Abstract
1697
Objectives: [11C](-)-4-methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetylpiperidine ([11C](-)GR103545) is the one and only kappa opioid receptor (KOR) agonist PET tracer currently used in human imaging studies. Although [11C](-)GR103545 is a very well validated radiotracer, there are conflicting data available about the off target binding and functional pharmacological properties of (-)GR103545. Occupancy measurement studies of nonselective KOR agents (-)-naloxone, and naltrexone using [11C](-)GR103545, provided 60-86% displacement of activity in brain regions. However, a PET experiment in mice using Salvinorin A, a selective KOR agonist, showed only marginal (37%) displacement at standard uptake value (SUV) at KOR dense regions with >50% occupancies using [11C](-)GR103545. This indicates the possibility of significant in vivo affinity of (-)GR103545 to off targets, likely mu opioid receptor (MOR), and this may interfere with the PET imaging outcome of the radiotracer for receptor quantification. In this presentation, we report binding affinities and cross selectivity of (-)GR103545 to various brain targets and its functional properties at KOR, DOR and MOR targets.
Methods: (-)GR103545 was obtained from NIMH drug supply program. Binding affinity (Ki) measurements of (-)GR103545 were performed stable HEK cells using [3H]U69593 as radioligand and Salvinorin A as reference standard for KOR; [3H]DAMGO and morphine for MOR, and [3H]DADLE and naltrindole for delta opioid receptor (DOR) respectively. Agonistic and antagonistic assays of (-)GR103545 at KOR, MOR and DOR were performed via GTPγS stimulation and cAMP formation in membranes from HEK cells or hMOR cells. Kis and cross selectivity to other targets were performed by the service of National Institute of Mental Health- Psychoactive Drug Screening program (NIMH-PDSP). Experimental data were analyzed and graphically processed using the GraphPad Prism Software. All values are expressed as the mean ± SEM (n = 3 or 4). Results: (-)GR103545 exhibits 0.13+0.03 nM, 2.1+0.4 nM and 80.6+31 nM Kis for KOR, MOR and DOR targets respectively. Apart from MOR, DOR, (-)GR103545 did not show significant affinity to other brain targets (Ki>10 μM). Functional assay measurements reveal that (-)GR103545 is a full agonist at KOR with EC50 26 pM and agonist at DOR with EC50 650 nM respectively. However, (-)GR103545 was not sensitive to the agonistic assays at MOR site, instead it exhibited antagonistic behavior in both MOR tango and Gi assays.
Conclusions: (-)GR103545 is a high affinity KOR agonist ligand and it shows a significant affinity to MOR. The antagonistic behavior of [11C](-)GR103545 with a Ki of 2.1 nM at MOR may provide sufficient binding in vivo with PET to MOR enriched brain regions. Further independent pharmacology evaluations of (-)GR103545 and occupancy experiments with KOR selective agonists are required to thoroughly establish the receptor selectivity of (-)GR103545. Details of the radioligand binding and functional assay measurements of (-) GR103545 will be presented. Grant support: Diane Goldberg Foundation (NYSPI/CUMC).