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High-dose rifampicin improved bactericidal activity and culture conversion in early-phase tuberculosis (TB) trials, done mainly in Africa. We performed a whole-blood bactericidal activity (WBA) study to determine whether the effects of high-dose rifampicin differ across globally relevant TB strains and whether effects are similar in dormant bacilli that will be required for enhancing cure. Whole blood from healthy volunteers was spiked with rifampicin (range, 0.63 to 60 mg/L) and incubated with one of four Mycobacterium tuberculosis clinical strains (Haarlem, Latin American-Mediterranean [LAM], East African-Indian [EAI], and Beijing lineages) or a dormant strain (streptomycin-starved 18b [ss18b]). Change in bacterial CFU was estimated after inoculation of WBA cultures in MGIT. WBA increased with higher concentrations of rifampicin in all strains. At rifampicin concentrations up to 5 mg/L, the rates of increase in WBA per unit increase in rifampicin concentration were similar in all 4 clinical strains ( P  > 0.51). Above 5 mg/L, EAI ( P  < 0.001) and Beijing ( P  = 0.007) strains showed greater increases in WBA than did LAM; Haarlem was similar to LAM. The dormant strain showed a lower rate of increase in WBA than clinical strains at rifampicin concentrations up to 5 mg/L; above 5 mg/L, the rate of increase was similar to those in the LAM, Beijing, and Haarlem strains. Increasing rifampicin concentration enhanced WBA in all strains; the greatest effects were seen in strains common in Asia, suggesting that early-phase trial findings may be generalizable beyond Africa. Similar effects of high concentrations of rifampicin on the dormant strain support the concept that this intervention may enhance sterilizing activity.

COX-2 inhibition may be of benefit in the treatment of tuberculosis (TB) through a number of pathways including efflux pump inhibition (increasing intracellular TB drug levels) and diverse effects on inflammation and the immune response. We investigated celecoxib (a COX-2 inhibitor) alone and with standard anti-tuberculosis drugs in the whole-blood bactericidal activity (WBA) model. Healthy volunteers took a single dose of celecoxib (400 mg), followed (after 1 week) by a single dose of either rifampicin (10 mg/kg) or pyrazinamide (25 mg/kg), followed (after 2 or 7 days respectively) by the same anti-tuberculosis drug with celecoxib. WBA was measured at intervals until 8 hours post-dose (by inoculating blood samples with Mycobacterium tuberculosis and estimating the change in bacterial colony forming units after 72 hours incubation). Celecoxib had no activity alone in the WBA assay (cumulative WBA over 8 hours post-dose: 0.03 ± 0.01ΔlogCFU, p = 1.00 versus zero). Celecoxib did not increase cumulative WBA of standard TB drugs (mean cumulative WBA -0.10 ± 0.13ΔlogCFU versus -0.10 ± 0.12ΔlogCFU for TB drugs alone versus TB drugs and celecoxib; mean difference -0.01, 95% CI -0.02 to 0.00; p = 0.16). The lack of benefit of celecoxib suggests that efflux pump inhibition or eicosanoid pathway-related responses are of limited importance in mycobacterial killing in the WBA assay.

Coadministering pyrazinamide (PZA) with the xanthine oxidase inhibitor allopurinol increases systemic levels of the active metabolite, pyrazinoic acid (POA), but the effects on bactericidal activity against tuberculosis are unknown. We randomized healthy volunteers to take a single dose of PZA (either 10 or 25 mg/kg of body weight) at the first visit and the same dose 7 days later, coadministered with allopurinol (100 mg daily; 2 days before to 1 day after the PZA dose). Blood was drawn at intervals until 48 h after each PZA dose, and drug levels were measured using liquid chromatography-tandem mass spectrometry. Whole-blood bactericidal activity (WBA) was measured by inoculating blood samples with Mycobacterium tuberculosis and estimating the change in bacterial CFU after 72 h of incubation. Allopurinol increased the POA area under the concentration-time curve from 0 to 8 h (AUC 0-8 ) (18.32 h · μg/ml versus 24.63 h · μg/ml for PZA alone versus PZA plus allopurinol) ( P < 0.001) and its peak plasma concentration ( C max ) (2.81 μg/ml versus 4.00 μg/ml) ( P < 0.001). There was no effect of allopurinol on mean cumulative WBA (0.01 ± 0.02 ΔlogCFU versus 0.00 ± 0.02 ΔlogCFU for PZA alone versus PZA plus allopurinol) ( P = 0.49). Higher systemic POA levels were associated with greater WBA levels ( P < 0.001), but the relationship was evident only at low POA concentrations. The lack of an effect of allopurinol on WBA despite a significant increase in blood POA levels suggests that host-generated POA may be less effective than POA generated inside bacteria. Coadministration of allopurinol does not appear to be a useful strategy for increasing the efficacy of PZA in clinical practice. (This study has been registered at ClinicalTrials.gov under registration no. NCT02700347.)., (Copyright © 2017 American Society for Microbiology.)

