α‐Spinasterol: a COX inhibitor and a transient receptor potential vanilloid 1 antagonist presents an antinociceptive effect in clinically relevant models of pain in mice

Abstract

Background and Purpose

Postoperative pain is one of the most common manifestations of acute pain and is an important problem faced by patients after surgery. Moreover, neuronal trauma or chemotherapeutic treatment often causes neuropathic pain, which induces disabling and distressing symptoms. At present, treatments of both painful conditions are inadequate. α‐Spinasterol, which is well characterized as a transient receptor potential vanilloid 1 antagonist, has anti‐inflammatory, antioxidant and antinociceptive effects. Therefore, we investigated its antinociceptive potential on postoperative and neuropathic pain, as well as its effect on COX‐1 and COX‐2 activities.

Experimental Approach

Nociceptive responses in a postoperative pain model (surgical incision‐induced) or different neuropathic pain models (trauma or chemotherapy‐induced) were investigated in mice.

Key Results

Oral administration of α‐spinasterol reduced postoperative pain, when given as a pre‐ (0.5 h before incision) or post‐treatment (0.5 h after incision), and reduced cell infiltration in the injured tissue. α‐Spinasterol also reduced the mechanical allodynia induced by partial sciatic nerve ligation and the mechanical and cold allodynia induced by paclitaxel. Moreover, α‐spinasterol inhibited COX‐1 and COX‐2 enzyme activities without altering the body temperature of animals. Importantly, α‐spinasterol did not alter spontaneous or forced locomotor activity. Furthermore, it did not cause gastric damage or liver and kidney changes, nor did it alter cell viability in the cerebral cortex and spinal cord slices of mice.

Conclusion and Implications

α‐Spinasterol is an effective and safe COX inhibitor with antinociceptive effects in postoperative and neuropathic pain models. Therefore, it is an interesting prototype for the development of novel analgesic drugs.

Abbreviations

aCFS

artificial CSF

ALT

alanine aminotransferase

AMG9810

2E)‐N‐(2,3‐dihydro‐1,4‐benzodioxin‐6‐yl)‐3‐[4‐(1,1dimethylethyl)phenyl]‐2‐propenamide

AST

aspartate aminotransferase

B

baseline

ID₅₀

inhibitory dose 50%

MTT

3‐(4,5‐dimethylthiazol‐2‐yl‐diphenyltetrazolium bromide

NaCl

sodium chloride

PWT

paw withdrawal threshold

TRP

transient receptor potential

TRPV1

transient receptor potential vanilloid 1.

Introduction

Pain is one of the most significant causes of suffering and disability worldwide, impairing the quality of life of affected individuals (King and Fraser, 2013; Taylor et al., 2016). Acute postoperative pain, which is a type of inflammatory pain that is caused by surgical procedures, affects a majority of patients, with less than 50% of them reporting adequate relief. Moreover, the severity of this pain is characterized as moderate, severe or extreme (Kehlet et al., 2006; Pogatzki‐Zahn et al., 2007; Gan et al., 2014; Chou et al., 2016). Satisfactory treatment of postoperative pain reduces cognitive impairment and the risk of chronic or persistent postsurgical pain (Kehlet et al., 2006; Vaurio et al., 2006; Pogatzki‐Zahn et al., 2007; Wu and Raja, 2011; Pogatzki‐Zahn et al., 2017). However, the analgesic agents commonly used to treat postoperative pain [opioids and nonsteroidal anti‐inflammatory drugs (NSAIDs)] can cause adverse effects, limiting their use (Wu et al., 2011; Gan et al., 2014; Chou et al., 2016). Thus, regardless of many advances in this field, the treatments of postoperative pain remain inadequate (Wu et al., 2011; Chou et al., 2016; Pogatzki‐Zahn et al., 2017).

Neuropathic pain is also a painful clinical condition, which results from a lesion (trauma) or disease (such as the neuropathies associated with a tumour or the use of chemotherapy). It affects the somatosensory system, altering its structure and function (Sisignano et al., 2014; Finnerup et al., 2015; Gilron et al., 2015). The incidence of chemotherapy‐induced peripheral neuropathy occurs in up to 80% of patients who receive chemotherapy (Sisignano et al., 2014). However, the treatment of neuropathic pain remains a challenge. The therapies available are often inadequate, their efficacy is unpredictable and they often cause adverse effects (Dworkin et al., 2007; Kamerman et al., 2015).

Therefore, the development of therapeutic alternatives, which are more effective and can safely treat postoperative and neuropathic pain, is urgently needed. In this regard, natural products have been an important source of new therapeutic agents, including analgesic molecules (Calixto, 2005; Mishra and Tiwari, 2011). α‐Spinasterol is a steroid compound that is found in a variety of plants (Trevisan et al., 2012; Borges et al., 2014). It exhibits antioxidant (Coballase‐Urrutia et al., 2010), anticonvulsant (Socała et al., 2015), antidepressant (Socała and Wlaź, 2016) and anti‐inflammatory (Boller et al., 2010; Borges et al., 2014) pharmacological effects. Moreover, α‐spinasterol also elicits antinociceptive effects, through its action as a transient receptor potential vanilloid 1 (TRPV1) antagonist. TRPV1 is a receptor for noxious stimuli and a therapeutic target for new analgesics (Trevisan et al., 2012).

Compounds with a multi‐target action, for instance, on both TRPV1 receptor and COX enzymes, might improve the efficacy and minimize the adverse effects caused by TRPV1 antagonism or COX inhibition (Tributino et al., 2010). Several COX inhibitors and TRPV1 antagonists have been shown attenuate postoperative pain (Whiteside et al., 2004; Uchytilova et al., 2014). Moreover, inflammatory mediators, such as PGE₂, the COX end‐product, drive neuropathy and sensitize TRPV1 channels (Lau et al., 2010). Either the direct modulation of TRPV1 or the inhibition of the COX pathway can relieve neuropathic pain (Marwaha et al., 2016). Therefore, COX inhibitors and TRPV1 antagonists can also reduce the neuropathy induced by trauma or chemotherapy (Guindon and Beaulieu, 2006; Ito et al., 2012; Lima et al., 2014; Li et al., 2015). Likewise, a TRPV1 antagonist with additional anti‐inflammatory properties would be a promising prototype for the treatment of neuropathies (Sisignano et al., 2014).

We investigated the antinociceptive potential and any possible adverse effects of α‐spinasterol in different clinically relevant pain models, including those of postoperative and neuropathic pain. We also evaluated the effect of α‐spinasterol on the activities of the enzymes COX‐1 and COX‐2.

Methods

Animals

Adult male Swiss mice (25–30 g) were kept at a controlled temperature (22 ± 1°C) in individually ventilated cages, on a 12 h light/12 h dark cycle, with food and water available ad libitum. All experiments were conducted in accordance with the national and international legislation (guidelines of Brazilian Council of Animal Experimentation and of the U.S. Public Health Service's Policy on Humane care and Use of Laboratory Animals‐PHS Policy), under the ethical guidelines established for investigations of experimental pain in conscious animals (Zimmermann, 1983). The study was approved by the Committee on the Ethical Use of Animals of the Federal University of Santa Maria (process number 3652150416/2016).

