Antinociceptive, antiallodynic and antihyperalgesic effects of the 5-HT1A receptor selective agonist, NLX-112 in mouse models of pain

Accepted Manuscript Antinociceptive, antiallodynic and antihyperalgesic effects of the 5-HT1A receptor selective agonist, NLX-112 in mouse models of pain Kinga Sałat, Marcin Kołaczkowski, Anna Furgała, Adriana Rojek, Joanna Śniecikowska, Mark A. Varney, Adrian Newman-Tancredi PII:

S0028-3908(17)30353-2

DOI:

10.1016/j.neuropharm.2017.07.022

Reference:

NP 6791

To appear in:

Neuropharmacology

Received Date: 11 April 2017 Revised Date:

11 June 2017

Accepted Date: 22 July 2017

Please cite this article as: Sałat, K., Kołaczkowski, M., Furgała, A., Rojek, A., Śniecikowska, J., Varney, M.A., Newman-Tancredi, A., Antinociceptive, antiallodynic and antihyperalgesic effects of the 5-HT1A receptor selective agonist, NLX-112 in mouse models of pain, Neuropharmacology (2017), doi: 10.1016/ j.neuropharm.2017.07.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Antinociceptive, antiallodynic and antihyperalgesic effects of the 5-HT1A receptor selective agonist, NLX-112 in mouse models of pain Kinga Sałat1*, Marcin Kołaczkowski2, Anna Furgała1, Adriana Rojek1, Joanna Śniecikowska2, Mark A.

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Varney3, Adrian Newman-Tancredi3

Chair of Pharmacodynamics, Jagiellonian University, Medical College, Medyczna 9 St., 30-688

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Krakow, Poland

Chair of Pharmaceutical Chemistry, Jagiellonian University, Medical College, Medyczna 9 St., 30-688

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Krakow, Poland

Neurolixis Inc. 34145 Pacific Coast Highway #504, Dana Point, CA 92629 USA

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*corresponding author: [email protected] (Kinga Sałat)

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Abstract Background and purpose: NLX-112 (a.k.a. befiradol, F13640) is a drug candidate intended for the treatment of L-DOPA-induced dyskinesia. It is a highly selective serotonin 5-HT1A receptor full agonist which has been previously tested in a variety of models of CNS effects including analgesic activity in rat. Its activity in mouse models of pain has not been previously investigated.

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Experimental approach: The activity of NLX-112 was tested in mouse models of acute pain (hot plate), tonic pain (intraplantar formalin test), in the oxaliplatin-induced neuropathic pain model of chemotherapy-induced peripheral neuropathy and in the streptozotocin (STZ)-induced model of painful diabetic neuropathy.

Key results: The main findings indicate that (i) NLX-112 was markedly active in the formalin test with

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potent reduction of paw licking in both phases of the test (minimal effective dose (MED) 0.5 mg/kg i.p. and p.o. in acute phase, and 0.1 mg/kg i.p. and 1 mg/kg p.o. in late phase). The effects of NLX-

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112 in this test were completely abolished by the selective 5-HT1A receptor antagonist, WAY100635; (ii) NLX-112 was active in the hot plate test and in the oxaliplatin-induced neuropathic pain model of chemotherapy-induced peripheral neuropathy, but at markedly higher doses (MED 2.5 mg/kg i.p.); (iii) NLX-112 was least active in the STZ-induced model of painful diabetic neuropathy (MED 5 mg/kg i.p.); (iv) NLX-112 did not affect locomotor activity.

Conclusions and implications: NLX-112 may have significant potential for treatment of tonic pain but

neuropathic pain.

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may be less promising as a candidate for treatment of chemotherapy-induced or diabetic

Keywords: pain models; NLX-112;

tactile allodynia; thermal hyperalgesia; painful diabetic

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neuropathy; chemotherapy-induced peripheral neuropathy.

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Chemical compounds studied in this article: Befiradol (PubChem CID: 9865384); formalin (PubChem CID: 712); WAY100635 (PubChem CID: 5684); streptozotocin (PubChem CID: 29327); oxaliplatin (PubChem CID: 9887054)

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1. Introduction Although painful conditions, including acute, tonic and neuropathic pains, constitute a major public health challenge and a large market opportunity, there remain large unmet medical needs for improved therapeutic options. For example, although morphine is often indicated for the relief of severe pain (e.g. post-operatively, in myocardial infarction, severe injury or in severe chronic pain

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associated with terminal cancer after non-narcotic analgesics have failed) it is associated with substantial limitations, including psychological and physical tolerance and dependence, respiratory depression, lethargy, euphoria and a short plasma half-life of 2 to 3 hours (Trescot et al., 2008).

In the case of neuropathic pain, which affects between 3-8% of the world’s population, tricyclic antidepressants, serotonin-noradrenaline reuptake inhibitors, pregabalin, gabapentin and tramadol,

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as well as lidocaine or high-concentration capsaicin patches, often have undesirable and doselimiting adverse effects which makes pharmacotherapy of neuropathic pain even more challenging,

al., 2003; Gilron et al., 2009).

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and are ineffective in approximately 40% of neuropathic patients (Finnerup et al., 2005; Gagnon et

There is, therefore, a strong medical need to explore novel drug targets and identify new, active analgesic compounds (Brederson et al., 2013). In this context, serotonergic mechanisms have been implicated in the control of pain states and specifically, 5-HT1A receptors are attractive candidates because they are expressed throughout the pain neuroaxis. For example, 5-HT1A receptors are

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expressed at high levels in the dorsal horn of the spinal cord (Daval et al., 1987), both centrally, where they regulate 5-HT release and inhibit descending serotonergic neurons, as well as on spinal cord spinothalamic neurons and on inhibitory interneurons where they regulate transmission of nociceptive signals (Bardin, 2011; Perrin et al., 2011). The prototypical 5-HT1A receptor agonist, such

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as 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) influenced spinal-cord regulated pain responses, but with discordant results. Thus, intrathecal administration of 8-OH-DPAT was found to

