Vitex cymosa Bertero is a small tree widely distributed in the Central and Amazon regions of Brazil. It is used by the native populations to treat rheumatic pains. From this species, the compound (±)-trans-4-hydroxy-6-propyl-1-oxocyclohexan-2-one [(±)-δ-lactone] was isolated and determined to be the active principle.
The diffusible molecule nitric oxide (NO) acts as a major intracellular and intercellular messenger, and has been implicated in many physiological pathways throughout the body (Knowles and Moncada, 1994). NO is an important mediator of nociception and it is unequivocally involved in central sensitization; it is also a signalling molecule that plays an important role in acute (Toriyabe et al., 2004) and chronic (Chen et al., 2010) pain states at both the central (Freire et al., 2009) and the peripheral (Omote et al., 2001) levels. One of the mechanisms involved in central sensitization is the activation of the NMDA (N-methyl-D-aspartic acid) receptors by glutamate. Activation of these receptors generates NO, which diffuses out of the neuron to act on nerve endings and astrocyte processes, acting as a neurotransmitter (Pace et al., 2006). The central sensitization plays a major role in the generation of hypersensitivity, resulting in the phenomena of hyperalgesia and allodynia.
There is a large body of evidence supporting a role for NO in both the development and the maintenance of hyperalgesia. Inhibition of NOS has been shown to decrease hyperalgesia in models of both acute and chronic pain (Meller et al., 1992). Thus, the development of drugs acting on the nitrergic system is an important pharmacological target in the management of pain, mainly because the analgesics currently used exert significant adverse effects. For example, the opioids are among the most powerful analgesics in clinical use for the treatment of pain. Nevertheless, opioid treatment of pain is often discouraged because of concerns related to the development of analgesic tolerance and other debilitating adverse effects including constipation and nausea (Munro et al., 2008). Efforts to eliminate or minimize the undesired adverse effects have led to the synthesis of numerous compounds (Cowan et al., 2002; Gilson et al., 2004).
In our continuing search for bioactive substances, we recently reported the synthesis of (±)-δ-lactone which showed antinociceptive activity in preliminary experiments (Miranda et al., 2010). The purpose of this study was to evaluate the antinociceptive profile of this new substance to elucidate its mechanism of action.
The synthesis of (±)-trans-4-hydroxy-6-propyl-1-oxocyclohexan-2-one
(±)-δ-lactone (Fig. 1), referred to here as (±)-δ-lactone, was synthesized as described by Miranda et al. (2010). The purity of (±)-δ-lactone was greater than 95% and the other compounds present did not interfere with pharmacological activity, as will be shown in the results section.
Male Swiss mice (20–22 g) were obtained from our own animal facility. The animals were maintained in a room with a controlled temperature (22±2°C) and a 12-h light/dark cycle, with free access to food and water. Twelve hours before each experiment, animals received only water, to avoid the interference of food with drug absorption. The animal care and research protocols were performed in accordance with the principles and guidelines adopted by the Ethics Committee on Animal Use, and were approved by the Biomedical Science Institute/Federal University of Rio de Janeiro under number ICBDFBC-015.
The acetic acid-induced abdominal writhing test
Mice were used as described previously (Whittle, 1964). In brief, the total number of writhes after the intraperitoneal (i.p.) administration of 2% (v/v) acetic acid was recorded over a period of 20 min, beginning 5 min after the acetic acid injection. Vehicle, morphine or (±)-δ-lactone was administered orally 60 min before the i.p. injection of acetic acid.
The formalin test
Formalin-induced behaviour was assessed as described previously (Hunskaar et al., 1986). Mice received an injection of 0.02 ml formalin (2.5% v/v) below the dorsal surface of the left hind paw. They were immediately placed individually in an observation chamber, and the period (s) that the animal spent licking the injected paw was recorded. The nociceptive response includes two phases. The first phase is the neurogenic pain response, and it was recorded in the first 5 min after the formalin injection. The second phase is the inflammatory response, and it was recorded 15–30 min after the formalin injection. Vehicle, morphine or (±)-δ-lactone was administered 60 min (oral administration) or 15 min [intrathecal (i.t.) and subplantar (s.p.) injections] before the injection of formalin. When administered by an s.p. injection, (±)-δ-lactone was administered to the ipsilateral and contralateral paws relative to the injection site of the formalin solution. This procedure was performed to confirm the local effect of an s.p. application of (±)-δ-lactone.
