Development of Novel PET Ligands for Imaging Orexin 2 Receptor

Introduction: The orexin 2 receptor (OX₂R) is a G-protein coupled receptor expressed in brain that binds orexin neuropeptides A and B. OX₂R is involved in motivation, feeding behaviour, and sleep-wake regulation. Modulation of OX₂R with agonists has been found to be a potential treatment towards sleep related disorders. Herein we report [¹¹C]1 and [¹¹C]2 as novel and potential PET ligands for imaging orexin 2 receptor and explore their applications in small animals.

Methods: Compounds 1 and 2 were prepared according to a published patent.¹ The phenolic precursors were synthesized via demethylation of compounds 1 and 2. The radiosynthesis of [¹¹C]1 and [¹¹C]2 were performed by ¹¹C-methylation of phenolic precursors with [¹¹C]CH₃I and Cs₂CO₃ as the base in DMF at 80 ^oC for 5 min. The in vitro autoradiography was performed with [¹¹C]1 and [¹¹C]2 on Sprague Dawley rat brain slices. Dynamic PET imaging studies with [¹¹C]1 and [¹¹C]2 were carried out on Sprague Dawley rats with 60 min scans.

Results: Compounds 1 (OX₂: IC₅₀ = 4 nM, OX₁: IC₅₀ > 4000 nM) and 2 (OX₂: IC₅₀ = 4 nM, OX₁: IC₅₀ > 5000 nM) are selective antagonists at the OX₂.¹ Compounds 1 and 2 were prepared in 2% and 20% overall yields over four steps, respectively. The phenolic precursors were synthesized via demethylation of compounds 1 and 2 in 41% and 45% yields, respectively. The radiosynthesis of PET ligands [¹¹C]1 and [¹¹C]2 were via ¹¹C-methylation of phenolic precursors with [¹¹C]CH₃I and both [¹¹C]1 and [¹¹C]2 were obtained in more than 10% radiochemical yields (decay corrected). Both [¹¹C]1 and [¹¹C]2 had radiochemical purities greater than 99%, and no obvious decompositions of [¹¹C]1 and [¹¹C]2 were found in saline in 90 min. In in vitro autoradiography studies with [¹¹C]1 and [¹¹C]2, high radioactivity accumulation was observed in the hippocampus and cortex. In blocking studies of autoradiography, EMPA (10 μM) rendered diminished radioactivity in OX₂R-rich brain regions. In dynamic PET imaging studies with [¹¹C]1, the standard uptake value (SUV) of [¹¹C]1 in hippocampus and cerebellum reached the max value of 0.2 at 2 min and washed out rapidly, and finally reduced to 0.1 at 60 min. PET imaging with [¹¹C]2 showed a similar result. In blocking studies of PET imaging, no significant difference in uptake in the hippocampus and cerebellum was observed for both [¹¹C]1 and [¹¹C]2. Pretreatment with elacridar (3 mg/kg) led to a significant increase of uptake in hippocampus and cerebellum for both [¹¹C]1 and [¹¹C]2.

Conclusions: We have developed two novel PET ligand [¹¹C]1 and [¹¹C]2 for imaging OX₂R. The PET ligands [¹¹C]1 and [¹¹C]2 were both prepared in more than 10% radiochemical yield (decay corrected). In autoradiography studies, both [¹¹C]1 and [¹¹C]2 showed high binding specificities in hippocampus and cortex in vitro. While in vivo PET imaging with [¹¹C]1 and [¹¹C]2 demonstrated moderate binding affinities towards OX₂R, their poor blood-brain barrier permeabilities and rapid clearance from the brain prevent further evaluation.

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