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Expert Review of Neurotherapeutics

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New advances in the treatment of generalizedanxiety disorder: the multimodal antidepressantvortioxetine

Laura Orsolini, Carmine Tomasetti, Alessandro Valchera, Felice Iasevoli,Elisabetta Filomena Buonaguro, Federica Vellante, Michele Fornaro,Annastasia Fiengo, Monica Mazza, Roberta Vecchiotti, Giampaolo Perna,Andrea de Bartolomeis, Giovanni Martinotti, Massimo Di Giannantonio &Domenico De Berardis

To cite this article: Laura Orsolini, Carmine Tomasetti, Alessandro Valchera, Felice Iasevoli,Elisabetta Filomena Buonaguro, Federica Vellante, Michele Fornaro, Annastasia Fiengo, MonicaMazza, Roberta Vecchiotti, Giampaolo Perna, Andrea de Bartolomeis, Giovanni Martinotti,Massimo Di Giannantonio & Domenico De Berardis (2016) New advances in the treatment ofgeneralized anxiety disorder: the multimodal antidepressant vortioxetine, Expert Review ofNeurotherapeutics, 16:5, 483-495, DOI: 10.1586/14737175.2016.1173545

To link to this article: http://dx.doi.org/10.1586/14737175.2016.1173545

Accepted author version posted online: 06Apr 2016.Published online: 18 Apr 2016.

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New advances in the treatment of generalized anxiety disorder: the multimodalantidepressant vortioxetineLaura Orsolinia,b,c,d, Carmine Tomasettic,e,f, Alessandro Valcherab,c, Felice Iasevolic,f, Elisabetta Filomena Buonaguroc,f,Federica Vellantec,g,h, Michele Fornaroc,i, Annastasia Fiengoc, Monica Mazzac,j, Roberta Vecchiottib,c,d,Giampaolo Pernad,k,l, Andrea de Bartolomeisf, Giovanni Martinottii, Massimo Di Giannantonioi

and Domenico De Berardisc,g,i

aSchool of Life and Medical Sciences, University of Hertfordshire, Hatfield, UK; bVilla San Giuseppe Hospital, Hermanas Hospitalarias, Ascoli Piceno,Italy; cPolyedra Research Group, Teramo, Italy; dDepartment of Psychiatry and Neuropsychology, University of Maastricht, Maastricht, TheNetherlands; eNHS, Department of Mental Health ASL Teramo, Psychiatric Service of Diagnosis and Treatment, Hospital ‘Maria SS dello Splendore’,Giulianova, Italy; fLaboratory of Molecular and Translational Psychiatry, Department of Neuroscience, Reproductive and OdontostomatogicalSciences, University of Naples ‘Federico II’, Napoli, Italy; gNHS, Department of Mental Health ASL Teramo, Psychiatric Service of Diagnosis andTreatment, Hospital ‘G. Mazzini’, Teramo, Italy; hDepartment of Neuroscience and Imaging, University ‘G. d’Annunzio’, Chieti, Italy; iNew YorkPsychiatric Institute, Columbia University, New York, NY, USA; jDepartment of Life, Health and Environmental Sciences, University of L’Aquila,L’Aquila, Italy; kHermanas Hospitalarias, FoRiPsi, Department of Clinical Neurosciences, Villa San Benedetto Menni, Albese con Cassano, Como, Italy;lDepartment of Psychiatry and Behavioral Sciences, Leonard Miller School of Medicine, University of Miami, Coral Gables, Florida, USA

ABSTRACTGeneralized Anxiety Disorder (GAD) is a persistent condition characterized by chronic anxiety, exag-gerated worry and tension, mainly comorbid with Major Depressive Disorder (MDD). Currently, selectiveserotonin reuptake inhibitors and serotonin-norepinephrine reuptake inhibitors are recommended asfirst-line treatment of GAD. However, some patients may not respond to the treatment or discontinuedue to adverse effects. Vortioxetine (VRX) is a multimodal antidepressant with a unique mechanism ofaction, by acting as 5-HT3A, 5-HT1D and 5-HT7 receptor antagonist, partial agonist at the 5-HT1A and 5-HT1B receptors and inhibitor at the 5-HT transporter. Preliminary clinical trials showed contrastingfindings in terms of improvement of the anxiety symptomatology and/or cognitive impairment. Here,we aim to systematically review the evidence currently available on the efficacy, safety and tolerabilityof VRX in the treatment of GAD. The generalizability of results on the efficacy of VRX in patients withanxiety symptomatology and GAD is limited due to few and contrasting RCTs so far available. Only twostudies, of which one prevention relapse trial, reported a significant improvement in anxiety sympto-matology compared to three with negative findings.

ARTICLE HISTORYReceived 26 December 2015Accepted 30 March 2016Published online19 April 2016

KEYWORDSVortioxetine; antidepressant;Lu AA21004; brintellix;anxiety; generalized anxietydisorder

Introduction

Generalized anxiety disorder (GAD) is a common mental dis-order marked by persistent, distressing, and excessive anxietyand worries (apprehensive expectation) about several events/situations/activities (i.e. work, school performance, etc.) [1].The intensity, duration, and frequency of the anxiety andworry are out of control and may be associated with irritabil-ity, muscle tension, sleep disturbance (i.e. restlessness, stayingasleep, etc.), fatigue, difficult concentration, and restlessness[1]. Subjects affected with GAD may have a significant impair-ment in work, social and family functioning, as well as aworsening in health-related quality of life [2,3]. GAD usuallyhas a chronic course [4]. The 12-month prevalence of GADranges from 0.4% to 3.6%, depending on the countries [1].Lifetime prevalence of GAD is approximately 6% [5]. GAD ishighly associated with comorbid psychiatric conditions, parti-cularly with major depressive disorder (MDD), with a rateranging 28–55% [6,7]. Given the aforementioned findings,proper and effective treatment of GAD is fundamental [8].

According to the most recent guidelines, the first-line psycho-pharmacological strategies in the treatment of GAD compriseselective serotonin reuptake inhibitors (SSRIs) and serotonin–norepinephrine reuptake inhibitors (SNRIs) [9,10]. Promisingresults have also been obtained with pregabalin and quetia-pine [10]. Further useful alternative treatments include buspir-one, benzodiazepines, and hydroxyzine [11]. However, mostpatients affected with GAD do not receive an adequateresponse to initial treatment with an SSRI/SNRI, with differencein the response rate between SSRI/SNRI and placebo rangingfrom 15% to 20% [12].

