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Background Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. These studies evaluated oral doses (PO) of EBO against 5 M. avium complex (MAC) strains in a chronic mouse infection model either as monotherapy or in combination with standard of care [SOC; clarithromycin (CLR), rifabutin (RFB), ethambutol (EMB)]. Methods A pilot chronic efficacy study against M. avium 2285R evaluated EBO at 1, 10, 30, 100, 300 and 500 mg/kg PO once daily (QD) compared to 250 mg/kg CLR PO QD. C57BL/6 mice were infected with a pulmonary aerosol of 1x1011 CFU. Treatment was administered for 56 days starting on day 28 post-infection. The bacterial burden (CFU) in lungs was evaluated on days 1, 28 and 84 post-infection by plating serial dilutions of homogenates on Middlebrook 7H11 charcoal agar plates. An additional 4 strains of MAC were evaluated with EBO doses of 100, 200, 300 or 400 mg/kg QD compared with the SOC therapy for MAC (CLR 250 mg/kg, RFB 100 mg/kg, EMB 100 mg/kg) QD and SOC plus EBO 200mg/kg QD. Oral exposures of EBO were determined in a group of uninfected mice (Table 1).Table 1:C57BL/6 Murine Pharmacokinetic Parameters Results In a study with M. avium 2285R, a biofilm-forming strain, EBO at all doses tested was significantly better than CLR dosed at 250 mg/kg (Figure 1), and no CFU were detected on agar plates containing EBO (16 mg/L). In subsequent studies, SOC was compared to EBO in 4 additional MAC strains (Figure 2). Efficacy of EBO monotherapy was better than SOC against M. avium ATCC 700898, while it was as good as SOC with M. intracellulare 1956, M. intracellulareDNA00055, and M. intracellulare DNA00111 with CFU reductions ranging from 2 - 4.8 log10 compared to day 28 controls. In all four strains tested, 200 mg/kg EBO, which approximates the human oral equivalent dose of 500 mg, combined with SOC increased bacterial killing from 1.4 - 3.0 log10 CFU compared to SOC alone resulting in total lung CFU reductions of 4.6 - 5.6 log10. Figure 1Figure 2 Conclusion In this chronic mouse lung infection model, no EBO resistance development was detected with M. avium 2285R at day 84. EBO demonstrated potent in vivo efficacy against 5 MAC strains and significantly improved efficacy when combined with SOC, supporting further clinical development for EBO. Disclosures Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn Shoen, PhD, AN2 Therapeutics: Grant/Research Support Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds.

Background Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria (NTM). The standard of care therapy for Mycobacterium avium complex (MAC) lung disease consists of a combination of a macrolide, ethambutol (EMB) and a rifamycin. A total of 5 strains of MAC, M. abscessus ATCC 19977 and M. peregrinum ATCC 700686 were tested in checkerboard assays to evaluate the effects of combining EBO with other agents. The spontaneous resistance frequency (RF) was determined for EBO singly and in combination against M. avium ATCC 700898. Methods The effects of combining EBO with clarithromycin (CLR), rifabutin (RBT), EMB, amikacin (AMK) or bedaquiline (BDQ) were evaluated in 7 strains of NTM. Synergy, additive effects, indifference or antagonism was characterized in the checkerboard assay using EUCAST criteria. The RF of M. avium ATCC 700898 at 2x, 4x and 8x the MIC (8 mg/L) of EBO was determined, as was the RF of EBO combined with CLR, RBT, AMK or EMB. MICs of selected EBO mutants were determined against AMK, BDQ, CLR, RBT, EMB, and clofazimine (CFZ) and the mutants were further characterized. Results The RF of EBO ranged from 1.58x10-7 to 8.48x10-9 when selected on 2 - 8x agar MIC. The MIC for EBO increased 32- > 256-fold for the resistant mutants; however, the MICs for the other drugs tested against EBO-resistant strains did not change significantly. The activity of EBO was not antagonized, and was mainly indifferent to the addition of CLR, RBT, AMK, or BDQ for all the NTM strains tested. Synergy with EMB was observed with 2 strains and additivity with 2 additional strains of MAC. The addition of EMB (24 mg/L), CLR (32 mg/L), RFB (2 mg/L) or AMK (128 mg/L) to agar plates containing 2x MIC of EBO lowered the RF to at least < 2.13x10-10 (Table 1). Table 1.In Vitro Resistance Frequency for the Components of SOC with and without EBO in Mycobacterium avium ATCC 700898 Conclusion In the checkerboard studies, no evidence of antagonism was observed with any strain or EBO combination; interactions were largely indifferent. EBO combined with EMB in MAC resulted in synergy or additive effects in the checkerboard assay. The addition of EMB, CLR, RFB or AMK to EBO led to a > 700-fold reduction in RF. Activity of other drugs was not impacted by EBO resistance suggesting that cross-resistance did not occur. Disclosures Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn M. Shoen, PhD, AN2 Therapeutics: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support.

