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Background and purpose: The histamine H3 receptor antagonist radioligand [3H]-A-349821 was characterized as a radiotracer for assessing in vivo receptor occupancy by H3 receptor antagonists that affect behaviour. This model was established as an alternative to ex vivo binding methods, for relating antagonist H3 receptor occupancy to blood levels and efficacy in preclinical models. Experimental approach: In vivo cerebral cortical H3 receptor occupancy by [3H]-A-349821 was determined in rats from differences in [3H]-A-349821 levels in the isolated cortex and cerebellum, a brain region with low levels of H3 receptors. Comparisons were made to relate antagonist H3 receptor occupancy to blood levels and efficacy in a preclinical model of cognition, the five-trial inhibitory avoidance response in rat pups. Key results: In adult rats, [3H]-A-349821, 1.5 µg·kg−1, penetrated into the brain and cleared more rapidly from cerebellum than cortex; optimally, [3H]-A-349821 levels were twofold higher in the latter. With increasing [3H]-A-349821 doses, cortical H3 receptor occupancy was saturable with a binding capacity consistent with in vitro binding in cortex membranes. In studies using tracer [3H]-A-349821 doses, ABT-239 and other H3 receptor antagonists inhibited H3 receptor occupancy by [3H]-A-349821 in a dose-dependent manner. Blood levels of the antagonists corresponding to H3 receptor occupancy were consistent with blood levels associated with efficacy in the five-trial inhibitory avoidance response. Conclusions and implications: When employed as an occupancy radiotracer, [3H]-A-349821 provided valid measurements of in vivo H3 receptor occupancy, which may be helpful in guiding and interpreting clinical studies of H3 receptor antagonists.

Background and purpose: The histamine H4 receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In this report, we describe the in vitro and in vivo anti-inflammatory properties of a potent histamine H4 receptor antagonist, A-940894 (4-piperazin-1-yl-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-d]pyrimidin-2-ylamine). Experimental approach: We have analysed the pharmacological profile of A-940894 at mouse native, rat recombinant and human recombinant and native, histamine H4 receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan-induced peritonitis. Key results: A-940894 potently binds to both human and rat histamine H4 receptors and exhibits considerably lower affinity for the human histamine H1, H2 or H3 receptors. It potently blocked histamine-evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine-induced shape change of mouse bone marrow-derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell-dependent model of zymosan-induced peritonitis, A-940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D2 levels. Finally, A-940894 has good pharmacokinetic properties, including half-life and oral bioavailability in rats and mice. Conclusions and Implications: These data suggest that A-940894 is a potent and selective histamine H4 receptor antagonist with pharmacokinetic properties suitable for long-term in vivo testing and could serve as a useful tool for the further characterization of histamine H4 receptor pharmacology.