The hot-plate test
The procedure used was as described previously (Sahley and Berntson, 1979). The animals were placed on a hot-plate set at 55±1°C. The reaction time (RT) was recorded when the animals licked their hind paws or jumped. Two measurements were taken before the administration of substances (morphine, vehicle or (±)-δ-lactone) and six measurements were taken after the application of substances at 30-min intervals. The mean of RTs obtained before the administration of the substances is called the baseline. Antinociception was quantified as either the RT obtained at each measurement time or the area under the curve (AUC) of the responses from 30 to 180 min after drug administration, calculated according to the following formula on the basis of the trapezoid rule: AUC=30×RT [(30 min)+(60 min)+[Midline Horizontal Ellipsis]+(180 min)/2]. The cutoff, which is considered the longest possible period during which the animal may be subjected to a noxious stimulus without producing tissue damage, was set to 18 s, calculated by multiplying the baseline by 3.
The open-field test
This model was used to evaluate motor activity. Five days before behavioural testing, each animal was handled daily for a few minutes. The procedure was similar to the method described by Barros et al. (1991). The mice received vehicle, morphine or (±)-δ-lactone by oral administration and were immediately individually placed for observation in a box whose floor was divided into 50 squares (5×5 cm). Measurements were performed at intervals of 30 min for 180 min. The total number of squares covered by the animals in 5 min was counted.
(±)-δ-Lactone was administered orally (6, 60, 300, 600 and 900 µmol/kg) in a final volume of 0.1 ml. (±)-δ-Lactone was administered by i.t. and s.p. injections at concentrations of 10, 30, 100, 300 and 600 nmol in a final volume of 0.005 ml. The EC50 and ED50 values for (±)-δ-lactone and morphine were calculated in various tests using the various routes of administration (oral, i.t. and s.p.). These values were used in the administration of morphine as a reference drug. The vehicle group received PBS with the same concentration of DMSO (dimethyl sulphoxide) as that used with all the doses of (±)-δ-lactone (1%). At this percentage, the vehicle had no significant effect. All tests were carried out in a blinded manner.
The i.t. injection of (±)-δ-lactone consisted of 0.005 ml injected by a percutaneous puncture through an intervertebral space at the level of the fifth or sixth lumbar vertebra, according to a previously reported procedure (Hylden and Wilcox, 1980), using a 0.025-ml Hamilton microsyringe with a 30-G needle. The s.p. injection of (±)-δ-lactone consisted of 0.005 ml injected below the dorsal surface of the paw (ipsilateral and contralateral relative to the injection site of formalin). The mice were not anaesthetized during the i.t. and s.p. injections.
To evaluate the involvement of some systems (opioid, adrenergic, muscarinic, nicotinic and nitrergic) on the effect shown by (±)-δ-lactone, naloxone (3 µmol/kg), yohimbine (0.5 µmol/kg), atropine (4 µmol/kg), mecamylamine (10 µmol/kg), L-NAME (10 µmol/kg), L-arginine (20 µmol/kg) and glibenclamide (10 µmol/kg) were administered i.p. 15 min before the i.t. or s.p. administration of (±)-δ-lactone. The doses of antagonists were chosen from the administration of increasing doses of the antagonist compared with a single concentration of (±)-δ-lactone, as can be seen in Fig. 5. These antagonists were administered before standard reference drugs to verify the specificity and selectivity of these substances to the test and doses used. The doses were chosen to have the highest ability to alter the antinociceptive effects produced by (±)-δ-lactone without producing activity.
Calculation of ED50 and EC50
For the calculation of ED50 and EC50, (±)-δ-lactone and morphine were administered by different routes of administration (oral, i.t. and s.p.) at increasing doses or concentrations in different models. From the results obtained in these models, graphs were constructed correlating effect with these doses or concentrations.
