GSK2556286 Is a Novel Antitubercular Drug Candidate Effective In Vivo with the Potential To Shorten Tuberculosis Treatment.

As a result of a high-throughput compound screening campaign using Mycobacterium tuberculosis-infected macrophages, a new drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (50% inhibitory concentration [IC50] = 0.07 µM), is active against extracellular bacteria in cholesterol-containing culture medium, and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug-resistant (MDR) or extensively drug-resistant (XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens. (This study has been registered at ClinicalTrials.gov under identifier NCT04472897).

Contribution of Pretomanid to Novel Regimens Containing Bedaquiline with either Linezolid or Moxifloxacin and Pyrazinamide in Murine Models of Tuberculosis.

Novel regimens combining bedaquiline and pretomanid with either linezolid (BPaL regimen) or moxifloxacin and pyrazinamide (BPaMZ regimen) shorten the treatment duration needed to cure tuberculosis (TB) in BALB/c mice compared to that of the first-line regimen and have yielded promising results in initial clinical trials. However, the independent contribution of the investigational new drug pretomanid to the efficacy of BPaMZ has not been examined, and its contribution to BPaL has been examined only over the first 2 months of treatment. In the present study, the addition of pretomanid to BL increased bactericidal activity, prevented emergence of bedaquiline resistance, and shortened the duration needed to prevent relapse with drug-susceptible isolates by at least 2 months in BALB/c mice. Addition of pretomanid to bedaquiline, moxifloxacin, and pyrazinamide (BMZ) resulted in a 1-log10 greater CFU reduction after 1 month of treatment and/or reduced the number of mice relapsing in each of 2 experiments in BALB/c mice and in immunocompromised nude mice. Bedaquiline-resistant isolates were found at relapse in only one BMZ-treated nude mouse. Treatment of infection with a pyrazinamide-resistant mutant in BALB/c mice with BPaMZ prevented selection of bedaquiline-resistant mutants and reduced the proportion of mice relapsing compared to that for BMZ treatment alone. Among severely ill C3HeB/FeJ mice with caseous pneumonia and cavitation, BPaMZ increased median survival (≥60 versus 21 days) and reduced median lung CFU by 2.4 log10 at 1 month compared to the level for BMZ. In conclusion, in 3 different mouse models, pretomanid contributed significantly to the efficacy of the BPaMZ and BPaL regimens, including restricting the selection of bedaquiline-resistant mutants.

Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis.

The novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, ≥4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance in Mycobacterium tuberculosis: loss-of-function mutations in pepQ (Rv2535c), which corresponds to a putative Xaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression of Rv0678, mmpS5, or mmpL5 between mutant and parent strains. Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene.

The tuberculosis drug discovery and development pipeline and emerging drug targets.

The recent accelerated approval for use in extensively drug-resistant and multidrug-resistant-tuberculosis (MDR-TB) of two first-in-class TB drugs, bedaquiline and delamanid, has reinvigorated the TB drug discovery and development field. However, although several promising clinical development programs are ongoing to evaluate new TB drugs and regimens, the number of novel series represented is few. The global early-development pipeline is also woefully thin. To have a chance of achieving the goal of better, shorter, safer TB drug regimens with utility against drug-sensitive and drug-resistant disease, a robust and diverse global TB drug discovery pipeline is key, including innovative approaches that make use of recently acquired knowledge on the biology of TB. Fortunately, drug discovery for TB has resurged in recent years, generating compounds with varying potential for progression into developable leads. In parallel, advances have been made in understanding TB pathogenesis. It is now possible to apply the lessons learned from recent TB hit generation efforts and newly validated TB drug targets to generate the next wave of TB drug leads. Use of currently underexploited sources of chemical matter and lead-optimization strategies may also improve the efficiency of future TB drug discovery. Novel TB drug regimens with shorter treatment durations must target all subpopulations of Mycobacterium tuberculosis existing in an infection, including those responsible for the protracted TB treatment duration. This review summarizes the current TB drug development pipeline and proposes strategies for generating improved hits and leads in the discovery phase that could help achieve this goal.

Contribution of the nitroimidazoles PA-824 and TBA-354 to the activity of novel regimens in murine models of tuberculosis.

