Gabapentin and Duloxetine Prevent Oxaliplatin- and Paclitaxel-Induced

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Article Gabapentin and Duloxetine Prevent Oxaliplatin- and Paclitaxel-Induced Peripheral Neuropathy by Inhibiting Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Phosphorylation in Spinal Cords of Mice

Natsuki Kato 1,†, Keisuke Tateishi 1,†, Masanobu Tsubaki 1, Tomoya Takeda 1, Mikihiro Matsumoto 1, Katsumasa Tsurushima 1,2, Toshihiko Ishizaka 2 and Shozo Nishida 1,*

1 Division of Pharmacotherapy, Kindai University Faculty of Pharmacy, Kowakae, Higashi-Osaka 577-8502, Japan; [email protected] (N.K.); [email protected] (K.T.); [email protected] (M.T.); [email protected] (T.T.); [email protected] (M.M.); [email protected] (K.T.) 2 Department of Pharmacy, Sakai City Medical Center, Sakai 593-8304, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-6-6721-2332 † These authors contribute equally to this paper.

Abstract: Chemotherapy-induced peripheral neuropathy is a common factor in limiting therapy which can result in therapy cessation or dose reduction. Gabapentin, a calcium channel inhibitor, and duloxetine, a serotonin noradrenaline reuptake inhibitor, are used to treat a variety of pain conditions such as chronic low back pain, postherpetic neuralgia, and diabetic neuropathy. It has been reported that administration of gabapentin suppressed oxaliplatin- and paclitaxel-induced Citation: Kato, N.; Tateishi, K.; mechanical hyperalgesia in rats. Moreover, duloxetine has been shown to suppress oxaliplatin- Tsubaki, M.; Takeda, T.; Matsumoto, induced cold allodynia in rats. However, the mechanisms by which these drugs prevent oxaliplatin- M.; Tsurushima, K.; Ishizaka, T.; Nishida, S. Gabapentin and and paclitaxel-induced neuropathy remain unknown. Behavioral assays were performed using Duloxetine Prevent Oxaliplatin- and cold plate and the von Frey test. The expression levels of proteins were examined using western Paclitaxel-Induced Peripheral blot analysis. In this study, we investigated the mechanisms by which gabapentin and duloxetine Neuropathy by Inhibiting prevent oxaliplatin- and paclitaxel-induced neuropathy in mice. We found that gabapentin and Extracellular Signal-Regulated Kinase duloxetine prevented the development of oxaliplatin- and paclitaxel-induced cold and mechanical 1/2 (ERK1/2) Phosphorylation in allodynia. In addition, our results revealed that gabapentin and duloxetine suppressed extracellular Spinal Cords of Mice. Pharmaceuticals signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation in the spinal cord of mice. Moreover, 2021, 14, 30. https://doi.org/ PD0325901 prevented the development of oxaliplatin- and paclitaxel-induced neuropathic-like pain 10.3390/ph14010030 behavior by inhibiting ERK1/2 activation in the spinal cord of mice. In summary, our ﬁndings suggest that gabapentin, duloxetine, and PD0325901 prevent the development of oxaliplatin- and Received: 26 October 2020 paclitaxel-induced neuropathic-like pain behavior by inhibiting ERK1/2 phosphorylation in mice. Accepted: 28 December 2020 Therefore, inhibiting ERK1/2 phosphorylation could be an effective preventive strategy against Published: 31 December 2020 oxaliplatin- and paclitaxel-induced neuropathy.

Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- Keywords: chemotherapy-induced peripheral neuropathy; gabapentin; duloxetine; ERK1/2; oxali- ms in published maps and institutio- platin; paclitaxel nal afﬁliations.

1. Introduction Copyright: © 2020 by the authors. Li- censee MDPI, Basel, Switzerland. Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting This article is an open access article adverse effect of multiple chemotherapeutic agents, including oxaliplatin and paclitaxel, distributed under the terms and con- which can result in therapy cessation or dose reduction [1–3]. In addition, neuropathy ditions of the Creative Commons At- causes a signiﬁcant loss of functional abilities and decreases the quality of life of patients. tribution (CC BY) license (https:// Among chemotherapeutic agents, platinum drugs (oxaliplatin and cisplatin), vinca al- creativecommons.org/licenses/by/ kaloids (vincristine and vinblastine), taxanes (paclitaxel and docetaxel), and bortezomib 4.0/). induce the most severe effects on the peripheral nervous system [4–7]. Since little is known

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about the mechanisms involved in the development of CIPN, no drugs are currently available to prevent or treat CIPN. Thus, the identiﬁcation of drugs that effectively prevent or treat CIPN is important. The activation of protein kinase C (PKC)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway contributes to the development of neuropathic pain. Administra- tion of oxaliplatin to rats evoked thermal and mechanical hypersensitivity via increased PKC/ERK1/2 activation in the spinal cord and cortical areas [8–10]. Treatment with paclitaxel induced mechanical hypersensitivity via increased ERK1/2 activation in the dorsal root ganglion (DRG) of rats [11]. In addition, PKC-induced ERK1/2 phosphorylation has been associated with neuropathic pain in rats [12,13]. Moreover, it has been reported that PKC mediates a positive feedback mechanism regulating calcium channel activity, which may affect nerve sensitivity during chemotherapy [14,15]. Gabapentin, an anticonvulsant, has been used to treat a variety of non-epileptic conditions such as chronic pain, psychiatric disorders, and movement disorders. It is thought to act by binding to the α2δ subunit of voltage-dependent calcium channels, thus inhibiting the release of excitatory neurotransmitters [16,17]. Duloxetine, a serotonin-noradrenaline reuptake inhibitor, is used to treat painful diabetic neuropathy, osteoarthritis-related pain, and chronic low back pain [18–21]. A number of studies have reported that these drugs suppress oxaliplatin- and paclitaxel-induced mechanical hyperalgesia and cold allodynia in rat and mice models [22–24]. However, the mechanisms underlying this analgesic effect remains unclear. Therefore, the present study investigated the mechanism by which gabapentin and duloxetine suppress oxaliplatin- and paclitaxel-induced neuropathic pain.