Vortioxetine (LU-AA21004; 1-[2-(2, 4-dimethylphenyl-sulfa-nyl)-phenyl]-piperazine; Brintellix®; VRX) is a multimodal-act-ing antidepressant agent, recently identified in a designedmultiple ligands program by H. LundBeck A/S and co-mar-keted by Lundbeck® and Takeda® [13,14]. In September2013, VRX was approved in North America by the US FDA forthe once-daily treatment of adults with MDD [14,15]. Recently,it was also approved in Europe by the European Medicines

CONTACT Domenico De Berardis dodebera@aliceposta.it Department of Mental Health, Psychiatric Service of Diagnosis and Treatment, ‘G. Mazzini’Hospital, p.zza Italia 1, 64100 Teramo, Italy.

EXPERT REVIEW OF NEUROTHERAPEUTICS, 2016VOL. 16, NO. 5, 483–495http://dx.doi.org/10.1586/14737175.2016.1173545

© 2016 Informa UK Limited, trading as Taylor & Francis Group

Agency for the same indication [16]. VRX is in phase III devel-opment for the treatment of GAD and in phase II developmentfor anxiety and depressive disorders in children and adoles-cents (NCT01491035), as well as attention-deficit hyperactivitydisorder. A further phase IV, open-label study is still evaluatingits efficacy and safety for panic disorder (PD; NCT02395510).

Chemistry

It belongs to the chemical class of the bisarylsulfanyl amines. Itis structurally related to citalopram, ondansetron, and buspir-one [17]. Figure 1 shows the chemical structure of VRX.

Pharmacodynamics

The structure–activity relationship studies, made by in vitrobinding affinity assays, revealed the substantial activity ashuman serotonin (5-HT) transporter (SERT) inhibitor by VRX(Ki = 1.6 nmol/l in human receptors), as well as a discrete actionas norepinephrine transporter (NET) blocker (Ki = 113 nmol/l)[18,19]. Its SERT occupancy rates range from 50% at 5 mg/day,53–65% at 10 mg/day, to >80% at 20 mg/day [20,21].Furthermore, VRX has been demonstrated to act as a partialagonist (with near full agonist activity) at 5-HT1A receptors(Ki = 15 nmol/l), partial agonist at 5-HT1B (Ki = 33 nmol/l),antagonist at 5-HT1D (Ki = 54 nmol/l), antagonist at 5-HT3A(Ki = 3.7 nmol/l), antagonist at 5-HT7 (Ki = 19 nmol/l), as wellas to bind with high affinity, but not obvious in vivo pharma-cology, at noradrenergic β1 receptors (Ki = 46 nmol/l) [22–25].These pharmacological properties induced discoverers to defineVRX as a new serotonergic multimodal agent with pleiotropicactivity on multiple serotonin-related neurotransmissions andthereby with potential benefits in the treatment of MDD.Indeed, several studies have pointed out the role of specificserotonin receptors modulation in the management of coredepressive symptoms.

In particular, attention has been progressively focusing on5-HT1A receptors. These subclasses of seven-transmembranepassage serotonin receptors are ubiquitously distributed inthe brain, with scarce differences among species (i.e. fromrat to human) [26–29]. 5-HT1A receptors may be both presy-naptically and postsynaptically localized. Peculiarly, 5-HT1Apresynaptic autoreceptors are mainly located on serotonergicneurons of the raphe nuclei [30], whereas postsynaptic 5-HT1Areceptors are principally distributed in the cortex, as well as inthe hippocampus, septum, amygdala, and hypothalamus[26,27]. Both types of 5-HT1A receptors act as G-protein-

coupled receptors, whose principal effect is the modulationof inward rectifier K+ channels through cAMP and phospholi-pase C, thereby causing the final reduction of firing by neu-rons involved [31,32]. The potential antidepressant effects of5-HT1A modulation were already suggested in early studies, inwhich some authors reported that the add-on of pindolol (a β-adrenergic receptors blocking agent, with partial agonist prop-erties at 5-HT1A, used to treat hypertension) to paroxetinecould accelerate the improvement of depressed patients trea-ted with the serotonin reuptake inhibitor [33,34]. Pindolol waspreviously known to exert antagonistic activity at 5-HT1Areceptors. However, successive studies have demonstratedthat actually, pindolol may possess agonist-like activity atsomatodendritic 5-HT1A receptors, with weak antagonisticproperties [35]. Successive studies determined and confirmedthe partial agonist properties of pindolol but somewhat statedthat other 5-HT receptors should be involved in the antide-pressant-potentiation effects by this drug [36]. Recent reportsalso demonstrated the involvement of 5-HT1A receptors inanxiety-like behaviors and in the susceptibility to anxiety dis-orders [37]. Moreover, drugs with antagonistic actions at 5-HT1A receptors have been reported to display anxiolyticeffects [38].

Its partial agonist activity at 5-HT1B receptors may exerteffects in modulating stress sensitivity, mood, anxiety, andaggression by inhibiting the release of 5-HT, NE, GABA, acet-ylcholine, and glutamate [39]. In fact, 5-HT1B receptors have alsobeen associated to both anxiety and depression in animals aswell as in human studies [40,41]. These are G-coupled receptorsprincipally localized on the serotonergic terminals of dorsalraphe nucleus efferent fibers, acting as autoreceptors thatrespond to serotonin extracellular elevation by feedback inhi-biting further release and synthesis and enhancing serotoninreuptake [42–44]. Animal models of learned helplessnessinduced by stress have been reported to express high levelsof 5-HT1B receptors in the dorsal raphe nucleus [45], as well asthe viral-induced overexpression of 5-HT1B receptors in thesame nucleus may induce anxiety behaviors [46]. However,other studies revealed a substantial increase in 5-HT1B receptorspercentage in the dorsal raphe nucleus in animal models ofstress resistance [47], thus defining a complex role of thesereceptors in anxiety. SSRIs have been demonstrated to reduce5-HT1B expression in the dorsal raphe nucleus in a time- anddose-dependent manner [48]. Recent studies, moreover,demonstrated that 5-HT1B agonists may counteract the onsetof anxiogenic behaviors in animals exposed to fear contexts[49]. By contrast, anxiogenic responses may be induced inanimals by selectively injecting 5-HT1B agonist drugs in themedial prefrontal cortex, a brain region crucially involved inprocessing emotional behaviors [50]. 5-HT1B antagonists mayalso display anxiolytic effects when systemically administered[51]. Given the composite action of 5-HT1B receptors on anxietybehaviors, drugs showing partial agonism, such as VRX, areexpected to balance serotonergic action upon these receptorsin order to facilitate anxiolytic and antidepressant effects.

