Identification of potent, nonabsorbable agonists of the calcium-sensing receptor for GI-specific administration.

Modulation of gastrointestinal nutrient sensing pathways provides a promising a new approach for the treatment of metabolic diseases including diabetes and obesity. The calcium-sensing receptor has been identified as a key receptor involved in mineral and amino acid nutrient sensing and thus is an attractive target for modulation in the intestine. Herein we describe the optimization of gastrointestinally restricted calcium-sensing receptor agonists starting from a 3-aminopyrrolidine-containing template leading to the identification of GI-restricted agonist 19 (GSK3004774).

Exploration of phenylpropanoic acids as agonists of the free fatty acid receptor 4 (FFA4): Identification of an orally efficacious FFA4 agonist.

The long chain free fatty acid receptor 4 (FFA4/GPR120) has recently been recognized as lipid sensor playing important roles in nutrient sensing and inflammation and thus holds potential as a therapeutic target for type 2 diabetes and metabolic syndrome. To explore the effects of stimulating this receptor in animal models of metabolic disease, we initiated work to identify agonists with appropriate pharmacokinetic properties to support progression into in vivo studies. Extensive SAR studies of a series of phenylpropanoic acids led to the identification of compound 29, a FFA4 agonist which lowers plasma glucose in two preclinical models of type 2 diabetes.

Identification of nonabsorbable inhibitors of the scavenger receptor-BI (SR-BI) for tissue-specific administration.

The identification of a low-permeability scavenger receptor BI (SR-BI) inhibitor starting from the ITX-5061 template is described. Structure-activity and structure-permeability relationships were assessed for analogs leading to the identification of compound 8 as a potent and nonabsorbable SR-BI inhibitor.

Identification of diarylsulfonamides as agonists of the free fatty acid receptor 4 (FFA4/GPR120).

The exploration of a diarylsulfonamide series of free fatty acid receptor 4 (FFA4/GPR120) agonists is described. This work led to the identification of selective FFA4 agonist 8 (GSK137647A) and selective FFA4 antagonist 39. The in vitro profile of compounds 8 and 39 is presented herein.

Discovery of 3-aryl-4-isoxazolecarboxamides as TGR5 receptor agonists.

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent small molecule agonists of the human TGR5 G-protein coupled receptor is described. Subsequent optimization resulted in the rapid identification of potent exemplars 6 and 7 which demonstrated improved GLP-1 secretion in vivo via an intracolonic dose coadministered with glucose challenge in a canine model. These novel TGR5 receptor agonists are potentially useful therapeutics for metabolic disorders such as type II diabetes and its associated complications.

Functional screening in the melanophore bioassay.

The melanophore bioassay is a robust, sensitive, and versatile procedure for screening G protein-coupled receptors in a variety of formats. Because melanophores contain a wide variety of G proteins, they can be employed as a sensitive, real-time response system for studying transfected receptors and for defining equilibria for drug effects. This assay can be run in 96-well microtiter plates or in open-lawn 1536 format, and can yield conventional agonist-antagonist as well as constitutive assays.

In vitro characterization of a human calcitonin receptor gene polymorphism.

Calcitonin is a 32 amino acid peptide hormone that inhibits bone resorption by stimulating calcitonin receptors (CTR) located on the surfaces of osteoclasts. A polymorphism at nucleotide 1340 of the human calcitonin receptor gene (CALCR) lies within the coding region and has the potential to change the amino acid at codon 447 from leucine to proline. In the present study, we scanned the coding region, portions of the 5'-flanking and 3'-flanking sequences, and the intron-exon boundaries of the human CALCR gene for additional polymorphisms, and determined the frequency of the codon 447 polymorphism in several ethnic groups.

1,4-Benzodiazepine peripheral cholecystokinin (CCK-A) receptor agonists.

A series of 1,4-benzodiazepines, N-1-substituted with an N-isopropyl-N-phenylacetamide moiety, was synthesized and screened for CCK-A agonist activity. In vitro agonist activity on isolated guinea pig gallbladder along with in vivo induction of satiety following intraperitoneal administration in a rat feeding assay was demonstrated.

Use of a cell-based, lawn format assay to rapidly screen a 442,368 bead-based peptide library.

A cell-based, lawn format assay utilizing an in situ photocleavage method has been developed that allows the rapid examination of large bead-based compound libraries as discrete molecules. The format uses frog melanophore cells in a contiguous, adherent, confluent layer in small petri dishes covered with a 0.5-1-mm layer of agarose containing 130 micron diameter TentaGel beads at a density of 2-20 beads/mm2.

