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) developing structural and functional imaging techniques using CT/PET for use in live, M. tuberculosis (Mtb) infected animals, (2) development of advanced animal models for predicting drug efficacy under conditions that exactly mimic those experienced by TB patients, (3) understanding the activity of various drugs in animal models of tuberculosis therapy, (4) correlating responses seen in animal models with the pathology and response to therapy observed in human TB, and (5) developing techniques for assessing drug distribution, penetration, and pharmacokinetics in vivo. We have continued developing a new, non-human primate (NHP) model for tuberculosis - the common marmoset. In the past we explored 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 have continued to study 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. These new oxazolidinones have vastly different activities in the marmoset model of tuberculosis and two mouse models of tuberculosis that appear to be related to lesion type and physical distribution of the agents into the the lesions. Together with scientists at Merck we have been engaged in developing novel oxazolidinones that are TB-selective and less toxic than linezolid and throughout the reporting period we have been actively involved in testing the PK and ADME of novel candidates as well as the efficacy of advanced lead candidates. In addition, we are also studying other classes of antibiotics from partners engaged in developing these for TB through the Gates Foundation's TB Drug Accelerator program including diarylquinolines, nitroimidazoles, and benzothiazinones amoung others. These classes of antibiotics are being explored with an eye toward composing new regimens for treatment of MTB and understanding the specific contibution of each one to activity including consideration of spatial distribution and the kinetics of accumulation in lesions to avoid temporal and spatial black holes of monotherapy. Using non-compartmental and population pharmacokinetic approaches, we have modeled the rate and extent of distribution of isoniazid, rifampicin, pyrazinamide and moxifloxacin in rabbit tissues and human lesions in the past finding that penetration of antibiotics in necrotic tuberculosis lesions is heterogeneous and drug-specific. In 2017 together with colleagues, we hypothesized that drug binding to macromolecules in necrotic tissue or caseum prevents passive drug diffusion through the critical site of infection. Using a caseum binding assay and MALDI mass spectrometry imaging of tuberculosis drugs, colleagues showed that binding to caseum inversely correlated with passive diffusion into the necrotic core. A high-throughput assay relying on rapid equilibrium dialysis and a caseum surrogate designed to mimic the composition of native caseum was developed and a set of 279 compounds was profiled in this assay to generate a large data set and explore the physicochemical drivers of free diffusion into caseum (PMID:27626295). To provide simple guidance in the property-based design of new compounds, a rule of thumb was derived whereby the sum of the hydrophobicity (clogP) and aromatic ring count is proportional to caseum binding. These tools can be used to ensure desirable lesion partitioning and guide the selection of optimal regimens against tuberculosis. The method of generating the matrix and conducting the assay was published (PMID:28518128) recently. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases. We continue to explore host-directed therapy as a method to increase drug efficacy by increasing agent delivery to the site of infection. 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. 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.
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