Chagas, Pietro M.a; Bortolatto, Cristiani F.a; Wilhelm, Ethel A.b; Roehrs, Juliano A.a; Nogueira, Cristina W.a
Unrelieved acute or chronic pain is often related to negative consequences in various aspects of patients’ health-related quality of life (Miller and Cano, 2009; Sinatra, 2010). The analgesic drugs currently available for pain relief, albeit efficacious, still raise several concerns in terms of their safety and side-effects (Steinmeyer, 2000; Jage, 2005). Hence, there is a significant need for exploring more promising and well-tolerated analgesic drugs.
Convincing evidence now indicates that the endogenous antioxidant system plays an important role in the regulation of pain pathways (Rossato et al., 2010). Moreover, pathologies related to pain and inflammation processes, such as fibromyalgia and rheumatoid arthritis, are also believed to involve free radicals and oxidative stress generation (Arranz et al., 2010; Wruck et al., 2011). Some natural and synthetic antioxidants are reported to have antinociceptive activities, including organoselenium compounds, which show anti-inflammatory, antinociceptive and antiallodynic effects in different animal models of nociception (Shin et al., 2009; Valério et al., 2009; Wilhelm et al., 2009).
In this context, antioxidants diaryl diselenides and their derivatives deserve special attention. Diphenyl diselenide (PhSe)2 exerts an antinociceptive action when assessed in models of licking behaviour induced by an intraperitoneal injection of glutamate, formalin or capsaicin, and visceral pain induced by acetic acid, and these effects are not influenced by opioidergic mechanisms, but rather seem to be related to serotonergic, nitrergic and glutamatergic pathways (Nogueira et al., 2003; Zasso et al., 2005; Savegnago et al., 2007). There is evidence supporting the idea that p-methoxy-diphenyl diselenide (MeOPhSe)2 and m-trifluoromethyl-diphenyl diselenide (F3CPhSe)2, disubstituted diaryl diselenides, produce antinociception similar to that of (PhSe)2, and these effects involve GABAergic (Pinto et al., 2008) and opioidergic mechanisms (Brüning et al., 2010), respectively.
Considering that slight changes in molecular structures could partially or completely modify the effect of a drug, novel molecules based on the structure of diaryl diselenides have been synthesized. The compound bis(phenylimidazoselenazolyl) diselenide (BPIS) is a novel diaryl diselenide derivative and it has been shown to be a promising antioxidant in vitro (P.M. Chagas, E.A. Wilhelm, J.A. Roehrs, C.W. Nogueira, unpublished data) compared with other diselenides. Therefore, on the basis of the antinociceptive property already described for antioxidant diaryl diselenides, the aims of the present study were to examine (a) the antinociceptive action of BPIS in different models of acute nociception; (b) the possible involvement of the opioid system in the antinociceptive action of BPIS; (c) possible nonspecific disturbances in the locomotor activity of mice treated with BPIS; and (d) the potential acute toxicity caused by BPIS in mice.
The experiments were conducted using male adult Swiss mice (25–35 g) from our own breeding colony. The animals were kept in a separate animal room, on a 12-h light/dark cycle with lights on at 07:00 h, at room temperature (22±1°C) with free access to water and food. All manipulations were carried out between 08:00 and 16:00 h. All experiments were conducted on separate groups of animals and each animal was used only once and only in a single test to avoid interference between tests. Experiments were conducted according to the guidelines of the Committee on Care and Use of Experimental Animal Resources, the Federal University of Santa Maria, Brazil. All efforts were made to minimize suffering and to reduce the number of animals used in the experiments. Animals were divided randomly into groups of seven to nine animals each.
Thermal models of nociception
The tail-immersion test was carried out as described by Janssen et al. (1963). The test was performed by immersing the lower 3.5 cm of the tail into a cup freshly filled with water from a large constant-temperature (55°C) bath until the typical tail withdrawal response was observed. A 7 s cut-off was imposed. Changes in tail-immersion latency, Δt (s), were calculated for each animal according to the formula [Δt (s)=postdrug latency−predrug latency] (Pinardi et al., 2003).
To assess the time course of the antinociceptive effect of BPIS, mice were pretreated with BPIS (50 mg/kg, orally) or vehicle (canola oil, 10 ml/kg, orally) at 15, 30 min, 1, 2, 4 and 8 h before the test; the control group received canola oil 30 min before the test. To examine the dose–response effect of BPIS, the compound was administered (10–100 mg/kg, orally) 30 min before the test.
