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

Abstract

Background: Viloxazine was historically described as a norepinephrine reuptake inhibitor (NRI). Since NRIs have previously demonstrated efficacy in attention deficit/hyperactivity disorder (ADHD), viloxazine underwent contemporary investigation in the treatment of ADHD. Its clinical and safety profile, however, was found to be distinct from other ADHD medications targeting norepinephrine reuptake. Considering the complexity of neuropsychiatric disorders, understanding the mechanism of action (MoA) is an important differentiating point between viloxazine and other ADHD medications and provides pharmacology-based rationale for physicians prescribing appropriate therapy.

Methods: Viloxazine was evaluated in a series of in vitro binding and functional assays. Its effect on neurotransmitter levels in the brain was evaluated using microdialysis in freely moving rats.

Results: We report the effects of viloxazine on serotoninergic (5-HT) system. In vitro, viloxazine demonstrated antagonistic activity at 5-HT_2B and agonistic activity at 5-HT_2C receptors, along with predicted high receptor occupancy at clinical doses. In vivo, viloxazine increased extracellular 5-HT levels in the prefrontal cortex (PFC), a brain area implicated in ADHD. Viloxazine also exhibited moderate inhibitory effects on the norepinephrine transporter (NET) in vitro and in vivo, and elicited moderate activity at noradrenergic and dopaminergic systems.

Conclusion: Viloxazine's ability to increase 5-HT levels in the PFC and its agonistic and antagonistic effects on certain 5-HT receptor subtypes, which were previously shown to suppress hyperlocomotion in animals, indicate that 5-HT modulating activity of viloxazine is an important (if not the predominant) component of its MoA, complemented by moderate NET inhibition. Supported by clinical data, these findings suggest the updated psychopharmacological profile of viloxazine can be best explained by its action as a serotonin norepinephrine modulating agent (SNMA).

Keywords: ADHD; SPN-812; mechanism of action; norepinephrine transporter; serotonin norepinephrine modulating agent; viloxazine.

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.

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.

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.

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.

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.