ABSTRACT
Inhibition of H(+),K(+)-ATPase is accepted as the most effective way of controlling gastric acid secretion. However, current acid suppressant therapy for gastroesophageal reflux disease, using histamine H(2) receptor antagonists and proton pump inhibitors, does not fully meet the needs of all patients because of their mechanism of action. This study sought to characterize the in vitro and in vivo pharmacology of a novel acid pump antagonist, N-(2-Hydroxyethyl)-N,2-dimethyl-8-{[(4R)-5-methyl-3,4-dihydro-2H-chromen-4-yl]amino}imidazo[1,2-a]pyridine-6-carboxamide (PF-03716556), and to compare it with other acid suppressants. Porcine, canine, and human recombinant gastric H(+),K(+)-ATPase activities were measured by ion-leaky and ion-tight assay. The affinities for a range of receptors, ion channels, and enzymes were determined to analyze selectivity profile. Acid secretion in Ghosh-Schild rats and Heidenhain pouch dogs were measured by titrating perfusate and gastric juice samples. PF-03716556 demonstrated 3-fold greater inhibitory activity than 5,6-dimethyl-2-(4-fluorophenylamino)-4-(1-methyl-1,2,3,4-tetrahydroisoquinoline-2-yl)pyrimidine (revaprazan), the only acid pump antagonist that has been available on the market, in ion-tight assay. The compound did not display any species differences, exhibiting highly selective profile including the canine kidney Na(+),K(+)-ATPase. Kinetics experiments revealed that PF-03716556 has a competitive and reversible mode of action. More rapid onset of action than 5-methoxy-2-{[(4-methoxy-3,5-dimethyl-2-pyridyl)methyl]-sulfinyl}-benzimidazole (omeprazole) and 3-fold greater potency than revaprazan were observed in Ghosh-Schild rats and Heidenhain pouch dogs. PF-03716556, a novel acid pump antagonist, could improve upon or even replace current pharmacological treatment for gastroesophageal reflux disease.
Subject(s)
Aminopyridines/therapeutic use , Benzopyrans/therapeutic use , Gastroesophageal Reflux/drug therapy , Proton Pump Inhibitors/therapeutic use , Aminopyridines/pharmacology , Animals , Anti-Ulcer Agents/pharmacology , Anti-Ulcer Agents/therapeutic use , Benzopyrans/pharmacology , Disease Models, Animal , Dogs , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Humans , Omeprazole/pharmacology , Omeprazole/therapeutic use , Proton Pump Inhibitors/pharmacology , Rats , Rats, Sprague-Dawley , Stomach/physiopathology , SwineABSTRACT
GPR35 is a Gi/o- and G16-coupled receptor abundantly expressed in gastrointestinal tissues and immune cells. Kynurenic acid (a tryptophan metabolite and ionotropic glutamate receptor antagonist) and zaprinast (a phosphodiesterase inhibitor) are GPR35 agonists. Here, we show that the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) is also a GPR35 agonist. NPPB activates the GPR35-Gi/o and GPR35-G16 pathways in human embryonic kidney 293 (HEK293) cells and induces intracellular calcium mobilization in a concentration-dependent manner in HEK293 cells coexpressing human, rat or mouse GPR35 and the chimeric G protein G(qi5). These results suggest a novel pharmacological activity of NPPB and will provide useful information to search for more potent and selective GPR35 agonists.
Subject(s)
Nitrobenzoates/pharmacology , Receptors, G-Protein-Coupled/agonists , Animals , Cell Line , Chloride Channels/antagonists & inhibitors , Dose-Response Relationship, Drug , GTP-Binding Protein alpha Subunits, Gi-Go/drug effects , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/drug effects , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Humans , Kidney/cytology , Kidney/metabolism , Mice , Nitrobenzoates/administration & dosage , Purinones/administration & dosage , Purinones/pharmacology , RatsABSTRACT
GPR35, previously an orphan G-protein coupled receptor, is a receptor for kynurenic acid. Here we examine the distribution of GPR35 in the rat dorsal root ganglion (DRG) and the effects of its selective activation. GPR35 was expressed predominantly by small- to medium-diameter neurons of the DRG. Many of these same neurons also expressed the transient receptor potential vanilloid 1 channel, a nociceptive neuronal marker. The GPR35 agonists kynurenic acid and zaprinast inhibited forskolin-stimulated cAMP production by cultured rat DRG neurons. Inhibition required G(i/o) proteins as the effect was completely abolished by pretreatment with pertussis toxin. This is the first study to report the expression and function of GPR35 in rat nociceptive DRG neurons. We propose that GPR35 modulates nociception and that continued study of this receptor will provide additional insight into the role of kynurenic acid in pain perception.