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increase the A-delta and C-fiber responses and decrease tail-flick latency (Alhaider and Wilcox, 1993; Ali et al., 1994; Zhang et al., 2001), whereas other studies found that spinal administration of 5-HT1A receptor agonists induced analgesia (Bardin and Colpaert, 2004; Nadeson and Goodchild, 2002; Xu et al., 1994). An important consideration is that 5-HT1A receptor agonists may differentially influence pain responses depending on the type of nociceptive stimulus: when administered intrathecally, 8OH-DPAT exhibited pronociceptive activity in the paw pressure test but analgesic activity in the formalin test (Bardin and Colpaert, 2004), for review see Bardin (2011). Taken together, these considerations suggest that 5-HT1A receptor agonists could constitute useful therapeutic agents, if their mechanisms could be better elucidated and if suitably selective and efficacious compounds were available. Indeed, investigation of the relevance of such findings to human subjects has been hampered by the lack of clinically-available serotonin 5-HT1A agonists. The Page 3 of 23

ACCEPTED MANUSCRIPT only approved drugs that possess 5-HT1A receptor agonist properties are non-selective partial agonists such as buspirone (which also acts as a dopamine D2 receptor antagonist (Peroutka, 1985)), antidepressants such as vilazodone and vortioxetine (which also inhibit 5-HT reuptake and interact with various other receptors) or some antipsychotics such as aripiprazole or cariprazine, which are ‘promiscuous’ drugs interacting with multiple targets (Newman-Tancredi and Kleven, 2011). In contrast to these drugs, the novel, serotonergic ligand, NLX-112 (a.k.a. befiradol or F13640)

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exhibits a different profile. NLX-112 specifically targets 5-HT1A receptors, with a ‘full agonist’ activity, and exhibits no measurable affinity at a wide range of other receptors, binding sites or enzymes (Colpaert et al., 2002). The properties of NLX-112 have previously been investigated in a variety of rat models of pain with pronounced activity in the formalin test (Bardin et al., 2003), in models of

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intraoperative and postoperative pains (Kiss et al., 2005) and in models of chronic pain, such as ischemic injury of spinal cord or ligature of the infraorbital nerve (Deseure et al., 2002, 2004; Wu et

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al., 2003). These data suggest that NLX-112 may be a promising pharmacotherapeutic agent for treatment of a variety of chronic pain conditions. However, (i) information is not available concerning the analgesic activity of NLX-112 in other species, notably mouse, which is commonly used in preclinical testing; and (ii) no preclinical data are available characterizing the activity of NLX-112 in models of some common chronic pain conditions, such as chemotherapy-induced peripheral neuropathy (CIPN) and painful diabetic neuropathy (PDN). Lack of preclinical data supporting activity

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of NLX-112 in PDN is surprising because NLX-112 was previously tested in an 8-week, multicenter, randomized, double-blind and placebo-controlled Phase 2 clinical trial for this indication. The trial was completed in 2010 and it did not confirm efficacy of NLX-112 in reducing pain related to PDN (https://www.clinicaltrialsregister.eu).

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The present study therefore used mouse models of pain to assess the antinociceptive, antiallodynic and antihyperalgesic properties of NLX-112 as a potential new analgesic to treat pain of various

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origins, including acute pain, tonic pain and neuropathic pain related to PDN and CIPN.

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2. Materials and methods 2.1. Animals Adult male Albino Swiss (CD-1) mice weighing between 18–22 g were used in this study. Mice were housed in groups of 10 per cage at constant ambient temperature of 22±2 °C, and humidity (50 ± 10%) under a light/dark (12:12) cycle. The animals had free access to food and water before

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experiments. Each experimental group consisted of 8-10 animals selected randomly. The experiments were performed between 8 AM and 2 PM and immediately after completion of the assay the animals were euthanized via cervical dislocation. All procedures for animal maintenance and treatment were performed in compliance with the European Union Directive 2010/63/EU on the

of the Jagiellonian University in Krakow (39/2016).

2.2. Chemicals used in in vivo tests

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protection of animals used for scientific purposes and were approved by the Local Ethics Committee

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NLX-112 (a gift from Neurolixis, Inc., California, US) was dissolved in 0.9% saline (Polfa Kutno, Poland). STZ and oxaliplatin were purchased from Sigma Aldrich (Poland) and (Activate Scientific GmbH, Germany), respectively. NLX-112, STZ and oxaliplatin were administered at a constant volume of 10 ml/kg. For the experiments STZ was prepared by dissolving in 0.1 N citrate buffer (Polskie Odczynniki Chemiczne, Poland). Oxaliplatin was prepared in a 5% glucose solution (Polfa Kutno, Poland). Formalin (37% formaldehyde solution) was purchased from Polskie Odczynniki Chemiczne

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(Poland). For pain tests, it was diluted in distilled water (Polskie Odczynniki Chemiczne, Poland) to obtain 5% solution. The selective 5-HT1A receptor antagonist, WAY100635, (N-[2-[4-(2methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide maleate, Tocris UK),

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was dissolved in distilled water immediately before administration in a volume of 2 ml/kg.

2.3. Streptozotocin-induced diabetic neuropathic pain model

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2.3.1. Induction of diabetes and painful diabetic neuropathy

To induce type I diabetes and diabetic neuropathic pain, mice were administered streptozotocin (STZ), an alkylating antitumor drug which destroys insulin-secreting islet cells. Mice were administered a single injection of STZ (200 mg/kg i.p.). Age-matched control mice received an equal volume of citrate buffer. Blood glucose levels were measured 1 day before (referred to as ‘day 0’) and repeatedly 1, 2 and 3 weeks after STZ injection using a blood glucose monitoring system (AccuChek Active, Roche, France). Blood samples (5 µl) for the measurement of glucose concentration were obtained from the tail vein of the mice. The animals were considered to be diabetic when their blood glucose concentration exceeded 300 mg/dl (Tanabe et al., 2008). Approx. 70% of mice developed diabetes and were used in subsequent pain tests. Page 5 of 23

ACCEPTED MANUSCRIPT 2.3.2. Assessment of tactile allodynia in STZ-treated mice – von Frey test The electronic von Frey unit (Bioseb, France) is supplied with a single flexible filament applying increasing force (from 0 to 10 g) against the plantar surface of the hind paw. In this assay the nocifensive paw withdrawal response automatically turns off the stimulus and the mechanical pressure that evoked the response was recorded. On the day of the experiment, the mice were placed individually in test compartments with a wire mesh bottom and were allowed to habituate for

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1 h. After the habituation period, in order to obtain baseline values, each mouse was tested 3 times alternately in each hind paw, allowing at least 30 s between each measurement. Then, the mice were intraperitoneally pretreated with NLX-112 (2.5 and 5 mg/kg) or vehicle and 60 min later the animals were tested again and 3 measures were taken and averaged to obtain mean post-drug values for

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each mouse (Sałat et al., 2013). The test lasted approx. 15 min.

