Prediction of Drug Penetration in Tuberculosis Lesions

Sarathy, Jansy; Zuccotto, Fabio; Ho, Hsinpin; Sandberg, Lars; Via, Laura E.; Marriner, Gwendolyn A.; Masquelin, Thierry; Wyatt, Paul G.; Ray, Peter C.; Dartois, Véronique

doi:10.1021/acsinfecdis.6b00051

Cited by 115 publications

(160 citation statements)

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“…In contrast, under conditions mimicking caseous granulomas (hypoxia at pH 7.3), only the rifamycins RIF and RFP rapidly killed NR M. tuberculosis, while all other lipophilic and Activity of Anti-TB Drugs in Hypoxia at Neutral pH Antimicrobial Agents and Chemotherapy hydrophilic drugs tested had no or little effect. RIF is known to accumulate and maintain therapeutic levels in caseum, where dormant M. tuberculosis resides (12,13), and a 3-month treatment of latent TB with RFP and INH was found to be noninferior to 9 months of INH treatment alone (3,14). Overall, our Wayne model experiment in hypoxia at pH 7.3 mimicking caseum may be important for testing the activity of new drugs and/or combinations against NR M. tuberculosis and guiding the selection of future therapies inhibiting the reactivation of latent TB to active TB.…”

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confidence: 99%

Mycobacterium tuberculosis Is Selectively Killed by Rifampin and Rifapentine in Hypoxia at Neutral pH

Iacobino

Piccaro

Giannoni

et al. 2017

Antimicrob Agents Chemother

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The activities of rifampin, rifapentine, bedaquiline, PA-824, clofazimine, nitazoxanide, isoniazid, amikacin, moxifloxacin, niclosamide, thioridazine, and pyrazinamide were tested against nonreplicating (dormant) Mycobacterium tuberculosis H37Rv under conditions of hypoxia at pHs 5.8 and 7.3, mimicking environments of cellular granulomas and caseous granulomas, respectively. At pH 5.8, several drugs killed dormant bacilli, with the best being rifampin and rifapentine. At pH 7.3, only rifampin and rifapentine efficiently killed dormant bacilli, while all other drugs showed little activity.KEYWORDS Mycobacterium tuberculosis, dormancy, hypoxia, killing, pH, rifampin, rifapentine, tuberculosis T he etiologic agent of tuberculosis (TB) is Mycobacterium tuberculosis. Two billion people are latently infected with this organism, and in 10% of them, it reactivates to active TB in their lifetime (1). Currently, treatments require 6 months of combination therapy with isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol for active TB and 9 months of INH or 3 months of rifapentine (RFP) and INH for latent TB (2, 3). Active and latent TB infections comprise mixtures of cellular and caseous granulomas, with tubercle bacilli ranging from actively replicating (AR) to dormant nonreplicating (NR) stages (4). In cellular granulomas, replicating bacilli are killed by current therapy, while in low vascularized caseous granulomas, low oxygen pressure restricts the growth from the AR to the NR stage in their hypoxic centers, enabling M. tuberculosis to move into a dormant drug-refractory state.The pH of the phagolysosomes of activated macrophages present in cellular granulomas is acidic (5), while in necrotic centers of hypoxic cholesterol/triacylglycerol-rich caseous granulomas, where dormant M. tuberculosis lives, the pH is 7.2 to 7.5 (6-8). No information on drug activity against NR M. tuberculosis grown in hypoxic neutral conditions is known. In this study, we investigated the growth patterns of NR M. tuberculosis at different pHs and measured the activities of 12 drugs against NR cells under hypoxic conditions at pHs 5.8 and 7.3, mimicking the environments of cellular granulomas and caseous granulomas, respectively.Briefly, M. tuberculosis strain H37Rv was grown at 37°C for 40 days in 20-by 125-mm screw-cap tubes containing Dubos-Tween-albumin broth (DTAB) as previously described (9-11), but in the present study, the DTAB was adjusted to pHs 5. 8, 6.6, 7.0, 7.4, and 7.6. For preparation of NR cells, log-phase cultures were diluted in DTAB and incubated in tubes with the caps tightly screwed and tight rubber caps put under the screw caps. Growth in the tubes was monitored by measuring pH, optical density at 600 nm, and CFU/ml on Middlebrook 7H10 (7H10) agar plates incubated at 37°C for 3 weeks. To determine drug activity against NR cells, 12-and 19-day-old hypoxic cultures (H12 and H19, respectively) were incubated with drugs for 7, 14, and 21 days. Drugs