A-349821 is a selective histamine H3 receptor antagonist/inverse agonist. Herein, binding of the novel non-imidazole H3 receptor radioligand [3H]A-349821 to membranes expressing native or recombinant H3 receptors from rat or human sources was characterized and compared with the binding of the agonist [3H]N-α-methylhistamine ([3H]NαMH). [3H]A-349821 bound with high affinity and specificity to an apparent single class of saturable sites and recognized human H3 receptors with 10-fold higher affinity compared to rat H3 receptors. [3H]A-349821 detected larger populations of receptors compared to [3H]NαMH. Displacement of [3H]A-349821 binding by H3 receptor antagonists/inverse agonists was monophasic, suggesting recognition of a single binding site, while that of H3 receptor agonists was biphasic, suggesting recognition of both high- and low-affinity H3 receptor sites. pKi values of high-affinity binding sites for H3 receptor competitors utilizing [3H]A-349821 were highly correlated with pKi values obtained with [3H]NαMH, consistent with labelling of H3 receptors by [3H]A-349821. Unlike assays utilizing [3H]NαMH, addition of GDP had no effect on saturation parameters measured with [3H]A-349821, while displacement of [3H]A-349821 binding by the H3 receptor agonist histamine was sensitive to GDP. In conclusion, [3H]A-349821 labels interconvertible high- and low-affinity states of the H3 receptor, and displays improved selectivity over imidazole-containing H3 receptor antagonist radioligands. [3H]A-349821 competition studies showed significant differences in the proportions and potencies of high- and low-affinity sites across species, providing new information about the fundamental pharmacological nature of H3 receptors. Keywords: Histamine, radioligand binding, H3-receptor binding, radiolabelled H3-receptor antagonist, radiolabelled H3-receptor inverse agonist, [3H]A-349821, guanine nucleotide shift, G-protein, G-protein-coupled receptor Introduction To date, a majority of histamine H3 receptor-binding studies have relied on the H3 receptor-selective agonists [3H]RαMH and [3H]NαMH as radiolabels (van der Goot & Timmerman, 2000). While having high selectivity and specific binding along with favorable signal to noise ratios, binding of these agonist radioligands to H3 receptors is sensitive to guanine nucleotides, suggesting that H3 receptor agonist radioligands recognize both high- and low-affinity receptor sites (Arrang et al., 1990; West et al., 1990a; Kilpatrick & Michel, 1991; Clark et al., 1993). This makes characterization of H3 receptors difficult because of assay-dependent variables, such as membrane preparation procedures (Childers & Lariviere, 1984; Kim & Neubig, 1987) and ionic composition of assay buffers that can alter receptor–G-protein interactions and affect the apparent affinity state of the receptor (Hamblin & Creese, 1982; Kilpatrick & Michel, 1991; Parkinson & Fredholm, 1992). Several radiolabelled H3-antagonists have been described previously, such as [3H]S-methylthioperamide, [125I]iodophenpropit, [125I]iodoproxyfan, [3H]thioperamide, [3H]{"type":"entrez-nucleotide","attrs":{"text":"GR168320","term_id":"238436299","term_text":"GR168320"}}GR168320 and [3H]clobenpropit (Jansen et al., 1992; Ligneau et al., 1994; Yanai et al., 1994; Alves-Rodrigues et al., 1996; Brown et al., 1996; Harper et al., 1999), but they have not been widely used. This is attributable to a variety of limiting factors, such as low specific activity, poor H3 receptor specificity, equivocal functional properties or lack of commercial availability. Moreover, all of the previously referenced H3 receptor antagonist radiolabels contain an imidazole moiety, which may be responsible for off-target binding to 5-HT3 receptors, H4 receptors and cytochrome P450 isoenzymes (Labella et al., 1992; Leurs et al., 1995; Schlicker et al., 1995; Alves-Rodrigues et al., 1996; Harper et al., 1999; Yang et al., 2002; Esbenshade et al., 2005a; Kitbunnadaj et al., 2005). More recently, we described the first non-imidazole, H3 receptor antagonist, [3H]A-317920 (Esbenshade et al., 2005b; Yao et al., 2005). [3H]A-317920 is a highly selective and potent radiolabel useful for detection and characterization of recombinant rat H3 receptors expressed in C6 cells, a rat glioma cell line, or native H3 receptors expressed in rat cortical tissue. However, this radioligand does not recognize human H3 receptors with high affinity, and as such has limited utility. Subsequent medicinal chemistry efforts revealed a novel non-imidazole H3 receptor ligand, A-349821, having high potency and selectivity for both rat and human H3 receptors in competition-binding assays and potent antagonist activity in a variety of in vitro functional assays as well as H3 receptor-mediated in vivo models (Esbenshade et al., 2004). A-349821 also displayed highly efficacious and potent inverse agonist properties in [35S]GTPγS-binding assays across species (Esbenshade et al., 2004). Competition-binding studies carried out with ∼75 G-protein coupled receptors (GPCRs) and ligand-gated ion channels showed minimal crossreactivity (IC50>1 μM), with weak affinity (IC50=250 nM) observed for α2c adrenergic receptors (Esbenshade et al., 2004). With these promising binding and pharmacological properties, [3H]A-349821 was prepared and evaluated as a radiolabel for the detection of H3 receptors in membranes expressing recombinant or native rat or human H3 receptors. Like the agonist radioligand [3H]NαMH, [3H]A-349821 exhibited specific and saturable binding to tissues or cell lines expressing either rat or human H3 receptors. Detailed competition-binding studies using a large panel of H3 receptor ligands were performed to compare the pharmacology of [3H]A-349821 with that of [3H]NαMH. The effects of guanosine 5′-diphosphate (GDP) on binding were determined to investigate the contribution of low- and high-affinity binding states to the observed pharmacology. Herein, we describe the use of [3H]A-349821 as the first non-imidazole radioligand for the highly specific detection and characterization of both rat and human H3 receptors.

1. This study reports on the identification and characterization of a 1,4-dihydropyridine analogue, 9-(3,4-dichlorophenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione (A-184209) as a novel inhibitor of ATP-sensitive K(+) channels. 2. A-184209 inhibited membrane potential changes evoked by the prototypical cyanoguanidine ATP-sensitive K(+) channel opener (KCO) P1075 in both vascular (A10) and urinary bladder smooth muscle cells with IC(50) values of 1.44 and 2.24 micro M respectively. 3. P1075-evoked relaxation of 25 mM K(+) stimulated aortic strips was inhibited by A-184209 in an apparently competitive fashion with a pA(2) value of 6.34. 4. The potencies of A-184209 to inhibit P1075-evoked decreases in membrane potential responses in cardiac myocytes (IC(50)=0.53 micro M) and to inhibit 2-deoxyglucose-evoked cation efflux pancreatic RINm5F cells (IC(50)=0.52 micro M) were comparable to the values for inhibition of smooth muscle K(ATP) channels. 5. On the other hand, a structural analogue of A-184209 that lacked the gem-dimethyl substituent, 9-(3,4-dichlorophenyl)-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione (A-184208), was found to be a K(ATP) channel opener, evoking membrane potential responses in A10 smooth muscle cells (EC(50)=385 nM) and relaxing aortic smooth muscle strips (IC(50)=101 nM) in a glyburide-sensitive manner. 6. Radioligand binding studies demonstrated that A-184209 displaced SUR1 binding defined by [(3)H]glyburide binding to RINm5F cell membranes with a K(i) value of 0.11 micro M whereas A-184208 was ineffective. On the other hand, both A-184209 (K(i)=1.34 micro M) and A-184208 (K(i)=1.14 micro M) displaced binding of the KCO radioligand, [(125)I]A-312110 in guinea-pig bladder membranes with similar affinities. 7. These studies demonstrate that A-184209 is a novel and structurally distinct compound that inhibits K(ATP) channels in smooth muscle with potencies comparable to glyburide. The structural overlap between DHP openers and blockers, together with their differential interaction with ligand binding sites, support the notion that both openers and blockers bind to similar or very closely coupled sites on the sulfonylurea receptor and that subtle changes in the pharmacophore itself could switch functional properties from K(ATP) channel activation to inhibition.