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抗结核化疗如何使用最现代的技术发挥作用以及开发新的和改进的疗法和治疗方法的方法。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。 2020 年，我们在 Mediso PET/CT 扫描仪中开发了有限的小鼠成像能力，使用的通道每次成像可容纳 4 只小鼠。 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的真核细胞的代谢进行成像。然而，我们更喜欢一种小分子，它可以作为 PET 放射性示踪剂，在体内特异性地、内源性地标记 Mtb。我们重点关注在 MTb 细胞壁外部表达的 MTb 抗原 85 酶，它们可以将外源海藻糖（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）作为细胞壁中的单霉菌酸或二霉菌酸掺入。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。 2020 年，我们已经证明，无热原生物催化工艺可以立即生产 2-18F氟-2- 脱氧海藻糖 (18FFDT) 作为结核分枝杆菌二糖海藻糖的 PET 活性模拟物。使用 FDT 在包括非人类灵长类动物在内的多种模型中对 Mtb 进行成像，成功地利用了 Mtb 特异性海藻糖处理，从而实现了 TB 相关病变的特异性成像 并监测狨猴的治疗效果。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。 我们继续开发一种新的非人类灵长类 (NHP) 结核病模型 - 普通狨猴。过去，我们通过向受感染的症状狨猴提供标准结核病治疗（RIFR、INHH、PZAZ 和 EMBE）来探索狨猴模型是否准确反映了对治疗的反应，并证明狨猴表现出与人类相似的治疗结果。 NexGen EBA 与在南非进行的早期杀菌活性 (EBA) 和配对 PET/CT 临床试验相对应； NCT02371681，2018-2020 年，我们在随机 Mtb 感染的狨猴中复制了治疗组和观察结果。在该研究中，标准方案被解构，每种药物单独或成对组合施用，以测量药物对微生物和放射学标记物以及两种4药物方案HRZE和MRZE的影响，其中M是莫西沙星。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。作为第一个评估，基于简单的疾病体积分析，MRZE 和 HRZE 组之间的治疗反应似乎存在差异。当应用 2020 年开发的 PET/CT 图像评分系统时，我们发现 HRZE 治疗的狨猴的病理学解决速度明显更快，但 FDG 摄取的减少没有不同。治疗 2 个月后，解决率的提高导致 HRZE 组与 MRZE 组的疾病体积显着不同。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。对这些新药物的 PET/CT 的后续 EBA 研究正在进行中，我们现在有狨猴数据可用于其中的一部分，并将于 2021 年添加到数据集中。 我们继续研究恶唑烷酮 (OXA) 抗生素，例如利奈唑胺，尽管在啮齿类动物中效果不大，并且在人体 2 期试验中没有 EBA，但这些药物对广泛耐药 (XDR) 结核病患者显示出显着的治疗效果。这些新的 OXA 在狨猴结核病模型中具有不同的活性，这些活性似乎与病变类型和病变的物理分布有关。我们与盖茨基金会的结核病药物加速器科学家一起致力于开发新型 OXA，该药物具有结核病选择性且比利奈唑胺 (LIN) 毒性更低。 2020 年全年，我们一直在分析此类药物狨猴的 PET/CT 成像数据。例如，Oxas AZD-5847 (AZD)、sutezolid (SUT) 和 LIN 都可以促进病理学的减少以及 FDG 摄取或炎症的减少。与雷德佐利德 (RAD) 相比，AZD 治疗随着时间的推移显着降低了炎症发生率。此外，AZD治疗组的残余炎症明显低于RAD治疗组。一些 OXA 的另一个特征是，在治疗开始后，疾病体积和/或 FDG 摄取量在开始下降轨迹之前短暂增加。大多数接受 LIN 治疗的动物 (75%) 都有一些遵循这种模式的病变，而 SUT 和 AZD 则很少见。这可能是因为治疗需要时间来减缓导致病变扩大的细菌生长，或者化合物导致细菌溶解，从而将更多的炎症细胞招募到病变处，导致疾病短暂发作。这种增加是可逆的，人们可以假设炎症的增加最终有利于恢复。 我们还在研究 TBDA 项目合作伙伴提供的其他类别的抗生素，包括二芳基喹啉、喹啉、咪唑并吡啶、硝基咪唑等。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。对于每一种新候选药物，我们与学术和行业合作伙伴一起测试其体内功效，并继续评估候选药物对模型动物肉芽肿和空腔的渗透情况，将信息与任何观察到的功效相关联。通过 2019-20 年收集的样本，我们现在知道二芳基喹啉贝达喹啉 (BDQ) 在稳定状态下在狨猴和兔子结核病病变中显着积累，包括在空洞中，这或许解释了其在难以治疗的广泛耐药结核病患者中的出色疗效。我们继续探索宿主定向治疗（HDT）作为一种通过增加结核分枝杆菌兔模型中药物递送至感染部位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。在2020年的结果中，对照和HDT治疗的兔子在稳态时BDQ渗透在病变中增加，因此用莫西沙星再次进行了这些实验，莫西沙星在兔子模型中稳态时不会积累，并通过CT成像监测结果，病变组织学、药物定量和细菌载量。