Abstract
P487
Introduction: The orexin receptor subtype, OX1R, is involved in the regulation of reward processing, emotion, pain, feeding, addiction, and the sleep-wake cycle. A PET radioligand for imaging OX1R could be beneficial for investigating these CNS functions in pathophysiology. In addition, such a radioligand could aid in developing drugs targeting OX1R. Currently, no effective PET radioligand exists for OX1R. GSK1059865 (1) has had a high affinity for OX1R (KB = 2 nM) and more than 100-fold selectivity over OX2R and a panel of 113 other receptors.[1] Compound 1 displays other desirable CNS PET radioligand properties, such as low molecular weight (436 Da), moderate lipophilicity (clogD7.4, 3.18), and low polarity (tPSA, 53.9), which are all conducive to good brain entry. We aimed to label 1 with carbon-11 (t1/2 = 20.4 min) for evaluation as a candidate PET radioligand for imaging brain OX1R in monkey and mouse.
Methods: Compound 1 and precursor for 11C-labeling (2) were obtained commercially. [11C]1 was prepared by treating 2 with [11C]iodomethane for 5 minutes in the presence of t-BuOK at room temperature followed by acidic (TFA) removal of the Boc protecting group at 80 °C (Fig. 1). The in vitro stability and logD7.4 values of [11C]1 with respect to octanol and cyclohexane were determined.[2] Two male rhesus monkeys (~ 9 kg) were administered with [11C]1 (0.27 GBq) and PET scans were acquired at baseline for up to 120 minutes. In OX1R blocking experiments, suvorexant (0.5 mg/kg, i.v.) was administered 10 minutes before [11C]1. Concurrent arterial blood sampling was performed to obtain the radiometabolite-corrected arterial input function and to enable measurement of total volumes of distribution (VT). [11C]1 was also injected intravenously into male C57BL mice (3.7-7.4 MBq/mouse) to acquire dynamic PET scans over 60 minutes. Mice were administered with unlabeled 1 (0.2 mg/kg or 2 mg/kg, i.v.) 5 minutes before [11C]1 to test for specific binding.
Results: [11C]1 was obtained in 10-15% overall formulated yields with high molar activity (60-200 GBq/µmol) in about 50 minutes of radiosynthesis time. [11C]1 was stable in phosphate buffer for at least 2.5 h. The measured logD7.4 values of [11C]1 were 3.69 ± 0.06 (for octanol) and 2.12 ± 0.07 (for cyclohexane) with a difference (ΔlogD7.4) of 1.58 ± 0.01. After administration of [11C]1 at baseline, radioactivity in monkey brain rapidly peaked at about 1.2 SUV and then declined slowly (Fig. 2A). At 60 minutes, brain radioactivity slightly increased indicating the possibility of radiometabolite accumulation. Parent plasma concentrations were similar under both baseline and receptor pre-block conditions. No OX1R-specific binding was detected in any monkey brain region. VT could not be accurately quantified by compartmental modeling due to slow radioactivity washout from the brain (k4 ~ 0). VT values calculated from Logan plots were >1 mL/cm3 in all brain regions under baseline and receptor pre-block conditions. The whole brain time‒activity curves in the mouse brain were similar to those in the monkey brain, apart from higher initial peak radioactivity. No specific binding was detected (Fig. 2B).
Conclusions: [11C]1 was successfully synthesized and evaluated with PET in monkey and mouse. Despite favorable in vitro pharmacological properties, [11C]1 did not prove to be a promising PET radioligand, showing only moderate brain uptake and no OX1R-specific signal. Strong H-bonding capacity, indicated by a high ΔlogD7.4 value, may be an important factor in low brain entry. A higher-affinity ligand with improved brain entry is likely needed for successful PET imaging of brain OX1R. We infer that brain OX1R exist in monkey and mouse brain at very low density.
Acknowledgments: The Intramural Research Program of NIH (NIMH).
References:
[1] A. Gozzi et al., PLoS One, 2011, 6, e16406,
[2] R. C. Young et al., J. Med. Chem. 1988, 31, 656.
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