The S-enantiomer of R,S-citalopram, increases inhibitor binding to the human serotonin transporter by an allosteric mechanism. Comparison with other serotonin transporter inhibitors

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Abstract

The interaction of the S- and R-enantiomers (escitalopram and R-citalopram) of citalopram, with high- and low-affinity binding sites in COS-1 cell membranes expressing human SERT (hSERT) were investigated. Escitalopram affinity for hSERT and its 5-HT uptake inhibitory potency was in the nanomolar range and approximately 40-fold more potent than R-citalopram. Escitalopram considerably stabilised the [3H]-escitalopram/SERT complex via an allosteric effect at a low-affinity binding site. The stereoselectivity between escitalopram and R-citalopram was approximately 3:1 for the [3H]-escitalopram/hSERT complex. The combined effect of escitalopram and R-citalopram was additive. Paroxetine and sertraline mainly stabilised the [3H]-paroxetine/hSERT complex. Fluoxetine, duloxetine and venlafaxine have only minor effects. 5-HT stabilised the [125I]-RTI-55, [3H]-MADAM, [3H]-paroxetine, [3H]-fluoxetine and [3H]-venlafaxine/SERT complex to some extent. Thus, escitalopram shows a unique interaction with the hSERT compared with other 5-HT reuptake inhibitors (SSRIs) and, in addition to its 5-HT reuptake inhibitory properties, displays a pronounced effect via an affinity-modulating allosteric site.

Introduction

The selective serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitors (SSRIs) have gained extensive clinical use in treatment of depressive and anxiety disorders. The SSRI, citalopram (R,S-citalopram), is a racemic 1:1 mixture of S(+)- and R(−)-enantiomers (escitalopram and R-citalopram). The 5-HT reuptake inhibitory activity of R,S-citalopram has previously been reported to reside in the S-enantiomer while the R-enantiomer is practically devoid of 5-HT reuptake inhibitory activity (Hyttel et al., 1992). However, recent pharmacological studies have shown that R-citalopram dose-dependently counteracts escitalopram-induced increase in extracellular 5-HT levels in the frontal cortex of freely moving rats (Mørk et al., 2003) and anxiolytic- and antidepressant-like effects of escitalopram in animal models (Sánchez, 2003, Sánchez et al., 2003a, Sánchez et al., 2003b). The interaction is a pharmacodynamic effect most likely taking place at the serotonin transporter (SERT) level, but the exact molecular mechanism is currently unknown.

In vitro binding kinetics using brain neuronal membranes and platelet membranes have demonstrated the existence of affinity-modulating allosteric binding sites on the SERT protein, which modulate the association and/or dissociation rates of SERT ligands. It is a low-affinity binding site and some but not all serotonergic antidepressants exert an allosteric effect via this mechanism, which is not correlated to their affinity for the primary high affinity-binding site and 5-HT reuptake inhibitory potencies (e.g., Plenge et al., 1991). Studies of [3H]-citalopram dissociation in rat brain homogenates and human platelets showed that high concentrations of citalopram in the dissociation buffer stabilises the binding of [3H]-citalopram considerably resulting in a very low off-rate (Plenge et al., 1991, Plenge and Mellerup, 1985). Furthermore, the binding of citalopram to the allosteric binding site may show some degree of stereoselectivity as the dissociation half-life of [3H]-citalopram was increased from 20 min in buffer control to 59 and 44 min, respectively, with 10 μM S- and R-enantiomer, respectively, being added (Plenge and Mellerup, 1997).

The aim of the present study was to investigate if this phenomenon can explain the in vivo inhibitory action of R-citalopram on escitalopram. Firstly, we determined binding affinities at the primary high-affinity binding site and 5-HT uptake inhibitory potencies in COS-1 cell membranes expressing human SERT (hSERT). Secondly, we determined the dissociation rates for hSERT in complex with [3H]-escitalopram in buffer and in presence of various concentrations of escitalopram, R-citalopram and escitalopram+R-citalopram (ratio 1:1, 1:2 and 1:4). Thirdly, we compared the effects of escitalopram, R-citalopram, fluoxetine, paroxetine, sertraline, venlafaxine, duloxetine and 5-HT on the dissociation rate for hSERT in complex with [3H]-S-citalopram, [3H]-paroxetine, [3H]-fluoxetine, [3H]-sertraline, [3H]-venlafaxine, the cocaine analogue, [125I]-RTI-55, or the new positron emission tomography ligand, [3H]-MADAM (Chalon et al., 2002).

Section snippets

Inhibition of [3H]-5-HT uptake and [125I]-RTI-55 binding in COS-1 cells expressing hSERT

These studies were performed in a COS-1 cell line stably transfected with hSERT. Assays were carried out in confluent 96-well plates.

Inhibition of [3H]-5-HT uptake and [125I]-RTI binding in COS-1 cells expressing hSERT

The 5-HT uptake inhibitory potency of escitalopram was in the nanomolar range and approximately twice that of R,S-citalopram and at least 40 times higher than the corresponding values for R-citalopram (Table 2). Similar potency ratios were found for inhibition of [125I]-RTI binding (Table 2).

Dissociation studies

The dissociation rate of [3H]-escitalopram from hSERT deceased with increasing escitalopram concentration in the dissociation buffer (Fig. 1). R-Citalopram produced a similar effect although with less

Discussion

Consistent with previous findings (Hyttel et al., 1992, Owens et al., 2001), escitalopram was found to mediate in vitro 5-HT uptake inhibitory activity of R,S-citalopram (Table 1). R-Citalopram has only weak 5-HT reuptake inhibitory activity, while escitalopram was approximately twice as potent as R,S-citalopram at the primary, high-affinity binding site on the hSERT.

Escitalopram considerably stabilises the binding of [3H]-escitalopram to hSERT. This is consistent with a pronounced

Acknowledgement

Mikael Bols, University of Aarhus, is gratefully acknowledged for synthesis of [3H]-venlafaxine. David J. Simpson, H. Lundbeck, is gratefully acknowledged for suggestions and discussions during the preparation of this manuscript.

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    The pure S-enantiomer is known as escitalopram, while the racemic mixture is referred to as citalopram (Moore et al., 2005). The S-enantiomer is responsible for the high selectivity and affinity for hSERT (Hyttel et al., 1992), while R-citalopram has been shown to be capable of allosterically affecting the dissociation of other hSERT inhibitors (Chen et al., 2005a, 2005b; Plenge et al., 2007). Multiple in vitro experiments (Andersen et al., 2010, 2011; Banala et al., 2013; Barker et al., 1998; Henry et al., 2006; Larsen et al., 2004; Mortensen et al., 2001; Zhang et al., 2010) and docking studies (Andersen et al., 2009b; Jørgensen et al., 2007; Koldsø et al., 2010) have been performed in order to identify how escitalopram binds to hSERT.

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