Introduction: Internalisation is by its nature kinetic. Hence, application of the standard assumption of equilibrium conditions used by pharmacologists to determine the equilibrium constant (KD) is not obvious for either the ligand-mediated internalisation pathway or other functional assays that occur over the same timeframe as internalisation. However, it is also difficult or impossible to estimate receptor-binding micro-constants from typical functional assays. Hence, there is a need to develop a quasi-equilibrium model that contains the essential features of the full kinetic model. The aim of current the study is to develop a kinetic model of ligand mediated internalisation under quasi-equilibrium assumption .
Methods: A kinetic model of internalisation was developed under an assumption of quasi-equilibrium conditions based on constitutive and ligand-mediated CB1 receptor internalisation data determined utilising quantitative immunocytochemistry protocols. Data were analysed using the nonlinear regression module as implemented in GraphPad Prism (the standard in experimental pharmacology).
Results: The developed model could adequately describe the constitutive and ligand-mediated internalisation of CB1 receptor overtime. The equilibrium constants estimated from non-equilibrium internalisation assay had the same rank order as those estimated from equilibrium competition-displacement radio ligand binding assays.
Discussion: It was demonstrated in our case that with certain assumptions the equilibrium constant can be estimated from non-equilibrium data. A kinetic internalisation model that describes the dynamic change of surface receptor will be valuable for the future work when quantifying of other functional assays that occur over the same timeframe as internalisation.
Mager DE et al (2005). Quasi-equilibrium pharmacokinetic model for drugs exhibiting target-mediated drug disposition. Pharm Res 22: 1589-1596.