Similar to 5-HT1B receptors, 5-HT1D serotonin receptors arelocalized on presynaptic terminals of dorsal raphe nucleus fibers,although they are slightly less abundant [52]. Characteristically, 5-HT1D receptors are expressed on trigeminovascular nerveFigure 1. The chemical structure of VRX.

484 L. ORSOLINI ET AL.

endings and are targeted by antimigraine drugs (e.g. sumatrip-tan), in order to regulate the activity of cerebral vascular smoothmuscle contraction to counteract vascular-generated cephalalgia[53]. Some studies, however, reported 5-HT1D central localizationsin the globus pallidus and substantia nigra pars reticulata, wherethey appear to be located on GABAergic striatal terminals, as wellas in nonpyramidal neuron of the neocortex [54,55]. According tothese data, 5-HT1D agonists have been described to inhibit GABAcortical release, which may be prevented by antagonists, bothactions possibly having physiological relevance in anxiety disor-ders [56]. A specific increase of 5-HT1D receptors expression(together with 5-HT1A, 5-HT1B, 5-HT2A, and 5-HT4 receptors) hasbeen described in animal models of heightened stress resilience[57]. According to some contrasting studies in which 5-HT1Dagonists could/could not exacerbate obsessive-compulsivesymptoms in patients [58,59], an implication in the pathophysiol-ogy of obsessive-compulsive disorder has been suggested forthese receptors [60]. Moreover, sumatriptan administration hasbeen reported to aggravate anxiety symptoms in patients suffer-ing from panic disorder [61]. Therefore, antagonism at 5-HT1Dreceptors, as displayed by VRX, may have attenuating effects onanxiety symptoms.

Differently from all the other 5-HT receptors, which areseven-transmembrane passages-metabotropic receptors, 5-HT3receptors may be considered members of the cys-loop family ofligand-activated ion channels, which also includes nicotinicacetylcholine receptors and GABA-A receptors [62]. The 5-HT3receptor ion channel is composed of five subunits among sixidentified cDNA encoded units (A, B, C, D, E, and F) [63], whichhave been characterized as possessing multiple splicing variantsoriginating several channel combinations. To date, homomeric5-HT3A subunit-formed receptors have been described [64], aswell as heteromeric complex formed by all the other subunitstogether with the 5-HT3A subunit [65–67]. However, currentfunctional studies principally refer to two isoforms of 5-HT3receptors, the 5-HT3A and 5-HT3AB receptors, which representthe most diffused ones, both characterized as cation-permeable(Na+, K+, and Ca2+) channels with different conductances andCa2+ permeabilities [68,69]. Early studies described a broaddistribution of 5-HT3 receptors in the brain, with high densitiesin the brainstem areas (nucleus tractus solitarius, area postrema,and spinal trigeminal nucleus) that are mainly involved inemesis and migraine [70,71], as well as in the vagal afferentsof the peripheral nervous system that control the Bezold–Jarisch reflex [72]. More recent reports demonstrated specificdistribution of 5-HT3 receptors in limbic regions (hippocampus,amygdala, nucleus accumbens, and caudate-putamen), wherethey might have peculiar role in neuron–neuron synchronicintegration through gamma oscillations [73], in glutamate-dependent synaptic plasticity [74], and in the formation ofemotional memories [75]. Furthermore, 5-HT3 receptors havepeculiar presynaptic subcellular localization in the most part ofbrain regions, thereby controlling neurotransmitters release,except for the hippocampus, where they have been mainlydetected postsynaptically in dendrites [76]. Last but not least,the broad distribution of 5-HT3 receptors in the gastroenterictract accounts for the essential role of these receptors in con-trolling gastrointestinal peristalsis and motility, as well as in thepathophysiology of functional and somatoform anxiety-linked

gastroenteric disorders, such as the irritable bowel syndrome[77]. Extensive evidence points out the crucial involvement of 5-HT3 receptors in the pathophysiology of anxiety behaviors,alone or included in broad syndromes, such as depression orgut comorbidities [78]. Indeed, animal studies described power-ful effects of 5-HT3 blockade in the inhibition of limbic hyper-activity responses generating anxiety behaviors [79], and 5-HT3knock-out animals have been demonstrated to display anxioly-tic phenotypes [80]. Based on these data, several drugs withantagonist activity at 5-HT3 receptors have been demonstratedto be effective in treating anxiety disorders [81,82].

5-HT7 receptors have been the last cAMP-activating, seven-transmembrane passages, G-protein-coupled serotonin recep-tors to be discovered by targeted DNA analysis [83]. Foursplice variants of 5-HT7 receptor (namely 5-HT7A, 5-HT7B, 5-HT7C, and 5-HT7D) have been detected in humans, with differ-ent lengths of C-terminals [84], and different polymorphicvariants, among which some having pharmacological interestbecause of altered binding to agonists [85]. 5-HT7 receptorshave high expression in the central nervous system, particu-larly in the hypothalamus (suprachiasmatic nucleus), thalamus,hippocampus, and cerebral cortex [86–88], as well as in theileum, spleen, blood vessels and lymphocytes, and endocrineglands [89]. Also, some distribution of 5-HT7 receptors hasbeen detected in the raphe nucleus [90], with probable func-tion of controlling serotonin release. In the hippocampus, 5-HT7 receptors appear to be specifically located in cell bodies ofCA1 pyramidal neurons [91]. By means of distribution- andligand-based studies, it has been possible to implicate 5-HT7receptors in specific brain functions, such as circadian rhythmsregulation, thermoregulation, endocrine regulation due tohypothalamus localizations, as well as sleep and mood regula-tion due to thalamic localization [92], and memory and learn-ing due to hippocampal detection [93]. Animal 5-HT7 knock-out models have been demonstrated to display anxiolyticbehaviors [94], as well as 5-HT7 antagonists have beendescribed to induce anxiolysis in animal behavioral tests [95]and to modulate the formation of emotional learning [96].Thus, 5-HT7 antagonists could be considered valuable com-pounds for the treatment of anxiety disorders.