The ED50 and EC50 values (the dose and concentration producing 50% of the maximal effect) for the antinociceptive action were obtained by fitting the data points representing the antinociceptive effect shown in each model by nonlinear regression (sigmoidal dose or concentration response) using the GraphPad Prism software, version 3.0 (San Diego, California, USA). The maximal effect is equal to the cutoff calculated from the results obtained previously in the hot-plate test. The maximal effect is equivalent to a suppression of all licking in the formalin test and all writhing in the acetic acid-induced abdominal-writhing test.
All the experimental groups included seven to 10 animals. The results are presented as the mean±SD. The statistical significance of differences between the groups was determined by one-way analyses of variance, followed by Bonferroni’s test. A P value of less than 0.05 was considered statistically significant.
The effect of oral (±)-δ-lactone on performance in the acetic acid-induced writhing, formalin and hot-plate tests
The i.p. injection of acetic acid (2%) induced 100.5±8.7 writhes over a period of 20 min. (±)-δ-Lactone exerted a dose-dependent effect. Only the 6 µmol/kg dose did not reduce the number of writhes significantly. The higher doses inhibited writhing by 24.8, 68.7, 84.5 and 87% at doses of 60, 300, 600 and 900 µmol/kg, respectively. Morphine inhibited the number of writhes by ∼50% (Fig. 2a).
Pretreatment with (±)-δ-lactone significantly reduced the period that the animals spent licking the injected paw during both response phases after the formalin injection. During the first response phase, the inhibitory effect was observed at higher doses (300, 600 and 900 µmol/kg), whereas the inhibition was observed during the second response phase at all doses except 6 µmol/kg (Fig. 2b).
Figure 2c shows the dose-dependent antinociceptive effect of (±)-δ-lactone in the hot-plate test. The antinociceptive effect of (±)-δ-lactone had a different profile of action than morphine. Morphine was active early (at 30 min), whereas (±)-δ-lactone showed activity later (90 min). The effects of the 600 and 900 µmol/kg doses were greater over time than the effects of morphine. (±)-δ-Lactone resulted in the maximum percentage increase in latency time in relation to baseline (basal latency period of the animals) of ∼15% at a dose of 6 µmol/kg, 17% at 60 µmol/kg, 50% at 300 µmol/kg, 70% at 600µmol/kg and 80% at 900 µmol/kg. Figure 2d shows that, in terms of the AUC, the effects of 300 µmol/kg were similar to those of morphine, administered at its ED50. The ED50 values for morphine and (±)-δ-lactone are shown in Table 1.
The effect of (±)-δ-lactone in the open-field test
(±)-δ-Lactone exerted no significant effect on locomotor activity, relative to vehicle, at a dose of 900 µmol/kg (Fig. 3) or at any other dose tested (data not shown). Morphine significantly decreased locomotor activity from 30 min to the end of the experiment (Fig. 3).
The effect of (±)-δ-lactone by i.t. and s.p. injections on formalin-induced licking
(±)-δ-Lactone administered i.t. showed significant inhibitory activity during the first phase of the model at the two highest concentrations (300 and 600 nmol), reducing the licking period by 35.1 and 51.3% compared with the vehicle, respectively. During the second phase, inhibitory activity was observed at concentrations of 100, 300 and 600 nmol, with the licking period reduced by 53.9, 56.3 and 61% compared with the vehicle, respectively (Table 2).
s.p. administration of (±)-δ-lactone was only antinociceptive during the second phase of the model. The results were concentration dependent: responding was reduced relative to that observed in the vehicle group by 30.7, 48.4, 63.5, 77.1 and 87.5% at doses of 10, 30, 100, 300 and 600 nmol, respectively. Morphine exerted an effect during both phases (Table 3). These results were obtained using an ipsilateral injection of (±)-δ-lactone. Contralateral administration did not produce an antinociceptive effect (data not shown). The EC50 values for morphine and (±)-δ-lactone are shown in Table 1.
