Adrenergic and Opioidergic Receptors Mediate the Analgesic Efﬁcacy of Vortioxetine in Mice

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Article 5-HT1A Serotonergic, α-Adrenergic and Opioidergic Receptors Mediate the Analgesic Efﬁcacy of Vortioxetine in Mice

Nazlı Turan Yücel 1 , Ümmühan Kandemir 2 , Ümide Demir Özkay 1 and Özgür Devrim Can 1,*

1 Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eski¸sehir26470, Turkey; [email protected] (N.T.Y.); [email protected] (Ü.D.Ö.) 2 Department of Pharmacology, Institute of Health Sciences, Anadolu University, Eski¸sehir26470, Turkey; [email protected] * Correspondence: [email protected]

Abstract: Vortioxetine is a multimodal antidepressant drug that affects several brain neurochemicals and has the potential to induce various pharmacological effects on the central nervous system. Therefore, we investigated the centrally mediated analgesic efficacy of this drug and the mechanisms underlying this effect. Analgesic activity of vortioxetine (5, 10 and 20 mg/kg, p.o.) was examined by tail-clip, tail-immersion and hot-plate tests. Motor performance of animals was evaluated using Rota-rod device. Time course measurements (30–180 min) showed that vortioxetine (10 and 20 mg/kg) administrations significantly increased the response latency, percent maximum possible effect and area under the curve values in all of the nociceptive tests. These data pointed out the analgesic effect of vortioxetine on central pathways carrying acute thermal and mechanical nociceptive stimuli. Vortioxetine did not alter the motor coordination of mice indicating that the analgesic activity of this drug was specific. In mechanistic studies, pre-treatments with p-chlorophenylalanine

(serotonin-synthesis inhibitor), NAN-190 (serotonin 5-HT1A receptor antagonist), α-methyl-para- tyrosine (catecholamine-synthesis inhibitor), phentolamine (non-selective α-adrenoceptor blocker),

Citation: Turan Yücel, N.; Kandemir, and naloxone (non-selective opioid receptor blocker) antagonised the vortioxetine-induced analgesia. Ü.; Demir Özkay, Ü.; Can, Ö.D. Obtained ﬁndings indicated that vortioxetine-induced analgesia is mediated by 5-HT1A serotonergic,

5-HT1A Serotonergic, α-Adrenergic α-adrenergic and opioidergic receptors, and contributions of central serotonergic and catecholamin- and Opioidergic Receptors Mediate ergic neurotransmissions are critical for this effect. the Analgesic Efﬁcacy of Vortioxetine in Mice. Molecules 2021, 26, 3242. Keywords: vortioxetine; hot-plate test; tail-clip test; tail-immersion test; monoamines; opioid https://doi.org/10.3390/ molecules26113242

Received: 6 April 2021 1. Introduction Accepted: 25 May 2021 Published: 28 May 2021 Vortioxetine, an antidepressant drug, was licensed by the Food and Drug Adminis- tration (FDA) in September 2013 and the European Medicines Agency (EMA) in October

Publisher’s Note: MDPI stays neutral 2013. This drug has been approved for treating major depressive disorder in adults under ® ® with regard to jurisdictional claims in the trade names Brintellix and Trintellix [1,2]. Vortioxetine is a serotonergic modulator published maps and institutional afﬁl- with antagonistic effects on serotonergic 5-HT3, 5-HT7, and 5-HT1D, agonistic effects on iations. serotonergic 5-HT1A, and partial agonistic effects on serotonergic 5-HT1B receptor subtypes. It also has a potent inhibitory effect on serotonin transporters [2]. Vortioxetine alters neurotransmitter levels in several brain regions. In microdialysis experiments, this drug was found to enhance extracellular serotonin, dopamine, and noradrenaline levels in the medial prefrontal cortex and ventral hippocampus of the rat Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. brain [3]. Moreover acute vortioxetine administration increases serotonin levels in the This article is an open access article nucleus accumbens [4]. The vortioxetine-induced increase in neurotransmitter levels is distributed under the terms and associated with the 5-HT3 receptor antagonistic and 5-HT1A agonistic activities of this drug conditions of the Creative Commons because these receptors are involved in regulating neurotransmitter release in multiple Attribution (CC BY) license (https:// brain regions [5]. In addition to the serotonergic and catecholaminergic systems, vortiox- creativecommons.org/licenses/by/ etine also affects some other neurotransmitter systems in the brain. This drug has been 4.0/). suggested to increase extracellular acetylcholine and histamine levels in the rat medial

Molecules 2021, 26, 3242. https://doi.org/10.3390/molecules26113242 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 21

increase extracellular acetylcholine and histamine levels in the rat medial prefrontal cortex [6] and modulate GABAergic and glutamatergic neurotransmissions in the brain [7]. As a drug affecting several brain neurochemicals, vortioxetine is expected to have a broad pharmacological activity spectrum in the central nervous system (CNS). Recent studies have shown that this molecule does indeed have anxiolytic [8], anti-panic [9], cognitive enhancing [8] and anti-epileptic [10] activities in association with the CNS. Moreo- ver, as an antidepressant drug, vortioxetine has also the potential to block nociceptive Moleculessignals2021, 26 by, 3242 enhancing the pain suppression pathways of the CNS [11,12]. This knowledge2 of 19 suggests that a multimodal antidepressant drug as vortioxetine could has an analgesic activity mediated by central mechanisms. However, the analgesic efficacy of this drug on acute mechanical andprefrontal thermal cortex nociceptive [6] and modulate stimuli GABAergic together and with glutamatergic the underlying neurotransmissions pharma- in the brain [7]. cological mechanisms hasAs anot drug been affecting demonstrated, several brain neurochemicals, so far. Therefore, vortioxetine in this is expectedstudy, towe have investigated whether vortioxetinea broad pharmacological has central activity analgesic spectrum activity. in the central Moreover, nervous systempossible (CNS). involve- Recent ments of serotonergic,studies catecholaminergic have shown that this and molecule opioidergic does indeed systems have anxiolytic in the analgesic [8], anti-panic effect [9], cognitive enhancing [8] and anti-epileptic [10] activities in association with the CNS. More- of this drug were elucidatedover, as an antidepressantby mechanism drug, studies vortioxetine performed has also the with potential p-chlorophenylala- to block nociceptive nine (PCPA, serotonin-synthesissignals by enhancing inhibitor), the pain suppressionNAN-190 pathways (serotonin of the 5-HT CNS [1A11 ,receptor12]. This knowledge antagonist), α-methyl-para-tyrosinesuggests that (AMPT, a multimodal catecholamine-synthesis antidepressant drug as vortioxetine inhibitor), could phentolamine has an analgesic activity mediated by central mechanisms. However, the analgesic efﬁcacy of this drug (non-selective α-adrenoceptoron acute mechanical blocker), and thermal propranolol nociceptive stimuli (non-selective together with theβ-adrenoceptor underlying phar- blocker), and naloxonemacological (non-selective mechanisms opioid has not receptor been demonstrated, blocker). so far. Therefore, in this study, we investigated whether vortioxetine has central analgesic activity. Moreover, possible involve- ments of serotonergic, catecholaminergic and opioidergic systems in the analgesic effect of 2. Results this drug were elucidated by mechanism studies performed with p-chlorophenylalanine 2.1. Motor Coordination(PCPA, serotonin-synthesis inhibitor), NAN-190 (serotonin 5-HT1A receptor antagonist), α-methyl-para-tyrosine (AMPT, catecholamine-synthesis inhibitor), phentolamine (non- Rota-Rod Test selective α-adrenoceptor blocker), propranolol (non-selective β-adrenoceptor blocker), and The effects of acutenaloxone administrations (non-selective opioid of vortioxetine receptor blocker). (5, 10 and 20 mg/kg), diazepam, PCPA, NAN-190, AMPT,2. Results phentolamine, propranolol, and naloxone on the falling latencies of the mice, as assessed2.1. Motor Coordinationusing the Rota-rod device, are shown in Figure 1 [F (10, 66) = Rota-Rod Test 14.48, p < 0.001]. Tukey’s HSD test for multiple comparisons showed that the motor-coor- The effects of acute administrations of vortioxetine (5, 10 and 20 mg/kg), diazepam, dination parametersPCPA, of the NAN-190, mice AMPT,administered phentolamine, vortioxetine, propranolol, and PCPA, naloxone NAN-190, on the falling AMPT, latencies phentolamine, propranolol,of the mice, and as assessed naloxone using thedid Rota-rod not significantly device, are shown differ in Figure from1 [F (10,those 66) =of 14.48, the control group mice. Diazepamp < 0.001]. Tukey’s at 2 HSDmg/kg test fordose multiple impaired comparisons the motor showed performances that the motor-coordination of mice, parameters of the mice administered vortioxetine, PCPA, NAN-190, AMPT, phentolamine, as expected. propranolol, and naloxone did not signiﬁcantly differ from those of the control group mice. Diazepam at 2 mg/kg dose impaired the motor performances of mice, as expected.

