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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2018 Sep 9;175(20):3911–3927. doi: 10.1111/bph.14461

GpTx‐1 and [Ala5, Phe6, Leu26, Arg28]GpTx‐1, two peptide NaV1.7 inhibitors: analgesic and tolerance properties at the spinal level

Chao Chen 1,†, Biao Xu 2,†, Xuerui Shi 2, Mengna Zhang 2, Qinqin Zhang 2, Ting Zhang 2, Weidong Zhao 2, Run Zhang 2, Zilong Wang 2, Ning Li 2, Quan Fang 2,✉
PMCID: PMC6151339  PMID: 30076786

Abstract

Background and Purpose

The voltage‐gated sodium channel NaV1.7 is considered a therapeutic target for pain treatment based on human genetic evidence. GpTx‐1 and its potent analogue [Ala5, Phe6, Leu26, Arg28]GpTx‐1 (GpTx‐1‐71) were recently characterized as NaV1.7 inhibitors in vitro. Furthermore, the present work was conducted to investigate the analgesic properties of these two peptides in different pain models after spinal administration.

Experimental Approach

The antinociceptive activities of both GpTx‐1 and GpTx‐1‐71 were investigated in mouse models of acute, visceral, inflammatory and neuropathic pain. Furthermore, the side effects of GpTx‐1 and GpTx‐1‐71 were evaluated in rotarod, antinociceptive tolerance, acute hyperlocomotion and gastrointestinal transit tests.

Key Results

The i.t. administration of both GpTx‐1 and GpTx‐1‐71 dose‐dependently produced powerful antinociception in the different pain models. This effect was attenuated by the opioid receptor antagonist naloxone, suggesting the involvement of the opioid system. In this study, repeated administration of these two_peptides produced spinal analgesia without a loss of potency over 8 days in mouse models of acute, inflammatory and neuropathic pain. Moreover, spinal administration of GpTx‐1 and GpTx‐1‐71 did not induce significant effects on motor coordination, evoke acute hyperlocomotion or increase gastrointestinal transit time.

Conclusions and Implications

Our data indicate that the NaV1.7 peptide inhibitors GpTx‐1 and GpTx‐1‐71 produce powerful, nontolerance‐forming analgesia in preclinical pain models, which might be dependent on the endogenous opioid system. In addition, at the spinal level, the limited side effects imply that these NaV1.7 peptide inhibitors could be potentially developed as GpTx‐1‐based drugs for pain relief.

Abbreviations

CFA

complete Freund's adjuvant

MPE

maximum possible effect

Introduction

http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=82 have important roles in electrical excitability in the excitable cells of mammalian nerves and muscles. The NaV channel family contains nine known subtypes, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=578–http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=586 (Catterall et al., 2005), and each particular isoform of the NaV channels might be associated with specialized functions in different physiological processes (Eijkelkamp et al., 2012). Recently, the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=584 channel has emerged as a therapeutic target for pain control based on human genetic evidence (Dib‐Hajj et al., 2008; Dib‐Hajj et al., 2010). Clinical gene‐linkage analyses have shown that gain‐of‐function mutations of the SCN9A gene that encodes the NaV1.7 channel contribute to painful inherited neuropathies (Faber et al., 2012), whereas the loss of NaV1.7 function results in insensitivity to any form of pain (Nassar et al., 2004; Cox et al., 2006). Therefore, the NaV1.7 channel is considered an essential and non redundant requirement for pain signalling and generation in humans.

Clinically, nonselective sodium channel blockers are useful drugs for pain, but their utility is constrained by unwanted side effects induced by their lack of subtype selectivity (Wallace, 2000; Cummins and Rush, 2007). Thus, subtype‐selective inhibitors of NaV1.7 have emerged as promising analgesics to relieve a broad range of pain syndromes. Currently, NaV1.7 inhibitors are classified into small molecular inhibitors and peptide inhibitors according to their chemical structure. Several compounds, including benzazepinones, amino‐thiazoles, amino‐pyridines and isoxazoles, were found to be NaV1.7 blockers (Hoyt et al., 2007; Macsari et al., 2011). CNV‐1014802 (GSK‐1014802, BIIB074), PF‐04856264 and PF‐05089771 were also found to be selective inhibitors of NaV1.7 channels in previous studies (McCormack et al., 2013; Alexandrou et al., 2016). CNV‐1014802 produced well‐tolerated analgesic effects on trigeminal neuralgia without major side effects in humans, and this blocker has been tested in a phase II clinical trial (Zakrzewska et al., 2017). In contrast, NaV1.7 peptide blockers isolated from venom toxins of different species, such as snake, spider, wasp, scorpion and cone snail, are typically cysteine‐rich peptides containing three interlinked disulfide bonds that are crucial for their biological activity (Wright et al., 2017). Huwentoxin‐IV, derived from the venom of the Chinese spider Selenocosmia huwena, is a potent blocker of human NaV1.7 channels, which showed antinociceptive effects on inflammatory and neuropathic pain (Liu et al., 2014b). In addition, Protoxin‐II was isolated from the toxin of the spider Thrixopelma pruriens (Sokolov et al., 2008), and μ‐SLPTX‐Ssm6a was derived from centipede venom (Yang et al., 2013). Recently, the novel NaV1.7 selective inhibitor μ‐theraphotoxin‐Pn3a (μ‐TRTX‐Pn3a) was pharmacologically characterized from venom of the tarantula Pamphobeteus nigricolor (Deuis et al., 2017).

GpTx‐1 is a voltage‐dependent calcium (http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=535) channel blocker originally isolated from tarantula venom in 2011 (Ono et al., 2011). Interestingly, the structure of GpTx‐1 exhibited a high similarity to those of the sodium channel blockers PaurTx3 (76.5%), HnTx‐IV (60.0%) and CcoTx2 (55.9%) (Bosmans et al., 2006; Ono et al., 2011). Furthermore, GpTx‐1 was identified to possess inhibitory effects on the NaV1.7 channel (Cherki et al., 2014). In fluorometric imaging plate reader (FLIPR) membrane potential assays, GpTx‐1 functioned as a NaV1.7 blocker with 100‐fold selectivity for this channel compared to http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=581, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=582 and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=585 channels (Deuis et al., 2016). Recently, [Ala5, Phe6, Leu26, Arg28]GpTx‐1(GpTx‐1‐71) was screened from hundreds of GpTx‐1 analogues by Amgen and was characterized as a potent NaV1.7 blocker (Murray et al., 2015). In vitro studies indicated that the NaV1.7 IC50 values of GpTx‐1 and GpTx‐1‐71 were 10 and 1.6 nM respectively. It is noteworthy that GpTx‐1‐71 is the only confirmed peptide sequence with single‐digit nanomolar NaV1.7 inhibitory activity and >1000‐fold selectivity for this channel relative to NaV1.4 and NaV1.5 channels, which suggests GpTx‐1‐71 is a promising compound for pain treatment. To further evaluate the in vivo properties of GpTx‐1 and related peptides, their analgesic properties and side effects were investigated in mice after i.t. injections. Our data demonstrated that both GpTx‐1 and GpTx‐1‐71 reduced nociceptive, visceral, inflammatory and neuropathic pain with no major side effects, including tolerance, acute hyperlocomotion, constipation and motor impairment.

