The affinity, intrinsic activity and selectivity of a structurally novel EP2 receptor agonist at human prostanoid receptors.

BACKGROUND AND PURPOSE: Prostanoid EP2 receptor agonists exhibit several activities including ocular hypotension, tocolysis and anti-inflammatory activity. This report describes the affinity and selectivity of a structurally novel, non-prostanoid EP2 receptor agonist, PGN-9856, and its therapeutic potential. EXPERIMENTAL APPROACH: The pharmacology of a series of non-prostanoid EP2 receptor agonists was determined according to functional and radioligand binding studies, mostly using human recombinant prostanoid receptor transfectants. The selectivity of PGN-9856, as the preferred compound, was subsequently determined by using a diverse variety of non-prostanoid target proteins. The therapeutic potential of PGN-9856 was addressed by determining its activity in relevant primate cell, tissue and disease models. KEY RESULTS: PGN-9856 was a selective and high affinity (pKi ≥ 8.3) ligand at human recombinant EP2 receptors. In addition to high affinity binding, it was a potent and full EP2 receptor agonist with a high level of selectivity at EP1 , EP3 , EP4 , DP, FP, IP and TP receptors. In cells overexpressing human recombinant EP2 receptors, PGN-9856 displayed a potency (pEC50 ≥ 8.5) and a maximal response (increase in cAMP) comparable to that of the endogenous agonist PGE2 . PGN-9856 exhibited no appreciable affinity (up 10 µM) for a range of 53 other receptors, ion channels and enzymes. Finally, PGN-9856 exhibited tocolytic, anti-inflammatory and long-acting ocular hypotensive properties consistent with its potent EP2 receptor agonist properties. CONCLUSIONS AND IMPLICATIONS: PGN-9856 is a potent, selective and efficacious prostanoid EP2 receptor agonist with diverse potential therapeutic applications: tocolytic, anti-inflammatory and notably anti-glaucoma.

A comparison of the expression and metabolizing activities of phase I and II enzymes in freshly isolated human lung parenchymal cells and cryopreserved human hepatocytes.

The pulmonary and hepatic expression and catalytic activities of phase I and II drug-metabolizing enzymes were compared using human lung and liver tissue, and lung parenchymal cells (LPCs) and cryopreserved hepatocytes. Cytochrome P450 gene expression was generally lower in lung than in liver and CYP3A4 expression in lung was negligible. Esterase gene expression was similar in lung and liver. Expression of all sulfotransferase isoforms in lung was similar to or higher than that in liver. Lung tissue expressed low levels of UGT. However, the expression of UGT2A1 in lung was higher than that in liver. There was a range of catalytic activities in LPCs, including cytochrome P450, esterase, and sulfation pathways. Phase I activities were generally less than 10% of those determined in hepatocytes. Rates of ester hydrolysis and sulfation in LPCs were similar to those in hepatocytes. When measurable, glucuronidation in LPCs was present at very low levels, reflecting the gene expression data. The metabolism of salbutamol, formoterol, and budesonide was also investigated. Production of salbutamol-4-O-sulfate and budesonide oleate was observed in LPCs from at least two of three donor preparations studied. Formoterol sulfate and low levels of formoterol glucuronide were detected in one of three donors. In general, drug-metabolizing capability of LPCs is low compared with liver, although some evidence for substantial sulfation and deesterification capacity was observed. Therefore, these data support the use of this cell-based system for the investigation of key routes of xenobiotic metabolism in human lung parenchyma.

Cytochrome P450 activity in control and induced long-term cultures of rat hepatocyte spheroids.

Long-term events such as enzyme induction or chronic toxicity require long-term liver culture models that maintain activity of xenobiotic metabolising enzymes. The levels of these enzyme activities and their responsiveness to chemical induction was studied in rat hepatocyte spheroids, a potential long-term hepatocyte culture model. In comparison with other long-term liver culture models, the basal metabolic activity of spheroids has not been well studied. Additionally, no existing data on the induction of CYP3A activity in spheroids could be found. The basal xenobiotic metabolising activity of rat hepatocyte spheroids was monitored over 14 days in culture, using testosterone as a probe substrate. When spheroids from days 2-14 in culture were compared to 24-h control spheroids, there was a differential maintenance of basal CYP activity. CYP2A and CYP3A activities were maintained over the culture period, while there were time-related decreases in CYP2C11 and CYP2C/CYP2B1/2 activities. The responsiveness of rat hepatocyte spheroids to chemical induction was studied following treatment with phenobarbitone (PB) or dexamethasone (DEX). PB treatment induced CYP2A, CYP2C, CYP2B1/2 and CYP3A activities. DEX treatment resulted in an induction of CYP3A and CYP2C11 activities. The results demonstrate that rat hepatocyte spheroids retained some of the liver-specific functions essential in a long-term hepatocyte culture model, thus making spheroids comparable to other long-term culture models available.