Figure 1. The effects of diazepam (2 mg/kg, p.o.), vortioxetine (5, 10 and 20 mg/kg, p.o.), PCPA (100 mg/kg, 4 day, i.p.), FigureNAN-190 1. (0.5The mg/kg, effectsi.p. ),of AMPT diazepam (100 mg/kg, (2 mg/kg,i.p.), phentolamine p.o.), vortioxetine (4 mg/kg, i.p. ),(5, propranolol 10 and 20 (2 mg/kg,mg/kg,i.p. p.o.), and), naloxonePCPA (100 mg/kg,(5.48 mg/kg, 4 day,i.p. )i.p. administrations), NAN-190 on (0.5 motor mg/kg, coordination i.p.), parametersAMPT (100 of mice mg/kg, in the i.p. Rota-Rod), phentolamine Test. Signiﬁcant (4 differencemg/kg, i.p.against), propranolol control group (2 *** mg/kg,p < 0.001, i.p. One), wayand ANOVA, naloxone post-hoc (5.48 Tukey mg/kg, test, ni.p. = 7.) administrations on motor coordination parameters of mice in the Rota-Rod Test. Significant difference against control group *** p < 0.001, One way ANOVA, post-hoc Tukey test, n = 7.

Molecules 2021, 26, 3242 3 of 19

2.2. Analgesic Activity 2.2.1. Analgesic Effect of Vortioxetine on Mechanical Noxious Stimuli-Induced Pain Behavior Molecules 2021, 26, x FOR PEER REVIEW 4 of 21 The effects of acute vortioxetine (5, 10 and 20 mg/kg) administrations on response latency, MPE% and AUC values obtained from the tail-clip tests are shown in Figure2.

Figure 2. The effects of morphine (10 mg/kg, i.p.) and vortioxetine (5, 10 and 20 mg/kg, p.o.) Figure 2. The effects of morphine (10 mg/kg, i.p.) and vortioxetine (5, 10 and 20 mg/kg, p.o.) admin- istrationsadministrations on latency (A), onMPE% latency (B)( Aand), MPE% AUC (C (B)) values and AUC of mice (C) valuesin the oftail-clip mice intest the (mechanical tail-clip test (mechanical nociception).nociception). (A) and ( BA):) andSignificant (B): Signiﬁcant difference difference against control against group control at group 30th min at 30th * p min< 0.05, * p **< 0.05,p < ** p < 0.01, 0.01, *** ***p < p0.001;< 0.001; at 60that 60th min min & p <& 0.05,p < 0.05, && p &&< 0.01,p < &&& 0.01, p &&&< 0.001;p < at 0.001; 90that min 90th éé p min < 0.01,éé p ééé< 0.01,p < 0.001;ééé p < 0.001; at at 120th 120thmin ^ p min < 0.05,ˆ p <^^ 0.05,p < 0.01,ˆˆ p ^^^< 0.01,p < 0.001;ˆˆˆ p < at 0.001; 180th atmin 180th + p < min 0.05;+ ++p p< < 0.05; 0.01.++ Two-wayp < 0.01. repeated Two-way repeated ANOVA,ANOVA, post-hoc post-hocTukey test, Tukey n = 7. test, (C): nSignificant = 7. (C): Signiﬁcantdifference against difference control against group control ** p < 0.01, group *** ** p < 0.01, p < 0.001;*** One-wayp < 0.001; ANOVA, One-way post-hoc ANOVA, Tukey post-hoc test, n Tukey = 7. test, n = 7.

Molecules 2021, 26, 3242 4 of 19

Two-way repeated ANOVA analysis indicated that both of the treatment ([F (4, 30) = 16.95, p < 0.001]) and time ([F (3.084, 92.51) = 83.55, p < 0.001]) factors affected the response latency values in the tail-clip tests. There was a significant interaction between treatment and time factors ([F (20, 150) = 11.33, p < 0.001]) (Figure2A). Treatment ([F (4, 30) = 42.94, p < 0.001]) and time ([F (2.879, 86.37) = 39.47, p < 0.001]) factors also affected the MPE% values in the same test with a significant interaction between them ([F (16, 120) = 6.85, p < 0.001]) (Figure2B ). Besides, vortioxetine administrated at 10 mg/kg (p < 0.01) and 20 mg/kg (p < 0.001) doses caused significant increase in the AUC values compared to the control group ([F (4, 150) = 18.54, p < 0.001]) (Figure2C).

2.2.2. Spinally Mediated Analgesic Effect of Vortioxetine on Thermal Noxious Stimuli-Induced Pain Behavior The effects of acute vortioxetine (5, 10 and 20 mg/kg) administration on response latency, MPE% and AUC values obtained from the tail-immersion tests are shown in Figure3. Two-way repeated ANOVA analysis indicated that both of the treatment ([F (4, 30) = 51.42, p < 0.001]) and time ([F (3.154, 94.63) = 64.26, p < 0.001]) factors affected the response latency values in the tail-immersion tests. There was a significant interaction between treatment and time factors ([F (20, 150) = 8.925, p < 0.001]) (Figure3A). Treatment ([ F (4, 30) = 38.0, p < 0.001]) and time ([F (2.858, 85.74) = 29.5, p < 0.001]) factors also affected the MPE% values in the same test with a significant interaction between them ([F (16, 120) = 6.716, p < 0.001]) (Figure3B). Besides, vortioxetine administrated at 10 mg/kg ( p < 0.01) and 20 mg/kg (p < 0.001) doses caused significant increase in the AUC values compared to the control group ([F (4, 150) = 13.98, p < 0.001]) (Figure3C).

2.2.3. Supraspinally Mediated Analgesic Effect of Vortioxetine on Thermal Noxious Stimuli-Induced Pain Behavior The effects of acute vortioxetine (5, 10 and 20 mg/kg) administration on response latency, MPE% and AUC values obtained from the hot-plate tests are shown in Figure4. Two-way repeated ANOVA analysis indicated that both of the treatment ([F (4, 30) = 12.53, p < 0.001]) and time ([F (3.958, 118.8) = 68.46, p < 0.001]) factors affected the response latency values in the hot-plate tests. There was a significant interaction between treatment and time factors ([F (20, 150) = 5.304, p < 0.001]) (Figure4A). Treatment ([F (4, 30) = 23.57, p < 0.001]) and time ([F (3.366, 101.0) = 28.08, p < 0.001]) factors also affected the MPE% values in the same test with a significant interaction between them ([F (16, 120) = 2.986, p < 0.001]) (Figure4B). Besides, vortioxetine administrated at 10 mg/kg ( p < 0.05) and 20 mg/kg (p < 0.001) doses caused significant increase in the AUC values compared to the control group ([F (4, 150) = 10.71, p < 0.001]) (Figure4C). In all of the three nociceptive tests, results of the multiple comparison analysis showed that vortioxetine at its 20 mg/kg dose, significantly increased the response latencies and MPE% values with respect to the corresponding control levels, in all of the time points. Although this drug showed significant efficacy up to 180 min when administered at a dose of 10 mg/kg, it was effective only in the 60th minute at a dose of 5 mg/kg. Reference drug morphine (10 mg/kg, i.p.) was exhibited its analgesic effect, as expected (Figures2–4).

