The epithelium regulates the duration of action, but not the potency of the histamine H1-receptor antagonist azelastine in guinea-pig isolated trachea

Adam Hart, Kenneth Clark, Malcolm Begg
GlaxoSmithKline, Stevenage, Hertfordshire, United Kingdom

Azelastine is a histamine-H1 receptor antagonist used intranasally in patients for the treatment of allergic-rhinitis (eg hayfever). The aim of this study was to investigate the role of the epithelium in regulating the duration of action of azelastine using a guinea-pig isolated trachea assay.

Briefly, tracheae from male Dunkin-Hartley guinea pigs (0.25-0.6kg) were removed and placed in Kreb’s solution containing (mM): NaCl (113.0), KCl (4.8), CaCl2 (2.5), KH2PO4 (1.2), MgSO4 (1.2), NaHCO3 (25.0), glucose (11.0), the COX inhibitor meclofenamic acid (1μM), the H2-receptor antagonist cimetidine (1μM) and the H3-receptor antagonist clobenpropit (1μM). Tracheal strips, 2-3 cartilage rings wide were suspended in immersion or superfusion tissue baths at an initial tension of 1g and maintained with Krebs at 37°C, gassed with 95% O2/5% CO2.

Histamine evoked a concentration-dependent contraction of the guinea-pig trachea (EC50=1.2±0.2μM, n=9). Under immersion conditions, azelastine (3, 10, 30nM) evoked concentration-dependent rightward shifts of the histamine concentration-response curve. The pA2 of azelastine was determined to be 9.8±0.3 (n=3). Under immersion conditions, using tissues with intact epithelium, a 1hour incubation with azelastine (30nM, n=9) evoked a 50±9 fold rightward shift of the histamine concentration-response curve. Following constant washing (2ml/min) for an 18 hour period no significant recovery was observed, determined as the fold leftward shift of the histamine concentration response curve (0.6±0.3). In tissues where the epithelium was removed, a 1hour incubation with azelastine (30nM, n=5) evoked a 60±10 fold rightward shift of the histamine concentration-response curve. Following constant washing (2ml/min) for an 18 hour period a 78±29 fold recovery was observed. In the superfusion experiments the EC50 of histamine was 1.4±0.2μM with epithelium, and 1.1±0.1μM without, while the pA2 of azelastine at 30nM was 9.6±0.1 with epithelium present and 9.7±0.1 without epithelium. The repeated concentration-response curves over a prolonged period had no effect on histamine potency in vehicle treated tissues.

In conclusion, the pA2 of azelastine was initially determined to be 9.8±0.3, and hence 30nM was used in experiments to assess its duration of action. With intact epithelium azelastine continued to antagonise the effects of histamine following washout suggesting that azelastine has a duration similar to that previously observed in man (Greiff et al., 1997). However, removal of epithelium layer resulted in a complete reversal of antagonism by azelastine following washout suggesting the compound had been entirely removed. Importantly, the removal of the epithelium had no effect on the potency of histamine, nor the degree of antagonism achieved with azelastine, which agrees with previously published observations (Kamikawa, 1993). In conclusion, the epithelial layer in the guinea-pig trachea contributes to the duration of action of the H1-receptor antagonist azelastine

Grieff et al., (1997) Clin. And Exp. Allergy 27:438-444
Kamikawa , K., (1993) Agents Actions 40:135-40