Experimental Animal Models of TB: Chemotherapeutics and Imaging

National Institute Of Allergy And Infectious Diseases

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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. 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. In 2021 we developed moderate capacity to image mice in the Mediso PET/CT scanner using a chamber that holds 4 mice per imaging session. In 2022, we used this capacity to characterize murine lesions that were stimulated by cytokine treatment to recruit neutrophils and test their susceptibility to pyrazinamide. 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. Yet, we would prefer a small molecule that could be used to label Mtb in vivo endogenously as a PET radiotracer. For several years we have focused employing Mtb antigen 85 enzymes that are expressed on the exterior of Mtb cell walls to incorporate exogenous-provided 18F 2-deoxy trehalose (FDT) as either the mono- or dimycolate in or near the cell wall. Use of FDT in the imaging of Mtb in non-human primates, successfully allows the specific imaging of TB-associated lesions and to monitor the effects of treatment in marmosets. This is a promising sign that the FDT will be able to give a more accurate indication of treatment success or failure compared to FDG. We are continuing to develop this probe in the 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 and demonstrated that marmosets show similar treatment results as humans. As a counterpart to an early bactericidal activity (EBA) and paired PET/CT clinical trial, NexGen EBA conducted in South Africa; NCT02371681, in 2018-2020 we have replicated the treatment groups and observations in randomized Mtb infected marmosets. In that study, the standard regimen was deconstructed, and each drug was administered by itself or in pair-wise combinations to measure the effect of the drugs on the microbiological and radiographic markers as well as two 4 drug regimens HRZE and MRZE where M is moxifloxacin. In 2022, we determined that there were significant differences in treatment response between the MRZE and HRZE groups based on the lesion volumetric disease in both the CT and FDG-PET data at 2 months, but not 2 weeks (the duration of the typical EBA study). When the PET/CT image scoring system was applied, we found that the pathology in HRZE-treated marmosets resolved significantly faster and the increased resolution rate led to a significantly different residual volume of disease. In 2021-2022 we have deconstructed the Nix-TB regimen of Bedaquiline (BDQ, B), Pretomenid (Pa) and LZD (L) with all the drug combinations in roughly 35 marmosets. Previously we, found that BDQ accumulated significantly in both marmoset (and rabbit) TB lesions at steady state, including in cavity caseum, possibly explaining its outstanding efficacy in hard-to-treat patients with XDR TB. The lower more human-like dose of BDQ modeled by collaborators gave a very good approximation of the Cavg seen in humans but was less effective in sterilizing lung tissue than our original dose indicating that a higher BDQ does in humans might be even more effective if it was tolerable. Each of the individual agents are active against MTB in the marmoset although BDQ is more active alone that LZD. We have found that the BPaL regimen is very active in the marmoset but no more so than BPa. In fact, adding LZD to Pa appears antagonistic by some measures and adding LZD to BDQ is like BDQ alone although there is a trend toward fewer necrotic lesions in the BL combination. The 3-drug regimen is similar to HRZE in activity in the marmoset, but not superior to it. As with the BPaL regimen, we have investigated another drug combination that is likely headed for a human phase 2 trial this summer. The combination of Delamanid (D), BDQ (B), and an DprE2 inhibitor OPC-167832 (O) was tested in the marmosets in single, double and the triple drug combination. Each individual agent was highly effective promoting survival, reducing pathology, inflammation, and bacterial burdens in the lung and extrapulmonary organs. The combination of DO reduces residual pathology significantly more than either agent alone and the 3-drug combination DBO is significantly better than the individual agents in reducing bacterial burden and organ culture positivity rates. The analysis of the two-agent combinations and pathology measures are ongoing. We continue to study the oxazolidinone (OXA) antibiotics such as linezolid (LZD) which have shown significant therapeutic effects in patients with extensively drug-resistant (XDR) TB despite modest effects in rodents. One of these new amino-OXAs causes a significant rapid reduction in lesion pathology and inflammation compared with LZD as measured by FDG PET/CT. This activity was associated with lesion type and physical distribution into the lesions. Together with the Gates Foundation's TB Drug Accelerator scientists we have engaged in developing novel OXAs that are TB-selective and less toxic than LZD. We continue to evaluate the PET/CT imaging data from marmosets from treated with other classes of antibiotics from partners in the TBDA program including diarylquinolines, quinolines, imidazopyridines, nitroimidazoles, 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. With each new drug candidate, we test for in vivo efficacy with our academic and industry partners, we continue to assess the candidates penetration into granulomas and cavities in our model animals to correlate the information with any observed efficacy. 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. In 2022 modeling results from the Savic lab, BDQ penetration is increased in fibrotic lesions at steady state in HDT-treated rabbits but not in other lesion types. These experiments have also been conducted with moxifloxacin, but the results of the modeling are not yet available. In host RNA analysis (2022) from the control and HDT treatment indicates that signaling is changed during the HDT treatment with significant changes in expression of extracellular matrix components as predicted as well as changes in regulation of signal transduction, cell communication and signaling. These data are being explored to see what effect the HDT treatment had on the TB lesions.