2.3. Mechanistic Studies After the acute analgesic efficacy profile of vortioxetine was determined, mechanistic studies were conducted to elucidate the mechanism of this effect. In time course studies conducted with this drug, it was observed that vortioxetine showed its highest activity in the 60th minute at every dose administrated; then mechanistic studies were carried out by comparing MPE% values at 60th minute. This drug was administrated at 10 mg/kg dose in the mechanistic studies, since there were no statistically significant difference between the MPE% values of 10 and 20 mg/kg doses 60 min after the administrations. MoleculesMolecules 20212021, 26, ,26 x ,FOR 3242 PEER REVIEW 5 of5 of21 19

2.2.2.2.3.1. Spinally Involvement Mediated of Serotonergic Analgesic Effect System of Vortioxetine in the Analgesic on Thermal Effect of Noxious Vortioxetine Stimuli- Induced Pain Behavior The effects of PCPA pre-treatment on vortioxetine (10 mg/kg)-induced analgesic responsesThe effects in the of acute tail-clip, vortioxetine tail-immersion (5, 10 and 20 hot-plate mg/kg) tests administrati are shownon inon Figures response5A, la-5B tency,and 5MPE%C, respectively. and AUC values obtained from the tail-immersion tests are shown in Figure 3.

FigureFigure 3. The 3. The effects effects of morphine of morphine (10 mg/kg, (10 mg/kg, i.p.) andi.p. vortioxetine) and vortioxetine (5, 10 and (5, 20 10 mg/kg, and 20 p.o. mg/kg,) admin-p.o.) istrationsadministrations on latency on (A latency), MPE% (A (),B MPE%) and AUC (B) and(C) values AUC (ofC) mice values in ofthe mice tail-immersion in the tail-immersion test (ther- test mal(thermal nociception). nociception). (A) and (A (B)): and Significant (B): Signiﬁcant difference difference against against control control group groupat 30th at min 30th * minp < 0.05,* p < ** 0.05, p <** 0.01,p < 0.01,*** p ***< 0.001;p < 0.001; at 60th at min 60th & minp < 0.05,& p <&& 0.05, p < 0.01,&& p &&&< 0.01,p < 0.001;&&& pat< 90th 0.001; min at éé 90th p < 0.01; min ééééé p < 0.01; 0.001ééé pat< 120th 0.001 minat 120th ^^ p < min0.01, ˆˆ^^^p p< < 0.01,0.001;ˆˆˆ atp 180th< 0.001; minat + p 180th < 0.05, min ++ p +< p0.01.< 0.05, Two-way++ p < repeated 0.01. Two-way ANOVA,repeated post-hoc ANOVA, Tukey post-hoc test, n Tukey= 7. (C test,): Significant n = 7. (differenceC): Signiﬁcant against difference control group against ** controlp < 0.01, group*** p <** 0.001;p < 0.01, One-way *** p < ANOVA, 0.001; One-way post-hoc ANOVA, Tukey test, post-hoc n = 7. Tukey test, n = 7.

Two-way repeated ANOVA analysis indicated that both of the treatment ([F (4, 30) = 51.42, p < 0.001]) and time ([F (3.154, 94.63) = 64.26, p < 0.001]) factors affected the response latency values in the tail-immersion tests. There was a significant interaction between

Molecules 2021, 26, x FOR PEER REVIEW 6 of 21

treatment and time factors ([F (20, 150) = 8.925, p < 0.001]) (Figure 3A). Treatment ([F (4, 30) = 38.0, p < 0.001]) and time ([F (2.858, 85.74) = 29.5, p < 0.001]) factors also affected the MPE% values in the same test with a significant interaction between them ([F (16, 120) = 6.716, p < 0.001]) (Figure 3B). Besides, vortioxetine administrated at 10 mg/kg (p < 0.01) and 20 mg/kg (p < 0.001) doses caused significant increase in the AUC values compared to the control group ([F (4, 150) = 13.98, p < 0.001]) (Figure 3C).

2.2.3. Supraspinally Mediated Analgesic Effect of Vortioxetine on Thermal Noxious Stimuli-Induced Pain Behavior Molecules 2021, 26, 3242 The effects of acute vortioxetine (5, 10 and 20 mg/kg) administration on response 6la- of 19 tency, MPE% and AUC values obtained from the hot-plate tests are shown in Figure 4.

Figure 4. The effects of morphine (10 mg/kg, i.p.) and vortioxetine (5, 10 and 20 mg/kg, p.o.) administrations on latency (A), MPE% (B) and AUC (C) values of mice in the hot-plate test (thermal nociception). (A) and (B): Signiﬁcant difference against control group at 30th min * p < 0.05, ** p < 0.01, *** p < 0.001; at 60th min & p < 0.05, && p < 0.01, &&& p < 0.001; at 90th min é p < 0.05, éé p < 0.01; at 120th min ˆ p < 0.05, ˆˆ p < 0.01, ˆˆˆ p < 0.001; at 180th min + p < 0.05. Two-way repeated ANOVA,

post-hoc Tukey test, n = 7. (C): Signiﬁcant difference against control group * p < 0.05, *** p < 0.001; One-way ANOVA, post-hoc Tukey test, n = 7.

Two-way ANOVA indicated that the treatment (tail-clip test: [F (1, 24) = 126.0, p < 0.001]; tail-immersion test: [F (1, 24) = 71.62, p < 0.001]; hot-plate test: [F (1, 24) = 63.30, p < 0.001]) and antagonist (tail-clip test: [F (1, 24) = 38.51, p < 0.001]; tail-immersion test: [F (1, 24) = 19.26, p < 0.001]; hot-plate test: [F (1, 24) = 11.77, p < 0.01]) factors affected the MPE% values. Furthermore, there was a statistically signiﬁcant interaction (tail-clip test: [F (1, 24) = 41.50, p < 0.001]; tail-immersion test: [F (1, 24) = 18.09, p < 0.001]; hot-plate test: Molecules 2021, 26, 3242 7 of 19

[F (1, 24) = 14.40, p < 0.001]) between these two factors. Bonferroni analysis revealed that Molecules 2021, 26, x FOR PEER REVIEW administration of PCPA at 100 mg/kg for 4 consecutive days antagonized the analgesic8 of 21

activity of vortioxetine in the tail-clip (p < 0.001), tail-immersion (p < 0.001) and hot-plate (p < 0.001) tests.

Figure 5. Involvement of serotonergic system in the analgesic effects of vortioxetine in the tail-clip (A), Figure 5.tail-immersion Involvement (B of) and serotonergic hot-plate (C system) tests. Significantin the anal differencegesic effects against of vehiclevortioxetine treated in control the tail-clip groups (A), tail-immersion*** p < 0.001; Significant(B) and hot-plate difference (C against) tests. vortioxetineSignificant difference administrated against control vehicle groups treated&&& p < control 0.001; &&& groups ***Significant p < 0.001; difference Significant against difference PCPA administrated against vortioxetine control groups administratedé p < 0.05, controléé p < 0.01. groups Two-way p < 0.001; Significant difference against PCPA administrated control groups é p < 0.05, éé p < 0.01. ANOVA, post-hoc Bonferroni test, n = 7. Two-way ANOVA, post-hoc Bonferroni test, n = 7.

2.3.2. Involvement of Serotonergic 5HT1A Receptors in the Analgesic Effect of Vortioxetine Two-wayThe ANOVA effects of NAN-190 indicated pre-treatment that the treatment on vortioxetine (tail-clip (10 mg/kg)-induced test: [F (1, 24) analgesic= 126.0, re-p < 0.001]; tail-immersionsponses in the tail-clip, test: [F tail-immersion (1, 24) = 71.62, and hot-platep < 0.001]; tests hot-plate are shown test: in Figures [F (1, 24)6A, 6=B 63.30, and6C p < 0.001]) respectively.and antagonist Two-way (tail-clip ANOVA test: indicated[F (1, 24) that = 38.51, the treatment p < 0.001]; (tail-clip tail-immersion test: [F (1, 24) test: = 160.9, [F (1, 24) = 19.26,p < 0.001]; p < 0.001]; tail-immersion hot-plate test: test: [F [F (1, (1, 24) 24) = 126.3,= 11.77,p < p 0.001]; < 0.01]) hot-plate factors test: affected [F (1, 24)the = MPE% 46.00, values. pFurthermore,< 0.001]) and antagonistthere was (tail-clipa statistically test: [F significant (1, 24) = 32.21, interactionp < 0.001]; (tail-clip tail-immersion test: [F (1, test: 24) = 41.50,[F p (1,< 0.001]; 24) = 37.33, tail-immersionp < 0.001]; hot-platetest: [F (1, test: 24) F = (1, 18.09, 24) = p 9.03, < 0.001];p < 0.01]) hot-plate factors test: affected [F (1, the 24) = 14.40,MPE% p < 0.001]) values between of mice. Furthermore, these two factors. there was Bo anferroni statistically analysis signiﬁcant revealed interaction that (tail-clip admin- test: [F (1, 24) = 38.86, p < 0.001]; tail-immersion test: [F (1, 24) = 37.46, p < 0.001]; hot-plate istration of PCPA at 100 mg/kg for 4 consecutive days antagonized the analgesic activity test: [F (1, 24) = 11.39, p < 0.01]) between these two factors. Bonferroni analysis revealed of vortioxetinethat NAN-190 in the antagonizedtail-clip (p < 0.001), the analgesic tail-immersion activity of ( vortioxetinep < 0.001) and in hot-plate tail-clip (p (p< < 0.001), 0.001) tests. tail-immersion (p < 0.001) and hot-plate (p < 0.001) tests.

