Antinociceptive effects of sinomenine in a rat model of postoperative pain

Abstract

Background and Purpose

This study examined the antinociceptive effects of sinomenine in a rat model of postoperative pain.

Experimental Approach

Male and female rats were subjected to a surgical incision in the right hind paw, and the von Frey filament test was used to measure mechanical hypersensitivity after drug or vehicle treatment (p.o. or i.p.). Rats were treated daily with sinomenine before or after the surgery and the AUCs of the antinociceptive effects measured during a 4 h period were calculated to determine the ED₅₀ values of sinomenine. The anti‐hyperalgesic effects of different doses of a combination of sinomenine and acetaminophen (paracetamol) were assessed in another group of rats. Dose combinations were determined by using a fixed ratio dose‐addition analysis method.

Key Results

Sinomenine (5–80 mg·kg⁻¹) produced dose‐dependent antinociceptive effects in rats that had been subjected to surgery and this effect lasted for 4 h. The potency of sinomenine, given i.p. or p.o., did not differ between male and female rats. However, sinomenine was fourfold more potent when given i.p. than p.o. The GABA_A receptor antagonist bicuculline blocked the antinociceptive effects of sinomenine. The antinociceptive effect of a daily treatment with sinomenine remained stable throughout the course of postoperative pain. Pretreatment with sinomenine did not alter the mechanical hypersensitivity post‐surgery. The combination of sinomenine with acetaminophen produced an infra‐additive interaction.

Conclusions and Implications

Sinomenine demonstrated significant antinociceptive activity against postoperative pain and may be a useful novel pharmacotherapy for the management of postoperative pain.

Abbreviations

CL

confidence limits

PWT

paw withdrawal threshold

Tables of Links

TARGETS
GPCRs a [Link]	Ligand‐gated ion channels b [Link]
5‐HT1A receptor	GABAA receptor
Opioid receptors

LIGANDS
Acetaminophen (paracetamol)
Bicuculline

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (^a,bAlexander et al., 2015a, 2015b).

Introduction

Postoperative pain remains a major clinical problem after many surgical procedures (Apfelbaum et al., 2003). The mismanagement of postoperative pain could lead to a variety of negative consequences, such as chronic postsurgical pain and delayed rehabilitation (Argoff, 2014). Despite advances in the understanding of the pathophysiological mechanism of postoperative pain and the use of preventative strategies and analgesic agents, including opioids, non‐steroidal anti‐inflammatory drugs (NSAIDs), adjuvant drugs and topical anaesthesia, postoperative pain continues to be under‐treated, and most surgical patients still suffer from moderate to severe postoperative pain (Apfelbaum et al., 2003; Lovich‐Sapola et al., 2015). The analgesic agents widely used in clinical treatment usually have limited effectiveness and safety profiles. For example, NSAIDs are associated with serious gastrointestinal lesions or renal and liver failure. The opioids remain the most effective analgesics for pain management, but their use is controversial and restricted due to their perceived side effects and fears of addiction and tolerance (Argoff, 2014). Therefore, further studies are required to develop novel and effective alternative medicine for pain treatment and preventative strategies.

Sinomenine, (+)‐4‐hydroxy‐3,7‐dimethoxy‐17‐methylmorphin‐7‐en‐6‐one, is the major active ingredient of the traditional Chinese medicine Sinomenium acutum. There are an increasing number of preclinical and clinical studies reporting that sinomenine is effective against rheumatoid arthritis (Yamasaki, 1976; Xu et al., 2008) and glomerular diseases, which may be due to its ability to modulate the immune system (Zhao et al., 2012). However, there is also limited evidence suggesting that sinomenine may be able to alleviate pain (Gao et al., 2013). Sinomenine only exerts modest antinociceptive effects in animal models of acute nociception including the hot plate test and radiant tail flick test. In a mice model of carrageenan‐induced inflammatory pain, sinomenine reduces mechanical allodynia and heat hyperalgesia (Gao et al., 2013). We recently reported that sinomenine is effective against mechanical hyperalgesia in a rat model of chronic constriction injury‐induced neuropathic pain (Zhu et al., 2014). Importantly, repeated treatment with sinomenine does not appear to induce antinociceptive tolerance (Zhu et al., 2014), suggesting that it has therapeutic potential as a treatment for neuropathy‐related painful conditions.