Finally, its activity as NET may play a role in enhancing depres-sive symptoms both in patients with anxiety in comorbid MDDand in those patients affected with ADHD, frequently comorbidwith anxiety disorders [25]. In addition, NET inhibition may reducenoradrenergic hyperactivity often responsible for the onset ofnumerous central symptoms of anxiety, such as nightmares,hyperarousal states, flashbacks, and panic attacks [25]. This activitymay be explained due in part to its activity in desensitizing post-synaptic β and α−1 noradrenergic receptors [18,19].

Additional pharmacodynamic profiling of VRX revealed a mod-est binding affinity to noradrenergic β2 receptors but a potentbinding affinity at noradrenergic β1 receptors (Ki = 46 nmol/l).However, the affinity for β1 receptor could not be at presenttranslated to an obvious pharmacology in vivo [97].

Pharmacokinetics and metabolism

VRX has a favorable pharmacokinetic profile with dose-propor-tional and linear exposure, moderate oral bioavailability

EXPERT REVIEW OF NEUROTHERAPEUTICS 485

(around 75%), being 98% protein bound. There was no clini-cally meaningful effect of food on the pharmacokinetics ofVRX, suggesting that it can be administered with or withoutfood [98,99]. After oral intake, VRX is absorbed in the gastro-intestinal tract, by showing an extended absorption phase anda peak plasma concentration in about 3–16 h (Tmax) [98]. Theexposure (Cmax and area under curve) increased linearly withdose (2.5–60 mg). The steady-state concentration is achievedin about 2 weeks. It exerts a medium clearance, a large volumeof distribution (approximately 2600 l), and a long eliminationhalf-life of around 57–66 h [100]. VRX undergoes extensivemetabolism, primarily through oxidation and subsequent glu-curonic acid conjugation. It is mainly metabolized in the liverby at least five cytochrome P450 enzymes: e.g. CYP2D6(IC50 = 34 μM), CYP1A2 (IC50 > 40 μM), CYP2C9 (IC50 = 15μM), CYP3A4 (IC50 > 40 μM), and CYP2C19 (IC50 > 40 μM) [100–102]. VRX is metabolized, mainly through the CYP2D6 pathway,to its major carboxylic acid metabolite, LuAA34443 (3-methyl-4-(2-piperazine-1-yl-phenylsulfanyl)-benzoic acid), which isconsidered to be much less pharmacologically active andincapable of crossing the blood–brain barrier. LuAA34443shows a similar half-life as VRX but a lower accumulation atsteady state. A minor metabolite, LuAA39835, showed similar5-HT transporter inhibition to the parent compound, althoughit is not expected to penetrate the blood–brain barrier. One-third of the drug is excreted in the feces (around 26%) whiletwo-thirds in the urine (approximately 59%), mainly asLuAA34443 and its glucuronide [103]. VRX demonstrated alow drug–drug interaction potential. In fact, it has no signifi-cant effect on the pharmacokinetics of ethinyl estradiol/levo-norgestrel, bupropion, or omeprazole. In addition,coadministration of VRX did not alter the pharmacokineticsof warfarin or aspirin [104].

Dosage and administration

VRX is marketed in pink, yellow, orange, and, red oval, film-coated tablets, based on the strength, as it is available as 5, 10,15 (not available in USA), and 20 mg immediate releasetablets. The recommended initial dose is usually 10 mg admi-nistered orally once daily without regards to meals. Thedosage can be increased gradually to a maximum of 20 mgdaily if tolerated. The dosage should be decreased by 50% forCYP4502D6 poor metabolizers. Discontinuation of higher dosesshould be gradual, but abrupt discontinuation of 10 mg dailyhas been recommended.

The present systematic review aims here to provide anupdated review on clinical studies on VRX in the treatmentof GAD.

Methodology

Search sources and strategies

A systematic review was here conducted according to themethods recommended by the Cochrane Collaboration [105]and documented the process and results in accordance withthe Preferred Reporting Items for Systematic Reviews andMeta-Analyses (PRISMA) guidelines [106]. Literature searches

were performed by using the following electronic databases(last update: December 2015): MEDLINE, SCOPUS,ScienceDirect, and PubMed. We combined the search strategyof free text terms and exploded MESH headings for the topicsof Vortioxetine, Generalized Anxiety Disorder, and Anxiety asfollowing: ((Vortioxetine[Title/Abstract] OR Lu AA21004[Title/Abstract] OR brintellix[Title/Abstract]) AND (antidepressant[Title/Abstract] OR anxiety [Title/Abstract]). The strategy wasfirst developed in MEDLINE and then adapted for use in theother databases. No restrictions by year of publication orlanguage were applied to any search. Studies publishedthrough 20 December 2015 were included. Further studieswere retrieved from reference listing of relevant articles andconsultation with experts in the field and/or manual search. Inaddition, in order to include unpublished studies, within thetime frame of the electronic searches, additional handsearches were performed on websites of pharmaceutical com-panies, clinical trials repositories and registers, and regulatoryagencies (e.g. www.clinicaltrials.gov, US FDA website), as wellas on GoogleScholar.

Study selection

We considered studies that included VRX treatment for GAD.We examined all titles and abstracts and obtained full texts ofpotentially relevant papers. Working independently and induplicate, two reviewers (L.O. and C.T.) read the papers anddetermined whether they met inclusion criteria. Duplicatepublications were properly excluded. All English-language arti-cles identified by the data sources, reporting original datarelated to VRX, were evaluated in the present review. Allexperimental and observational study designs were includedapart from case reports. Randomized, controlled clinical trialsinvolving humans were prioritized in the present systematicreview. Narrative and systematic reviews, letters to the editor,and book chapters were excluded.

Data extraction and management

L.O. and C.T. independently extracted the data on participantcharacteristics, intervention details, and outcome measures.Disagreements were resolved by discussion and consensuswith a third member of the team (D.D.). Data were collectedusing an ad hoc-developed data extraction spreadsheet.

Characteristics of included studies

From 442 potentially relevant records from the search ofdatabases and additional sources, 171 were excluded on thebasis of title or abstract, and 262 were excluded becauseduplicates. The remaining nine studies were retrieved formore detailed evaluation. Overall, five studies, of which fourwere short-term randomized clinical trials (RCTs) and onelong-term relapse prevention study, met the inclusion criteria.Furthermore, of the 54 records obtained from ClinicalTrials.gov, among the seven trials retrieved here, five were dupli-cates of those identified by the aforementioned search. Themain characteristics of the seven studies retrieved here arereported in Table 1.