2. Results 2.1. Effects of Gabapentin on Oxaliplatin- and Paclitaxel-Induced Cold and Mechanical Allodynia To evaluate the effect of gabapentin on oxaliplatin- and paclitaxel-induced cold sensitivity, mice received oxaliplatin or paclitaxel on days 0 and 7 and were administered gabapentin (30 or 100 mg/kg/day, p.o.) daily. Oxaliplatin and paclitaxel induced a significant progressive reduction in withdrawal thresholds at 10 ◦C. Oral administration of gabapentin prevented the development of oxaliplatin- and paclitaxel-induced cold allodynia (Figures1A and2A). No significant differences were observed in withdrawal latency at any time point by the combination treatment with 30 mg/kg gabapentin and 6 mg/kg oxaliplatin or 6 mg/kg paclitaxel compared with the combination treatment with 100 mg/kg gabapentin and 6 mg/kg oxaliplatin or 6 mg/kg paclitaxel (Figures1A and2A). In addition, we investigated the effect of gabapentin on the development of oxaliplatin- and paclitaxel- induced mechanical allodynia using the following von Frey filaments: 0.16 g (mechanical allodynia), 0.4 g (intermediate), and 1.4 g (mechanical hyperalgesia). Administration of 30 mg/kg gabapentin prevented the development of oxaliplatin- and paclitaxel-induced mechanical allodynia and hyperalgesia, although the difference was more significant than that in the vehicle group in a few days (Figures1B–D and2B–D). In addition, treatment with 100 mg/kg gabapentin significantly prevented the development of oxaliplatin- and paclitaxel-induced mechanical allodynia and hyperalgesia compared with the combination treatment with 30 mg/kg gabapentin and oxaliplatin or paclitaxel (Figures1B–D and2B–D). Although treatment of mice with 6 mg/kg oxaliplatin, 6 mg/kg oxaliplatin plus 30 mg/kg gabapentin, 6 mg/kg oxaliplatin plus 100 mg/kg gabapentin, and 6 mg/kg paclitaxel resulted in weight loss in a few days, no dramatic changes were observed (Supplementary Materials, Figure S1). Moreover, no significant differences in weight gain were observed at any time point in the gabapentin group compared with the vehicle group (Supplementary Materials, Figure S1). These observations indicated that gabapentin prevented oxaliplatin- and paclitaxel-induced neuropathic-like pain behavior in a dose-dependent manner. Pharmaceuticals 2021, 14, 30 3 of 16

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Figure 1. InhibitoryInhibitory effect effect of of gabapentin gabapentin on on oxaliplatin-induced oxaliplatin-induced cold cold allodynia allodynia in inmice. mice. Oxaliplatin Oxaliplatin (6 mg/kg/i.v.) (6 mg/kg/i.v.) was wasadministered administered on days on days0 and 0 and7, and 7, andgabapentin gabapentin (30 or (30 100 or 100mg/kg, mg/kg, p.o.) p.o.)was administered was administered daily dailyfrom fromdays days0 to 014. to (A 14.) (WithdrawalA) Withdrawal latencies, latencies, presented presented as means as means ± standard± standard deviations deviations (S.D.), (S.D.), represent represent the time the timetaken taken by mice by miceto withdraw to withdraw their hindtheir hindpaws pawsfollowing following cold stimulation cold stimulation (10 °C) (10 (n◦ C)= 7 ( nper= 7group). per group). * p < *0.05,p < 0.05,** p < ** 0.01p < vs. 0.01 vehicles vs. vehicles (Shapiro–Wilk (Shapiro–Wilk test and test Kruskal–Wallisand Kruskal–Wallis test testfollowed followed by the by theScheffe Scheffe test). test). (B– (BD–)D The) The number number of of paw paw lifts lifts elicited elicited by by five ﬁve mechanical mechanical stimulations stimulations using von Frey filaments corresponding to (B) innocuous (0.16 g), (C) intermediate (0.4 g), and (D) noxious (1.4 g) bending using von Frey ﬁlaments corresponding to (B) innocuous (0.16 g), (C) intermediate (0.4 g), and (D) noxious (1.4 g) bending forces. The aversive response score was calculated based on two paw lifts (n = 7 per group). * p < 0.05, ** p < 0.01 vs. forces. The aversive response score was calculated based on two paw lifts (n = 7 per group). * p < 0.05, ** p < 0.01 vs. vehicles, vehicles, # p < 0.05, ## p < 0.01 vs. 6 mg/kg oxaliplatin + 30 mg/kg gabapentin (Shapiro–Wilk test and one-way analysis of # ## variancep < 0.05, (ANOVA)p < 0.01 with vs. 6the mg/kg Tukey oxaliplatin test). + 30 mg/kg gabapentin (Shapiro–Wilk test and one-way analysis of variance (ANOVA) with the Tukey test).