Methods

Animals

Male and female Kunming strain mice (Experimental Animal Centre of Lanzhou University, Lanzhou, China) weighing approximately 22 g were housed in groups (4–6 per cage) in an environment with a controlled temperature (22 ± 1°C) and a 12 h light/dark cycle. All the animals had access to food and water ad libitum. All the experimental protocols were approved by the Ethics Committee of Lanzhou University and followed the guidelines formulated by the European Community (2010/63/EU). Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010). The mice were divided randomly into groups on the day of the experiment. The studies were blinded to treatment assignment and outcome assessment.

Intrathecal injection

I.t. injections were performed in awake mice as described previously (Hylden and Wilcox, 1980; Liu et al., 2014a). Briefly, all the reagents were injected between the L5 and L6 vertebral segments using a 30‐gauge needle attached to a 10 μL Hamilton syringe. Each mouse was injected with a volume of 5 μL at a constant rate of 20 μL·min−1. Successful i.t. injection was determined by a quick tail‐flick or an ‘S’ shape of the tail. In addition, to investigate the analgesic mechanisms, the opioid antagonist http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1638 (5 nmol) was i.t. injected 10 min prior to the inhibitors.

Intracerebroventricular injection

I.c.v. injections were performed via a permanent guide cannula embedded into the lateral ventricle (Li et al., 2016a). Briefly, the mice were anaesthetized with pentobarbital sodium (80 mg·kg−1, i.p.) and were stabilized in stereotaxic equipment (68001, RWD Life Science, Shenzhen, China). The scalp covering the incision area was shaved, and an incision was made to expose the skull. A single hole was made 3 mm posterior and 1 mm lateral from bregma. A stainless steel cannula (inner diameter 0.25 mm, outer diameter 0.50 mm) was inserted 3 mm into the skull surface (lateral ventricle) via the hole. All the animals were used at least 4 days after surgery in this study. At the end of the study, the mice were injected with methylene blue dye, and only the data obtained from the animal with the dye throughout the ventricles were used.

The mice were injected with 4 μL of the inhibitors followed by 1 μL of saline at a rate of 10 μL·min−1 using a 25 μL microsyringe. In addition, to investigate the analgesic mechanisms of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2536 (ω‐conotoxin MVIIA), the opioid antagonist naloxone was i.c.v. injected 10 min prior to administration of saline or ziconotide.

Tail‐flick test

Nociceptive responses in mice were confirmed using the tail‐flick test as previously described (Li et al., 2016a). Briefly, the tail of the mouse was positioned in a groove underneath a radiant heat source. The intensity of the heat source was adjusted to the baseline of naïve mice between 3–5 s, and a cut‐off time of 10 s was set in this study. The latency was recorded before and at 15, 30, 45, 60, 90, 120, 240, 480 and 1440 min post‐injection. Maximal possible effect (MPE) was used to quantify the antinociceptive effects as follows: MPE (%) = 100 × [(post‐drug response‐baseline response)/(cut‐off response‐baseline response)].

Carrageenan‐ or complete Freund's adjuvant (CFA)‐induced inflammatory pain model

Chronic inflammatory pain was induced as previously reported (Zhang et al., 2017). In this study, the mice were injected with 20 μL of carrageenan (2% in sterile water, Sigma, for antinociceptive study) or CFA (Sigma‐Aldrich, St. Louis, MO, USA, for tolerance evaluation) in the right hindpaw of mice anaesthetized with 1.4% isoflurane. The baseline responses of the mice to mechanical stimulation were determined before injection. All the animals were used 1 day after carrageenan injection or 4 days after CFA treatment.

Neuropathic pain model

Peripheral neuropathic pain was induced by chronic constriction nerve injury (CCI) (Narita et al., 2005). Prior to surgery, the baseline latency to mechanical and thermal stimuli was determined. All the surgical procedures were carried out in mice anaesthetized with pentobarbital sodium (i.p., 20 mg·kg−1). The sciatic nerve of the right hindpaw was exposed via a small lateral incision. Three loose surgical chorda chirurgicalis (8/0 silk) were constricted around the dissected nerve, with approximately 1 mm spacing between each ligature. The ligatures were tightened carefully until they caused a slight twitch in the ipsilateral hindlimb. The wound was closed with 4‐0 surgical sutures, and erythromycin ointment was applied externally. All the mice in the study were used 7 days after surgery.

Mechanical allodynia

Mechanical allodynia was tested using an electronic von Frey filament (IITC, Woodland Hills, CA, USA). The mice were placed individually in Plexiglas cages (11 × 15 × 18 cm) with a wire‐mesh floor and allowed to acclimatize for at least 20 min. The central part of the right hindpaw was stimulated by the von Frey filament, and the withdrawal threshold was defined as the maximal force that elicited paw withdrawal, flinching or licking. The stimulus was applied three times at the same time point at intervals of 2 min. The withdrawal threshold of the mice was determined before and at 15, 30, 45, 60, 90, 120, 240, 480 and 1440 min post‐injection.

Thermal hyperalgesia

Thermal hyperalgesia in mice was evaluated by the Hargreaves test. Prior to the behavioural test, the mice were placed individually in the chambers and allowed to habituate for at least 30 min. The intensity of the radiant beam was adjusted to the withdrawal latency of naïve mice after approximately 15 s (PL‐200, Chengdu Technology & Market Co., Ltd., China). The paw withdrawal threshold was recorded as the time from the start of the thermal stimulus to the quick movement of the hindpaw from the radiant source. To avoid any tissue damage, the cut‐off time was set at 25 s. The paw withdrawal threshold of the mice was determined before and at 15, 30, 45, 60, 90, 120, 240, 480 and 1440 min post‐injection.

Writhing test

An acetic acid‐induced writhing test was selected as a visceral pain model. Briefly, the mice were placed in individual plastic boxes to acclimatize them to the environment for 30 min. Then, the animals were treated with saline (i.t.) or the two different NaV1.7 inhibitors (i.t.). After 15 min, each mouse was injected i.p. with 0.6% acetic acid (10 mL·kg−1 body weight). The number of writhing movements was recorded during a 10 min period, beginning 5 min after the injection of acetic acid. A writhe was characterized as an abdominal muscle contraction following the stretching of the hindlimbs.

Formalin test

The procedure of the formalin test was performed as in our previous study (Zheng et al., 2018). The test apparatus consisted of three Plexiglas chambers with a mirror placed under the floor at an angel of approximately 45°. Briefly, the mice were placed individually in the chamber to acclimatize them for at least 15 min. Saline or the two NaV1.7 inhibitors were administered i.t., and after another 5 min, 20 μL 2% formalin was injected into the dorsal side of the right hindpaw. Then, the total time the animals spent licking and biting the injected paw was recorded during the periods of 0–5 (phase I) and 15–30 min (phase II) after the formalin injection respectively.