This study was designed to examine the antinociceptive effects of sinomenine in a rat model of postoperative pain. Because clear antinociception was observed, we further examined the potential utility of combining sinomenine with the widely used non‐opioid analgesic acetaminophen (paracetamol). Combination therapy is a viable therapeutic strategy, which combines two or more medications with different mechanisms of action to achieve better therapeutic effectiveness and/or reduce adverse effects. It was expected that the combination of sinomenine and acetaminophen would produce a synergistic interaction compared with the individual drugs alone against mechanical hyperalgesia in the rat postoperative pain model.

Methods

Animals

Male and female adult Sprague–Dawley rats with initial weights of 250–300 g (Laboratory Animal Center, Nantong University, China) were habituated to the temperature, humidity and lighting (12 h light/dark cycle, lights on at 07:00 h) controlled housing facility and group housed for at least 3 days before the behavioural studies began. The animals had free access to regular rodent chow and water except during the test sessions. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee, Nantong University. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition, Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington DC). All testing was performed in accordance with the recommendations of the International Association for the Study of Pain (Zimmermann, 1983). All studies involving animals were in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath & Lilley, 2015).

Incisional surgery

Incisional surgery was performed as previously described (Brennan et al., 1996), with minor modifications. Briefly, rats were anaesthetized with isoflurane (2% isoflurane mixed with 100% oxygen at a flow rate of 5 L·min⁻¹), and the plantar surface of the right hind paw was prepared under sterile conditions. A 1 cm longitudinal incision was cut with a number 11 scalpel, through skin and fascia of the plantar aspect of the paw, starting 0.5 cm from the proximal end of the heel and extending towards the toes. The plantaris muscle was elevated and incised longitudinally. After bleeding was stopped through gentle pressure, the skin was sutured with two single stitches using 5–0 nylon. The animals were allowed to recover in their home cages.

The von Frey filament test

The mechanical hyperalgesia was measured by assessing paw withdrawal thresholds (PWTs) to mechanical stimuli using a series of von Frey filaments (Stoelting, Kiel, WI, USA). Ten von Frey filaments, with approximately equal logarithmic incremental bending forces (equivalent to 1, 1.4, 2, 4, 6, 8, 10, 15, 26, 60 g force respectively), were used. Rats were placed in custom‐made individual transparent Perspex cubicles with a wire mesh floor and allowed to acclimatize for at least 15 min before testing began. The filaments were applied to the plantar surface of each hind paw in a series of ascending forces. Each filament was tested three times per paw, and the mechanical threshold was defined as the minimal force that caused at least two withdrawals to be observed in three consecutive trials (Yalcin et al., 2009).

Experimental design

For the measurement of mechanical hyperalgesia, the PWT was measured daily for 7 days. Daily baseline measures prior to surgery were performed for 3 days to allow rats to habituate to the procedure and experimenters. For acute effects of sinomenine, the drug was administered i.p. or p.o. 1 day post‐surgery. After treatment, the PWT was measured every 30 min for 4 h. Studies with acute acetaminophen treatment were similar except that the measurement lasted for 6 h. For the repeated treatment study, the drug was administered to rats once daily for 7 days, and the PWT was measured 2 h after the administration of the drug [results from acute treatment tests indicated that 2 h after sinomenine administration the antinociceptive effect approached maximum (Figure 2)]. For determining the preventive effects of sinomenine, rats received sinomenine once daily for 7 days prior to surgery, and then the PWT of these rats was measured post‐surgery once daily for 6 days. For antagonist studies, 1 day after surgery, the GABA_A receptor antagonist bicuculline was administered 10 min prior to 40 mg·kg⁻¹ sinomenine and the PWT was measured thereafter. For drug combination studies, acetaminophen and sinomenine were administered as a mixture, and the PWT was measured for 6 h. A blind design was strictly followed for all the studies such that different experimenters performed drug treatments and behavioural measures. Additional studies that examined the antinociceptive effects of sinomenine alone or in combination with acetaminophen and the GABA_A receptor agonist muscimol are presented in the Supporting Information.

Figure 2.

Anti‐hyperalgesic effects of acute sinomenine treatment in male (left) and female (right) rats. Top panels: sinomenine was administered p.o.; bottom panels: sinomenine was administered i.p. Filled symbols indicated significant difference in sinomenine‐treated groups as compared with the vehicle‐treated group. n = 7–8 rats per group. See Figure 1 for other details.

