Pharmacological approach to mechanism of action of tramadol in murine nociception and inflammation assays

The analgesic activity of tramadol has been recognized both in man and in several animal models of pain. However an extensive characterization of the opioid mechanism of action of tramadol of pain has not been reported. The objective of the present study was to evaluate the antinociceptive and anti-inflammatory activity of tramadol in different animal pain models and to determine the effect of the selective opioid antagonist: naltrexone, naltrindole and norbinaltorphimine. The i.p. administration of tramadol induced a dose-dependent with the following order of potency: formalin hind paw, phase II > formalin hind paw, phase I> acetic acid writhing > tail flick > hot plate. Pretreatment of the mice with naltrexone (1 mg/kg i.p.) antagonized tramadol activity in the acetic acid writhing test, in the hot plate and the tail flick assays, however lack of effect in the formalin hind paw assays. Naltrindole (1 mg/kg i.p.) did not induce a significant change in all the murine assays. However, the mice pretreated with nor-binaltorphimine (1 mg/kg, i.p.) did not modified the tramadol antinociception in the acetic acid writhing and in the hot plate assays. Besides, norbinaltorphimine pretreatment reversed significantly the tramadol effect in the tail flick and in the formalin hind paw assays. This findings suggests that tramadol effect is mediated by MOR and KOR rather DOR receptors.


Introduction
The IASP (International Association for the Study of Pain) approved the following definition of pain as "An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage." [1]. The IASP definition of pain recognizes its different components. In sensory aspect, it activates the nociceptors and the different routes and mechanisms of transmission of the painful stimulus. . On the other hand, cognitive and behavioral perception and experience represent individual and psychological personal experience [2]. The classification of pain can be done according to different variables, including duration (acute or chronic), pathogenesis (nociceptive or neuropathic), location (somatic or visceral), and others. To control pain, it is necessary to have drugs that modify its origin, can alter its perception at the central level and are capable of blocking its transmission to the central nervous system. There are no drugs that meet all the previously stated objectives, however the most frequently used in the treatment of pain are NSAIDs and opioids.
Opioids constitute a group of natural and synthetic medications, highlighting morphine, codeine, heroin, methadone, fentanyl, tramadol and others. Opioids are highly powerful and effective pain relievers, but it produces a high potential for side effects, as a consequence of the repeated use that causes neuron adaptation, such as tolerance, dependence and addiction [5].
Tramadol is a synthetic chiral MOR opioid drug formed for two enantiomers, (+) tramadol is a selective agonist of μ receptors and preferentially inhibits serotonin reuptake, whereas (−) tramadol mainly inhibits noradrenaline reuptake. The synergistic actions of the two enantiomers increase the analgesic efficacy and tolerability profile of the racemic tramadol [6,7] The antinociceptive activity of tramadol has been demonstrated in both man and animal assays of pain; however an exhaustive characterization of the opioid mechanism of action of tramadol, in animal models of pain, has not been reported. The objective of the present study was to evaluate the antinociceptive and anti-inflammatory activity of tramadol in different animal pain models and to determine the effect of the selective opioid antagonist: naltrexone, naltrindole and nor-binaltorphimine.

Animals
Male CF-1 mice (25-28 g), housed on a 12 h light-dark cycle at 22±1 °C with ccess to food and water ad libitum, were used. All animal procedures were approved by the Animal Care and Use Committee at the Faculty of Medicine, University of Chile (Protocol CBA 0852/FMUCH/2018). Animals were acclimatized to the laboratory for at least 1 h before testing, used only once during the protocol, and euthanized immediately after the algesiometric test by one intraperitoneal (i.p.) injection of 60 mg/kg of pentobarbital. The number of animals was kept at a minimum, compatible with consistent effects of the drug treatment.

Measurement of antinociceptive activity
Antinociception was assessed by the following murine tests: (A) Acetic acid abdominal contraction (writhing test), as previously described [8]. Antinociception, expressed in % of maximum possible effect (% MPE), was calculated as percent inhibition of the saline control writhes (20.78±0.74, n=12).
(B) tail-flick as described previously [8]. Tail flick latencies control were 2.49±0.07 (n=12) and converted to %MPE as follows: %MPE= (latency postdrug -latency control)/ (cut-off time − predrug latency) x 100 (C) The formalin hind paw test described previously was used [9]. The test show 2 clear cut-periods: phase I corresponding to the 5 min immediately after formalin injection and phase II, chronicled by 10 min, a period starting 20 min after formalin injection. The control licking or biting, in sec, of the injected paw were, phase I: 133.05 ± 7.04 (n =12) and phase II: 157.83 ± 9.10 (n=12). Licking time was converted to % MPE as follow: (D) The hot plate test as previously described [10]. The control licking of the forelegs were 18.96 ± 0.57 (n=12). Hot plate latencies, with a cutoff time of 30 sec to elude skin damage, were converted to % MPE as follow: % MPE= [(post drug latency -control latency) / cutoff -control latency] x 100 For each NSAIDs the DE50, dose that induce 50% of MPE was calculated from lineal regression of dose-response curves.

Experimental design
In order to determine the antinociceptive potency of i.p. tramadol a dose-response curves produced from 0.3 to 100 mg/kg was obtained in the writhing, tail flick, formalin hind paw and hot plate tests using at least 6 animals for each at least 4 doses. To identify the participation of the opioid antagonist receptors, in the tramadol antinociception, mice were pretreated with either 1 mg/kg i.p. of naltrexone or naltrindole or nor-binaltorphimine, which are doses uses in the literature.

Drugs
Drugs were freshly dissolved in sterile physiological salt solution of 10 mL/Kg, for intraperitoneal administration. Tramadol hydrochloride, naltrexone hydrochloride, naltrindole hydrochloride and Nor-binaltorphimine dihydrochloride were purchased from Sigma-Aldrich Chemical Co, St.Louis, Mo, USA.

