Pharmacological characterisation of adenosine A2a receptor in response to changes in extracellular pH

Carrie Larner, Giles Brown, Edward Savory, Goran Selén, Peter Richardson

1Cambridge Biotechnology Ltd, Cambridge, UK, 2Biovitrum, Stockholm, Sweden

Adenosine is a ubiquitous nucleoside produced in response to cell activation, exercise and disease. Adenosine is upregulated during inflammation and much of its anti-inflammatory effects are mediated via the A2a receptor. Animals lacking this receptor show exaggerated inflammatory reactions whilst selective A2a receptor agonists such as CGS21680 are able to reverse inflammatory damage in multiple animal models. Whilst superficially this provides for an attractive therapeutic target for many inflammatory conditions, agonism of the A2a receptor in the vascular endothelium results in severe side-effects, namely, a rapid catastrophic vasodilation and resulting hypotension. Inflammatory conditions and ischaemia are associated with a reduction in the local tissue pH. Similar changes in pH are not observed within the vasculature, as even minor pH fluctuations would be detrimental leading to rapid cardiovascular (CV) failure, tissue acidosis and death. Therefore, there is a fundamental difference in the environment surrounding A2a receptors in diseased areas compared to those in the vascularture.

A2a receptor agonists that exploit differential pH environments, and selectively activate receptors only in areas of lowered pH, have been identified and characterised. These agonists, as predicted, exhibit anti-inflammatory properties, but are not associated with the detrimental A2a mediated CV side-effects.

The effects of pH on ligand affinity was explored using [3H]CGS21680 equilibrium binding in rat (male Sprague Dawley) striatal membrane preparations over a pH range (pH5.5, pH6.0, pH6.5, pH7.0 and pH7.4 (n=≥4 represented as mean Ki ± s.e.m)). The therapeutic potential of CBT0002 was explored in the Carrageenan (Cgn) model of inflammation where 0.6% Cgn was injected into the rat (male Sprague Dawley) hind paw and paw volume measured over four hours using a plethysmometer. A pH electrode passing through a twenty gauge needle was used to measure paw pH at the four hour time point, five seconds after insertion into the paw, and less than two minutes post mortem. The effects of orally administered A2a receptor agonists on blood pressure and heart rate were measured in conscious, freely moving rats (Sprague Dawley) with implanted telemetry devices.

Binding results show both CGS21680 and CBT0002 demonstrate a pH selective shift in affinity for the A2a receptor, whereby as pH is reduced the affinity of the ligand for the A2a receptor is increased. While CGS21680 shows weak pH selective binding with only a 7.7 fold increase in affinity from pH7.4 (Ki 10 ± 0.25nM) to pH5.5 (Ki 1.3 ± 0.25nM), CBT0002 shows a 40.8 fold increase in binding affinity in these conditions (pH7.4 Ki 6.3 ± 0.88nM, pH5.5 Ki 257.3 ± 70nM) . A reduction in pH was observed in the Cgn induced inflammatory paw (treated paw pH7.12 ± 0.037, untreated paw pH7.44 ± 0.018 (mean ± s.e.m, n=20)). CBT0002 working at low pH was efficacious at reducing Cgn induced paw oedema at doses which did not produce CV side effects (no effect on mean arterial blood pressure or heart rate).

CBT0002 is a characterised example of an agonist which selectively activates A2a receptors only in damaged tissues where the pH is lowered. This novel approach allows pathology-targeted drug activity, minimising detrimental drug side-effects, allowing for the development of safer medicines.