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2.3.3. Assessment of heat hyperalgesia in STZ-treated mice – hot plate test The effect of NLX-112 on heat hyperalgesia was assessed using the hot plate test. In this assay after the establishment of pre-drug latency to pain reaction for each animal, the mice were treated with NLX-112 at doses 2.5 and 5 mg/kg and 60 min later they were placed on the hot plate apparatus (Hot/cold plate, Bioseb, France) set at 55-56 °C and were observed to the appearance of a nocifensive response (hind paw licking or jumping). A cut-off time of 60 s was established to avoid

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paw tissue damage, and mice not responding within 60 s were removed from the apparatus and assigned a score of 60 s. The test lasted approx. 10 min.



2.4. Oxaliplatin-induced CIPN neuropathic pain model

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2.4.1. Induction of CIPN

In order to induce CIPN oxaliplatin was administered to mice as a single dose of 10 mg/kg i.p. Pain

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tests on oxaliplatin-treated mice and naïve mice were performed 3 h and 7 days later.

2.4.2. Assessment of tactile allodynia in oxaliplatin-treated mice – von Frey test

This test was performed according to a method described in Section 2.3.2. Acute-phase tactile allodynia in oxaliplatin-treated mice was assessed 3 h after oxaliplatin, whereas late-phase allodynia was tested 7 days later. Antiallodynic efficacy of NLX-112 was established for doses 1.25, 2.5 and 5 mg/kg.

2.4.3. Assessment of cold allodynia in oxaliplatin-treated mice – cold plate test The effect of NLX-112 on cold allodynia was assessed using the cold plate test. In this assay after the establishment of pre-drug latency to pain reaction for each animal, the mice were treated Page 6 of 23

ACCEPTED MANUSCRIPT intraperitoneally with NLX-112 at doses 1.25, 2.5 and 5 mg/kg and 60 min later they were placed on the cold plate apparatus (Hot/cold plate, Bioseb, France) set at 4°C and were observed to the appearance of a nocifensive response (hind paw licking, shaking, jumping or abnormal movements). A cut-off time of 60 s was established to avoid paw tissue damage, and mice not responding within 60 s were removed from the apparatus and assigned a score of 60 s. The test lasted approx. 10 min.

2.5. Tonic pain model - formalin test

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In mice, the intraplantar injection of diluted formalin produces a biphasic nocifensive behavioral response (i.e. licking or biting the injected paw). The acute nociceptive (neurogenic) phase lasts for the first 5 min, whereas the second (inflammatory) phase occurs between 15-30 min after formalin injection. The formalin test in mice was performed according to (Laughlin et al., 2002). The mice

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were pretreated intraperitoneally or orally with NLX-112 (doses: 0.1 - 5 mg/kg, or 0.5 – 5 mg/kg, respectively), or vehicle and were allowed to habituate in Plexiglas observation chambers (20 x 30 x

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15 cm) for 60 min before the test. Then, 20 μl of a 5% formalin solution was injected intraplantarly into the right hind paw using a 26-gauge needle. Immediately after formalin injection, the animals were placed individually into glass beakers and were observed over the next 30 min. Time (in seconds, recorded with a stopwatch (Q&Q HS-46, Japan, precision: 1/100 s) spent on licking or biting the injected paw provided an indicator of nociceptive behavior and was determined during selected intervals: 0-5 min, 15-20, 20-25 and 25-30 min post-formalin injection.

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For experiments with the selective 5-HT1A receptor antagonist, WAY100635 (0.63 mg/kg i.p.), the latter was administered 15 min before the NLX-112 and 75 min before formalin injection. The dose of WAY100635 was chosen based on our previous studies that utilized this compound (for example, see

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Assié et al., 2010).

2.6. Acute, thermally-induced pain model - hot plate test

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Antinociceptive properties of NLX-112 in the hot plate test were assessed as described previously (Eddy and Leimbach, 1953) with some minor modification. One day before the pharmacological experiment, the animals were tested for their baseline pain sensitivity threshold (baseline latency). For further pain tests, only mice with baseline latencies ≤ 20 s were used. On the test day, the mice were intraperitoneally treated either with NLX-112 (doses: 0.5, 1, 2.5 and 5 mg/kg) or vehicle 60 min before placing the animal on an apparatus from Bioseb, France. This can generate heat or cold and has a temperature-controller that can maintain surface temperature to a set point. Herein, the temperature was set at 55-56◦C. Latency time to pain reaction, i.e., the time until the animal licked its hind paws or jumped was recorded by means of a stop-watch (Q&Q HS-46, Japan, precision: 1/100 s). In this assay, a cut-off time (60 s) was enforced to avoid paw tissue damage, and mice not responding

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ACCEPTED MANUSCRIPT within 60 s were removed from the apparatus and assigned a score of 60 s. The test lasted approx. 10 min.

2.7. Locomotor activity test The locomotor activity test was performed as described (Sałat et al., 2014) using activity cages (40 x 40 x 31 cm, L x W x H) supplied with infrared beams (Activity Cage 7441, Ugo Basile, Italy) connected to a counter for the recording of beam interrupts. Sixty min before the experiment the mice were

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intraperitoneally pretreated with NLX-112 (5 mg/kg) or vehicle, then being individually placed in the activity cages in a sound-attenuated room. The animals' locomotor activity (i.e. the number of lightbeam interrupts) was counted during 0-5, 5-10, 10-15, 15-20, 20-25 and 25-30 min intervals.

2.8. Data analysis

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Data analysis was carried out using GraphPad Prism software (v.6.0, California, US). Numerical results are expressed as the mean ± SEM. Statistical analysis was carried out by one-way analysis of variance

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(ANOVA), followed by Tukey’s or Dunnett’s post-hoc comparisons to compare all pairs of groups, or to compare drug-treated groups vs. the control group, respectively. Repeated measures ANOVA, followed by Bonferroni’s multiple comparison was used for group comparisons made repeatedly at

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different time-points (oxaliplatin model and locomotor activity test).