2.2. Effects of Duloxetine on Oxaliplatin- and Paclitaxel-Induced Cold and Mechanical Allodynia To investigate the effect of duloxetine on oxaliplatin- and paclitaxel-induced neuropathic-like pain behavior, mice received oxaliplatin or paclitaxel on days 0 and 7 and were administered duloxetine (10 or 30 mg/kg/day, p.o.) daily. Oral administration of duloxetine prevented the development of oxaliplatin- and paclitaxel-induced cold allodynia (Figures3A and4A). No significant differences were observed in withdrawal latency at any time point by the combination treatment with 10 mg/kg duloxetine, 6 mg/kg oxaliplatin, or 6 mg/kg paclitaxel compared with the combination treatment with 30 mg/kg duloxetine and 6 mg/kg oxaliplatin or 6 mg/kg paclitaxel (Figures3A and4A). In addition, we investigated the effect of duloxetine on the development of oxaliplatin- and paclitaxel- induced mechanical allodynia. Administration of 10 mg/kg duloxetine prevented the development of oxaliplatin- and paclitaxel-induced mechanical allodynia and hyperalgesia, although the difference was more significant than that in the vehicle group in a few days (Figures3B–D and4B–D). In addition, treatment with 30 mg/kg duloxetine significantly

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prevented the development of oxaliplatin- and paclitaxel-induced mechanical allodynia and hyperalgesia compared with the combination treatment with 30 mg/kg duloxetine and oxaliplatin or paclitaxel (Figures3B–D and4B–D). Although treatment of mice with 6 mg/kg oxaliplatin, 6 mg/kg oxaliplatin plus 10 mg/kg duloxetine, 6 mg/kg oxaliplatin plus 30 mg/kg duloxetine, and 6 mg/kg paclitaxel in mice resulted in a little weight loss in a few days, no dramatic changes were observed (Supplementary Materials, Figure S2). Moreover, no signiﬁcant differences in weight gain were observed at any time point in the gabapentin group compared with the vehicle group (Supplementary Materials, Figure S2). These observations indicated that duloxetine prevented oxaliplatin- and paclitaxel-induced Pharmaceuticals 2020, 13, x FOR PEER neuropathic-likeREVIEW pain behavior in a dose-dependent manner. 4 of 16

Figure 2. InhibitoryInhibitory effect effect of of gabapent gabapentinin on on paclitaxel-induced paclitaxel-induced mechanical mechanical allodyni allodyniaa in mice. in mice. Paclitaxel Paclitaxel (6 mg/kg, (6 mg/kg, i.p.) was i.p.) wasadministered administered on days on days0 and 0 and7, and 7, andgabapentin gabapentin (30 or (30 100 or 100mg/kg, mg/kg, p.o.) p.o.)was administered was administered daily dailyfrom fromdays days0 to 014. to (A 14.) (WithdrawalA) Withdrawal latencies, latencies, presented presented as means as means ± standard± standard deviations deviations (S.D.), (S.D.), represent represent the time the timetaken taken by mice by miceto withdraw to withdraw their hindtheir hindpaws pawsfollowing following cold stimulation cold stimulation (10 °C) (10 (n◦ C)= 7 ( nper= 7group). per group). * p < *0.05,p < 0.05,** p < ** 0.01p < vs. 0.01 vehicles vs. vehicles (Shapiro–Wilk (Shapiro–Wilk test and test Kruskal–Wallis test followed by the Scheffe test). (B–D) The number of paw lifts elicited by five mechanical stimulations and Kruskal–Wallis test followed by the Scheffe test). (B–D) The number of paw lifts elicited by ﬁve mechanical stimulations using von Frey filaments corresponding to (B) innocuous (0.16 g), (C) intermediate (0.4 g), and (D) noxious (1.4 g) bending using von Frey ﬁlaments corresponding to (B) innocuous (0.16 g), (C) intermediate (0.4 g), and (D) noxious (1.4 g) bending forces. The aversive response score was calculated based on two paw lifts (n = 7 per group). * p < 0.05, ** p < 0.01 vs. vehicles,forces. The # p aversive < 0.05, ## response p < 0.01 score vs. 6 wasmg/kg calculated paclitaxel based + 30 on mg/kg two paw gabapent lifts (nin= 7(Shapiro–Wilk per group). * p test< 0.05, and ** one-wayp < 0.01 vs.analysis vehicles, of # ## variancep < 0.05, (ANOVA)p < 0.01 with vs. 6the mg/kg Tukey paclitaxel test). + 30 mg/kg gabapentin (Shapiro–Wilk test and one-way analysis of variance (ANOVA) with the Tukey test). 2.2. Effects of Duloxetine on Oxaliplatin- and Paclitaxel-Induced Cold and Mechanical Allodynia To investigate the effect of duloxetine on oxaliplatin- and paclitaxel-induced neuropathic-like pain behavior, mice received oxaliplatin or paclitaxel on days 0 and 7 and were administered duloxetine (10 or 30 mg/kg/day, p.o.) daily. Oral administration of duloxetine prevented the development of oxaliplatin- and paclitaxel-induced cold allodynia (Fig- ures 3A and 4A). No significant differences were observed in withdrawal latency at any time point by the combination treatment with 10 mg/kg duloxetine, 6 mg/kg oxaliplatin, or 6 mg/kg paclitaxel compared with the combination treatment with 30 mg/kg duloxetine and 6 mg/kg oxaliplatin or 6 mg/kg paclitaxel (Figures 3A and 4A). In addition, we investigated the effect of duloxetine on the development of oxaliplatin- and paclitaxel-induced mechanical allodynia. Administration of 10 mg/kg duloxetine prevented the development of oxaliplatin- and paclitaxel-induced mechanical allodynia and hyperalgesia, although the difference was more significant than that in the vehicle group in a few days (Figures 3B–D and 4B–D). In addition, treatment with 30 mg/kg duloxetine significantly prevented the development of oxaliplatin- and paclitaxel-induced mechanical allodynia and hyperalgesia compared with the combination treatment with 30 mg/kg duloxetine and oxaliplatin or paclitaxel (Figures 3B–D and 4B–D). Although treatment of mice with 6 mg/kg

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oxaliplatin, 6 mg/kg oxaliplatin plus 10 mg/kg duloxetine, 6 mg/kg oxaliplatin plus 30 mg/kg duloxetine, and 6 mg/kg paclitaxel in mice resulted in a little weight loss in a few days, no dramatic changes were observed (Supplementary Materials, Figure S2). Moreo- ver, no significant differences in weight gain were observed at any time point in the Pharmaceuticals 2021, 14, 30 gabapentin group compared with the vehicle group (Supplementary Materials, Figure5 of 16 S2). These observations indicated that duloxetine prevented oxaliplatin- and paclitaxel- induced neuropathic-like pain behavior in a dose-dependent manner.

