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In vitro characterisation of the duration of action of the Histamine-1 receptor antagonist azelastine Azelastine is a selective antagonist at the human histamine-1 (H1) receptor and is used clinically in the treatment of allergic rhinitis (Greiff et al., 1997). In this study we have investigated its duration of action in vitro in an effort to characterise the receptor and tissue components involved. Chinese hamster ovary cells expressing the human recombinant H1 receptor were used to generate membrane fragments to investigate the nature of antagonism and receptor binding kinetics of azelastine in a filtration radioligand binding assay at 37ºC (Slack et al., 2009). Further duration of action studies were completed in tissue preparations using superfused guinea-pig isolated tracheal strips (Hart et al., 2007) and human isolated bronchial strips (methods adapted from Coleman and Nials, 1989). Azelastine reached equilibrium at the H1 receptor after 41 min with a koff of 0.042 ± 0.009 min-1 (mean ± SEM, n = 4). The koff for azelastine was significantly slower than that for the first generation antihistamine mepyramine (0.817 ± 0.092 min-1, mean ± SEM, n = 4) (p<0.0001, Student’s t-test). In superfused guinea-pig tracheal strips with intact epithelium, 30nM azelastine evoked a 50 ± 9 (mean ± SEM, n = 9) fold rightward shift of the histamine CRC. Constant washing of epithelial intact trachea for 18 h resulted in no recovery of the azelastine antagonism, determined as the fold leftward shift (0.6 ± 0.3, mean ± SEM, n = 9) of the histamine CRC. In tissues where the epithelium was removed, 30nM azelastine evoked a 61 ± 11 (mean ± SEM, n = 5) fold rightward shift of the histamine CRC. In contrast to epithelial intact trachea, constant washing for 18 h resulted in a 78 ± 29 (mean ± SEM, n = 5) fold recovery (leftward shift) of the histamine CRC. In superfused human bronchial strips treated with 30 and 100nM azelastine, ∼3 and 10 fold rightward shifts of the histamine CRC respectively, were observed following a 21 h washout. In summary, azelastine has a moderately slow dissocation half-life (16 min) from the H1 receptor at 37ºC. In contrast, washout studies completed in guinea-pig and human bronchus showed azelastine continued to antagonise the effects of histamine at the H1 receptor for at least 18 h post-washout of the antagonist. This outcome was reversed following removal of the epithelium from guinea-pig isolated tracheal strips. These studies indicate that tissue kinetics are the main contributor to azelastine’s duration of action, with evidence suggesting a key role for the epithelium layer.
Coleman and Nials, (1989), J. Pharmacol. Meth. 21, 71-86. Greiff et al., (1997), Clin. Exp. Allergy 27, 438-444. Hart et al., (2007), Proc. Brit. Pharmacol. Soc. http://www.pa2online.org/abstracts/Vol5Issue2abst078P.pdf Slack et al., (2009), Proc. Brit. Pharmacol. Soc. http://www.pa2online.org/abstracts/Vol7Issue4abst152P.pdf
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