Animal models for postoperative and neuropathic pain were used, since both cause incapacitating symptoms in patients and are often inadequately treated (Gan et al., 2014; Gilron et al., 2015; Taylor et al., 2016). Therefore, the use of an intact organism was important to obtain an adequate response to the experimental models used here. In this context, Swiss mice have been used previously in experiments involving postoperative pain (Oliveira et al., 2011; 2013), partial sciatic nerve ligation‐induced neuropathic pain (Villarinho et al., 2012; Oliveira et al., 2014) and paclitaxel‐induced neuropathic pain (Brusco et al., 2016).

Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). The group size used for each experiment was based on studies that used protocols similar to those proposed here. Therefore, we estimated a group size of six mice for each experimental group (Villarinho et al., 2012; Oliveira et al., 2014; Brusco et al., 2016). The number of animals and intensities of noxious stimuli used were the minimum needed to demonstrate the consistent effects of the treatments. Allocation concealment was performed using a randomization procedure (http://www.randomizer.org/). Behavioural evaluations were performed blindly on drug administration. Each experiment was repeated two to three times (using two or three animals for each repetition) and experiments were carried out between 08:00 and 17:00 h.

Treatment

To evaluate the antinociceptive effect of α‐spinasterol in the postoperative pain model, the animals were treated with vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or indomethacin (10 mg·kg⁻¹, p.o.; positive control), 0.5 h before the incision (pretreatment) or 0.5 h after the incision (post‐treatment). The antinociceptive effect in the postoperative pain model (pretreatment) was also evaluated, using a dose–response curve for α‐spinasterol (0.1–1 mg·kg⁻¹, p.o.) or indomethacin (1–10 mg·kg⁻¹, p.o.).

In the partial sciatic nerve ligation‐induced neuropathy model, the animals received vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or celecoxib (100 mg·kg⁻¹, p.o.; positive control), 7 days after sciatic nerve ligation. To assess the chemotherapy‐induced neuropathy, the animals were treated orally with vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or acetaminophen (100 mg·kg⁻¹, p.o.; positive control), 1 day after a single dose of paclitaxel administration (acute treatment) or 21 days after the first repeated paclitaxel dose (chronic treatment).

Behavioural tests

Postoperative pain model

The postoperative pain model was carried out as previously described (Oliveira et al., 2011, 2013). Animals were anaesthetised with 2% isoflurane via a nose cone. After antiseptic preparation, a 5 mm longitudinal incision was made in the skin and the fascia of the right hind paw, which started 2 mm from the proximal edge of the heel and extended to the toes. The underlying muscle was elevated with forceps, leaving the muscle origin and insertion intact. The skin was sutured with 6.0 nylon wire. The animals were pretreated (0.5 h before incision) or post‐treated (0.5 h after incision) with vehicle (10 mL·kg⁻¹, p.o), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or indomethacin (10 mg·kg⁻¹, p.o.; positive control). The mechanical paw withdrawal threshold (PWT) was measured using a time‐response curve from 0.5 to 24 h after treatment. The combined effect of α‐spinasterol (0.3 mg·kg⁻¹, p.o.) plus indomethacin (10 mg·kg⁻¹, p.o.) versus α‐spinasterol or indomethacin alone was also evaluated in the pretreatment protocol. The mechanical PWT was also evaluated in the pretreatment protocol using a dose–response curve, with different doses of α‐Spinasterol (0.1–1 mg·kg⁻¹, p.o.) or indomethacin (1–10 mg·kg⁻¹, p.o.), at 1 h after its administration. The reduction in the mechanical PWT after the surgical incision was characterized as mechanical allodynia.

Partial sciatic nerve ligation‐induced neuropathy model

For the partial sciatic nerve ligation‐induced neuropathy model, animals were first anaesthetised using an injection of ketamine (90 mg·kg⁻¹, i.p.) plus xylazine hydrochloride (3 mg·kg⁻¹, i.p.). The depth of anesthesia was considered when the animal presented severe muscle relaxation, pupils dilated and no reflexes palpebral or corneal. Posteriorly, a partial ligation of the right sciatic nerve was made by tying one‐third to one‐half of the dorsal portion of the sciatic nerve (Villarinho et al., 2012; Oliveira et al., 2014). In another group of animals, the nerve was exposed without ligation (sham‐operated group). The mechanical PWT was measured before the nerve ligation and then 7 days after the procedure. Mice that presented a reduction in the mechanical PWT after injury, characterized as mechanical allodynia, were treated with vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or celecoxib (100 mg·kg⁻¹, p.o.; positive control). Thereafter, the mechanical PWT was assessed again from 0.5 to 6 h after treatments.

Chemotherapy‐induced neuropathic pain model

For the model chemotherapy‐induced neuropathic pain, mice received one dose (acute treatment; 1 mg·kg⁻¹, i.p.) or four doses (chronic treatment; 4 × 1 mg·kg⁻¹, i.p.; on alternate days, i.e. on days 1, 3, 5 and 7, resulting in a cumulative dose of 4 mg·kg⁻¹) of paclitaxel (Brusco et al., 2016). The animals were then administered with vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or acetaminophen (100 mg·kg⁻¹, p.o.; positive control), 1 day after acute treatment or 21 days after the first administration of the chronic treatment of paclitaxel. Thereafter, the mechanical PWT was assessed from 0.5 up to 24 h. Cold allodynia was evaluated from 1 up to 6 h after the administration of treatments.

Assessment of mechanical allodynia

The mechanical PWT was determined before and after the induction of all pain models or before and after the treatments, using a series of flexible nylon von Frey calibrated filaments of increasing stiffness (i.e. 0.02, 0.07, 0.16, 0.4, 1.4, 4.0 and 10.0 g) using the up‐and‐down method (Oliveira et al., 2011, 2013). Mice were acclimatized in individual Plexiglas boxes (9 × 7 × 11 cm) on an elevated, wire mesh platform to allow access to the plantar surface of the right hind paw. Calibrated filaments of von Frey were applied to the plantar surface, using pressure to bend the filament. Six measurements were performed. An absence of a paw‐lifting response led to the use of the next filament with increasing weight, while a paw‐lifting response led to the use of the next weaker filament. The mechanical PWT response was then calculated as previously described by Dixon (1980). A significant decrease in mechanical PWT compared to the baseline value was considered mechanical allodynia.