Tolerance evaluation

In this study, the development of tolerance to these two NaV1.7 inhibitors was evaluated in different models of acute, inflammatory and neuropathic pain. Briefly, the mice were i.t. injected with saline, GpTx‐1 (5 pmol) or GpTx‐1‐71 (1 pmol) once daily for 8 days. The tail‐flick latency was determined 60 min after GpTx‐1 administration and 45 min after GpTx‐1‐71 administration because antinociception peaks at 60 and 45 min post‐injection respectively. In addition, paw withdrawal thresholds to mechanical and thermal stimuli were determined at the peak time points for maximum antinociception.

Rotarod test

The motor coordination and equilibrium of mice were measured following the procedures of our previous study (Wang et al., 2016). Prior to the injection, the mice were trained on a rotarod apparatus (ZB‐200, Chengdu Technology & Market Co., Ltd.) at a speed of 16 rpm for two consecutive days, and each day they were trained for three sessions at 5 min intervals. The cut‐off time was set as 300 s in this study. On the post‐conditioning day, the mice were i.t. injected with GpTx‐1 (10 pmol) or GpTx‐1‐71 (10 pmol), and the latency to fall off the rotarod was tested at 10, 20, 30 and 40 min post‐injection.

Open field test

The locomotor activities of mice were evaluated using the open field test (Manglik et al., 2016). The apparatus consisted of an uncovered black Plexiglass arena (50 cm × 50 cm × 40 cm) and a video tracking system (PMT‐100, Chengdu Technology & Market Co., Ltd.) to track the activity of the mouse. At the beginning of the test, the mice were individually placed in the centre of the arena and allowed to explore the environment freely for 30 min. Then, the animals were i.t. treated with saline or one of the two inhibitors, and locomotor activity was monitored for another 150 min. The arena was cleaned with 75% ethanol before and after each test to eliminate scents left by other mice.

Gastrointestinal transit test

Gastrointestinal transit was evaluated according to the procedures described in a previous study (Li et al., 2016b). The mice were individually placed in cages and fasted for 16 h with water available ad libitum. A charcoal meal (5% charcoal and 10% gum arable in water) was given p.o. at a volume of 0.1 mL.10 g‐1 body weight 15 min after i.t. administration of the reagents. After 30 min, the animals were killed by cervical dislocation, and the small intestine (from the pyloric junction to the caecum) was carefully removed. The total length of the small intestine and the distance the charcoal meal travelled were measured. The results are expressed as % gastrointestinal transit (GIT%), which was calculated with the formula GIT% = (the distance travelled by the charcoal/the total distance of small intestine) × 100%.

Statistical analysis

The data and statistical analysis comply with the recommendations of experimental design and analysis in pharmacology (Curtis et al., 2018). Two‐way ANOVA was used to analyse the time courses for the effects of these two blockers. The results obtained from the tolerance tests were analysed by one‐way ANOVA followed by Tukey's HSD test, and the other data were analysed using one‐way ANOVA followed by Dunnett's or Bonferroni's post hoc test, as appropriate. The post hoc test was only conduced when F achieved the necessary level (P < 0.05). SPSS (version 20; SPSS Inc., USA) and GraphPad Prism (version 7.00; GraphPad Software Inc., USA) were used for the statistical analyses. The data obtained from this study are expressed as the mean ± SEM, and P < 0.05 was considered statistically significant.

Materials

GpTx‐1 and GpTx‐1‐71 were synthesized using N α‐Fmoc solid‐phase peptide synthesis with appropriate orthogonal protection and supporting matrix strategies (Murray et al., 2015). N α‐Fmoc protected amino acids were purchased from CD CHEMPEP (Chengdu, China) and ChengDu ChengNuo New‐Tech Co., Ltd. (Chengdu, China). Rink amide MA resin was purchased from XIANSUNRESIN (Xi'an, China). These two peptides were assembled on AM resin (100–200 mesh, 1% DVB, 1.0–1.2 mmol·g−1 initial loading) on a 3 mmol scale. The substitution of Fmoc‐Phe‐AM resin was controlled to 0.25–0.55 mmol·g−1. The coupling reactions were performed in the presence of amino acids (2.0–3.0 equiv), HOBt (3.0–3.5 equiv) and DIC (4.0–5.0 equiv) in DMF/DCM mixed solvents and were stirred with N2 bubbles for 1–3 h. After coupling, the resin was washed with DMF three times, and 20% piperidine in DMF was employed to remove the Fmoc groups. Subsequently, the reaction mixture was drained and washed with DMF six times. The reaction cycle was repeated until the entire linear peptide resin was acquired. Amino acid side chains were protected as Asn (Trt), Tyr (tBu), Lys (N ϵ‐Boc), Cys (Trt), Trp (Boc), His (Trt), Thr (tBu), Asp (OtBu) and Ser (tBu). Cleavage of the peptides from the resin was performed by treatment with TIS/DODT/H2O/TFA (5.0:2.5:2.5:9.0, v.v‐1) at room temperature for 1.5–2.0 h. Then, the crude linear peptide was precipitated by adding the cleavage mixture to a low polarity solvent methyl tert‐butyl ether (MTBE). The peptide was then washed three times using MTBE and dried under a vacuum. The electrospray ionization mass spectrometer (LCMS‐8030, Shimadzu, Tokyo, Japan) results indicated that the peaks with m/z ratios of 1360.60, 1020.90, 816.95, 681.05 and 583.90 represent the [M + 3H]3+, [M + 4H]4+, [M + 5H]5+, [M + 6H]6+ and [M + 7H]7+ products of the linear targeting product GpTx‐1, respectively, and that ratios of 1354.50, 1015.70, 813.05, 677.55 and 581.05 represent the [M + 3H]3+, [M + 4H]4+, [M + 5H]5+, [M + 6H]6+ and [M + 7H]7+ products of the linear targeting product GpTx‐1‐71 respectively. Oxidative folding of the two linear peptides was performed as described previously (Murray et al., 2015). After the linear peptide was completely depleted, as detected by HPLC, glacial acetic acid was added to quench the reaction. The crude peptide was purified by RP‐HPLC and lyophilized to obtain white solid products with the HPLC purity of both peptides being higher than 95%. The high‐resolution MS of the two targeting compounds matched the calculated values (ESI‐Q‐TOF maXis‐4G; Bruker Daltonics, Bremen, Germany).

In addition, ziconotide was obtained from Hybio Pharmaceutical Co., Ltd. (Shenzhen, China). The non‐selective opioid antagonist naloxone was purchased from Sigma‐Aldrich (St. Louis, MO, USA). http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1627 hydrochloride was produced by Shenyang First Pharmaceutical Factory (Shenyang, China). All the test compounds were dissolved in physiological saline (0.9% NaCl) to the required concentrations and stored at −20°C.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a,b).