The effect of (±)-δ-lactone by intrathecal injection in the hot-plate test
i.t. administration of (±)-δ-lactone in the hot-plate test showed a significant effect at the highest concentrations (100, 300 and 600 nmol). (±)-δ-Lactone showed activity later (60 min) than morphine (30 min). The antinociception caused by higher concentrations (100, 300 and 600 nmol) of (±)-δ-lactone persisted until the end of the experiment (Fig. 4a). In Fig. 4b, a concentration-dependent activity is clear and the results of administering 300 nmol of either (±)-δ-lactone or morphine are similar. The EC50 values for morphine and (±)-δ-lactone are shown in Table 1.
Evaluation of the interaction between intrathecal-injected (±)-δ-lactone and antagonists in the formalin test
In Fig. 5, (±)-δ-lactone was administered in association with increasing doses of L-NAME, L-arginine and atropine to determine the lowest dose capable of producing a change in the antinociceptive activity of (±)-δ-lactone. The same procedure was adopted for glibenclamide, yohimbine, mecamylamine and naloxone (data not shown).
The antagonists were administered before standard reference drugs, where antagonists have proven their ability to alter the biological effects produced by these drugs (Fig. 5). Figure 5a shows that atropine (4 μmol/kg) completely reversed the antinociceptive effect induced by carbachol administered i.t. Figure 5b shows that L-arginine (20 μmol/kg) completely reversed the antinociceptive effect induced by L-NAME administered i.t.
In both phases of the model shown in Table 2, the isolated use of the antagonists naloxone, yohimbine and glibenclamide produced results similar to those obtained with the vehicle, whereas the concomitant use of these antagonists with (±)-δ-lactone produced results similar to those obtained with (±)-δ-lactone alone.
The antagonists mecamylamine and atropine administered alone did not produce significant effects relative to the vehicle. The effect of administration of mecamylamine with (±)-δ-lactone was not significantly different from that of (±)-δ-lactone administered alone. However, the administration of atropine with (±)-δ-lactone partially reduced the antinociceptive effect relative to that produced by (±)-δ-lactone in both phases (Table 2).
Table 2 shows that L-arginine and L-NAME, administered alone, did not produce significant effects relative to the vehicle. However, L-NAME administered with (±)-δ-lactone enhanced the effect of (±)-δ-lactone, and L-arginine reduced the antinociceptive effect of (±)-δ-lactone, in both phases.
Evaluation of the interaction of intrathecal-injected (±)-δ-lactone with antagonists in the hot-plate test
In this model, the effects of naloxone, yohimbine and glibenclamide, used alone, were not significantly different from those of vehicle and when combined with (±)-δ-lactone, did not influence its effects of (data not shown). The administration of atropine before the administration of (±)-δ-lactone partially reduced the antinociceptive effect of (±)-δ-lactone (Fig. 6a and b).
The previous administration of L-arginine also partially reduced the effect of (±)-δ-lactone, whereas the previous administration of L-NAME increased the antinociceptive effect of (±)-δ-lactone (Fig. 6c and d).
Evaluation of interaction between (±)-δ-lactone administered by an subplantar injection and antagonists in the formalin test
L-Arginine and L-NAME, administered alone, had no significant effects relative to the vehicle during either phase of the formalin test. L-NAME administered with (±)-δ-lactone enhanced the effect of (±)-δ-lactone, whereas L-arginine reduced the antinociceptive effect of (±)-δ-lactone. These effects were observed only during the second phase (Table 3).
In the acetic acid-induced abdominal-writhing test, oral administration of (±)-δ-lactone produced marked and dose-related antinociception. This test, described as a typical model of visceral inflammatory pain, has been used as a screening tool for the assessment of analgesic or anti-inflammatory agents (Collier et al., 1968). However, it has been discovered previously that the constriction induced by acetic acid is considered to be nonselective (Bighetti et al., 1999; Sánchez-Mateo et al., 2006). Our results indicated that (±)-δ-lactone reduced the number of writhes, implying that it produced a significant antinociceptive effect.