486 L. ORSOLINI ET AL.

Table1.

Vortioxetin

ein

GAD

:sum

maryof

completed

andon

goingclinicaltrials.

Outcomes

Clinicaltrials.gov

IDnu

mber

Peer-

review

edpu

blication

and/or

abstract

conference

1Stud

ydesign

Endpo

int

classification

Aim/hypothesis

Sample

characteristics

Stud

ydescrip

tion

Primary

Second

ary

Find

ings

NCT00744627

(com

pleted)

Augu

st2008–

Octob

er2013

[107]

Dou

ble-blind,

placebo-controlled,

parallel-g

roup

,fixed-dosestud

y–

phaseIII

Efficacy/

safety

stud

y

Stud

yevaluatin

gefficacy

andsafety

ofasing

ledo

seof

VRXin

acute

treatm

entof

adultswith

GAD

N:3

01Ag

e:≥18

years

Diagn

osis:

GAD

Leng

th:8

weeks

Experim

ental:

fixed

dose

(5mg/day)

oforalVR

X(n

=150)

Comparator(s):

PBO(n

=151)

HAM

-AHAD

CGI-I

SDS

SF-36

Percentage

ofrespon

dersin

HAM

-Atotalscore

atweek8

Change

from

baselinein

HAM

-Atotalscore

atweek8in

participants

with

baselineHAM

-A≥25

CGI-S

Health

Care

Resource

UtilizationAssessed

bytheHealth

Econ

omic

AssessmentQuestionn

aire

Astatisticallysign

ificant

improvem

entfrom

baseline

toweek8in

primaryand

second

aryefficacyend

pointsin

VRXvs.P

BO(p

<0.001)

NCT00731120

(com

pleted)

Augu

st2008–

Janu

ary2009

[108]

Dou

ble-blind,

placebo-controlled,

fixed-dosestud

y–

phaseIII

Safety/

efficacy

stud

y

Stud

yevaluatin

gsafety

and

efficacyof

VRXin

the

treatm

entof

GAD

N:4

57Ag

e:≥18

years

Diagn

osis:

GAD

Leng

th:8

weeks

Experim

ental:

2.5mg/day

(n=152);

10mg/day

(n=152)

Comparator(s):

PBO(n

=153)

HAM

-APercentage

ofrespon

dersin

HAM

-Atotalscore

atweek8

Percentage

ofparticipantsin

HAM

-Aremission

atweek

8CG

I-ICG

I-SHAD

Scales

SDS

SF-36

Health

Care

Resource

UtilizationAssessed

bytheHealth

Econ

omic

AssessmentQuestionn

aire

Nostatisticallysign

ificant

improvem

entneith

erto

the

primaryno

rto

second

ary

endpo

ints

NCT00734071

(com

pleted)

June

2008–

February

2009

[109]

Dou

ble-blind,

placebo-controlled,

parallel-g

roup

,fixed-dosestud

y–

phaseIII

Efficacy/

safety

stud

y

Stud

yevaluatin

gefficacy

andsafety

ofafixed

dose

ofVR

Xin

acutetreatm

ent

ofadultswith

GAD

N:3

04Ag

e:≥18

years

Diagn

osis:

GAD

Leng

th:8

weeks

Experim

ental:

fixed

dose

(5mg/day)

oforalVR

X(n

=152)

Comparator(s):

PBO(n

=152)

HAM

-AHAD

CGI-I

SDS

SF-36

Percentage

ofrespon

dersin

HAM

-Atotalscore

atweek8

Change

from

baselinein

HAM

-Atotalscore

atweek8in

participants

with

baselineHAM

-A≥25

Nostatisticallysign

ificant

improvem

entneith

erto

the

primaryno

rto

second

ary

endpo

ints

(Con

tinued)

EXPERT REVIEW OF NEUROTHERAPEUTICS 487

Table1.

(Con

tinued).

Outcomes

Clinicaltrials.gov

IDnu

mber

Peer-

review

edpu

blication

and/or

abstract

conference

1Stud

ydesign

Endpo

int

classification

Aim/hypothesis

Sample

characteristics

Stud

ydescrip

tion

Primary

Second

ary

Find

ings

NCT00788034

(com

pleted)

Octob

er2008–July

2010

[110]

Dou

ble-blind,

placebo-controlled,

parallel-g

roup

,stud

y–ph

aseIII

Safety/

efficacy

stud

y

Stud

yevaluatin

gefficacyof

VRXin

preventin

grelapse

inpatientswith

GAD

who

hadrespon

dedto

acute

VRXtreatm

ent

N:4

59Ag

e:≥18

years

Diagn

osis:

GAD

Leng

th:2

4weeks

Experim

ental:5or

10mg/day

(n=229)

Comparator(s):

PBO(n

=230)

Timeto

relapse

after24-

week

treatm

ent

Percentage

ofrelapse

HAM

-ACG

ISD

SSF-36

Percentage

ofAE

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488 L. ORSOLINI ET AL.

Preclinical studies

As mentioned previously, most of the pharmacodynamicsproperties displayed by VRX suggest possible anxiolyticeffects. Indeed, although the most part of preclinical studiesfocused on antidepressant features of VRX, some demon-strated peculiar anxiolytic potential of the compound. In arecent study by Guilloux et al. [112], VRX has been demon-strated to counteract both spontaneous and novelty-relatedanxiogenic stimuli, respectively, in the open-field and thenovelty-suppressed feeding behavioral tests, when acutelyadministered. Notably, these effects were displayed at dosescorresponding to 60–70% of SERT occupancy, thereby sug-gesting a mechanism not relying on it, and disappeared athigh dosages, thus paralleling the biphasic effects of 5-HT7receptors antagonists in as well as the dose-dependent effectsof 5-HT3 antagonists in preclinical anxiety paradigms [95].Moreover, in the same study, the anxiolytic effects displayedby VRX persisted in chronic paradigm, with the same peculiardose- and time-dependent features.

The antagonist action at 5-HT7 receptors by VRX has alsobeen demonstrated to play a crucial role in the modulation ofcircadian rhythms, whose dysfunction is considered essentiallyrelevant in the pathophysiology of anxiety disorders. Indeed,the acute administration of VRX has been reported to induce aphase shift in circadian rhythms in animal models (i.e. a delayedtiming), which could be abolished by 5-HT7 agonists [113].Moreover, the circadian delay induced by VRX might bemimicked by the simultaneous administration of 5-HT1a partialagonists plus 5-HT7 antagonists, but not by each compoundsadministered alone, thus suggesting that the effects of VRX oncircadian rhythms may derive by a complex interplay amongmultiple 5-HT receptors modulation exerted by this compound.