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3. Results 3.1. STZ-induced diabetic neuropathy: Influence on tactile allodynia (von Frey test) In this test an overall effect of treatment was observed (F[4,45]=34.10; p < 0.0001). STZ significantly (p < 0.001) reduced mechanical nociceptive threshold and the treatment with intraperitoneal NLX-

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112 at the dose of 5 mg/kg significantly (p < 0.001) attenuated this effect (Fig. 1A).

Fig. 1

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3.2. STZ-induced diabetic neuropathy: Influence on heat hyperalgesia (hot plate test) The hot plate test used to assess the impact of STZ and NLX-112 on heat hyperalgesia did not reveal

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any significant effect of treatment on heat nociceptive threshold (F[4,35]=1.394; p > 0.05; Fig. 1B). In STZ-treated diabetic mice compared to not treated controls (i.e. non-diabetic mice) we did not observe the development of heat hyperalgesia as in these groups the baseline latencies to pain reaction were similar: 20.7±2.2 s (normoglycemic control group), 23.4±2.0 s (pre-drug latency of NLX112 2.5 mg/kg group) and 23.3±2.8 s (pre-drug latency of NLX-112 5 mg/kg group). The analysis of post-drug latencies of NLX-112-treated mice showed that in contrast to its statistically significant

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antiallodynic activity in the von Frey test, NLX-112 was not effective in this assay.

3.3. Oxaliplatin-induced peripheral neuropathy: Influence on tactile allodynia (von Frey test)

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In this neuropathic pain model an overall effect of treatment was demonstrated (F[12,117]=37.89; p < 0.0001). The injection of oxaliplatin significantly (p < 0.001) decreased the paw withdrawal force, i.e. increased pain sensitivity of mice both in the acute and in the late phases (Fig. 2A).

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Intraperitoneal NLX-112, at 2.5 and 5 mg/kg effectively reduced acute and late phase tactile allodynia (significant at p < 0.001). In contrast, the low dose of 1.25 mg/kg i.p. was not active in either phases.

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3.4. Oxaliplatin-induced peripheral neuropathy: Influence on cold allodynia (cold plate test) In the cold plate test an overall effect of treatment was observed (F[12,99]=2.328; p < 0.05). In this assay mice not treated with oxaliplatin (i.e. mice without CIPN) were able to remain on the cold plate during the whole observation period (60 s). In contrast, oxaliplatin-treated mice developed cold allodynia and they showed nocifensive behavior in response to cold. In these animals, we observed Page 9 of 23

ACCEPTED MANUSCRIPT significantly reduced (p < 0.05 vs. control mice not treated with oxaliplatin) latency time to pain reaction that ranged from 30-40 s. The use of NLX-112 in oxaliplatin-treated mice did not significantly influence the cold nociceptive threshold (Fig. 2B).

3.5. Tonic pain model (formalin test) Intraperitoneal administration of NLX-112 resulted in a marked and dose-dependent antinociceptive

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effect in both phases of the formalin test (drug effect: F[5,120]=26.25; p < 0.0001; time effect: F[3,20]=4.09; p < 0.01; drug x time interaction: F[15,120]=0.82; p > 0.05) (Fig 3A). That was true for both the first (neurogenic, from 0-5 min post formalin injection) and the second (inflammatory, from 15-30 min) phases.

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NLX-112 also showed strong antinociceptive activity when administered orally (drug effect: F[4,108]=11.11; p < 0.0001; time effect: F[3,108]=3.10; p < 0.05; drug x time interaction:

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F[12,108]=2.14; p < 0.05). In the early phase, it significantly reduced nocifensive response at all doses tested, whereas in the late phase it displayed antinociceptive properties mostly from 2.5 mg/kg p.o. (Fig. 3B). In all cases, the Minimal Effective Dose (MED) was 0.5 mg/kg i.p. and p.o. in the acute phase, and 0.1 mg/kg i.p. and 1 mg/kg p.o. in the late phase.

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Fig. 3

3.6. Blockade of the effects of NLX-112 with WAY100635 in the formalin test The involvement of 5-HT1A receptors in the analgesic efficacy of NLX-112 (0.5 mg/kg i.p.) in the formalin test was tested by interaction with the selective antagonist, WAY100635 (0.63 mg/kg, i.p.).

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There was a significant overall effect of treatment in both the early and late phases (early phase: F[3,26]=14.19; p < 0.0001), late phase: F[3,26]=19.56; p < 0.0001; Fig. 4). NLX-112 efficaciously

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decreased paw licking in both phases of the test (significant at p < 0.01 vs. vehicle-treated mice) whereas WAY100635 did not. WAY100635 abolished the analgesic effect of NLX-112 in the early phase (p < 0.001), and significantly reduced it in the late phase (p < 0.01) of the formalin test. Fig. 4

3.7. Acute thermally-induced pain model (hot plate test) In the hot plate test an overall effect of treatment with intraperitoneal NLX-112 was observed (F[4,33]=14.32; p < 0.0001). A statistically significant prolongation of latency time to pain reaction was shown for doses of 2.5 and 5 mg/kg (Fig. 5).

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3.8. Locomotor activity In this test, only the highest previously tested in pain tests dose of NLX-112 (5 mg/kg; i.p.) was assessed for its influence on locomotor activity (Fig. 6). NLX-112 did not affect locomotor activity as measured in an actimeter (drug effect: F[1,65]=0.22; p > 0.05; time effect: F[5,65]=1.33; p > 0.05;

tests were not due to behavioral interference on motor responses.

Fig. 6

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4. Discussion

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drug x time interaction: F[5,65]=2.07; p > 0.05), which confirmed that the activities observed in pain

4.1. Effects of NLX-112 in a model of painful diabetic neuropathy

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Although NLX-112 has previously been characterized in a variety of tests of analgesic activity in rat models, it has not been characterized in mouse, a species which is commonly used for testing potential analgesic agents (Barrett, 2015; Mogil, 2009). In addition, NLX-112 has not been previously tested in models of two common forms of chronic (neuropathic) pain, i.e. CIPN and PDN. The latter is of particular relevance because NLX-112 was previously developed as a potential clinical therapeutic for PDN and clinical trials for this indication were discontinued (see comments in Introduction).