Assessment of spontaneous nociception

The spontaneous nociception score was observed in the postoperative pain model, immediately before starting the measurements of mechanical allodynia following the surgical procedure in the paw tissue (Oliveira et al., 2013; Silva et al., 2016). The animals remained in the Plexiglas boxes on an elevated, wire mesh platform, to allow observation of the plantar surface of the right hind paw. The amount of weight that the animals were willing to put on their paw after being submitted to an incision was estimated by qualitative visual analysis, using the following scale: score 0, normal paw pressure, with equal weight distribution on both hind paws; score 1, slightly reduced paw pressure, with the hind paw completely on the floor, but the toes are not spread; score 2, moderately reduced hind paw pressure, with only some parts of the paw slightly touching the floor; score 3, severely reduced hind paw pressure, with the hind paw completely removed from the floor. The results are expressed as the sum of scores, from 0.5 to 4 h and 0.5 to 6 h after treatments, for the pre‐ and postoperative respectively.

Assessment of cold allodynia

Cold allodynia was assessed in the chemotherapy‐induced neuropathy model using the acetone drop method (Zheng et al., 2012), which was adapted to mice. The score for cold allodynia was evaluated immediately after the measurements of mechanical allodynia (following the development of paclitaxel‐induced neuropathic pain). The animals remained in the Plexiglas boxes on an elevated, wire mesh platform, to allow access to the plantar surface of the right hind paw where a drop (20 μL) of acetone was then applied three times. Cumulative scores were generated for each mouse using the following 4‐point scale: 0, no response; 1, quick withdrawal, flick or stamp of the paw; 2, prolonged with withdrawal or repeated flicking; and 3, repeated flicking of the paw with licking directed at the ventral side of the paw. The results are expressed as the sum of three scores that were evaluated for each animal.

Leukocyte infiltration assessment

To assess cell infiltration in the paws of the animals subjected to the postoperative pain, mice were pretreated (0.5 h before incision) with vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or indomethacin (10 mg·kg⁻¹, p.o.). The mice were then killed 2 h after the treatment. The plantar surface of the hind paw was removed and fixed for 12 h in an alfac solution (16:2:1 mixture of ethanol 80%, formaldehyde 40%, and acetic acid) in a volume that was 5–10 times greater than the volume of the sample. The sections were then fixed and transferred to containers containing 70% alcohol to preserve the tissue. Thereafter, the paws were embedded in paraffin wax to prepare the histological lamina, sectioned at 5 μm and stained with haematoxylin–eosin. The prepared histological lamina were analysed using a light microscopic, with 20× and 40× objectives. Representative areas were selected for the quantitative count of the cellular infiltration (Oliveira et al., 2011; 2014).

COX‐1 and COX‐2 enzyme activities in vitro

α‐Spinasterol has been already characterized as a TRPV1 antagonist (Trevisan et al., 2012), and we also evaluated whether this compound inhibits the activity of the enzymes COX‐1 and COX‐2. A COX screening assay kit (Kit No. 560101, Cayman), was used according to the manufacturer's instructions and as previously described (Oliveira et al., 2014), for the evaluation of COX activity in two experiments. The α‐Spinasterol concentration (i.e. 3–100 μM) was chosen from pilot experiments. The COX inhibitory activities were quantified from the absorption at 405 nm. The results are presented as prostaglandin levels (in ng·mL⁻¹).

Investigation of possible adverse effects α‐Spinasterol‐induced

Evaluation of cell viability on the cerebral cortex and spinal cord slices

Preparation and incubation of slices

Animals were killed and the cerebral cortex and spinal cord were immediately removed and used for the preparation of slices (400 μm thick, with a Mcllwain tissue chopper) as previously described (Carvalho et al., 2012). Then, the slices were placed in a pre‐gassed artificial CSF (aCFS) containing the following (in mM): NaH₂PO4 (1.25); NaH₂CO₃ (22); MgCl₂ (1.8); NaCl (129); CaCl₂ (1.8); KCl (3.5); and D‐glucose, pH 7.4 (10). Before the experiment, the aCFS was pre‐conditioned for 2 h in the incubator in a 95% air, 5% CO₂ atmosphere. The cerebral cortex and spinal cord slices were treated with α‐spinasterol (1–1000 nM), for 6 h at 37°C, in a 95% air, 5% CO₂ atmosphere.

LDH release assay

LDH is an intracellular enzyme. Its extracellular release serves as a useful marker of cell membrane damage (Zeni et al., 2014). The integrity of cells after 6 h of treatment with α‐spinasterol (1–1000 nM) in the cerebral cortex and spinal cord slices was evaluated through an LDH release assay, using a colourimetric kit (Labtest Diagnostica SA, Lagoa Santa, MG, Brazil). The results are expressed as % LDH released^. g^‐1 of tissue.

MTT reduction

Cell viability was determined by the ability of cells to reduce MTT (Mozes et al., 2012); viable cells reduce the water‐soluble yellow MTT to water‐insoluble blue MTT formazan. Cerebral cortex and spinal cord cell slices pretreated for 6 h with α‐spinasterol (1–1000 nM) were incubated with MTT (0.5 mg·mL⁻¹) dissolved in aCFS pre‐conditioned for 30 min at 37°C (in 95% air, 5% CO₂ atmosphere). Then, the medium was discarded, and the formazan produced was solubilized with 300 μL of DMSO. Following a 30 min incubation, in the dark, at room temperature, the slices produced a coloured compound whose OD was measured at 550 and 620 nm. Data were standardized through the following formula: (OD550‐OD620) · g⁻¹ of tissue − 100% in relation to control group (only aCFS).

Gastric lesion assessment

Mice were fasted for 18 h before treatments to evaluate the gastric tolerability of animals after the oral administration of α‐spinasterol. The animals were then treated with vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (10 mg·kg⁻¹, p.o.) or indomethacin (100 mg·kg⁻¹, p.o) and were killed after 4 h. The stomachs were removed, opened and washed with saline (NaCl 0.9%) at 4°C. The lesion index was visually evaluated using a magnifying glass. Gastric mucosal lesions were quantified using a score of 0–6 according to the size and number of lesions (Oliveira et al., 2009).

Biochemical markers

The occurrence of changes in the liver or kidney was also evaluated in the animals treated with vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (10 mg·kg⁻¹, p.o.) or indomethacin (100 mg·kg⁻¹, p.o.). Blood was collected, 4 h later, via a cardiac puncture, and the mice were killed. The blood was then centrifuged to obtain the serum samples. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the level of urea and creatinine, which are sensitive indicators of liver and kidney injury, respectively, were assessed in the serum samples, using the appropriate Labtest® kit for each biochemical analysis, according to the manufacturer's specifications (Labtest Diagnostica, Brazil) (Oliveira et al., 2014).

Evaluation of locomotor activity

Locomotor activity was evaluated 1 h after vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.), indomethacin (10 mg·kg⁻¹, p.o.) or AMG9810 (30 mg·kg⁻¹, p.o.) administration, as previously described by Trevisan et al. (2012). Briefly, the spontaneous locomotor activity of the animals was evaluated using an open‐field test, which consists of a glass box measuring 40 × 60 × 50 cm, wherein the floor of the box is divided into 12 equal squares. The forced locomotor activity of each animal was evaluated using the rotarod test (3.7 cm in diameter, 8 rpm). The animals were first trained to remain on the apparatus for 60 s without falling. The results are expressed as the number of crossings and rearings during 5 min in the open‐field test and as the number of falls during 4 min in the rotarod test.