Background Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. We evaluated the effects of select culture conditions on MIC determinations of EBO against isolates of M. avium complex (MAC), as well as EBO MIC90 results with Middlebrook 7H9 broth compared to those with cation-adjusted Mueller Hinton Broth (CAMHB) for 51 MAC isolates. Methods Six strains of MAC were used to test the in vitro activity of EBO in different conditions in a broth microdilution (BMD) assay. Activity was compared in Middlebrook 7H9 and CAMHB with 5% OADC from different manufacturers. The effects of glycerol, cations, oxyrase, varying pH levels, and increasing inoculum sizes were tested. Finally, EBO in vitro activity was tested for 51 MAC isolates in a BMD assay in both Middlebrook 7H9 and CAMHB with 5% OADC. Results In general, manipulation of select culture conditions caused very little variation in EBO MIC values for the 6 MAC strains except for increasing the inoculum from ∼105 to 107 CFU/mL, which caused an approximately 64x increase in the MIC. Since 1 MAC isolate out of 6 was affected by the addition of casitone, we tested 51 MAC isolates in both the minimal media Middlebrook 7H9 and the complex media CAMHB. EBO had a narrow MIC range in both broths, 0.25-8 mg/L for all isolates. The EBO modal MIC, MIC50 and MIC90 for the entire MAC panel of 51 isolates was 2 mg/L, 2 mg/L, and 8 mg/L for CAMHB and 1 mg/L, 1 mg/L, and 4 mg/L for Middlebrook 7H9, respectively (Table 1). Three clarithromycin-resistant isolates had EBO MIC values of 0.5 mg/L, 1 mg/L, and 2 mg/L suggesting that clarithromycin resistance does not affect EBO in vitro activity. In addition, amikacin resistance as determined using the Clinical Laboratory Standards Institute (CLSI) IV amikacin breakpoint (MIC ≥ 64 mg/L) had no noticeable effect on EBO MIC values. Table 1EBO In Vitro Activity Against 51 Isolates of Mycobacterium avium complex Conclusion The MIC distribution for the 51 MAC isolates tested was similar in both media types, indicating that CAMHB can be used to test EBO MAC susceptibilities per CLSI guidelines. Clarithromycin- and amikacin-resistant isolates demonstrated no cross-resistance with EBO. Disclosures Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn M. Shoen, PhD, AN2 Therapeutics: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support.

The current standard of care therapy for pulmonary Mycobacterium kansasii infection is isoniazid (300 mg/day), rifampin (600 mg/day), and ethambutol (15 mg/kg/day) for 12 months after achieving sputum culture negativity. Rifampin is the key drug in this regimen. The contribution of isoniazid is unclear since its in vitro MICs against M. kansasii are near the peak achievable serum levels and more than 100-fold greater than the MICs for Mycobacterium tuberculosis. Ethambutol likely decreases the emergence of rifampin resistant organisms. There are several new drug classes (e.g., quinolones, macrolides, nitroimidazoles, diarylquinolines, and clofazimine) that exhibit antimycobacterial activities against M. tuberculosis but have not yet been adequately studied against M. kansasii infections. The evaluation of in vitro activities of these agents as well as their study in new regimens in comparison to the standard of care regimen in mouse infection models should be undertaken. This knowledge will inform development of human clinical trials of new regimens in comparison to the current standard of care regimen. It is likely that shorter and more effective therapy is achievable with currently available drugs.

