A previous study has reported that the β-adrenoceptor antagonist ICI 118551 elicits a depression in the maximum cAMP response to isoprenaline (Hopkinson et al., 2000) in a cell-based functional assay. This observation was hypothesized to be the result of slow dissociation of ICI 118551 from the receptor. The aim of the present study was to test this hypothesis by determining apparent off-rates for ICI 118551 and a range of other β-adrenoceptor antagonists. Additionally this data would provide insight into whether a relationship exists between the level of depression observed in a functional assay and the off-rate of the antagonist.

To determine the % depression in maximal cAMP production A431 cells, which endogenously express β₂ -adrenoceptors (350 fmol mg-1 protein), were pre-incubated with antagonist (10xK_i) for 2 hours. Formoterol (1µM) was then added and incubated for a further 1 hour, before the cells were lysed and the levels of cAMP detected using an AlphaScreen^TM  assay. Apparent off-rates were measured by radioligand binding, using membranes prepared from Sf9 insect cells over-expressing the human β₂-adrenoceptor (1.5 pmol mg-1 protein), pre-incubated with antagonist (10xK_i) for 2 hours, followed by the addition of a saturating concentration of [³H]-propranolol (100xK_D). At time points varying between 2 minutes and 24 hours, the membranes were filtered onto Whatman GF-C filter plates and bound [³H]-propranolol was measured.

Table 1: Depression of cAMP response and apparent off-rates for a range of β-adrenoceptor antagonists. Data expressed as mean ± s.e.m (n given in parentheses). ⁺ = % total binding.

The data summarised in Table 1 show that ICI 118551 and timolol have slower off-rates than propranolol and alprenolol, and that the off-rates broadly correlate to the level of depression observed in the functional assay. Interestingly, using this methodology CGP12177A could not be fully displaced by [³H]-propranolol, unless much higher competing concentrations (500xK_D) were employed. In conclusion, ICI 118551 and timolol have slow apparent off-rates, whilst as expected BAAM (Bromoacetyl alprenolol menthane) binds irreversibly (Liptak et al., 1985). These data also suggest that the functional assay described can be employed to provide qualitative comparisons of compound off-rates.

Hopkinson, H.E. et al., (2000) Br J Pharmacol. 131,124-130.
Liptak, A. et al., (1985) J Med Chem. 28, 1699-1703.