Combination Therapy to Kill Mycobacterium tuberculosis in Its Nonreplicating Persister Phenotype.

Mycobacterium tuberculosis (Mtb) exists in various metabolic states, including a nonreplicating persister (NRP) phenotype which may affect response to therapy. We have adopted a model-informed strategy to accelerate discovery of effective Mtb treatment regimens and previously found pretomanid (PMD), moxifloxacin (MXF), and bedaquiline (BDQ) to readily kill logarithmic- and acid-phase Mtb. Here, we studied multiple concentrations of each drug in flask-based, time-kill studies against NRP Mtb in single-, two- and three-drug combinations, including the active M2 metabolite of BDQ. We used nonparametric population algorithms in the Pmetrics package for R to model the data and to simulate the 95% confidence interval of bacterial population decline due to the two-drug combination regimen of PMD + MXF and compared this to observed declines with three-drug regimens. PMD + MXF at concentrations equivalent to average or peak human concentrations effectively eradicated Mtb. Unlike other states for Mtb, we observed no sustained emergence of less susceptible isolates for any regimen. The addition of BDQ as a third drug significantly (P < 0.05) shortened time to total bacterial suppression by 3 days compared to the two-drug regimen, similar to our findings for Mtb in logarithmic or acid growth phases.

Polymyxin B Pharmacodynamics in the Hollow-Fiber Infection Model: What You See May Not Be What You Get.

Dose range studies for polymyxin B (PMB) regimens of 0.75 to 12 mg/kg given every 12 h (q12h) were evaluated for bacterial killing and resistance prevention against an AmpC-overexpressing Pseudomonas aeruginosa and a blaKPC-3-harboring Klebsiella pneumoniae in 10-day in vitro hollow-fiber models. An exposure-response was observed. But all regimens failed due to regrowth. Lower-dose regimens amplified isolates that expressed transient, lower-level adaptive resistance to PMB (MICs ≤ 4 mg/liter). Higher PMB dosages amplified isolates that expressed this resistance mechanism, a higher-MIC "moderately stable" adaptive resistance, and a higher-MIC stable resistance to PMB. Failure of the highest dose regimens was solely due to subpopulations that expressed the two higher-level resistances. Total and bioactive PMB concentrations in broth declined below targeted PK profiles within hours of treatment initiation and prior to bacterial regrowth. With treatment failure, the total PMB measured in bacteria was substantially higher than in broth. But the bioactive PMB in broth and bacteria were low to nondetectable. Together, these findings suggest a sequence of events for treatment failure of the clinical regimen. First, PMB concentrations in broth are diluted as PMB binds to bacteria, resulting in total and bioactive PMB in broth that is lower than targeted. Bacterial regrowth and treatment failure follow, with emergence of subpopulations that express transient lower-level adaptive resistance to PMB and possibly higher-level adaptive and stable resistances. Higher-dose PMB regimens can prevent the emergence of transient lower-level adaptive resistance, but they do not prevent treatment failure due to isolates that express higher-level resistance mechanisms.

The Funnel: a Screening Technique for Identifying Optimal Two-Drug Combination Chemotherapy Regimens.

The Mycobacterium tuberculosis drug discovery effort has generated a substantial number of new/repurposed drugs for therapy for this pathogen. The arrival of these drugs is welcome, but another layer of difficulty has emerged. Single agent therapy is insufficient for patients with late-stage tuberculosis because of resistance emergence. To achieve our therapeutic ends, it is requisite to identify optimal combination regimens. These regimens go through a lengthy and expensive evaluative process. If we have a modest group of 6 to 8 new or repurposed agents, this translates into 15 to 28 possible 2-drug combinations. There is neither time nor resources to give an extensive evaluation for all combinations. We sought a screening procedure that would identify combinations that had a high likelihood of achieving good bacterial burden decline. We examined pretomanid, moxifloxacin, linezolid, and bedaquiline in log-phase growth, acid-phase growth, and nonreplicative persister (NRP) phase in the Greco interaction model. We employed the interaction term α and the calculated bacterial burden decline as metrics to rank different regimens in different metabolic states. No relationship was found between α and bacterial kill. We chose bacterial kill as the prime metric. The combination of pretomanid plus moxifloxacin emerged as the clear frontrunner, as the largest bacterial declines were seen in log phase and acid phase with this regimen and it was second best in NRP phase. Bedaquiline also produced good kill. This screening process may identify optimal combinations that can be further evaluated in both the hollow-fiber infection model and in animal models of Mycobacterium tuberculosis infection.

