Tc-99m-Labled Cytokine Ligands and SPECT Inflammation Imaging of Ischemic Hearts

描述(由申请人提供)：心肌梗死和再灌注后的强烈炎症反应被认为是一个关键因素，最终导致心室重构、心力衰竭和心血管死亡。我们研究的长期目标是开发高效的SPECT成像技术，以筛查和诊断心血管疾病患者，并指导他们的治疗。本提案的目的是验证99mTC标记的双特异性细胞因子配体用于心肌缺血和再灌流引起的炎症的SPECT显像。肿瘤坏死因子(TNF-1)、白介素1(IL-12)和白介素18(IL-18)是参与心肌缺血再灌注和心室重构过程中最强的促炎细胞因子，它们的作用分别受可溶型I/II型肿瘤坏死因子受体(TNFR1/TNFR2)、IL-1受体拮抗剂(IL-1ra)和IL-18结合蛋白(IL-18bp)的调节。本实验室最近开发了两种99mTC标记的重组双特异性蛋白[99mTC]IL-18BP-Fc-IL-1Ra(99mTC-IL181)和[99mTC]TNFR2-Fc-IL-1Ra(99mTC-TR21)，每一种蛋白都含有与TNFR2或IL-18特异结合的氨基末端片段、人IgG1的Fc片段和含有IL-1ra序列的羧基末端片段，用于通过双细胞因子途径高亲和力靶向炎症部位。在这项建议中，我们的目标是：(1)确定这两个双特异性放射性配体的炎症靶向性和放射性药物动力学，并与99mTC标记的单个细胞因子配体99mTC-TNFR2-Fc、99mTC-IL-1Ra-Fc和99mTC-IL-18BP-Fc进行体内外比较；(2)在急性梗死区和非梗死区摄取99mTC-IL181和99mTC-TR21与炎症强度和心肌损伤进展的相关性；以及(3)评估99mTC-IL181和99mTC-TR21摄取与慢性左心室重塑和抗炎治疗期间进行性左心室扩张和病理生理改变的关系。为了确定99mTC-IL181和99mTC-TR21对特定细胞因子的靶向能力，我们将在依赖IL-1的小鼠D10.G4.1(D10)细胞中进行细胞结合实验。我们将探索细胞因子放射性配基与心肌细胞(心肌细胞、内皮细胞和成纤维细胞)的相互作用。此外，我们建议使用最先进的小动物SPECT成像仪FastSPECT II来收集大鼠缺血再灌流心脏的动态心脏成像数据。炎症反应的动力学数据将与心肌梗死的范围和心功能的变化相关联。这项拟议的研究将应用SPECT成像技术来开发一种创新的特定工具，以从根本上了解缺血再灌注心脏的炎症反应，提供对促炎症细胞因子途径在不同阶段的集成的见解，并选择治疗方法，以在严重心功能衰竭之前限制强烈的炎症反应。公共卫生相关性：开发高度特异的显像剂，用于非侵入性检测心脏病发作引起的炎症。

DESCRIPTION (provided by applicant): The intense inflammatory reaction following myocardial infarction and reperfusion has been implicated as a crucial factor, which ultimately leads to ventricular remodeling, heart failure, and cardiovascular death. Our long-term goal in our research is to develop highly effective SPECT imaging techniques to screen and diagnose patients with cardiovascular diseases and to guide their therapy. The objective of the present proposal toward this goal is to validate 99mTc-labeled bispecific cytokine ligands for SPECT imaging of inflammation induced by myocardial ischemia and reperfusion. Tumor necrosis factor (TNF-1), Interleukin-1 (IL-12), and Interleukin-18 (IL-18) are the most potent proinflammatory cytokines involved in the progress of ischemia- reperfusion and ventricular remodeling; their actions are regulated by soluble Type I/Type II TNF receptor (TNFR1/TNFR2), IL-1 receptor antagonist (IL-1ra), and IL-18 binding protein (IL-18bp), respectively. Two 99mTc-labeled recombinant bispecific proteins, [99mTc]IL-18bp-Fc-IL-1ra (99mTc-IL181) and [99mTc]TNFR2-Fc- IL-1ra (99mTc-TR21), each containing an amino-terminal segment that specifically binds to TNFR2 or IL-18, the Fc portion of human IgG1, and a carboxy-terminal segment with the sequence of IL-1ra, have been recently developed in our laboratory for high-affinity targeting inflammatory sites via dual-cytokine pathways. In this proposal, we aim to: (1) determine the inflammation-targeting properties and radiopharmaceutical kinetics of these two bispecific radioligands in comparison with 99mTc-labeled individual cytokine ligands, 99mTc-TNFR2- Fc, 99mTc-IL-1ra-Fc and 99mTc-IL-18bp-Fc, in vitro and in vivo; (2) correlate the uptake of 99mTc-IL181 and 99mTc-TR21 in acute infarct and non-infarct regions with inflammatory intensity and progression of myocardial injury; and (3) evaluate 99mTc-IL181 and 99mTc-ILTR21 uptake associated with progressive left ventricular dilatation and pathophysiological alterations during the chronic remodeling phase and anti-inflammation therapy. To ascertain the abilities of 99mTc-IL181 and 99mTc-TR21 for specific cytokine targeting, cell-based binding assay will be carried out in IL-1-dependent murine D10.G4.1 (D10) cells. We will explore the interactions of the cytokine radioligands and cardiac cells (myocytes, endothelium and fibroblasts). Furthermore, we propose using a state-of-the-art small-animal SPECT imager, FastSPECT II, to collect dynamic cardiac imaging data in ischemic-reperfused rat hearts. Kinetic data of inflammatory reactions will be correlated with the extent of myocardial infarct and changes in cardiac function. The proposed research will apply SPECT imaging techniques toward the development of an innovative specific tool for fundamentally understanding inflammatory response in ischemic-reperfused hearts, providing insights into the intergradations of the proinflammatory cytokine pathway at different phases, and timing therapeutic modalities to limit the intense inflammatory reaction before severe cardiac function failure. PUBLIC HEALTH RELEVANCE: Development of highly specific imaging agents for noninvasive detection of inflammation induced by heart attacks.