Theophylline Inhibits Cough In A Pre-Clinical Model By Decreasing The Excitatory Threshold Of Airway Sensory Nerves

Eric D. Dubuis, Michael Wortley, Megan Grace, Sarah A. Maher, Mark A. Birrell, Maria G. Belvisi. Imperial College London, London, UK

Cough is an important protective mechanism in the airway, however excessive coughing is the most common reason people visit their GP and/or go to A&E (Armon et al., 2001; Sands et al., 2011). Coughing is often a symptom of respiratory diseases, but can also be idiopathic in nature, and currently there are no effective treatments that have acceptable safety profiles (Nasra et al., 2009). We have previously shown that a methylxanthine derivate, theobromine (TB), reduced capsaicin-induced isolated sensory nerve depolarisation and cough in guinea pigs and man (Usmani et al., 2005). Our hypothesis is therefore that compounds of this class may possess useful anti-tussive properties; however their mechanism of action is currently unknown.

The aim of this study is to use 2 methylxanthine derivatives, TB and theophylline (TP), in a range of in vitro and in vivo assays, to determine the mechanism by which they suppress sensory nerve activation and therefore cough.

To demonstrate that the anti-tussive properties of TB were shared by TP, male Dunkin–Hartley guinea pigs (300-400g) were treated with vehicle (0.5% methylcellulose, 0.2% Tween80 v/v in saline, 10 ml/kg, i.p.) or increasing doses of TP one hour prior to challenge with aerosolised capsaicin (30μM for 5 minutes). The numbers of coughs were counted by a trained observer (blinded to treatment received) for 10 minutes. TP dose-dependently inhibited capsaicin-evoked in vivo cough, down to 1.5±0.4 coughs at 100mg.kg-1 compared to 10.3±2.4 coughs in the vehicle control (Figure 1).

Using a preparation of isolated vagus nerve, dissected from schedule one culled male guinea pigs (as described in Usmani et al, 2005), at 37°C we demonstrated that pre-incubation with either TB or TP for 10 minutes blocked sensory nerve depolarisation elicited by 1 µM capsaicin in a concentration-dependent manner (non cumulative dose response, vehicle 0.1%v/v DMSO) (Figure 2). Furthermore, a selected sub-maximal concentration (10 µM) of TB or TP inhibited (40-80%) sensory nerve depolarisation induced by a wider range of cough inducers: bradykinin, acrolein, pH5, resiniferatoxin, and prostaglandin E2 (Figure 3).

We next began to probe the mechanisms by which the methylxanthine compounds exert their inhibitory effect by utilising several potassium channel blockers. Pre-incubation (10min) with apamin and clotrimazole, blockers of low-conductance-calcium-activated potassium channels (SK) and intermediate-conductance-calcium-activated potassium channels (IKCa) respectively, were found to abrogate the inhibitory effect of TP (10µM) on capsaicin-induced sensory nerve depolarisation (Figure 4).

These data suggest that TP activates both SKCa and IKCa channels, and the resulting outward potassium current blocks the capsaicin-induced depolarisation of sensory nerves.

Although the mechanism by which TP activates SKCa and IKCa remains to be elucidated, this study provides a better understanding of how methylxanthines can block sensory nerve depolarisation for such a wide range of agonists. In addition, it further highlights that this class of compound could be a truly effective treatment against cough.

Armon, K., Stephenson, T., Gabriel, V. et al. (2001). Arch Dis Child, 84, 390-2.

Nasra, J. & Belvisi, M.G. (2009). Pharmacol Ther, 124, 354-75.

Sands, R., Shanmugavadivel, D., Stephenson, T. et al. (2011). Emerg Med J, Advanced online.

Usmani, O.S., Belvisi, M.G., Patel, H.J. et al. (2005). Faseb J, 19, 231-3.