A Novel PET Radiotracer for Imaging Alpha Synuclein Fibrils in Multiple System Atrophy (MSA)

Introduction: Abnormal aggregation of α-synuclein (Asyn) in the CNS is well known to be associated with neurodegenerative diseases such as Parkinson’s disease (PD) and Multiple System Atrophy (MSA). Currently, many PET radiotracers for imaging Asyn are under development but there is no clear candidate for PET imaging studies in PD and MSA patients. In this study, we describe the radiosynthesis, in vitro binding, and in vivo evaluation of [³H/¹¹C]HY-2-15, a compound that binds with high affinity to Asyn (Figure).

Methods: HY-2-15 was identified from structure-activity relationship studies based on BF2846, a ligand that binds to both Asyn and Ab. Competition binding assays were performed with [³H]BF2846 and [³H]PIB to measure affinity to Asyn and Ab. HY-2-15 was radiolabeled with tritium and C-11 for in vitro binding and in vivo studies. K_d and Bmax values of [³H]HY-2-15 were obtained from a panel of postmortem brain samples (e.g., PD, MSA, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Alzheimer's disease (AD)). Nuclear emulsion autoradiography identified the distribution of [³H]HY-2-15 on PD and MSA tissues for aggregated Asyn, which was confirmed by antibody staining. Dynamic whole body PET images of [¹¹C]HY-2-15 in non-human primate were obtained over 90 min.

Results: In in vitro binding assays, HY-2-15 exhibited high binding affinity for Asyn (6.1 ± 2.1 nM) and low binding affinity at Aβ fibrils (33 – 115 nM). In the tissue binding study, [³H]HY-2-15 exhibited high specific binding to all the investigated tissues (Kd = 5.1 nM for PD tissue; 4.5 nM for MSA; 18 nM for CBD, and 7.1 nM for PSP and 3.2 for AD). Interestingly, [³H]HY-2-15 had higher binding to pathologic Asyn in MSA compared to PD brain sections in autoradiography, suggesting [³H]HY-2-15 may be specific for MSA Asyn. In AD tissue, HY-2-15 competed poorly with [³H]PiB (Ki=120 nM) for Aβ, indicating high affinity binding of HY-2-15 to AD tau only. The high affinity of [³H]HY-2-15 to AD, CBD and PSP may reflect binding to a novel binding site on aggregated tau since it could not be blocked with the tau ligands PI-2620, JNJ-067, PM-PBB3, and GTP-1 in homogenate binding and autoradiography studies. The non-human primate PET study confirmed that [¹¹C]HY-2-15 had good brain uptake (SUV ~1.6) and rapid washout (2 : 60 min ratio ~3.0).

Conclusions: [¹¹C]HY-2-15 displayed binding to Asyn fibrils with low nM binding affinity. Tissue binding and autoradiography studies demonstrated [³H]HY-2-15 can bind to Asyn in postmortem tissue, particularly Asyn aggregates in MSA. Binding in CBD and PSP may indicate binding to a new site in aggregated tau. Whole body PET confirmed the favorable in vivoproperties of [¹¹C]HY-2-15 for brain imaging. In conclusion, [¹¹C]HY-2-15 may be a useful radiotracer for imaging Asyn in MSA patients.

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