Toward Novel [18F]Fluorine-Labeled Radiotracers for the Imaging of α-Synuclein Fibrils

Bright C Uzuegbunam; Junhao Li; Wojciech Paslawski; Wolfgang Weber; Per Svenningsson; Hans Ågren; Behrooz Hooshyar Yousefi

doi:10.3389/fnagi.2022.830704

Toward Novel [¹⁸F]Fluorine-Labeled Radiotracers for the Imaging of α-Synuclein Fibrils

Front Aging Neurosci. 2022 Apr 29:14:830704. doi: 10.3389/fnagi.2022.830704. eCollection 2022.

Authors

Bright C Uzuegbunam¹, Junhao Li², Wojciech Paslawski³, Wolfgang Weber¹, Per Svenningsson³, Hans Ågren², Behrooz Hooshyar Yousefi⁴

Affiliations

¹ Department of Nuclear Medicine, Technical University of Munich, Munich, Germany.
² Department of Physics and Astronomy, Uppsala University, Uppsala, Sweden.
³ Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden.
⁴ Department of Nuclear Medicine, Philipps University of Marburg, Marburg, Germany.

Abstract

The accumulation of α-synuclein aggregates (α-syn) in the human brain is an occurrence common to all α-synucleinopathies. Non-invasive detection of these aggregates in a living brain with a target-specific radiotracer is not yet possible. We have recently discovered that the inclusion of a methylenedioxy group in the structure of diarylbisthiazole (DABTA)-based tracers improves binding affinity and selectivity to α-syn. Subsequently, complementary in silico modeling and machine learning (ML) of tracer-protein interactions were employed to predict surface sites and structure-property relations for the binding of the ligands. Based on this observation, we developed a small focused library of DABTAs from which 4-(benzo[d][1,3]dioxol-5-yl)-4'-(3-[¹⁸F]fluoro-4-methoxyphenyl)-2,2'-bithiazole [ ¹⁸ F]d ₂, 6-(4'-(3-[¹⁸F]fluoro-4-methoxyphenyl)-[2,2'-bithiazol]-4-yl)-[1,3]dioxolo[4,5-b]pyridine [ ¹⁸ F]d ₄, 4-(benzo [d][1,3]dioxol-5-yl)-4'-(6-[¹⁸F]fluoropyridin-3-yl)-2,2'-bithiazole [ ¹⁸ F]d ₆, and 6-(4'-(6-[¹⁸F]fluoropyridin-3-yl)-[2,2'-bithiazol]-4-yl)-[1,3]dioxolo[4,5-b]pyridine [ ¹⁸ F]d ₈ were selected based on their high binding affinity to α-syn and were further evaluated. Binding assay experiments carried out with the non-radioactive versions of the above tracers d ₂, d ₄, d ₆, and d ₈ showed high binding affinity of the ligands to α-syn: 1.22, 0.66, 1.21, and 0.10 nM, respectively, as well as excellent selectivity over β-amyloid plaques (Aβ) and microtubular tau aggregates (>200-fold selectivity). To obtain the tracers, their precursors were radiolabeled either via an innovative ruthenium-mediated (S_NAr) reaction ([ ¹⁸ F]d ₂ and [ ¹⁸ F]d ₄) or typical S_NAr reaction ([ ¹⁸ F]d ₆ and [ ¹⁸ F]d ₈) with moderate-to-high radiochemical yields (13% - 40%), and high molar activity > 60 GBq/μmol. Biodistribution experiments carried out with the tracers in healthy mice revealed that [ ¹⁸ F]d ₂ and [ ¹⁸ F]d ₄ showed suboptimal brain pharmacokinetics: 1.58 and 4.63 %ID/g at 5 min post-injection (p.i.), and 1.93 and 3.86 %ID/g at 60 min p.i., respectively. However, [ ¹⁸ F]d ₆ and [ ¹⁸ F]d ₈ showed improved brain pharmacokinetics: 5.79 and 5.13 %ID/g at 5 min p.i.; 1.75 and 1.07 %ID/g at 60 min p.i.; and 1.04 and 0.58 %ID/g at 120 min p.i., respectively. The brain uptake kinetics of [ ¹⁸ F]d ₆ and [ ¹⁸ F]d ₈ were confirmed in a dynamic PET study. Both tracers also showed no brain radiometabolites at 20 min p.i. in initial in vivo stability experiments carried out in healthy mice. [ ¹⁸ F]d ₈ seems very promising based on its binding properties and in vivo stability, thus encouraging further validation of its usefulness as a radiotracer for the in vivo visualization of α-syn in preclinical and clinical settings. Additionally, in silico and ML-predicted values correlated with the experimental binding affinity of the ligands.

Keywords: PET tracer development; disarylbisthiazole (DABTA); positron emission tomography (PET); ruthenium-mediated deoxyfluorination; α-synuclein aggregates; α-synucleinopathies.