Statistical analyses

All data are presented as mean ± SEM and were analysed using the graphpad prism 5.01 software (San Diego, CA, USA). For surgery‐induced mechanical hyperalgesia and for anti‐hyperalgesia studies, the PWT (g) was plotted as a function of time (h or days). Statistical differences among groups were analysed by a two‐way ANOVA with repeated measures using time as within group factor and treatment (doses or dose combinations) as between group factor, which was followed by Bonferroni post hoc analysis. For all analyses, the statistical significance level was set at P < 0.05. AUC was calculated using graphpad prism 5.01. The AUC data were further used to analyse data from acute sinomenine and acetaminophen treatment, which were fitted using nonlinear regression as described previously (Wang and Pang, 1993) to obtain the estimated ED₅₀ [±95% confidence limits (CL)] values. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015).

For the sinomenine–acetaminophen combination studies, the fixed ratio dose‐addition analysis method was used to examine their interactions, as described in previous studies (Li et al., 2014; Thorn et al., 2015). For this analysis, two drugs were combined in fixed ratios (1:3, 1:1 and 3:1) and administered as one dose of a combination of the two drugs per test. The actual doses of the individual drugs were determined based on the relative potency of the drugs and the fixed ratios used. The dose–effect curves of the drug combinations were constructed using the AUC of each dose combination as described previously. For drug combinations, the dose–response curves were determined and the individual AUC ED₅₀ values of the two drugs in the combination were calculated based upon the shared dose–response curves. Finally, an isobolographical plot was constructed to visually reveal the drug interactions as supra‐additive, additive or infra‐additive. The isobolographical plot was constructed by connecting the ED₅₀ values of sinomenine plotted on the abscissa scale with the ED₅₀ values of acetaminophen plotted on the ordinate scale to obtain the line of additivity. The 95% CLs of ED₅₀ for sinomenine and acetaminophen alone were also connected to obtain the global 95% confidence boundaries, indicating the limits of the additive line. If the effects of the two drugs are additive, the ED₅₀ values (±95% CL) for the drug combination should fall within the limits of the additive line. If the ED₅₀ values (±95% CL) fall below the limits of the line of additivity, the effects of the two drugs are considered to be supra‐additive or synergistic. If the ED₅₀ values (±95% CL) fall above the limits of the additive line, then the effects of the two drugs are considered to be infra‐additive.

Drugs

Sinomenine [(+)‐4‐hydroxy‐3,7‐dimethoxy‐17‐methylmorphin‐7‐en‐6‐one] was purchased from Aladdin Reagents (Shanghai, China). Bicuculline was purchased from Selleck Chemicals (Houston, TX, USA). Both drugs were dissolved in 0.9% physiological saline. Drugs were administered i.p. or p.o. in a volume of 1 mL·kg⁻¹ of body weight.

Results

Before the incisional surgery, there were no significant differences in baseline mechanical PWT between the control group and the model group (19.1 ± 2.0 vs. 18.5 ± 2.3 g, P > 0.05). One day after surgery, the PWT in the model group was markedly decreased, and then gradually recovered to the pre‐surgery baseline level 6 days after surgery (Figure 1). Two‐way ANOVA analysis showed significant main effects of surgery [F(1, 14) = 34.77, P < 0.0001], time [F(6, 84) = 16.48, P < 0.0001], and interaction between time and surgery [F(6, 84) = 14.28, P < 0.0001]. Post hoc analyses revealed that the PWT was markedly lower in the model group as compared with control group from 1 to 5 days after surgery (P < 0.05). However, the PWT in control group remained at the baseline level with repeated measurement throughout the experimental period (Figure 1).

Figure 1.

Duration of incisional surgery‐induced mechanical hyperalgesia. Control: control group that did not receive the incisional surgery. Model: model group that received the incisional surgery. Abscissae: time (days); ordinate: paw withdrawal threshold (g) as measured by von Frey filaments. Filled symbols indicated significant difference in model group as compared with the control group (right hind paw). n = 8 male rats per group.

