Fully automated radiosynthesis of [(18)F]FCPPC for imaging microglia with PET.

Am J Nucl Med Mol Imaging 2024;14(6):351-356 www.ajnmmi.us /ISSN:2160-8407/ajnmmi0160149 Original Article Fully automated radiosynthesis of [18F]FCPPC for imaging microglia with PET Pritam Roy1,2,3, Yan Guo1,2,3, Otto Muzik2,4, Eric A Woodcock5, Huailei Jiang1,2,3 Cyclotron and Radiochemistry Core, Karmanos Cancer Institute, Detroit, MI, USA; 2PET Center, Karmanos Cancer Institute, Detroit, MI, USA; 3Department of Oncology, Wayne State University, Detroit, MI, USA; 4Department of Pediatrics and Neurology, Wayne State University, Detroit, MI, USA; 5Department of Psychiatry and Behavioral Neurosciences and Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA 1 Received August 28, 2024; Accepted December 9, 2024; Epub December 15, 2024; Published December 30, 2024 Abstract: Colony-stimulating factor 1 receptor (CSF1R) is almost exclusively expressed on microglia in the human brain and thus, has promise as a biomarker for imaging microglia density as a proxy for neuroinflammation. [11C]CPPC is a radiotracer with selective affinity to CSF1R, and has been evaluated for in-human microglia PET imaging. The flourine-18 labeled CPPC derivative, 5-cyano-N-(4-(4-(2-[18F] fluoroethyl)piperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide ([18F]FCPPC), was previously synthesized, however, with a low radiochemical yield using manual radiosynthesis. In this work, we report a fully automated radiosynthesis of [18F]FCPPC on a Synthra RNplus research module. In a total synthesis time of 50 min, [18F]FCPPC was obtained in decay corrected radiochemical yields of 26.8 ± 0.1% (n = 3) with >99% radiochemical purities. Quality control testing showed that [18F]FCPPC met all release criteria. In sum, we report the first fully automated radiosynthesis of [18F]FCPPC, a promising radiopharmaceutical for imaging microglia in humans. Keywords: Colony-stimulating factor 1 receptor, [18F]FCPPC, radiosynthesis, automation, PET imaging, radiopharmaceutical Introduction Neuroinflammation is an immune response within the Central Nervous System (CNS) associated with activation and proliferation of glial cells, especially microglia. Microglia are the resident macrophages in the human brain and play a crucial role in the development and homeostasis of the CNS [1, 2]. Upon detection of inflammatory stimuli, microglia activation is critical for the initiation of a neuroinflammatory response and restoration of homeostasis [3, 4]. Neurodegenerative diseases, such as Alzheimer’s disease (AD), are associated with chronic neuroinflammation and microglial activation [5, 6]. Hence, improved understanding of the role of neuroinflammatory signaling in psychiatric and neurological disorders, including neurodegenerative diseases, may lead to novel therapeutics and improved clinical outcomes. Colony-stimulating factor 1 receptor (CSF1R) is a subfamily of tyrosine kinase receptor activated by binding to colony-stimulating factor 1 or interleukin 34, and plays a significant role in survival, homeostatic functions, and proliferation of microglia. CSF1R is almost exclusively expressed on microglia in the human brain, and thus, holds considerable potential as a cell-specific imaging biomarker of neuroimmune state in humans [7-9]. Many lead compounds targeting CSF1R have been developed and evaluated as CSF1R inhibitors, and showed promising profiles as potential therapeutic agents to prevent neurodegeneration. As such, Positron Emission Tomography (PET) imaging of the CSF1R has been proposed as an in vivo technique for quantifying human microglial activation and proliferation [10-12]. Several CSF1R-targeting radiotracers have been previously developed (Figure 1) [12-19]. One of these radiotracers is [11C]5-cyano-N-(4-(4methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide ([11C]CPPC), which has been shown to exhibit selective CSF1R-affinity as well as suitable kinetic properties for PET imaging in humans [20]. Despite its promising biological properties for CSF1R imaging, the use of [11C] CPPC is limited by the short half-life of carbon-11 (t1/2 = 20.4 min). In 2022, Lee and co-workers developed a fluoride-18 labeled CPPC derivative, 5-cyano-N-(4-(4-(2-[18F] fluoroethyl)piperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan2-carboxamide ([18F]FCPPC) for CSF1R imaging [21]. In the preclinical evaluations, [19F]FCPPC showed high CSF1R binding affinity with IC50 of 3.42 ± 0.33 nM, and [18F]FCPPC exhibited significantly increased brain uptake over the control mice. The radiosynthesis of [18F]FCPPC was performed manually with non-decay corrected radiochemical yields of 8-8.5%, providing an opportunity for further optimization and automated radiosynthesis. Previously, we reported an high-yield radiosynthesis of [11C]CPPC for in-human microglia PET imaging [22]. In the present study, we report an automated radiosynthesis of [18F]FCPPC with improved yield over the reported manual synthesis method (Figure 2), and three validation runs to support its clinical translation for CSF1R PET imaging. Synthra RNPlus research module is designed to allow multi-step radiosyntheses of research radiotracers and our approach utilized its versatile functions, which https://doi.org/10.62347/QFGP5253 Fully automated radiosynthesis of [18F]FCPPC Figure 1. Representative chemical structures of CSF1R-targeting radiotracers. Figure 2. Scheme of automated radiosynthesis of [18F]FCPPC from its Cl-form precursor. Radiosynthesis conditions: MeCN, 100°C, 5 min. Decay-corrected yield at EOS: 26.8%. enabled a streamlined process encompassing one-pot radiofluorination, semi-preparative HPLC purification, and solid phase extraction (SPE) assisted formulation. Materials and methods Chemicals and supplies Unless otherwise stated, reagents, solvents, and chemicals were purchased from commercially available vendors and used without further purification. The 5-cyano-N(4-(4-(2-fluoroethyl)piperazin-1-yl)-2-(piperidin-1-yl)phenyl) furan-2-carboxamide (FCPPC) reference standard and 5-cyano-N-(4-(4-(2-chloroethyl)piperazin-1-yl)-2-(piperidin1-yl)phenyl)furan-2-carboxamide (pre-FCPPC) precursor were synthesized in-house following the reported method [21]. Acetonitrile (MeCN; anhydrous 99.8%), potassium carbonate (K2CO3; 99.995% trace metals basis), and 0.22 µm Millex-GV syringe-driven filter unit were purchased from Millipore Sigma (St. Louis, MO, USA). Acetonitrile (MeCN; HPLC grade) was purchased from Fisher Scientific (Hampton, NH, USA). Kryptofix 2.2.2 (K2.2.2; chemical grade) and [18O]H2O (≥98%) was purchased from ABX (Radeberg, Germany). QMA carbonate plus light cartridge (46 mg sorbent per cartridge; 40 μm), alumina N Plus Light cartridge, and tC18 plus short cartridge (400 mg sorbent per cartridge; 37-55 μm) were purchased from Waters (Milford, MA, USA). Absolute ethanol (EtOH; USP grade) was purchased from Greenfield Global USA Inc. (Shelbyville, KY, USA). Sterile water for injection, USP and 0.9% sodium chloride (NaCl) for in (...truncated)