Body temperature

Since severe hyperthermia is a well‐known adverse effect of TRPV1 receptor antagonists, such as AMG9810 (Trevisan et al., 2012), we investigated the effect of α‐spinasterol on body temperature. Animals were treated with vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.), indomethacin (10 mg·kg⁻¹, p.o.) or AMG9810 (30 mg·kg⁻¹, p.o.; TRPV1 antagonist, used as a positive control). The animals' rectal temperature was then determined 1 h later. The results are expressed as the difference in the temperature (°C) after treatments, from the baseline measurement (Trevisan et al., 2012).

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Results are expressed as the mean and SEM, except for the inhibitory dose (ID₅₀) values (i.e. the α‐spinasterol or indomethacin dose that reduces mechanical allodynia to the order of 50% relative to the control value), which are expressed as geometric means accompanied by their respective 95% confidence limits, and gastric lesion scores, spontaneous nociception scores and cold allodynia, which are reported as medians followed by their 25th and 75th percentiles. Statistical analysis was performed using GraphPad Prism 6.0 software (San Diego, CA, USA). The significance of the differences between groups was evaluated by one‐way or two‐way ANOVA, followed by Tukey's post hoc test or Bonferroni's post hoc test for parametric data. The data of mechanical threshold and cold allodynia scores were log transformed before analysis to meet the parametric assumptions. Non‐parametric data were evaluated using the Kruskal–Wallis test, followed by Dunn's test. Post hoc tests were performed only when the F‐value achieved the necessary level of statistical significance (P < 0.05) and when there was no significant variance in homogeneity (Curtis et al., 2015).

Materials

Indomethacin and celecoxib were purchased from Sigma Chemical Co (St. Louis, MO, USA). Both were dissolved in DMSO (10%), Tween 80 (10%) and 0.9% NaCl (80%). Acetaminophen and α‐spinasterol were purchased from local suppliers and Tocris (Bristol, UK) respectively. Both were dissolved in Tween 80 (5%), polyethylene glycol (20%) and 0.9% NaCl (75%). Paclitaxel (i.e. 6 mg·mL⁻¹ paclitaxel in Cremophor EL and dehydrated ethanol) was purchased from Glenmark (Buenos Aires, Argentina) and was dissolved in NaCl (0.9%). The AMG9810 was purchased from Sigma Chemical Co (St. Louis, MO, USA) and dissolved in DMSO (5%), Tween 80 (5%) and 0.9% NaCl (90%). All drugs (10 mL·kg⁻¹), except paclitaxel (10 mL·kg⁻¹; which was administered i.p.), were administered by gavage (p.o.). Doses of the drugs used were based on those used in previous studies (Trevisan et al., 2012; Brusco et al., 2016). The von Frey filaments and 6.0 nylon wire were purchased from North Coast Medical (CA, USA) and Huaiyin Medical Instruments Co., Ltd. (Jiangsu, China) respectively.

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 (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015a,b).

Results

Effect of α‐spinasterol on postoperative pain

α‐Spinasterol reduces the surgical incision‐induced nociception

The right hind paw surgical incision produced a decrease in the PWT in response to mechanical stimuli of von Frey filaments compared to the baseline (B), characterized as mechanical allodynia (Figure 1A–D). Post‐treatment (0.5 h after incision) with α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or indomethacin (10 mg·kg⁻¹, p.o.) reversed this mechanical allodynia from 0.5 up to 6 h after treatments, with maximum inhibitions of 76 ± 5 and 82 ± 3%, respectively, at 2 h after its administration when compared to the vehicle group (Figure 1A). Pretreatment (0.5 h before incision) with α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or indomethacin (10 mg·kg⁻¹, p.o.) was also effective in preventing the mechanical allodynia induced by surgical incision from 0.5 up to 6 h after the treatments, with maximum inhibition of 60 ± 5 and 59 ± 5% at 1 h and of 64 ± 6 and 54 ± 8% at 2 h after its administration, respectively, when compared to the vehicle group (Figure 1B). The combined treatment with α‐spinasterol (0.3 mg·kg⁻¹, p.o.) and indomethacin (10 mg·kg⁻¹, p.o.) was more effective in preventing mechanical allodynia induced by surgical incision at 0.5 and 1 h after the treatments, with a maximum inhibition of 79 ± 5% at 1 h, compared to the individual treatment with indomethacin (Figure 1B).

Figure 1.

Effects of α‐spinasterol and indomethacin on mechanical allodynia induced by surgical incision. Time‐response curves in animals that received vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or indomethacin (10 mg·kg⁻¹, p.o.), 0.5 h after surgical incision (post‐treatment) (A) or 0.5 h before surgical incision (pretreatment) (B). Dose–response curves in animals that received vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.1–3 mg·kg⁻¹, p.o.) (C) or indomethacin (1–10 mg·kg⁻¹, p.o.) (D) 0.5 h before incision (pretreatment). B denotes the basal mechanical threshold before surgical incision. 0 indicates the basal mechanical threshold after surgical incision and before of treatments. Data are expressed as the mean ± SEM of six animals per group. *P < 0.05 when compared with the vehicle group; two‐way ANOVA followed by Bonferroni's post hoc test. ^& P < 0.05 when compared with indomethacin group; two‐way ANOVA followed by Tukey's post hoc test. ^# P < 0.05 when compared with basal mechanical threshold, B; one‐way ANOVA followed by Bonferroni's post hoc test.

We also evaluated the effect of the pretreatment with α‐spinasterol using a dose–response curve. The pretreatment (0.5 h before incision) with α‐spinasterol at doses of 0.1, 0.3 and 1 mg·kg⁻¹ was effective in preventing mechanical allodynia induced by surgical incision compared to the vehicle group. The calculated ID₅₀ value was 1.85 (0.19–17.30) mg·kg⁻¹, with a maximum inhibition of 58 ± 5% (at 1 mg·kg⁻¹), at 1 h after the treatment (Figure 1C). Likewise, the anti‐allodynic effect of indomethacin occurred at the three doses tested (1, 3 and 10 mg·kg⁻¹, p.o.), and the calculated inhibitory dose value was 6.53 (3.16–13.49) mg·kg⁻¹, with a maximum inhibition of 59 ± 3% (at 10 mg·kg⁻¹), at 1 h after its administration (Figure 1D).

Both the post‐treatment and pretreatment with α‐spinasterol (0.3 mg·kg⁻¹, p.o.) did not alter the spontaneous nociception score induced by surgical incision, when evaluated from 0.5 up to 6 h or from 0.5 up to 4 h after treatments, respectively, compared to the vehicle group. Post‐treatment with indomethacin (10 mg·kg⁻¹, p.o.) also did not alter the spontaneous nociception score of animals. Pretreatment with indomethacin, however, was effective in decreasing the spontaneous nociception score induced by surgical incision, when compared to the vehicle group, with a maximum inhibition of 77 ± 11% (Figure 2).

