture medium alone. The cells were lysed with 0.05% sodium dodecyl sulfate at various times postinfection, and the numbers of CFU were quantified by plating serial dilutions of lysates on M7H10 agar plates. Cell viability was evaluated by trypan blue exclusion before and after infection and/or drug treatment at each time point. Peritoneal macrophages were 95% viable at 48 h postinfection; drug exposure did not affect cell viability (data not shown).
Animal experiments. Studies were conducted using an immunocompromised mouse model modified to mimic respiratory infection (36). Briefly, 24 6-week-old male BALB/c mice (20 to 22 g) were rendered neutropenic by intraperitoneal injection of cyclophosphamide at 150 mg/kg administered 4 days and 1 days prior to infection. The mice were then infected intranasally with ϳ4 ϫ 10 7 CFU of M. abscessus ATCC 19977. On day 3 postinfection, three mice were sacrificed, and the CFU/lung were determined to evaluate the model's efficacy. The remaining 21 infected mice were divided randomly into three groups: group 1 (control administered PBS with 0.5% methylcellulose-0.1% Tween 80 by daily oral gavage for 2 weeks), group 2 (treated with 50 mg/kg AR-12 in PBS with 0.5% methylcellulose-0.1% Tween 80 administered by daily oral gavage for 2 weeks), and group 3 (administered 50 mg/kg amikacin intramuscularly for 2 weeks daily). The dosage of amikacin was selected based upon the report of Das et al. (36). The same AR-12 dose was chosen to facilitate a comparison of antibacterial effects. The mice were sacrificed on day 17 postinfection, and the upper lobes of the right lungs of mice in each group were dissected, fixed with 4% paraformaldehyde for 24 h, and sectioned. The tissue sections were subjected to hematoxylin and eosin (H&E) and acid-fast staining, and the bacterial loads were compared by histologic examination. The remaining portions of the lungs were homogenized, and 10-fold serial dilutions of the homogenates were plated on M7H10 agar supplemented with 10% Middlebrook OADC enrichment. The bacterial content of the lungs was estimated from the colonies that grew on plates incubated for 5 days at 37°C. The animal experiments were approved by Tongji University School of Medicine review boards and performed according to established safety protocols in a biosafety level 2 laboratory at the Shanghai Public Health Clinical Center.
Statistical analysis. Statistical differences between study groups were determined by a nonpaired Student t test; a P value of Ͻ0.05 was considered significant. Computations were performed using Prism 7 (GraphPad Software, San Diego, CA).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.2 MB.