Morphine (2.5 mg/kg, subcutaneously), administered 30 min before the test, was used as a positive control (Brüning et al., 2010).
The hot-plate test was carried out according to the method described previously (Woolfre and MacDonald, 1944). In this experiment, the hot-plate apparatus was maintained at 55±0.1°C. Animals were placed in an acrylic cylinder (20 cm in diameter) on the heated surface, and the time (s) between placement and licking of their hind paws or jumping was recorded as the response latency. A 60 s cut-off was used to prevent tissue damage. Twenty-four hours before the experiment, all mice were habituated to the experimental procedure to minimize novelty-induced antinociception (Siegfried et al., 1987). Animals presenting training latencies higher than 20 s were excluded. On the day of the experiment, animals were treated with BPIS (10–100 mg/kg, orally) or vehicle and subjected to the hot-plate test 30 min thereafter. Morphine (2.5 mg/kg, subcutaneously), administered 30 min before the test, was used as a positive control (Khazaeli et al., 2010).
Chemical models of nociception
Acetic acid-induced abdominal writhing
Abdominal constrictions were induced by acetic acid (1.6%, intraperitoneally) according to the procedure described previously (Corrêa et al., 1996). After the injection of acetic acid, mice were individually placed in separate boxes and the abdominal constrictions were counted cumulatively over a period of 20 min. Mice were pretreated with BPIS (10–50 mg/kg, orally) 30 min before the injection of acetic acid. Control animals received a similar volume of vehicle, and as a positive control, morphine (2.5 mg/kg, subcutaneously) was administered 30 min before the test (Savegnago et al., 2007).
The formalin test was carried out as described previously (Hunskaar and Hole, 1987; Okuda et al., 2001). Animals received an intraperitoneal administration of formalin (2.5%, v/v; 20 μl/paw) into the ventral surface of the right hind paw. After formalin injection, mice were returned to the experimental cage and the time spent licking the injected paw was recorded during the periods of 0–5 min (early neurogenic phase) and 15–30 min (late inflammatory phase). BPIS (10–50 mg/kg, orally) or vehicle were administered 30 min before the injection of formalin into the ventral right (ipsilateral) hind paw. Morphine (2.5 mg/kg, subcutaneously) (Khazaeli et al., 2010) or diclofenac sodium (10, 20 and 40 mg/kg, intraperitoneally) (Cristiano et al., 2008), administered 30 min before the test, were used as a positive control.
The procedure used was similar to that described previously (Meotti et al., 2010). Mice were treated with BPIS (10–50 mg/kg, orally) or vehicle 30 min before an intraperitoneal injection of glutamate (20 μmol, 20 μl/paw) on the ventral surface of the right hind paw. Mice were observed individually for 15 min following an injection of glutamate and the amount of time spent licking the injected paw was recorded using a chronometer. Morphine (2.5 mg/kg, subcutaneously), administered 30 min before the test, was used as a positive control (Freitas et al., 2009).
The procedure used was similar to that described previously (Santos et al., 1999). Capsaicin (1.6 μg, 20 μl/paw) was injected into the ventral surface of the right hindpaw. Animals were observed individually for 5 min following capsaicin injection. The amount of time spent licking the injected paw was recorded using a chronometer. Animals were treated with BPIS (1–50 mg/kg, orally) or vehicle 30 min before capsaicin injection.
Role of the opioid system in the antinociceptive effect of bis(phenylimidazoselenazolyl) diselenide in the tail-immersion test
To determine the role of the opioid system in the antinociceptive effect of BPIS, the tail-immersion test was chosen. Mice were pretreated with naloxone, a nonselective opioid antagonist (1 or 10 mg/kg, subcutaneously), or saline (vehicle) (Savegnago et al., 2007) 15 min before BPIS (50 mg/kg, orally), morphine (2.5 mg/kg, subcutaneously, as a positive control) or their respective vehicles. The tail-immersion test was carried out 30 min later.
Spontaneous exploratory behaviour was assessed in the open-field test to rule out any motor disturbance related to the administration of BPIS. The open field was made of plywood and surrounded by walls 30 cm in height. The floor of the open field, 45 cm in length and 45 cm in width, was divided by masking tape markers into nine squares (three rows of three). Animals were evaluated 30 min after a single oral dose of vehicle or BPIS (10–50 mg/kg). Each animal was placed individually at the centre of the apparatus and observed for 6 min to record the number of segments crossed with the fore-paws and the number of rears on the hind limbs (Walsh and Cummins, 1976).
