Radiosynthesis and in vitro evaluation of [18F]N-decyl-6-fluoronicotinamide-TEMPO as a PET/MR probe of oxidative stress in cell membranes

6 Objectives: Nitroxide radicals such as tetramethylpiperidine (TEMPO) have been investigated as functional contrast agents in electron paramagnetic resonance (EPR) and magnetic resonance imaging (MRI). In its oxidized form, the free radical group that imparts a spectroscopic EPR signal also confers T1 MRI contrast whereas these signals are lost in the reduced state. A fundamental limitation is that T1 signal changes are ambiguous if the pharmacokinetics are unknown. To address this, we proposed radiolabeling derivatives of TEMPO and imaging with PET/MR, using the PET data to monitor uptake and clearance thereby permitting unambiguous estimation of redox processes from the MR data. Because the T1 contrast depends only on the immediate microenvironment, molecular targeting permits measurement of redox status within specific organelles or subcellular compartments. In this work, we developed an F-18 labeled derivative of TEMPO with an alkyl chain as a lipid bilayer targeting moiety for the measurement of oxidative stress in cellular membranes. Methods: [18F]Fluoride was trapped on a QMA cartridge and eluted into a reaction vessel using a solution of Kryptofix 2.2.2 (8.0 mg) and potassium carbonate (2.0 mg) in CH3CN/H2O (0.8 mL/0.2 mL). Solvent was removed at 115°C under a stream of nitrogen and the residue was azeotropically dried three times with 1 mL of anhydrous acetonitrile each at 115°C, followed by adding a solution of N-decyl-6-nitronicotinamide-TEMPO precursor (2.5 mg) in DMSO (0.5 mL) was added to the reaction vessel and heated at 140°C for 10 min. The reaction mixture was diluted with 1mL of acetonitrile/0.1 M PBS (1:9) and injected onto a reverse phase HPLC column for purification. To measure cell uptake and washout, MDA-MB-231 tumor cells were plated to confluency on a culture plate and either membrane targeting [18F]N-decyl-6-fluoronicotinamide-TEMPO or untargeted [18F]6-fluoronicotinamide-4-nitroTEMPO was added to the media. A LigandTracer White made times series measurements of cell uptake for 15 minutes then radioactive media was replaced with clean media and washout was measured. To confirm membrane targeting, MD-MB-231 and HT520 cells were incubated with targeted or untargeted radiotracer then lysed with ultrapure water and immediately centrifuged. The membranous pellet was measured on a well counter. An MRI phantom was constructed containing various concentrations of targeted TEMPO and unmodified TEMPO structure at full oxidation. Imaging was performed on a 3T scanner using a T1-mapping sequence. Results: Precursor was obtained with 28% overall yield and the radiolabeled product [18F]N-decyl-6-fluoronicotinamide-TEMPO was obtained with 8.5% radiochemical yield. In the in vitro cell uptake and washout assay, the targeted compound [18F]N-decyl-6-fluoronicotinamide-TEMPO showed 60% greater uptake and washout was considerably slowed relative to the untargeted [18F]6-fluoronicotinamide-4-amino-TEMPO. Lysed cells incubated with targeted tracer had ~5-fold more activity in the membranous pellet than those incubated with untargeted tracer. In the MRI phantom, [18F]N-decyl-6-fluoronicotinamide-TEMPO produced a dose-dependent T1 signal which matched the relaxation rates of unmodified TEMPO at the same molar concentrations. Conclusions: [18F]N-decyl-6-fluoronicotinamide-TEMPO was synthesized as a potential multimodal PET/MR agent for imaging redox status in cellular membranes. Cell studies demonstrated enhanced uptake and protracted washout with lysis confirming accumulation in membranes. MRI phantom experiments demonstrated that the T1 contrast properties do not differ from unmodified TEMPO. Together, these results suggest that [18F]N-decyl-6-fluoronicotinamide-TEMPO has potential as a PET/MR multimodal agent for quantitative imaging of redox status in cellular membranes. Acknowledgements: NIH R01MH100350, P41EB022544, and S10OD018035