FigureFigure 3.3.Inhibitory Inhibitory effect effect of duloxetineof duloxetine on oxaliplatin-inducedon oxaliplatin-induced cold andcold mechanical and mechanical allodynia allodynia in mice. in Oxaliplatin mice. Oxaliplatin (6 mg/kg, (6 i.v.)mg/kg, was i.v.) administered was administered on days on 0 and days 7, 0 and and duloxetine 7, and duloxetine (10 or 30mg/kg, (10 or 30mg/kg, p.o.) was p.o.) administered was administered daily fromdaily days from 0 days to 14. 0 (Ato) 14. Withdrawal (A) Withdrawal latencies, latencies, presented presented as means as± meansstandard ± standard deviations deviations (S.D.), represent (S.D.), represent the time takenthe time by mice taken to by withdraw mice to theirwithdraw hind pawstheir hind following paws cold following stimulation cold stimulation (10 ◦C) (n = (10 7 per°C) group).(n = 7 per * p group).< 0.05, *** pp << 0.05, 0.01 ** vs. p < vehicles 0.01 vs. (Shapiro–Wilk vehicles (Shapiro– test Wilk test and Kruskal–Wallis test followed by the Scheffe test). (B–D) The number of paw lifts elicited by five mechanical and Kruskal–Wallis test followed by the Scheffe test). (B–D) The number of paw lifts elicited by ﬁve mechanical stimulations stimulations using von Frey filaments corresponding to (B) innocuous (0.16 g), (C) intermediate (0.4 g), and (D) noxious using von Frey ﬁlaments corresponding to (B) innocuous (0.16 g), (C) intermediate (0.4 g), and (D) noxious (1.4 g) bending (1.4 g) bending forces. The aversive response score was calculated based on two paw lifts (n = 7 per group). * p < 0.05, ** p n p p forces.< 0.01 Thevs. vehicles, aversive response# p < 0.05, score ## p was< 0.01 calculated vs. 6 mg/kg based oxaliplatin on two paw + 10 lifts mg/kg ( = 7 duloxetine per group). (Shapiro–Wilk * < 0.05, ** < test 0.01 and vs. vehicles,one-way # ## analysisp < 0.05, of variancep < 0.01 vs.[ANO 6 mg/kgVA] with oxaliplatin the Tukey + 10test). mg/kg duloxetine (Shapiro–Wilk test and one-way analysis of variance [ANOVA] with the Tukey test).

2.3. Gabapentin and Duloxetine Inhibited the Expression of Phosphorylated ERK1/2 in the Lumbar Spinal Cord We examined whether gabapentin and duloxetine altered the expression of phosphorylated ERK1/2 in the lumbar spinal cord (lumbar segments 4–6) using western blotting. A marked increase in the expression of phosphorylated ERK1/2 was observed in mice treated with oxaliplatin or paclitaxel. Mice treated with gabapentin in addition to oxaliplatin or paclitaxel exhibited signiﬁcantly reduced phosphorylated ERK1/2 expression compared to those treated with oxaliplatin or paclitaxel alone (Figure5). Similarly, mice treated with duloxetine in addition to oxaliplatin or paclitaxel exhibited signiﬁcantly reduced phosphorylated ERK1/2 expression compared to those treated with oxaliplatin or paclitaxel alone. These results indicate that gabapentin and duloxetine suppress oxaliplatin- and paclitaxel-induced neuropathy by inhibiting the expression of phosphorylated ERK1/2. Pharmaceuticals 2021, 14, 30 6 of 16

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Figure 4. Inhibitory effect of duloxetine onon paclitaxel-inducedpaclitaxel-induced coldcold andand mechanicalmechanical allodyniaallodynia inin mice.mice. PaclitaxelPaclitaxel (6(6 mg/kg,mg/kg, i.p.) was was administered administered on on days days 0 0 and and 7, 7, and and duloxetine duloxetine (10 (10 or or 30mg/kg, 30mg/kg, p.o.) p.o.) was was administered administered daily daily from from days days 0 to 014. to ( 14.A) (WithdrawalA) Withdrawal latencies, latencies, presented presented as means as means ± standard± standard deviations deviations (S.D.), (S.D.), represent represent the time the timetaken taken by mice by miceto withdraw to withdraw their theirhind hindpaws paws following following cold stimulation cold stimulation (10 °C) (10 (n◦C) = 7 ( nper=7 group). per group). * p < *0.05,p < 0.05,** p < ** 0.01p < vs. 0.01 vehicles vs. vehicles (Shapiro–Wilk (Shapiro–Wilk test and test Kruskal–Wallis test followed by the Scheffe test). (B–D) The number of paw lifts elicited by five mechanical stimulations and Kruskal–Wallis test followed by the Scheffe test). (B–D) The number of paw lifts elicited by ﬁve mechanical stimulations using von Frey filaments corresponding to (B) innocuous (0.16 g), (C) intermediate (0.4 g), and (D) noxious (1.4 g) bending using von Frey ﬁlaments corresponding to (B) innocuous (0.16 g), (C) intermediate (0.4 g), and (D) noxious (1.4 g) bending forces. The aversive response score calculated based on two paw lifts (n = 7 per group). * p < 0.05, ** p < 0.01 vs. vehicles, # forces.p < 0.05, The ## p aversive < 0.01 vs. response 6 mg/kg score paclitaxel calculated + 10 based mg/kg on duloxetine two paw lifts (Shapiro–Wilk (n = 7 per group). test and * p

