Inhibition of viral and bacterial trigger-stimulated prostaglandin E2 by a throat lozenge containing flurbiprofen: An in vitro study using a human respiratory epithelial cell line

Abstract

Objectives: Symptoms of sore throat result from oropharyngeal inflammation, for which prostaglandin E₂ is a key mediator. Flurbiprofen is a non-steroidal anti-inflammatory that provides sore throat relief. The preliminary objective of this study was to develop an in vitro model for assessing prostaglandin E₂ stimulation by viral and bacterial triggers. The primary objective was to investigate the effect of diluted flurbiprofen-containing lozenges on prostaglandin E₂ concentrations in stimulated cells.

Methods: Prostaglandin E₂ production was stimulated in three epithelial cell lines (A549, HEp2, and clonetics bronchial/tracheal epithelial) with influenza A virus (4.5 log₁₀ tissue culture infectious dose₅₀/mL), or bacterial lipopolysaccharide (10µ g/mL) and peptidoglycan (3µ g/mL) and incubated overnight. Prostaglandin E₂ levels were assessed by enzyme-linked immunosorbent assay up to 24 h after stimulation. The effect of flurbiprofen 8.75 mg lozenges (diluted to 0.44 mg/mL) on PGE₂ production in stimulated cells was assessed in parallel; prior to viral/LPS/PEP stimulation of cells, 300 μL of test product or control was added and incubated for 30 s, 2 and 5 min (and 10 min for bacterial trigger). Prostaglandin E₂ levels were measured following stimulation.

Results: Viral and lipopolysaccharide/peptidoglycan infection did not consistently stimulate HEp2 cells and bronchial/tracheal epithelial cells to produce prostaglandin E₂. Influenza virus, and lipopolysaccharide/peptidoglycan stimulated high prostaglandin E₂ concentrations in A549: mean prostaglandin E₂ concentration 106.48 pg/mL with viral stimulation vs 33.82 pg/mL for uninfected cells; 83.84 pg/mL with lipopolysaccharide/peptidoglycan vs 71.96 pg/mL for uninfected cells. Flurbiprofen produced significant reductions in virus-stimulated prostaglandin E₂ vs stimulated untreated cells at 2 min (p = 0.03). Flurbiprofen produced significant reductions in lipopolysaccharide/peptidoglycan-stimulated prostaglandin E₂ concentrations from 30 s (p = 0.02), and at 2, 5 and 10 min (all p < 0.005) vs stimulated untreated cells.

Conclusions: A549 cells provide a suitable model for assessment of prostaglandin E₂ stimulation by viral and bacterial triggers. Diluted flurbiprofen-containing lozenges demonstrated rapid anti-inflammatory activity in viral- and lipopolysaccharide/peptidoglycan-stimulated A549 cells.

Keywords: Pharmaceutical analysis; bioassay approaches; development of novel analytical techniques; drug characterisation studies; upper respiratory tract infection.

Figure 1.

Concentrations of PGE₂ (pg/mL; 95% CI)^a in A549 cells after infection with influenza virus or bacterial LPS/PEP compared with controls (unstimulated cells only or citrate buffer). PGE₂: prostaglandin E₂; CI: confidence interval; LPS: lipopolysaccharide; PEP: peptidoglycan. ^aAverage of duplicate tests.

Figure 2.

PGE₂ concentrations (pg/mL; 95% CI)^a for influenza virus- and LPS/PEP-infected A549 cells incubated with diluted flurbiprofen lozenges at 0.5-, 2-, 5- and 10-min^b timepoints. PGE₂: prostaglandin E₂; CI: confidence interval; LPS: lipopolysaccharide; PEP: peptidoglycan. ^aAverage of duplicate tests. ^b10 min timepoint assessed for LPS/PEP-infected cells only. *p = 0.03, vs untreated influenza A virus-infected cells (PGE₂ concentrations 106.48 pg/mL); **p = 0.02, ^†p = 0.002, ^‡p = 0.004, ^§p = 0.003, vs untreated LPS/PEP-infected cells (PGE₂ concentration 83.84 pg/mL).