Experimental Animal Models of TB: Chemotherapeutics and Imaging

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

This project encompasses approaches to understand how current anti-tubercular chemotherapy works using the most modern technologies and to develop new and improved therapies and therapeutic approaches for Tuberculosis (TB). Individual projects within this framework are (1) developing structural and functional imaging techniques using PET/CT 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. Most of our PET/CT studies have used 18F-2-fluoro-2-deoxyglucose (FDG) to image the metabolism of the eukaryotic cells in TB lesions in our animal models of tuberculosis. We are also identifying small molecules that could be used to specifically and endogenously label Mtb in vivo to be used as PET radiotracers. We are focusing on Mtb antigen 85 enzymes that are located on the exterior of Mtbs cell wall and can incorporate exogenous trehalose (a nonmammalian disaccharide consisting of a two 1-1 ,-linked glucose monomers) as either the mono- or dimycolate into the cell wall. We used these enzymes to chemically incorporate 18F trehalose (FDT) into bacteria in the lesions of infected rabbits and marmosets. FDT PET-CT scans seem to accurately reflect low and high bacterial burden in marmoset lesions assayed for bacterial load. This is a promising sign that the FDT will be able to give an earlier indication of treatment success or failure compared to FDG. In 2019 dose and duration optimization studies, prolonging uptake time beyond 90 minutes did not seem to increase the signal to noise ratio in the collected images. In blocking studies, 18F FDT uptake was blocked by pre-dosing the marmosets with stable FDT produced by our partners in Oxford. We have performed a biodistribution study via PET-CT in 3 naive macaques to provide organ exposure estimates and used the stable form of FDT in GLP toxicity studies in rats and dogs to provide data for first in human dosing studies. Recent automation characterization studies by the Imaging Probe Development Center to have been completed to demonstrate automated synthesis suitable for GMP production within 50 minutes with adequate radiochemical yield to support an IND application. We have continued developing the Common marmoset (Callithrix jacchus) non-human primate (NHP) model for TB. 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 and showed the marmoset shows similar treatment results with humans including demonstrating the superior activity of standard therapy to the early regimen containing INH and streptomycin that lead to > 30% relapse rates. As a counterpart to an early bactericidal activity and paired PET/CT clinical trial we are conducting in South Africa, NexGen EBA Radiologic and Immunologic Biomarkers of Sterilizing Drug Activity in TB; NCT02371681 we are replicating the treatment groups and observations in randomized Mtb-infected marmosets. In the study, the standard regimen is deconstructed and each drug is administered by itself or in pair-wise combinations to measure the effect of the drugs on the microbiological, immunological and radiographic markers. We are looking for unique drug signatures in the radiologic features of the animals on treatment and comparing those to the histological presentation of the lesions upon necropsy. We hypothesize that understanding the specific contributions of each drug to the disease resolution will assist in the pairing of future agents into more successful and rapidly acting regimens. In 2019, we completed the first phase of the paired study, observing that the human radiological outcomes and those of the marmoset where highly correlated including differences in the performance of the four-drug regimens. So that all groups could be represented initially, only 2 or 3 marmosets were included per arm to date, additional animals are be added to each arm to both confirm the observations and benchmark the CT and PET readouts on a new PET/CT. In order to complete the paired study, we needed to add an immune component to the marmoset characterization. We tested antibody reactivities to various marmoset immune cells and successfully developed a panel of monoclonal antibodies, which shows clear reactivity to the common marmoset. We optimized conditions of clearing-enhanced 3D (Ce3D) tissue clearing and antibody-based immunolabeling in marmoset lung tissue in preparation for the next phase of the NexGen study. We continue to study another drug class, the oxazolidinone antibiotics that are under intense investigation by consortia hoping to develop TB regimens. The oxazolidinone linezolid has shown significant therapeutic effects in patients with extensively drug-resistant (XDR) TB in human phase 2 trials while having only weak activity in mouse models. These new oxazolidinone compounds have vastly different activities in the marmoset model of TB that appear to be related to lesion type, physical distribution of the agents into the lesions, and caseum binding. Together with scientists at Merck we have been engaged in developing novel oxazolidinones that are TB-selective and less toxic than linezolid. Throughout the 2019 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 with others working on this drug class. 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, quinolines, imidazopyridines, nitroimidazoles, and benzothiazinones among others. These classes of antibiotics are being explored as composing new regimens for treatment of MTB and understanding the specific contribution 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. We tested a partners unique DprE1 inhibitor promoted reduction in both structural and functional radiologic markers of disease and showed significant sidal activity against Mtb. In 2019, we have begun testing combinations of this inhibitor with other new tuberculocidal agents to evaluate its contribution to new regimens. We continue to explore host-directed therapy (HDT) as a method to increase drug efficacy by increasing agent delivery to the site of infection in the rabbit model of Mtb. 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 increased could improve drug access to the lesion. In 2019 results, penetration of Bedaquiline and a drug mimic are conditionally increased in lesions exposed HDT. These experiments, with other anti-tubercular agents with varying tissue penetration and clearance profiles are ongoing in the rabbit model with results monitored by FDG-PET/CT imaging, lesion histology, drug quantification and bacterial load.