To examine the potential acute toxicity caused by BPIS, mice received a single oral dose of BPIS (10–50 mg/kg) or vehicle. After drug administration, animals were observed up to 72 h to determine the lethal potential of BPIS. The animals were separated four to five per cage and all mice in the same cage received the same treatment (n=8–9). The individual gain in body weight was recorded and calculated according to the formula: [baseline body weight (obtained before the beginning of treatment)−body weight at the end of the experiment]. Water and food consumption was measured daily in mice exposed to BPIS. The average weight of water and food consumed was calculated according to the formula: [water and food intake (g)/number of animal per cage].
After 72 h, mice were anaesthetized and blood was collected by heart puncture in tubes containing heparin. Plasma was obtained by centrifugation at 2000g for 10 min (haemolysed plasma was discarded) and used for biochemical assays, which were performed using commercial test kits. Plasma aspartate aminotransferase and alanine aminotransferase activities, used as the biochemical markers for early acute hepatic damage, were determined using the colorimetric method of Reitman and Frankel (1957). Renal function was analysed by determining plasma urea (Makay and Mackay, 1927) and creatinine levels (Jaffe, 1886).
BPIS (Fig. 1) was prepared and characterized according to Roehrs et al. (2012). Analysis of the 1H NMR and 13C NMR spectra showed analytical and spectroscopic data in full agreement with its assigned structure (Roehrs et al., 2012). The chemical purity of BPIS (99.9%) was determined by GC/MS. Capsaicin and naloxone hydrochloride were purchased from the Sigma Chemical Co. (St Louis, Missouri, USA). All other chemicals were of analytical grade and obtained from standard commercial suppliers. All drugs were dissolved in saline, except BPIS, which was dissolved in canola oil. The mice received drugs by the oral (administered by an intragastric gavage), subcutaneous and intraperitoneal routes at a constant volume of 10 ml/kg body weight. Appropriate vehicle-treated groups were also assessed simultaneously.
The results are presented as mean±SEM. Differences between groups were analysed by means of one-way (tail-immersion, hot-plate, acetic acid-induced abdominal writhing, formalin, glutamate, capsaicin, and open-field tests, and parameters of acute toxicity) or two-way (investigation of the possible involvement of the opioid system in the antinociceptive effect of BPIS) analysis of variance, followed by the Newman–Keuls test when appropriate. The criterion for statistical significance was P value less than 0.05, which was considered as statistically significant. Effective dose 50 (ED50) values (i.e. the dose of BPIS that reduced the pain response by 50% in relation to control group values or the dose with the maximal effect in the thermal tests) were determined by linear regression GraphPad Software (GraphPad Software Inc., San Diego, California, USA), and are reported as the median effective dose accompanied by their respective 95% confidence limits.
Thermal models of nociception
The antinociceptive effect of BPIS, at a dose of 50 mg/kg, reached its peak 30 min after oral administration and remained significant up to 4 h [F(6,50)=6.03, P<0.001] (Fig. 2a). Thus, the time point of the maximum effect (30 min) was chosen for all further studies.
BPIS, at doses of 25, 50 and 100 mg/kg, and the positive control morphine, administered 30 min earlier, led to a significant increase in the tail-immersion response latency relative to the control group [F(5,41)=7.90; P<0.001] (Fig. 2b). The ED50 for BPIS was 14.49 (6.48–32.41) mg/kg. No significant effect of BPIS was observed at the lowest dose (10 mg/kg).
The mean baseline latencies were 2.13±0.12 s [F(6,50)=1.09; NS] in the time-course study and 1.54±0.10 s [F(5,41)=1.07; NS] in the dose–response study, and were not significantly different between groups.
In the hot-plate test, treatment with BPIS, at doses of 25, 50 and 100 mg/kg, or morphine, increased response latency to thermal stimuli, compared with the control group [F(5,41)=8.80; P<0.001] (Fig. 2c). No significant effect was observed at the lowest dose of BPIS (10 mg/kg). The ED50 was 26.97 (19.93–36.49) mg/kg. A dose of 100 mg/kg did not cause any further increase in the antinociceptive effect observed at 50 mg/kg BPIS. As no dose-dependent effect was found, the 100 mg/kg dose was not used in subsequent experiments. The mean baseline latency was 5.86±0.21 s [F(5,41)=0.25; NS], and did not differ significantly between groups.