Results

Antinociceptive effects of GpTx‐1 and GpTx‐1‐71

GpTx‐1 and GpTx‐1‐71 prevented nociceptive responses in the acute pain model

In Figure 1A, B, the data obtained from the tail‐flick assay showed the time course of analgesia produced by i.t. injection of GpTx‐1 and GpTx‐1‐71 in male mice. Both of these peptides dose‐dependently produced potent antinociceptive effects (GpTx‐1: F 3, 31 = 260, P < 0.05; GpTx‐1‐71: F 3, 31 = 570, P < 0.05). Two‐way ANOVA showed significant differences for dose (GpTx‐1: F 3, 280 = 950, P < 0.05; GpTx‐1‐71: F 3, 280 = 1100, P < 0.05), time (GpTx‐1: F 9, 280 = 270, P < 0.05; GpTx‐1‐71: F 9, 280 = 250, P < 0.05) and interaction between dose and time (GpTx‐1: F 27, 280 = 43, P < 0.05; GpTx‐1‐71: F 27, 280 = 42, P < 0.05) in this study. The analgesic effects of GpTx‐1 and GpTx‐1‐71 peaked at 60 and 45 min post‐injection, respectively, and the duration of the antinociceptive effects was approximately 24 h. The antinociceptive ED50 values for GpTx‐1 and GpTx‐1‐71 were 0.33 (0.27–0.41) and 0.024 (0.020–0.029) pmol respectively.

Figure 1.

Figure 1

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Effects of GpTx‐1, GpTx‐1‐71 and ziconotide on nociceptive responses in a mouse tail‐flick test. Dose‐ and time‐related antinociception of GpTx‐1 (0.2, 1 and 5 pmol) (A) and GpTx‐1‐71 (0.01, 0.1 and 1 pmol) (B, male mice; C, female mice) after i.t. injection. The highest MPE induced by these two inhibitors are presented in the inserts. The antinociceptive effects of the reference compound ziconotide (50 pmol) after i.t. injection (G) and the antinociceptive effects of GpTx‐1 (10 pmol), GpTx‐1‐71 (10 pmol) and ziconotide (10 pmol) after i.c.v. injection (H). *P < 0.05 compared to saline group (one‐way ANOVA followed by Dunnett's post hoc test). Effects of central pretreatment with the non‐selective opioid antagonist naloxone on the antinociceptive effects of GpTx‐1 (D), GpTx‐1‐71 (E, male mice; F, female mice) and ziconotide (G and H). *P < 0.05 compared to saline + saline group, # P < 0.05 compared to saline + drug group (one‐way ANOVA followed by Bonferroni's post hoc test). Data points and columns are mean ± SEM, n = 7–9 per group.

In addition, to investigate the sexual dimorphism of GpTx‐1‐related peptides in pain studies, as shown in Figure 1C, i.t. injection of GpTx‐1‐71 similarly produced potent antinociceptive effects in female mice (F 3, 29 = 190, P < 0.05), with an ED50 value of 0.027 (0.019–0.037) pmol. Two‐way ANOVA showed significantly differences for dose (F 3, 260 = 700, P < 0.05), time (F 9, 260 = 160, P < 0.05) and interaction between dose and time (F 27, 260 = 29, P < 0.05).

Figure 1D–F shows that i.t. pretreatment with the non‐selective opioid antagonist naloxone (5 nmol) significantly reduced the antinociceptive effects of these two peptides (GpTx‐1: F 3, 31 = 95, P < 0.05; GpTx‐1‐71: F 3, 32 = 110 for male mice and F 3, 28 = 320 for female mice, P < 0.05), which suggests that the opioid system is involved in the central analgesia of GpTx‐1 and related peptide.

In this study, the N‐type calcium channel (http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=533) blocker ziconotide was used as a reference compound in the mouse tail‐flick test. Figure 1G shows that i.t. injection of ziconotide (50 pmol) induced potent antinociceptive effects, which were not reduced by the opioid receptor antagonist naloxone. At the supraspinal level, a high dose of GpTx‐1 (10 pmol, i.c.v.) or GpTx‐1‐71 (10 pmol, i.c.v.) produced no significant influences on tail‐flick latency, while ziconotide (10 pmol, i.c.v.) induced powerful antinociceptive effects, and this effect was insensitive to the opioid receptor antagonist naloxone (Figure 1H).

GpTx‐1 and GpTx‐1‐71 prevented mechanical allodynia in carrageenan‐induced inflammatory pain

In Figure 2A, B, both GpTx‐1 and GpTx‐1‐71 exhibited dose‐dependent anti‐allodynic activities in the carrageenan‐induced inflammatory pain model compared with saline (GpTx‐1: F 4, 39 = 120, P < 0.05; GpTx‐1‐71: F 3, 31 = 84, P < 0.05). These effects exhibited significant differences for dose (GpTx‐1: F 4, 350 = 190, P < 0.05; GpTx‐1‐71: F 3, 280 = 300, P < 0.05), time (GpTx‐1: F 9, 350 = 100, P < 0.05; GpTx‐1‐71: F 9, 280 = 75, P < 0.05) and the interaction between dose and time (GpTx‐1: F 36, 350 = 9.6, P < 0.05; GpTx‐1‐71: F 27, 280 = 11, P < 0.05). The anti‐allodynic activities of GpTx‐1 and GpTx‐1‐71 lasted for up to 24 h (Figure 2A, B), with a peak time effect of 45 and 60 min post‐injection respectively. The anti‐allodynic ED50 values of GpTx‐1 and GpTx‐1‐71 were 0.38 (0.29–0.49) and 0.021 (0.012–0.034) pmol respectively.

Figure 2.

Figure 2

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Effects of GpTx‐1 and GpTx‐1‐71 on inflammatory pain induced by carrageenan in mice. Dose‐ and time‐related anti‐allodynia of GpTx‐1 (0.1, 0.5, 1 and 5 pmol) (A) and GpTx‐1‐71 (0.01, 0.1 and 1 pmol) (B) after i.t. injection. The highest MPE induced by these two inhibitors are presented in the inserts. *P < 0.05 compared to saline group (one‐way ANOVA followed by Dunnett's post hoc test). Effects of central (i.t.) pretreatment with the non‐selective opioid antagonist naloxone on the anti‐allodynic effects of GpTx‐1 (C) and GpTx‐1‐71 (D). *P < 0.05 compared to saline + saline group, # P < 0.05 compared to saline + drug group (one‐way ANOVA followed by Bonferroni's post hoc test). Data points and columns are mean ± SEM, n = 7–8 per group.

As illustrated in Figure 2C, D, the analgesic activities of GpTx‐1 and GpTx‐1‐71 were completely abolished in naloxone‐pretreated animals (GpTx‐1: F 3, 31 = 110, P < 0.05; GpTx‐1‐71: F 3, 30 = 130, P < 0.05). In addition, there were no significant differences in the paw withdrawal thresholds observed in the naloxone + saline and saline + saline groups.

