Estimation of binding rate constants using a simultaneous mixed-effects method: Application to monoamine transporter reuptake inhibitor reboxetine

Abstract

Background and purpose: Reboxetine is a clinically used antidepressant and is a racemic mixture of two enantiomers, SS- and RR-reboxetine. The aim of the work described in this manuscript was to determine the kinetics of binding of the RR- and SS-reboxetine to the human noradrenaline transporter (hNET). Experimental approach: We have applied a simultaneous mixed-effects method to the analysis of the transient kinetics of binding of SS-, RR- and racemic reboxetine to hNET. This method allowed simultaneous modelling of multiple datasets, taking into account inter-experiment variability, thereby facilitating robust parameter estimation and minimizing the assumptions made. Key results: The mixed-effects method proved simple and robust. SS-reboxetine bound to hNET according to a one-step binding model with the SS-enantiomer having 130-fold higher steady state affinity than the RR-enantiomer (K d = 0.076 ± 0.009 nM vs. 9.7 ± 0.8 nM respectively). The k on for SS-reboxetine was c. 1.4 × 10 5 M -1·s -1 and k off 1.05 × 10 -5 s -1 (t 1/2 ∼18 h). The k on for RR-reboxetine was c. 4.3 × 10 5 M -1·s -1 and k off 4.2 × 10 -3 s -1 (t 1/2 ∼3 min). The racemate behaved as expected for an equimolar mixture of RR- and SS-reboxetine, assuming mutually exclusive binding. Conclusions and implications: These data will be useful for the interpretation of the behaviour of reboxetine and its enantiomers in man and the method used could be applied to other candidate drugs. © 2010 The British Pharmacological Society.