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 pharmacokinetics 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. In 2010, 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. These results have been confirmed in steady state models and moxifloxacin was found to remain above the MIC over 24 hours. Spatial 2-D distribution of rifampicin and isoniazid within lesions is also being studied in collaboration with researchers at the University of Vanderbilt using matrix-assisted laser desorption/ionization mass spectrometry. A similar study is underway in collaboration with Novartis Pharma AG for pyrazinamide and 5 fluoroquinolones. 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. In one such effort, the 3 TB enzymes that function to incorporate trehalose (a sugar) into their cell surface are being harnessed to incorporate 18F trehalose into bacteria in the lesions of infected rabbits. A series of different positions and methods for attaching the 18F to the sugar are being explored to see which is most efficiently incorporated. In addition, the section is developing a new, non-human primate model for tuberculosis - the common marmoset. The motivation for this was four fold: i) the animal is very small and experiments with new agents can be accomplished with as little as 4 grams of the compound, ii) the animal is used for toxicology studies and so both efficacy and toxicology data could be gained from the same animals, iii) the animals are known to tolerate anesthesia well and were expected to be amenable to prolonged PET-CT studies without morbidity or mortality, and iv) over 80% of live births result in identical twins, allowing us to significantly reduce variability in outcome using geneticall identical controls and treated animals. In 2010 three quite different strains of Mtb were used to infect groups of Marmosets and FDG PET-CT scanning was used for monitoring disease prgression until the animal became symptomatic for tuberculosis. The progression of disease in animals infected with the Beijing clade clinical isolate of Mtb expressing the phenolic glycolipid (a molecule known to depress the TH1 response in mice) was more rapid than with the remaining Mtb strains (4 weeks vs. 8 weeks). The bacilli burden was also greater in these animals at sacrifice. The histopathology analysis is ongoing, but preliminary reports suggest that the disease displayed by the marmoset is as similar to human disease as that of the larger primate models. In preparation for the first drug treatment study, the pharmacokinetics of the standard first line drugs is being evaluated to determine a dose that will provide a similar drug exposure to that used in humans.
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