KINETICS OF REBOXETINE AT THE [3H]NISOXETINE BINDING SITE IN RECOMBINANT CELL MEMBRANES

Filippo Andreetta1, Keri Hildick2, John S. Kelly2, Michele Negri1, Elisa Simeoni1, Paul Wren1
1GlaxoSmithKline, Psychiatry Centre of Excellence for Drug Discovery, Verona, Italy, 2Division of Neuroscience, University of Edinburgh, Edinburgh, United Kingdom

Reboxetine is a selective noradrenaline reuptake inhibitor marketed for the treatment of major depression (Hajos et al., 2004). However, the occupancy of the noradrenaline transporter (NET) that is required for efficacy of the drug is poorly understood due in part to the lack of suitable PET ligands (Ding et al., 2006) and information regarding the kinetics of reboxetine binding to the transporter. Methodologies to determine the binding kinetics of unlabelled compounds have recently been validated for the serotonin transporter (Andreetta et al., 2006) using a method decribed by Motulsky & Mahan (1984). The aim of the present study was therefore to use a similar approach to investigate the kinetics of binding of reboxetine to recombinant human NET stably expressed in LLCPK cells. Experiments were conducted with [3H]nisoxetine, unlabelled nisoxetine, and reboxetine.

The association and dissociation rates of unlabelled nisoxetine were faster than those of the radiolabel (Table 1, p<0.001, one way ANOVA). Despite this, pKi values derived from the association and dissociation rates for unlabelled nisoxetine and reboxetine were in good agreement with competition binding data (Table 1). Reboxetine was found to associate and dissociate much more slowly than nisoxetine, despite the two compounds having similar affinities (Table 1).

Data shows the mean ± s.e.m of at least 3 independent experiments at 24°C. * = pKD value determined from Scatchard analysis of saturation experiments. Kinetic parameters for the unlabelled compounds were determined using the Motulsky & Mahan (1984) method.

The displacement of [3H]nisoxetine binding has been shown to be sensitive to temperature in a compound-specific manner (Reith et al., 2005). In the present study, we found that association rates increased for both reboxetine and nisoxetine at 4°C; the dissociation rate for reboxetine also increased, but was decreased for nisoxetine. Affinities derived from these rates did not match those derived from competition experiments conducted at 4°C (reboxetine pKi = 7.80 ± 0.02; nisoxetine pKi = 8.48 ± 0.02).

These studies have shown that the Motulsky & Mahan approach can be applied to human NET to investigate the binding kinetics of unlabelled ligands at 24°C, and that the apparent affinities derived from these studies are in line with standard competition binding studies. Divergent results at 4°C, suggest that reboxetine and nisoxetine may bind to distinct, but overlapping differentially temperature sensitive NET binding sites. The slower binding kinetics of reboxetine compared to nisoxetine, despite similar apparent affinities, may be an important factor in determining the efficacy of the drug in vivo. Further studies with clinically active NET inhibitors are warranted.

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