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，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）中的炎症通路可能有助于预防或延迟AD。环氧合酶-2（考克斯-2）响应于炎性刺激而被诱导，并且其抑制是许多非甾体抗炎药（NSAID）的治疗功效的基础。考克斯-2的表达在AD患者的脑中显著升高，并且这种升高与脑淀粉样斑块病理学的严重程度相关。因此，脑中考克斯-2水平的体内和非侵入性监测可以追踪AD过程中的考克斯-2诱导，并且还检查AD中考克斯-2抑制的临床益处。正电子发射断层扫描（PET）配体定量功能考克斯-2在大脑中的水平将允许直接测量AD中的神经炎症，从而使疾病分期和治疗评估。然而，目前还没有这样的PET配体可用。为了建立一个临床上有用的PET示踪剂的考克斯-2成像在AD脑，我们建议评估高度有效的[18 F]标记的考克斯-2抑制剂，由于与110分钟的半衰期相关的优势。[18 F]-标记的示踪剂允许扫描更长的持续时间，从而能够平衡扫描并促进稳健的动力学研究，导致考克斯-2的准确定量。此外，它们可以从合成中心运输到附近的PET中心，这些中心缺乏回旋加速器，并有助于成本效益的PET研究。我们鉴定了MTP，一种高亲和力考克斯-2抑制剂（IC 50 = 2.2 nM;图2），具有用于引入[18 F]-标记的芳香族位点，其对去甲肾上腺素不太敏感。MTP还具有足够的亲脂性（LogP = 2.7）以被动穿过血脑屏障（BBB）。我们合成了[18 F]MTP，并成功证明了其在考克斯-2阳性BxPC-3细胞系中的特异性结合（图3，112）。在小鼠中进行体内PET成像，用脂多糖（LPS）诱导，与对照小鼠相比，在脑中获得显著更高的结合，没有可见的骨骼摄取（图4，150）。随后对收获的脑的载玻片切片进行体外放射自显影，建立了示踪剂在LPS诱导的神经炎症中的特异性结合（图5和6）。鉴于上述支持证据表明BBB渗透性、神经炎症中的更高结合以及与考克斯-2的特异性结合，[18 F]MTP将与两种备用配体2和3一起沿着进行进一步评估（图2），在神经炎症以及AD神经病理学的小鼠模型中使用额外的PET成像（目的1和2）。将在雄性和雌性麻醉猴中对识别出的最合格示踪剂进行重测动态PET定量评价，同时进行动脉血样采集，以定量结合为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成功的[18 F]-考克斯-2 PET示踪剂，其特征在于过渡到AD的临床研究，用于早期诊断、监测治疗反应和辅助新药开发。

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.