Expression cloning and receptor pharmacology of human calcitonin receptors from MCF-7 cells and their relationship to amylin receptors.

Human breast cell carcinoma MCF-7 cells were found to bind 125I-labeled rat amylin (rAmylin) and the peptide amylin antagonist radioligand 125I-AC512 with high affinity. This high affinity binding possessed characteristics unique to the already defined high affinity binding site for amylin in the rat nucleus accumbens [Mol. Pharmacol.

A mathematical model for analysis of pharmacologically induced changes in the kinetics of cardiac muscle.

A mathematical model of the isometric contraction of cardiac muscle is developed and utilized to characterize the inotropic and lusitropic effects of cardioactive compounds in isolated guinea pig left atria. In contrast to metrics that are based on minima and maxima of an isometric twitch and its derivative function, the entire time course of the twitch is used to quantify the kinetics of the contraction-relaxation cycle. The model relates observed tension to a time-dependent activation function that describes generation of internal force and a coupling function that determines mechanical response to the activation function.

3-[2-(N-phenylacetamide)]-1,5-benzodiazepines: orally active, binding selective CCK-A agonists.

A series of modifications were made to the C-3 substituent of the 1,5-benzodiazepine CCK-A agonist 1. Replacement of the inner urea NH and addition of a methyl group to generate a C-3 quaternary carbon resulted in acetamide 6, which showed CCK-A receptor binding selectivity and sub-micromolar agonist activity in vitro. Benzodiazepine 6 was active in an in vivo mouse gallbladder emptying assay and represents a novel orally active, binding selective CCK-A agonist.

Structure activity relationships of a series of buspirone analogs at alpha-1 adrenoceptors: further evidence that rat aorta alpha-1 adrenoceptors are of the alpha-1D-subtype.

The activity of a series of busprione analogs at recombinant and rat thoracic aorta alpha-1 adrenoceptors was investigated. Compound affinity for recombinant alpha-1A, alpha-1B and alpha-1D adrenoceptors from human and animal sources was determined by radioligand binding assays using membranes prepared from rat-1 fibroblasts expressing recombinant receptors with ( +/- )-[125l]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone as the radioligand. Compound affinity and functional activity at rat aortic alpha-1 adrenoceptors were determined using endothelium denuded rings contracted with phenylephrine.

Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity. 1. Optimization of the agonist "trigger".

Directed screening of compounds selected from the Glaxo registry file for contractile activity on the isolated guinea pig gallbladder (GPGB) identified a series of 1,5-benzodiazepines with peripheral cholecystokinin (CCK) receptor agonist activity. Agonist efficacy within this series was modulated by variation of substituents on the N1-anilinoacetamide moiety. Remarkably, a single methyl group confers agonist activity, with an N-isopropyl substituent providing optimal efficacy.

Use of resampling techniques to estimate the variance of parameters in pharmacological assays when experimental protocols preclude independent replication: an example using Schild regressions.

Estimates of variance in pharmacological assays are usually made by repeating the experiment with different tissues. Biological factors, such as the inability to wash a drug from tissue, may preclude the type of replication that is appropriate for the statistics of interest. For example, in Schild regressions, replication is usually done at each concentration of antagonist.

Secretion of cyclic GMP by cultured epithelial and fibroblast cell lines in response to nitric oxide.

LLC-PK1 epithelial cells and RFL-6 fibroblasts secreted both cyclic AMP (cAMP) and cyclic GMP (cGMP) when costimulated with forskolin and 3-morpholinosydnonimine (a chemical nitric oxide generator). Intracellular cAMP levels as high as 1100 and 12,000 pmol/10(6) cells were achieved for the two cell types, respectively. These levels were high enough to reach approximately 50% saturation of the cAMP transporter and inhibited transport of cGMP to an equal extent, suggesting that the two cyclic nucleotides compete for a common transport system.

BMY 7378 is a selective antagonist of the D subtype of alpha 1-adrenoceptors.

BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8- azaspiro[4.5]decane-7,9-dione dihydrochloride), a 5-HT1A receptor partial agonist, also binds to alpha 1-adrenoceptors. Competition assays were performed using (+/-)-beta-([125I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone ([125I]HEAT), and membranes prepared from Rat-1 fibroblasts expressing hamster alpha 1b-, bovine alpha 1c-, or rat alpha 1d-adrenoceptor, or their respective human homologues.

