New Insights into the Mechanism of Action of Viloxazine: Serotonin and Norepinephrine Modulating Properties

Figures & data

Table 1 Monoamine Uptake Inhibition, Transporter Binding Affinity, and Selectivity for Viloxazine, Atomoxetine, and Reboxetine.

	Viloxazine	Atomoxetine	Reboxetine
[^3H]-NE uptake	2300^a	3.4^b	8.2^c
[^3H]-5-HT uptake	>10,000^a	390^b	1070^c
[^3H]-DA uptake	NC^a	1750^b	NC^c
NET binding	155^d	2^d–5^e	1.1^e–11^f
SERT binding	17,300^d	9^d–77^e	129^e–440^f
DAT binding	>100,000^d	1080^d–1451^e	>10,000^f
KD (SERT)/KD (NET)	111	4
Ki (SERT)/Ki (NET)		15	40–117

Notes: All values in 10⁻⁹ M. ^aK_i calculated from rat hypothalamic synaptosomes uptake assay. ^bK_i calculated from rat cerebral cortex synaptosomes uptake assay. ^cK_i calculated from rat striatal synaptosomes uptake assay. ^dK_D calculated from competition assays using [³H]-nisoxetine for hNET, [³H]-imipramine for hSERT, and [³H]-WIN35428 for hDAT. ^eK_i calculated using specific radiolabeled ligands, [³H]-nisoxetine for NET, [³H]-paroxetine for SERT, and [³H]-WIN35428 for DAT. ^fK_i calculated using specific radiolabeled ligands, [³H]-nisoxetine for NE, [³H]-citalopram for 5-HT, and [³H]-WIN35428 for DA uptake sites. Data from these studies.^15–18

Abbreviations: 5-HT, serotonin; DA, dopamine; DAT, dopamine transporter; K_D, dissociation constant; K_i, inhibition constant; NC, not calculated; NE, norepinephrine; NET, norepinephrine transporter; SERT, serotonin transporter.

Figure 1 Neurotransmitter transporters and receptors binding profile of viloxazine. Representative results of binding competition assays of viloxazine (10 µM). Viloxazine binding was calculated as a percent inhibition of the binding of a radiolabeled ligand specific for each target (mean ± SEM, n = 2). Targets that presented an inhibition higher than 50% were considered a significant effect for viloxazine binding.

Abbreviations: 5-HT, serotonin; CHT-1, choline transporter; DAT, dopamine transporter; GAT, GABA transporter; NET, norepinephrine transporter; SEM, standard error of the mean; SERT, serotonin transporter.

Figure 2 Effects of viloxazine on uptake and cellular functional activity in vitro. (A) Dose-dependent inhibition of NET-mediated [³H]-NE uptake (n = 3) measured in rat hypothalamic synaptosomes and [³H]-5-HT uptake (n = 3) measured in HEK293 cells expressing hSERT. (B) Viloxazine activated the response of 5-HT_2C (agonist) in a CHO cell-based IP₁ HTRF^® assay (n = 2). (C) Viloxazine antagonized the activity of 5-HT_2B after stimulation with 5-HT in a CHO cell-based IP₁ HTRF^® assay (n = 2). Data presented as mean ± SEM.

Abbreviations: 5-HT, serotonin; CHO, Chinese hamster ovary; DA, dopamine; EC₅₀, concentration producing a half-maximal response; HEK, human embryonic kidney; hSERT, human serotonin transporter gene; HTRF, homogeneous time-resolved fluorescence; IC₅₀, concentration causing a half-maximal inhibition of control response; IP₁, inositol phosphate-1; NE, norepinephrine; NET, norepinephrine transporter; SEM, standard error of the mean; SERT, serotonin transporter.

