CAP: Development of a Tuberculosis-Specific PET Imaging Agent

CAP：结核病特异性 PET 显像剂的开发

• 批准号: 9161705
• 负责人: Clifton Barry
• 金额: $ 25.22万
• 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/9161705
• 关键词: 2-Fluoro-2-deoxyglucose; Adenosine Triphosphate; Alcohols; Animals; Antigens; Area; Autopsy; Back; Bacteria; Biodistribution; Biological Assay; Biomimetics; Callithrix; Cell Wall; Colony-forming units; Complex; Cyanogen Bromide; Data; Deoxyglucose; Development; Diagnosis; Disaccharides; Disease; Dose; Enzymes; Equipment; Ethambutol; Eukaryotic Cell; Failure; Feedback; Fibrosis; Fluorine; Glucose; Goals; Human; Image; Immobilization; Immobilized Enzymes; Inflammation; Kidney; Label; Lesion; Link; Location; Macaca; Mammalian Cell; Metabolic; Metabolism; Methods; Modeling; Modification; Monitor; Mono-S; Mycobacterium tuberculosis; Noise; Organ; Oryctolagus cuniculus; Performance; Plant Resins; Positioning Attribute; Positron-Emission Tomography; Preparation; Procedures; Process; Production; Protein Dephosphorylation; Proteins; Pyrazinamide; Reaction; Regimen; Rifampin; Route; Scanning; Schedule; Sepharose; Series; Solid; Solutions; System; Thick; Time; Tissue Sample; Tissues; Tracer; Training; Treatment Efficacy; Trehalase; Trehalose; Tuberculosis; Uridine Diphosphate Glucose; X-Ray Computed Tomography; analog; burden of illness; design; dosimetry; extracellular; falls; hexokinase; human disease; imaging agent; improved; in vivo; inorganic phosphate; mRNA Differential Displays; monomer; mycolate; nonhuman primate; premature; radiotracer; small molecule; success; sugar; uptake

Most of our PET-CT studies to date have used 18F-2-fluoro-2-deoxyglucose (FDG) to image the metabolism of the eukaryotic cells in TB lesions but we are also making attempts to identify the location, abundance and metabolic state of the bacteria in lesions. In an effort to identify small molecules that could be used to specifically label MTb in vivo, we capitalized on the unusually broad substrate tolerance of the MTb antigen 85 enzymes, which transfer mycolates onto structurally diverse sugars to form part of MTbs cell wall. Antigen-85 enzymes are expressed on the exterior of MTbs cell wall and incorporate exogenous trehalose (a nonmammalian disaccharide consisting of a two 1-1 α,α -linked glucose monomers) as either the mono- or dimycolate, even tolerating trehalose molecules containing bulky modifications. We have used this system to incorporate 18F trehalose into bacteria in the lesions of infected rabbits and 18F activity has been detected in lesions of infected rabbits by PET-CT imaging. A series of different positions and methods for attaching the 18F to the sugar are being explored to see which is most efficiently incorporated. Using trehalose should afford an improvement over the currently used 18F-FDG, as glucose is used by mammalian cells as well as bacterial, causing noise in the scans due to increased metabolism or inflammation in the host. Use of trehalose is unique to bacteria; it is not absorbed by mammalian cells, which should limit noise in PET scans. The Davis group has designed three trehalose analogs incorporating fluorine at the 