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In the present study, mouse PDN was investigated using the STZ model, with tests performed 21 days after STZ administration. At this time-point, diabetic mice exhibit a significant decrease of mechanical nociceptive threshold that results in the development of tactile allodynia, heat hyper- or hypoalgesia, with the latter more often observed at more advanced stages of diabetes (i.e., ≥12 weeks after STZ

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treatment) (Obrosova, 2009; Ulugol et al., 2012). Under the protocol used in the present study, STZtreated diabetic mice exhibited significantly increased blood glucose levels, urine output and

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decreased body weight gain compared to their normoglycemic littermates (Sałat et al., 2013). Under these conditions, NLX-112 exhibited a significant reduction of tactile allodynia in diabetic neuropathic mice (MED 5 mg/kg i.p.). In fact, NLX-112 almost returned the mechanical nociceptive threshold of the mice in the von Frey test to levels similar to those in control mice. In contrast, unlike reference drugs such as pregabalin tested in our laboratory under the same conditions (Sałat et al., 2013), NLX-112 did not influence pain responses of diabetic mice in the hot plate test at the doses tested (2.5 and 5 mg/kg i.p.) even though NLX-112 did show activity in the hot plate test in normoglycemic mice at these doses. This suggests that some changes may have taken place in the sensitivity of 5-HT1A receptor signaling in PDN such that they are less responsive than under normal conditions – it would be interesting to test this hypothesis in appropriately-designed studies. Page 11 of 23

ACCEPTED MANUSCRIPT Taken together these observations indicate that although NLX-112 can reduce some aspects of PDN in mice (i.e. tactile allodynia) this does not extend to other aspects, notably thermal hyperalgesia. The implications of these data remain to be clarified but a lack of influence on thermal nociceptive threshold could limit the potential use of NLX-112 as a treatment for neuropathic pain states, in particular diabetic neuropathic pain. In addition, it is notable that the potency of NLX-112 for reducing allodynia in the PDN model is markedly less pronounced than in other pain tests (see

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below), suggesting that the latter may be more amenable to treatment with 5-HT1A receptor agonists.

4.2. Effects of NLX-112 in oxaliplatin-induced painful neuropathy

NLX-112 was more active in another model of neuropathic pain, i.e. painful neuropathy caused by

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chemotherapy treatment (CIPN) than in the PDN model. In the CIPN model, a single injection of oxaliplatin induces painful peripheral neuropathy accompanied by mechanical (tactile) and cold

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allodynia (but not heat hyperalgesia) (Xiao et al., 2012). Intraperitoneal NLX-112 attenuated tactile allodynia in both the acute-phase and in the late-phase neuropathy at doses of 2.5 and 5 mg/kg. As for the PDN model, NLX-112 at 5 mg/kg almost returned the mechanical nociceptive threshold of the mice in the von Frey test to levels similar to those in control mice. In contrast, NLX-112 failed to modify responses in the cold plate test, although reference drugs such as pregabalin are known to be active in this system (Aoki et al., 2014). The present data raises the question of the potential use of 5-

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HT1A receptor agonists as potential treatments for CIPN-related neuropathic pain. Although their role in oxaliplatin-induced neuropathy remains to be fully elucidated, a study showed that 5-HT1A receptor activation is important for the analgesic action of xaliproden, an agent which has been shown to effectively attenuate CIPN (Martel et al., 2009). Another study demonstrated a protective

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role of 5-HT1A receptor stimulation in CIPN induced by paclitaxel (Ward et al., 2014). These observations suggest that targeting 5-HT1A receptors can be a useful strategy for treatment of CIPN if

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suitably potent and efficacious agonists can be identified. In the case of NLX-112, other painful conditions, such as tonic pain or spinal cord-mediated nociception may respond better to its profile of 5-HT1A receptor activation (see next section).

4.3. Effects of NLX-112 in the formalin model of tonic pain In comparison with the tests described above, NLX-112 exhibited striking activity in the intraplantar formalin model of chemogenic pain both upon intraperitoneal and oral administration. In rodents, this model reflects persistent (tonic) pain comprising an early, neurogenic phase dependent on activation of sensory C-fibers and a late phase elicited by peripheral inflammation, particularly at the 5% formalin solution used here (Hunskaar and Hole, 1987; Tjølsen et al., 1992; Yashpal and Coderre, 1998). NLX-112 was highly effective, completely abolishing paw licking behavior in both the early (neurogenic), and late (inflammatory) phases: in comparison, morphine did not completely reverse Page 12 of 23

ACCEPTED MANUSCRIPT paw licking in the early phase, even when administered at 5 mg/kg i.p., although it did abolish paw licking in the late phase (Sałat et al., 2013). It is also notable that the effective doses of NLX-112 in the formalin test were 25-50-fold lower than the minimal effective doses (MEDs) determined for the other pain tests discussed above (i.e. 0.1 mg/kg i.p., compared with 2.5 to 5 mg/kg i.p. for other tests). The neurobiological basis for this is unclear but the present data might indicate that NLX-112 preferentially targets pain controlled by peripheral nociceptive mechanisms (i.e., stimulation of

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sensory C-fibers or peripheral inflammation) rather than the supra-spinal or centrally-mediated pain responses measured in the hot plate test or the PDN or CIPN neuropathic pain models. In fact, it is known that activation of spinal cord 5-HT1A receptors by intrathecal administration of 8-OH-DPAT abolishes paw licking in the rat formalin test (Bardin and Colpaert, 2004). NLX-112 may therefore

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preferentially activate spinal cord-located 5-HT1A receptors, rather than supraspinal 5-HT1A receptors, to mediate its analgesic effects. This interpretation is supported by the known capacity of NLX-112 to

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oppose formalin-induced c-Fos expression in dorsal horn laminae (Buritova et al., 2005), to inhibit electrophysiological C-fiber-mediated responses of spinal dorsal horn wide-dynamic range neurons (You et al., 2005) and to protect against allodynia in rats with spinal cord injury (Colpaert et al., 2004).

