Development of Novel PET Radiotracers for Neuroinflammation and COVID-19: Targeting FPR2, I2BS and ACE2

Abstract

Positron Emission Tomography (PET) is a non-invasive bioimaging technique that applies highly selective and sensitive radiotracers to biological markers. Imaging with PET radiotracers is a powerful means for the assessment and early detection of various diseases such as neurological diseases, cardiovascular diseases and cancer. Neuroinflammation is an inflammatory response to insults such as injury, infection or toxic protein deposits in the central nervous system (CNS). Chronic neuroinflammation leads to disruptive brain functioning and can cause neurodegenerative diseases. Coronavirus disease 2019 (COVID-19) is an infectious disease spread rapidly across the world, threatening global health and causing huge economic losses. This work involves the development of novel small organic molecular probes that have the potential for being applied as radiotracers for imaging N-formyl peptide receptor 2 (FPR2), imidazoline-2 binding sites (I2BS) and angiotensin-converting enzyme 2 (ACE2) via PET. These molecular targets are important to understand the pathological processes in neuroinflammation and COVID-19. Over the course of project 1, automated radiosynthetic methods were developed to produce [11C]Quin C1 and [18F]Quin C1-click-F, two potential FPR2-targeting PET radioligands identified through collaboration with Imperial College London. Radiotracer [11C]Quin C1 was produced with decay-corrected radiochemical yield (d.c.RCY) (decay corrected to the start of synthesis) of 12% (n = 1) at the end of synthesis (EOS) from [11C]methyl iodide ([11C]CH3I), radiochemical purity (RCP) of 94% (n = 1) and molar activity (Am) of 6.09 GBq/µmol at EOS (n = 1). Radiotracer [18F]Quin C1-click-F was produced with d.c.RCYs (decay corrected to the start of synthesis) of 8 ± 6% (n = 7) at EOS from [18F]fluoride, RCPs of 97 ± 2% (n = 7) and the highest molar activity achieved of 27.74 GBq/µmol at EOS. The logD7.4 value of [18F]Quin C1-click-F was 1.5 (n = 1). The radiotracer [18F]Quin C1-click-F was then evaluated in preliminary in vitro autoradiography and in vivo microPET (µPET) study using healthy rats. Fluorescence imaging using a Rhodamine B-Quin C1 fluorescent probe was conducted in parallel. In vitro fluorescence imaging studies showed increased binding of the Rhodamine B-Quin C1 fluorescent probe in the stroke lesion compared to the contralateral cortex in stroke rat brain tissues. In vitro autoradiography showed high nonspecific binding of [18F]Quin C1-click-F in healthy and stroke rat brain tissues. In vivo µPET study in a healthy Wistar rat showed [18F]Quin C1-click-F’s low brain penetration (n = 1). For project 2, analogues of known I2BS ligands were synthesised following published protocols. One lead compound, BU-5, was selected to proceed with radiosynthesis. A model compound was first employed to test the copper-mediated radiofluorination. An automated radiosynthetic method was developed to afford [18F]BU-5 via an alcohol-enhanced copper-mediated 18F-radiolabelling approach. Radiotracer [18F]BU-5 was produced with d.c.RCYs (decay corrected to the start of synthesis) of 0.2-0.8 % (n = 4) at EOS from [18F]fluoride, RCPs of 97 ± 2% (n = 4) and the highest molar activity achieved of 5.08 GBq/µmol at EOS. The logD7.4 value of [18F]BU-5 was 1.0 (n = 1). Preliminary in vitro autoradiography, immunofluorescence and in vivo µPET studies have been performed with this tracer. In vitro immunofluorescence imaging study showed presence of activated astrocytes in the stroke rat brain tissues, which are suitable for using in [18F]BU-5 autoradiography studies. In vitro autoradiography showed heterogeneous binding of [18F]BU-5 in healthy rat brain tissues and in stroke rat brain tissues, [18F]BU-5 bindings were partially blocked by BU-5. In vivo µPET in one healthy female Sprague Dawley (SD) rat showed [18F]BU-5 crossed the blood-brain barrier (BBB) with high brain uptake and stable retention in I2BS-rich regions like the hypothalamus. For project 3, in collaboration with Professor Andreas Bender’s research group (University of Cambridge) and Prof Véronique Gouverneur’s research group (University of Oxford), a library of ACE2 inhibitors based on MLN-4760 was screened by in silico methods and the best performing analogues were synthesised. Subsequently, the synthesised compounds were evaluated using a commercially available ACE2 inhibition assay to identify promising candidates for further development and future radiolabelling studies. In conclusion, three PET radiotracers have been developed: [11C]Quin C1, [18F]Quin C1-click-F, and [18F]BU-5. Preliminary in vitro and in vivo data suggest that [18F]BU-5 warrants further investigation as a brain PET tracer for activated astrocytes. In contrast, [18F]Quin C1-click-F is not suitable for microglial imaging due to its insufficient BBB penetration. Radiotracer [11C]Quin C1 remains a potential FPR2 PET tracer, but further biological evaluation is required. For ACE2 PET imaging, lead compounds identified with high inhibitory potency should be radiolabelled and further biological studies performed.