Chemistry, Radiochemistry and Preliminary Biological Evaluations of the First Brain-penetrant GPR119-based Radiotracer in Rodents and Non-human primates

Introduction: Reduction of a few important G-protein-coupled receptors (GPCRs) is implicated in AD pathogenesis associated with metabolic dysfunctions. One such GPCR is GPR119, which plays a key role in directly stimulating glucagon-like peptide-1 (GLP1) secretion to regulate insulin release in glucose metabolism. While GLP1/GPR119 agonists primarily regulate glucose metabolism, they also showed promising activity on improving neurologic and cognitive functions in AD subjects with type-2 diabetes mellitus (T2DM). However, the neuroprotective effects of these agonists remain largely unknown and PET imaging of GPR119 levels will significantly benefit these clinical therapeutic interventions. Our lab synthesized a series of novel piperidine analogs as GPR119 agonists and identified two analogs (T1 and T2) with high GPR119 binding potency (2-5 nM) for [¹⁸F]-radiochemistry. Here we present the chemistry, radiochemistry, initial in vitro cell uptake, and in vivo imaging in rodents and vervets.

Methods: T1 and T2 were synthesized from 4-substituted aminophenoxy ethyl substituents and piperidine derivatives following the NEt₃-assisted coupling reactions. BBB penetration of T1 and T2 were tested along with the commercially available GPR119 agonist AR231453, using artificial membrane-based PAMPA assays. [¹⁸F]T2 radiochemistry was carried out following [¹⁸F]¯-based nucleophilic substitution of corresponding tosylates at 100 °C for 15 min. Radiotracers were obtained through column chromatography separation and C18-based SepPak formulation with 10% EtOH in saline. Cell-binding assays were performed in three cell lines with different GPR119 expressions: MDM-MD-231 < NCI-H1716 < HepG2 with [¹⁸F]T2, with and without AR231453 and nonradioactive T1/T2. Dynamic 0-60 min brain microPET imaging and post-PET biodistribution in normal balb/c mice (n=4), and 0-90 min brain PET imaging were performed in 6 healthy female vervets (13-19 y, 4.7-6.5 kg) with [¹⁸F]T2. Standard uptake values (SUVs) and time-activity curves (TACs) were calculated for the whole brain.

Results: T1 and T2 were synthesized in 22% and 30% chemical yields. All the intermediates and final compounds were characterized with ¹H NMR. T1 and T2 demonstrated a permeability rate of 12.2 and 30.2 nm/s, respectively using the PAMPA assays. While both acceptable, T2 and reference compound AR231453 showed similar P_e suggesting T2’s high brain permeability, so T2 was selected for the next steps. [¹⁸F]T2 was produced in high radiochemical purity (>98%) and specific activity (~3800-4500 mCi/µmol), decay corrected to end of synthesis. [¹⁸F]T2 cell uptake was lower in MDM-MD-231 and higher in HepG2 cells, demonstrating the direct relationship between [¹⁸F]T2 uptake to GPR119 expression. Additionally, uptake increased significantly with AR231452 and T1/T2 treatments compared to baseline. SUVs (Avg SUV_max=0.5±0.1 (mice) and 2.4±0.1g/mL) showed high brain uptake, and TACs demonstrated rapid BBB penetration within the first 10 min of tracer injection, followed by favorable washout kinetics (Fig 1). Biodistribution results also showed high brain uptake; corroborating in vivo imaging results.

Conclusions: We report the synthesis and radiochemistry of [¹⁸F]T2 with high radiochemical purity, and specific activity. In vitro studies shows selectivity and specificity of the radiotracer towards GPR119 expressions and in vivo imaging studies in rodents and monkeys indicate BBB penetration for the first time. The combination of selective binding and favorable pharmacokinetics with excellent brain uptake makes [¹⁸F]T2 a suitable PET ligand for imaging GPR119.

• Download figure
• Open in new tab
• Download powerpoint