Effect of Moxifloxacin plus Pretomanid against Mycobacterium tuberculosis in Log Phase, Acid Phase, and Nonreplicating-Persister Phase in an In Vitro Assay.

Combination therapy is a successful approach to treat tuberculosis in patients with susceptible strains of Mycobacterium tuberculosis However, the emergence of resistant strains requires identification of new, effective therapies. Pretomanid (PA824) and moxifloxacin (MXF) are promising options currently under evaluation in clinical trials for the treatment of susceptible and resistant mycobacteria. We applied our recently described screening strategy to characterize the interaction between PA824 and MXF toward the killing of M. tuberculosis in logarithmic growth phase (log phase), acid phase, and nonreplicating-persister (NRP) phase. Respective in vitro data generated for the H37Rv and 18b strains were evaluated in a microdilution plate system containing both drugs in combination. The Universal Response Surface Approach model from Greco et al. (W. R. Greco, G. Bravo, and J. C. Parsons, Pharmacol Rev 47:331-385, 1995) was used to characterize the nature of the interaction between both drugs; synergistic or additive combinations would prompt additional evaluation in the hollow-fiber infection model (HFIM) and in animal studies. The interaction between MXF and PA824 was additive against M. tuberculosis organisms in acid phase (interaction parameter [α] = 5.56e-8 [95% confidence interval {CI} = -0.278 to 0.278] and α = 0.408 [95% CI = 0.105 to 0.711], respectively), NRP phase (α = 0.625 [95% CI = -0.556 to 1.81] and α = 2.92 [95% CI = 0.215 to 5.63], respectively), and log phase (α = 1.57e-6 [95% CI = -0.930 to 0.930] and α = 1.83e-6 [95% CI = -0.929 and 0.929], respectively), prompting further testing of this promising combination for the treatment of tuberculosis in the HFIM and in animal studies.

Effect of Linezolid plus Bedaquiline against Mycobacterium tuberculosis in Log Phase, Acid Phase, and Nonreplicating-Persister Phase in an In Vitro Assay.

Tuberculosis is the ninth-leading cause of death worldwide. Treatment success is approximately 80% for susceptible strains and decreases to 30% for extensively resistant strains. Shortening the therapy duration for Mycobacterium tuberculosis is a major goal, which can be attained with the use of combination therapy. However, the identification of the most promising combination is a challenge given the quantity of older and newer agents available. Our objective was to identify promising 2-drug combinations using an in vitro strategy to ultimately be tested in an in vitro hollow fiber infection model (HFIM) and in animal models. We studied the effect of the combination of linezolid (LZD) and bedaquiline (BDQ) on M. tuberculosis strain H37Rv in log- and acid-phase growth and M. tuberculosis strain 18b in log- and nonreplicating-persister-phase growth in a plate system containing a 9-by-8 matrix of concentrations of both drugs alone and in combinations. A characterization of the interaction as antagonistic, additive, or synergistic was performed using the Greco universal response surface approach (URSA) model. Our results indicate that the interaction between LZD and BDQ is additive for bacterial killing in both strains for both of the metabolic states tested. This prescreen strategy was suitable to identify LZD and BDQ as a promising combination to be further tested in the HFIM. The presence of nonoverlapping mechanisms of drug action suggests each drug in the combination will likely be effective in suppressing the emergence of resistance by M. tuberculosis to the companion drug, which holds promise in improving treatment outcomes for tuberculosis.

Linezolid Kills Acid-Phase and Nonreplicative-Persister-Phase Mycobacterium tuberculosis in a Hollow-Fiber Infection Model.

