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Abstract: NK-1 receptor antagonists have shown potential for the clinical treatment of chemotherapy-induced nausea and vomiting, depression and alcoholism. In a recent study, we disclosed the potential for the NK-1 antagonist, LY686017, to treat alcoholism in a clinical population. To assess whether this compound could be utilized as a platform for a brain imaging ligand, we evaluated the binding of [3H]-LY686017 to sections of guinea pig in vitro. In these studies, [3H]-LY686017 bound with a distribution and pharmacology consistent with the NK-1 receptor. Using sections through the region of the caudate nucleus, we obtained a K d of 0.34nM and a B max of 31.37fmoles/mg tissue. Based on its high potency and low nonspecific binding in vitro, we initiated studies to evaluate the radioligand as a tool to measure in vivo receptor occupancy. In initial studies, 25microCi of [3H]-LY686017 was administered via an indwelling jugular catheter and accumulation of radioactivity in the caudate (NK-1 containing tissue) and cerebellum (low NK-1 expression) were assessed. The ratios of caudate to cerebellum radioactivity were optimal 2h after radioligand administration so this time point was used for subsequent studies. To assess the pharmacological specificity of the radioactivity accumulation, we administered various doses of Aprepitant, a potent NK-1 antagonists 1h prior to intravenous administration of [3H]-LY686017. Aprepitant produced a dose-dependent reduction in radioactivity in the caudate with an approximate 70% reduction at 10mg/kg. To image NK-1 receptors, 100microCi of [3H]-LY686017 was administered and the brains sectioned for autoradiography. In these studies, a characteristic distribution on NK-1 receptors was observed. Based on these results, LY686017 should serve as a suitable chemical platform for future imaging ligand development. [Copyright &y& Elsevier]

Abstract: The peptide, nociceptin, was discovered as the endogenous ligand for the opioid-like receptor, ORL1. Since its discovery, this peptide has been shown to modulate the perception of pain, modulate feeding and produce behavioral effects in rodent models of mood disorders. Recently, the non-peptide agonist {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one} (Ro64-6198) of the ORL1 receptor has been reported in the literature. In the present study, we compared the distribution and potency of Ro64-6198 with nociceptin for their ability to stimulate [35S]-GTPγS binding to sections of rat brain. In initial studies, Ro64-6198 inhibited 125I-nociceptin binding to the hORL1 receptors with a K i of 1.75nM compared with 0.20nM for nociceptin. To assess agonist potency in a whole cell assay, a cell line expressing the hORL1 receptor and Gα15 was created and used for calcium mobilization studies. In this assay system, Ro64-6198 increased intracellular calcium with an EC50 of 52nM compared with 24nM for nociceptin. Having verified the agonist properties of Ro64-6198, we then assessed the potency and distribution of ORL1 receptor activation in rat brain sections. In dose–response studies, Ro64-6198 increased [35S]-GTPγS binding to a variety of brain regions with EC50 values ranging from 84.9 to 2143nM depending on the brain regions evaluated. These potencies were similar to that seen for nociceptin, but substantially lower than values established using [125I] nociceptin binding to the cloned human ORL1 receptor. In general, the brain distribution of agonist stimulated [35S]-GTPγS binding was similar when either Ro64-6198 or nociceptin were used. Using these techniques, we have demonstrated, for the first time that Ro64-6198 activates [35S]-GTPγS binding to rat brain sections and this compound stimulates a similar population of receptors as nociceptin. [Copyright &y& Elsevier]

Neuropeptide Y, one of the most abundant brain peptides, has been found to modulate several important biological functions via a family of G-protein coupled receptors. To investigate the localization of functional NPY receptor subtypes in the rat brain, we performed agonist-induced [35S]GTPγS autoradiography. The Y1/Y4/Y5 agonist Leu31, Pro34-NPY increased [35S]GTPγS binding in several brain areas with a regional distribution consistent with that produced when labeling adjacent sections with [125I]-Leu31, Pro34-PYY. The Y1 selective antagonist BIBP3226 antagonized the Leu31, Pro34-NPY stimulated increase in [35S]GTPγS binding in all areas examined. The Y2 agonist C2-NPY stimulated [35S]GTPγ binding in numerous brain areas with a regional distribution similar to the binding observed with [125I]-PYY 3-36. No increase in [35S]GTPγS binding above basal was observed in any brain area evaluated using Y4 and Y5 selective agonists. This study demonstrates abundant Y1 and Y2 receptor activation in the rat brain, while evidence for functional Y4 and Y5 receptors was not observed. [Copyright &y& Elsevier]

Analogues of 3,4-dimethyl-4-(3-hydroxyphenyl)piperidines are high affinity inverse agonists for μ-, δ- and κ-opioid receptors. To characterize inverse agonist binding, we synthesized a high specific activity radioligand from this series, [3H]LY515300 (3-[1-((3-cyclohexyl-[3,4-3H2])-3(R,S)-hydroxypropyl)-3(R),4(R)-dimethylpiperidin-4-yl]phenol). In membranes expressing cloned human opioid receptors, [3H]LY515300 binding was saturable and exhibited low nonspecific binding. [3H]LY515300 bound with high affinity to the μ- (Kd=0.07 nM), δ- (Kd=0.92 nM) and κ-(Kd=0.45 nM) opioid receptors. High affinity [3H]LY515300 binding to all opioid receptors was Na+-dependent, a characteristic of inverse agonists. Displacement by standard opioid compounds yielded Ki values consistent with their known opioid receptor affinities. Autoradiographic localization of specific [3H]LY515300 binding in rat and guinea pig brain was high in areas known to express high levels of opioid (particularly μ-opioid receptor) binding sites including the caudate, nucleus accumbens, and nucleus tractus solitarius. Thus, [3H]LY515300 is the first radiolabeled opioid receptor inverse agonist useful for the study of opioid receptors in cell lines and native tissues. [Copyright &y& Elsevier]

Abstract: Corticotropin releasing factor (CRF) and Urocortin are important neurotransmitters in the regulation of physiological and behavioral responses to stress. Centrally administered CRF or Urocortin produces anxiety-like responses in numerous animal models of anxiety disorders. Previous studies in our lab have shown that Urocortin infused into the basolateral nucleus of the amygdala produces anxiety-like responses in the social interaction test. Subsequently, in the current study we prepared a specific CRF1 receptor antagonist (N-Cyclopropylmethyl-2,5-dimethyl-N-propyl-N''-(2,4,6-trichloro-phenyl)-pyrimidine-4,6-diamine, NBI3b1996) to examine in this paradigm. This CRF1 receptor antagonist inhibited the ex vivo binding of 125I-sauvagine to rat cerebellum with an ED50 of 6 mg/kg, i.p. NBI3b1996 produced a dose-dependent antagonism of Urocortin-induced anxiety-like behavior in Social Interaction test with an ED50 of 6 mg/kg, i.p. The compound had no effect on baseline social interaction. In addition, the CRF1 receptor antagonist prevented the stress-induced decrease in social interaction. These results provide further support for the CRF1 receptor in anxiety-like behavior and suggest this pathway is quiescent in unstressed animals. [Copyright &y& Elsevier]