2.3.2. Involvement2.3.3. Involvement of Serotonergic of Catecholaminergic 5HT1A Receptors System inin the the Analgesic Analgesic Effect Effect of of Vortioxetine Vortioxetine The effectsThe effects of NAN-190 of AMPT pre-tr pre-treatmenteatment on on the vortioxetine vortioxetine (10(10 mg/kg)-inducedmg/kg)-induced analgesic analgesic responses in the tail-clip, tail-immersion and hot-plate tests are shown in Figures7A,7B and7C , responses in the tail-clip, tail-immersion and hot-plate tests are shown in Figure 6A, Fig- respectively. Two-way ANOVA indicated that the treatment (tail-clip test: [F (1, 24) = 62.75, ure 6B pand< 0.001]; Figure tail-immersion 6C respectively. test: [F Two-wa (1, 24) =y 49.62, ANOVAp < 0.001]; indicated hot-plate that test: the [F treatment (1, 24) = 48.15, (tail- clip test:p <[F 0.001]) (1, 24) and = 160.9, antagonist p < 0.001]; (tail-clip tail-immersion test: [F (1, 24) test: = 44.15, [F (1,p < 24) 0.001]; = 126.3, tail-immersion p < 0.001]; test:hot- plate test:[F (1, [F 24)(1, =24) 31.41, = 46.00,p < 0.001]; p < 0.001]) hot-plate and test: antagonist F (1, 24) = (tail-clip 27.83, p < test: 0.001]) [F factors(1, 24) affected= 32.21, the p < 0.001]; MPE%tail-immersion values. Furthermore, test: [F (1, 24) there = 37.33, was a statisticallyp < 0.001]; signiﬁcanthot-plate test: interaction F (1, 24) (tail-clip = 9.03, test: p < 0.01]) factors[F (1, 24)affected = 37.78, thep MPE%< 0.001]; values tail-immersion of mice. Furthermore, test: [F (1, 24) =there 30.26, wasp < a 0.001];statistically hot-plate significant interaction (tail-clip test: [F (1, 24) = 38.86, p < 0.001]; tail-immersion test: [F (1, 24) = 37.46, p < 0.001]; hot-plate test: [F (1, 24) = 11.39, p < 0.01]) between these two factors. Bonferroni analysis revealed that NAN-190 antagonized the analgesic activity of vortioxetine in tail-clip (p < 0.001), tail-immersion (p < 0.001) and hot-plate (p < 0.001) tests.

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Molecules 2021, 26, x FOR PEER REVIEW test: [F (1, 24) = 31.61, p < 0.001]) between these two factors. Bonferroni analysis revealed9 of 21

that AMPT antagonized the analgesic activity of vortioxetine in the tail-clip (p < 0.001), tail-immersion (p < 0.001) and hot-plate (p < 0.001) tests.

Figure 6. Involvement of serotonergic 5HT1A receptors in the antinociceptive effects of vortiox-etine Figure 6.in Involvement the tail-clip (A of), tail-immersionserotonergic 5HT (B) and1A receptors hot-plate in (C )the tests. antinociceptive Significant difference effects againstof vortiox- vehicle etine in treatedthe tail-clip control (A groups), tail-immersion *** p < 0.001; ( SignificantB) and hot-plate difference (C against) tests. vortioxetineSignificant administrateddifference against control vehicle groupstreated&&& controlp < 0.001; groups Significant *** p < difference0.001; Significant against NAN-190 difference administrated against vortioxetine control groups adminis-é p < 0.05, &&& trated controléé p < 0.01, groupsééé p < 0.001. p < 0.001; Two-way Significant ANOVA, difference post-hoc Bonferroni against NAN-190 test, n = 7. administrated control groups é p < 0.05, éé p < 0.01, ééé p < 0.001. Two-way ANOVA, post-hoc Bonferroni test, n = 7. 2.3.4. Involvement of α-Adrenergic Receptors in the Analgesic Effect of Vortioxetine 2.3.3. InvolvementThe effects of Catecholaminergic of phentolamine pre-treatment System in the on Analgesic vortioxetine Effect (10 of mg/kg)-induced Vortioxetine Theanalgesic effects of responses AMPT pre-treatment in the tail-clip, on tail-immersion the vortioxetine and (10 hot-plate mg/kg)-induced tests are shown analgesic in responsesFigures in the8A, 8tail-clip,B and8C tail-immersion, respectively. Two-way and hot-plate ANOVA tests indicated are shown that thein Figure treatment 7A, (tail- Fig- ure 7B clipand test:Figure [F (1,7C, 24) respectively. = 208.6, p < 0.001];Two-wa tail-immersiony ANOVA indicated test: [F (1, that 24) the = 94.27, treatmentp < 0.001]; (tail- hot-plate test: [F (1, 24) = 53.11, p < 0.001]) and antagonist (tail-clip test: [F (1, 24) = 77.62, clip test: [F (1, 24) = 62.75, p < 0.001]; tail-immersion test: [F (1, 24) = 49.62, p < 0.001]; hot- p < 0.001]; tail-immersion test: [F (1, 24) = 32.77, p < 0.001]; hot-plate test: F (1, 24) = 18.71, plate test:p < [F 0.001]) (1, 24) factors = 48.15, affected p < 0.001]) the MPE% and antagonist values. Furthermore, (tail-clip test: there [F was(1, 24) a statistically= 44.15, p < 0.001]; significanttail-immersion interaction test: [F (tail-clip (1, 24) test:= 31.41, [F (1,p < 24) 0.001]; = 80.75, hot-platep < 0.001]; test: F tail-immersion (1, 24) = 27.83, test: p < 0.001]) [factorsF (1, 24) affected = 39.06, pthe< 0.001];MPE% hot-plate values. test:Furthermore, [F (1, 24) = there 13.75, wasp < 0.01])a statistically between significant these two interactionfactors. (tail-clip Bonferroni test: [F analysis (1, 24) revealed = 37.78, thatp < 0.001]; administration tail-immersion of phentolamine test: [F (1, at 24) 4mg/kg = 30.26, p < 0.001];(i.p. )hot-plate led to antagonism test: [F (1, of 24) the = analgesic 31.61, p < activity 0.001]) of between vortioxetine these in two the tail-clipfactors. (Bonferronip < 0.001), analysistail-immersion revealed that (p AMPT< 0.001) antagonized and hot-plate the (p < analgesic 0.001) tests. activity of vortioxetine in the tail- clip (p <2.3.5. 0.001), Involvement tail-immersion of β-Adrenergic (p < 0.001) Receptors and hot-plate in the Analgesic (p < 0.001) Effect tests. of Vortioxetine The effects of propranolol pre-treatment on the vortioxetine (10 mg/kg)-induced analgesic responses in tail-clip, tail-immersion and hot-plate tests are shown in Figures9A, 9B and9C, respectively.

