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

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 x 10(5) M(-1).s(-1) and k(off) 1.05 x 10(-5) s(-1) (t(1/2) approximately 18 h). The k(on) for RR-reboxetine was c. 4.3 x 10(5) M(-1).s(-1) and k(off) 4.2 x 10(-3) s(-1) (t(1/2) approximately 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.