The interaction of escitalopram and R-citalopram at the human serotonin transporter investigated in the mouse

Abstract

Rationale Escitalopram is a superior antidepressant to racemic citalopram. It has been hypothesized that binding of R-citalopram to the serotonin transporter (SERT) antagonizes escitalopram binding to and inhibition of the SERT, curtailing the elevation of extracellular 5-hydroxytryptamine (5-HTExt), and antidepressant efficacy. Further, it has been suggested that a putative allosteric binding site is important for binding of escitalopram to the primary, orthosteric, site, and for R-citalopram’s inhibition hereof. Objectives Primary: Investigate at the human (h)SERT, at clinical relevant doses, whether R-citalopram antagonizes escitalopram-induced 5-HTExt elevation. Secondary: Investigate whether abolishing the putative allosteric site affects escitalopram-induced 5-HTExt elevation and/or modulates the effect of R-citalopram. Methods Recombinant technology; in vivo microdialysis; receptor binding; pharmacokinetics; 5-HT sensitive behaviors (tail suspension, marble burying). Results We generated mice expressing either the wild-type human SERT (hSERTWT) or hSERT carrying amino acid substitutions (A505V, L506F, I507L, S574T and I575T) collectively abolishing the putative allosteric site (hSERTALI/VFL+SI/TT). One mg/kg escitalopram yielded clinical relevant plasma levels and brain levels consistent with therapeutic SERT occupancy. Importantly, escitalopram-induced 5-HTExt elevation was not decreased by R-citalopram co-treatment. Further, escitalopram-induced 5-HTExt elevation was not affected by loss of the allosteric site. The behavioral effects of the clinically relevant escitalopram dose were small, tending to be enhanced by R-citalopram co-administration. Conclusions We find no evidence that R-citalopram directly antagonizes escitalopram or that the putative allosteric site is important for hSERT inhibition by escitalopram. Our findings points to mechanisms for R-citalopram antagonism of escitalopram’s antidepressant action other than direct antagonistic binding interactions at the hSERT.

X-ray structures and mechanism of the human serotonin transporter

“…5c) with prominent electron density (>5 σ in F o -F c omit maps) present in this region for crystals soaked with ( S )-citalopram. Interestingly, mutagenesis of residues proximal to the allosteric site has been reported to severely alter allosteric potency 46 yet the physiological role of this site is not well established 47 . Residues of the extracellular gate, Glu494 and Arg104, are located 4.1 and 4.8 Å from the aminopropyl group, while Asp328 is 6.8 Å away.…”

In vivo investigation of escitalopram's allosteric site on the serotonin transporter

“…Mice with either the hSERT protein (hSERT-wt) or mutant allosteric-null hSERT protein with 6 amino acid substitutions: I522V, I553T, M558S, S559N, S574T, I575T (hSERT-mut) were generated via homologous recombination and established on a 129S6/SvEv background as previously described for the hSERT-wt mice and similar, albeit distinct, allosteric-null mutant (Jacobsen et al , 2014). The substitutions on the hSERT-mut protein significantly reduce escitalopram’s in vitro allosteric activity, and it is important to note that this mutation is more effective at eliminating the allosteric site that that of the Jacobsen group as that these mutated amino acids are key to allosteric binding (Neubauer et al , 2006).…”

“…Very recent work using another allosteric-null hSERT knockin mouse investigated the interaction of escitalopram and R -citalopram at the allosteric site. That work concluded that the allosteric site is not a locus of R -citalopram antagonism of escitalopram (Jacobsen et al , 2014). Here the role of escitalopram alone at the allosteric site is investigated to identify if the presence or absence of the allosteric site alone is enough to alter escitalopram’s potency.…”

Pharmacometabolomics informs pharmacogenomics

“…As a follow-up to the initial SSRI pharmacometabolomics-informed pharmacogenomics study that implicated glycine in SSRI response, plasma samples from 290 of the PGRN-AMPS patients were assayed using a targeted liquid chromatography electrochemical coulometric array (LCECA) metabolomics platform which identified and quantified metabolites primarily from the tryptophan, tyrosine and tocopherol pathways, metabolites that included serotonin – a metabolite that is effectively the target for SSRIs which block the reuptake of serotonin by the monoamine transporter encoded by the SLC6A4 gene (Jacobsen et al, 2014, Zhong et al, 2009). Of the 31 metabolites identified by use of this platform, plasma serotonin was the metabolite most highly associated with the SSRI clinical outcomes.…”