GpTx‐1 and GpTx‐1‐71 prevented mechanical allodynia and thermal hyperalgesia in CCI‐induced neuropathic pain

A painful state of CCI‐induced neuropathic pain model developed 7 days after surgery and lasted for up to 21 days (Narita et al., 2008; Osikowicz et al., 2008). The analgesic activities of these two peptides were determined using both mechanical and thermal stimuli. As reported previously, CCI induced significant mechanical allodynia (1.6 ± 0.08 g) and thermal hyperalgesia (5.6 ± 0.2 s) in comparison to the baseline latency (4.4 ± 0.08 g and 14.4 ± 0.2 s, Figures 3 and 4). As shown in Figure 3A, B, i.t. injection of both GpTx‐1 and GpTx‐1‐71 dose‐dependently produced potent anti‐allodynic activities (GpTx‐1: F 3, 30 = 160, P < 0.05; GpTx‐1‐71: F 3, 29 = 75, P < 0.05), with a significant effect starting at the doses of 0.2 and 0.01 pmol, respectively, and with a peak anti‐allodynic effect 45 min post‐injection. The ED50 values for the anti‐allodynia effects of GpTx‐1 and GpTx‐1‐71 were 0.68 (0.50–0.90) and 0.012 (0.0046–0.023) pmol respectively. Figure 4A, B depicts that i.t. injection of GpTx‐1 (0.2, 1 and 5 pmol) and GpTx‐1‐71 (0.01, 0.1 and 1 pmol) dose‐dependently produced anti‐hyperalgesic effects (GpTx‐1: F 3, 28 = 120, P < 0.05; GpTx‐1‐71: F 3, 28 = 96, P < 0.05), with a peak effect 45 min post‐injection. The ED50 values for the analgesia effects of GpTx‐1 and GpTx‐1‐71 were 0.48 (0.31–0.69) and 0.026 (0.014–0.044) pmol, respectively, and the duration time for these two inhibitors was approximately 24 h. Two‐way ANOVA also revealed significant differences for the anti‐allodynia (dose: F 3, 270 = 200 for GpTx‐1 and F 3, 260 = 300 for GpTx‐1‐71, P < 0.05; time: F 9, 270 = 64 for GpTx‐1 and F 9, 260 = 75 for GpTx‐1‐71, P < 0.05; the interaction between dose and time: F 27, 270 = 14 for GpTx‐1 and F 27, 260 = 12 for GpTx‐1‐71, P < 0.05) and anti‐hyperalgesia (dose: F 3, 250 = 200 for GpTx‐1 and F 3, 250 = 230 for GpTx‐1‐71, P < 0.05; time: F 9, 250 = 65 for GpTx‐1 and F 9, 250 = 61 for GpTx‐1‐71, P < 0.05; the interaction between dose and time: F 27, 250 = 10 for GpTx‐1 and F 27, 250 = 11 for GpTx‐1‐71, P < 0.05) properties.

Figure 3.

Figure 3

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Effects of GpTx‐1 and GpTx‐1‐71 on neuropathic pain induced by CCI under mechanical stimulation. Dose‐ and time‐related anti‐allodynia of GpTx‐1 (0.2, 1 and 5 pmol) (A) and GpTx‐1‐71 (0.01, 0.1 and 1 pmol) (B) after i.t. injection. The highest MPE induced by these two inhibitors are presented in the inserts. *P < 0.05 compared to saline group (one‐way ANOVA followed by Dunnett's post hoc test). Effects of central (i.t.) pretreatment with the non‐selective opioid antagonist naloxone on the anti‐allodynic effects of GpTx‐1 (C) and GpTx‐1‐71 (D). *P < 0.05 compared to saline + saline group, # P < 0.05 compared to saline + drug group (one‐way ANOVA followed by Bonferroni's post hoc test). Data points and columns are mean ± SEM, n = 7–8 per group.

Figure 4.

Figure 4

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Effects of GpTx‐1 and GpTx‐1‐71 on neuropathic pain induced by CCI under thermal stimulation. Dose‐ and time‐related anti‐hyperalgesia of GpTx‐1 (0.2, 1 and 5 pmol) (A) and GpTx‐1‐71 (0.01, 0.1 and 1 pmol) (B) after i.t. injection. The highest MPE induced by these two inhibitors are presented in the inserts. *P < 0.05 compared to saline group (one‐way ANOVA followed by Dunnett's post hoc test). Effects of central (i.t.) pretreatment with the non‐selective opioid antagonist naloxone on the anti‐hyperalgesic effects of GpTx‐1 (C) and GpTx‐1‐71 (D). *P < 0.05 compared to saline + saline group, # P < 0.05 compared to saline + drug group (one‐way ANOVA followed by Bonferroni's post hoc test). Data points and columns are mean ± SEM, n = 7–8 per group.

In Figure 3C, D, naloxone was injected i.t. 10 min prior to administration of GpTx‐1 and the related peptide. We found that naloxone completely reduced the anti‐allodynia (GpTx‐1: F 3, 29 = 91, P < 0.05; GpTx‐1‐71: F 3, 29 = 51, P < 0.05) and anti‐hyperalgesia (GpTx‐1: F 3, 28 = 39, P < 0.05; GpTx‐1‐71: F 3, 28 = 76, P < 0.05) effects induced by GpTx‐1 and GpTx‐1‐71 (Figure 4C, D). However, naloxone itself had no influence on the withdrawal threshold.

GpTx‐1 and GpTx‐1‐71 produced potent analgesic activities in the acetic acid‐induced visceral pain

An acetic acid‐induced writhing test was used to evaluate the analgesic activities of GpTx‐1 and GpTx‐1‐71 in this visceral pain model. As shown in Figure 5A, B, we found that i.t. administration of both GpTx‐1 (0.05, 0.5 and 5 pmol) and GpTx‐1‐71 (0.001, 0.01, 0.1, 1 and 10 pmol) dose‐dependently exerted potent analgesic effects compared with the vehicle treatment (F 3, 31 = 130, P < 0.05; F 5, 48 = 87, P < 0.05 respectively). The ED50 values for GpTx‐1 and GpTx‐1‐71 analgesia were 0.30 (0.22–0.40) and 0.015 (0.0086–0.025) pmol, respectively, in this visceral pain model (Table 1).

Figure 5.

Figure 5

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Effects of GpTx‐1 and GpTx‐1‐71 on visceral pain induced by acetic acid. Dose‐related antinociceptive effects of GpTx‐1 (0.05, 0.5 and 5 pmol) (A) and GpTx‐1‐71 (0.001, 0.01, 0.1, 1 and 10 pmol) (B) after i.t. injection. *P < 0.05 compared to saline group (one‐way ANOVA followed by Dunnett's post hoc test). Effects of central (i.t.) pretreatment with the non‐selective opioid antagonist naloxone on the antinociceptive effects of GpTx‐1 (C) and GpTx‐1‐71 (D). *P < 0.05 compared to saline + saline group, # P < 0.05 compared to saline + drug group (one‐way ANOVA followed by Bonferroni's post hoc test). Data columns are mean ± SEM, n = 8–10 per group.

Table 1.

