Do Anti-tuberculosis Drugs Reach Their Target?─High-Resolution Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Provides Information on Drug Penetration into Necrotic Granulomas

Abstract

Tuberculosis (TB) is characterized by mycobacteria-harboring centrally necrotizing granulomas. The efficacy of anti-TB drugs depends on their ability to reach the bacteria in the center of these lesions. Therefore, we developed a mass spectrometry (MS) imaging workflow to evaluate drug penetration in tissue. We employed a specific mouse model thatin contrast to regular inbred micestrongly resembles human TB pathology. Mycobacterium tuberculosis was inactivated in lung sections of these mice by γ-irradiation using a protocol that was optimized to be compatible with high spatial resolution MS imaging. Different distributions in necrotic granulomas could be observed for the anti-TB drugs clofazimine, pyrazinamide, and rifampicin at a pixel size of 30 μm. Clofazimine, imaged here for the first time in necrotic granulomas of mice, showed higher intensities in the surrounding tissue than in necrotic granulomas, confirming data observed in TB patients. Using high spatial resolution drug and lipid imaging (5 μm pixel size) in combination with a newly developed data analysis tool, we found that clofazimine does penetrate to some extent into necrotic granulomas and accumulates in the macrophages inside the granulomas. These results demonstrate that our imaging platform improves the predictive power of preclinical animal models. Our workflow is currently being applied in preclinical studies for novel anti-TB drugs within the German Center for Infection Research (DZIF). It can also be extended to other applications in drug development and beyond. In particular, our data analysis approach can be used to investigate diffusion processes by MS imaging in general.

Interleukin-13-Overexpressing Mice Represent an Advanced Preclinical Model for Detecting the Distribution of Antimycobacterial Drugs within Centrally Necrotizing Granulomas

“…Importantly, regarding the distribution of CFZ, no irradiation-induced changes were observed by MALDI-MS imaging. Furthermore, as we described elsewhere ( 82 ), by applying the identical procedure, we also observed for the two first-line drugs PZA and RIF similar distributions within lung cryosections irrespective of irradiation. Even though gamma irradiation was previously reported for the inactivation of M. tuberculosis for subsequent LC/MS-MS or MALDI-MS imaging analyses ( 7 , 13 , 30 , 31 ), this is the first study to our knowledge that comprehensively investigated the effect of gamma irradiation on tissue morphology, including cellular integrity, drug concentration, and drug distribution.…”

“…Most importantly, the CFZ distribution within necrotic granulomas of BALB/c IL-13 tg mice resembles its penetration observed in human necrotic lesions, where CFZ showed a clear partitioning between the cellular rim and the necrotic core of lesions with an accumulation in cellular layers compared to the necrotic caseum ( 17 ). Moreover, the herein-detected distribution patterns for CFZ, PZA, and RIF within lesions of BALB/c IL-13 tg mice were confirmed by another study conducted by us ( 82 ). Remarkably, the CFZ accumulation in cellular granulomas of BALB/c mice and the poor penetration into centrally necrotizing granulomas developing in BALB/c IL-13 tg mice provide an explanation for the striking discrepancy regarding its efficacy in C3HeB/FeJ and BALB/c mouse strains.…”

“…However, in the present study, CFZ was imaged at a low spatial resolution (35-μm pixel size) so that an adequate correlation of immune cells and drug distribution was not possible. Elsewhere, we revealed, by high-resolution mapping of CFZ in centrally necrotizing lesions of BALB/c IL-13 tg mice in combination with macrophage detection, an accumulation of the drug within the rim of foamy macrophages ( 82 ). In striking contrast, CFZ failed to efficiently diffuse into the necrotic core of granulomas, which, however, is a niche for extracellular mycobacteria, as demonstrated by acid-fast staining.…”

“…Compound identification was based on accurate mass. To confirm the identification of the drugs, lung sections of BALB/c IL-13 tg mice, which were not dosed with the drug combination (CFZ/PZA/RIF), were analyzed as negative control samples as we described elsewhere ( 82 ).…”

“…To validate IL-13 tg mice as an appropriate model for determining the antibiotic distribution within granulomatous lesions, we infected these animals with M. tuberculosis , treated them with PZA, CFZ, and RIF, antibiotics whose penetration into the centrally necrotizing granulomas is known ( 17 ), and measured the drug distribution by an optimized MALDI-MS imaging method ( 12 ). While the present manuscript focuses on the validation of the preclinical IL-13 tg model and the inactivation of M. tuberculosis for subsequent drug detection, we describe in a separate analytical study the MALDI-MS imaging acquisition and data analysis in further detail ( 82 ).…”