129P Brighton
Winter Meeting December 2008 |
Characterisation of the binding kinetics of β2 adrenoceptor agonists
David Sykes, Steven Charlton
Novartis Institutes for Biomedical Research, Horsham, UK
β2 adrenoceptor agonists are important bronchodilators used for the treatment of chronic obstructive pulmonary disease (COPD) and asthma. Clinical data on several β2 agonists show a range of onset and duration of action, summarised in Table 1. One characteristic that may influence both these elements is the binding association and dissociation rates of the agonist and receptor. We have investigated whether the kinetic rate constants of β2 agonists influence the observed onset of action and duration of effect in the clinic.
[3H]-dihydroalprenolol (DHA) was used to label the β2 receptor expressed in CHO cell membranes. Competition kinetic experiments were carried out in the presence of unlabelled β2 agonists as previously described by Dowling & Charlton, 2006. All experiments were run at 37°C in HBSS and used membranes prepared from CHO cells stably expressing the human β2 receptor. Using equilibrium binding assays [3H]-DHA was shown to have a Kd of 0.084 nM. To determine the kinetic parameters of the unlabelled compounds 3 concentrations (30, 10 & 3 x Ki) were employed against a fixed concentration of [3H]-DHA (0.6 nM). The assay was initiated by addition of membranes at specific time points and the plate was harvested at time point 0 using a manual Packard harvester. Data was analysed using GraphPad Prism software to give the kon and koff of the unlabelled compound. The kinetics of 6 β2 agonists were examined using this method, the results of which are shown in table 1.
Table 1. Comparing the kinetic parameters of β2 agonists with their clinical onset and duration of action. Data are expressed as mean ± s.e.mean (n ≥ 3). * from Brookman et al,(2007) and Wegener et al, (1992)
| Compound | Clinical data* | Kinetically –derived parameters | pKi (M) |
| Onset | Duration | kon (μM-1 min-1) | koff (min-1) | pKd (M) |
| Isoprenaline |
- |
- |
24.7 ± 13.9 |
3.06 ± 1.53 |
6.89 ± 0.08 |
6.72 ± 0.15 |
| Salmeterol |
15 min |
<24 h |
4310 ± 1340 |
0.76 ± 0.06 |
9.70 ± 0.20 |
9.21 ± 0.06 |
| Salbutamol |
5 min |
4-6 h |
20.5 ± 10.3 |
4.06 ± 1.19 |
6.65 ± 0.14 |
6.54 ± 0.07 |
| Formoterol |
3 min |
12 h |
215 ± 45 |
3.29 ± 0.79 |
7.83 ± 0.09 |
7.28 ± 0.10 |
| Indacaterol |
5 min |
>24 h |
87.4 ± 21.2 |
3.48 ± 0.42 |
7.37 ± 0.11 |
6.95 ± 0.13 |
| Adrenaline |
- |
- |
3.15 ± 0.61 |
5.12 ± 1.39 |
5.85 ± 0.10 |
5.82 ± 0.05 |
The β2 agonists tested here exhibited similar dissociation kinetics with the exception of salmeterol which had the slowest off-rate. It does not, however, appear that dissociation rate is a key determinant of duration of action in this class of molecules as indacaterol has a longer duration of action but shorter residency receptor time than salmeterol. Modelled kobs values at EC50 concentrations were also similar, so it is unlikely that kinetic parameters determine the slow onset of salmeterol in the clinic.
Brookman LJ, Knowles LJ, Barbier M et al (2007) Current Medical Research and Opinion 23:3113
Dowling MR & Charlton SJ (2006). British Journal of Pharmacology 148, 927-937.
Wegener T, Hedenstrom H and Melander B (1992) Chest 102:535-538
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