It should be noted that NLX-112 also exhibited potent analgesic activity in the formalin test in rat, at doses which are comparable to those which were found to be active here (Bardin et al., 2003). In

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addition, in both rat and in mouse, the activity of NLX-112 in this test was blocked by the selective 5HT1A receptor antagonist, WAY100635 (Bardin et al., 2003), indicating that the effects of NLX-112 are specifically mediated by these receptors.

It should be noted that the effects of NLX-112 herein do not appear to be attributable to sedation or

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motor dysfunction. In fact, NLX-112, at the highest and maximally effective dose in all pain tests of this study, did not elicit any significant change in the mice’s locomotor activity, suggesting that its

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effects in the pain models were indeed due to antinociceptive activity.

4.4. Perspectives and Conclusions The above considerations raise several perspectives. Firstly, the present data from acute administration experiments raise the question of potential changes in 5-HT1A receptors that may take place if NLX-112 is administered in chronic pain conditions. Indeed, previous studies with NLX-112 in rat found that its analgesic activity is amplified upon protracted subcutaneous minipump administration (Bruins Slot et al., 2003). This may reflect neuroadaptive changes or alterations in 5HT1A receptor signaling that may lead to increased potency of NLX-112, notably in the neuropathic pain tests. Secondly, the present data raise the question of the best choice of clinical pain indication for NLX-112. Indeed, the modest activity of NLX-112 in the PDN test suggests that other pain indications may constitute more appropriate options for pharmacotherapy. Thirdly, NLX-112 is Page 13 of 23

ACCEPTED MANUSCRIPT currently undergoing clinical development as a treatment for L-DOPA-induced dyskinesia in Parkinson’s disease (PD). Given that pain is the most prominent non-motor symptom observed in patients with PD (Park et al., 2015) and PD patients often suffer from co-morbid chronic pain, it will be interesting to determine whether NLX-112 could alleviate some of their pain symptoms as well as their motor symptoms. In conclusion, the present study demonstrated that NLX-112 possesses antinociceptive and

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antiallodynic activity in several mouse models of acute, tonic and neuropathic pain. The results suggest that selective, high efficacy activation of 5-HT1A receptors could be beneficial for treatment of various pain conditions but NLX-112 may be particularly suited for treatment of some specific types of pain. This is suggested by the fact that it was not very active in some tests, notably the STZ-

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induced model of diabetic neuropathy, which had been the focus of its previous clinical development, but highly efficacious in the intraplantar formalin test. It is possible that NLX-112 may

activity in this test of tonic pain analgesia.

Acknowledgements

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preferentially target subpopulations of 5-HT1A receptors in the spinal cord to achieve its potent

Disclosures

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This study was financially supported by the National Science Centre grant No. DEC015/19/B/NZ7/03543.

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AN-T and MAV are employees and stockholders of Neurolixis Inc.

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References

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Alhaider, A.A., Wilcox, G.L., 1993. Differential roles of 5-hydroxytryptamine1A and 5hydroxytryptamine1B receptor subtypes in modulating spinal nociceptive transmission in mice. J. Pharmacol. Exp. Ther. 265, 378–385. Ali, Z., Wu, G., Kozlov, A., Barasi, S., 1994. The actions of 5-HT1 agonists and antagonists on nociceptive processing in the rat spinal cord: results from behavioural and electrophysiological studies. Brain Res. 661, 83–90. Aoki, M., Kurauchi, Y., Mori, A., Nakahara, T., Sakamoto, K., Ishii, K., 2014. Comparison of the effects of single doses of elcatonin and pregabalin on oxaliplatin-induced cold and mechanical allodynia in rats. Biol. Pharm. Bull. 37, 322–326. Assié, M.B., Bardin, L., Auclair, A.L., Carilla-Durand, E., Depoortère, R., Koek, W., Kleven, M.S., Colpaert, F., Vacher, B., Newman-Tancredi, A., 2010. F15599, a highly selective post-synaptic 5-HT1A receptor agonist: in-vivo profile in behavioural models of antidepressant and serotonergic activity. Int. J. Neuropsychopharmacol. 13(10):1285-1298. doi:10.1017/S1461145709991222; Bardin, L., 2011. The complex role of serotonin and 5-HT receptors in chronic pain. Behav. Pharmacol. 22, 390–404. doi:10.1097/FBP.0b013e328349aae4 Bardin, L., Colpaert, F.C., 2004. Role of spinal 5-HT(1A) receptors in morphine analgesia and tolerance in rats. Eur. J. Pain Lond. Engl. 8, 253–261. doi:10.1016/j.ejpain.2003.09.002 Bardin, L., Tarayre, J.P., Malfetes, N., Koek, W., Colpaert, F.C., 2003. Profound, non-opioid analgesia produced by the high-efficacy 5-HT(1A) agonist F 13640 in the formalin model of tonic nociceptive pain. Pharmacology 67, 182–194. doi:68404 Barrett, J.E., 2015. The pain of pain: challenges of animal behavior models. Eur. J. Pharmacol. 753, 183–190. doi:10.1016/j.ejphar.2014.11.046 Brederson, J.-D., Kym, P.R., Szallasi, A., 2013. Targeting TRP channels for pain relief. Eur. J. Pharmacol. 716, 61–76. doi:10.1016/j.ejphar.2013.03.003 Bruins Slot, L.A., Koek, W., Tarayre, J.-P., Colpaert, F.C., 2003. Tolerance and inverse tolerance to the hyperalgesic and analgesic actions, respectively, of the novel analgesic, F 13640. Eur. J. Pharmacol. 466, 271–279. Buritova, J., Larrue, S., Aliaga, M., Besson, J.-M., Colpaert, F., 2005. Effects of the high-efficacy 5HT1A receptor agonist, F 13640 in the formalin pain model: a c-Fos study. Eur. J. Pharmacol. 514, 121–130. doi:10.1016/j.ejphar.2005.03.016 Colpaert, F.C., Tarayre, J.P., Koek, W., Pauwels, P.J., Bardin, L., Xu, X.-J., Wiesenfeld-Hallin, Z., Cosi, C., Carilla-Durand, E., Assié, M.B., Vacher, B., 2002. Large-amplitude 5-HT1A receptor activation: a new mechanism of profound, central analgesia. Neuropharmacology 43, 945–958. Colpaert, F.C., Wu, W.-P., Hao, J.-X., Royer, I., Sautel, F., Wiesenfeld-Hallin, Z., Xu, X.-J., 2004. Highefficacy 5-HT1A receptor activation causes a curative-like action on allodynia in rats with spinal cord injury. Eur. J. Pharmacol. 497, 29–33. doi:10.1016/j.ejphar.2004.06.026 Daval, G., Vergé, D., Basbaum, A.I., Bourgoin, S., Hamon, M., 1987. Autoradiographic evidence of serotonin1 binding sites on primary afferent fibres in the dorsal horn of the rat spinal cord. Neurosci. Lett. 83, 71–76. Deseure, K., Koek, W., Colpaert, F.C., Adriaensen, H., 2002. The 5-HT(1A) receptor agonist F 13640 attenuates mechanical allodynia in a rat model of trigeminal neuropathic pain. Eur. J. Pharmacol. 456, 51–57. Deseure, K.R., Adriaensen, H.F., Colpaert, F.C., 2004. Effects of the combined continuous administration of morphine and the high-efficacy 5-HT1A agonist, F 13640 in a rat model of trigeminal neuropathic pain. Eur. J. Pain Lond. Engl. 8, 547–554. doi:10.1016/j.ejpain.2004.01.002 Eddy, N.B., Leimbach, D., 1953. Synthetic analgesics. II. Dithienylbutenyl- and dithienylbutylamines. J. Pharmacol. Exp. Ther. 107, 385–393. Eu Clinical Trials Register, 2017. https://www.clinicaltrialsregister.eu (accessed 24.05.17). Page 15 of 23