Both acute p.o. and acute i.p. administration of sinomenine dose‐dependently increased the PWT in both male and in female rats after surgery (Figure 2). In male rats (left panels, Figure 2), both p.o. (top left panel, Figure 2) and i.p. (bottom left panel, Figure 2) administration of different doses of sinomenine gradually produced antinociceptive effects; the effect was maximum (ie reached a peak) at 2.5 h and subsequently decreased to the pretreatment level at 4 h post‐treatment. Two‐way ANOVA revealed significant main effects of time [p.o.: F(7, 196) = 22.70, P < 0.0001; i.p.: F(7, 196) = 32.44, P < 0.0001] and sinomenine treatment [p.o.: F(3, 28) = 20.57, P < 0.0001; i.p.: F(3, 28) = 35.81, P < 0.0001], and interaction between time and sinomenine treatment [p.o.: F(21, 196) = 3.18, P < 0.0001; i.p.: F(21, 196) = 5.41, P < 0.0001]. For p.o. administration, post hoc analyses indicated that the PWT was significantly increased at 2.5 h after 20 mg·kg⁻¹, between 1.5 and 3 h after 40 mg·kg⁻¹ and between 1 and 3 h after 80 mg·kg⁻¹ (P < 0.05). For i.p. administration, post hoc analyses indicated that the PWT was significantly increased at 2.5 h after 5 mg·kg⁻¹, between 1.5 and 3 h after 10 mg·kg^‐1, between 0.5 and 3.5 h after 20 mg·kg⁻¹, and between 0.5 and 3 h after 40 mg·kg⁻¹ (P < 0.05). In female rats (right panels, Figure 2), both p.o. (top right panel, Figure 2) and i.p. (bottom right panel, Figure 2) administration of different doses of sinomenine gradually produced the antinociceptive effects, the peak was reached at 2–2.5 h and subsequently decreased to pretreatment level at 4 h post‐treatment. Two‐way ANOVA analysis revealed significant main effects of time [p.o.: F(7, 189) = 21.92, P < 0.0001; i.p.: F(7, 224) = 24.38, P < 0.0001] and sinomenine treatment [p.o.: F(3, 27) = 36.45, P < 0.0001; i.p.: F(4, 32) = 43.07, P < 0.0001], and interaction between time and sinomenine treatment (p.o.: F(28, 224) = 4.07, P < 0.0001; i.p.: F(21, 189) = 3.20, P < 0.0001]. For p.o. administration, post hoc analyses indicated that the PWT was significantly increased at 2–2.5 h after 20 mg·kg⁻¹, between 1.5 and 3 h after 40 mg·kg⁻¹, and between 1 and 3.5 h after 80 mg·kg⁻¹ (P < 0.05). For i.p. administration, post hoc analyses indicated that the PWT was significantly increased between 1.5 and 3 h after 10 and 20 mg·kg⁻¹, and between 0.5 and 3 h after 40 mg·kg⁻¹ (P < 0.05). In male rats, intrathecal administration of 0.4 mg·kg⁻¹ sinomenine significantly increased PWT while intraplantar administration of 20 mg·kg⁻¹ sinomenine failed to alter the pain hypersensitivity (Supporting Information Fig. S2).

The AUC dose–effect curves are shown in Figure 3. Nonlinear regression analyses revealed that the dose–effect curves of p.o. sinomenine administration in male and female rats were not significantly different from each other and can be fitted with one curve [F(2, 43) = 0.42, P > 0.05]. The ED₅₀ values (95% CL) of sinomenine in male and female rats were 37.95 (30.63, 47.01) mg·kg⁻¹ and 43.01 (35.54, 52.06) mg·kg⁻¹ respectively for reducing mechanical hyperalgesia when the drug was administered p.o., and, therefore, were not significantly different. Similarly, the dose–effect curves of i.p. sinomenine administration in male and female rats were also not significantly different from each other and can be fitted with one curve [F(2, 60) = 2.95, P > 0.05]. In this case, the ED₅₀ values (95% CL) of sinomenine in male and female rats were 9.66 (7.94, 11.76) mg·kg⁻¹ and 13.48 (10.90, 16.68) mg·kg⁻¹ respectively. Thus, the potency of sinomenine for reducing surgical hyperalgesia was not different between male and female rats when the drug was administered either p.o. or i.p. In addition, sinomenine was about fourfold more potent when the drug was given i.p. compared with when given p.o. in both male and female rats. In all subsequent studies, only male rats were used and drugs were administered i.p. for convenience.

Figure 3.

AUC dose–effect curves of sinomenine in rats. Abscissae: sinomenine dose (mg·kg⁻¹); ordinate scale: PWT AUC between 0–4 h post sinomenine treatment. M, male; F, female. Data above ’V’ represents data from vehicle‐treated rats.

