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The lack of high-affinity and selective ligands for α-synuclein (α-syn) is a major limitation to the development of positron emission tomography (PET) imaging agents for the in vivo quantification of pathological α-syn aggregates. PET imaging would be invaluable for early and non-invasive diagnosis of α-synucleinopathies, such as Parkinson’s disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), as well as dramatically increasing the understanding of the field and facilitating the development of new therapeutic strategies.
Despite numerous ongoing efforts, no candidate has reached clinical routine application. Indeed, several challenges remain due to the low abundance and structural difference of the target in the brain and, in general, the need for adequate selectivity with respect to other amyloid proteins, such as amyloid-β (Aβ) and tau, which often co-exist with α-syn pathology. Furthermore, in order to develop a central nervous system PET tracer, a tracer must be able to cross the blood-brain barrier and exhibit low non-specific binding (NSB) to brain tissue.
Here we report the design, synthesis, radiosynthesis, in vitro and in vivo evaluation of a series of novel arylpyrazolethiazole (APT) and diarylpyrazolethiazole (DAPT) derivatives, and the 11C-radiolabeling of a recent diphenylpyrazole (DPP), [11C]MODAG-005, as promising new α-syn PET tracers.
A series of arylpyrazolethiazole (APT) derivatives was rationally designed on the basis of known ligands for α-syn fibrils, with the goal of improving selectivity towards α-syn fibrils and controlling physicochemical properties favorable for a CNS PET tracer. In vitro competition binding assays performed against [3H]MODAG-001 using recombinant α-syn and Aβ1-42 fibrils enabled a structureactivity relationship study to design APT-13 with an inhibition constant (Ki) of 27.8 ± 9.7 nM and a more than 3.3-fold selectivity on Aβ fibrils. Radiolabeled [11C]APT-13 showed excellent brain penetration in healthy mice, with a peak standardized uptake value (SUV) of 1.94 ± 0.29 and rapid washout from the brain (t1/2 = 9 ± 1 min). Moreover, in vitro autoradiography (AR) in human brain sections with α-syn, Aβ, and tau pathology displayed specific binding (SB) to the area where α-syn pathology was also found
by immunohistochemistry, although it showed a high NSB in the brain white matter.
This study highlights the potential of APT-13 as a lead compound for the development of PET tracers to detect α-syn aggregates in vivo. In addition, a library of DAPT was designed via the rational drug design technique of molecular hybridization based on the fusion of two known lead compounds, with the aim of enhancing their binding affinity. In vitro competition binding assays performed against [3H]MODAG-001 using human α-syn fibrils revealed moderate to high binding affinity to α-syn fibrils, with an overall range Ki between 5.7-221.3 nM. Competition binding assays using amyloid-β1-42 (Aβ1-42) fibrils revealed a generally higher
selectivity of DAPT derivatives compared to DPP compounds (range Ki = 18.1 – 404.6 nM). The most promising compounds were further evaluated in in vitro competition binding assays against [3H]PiB, resulting in a range Ki of 40.7 – 1204 nM for α-syn and 86.7 – 607.4 nM for Aβ1-42 fibrils. Based on its performance on fibrils binding assays, DAPT-23 was chosen to be 11C-radiolabeled. [11C]DAPT-23 was able to detect SB in the area where pathological aggregates were found that could be effectively blocked with cold DAPT-23 in in vitro AR studies on pathological human brain tissues from two different MSA cases.
[11C]DAPT-23 demonstrated brain penetration in healthy mice with a peak SUV of 0.79 ± 0.1 at 31.7 seconds. Thus, our results revealed that the new class of DAPT derivatives binds to α-syn fibrils with desirable affinity and selectivity, with promising potential as new leads for the development of imaging agents for the selective biological detection of α-synucleinopathies.
Furthermore, MODAG-005 was shown to be an effective ligand for α-syn fibrils and demonstrated promising binding to MSA tissue in in vitro AR. This thesis reports the radiolabeling of [11C]MODAG-005 using a direct methylation approach of its unprotected precursor, anle190214, with [11C]methyltriflate ([11C]MeOTf) in an overall highly reliable radiosynthesis process. The study revealed the best radiolabeling conditions and yielded [11C]MODAG-005 with a radiochemical yield (RCY) of 15.9 and a molar activity (Am) of 149 GBq/µmol. |
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