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: 10014061
• 负责人: Clifton Barry
• 金额: $ 144.41万
• 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/10014061
• 关键词: 3-Dimensional; Advanced Development; Animal Model; Animals; Antibiotics; Antibodies; Antigens; Antitubercular Agents; Automation; Autopsy; Bacteria; Benchmarking; Binding; Biodistribution; Biological Assay; Blood Vessels; Callithrix; Callithrix jacchus jacchus; Canis familiaris; Cell Wall; Cells; Chemicals; Clinical Trials; Conduct Clinical Trials; Data; Deoxyglucose; Development; Diagnostic radiologic examination; Disaccharides; Disease; Disease Marker; Disease Progression; Dose; Drug Kinetics; Emission-Computed Tomography; Enzymes; Eukaryotic Cell; Experimental Animal Model; Extreme drug resistant tuberculosis; Failure; Foundations; Functional Imaging; Future; Glucose; Histologic; Histology; Human; Hypoxia; Image; Imaging Techniques; Immune; Immunologic Markers; Immunologics; Individual; Infection; Investigation; Kinetics; Knowledge; Label; Lead; Lesion; Linezolid; Link; Macaca; Measures; Metabolism; Methods; Microbiology; Modeling; Modernization; Monitor; Monoclonal Antibodies; Mus; Mycobacterium tuberculosis; Nitroimidazoles; Noise; Organ; Oryctolagus cuniculus; Outcome; Oxazolidinones; Pathology; Patients; Penetration; Performance; Pharmaceutical Preparations; Phase; Phase II Clinical Trials; Positron-Emission Tomography; Pre-Clinical Model; Preparation; Production; Radiology Specialty; Randomized; Rattus; Regimen; Relapse; Reporting; Resolution; Scanning; Scientist; Series; Side; Signal Transduction; Site; South Africa; Spatial Distribution; Streptomycin; Structure; Structure of parenchyma of lung; Techniques; Technology; Testing; Therapeutic; Therapeutic Effect; Time; Tissues; Toxic effect; Treatment Protocols; Trehalose; Tuberculosis; Work; X-Ray Computed Tomography; arm; bacterial metabolism; bactericide; base; black hole; chemotherapy; design; drug distribution; drug efficacy; drug testing; experience; experimental study; extensive drug resistance; first-in-human; fluorodeoxyglucose; human data; imaging probe; imaging study; improved; in vivo; inflammatory marker; inhibitor/antagonist; lead candidate; monomer; mouse model; nonhuman primate; novel; predictive modeling; programs; quinoline; radiochemical; radiotracer; response; small molecule; success; 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 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.