CCK-A receptor selective antagonists derived from the CCK-A receptor selective tetrapeptide agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (A-71623).

Analogs of the CCK-A receptor selective agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (A-71623) were prepared in which the lysine residue was replaced with L-4-aminophenylalanine and D-or L-3-aminophenylalanine. These new analogs were moderately potent antagonists of CCK-8 in the isolated guinea pig gallbladder with exceptional CCK-A receptor selectivity as evaluated in membrane preparations from CHO K1 cells stably transfected with human CCK-A and CCK-B receptors.

Characterization of alpha-1 adrenoceptor subtypes in human and canine prostate membranes.

Experiments were designed to characterize the predominant subtype of alpha-1 adrenoceptors in human and canine prostate tissue. The chemical (+/-)-beta-([125I]iodo-4-hydroxyphenyl)-ethyl- aminomethyl-tetralone bound in a specific, saturable manner to a single class of binding sites in membranes that expressed recombinant hamster alpha-1B, bovine alpha-1C and rat alpha-1D receptors expressed in rat-1 fibroblasts and to those from prostate tissue. Competition assays with human and canine prostate membranes revealed only a single class of binding sites.

Rat peritoneal neutrophils selectively relax vascular smooth muscle.

A vascular relaxing factor from oyster glycogen-elicited rat peritoneal neutrophils has been shown previously to possess a pharmacologic profile similar to that described for endothelium-derived-relaxing factor. The present experiments were designed to determine the in vitro tissue and species selectivity effects of the neutrophil-derived nitric oxide. Neutrophils (1 x 10(5) to 1 x 10(8) cells/10-ml organ chamber) were added to organ chambers filled with a physiological salt solution (37 degrees C; pH 7.

Neutrophil-derived relaxing factor relaxes vascular smooth muscle through a cGMP-mediated mechanism.

Neutrophils harvested from the peritoneal cavities of rats have been shown to release a factor that relaxes precontracted aorta and has a pharmacologic profile similar to that previously reported for endothelium-derived relaxing factor (EDRF). The present study was designed to determine if this neutrophil-derived relaxing factor (NDRF) relaxes rat aortic smooth muscle by affecting the intracellular cGMP levels. Aortic sheets (endothelium removed) were incubated in organ chambers in a physiological salt solution containing phenylephrine (1 x 10(-7) M) and superoxide dismutase (10 or 100 U/ml).

Potential regulatory role of inflammatory cells on local vascular smooth muscle tone.

A neutrophil-derived relaxing factor (NDRF) from oyster glycogen (OG)-elicited rat PMN, which causes an endothelium independent relaxation of rat aorta, and which is pharmacologically indistinguishable from endothelium-derived relaxing factor (EDRF) has been described. Experiments were designed to evaluate the presence of NDRF in PMN from rat -whole blood, -carrageenan pleurisy, -OG peritonitis, and guinea pig (GP) -OG peritonitis, as well as in OG-elicited rat macrophages (M phi). Significant vascular relaxing activity was found using rat PMN from OG peritonitis and carrageenan pleurisy, as well as from OG-M phi.

Structure-activity relationships of 1H-indole-7-carboxamides as a novel series of selective alpha 1-adrenoceptor agonists.

The present isolated tissue study was designed to quantitate the alpha-adrenoceptor agonist activity of AY- 30,191 (5-(1-hydroxy-2-amino-ethyl)-1H-indole-7-carboxamide) and a series of related compounds. AY-30,191 induced contractions in the rabbit aorta, which were blocked by prazosin. In the rat vas deferens, while clonidine inhibited the electrically induced twitch response, AY-30,191 caused a prazosin-sensitive augmentation.

Comparison of the beta-adrenoceptor affinity and selectivity of cetamolol, atenolol, betaxolol, and ICI-118,551.

The objective of the present study was to compare the quantitative differences in the beta 1- vs. beta 2-adrenoceptor affinity and selectivity of cetamolol and its enantiomers to the reference compounds atenolol, betaxolol, and ICI-118,551, using isolated tissues obtained from the dog, guinea pig, and rat. Cetamolol antagonized the beta-adrenoceptor-mediated responses induced by isoproterenol, epinephrine, norepinephrine, and salbutamol, in tissues from both the dog and guinea pig, in a concentration-dependent manner.