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confidence: 99%

Mycobacterium tuberculosis Is Selectively Killed by Rifampin and Rifapentine in Hypoxia at Neutral pH

Iacobino

Piccaro

Giannoni

et al. 2017

Antimicrob Agents Chemother

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“…This fraction has been shown to directly correlate with drug penetration in caseum in vivo . 7 As Figure 2 illustrates using MALDI (Matrix-assisted laser desorption/ionization) mass spectrometry images, the higher the f u , the more extensively the drug diffuses into caseum. Hence, this agrees with the hypothesis that as drug molecules diffuse into the outer rim of caseum from its interface with cellular layers of the lesion, binding to caseous macromolecules sequesters the drug, preventing it from further diffusing into the caseous core.…”

Section: Representative Resultsmentioning

confidence: 99%

“…Centrifuge at 10,000 × g for 5 min to sediment the precipitate and transfer supernatants into 96-well deep-well plates for liquid chromatography-mass spectrometry (LCMS) analysis. 7 …”

Section: Protocolmentioning

confidence: 99%

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An <em>In Vitro</em> Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Sarathy

Liang

Weiner

et al. 2017

JoVE

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LONG ABSTRACT: The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

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“…In addition to the phenotypic and genetic characterization of strains, other in vivo factors, such as the population structure [26, 27], pharmacokinetic/pharmacodynamic parameters [28], permeability of the tuberculosis lesions [29], and adverse reactions, also affect the clinical outcome.…”

Section: Discussionmentioning

confidence: 99%

A Comparison of the Sensititre MycoTB Plate, the Bactec MGIT 960, and a Microarray-Based Molecular Assay for the Detection of Drug Resistance in Clinical Mycobacterium tuberculosis Isolates in Moscow, Russia

Nosova¹,

Zimenkov

Khakhalina³

et al. 2016

PLoS ONE

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BackgroundThe goal of this study was to compare the consistency of three assays for the determination of the drug resistance of Mycobacterium tuberculosis (MTB) strains with various resistance profiles isolated from the Moscow region.MethodsA total of 144 MTB clinical isolates with a strong bias toward drug resistance were examined using Bactec MGIT 960, Sensititre MycoTB, and a microarray-based molecular assay TB-TEST to detect substitutions in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis, and embB genes that are associated with resistance to rifampin, isoniazid, fluoroquinolones, second-line injectable drugs and ethambutol.ResultsThe average correlation for the identification of resistant and susceptible isolates using the three methods was approximately 94%. An association of mutations detected with variable resistance levels was shown. We propose a change in the breakpoint minimal inhibitory concentration for kanamycin to less than 5 μg/ml in the Sensititre MycoTB system. A pairwise comparison of the minimal inhibitory concentrations (MICs) of two different drugs revealed an increased correlation in the first-line drug group and a partial correlation in the second-line drug group, reflecting the history of the preferential simultaneous use of drugs from these groups. An increased correlation with the MICs was also observed for drugs sharing common resistance mechanisms.ConclusionsThe quantitative measures of phenotypic drug resistance produced by the Sensititre MycoTB and the timely detection of mutations using the TB-TEST assay provide guidance for clinicians for the choice of the appropriate drug regimen.

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Prediction of Drug Penetration in Tuberculosis Lesions

Cited by 115 publications

References 40 publications

Mycobacterium tuberculosis Is Selectively Killed by Rifampin and Rifapentine in Hypoxia at Neutral pH

Mycobacterium tuberculosis Is Selectively Killed by Rifampin and Rifapentine in Hypoxia at Neutral pH

An <em>In Vitro</em> Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

A Comparison of the Sensititre MycoTB Plate, the Bactec MGIT 960, and a Microarray-Based Molecular Assay for the Detection of Drug Resistance in Clinical Mycobacterium tuberculosis Isolates in Moscow, Russia

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