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，后者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缓AD的发生。环氧合酶-2(COX-2)是对炎症刺激的反应，它的抑制是许多非类固醇抗炎药(NSAIDs)治疗效果的基础。环氧合酶-2在AD患者脑组织中的表达显著升高，并且这种升高与脑淀粉样斑块的严重程度相关。因此，活体和非侵入性监测脑内COX-2水平可以追踪AD病程中COX-2的诱导，也可以检验抑制COX-2在AD中的临床益处。正电子发射断层扫描(PET)配基可以量化大脑中功能性COX-2的水平，从而可以直接测量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配体可用。为了建立一种临床上有用的用于AD脑内COX-2显像的PET示踪剂，我们建议评估高效的[18F]标记的COX-2抑制剂，因为与110分钟的半衰期相关的优点。[18F]标记的示踪剂允许进行更长时间的扫描，以实现平衡扫描并促进稳健的动力学研究，从而准确地定量COX-2。此外，它们可以从合成中心运输到附近没有回旋加速器的PET中心，从而帮助进行具有成本效益的PET研究。我们鉴定了MTP，一种高亲和力的COX-2抑制剂(IC50=2.2 nM；图2)，具有引入[18F]标记的芳香族位点，对脱氟较不敏感。MTP也有足够的亲脂性(LogP=2.7)来被动穿越血脑屏障(BBB)。我们合成了[18F]MTP，并成功地在COX-2阳性的BxPC-3细胞系中证明了其特异性结合(图3,112)。在小鼠体内进行了PET成像，在脂多糖(LPS)诱导下，与对照组小鼠相比，大脑中的结合显著增加，没有明显的骨骼摄取(图4,150)。随后，对收集的脑组织切片进行体外放射自显影，证实示踪剂在脂多糖诱导的神经炎症中具有特异性结合(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与COX-2的特异性结合，[18F]MTP将与两个备份配体2和3(图2)一起进一步评估，使用额外的PET成像在小鼠神经炎症和AD神经病理模型中(目标1和2)。确定的最合格示踪剂将在雄性和雌性麻醉猴子的伴随动脉血液样本中进行测试-重新测试动态PET定量评估，以量化结合在不同大脑区域的总分布体积(目标3)。我们相信，拟议的实验将导致选择一种成功的[18F]-COX-2 PET示踪剂，该示踪剂的特点是过渡到AD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.