Molecules 2021, 26, x FOR PEER REVIEW 10 of 21 Molecules 2021, 26, 3242 9 of 19

Figure 7. Involvement of catecholaminergic system in the analgesic effects of vortioxetine in the Figure 7.tail-clip Involvement (A), tail-immersion of catecholaminergic (B) and hot-plate system (C) tests. in th Signiﬁcante analgesic difference effects against of vortioxetine vehicle treated in the Molecules 2021, 26, x FOR PEER REVIEW 11 of 21 tail-clipcontrol (A), tail-immersion groups *** p < 0.001; (B) and Signiﬁcant hot-plate difference (C) tests. against Significant vortioxetine di administratedfference against control vehicle groups treated &&&controlp < 0.001.groups Two-way *** p < ANOVA,0.001; Significant post-hoc Bonferroni difference test, agains n = 7. t vortioxetine administrated control groups &&& p < 0.001. Two-way ANOVA, post-hoc Bonferroni test, n = 7.

2.3.4. Involvement of α-Adrenergic Receptors in the Analgesic Effect of Vortioxetine The effects of phentolamine pre-treatment on vortioxetine (10 mg/kg)-induced analgesic responses in the tail-clip, tail-immersion and hot-plate tests are shown in Figure 8A, Figure 8B and Figure 8C, respectively. Two-way ANOVA indicated that the treatment (tail-clip test: [F (1, 24) = 208.6, p < 0.001]; tail-immersion test: [F (1, 24) = 94.27, p < 0.001]; hot-plate test: [F (1, 24) = 53.11, p < 0.001]) and antagonist (tail-clip test: [F (1, 24) = 77.62, p < 0.001]; tail-immersion test: [F (1, 24) = 32.77, p < 0.001]; hot-plate test: F (1, 24) = 18.71, p < 0.001]) factors affected the MPE% values. Furthermore, there was a statistically significant interaction (tail-clip test: [F (1, 24) = 80.75, p < 0.001]; tail-immersion test: [F (1, 24) = 39.06, p < 0.001]; hot-plate test: [F (1, 24) = 13.75, p < 0.01]) between these two factors. Bon- ferroni analysis revealed that administration of phentolamine at 4 mg/kg (i.p.) led to antagonism of the analgesic activity of vortioxetine in the tail-clip (p < 0.001), tail-immersion (p < 0.001) and hot-plate (p < 0.001) tests.

Figure 8. Involvement of α-adrenergic receptors in the analgesic effects of vortioxetine in the tail- Figure 8. Involvement of α-adrenergic receptors in the analgesic effects of vortioxetine in the tail- clip (A), tail-immersion (B) and hot-plate (C) tests. Significant difference against vehicle treated clip (A), tail-immersion (B) and hot-plate (C) tests. Significant difference against vehicle treated p controlcontrol groups groups *** *** p < 0.001;< 0.001; Significant Significant difference difference against against vortioxetine vortioxetine administrated administrated control control groups &&& é groupsp &&&<0.001; p < 0.001;Significant Significant difference difference against against phentolamine phentolamine administrated administrated control control groups groupsp <é p 0.05, éé < 0.05,p < éé 0.01.p < 0.01. Two-way Two-way ANOVA, ANOVA, post-hoc post-hoc Bonferroni Bonferroni test, test, n =7. n = 7.

2.3.5. Involvement of β-Adrenergic Receptors in the Analgesic Effect of Vortioxetine The effects of propranolol pre-treatment on the vortioxetine (10 mg/kg)-induced analgesic responses in tail-clip, tail-immersion and hot-plate tests are shown in Figure 9A, Figure 9B and Figure 9C, respectively.

Figure 9. Involvement of β-adrenergic receptors in the analgesic effects of vortioxetine in the tail- clip (A), tail-immersion (B) and hot-plate (C) tests. Significant difference against vehicle treated control groups *** p < 0.001. Two-way ANOVA, post-hoc Bonferroni test, n = 7.

Molecules 2021, 26, x FOR PEER REVIEW 11 of 21

Figure 8. Involvement of α-adrenergic receptors in the analgesic effects of vortioxetine in the tail- clip (A), tail-immersion (B) and hot-plate (C) tests. Significant difference against vehicle treated control groups *** p < 0.001; Significant difference against vortioxetine administrated control groups &&& p < 0.001; Significant difference against phentolamine administrated control groups é p < 0.05, éé p < 0.01. Two-way ANOVA, post-hoc Bonferroni test, n = 7.

2.3.5. Involvement of β-Adrenergic Receptors in the Analgesic Effect of Vortioxetine The effects of propranolol pre-treatment on the vortioxetine (10 mg/kg)-induced an- Molecules 2021, 26, 3242 algesic responses in tail-clip, tail-immersion and hot-plate tests are shown in10 Figure of 19 9A, Figure 9B and Figure 9C, respectively.

Figure 9. Involvement of β-adrenergic receptors in the analgesic effects of vortioxetine in the tail- Figureclip ( A9.), Involvement tail-immersion of ( Bβ)-adrenergic and hot-plate receptors (C) tests. in Signiﬁcant the analgesic difference effects against of vortioxetine vehicle treated in the tail- clipcontrol (A), tail-immersion groups *** p < 0.001. (B) and Two-way hot-plate ANOVA, (C) tests. post-hoc Significant Bonferroni difference test, n = 7. against vehicle treated control groups *** p < 0.001. Two-way ANOVA, post-hoc Bonferroni test, n = 7. Two-way ANOVA indicated that the treatment (tail-clip test: [F (1, 24) = 166.4, p < 0.001]; tail-immersion: [F (1, 24) = 185.3, p < 0.001]; hot-plate: [F (1, 24) = 108.5, p < 0.001]) factors affected the MPE% values, whereas the antagonist factor did not (tail- clip test: [F (1, 24) = 0.002, p > 0.05]; tail-immersion: [F (1, 24) = 0.01, p > 0.05]; hot- plate: F (1, 24) = 0.07, p > 0.05]). In addition, there was no signiﬁcant interaction (tail- clip test: [F (1, 24) = 0.03, p > 0.05]; tail-immersion: [F (1, 24) = 0.09, p > 0.05]; hot-plate: [F (1, 24) = 0.01, p > 0.05]) between these two factors. Bonferroni analysis revealed that administration of propranolol at 2 mg/kg (i.p.) did not cause antagonism of the analgesic activity of vortioxetine in the tail-clip (p > 0.05), tail-immersion (p > 0.05) and hot-plate tests (p > 0.05).

2.3.6. Involvement of Opioid Receptors in the Analgesic Effect of Vortioxetine The effects of naloxone pre-treatment on vortioxetine (10 mg/kg)-induced analgesic responses in the tail-clip, tail-immersion and hot-plate tests are shown Figures 10A, 10B and 10C, respectively. Two-way ANOVA indicated that the treatment (tail-clip test: [F (1, 24) = 68.51, p < 0.001]; tail-immersion test: [F (1, 24) = 65.43, p < 0.001]; hot-plate test: [F (1, 24) = 26.23, p < 0.001]) and antagonist (tail-clip test: [F (1, 24) = 31.57, p < 0.001]; tail-immersion test: [F (1, 24) = 34.47, p < 0.001]; hot-plate test: [F (1, 24) = 10.65, p < 0.01]) factors affected the MPE% values. Furthermore, there was a statistically signiﬁcant interaction (tail-clip test: [F (1, 24) = 29.63, p < 0.001]; tail-immersion test: [F (1, 24) = 30.73, p < 0.001]; hot-plate test: [F (1, 24) = 10.36, p < 0.01]) between these two factors. Multiple comparison Bonferroni analysis revealed that pre-treatment with naloxone (5.48 mg/kg; single dose) led to antagonism of the analgesic activity of vortioxetine in the tail-clip (p < 0.001), tail-immersion (p < 0.001) and hot-plate (p < 0.001) tests. Molecules 2021, 26, x FOR PEER REVIEW 12 of 21

Two-way ANOVA indicated that the treatment (tail-clip test: [F (1, 24) = 166.4, p < 0.001]; tail-immersion: [F (1, 24) = 185.3, p < 0.001]; hot-plate: [F (1, 24) = 108.5, p < 0.001]) factors affected the MPE% values, whereas the antagonist factor did not (tail-clip test: [F (1, 24) = 0.002, p > 0.05]; tail-immersion: [F (1, 24) = 0.01, p > 0.05]; hot-plate: F (1, 24) = 0.07, p > 0.05]). In addition, there was no significant interaction (tail-clip test: [F (1, 24) = 0.03, p > 0.05]; tail-immersion: [F (1, 24) = 0.09, p > 0.05]; hot-plate: [F (1, 24) = 0.01, p > 0.05]) between these two factors. Bonferroni analysis revealed that administration of propranolol at 2 mg/kg (i.p.) did not cause antagonism of the analgesic activity of vortioxetine in the tail-clip (p > 0.05), tail- immersion (p > 0.05) and hot-plate tests (p > 0.05).