The advantage of the formalin test is that it can discriminate between central and/or peripheral components of pain. Persistent pain induced in the mice paws by formalin comprises a distinct biphasic nociception (Hunskaar and Hole, 1987; Tjolsen et al., 1992). The neurogenic phase is elicited by the direct activation of nociceptive terminals; in contrast, the inflammatory phase is mediated by a combination of peripheral and central mechanisms (Hunskaar and Hole, 1987). (±)-δ-Lactone, administered orally, was effective in inhibiting both phases (neurogenic and inflammatory) of the formalin-induced nociception, showing a potential central antinociceptive effect.
To confirm the central antinociceptive activity of (±)-δ-lactone, the compound was evaluated in the hot-plate test. In this test, the thermal stimulus activates supraspinal structures, where pain is perceived and the subject protects itself from the noxious stimuli (Yaksh and Rudy, 1977). (±)-δ-Lactone showed activity in this test in a dose-dependent manner, thus confirming its central antinociceptive effect.
The antinociception caused by (±)-δ-lactone seemed unrelated to motor impairment because the compound had no significant effect on behaviour in the open field at the same doses as those used in the hot-plate test. The bioavailability of oral morphine is one-fourth to one-sixth of that obtained with parenteral administration, owing to incomplete and irregular enteric absorption and hepatic first-pass metabolism (Tassinari et al., 1995). On the basis of this information, the reduction in motor activity elicited by oral morphine in the open-field test may be related to the time window and the route of administration used, given that morphine can stimulate motor activity in mice on parenteral administration (Patti et al., 2005).
To evaluate the effect of (±)-δ-lactone at the spinal and peripheral levels, i.t. and s.p. administrations of (±)-δ-lactone were performed, respectively. After the i.t. administration, a reduction in the licking period during both phases of the formalin test was observed, indicating that (±)-δ-lactone has spinal antinociceptive activity.
After s.p. administration, (±)-δ-lactone had a significant effect only during the second phase when it had been administered to the ipsilateral paw; administration to the contralateral paw exerted no antinociceptive effect, showing the peripheral action of the compound. The action of the compound during the second phase of the test shows its ability to act on the production and/or release of postformed inflammatory mediators (Tassorelli et al., 2006). The hot-plate test was performed after i.t. administration and confirmed the spinal action of the compound.
To evaluate the contribution of the opioid receptors towards the effect of the compound, naloxone was administered i.p. before the i.t. administration of (±)-δ-lactone in the formalin test. Naloxone is the N-allyl derivative of oxymorphone. Similar to the other opioid antagonists, it acts by competitively binding to opiate receptors. Naloxone shows the maximum affinity towards the μ receptor, but also shows antagonistic activity at the κ and δ receptors (Handal et al., 1983). The previous administration of naloxone did not alter the effect produced by (±)-δ-lactone, precluding the participation of opioid receptors.
The participation of α2 adrenoreceptors was assessed by pretreatment with yohimbine in the formalin test. Yohimbine is classified as an α2 adrenergic receptor antagonist (Kim et al., 2005). In fact, the selective interaction of yohimbine with α2 receptors provides the basis for determining the function of these receptors; any effects induced or inhibited by yohimbine are considered to be mediated by α2 adrenoreceptors (Kim et al., 2005). In the present study, we observed that α2 adrenergic receptors do not appear to be involved in the antinociception induced by i.t.-administered (±)-δ-lactone.
Several lines of evidence show that the cholinergic system has broad therapeutic potential in the treatment of many clinically relevant pain states including inflammatory, neuropathic, visceral pain and pain because of arthritis (Jones and Dunlop, 2007). Furthermore, the effects of acetylcholine are mediated through both nicotinic acetylcholine receptors and the G protein-coupled muscarinic receptors (Jones and Dunlop, 2007). The previous administration of mecamylamine (a nonselective nicotinic acetylcholine receptor) and atropine (a nonselective muscarinic acetylcholine receptor) was used to evaluate the role of the nicotinic and muscarinic receptors, respectively, in the antinociceptive activity shown by (±)-δ-lactone. The results show the involvement of muscarinic receptors in the effect of the compound, because pretreatment with atropine reduced the antinociceptive effect of (±)-δ-lactone, whereas mecamylamine pretreatment did not alter the activity of (±)-δ-lactone.
