Development of class-IIa HDAC targeting PET probes for molecular imaging of disorders of the CNS

摘要。阿尔茨海默病（AD）是一种毁灭性的脑神经退行性疾病，目前尚无治愈方法。AD与进行性痴呆和认知能力下降有关，这突出了对新的基于生物标志物的工具的迫切需求，以准确检测早期AD并选择患者进行靶向治疗。表观遗传机制的发现及其对AD认知功能下降的实质性贡献激发了广泛的研究，试图描述组蛋白脱乙酰酶（HDAC）在记忆功能中的作用。因此，已经研究了用panHDAC抑制剂阻断HDAC以减轻AD脑中的记忆衰退。然而，最近两项独立的人体成像研究报告，I类HDAC水平随着AD进展而降低，这强调了AD大脑中HDAC特异性靶向的必要性。IIa类HDAC成员HDAC 4和HDAC 5在神经元突触可塑性和记忆形成中起关键作用。安德森KW等人的定量研究发现，AD人类额叶皮质中IIa类HDAC 5表达增加47%，而AD中I类HDAC下调（HDAC 1，2降低32%），这与最近用11 C-马丁诺司特获得的临床数据一致。这些发现为我们定量人类AD大脑中IIa类HDAC表达变化的方法提供了强有力的证据，并验证了IIa类HDAC作为AD的治疗靶点;这将对AD患者产生显著的积极影响。因此，我们的目标是开发和验证用于正电子发射断层扫描（PET）的放射性示踪剂，以非侵入性和定量地绘制整个大脑中IIa类HDAC的表达和定位，从而提供整个健康大脑与AD大脑的对比视图。我们的初步结果支持拟议研究的适用性和可行性，其中一种示踪剂（18F-26）证明了有用的CNS PET示踪剂的特性。18 F-26显示出出色的药代动力学和成像特征，因为灰质区域的脑摄取很高，从而产生高质量的PET图像;组织动力学适合18 F示踪剂，并且通过自封闭和SAHA（panHDAC抑制剂）证明了与IIa类HDAC的特异性结合。因此，我们提出了这三个具体目标：（1）合成和筛选30个候选抑制剂的集中库，（2）使用我们的新的放射合成途径和其他已建立的放射化学方法放射性标记来自aim 1的选定示踪剂候选物（N=6），以及（3）验证用于健康大鼠脑中IIa类HDAC表达的体内PET/CT成像的最佳三种放射性示踪剂。我们将使用我们的铅示踪剂进行定量体外放射自显影，以测量死于AD的患者的死后脑切片中的IIa类HDAC表达，并将这些样品与健康对照（无AD）的切片进行比较。通过IIa类HDAC PET成像早期检测AD将允许使用IIa类HDAC抑制剂进行早期干预以减缓或停止疾病进展。此外，IIa类HDAC PET成像与其它疾病（例如，亨廷顿氏病、缺血性中风、创伤性脑损伤和癌症）具有很大的相关性。

ABSTRACT. Alzheimer’s diseases (AD) is a devastating brain neurodegenerative disease with no cure currently available. AD is associated with progressive dementia and cognitive decline, which highlight the glaring need for new biomarker-based tools to accurately detect early AD and select patients for targeted therapies. The discovery of epigenetic mechanisms and their substantial contribution to cognitive decline in AD inspired extensive research in an attempt to delineate the role of Histone Deacetylases (HDACs) in memory function. As such, HDAC blockade with panHDAC inhibitors have been perused to alleviate the memory decline in the AD brain. However, two recent and independent human imaging studies reported that class-I HDAC levels decrease as AD advances, which emphasizes the need for specific targeting of HDACs in the AD brain. The class-IIa HDAC members HDAC4 and HDAC5 play a key role in neuronal synaptic plasticity and memory formation. A quantitative study by Anderson KW, et al. found a 47% increase in the class-IIa HDAC5 expression in the AD human frontal cortex, while class-I HDACs were downregulated in AD (HDAC1,2 decreased 32%), which is consistent with recent clinical data obtained with 11C-Martinostate. These findings establish strong evidence for our approach to quantitate changes in class-IIa HDAC expression in the human AD brain and validate class-IIa HDACs as a therapeutic target in AD; this will have a significant positive impact for AD patients. Therefore, we aim to develop and validate radiotracers for positron emission tomography (PET) to non-invasively and quantitatively map the expression and localization of class-IIa HDACs in the brain in its entirety, thus providing a contrast view of the whole healthy brain versus the AD brain. Our preliminary results support the suitability and feasibility of the proposed studies, with one tracer (18F-26) demonstrating the characteristics of a useful CNS PET tracer. 18F-26 displays excellent pharmacokinetic and imaging characteristics, since brain uptake is high in gray matter regions, leading to high-quality PET images; tissue kinetics are appropriate for an 18F tracer, and specific binding for class-IIa HDAC is demonstrated by self-blockade and with SAHA (panHDAC inhibitor). Therefore, we propose these three specific aims: (1) to synthesize and screen a focused library of 30 candidate inhibitors, (2) to radiolabel the selected tracer candidates (N=6) from aim1 using our novel radiosynthetic route and other established radiochemical methods, and (3) to validate the best three radiotracers for in vivo PET/CT imaging of class-IIa HDAC expression in the brain of healthy rats. We will perform quantitative in vitro autoradiography with our lead tracer to measure class-IIa HDAC expression in postmortem brain sections from patients who died with AD and compare those samples with sections obtained from healthy controls (sans AD). Early detection of AD by class-IIa HDAC PET imaging will allow early intervention to slow or halt disease progression with class-IIa HDAC inhibitors. Further, class-IIa HDAC PET imaging is of great relevance to other diseases, such as Huntington’s disease, ischemic stroke, traumatic brain injuries, and cancer.