Development of a native translatable assay for the EP4 prostanoid receptor

S Pritchard, J Rhee, M Swarbrick, G Gerrard, A Medhurst, M Kay

Glaxo SmithKline, Harlow Essex, UK

Human native tissue assays are important tools in determining activity of compounds at receptors and are increasingly recognised as a prime decision making tool in drug development, for example in providing important information for predicted efficacy and dose selection in clinical studies.

The prostanoid EP4 receptor is expressed on human blood cells including monocytes and neutrophils (Meja et al ,1997; Mita et al, 2002) and ligands at this receptor have been reported to demonstrate anti-inflammatory activity (Kabashima et al, 2002). In this study, we have identified and validated a native tissue assay for EP4 receptor activation using human whole blood based on inhibition of lipopolysaccharide (LPS)-induced cytokine/chemokine formation.

In whole blood, LPS (0.01 pg/ml - 10 ug/ml) stimulated a concentration-dependent increase in the production of cytokines and chemokines which could be measured in the plasma using the MSD multi-cytokine analysis system. The time-course of cytokine/chemokine release was studied using a submaximal LPS concentration (100 ng/ml) and sampling at 0-48 hrs post stimulation in order to determine optimal assay conditions. The effects of the non-selective prostanoid agonist PGE2 and selective EP4 agonists (compounds A-C) were investigated using a 2 hr preincubation with compound and the optimal conditions of a 24 hr stimulation with 100 ng/ml LPS. PGE2 and selective EP4 agonists inhibited LPS-induced TNF-alpha, interferon-gamma and IP-10 production in whole blood.

Finally in order to validate this system fully as being EP4 selective, the effects of cmpd C were evaluated in the presence of a selective EP4 antagonist, CJ-023,423, and a reversal of inhibition was seen. Data are the mean+ S.E.M of 4 experiments.

This study has identified a suitable native tissue assay for measuring EP4 prostanoid receptor activity in human blood that may be applied to the clinic

Meja KK, Barnes PJ, Giembycz MA (1997) British Journal of Pharmacology 122:149-157

Mita H, Hasegawa M, Higashi N, Akiyama K (2002). Journal of Allergy and Clinical Immunology 110:457-459

Kabashima K, Saji T, Murata T, Nagamachi M, Matsuoka T, Segi E (2002) Journal of Clinical Investigation 109:888-893