The multimodal regulation of serotonin receptors by VRXhas also been correlated to the improvement in memoryformation, whose impairment in fear-elaborating neurocircuitsis considered at the basis of anxiety pathophysiology [114].Indeed, the administration of VRX to rats 1 hour before theadministration of a contextual fear conditioning test has beendemonstrated to improve memory acquisition in the retentiontest [115]. Moreover, successive studies have also demon-strated that VRX, but not escitalopram or duloxetine, mayrestore memory impairment caused by serotonin depletion[116], and these effects are dose dependent and most likelyrely on VRX modulation of 5-HT1a and 5-HT3 receptors [117].

To further corroborate the peculiar characteristics of VRX asantianxiety drug, a recent study has also demonstrated thatonly this compound, as well as amitriptyline, may attenuateanxiety behaviors in animal models of progesterone withdra-wal (which is a classical model of hormone-related dysphoricbehaviors characterized by anxiety, irritability, anhedonia, anddepression), whereas duloxetine and fluoxetine have beenreported as ineffective [118]. Again, these effects might beattributed to the peculiar 5-HT3 receptors antagonist and 5-HT1a partial agonist action.

Therefore, although more specific studies are needed, pre-clinical studies demonstrate a peculiar anxiolytic action of VRX,which could be possibly due to the specific multimodal inter-action with serotonin receptors exerted by this compound.

Clinical efficacy

Phase II studies

A phase II, open-label study (NCT01491035) whose results arestill ongoing evaluated the pharmacokinetics of VRX and itsmetabolites in connection with multiple oral dosing in childand adolescent patients with a diagnosis of depressive oranxiety disorders. There are not still available results onoutcomes.

Phase III studies

RCT – placebo controlledA multinational, randomized, parallel-group, placebo-controlled,fixed-dose (5 mg/day), 8-week study [107] (NCT00744627) wasconducted on 301 patients with a primary diagnosis of GADand an Hamilton Anxiety Rating Scale (HAM-A) total score ≥20at baseline. Subjects were randomized to placebo (n = 151) or5 mg/day of VRX (n = 150). A statistically significant differencebetween VRX and placebo was reported from baseline to week8 in HAM-A total score (p < 0.001) and Clinical GlobalImpression of Improvement (CGI-I) score (p < 0.001). In addition,subjects who received VRX had statistically significant improve-ment (vs. placebo) from baseline to week 8 in all key secondaryefficacy end points (change from baseline at week 8 in HADanxiety subscore, CGI-I, Sheehan Disability Scale [SDS] totalscore, HAM-A response rate, HAM-A total score, SF-36 socialfunctioning subscore; p < 0.05). HAM-A response rates at week8 in the VRX group was higher than in the placebo group (oddsratio [OR] = 2.393; p < 0.001). The response rate was 61.7% inVRX group (vs. 39.9% in placebo group). Subjects treated withVRX were more likely to achieve remission by week 8(OR = 1.958). The remission rate was 30% in VRX group (vs.18% in placebo group).

A randomized, double-blind, fixed-dose study (NCT00731120)[108] comparing the efficacy and tolerability of VRX 2.5 and10 mg in acute treatment of GAD assessed 457 subjects, ran-domly assigned to 2.5 mg/day vs. 10 mg/day vs. placebo. Nostatistically significant improvement was achieved concerningVRX over placebo on the primary and secondary end points(respectively, p = 0.279 for 2.5 mg/day and 0.306 for 10 mg/day).

Another multicenter, placebo-controlled, parallel-group,double-blind trial evaluated the safety and efficacy of a fixeddose of 5 mg VRX on 304 subjects with a primary diagnosis ofGAD [109] (NCT00734071). No statistically significant differ-ences in HAM-A total score changes from baseline to week 8were observed in the VRX group vs. placebo (p = 0.518). Inaddition, no statistically significant differences were observedfor any secondary end point. HAM-A response rates at week 8were 50% in the placebo group (vs. 53% in VRX group;p = 0.602). HAM-A remission rates at week 8 were 22 vs.26%, respectively (p = 0.517). The authors did not describeany clinical advantage for the 5-mg dose of VRX for the treat-ment of subjects with GAD compared to placebo.

A long-term (24–56 weeks), double-blind, randomized,fixed-dose, placebo-controlled relapse prevention study [110](NCT00788034) evaluating the efficacy of VRX in the preven-tion of relapse in patients with GAD who had previously

EXPERT REVIEW OF NEUROTHERAPEUTICS 489

responded to an initial 20-week, open-label, 8-week, flexible-dose (5–10 mg/day) treatment period with VRX (HAM-A totalscore ≤10 at both weeks 16 and 20). The study initiallyrecruited 687 outpatients from 80 centers in 10 countriesand included in the first acute treatment (20 weeks, openlabel). Subsequently, 459 patients who responded were ran-domized to placebo (n = 230) or fixed dose (5 or 10 mg/day)of VRX (n = 229) in the second phase of study (relapse pre-vention, 24–56 weeks, double blind). All patients were thenmonitored and assessed during a 4-week safety follow-up.During the open-label period, the mean HAM-A total scoreand Clinical Global Impression of Severity (CGI-S) decreased atweek 20. At week 20, 79.9% of patients had responded (HAM-A ≥ 50% decrease from baseline), and 60.5% had achievedremission (HAM-A total score ≤7). A statistically significanteffect of VRX compared to placebo was reported on the timeto relapse in the double-blind period (p < 0.0001). The risk ofrelapse in the placebo group was almost 3 times that forpatients in the VRX group (p < 0.0001). The proportion ofpatients who relapsed was significantly lower in the VRXgroup compared to placebo group (15 vs. 24%; p < 0.0001).A statistically significant effect of VRX was reported in stableresponders on the time to relapse in VRX-treated patients vs.placebo-treated (p < 0.001). Stable responders who relapsedwas lower in VRX group vs. placebo one (13 vs. 33%).