Antinociceptive effects of GpTx‐1 and GpTx‐1‐71 in different pain models

Pain models Acute pain Inflammatory pain Neuropathic pain Visceral pain Formalin induced pain
Mechanical Thermal Phase I Phase II
ED50 for GpTx‐1 (pmol) 0.33 (0.27–0.41) 0.38 (0.29–0.49) 0.68 (0.50–0.90) 0.48 (0.31–0.69) 0.30 (0.22–0.40) 0.44 (0.17–0.89) 1.1 (0.53–2.4)
ED50 for GpTx‐1‐71 (pmol) 0.024 (0.020–0.029) 0.021 (0.012–0.034) 0.012 (0.0046–0.023) 0.026 (0.014–0.044) 0.015 (0.0086–0.025) 0.86 (0.061–5.4) N.D
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N.D indicated that the data were not determined. Both GpTx‐1 and GpTx‐1‐71 were i.t. injected in mice. The ED50 (dose of the inhibitors that produced half of the maximal antinociception) values with their 95% confidence intervals were calculated from their dose–response curves using nonlinear regression analysis with the GraphPad Prism version 7.00 Programme.

Furthermore, spinal administration of naloxone significantly reduced the antinociceptive effects induced by both GpTx‐1 (F 3, 33 = 92, P < 0.05) and GpTx‐1‐71 (F 3, 31 = 93, P < 0.05) in this visceral pain model (Figure 5C, D). Compared with the saline group, i.t. naloxone alone did not alter the number of writhes in the writhing test (Figure 5C, D).

GpTx‐1 and GpTx‐1‐71 produced potent analgesic activities in the formalin test

The mice displayed a biphasic nociceptive response (phase I: 0–5 min and phase II: 15–30 min) after formalin injection. Figure 6A, B shows that both GpTx‐1 and GpTx‐1‐71 dose‐dependently exerted antinociceptive effects in phase I of formalin pain (GpTx‐1: F 3, 31 = 34, P < 0.05; GpTx‐1‐71: F 3, 32 = 6.2, P < 0.05), with ED50 values of 0.44 (0.17–0.89) and 0.86 (0.061–5.4) pmol respectively. Furthermore, GpTx‐1 reduced the flicking response in phase II in a dose‐related manner (GpTx‐1: F 3, 31 = 23, P < 0.05), with an ED50 value of 1.1 (0.53–2.4) pmol (Figure 6A). However, spinal administration of a high dose of GpTx‐1‐71 (10 pmol) decreased ongoing nociception time by approximately 43% (Figure 6B).

Figure 6.

Figure 6

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Effects of GpTx‐1 and GpTx‐1‐71 on formalin induced pain. Dose‐related antinociceptive effects of GpTx‐1 (0.1, 1 and 10 pmol) (A) and GpTx‐1‐71 (0.1, 1 and 10 pmol) (B) after i.t. injection. *P < 0.05 compared to saline group (one‐way ANOVA followed by Dunnett's post hoc test). Effects of central (i.t.) pretreatment with the non‐selective opioid antagonist naloxone on the antinociceptive effects of GpTx‐1 (C) and GpTx‐1‐71 (D). *P < 0.05 compared to saline + saline group, # P < 0.05 compared to saline + drug group (one‐way ANOVA followed by Bonferroni's post hoc test). Data columns are mean ± SEM, n = 7–10 per group.

In line with the inhibitory results mentioned above, naloxone significantly inhibited the antinociceptive effects of GpTx‐1 (phase I: F 3, 29 = 25, P < 0.05; phase II: F 3, 29 = 16, P < 0.05) and GpTx‐1‐71 (phase I: F 3, 29 = 21, P < 0.05; phase II: F 3, 29 = 18, P < 0.05) in the formalin test (Figure 6C, D).

GpTx‐1 and GpTx‐1‐71 produced non‐tolerance‐forming antinociception

In this study, the development of tolerance to antinociceptive effects of both GpTx‐1 and GpTx‐1‐71 was determined in acute, inflammatory and neuropathic pain models. As shown in Figure 7, the mice were i.t. injected with saline, GpTx‐1 or GpTx‐1‐71 once daily for eight consecutive days. The tail‐flick latency was confirmed 60 min post‐injection for GpTx‐1 or 45 min post‐injection for GpTx‐1‐71 because maximum effects were seen at 60 and 45 min post‐injection respectively. Our data show that antinociceptive tolerance developed to the i.t. injection of morphine (2 nmol) on day 4 based on a tail‐flick test (Li et al., 2016a). In contrast, the increased flick latency induced by both GpTx‐1 and GpTx‐1‐71 remained unchanged during the observation period (Figure 7A).

Figure 7.

Figure 7

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Tolerance evaluation of GpTx‐1 and GpTx‐1‐71 in different pain models. The antinociceptive effects were evaluated in acute (A), inflammatory (B) and neuropathic pain (C, anti‐allodynia; D, anti‐hyperalgesia). No significant difference was observed according to one‐way ANOVA followed by Tukey's HSD test. Data points are mean ± SEM, n = 7–10 per groups.

In the mouse models of CFA‐induced inflammatory pain and CCI‐induced neuropathic pain, repeated i.t. administration with GpTx‐1 (1 pmol) and GpTx‐1‐71 (5 pmol) produced equivalent analgesic effects over 8 days (Figure 7B–D). The mice treated with saline had no differences in paw withdrawal thresholds during the experimental time period.

Effects of i.t. injections of GpTx‐1 and GpTx‐1‐71 on motor coordination

The spinal effects of both GpTx‐1 and GpTx‐1‐71 on motor coordination in mice were evaluated using a rotarod test. Figure 8 shows that, at the highest dose of 10 pmol, neither GpTx‐1 nor GpTx‐1‐71 modified the time mice spent on a rotarod.

Figure 8.

Figure 8

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Effects of GpTx‐1 and GpTx‐1‐71 on motor coordination in mice. No significant difference was observed according to one‐way ANOVA followed by Dunnett's post hoc test. Data columns are mean ± SEM, n = 7–9 per group.

GpTx‐1 and GpTx‐1‐71 did not induce an acute hyperlocomotive response

A major liability of current opioid analgesics is reinforcement and addiction, which are both postulated to be mediated, at least in part, by the activation of the dopaminergic reward circuit (Siegfried et al., 1982; Funada et al., 1993). A biomarker for such activation is an acute hyperlocomotive response in mice, reflecting mesolimbic dopaminergic activation.

Figure 9A shows the mean (±SEM) distance (m) travelled during the observed period (180 min). The baseline activity was determined beginning 30 min prior to saline or peptide administration. Figure 9B shows the total distance travelled by mice after administration of the NaV1.7 inhibitors. As shown in Figure 9A, B, i.t. injection of either GpTx‐1 (5 pmol) or GpTx‐1‐71 (1 pmol) had no influence on the locomotor activity of mice in comparison to the saline group (P > 0.05).

Figure 9.

Figure 9

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Effects of GpTx‐1 and GpTx‐1‐71 on locomotor activity in mice. (A) Time‐response curve of GpTx‐1 and GpTx‐1‐71 after i.t. injection. (B) The total distance travelled in 180 min. No significant difference was observed according to one‐way ANOVA followed by Bonferroni's post hoc test. Data points are mean ± SEM, n = 8–9 per groups.

