Experimental Animal Models of TB: Chemotherapeutics and Imaging

结核病实验动物模型：化疗和影像学

• 批准号: 10272059
• 负责人: Clifton Barry
• 金额: $ 146.95万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10272059
• 关键词: Advanced Development; Aftercare; Animal Model; Animals; Antibiotics; Antigens; Autopsy; Bacteria; Benchmarking; Biological Assay; Blood Vessels; Callithrix; Callithrix jacchus jacchus; Cell Wall; Cells; Chemicals; Clinical Trials; Computed Tomography Scanners; Conduct Clinical Trials; Cytolysis; Data; Data Set; Deoxyglucose; Development; Diagnostic radiologic examination; Disaccharides; Disease; Disease Progression; Drug Kinetics; Emission-Computed Tomography; Enzymes; Eukaryotic Cell; Experimental Animal Model; Extreme drug resistant tuberculosis; Failure; Flare; Foundations; Functional Imaging; Future; Glucose; Granuloma; Growth; Histologic; Histology; Human; Hypoxia; Image; Imaging Techniques; Individual; Infection; Inflammation; Inflammatory; Kinetics; Knowledge; Label; Lead; Lesion; Linezolid; Link; Measures; Metabolism; Methods; Microbiology; Modeling; Modernization; Monitor; Moxifloxacin; Mus; Mycobacterium tuberculosis; New Agents; Nitroimidazoles; Oryctolagus cuniculus; Outcome; Oxazolidinones; Pathology; Patients; Pattern; Penetration; Pharmaceutical Preparations; Positron-Emission Tomography; Pre-Clinical Model; Process; Production; Pyrogens; Radiology Specialty; Randomized; Recovery; Regimen; Residual state; Resolution; Rodent; Sampling; Scanning; Scientist; Series; Site; South Africa; Spatial Distribution; Structure; System; Techniques; Technology; Therapeutic; Therapeutic Effect; Time; Treatment Protocols; Trehalose; Tuberculosis; Work; X-Ray Computed Tomography; bacterial metabolism; bactericide; base; black hole; chemotherapy; design; drug candidate; drug distribution; drug efficacy; drug testing; experience; experimental study; extensive drug resistance; fluorodeoxyglucose; human data; improved; in vivo; in vivo evaluation; industry partner; inflammatory marker; monomer; nonhuman primate; novel; novel therapeutics; phase II trial; predictive modeling; programs; quinoline; radiotracer; recruit; response; success; treatment effect; treatment group; treatment response; tuberculosis chemotherapy; tuberculosis drugs; tuberculosis treatment; uptake

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 2020 we developed limited capacity to image mice in the Mediso PET/CT scanner using a chanber that holds 4 mice per imaging session. 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 asmall molecule that could be used to specifically and endogenously label Mtb in vivo as a PET radiotracer. We focused on MTb antigen 85 enzymes that are expressed 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 in 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 seems to accurately reflect low and high bacterial burden in marmoset lesions assayed for bacterial load. In 2020 we have shown that a pyrogen-free, biocatalytic process allows the ready production of 2-18Ffluoro-2- deoxytrehalose (18FFDT) as a PET-active mimic of the Mtb disaccharide trehalose. Use of FDT in the imaging of Mtb in diverse models, including non-human primates, successfully co-opts Mtb-specific processing of trehalose to allow 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 an earlier indication of treatment success or failure compared to FDG. 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 (RIFR, INHH, PZAZ, and EMBE) 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 conducted in South Africa, NexGen EBA; NCT02371681, in 2018-2020 we have 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 and radiographic markers as well as two 4 drug regimens HRZE amd MRZE where M is moxifloxacin. 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. As a first assessment there appeared to differences in treatment response between the MRZE and HRZE groups based on the simple disease volumetric analysis. When the PET/CT image scoring system developed in 2020 was applied, we found that the pathology in HRZE-treated marmosets resolved significantly faster but the reduction in uptake of FDG was not different. The increased resolution rate lead to a significantly different volume of disease in the HRZE vs. MRZE groups after 2 months of treatment. 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. Follow-on EBA studies with PET/CTs of these new agents are in progress and we have marmoset data to use with a subset of them now and will be adding to dataset in 2021. We continue to study the oxazolidinone (OXA) antibiotics such as linezolid which have shown significant therapeutic effects in patients with extensively drug-resistant (XDR) TB despite modest effects in rodents and no EBA in human phase 2 trials. These new OXAs have different activities in the marmoset TB model that appear to be related to 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 linezolid (LIN). Throughout 2020 we have been analyizing the PET/CT imaging data from marmosets from this drug class. As an example, Oxas AZD-5847 (AZD), sutezolid (SUT) and LIN, all promoted a reduction in pathology and reduction FDG uptake or inflammation. AZD treatment reduced the rate inflammation over time significantly compared to radezolid (RAD). In addition, the residual inflammation of the group treated with AZD was significantly lower than the group treated with RAD. Another feature of some OXAs is that disease volume and/or FDG uptake increases briefly after treatment start, prior to beginning a downward trajectory. Most of the LIN-treated animals (75%) animals had some lesions that followed this pattern, where it was rare with SUT and AZD. This could be because the treatment takes time to slow the bacterial growth that drives lesion expansion or that the compounds cause bacterial lysis thus recruiting more inflammatory cells to the lesion causing a brief flare up in disease. This increase is reversable, and one could hypothesize that the increase in inflammation eventually facilitates recovery. We are also studying 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. Through samples collected in 2019-20, we now know that the diarylquinoline Bedaquiline (BDQ) accumulatates significantly in both marmoset and rabbit TB lesions at steady state, including in cavity caseum, perhaps explaining its outstanding effacicy in hard to treat patients with XDR TB. 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 2020 results, BDQ penetration is increased in lesions at steady state in both control and HDT -treated rabbits, so these experiments have been conducted again with moxifloxacin which does not accumulate at steady state in the rabbit model with results monitored by CT imaging, lesion histology, drug quantification and bacterial load.

该项目包括了解当前使用最现代技术的抗结核化疗如何起作用的方法，以及开发新的和改进的疗法和治疗方法。该框架下的个别项目包括：(1)开发用于活的结核分枝杆菌感染动物的PET/CT结构和功能成像技术；(2)开发先进的动物模型，用于在完全模仿结核病患者所经历的条件下预测药物疗效；(3)了解结核病治疗动物模型中各种药物的活性。(4)将动物模型中观察到的反应与人类结核病的病理和治疗反应联系起来；(5)开发评估药物在体内分布、渗透和药代动力学的技术。2020年，我们开发了有限的能力，在Mediso PET/CT扫描仪中对小鼠进行成像，使用每个成像会话可容纳4只小鼠的腔室。我们的大多数PET/CT研究都使用18f -2-氟-2-脱氧葡萄糖（FDG）来成像结核病动物模型中结核病病变真核细胞的代谢。然而，我们更倾向于小分子，可以用来特异性和内源性标记Mtb在体内作为PET放射性示踪剂。我们重点研究了在MTb细胞壁外表达的MTb抗原85酶，这些酶可以将外源性海藻糖（一种由两个1-1连接的葡萄糖单体组成的非哺乳动物双糖）作为细胞壁中的单或二真菌酸盐。我们使用这些酶将18F海藻糖（FDT）化学结合到感染兔子和狨猴病变的细菌中。FDT PET/CT扫描似乎准确地反映了绒猴病变中细菌负荷的高低。在2020年，我们已经证明了一种无热原的生物催化工艺可以立即生产2- 18fluoro -2-