Table 2 Statistical Analysis of the Effects of Viloxazine Administration on Monoamine Levels in the Three Brain Regions as Measured by Microdialysis in Freely Moving Rats

	PFC				Acb				Amg
	Peak (%)	Effect of Treatments	Effect of Time	Interaction of Treatment and Time	Peak (%)	Effect of Treatments	Effect of Time	Interaction of Treatment and Time	Peak (%)	Effect of Treatments	Effect of Time	Interaction of Treatment and Time
5-HT	506 ± 133	F1,10=38.28 p<0.001 n=5	F1,10=3.971 p<0.001 n=5	F1,10=2.850 p=0.05 n=5	365 ± 48	F1,8=118.401 p<0.001 n=5	F1,8=12.168 p<0.001 n=5	F1,10=10.434 p<0.001 n=5	312 ± 15	F1,8=118.061 p<0.001 n=5	F1,8=13.675 p<0.001 n=5	F1,8=9.135 p<0.001 n=5
NE	649 ± 57	F1,10=448.561 p<0.001 n=5	F1,10=14.600 p<0.001 n=5	F1,10=12.519 p<0.001 n=5	187 ± 28	F1,8=48.634 p<0.001 n=5	F1,8=1.372 p=0.222 n=5	F1,8=1.185 P=0.319 n=5	571 ± 82	F1,8=249.935 p<0.001 n=5	F1,8=10.919 p<0.001 n=5	F1,10=8.485 p<0.001 n=5
DA	670 ± 105	F1,10=122.245 p<0.001 n=3	F1,10=4.496 p<0.001 n=3	F1,10=4.131 p<0.001 n=3	186 ± 18	F1,8=14.316 p<0.001 n=5	F1,8=3.158 p=0.003 n=5	F1,8=2.777 p=0.009 n=5	254 ± 19	F1,8=129.811 p<0.001 n=5	F1,8=10.847 p<0.001 n=5	F1,8=5.697 p<0.001 n=5

Notes: Statistical evaluation was performed using SigmaStat for Windows, Version 4 (Systat Software, Inc., 2011–2012). Serial samples were evaluated using a two-way ANOVA statistical test with treatment as the independent value and time as the repeated measurement. The level of statistical significance was set at p<0.05 (shadowed). Peak (%) values correspond to the maximum value relative to baseline expressed as Mean ± S.E.M., n is the number of animals included in the analysis.

Figure 3 Effects of viloxazine on neurotransmitter levels in the PFC, Acb, and Amg of freely moving rats. Administration of viloxazine (50 mg/kg, IP) at time 0 is indicated by the arrow. (A) In the PFC, viloxazine significantly increased extracellular levels of NE and DA throughout the 4 h period (**p < 0.001, Dunnett’s post hoc test) and 5-HT from 30 to 120 minutes (^#p < 0.05 Dunnett’s post hoc test) in comparison to vehicle treated groups. (B) In the Acb, extracellular levels of 5-HT and NE increased throughout the 4 h period (**p < 0.001, Dunnett’s post hoc test) and DA from 30 to 60 min (^#p < 0.05, Dunnett’s post hoc test). (C) In the Amg, extracellular levels of 5-HT, NE, and DA increased throughout the 4 h period (**p < 0.001, Dunnett’s post hoc test). Measured neurotransmitter levels (mean ± SEM) are reported as the percent of pre-treatment baseline. The statistical post-hoc analyses of vehicle vs viloxazine at each time point (T = 0 to T = 240) and significant interactions (two-way ANOVA with p<0.05) are presented in the Table 2.

Abbreviations: 5-HT, serotonin; Acb, nucleus accumbens; ACh, acetylcholine; Amg, amygdala; ANOVA, analysis of variance; DA, dopamine; GABA, gamma-aminobutyric acid; Glu, glutamate; His, histamine; IP, intraperitoneal; NE, norepinephrine; PFC, prefrontal cortex; SEM, standard error of the mean.

Table 3 Receptor Occupancy Calculated Based on Obtained C_unbound Brain Concentration Estimated from C_max Plasma After 100, 200, 400 and 600 mg/day Doses in Pediatric ADHD Patients and K_i Values of 630, 3900 and 6400 nM for NET, 5-HT_2B and 5-HT_2C, Respectively

Dose (mg/kg)	Estimated Receptor Occupancy (%)
	NET	5-HT2B	5-HT2C
100	96.5	81.4	72.8
200	98.6	91.7	87.1
400	99.3	95.7	93.2
600	99.3	96.1	93.7

Figure 4 Dose – Calculated Receptor Occupancy Curves. Relationship between the dose of administered viloxazine to pediatric ADHD patients (31.5 kg mean body weight) and the estimated receptor occupancy for NET, 5-HT_2B, and 5-HT_2C based on the estimated unbound brain concentration of viloxazine at target receptor and its binding affinity (K_i) at clinical doses of 100, 200, 400, and 600 mg/day.