Subject(s)
Ganglia, Spinal/metabolism , Kynurenic Acid/administration & dosage , Posterior Horn Cells/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Cells, Cultured , Ganglia, Spinal/drug effects , Posterior Horn Cells/drug effects , RatsABSTRACT
Activation of the prostaglandin E(2) (PGE(2)) EP(4) receptor, a G-protein-coupled receptor (GPCR), results in increases in intracellular cyclic AMP (cAMP) levels via stimulation of adenylate cyclase. Here we describe the in vitro pharmacological characterization of a novel EP(4) receptor antagonist, CJ-042794 (4-{(1S)-1-[({5-chloro-2-[(4-fluorophenyl)oxy]phenyl}carbonyl)amino]ethyl}benzoic acid). CJ-042794 inhibited [(3)H]-PGE(2) binding to the human EP(4) receptor with a mean pK(i) of 8.5, a binding affinity that was at least 200-fold more selective for the human EP(4) receptor than other human EP receptor subtypes (EP(1), EP(2), and EP(3)). CJ-042794 did not exhibit any remarkable binding to 65 additional proteins, including GPCRs, enzymes, and ion channels, suggesting that CJ-042794 is highly selective for the EP(4) receptor. CJ-042794 competitively inhibited PGE(2)-evoked elevations of intracellular cAMP levels in HEK293 cells overexpressing human EP(4) receptor with a mean pA(2) value of 8.6. PGE(2) inhibited the lipopolysaccharide (LPS)-induced production of tumor necrosis factor alpha (TNFalpha) in human whole blood (HWB); CJ-042794 reversed the inhibitory effects of PGE(2) on LPS-induced TNFalpha production in a concentration-dependent manner. These results suggest that CJ-042794, a novel, potent, and selective EP(4) receptor antagonist, has excellent pharmacological properties that make it a useful tool for exploring the physiological role of EP(4) receptors.
Subject(s)
Analgesics/pharmacology , Benzoates/pharmacology , Dinoprostone/metabolism , Epithelial Cells/drug effects , Receptors, Prostaglandin E/antagonists & inhibitors , Benzamides , Binding Sites , Binding, Competitive , Cell Line , Cyclic AMP/antagonists & inhibitors , Cyclic AMP/metabolism , Dinoprostone/pharmacology , Dose-Response Relationship, Drug , Epithelial Cells/metabolism , Humans , Kidney/embryology , Lipopolysaccharides/pharmacology , Protein Binding , Receptors, Prostaglandin E/metabolism , Receptors, Prostaglandin E, EP4 Subtype , Tumor Necrosis Factor-alpha/bloodABSTRACT
Recent study suggests that the proinflammatory and nociceptive effects of prostaglandin E(2) are mediated by prostanoid receptor subtype EP(4) and prostanoid EP(4) receptor may be a potential target for the treatment of inflammatory pain. Here we describe pharmacological characterization of a novel prostanoid EP(4) receptor antagonist, CJ-042,794 (4-{(1S)-1-[({5-chloro-2-[(4-fluorophenyl) oxy] phenyl} carbonyl) amino] ethyl} benzoic acid) in comparison with piroxicam (non-steroidal anti-inflammatory drug) or rofecoxib (cyclooxygenase-2 inhibitor). CJ-042,794 competitively antagonized cAMP accumulation with a pA(2) value of 8.7 in HEK293 cells overexpressing rat prostanoid EP(4) receptors. Orally administered CJ-042,794 dose-dependently inhibited carrageenan-induced mechanical hyperalgesia with an ED(50) value of 4.7 mg/kg (11 micromol/kg) and its maximal activity was somewhat less effective than that of 10 mg/kg piroxicam (30 micromol/kg p.o.). When CJ-042,794 and rofecoxib were administered to adjuvant-induced arthritis rats on Days 12-22 twice daily, both compounds reversed paw swelling to normal levels. These results suggest that a pharmacological blockade of the prostanoid EP(4) receptor may represent a new therapeutic strategy in signs and symptomatic relief of osteoarthritis and/or rheumatoid arthritis.