2, epi-4, or 6-position of trehalose for use as PET radiotracers. The 2-fluorotrehalose (FDT) synthesis is a biomimetic process, inpired by bacterial synthesis of trehalose from glucose. Chemoenzymatic synthesis of FDT occurs as a one-pot cascade reaction in which hexokinase transfers a phosphate from adenosine triphosphate (ATP) to FDG (normally glucose in bacterial trehalose synthesis). OtsA then transfers the glucose from the donor UDP-glucose to the acceptor phosphorylated FDG. Dephosphorylation to give the desired product is effected by OtsB. The entire one-pot process is complete in 45 min. The advantage here is that a relatively technically facile manipulation would convert a commercially available radiotracer to a TB-specific one. We have acquired some preliminary PET-CT scan data in rabbits using the FDT probe as well, one healthy, three infected with HN878 MTb. In the first infected animal, four lesions were present. Two were not PET-active, and two were, although all four had similar amounts of colony forming units (CFU). Upon necropsy, the two PET-inactive lesions were extremely rigid and thick-walled, implying that uptake is related more to accessibility (i.e. vasculature) than amount of bacteria present. Intriguingly, the second two rabbits showed more complex lesions which clearly displayed differential labeling between the 18F-FDG and FDT. Metabolism data was inconclusive but suggested some metabolism (as much as 20%) of the probe from FDT back info FDG. It is assumed this happens via trehalase natively expressed by the rabbits. We then evaluated FDT in Mtb-infected marmosets, as marmosets should express lower levels of trehalase and are a more physiologically relevant model to human disease. The marmosets showed no metabolism of the FDT back to FDG. The FDT continued to show differential labeling compared to FDG. We were able to see the differential labeling both qualitatively and quantitatively. Areas in the top 25% of FDG uptake show disproportionately lower FDT uptake, and the areas in the top 25% of FDT uptake show disproportionately lower FDG uptake. The intriguing finding suggestes that bacterial are actively replicating in areas of lower inflammation than they are in areas with high levels of inflammation. It is premature to draw correlations between bacterial load and tracer uptake; however, it appears that the FDT may be a more reliable predictor of treatment success or failure. In one marmoset, we treated with the first-line regimen of izoniazid, pyrazinamide, rifampicin, and ethambutol for five weeks, with dosing five days a week. The animal showed very low disease burden upon necropsy as judged by CFU. The FDT PET-CT scans reflected this low burden, showing a clear reduction in uptake compared to pre-treatment scans. The FDG did not show a significant decrease in uptake and in some lesions displayed increase uptake. This is an extremely promising sign that the FDT will be able to give an earlier indication of treatment success or failure as compared to FDG. We have performed biodistribution studies by analyzing tissue samples from relevant organs in two marmosets, which showed little uptake in uninfected