2.3.6. Involvement of Opioid Receptors in the Analgesic Effect of Vortioxetine The effects of naloxone pre-treatment on vortioxetine (10 mg/kg)-induced analgesic responses in the tail-clip, tail-immersion and hot-plate tests are shown Figure 10A, Figure 10B and Figure 10C, respectively. Two-way ANOVA indicated that the treatment (tail- clip test: [F (1, 24) = 68.51, p < 0.001]; tail-immersion test: [F (1, 24) = 65.43, p < 0.001]; hot- plate test: [F (1, 24) = 26.23, p < 0.001]) and antagonist (tail-clip test: [F (1, 24) = 31.57, p < 0.001]; tail-immersion test: [F (1, 24) = 34.47, p < 0.001]; hot-plate test: [F (1, 24) = 10.65, p < 0.01]) factors affected the MPE% values. Furthermore, there was a statistically significant interaction (tail-clip test: [F (1, 24) = 29.63, p < 0.001]; tail-immersion test: [F (1, 24) = 30.73, p < 0.001]; hot-plate test: [F (1, 24) = 10.36, p < 0.01]) between these two factors. Multiple comparison Bonferroni analysis revealed that pre-treatment with naloxone (5.48 mg/kg; Molecules 2021, 26, 3242 single dose) led to antagonism of the analgesic activity of vortioxetine in the tail-clip11 (p of < 19 0.001), tail-immersion (p < 0.001) and hot-plate (p < 0.001) tests.

Figure 10. The effects of naloxone pretreatments on vortioxetine-induced analgesia in the tail-clip (A), Figuretail-immersion 10. The effects (B) and of hot-platenaloxone (pretreatmentsC) tests. Significant on vortioxetine-induced difference against vehicle analgesia treated in the control tail-clip groups (A), tail-immersion (B) and hot-plate (C) tests. Significant difference against vehicle treated control *** p < 0.001; Significant difference against vortioxetine administrated control groups &&& p < 0.001. groups *** p < 0.001; Significant difference against vortioxetine administrated control groups &&& p Two-way ANOVA, post-hoc Bonferroni test, n = 7. < 0.001. Two-way ANOVA, post-hoc Bonferroni test, n = 7. 3. Discussion 3. Discussion In the current study, we investigated the potential analgesic efficacy of vortioxetine basedIn the on itscurrent multimodal study, we activity investigated profile in the the potential CNS. The analgesic use of agents efficacy that of disruptvortioxetine motor basedactivity on inits experimentalmultimodal activity animals profile can lead in tothe misleading CNS. The resultsuse of agents in pain that experiments disrupt motor [13,14 ]. activityTherefore, in experimental before investigating animals thecan analgesic lead to efficacymisleading of vortioxetine, results in pain the possibleexperiments effect of this drug on the motor coordination of the mice was investigated using Rota-rod tests. We found that vortioxetine did not significantly affect motor activities at any of the doses administered (5, 10 and 20 mg/kg). These findings, in line with previous reports [15,16], is important because it showed that the data obtained from further analgesic activity tests were specific. The analgesic activity of vortioxetine was evaluated by the tail-clip, tail-immersion and hot-plate tests. In the tail-clip tests, animals administered vortioxetine at 10 and 20 mg/kg had significantly longer reaction time than saline-administered control mice. Moreover, MPE% and AUC values were also significantly higher with respect to the control groups. 5 mg/kg dose of this drug was only effective at 60th minute (Figure2). In the tail-clip method, the clamp-biting reaction of animals is known to be associated with spinal transmission of nociception [14,17]. Therefore, our findings suggest that the analgesic activity of vortioxetine is related to its effect on spinal nociceptive pathways that carry painful mechanical stimuli. The tail-immersion method [18] was another procedure used to evaluate spinally mediated acute analgesic effect, in this study. In these tests, vortioxetine at doses of 10 and 20 mg/kg significantly increased the reaction times of the mice as well as the MPE% and AUC values compared to the saline-treated control groups. A 5 mg/kg dose was effective only at the 60th minute (Figure3). Findings from tail-immersion tests showed that vortioxetine affects also spinal nociceptive pathways that carry thermal painful stimuli along with mechanical ones. Similar to the findings for the tail-clip and tail-immersion tests, in the hot-plate tests, vortioxetine administrated at 10 and 20 mg/kg prolonged the response latencies of mice and enhanced the related MPE% and AUC values, significantly (Figure4). However, different from the tail-clip and tail-immersion tests, which measure spinal reflexes, the Molecules 2021, 26, 3242 12 of 19

nociceptive stimulus-induced paw-licking or jumping behaviors in hot-plate test are known to be related to the supraspinal pathways [14,17]. Therefore, it can be suggested that vortioxetine-induced analgesia is mediated by supraspinal mechanisms in addition to spinal ones. In parallel to our results, 10 mg/kg/day i.p. injection of vortioxetine for 27 days has been shown to reduce tactile allodynia in mice with chronic constriction damage [16]. Similarly, in oxaliplatin-induced neuropathic mice, vortioxetine has been shown to reduce mechanical allodynia in the Von Frey test and cold allodynia in acetone tests at 1–10 mg/kg p.o. doses [19]. Moreover, studies investigating the efficacy of vortioxetine on chemically- induced acute pain have revealed that this drug is effective in both phases of formalin test [20,21]. Our study is different from these previous studies since we used direct mechanical and thermal nociceptive stimulus (but not chemical) to induce pain instead of allodynia. Moreover, we administrated vortioxetine at 20 mg/kg p.o. dose, which has not been tested in nociceptive setups before. On the other hand, the only comparable study was conducted by Mørk et al., who tested the acute effect of vortioxetine (2.5, 5, and 10 mg/kg, s.c.) in hot-plate method. The results of the mentioned paper, showing that the 10 mg/kg dose of vortioxetine causes a significant increase in the reaction times of the animals, support our findings in this study [6]. After presenting the acute analgesic efficacy of vortioxetine against mechanical and thermal nociceptive stimuli; the possible mechanisms underlying this analgesic activity were examined, in this study. As serotonergic, catecholaminergic and opioidergic systems play critical roles in the nociception and analgesia processes of the CNS [22–24], we searched the possible contribution of these systems to the analgesic effect of vortioxetine. The participation of the endogenous serotonergic system in the analgesic effect of vortioxetine was examined using PCPA, a serotonin-depleting agent. PCPA administration (100 mg/kg; four consecutive days) was previously found to block serotonin synthesis by inhibiting the tryptophan hydroxylase enzyme; furthermore, this process depleted 60–90% of the endogenous serotonin stores in the CNS without affecting the central noradrenaline and dopamine levels [25]. Results of the nociceptive tests indicated that PCPA pre-treatment antagonized the analgesic activity of vortioxetine (Figure5). These findings revealed that the central serotonin level, which is probably modulated by the serotonin reuptake inhibitory effect of this drug [2], is critical for vortioxetine-induced analgesia. Serotonin has extremely complex effects on pain transmission and modulation; it can induce both analgesic and pronociceptive effects in the CNS depending on receptor availability and affinity, ligand concentration, and the neural network involved [26]. 5-HT1A subtypes of serotonergic receptors have been shown to densely localized in the dorsal horn of the spinal cord and in brain regions that related to pain processing or modulation [27–29]. These receptor subtypes have been revealed to play critical roles in analgesia [30–33] and ligands that activate them have been demonstrated to possess notable analgesic activities [34–38]. Based on the prominent agonistic effect of vortioxetine on 5-HT1A receptors, we examined the possible participation of this receptor subtype to the analgesic effect of this drug. The fact that pre-treatment with NAN-190, a selective 5-HT1A receptor antagonist, attenuated the analgesic activity of vortioxetine (Figure6), suggests that 5-HT 1A receptors contribute to the acute analgesic effect of this drug. On the other hand, along with the 5-HT1A, possible modulatory effects of other serotonergic receptors, especially the 5-HT1B, 5-HT1D, 5-HT3, and 5-HT7 subtypes [23,27], on vortioxetine-induced analgesia should be clarified in further studies. We investigated the involvement of the catecholaminergic system in the analgesic activity of vortioxetine by using AMPT, a catecholamine-depleting agent. AMPT pre- treatment at a dose of 100 mg/kg inhibits tyrosine hydroxylase, a rate-limiting enzyme in noradrenaline and dopamine syntheses [39]. AMPT pre-treatment lowers central dopamine and noradrenaline levels by 57% and 53%, respectively, but does not affect central serotonin levels [40]. The AMPT results indicated that catecholamine depletion abolished the analgesic activity of vortioxetine in all of the nociceptive tests (Figure7). These findings Molecules 2021, 26, 3242 13 of 19