Chemical models of nociception
Acetic acid-induced abdominal writhing
As shown in Fig. 3a, BPIS, at doses of 25 and 50 mg/kg, but not at 10 mg/kg, caused a significant decrease in the number of writhes [F(4,33)=29.75; P<0.001]. The ED50 was 37.26 (31.77–43.71) mg/kg.
The effect of BPIS on the time spent licking the formalin-injected paw during the first (0–5 min) and second phases (15–30 min) of the test is shown in Fig. 3b and c, respectively. BPIS, at doses of 10 mg/kg or higher, decreased the time spent licking the hind paw in the first [F(7,52)=9.67; P<0.001] and second phases [F(7,52)=7.29; P<0.001] of the formalin test. Morphine (2.5 mg/kg; subcutaneously) and diclofenac at doses of 20 and 40 mg/kg, but not at 10 mg/kg (intraperitoneally), also decreased the time spent licking the hind paw, in both phases. The ED50 values for BPIS were 25.60 (17.42–37.61) mg/kg for the first phase and 34.23 (27.94–41.93) mg/kg for the second phase.
Figure 3d shows that treatment with BPIS (at 25 and 50 mg/kg, but not at 10 mg/kg) resulted in a significant inhibition of glutamate-induced nociception [F(4,40)=8.77; P<0.001]. The ED50 was 27.13 (16.77–43.89) mg/kg.
BPIS, at all tested doses, caused a significant reduction in the capsaicin-induced licking response [F(3,28)=13.50; P<0.001] (Fig. 3e). The ED50 was 18.62 (10.45–33.16) mg/kg.
The role of the opioid system in the antinociceptive effect of bis(phenylimidazoselenazolyl) diselenide in the tail-immersion test
Pretreatment with naloxone at a dose of 1 mg/kg (subcutaneously) abolished the antinociceptive effect of morphine (positive control) at a dose of 2.5 mg/kg (subcutaneously) [F(1,24)=12.71; P<0.001]. By contrast, the antinociceptive effect of BPIS (50 mg/kg, orally) was not blocked by pretreatment with naloxone at doses of 1 mg/kg [F(1,25)=3.33; NS] or 10 mg/kg [F(1,26)=0.07; NS] (Fig. 4). The mean baseline latency was 1.78±0.07 s [F(7,50)=0.32; NS] and did not differ significantly between groups.
Treatment of mice with BPIS (10–50 mg/kg, orally) did not cause any significant change in the number of crossings [F(3,28)=0.2450; NS] or rears [F(3,28)=0.17; NS] in the open-field test (Table 1).
A single oral administration of BPIS (10–50 mg/kg) reduced neither body weight gain [F(3,32)=1.90; NS] nor food intake [F(3,32)=0.17; NS] (Table 2), and did not cause any fatalities. Only water intake was reduced in animals exposed to BPIS at a dose of 50 mg/kg [F(3,32)=9.03; P<0.001] (Table 2).
The activities of plasma alanine aminotransferase [F(3,32)=0.85; NS] and aspartate aminotransferase [F(3,32)=0.47; NS] were unchanged by BPIS treatment, relative to the control group (Table 3). There was also no change in the levels of urea [F(3,32)=1.09; NS] and creatinine [F(3,32)=0.82; NS] in animals treated with BPIS (Table 3).
In the present study, we found that BPIS elicited an antinociceptive action in chemical and thermal models of pain in mice, and that this action seems not to be related to opioidergic mechanisms. The results also indicate that the administration of BPIS to mice caused neither acute toxicity nor nonspecific locomotor disturbances.
Administration of BPIS to mice caused a significant prolongation of response latency in the tail-immersion and hot-plate tests, indicating an increase in the nociceptive threshold similar to that caused by morphine, the positive control (administered at a different dose and by a different route). These results indicate that BPIS was effective in inhibiting thermal nociception at different levels of the central nervous system, as the hot-plate test mainly reflects supraspinal responses and the tail-immersion test reflects spinal responses (Langerman et al., 1995).
The time-course study in the tail-immersion test yielded the interesting finding that the antinociceptive effect of BPIS remained significant for up to 4 h after administration. Studies have shown that the effects of other organoselenium compounds, such as (PhSe)2 and bis-selenide derivatives, remain significant up to 2 h and 45 min, respectively (Savegnago et al., 2006; 2007), and that effects of morphine, diacetylmorphine and 6-acetylmorphine, known analgesic drugs, last 2.5, 1.8 and 1.7 h, respectively (Umans and Inturrisi, 1981).