In an effort to examine the receptor mechanism mediating the antinociceptive action of sinomenine in postoperative pain, the animals were pretreated with a GABA_A receptor antagonist, bicuculline, before the administration of 40 mg·kg⁻¹ sinomenine (Figure 4). Although bicuculline at a dose of 2 mg·kg⁻¹ alone did not alter the pain hypersensitivity, it nearly completely blocked the antinociceptive effects of sinomenine. Two‐way ANOVA revealed significant main effects of time [F(7, 91) = 8.98, P < 0.0001], bicuculline treatment [F(1, 13) = 42.14, P < 0.0001], and an interaction between time and bicuculline treatment [F(7, 9) = 7.09, P < 0.0001). Post hoc analyses indicated that the PWT was significantly lower at 0.5–2.5 h post‐sinomenine administration between sinomenine treatment and combination treatment conditions, suggesting significant blockade. In addition, the GABA_A receptor agonist muscimol significantly increased PWT when administered i.t. and potentiated the effect of sinomenine (Supporting Information Fig. S3).

Figure 4.

Blockade of sinomenine‐induced anti‐hyperalgesic effects by the GABA_A receptor antagonist bicuculline. Filled symbols indicated significant difference in bicuculline‐treated and sinomenine‐treated group as compared with the sinomenine alone group. n = 7–8 male rats per group. See Figure 1 for other details.

Daily treatment with sinomenine improved incision‐induced mechanical hyperalgesia in rats in a dose‐dependent manner (Figure 5). Two‐way ANOVA revealed significant main effects of time and sinomenine treatment [F(6, 168) = 45.48, P < 0.0001; F(3, 28) = 9.00, P < 0.001, respectively], although no significant interaction was found [F(18, 168) = 0.78, P > 0.05). Post hoc analyses indicated that daily 40 mg·kg⁻¹ sinomenine administration significantly increased the PWT from 1 to 3 days post‐surgery (P < 0.05). In an effort to examine whether repeated pretreatment with sinomenine has a preventive effect against postoperative pain, we treated rats with daily sinomenine for 7 days prior to incisional surgery. Such a treatment did not alter the PWT and produced no preventive effect against postoperative pain (Figure 6). Two‐way ANOVA did not reveal any statistically significant difference among rats with or without sinomenine treatment.

Figure 5.

Anti‐hyperalgesic effects of repeated sinomenine treatment after the incisional surgery. Filled symbols indicated significant difference in sinomenine‐treated group as compared with the vehicle‐treated group. n = 8 male rats per group. See Figure 1 for other details.

Figure 6.

Effects of pretreatment with sinomenine before surgery on the duration of postoperative mechanical hypersensitivity. n = 7–8 male rats per group. See Figure 1 for other details.

In an effort to examine whether sinomenine could be used as an effective combination therapy with the widely used analgesic acetaminophen, the antinociceptive action of acetaminophen alone was first examined in this model. Acetaminophen dose‐dependently increased the PWT with the effect reaching a peak at 3–3.5 h and dissipating 6 h after drug administration (top left panel, Figure 7). Nonlinear regression analysis with the AUC dose–effect curve of acetaminophen led to an estimated ED₅₀ (±95% CL) value being 57.09 (49.26. 66.17) mg·kg⁻¹. Thus, the potency ratio between acetaminophen and sinomenine in male rats was 5.90 when both drugs were administered i.p. In a subsequent study, different groups of rats were tested with the combination of sinomenine and acetaminophen for which the drugs were administered as a mixture at different fixed ratios. For example, when a 1:1 ratio was studied (bottom left panel, Figure 7), each of the four group of rats received the combination of a proportion of ED₅₀ values of sinomenine together with that of acetaminophen (i.e. 1/4, 1/2, 1, 2 ED₅₀s), and the dose ratio of the two drugs in each combination test remained constant (i.e. 5.90). In each case of the three studied fixed ratios, the drug combination dose‐dependently increased the PWT, which reached peak at 3–3.5 h and dissipated 6 h after drug administration (Figure 7). Two‐way ANOVA results were as follows: 1:3 ratio, significant main effects of time [F(10, 320) = 12.3, P < 0.0001), drug treatment [F(4, 32) = 11.4, P < 0.0001] and time × treatment interaction [F(40, 320) = 2.2, P < 0.001]; 1:1 ratio, significant main effects of time [F(10, 350) = 30.2, P < 0.0001], drug treatment [F(4, 35) = 21.4, P < 0.0001) and time × treatment interaction [F(40, 350) = 4.7, P < 0.0001); 3:1 ratio, significant main effects of time [F(10, 330) = 24.3, P < 0.0001), drug treatment [F(4, 33) = 14.9, P < 0.0001) and time × treatment interaction [F(40, 330) = 2.4, P < 0.0001]. Post hoc analyses indicated that the drug combinations significantly increased the PWT at different times (Figure 7).

Figure 7.