Figure 5 Complex regulation of 5-HT projections to prefrontal cortex. 1. “Proximal” regulatory loop includes feedback collateral branches and 5-HT_1A autoreceptors, whose activation by excessive neurotransmitter results in reduced 5-HT synthesis and neuronal firing rate. Feedback collaterals also synapse with 5-HT_2B receptors on DNR GABA interneurons. Inhibition of these receptors may be one of the principal regulators of 5-HT tonic neurotransmission. Viloxazine, for instance, may inhibit 5-HT_2B receptors leading to inhibition of GABAergic transmission and consequently to upregulation of 5-HT tonic neurotransmission in mPFC, which may explain the increase of 5-HT in PFC observed in current microdialysis study. 2. At the terminal end 5-HT_1B/1D autoreceptors regulate the 5-HT release. Serotoninergic projections in mPFC interface with SST-expressing and PV-expressing GABA interneurons as well as directly modulate glutamatergic pyramidal neurons via postsynaptic inhibitory 5-HT_1A receptors and excitatory 5-HT_2A receptors. SST GABA interneurons have a primary role in “signal-to-noise” gating of pyramidal neurons, while PV interneurons via axonal receptors regulate “volume” of glutamate transmission. 3. Serotoninergic input, indirectly, via mPFC GABA interneurons, and directly, via pyramidal neurons, regulates initiation of the “distal” regulatory loop. Additionally, the balance between activation of 5-HT_2C receptors on GABA interneurons vs direct stimulation of 5-HT_2C receptors located on pyramidal neurons, ultimately tunes glutamatergic output from mPFC. 4. Glutamatergic projections from mPFC to LC noradrenergic neurons, VTA dopaminergic neurons, and DNR/MNR serotoninergic neurons, as well as their corresponding GABA interneurons, directly and indirectly regulate function of the brainstem monoaminergic nuclei. Local balance between stimulation of 5-HT_2C receptors on GABA interneurons vs ones located directly on VTA DA neurons, modulates DA output from the nucleus. At the termination point of the “distal” loop, glutamate fibers via excitatory AMPA and NMDA receptors located on 5-HT neurons and adjacent GABA interneurons, provide balanced regulatory input to serotoninergic nuclei.

Notes: Artigas F. 2013, Pharmacol Ther. 137:119–131. Cathala et al, 2019, Experimental Neurology, 311:57–66. Leggio et al, 2009, Neuropharmacology, 56: 507–513.

Abbreviations: 5-HT, 5-hydroxytryptamine, AMPA, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid; DNR, dorsal nuclei raphe; GABA, gamma-aminobutyric acid; Glu, glutamate; LC, locus coeruleus; mGluR, metabotropic glutamate receptor; MnR, median raphe; mPFC, medial prefrontal cortex; NMDA, N-methyl-D-aspartate; PV, parvalbumin; SST, somatostatin; VTA, ventral tegmental area.

Figure 6 Proposed dual mechanism of action of viloxazine. The schematic shows purported clinical implications (dashed lines) of the observed neurotransmitter modulating effects of viloxazine. Microdialysis and in vitro studies demonstrate that viloxazine increases 5-HT, NE, and DA levels in the PFC of awake rats, as well as exhibits antagonistic activity towards 5-HT_2B receptors and agonistic activity towards 5-HT_2C receptors. These effects of viloxazine on modulating both the 5-HT and NE systems help explain why viloxazine is therapeutically relevant to neuropsychiatric disorders, such as the core symptoms of ADHD and depression.

Abbreviations: 5-HT, serotonin; ADHD, attention-deficit/hyperactivity disorder; DA, dopamine; NE, norepinephrine; NET, norepinephrine transporter; PFC, prefrontal cortex.

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