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Figure 5. FigureGabapentin 5. Gabapentin and duloxetine and duloxetine inhibited oxaliplatin-inhibited oxalipla and paclitaxel-inducedtin- and paclitaxel-induced extracellular signal-regulatedextracellular kinase 1/2 (ERK1/2)signal-regulated activation. Western kinase 1/2 blotting (ERK was1/2) conducted activation. to Western analyze phosphorylatedblotting was conducted ERK1/2 (phospho-ERK1/2) to analyze phos- protein expressionphorylated in the spinal ERK1/2 cord (L4–L6)(phospho-ERK1/2) obtained from protein mice on expre day 14ssion after in treatment the spinal with cord oxaliplatin, (L4–L6) paclitaxel,obtained from gabapentin, or duloxetine.mice onβ-actin day 14 was after used treatment as a protein with loading oxalipla control.tin, paclitaxel, Quantiﬁcation gabapentin, of phospho-ERK1/2 or duloxetine. and β ERK1/2-actin was expression, normalizedused against as a protein that of βloading-actin. Thecontro resultsl. Quantification show four independent of phospho-ER experiments.K1/2 and ** p ERK1/2< 0.01 vs. expression, controls (Shapiro–Wilk nor- test andmalized ANOVA against with Dunnett’s that of test).β-actin. The results show four independent experiments. ** p < 0.01 vs. controls (Shapiro–Wilk test and ANOVA with Dunnett’s test).

2.4. PD0325901 Prevented Oxaliplatin- and Paclitaxel-Induced Neuropathic-Like Pain Behavior Our results indicated that gabapentin and duloxetine prevented oxaliplatin- and paclitaxel-induced neuropathic-like pain behavior by suppressing ERK1/2 activation in the lumbar spinal cord (lumbar segments 4–6) of mice. Therefore, we investigated whether PD0325901, a MEK1/2 inhibitor, prevented oxaliplatin- and paclitaxel-induced neuropa-

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thy in mice. Treatment with 30 mg/kg PD032501 prevented the development of oxaliplatin- and paclitaxel-induced cold allodynia, mechanical allodynia, and hyperalgesia (Figures 6 and 7). In addition, PD0325901 suppressed oxaliplatin- and paclitaxel-induced ERK1/2 activation in the lumbar spinal cord (lumbar segments 4–6) of mice (Figure 8).

Pharmaceuticals 2021, 14, 30 These observations indicate that inhibition of ERK1/2 activation by a MEK inhibitor8 of 16 cor- relate with the prevention oxaliplatin- and paclitaxel-induced neuropathic-like pain by pre-treatment with gabapentin, and duloxetine.

FigureFigure 6. 6.InhibitoryInhibitory effect effect of of PD0325901 PD0325901 on on oxaliplatin-induced oxaliplatin-induced cold cold and and mechanical allodyniaallodynia inin mice.mice. OxaliplatinOxaliplatin (6 mg/kg,(6 mg/kg, i.v.) was i.v.) administered was administered on days on days0 and 0 7, and and 7, PD0325901 and PD0325901 (30 mg/kg, (30 mg/kg, p.o.) p.o.)was administered was administered daily daily from from days days 0 to 14. (A0) Withdrawal to 14. (A) Withdrawal latencies, latencies,presented presented as means as ± meansstandard± standard deviations deviations (S.D.), represen (S.D.), representt the time the taken time by taken mice by to micewithdraw to theirwithdraw hind paws their following hind paws cold following stimulation cold stimulation(10 °C) (n = (107 per◦C) group). (n = 7 per** p group).< 0.01 vs. ** vehiclesp < 0.01 vs.(Shapiro–Wilk vehicles (Shapiro–Wilk test and Kruskal– test Wallisand Kruskal–Wallistest followed by test the followed Scheffe by test). the Scheffe(B–D) The test). number (B–D) The of paw number lifts of elicited paw lifts by elicited five mechanical by ﬁve mechanical stimulations stimulations using von Freyusing filaments von Frey corresponding ﬁlaments corresponding to (B) innocuous to (B) innocuous (0.16 g), (0.16(C) intermediate g), (C) intermediate (0.4 g), (0.4and g), (D and) noxious (D) noxious (1.4 g) (1.4 bending g) bending forces. Theforces. aversive The response aversive score response was score calculated was calculated based on basedtwo paw on twolifts ( pawn = 7 lifts per (group).n = 7 per ** group).p < 0.01 vs. ** p vehicles< 0.01 vs. (Shapiro-Wilk vehicles test(Shapiro-Wilk and one-way test analysis and one-way of vari analysisance (ANOVA) of variance with (ANOVA) Tukey test). with Tukey test).

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FigureFigure 7. 7.InhibitoryInhibitory effect effect of of PD0325901 PD0325901 on on paclitaxel-induced paclitaxel-induced coldcold and and mechanical mechanical allodynia allodynia in in mice. mice. Paclitaxel Paclitaxel (6 mg/kg, (6 mg/kg, i.p.)i.p.) was was administered administered on on days days 0 and 0 and 7, 7,and and PD0325901 PD0325901 (30 (30 mg/k mg/kg,g, p.o.) p.o.) was was administered administered daily daily from from days days 0 0to to 14. 14. (A) Withdrawal(A) Withdrawal latencies, latencies, presented presented as means as means ± standard± standard deviations deviations (S.D.), (S.D.), represent represent the thetime time taken taken by bymice mice to withdraw to withdraw their hindtheir paws hind following paws following cold stimulation cold stimulation (10 °C) (10(n =◦C) 7 per (n = group). 7 per group). ** p < 0.01 ** p vs.< 0.01 vehicles vs. vehicles (Shapiro–Wilk (Shapiro–Wilk test and test Kruskal– and WallisKruskal–Wallis test followed test by followed the Scheffe by the test). Scheffe (B–D test).) The ( Bnumber–D) The of number paw lifts of pawelicited lifts by elicited five mechanical by ﬁve mechanical stimulations stimulations using von Freyusing filaments von Frey corresponding ﬁlaments corresponding to (B) innocuous to (B) innocuous (0.16 g), ( (0.16C) intermediate g), (C) intermediate (0.4 g), (0.4and g), (D and) noxious (D) noxious (1.4 g) (1.4 bending g) bending forces. Theforces. aversive The response aversive score response was scorecalculated was calculated based on two based paw on lifts two ( pawn = 7lifts per (group).n = 7 per ** group).p < 0.01 vs. ** p vehicles< 0.01vs. (Shapiro-Wilk vehicles test(Shapiro-Wilk and one-way test analysis and one-way of variance analysis (ANOVA) of variance with (ANOVA) Tukey test). with Tukey test).

