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Cannabinoid CB(1) receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB(1) receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB(1) receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB(1) receptors in both binding (K(i)=0.7 nM) and functional assays (K(i)=0.2 nM). The compound has low affinity (K(i)=7600 nM) for human CB(2) receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB(1) receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB(1) receptor competitive antagonist that may further our understanding of the endocannabinoid system., (2010 Elsevier Inc. All rights reserved.)

To determine which residues within the rat somatostatin receptor subtype SSTR2 may be interacting with the lys9 of somatostatin-14 (S-14), mutant SSTR2 receptors were created by mutating asp89 or asp122. [125I Tyr11]S-14 binding to D89A and D89E mutants suggests that asp89 is not directly involved in S-14 binding. Binding studies with the charge switch mutants, asp9S-14, and D122K, suggest that asp122 may be interacting with the lys9 of S-14. [125I Tyr11]asp9S-14 displayed saturable binding to D122K with an affinity comparable to that seen with [125I Tyr11]S-14 and WT SSTR2. These data suggest that the interaction between lys9 of S-14 and the TM3 asp122 of SSTR2 represents one contact site between S-14 and SSTR2.

The rat somatostatin receptor SSTR2 subtype has been cloned and expressed in Chinese Hamster Ovary (CHO) cells. Four different radioligands were used to determine the pharmacological properties of this somatostatin receptor subtype. [125ITyr11]S-14, [125ITyr25]S-28, and cyclo (D-Trp-Lys-Abu-Phe-MeAla-[125ITyr]) displayed comparable affinities for the SSTR2 subtype (approximately 100 pM). The affinity of a fourth radioligand, D-beta Nal-cyclo (Cys-[125ITyr]-DTrp-Lys-Val-Cys)-Thr-H2N, was approximately 10-fold lower (approximately 1000 pM) than the three other radioligands. Competition of [125I]S-14 with either S-14 or S-28 also revealed comparable IC50 values (250 pM). In CHO cells transfected with the SSTR2 cDNA, S-14 inhibited forskolin-stimulated cAMP accumulation by 75% in a dose-dependent fashion (EC50 = 350 pM).

Glucocorticoids and thyroid hormones regulate the expression of G-protein-linked receptors, typified by the beta-adrenergic receptor. The influence of glucocorticoids on the steady-state levels of receptor and receptor mRNA were examined in Chinese hamster ovary (CHO) cells in culture. Incubation of wild-type CHO cells with dexamethasone, a potent glucocorticoid, elevated both receptor number (as measured by radioligand binding) and receptor mRNA levels (as measured by DNA-excess solution hybridization). Both responses were time- and dose-dependent. Stable transfectant CHO clones harboring the hamster beta 2-adrenergic receptor cDNA under the control of the SV40 early promoter were investigated for their ability to respond to glucocorticoid stimulation. The cDNA employed in these studies displays glucocorticoid-response elements (GREs) in the coding and 3'-noncoding regions, but lacks GREs identified in the 5'-noncoding regions of the gene. Transfectant clones expressing 200 (X13), 600 (32E), or 1580 (32A) fmol of receptor/10(6) cells, as compared to wild-type CHO clones expressing 15 fmol/10(6) cells, displayed elevated steady-state levels of receptor mRNA. Incubating the transfectant CHO clones with glucocorticoids, however, failed to enhance the level of receptor or receptor mRNA derived from the expression vector employed in the transfection. These data demonstrate for wild-type CHO cells that glucocorticoids stimulate an increase in receptor mRNA and receptor expression, but that in CHO clones stably transfected with a cDNA lacking specifically the GREs in the 5'-noncoding region of the gene, the glucocorticoid-stimulated increase in beta-adrenergic receptor mRNA is not evident.

The relationship between hormone receptor number and hormone-stimulated cAMP accumulation was probed in CHO cells that were transfected with the cDNA encoding the beta-adrenergic receptor under the control of the SV40 early promoter (expression vector pSV2BAR). CHO cells were cotransfected with pSV2BAR and expression vector pHOMER that directs the expression of a neomycin-resistance gene, and stable transfectants were selected. Clones expressing receptor at levels from 30 (wild-type) to 6000 fmol/mg membrane protein were isolated and further characterized for receptor mRNA content (measured by solution hybridization with a single-stranded cDNA probe), steady-state expression of receptor (measured by immunoblotting and indirect immunofluorescence), and their ability to accumulate intracellular cAMP in response to a beta-adrenergic agonist. Receptor mRNA content and the steady-state level of receptor protein and its expression at the cell surface were found to increase with receptor density as measured by radioligand binding. Over a 200-fold range of receptor expression, CHO transfectants displayed increasing efficacy of agonist-stimulated cAMP accumulation and increasing maximal cAMP accumulation in response to agonist. These data provide for the first time an analysis of the relationship between the density of a G-protein-linked receptor and a receptor-mediated response under conditions where the levels of G-proteins and adenylate cyclase are unaltered.