Anti‐hyperalgesic effects of acetaminophen alone (top left) or in combination with sinomenine. The individual doses of sinomenine and acetaminophen were determined by the respective ED₅₀ values and given at different fixed ratios. Filled symbols indicated significant difference in drug‐treated group as compared with the vehicle‐treated group. n = 7–8 male rats per group. See Figure 1 for other details.

The combined drug AUC dose–effect curves are shown in Figure 8 (left panel). At the fixed ratio of 1:1, the ED₅₀ value (95% CL) of sinomenine was 8.36 (6.64, 10.51) and that of acetaminophen was 49.37 (39.23, 62.13). At the fixed ratio of 3:1, the ED₅₀ value (95% CL) of sinomenine was 12.71 (9.78, 16.50) and that of acetaminophen was 25.10 (19.31, 32.62). Isobolographical plot (right panel, Figure 8) showed that the ED₅₀ values of the drug combinations fell above the line of additivity at both fixed ratios, suggesting a significant infra‐additive interaction between sinomenine and acetaminophen. At the fixed ratio of 1:3, the drug combination produced an antinociceptive effect that was lower than 50% of the maximal effect; thus, the ED₅₀ value could not be accurately estimated.

Figure 8.

Left: combined AUC dose–effect curves of sinomenine and acetaminophen alone or in combination; Right: Isobolographical plot of the ED₅₀ values (95% CL) of sinomenine (abscissae), acetaminophen (ordinates) and the combinations. Dash lines indicate the range of 95% CL.

Discussion

The primary findings of the present study are that sinomenine demonstrated significant antinociceptive activity in a rat model of postoperative pain in both male and female rats. The antinociceptive effect showed no sex difference but there was a significant pharmacokinetic difference when sinomenine was administered through the different routes of administration. In addition, the results showed that the antinociceptive effect of sinomenine was primarily mediated through GABA_A receptors, persisted after repeated treatments and did not have a prophylactic effect in this model. Because acetaminophen is often used to control mild to moderate pain, including postsurgical pain, we also examined the interaction between sinomenine and acetaminophen. The results clearly show that sinomenine and acetaminophen produced an infra‐additive interaction, suggesting that the two drugs are unlikely to produce a favourable therapeutic interaction if used for pain control. Taken together, the results of the present study substantially extend those in the existing literature and potentially expand the clinical utility of sinomenine for the control of postoperative pain.

Acute postoperative pain remains a clinical challenge. Patients undergoing outpatient ambulatory surgery have clinically significant postoperative pain, even when optimal pharmacotherapy including p.o. opioids and non‐opioid adjuncts are used (Apfelbaum et al., 2003). Thus, the need to develop simple, safe and effective therapies remains high. Sinomenine is a principal ingredient of traditional Chinese medicine, Sinomenium acutum, and has been used for the treatment of rheumatoid arthritis for decades. Meta analyses of clinical data have confirmed the efficacy of sinomenine for the clinical control of rheumatoid arthritis, which show that it is more efficacious and safer than NSAIDs (Xu et al., 2008). Although pain relief is often not the main endpoints of the clinical studies, the improvement of arthritis symptoms is certainly accompanied by the amelioration of joint pain. Surprisingly, the analgesic potential of sinomenine has only recently been investigated. Sinomenine does not seem to be efficacious for dampening acute nociception in assays such as the hot plate test and radiant tail flick test (Gao et al., 2013). However, in two preparations of neuropathic pain, sinomenine exerts significant anti‐hyperalgesic activity (Gao et al., 2013; Zhu et al., 2014), suggesting that sinomenine may be particularly effective against chronic pathological pain. Because postoperative pain has a distinct pathophysiological mechanism that includes both inflammatory and neurogenic components (Apfelbaum et al., 2003; Whiteside et al., 2004), here, we hypothesized that sinomenine could alleviate postoperative pain. Indeed, we found that sinomenine exerted significant effects against mechanical hyperalgesia. These effects are selective because the doses used here do not produce sedation or impair spontaneous activity (Zhu et al., 2014). In a previous study, we found that the antinociceptive effect of sinomenine in a rat model of chronic constriction injury‐induced neuropathic pain is primarily mediated through GABA_A receptors, but not through opioid receptors or 5‐HT_1A receptors (Zhu et al., 2014). Here, we confirmed, using a selective GABA_A receptor antagonist bicuculline, that the antinociceptive action of sinomenine against postoperative pain is also predominantly mediated through GABA_A receptors. In agreement with this, we found that the GABA_A receptor agonist muscimol when given i.t. produced a significant antinociceptive effect in this incisional pain model and potentiated the effect of sinomenine. Although it remains to be seen whether sinomenine produced the antinociceptive effect by acting on the GABA_A receptor as an agonist, which can be confirmed by a receptor binding assay and in vitro GABA_a receptor functional assay, the finding that a GABA_A receptor antagonist completely blocked while a GABA_A receptor agonist potentiated the antinociceptive effect of sinomenine does strongly suggest that GABA_A receptors play a critical role in mediating the antinociceptive activity of sinomenine. Interestingly, although sinomenine was effective when it was administered through the p.o. and i.p. routes, intraplantar drug administration failed to produce antinociception while i.t. administration produced a significant effect. These findings suggest that sinomenine‐induced antinociception was a centrally‐, not peripherally‐mediated effect, and the effect probably occurred at the spinal level.

