Preclinical autoradiography and PET studies reveal [18F]asyn-44 and ACI-12589 target different α‑synuclein sites, but asyn-44 shows low specificity and species-dependent rapid metabolism (problematic in NHPs) and ACI-12589 exhibits off-target amyloid‑β binding.

Although neither tracer is immediately translatable, the negative and comparative data clarify tracer selectivity, off-target liabilities, and species metabolic differences that are directly actionable for designing better α‑synuclein PET ligands to support Parkinson's therapeutic development and…

BackgroundParkinson's disease (PD) and multiple system atrophy (MSA) are considered α-synucleinopathies, characterized by the presence of pathological α-synuclein (α-syn) aggregates. A positron emission tomography (PET) tracer for imaging α-syn aggregates in vivo is highly sought after, as disease progression correlates with the accumulation of aggregated α-syn. We recently reported [18F]asyn-44 as a radiotracer for α-syn, worthy of evaluation in higher species, based on in vitro binding data from human brain tissues and in vivo PET imaging studies in rodents.Objective[3H]ACI-12589 is a promising α-syn PET tracer which recently showed binding in MSA patients but appears to have limited utility in other α-synucleinopathies. Objective 1) compare the in vitro binding properties of our lead, [3H]asyn-44, to [3H]ACI-12589; Objective 2) evaluate [18F]asyn-44 and [18F]ACI-12589 kinetics by in vivo PET imaging in normal rodents; Objective 3) assess pharmacokinetic properties and metabolism of [18F]asyn-44 in normal pig and non-human primate (NHP).MethodsIn vitro autoradiography with [3H]asyn-44 and [3H]ACI-12589 was performed to compare radiotracer binding in PD, MSA, Alzheimer's disease and healthy control post-mortem brain tissue. Additionally, preclinical PET imaging was performed in rats with [18F]ACI-12589 to compare with our previously reported [18F]asyn-44 data. Further evaluation of [18F]asyn-44 in higher species was carried out by preclinical PET imaging in pig and NHP with metabolite analysis. Liver microsome assays and mass spectrometry were performed to identify the metabolites formed in NHP.Results[3H]Asyn-44 and [3H]ACI-12589 displayed different binding properties in both PD and MSA tissue, suggesting that the tracers target different binding sites and asyn-44 might therefore be more suited for PD imaging. In the pig, [18F]asyn-44 readily entered the brain and no brain penetrant metabolites were observed in arterial blood samples. In the NHP, [18F]asyn-44 readily entered the brain but was rapidly metabolized. Radiolabeled metabolites of asyn-44 were proposed and will be considered in the design of future derivatives.ConclusionsSpecies differences in metabolism of [18F]asyn-44 are observed between pig and NHP, and do not support the further translation of [18F]asyn-44. Additionally, autoradiography with [3H]asyn-44 revealed low signal specificity and high non-displaceable binding. We report evidence for off-target binding of [3H]ACI-12589 to amyloid-β plaques. The limitations of both [3H]asyn-44 and [3H]ACI-12589 reported here support the development of additional derivatives and structural scaffolds of asyn-44 with the potential to improve radiotracer specificity and selectivity towards α-syn.