Figure 2.

Effects of α‐spinasterol and indomethacin on spontaneous nociception induced by surgical incision. Spontaneous nociception score in animals that received vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or indomethacin (10 mg·kg⁻¹, p.o.), 0.5 h after incision (post‐treatment) (A) or 0.5 h before incision (pretreatment) (B). Data are expressed as the medians followed by their 25th and 75th percentiles of six animals per group. *P < 0.05 when compared with the vehicle group; Kruskal–Wallis test followed by Dunn's test.

α‐Spinasterol reduces the infiltration of cells after surgical incision

A histological analysis was conducted to measure the infiltration of inflammatory cells into the paw tissue of mice subjected to postoperative pain (pretreatment). The surgical incision induced a significant increase in cell infiltration in the vehicle group, compared to the sham group. Pretreatment with α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or indomethacin (10 mg·kg⁻¹, p.o.) reduced the number of inflammatory cells in the paw tissue compared to the vehicle group (Figure 3A, B).

Figure 3.

Effects of α‐spinasterol and indomethacin on cell infiltration induced by surgical incision. Representative light microphotograph of the right hind paw (A; arrows indicate polymorphonuclear cells) 2 h after treatments in animals that received vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or indomethacin (10 mg·kg⁻¹, p.o.), 0.5 h before the incision (pretreatment) or sham group. Quantification of polymorphonuclear cells per field (B) of the paw tissue of mice. Data are presented as the mean ± SEM (n = 6 per group). ^# P < 0.05 when compared to the sham group; *P < 0.05 when compared to the vehicle group; one‐way ANOVA, followed by Bonferroni's post hoc test.

α‐Spinasterol reduces the partial sciatic nerve ligation‐induced neuropathic pain

Treatment with α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or celecoxib (100 mg·kg⁻¹, p.o.) did not alter the mechanical PWT in sham‐operated animals compared to the vehicle group (Figure 4A). Moreover, in another group of animals, partial sciatic nerve ligation produced a significant decrease in the PWT in response to mechanical stimuli with von Frey filaments compared to the baseline (B), 7 days after the surgery, apparent as mechanical allodynia (Figure 4B). Treatment with α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or celecoxib (100 mg·kg⁻¹, p.o.) reversed the mechanical allodynia induced by partial sciatic nerve ligation, from 1 up to 2 h, after its treatment, compared to the vehicle group, with maximum inhibition of 50 ± 9 and 63 ± 11%, respectively, at 1 h after administration (Figure 4B).

Figure 4.

Effects of α‐spinasterol and celecoxib on mechanical allodynia induced by partial sciatic nerve ligation. Time‐response curves in animals that received vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or celecoxib (100 mg·kg⁻¹, p.o.), 7 days after the surgical procedure. (A) Sham‐operated mice. (B) Mice subjected to partial sciatic nerve ligation. B denotes the basal mechanical threshold before surgical incision, while 0 indicates the basal mechanical threshold 7 day after surgical incision. Data are expressed as the mean ± SEM of six animals per group. *P < 0.05 when compared with the vehicle group; two‐way ANOVA, followed by Bonferroni's post hoc test. ^# P < 0.05 when compared with basal mechanical threshold (B); one‐way ANOVA followed by Tukey's post hoc test.

α‐Spinasterol reduces chemotherapy‐induced neuropathic pain

Single (acute) or repeated (chronic) paclitaxel administration decreased the PWT in response to mechanical stimuli of von Frey filaments compared to the baseline (B), which was apparent as mechanical allodynia (Figure 5A, B). Treatment with α‐Spinasterol (0.3 mg·kg⁻¹, p.o.) or acetaminophen (100 mg·kg⁻¹, p.o.) reversed the mechanical allodynia induced by acute paclitaxel administration, from 2 up to 4 h or from 0.5 up to 4 h, after treatments, respectively, with maximal inhibitions of 39 ± 9% for α‐spinasterol at 2 h and 51 ± 5% for acetaminophen at 1 h after treatments (Figure 5A). Moreover, α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or acetaminophen (100 mg·kg⁻¹, p.o.) also reversed the mechanical allodynia induced by chronic paclitaxel administration, from 1 up to 4 h and from 1 up to 2 h, following treatments with maximum inhibitions of 38 ± 4 and 44 ± 9% at 2 h after treatments respectively (Figure 5B).

Figure 5.

Effects of α‐spinasterol and acetaminophen on paclitaxel‐induced mechanical allodynia. Time‐response curves in animals that received vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or acetaminophen (100 mg·kg⁻¹, p.o.), 1 day after acute paclitaxel administration (1 mg·kg⁻¹, i.p.) (A) or 21 days after the first repeated paclitaxel administration (4 × 1 mg·kg⁻¹, i.p.) (B). B denotes the basal mechanical threshold before paclitaxel administration, while 0 indicates the basal mechanical threshold after paclitaxel administration and before treatments. Data are expressed as the mean ± SEM of six animals per group. *P < 0.05 when compared with the vehicle group; two‐way ANOVA followed by Bonferroni's post hoc test. ^# P < 0.05 when compared with basal mechanical threshold (B); one‐way ANOVA followed by Bonferroni's post hoc test.

Apart from mechanical allodynia, paclitaxel is also known to cause cold allodynia in patients. Therefore, we evaluated the effect of α‐spinasterol on acetone‐induced cold allodynia after acute or chronic paclitaxel injections. Treatment with α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or acetaminophen (100 mg·kg⁻¹, p.o.) reversed the acetone‐induced cold allodynia following acute paclitaxel administration, from 1 up to 2 h or at 1 and 4 h after treatments respectively. The maximal inhibition observed at 1 h after treatments was 55 ± 6 and 56 ± 10% respectively (Figure 6A, B). Treatments with α‐spinasterol or acetaminophen, however, were not able to reverse the acetone‐induced cold allodynia following chronic paclitaxel administration (data not shown).

Figure 6.

Effects of α‐spinasterol and acetaminophen on acetone‐induced cold‐allodynia after paclitaxel administration. Time‐response curves in animals that received vehicle (10 mL·kg⁻¹, p.o.), α‐spinasterol (0.3 mg·kg⁻¹, p.o.) (A) or acetaminophen (100 mg·kg⁻¹, p.o.) (B), 1 day after acute paclitaxel administration (1 mg·kg⁻¹, i.p.). Data are expressed as the medians followed by their 25th and 75th percentiles of six animals per group. *P < 0.05 when compared with the vehicle group; two‐way ANOVA followed by Bonferroni's post hoc test.