together with the results of PCPA studies revealed that the central analgesic activity of vortioxetine is related to catecholamine and serotonin levels in the CNS. The noradrenergic system modulates pain via α- and β-adrenergic receptors [23,41–45]. Therefore, we investigated the participation of adrenoceptors in the analgesic effect of vortioxetine. Our phentolamine studies showed that pre-treatment with this non-selective α-adrenoceptor blocker antagonised the analgesic effect of vortioxetine (Figure8). These data indicate that α-adrenoceptors contribute to vortioxetine-induced analgesia. On the other hand, blocking β-adrenergic receptors with propranolol did not alter the analgesic effect (Figure9), revealing that β-adrenoceptors do not mediate vortioxetine-induced analgesia, at least in healthy mice. The involvement of opioid receptors in the analgesic effect of vortioxetine was examined using naloxone, a non-selective antagonist of opioid receptors. Naloxone pre- treatment eliminated the analgesic activity induced by 10 mg/kg vortioxetine in all of the nociceptive tests (Figure 10), suggesting that the opioidergic system mediates the acute analgesic effect of this drug at the supraspinal and spinal levels. Vortioxetine has been reported to increase the levels of noradrenaline in various areas of brain [3]. Therefore, it is possible that this drug increase noradrenaline levels in pain- related regions of CNS, such as the descending noradrenergic pathway. The results of our AMPT studies pointing out the critical role of central noradrenaline level in the analgesic effect of vortioxetine are supportive for this idea. It has been shown that noradrenaline releasing from descending inhibitory pathway acting predominantly at the α2-adrenoceptors to induce analgesia [23]. Moreover, there is a well-described interaction between the opioidergic and α2 adrenergic receptors. Namely, co-administration of their agonists are known to produce synergistic analgesia, besides, α2 adrenoceptor agonists-induced analgesia can be reversed by naloxone administration [46]. In addition to the noradrenergic system, serotonergic system is known to interact with the opioid-mediated pain modulatory cir- cuit [47]. It has been reported that the analgesic effects of µ and δ opioid receptor agonists at spinal and supraspinal levels were significantly weakened in Lmx1bf/f/p mice, which are genetically lack of central serotonergic neurons, while supraspinal analgesic efficacy of k receptor agonists completely disappears. Central serotonergic system has been reported as a key component of supraspinal pain modulatory circuitry mediating opioid analgesia [48]. Our experimental data demonstrating the involvement of the serotonergic, noradrenergic, and opioidergic systems in vortioxetine-induced analgesia are consistent with these previous studies. Taken together, the analgesic effect of vortioxetine seems to arise because of interaction between the main endogenous pathways of the CNS, similar to its multimodal antidepressant effect. One limitation of this study is that the antagonists used in the mechanism studies were administered in single doses and these agents have potential to act on other receptors than the targeted ones. For example, phentolamine, which shows its primary pharmacological effect by antagonizing α-adrenergic receptors, has also been reported to block serotonergic receptors and potassium channels and to inhibit histamine release from mast cells [49–52]. Moreover, NAN-190, a selective 5-HT1A receptor antagonist, has been shown to block α2-adrenergic receptors in rodents [53]. Finally, the non-selective beta-adrenergic antagonist propranolol has been demonstrated to bind to serotonergic 5HT1 and 5HT2 receptors in brain membranes. Furthermore there is a pharmacological evidence that propranolol can behave as a 5-HT1A receptor antagonist and a 5-HT1B agonist in rat brain cortex [54]. Although the pharmacological significance of these activities is ambiguous, they may interfere with experimental results. Therefore, special attention was paid to dose selections and these agents were administrated in previously used doses [14,55–65], at which disappearance of the pharmacological effect have been associated to the involvement of targeted receptors in the activity. On the other hand, it is clear that further mechanical studies with various doses of antagonists would be useful to confirm the receptors that mediate the central analgesic effect of vortioxetine. Molecules 2021, 26, 3242 14 of 19

4. Materials and Methods 4.1. Animals Adult male BALB/c mice (aged 12–15 weeks, body weight, 30–35 g) were used in this study. The animals were housed in a well-ventilated room at a controlled temperature of 24 ± 1 ◦C with a 12-h light/dark cycle (08:00–20:00). Water and food were provided ad libitum. The animals were obtained from the Anadolu University Research Unit for Exper- imental Animals (Eski¸sehir, Turkey). The experimental protocol of the study approved by the Local Ethical Committee on Animal Experimentation of Anadolu University, Es- ki¸sehir, Turkey (Protocol code 2020-30 and date of approval: 14 July 2020). The relevant law of the Republic of Turkey (Regulation on the welfare and protection of animals used for experimental and other scientiﬁc purposes No. 28141, 15 February 2014) has been strictly followed.

4.2. Drugs and Administration Protocol Vortioxetine hydrobromide (Brintellix®) was purchased from Lundbeck (North Ryde, NSW, Australia), while PCPA, NAN-190 hydrobromide, morphine sulphate (authorization date and number: 23 May 2018; 26/15), diazepam, AMPT, phentolamine hydrochloride, propranolol hydrochloride, and naloxone hydrochloride dehydrate were acquired from Sigma-Aldrich (St. Louis, MO, USA). Vortioxetine, morphine sulphate and all the other chemicals, except AMPT and diazepam, were dissolved in physiological saline (0.9% NaCl) immediately before use. AMPT and diazepam was dissolved in saline with 10% Tween 80. Mice were randomly assigned to the experimental groups. Randomisation was performed by an online software QuickCalcs (GraphPad Software, San Diego, CA, USA). Randomization, drug/agent administrations (Ü.K.), conduction of the experiments (N.T.Y.), outcome assessment and data analysis (Ö.D.C. and Ü.D.Ö.) were performed by the stated researchers. Investigators were blinded to group allocation during the conduction of the experiments, outcome assessment and data analysis. Vortioxetine was orally administered to the mice at doses of 5, 10 and 20 mg/kg [15]. The reference drugs, morphine and diazepam, were administrated at 10 mg/kg (i.p.) and 2 mg/kg (p.o.) doses, respectively [14,66]. Experiments were initiated 30 min after the i.p. morphine injection and 60 min after the p.o. saline, diazepam and vortioxetine administrations. For mechanistic studies, the mice were pre-treated with PCPA (serotonin-synthesis inhibitor, 100 mg/kg i.p. once a day, four consecutive days) [67], NAN-190 (serotonin 5-HT1A receptor antagonist, 0.5 mg/kg, i.p.)[57,59], AMPT (catecholamine-synthesis inhibitor, 100 mg/kg, i.p., 4 h prior to saline or vortioxetine administrations) [68], phentolamine (non-selective α-adrenoceptor blocker, 4 mg/kg, i.p.)[60], propranolol (non-selective β- adrenergic receptor blocker, 2 mg/kg, i.p.)[64] and naloxone (non-selective opioid receptor blocker, 5.48 mg/kg, i.p.)[14]. Except for PCPA and AMPT, the antagonists were administered 15 min before saline or vortioxetine administration. The doses and ways of administration were chosen according to the previous experi- ences of our laboratory and to data previously reported for mice [14,15,57,59,60,64,67,68]. Details of the experimental settings and treatment protocols are presented in Figure 11.