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Figure 8. PD0325901PD0325901 inhibited inhibited oxaliplatinoxaliplatin- and and paclitaxel paclitaxel-induced-induced extracellula extracellularr signal-regulated signal-regulated kinase 1/2 (ERK1/2) (ERK1/2) activation. activation. Western Western blotti blottingng was conducted to analyze phosphorylatedphosphorylated ERK1/2 ERK1/2(phospho-ERK1/2) (phospho-ERK1/2) protein protein expression expression in the spinal in the cord spinal (L4–L6) cord obtained(L4–L6) obtained from mice from on daymice 14 on after daytreatment 14 after with treatment oxaliplatin, with paclitaxel,oxaliplatin, or paclitaxel, PD0325901. or PD0325901.β-actin was β used-actin as was a protein used as loading a protein control. loadingQuantiﬁcation control. of Quantification phospho-ERK1/2 of phospho-ERK1 and ERK1/2 expression,/2 and ERK1/2 normalized expressi againston, normalized that of β against-actin. The thatresults of β show-actin. four The independent results show experiments. four independent ** p < 0.01experiments. vs. controls ** p (Shapiro–Wilk < 0.01 vs. controls test and(Shapiro– ANOVA Wilkwith Dunnett’stest and ANOVA test). with Dunnett’s test).

3. Discussion Discussion In this study, study, we demonstrated that gaba gabapentinpentin and duloxetine prevented prevented the the devel- devel- opment of oxaliplatin- and paclitaxel-induced cold and mechanical allodynia. In In addition, addition, 100 mg/kg mg/kg gabapentin gabapentin and and 30 30 mg/kg mg/kg duloxetine duloxetine significantly significantly suppressed suppressed the development the develop- ofment oxaliplatin- of oxaliplatin- and andpaclitaxel-induced paclitaxel-induced neuropathic-like neuropathic-like pain pain behavior. behavior. Moreover, Moreover, gabapentin andand duloxetineduloxetine treatment treatment did did not no affectt affect body body weight weight when when administered administered alone, alone,and did and not did have not anhave effect an effect on the on transient the transi lossent ofloss body of body weight weight in mice in mice treated treated with with ox- oxaliplatinaliplatin or or paclitaxel. paclitaxel. Western Western blot blot analysis analysis showed showed that that micemice treatedtreated withwith gabapentingabapentin or duloxetine in addition addition to to oxaliplatin oxaliplatin or or paclitaxel paclitaxel exhibited exhibited significantly significantly reduced reduced phos- phos- phorylated ERK1/2 expression expression compared compared to to th thoseose treated with oxaliplatin or paclitaxelpaclitaxel alone. Furthermore, Furthermore, we we observed that PD0 PD0325901325901 prevented oxaliplatin- and paclitaxel- induced neuropathic-like pain behaviorbehavior byby inhibitinginhibiting ERK1/2.ERK1/2. These findings findings suggested that gabapentingabapentin and and duloxetine duloxetine exerted exerted their thei analgesicr analgesic effects effects of suppressing of suppressing ERK1/2 ERK1/2 phos- phosphorylationphorylation in the in spinal the spinal cord. Ourcord. previous Our previous study showed study showed that trametinib, that trametinib, a MEK inhibitor, a MEK inhibitor,inhibited inhibited oxaliplatin-, oxaliplatin-, paclitaxel-, paclitaxel-, vincristine-, vincristine-, and bortezomib-induced and bortezomib-induced neuropathy neurop- by athyrepressing by repressing chemotherapy-induced chemotherapy-induced ERK1/2 activationERK1/2 activation in the lumbar in the spinal lumbar cord spinal of mice cord [25 of]. miceTherefore, [25]. Therefore, ERK1/2 phosphorylation ERK1/2 phosphorylation appears to appears be involved to be ininvolved CIPN, andin CIPN, its inhibition and its inhibitionby gabapentin by gabapentin and duloxetine and duloxetine effectively effectively prevented prevented oxaliplatinoxaliplatin- and paclitaxel-induced and paclitaxel- inducedneuropathic-like neuropathic-like pain behavior. pain behavior. We found that 6 mg/kg mg/kg oxaliplatin oxaliplatin and and paclitaxel paclitaxel induced induced neuropathy neuropathy in in mice. mice. Oxal- Ox- aliplatin is injected at 85 mg/m2 every 2 weeks or 130 mg/m2 every 3 weeks in humans, iplatin is injected at 85 mg/m2 every 2 weeks or 130 mg/m2 every 3 weeks in humans, which corresponds to approximately 6 mg/kg in mice [24]. In addition, several studies have which corresponds to approximately 6 mg/kg in mice [24]. In addition, several studies shown that treatment with oxaliplatin (6 or 10 mg/kg) induced neuropathy in mice [26–29]. have shown that treatment with oxaliplatin (6 or 10 mg/kg) induced neuropathy in mice In the clinical setting, the recommended paclitaxel dose is 210 mg/m2. For an adult human [26–29]. In the clinical setting, the recommended paclitaxel dose is 210 mg/m2. For an adult body weighing 60 kg, the paclitaxel dose is 5.9 mg/kg [30]. Moreover, the dose required to human body weighing 60 kg, the paclitaxel dose is 5.9 mg/kg [30]. Moreover, the dose induce peripheral neuropathy with paclitaxel is 4–10 mg/kg in mice [31–35]. Therefore, required to induce peripheral neuropathy with paclitaxel is 4–10 mg/kg in mice [31–35]. we selected a dose of 6 mg/kg oxaliplatin and paclitaxel in mice. In addition, the equiv- Therefore, we selected a dose of 6 mg/kg oxaliplatin and paclitaxel in mice. In addition, the equivalent dose (mg/kg) of gabapentin or duloxetine for mice was calculated by multiplying human equivalent doses by a factor of 12.3 [36,37]. The resultant therapeutic

