1. Serotonin Transporter-Independent Actions of the Antidepressant Vortioxetine Revealed by the SERT M172 Mouse
A.G. Nackenoff1, L.D. Simmler1, N.L. Baganz1, A.L. Pehrson4, C. Sánchez4, R.D. Blakely1,2
Department of 1Pharmacology, 2Psychiatry, Vanderbilt University Medical Center, Nashville, TN;
4Lundbeck Research USA, Paramus, NJ
Vortioxetine at SERT M172
5-HT1A
5-HT1B
WT SERT
5-HT1D
5-HT3
5-HT7
SERT M172 WT
: Vortioxetine
Introduction
Depression is one of the most common and burdensome disorders worldwide. The most widely prescribed antidepressants are serotonin (5-HT) selective reuptake inhibitors (SSRIs), believed to provide therapeutic benefit by antagonizing the 5-HT transporter (SERT) and elevating extracellular 5-HT levels. To improve efficacy, a number of drugs have been developed that offer additional target engagements, including other transporters and receptors. The efficacy of such agents raises important questions as the degree to which one (e.g. SERT) or more of the anticipated targets accounts for therapeutic efficacy. In the present report, we describe our efforts to investigate antidepressant targeting specificity using a novel transgenic mouse model wherein high-affinity interactions at SERT of many antidepressants has been disabled without alteration of 5-HT transport function (SERT Met172). In this effort, we seek to understand the degree to which vortioxetine, an FDA-approved antidepressant, requires SERT antagonism given that the drug also has significant interactions with 5-HT1A, 5-HT1B, 5-HT1D, 5-HT3 and 5-HT7 receptors at clinically relevant doses. By selectively removing the pharmacological actions of vortioxetine at SERT utilizing the SERT M172 mouse model, we examine whether vortioxetine retains its behavioral efficacy in preclinical antidepressant assays.
In Vitro Vortioxetine PD
Target
Function
Binding affinity (nM) (Human)
IC/EC50 (nM) (Human)
SERT
Inhib.
1.6
5.4
5-HT1A
Agonist 15
200
5-HT1B
Partial Ag. 33
120
5-HT1D
Antag. 54
370
5-HT3
3.7 12
5-HT7 19
450
SERT Sequence Alignment
Adapted from: Sánchez et al. Pharmacology & Therapeutics. 2015
Modified from: Henry, L. K. et al. J. Biol. Chem. 2006;281:
Ex Vivo Synaptosomal Uptake
Modified from Yamashita, A. et al, Nature. 2005
I172M
SERT M172 Homology Model
Combs et al. ACS Chem Neuro. 2011
5-HT Uptake Kinetics
S-Citalopram
SERT Protein Expression
Protein was isolated from brain regions from SERT I172 and SERT M172 mice (n = 6) and subjected to SDS-PAGE (right). SERT expression was quantified via image densitometry using ImageJ and normalized to β-actin and expressed as percentage of SERT I172 (WT) levels within brain regions (left).
Competition uptake analysis of midbrain-derived synaptosomes. Various SERT blockers were assayed for their ability to compete with 50 nM [3H]5-HT uptake (n = 6-8 per drug). Fluoxetine, citalopram, and vortioxetine demonstrated significantly reduced inhibition efficacy in SERT M172 relative to SERT I172 synaptosomes, whereas paroxetine displayed no significant reduction in inhibition efficacy.
Whole brain derived synaptosomes were assayed for specific SERT uptake across various concentrations of [3H]5-HT. Kinetic parameters Km and Vmax do not differ between SERT I172 and SERT M172 (n = 8-10)
In Vivo Microdialysis
Extracellular 5-HT in dorsal hippocampus measured via in vivo microdialysis. Arrow indicates when vortioxetine was administered 10 mg/kg i.p. (n = 3-4/genotype)
Tail Suspension Test
Forced Swim Test
Mice were injected i.p. with the treatment listed 60 minutes prior to behavioral analysis. Mice were then suspended by their tail (TST, above) or placed in inescapable water bath (FST, right) and their immobility behavior was observed for 6 minutes. Vortioxetine maintains its behavioral efficacy in both WT and SERT M172 mice. * indicates significant difference from vehicle following planned Student’s t-test (n = 8-12/condition, p < 0.05). Citalopram and Fluoxetine groups are superimposed from separate datasets and provided for comparative purposes.
Conclusions
The antidepressant activity of vortioxetine, as shown in the TST, FST and NIH, does not require SERT antagonism
Results imply clinical viability of future 5-HT receptor directed antidepressants
Novelty Induced Hypophagia
Hippocampal Neurogenesis * * * * *
Future Directions
Investigate serotonin-specific gene expression changes following chronic vortioxetine to identify downstream targets for future antidepressants * * * * * * *
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Mice were injected with proliferation marker BrdU either after (Proliferation, 24 hrs before sacrifice) or before (Survival, 28 days before sacrifice) 28 days of drug administration. BrdU incorporation was measured in the dentate gyrus of the Hippocampus. Vortioxetine maintains hippocampal neurogenesis in both WT and SERT M172 mice. * indicates significant difference from vehicle following Two Way ANOVA and Bonferroni post-hoc test (n = 15-20/condition, p < 0.05).
Mice consumed ~10 mg/kg/day vortioxetine in chow for 28 days prior to NIH test. Paroxetine delivered in drinking water at 160 mg/L. On test day, mice are placed in a new cage in high light and no bedding, and latency to consume vanilla Ensure was measured. Vortioxetine maintains its behavioral efficacy in both WT and SERT M172 mice. * indicates significant difference from vehicle following Two Way ANOVA and Bonferroni post-hoc test (n = 15-20/condition, p < 0.05).
Acknowledgements & Disclosure: This work is supported by Lundbeck Research USA & NIH/NIMH grant R01 MH (RDB).