Characterization of an 18F COX-2 PET ligand for in vivo brain imaging

阿尔茨海默病（AD）的发病机制与早期神经炎症有关，这被认为有助于疾病的进展和严重程度。因此，了解和调节中枢神经系统（CNS）的炎症通路可能有助于预防或延缓AD的发生。环氧合酶-2 （COX-2）在炎症刺激下被诱导，其抑制作用是许多非甾体抗炎药（NSAIDs）治疗效果的基础。AD患者脑内COX-2表达显著升高，且与脑淀粉样斑块病理的严重程度相关。因此，体内和无创监测大脑中COX-2水平可以跟踪AD过程中COX-2的诱导，也可以检查抑制COX-2在AD中的临床益处。正电子发射断层扫描（PET）配体量化大脑中功能性COX-2的水平将允许直接测量AD的神经炎症，从而使疾病分期和治疗评估成为可能。然而，目前还没有这样的PET配体。为了建立一种临床有用的PET示踪剂，用于AD脑内COX-2成像，我们建议评估高效的[18F]标记的COX-2抑制剂，因为它具有110分钟半衰期的优势。[18F]标记的示踪剂允许更长时间的扫描，从而实现平衡扫描，并促进稳健的动力学研究，从而精确定量COX-2。此外，它们可以从合成中心运送到附近缺乏回旋加速器的PET中心，并有助于成本效益高的PET研究。我们发现了MTP，一种高亲和力的COX-2抑制剂（IC50 = 2.2 nM；图2），具有引入[18F]标签的芳香位点，对除氟不太敏感。MTP还具有足够的亲脂性（LogP = 2.7），可以被动地穿过血脑屏障（BBB）。我们合成了[18F]MTP，并成功证明了其在COX-2阳性的BxPC-3细胞系中的特异性结合（图3,112）。用脂多糖（LPS）诱导小鼠进行体内PET成像，与对照小鼠相比，在大脑中获得了明显更高的结合，没有可见的骨骼摄取（图4,150）。随后对收获的脑组织切片进行体外放射自显影，确定了示踪剂在lps诱导的神经炎症中的特异性结合（图5和图6）。鉴于上述支持证据表明血脑屏障通透性，神经炎症中更高的结合，以及对COX-2的特异性结合，[18F]MTP将与两个备用配体2和3一起进一步评估（图2），在神经炎症和AD神经病理小鼠模型中使用额外的PET成像（目的1和2）。确定的最合格的示踪剂将在麻醉的雄性和雌性猴子中进行反复测试的动态PET定量评估，并同时采集动脉血液，以量化不同脑区的总分布体积（目标3）。我们相信，所提出的实验将导致选择一种成功的[18F]- COX-2 PET示踪剂，其特点是过渡到阿尔茨海默病的临床研究，用于早期诊断，监测治疗反应，并帮助开发新药。

Alzheimer’s disease (AD) pathogenesis is associated with early neuroinflammation, which is considered to contribute to disease progression and severity. Therefore, understanding and regulating inflammatory pathways in the central nervous system (CNS) may contribute to prevention or delay of AD. Cyclooxygenase-2 (COX-2) is induced in response to inflammatory stimuli, and its inhibition underlies the therapeutic efficacy of many non- steroidal anti-inflammatory drugs (NSAIDs). COX-2 expression is significantly elevated in brain in AD and this elevation correlates with the severity of brain amyloid plaque pathology. Hence, in vivo and non-invasive monitoring of COX-2 level in the brain can track COX-2 induction during the course of AD and also examine the clinical benefits of COX-2 inhibition in AD. A positron emission tomography (PET) ligand to quantify the level of functional COX-2 in the brain would allow direct measurement of neuroinflammation in AD, and thereby enable disease staging and therapy evaluation. However, there is no such PET ligand is currently available. In order to establish a clinically useful PET tracer for COX-2 imaging in AD brain, we propose to evaluate highly potent [18F]- labeled COX-2 inhibitors due to the advantages associated with the 110-minute half-life. [18F]-labeled tracers allow scanning for longer duration so as to enable equilibrium scanning and facilitate robust kinetic studies leading to accurate quantification of COX-2. Moreover, they can be transported from a synthesis hub to nearby PET centers that lack a cyclotron and aid cost-effective PET studies. We identified MTP, a high affinity COX-2 inhibitor (IC50 = 2.2 nM; Figure 2), possessing an aromatic site for introducing [18F]-label that is less susceptible for defluorination. MTP also has adequate lipophilicity (LogP = 2.7) to passively traverse the blood-brain barrier (BBB). We synthesized [18F]MTP and successfully demonstrated its specific binding in COX-2 positive BxPC-3 cell lines (Figure 3, 112). In vivo PET imaging was performed in mice, induced with lipopolysaccharide (LPS), and obtained a significantly higher binding in the brain compared to control mice, with no visible skeletal uptake (Figure 4, 150). Subsequent in vitro autoradiography of slide-mounted sections of the harvested brain, established specific binding of the tracer in LPS-induced neuroinflammation (Figures 5 & 6). In light of the above supporting evidence demonstrating BBB permeability, higher binding in neuroinflammation, and specific binding to COX-2, [18F]MTP will be further evaluated along with two backup ligands 2 & 3 (Figure 2), using additional PET imaging in mice models of neuroinflammation as well as AD neuropathology (Aims 1 & 2). The most qualified tracer identified will undergo test-retest dynamic PET quantitative evaluations with concomitant arterial blood sampling in male and female anesthetized monkeys to quantify binding as total distribution volume in various brain regions (Aim 3). We believe, the proposed experiments would lead to the selection of a successful [18F]- COX-2 PET tracer, characterized for the transition to clinical studies in AD for early diagnosis, monitoring therapeutic response, and aid development of new medications.