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alent dose (mg/kg) of gabapentin or duloxetine for mice was calculated by multiplying human equivalent doses by a factor of 12.3 [36,37]. The resultant therapeutic equivalent doses of duloxetine and gabapentin were found to be 140 and 28 mg/kg, respectively. Thus, 100 mg/kg (round off) of gabapentin or 30 mg/kg (round up) of duloxetine was administered as the maximum dose to mice. Oxaliplatin administration induces PKCγ up-regulation and PKCγ and PKCε phosphorylation within the thalamus and periaqueductal gray area in rats [10,38]. In mouse model, treatment with paclitaxel induced persistent activation of PKCβII, PKCδ, and PKCε in the DRG and spinal cord [11,39–41]. In addition, PKC-induced ERK1/2 phosphorylation has been associated with chronic pain [10,42]. Moreover, ERKs act as regulators of nociceptive sensitivity in various models of inflammatory pain and mechanical and thermal hyperalgesia, and have been used as measurable marker for sensitization in pain studies [43–48]. Therefore, inhibiting ERK signaling may be an effective strategy for preventing chemotherapy-induced neuropathy. A number of studies have suggested that transient receptor potential (TRP) channels are involved in chemotherapy-induced neuropathic pain [49,50]. Treatment with oxaliplatin increases the expression of TRPV1, TRPV4, TRPA1, and TRPM8, and the upregulation of TRPA1 and TRPM8 in mice and rats is associated with oxaliplatin-induced cold allodynia [51–53]. Furthermore, TRPV1 and TRPV4 are involved in paclitaxel-induced cold and mechanical allodynia in rats and mice [54–56]. In addition, ERK activation in DRG nociceptive neurons promotes TRPV1 expression [57]. The activation of TRP channels activates several signaling pathways such as the nuclear factor-kappa B pathway and mitogen-activated protein kinase pathways, including ERK and p38 pathways [58–60]. Thus, this evidence indicates that ERK1/2 and members of the TRP family interact with each other, and together may be involved in the pathophysiology of neuropathic pain. It has been reported that administration with 40 mg/kg duloxetine improved the chemotherapy (oxaliplatin-based chemotherapy: FOLFOX therapy, paclitaxel, and bortezomib)-induced peripheral neuropathic pain in patients with multiple myeloma, colon, and breast cancer [61]. It has also indicated that treatment with duloxetine for chemotherapy-induced peripheral neuropathy was moderate recommendation in the American Society of Clinical Oncology guidelines [62]. In addition, administration with gabapentin was improved paclitaxel-induced neuropathy symptoms, neuropathic pain, neurologic deficit, and quality of life in ovarian cancer patients [63]. Our results showed that gabapentin and duloxetine prevented the development of oxaliplatin- and paclitaxel- induced neuropathy via inhibition of ERK1/2 activation in spinal cord of mice. Therefore, the present study showed part of the mechanism of action of gabapentin and duloxetine, which has been shown to be useful in clinical trials. There is one limitation in our study. Although we used male mice to investigate the preventive effects of gabapentin, duloxetine, and PD0325901 on oxaliplatin- and paclitaxel- induced neuropathic-like pain behavior, did not investigated female mice. It has been reported that sphingosine-1-phosphate receptor subtype 1 (S1PR1) antagonists and A3 adenosine receptor subtype (A3AR) agonists suppressed the oxaliplatin- and paclitaxel- induced neuropathy in male and female rats, but S1PR1 antagonists and A3AR agonists only inhibited the bortezomib-induced neuropathy in male rats. [64]. It has also indicated that resolvin D5 inhibited the paclitaxel-induced mechanical allodynia in male mice, but did not affect female mice [65]. These findings suggest that the effectiveness of drugs to suppress pain-like behavior present sex differences. Thus, future studies will examine the preventive effects of gabapentin, duloxetine, and PD0325901 on oxaliplatin- and paclitaxel- induced neuropathic-like pain behavior in female mice.

4. Materials and Methods 4.1. Animals Male 5-week-old BALB/c mice were purchased from Shimizu Laboratory Animals (Kyoto, Japan). The mice were housed in standard cages maintained at 25 ◦C, kept under a Pharmaceuticals 2021, 14, 30 12 of 16

12 h light/12 h dark cycle, and allowed free access to water and food pellets. All animal experiments were approved by the Animal Care and Use Committee of Kindai University (project identiﬁcation code KAPS-2020-011, 1 April 2020).

4.2. Drugs Oxaliplatin and PD0325901 were purchased from LC Laboratories (Woburn, MA, USA). Paclitaxel was purchased from FUJIFILM Wako (Tokyo, Japan). Gabapentin was purchased from Sigma (St Louis, MO, USA). Duloxetine was purchased from LKT Laboratories, Inc. (St Paul, MN, USA). Oxaliplatin was dissolved in 5% glucose solution. Paclitaxel was dissolved in cremophor/ethanol/saline (1:1:18, Sigma). Gabapentin, duloxetine, and PD0325901 were dissolved in dimethyl sulfoxide (DMSO), then diluted to 0.5% DMSO in phosphate buffered saline.

