Development and biological evaluation of[¹⁸F]FMN3PA & [¹⁸F]FMN3PU for leucine-rich repeat kinase 2 (LRRK2) in vivo...

Among all genetic mutations of LRRK2, the G2019S mutation is the most commonly associated with the late-onset of Parkinson's disease (PD). Hence, one potential therapeutic approach is to block the hyperactivity of mutated LRRK2 induced by kinase inhibition. To date, only a few LRRK2 kinase inhibitors have been tested for in vivo quantification of target engagement by positron emission tomography (PET). In this study, we performed biological evaluations of two radiolabeled kinase inhibitors i.e. [¹⁸F]FMN3PA (14) and [¹⁸F]FMN3PU for LRRK2 (15).

Radiosyntheses of [¹⁸F]FMN3PA (14) and [¹⁸F]FMN3PU (15) were performed using K[¹⁸F]-F-K222 complex in a TRACERlab FXN module and purification was carried out via C18 plus (Sep-Pak) cartridges. In vitro specific binding assays were performed in rat brain striatum and kidney tissues using GNE-0877 as a blocking agent (K_i = 0.7 nM). For in vivo blocking, 3 mg/kg of GNE-0877 was injected 30 min before radiotracer injection via tail vein in wild-type (WT) mice (n = 4). Dynamic scans by PET/CT (Siemens Inveon) were performed in WT mice (n = 3).

Radiofluorinations resulted in radiochemical yields (RCYs) of 25 ± 1.3% (n = 6) ([¹⁸F]FMN3PU, 15) and 37 ± 1.6% (n = 6) ([¹⁸F]FMN3PA, 14) with ≥96% radiochemical purity (RCP) and a molar activity (MA) of 3.55 ± 1.6 Ci/μmol (131 ± 56 GBq/μmol) for [¹⁸F]FMN3PU (15) and 4.57 ± 1.7 Ci/μmol (169 ± 63 GBq/μmol) for [¹⁸F]FMN3PA (14), respectively. Saturation assays showed high specific binding for rat brain striatum with K_d 20 ± 1.3 nM ([¹⁸F]FMN3PA, 14) and 23.6 ± 4.0 nM ([¹⁸F]FMN3PU, 15). In vivo blocking data for [¹⁸F]FMN3PA (14) was significant for brain (p < 0.0001, 77% blocking) and kidney (p = 0.0041, 65% blocking). PET images showed uptake in mouse brain striatum.

In the presence of GNE-0877 as a blocking agent, the specific binding of [¹⁸F]FMN3PA (14) and [¹⁸F]FMN3PU (15) was significant in vitro. [¹⁸F]FMN3PA (14) showed good brain uptake in vivo, though fast clearance from brain was observed (within 10-15 min).