α‐Spinasterol inhibits the activities of COX‐1 and COX‐2

Since the antinociceptive effects of α‐spinasterol were similar to known COX inhibitors (i.e. indomethacin, celecoxib and acetaminophen), we evaluated the possible action of α‐spinasterol on COX‐1 and COX‐2 enzyme activities. α‐Spinasterol inhibited COX‐1 and COX‐2 enzyme activities, with IC₅₀ values of 16.17 (15.12–17.30) μM and 7.76 (1.27–47.52) μM respectively (Figure 7).

Figure 7.

Effects of α‐spinasterol on COX‐1 and COX‐2 enzyme activities. COX‐1 activity (A) and COX‐2 activity (B) following the administration of different concentrations of α‐spinasterol (3–100 μM). Data are expressed as mean ± SEM (n = 4 measurements).

α‐Spinasterol did not cause adverse effects

α‐Spinasterol did not alter the cell viability in the cerebral cortex and spinal cord slices

Cerebral cortex and spinal cord sections were treated with different concentrations of α‐spinasterol (1–1000 nM) to assess its effects on cell viability. From exploratory data, it was observed that α‐spinasterol did not damage these tissues or alter cell viability, as evaluated by LDH and MTT assays, respectively, compared to the vehicle group (Table 1).

Table 1.

Effects of α‐spinasterol on cell viability in cerebral cortex and spinal cord slices

	Cerebral cortex slices		Spinal cord slices
Treatments	MTT (% of control)	LDH (% released)	MTT (% of control)	LDH (% released)
Vehicle	105.2 ± 8	19.9 ± 7	124.4 ± 28	28.3 ± 4
α‐Spinasterol 1 nM	90.1 ± 36	10.9 ± 2	111.3 ± 10	34.3 ± 4
α‐Spinasterol 10 nM	135.0 ± 27	18.9 ± 4	87.09 ± 36	30.7 ± 0.4
α‐Spinasterol 100 nM	119.9 ± 4	23.2 ± 2	136.5 ± 7	29.2 ± 5
α‐Spinasterol 1000 nM	136.9 ± 8	33.1 ± 11	106.5 ± 6	45.3 ± 5

Slices were pre‐incubated for 6 h in the presence of α‐Spinasterol (1–1000 nM). Thereafter, LDH release and MTT reduction were measured. These exploratory data represent mean ± SEM of four animals per group.

α‐Spinasterol did not cause gastric lesions

α‐Spinasterol treatment (10 mg·kg⁻¹, p.o.) did not induce significant gastric mucosal lesions in the animals, 4 h after its administration, compared to the vehicle (10 mL·kg⁻¹, p.o.). However, in comparison to the vehicle, the positive control indomethacin (100 mg·kg⁻¹, p.o.) induced gastric lesions in the stomach mucosa of the mice 4 h after its administration (Table 2).

Table 2.

Effects of α‐spinasterol and indomethacin on gastric damage score, urea and creatinine levels, or ALT and AST activity, 4 h after their administration

Treatments	Gastric damage (score)	Urea (mg·dL−1)	Creatinine (mg·dL−1)	ALT (U·L−1)	AST (U·L−1)
Vehicle (10 mL·kg−1, p.o.)	0 (0.0–2.0)	29.2 ± 1.7	1.19 ± 0.24	30.7 ± 1.4	17.9 ± 2.5
α‐Spinasterol (10 mg·kg−1, p.o.)	0 (0.0–1.5)	31.2 ± 2.6	0.7 ± 0.10	39.6 ± 7.7	23.6 ± 9.0
Indomethacin (100 mg·kg−1, p.o.)	4 (2.3–4.5)*	30.4 ± 2.8	1.30 ± 0.29	37.0 ± 3.8	20.7 ± 3.4

Data are expressed as the mean ± SEM of six animals per group, except for the gastric damage scores, which are expressed as medians followed by their 25th and 75th percentiles.

^* P < 0.05 when compared to vehicle group; one‐way ANOVA followed by Bonferroni's post hoc test.

α‐Spinasterol did not cause changes in the kidney or liver

In comparison to the vehicle, α‐spinasterol (10 mg·kg⁻¹, p.o.) or indomethacin (100 mg·kg⁻¹, p.o.) caused no significant changes in the levels of urea and creatinine or in activity of ALT and AST, which are biochemical markers of changes in the kidney and liver, respectively, 4 h after its administration (Table 2).

α‐Spinasterol did not alter the locomotor activity of animals

The treatment with α‐spinasterol (0.3 mg·kg⁻¹, p.o.), indomethacin (10 mg·kg⁻¹, p.o.) or AMG9810 (30 mg·kg⁻¹, p.o.) had no significant effect on the number of crossings or rearing in the open‐field test and the number of falls in the rotarod test when compared to the vehicle group (Table 3).

Table 3.

Effects of α‐spinasterol, indomethacin and AMG9810 on locomotor activity and body temperature, 1 h after their administration

Treatments	Crossings (number)	Rearing (number)	Falls (number)	Body temperature change (°C)
Vehicle (10 mL·kg−1, p.o.)	63.0 ± 8	30.3 ± 3	0.6 ± 0.5	0.08 ± 0.07
α‐Spinasterol (0.3 mg·kg−1, p.o.)	65.6 ± 4	32.2 ± 6	0.4 ± 0.2	−0.04 ± 0.04
Indomethacin (10 mg·kg−1, p.o.)	53.0 ± 3	30.4 ± 2	0.2 ± 0.2	0.18 ± 0.17
AMG9810 (30 mg·kg−1, p.o.)	69.0 ± 5	44.6 ± 3	0.2 ± 0.2	0.75 ± 0.08*

Data are expressed as mean ± SEM of six animals per group;

^* P < 0.05 when compared to the vehicle group; one‐way ANOVA followed by Bonferroni's post hoc test.

α‐Spinasterol did not alter the body temperature of the animals

Treatment with α‐spinasterol (0.3 mg·kg⁻¹, p.o.) or indomethacin (10 mg·kg⁻¹, p.o.) did not change the body temperature 1 h after its administration. The TRPV1 antagonist AMG9810 (30 mg·kg⁻¹, p.o.), however, significantly increased the rectal temperature compared to the vehicle group (Table 3).

Discussion

Pain is a major health problem, which generates socio‐economic losses and substantially reduces patients' quality of life (Kamerman et al., 2015). A common form of acute inflammatory pain is the postoperative pain that is experienced by most patients who undergo surgical procedures (Chou et al., 2016; Pogatzki‐Zahn et al., 2017). Another type of pain is neuropathic pain, which results from a lesion or abnormal functioning of the CNS and presents distinct pathological characteristics (Grace et al., 2014). Both postoperative and neuropathic pain cause incapacitating symptoms, but remain inadequately treated since most analgesics that are currently available are relatively ineffective and have adverse effects (Gan et al., 2014; Gilron et al., 2015; Taylor et al., 2016; Pogatzki‐Zahn et al., 2017). Moreover, despite the investment by the pharmaceutical industry, there has been little progress in developing safe and effective analgesics (Woolf, 2010).