Subject(s)
Analgesics/pharmacology , Benzoates/pharmacology , Inflammation/drug therapy , Pain/drug therapy , Receptors, Prostaglandin E/antagonists & inhibitors , Administration, Oral , Analgesics/administration & dosage , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/pharmacology , Benzamides , Benzoates/administration & dosage , Cell Line , Cyclic AMP/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , In Vitro Techniques , Lactones/pharmacology , Male , Pain Measurement , Piroxicam/pharmacology , Rats , Rats, Inbred Lew , Receptors, Prostaglandin E, EP4 Subtype , Sulfones/pharmacologyABSTRACT
We found that zaprinast, a well-known cyclic guanosine monophosphate-specific phosphodiesterase inhibitor, acted as an agonist for a G protein-coupled receptor, GPR35. In our intracellular calcium mobilization assay, zaprinast activated rat GPR35 strongly (geometric mean EC(50) value of 16nM), whereas it activated human GPR35 moderately (geometric mean EC(50) value of 840nM). We also demonstrated that GPR35 acted as a Galpha(i/o)- and Galpha(16)-coupled receptor for zaprinast when heterologously expressed in human embryonic kidney 293 (HEK 293) cells. These findings will facilitate the research on GPR35 and the drug discovery of the GPR35 modulators.
Subject(s)
Phosphodiesterase Inhibitors/pharmacology , Purinones/pharmacology , Receptors, G-Protein-Coupled/agonists , Amino Acid Sequence , Animals , Cells, Cultured , Cloning, Molecular , Gene Expression Profiling , Humans , Mice , Molecular Sequence Data , Phosphodiesterase Inhibitors/chemistry , Purinones/chemistry , Rats , Receptors, G-Protein-Coupled/chemistry , Sequence AlignmentABSTRACT
A new D-glucose-6-phosphate phosphohydrolase (G6Pase) inhibitor, CJ-21,164 (1) was isolated from the fermentation broth of the fungus Chloridium sp. CL48903. The structure was elucidated to be a novel tetramer of the salicylic acid derivatives by spectroscopic analyses. Compound I inhibited G6Pase in rat liver microsomes with an IC50 of 1.6 microM. Glucose output from hepatocytes isolated from rat liver was inhibited when I was present in the incubation medium, consistent with the role of I as a G6Pase inhibitor.
Subject(s)
Benzoates/isolation & purification , Enzyme Inhibitors/isolation & purification , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Animals , Benzoates/chemistry , Benzoates/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Fermentation , Glucose/metabolism , Male , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Molecular Structure , Rats , Rats, Sprague-DawleyABSTRACT
High-throughput screening of microbial extracts using rat hepatic microsomal glucose-6-phosphatase (G6Pase) led us to find thielavin B as a G6Pase inhibitor with inhibition of glucose output from glucagon-stimulated hepatocytes. Further searching for more potent analogs identified 11 new thielavins F-P in addition to the known thielavins A and B from a fungus Chaetomium carinthiacum ATCC 46463. Thielavin G showed the strongest activity as a G6Pase inhibitor (IC50=0.33 microM), while the IC50 of thielavin B was 5.5 microM. According to the structure-activity relationship, including authentic thielavins C, D and 3 partial hydrolysates from thielavins A and B, 3 benzoic acid-units and carboxylic acid functions are essential for G6Pase inhibition.
