This research project encompasses a number of different approaches to both understand how current anti-tubercular chemotherapy works using the most modern technologies and to use this information to develop new and improved therapies and therapeutic approaches. Individual projects within this framework are; (1) understanding the activity of various drugs in animal models of tuberculosis therapy, (2) development of advanced animal models for predicting drug efficacy under conditions that exactly mimic those experienced by TB patients (3) correlating responses seen in animal models with the pathology and response to therapy observed in human TB, (4) developing structural and functional imaging techniques using CT/PET for use in live, M. tuberculosis (Mtb) infected animals, and (5) developing techniques for assessing drug distribution, penetration, and pharmacokinetics in vivo. Using non-compartmental and population pharmacokinetic approaches, we have modeled the rate and extent of distribution of isoniazid, rifampicin, pyrazinamide and moxifloxacin in rabbit tissues and lesions in preparation of doing the same analysis in human tissues. In 2016 together with colleagues, we showed that the key sterilizing drugs rifampicin and pyrazinamide efficiently penetrate the sites of TB infection in human lung lesions. Rifampicin even accumulates in necrotic caseum, a critical lesion site where persisting tubercle bacilli reside. In contrast, moxifloxacin, which is active in vitro against a subpopulation of M. tuberculosis that persists in specific niches under drug pressure and has achieved treatment shortening in mice, does not diffuse well in caseum, concordant with its failure to shorten therapy in recent clinical trials. This differential spatial distribution and the kinetics of accumulation in lesions may create temporal and spatial black holes of monotherapy, allowing development of multidrug-resistant TB. The finding that lesion penetration may contribute to treatment outcome has wide implications for TB (PMID:26343800). In addition, colleagues have developed a tissue model for quantification of rifampicin (RIF), an antibiotic used to treat tuberculosis, and have tested different methods of applying an isotopically labeled internal standard for MALDI IMS analysis. The application of the standard and subsequently the matrix onto rabbit tissue sections resulted in quantitation that comparable (>90% similarity) to HPLC-MS/MS results obtained from extracts of the same tissue (PMID:26814665). We continue to study the lesion penetration of the modified analogues of PA 824, linezolid and new oxazolidinones in development, bedaquiline and other compounds of potential use in treating TB. The objective of these studies is to compare the lesion penetration of similar analogues and several members of each class of drugs and use lesion penetration as a factor in the selection of better candidates for future preclinical studies. As our results suggest that increasing delivery to the site of infection may increase drug efficacy, we have been performing a series of experiments to determine if treatment with an agent that promotes normalization of blood vessel structure such that hypoxia is decreased and drug penetration is increased could improve drug access to the lesion. These host-directed therapy experiments are ongoing in the rabbit model with results monitored by FDG-PET/CT imaging, lesion histology, drug quantification and bacterial load. We have continued developing a new, non-human primate (NHP) model for tuberculosis - the common marmoset. Quantitative assessment of disease burden by FDG-PET/CT allowed an accurate assessment of disease progression in these animals that was highly correlated with pathology findings at necropsy. Encouraged by these results, we began exploring if the marmoset model accurately reflects the response to treatment by providing standard TB treatment (RIF, INH, PZA, and EMB) to infected symptomatic marmosets. The results reflected findings in humans suggesting cavities are the most difficult treat compartment and the HRZE is highly effective in treating these compartments compared to other regimens and support our previous results in the rabbit model suggesting that PET/CT may be an important early correlate of efficacy of novel combinations of new drugs that can be directly translated to human clinical trials. We are studying another drug class, the oxazolidinones antibiotics such as linezolid which have shown significant therapeutic effects in patients with extensively drug-resistant (XDR) tuberculosis (TB) despite modest effects in rodents and no demonstrable early bactericidal activity in human phase 2 trials. We are studying additional new oxazolidinones in the marmoset model of tuberculosis and two mouse models of tuberculosis. We are also studying other classes of antibiotics with targets that should be susceptible to inhibition in vivo including the ATPase and other vulnerable bacterial targets. Most of our PET-CT studies have used 18F fluorodeoxyglucose to image the metabolism of the eukaryotic cells in TB lesions but we are also making attempts to identify the location, abundance and metabolic state of the bacteria in lesions using bacteria-specific probes. To delineate the function of the tuberculous granuloma, colleagues and we analyzed the proteomes of granulomas from subjects with tuberculosis using laser-capture microdissection, mass spectrometry and confocal microscopy generating detailed molecular maps. We found that the centers of granulomas have a pro-inflammatory environment. Conversely, the cell layers surrounding the central necrotic core have a comparatively anti-inflammatory signature. Across a set of six human subjects and in M. tuberculosis infected rabbits we found that these signals are consistently physically segregated within each granuloma. From the protein and lipid snapshots of human and rabbit lesions analyzed, we hypothesize that the pathologic response to TB is shaped by the precise anatomical localization of these inflammatory pathways during granuloma formation (PMID:27043495).
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