In the current study, α‐spinasterol, which has already been characterized as a TRPV1 antagonist, had an antinociceptive effect on postoperative and neuropathic pain models, without impairing the locomotor activity of animals. Moreover, α‐spinasterol inhibited COX‐1 and COX‐2 enzyme activities, reduced the infiltration of inflammatory cells into the injured paw and did not alter cell viability. Importantly, α‐spinasterol did not cause the adverse effects that are commonly observed with COX inhibitors or induce severe hyperthermia, which is a known adverse effect of TRPV1 antagonists like AMG9810 (Trevisan et al., 2012; Migliore et al., 2016).

Acute postoperative pain is characterized by spontaneous nociception, hyperalgesia (increased pain in response to noxious stimuli) and allodynia (pain in response to innocuous stimuli) in the region of the incision. This occurs due sensitization of peripheral nociceptors mediated by the infiltration of inflammatory cells and release of inflammatory mediators (Kehlet et al., 2006; Pogatzki‐Zahn et al., 2007; Ren and Dubner, 2010; Pogatzki‐Zahn et al., 2017). In the current study, α‐spinasterol and indomethacin presented preventive (treatment before surgical incision) and curative (treatment after surgical incision) effects on the plantar incision‐induced mechanical allodynia. Our results corroborate those of Trevisan et al. (2012), who demonstrated that α‐spinasterol exerts an antinociceptive effect in an inflammatory pain model. Given the reduction in the number of polymorphonuclear cells in the paw tissue subjected to a surgical incision, this antinociceptive effect can be attributed to the ability α‐spinasterol to reduce the number of inflammatory cells infiltrating into the injured paw. This result is in agreement with the study conducted by Borges et al. (2014) where α‐spinasterol reduced the inflammatory cell infiltration induced by LPS. Moreover, it is likely that TRPV1 antagonism also contributes to the antinociceptive effect of α‐spinasterol, since previous studies demonstrated that the TRPV1 inhibition attenuates postoperative pain (Honore et al., 2009; Uchytilova et al., 2014).

The antinociceptive action of α‐spinasterol is also similar to that of classical analgesic drugs, such as nonselective COX inhibitors, since α‐spinasterol inhibited COX‐1 and COX‐2 enzyme activities. These results corroborate those observed in a study conducted by Jeong et al. (2010), where α‐spinasterol suppressed the LPS‐induced expression of COX‐2. Since COX‐1 plays an important role in pain processing and sensitization in the spinal cord after surgery, COX inhibitors may be effective in treating postoperative pain (Zhu et al., 2003). Moreover, COX‐1 and COX‐2 are up‐regulated in the spinal cord following surgical incision (Zhu et al., 2003; Kroin et al., 2004). Therefore, the inhibition of COX by α‐spinasterol could also contribute to its ability to attenuate postoperative pain, since NSAIDs alone, or in combination with other analgesics (multimodal analgesia), are one of the main recommended therapies for the clinical management of postoperative pain (Chou et al., 2016; Pogatzki‐Zahn et al., 2017). Furthermore, it was found that the combined therapy of α‐spinasterol and indomethacin increased the analgesic effect of either α‐spinasterol (19 ± 5%) or indomethacin (20 ± 5%) administered alone. This may have occurred as indomethacin inhibits the COX enzymes whereas α‐spinasterol antagonizes TRPV1, in addition to inhibiting COX‐1 and COX‐2.

Indomethacin and other COX inhibitors are effective at reducing incision‐induced mechanical allodynia (Whiteside et al., 2004; Oliveira et al., 2014; Pogatzki‐Zahn et al., 2017). However, although effective against mechanical allodynia, α‐spinasterol was not able to reduce incision‐induced spontaneous nociception while indomethacin was only effective when administered before the surgical procedure. In fact, results from previous studies about the efficacy of COX inhibitors are controversial (Gilron et al., 2000; Eisenach et al., 2010) as are the ability of TRPV1 antagonists (Honore et al., 2009; Okun et al., 2012) to reduce spontaneous pain in painful inflammatory conditions. Moreover, compared with other analgesics, spontaneous pain is more sensitive to morphine and buprenorphine in animal models of acute postoperative pain (Kabadi et al., 2015). Hyperalgesia and allodynia are more severe and longer lasting in patients, while spontaneous nociception is usually short lasting and moderate (Pogatzki‐Zahn et al., 2007; 2017). Thus, it is important to note that the efficacies of both α‐spinasterol and indomethacin at reducing surgical incision‐induced mechanical allodynia are similar.

Neuropathic pain, like postoperative pain, may also occur spontaneously, or be amplified, in turn causing hyperalgesia and allodynia (Costigan et al., 2009; Gilron et al., 2015). α‐Spinasterol was also effective in reversing mechanical allodynia in a neuropathic pain model induced by trauma. Although celecoxib, a COX‐2 inhibitor used as reference drug, and α‐spinasterol showed similar efficacy in reducing neuropathic pain, α‐spinasterol was much more potent than celecoxib. The higher potency of α‐spinasterol improves its therapeutic index since it minimizes its circulating concentrations, and thereby reduces the likelihood of adverse effects (Daley‐Yates, 2015). Thus, α‐spinasterol is a more favourable treatment. Moreover, the antinociceptive effect of α‐spinasterol in traumatic neuropathy can be attributed to its ability to antagonize TRPV1 and inhibit COXs. Although the topical application of a high‐concentration capsaicin patch (8%) is considered as second line of treatment for peripheral neuropathic pain (post‐traumatic painful neuropathies and painful polyneuropathies), TRPV1 antagonists have been shown to reduce the nociception in models of nerve injury‐induced neuropathy (Tributino et al., 2010; Lima et al., 2014; Finnerup et al., 2015).

Although generally ineffective against neuropathic pain, celecoxib and other COX inhibitors can reduce trauma‐induced neuropathy (Guindon and Beaulieu, 2006; Ma et al., 2012; Oliveira et al., 2014). COX‐2 and its end‐product, PGE₂, are up‐regulated in injured nerves cells and spinal dorsal horn after the development of neuropathy following nerve ligation (Lau et al., 2010; Ma et al., 2012). Moreover, they facilitate the production of other pain mediators in inflammatory cells of injured nerves and dorsal root ganglion (DRG) neurons, which contributes to the establishment of neuropathic pain (Ma et al., 2012).

