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成像技术来筛查和诊断心血管疾病患者，并指导他们的治疗。本研究的目的是验证99mtc标记的双特异性细胞因子配体在心肌缺血和再灌注引起的炎症的SPECT成像中的应用。肿瘤坏死因子（TNF-1）、白细胞介素-1 （IL-12）和白细胞介素-18 （IL-18）是参与缺血再灌注和心室重构过程中最有效的促炎细胞因子；它们的作用分别受可溶性I/ II型TNF受体（TNFR1/TNFR2）、IL-1受体拮抗剂（IL-1ra）和IL-18结合蛋白（IL-18bp）的调控。两个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)，每个都含有一个氨基末端片段，特异性结合TNFR2或IL-18，人类IgG1的Fc部分，以及一个具有IL-1ra序列的羧基末端片段，最近在我们的实验室开发了通过双细胞因子途径靶向炎症部位的高亲和力。在本研究中，我们的目标是：(1)在体外和体内，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进行比较，确定这两种双特异性放射性配体的炎症靶向特性和放射性药物动力学；(2)急性梗死区和非梗死区99mTc-IL181和99mTc-TR21的摄取与炎症强度和心肌损伤的进展相关；(3)评估99mTc-IL181和99mTc-ILTR21摄取与慢性重构期左室扩张和病理生理改变的相关性以及抗炎治疗。为了确定99mTc-IL181和99mTc-TR21特异性靶向细胞因子的能力，我们将在il -1依赖的小鼠D10. g4.1 （D10）细胞中进行基于细胞的结合实验。我们将探讨细胞因子放射配体与心肌细胞（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来收集缺血-再灌注大鼠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程度和心功能的变化相关。该研究将应用SPECT成像技术开发一种创新的特异性工具，从根本上了解缺血-再灌注心脏的炎症反应，为不同阶段的促炎细胞因子通路的整合提供见解，并确定治疗方式的时机，以限制严重心功能衰竭前的强烈炎症反应。公共卫生相关性：用于无创检测心脏病发作引起的炎症的高特异性显像剂的发展

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.