Pharmacological characterisation of the LPS challenge human whole blood assay demonstrates EP4 dominance as a target engagement marker
By Hannah Neale, Susan Brown, Kirstie Bennett, Jason Brown, Matt Barnes | Dec 18, 2019
The British Pharmacological Society
15-17 December 2019
Hannah Neale, Scientist, Molecular Pharmacology, attended Pharmacology 2019 organised by the British Pharmacological Society held on 15-17 December in Edinburgh, UK to present on Pharmacological characterisation of the LPS challenge human whole blood assay demonstrates EP4 dominance as a target engagement marker.
Background and purpose
Prostaglandin E2 (PGE2) expression correlates with the progression of many epithelial cancers1 and may play a role in reducing inflammatory cytokine release in the tumour microenvironment. The reversal of PGE2 inhibition of tumour necrosis factor α (TNFα) in lipopolysaccharide (LPS) stimulated human whole blood (LPS-HWB) has been used as a target engagement marker for antagonists of the prostaglandin receptor 4 (EP4)2, with the effect thought to be mediated primarily by monocytes3. However, there is conflicting data as to whether the LPS-HWB assay can also measure an EP2 response 4. The aim of this study was to characterise the PGE2 response in both an immortalised monocytic cell line (THP-1) and in an LPS-HWB assay.
In THP-1 cells, a Gs cAMP assay was performed to manufacturer’s instructions (CisBio); relative expression of prostaglandin receptors was characterised using qRT-PCR. The LPS-HWB was performed following literature methods1 , with antagonists pre-incubated (37˚C at 5% CO2) for 30 mins prior to agonist addition. 4 h after LPS addition (10 ug/mL), TNFα concentration was measured via ELISA following manufacturers conditions (DY210 R&D systems DuoSet). Concentration-response curves were fitted using a sigmoidal four-parameter fit (GraphPad Prism).
In THP-1 cells, EP4 expression was 50-fold higher than EP2 and the selective EP4 agonist, KAG308, was found to elicit a Gs cAMP response (pEC50 8.3±0.1; n=3), which could be dose dependently inhibited by the EP4 antagonist ONO-AE3-208 but not by EP2 antagonist PF-04418948. EP2 agonist AH13205 was found to be inactive at concentrations below 1 uM. In the LPS-HWB assay, PGE2 inhibition of TNFα release (pIC50 9.1±0.36, n=3) exhibited a shallow Hillslope (0.62±0.22) suggesting activity on multiple receptors. PGE2 inhibition of TNFα release could be reversed by the EP4 antagonist ONO-AE3-208 but not by the EP2 antagonist PF-04418948. EP2 agonist AH13205 was found to be inactive in the LPS-HWB assay at concentrations up to 1 uM.
Conclusion and implications
This data suggests in both the THP-1 cells and in the LPS-HWB assay, EP2 agonists and antagonists have little activity, in contrast to published data3. The LPS-HWB assay is an appropriate assay to measure EP4 responses.