The minimal inhibitory concentrations (MICs) of copper and cobalt based dimeric pyrophosphate complexes with capping 1,10-phenanthroline groups on clinical isolates of C. albicans (28 isolates), C. krusei (20 isolates) and C. tropicalis (20 isolates) are reported. C. albicans was inhibited by the cobalt complex better than by the copper complex, while C. krusei demonstrated the opposite results. C. tropicalis showed similar sensitivities to both metals in terms of calculated MIC50 values but was more sensitive to cobalt when MIC90 values were noted. Knockout strains of C. albicans that had the copper efflux protein P-type ATPase (CRP1), the copper binding metallothionein CUP1 or both CRP1/CUP1 removed clearly demonstrate that the origins of copper resistant in C. albicans lies primarily in the P-type ATPase, with the MT playing an important secondary role in the absence of the efflux protein. This study suggests that certain strains of Candida have evolved to protect against particular metal ions and that in the case of C. albicans, a primary invasive fungal species, cobalt may be a good starting-point for new therapeutic development.

The genus Mycobacterium includes non-pathogenic species such as M. smegmatis, and pathogenic species such as M. tuberculosis, the causative agent of tuberculosis (TB). Treatment of TB requires a lengthy regimen of several antibiotics, whose effectiveness has been compromised by the emergence of resistant strains. New antibiotics that can shorten the treatment course and those that have not been compromised by bacterial resistance are needed. In this study, we report that thiadiazolidinones, a relatively little-studied heterocyclic class, inhibit the activity of mycobacterial alanine racemase, an essential enzyme that converts L-alanine to D-alanine for peptidoglycan synthesis. Twelve members of the thiadiazolidinone family were evaluated for inhibition of M. tuberculosis and M. smegmatis alanine racemase activity and bacterial growth. Thiadiazolidinones inhibited M. tuberculosis and M. smegmatis alanine racemases to different extents with 50% inhibitory concentrations (IC50) ranging from 150 µM, respectively. The compounds also inhibited the growth of these bacteria, including multidrug resistant strains of M. tuberculosis. The minimal inhibitory concentrations (MIC) for drug-susceptible M. tuberculosis and M. smegmatis ranged from 6.25 µg/ml to 100 µg/ml, and from 1.56 to 6.25 µg/ml for drug-resistant M. tuberculosis. The in vitro activities of thiadiazolidinones suggest that this family of compounds might represent starting points for medicinal chemistry efforts aimed at developing novel antimycobacterial agents.

Previous experiments with rifalazil (RLZ) (also known as KRM-1648) in combination with isoniazid (INH) demonstrated its potential for short-course treatment of Mycobacterium tuberculosis infection. In this study we investigated the minimum RLZ-INH treatment time required to eradicate M. tuberculosis in a murine model. RLZ-INH treatment for 6 weeks or longer led to a nonculturable state. Groups of mice treated in parallel were killed following an observation period to evaluate regrowth. RLZ-INH treatment for a minimum of 10 weeks was necessary to maintain a nonculturable state through the observation period. Pyrazinamide (PZA) was added to this regimen to determine whether the treatment duration could be further reduced. In this model, the addition of PZA did not shorten the duration of RLZ-INH treatment required to eradicate M. tuberculosis from mice. The addition of PZA reduced the number of mice in which regrowth occurred, although the reduction was not statistically significant.

Tuberculosis (TB) causes up to 10 million incident cases worldwide per annum. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains are leading factors in the resurgence of TB cases and the need to produce new agents to combat such infection. Herein, we describe Co(II) and Cu(II) metal based complexes that feature the pyrophosphate ligand with notable selectivity and marked potency against Mycobacterium tuberculosis, including MDR strains. Such complexes are confirmed to be bacteriocidal and not affected by efflux inhibitors. Finally, while susceptibility to copper has recently been established for M. tuberculosis, the greater efficacy of cobalt observed herein is of considerable note and in line with the discovery of a copper metallothionein in M. tuberculosis.