Statistical analysis
Results are presented as means ± SEM. The statistical difference between the results were assessed by one-way ANOVA, followed by Tukey's post test for and p values less than 0.05 (p<0.05) were considered statistically significant. Statistical analyses were carried out using the program Pharm Tools Pro, version 1.27, Mc Cary Group Inc., PA, USA.

Antinociception induced by tramadol in the murine tests
The i.p. administration of tramadol induced a dose-dependent antinociceptive action in the acetic acid writhing, tail flick, formalin hind paw and hot plate tests. The corresponding dose-response of each assay is shown in Figure 1. The ED50 antinociceptive values with their respective SEM resulting from each test is presented in table 1. The order of tramadol potency range in each assay was: formalin hind paw, phase II > formalin hind paw, phase I> acetic acid writhing > tail flick > hot plate.

Effect of opioids antagonists in the tramadol antinociception
The pretreatment of the mice with naltrexone (1 mg/kg i.p.) induced a significant increment of the tramadol activity in the control writhes of the acetic acid writhing test, in the control licking of the forelegs of the hot plate and in the control latency of the tail flick assays, these changes means an antagonism in the tramadol antinociception (see figure 2). Otherwise, after pretreatment with naltrexone did not change the control licking in the formalin hind paw assays induced by tramadol. All these data are shown in figure 2. The pretreatment of the mice with naltrindole (1 mg/kg i.p.) did not induced a significant change of the antinociceptive activity of tramadol in the acetic acid writhing, in the hot plate assay, in tail flick and in the formalin hind paw tests. All these data are shown in figure 3. In the case of the mice pretreated with nor-binaltorphimine (1 mg/kg, i.p.) did not produced a significant increase of the tramadol antinociception in the number of writhes in the acetic acid writhing and in the licking of the foreleg in the hot plate assays. Besides, nor-binaltorphimine pretreatment reversed significantly the tramadol effect in the latency of the tail flick and in the licking in the formalin hind paw assays (see figure 4).

Discussion
Animal models are primarily used to understand the mechanism of action of pain relievers and anti-inflammatory drugs.
In the current study, tramadol was found to be able to induce a significant antinociceptive effect, demonstrated by the corresponding behavior of the pain test used. Thus, the administration of tramadol produced antinociception in the writhing, tail flick and hot plate. Furthermore, induce anti-inflammatory activity in the formalin hind paw. These findings are in agreement with previous reports of a similar effect of tramadol restricted to animal models of nociception such as those of hot plate [11] tail movement [12] and acetic acid writhing tests [13], nevertheless the effect of tramadol in animal inflammatory models such as formalin tests had not been reported.
To establish the role of different opioid receptors in the tramadol-induced nocifensive effect, opioid antagonists were used, which demonstrated that naltrexone reversed tramadol activity in the nociceptive pain (writhing, hot plate, and tail flickl). Furthermore, naltrexone had no effect in the tonic inflammatory pain assay (formalin hind paw). The use of naltrindole did not significantly change the tramadol control values in the different tests. However, the administration of nor-binaltorphimine did not modify the effect of tramadol in the writhing and hot plate tests, on the other hand, abolished the effect of the opioid on the tail flick and the hind paw formalin.
Tramadol is a prodrug that exists as 2 enantiomers with antinociceptive activity but with different mechanisms of action. Thus, (+)-Tramadol and it metabolite O-desmethyltramadol (M1) act as selective MOR receptor agonist and inhibits serotonin reuptake. The other enantiomer, (-) inhibits noradrenaline reuptake, both synaptic reuptake increase the inhibition of descending pathways connected with pain transmission. [14][15].
The results obtained in the present study are consistent with those previously reported, although in models other than pain, in which naltrexone significantly decreases the response of discriminatory behavior to tramadol [16], the inhibition in memory recovery [17]. However, It should be noted that the effect of opioid antagonists, in the antinociception of tramadol, has only been reported in a model of cerebral ischemia pain, in which the effect of the opioid was significantly cancelled by β-funaltrexamine (MOR selective opioid receptor antagonist), but not naltrindole or nor-binaltorphimine [18].
The present study showed that antinociception and anti-inflammatory activity of tramadol were mediated by the MOR and KOR receptor and not by the DOR receptor. This was validated by the change in tramadol activity by naltrexone and nor-binaltorphimine and the lack of antagonism developed by naltrindole in the tests used. The explanation for these tramadol findings could be justified with the argument that opioid analgesia being a complex process in which MOR receptors can be heteromerized with δ or κ opioid receptors. Based on the results of the present study, it is possible to speculate that MOR and KOR may play an important role in naltrexone antinociception. This finding is not consistent with the report by Choi et al. [19] suggesting that in the hind paw formalin trial, MOR, KOR and DOR are involved in the antinociception of naltrexone.
The mechanism of action proposed in this study for the analgesic action of tramadol is consistent with previous works in which it is proposed that O-desmethyltramadol (M1) has almost the same potency, as tapentadol and oxymorphone in MOR, KOR and DOR [20]. Nevertheless, it must be added that tramadol, described as an atypical opioid, with a special profile of opioidergic, noradrenergic and serotonergic actions, has other modulatory actions on pain, including mechanisms involved with substance P, in sodium ion channels, in adenosine, glutamate receptors, alpha-2 adrenoceptors, proinflammatory cytokines, and other modulatory effects that would regulate both central and peripheral neuronal excitability [21].

Conclusion
The findings of the present study demonstrated that the antinociceptive and anti-inflammatory activity of tramadol was blocked by naltrexone and nor-binaltorphimine but not naltrindole. This suggests that tramadol effect is mediated by MOR and KOR rather DOR receptors.