RCT – placebo controlled, active referenceA multicenter, randomized, double-blind, placebo-controlled,duloxetine-referenced, phase III study [111] (NCT00730691)was carried out on 781 patients randomized to placebogroup (n = 157), 2.5 mg/day VRX (n = 156), 5 mg/day VRX(n = 156), 10 mg/day VRX (n = 156), and 60 mg/day duloxetinegroup (n = 156). No statistically significant changes from base-line in HAM-A total score were reported in the three VRXtreatment groups. Similar results were reported among thethree VRX groups regarding to all key secondary efficacy endpoints. According to this study, the effects of VRX on mostefficacy outcome measures were not statistically significantlydifferent from that of placebo at daily doses of 2.5, 5, and10 mg in patients with GAD. However, duloxetine 60 mgsignificantly improved the primary end point (p < 0.05) vs.placebo.

Phase IV studies

A 10-week, open-label, flexible dose (5–20 mg/day) adaptivestudy is still evaluating the efficacy of VRX in subjects with PD(NCT02395510).

Safety and tolerability

Clinical studies suggest that VRX has a good safety andtolerability profile. During the open-label period of studyby Baldwin et al. [110], 8.7% of patients withdrew becauseof adverse events (AEs), mainly mild to moderate. Theseparticularly included nausea (27.1%) and headache(17.6%). Other AEs reported included diarrhea (7.4%), dizzi-ness (6.1%), insomnia (3.5%), and sexual dysfunction (2.6%),

i.e. decreased libido, erectile dysfunction, delayed ejacula-tion, loss of libido, anorgasmia, and decreased orgasmicsensation. Serious AEs were described in 1.5% of patients(n = 10), consisting of one committed suicide, suicidal idea-tion (n = 4). Whilst during the double-blind period of thesame study, the incidence of AEs was similar in VRX-treatedand placebo-treated patients (53.9 vs. 55.5%, respectively).Most AEs were mild to moderate. The most reported AEsstatistically significantly higher in VRX group vs. placebogroup were influenza (12 vs. 6%, respectively; p < 0.05)and accidental overdose (5.2 vs. 1.3%; p < 0.05). Three offour patients reported serious AEs in the VRX group vs.placebo. During both the open-label period and the dou-ble-blind period, no clinically significant mean changes inclinical safety laboratory values, vital signs, weight, or ECGvalues were described [110]. According to another phase IIIstudy [118], the most frequently reported treatment-emer-gent AEs were, respectively, in VRX and placebo group:nausea (11.3 vs. 3.3%), headache (6.7 vs. 6%), and dizziness(5.3 vs. 2.7%). The most common AEs in the VRX group vs.placebo included nausea, headache, dry mouth, diarrhea,dizziness, nasopharyngitis, somnolence, constipation, dys-pepsia, upper respiratory tract infection, vomiting, and fati-gue [111]. The most frequently reported AEs includednausea (25% in VRX-group vs. 4.6% in placebo-group),headache (10.1 vs. 8.6%), dizziness (8.1 vs. 3.3%), and drymouth (8.1 vs. 3.3%) [109]. A further study [108] describednausea (24.3% at 10 mg/day, 15.9% at 2.5 mg/day, and 8.5%with placebo), dry mouth (7.9% at 10 mg/day, 9.3% at2.5 mg/day, and 11.1% with placebo), headache (11.8% at10 mg/day, 13.2% at 2.5 mg/day, and 11.1% with placebo),diarrhea (11.2% at 10 mg/day, 8.6% at 2.5 mg/day, and 7.2%with placebo), constipation (5.9% at 10 mg/day, 2.6% at2.5 mg/day, and 3.9% with placebo), and vomiting (5.3%at 10 mg/day, 2.0% at 2.5 mg/day, and 2.6% with placebo),as the most common reported AEs.

A pooled study evaluating the incidence of treatment-emergent sexual dysfunction (TESD) during short-term(8 weeks) treatment with VRX compared to placebo, inseven trials carried out on subjects affected with MDD orGAD [119]. Overall, the incidence of TESD in VRX-treatedgroups combined (37.1%) was not significantly differentfrom placebo. The risk of the onset of TESD in the 5-mgVRX group was lower than in the placebo group and non-inferior to placebo. The effects of VRX treatment in patientswith pre-existing sexual dysfunction at baseline were similarto placebo. Overall, across the analyzed and pooled trials,VRX 5–20 mg was associated with an approximately 5%increase in incidence of TESD over placebo in patients with-out sexual dysfunction at baseline. However, the incidenceof TESD relative to placebo generally increased with increas-ing VRX dose in patients without sexual dysfunction atbaseline.

Overall, in clinical trials focusing on GAD patients, VRX isgenerally well tolerated at 5 mg/day [107,109]. Furthermore,according to Theunissen et al. [120], VRX does not appear toaffect driving, cognitive, and psychomotor performance in 24healthy subjects over a 15-day period.

490 L. ORSOLINI ET AL.

Conclusion

At writing time, a paucity of studies about VRX in the treat-ment of GAD has been published so far. The generalizability ofresults to the broad population of patients with anxiety symp-tomatology and GAD is limited because of few RCTs availableso far as well as sample characteristics, as patients aged<18 years or >65 years were not included, or patients at riskof suicidal behavior, or those with comorbid MDD. In addition,available findings are still contrasting in terms of the potentialefficacy in anxiety disorders and GAD [107–111]. Only twoclinical studies reported a significant improvement in anxietysymptomatology [107,110].

Furthermore, findings coming from preclinical studies arestill limited, most of them mainly exploring the antidepressanteffects of VRX, with only few ones specifically investigating itsanxiolytic efficacy [95,112–118]. In particular, data regarding tothe antianxiety properties of VRX are often extrapolated fromparts of preclinical antidepressant-specific paradigms. At thebest of our knowledge, although several behavioral models ofanxiety have been developed, the current ‘models’ may beaffected by the concept that anxiety is peculiarly a subjectivestatus. Overall, these models may be based on unconditioned(not needing training and owning a high ethological validity; i.e.open field and elevated plus mazes; social interaction, predatoravoidance, freezing test; etc.) or conditioned responses (influ-enced by cognitive processes and needing training; i.e. startleresponse, defensive burying, passive/active avoidance tests;etc.). Furthermore, recent advances in genetic technologiesfavored the development of specific animal strains that display‘anxiety’ behaviors that could be used to be tested with puta-tive anxiolytics drugs (i.e. HAB or high anxiety behavior rats;Syracuse rats; etc.). Therefore, based on the above-mentionedanxiety models, more specific paradigms could be applied tobetter evaluate the antianxiety effects of VRX.

