Soluble epoxide hydrolase (sEH) is a bifunctional enzyme located within cytosol and peroxisomes that converts epoxides to the corresponding diols and hydrolyzes phosphate monoesters. It serves to inactivate epoxyeicosatrienoic acids (EETs), which have vasoactive and anti-inflammatory properties. Inhibitors of sEH are pursued as agents to mitigate neuronal damage after stroke. We developed N-(3,3-diphenylpropyl)-6-18F-fluoronicotinamide (18F-FNDP), which proved highly specific for imaging of sEH in the mouse and non-human primate brain with PET.
Methods
18F-FNDP was synthesized from the corresponding bromo-precursor. sEH inhibitory activity of 18F-FNDP was measured using the sEH Inhibitor Screening Assay Kit. Biodistribution was undertaken in CD-1 mice. Binding specificity was assayed in CD-1 and sEH knock-out mice and Papio anubis (baboon) through pre-treatment with an sEH inhibitor to block sEH binding. Dynamic PET imaging with arterial blood sampling was performed in three baboons with regional tracer binding quantified using distribution volume (VT). Metabolism of 18F-FNDP in baboon was assessed using high performance liquid chromatography.
Results
18F-FNDP (Ki = 1.73 nM) was prepared in one step in radiochemical yield of 14 ± 7%, specific radioactivity in the range of 888 – 3,774 GBq/µmol and in radiochemical purity > 99% using an automatic radiosynthesis module. The time of preparation was about 75 min. In CD-1 mice regional uptake followed the pattern of striatum > cortex > hippocampus > cerebellum, consistent with the known brain distribution of sEH, with 5.2 percent injected dose per gram of tissue at peak uptake. Blockade of 80–90% was demonstrated in all brain regions. Minimal radiotracer uptake was present in sEH-KO mice. PET baboon brain distribution paralleled that seen in mouse with marked blockade (95%) noted in all regions indicating sEH-mediated uptake of 18F-FNDP. Two hydrophilic metabolites were identified with 20% parent compound present at 90 min post-injection in baboon plasma.
Conclusion
18F-FNDP can be synthesized in suitable radiochemical yield and high specific radioactivity and purity. In vivo imaging experiments demonstrated that 18F-FNDP targeted sEH in murine and non-human primate brain specifically. 18F-FNDP is a promising PET radiotracer likely to be useful for understanding the role of sEH in a variety of conditions affecting the central nervous system.