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) correlating responses seen in animal models of disease with the pathology and response to therapy observed in human TB patients, (3) developing structural and functional imaging techniques using CT/PET for use in live, infected animals, and (4) developing techniques for assessing drug penetration and pharmacokinitecs in vivo during laparoscopy and bronchoscopy. One important aspect of the project relies on the development of advanced animal models for predicting drug efficacy under conditions that exactly mimic those experienced by TB patients. In partnership with scientists at the University of Pittsburg, section scientists have been exploring the microenvironment of tuberculosis in both rabbits and non-human primates infected with Mtb. Understanding the physical characteristics of the local microenvironment in which Mycobacterium tuberculosis resides is an important goal that may allow targeting of metabolic processes to shorten drug regimens. Last year NIH installed a Ceretom CT and a Siemens MicroPET Focus 220 into t the ABSL-3 facility in Building 33 and scanning of infected rabbits had just begun. In 2009, we have completed imaging the disease development and treatment progress with two well known anti-TB drugs. Lesions were observed to be dynamic in both size and metabolic activity even when untreated. Treatment was observed to decrease both the volume and metabolic activity of lesions, but each lesion responded independently. In addition, the penetration of TB drugs into these lesions has been investigated using 4 different drugs. Rifampicin and Isoniazid were found in a 1:3 ratio in lesion to normal lung tissue comparisons, in contrast, pyrazinamide and moxifloxacin were found to concentrate in the lesions 2:1 and 8:1, respectively during single dose administration. Another group of infected rabbits were treated just long enough to achieve steady state drug levels and their tissues have been extracted and sent for analyses. Spatial 2-D distribution of these drugs within lesions is also being studied in collaboration with researchers at the University of Vanderbilt using matrix-assisted laser desorption/ionization mass spectrometry. These PET-CT studies used 18F fluorodeoxyglucose in order to image the metabolism of the TB lesions;we are also making attempts to identify the location, abundance and metabolic state of the bacteria in lesions. The TB bacterium has a set of 3 enzymes that function to incorporate trehalose (a sugar) into their cell surface. If the trehalose is labeled with an 18F bearing molecule, then infused into an infected rabbit, some lesions, especially those were pus filled cavities may label with the exogenous tehalose. The pilot experiment suggested that such an approach could work and the experiments are ongoing. In addition, the section decided to develop a new, non-human primate model for tuberculosis in the common marmoset. The motivation for this was three fold: the animal is very small and experiments with new agents can be accomplished with as little as 3 grams of the compound, the animal is used for toxicology studies and so both efficacy and toxicology data could be gained from the same animals, and finally the animals are known to tolerate anesthesia well and were expected to be amenable to prolonged PET-CT studies without. The first pilot experiment was successful in that the 6 animals exposed to the tuberculosis-laden aerosol became infected and developed progressive pulmonary TB. Additional bacterial strains are being tested and after a suitable one is found, a small drug treatment well be started.
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