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 was successful, now with collaborators at U. Pitt. using the macaque model we showed that addition of MTZ to INH/RIF effectively treated animals with active TB within 2 months. In addition, we have further shown that chemoprophylaxis of latently-infected macaques with MTZ alone was as effective as either six months of INH or 2 months of INH and RIF in preventing reactivation of latent infection. 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 infected rabbits by PET/CT imaging In addition, we have begun developing methods for 18F labeling of TB drugs to develop the tools to assess their localization to lesions and the bacteria themselves real-time. TRS scientists and collaborators have shown that a combination of meropenem and clavulanic acid effectively kills Mtb under both replicating and hypoxia-induced non-replicating conditions in vitro. The carbapenems imipenem and meropenem in combination with clavulanic acid reduced the bacterial burden in Mtb-infected macrophages. Despite poor stability in solution and a short half-life in rodents, treatment of chronically infected mice revealed significant reductions of bacterial burden, so studies in rabbits were initiated. The serum pharmacokinetics studies of meropenem in rabbits revealed a t1/2 of only 12.1 min and a total exposure AUC of only 15.2 mg-min/mL. Although co-administration of cilastatin to block the renal dihydropeptidase I improved exposure to 51.5 mg-min/mL, but treatment was associated with significant toxicity preventing evaluation of meropenem in the rabbit. Our results suggest that meropenem has activity in two in vivo systems but stability and pharmacokinetics of long-term administration will offer significant challenges to clinical evaluation. In addition, 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. The 2-D MALDI/MS data for both INH and RIF also indicate a lack of concentration of these drugs into lesion tissue. A similar study is underway in collaboration with UMDNJ and Novartis IBR for pyrazinamide and 5 fluoroquinolones. The 2-D MALDI/MS moxifloxacin images confirm the its favorable distribution in to lesion tissue and more specifically into the macrophage-rich region just distal to the necrotic core of the TB lesions. More recently we have begun exploring the lesion penetration of the 7-alkyl substituted PA 824 analogues and tail modifies analogues of PA 824. The objective of these studies is to compare the lesion penetration of the PA-824 analogues and 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. Attempts to normalize the vasculature of lesions will continue with and without anti-TB drugs, with results monitored by FDG-PET/CT imaging, lesion histology, drug quantification and bacterial load. 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. 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 and had a median survival time of only 37 days. 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 planning head to head studies of new potential anti-tubercular agents.
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