The therapy for treatment of Mycobacterium tuberculosis infections is long and arduous. It has been hypothesized that the therapy duration is driven primarily by populations of organisms in different metabolic states that replicate slowly or not at all (acid-phase and nonreplicative-persister [NRP]-phase organisms). Linezolid is an oxazolidinone antimicrobial with substantial activity against Log-phase M. tuberculosis Here, we examined organisms in acid-phase growth and nonreplicative-persister-phenotype growth and determined the effect of differing clinically relevant exposures to linezolid in a hollow-fiber infection model (HFIM). The endpoints measured were bacterial kill over 29 days and whether organisms that were less susceptible to linezolid could be recovered during that period. In addition, we evaluated the effect of administration schedule on linezolid activity, contrasting daily administration with administration of twice the daily dose every other day. Linezolid demonstrated robust activity when administered daily against both acid-phase and NRP-phase organisms. We demonstrated a clear dose response, with 900 mg of linezolid daily generating ≥3 Log(CFU/ml) killing of acid-phase and NRP-phase M. tuberculosis over 29 days. Amplification of a population less susceptible to linezolid was not seen. Activity was reduced with every 48-h dosing, indicating that the minimum concentration (Cmin)/MIC ratio drove the microbiological effect. We conclude that once-daily linezolid dosing has substantial activity against M. tuberculosis in acid-phase and NRP-phase metabolic states. Other studies have shown activity against Log-phase M. tuberculosis Linezolid is a valuable addition to the therapeutic armamentarium for M. tuberculosis and has the potential for substantially shortening therapy duration.

Activity of Moxifloxacin against Mycobacterium tuberculosis in Acid Phase and Nonreplicative-Persister Phenotype Phase in a Hollow-Fiber Infection Model.

A major goal for improving tuberculosis therapy is to identify drug regimens with improved efficacy and shorter treatment durations. Shorter therapies improve patient adherence to the antibiotic regimens, which, in turn, decreases resistance emergence. Mycobacterium tuberculosis exists in multiple metabolic states. At the initiation of therapy, the bulk of the population is in log-phase growth. Consequently, it is logical to focus initial therapy on those organisms. Moxifloxacin has good early bactericidal activity against log-phase bacteria and is a logical component of initial therapy. It would be optimal if this agent also possessed activity against acid-phase and nonreplicative-persister (NRP) phenotype organisms. In our hollow-fiber infection model, we studied multiple exposures to moxifloxacin (equivalent to 200 mg to 800 mg daily) against strain H37Rv in the acid phase and against strain 18b in streptomycin starvation, which is a model for NRP-phase organisms. Moxifloxacin possesses good activity against acid-phase organisms, generating cell killing of 3.75 log10(CFU/ml) (200 mg daily) to 5.16 log10(CFU/ml) (800 mg daily) over the 28 days of the experiment. Moxifloxacin also has activity against streptomycin-starved strain 18b. The 400- to 800-mg daily regimens achieved extinction at day 28, while the no-treatment control still had 1.96 log10(CFU/ml) culturable. The lowest dose (200 mg daily) still had 0.7 log10(CFU/ml) measurable at day 28, a net kill of 1.26 log10(CFU/ml). Moxifloxacin is an attractive agent for early therapy, because it possesses activity against three metabolic states of M. tuberculosis.

Evaluating the effect of clofazimine against Mycobacterium tuberculosis given alone or in combination with pretomanid, bedaquiline or linezolid.

In recent years, clofazimine (CFZ) has been regaining prominence for the treatment of tuberculosis. However, it shows limited efficacy as a single drug and optimal combination partners have not been identified. Therefore, the objective of our analysis was to evaluate the efficacy of CFZ-containing two-drug regimens with pretomanid (PMD), bedaquiline (BDQ) or linezolid (LZD) by: (i) determining their pharmacodynamic (PD) mode of interaction against Mycobacterium tuberculosis (Mtb) strain H37Rv in log- phase and acid-phase metabolic states, and against Mtb strain 18b in a non-replicating persister (NRP) metabolic state; (ii) predicting bacterial cell kill of the drugs alone and in combination; and (iii) evaluating the relationship between the interaction mode and the extent of bacterial cell kill. The results of our Greco universal response surface analysis showed that CFZ was at least additive with a clear trend towards synergy when combined with PMD, BDQ and LZD against Mtb in all explored metabolic states under in vitro checkerboard assay conditions. The results further showed that all two-drug combination regimens exerted greater bacterial kill than any of the drugs alone. CFZ alone showed the least antimicrobial efficacy amongst the evaluated drugs, and there was a lack of correlation between the mode of interaction and the extent of bacterial kill. However, we may underestimate the effect of CFZ in this screening approach owing to limited in vitro study duration and neglect of target site accumulation. Clofazimine; Pretomanid; Bedaquiline; Linezolid; Combination chemotherapy; Mycobacterium tuberculosis.