Antagonism of the prostaglandin D2 receptor CRTH2 attenuates asthma pathology in mouse eosinophilic airway inflammation

Abstract

Background: Mast cell-derived prostaglandin D2 (PGD2), may contribute to eosinophilic inflammation and mucus production in allergic asthma. Chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2), a high affinity receptor for prostaglandin D2, mediates trafficking of TH2-cells, mast cells, and eosinophils to inflammatory sites, and has recently attracted interest as target for treatment of allergic airway diseases. The present study involving mice explores the specificity of CRTH2 antagonism of TM30089, which is structurally closely related to the dual TP/CRTH2 antagonist ramatroban, and compares the ability of ramatroban and TM30089 to inhibit asthma-like pathology.

Methods: Affinity for and antagonistic potency of TM30089 on many mouse receptors including thromboxane A2 receptor mTP, CRTH2 receptor, and selected anaphylatoxin and chemokines receptors were determined in recombinant expression systems in vitro. In vivo effects of TM30089 and ramatroban on tissue eosinophilia and mucus cell histopathology were examined in a mouse asthma model.

Results: TM30089, displayed high selectivity for and antagonistic potency on mouse CRTH2 but lacked affinity to TP and many other receptors including the related anaphylatoxin C3a and C5a receptors, selected chemokine receptors and the cyclooxygenase isoforms 1 and 2 which are all recognized players in allergic diseases. Furthermore, TM30089 and ramatroban, the latter used as a reference herein, similarly inhibited asthma pathology in vivo by reducing peribronchial eosinophilia and mucus cell hyperplasia.

Conclusion: This is the first report to demonstrate anti-allergic efficacy in vivo of a highly selective small molecule CRTH2 antagonist. Our data suggest that CRTH2 antagonism alone is effective in mouse allergic airway inflammation even to the extent that this mechanism can explain the efficacy of ramatroban.

Figure 1

In vitro characterization of compounds on mouse CRTH2 (mCRTH2) and mouse TP (mTP) receptors. A, Competition binding analysis. B, inhibition of mCRTH2 and mTP receptor function. PGD₂- or U-46619-concentration response curves in the absence and presence of the indicated compounds in mCRTH2- or mTP-expressing cells. Inserts: Schild plots. Schild analysis show potent antagonism of mCRTH2 by TM30089 (pA₂ = 9.15 ± 0.11, Schild slope = 1.45 ± 0.08) whereas it does not interfere with signaling of mTP. Ramatroban is a potent antagonist on mCRTH2 (pA₂ = 8.08 ± 0.14, schild slope = 0.94 ± 0.05) and mTP (pA₂ = 9.36 ± 0.10, Schild slope = 1.35 ± 0.06). Experiments show mean ± SE from 5–7 independent experiments.

Figure 2

CRTH2 antagonism in vivo attenuates airway tissue eosinophilia and mucus cell hyperplasia. Above (A): OVA challenge produced marked eosinophilia and mucus cell hyperplasia (***, p < 0.001 compared to saline control). Treatment with ramatroban or the specific CRTH2 antagonist TM30089 significantly reduced airway tissue eosinophilia (black bars) and mucus production (white bars); ^#, p < 0.05 compared to vehicle treatment. Below (B-G): Light micrographs showing effects in particularly well-responding animals. Lung tissue eosinophilia in normal saline treated lung (B), OVA/vehicle treated lung (C), and OVA/TM30089 treated lung (D). Airway mucus cells are shown in normal saline treated lung (E), OVA/vehicle treated lung (F), and OVA/TM30089 treated lung (G). Scale bar = 100 μm.