Effects of GpTx‐1 and GpTx‐1‐71 on gastrointestinal transit

To determine the effects of GpTx‐1 and GpTx‐1‐71 on the intestinal tract, a charcoal meal test was used in this study (Li et al., 2016b). As shown in Figure 10, i.t. injection of different concentrations of GpTx‐1 (5, 10 and 25 pmol) and GpTx‐1‐71 (1, 10 and 25 pmol) produced no significant effect on gastrointestinal transit in comparison with the saline group (P > 0.05). However, at a high dose of 25 pmol, these two inhibitors caused mouse hindlimb paralysis. In contrast to the NaV1.7 inhibitors, i.t. pretreatment with a high dose of morphine (20 nmol) significantly inhibited the gastrointestinal transit (42% ± 4), which was in line with previous studies (Porreca and Burks, 1983; Jiang et al., 1987).

Figure 10.

Figure 10

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Effects of GpTx‐1 and GpTx‐1‐71 on gastrointestinal transit (GIT) in mice. *P < 0.05 compared to saline group according to one‐way ANOVA followed by Dunnett's post hoc test. Data columns are mean ± SEM, n = 7–8 per group.

Discussion

Human genetic evidence has shown that the voltage‐gated sodium channel NaV1.7 plays a critical role in pain control. Human and mouse loss‐of‐function mutations in the NaV1.7 gene SCN9A caused congenital insensitivity to pain in previous studies (Nassar et al., 2004; Cox et al., 2006). Based on this genetic evidence, NaV1.7 is considered a novel target for pain management. Recently, a monoclonal antibody SVmab1 from a hybridoma has been shown to selectively inhibit NaV1.7 and suppress inflammatory and neuropathic pain after spinal and systemic administrations (Lee et al., 2014; Bang et al., 2018). However, the recombinant monoclonal antibodies (arSVmab or rSVmab) that target NaV1.7 had no or weak binding to NaV1.7 and did not specifically inhibit NaV1.7 currents in HEK293 cells (Liu et al., 2016; Bang et al., 2018), implying that the sources of monoclonal antibodies is a critical requirement for their efficacy. In addition, to systemically evaluate this proof‐of‐concept of analgesics targeting NaV1.7, the antinociceptive profiles of potent NaV1.7 inhibitors were also investigated in different pain models. The NaV1.7 peptide inhibitor μ‐SLPTX‐Ssm6a produced powerful antinociception in the mouse models in tail‐flick, formalin and visceral pain assays (Yang et al., 2013). In the monosodium iodoacetate‐induced osteoarthritis pain model, the NaV1.7 peptide inhibitor ProTx‐II significantly attenuated neuronal responses evoked by mechanical and thermal stimuli after spinal injection (Rahman and Dickenson, 2015). In addition, the NaV1.7 selective inhibitor CNV1014802 (Convergence Pharmaceuticals, Cambridge, UK) is currently being developed as a clinical analgesic candidate for patients with trigeminal neuralgia (Zakrzewska et al., 2017). However, the clinical lead PF‐05089771 did not proceed to the second phase because of its modest efficacy for the treatment of painful diabetic peripheral neuropathy (Mc Donnell et al., 2018).

Recently, GpTx‐1 was identified as an inhibitor of the NaV1.7 sodium channel (Cherki et al., 2014). The structure activity relationship studies of GpTx‐1 further indicated that GpTx‐1‐71 was a more potent blocker of the NaV1.7 channel than its parent peptide (Murray et al., 2015). Previous studies have shown that NaV1.7 is important in pain modulation, but the roles of the novel NaV1.7 inhibitor GpTx‐1 and its analogues in pain inhibition remain unclear. To further characterize the central antinociception of GpTx‐1 and related peptides, their spinal analgesic profiles were evaluated in this study.

In the tail‐flick model of male mice, spinal administration of GpTx‐1 produced long‐acting analgesic effects, with an ED50 value of 0.33 pmol, while GpTx‐1‐71 produced spinal antinociception, with an ED50 value as low as 0.024 pmol. Our findings were consistent with recent in vitro data showing that GpTx‐1‐71 has sixfold higher potent activity towards NaV1.7 than GpTx‐1 (Murray et al., 2015). Furthermore, to investigate the sexual dimorphism of GpTx‐1‐related peptide in pain studies, GpTx‐1‐71 was also spinally administered in female mice. Our results demonstrated that, in the present acute pain model, GpTx‐1‐71 exhibited equivalent spinal antinociception in female and male mice, which was consistent with the report that both male and female NaV1.7 knockout mice caused mechanical and thermal sensory deficits (Minett et al., 2015). Thus, there is no sexual dimorphism in pain studies after genetic and pharmacological blockade of the NaV1.7 channel. In addition, our data show that supraspinal injection of GpTx‐1 and related peptides had no significant antinociception at a high dose, which might be explained by the fact that NaV1.7 is expressed predominantly in the primary afferent nociceptive neurons (Dib‐Hajj et al., 2008).

Moreover, both GpTx‐1 and GpTx‐1‐71 dose‐dependently produced spinal analgesia in different models of formalin, visceral, inflammatory and neuropathic pain. As shown in Table 1, the antinociceptive ED50 values of GpTx‐1 and GpTx‐1‐71 were 0.30–1.11 and 0.012–0.86 pmol in these preclinical pain models respectively. Taking into account their antinociceptive potency, GpTx‐1‐71 displayed more potent analgesia than GpTx‐1 in visceral, inflammatory and neuropathic pain models, supporting the higher potency of GpTx‐1‐71 than GpTx‐1 on NaV1.7 in vitro (Murray et al., 2015). To our surprise, in the formalin test, GpTx‐1‐71 displayed a decreased potency of antinociception compared with GpTx‐1. This difference in GpTx‐1 and GpTx‐1‐71 activity in the formalin assay is in contrast to other pain models, in which the potency of GpTx‐1 is lower than that of GpTx‐1‐71. The reason for this discrepancy is unknown, since GpTx‐1‐71 has more potent activity on NaV1.7 than GpTx‐1 in vitro. However, the paradoxical results might be explained by differences in the pain model applied. Indeed, the peptide μ‐SLPTX‐Ssm6a produced more potent antinociception than morphine in a formalin assay, whereas it caused equally effective analgesia in the tail‐flick assay and a visceral pain model (Yang et al., 2013).