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Finnerup, N.B., Otto, M., McQuay, H.J., Jensen, T.S., Sindrup, S.H., 2005. Algorithm for neuropathic pain treatment: an evidence based proposal. Pain 118, 289–305. doi:10.1016/j.pain.2005.08.013 Gagnon, B., Almahrezi, A., Schreier, G., 2003. Methadone in the Treatment of Neuropathic Pain. Pain Res. Manag. 8, 149–154. doi:10.1155/2003/236718 Gilron, I., Bailey, J.M., Tu, D., Holden, R.R., Jackson, A.C., Houlden, R.L., 2009. Nortriptyline and gabapentin, alone and in combination for neuropathic pain: a double-blind, randomised controlled crossover trial. Lancet Lond. Engl. 374, 1252–1261. doi:10.1016/S01406736(09)61081-3 Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between inflammatory and noninflammatory pain. Pain 30, 103–114. Kiss, I., Degryse, A.-D., Bardin, L., Gomez de Segura, I.A., Colpaert, F.C., 2005. The novel analgesic, F 13640, produces intra- and postoperative analgesia in a rat model of surgical pain. Eur. J. Pharmacol. 523, 29–39. doi:10.1016/j.ejphar.2005.09.003 Laughlin, T.M., Tram, K.V., Wilcox, G.L., Birnbaum, A.K., 2002. Comparison of antiepileptic drugs tiagabine, lamotrigine, and gabapentin in mouse models of acute, prolonged, and chronic nociception. J. Pharmacol. Exp. Ther. 302, 1168–1175. Martel, J.-C., Assié, M.-B., Bardin, L., Depoortère, R., Cussac, D., Newman-Tancredi, A., 2009. 5-HT1A receptors are involved in the effects of xaliproden on G-protein activation, neurotransmitter release and nociception. Br. J. Pharmacol. 158, 232–242. doi:10.1111/j.14765381.2009.00249.x Mogil, J.S., 2009. Animal models of pain: progress and challenges. Nat. Rev. Neurosci. 10, 283–294. doi:10.1038/nrn2606 Nadeson, R., Goodchild, C.S., 2002. Antinociceptive role of 5-HT1A receptors in rat spinal cord. Br. J. Anaesth. 88, 679–684. Newman-Tancredi, A., Kleven, M.S., 2011. Comparative pharmacology of antipsychotics possessing combined dopamine D2 and serotonin 5-HT1A receptor properties. Psychopharmacology (Berl.) 216, 451–473. doi:10.1007/s00213-011-2247-y Obrosova, I.G., 2009. Diabetic painful and insensate neuropathy: pathogenesis and potential treatments. Neurother. J. Am. Soc. Exp. Neurother. 6, 638–647. doi:10.1016/j.nurt.2009.07.004 Park, J., Lim, C.-S., Seo, H., Park, C.-A., Zhuo, M., Kaang, B.-K., Lee, K., 2015. Pain perception in acute model mice of Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Mol. Pain 11, 28. doi:10.1186/s12990-015-0026-1 Peroutka, S.J., 1985. Selective interaction of novel anxiolytics with 5-hydroxytryptamine1A receptors. Biol. Psychiatry 20, 971–979. Perrin, F.E., Gerber, Y.N., Teigell, M., Lonjon, N., Boniface, G., Bauchet, L., Rodriguez, J.J., Hugnot, J.P., Privat, A.M., 2011. Anatomical study of serotonergic innervation and 5-HT(1A) receptor in the human spinal cord. Cell Death Dis. 2, e218. doi:10.1038/cddis.2011.98 Sałat, K., Cios, A., Wyska, E., Sałat, R., Mogilski, S., Filipek, B., Więckowski, K., Malawska, B., 2014. Antiallodynic and antihyperalgesic activity of 3-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]dihydrofuran-2-one compared to pregabalin in chemotherapy-induced neuropathic pain in mice. Pharmacol. Biochem. Behav. 122, 173–181. doi:10.1016/j.pbb.2014.03.025 Sałat, K., Gawlik, K., Witalis, J., Pawlica-Gosiewska, D., Filipek, B., Solnica, B., Więckowski, K., Malawska, B., 2013. Evaluation of antinociceptive and antioxidant properties of 3-[4-(3trifluoromethyl-phenyl)-piperazin-1-yl]-dihydrofuran-2-one in mice. Naunyn. Schmiedebergs Arch. Pharmacol. 386, 493–505. doi:10.1007/s00210-013-0847-2 Tanabe, M., Murakami, T., Ono, H., 2008. Zonisamide suppresses pain symptoms of formalin-induced inflammatory and streptozotocin-induced diabetic neuropathy. J. Pharmacol. Sci. 107, 213– 220. Tjølsen, A., Berge, O.G., Hunskaar, S., Rosland, J.H., Hole, K., 1992. The formalin test: an evaluation of the method. Pain 51, 5–17. Page 16 of 23

