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. Understanding the characteristics of the local microenvironment in which Mtb resides is an important goal that may allow targeting of metabolic processes to shorten drug therapy. We have hypothesized that targeting Mtb bacilli in low oxygen microenvironments has this potential and to prevent reactivation of latent infection. Using the anaerobically-active drug Metronidazole (MTZ) to kill Mtb residing in caseous hypoxic lesions in the rabbit model and macaque model was successful. In our effort to correlate response to therapy in animal models and human disease we determined that chemoprophylaxis of humans with tuberculosis, MTZ decreased the time to sputum conversion to negative in early assessment of disease response although the effect was not maintained. Most of our PET-CT studies 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. In an effort to identify molecules that could be used to specifically label Mtb in vivo, we capitalized on the unusually broad substrate specificity of the Mtb antigen 85 enzymes, and we found could transfer mycolates to a variety of sugars including trehalose modified with bulky substituents, so now we are using these enzymes to incorporate 18F trehalose into bacteria in the lesions of infected rabbits and 18F activity has been detected in lesions of these rabbits by PET/CT imaging. Two approaches to chemically synthesize 18H trehaolse are also being explored. In another approach, we are targeting other molecules within the lesion, for example collagen that is deposited in the margin of the lesion by fiberblasts. 18F-Cis-4-fluoroproline has been suggested as a radiotracer for assessing the changes in collagen deposition so we are testing its accumulation in tubercular lesions at various stages of lesion development and in lesions in animals being treated with combination therapy targeted to reduce collagen and deliver anti-tubercular agents. Investigation of the penetration of both currently used and investigative TB drugs into Mtb rabbit lesions is ongoing. Using non-compartmental and population pharmacokinetic approaches, we modeled the rate and extent of distribution of isoniazid, rifampicin, pyrazinamide and moxifloxacin in rabbit lung and lesions. Moxifloxacin reproducibly showed favorable partitioning into lung and granulomas, while the exposure of isoniazid, rifampicin and pyrazinamide in lesions was markedly lower than in plasma. We intend to use lesion penetration as a factor in the selection of a better candidate for future preclinical studies. Our results suggest 3 of standard TB drugs, INH, RIF, PZA do not penetrate into lesion tissue well suggesting that increasing delivery to the site might increase 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. In collaboration with Mass General Hospital we have determined that TB lesion-associated vasculature is structurally and functionally abnormal, which might be the cause of poor oxygenation and reduced drug delivery in the rabbit model and human TB disease. Treatment with the normalization agent yielded transient increases in drug penetration and oxygenation of the TB lesions, but only for a short treatment window. Attempts to normalize the vasculature of lesions will continue with anti-TB drugs, with results monitored by FDG-PET/CT imaging, lesion histology, drug quantification and bacterial load. In a collaborative study with the Univ of Pitt., serial 2-deoxy -2-18F-D-deoxyglucose (FDG) positron emission tomography (PET) with computed tomography (CT) imaging was performed on cynomolgus macaques during Mtb infection and chemotherapy with individual agents or the four-drug combination therapy most widely used globally. Size and metabolic activity of lung granulomas varied among animals, and even within a single animal, during development of disease. Individual granulomas within untreated animals had highly local and independent outcomes illustrating the highly dynamic nature of active TB. A more marked reduction in overall metabolic activity in the lungs (decreased FDG uptake) was associated with effective treatment. Reduction in size of individual lesions correlated with lower bacterial burden at necropsy. Quadruple-drug therapy resulted in the highest decrease in FDG uptake. These results 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. Recently, the section began developing a new, non-human primate (NHP) model for tuberculosis - the common marmoset. To establish the susceptibility of this new world NHP to developing TB, we aerosol infected marmosets with one of three Mtb complex strains of diverse pathogenic potential. In the study published this year, we demonstrated that all three organisms were fully capable of producing fulminant disease in this NHP resulting in a spectrum of rates of progression and clinical presentation as monitored by 18F FDG-PET/CT. All three strains also resulted in pathology at necropsy that was highly similar to that observed in human TB patients. Animals infected with the recent Beijing isolate showed the most rapid progression. Animals infected with the Euro-American showed the slowest rate of disease progression and importantly, were the only animals to develop cavitary disease and substantial amounts of fibrosis. 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. Comparison of twin siblings with the same infecting strain or different strains allowed us to establish the reproducibility of the infection and the relative virulence of strains conclusively. 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. In two separate experiments we have succeeded in documenting response to treatment in both reduction in bacterial burden, reduction in lesion volume by PET/CT, and improved clinical score including weight gain. The analysis of these experiments is ongoing, but the models performance has encouraged us enough to begin comparing the activity of 5 oxazolidinones in early clinical development in order to help inform the community which of these agents should be taken forward in to phase 3 combination clinical trials for tuberculosis treatment.

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Mayer-Barber, Katrin D; Andrade, Bruno B; Oland, Sandra D et al. (2014) Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk. Nature 511:99-103
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