In previous studies, GpTx‐1 (i.p.) and the selective NaV1.7 inhibitor μ‐TRTX‐Pn3a (i.p.) significantly reversed spontaneous pain behaviours induced by intraplantar injection of the NaV1.7 activator OD1 (Deuis et al., 2016; Deuis et al., 2017), confirming on‐target NaV1.7 activity in vivo. However, previous studies have shown that the selective NaV1.7 inhibitors ProTx‐II and μ‐TRTX‐Pn3a did not produce significant antinociception in acute and inflammatory pain models (Schmalhofer et al., 2008; Deuis et al., 2017). At present, there are two potential explanations for the disparate findings as to why GpTx‐1 and related peptides were active, whereas why ProTx‐II and μ‐TRTX‐Pn3a failed to induced antinociceptive activities in acute and inflammatory pain models. It is possible that the antinociception of GpTx‐1 and GpTx‐1‐71 could be partially mediated by other ion channels in addition to NaV1.7. With the FLIPR membrane potential assay, Deuis et al. found that the GpTx‐1 rank‐order of potency was hNaV1.7 (IC50 = 0.58 μM) > hhttp://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=579 (IC50 = 5 μM) > hNaV1.1 (IC50 = 6 μM) > hhttp://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=583 (IC50 = 17 μM) > hhttp://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=580 (IC50 = 22 μM) and that GpTx‐1 inhibited NaV1.7 with 100‐fold selectivity over NaV1.4, NaV1.5 and NaV1.8 (Deuis et al., 2016). Both NaV1.1 and NaV1.6 play important roles in neuropathic and inflammatory pain respectively (Wang et al., 2011; Xie et al., 2016). However, NaV1.1 and NaV1.6 were reported to be widely expressed in the brain and spinal cord (Beckh et al., 1989). In contrast, it was notable that GpTx‐1 and related peptides exhibited different antinociceptive effects after supraspinal and spinal administrations in this work. Thus, further pharmacological studies are required to confirm the roles of other NaV channels in spinal antinociception of GpTx‐1‐related peptides. In addition, GpTx‐1 was first found as a CaV3.1 voltage‐dependent calcium channel blocker (Ono et al., 2011). Dogrul et al. demonstrated that i.t. administration of the T‐type calcium channel blocker mibefradil failed to induce antinociception under mechanical and thermal stimulation (Dogrul et al., 2003). The present work shows that the central antinociception induced by the N‐type calcium channel blocker ziconotide and GpTx‐1‐related peptide were mediated by different mechanisms. Collectively, these findings suggest that the antinociception of GpTx‐1 and GpTx‐1‐71 is independent from calcium channels. Another possibility is that GpTx‐1 and related peptide inhibit NaV1.7 channels via a different mechanism, distinct from that inhibited by the selective NaV1.7 inhibitors including μ‐TRTX‐Pn3a and ProTx‐II, the details of which will need further elaboration.

Interestingly, our results show that spinal analgesia induced by both GpTx‐1 and GpTx‐1‐71 were significantly reduced by the opioid antagonist naloxone in different pain models, which implies the involvement of the endogenous opioid system in spinal analgesia of these NaV1.7 blockers. These findings highlight the functional connection between NaV1.7 and opioid systems. In fact, a recent report demonstrated that mechanical and thermal insensitivities associated with NaV1.7 deletion were inhibited by systemic naloxone in mice, and the analgesia in a human NaV1.7‐null mutant was also reduced by naloxone (Minett et al., 2015). In addition, it has been shown that both preproenkephalin gene mRNA and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1614 levels increased in sensory neurons of NaV1.7‐null mice (Minett et al., 2015). Thus, genetic and pharmacological blockade of the NaV1.7 channel can result in the activation of the endogenous opioid system and then induce potent and naloxone‐sensitive antinociception. Indeed, our hypothesis is supported by the reported results that the subtype‐selective NaV1.7 inhibitors μ‐TRTX‐Pn3a, Phlotoxin 1 and PF‐04856264 produced analgesic synergy when co‐administered with subtherapeutic doses of opioids or the enkephalinase inhibitor http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?tab=biology&ligandId=5278 (Deuis et al., 2017). Therefore, we hypothesize that blockade of NaV1.7 might result in the activation of the endogenous opioid pathway and might then induce the analgesic effects of GpTx‐1 and related peptides. Recently, psalmotoxin 1, a blocker of the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=684 (ASIC1a channel), was reported to produce potent analgesic properties against inflammatory and neuropathic pain by stimulating release of endogenous enkephalin (Mazzuca et al., 2007).

It is notable that opioid analgesics usually cause several side effects after chronic treatment in the clinical setting, such as constipation, respiratory depression, tolerance and dependence (Inturrisi, 2002; Stein, 2013). Next, the side effects of GpTx‐1 and GpTx‐1‐71 were further investigated in the rotarod, antinociceptive tolerance, acute hyperlocomotion and gastrointestinal transit tests.

Previous data have shown that spinal morphine produces antinociceptive tolerance on day 4 in an acute pain model (Li et al., 2016a). In contrast to opioids, this study indicated that spinal administration of GpTx‐1 and GpTx‐1‐71 induced equivalent analgesia during the 8 days of dosing in acute, inflammatory or neuropathic pain models, which is consistent with the previous data that repeated i.v. injection of SVmab1, a monoclonal antibody targeting NaV1.7 channels, attenuated mechanical allodynia without tolerance development over 3 days in a neuropathic pain model (Lee et al., 2014). Moreover, our results show that both GpTx‐1 and GpTx‐1‐71 had no signs of acute hyperlocomotion at the spinal level. In addition, at high doses, both GpTx‐1 and GpTx‐1‐71 were devoid of motor effects, as assessed by a rotarod assay, suggesting that the doses of these two NaV1.7 peptide blockers that induce analgesia were clinically relevant. Finally, the present work showed that high doses of GpTx‐1 and the related peptide GpTx‐1‐71 had no effects on gastrointestinal transit in mice. However, spinal morphine has been reported to induce a significant decrease in the gastrointestinal transit (Porreca and Burks, 1983; Jiang et al., 1987; Tonello et al., 2014). Collectively, our results demonstrate that spinal GpTx‐1 and GpTx‐1‐71 provided powerful antinociception, notably, without inducing tolerance, acute hyperlocomotion, constipation and motor impairments.

In conclusion, our present study indicates that the NaV1.7 inhibitors GpTx‐1 and GpTx‐1‐71 produce powerful analgesia in a series of preclinical pain models, which might be associated with the activation of the endogenous opioid system. In addition, at the spinal level, both GpTx‐1‐ and GpTx‐1‐71‐treated mice showed no signs of tolerance, acute hyperlocomotion, constipation or motor impairments. Therefore, the NaV1.7 inhibitor GpTx‐1 and related peptides are potential clinical candidates for the development of novel analgesics without side effects.

Author contributions

C.C., B.X., X.S., M.Z., Q.Z., T.Z., W.Z., R.Z., Z.W. and N.L. performed the research. Q.F. designed the research study. B.X. and M.Z. analyzed the data. Q.F., C.C. and B.X. wrote the paper. Both C.C. and B.X. contributed equally to this work.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This http://onlinelibrary.wiley.com/doi/10.1111/bph.14461/abstract acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Acknowledgements

This study was supported by the grants from the National Natural Science Foundation of China (Nos. 81673282 and 21432003) and the Shenzhen Science and Technology Innovation Committee (JCYJ20170307100618421), Program for Changjiang Scholars and Innovative Research Team in University Foundation of China (IRT_15R27) and the Fundamental Research Funds for the Central Universities (lzujbky‐2017‐121).

Chen, C. , Xu, B. , Shi, X. , Zhang, M. , Zhang, Q. , Zhang, T. , Zhao, W. , Zhang, R. , Wang, Z. , Li, N. , and Fang, Q. (2018) GpTx‐1 and [Ala5, Phe6, Leu26, Arg28]GpTx‐1, two peptide NaV1.7 inhibitors: analgesic and tolerance properties at the spinal level. British Journal of Pharmacology, 175: 3911–3927. 10.1111/bph.14461.

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