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


METHODS
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.


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
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.
Benson
Neil
N
Pfizer Global Research & Development, Department of Pharmacokinetics Dynamics and Metabolism, Modelling & Simulation, Kent, UK. neil.benson@pfizer.com <neil.benson@pfizer.com>
Snelder
Nelleke
N
Ploeger
Bart
B
Napier
Carolyn
C
Sale
Harriet
H
Birdsall
Nigel J M
NJ
Butt
Richard P
RP
van der Graaf
Piet H
PH
D018759:Adrenergic Uptake Inhibitors / Q000378:metabolism*; D000928:Antidepressive Agents / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D008954:Models, Biological; D015233:Models, Statistical; D009025:Morpholines / Q000378:metabolism*; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000378:metabolism*; D011485:Protein Binding; D000077593:Reboxetine; D013237:Stereoisomerism
D018759:Adrenergic Uptake Inhibitors; D000928:Antidepressive Agents; D009025:Morpholines; D050484:Norepinephrine Plasma Membrane Transport Proteins; D000077593:Reboxetine