tissue other than kidneys, the route for clearance of the radiotracer. We have also successfully performed a biodistribution study via PET-CT in a macaque and have two additional scans scheduled for Fall 2015. These scans will give dosimetry data in preparation for moving the tracer forward for human use. We have optimized protein immobilization onto solid support. Each enzyme shows best performance on different resins. All perform acceptably using cyanogen-bromide linking onto agarose, but all three perform better on Enzyme Carrier Resins, with linker type optimized for each protein. Under optimized reaction conditions, these immobilized proteins provide good conversion which is complete in 1.5 h. With these immobilized enzymes, radiopharmacists could pass through commercial FDG and a solution of ATP and UDP-Glucose through the cartridge to produce the desired radiotracer with no specialized equipment or special training. The procedure would be operationally simple and allow TB-specific imaging to monitor course of treatment. Ideally, we would be able to rapidly assay treatment success or failure in a manner that relies only on abundance and metabolic state of the bacteria.

迄今为止，我们的大多数 PET-CT 研究均使用 18F-2-氟-2-脱氧葡萄糖 (FDG) 对结核病灶中真核细胞的代谢进行成像，但我们也在尝试确定病灶内的细菌。为了鉴定可用于体内特异性标记 MTb 的小分子，我们利用 MTb 抗原 85 酶异常广泛的底物耐受性，将霉菌酸盐转移到结构多样的糖上，形成 MTb 细胞壁的一部分。 抗原 85 酶在 MTb 细胞壁外部表达，并以单霉菌酸或二霉菌酸形式掺入外源海藻糖（一种非哺乳动物二糖，由两个 1-1 α,α 连接的葡萄糖单体组成），甚至可以耐受含有大体积的海藻糖分子。修改。我们利用该系统将 18F 海藻糖掺入感染兔病灶内的细菌中，并通过 PET-CT 成像在感染兔病灶中检测到 18F 活性。目前正在探索一系列将 18F 连接到糖上的不同位置和方法，以确定哪种位置和方法最有效地结合。使用海藻糖应该比目前使用的 18F-FDG 有所改进，因为葡萄糖被哺乳动物细胞和细菌使用，由于宿主新陈代谢或炎症的增加而导致扫描中出现噪音。海藻糖的使用是细菌所独有的；它不会被哺乳动物细胞吸收，这应该会限制 PET 扫描中的噪音。 Davis 小组设计了三种海藻糖类似物，在海藻糖的 2、epi-4 或 6 位上掺入氟，用作 PET 放射性示踪剂。 2-氟海藻糖 (FDT) 合成是一种仿生过程，其灵感来自细菌从葡萄糖合成海藻糖。 FDT 的化学酶合成作为一锅级联反应发生，其中己糖激酶将磷酸盐从三磷酸腺苷 (ATP) 转移到 FDG（通常是细菌海藻糖合成中的葡萄糖）。然后，OtsA 将葡萄糖从供体 UDP-葡萄糖转移到受体磷酸化 FDG。 OtsB 实现去磷酸化以得到所需产物。整个一锅过程在45分钟内完成。这里的优点是，技术上相对简单的操作可以将市售放射性示踪剂转换为结核病特异性示踪剂。 我们还使用 FDT 探针获得了一些兔子的初步 PET-CT 扫描数据，其中一只健康，三只感染 HN878 MTb。在第一只受感染的动物中，存在四个病变。其中两个不具有 PE​​T 活性，另外两个具有 PE​​T 活性，尽管所有四个都具有相似数量的集落形成单位 (CFU)。尸检后，两个 PET 不活跃的病变非常坚硬且壁厚，这意味着摄取更多地与可及性（即脉管系统）相关，而不是与存在的细菌数量相关。有趣的是，后两只兔子表现出更复杂的病变，清楚地显示 18F-FDG 和 FDT 之间的差异标记。代谢数据尚无定论，但表明来自 FDT 反馈信息 FDG 的探针有一些代谢（高达 20%）。假设这是通过兔子天然表达的海藻糖酶发生的。 然后，我们评估了感染 Mtb 的狨猴的 FDT，因为狨猴应该表达较低水平的海藻糖酶，并且是与人类疾病在生理上更相关的模型。狨猴没有表现出 FDT 代谢回 FDG。与 FDG 相比，FDT 继续显示出差异标记。我们能够定性和定量地看到差异标签。 FDG 吸收量前 25% 的区域显示 FDT 吸收量不成比例地降低，FDT 吸收量前 25% 的区域显示 FDG 吸收量不成比例地降低。这一有趣的发现表明，与炎症水平较高的区域相比，细菌在炎症程度较低的区域复制活跃。现在确定细菌负荷和示踪剂吸收之间的相关性还为时过早；然而，FDT 似乎是治疗成功或失败的更可靠的预测指标。在一只狨猴中，我们使用异烟肼、吡嗪酰胺、利福平和乙胺丁醇一线疗法治疗五周，每周给药五天。根据 CFU 判断，动物在尸检时显示出非常低的疾病负担。 FDT PET-CT 扫描反映了这种低负担，与治疗前扫描相比，摄取量明显减少。 FDG 并未显示出摄取显着减少，并且在一些病变中显示摄取增加。这是一个非常有希望的迹象，表明与 FDG 相比，FDT 将能够更早地指示治疗成功或失败。 我们通过分析两只狨猴相关器官的组织样本进行了生物分布研究，结果显示，除了肾脏（放射性示踪剂的清除途径）之外，未感染的组织几乎没有吸收。我们还通过 PET-CT 在猕猴身上成功进行了生物分布研究，并计划于 2015 年秋季进行两次额外的扫描。这些扫描将提供剂量测定数据，为将示踪剂推向人类使用做好准备。 我们优化了蛋白质在固体支持物上的固定。每种酶在不同的树脂上都表现出最佳性能。使用溴化氰连接到琼脂糖上时，所有这些都表现良好，但所有三种在酶载体树脂上都表现更好，并且连接体类型针对每种蛋白质进行了优化。在优化的反应条件下，这些固定化蛋白质提供了良好的转化，可在 1.5 小时内完成。 借助这些固定化酶，放射性药剂师可以将商业 FDG 以及 ATP 和 UDP-葡萄糖溶液通过试剂盒，从而产生所需的放射性示踪剂，无需专门设备或特殊培训。该程序操作简单，并允许结核病特异性成像来监测治疗过程。理想情况下，我们能够以仅依赖于细菌的丰度和代谢状态的方式快速测定治疗的成功或失败。