Two potential caveats deserve some discussion. Firstly, although the von Frey filament is traditionally used to assess the pain hypersensitivity status in animal models of persistent pain including incisional pain, relying exclusively on this measure may not fully capture the spectrum of incisional pain. For example, it is suggested to also evaluate the spontaneous pain after incisional surgery and incorporate presumptive measures of spontaneous pain such as exploratory activity and vocalization (Scholz and Yaksh, 2010). Combining both reflexive response‐based measures and spontaneous pain should be expected to improve the predictive validity of novel analgesics. Secondly, although effective, sinomenine could not completely reverse incisional pain as seen in this study. Opioids are very effective against postoperative pain and are widely used in the clinic to control this type of pain. In this regard, although sinomenine may not be as efficacious as opioids if used as a monotherapy to treat postoperative pain, it may still be useful as an adjuvant therapy to reduce opioid use and thus decrease opioid‐related adverse effects.

Postoperative pain has a well‐defined course, and in rats, the pain hypersensitivity lasts around 1 week (Whiteside et al., 2004; Zhu et al., 2014). We found that daily treatment with sinomenine could maintain its antinociceptive activity, and no indication of tolerance was found. This is consistent with our previous study that found that daily treatment with sinomenine for 2 weeks did not produce antinociceptive tolerance against chronic neuropathic pain (Zhu et al., 2014). This is important as in order for the drug to be useful to treat chronic pain (repeated dosing is often necessary); the analgesic activity has to remain relatively stable even during prolong drug use. Appropriate perioperative medication therapy could alleviate postoperative pain and facilitate recovery (Pyati and Gan, 2007). Prophylactic drug use that can reduce the magnitude of postoperative pain could be useful. In this regard, sinomenine does not seem to be effective, as daily treatment with sinomenine for a week pre‐incisional surgery failed to alleviate postoperative pain hypersensitivity.

Sex difference in pain is a well‐recognized phenomenon. Males and females not only perceive pain differently, they also respond to analgesics differently under certain conditions (Palmeira et al., 2011; Bartley and Fillingim, 2013). In order to better understand the antinociceptive effects of sinomenine, we used both male and female rats in this study. No significant difference was observed, which suggests that sinomenine can be equally effective against postoperative pain in male and female subjects, a favourable pharmacological property. As expected, sinomenine is much less potent when it is administered p.o. as compared with i.p., presumably due to significant first pass elimination. This could provide useful information when designing the dosing regimen for human use in pain management.

Acetaminophen is often used alone or as an adjuvant for postoperative pain to reduce opioid consumption and related side effects (Pyati and Gan, 2007; Moore et al., 2015). Combination therapy is a widely used strategy for treating various diseases including pain (Orru et al., 2014; Thorn et al., 2015). The overall aim of combination therapy is to increase the analgesic effectiveness of analgesics such as opioids and/or reduce unwanted effects as smaller doses of individual drugs may be needed. Therefore, we examined the possibility of combining sinomenine and acetaminophen for postoperative pain. Although acetaminophen alone is effective in this rat model of postoperative pain, consistent with clinical observations (Moore et al., 2015) and rat studies (Mickley et al., 2006), the combination of acetaminophen with sinomenine produced significant infra‐additive interactions. This finding is somewhat surprising, and the mechanism is unclear. In order to rule out the possibility that acetaminophen and sinomenine act on the same target and the observed infra‐additive interaction was in effect due to a pharmacological antagonism, we examined the antinociceptive effects of sinomenine and acetaminophen in a rat model of neuropathic pain (Supporting Information Fig. S1). Although sinomenine was effective, in accord with our previous report (Zhu et al., 2014), acetaminophen was ineffective. Importantly, the combination of sinomenine with acetaminophen produced an antinociceptive effect that was not different from that produced by sinomenine alone, suggesting acetaminophen did not antagonize sinomenine‐induced antinociception and the observed infra‐additive interaction was not a pharmacological antagonism that occurred at the same receptor or target. The antinociceptive action of acetaminophen is predominantly mediated through TRPA1 receptors (Andersson et al., 2011). It is speculated that the downstream signalling pathways of both drugs cross‐talk and produce counteractive interactions in the modulation of pain processing. Mechanistic studies are needed to reveal this functional antagonism for antinociception.