Early attempts to improve BCG have focused on increasing the expression of prominent antigens and adding recombinant toxins or cytokines to influence antigen presentation. One such modified BCG vaccine candidate has been withdrawn from human clinical trials due to adverse effects. BCG was derived from virulent Mycobacterium bovis and retains much of its capacity for suppressing host immune responses. Accordingly, we have used a different strategy for improving BCG based on reducing its immune suppressive capacity. We made four modifications to BCG Tice to produce 4dBCG and compared it to the parent vaccine in C57Bl/6 mice. The modifications included elimination of the oxidative stress sigma factor SigH, elimination of the SecA2 secretion channel, and reductions in the activity of iron co-factored superoxide dismutase and glutamine synthetase. After IV inoculation of 4dBCG, 95% of vaccine bacilli were eradicated from the spleens of mice within 60 days whereas the titer of BCG Tice was not significantly reduced. Subcutaneous vaccination with 4dBCG produced greater protection than vaccination with BCG against dissemination of an aerosolized challenge of M. tuberculosis to the spleen at 8 weeks post-challenge. At this time, 4dBCG-vaccinated mice also exhibited altered lung histopathology compared to BCG-vaccinated mice and control mice with less well-developed lymphohistiocytic nodules in the lung parenchyma. At 26 weeks post-challenge, 4dBCG-vaccinated mice but not BCG-vaccinated mice had significantly fewer challenge bacilli in the lungs than control mice. In conclusion, despite reduced persistence in mice a modified BCG vaccine with diminished antioxidants and glutamine synthetase is superior to the parent vaccine in conferring protection against M. tuberculosis. The targeting of multiple immune suppressive factors produced by BCG is a promising strategy for simultaneously improving vaccine safety and effectiveness.

Objective Designing a more rapid method to test antimycobacterial agents in a murine model would significantly improve the drug development process. We describe a short-course in vivo treatment model that could be used to screen potential antituberculous drugs. Methods In this model, C57BL/6 mice were infected intranasally with approximately 10(6) viable Mycobacterium tuberculosis organisms. Treatment began 1 day post-infection and was administered for 2 days. Mice were euthanized 3 days post-infection and their right lungs were removed and cell counts determined. Several antimycobacterial agents with superior in vivo activity in a 4 week treatment model were tested to evaluate the short-course treatment model. Results Two days of isoniazid (25 mg/kg), rifampicin (20 mg/kg), PNU-100480 (100 mg/kg), gatifloxacin (100 mg/kg), levofloxacin (100 mg/kg) and sparfloxacin (100 mg/kg) were all able to significantly reduce the mycobacterial load in the lungs compared with the untreated control mice. Conclusions Use of this model to screen potential chemotherapeutic agents will save time and resources.

Activities of clarithromycin alone and in combination with rifampicin, gatifloxacin or linezolid were evaluated against Mycobacterium kansasii in a murine infection model. Clarithromycin was the most active single agent. Rifampicin and gatifloxacin had similar activities, but were less active than clarithromycin. Clarithromycin in combination with rifampicin was the most active combination therapy.

Common treatments for nocardial infections include the sulfonamides used alone or in combination, e.g., trimethoprim-sulfamethoxazole, with amikacin, imipenem, or ceftriaxone. Treatment is especially difficult in cases involving the central nervous system or disseminated disease or with drug-resistant species such as Nocardia farcinica and Nocardia otitidiscaviarum (5). Sulfamethoxazole and dapsone inhibit dihydropteroate synthase and hence prevent the conversion of bacterial para-aminobenzoic acid to dihydrofolic acid. The active putative metabolite of PS-22 is a dihydrotriazine, a sister compound of WR99210 (1). WR99210 is a dihydrofolate reductase inhibitor that has demonstrated in vitro activity against Nocardia species (3). Theoretically, the combination of a dihydrofolate reductase inhibitor with a dihydropteroate synthase inhibitor should lead to enhanced activity, as both these enzymes lie on the folate biosynthesis pathway. This was evaluated in vitro. The active putative metabolite of PS-22 and dapsone was provided by Jacobus Pharmaceutical Co., Princeton, N.J.; sulfamethoxazole was purchased from Sigma Chemical Co., St. Louis, Mo. Twenty strains of Nocardia spp. from the American Type Culture Collection and clinical isolates provided by the Clinical Microbiology Laboratory, Upstate Medical University (Betty Ann Forbes), Syracuse, N.Y., were used in this study. Experimental in vitro procedures were modeled after those listed in the paper of Meyer et al. (4). Varying concentrations of the active putative metabolite of PS-22 were tested against fixed concentrations of sulfamethoxazole and dapsone. The concentration of sulfamethoxazole at 10 μg/ml was physiologically achievable. The concentration of dapsone was fixed at 1 μg/ml, as following the administration of 100 mg of the drug orally serum drug concentrations range from 0.4 to 1.2 μg/ml (2). Table ​Table11 shows the results of the drug combinations against various Nocardia species. The in vitro activity of the active putative metabolite of PS-22 was generally enhanced by combination with dapsone or sulfamethoxazole. These results agree with the findings of McNeil et al. (3) and demonstrate that the rational drug combination of a dihydrofolate reductase inhibitor combined with a dihydropteroate synthase inhibitor provides good targets for the evaluation of agents that work at these sites. TABLE 1. MICs (μg/ml) of a WR99210 analogue alone and in combination with dapsone or sulfamethoxazole against various strains of Nocardia spp.

