Extent of salmeterol‐mediated reassertion of relaxation in guinea‐pig trachea pretreated with aliphatic side chain structural analogues

1 Salmeterol is a potent, selective and long acting β 2 ‐adrenoceptor agonist. In vitro , salmeterol exerts ‘reassertion’ relaxation of airways smooth muscle. Reassertion relaxation refers to the capacity of salmeterol to cause repeated functional antagonism of induced contraction when airway smooth muscle is intermittently exposed to, then washed free from, β‐adrenoceptor antagonists such as sotalol. The mechanism(s) underlying reassertion relaxation are unknown but may relate to high affinity binding of the long aliphatic side chain of salmeterol to an accessory site, distinct from the agonist recognition site, in or near the β 2 ‐adrenoceptor (exosite binding hypothesis). 2 In order to test the exosite hypothesis, three pure analogues of salmeterol, each exactly preserving the molecular structure of the aliphatic side chain but with zero or low efficacy at the β 2 ‐adrenoceptor were synthesized. The effect of pre‐incubating guinea‐pig tracheal smooth muscle with these analogues on salmeterol‐induced reassertion relaxation was determined. 3 Computer Assisted Molecular Modelling of these molecules revealed that each of them exactly preserved the low energy linear conformation of the aliphatic side chain of salmeterol. Measurement of lipophilicity (octanol: water partition coefficient; log P) and direct partition into synthetic membranes (membrane partition coefficient; K pmem ) showed that all compounds had high affinity for lipids and membranes. In particular the biophysical properties of CGP 59162 (log P 1.89, K pmem 16500) were very similar to salmeterol (log P 1.73, K pmem 16800). 4 Two of the analogues, CGP 54103 and D 2543 (1 μ m ), which are structural mimics of the side chain of salmeterol, differing slightly in their length, did not prevent either the initial relaxation induced by salmeterol (0.1 μ m ) or the reassertion relaxation; however, it was not possible to determine whether either of these molecules occupied the β 2 ‐adrenoceptor. 5 The third analogue, CGP 59162, which has the substituents on the active saligenin head group of salmeterol in transposed positions, itself exerted a weak β 2 ‐adrenoceptor‐mediated relaxation antagonized by ICI 118551 (β 2 ‐selective antagonist) but not CGP 20712 (β 1 ‐selective antagonist) and, at higher concentrations CGP 59162 caused reassertion relaxation suggesting that it may occupy and activate the β 2 ‐adrenoceptor in a manner analogous to salmeterol. 6 CGP 59162, at concentrations up to ten fold molar excess, did not prevent or reduce salmeterol‐induced reassertion relaxation. 7 In conclusion these data are not consistent with the existence of a distinct ‘exosite’ recognising the aliphatic side chain of salmeterol mediating reassertion.