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

Abstract

Rationale: Escitalopram appears to be 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, there by curtailing the elevation of extracellular 5-hydroxytryptamine (5-HTExt), and hence anti-depressant 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 here of.

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 generation of hSERT transgenic mice; in vivo microdialysis; SERT binding; pharmacokinetics; 5-HT sensitive behaviors (tail suspension, marble burying).

Results: We generated mice expressing either the wild-type human SERT (hSERT(WT)) or hSERT carrying amino acid substitutions (A505V, L506F, I507L, S574T and I575T) collectively abolishing the putative allosteric site (hSERT(ALI/VFL+SI/TT)). One mg/kg escitalopram yielded clinical relevant plasma levels and brain levels consistent with therapeutic SERT occupancy. The hSERT mice showed normal basal 5-HTExt levels. Escitalopram-induced 5-HTExt elevation was not decreased by R-citalopram co-treatment and was unaffected by loss of the allosteric site. The behavioral effects of the clinically relevant escitalopram dose were small and tended 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.

Figure 1

Characterization of hSERT mice. (A, upper gel) Amplification of cDNA using primers recognizing both human and mouse SERT cDNA reveals brain expression of SERT mRNA in all genotypes. (A, lower gel) Restriction digests of the amplified fragments using Mfe1, which cleaves human, but not mouse, SERT cDNA confirm that mSERT is not present in hSERT^WT or hSERT^{ALI/VFL+SI/TT} mice. (B, upper gel). Amplification of cDNA using primers recognizing both human and mouse SERT cDNA reveals brain expression of SERT mRNA in all genotypes. (B, lower gel) Restriction digests of the amplified fragments using Afl2, which cleaves mouse, but not human, cDNA confirm that mSERT is not present in hSERT^WT or hSERT^{ALI/VFL+SI/TT} mice. (C) [³H]-escitalopram autoradiography reveals generally similar macroscopic anatomical distribution of SERT protein in mSERT, hSERT^WT or hSERT^{ALI/VFL+SI/TT} mice. Binding concentrations used of [³H]-escitalopram corresponding to K_D, as determined in (D), for the three types of SERT were used as indicated. (D) [³H]-escitalopram saturation binding (n = 3). (D, left) SERT protein levels as deduced from computed B_Max. (D, right) K_D (n = 3). (E) Frontal cortex tissue levels of 5-HT and 5-HIAA (n = 7). *, p < 0.05, vs. mSERT. ^#, p < 0.05 hSERT^WT vs. hSERT^{ALI/VFL+SI/TT}. (One-way ANOVA, Bonferroni’s post-hoc test).

Figure 2

Escitalopram dissociation binding. (A) Representative time-concentration plot of retardation of [³H]-escitalopram dissociation from hSERT^WT in neuronal membranes by increasing doses of unlabeled escitalopram. (B) Representative time-concentration plot of retardation of [³H]-escitalopram dissociation from hSERT^{ALI/VFL+SI/TT} in neuronal membranes by increasing doses of unlabeled escitalopram. (C) Retardation of [³H]-escitalopram dissociation from hSERT^WT in neuronal membranes by increasing doses of unlabeled escitalopram (n = 3). (D) Retardation of [³H]-escitalopram dissociation from hSERT^{ALI/VFL+SI/TT} in neuronal membranes by increasing doses of unlabeled escitalopram (n = 3). (E) Representative time-concentration plot of retardation of [³H]-escitalopram dissociation from hSERT^WT in neuronal membranes by increasing doses of unlabeled R-escitalopram added to 3 μM unlabeled escitalopram. (F) Retardation of [³H]-escitalopram dissociation from hSERT^WT in neuronal membranes by increasing doses of unlabeled R-escitalopram added to 3 μM unlabeled escitalopram (n = 3). *, p < 0.05 vs. control. ˆ, p < 0.05 vs. R-cit 0 μM (blue). (One-way ANONA, Dunnett’s post-hoc test).

Figure 3

Escitalopram plasma and brain pharmacokinetics. (A) Time-course of escitalopram plasma levels following acute dosing. Gray-shading delimits the concentration range encountered in the clinic (n = 3–5). (B) Maximal plasma escitalopram concentrations (at T = 30 min, all cases). (C) Time-course of escitalopram brain levels following acute dosing. (D) Time-course of escitalopram brain:plasma ratios following acute dosing. *, p < 0.05, 0.25 mg/kg vs. all other doses. (Two-way ANOVA, Bonferroni’s post-hoc test).

Figure 4

Frontal cortex 5-HT_Ext microdialysis. (A) Effect of escitalopram (1 mg/kg) in hSERT^WT and hSERT^{ALI/VFL+SI/TT} mice (n = 10). (B) Effect of escitalopram (0.5 mg/kg) in hSERT^WT and hSERT^{ALI/VFL+SI/TT} mice (n = 14). (C) Effect of addition of R-citalopram (2 mg/kg) to escitalopram (1mg/kg) in hSERT^WT mice (n = 12–16; saline n = 6). (D) Effect of addition of R-citalopram (2 mg/kg) to escitalopram (1mg/kg) in hSERT^{ALI/VFL+SI/TT} mice (n = 8–11; saline n = 7). (E) Effect of R-citalopram (2 mg/kg) alone in hSERT^WT mice (n = 4–6). (F) Effect of R-citalopram (2 mg/kg) alone in hSERT^{ALI/VFL+SI/TT} mice (n = 6–7). (G) Effect of R-citalopram (4 mg/kg) alone in hSERT^WT mice (n = 7). (H) Effect of R-citalopram (4 mg/kg) alone in hSERT^{ALI/VFL+SI/TT} mice (n = 6–12). ^#, p < 0.05, hSERT^WT vs. hSERT^{ALI/VFL+SI/TT}. *, p < 0.05, R-citalopram vs. no R-citalopram. (Two-way RM-ANOVA, Bonferroni’s post-hoc test).

Figure 5

5-HT_Ext sensitive behaviors. (A) Effect of escitalopram (1 mg/kg) alone or in combination with R-citalopram (2 mg/kg) on immobility in the tail suspension test in hSERT^WT and hSERT^{ALI/VFL+SI/TT} mice (n = 9–12). (B) Effect of escitalopram (1 mg/kg) alone or in combination with R-citalopram (2 mg/kg) on marble burying behavior (n = 13–16). *, p < 0.05, vs. saline. (T-test).