The present data indicated that BPIS inhibited the acetic acid-induced visceral nociceptive response in mice. Although the acetic acid-induced writhing is a test weakly predictive of antinociceptive activity because of its lack of specificity, showing false-positive responses to some drugs that have no analgesic action (Le Bars et al., 2001), this result indicates a potential antinociceptive action of BPIS in a chemical model of nociception.
In the formalin test, BPIS also inhibited both the first, or neurogenic, phase, which results basically from the direct activation of nociceptors, and the second phase, which involves a period of sensitization during which an inflammatory phenomenon occurs (McNamara et al., 2007). The effect of BPIS in this test is an important finding, because it indicates an antinociceptive effect of this compound against inflammatory pain. Also, this model is considered one of the most predictive of drugs used to treat acute pain (Le Bars et al., 2001).
On the basis of these antinociceptive effects of BPIS in different screening tests for analgesic drugs, we sought to determine whether BPIS inhibits the nociceptive response caused by an intraperitoneal injection of glutamate. BPIS was indeed effective in inhibiting glutamate-induced nociceptive behaviour. The neurotransmitter glutamate plays important roles in nociception: it is stored principally in C-fibres, released in the spinal cord or in peripheral nerves in response to noxious stimuli or inflammation, and activated ionotropic and/or metabotropic glutamatergic receptors (Bleakman et al., 2006). Studies have shown that the antinociceptive effect of some diaryl diselenides, such as (MeOPhSe)2 and (PhSe)2, involves glutamatergic mechanisms (Savegnago et al., 2007; Pinto et al., 2008).
BPIS was also effective in inhibiting the nociceptive behaviour induced by an intraperitoneal injection of capsaicin, a selective agonist of the vanilloid TRPV1 receptor. This receptor is activated by different chemical (capsaicin and acid) or physical (heat) stimuli and its activity can be heightened by agents involved in inflammation processes, such as nerve growth factor and bradykinin (Julius and Basbaum, 2001). The administration of capsaicin and consequent activation of TRPV1 receptors has been shown to lead to glutamate release in primary afferent fibres and to potentiate the glutamate input to areas such as the dorsolateral periaqueductal grey, an important area in pain modulation (Xing and Li, 2007; Jin et al., 2009). Although these results suggest that vanilloid and glutamatergic systems might be involved in the mechanism of BPIS-induced antinociception, we cannot confirm this hypothesis because chemical irritant pain models have a wide range of nociceptive mediators.
The opioid system is one of the most important systems involved in pain modulation. Drugs that activate different opioid receptors, such as morphine and its derivatives, have notable antinociceptive activity in different models of nociception (Jage, 2005). The results reported here show that the blockade of opioid receptors by the opioid antagonist naloxone was ineffective in antagonizing antinociception elicited by BPIS, suggesting that this effect is not directly related to an interaction with opioid receptors. Accordingly, studies from our research group have shown that in general, organoselenium compounds with antinociceptive action, other than (F3CPhSe)2, do not interact with the opioid system (Savegnago et al., 2006, 2007; Wilhelm et al., 2009; Brüning et al., 2010).
Changes in motor activity can interfere with the nociceptive response (Le Bars et al., 2001). No alteration in spontaneous exploratory behaviour was found in mice treated with BPIS, ruling out the possibility that locomotor changes caused a false-positive response in the nociceptive models.
To exclude a possible toxicity of BPIS, we examined the potential acute toxicity after oral administration of BPIS to mice. BPIS did not alter body weight gain, food consumption or biochemical markers of renal and hepatic damage, suggesting that there was no acute toxicity. Water consumption was the only toxicity parameter that was slightly reduced in mice treated with BPIS. In this context, results from our research group have shown that organoselenium compounds have an anorectic property, reducing water and food consumption in different species (Meotti et al., 2008; Savegnago et al., 2009).
Together, the present results indicate that BPIS might be of potential significance for the development of new clinically relevant drugs for the treatment of pain. Additional studies are, however, necessary to substantiate this proposition, especially evaluating BPIS in models of chronic afflictions related to pain, inflammation and oxidative stress, such as rheumatoid arthritis, which result in disability and impairment in the quality of life. The exact molecular mechanism by which BPIS exerts an antinociceptive action also requires investigation.
UFSM, CAPES, CNPq, FAPERGS/CNPq (PRONEX) research grant #10/0005-1 and FAPERGS research grant #10/0711-6.
Conflicts of interest
There are no conflicts of interest.
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