ABSTRACT
BACKGROUND AND PURPOSE: As baclofen is active in patients with anxiety disorders, GABAB receptors have been implicated in the modulation of anxiety. To avoid the side effects of baclofen, allosteric enhancers of GABAB receptors have been studied to provide an alternative therapeutic avenue for modulation of GABAB receptors. The aim of this study was to characterize derivatives of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) as enhancers of GABAB receptors. EXPERIMENTAL APPROACH: Enhancing properties of rac-BHFF were assessed in the Chinese hamster ovary (CHO)-Galpha16-hGABA(B1a,2a) cells by Fluorometric Imaging Plate Reader and GTPgamma[35S]-binding assays, and in rat hippocampal slices by population spike (PS) recordings. In vivo activities of rac-BHFF were assessed using the loss of righting reflex (LRR) and stress-induced hyperthermia (SIH) models. KEY RESULTS: In GTPgamma[35S]-binding assays, 0.3 microM rac-BHFF or its pure enantiomer (+)-BHFF shifted the GABA concentration-response curve to the left, an effect that resulted in a large increase in both GABA potency (by 15.3- and 87.3-fold) and efficacy (149% and 181%), respectively. In hippocampal slices, rac-BHFF enhanced baclofen-induced inhibition of PS of CA1 pyramidal cells. In an in vivo mechanism-based model in mice, rac-BHFF increased dose-dependently the LRR induced by baclofen with a minimum effective dose of 3 mg kg(-1) p.o. rac-BHFF (100 mg kg(-1) p.o.) tested alone had no effect on LRR nor on spontaneous locomotor activity, but exhibited anxiolytic-like activity in the SIH model in mice. CONCLUSIONS AND IMPLICATIONS: rac-BHFF derivatives may serve as valuable pharmacological tools to elucidate the pathophysiological roles played by GABAB receptors in the central and peripheral nervous systems.
Subject(s)
Anti-Anxiety Agents/pharmacology , Benzofurans/pharmacology , Receptors, GABA-B/drug effects , Allosteric Regulation/drug effects , Animals , Anti-Anxiety Agents/administration & dosage , Anti-Anxiety Agents/chemistry , Baclofen/adverse effects , Baclofen/pharmacology , Benzofurans/administration & dosage , Benzofurans/chemistry , CHO Cells , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , GABA Agonists/adverse effects , GABA Agonists/pharmacology , GTP-Binding Protein gamma Subunits/metabolism , Humans , Male , Mice , Mice, Inbred DBA , Pyramidal Cells/drug effects , Pyramidal Cells/metabolism , Rats , Rats, Wistar , Receptors, GABA-B/metabolism , Reflex/drug effects , StereoisomerismABSTRACT
1. The aim of this study was to establish the utility of a fluorometric imaging plate reader (FLIPR) assay to assess human adenosine A(2B) receptor function by characterizing its receptor pharmacology and comparing this profile to that obtained using a microphysiometer. 2. FLIPR was used, in conjunction with a Ca(2+)-sensitive dye (Fluo-3-AM), to measure rapid rises in intracellular calcium in a Chinese Hamster Ovary (CHO-K1) cell line stably transfected with both the human A(2B) receptor and a promiscuous G(alpha16) protein. Microphysiometry was used to measure rapid changes in the rate of extracellular acidification in a Human Embryonic Kidney (HEK-293) cell line also stably transfected with human A(2B) receptor. 3. Activation of A(2B) receptors by various ligands caused a concentration-dependent increase in both the intracellular calcium concentration and the extracellular acidification rate in the cells tested, with a similar rank order of potency for agonists: NECA > N(6)-Benzyl NECA > adenosine > or = R-PIA > CPA > S-PIA > CHA > CGS 21680. No comparable effects were observed in the non-transfected control cell lines. 4. The rank order of potency of the agonists examined was the same in all studies, whereas absolute potency and efficacy varied. Thus, all compounds exhibited greater potency in FLIPR than the microphysiometer and the efficacies obtained with CHO-K1 + G(alpha16) + A(2B) cell line and FLIPR were greater than those obtained with HEK-293 + A(2B) cell line in the microphysiometer. 5. ZM-241385 was the most potent of a range of adenosine antagonists tested with a pA(2) of 8.0 in both the FLIPR and microphysiometer assays. 6. In conclusion, the profile of the responses to both A(2B) receptor agonists and antagonists in FLIPR were similar to those obtained by the microphysiometer, although both potency and efficacy values were higher in the FLIPR assay. With this caveat in mind, this study shows that FLIPR coupled with a cell line transfected with both the human A(2B) receptor and a promiscuous G(alpha16) protein provides a useful, high throughput method for the assessment of A(2B) receptor function.
Subject(s)
Aniline Compounds/metabolism , Receptors, Purinergic P1/metabolism , Recombinant Proteins/metabolism , Xanthenes/metabolism , Animals , CHO Cells , Cell Line , Cricetinae , Dose-Response Relationship, Drug , Fluorescent Dyes/metabolism , Fluorometry/methods , Humans , Purinergic P1 Receptor Agonists , Receptor, Adenosine A2B , Receptors, Purinergic P1/genetics , Recombinant Proteins/agonists , Recombinant Proteins/geneticsABSTRACT
Synthesis and evaluation of the activity of new 4-methyl-1,2,3,4,10,10a-hexahydropyrazino[1,2-a]indoles as 5-HT(2C) receptor agonists are described. Appropriately substituted, several analogs displayed selectivity against the other 5-HT(2) receptor subtypes of 1 order of magnitude or more. Selectivity was improved for several compounds versus the lead 1, increasing the therapeutic interest in this series of 5-HT(2C) receptor agonists.