An Improved Antagonist Radiotracer for the κ-Opioid Receptor: Synthesis and Characterization of (11)C-LY2459989

Abstract

The κ-opioid receptors (KORs) are implicated in several neuropsychiatric diseases and addictive disorders. PET with radioligands provides a means to image the KOR in vivo and investigate its function in health and disease. The purpose of this study was to develop the selective KOR antagonist (11)C-LY2459989 as a PET radioligand and characterize its imaging performance in nonhuman primates.

Methods: LY2459989 was synthesized and assayed for in vitro binding to opioid receptors. Ex vivo studies in rodents were conducted to assess its potential as a tracer candidate. (11)C-LY2459989 was synthesized by reaction of its iodophenyl precursor with (11)C-cyanide, followed by partial hydrolysis of the resulting (11)C-cyanophenyl intermediate. Imaging experiments with (11)C-LY2459989 were performed in rhesus monkeys with arterial input function measurement. Imaging data were analyzed with kinetic models to derive in vivo binding parameters.

Results: LY2459989 is a full antagonist with high binding affinity and selectivity for KOR (0.18, 7.68, and 91.3 nM, respectively, for κ, μ, and δ receptors). Ex vivo studies in rats indicated LY2459989 as an appropriate tracer candidate with high specific binding signals and confirmed its KOR binding selectivity in vivo. (11)C-LY2459989 was synthesized in high radiochemical purity and good specific activity. In rhesus monkeys, (11)C-LY2459989 displayed a fast rate of peripheral metabolism. Similarly, (11)C-LY2459989 displayed fast uptake kinetics in the brain and an uptake pattern consistent with the distribution of KOR in primates. Pretreatment with naloxone (1 mg/kg, intravenously) resulted in a uniform distribution of radioactivity in the brain. Further, specific binding of (11)C-LY2459989 was dose-dependently reduced by the selective KOR antagonist LY2456302 and the unlabeled LY2459989. Regional binding potential values derived from the multilinear analysis-1 (MA1) method, as a measure of in vivo specific binding signal, were 2.18, 1.39, 1.08, 1.04, 1.03, 0.59, 0.51, and 0.50, respectively, for the globus pallidus, cingulate cortex, insula, caudate, putamen, frontal cortex, temporal cortex, and thalamus.

Conclusion: The novel PET radioligand (11)C-LY2459989 displayed favorable pharmacokinetic properties, a specific and KOR-selective binding profile, and high specific binding signals in vivo, thus making it a promising PET imaging agent for KOR.

Keywords: PET; antagonist; kappa opioid receptor; radioligand; synthesis and evaluation.

Figure 1

Structures of selected kappa opioid receptor ligands.

Figure 2

A: Ligand concentrations (ng/g of brain tissue) in the cerebellum (grey bar) and striatum (black bar) of Sprague-Dawley rats at 5, 20, 40 and 60 min (n = 4 rats per time point) following intravenous administration of LY2459989 (3 μg/g); B: Dose-receptor occupancy relationship of LY2459989 at KOR, MOR and DOR.

Figure 3

Radiosynthesis of ¹¹C-LY2459989. Reagents and conditions: a. H¹¹CN, KHCO₃, dppf, Pd₂dba₃, DMF, 80 °C, 5 min; b. NaOH, H₂O₂, 80 °C, 5 min.

Figure 4

Chiral HPLC chromatograms for the racemic compound (first row), the (S)-enantiomer LY2459989 (second row), the (R)-enantiomer (third row) and the radiolabeled compound ¹¹C-LY2459989 (last two rows). The (S)-enantiomer eluted first at 5.4 min, and the (R)-enantiomer eluted later at 7.5 min.

Figure 5

Analysis of ¹¹C-LY2459989 in the plasma: A. Total radioactivity (solid line) and metabolite-corrected parent activity (dotted line) over time; B. Time course of parent fraction from six baseline scans (mean ± SD).

Figure 6

PET images of ¹¹C-LY2459989 in transaxial (left), coronal (middle) and saggital (right n) views from a baseline scan (A), and blocking scans with 1 mg/kg of naloxone (B) or 0.3 mg/kg of LY2456302 (C). Images are summed from 30–45 min following ¹¹C-LY2459989 injection and presented in standard uptake value (SUV) unit.

Figure 7

Time-activity curves in selected brain regions from a ¹¹C-LY2459989 baseline scan (A), and blocking scans after naloxone (B), 0.3 mg/kg of LY2456302 (C), or 36 μg/kg of LY2459989 (D).