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This study presents a comprehensive lesion-centric analysis of disease progression in the rabbit model of tuberculosis, showing immunopathology and lesion heterogeneity similar to human and nonhuman primates. In this model, pyrazinamide sterilizes necrotic lesions, where persistent bacterial populations reside., In the 1970s, inclusion of pyrazinamide (PZA) in the drug regimen of tuberculosis (TB) patients for the first 2 mo achieved a drastic reduction of therapy duration. Until now, however, the mechanisms underlying PZA’s unique contribution to efficacy have remained controversial, and animal efficacy data vary across species. To understand how PZA kills bacterial populations present in critical lung lesion compartments, we first characterized a rabbit model of active TB, showing striking similarities in lesion types and fates to nonhuman primate models deemed the most appropriate surrogates of human TB. We next employed this model with lesion-centric molecular and bacteriology readouts to demonstrate that PZA exhibits potent activity against Mycobacterium tuberculosis residing in difficult-to-sterilize necrotic lesions. Our data also indicate that PZA is slow acting, suggesting that PZA administration beyond the first 2 mo may accelerate the cure. In conclusion, we provide a pharmacodynamic explanation for PZA’s treatment-shortening effect and deliver new tools to dissect the contribution of immune response versus drug at the lesion level.

Fluoroquinolones represent the pillar of multidrug-resistant tuberculosis (MDR-TB) treatment, with moxifloxacin, levofloxacin, or gatifloxacin being prescribed to MDR-TB patients. Recently, several clinical trials of “universal” drug regimens, aiming to treat drug-susceptible and drug-resistant TB, have included a fluoroquinolone., Fluoroquinolones represent the pillar of multidrug-resistant tuberculosis (MDR-TB) treatment, with moxifloxacin, levofloxacin, or gatifloxacin being prescribed to MDR-TB patients. Recently, several clinical trials of “universal” drug regimens, aiming to treat drug-susceptible and drug-resistant TB, have included a fluoroquinolone. In the absence of clinical data comparing their side-by-side efficacies in controlled MDR-TB trials, a pharmacological rationale is needed to guide the selection of the most efficacious fluoroquinolone. The present studies were designed to test the hypothesis that fluoroquinolone concentrations (pharmacokinetics) and activity (pharmacodynamics) at the site of infection are better predictors of efficacy than the plasma concentrations and potency measured in standard growth inhibition assays and are better suited to determinations of whether one of the fluoroquinolones outperforms the others in rabbits with active TB. We first measured the penetration of these fluoroquinolones in lung lesion compartments, and their potency against bacterial populations that reside in each compartment, to compute lesion-centric pharmacokinetic-pharmacodynamic (PK/PD) parameters. PK modeling methods were used to quantify drug penetration from plasma to tissues at human-equivalent doses. On the basis of these metrics, moxifloxacin emerged with a clear advantage, whereas plasma-based PK/PD favored levofloxacin (the ranges of the plasma AUC/MIC ratio [i.e., the area under the concentration-time curve over 24 h in the steady state divided by the MIC] are 46 to 86 for moxifloxacin and 74 to 258 for levofloxacin). A comparative efficacy trial in the rabbit model of active TB demonstrated the superiority of moxifloxacin in reducing bacterial burden at the lesion level and in sterilizing cellular and necrotic lesions. Collectively, these results show that PK/PD data obtained at the site of infection represent an adequate predictor of drug efficacy against TB and constitute the baseline required to explore synergies, antagonism, and drug-drug interactions in fluoroquinolone-containing regimens.

Clinical trials and practice have shown that ethambutol is an important component of the first-line tuberculosis (TB) regime. This contrasts the drug's rather modest potency and lack of activity against nongrowing persister mycobacteria. The standard plasma-based pharmacokinetic-pharmacodynamic profile of ethambutol suggests that the drug may be of limited clinical value. Here, we hypothesized that this apparent contradiction may be explained by favorable penetration of the drug into TB lesions. First, we utilized novel in vitro lesion pharmacokinetic assays and predicted good penetration of the drug into lesions. We then employed mass spectrometry imaging and laser capture microdissection coupled to liquid chromatography and tandem mass spectrometry (LCM and LC/MS-MS, respectively) to show that ethambutol, indeed, accumulates in diseased tissues and penetrates the major human-like lesion types represented in the rabbit model of TB disease with a lesion-to-plasma exposure ratio ranging from 9 to 12. In addition, ethambutol exhibits slow but sustained passive diffusion into caseum to reach concentrations markedly higher than those measured in plasma at steady state. The results explain why ethambutol has retained its place in the first-line regimen, validate our in vitro lesion penetration assays, and demonstrate the critical importance of effective lesion penetration for anti-TB drugs. Our findings suggest that in vitro and in vivo lesion penetration evaluation should be included in TB drug discovery programs. Finally, this is the first time that LCM with LC-MS/MS has been used to quantify a small molecule at high spatial resolution in infected tissues, a method that can easily be extended to other infectious diseases.