A novel probe for imaging apoptosis

描述（由申请人提供）：许多疾病与细胞死亡途径的改变有关。过度的细胞死亡导致进行性器官功能障碍，如缺血性疾病和神经变性。细胞死亡不足是癌症的标志，并有助于某些自身免疫性疾病。此外，许多药物的有益或有害作用可归因于它们对细胞死亡的影响。因此，评估细胞死亡的非侵入性方法将为临床医生提供有关疾病活动和治疗效果的关键信息。迄今为止，还没有探针被批准用于细胞凋亡的临床成像。我们意外地发现多种脱氢酶蛋白特异性地在细胞凋亡和坏死中积累。例如，重组乳酸脱氢酶（LDH），当荧光标记时，特异性地染色早期凋亡和晚期凋亡/坏死细胞。虽然已知LDH是一种从死亡细胞中泄漏出来的细胞内蛋白，但我们发现外源应用的LDH探针特异性地积聚在死亡细胞中。我们发现LDH探针与目前正在临床试验中用于细胞凋亡成像的Annexin V相比有几个优势，包括不需要钙，也不影响血清蛋白对染色的影响。这些特征表明，基于ldh的探针在细胞死亡的体内成像方面可能优于膜联蛋白V。在这项提议中，我们寻求建立一个优化的基于ldh的细胞凋亡成像探针。我们将首先建立全长LDH放射配体与凋亡细胞结合和体内成像的体外和体内特征。在第一个特异性目标中，我们将使用系统诱变来定义基于ldh的探针染色凋亡细胞所需和足够的结构元件。在第二个特定目标中，我们将开发一系列基于ldh的放射性配体探针，并表征它们在小鼠中的生物分布。在第三个特定目标中，我们将使用三种小鼠凋亡模型来确定一种优化的基于ldh的探针，用于细胞死亡的pet成像。最后，优化后的LDH探针的性能将直接与膜联蛋白V进行比较，用于细胞死亡的体内成像。总之，这些研究的成功将为使用新型基于ldh的探针成像细胞凋亡提供概念证明，并将为进行人体临床试验奠定基础。

DESCRIPTION (provided by applicant): A number of diseases are associated with alterations in cell death pathways. Excessive cell death leads to progressive organ dysfunction, such as ischemic diseases and neurodegeneration. Inadequate cell death is a hallmark of cancer, and contributes to certain autoimmune diseases. Furthermore, the beneficial or detrimental effects of many drugs can be attributed to their effects on cell death. Therefore, non-invasive methods for assessing cell death would provide clinicians with critical information on disease activity and therapeutic efficacy. To date, there are no probes approved for clinical imaging of apoptosis. We have unexpectedly found that a variety of dehydrogenase proteins specifically accumulate in cells undergoing apoptosis and necrosis. For example, recombinant lactate dehydrogenase (LDH), when fluorescently labeled, specifically stains both early apoptotic and late apoptotic/necrotic cells. While LDH is known to be an intracellular protein that leaks out of dying cells, we find that exogenously applied LDH- based probes specifically accumulate within dead cells. We have found that LDH probes have several advantages over Annexin V, which is currently in clinical trials for imaging of apoptosis, including no requirement for calcium and no effect of serum proteins on staining. These characteristics suggest that LDH-based probes may be superior to Annexin V for in vivo imaging of cell death. In this proposal, we seek to establish an optimized LDH-based probe for imaging apoptosis. We will first establish the in vitro and in vivo characteristics of a full length LDH radioligand for binding to apoptotic cells and in vivo imaging. In the first Specific Aim, we will then use systematic mutagenesis to define the structural elements necessary and sufficient for LDH-based probes to stain apoptotic cells. In the second Specific Aim, we will develop a series of LDH-based radioligand probes, and characterize their biodistribution in mice. In the third Specific Aim, we will use three mouse models of apoptosis to identify an optimized LDH-based probe for PET-imaging of cell death. Finally, the performance of an optimized LDH probe will be directly compared to that of Annexin V for iin vivoi imaging of cell death. Together, success in these studies would provide proof-of-concept for imaging apoptosis using novel LDH-based probes, and would establish the rationale for proceeding with human clinical testing. PUBLIC HEALTH RELEVANCE: The ability to non-invasively image cell death would provide a critical tool for diagnosing multiple diseases and monitoring the efficacy of drug treatments. No probes have yet been approved for the clinical imaging of apoptosis, underscoring the great unmet need for new sensitive and specific probes. The proposed studies seek to develop an entirely novel method for imaging apoptosis, and to bring this probe to readiness for human clinical testing.