The potassium channel regulated by ATP is an important channel in the NO-cGMP-ATP-sensitive K+ channel pathway. This pathway is responsible for the antinociceptive activity of various compounds (Ortiz et al., 2003). Previous application of glibenclamide (a specific ATP-dependent K+ channel blocker) did not alter the effect of (±)-δ-lactone, showing that these channels do not mediate the activity of the compound.
The s.p. injection of carrageenan and formalin causes the production and release of NO at the injured site (Omote et al., 2001). Previous administration of L-NAME (an NO synthase inhibitor) or L-arginine (a substrate of NO synthase) in the formalin test potentiated or inhibited the antinociceptive effect of (±)-δ-lactone, respectively. The use of an NO synthase inhibitor (L-NAME) enhances the effect of the compound by reducing the synthesis of NO, which acts as a retrograde messenger at the spinal level to increase the stimulatory neurotransmitters released (Oess et al., 2006). In contrast, L-arginine increases the concentration of NO at the spinal level and thus reduces the antinociceptive effect of the compound. These results show that the production and release of NO is important to the effect of the compound. On the basis of these results, we believe that (±)-δ-lactone can reduce the concentration of NO at the spinal level.
In the central nervous system, NO is concentrated in the dorsal horn of the spinal cord, where it derives from diverse sources (including glial cells). Neuronal NO synthase (nNOS) is the predominant form of NOS in the dorsal horn and plays a definite role in spinal cord circuits. In addition to nNOS, eNOS (endothelial NO synthase) is also found in some neuronal populations and in blood vessels (Freire et al., 2009). In terms of iNOS (inducible NO synthase), there is some disagreement on whether this isoform also participates in the central transmission of pain or whether it is expressed only peripherally during the inflammatory or neuropathic pain processes (Freire et al., 2009).
NO can diffuse from the neuron to act on nerve endings and astrocyte processes, acting as a neurotransmitter. NO can also enhance the release of substance P and calcitonin-gene-related peptide from C-fibre terminals (Miclescu and Gordh, 2009), contributing towards the development of secondary hyperalgesia. It has also been suggested that spinal NO participates in the glutamatergic mechanisms of descending facilitation triggered by illness and inflammation, weakening the influence of descending inhibition on dorsal horn neurons (Millan, 2002).
The same substances were administered before (±)-δ-lactone in the hot-plate test, showing results similar to those found in the formalin test. To evaluate the participation of NO in the peripheral action shown by the compound, L-NAME and L-arginine were administered before the s.p. administration of (±)-δ-lactone in the formalin test. Previous administration of L-NAME increased the antinociceptive effect, whereas previous administration of L-arginine reduced the antinociceptive effect, but these effects were observed only during the second phase. The second phase was characterized by an inflammatory response caused by the synthesis and release of mediators synthesized later, such as cytokines, eicosanoids, kinins, glutamate and NO (Chichorro et al., 2004; Tassorelli et al., 2006).
The participation of NO, derived from various NOS isoforms, as a key mediator of nociceptive phenomena has also been shown in the periphery, using various experimental approaches (Chen et al., 2010). The involvement of NO in peripheral nociception is corroborated by data showing the local release of NO by inflammatory stimuli (Toriyabe et al., 2004). Studies suggest that both nNOS, in peripheral nerves, and iNOS, in inflammatory cells, contribute towards the production of the NO peripherally released during inflammation (Omote et al., 2001).
A previous study has shown that the systemic administration of NO synthase inhibitors increased the antinociception induced by the administration of the muscarinic receptor agonist oxotremorine (Pavone et al., 1997). In contrast, our results show that the antinociceptive activity of (±)-δ-lactone was reduced by the previous administration of a muscarinic receptor antagonist, indicating an involvement of the L-arginine–NO pathway in the antinociceptive effects of cholinergic stimulation.
Collectively, these results indicate that the reduction of NO production/release represents a critical biochemical event in the mechanism of action of (±)-δ-lactone, most likely through mechanisms involving the cholinergic system. We believe that this new compound is a promising candidate for analgesia, but more studies are required to further elucidate its mechanism of action.
The study was funded by FAPERJ (Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).
Conflicts of interest
There are no conflicts of interest.
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