4.3. Oxaliplatin- and Paclitaxel-Induced Neuropathy Models After measuring the baseline nociceptive threshold, mice (6-week-old) were administered drugs as per the following schedule. Mice were administered oxaliplatin (6 mg/kg) by intravenous injection (i.v.), paclitaxel (6 mg/kg) by intraperitoneal injection (i.p.), 5% glucose solution, or cremophor/ethanol/saline (vehicle) on days 0 and 7 (n = 7 for each group). On day 0, 12 h after treatment with oxaliplatin or paclitaxel, mice were administered gabapentin, duloxetine, or PD0325901. Behavioral tests were performed daily from day 0 to 14. Gabapentin (30 or 100 mg/kg, p.o.), duloxetine (10 or 30 mg/kg, p.o.), PD0325901 (30 mg/kg, p.o.), or 0.5% DMSO (vehicle, p.o.) was administered daily from day 0 to 14 (n = 7 for each group) after conducting behavioral tests. The investigator was blinded to the experimental conditions while evaluating the antinociceptive effects of gabapentin, duloxetine, or PD0325901 on the behavioral features of mice.

4.4. Behavioral Tests Behavioral assays were performed as described in a previous study [25,66,67]. Sen- sitivity to cold was assessed using a hot/cold plate analgesiometer (Ugo Basile, Milan, Italy). Each mouse was placed at the center of a plate maintained at 10 ◦C (cold allodynia), following which oxaliplatin- and paclitaxel-induced pain-related behaviors, such as lifting and licking of the hind paw, were observed, and the time was recorded (cut-off time was 30 s). Mechanical allodynia and hyperalgesia were studied using 0.16, 0.4, and 1.4 g of von Frey filaments (Ugo Basile). For each filament, five stimuli were applied at an interval of 3–5 s, and mechanical sensitivity was scored as follows: 0, no response; 1, paw withdrawal; and 2, immediate flinching of the stimulated paw. Aversive response score of five trials from both hind paws of each mouse were averaged and recorded as mean ± S.D.

4.5. Western Blotting Mice were sacriﬁced, and the lumbar spinal cords were quickly dissected. The dissected tissue was homogenized in ice-cold buffer (20 mM Tris-HCl (Sigma, pH 7.4), 2% Triton X-100 (Sigma), 150 mM NaCl (FUJIFILM Wako), 1 mM EDTA (FUJIFILM Wako), 5 mM MgCl2 (FUJIFILM Wako), 10% anhydrous glycerol (FUJIFILM Wako), protease and phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA)) and centrifuged. The extracts (20 µg of protein) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene ﬂuoride membranes (GE Health- care, Buckinghamshire, UK). The membranes were blocked with a solution containing 3% skimmed milk and incubated overnight at 4 ◦C with anti-phospho-ERK1/2 (Thr202/Tyr204; 1:3000; #9101) (Cell Signaling Technology, Beverly, MA, USA), anti-ERK1/2 (1:3000; #9102, Cell Signaling Technology), and anti-β-actin (1:3000; AC-74, Sigma) antibodies. The membranes were incubated with horseradish peroxidase-coupled anti-rabbit IgG sheep antibodies (GE Healthcare) for 1 h at room temperature. The reactive proteins were visualized Pharmaceuticals 2021, 14, 30 13 of 16

using Luminata Forte HRP substrate (Merck Millipore, Nottingham, UK) according to the manufacturer’s instructions.

4.6. Statistics All results are expressed as the mean ± S.D. of at least 4 number of independent experiments. Following ANOVA test, multiple comparisons were conducted using the Tukey test, and the control group and various drug-treated groups were compared and analyzed using Dunnet’s test. Data were tested for normality using the Shapiro-Wilk test. When data were not normally distributed, these were analyzed using the Kruskal-Wallis test followed by the Scheffe test. p values < 0.05 were considered signiﬁcant.

5. Conclusions In summary, our study suggests that the analgesic effects of gabapentin, duloxetine, and PD0325901 against the development of oxaliplatin- and paclitaxel-induced neuropathic- like pain behavior in mice are mediated via the inhibition of ERK1/2 activation. In addition, we demonstrated that the activation of ERK1/2 in the spinal cord plays a critical role in the induction of mechanical and cold allodynia in oxaliplatin- and paclitaxel-induced neuropathic-like pain. Furthermore, these data implicate that inhibiting ERK1/2 activation using gabapentin, duloxetine, PD0325901, or other therapeutic agents could be an effective preventive strategy against oxaliplatin- and paclitaxel-induced neuropathy.

Supplementary Materials: The following are available online at https://www.mdpi.com/1424-8 247/14/1/30/s1, Figure S1: Safety of oxaliplatin, paclitaxel, and gabapentin administrated in vivo, Figure S2: Safety of oxaliplatin, paclitaxel, and duloxetine administrated in vivo, Figure S3: Safety of oxaliplatin, paclitaxel, and PD0325901 administrated in vivo, Figure S4: Full length of western blot analysis for Figures5 and8. Author Contributions: Investigation, N.K., K.T. (Keisuke Tateishi), M.T., T.T., M.M., and K.T. (Katsumasa Tsurushima); formal analysis, N.K., K.T. (Keisuke Tateishi), and T.I.; Conceptualization, S.N., writing—original draft preparation, N.K. and K.T. (Keisuke Tateishi); writing—review and editing, N.K., K.T. (Keisuke Tateishi), and S.N. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by a Grant-in-Aid for Scientific Research (C) (Grant number 20K07145 and 20K07168) from the Japan Society for the Promotion of Science (JSPS). Institutional Review Board Statement: All animal experiments were approved by the Animal Care and Use Committee of Kindai University (project identification code KAPS-2020-011, 1 April 2020). Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in the main text and the Supplementary Materials. Conflicts of Interest: The authors declare no conflict of interest.

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