New regimens based on two or more novel agents are sought in order to shorten or simplify the treatment of both drug-susceptible and drug-resistant forms of tuberculosis. PA-824 is a nitroimidazo-oxazine now in phase II trials and has shown significant early bactericidal activity alone and in combination with the newly approved agent bedaquiline or with pyrazinamide with or without moxifloxacin. While the development of PA-824 continues, a potential next-generation derivative, TBA-354, has been discovered to have in vitro potency superior to that of PA-824 and greater metabolic stability than that of the other nitroimidazole derivative in clinical development, delamanid. In the present study, we compared the activities of PA-824 and TBA-354 as monotherapies in murine models of the initial intensive and continuation phases of treatment, as well as in combination with bedaquiline plus pyrazinamide, sutezolid, and/or clofazimine. The monotherapy studies demonstrated that TBA-354 is 5 to 10 times more potent than PA-824, but selected mutants are cross-resistant to PA-824 and delamanid. The combination studies revealed that TBA-354 is 2 to 4 times more potent than PA-824 when combined with bedaquiline, and when administered at a dose equivalent to that of PA-824, TBA-354 demonstrated superior sterilizing efficacy. Perhaps most importantly, the addition of either nitroimidazole significantly improved the sterilizing activities of bedaquiline and sutezolid, with or without pyrazinamide, confirming the value of each agent in this potentially universally active short-course regimen.

Potent rifamycin-sparing regimen cures guinea pig tuberculosis as rapidly as the standard regimen.

Strategies involving new drug combinations, as well as new uses of existing drugs, are urgently needed to reduce the time required to cure patients with drug-sensitive or multidrug-resistant (MDR) tuberculosis (TB). We compared the sterilizing activity of the standard first-line antitubercular regimen, rifampin-isoniazid-pyrazinamide (RHZ), with that of the novel regimen PA-824-moxifloxacin-pyrazinamide (PaMZ), which is currently being studied in clinical trials (NCT01498419), in the guinea pig model of chronic TB infection, in which animals develop necrotic granulomas histologically resembling their human counterparts. Guinea pigs were aerosol infected with ~2 log10 bacilli of wild-type Mycobacterium tuberculosis H37Rv, and antibiotic treatment was initiated 6 weeks after infection. Separate groups of animals received RHZ, PaMZ, or single or two-drug components of the latter regimen administered at human-equivalent doses 5 days/week for a total of 8 weeks. Relapse rates were assessed 3 months after discontinuation of treatment to determine the sterilizing activity of each combination regimen. PaMZ given at human-equivalent doses was safe and well tolerated for the entire treatment period and rendered guinea pig lungs culture negative more rapidly than RHZ did. After 1 month of treatment, 80% and 50% of animals in the RHZ and PaMZ groups, respectively, had lung culture-positive relapse. Both combination regimens prevented microbiological relapse when administered for a total of 2 months. Our data support the use of PaMZ as a novel isoniazid- and rifamycin-sparing regimen suitable for treatment of both drug-sensitive TB and MDR-TB.

Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis.

In drug development, there are typically a series of preclinical studies that must be completed with new compounds or regimens before use in humans. A sequence of in vitro assays followed by in vivo testing in validated animal models to assess the activity against Mycobacterium tuberculosis, pharmacology and toxicity is generally used for advancing compounds against tuberculosis in a preclinical stage. A plethora of different assay systems and conditions are used to study the effect of drug candidates on the growth of M. tuberculosis, making it difficult to compare data from one laboratory to another. The Bill and Melinda Gates Foundation recognized the scientific gap to delineate the spectrum of variables in experimental protocols, identify which of these are biologically significant, and converge towards a rationally derived standard set of optimized assays for evaluating compounds. The goals of this document are to recommend protocols and hence accelerate the process of TB drug discovery and testing. Data gathered from preclinical in vitro and in vivo assays during personal visits to laboratories and an electronic survey of methodologies sent to investigators is reported. Comments, opinions, experiences as well as final recommendations from those currently engaged in such preclinical studies for TB drug testing are being presented. Certain in vitro assays and mouse efficacy models were re-evaluated in the laboratory as head-to-head experiments and a summary is provided on the results obtained. It is our hope that this information will be a valuable resource for investigators in the field to move forward in an efficient way and that key variables of assays are included to ensure accuracy of results which can then be used for designing human clinical trials. This document then concludes with remaining questions and critical gaps that are in need of further validation and experimentation.