The pharmacological profile of VRX is notably differentfrom that of other known antidepressants. VRX was specificallydesigned to combine a low occupancy of the 5-HT transporter,5-HT3 receptor antagonism, and 5-HT1A partial receptor agon-ism. Given its multimodal and peculiar pharmacological pro-file, VRX has been already approved for MDD in adults [120].Furthermore, some clinical trials carried out on MDD patientsshowed a significant improvement in comorbid anxiety symp-toms [121–124] by suggesting a promising research field alsofor the treatment of anxiety disorders. Despite the fact thatGAD and MDD may share some common biological under-pinnings and VRX owns a promising mechanism of action asalternative option for patients with GAD, findings available sofar are still contrasting [107–111]. Therefore, further RCTs areneeded in order to better evaluate its efficacy in GAD. Inparticular, active-reference RCTs comparing current clinicallyeffective anxiolytics vs. VRX should be considered. In addition,RCTs and open-label trials specifically evaluate short- andlong-term VRX efficacy among GAD patients and compareclinical samples with and/or without comorbid MDD, in orderto better discriminate the underlining substrates of the clinicalimprovement of anxiety symptomatology.

Overall, tolerability of VRX both in short- and in long-termstudies carried out on patients affected with GAD was similar

[107–111]. The tolerability of VRX was not different from thatof placebo in the long-term treatment of patients with GAD,despite the longer exposure of patients randomized toVRX [110].

VRX is well tolerated and appears to have relatively littlepotential for adverse drug interactions. Though there is nosuggestion of superiority over active comparators, most stu-dies suggest that there is a clinically meaningful advantage interms of tolerability [111]. The most common AEs reported(with an incidence ≥5% and at least twice the rate of placebo)comprised nausea (approximately ranging 11–27%, even atthe lowest dose), headache (ranging 6.7–17.6%), dry mouth(8–9%), dizziness (5.3–8.1%), diarrhea (7–8%), constipation(2.6–6%), vomiting (2–6%), and sexual dysfunction (22–34%among women vs. 16–29% among men). Rare but seriousside effects include serotonin syndrome (muscle tightness,fever, seizures, and death), increased risk of bleeding due toimpaired platelet aggregation, increased risk of syndrome ofinappropriate antidiuretic hormone secretion, hyponatremia(headache, weakness, difficulty concentrating, and remember-ing), induced manic episode in bipolar patients, and centralnervous system depression [107–111]. It should be suggestedfor patients who cannot tolerate psychomotor/cognitiveimpairment or cardiac side effects, and it may be helpful forolder patients [125,126]. However, further studies specificallyand directly comparing the efficacy and AEs of VRXwith current clinically effective anxiolytics should beperformed in order to better balance safety and tolerabilityVRX profile.

Expert commentary

It has been well established that GAD is a chronic and disablingdisease that may be associated with psychological and physicalsymptoms. Furthermore, it has also been documented thatresponse and remission rates in patients affected with GADare often disappointing. Therefore, the ‘ideal drug’ shouldhave a rapid onset of effects, with few/absence of drug inter-actions, be effective in achieving remission of symptomatologyand in enhancing quality of life, and should not affect psycho-logical/physical symptomatology, as well as determining dis-abling AEs. Moreover, it should be effective in preventingrelapse, not determining discontinuation symptoms and/ordependence and/or tolerance [11].

Expectations from this new multimodal antidepressant gobeyond its efficacy in the MDD patients, already hugelydemonstrated [127,128]. Although data coming from preclini-cal trials appear promising [112,114–117], clinical trials do notappear to confirm these findings.

Five-year view

In the next 5 years, we can expect that VRX will continue to betested and evaluated among patients affected with anxietysymptomatology with or without comorbid MDD. Futureresearch directions should provide more RCTs specifically tar-geted to clinical sample with GAD, in order to assess the efficacyof this new antidepressant in a larger sample, as well as evaluate

EXPERT REVIEW OF NEUROTHERAPEUTICS 491

the best therapeutic dosage. Then, we should also expect furtherstudies able to deepen its psychopharmacological mechanismsspecific for the anxiety and GAD, as well as discriminate how thetreatment of anxiety dimension may be influenced by the mooddimension in comorbid MDD patients. Furthermore, open-labeltrials should be provided to assess its safety and tolerabilityprofile in the subgroup of patients. In addition, longer-termstudies are needed to demonstrate that the beneficial effects ofVRX are maintained over time.

Finally, the peculiar multimodal pharmacological activity ofVRX may promote the development of newer drugs owningan intrinsic multimodal activity, able to improve both depres-sive and anxiety symptomatology.

Key issues

● Despite numerous pharmacological agents, response ratesto initial treatment with an SSRI or SNRI are still inadequatein the treatment of GAD

● VRX exerts reuptake inhibition on the serotonin transporter,increasing the level of serotonin in the neuronal synapse aswell as selectively binding to a variety of other serotoninreceptors (5-HT3, 5-HT1D, 5-HT7, 5-HT1B, 5-HT1A)

● The antianxiety effects of VRX have been consistentlyobserved in many MDD trials.

● VRX acts as partial agonist at 5-HT1A receptors, which areinvolved in anxiety-like behaviors and in the susceptibilityto anxiety disorders, displaying anxiolytic effects.

● Partial agonist activity at 5-HT1B receptors may exert effectsin modulating stress sensitivity, mood, anxiety and aggres-sion by inhibiting the release of serotonin, norepinephrine,GABA, acetylcholine and glutamate.

● VRX exerts antagonist activity at 5-HT1D which may attenu-ate effects on anxiety symptoms

● The antagonistic activity at 5-HT3 receptors, which have anessential role in controlling gastrointestinal peristalsis andmotility, may attenuate functional and somatoform anxiety-linked gastroenteric disorders

● Preclinical studies showed promising antianxiety effects ofVRX in the treatment of GAD animal models.

● VRX displays a favorable side effect pattern associated witha low risk of pharmacological interactions.

● However, RCT of GAD so far conducted showed inconsis-tent findings regarding the efficacy of VRX.

● Further clinical studies are needed in order to better estab-lish VRX efficacy in GAD and other anxiety disorders.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement withany organization or entity with a financial interest in or financial con-flict with the subject matter or materials discussed in the manuscript.This includes employment, consultancies, honoraria, stock ownership oroptions, expert testimony, grants or patents received or pending, orroyalties.

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