Superoxide dismutase (SOD) is a ubiquitous metalloenzyme in aerobic organisms that catalyzes the conversion of superoxide anion to hydrogen peroxide. Mycobacterium tuberculosis is unusual in that it secretes large quantities of iron-cofactored SOD. To determine the role of SOD in pathogenesis, we constructed mutants of M. tuberculosis H37Rv with reduced SOD production. Compared with controls, SOD-diminished isolates were more susceptible to killing by hydrogen peroxide. The isolates were markedly attenuated, exhibiting nearly 100,000-fold fewer bacilli than virulent control strains in the lungs and spleens of C57BL/6 mice 4 wk after intravenous inoculation. In the lung, SOD-attenuated M. tuberculosis induced robust interstitial mononuclear cell infiltration within 24 h and many cells were apoptotic by TUNEL staining, whereas virulent H37Rv exhibited minimal early inflammatory response and only rare interstitial mononuclear cell apoptosis. During prolonged infections, C57BL/6 mice tolerated SOD-attenuated M. tuberculosis better than BCG, exhibiting 68% greater weight gain, quicker eradication of bacilli from the spleen, and less alveolar lung infiltration. These results establish the importance of SOD in the pathogenesis of tuberculosis. Its effect appears to be mediated in part by inhibiting innate host immune responses, including early mononuclear cell infiltration of infected tissues and apoptosis.

Rifalazil (formerly known as KRM-1648) in combination with isoniazid has been found to be more active than rifampin/isoniazid. Administration of rifalazil/isoniazid for 12 weeks resulted in continued apparent sterilization of organs 6 months after cessation of therapy. In this study we evaluated the durability of rifalazil/isoniazid treatment. Female CD-1 mice were infected with Mycobacterium tuberculosis ATCC 35801 (strain Erdman). Rifalazil and isoniazid were given in combination for 6 and 12 weeks; no mycobacteria could be cultured from spleens and lungs at both the 6-week and 12-week time points. After completing treatment, groups of mice treated with rifalazil/isoniazid for 6 or 12 weeks were observed without any additional treatment. These observation groups were compared to groups of rifalazil/isoniazid-treated mice (6 and 12 weeks) given dexamethasone for 7 and 8 weeks, respectively. Modest regrowth was noted in the spleens and lungs of the group treated with rifalazil/isoniazid for 6 weeks. Regrowth in the 6-weeks group was enhanced slightly by treatment with dexamethasone. In contrast, no regrowth was noted in the 12-weeks rifalazil/isoniazid group, and treatment with dexamethasone did not result in any regrowth.

The activities of antituberculosis agents were evaluated in a murine tuberculosis model using a drug-resistant isolate. A multidrug-resistant clinical isolate from a recent outbreak of tuberculosis in the New York State correctional system was used for infection. Approximately 10(7) viable Mycobacterium tuberculosis ATCC 49967 (strain CNL) organisms were given intravenously to 4-week-old female outbred mice. Treatment was started 1 day after infection and given for 4 weeks. Spleens and lungs were homogenized, and viable cell counts were determined. Statistical analysis indicated that ethionamide, sparfloxacin, ofloxacin, capreomycin, clarithromycin, and clofazimine are active in the murine test system with this multidrug-resistant tuberculosis isolate. Sparfloxacin is the most active quinolone. Despite in vitro resistance, isoniazid has moderate activity. In vitro susceptibility data coupled with evaluation of agents against drug-resistant isolates in the murine system should provide information necessary to design clinical trials for treatment of infections with these organisms.