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. 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. 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 showed favorable partitioning into cellular regions of lesions but not the lipid-rich caseum. A similar study has been completed in collaboration with Rutgers Univ. for pyrazinamide (in humans and other species; ref 3). We showed that substantial host-mediated conversion of prodrug pyrazinamide (PZA) to the active form, pyrazinoic acid (POA) occurs in TB patients and in animal models. We also demonstrated favorable penetration of this pool of circulating POA from plasma into lung tissue and granulomas, where the pathogen resides. We further demonstrated good oral bioavailability and exposure in pharmacokinetic studies of oral POA in preclinical species, suggesting that the vascular supply of host-derived POA may contribute to the in vivo efficacy of PZA, thereby reducing the apparent discrepancy between in vitro and in vivo activity. However, the results also raise the possibility that subinhibitory concentrations of POA generated by the host could fuel the emergence of resistance to both PZA and POA. We continue to study the lesion penetration of the fluoroquinolones, modified analogues of PA 824, linezolid and 4 new oxazolidinones. 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. In collaboration with Mass General Hospital, we have determined that TB-infected rabbits have impaired small molecule distribution into lesions due to a functionally abnormal vasculature, with a low-molecular-weight tracer accumulating only in peripheral regions of granulomatous lesions (ref 2). Granuloma-associated vessels are morphologically and spatially heterogeneous, with poor vessel pericyte coverage in both human and rabbit TB granulomas. Moreover, we found enhanced VEGF expression in both species. In tumors, anti-VEGF, treatments can normalize their vasculature, reducing hypoxia and creating a window of opportunity for chemotherapy; thus, we investigated vessel normalization in rabbit TB granulomas. Treatment of TB-infected rabbits with the anti-VEGF antibody bevacizumab significantly decreased the total number of vessels while normalizing those vessels that remained. As a result, hypoxic fractions of these granulomas were reduced and small molecule tracer delivery was increased. Attempts to maintain that normalization for a relevant duration for treatment of tuberculosis will continue, but we are also identifying other agents that could modify the structure of the TB lesion at various stages of 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. We treated MTB-infected marmosets with two drug regimens known to differ in their relapse rates in human clinical trials: the standard four-drug combination of isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE) that has very low relapse rates and the combination of isoniazid and streptomycin that is associated with higher relapse rates (ref 1). As early as 2 weeks, the more sterilizing regimen significantly reduced the volume of lung disease by computed tomography (P = 0.035) and also significantly reduced uptake of (18)F-2-fluoro-2-deoxyglucose by positron emission tomography (P = 0.049). After 6 weeks of therapy, both treatments caused similar reductions in granuloma bacterial load, but the more sterilizing, four-drug regimen caused greater reduction in bacterial load in cavitary lesions (P = 0.009). These findings, combined with the association in humans between cavitary disease and relapse, suggest that the basis for improved sterilizing activity of the four-drug combination is both its faster disease volume resolution and its stronger sterilizing effect on cavitary lesions. The known association of cavitary disease with disease relapse suggests that this small non-human primate model of infection provides an early evaluation of the sterilizing potential of new antitubercular regimens, offering a powerful new methodology for prioritizing new combinations. 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. 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 showed that monotherapy with either linezolid or AZD5847, a second-generation oxazolidinone, reduced bacterial load at necropsy in MTB-infected cynomolgus macaques with active TB (ref 4). This effect coincided with a decline in 2-deoxy-2-(18)F-fluoro-d-glucose positron emission tomography (FDG PET) imaging avidity in the lungs of these animals and with reductions in pulmonary pathology measured by serial computed tomography (CT) scans over 2 months of monotherapy. Quantitative comparisons of PET/CT imaging changes in these human subjects were similar in magnitude to those observed in macaques, demonstrating that the therapeutic effect of these oxazolidinones can be reproduced in this model of experimental chemotherapy. We are further studying these and additional oxazolidinones in the marmoset model of tuberculosis and two mouse models of tuberculosis.
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