Background: Faropenem has in vitro activity against Mycobacterium tuberculosis (Mtb) and shows synergy with rifampicin. We tested this in a whole-blood bactericidal activity (WBA) trial., Methods: We randomized healthy volunteers to receive a single oral dose of faropenem (600 mg) with amoxicillin/clavulanic acid (500/125 mg) ( n  =   8), rifampicin (10 mg/kg) ( n  =   14) or the combination rifampicin + faropenem + amoxicillin/clavulanic acid ( n  =   14). Blood was drawn at intervals to 8 h post-dose. Drug levels were measured using LC-tandem MS. WBA was measured by inoculating blood samples with Mtb and estimating the change in bacterial cfu after 72 h. Trial registration: ClinicalTrials.gov (NCT02393586)., Results: There was no activity in the faropenem + amoxicillin/clavulanic acid group (cumulative WBA 0.02 Δlog cfu; P  =   0.99 versus zero change). There was a suggestion of a trend favouring the rifampicin + faropenem + amoxicillin/clavulanic acid group at 8 h (cumulative WBA -0.19 ± 0.03 and -0.26 ± 0.03 Δlog cfu in the rifampicin and rifampicin + faropenem + amoxicillin/clavulanic acid groups, respectively; P  =   0.180), which was significant in the first hour post-dose ( P  =   0.032). Faropenem C max and AUC were 5.4 mg/L and 16.2 mg·h/L, respectively, and MIC for Mtb H37Rv was 5-10 mg/L., Conclusions: Faropenem is not active when used alone, possibly due to inadequate plasma levels relative to MIC. However, there was a suggestion of modest synergy with rifampicin that may merit further testing in clinical trials., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mycobacterium tuberculosis (Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F(420) -dependent anti-oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F(420) -deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/NAD(+) ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F(420) (-) mutants are hypersensitive to mycobactericidal agents such as isoniazid, moxifloxacin and clofazimine that elevate oxidative stress. The Mtb deazaflavin-dependent nitroreductase (Ddn) and its two homologues Rv1261c and Rv1558 encode for an F(420) H(2) -dependent quinone reductase (Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F(420) H(2) -specific obligate two-electron reduction of endogenous quinones, thereby competing with the one-electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F(420) biosynthesis pathway or Fqr-class proteins as a mechanism to potentiate the action of bactericidal agents., (© 2012 Blackwell Publishing Ltd.)

Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F(420) deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F(420) and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding., (Journal compilation © 2011 FEBS. No claim to original US government works.)

PA-824 is a bicyclic 4-nitroimidazole, currently in phase II clinical trials for the treatment of tuberculosis. Dose fractionation pharmacokinetic-pharmacodynamic studies in mice indicated that the driver of PA-824 invivo efficacy is the time during which the free drug concentrations in plasma are above the MIC (fT>MIC). In this study, a panel of closely related potent bicyclic 4-nitroimidazoles was profiled in both invivo PK and efficacy studies. In an established murine TB model, the efficacy of diverse nitroimidazole analogs ranged between 0.5 and 2.3 log CFU reduction compared to untreated controls. Further, a retrospective analysis was performed for a set of seven nitroimidazole analogs to identify the PK parameters that correlate with invivo efficacy. Our findings show that the invivo efficacy of bicyclic 4-nitroimidazoles correlated better with lung PK than with plasma PK. Further, nitroimidazole analogs with moderate-to-high volume of distribution and Lung to plasma ratios of >2 showed good efficacy. Among all the PK-PD indices, total lung T>MIC correlated the best with invivo efficacy (rs = 0.88) followed by lung Cmax/MIC and AUC/MIC. Thus, lung drug distribution studies could potentially be exploited to guide the selection of compounds for efficacy studies, thereby accelerating the drug discovery efforts in finding new nitroimidazole analogs. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis ( Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F420-dependent anti-oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F420-deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/ NAD+ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F420− mutants are hypersensitive to mycobactericidal agents such as isoniazid, moxifloxacin and clofazimine that elevate oxidative stress. The Mtb deazaflavin-dependent nitroreductase ( Ddn) and its two homologues Rv1261c and Rv1558 encode for an F420H2-dependent quinone reductase ( Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F420H2-specific obligate two-electron reduction of endogenous quinones, thereby competing with the one-electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F420 biosynthesis pathway or Fqr-class proteins as a mechanism to potentiate the action of bactericidal agents. [ABSTRACT FROM AUTHOR]