该项目包括了解当前抗结核化疗如何使用最现代的技术发挥作用，并开发新的和改进的结核病（TB）疗法和治疗方法。该框架内的各个项目包括 (1) 使用 PET/CT 开发结构和功能成像技术，用于活体结核分枝杆菌 (Mtb) 感染动物，(2) 开发先进的动物模型，用于在完全模拟的条件下预测药物疗效结核病患者所经历的那些，（3）了解各种药物在结核病治疗动物模型中的活性，（4）将动物模型中观察到的反应与人类结核病中观察到的病理和治疗反应相关联，以及（5）开发用于结核病治疗的技术评估体内药物分布、渗透和药代动力学。 我们的大多数 PET/CT 研究均使用 18F-2-氟-2-脱氧葡萄糖 (FDG) 对结核病动物模型中结核病灶中真核细胞的代谢进行成像。我们还正在鉴定可用于特异性和内源性体内标记 Mtb 的小分子，以用作 PET 放射性示踪剂。我们关注的是 Mtb 抗原 85 酶，这些酶位于 Mtb 细胞壁的外部，可以将外源海藻糖（一种由两个 1-1 连接的葡萄糖单体组成的非哺乳动物二糖）作为单霉菌酸或二霉菌酸掺入细胞中墙。我们使用这些酶以化学方式将 18F 海藻糖 (FDT) 掺入受感染兔子和狨猴病变部位的细菌中。 FDT PET-CT 扫描似乎准确地反映了狨猴病变细菌负荷测定中的低和高细菌负荷。这是一个有希望的迹象，表明与 FDG 相比，FDT 将能够更早地指示治疗成功或失败。在 2019 年剂量和持续时间优化研究中，将摄取时间延长至 90 分钟以上似乎并没有增加收集图像中的信噪比。在阻断研究中，通过给狨猴预先注射我们牛津合作伙伴生产的稳定 FDT 来阻断 18F FDT 的吸收。我们通过 PET-CT 在 3 只幼猴身上进行了生物分布研究，以提供器官暴露估计，并在大鼠和狗的 GLP 毒性研究中使用稳定形式的 FDT，为首次人体剂量研究提供数据。成像探针开发中心最近完成的自动化表征研究已完成，以证明自动合成适用于 50 分钟内的 GMP 生产，并具有足够的放射化学产率来支持 IND 申请。 我们继续开发普通狨猴（Callithrix jacchus）非人类灵长类动物（NHP）结核病模型。过去，我们探索了狨猴模型是否通过向受感染的症状狨猴提供标准结核病治疗（RIF、INH、PZA 和 EMB）来准确反映对治疗的反应，并表明狨猴表现出与人类相似的治疗结果，包括证明了标准疗法包含 INH 和链霉素的早期治疗方案，导致复发率 > 30%。作为早期杀菌活性和配对 PET/CT 临床试验的对应项，我们正在南非进行 NexGen EBA 结核病灭菌药物活性的放射学和免疫学生物标志物； NCT02371681 我们正在随机 Mtb 感染的狨猴中复制治疗组和观察结果。在该研究中，标准方案被解构，每种药物单独或成对组合给药，以测量药物对微生物学、免疫学和放射学标记物的影响。我们正在治疗动物的放射学特征中寻找独特的药物特征，并将其与尸检时病变的组织学表现进行比较。我们假设，了解每种药物对疾病解决的具体贡献将有助于将未来的药物配对成更成功和快速起效的治疗方案。 2019 年，我们完成了配对研究的第一阶段，观察到人类放射学结果和狨猴的放射学结果高度相关，包括四种药物方案表现的差异。为了最初能够代表所有组，迄今为止，每只臂只包括 2 或 3 只狨猴，每只臂中添加了额外的动物，以确认观察结果并在新的 PET/CT 上对 CT 和 PET 读数进行基准测试。为了完成配对研究，我们需要在狨猴特征中添加免疫成分。 我们测试了抗体对各种狨猴免疫细胞的反应性，并成功开发了一组单克隆抗体，该抗体对普通狨猴表现出明显的反应性。我们优化了狨猴肺组织中透明增强 3D (Ce3D) 组织透明化和基于抗体的免疫标记的条件，为 NexGen 研究的下一阶段做好准备。 我们继续研究另一种药物，即恶唑烷酮抗生素，希望开发结核病治疗方案的联盟正在对此进行深入研究。恶唑烷酮利奈唑胺在人体 2 期试验中对广泛耐药 (XDR) 结核病患者显示出显着的治疗效果，而在小鼠模型中仅具有微弱的活性。这些新的恶唑烷酮化合物在狨猴结核病模型中具有截然不同的活性，这些活性似乎与病变类型、药物在病变中的物理分布以及酪蛋白结合有关。我们与默克公司的科学家一起致力于开发新型恶唑烷酮类药物，这种药物具有结核选择性且比利奈唑胺毒性更低。在整个 2019 年报告期内，我们一直积极参与测试新候选药物的 PK 和 ADME，以及与其他致力于该药物类别的其他先导候选药物的疗效。我们还正在研究通过盖茨基金会的结核病药物加速器计划参与开发结核病药物的合作伙伴的其他类别抗生素，包括二芳基喹啉、喹啉、咪唑并吡啶、硝基咪唑和苯并噻嗪酮等。正在探索这些类别的抗生素，以制定治疗 MTB 的新方案，并了解每种抗生素对活性的具体贡献，包括考虑空间分布和病灶中积累的动力学，以避免单一疗法的时空黑洞。我们测试了合作伙伴独特的 DprE1 抑制剂，可促进疾病的结构和功能放射标志物的减少，并显示出针对 Mtb 的显着 sidal 活性。 2019年，我们已开始测试该抑制剂与其他新型抗结核药物的组合，以评估其对新疗法的贡献。 我们继续探索宿主定向治疗 (HDT) 作为一种通过增加 Mtb 兔模型感染部位的药物递送来提高药物疗效的方法。我们一直在进行一系列实验，以确定使用促进血管结构正常化的药物治疗是否可以改善药物进入病变部位，从而减少缺氧并增加药物渗透。 2019 年的结果显示，在暴露于 HDT 的病灶中，贝达喹啉和药物模拟物的渗透有条件地增加。这些实验以及具有不同组织渗透和清除特性的其他抗结核药物正在兔子模型中进行，并通过 FDG-PET/CT 成像、病变组织学、药物定量和细菌负荷监测结果。