In summary, this study demonstrates that sinomenine is an effective analgesic against postoperative pain. The effect is mediated through GABA_A receptors and does not show sex difference. Also, daily treatment with sinomenine did not produce tolerance with the antinociceptive activity persisting the course of postoperative pain until natural healing. These properties seem ideal as a useful analgesic for the control of postoperative pain, a clinical condition that is often inadequately managed. The effort of characterizing combination therapy potential with sinomenine and acetaminophen yielded unfavourable results. Combining the two drugs clearly demonstrated infra‐additive antinociception, which suggests that the combined use of the two drugs at the same time is not recommended. Nevertheless, given that sinomenine has already been used clinically for decades and has a favourable safety profile, repurposing sinomenine to treat various pain conditions including postoperative pain seems reasonable and no significant foreseeable barriers are expected.

Author contributions

W.Z., Q.Z. and J.L. designed the research study; Q.Z., Y.S., L.M., C.L. and B.J. performed the study; J.L. prepared the manuscript; all authors approved the final version of the manuscript.

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 organizations engaged with supporting research.

Supporting information

Figure S1. Anti‐hyperalgesic effects of vehicle, 180 mg/kg acetaminophen, 40 mg/kg sinomenine or a combination of 180 mg/kg acetaminophen and 40 mg/kg sinomenine in different groups of adult male rats receiving CCI surgery (n = 7 per group). Repeated measures two‐way ANOVA revealed significant main effects of time (F [9, 54] =13.85, P < 0.0001), treatment (F [3, 18] =79.14, P < 0.0001) and time × treatment interaction (F [27, 162] =4.94, P < 0.0001). Post hoc analyses found that 40 mg/kg sinomenine and combination treatment groups produced significantly increased PWT between 1.5 and 6 hours post‐treatment.

Figure S2. Antinociceptive effects of intrathecal administration of 0.4 mg/kg sinomenine or intraplantar administration of 20 mg/kg sinomenine in rats receiving incisional surgery. Repeated measures two‐way ANOVA revealed significant main effects of time (F [7, 49] =5.70, P < 0.0001), treatment (F [1, 7] = 20.86, P < 0.01) and time × treatment interaction (F [7, 49] =3.92, P < 0.01). Post hoc analyses found that intrathecal administration of 0.4 mg/kg sinomenine produced significant antihyperalgesic effects between 2 and 4 hours post‐treatment. In contrast, intraplantar administration of 20 mg/kg sinomenine failed to significantly alter the paw withdrawal threshold.

Figure S3. Antinociceptive effects of intrathecal administration of 0.4 mg/kg sinomenine, 0.05 μg muscimol, or the combination in male rats receiving incisional surgery (n = 8 per group). Tests were conducted 1 day after the incisional surgery. See above for the details of intrathecal catheterization. The GABAa receptor agonist muscimol has been shown to produce antinociceptive effects when the drug was given intrathecally (Hama and Sagen, 2012). Repeated measures two‐way ANOVA revealed significant main effects of time (F [7, 49] =46.06, P < 0.0001), treatment (F [3, 21] = 96.63, P < 0.0001) and time × treatment interaction (F [21, 147] =5.73, P < 0.0001). Post hoc analyses found that intrathecal administration of the combined 0.4 mg/kg sinomenine and 0.05 μg muscimol produced significant antihyperalgesic effects than each drug alone. Statistical significance were seen between 2 and 4 hours post‐treatment when the combination group and muscimol group was compared. Note: the 0.4 mg/kg sinomenine data were replotted from Supporting Information Fig. S2.

Supporting info item

Zhu, Q. , Sun, Y. , Mao, L. , Liu, C. , Jiang, B. , Zhang, W. , and Li, J.‐X. (2016) Antinociceptive effects of sinomenine in a rat model of postoperative pain. British Journal of Pharmacology, 173: 1693–1702. doi: 10.1111/bph.13470.