In addition to trauma, neuropathic pain may originate from different aetiologies, such as chemotherapy‐induced neuropathy. Chemotherapy‐induced neuropathic pain is a severe dose‐ and therapy‐limiting adverse effect of antineoplastic pharmacotherapy (Sisignano et al., 2014). The use of the antineoplastic paclitaxel is associated with an acute pain syndrome, which develops during the first days after its administration, in addition to a peripheral sensory neuropathy that can begin weeks to months after initial treatment (Loprinzi et al., 2011; Pachman et al., 2011). This painful condition is characterized by paresthesia, sensory ataxia and mechanical and cold allodynia (Pachman et al., 2011; Sisignano et al., 2014). α‐Spinasterol and acetaminophen reversed mechanical allodynia in both the acute and neuropathic pain induced by paclitaxel and cold allodynia in the acute pain. Li et al. (2015) demonstrated that TRPV1 plays a role in paclitaxel‐induced acute pain and chronic painful neuropathy. Furthermore, TRPV1 antagonists may have therapeutic potential for treating paclitaxel‐induced neuropathy, since the paclitaxel may up‐regulate the expression of TRPV1 in small‐ and medium‐diameter DRG neurons (Hara et al., 2013; Sisignano et al., 2014).

Moreover, COX inhibitors also can relieve paclitaxel‐induced peripheral neuropathy (Ito et al., 2012) since inflammatory components of the spinal dorsal horn release pro‐algesic mediators, which increase the expression of COX‐2 (Souich et al., 2009; Sisignano et al., 2014). Inflammation usually occurs after the acute phase of paclitaxel treatment and might contribute to the neuropathic symptoms. Therefore, COX inhibition can be effective at the onset of the paclitaxel‐induced painful symptoms (Sisignano et al., 2014). Our results are particularly relevant, because α‐spinasterol is a compound that is able to inhibit TRPV1 as well as having additional anti‐inflammatory properties and, therefore, shows promise as a candidate for the monotherapy of paclitaxel‐induced pain (Sisignano et al., 2014).

α‐Spinasterol presents good oral absorption with high penetration into the brain and spinal cord of mice (Trevisan et al., 2012). Since α‐spinasterol is well distributed in the CNS, we assessed whether it could cause changes in cell viability in the cerebral cortex and spinal cord slices. As already reported by Jeong et al. (2010), α‐spinasterol neither altered cell viability in cerebral cortex nor spinal cord slices. Thus, the antinociceptive effects of α‐spinasterol observed here is associated with its ability to access the nervous system, where TRPV1 and COX‐1 and COX‐2 enzymes are expressed (Trevisan et al., 2012; Yagami et al., 2016). Moreover, the antioxidant (Coballase‐Urrutia et al., 2010) and anti‐inflammatory (Borges et al., 2014) properties of α‐spinasterol may indirectly contribute to its antinociceptive activity, since oxidants and inflammation could be the underlying mechanisms of postoperative and neuropathic pain (Oliveira et al., 2011; Ma et al., 2012; Sisignano et al., 2014; Saad et al., 2016).

The inhibitors of COX‐1 and COX‐2 enzymes that are used for pain treatment characteristically cause adverse effects, such as gastrointestinal and kidney alterations (Ingrasciotta et al., 2015; Migliore et al., 2016) and hepatoxicity (Unzueta and Vargas, 2013). Since α‐spinasterol presented antinociceptive effects and inhibited COX‐1 and COX‐2 activities, we also evaluated its possible adverse effects. Unlike indomethacin, a known inhibitor of COX that causes gastric damage, a high dose of α‐spinasterol did not induce gastric mucosal lesions in mice. Moreover, this high dose of α‐spinasterol also did not alter the levels of urea and creatinine or the activity of ALT and AST, which are biochemical markers of kidney or hepatic dysfunction respectively (Oliveira et al., 2014). In conclusion, α‐spinasterol did not elicit the adverse effects that are considered to be common with analgesic COX inhibitors. We also evaluated locomotor activity to rule out possible nonspecific muscle‐relaxant or sedative effects, which can cause false‐positive results in the nociceptive tests. According to Trevisan et al. (2012), α‐spinasterol does not cause motor deficits in animals.

Although COX inhibitors can cause gastrointestinal and renal changes, TRPV1 antagonists evoke hyperthermia (Trevisan et al., 2012). Thus, since α‐spinasterol is a TRPV1 antagonist, we also evaluated its effect on body temperature, but no change was found; this finding corroborates that of Trevisan et al. (2012). Since we characterized α‐spinasterol as a COX‐1 and COX‐2 inhibitor in the current study and Trevisan et al. (2012) characterized it as a TRPV1 antagonist, this compound can be considered a multi‐target agent. Therefore, due its multi‐target characteristics, α‐spinasterol could offset the hyperthermic effect induced by blocking TRPV1, by inhibiting COX‐2, which is an enzyme that is known to be involved in the regulation of body temperature (Yun et al., 2011). This counterbalancing effect on the temperature between targets such as TRPV1 and COX‐2 has also been attributed to another multi‐target agent (Lima et al., 2014). New approaches using drug combinations or multi‐target compounds to alleviate pain, especially chronic pain such as neuropathic pain, have been tested previously (Lima et al., 2014). Agents that modulate multiple targets simultaneously can be used to enhance the efficacy and safety of drugs; this trend has been mainly observed with COX inhibitors (Morphy and Rankovic, 2005).

Our results are particularly relevant for patients with chemotherapy‐induced neuropathy. Since these patients usually already receive a combination of different drugs, they are exposed to various adverse effects and cross reactions that can affect the efficacy of the antineoplastic drugs. Therefore, the administration of α‐spinasterol as a monotherapy would be critical for the management of chemotherapy‐induced neuropathy, since α‐spinasterol could reduce the number of adverse effects, compared to those caused by multiple therapies. In this sense, it has been suggested that lipid compounds, which exert a dual effect of blocking TRP ion channels and reducing glial activity and cytokine release, could be more useful for treating chemotherapy‐induced neuropathies, such as paclitaxel‐induced peripheral neuropathy (Ji et al., 2014; Sisignano et al., 2014).

In this study, we showed that α‐spinasterol is an efficacious and safe multi‐target compound with antinociceptive effects on postoperative and neuropathic pain. The inhibition of targets such as TRPV1, COX‐1 and COX‐2 could contribute to the efficacy of α‐spinasterol and reduce any adverse effects compared with drugs that act on only one of these targets. Our findings suggest that α‐spinasterol is a suitable drug prototype for treating pathological pain, such as postoperative and neuropathic pain, with no detectable adverse effects.

Author contributions

I.B. and S.M.O. conceived and designed the study. I.B., C.C., F.B.C. and S.M.O. carried out the acquisition, analysis and interpretation of data. I.B., C.C., F.B.C., M.R.C.S., M.S.O., G.T., J.F. and S.M.O. wrote the manuscript. S.M.O. supervised the study.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration 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.

Brusco, I. , Camponogara, C. , Carvalho, F. B. , Schetinger, M. R. C. , Oliveira, M. S. , Trevisan, G. , Ferreira, J. , and Oliveira, S. M. (2017) α‐Spinasterol: a COX inhibitor and a transient receptor potential vanilloid 1 antagonist presents an antinociceptive effect in clinically relevant models of pain in mice. British Journal of Pharmacology, 174: 4247–4262. doi: 10.1111/bph.13992.