4.3. Motor Coordination Analysis Rota-Rod Test Motor coordination of mice was evaluated using a Rota-rod device (Cat. no: 47600; Ugo Basile, Varese, Italy), as previously described [69]. The Rota-rod test is a two-stage test with training and experimental phases. In the training phase, the mice practised three times on the rotating rod, which was set at a constant speed of 16 rpm for three consecutive days. The mice that could not remain on the rod for more than 180 s were excluded from the experiments. In the experimental phase, the mice were placed on the rotating rod once Molecules 2021, 26, x FOR PEER REVIEW 16 of 21

(N.T.Y.), outcome assessment and data analysis (Ö.D.C. and Ü.D.Ö.) were performed by the stated researchers. Investigators were blinded to group allocation during the conduction of the experiments, outcome assessment and data analysis. Vortioxetine was orally administered to the mice at doses of 5, 10 and 20 mg/kg [15]. The reference drugs, morphine and diazepam, were administrated at 10 mg/kg (i.p.) and 2 mg/kg (p.o.) doses, respectively [14,66]. Experiments were initiated 30 min after the i.p.

Moleculesmorphine2021, 26, 3242 injection and 60 min after the p.o. saline, diazepam and vortioxetine administra-15 of 19 tions. For mechanistic studies, the mice were pre-treated with PCPA (serotonin-synthesis inhibitor, 100 mg/kgmore, i.p. and once the a falling day,time, four a consecutive parameter of motor days) coordination, [67], NAN-190 was automatically (serotonin recorded. 5- HT1A receptor antagonist,Diazepam 0.5 was mg/kg, used as i.p. a reference) [57,59], drug AMPT [66]. (catecholamine-synthesis inhibitor, 100 mg/kg, i.p., 4 h prior to saline or vortioxetine administrations) [68], phentolamine 4.4. Analgesic Activity Analysis (non-selective α-adrenoceptor blocker, 4 mg/kg, i.p.) [60], propranolol (non-selective β- The analgesic activity of vortioxetine was investigated using the following acute adrenergic receptornociception blocker, 2 tests: mg/kg, tail-clip, i.p. tail-immersion) [64] and naloxone and hot-plate (non-selective tests. opioid receptor blocker, 5.48 mg/kg, i.p.) [14]. Except for PCPA and AMPT, the antagonists were administered 15 min before4.4.1. Tail-Clip saline Testor vortioxetine administration. The doses and waysThe of tail-clip administration test was performed were chosen as previously according described to the [14]. previous A metal artery expe- clamp was applied to the tail of the mouse, and the latency of the nociceptive response (biting riences of our thelaboratory clamp) was recordedand usingto ada stopwatch.ta previously A sensitivity reported test was performed for mice before the [14,15,57,59,60,64,67,68].experiments, Details and of animals the experimental responding within settings 10 s wereand selectedtreatment for the protocols tests. Cut-off are time presented in Figurewas 11. chosen as 10 s to avoid possible tissue damage.

Figure 11. Brief summary Figureof the 11.experimentalBrief summary design. of the experimental design.

4.3. Motor Coordination4.4.2. Analysis Tail-Immersion Test The tail-immersion test was performed as described previously [18]. 1–2 cm of the tail Rota-Rod Test of each mouse was immersed into hot water maintained at 52 ± 1 ◦C, and the latency of the Motor coordinationnociceptive of mice response was (rapid evaluated ﬂick of theusing tail) a was Rota-rod recorded device using a stopwatch.(Cat. no: A47600; sensitivity Ugo Basile, Varese,test Italy), was performedas previously before desc theribed experiments, [69]. The and Rota-rod animals responding test is a two-stage within 4 s were selected for the tests. Cut-off time was chosen as 20 s to avoid possible tissue damage. test with training and experimental phases. In the training phase, the mice practised three times on the rotating4.4.3. rod, Hot-Plate which Test was set at a constant speed of 16 rpm for three consecutive days. The mice thatThe could hot-plate not testremain was performed on the rod as describedfor more earlier than [18014,70 s]. were In this excluded test, mice were from the experiments.individually In the experimental placed on the aluminiumphase, the plate mice of were the hot-plate placed deviceon the (Cat. rotating no: 7280; rod Ugo ± ◦ once more, and theBasile, falling Varese, time, Italy), a parameter which was set of at 55motor1.0 coordination,C. Paw-licking, shakingwas automatically or jumping latencies of each mouse were recorded. A sensitivity test was performed before the experiments and recorded. Diazepamanimals was used responding as a reference within 15 s drug were selected [66]. for the tests. Cut-off time was chosen as 30 s to avoid probable tissue damage. In each of the nociceptive tests, response latencies of mice were recorded at 0th (predrug latency), 30th, 60th, 90th, 120th and 180th min. following the vehicle, reference drug and vortioxetine administrations to obtain time course of drug effects.

Molecules 2021, 26, 3242 16 of 19

The following formula was used to convert tail-clip, tail-immersion, and hot-plate latencies to the percent maximum possible effect (MPE%):

MPE% = ((postdrug latency − predrug latency))/((cut off time − predrug latency)) × 100 (1) The MPE% values were plotted against time (0–180 min). Area under the MPE% versus time curves (AUC0–180) were calculated by using the GraphPad Prism ver. 8.4.3. (GraphPad Software, San Diego, CA, USA) based on the trapezoidal rule [18].

4.5. Statistical Analysis The data used in statistical analyses were acquired from seven animals for each group. Variables were first investigated for normality and homogeneity of variance using Shapiro–Wilk and Levene tests, respectively. GraphPad Prism ver. 8.4.3 was used for statistical evaluations. Experimental data obtained from Rota-rod tests were analysed using one-way analysis of variance (ANOVA) followed by the Tukey’s honestly significant difference (HSD) test for multiple comparisons. Data acquired from time course nociceptive studies were evaluated by two-way repeated ANOVA, followed by the Tukey’s multiple comparisons tests. AUC0–180 data were analysed with one-way ANOVA followed by the Tukey’s HSD tests. Antagonist study results were evaluated by two-way ANOVA, followed by the Bonferroni test for multiple comparisons. Experimental results have been provided in terms of mean ± standard error of the mean. p < 0.05 was considered significant.

5. Conclusions Findings of this research exhibited that vortioxetine exerts centrally mediated analgesic activity against acute mechanical and thermal nociceptive stimuli. Furthermore, this study demonstrated for the first time that serotonergic and catecholaminergic systems play critical roles in the vortioxetine-induced analgesia and that opioidergic, 5-HT1A serotonergic and α-adrenergic receptors mediate this effect. Since pain processing and modulation are highly complex functions regulated by various endogenous mechanisms in the nervous system, it may be beneficial to use more than one drug with different modes of action in pain clinics. This multi-drug analgesic approach can be advantageous due to the increased analgesic efficacy and reduced side effects. However, this time, problems related to patient compliance and drug-drug interaction risks may arise. On the other hand, inducing analgesia with a single drug acting on various pain pathways may make it possible to both strengthen the analgesic effect and avoid problems caused by polypharmacy [71,72]. Vortioxetine may meet these expectations as an analgesic drug with multimodal activity in the CNS [73]. However, further clinical studies are required to confirm this hypothesis.

Author Contributions: Conceptualization, Ö.D.C. and N.T.Y.; methodology, Ö.D.C. and N.T.Y.; software, Ö.D.C. and Ü.D.Ö.; validation, N.T.Y. and Ü.K.; formal analysis, Ö.D.C. and Ü.D.Ö.; investigation, N.T.Y. and Ü.K.; resources, Ü.D.Ö. and N.T.Y.; data curation, N.T.Y. and Ü.K.; writing—original draft preparation, Ö.D.C.; writing—review and editing, Ö.D.C. and Ü.D.Ö.; visualization, Ö.D.C. and Ü.D.Ö.; supervision, Ö.D.C.; project administration, N.T.Y.; funding acquisition, N.T.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The experimental protocol of the study approved by the Local Ethical Committee on Animal Experimentation of Anadolu University, Eski¸sehir, Turkey (Protocol code 2020-30 and date of approval: 14 July 2020). The relevant law of the Republic of Turkey (Regulation on the welfare and protection of animals used for experimental and other scientiﬁc purposes No. 28141, 15 February 2014) has been strictly followed. Informed Consent Statement: Not applicable. Data Availability Statement: All relevant data are included within the manuscript. The raw data are available on request from the corresponding author. Molecules 2021, 26, 3242 17 of 19

Acknowledgments: This research was supported from the Anadolu University Scientific Research Projects Commission (Project Numbers: 2006S079). Conflicts of Interest: The authors declare no conflict of interest.

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