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Trescot, A.M., Datta, S., Lee, M., Hansen, H., 2008. Opioid pharmacology. Pain Physician 11, S133153. Ulugol, A., Oltulu, C., Gunduz, O., Citak, C., Carrara, R., Shaqaqi, M.R., Sanchez, A.M., Dogrul, A., 2012. 5-HT7 receptor activation attenuates thermal hyperalgesia in streptozocin-induced diabetic mice. Pharmacol. Biochem. Behav. 102, 344–348. doi:10.1016/j.pbb.2012.05.006 Ward, S.J., McAllister, S.D., Kawamura, R., Murase, R., Neelakantan, H., Walker, E.A., 2014. Cannabidiol inhibits paclitaxel-induced neuropathic pain through 5-HT(1A) receptors without diminishing nervous system function or chemotherapy efficacy. Br. J. Pharmacol. 171, 636– 645. doi:10.1111/bph.12439 Wu, W.-P., Hao, J.-X., Xu, X.-J., Wiesenfeld-Hallin, Z., Koek, W., Colpaert, F.C., 2003. The very-highefficacy 5-HT1A receptor agonist, F 13640, preempts the development of allodynia-like behaviors in rats with spinal cord injury. Eur. J. Pharmacol. 478, 131–137. Xiao, W.H., Zheng, H., Bennett, G.J., 2012. Characterization of oxaliplatin-induced chronic painful peripheral neuropathy in the rat and comparison with the neuropathy induced by paclitaxel. Neuroscience 203, 194–206. doi:10.1016/j.neuroscience.2011.12.023 Xu, W., Qiu, X.C., Han, J.S., 1994. Serotonin receptor subtypes in spinal antinociception in the rat. J. Pharmacol. Exp. Ther. 269, 1182–1189. Yashpal, null, Coderre, null, 1998. Influence of formalin concentration on the antinociceptive effects of anti-inflammatory drugs in the formalin test in rats: separate mechanisms underlying the nociceptive effects of low- and high-concentration formalin. Eur. J. Pain Lond. Engl. 2, 63–68. You, H.-J., Colpaert, F.C., Arendt-Nielsen, L., 2005. The novel analgesic and high-efficacy 5-HT1A receptor agonist F 13640 inhibits nociceptive responses, wind-up, and after-discharges in spinal neurons and withdrawal reflexes. Exp. Neurol. 191, 174–183. doi:10.1016/j.expneurol.2004.08.031 Zhang, Y., Yang, Z., Gao, X., Wu, G., 2001. The role of 5-hydroxytryptamine1A and 5hydroxytryptamine1B receptors in modulating spinal nociceptive transmission in normal and carrageenan-injected rats. Pain 92, 201–211.

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Figures

Fig. 1 Antiallodynic activity of NLX-112 (2.5 and 5 mg/kg i.p.) in diabetic neuropathic pain model measured using the von Frey test (A), and anti-hyperalgesic effect of this compound assessed in

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diabetic mice measured using the hot plate test (B). Results are shown as the mean (+ SEM) force applied to elicit paw withdrawal (A), or latency time to pain reaction (B). Statistical analysis: one-way ANOVA, followed by Tukey’s post hoc comparison. Significance vs. normoglycemic control: ### p <

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0.001, and vs. pre-drug value: *** p < 0.001.

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Fig. 2 Antiallodynic activity of NLX-112 (1.25, 2.5 and 5 mg/kg i.p.) in oxaliplatin-induced neuropathic pain model measured using the von Frey test (A) and cold plate test (B). Results are shown as the mean (+ SEM) force applied to elicit paw withdrawal (A), or latency time to pain reaction (B).

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Statistical analysis: one-way ANOVA, followed by Tukey’s post hoc comparison. Significance vs. non-

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neuropathic, vehicle-treated mice: # p < 0.05, ### p < 0.001, and vs. pre-drug value: *** p < 0.001.

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Fig. 3 Antinociceptive activity of NLX-112 in tonic pain model (formalin test) after intraperitoneal (A) and oral (B) administration. Results are shown as the duration of licking/biting behavior in the first

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(neurogenic) phase (0-5 min after formalin injection), and in the second (inflammatory) phase (15-30 min after formalin injection). Statistical analysis: repeated measures ANOVA, followed by Bonferroni multiple comparison: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (vs. vehicle-treated mice

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at the respective time-point).

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Fig. 4 Antinociceptive activity of NLX-112 alone or in combination with WAY 100635 in the tonic pain model (formalin test) after intraperitoneal administration. Results are shown as the duration of

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licking/biting behavior in the first (neurogenic) phase (A), and in the second (inflammatory) phase (B). Statistical analysis: one-way ANOVA, followed by Tukey’s multiple comparison. Significance: # p < 0.05, ### p < 0.001 (vs. vehicle-treated group); ** p < 0.01, *** p < 0.001 (vs. NLX-112-treated

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group).

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Fig. 5 Antinociceptive activity of NLX-112 administered intraperitoneally in acute thermal pain model

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(hot plate test). Results are shown as mean latency time (± SEM) to pain reaction in response to thermal stimulus. Statistical analysis: one-way ANOVA, followed by Dunnett’s post- hoc test: *** p <

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0.001 (vs. vehicle-treated mice).

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Fig. 6 Influence of NLX-112 (5 mg/kg i.p.) on locomotor activity. Results are shown as mean number of light-beam crossings (+ SEM) measured at six 5-min intervals. Statistical analysis of the results was

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conducted using repeated measures ANOVA, followed by Bonferroni multiple comparison: p > 0.05.

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ACCEPTED MANUSCRIPT Highlights NLX-112 is a highly selective serotonin 5-HT1A receptor full agonist. NLX-112 potently reduces paw licking in the early and late phases of the intraplantar formalin test.

NLX-112 is least active in the diabetic neuropathic pain model.

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At higher doses it is active in the thermally-induced acute and oxaliplatin-induced neuropathic pain models.

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At the highest analgesic dose tested NLX-112 does not affect locomotor activity.