Radiosynthesis and evaluation of a novel (18)F-labeled tracer for PET imaging of glycogen synthase kinase 3.

Am J Nucl Med Mol Imaging 2024;14(5):327-336 www.ajnmmi.us /ISSN:2160-8407/ajnmmi0159323 Original Article Radiosynthesis and evaluation of a novel 18F-labeled tracer for PET imaging of glycogen synthase kinase 3 Zhiwei Xiao1,2*, Yinlong Li1,2*, Ahmed Haider1,2, Stefanie K Pfister2, Jian Rong1,2, Jiahui Chen1,2, Chunyu Zhao1,2, Xin Zhou1, Zhendong Song1, Yabiao Gao1, Jimmy S Patel1,3, Thomas L Collier1,2, Chongzhao Ran4, Chuangyan Zhai1, Hongjie Yuan5, Steven H Liang1,2 Department of Radiology and Imaging Sciences, Emory University, Atlanta, GA 30322, USA; 2Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital and Department of Radiology, Harvard Medical School, Boston, MA 02114, USA; 3 Department of Radiation Oncology, Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA; 4Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA; 5Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, GA 30322, USA. * Equal contributors. 1 Received July 19, 2024, Accepted September 10, 2024; Epub October 15, 2024; Published October 30, 2024 Abstract: Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase family that regulates diverse biological processes including glucose metabolism, insulin activity and energy homeostasis. Dysregulation of GSK3 is implicated in the development of several diseases such as type 2 diabetes mellitus, Alzheimer’s disease (AD), and various cancer types. In this study, we report the synthesis and evaluation of a novel positron emission tomography (PET) ligand compound 28 (codenamed [18F]GSK3-2209). The PET ligand [18F]28 was obtained via copper-mediated radiofluorination in more than 32% radiochemical yields, with high radiochemical purity and high molar activity. In vitro autoradiography studies in rodents demonstrated that this tracer exhibited a high specific binding to GSK3. Furthermore, PET imaging studies of [18F]28 revealed its ability to penetrate the blood-brain barrier (BBB). Keywords: Glycogen synthase kinase 3, Alzheimer’s disease, radiotracer, PET, 18F-labeled Introduction Glycogen synthase kinase 3 (GSK3) is an intracellular serine/threonine kinase family that phosphorylates and inactivates glycogen synthase [1, 2]. This multifunctional enzyme is widely distributed in numerous tissues with peak levels found in the central nervous system (CNS) [3-5]. GSK3 regulates diverse biological processes such as cell metabolism [6], proliferation/differentiation [7], and synaptic neurotransmission [8], and is implicated in many human diseases including neurodegenerative pathologies [9], cardiovascular disorders [10], and various cancer types [11]. GSK3 consists of two highly homologous isozymes termed GSK3α (51 kDa) and GSK3β (47 kDa). These two isozymes show 98% amino acid sequence identity within their kinase domains and 84% overall identity but share only 36% similarity in the last 76 C-terminal residues [3, 12, 13]. Previous studies have shown that both isoforms are ubiquitously expressed at high levels in the brain but particularly enriched in the hippocampus, cerebral cortex, and cerebellum [14, 15]. Given the key role of GSK3 in tau hyperphosphorylation and other signaling pathways, aberrant GSK3 activity is associated with the pathogenesis of Alzheimer’s disease (AD) [16], diabetes [17], and inflammation [18]. For instance, hyperactivity and/or overexpression of GSK3β has been observed in AD brains, leading to hyperphosphorylation of over 70% of potential phosphorylation sites on tau pro- teins, thereby disrupting their healthy association with microtubules [19]. Notably, GSK3 has emerged as a potential target for neurodegenerative and psychiatric drug development [20-22]. To date, despite among a variety of GSK3 inhibitors discovered some have reached clinical trials, including AZD1080 [23], Tideglusib [24], and LY2090314 [25], only lithium chloride (LiCl) [26] has been approved by the FDA. Notwithstanding the widespread use of lithium for bipolar disorders, limitations include a narrow therapeutic window, which required individual dose monitoring, as well as the potential to cause QT-prolongation. As such, there is an unmet medical need to provide alternative GSK3 inhibitors with an improved safety profile. Positron emission tomography (PET) is a noninvasive in vivo nuclear medicine imaging modality that utilizes radioligands to characterize, visualize, and quantify physiological processes by recording time-dependent distribution in living organs [27]. Specifically, PET serves as a powerful tool for brain imaging, capable of measuring the aberrant activity and levels of GSK3 in vivo using a suitable PET radiotracer [28]. Various classes of GSK3 radiotracers have been recently reported to quantify the distribution of GSK3 in healthy and diseased states (Figure 1). For example, [11C]AR-A014418 was the first reported radioligand for PET imaging of GSK3, but it exhibited limited bloodbrain barrier (BBB) permeability [29]. Similarly, [11C] https://doi.org/10.62347/OBZS8887 GSK3 PET tracer Figure 1. Representative radiotracers for GSK3 PET imaging. 328 Am J Nucl Med Mol Imaging 2024;14(5):327-336 GSK3 PET tracer PyrATP-1 (2) [30] and [11C]-oxadiazole-based radiotracers 3-5 demonstrated insufficient brain penetration in vivo [31]. Maleimide-derived tracers 6-7 showed promising preliminary results in BBB penetration and rodent brain uptake studies but require further evaluation in nonhuman primates [32-34]. [11C]PF-04802367 (8), one of the most potent and selective GSK-3 inhibitors, exhibited good uptake in brain regions with a homogeneous distribution [35]. Based on this finding, other 11C and 18F-labeled oxazole-4-carboxamide analogs 9-15 were developed, with [11C]OCM-44 showing promise for clinical translation [36, 37]. Recently, a series of isonicotinamide derivatives 17-19 were reported to have high affinity to GSK3β, but only [18F]19 showed reasonable brain uptake in GSK3βrich regions [38-41]. Although imaging data in rats revealed unfavorable in vivo stability and specificity, in view of the heterogeneous brain uptake, there is high interest in developing of GSK3β radiotracers based on the structure of isonicotinamide derivatives. With this objective, we designed the synthesis and evaluation of a novel 18F-labeled ligand [18F]28 for PET imaging of GSK3 in the brain of rodents. Preliminary physiochemical, in vitro binding properties, in vivo PET imaging, and metabolism studies were systematically investigated. Materials and methods General information Unless noted, all the commercial chemicals, solvents, and biological samples were purchased and used directly without further purification. Aluminum TLC plates, 60 F254, were employed for analytical thin-layer chromatography, visualizing with a 254 nm UV lamp. Flash column chromato (...truncated)