Previous studies have reported that the cannabinoid system is involved in pain inhibition, where CB₁ receptors are found in both presynaptic and postsynaptic terminals in the CNS, while CB₂ receptors are mainly located in the microglia and postsynaptic cells, identified in neuronal damage and inflammation [21]. In the peripheral areas, CB₁ receptors are located on nociceptors and CB₂ receptors in immune system cells involved in inflammation [22]. In therapy, the systemic administration of cannabinoids represents a clinical problem due to the adverse effects originating at the central level. Studies have reported that these undesirable effects depend on the activation of CB₁ receptors [23]. Therefore, a possible strategy for the treatment of pain to reduce these adverse effects is local administration.

It is known that the CB₁ and CB₂ receptors can be activated in pain diseases such as arthritis, gout, and musculoskeletal trauma. WIN 55,212-2 possesses a high affinity for both receptors [24]. In the present study, this non-selective agonist was administered to rats by the intraplantar route in the same extremity where nociception was generated to explore cannabinoid participation. Local injection was used to activate only peripheral cannabinoid receptors, avoiding their action in the CNS to prevent psychotropic effects. In our study, a maximal efficacy of 46.15 ± 1.87% was obtained after the administration of WIN. This result agrees with previous studies using the formalin test, where intrathecal administration showed the maximal efficacy of this drug in the inflammatory phase of approximately 50% [25]. In acute pain induced by thermal stimulus, a maximal efficacy of 34.6% was obtained after the intraperitoneal administration of WIN [26]. Meanwhile, for cancer pain, it has been reported that spontaneous activity decreases in C-fibers due to the activation of both the CB₁ and CB₂ receptors [22].

The great usefulness of ibuprofen in different pain states, due to the low incidence of adverse effects, has led us to think that there is a high probability that it can be co-administered with cannabinoids. Therefore, in this study, we evaluated different doses in monotherapy and combined with WIN and observed that it produced a maximal effect of 56.6 ± 2.52% using 5% formalin as a nociceptive agent, as previously reported in other studies [27]. The IBU + WIN combination reached a maximal efficacy of 63.56 ± 2.14%, demonstrating a greater effect than IBU alone. Doses of individual drugs were significantly reduced when combined, indicating that antinociceptive potency was increased. The administration of low doses may subsequently result in a possible decrease in adverse effects; thus, patients who undergo chronic treatment with analgesics might use this therapy for longer periods.

The isobolographic analysis showed that COMB generated a potentiation synergism with an interaction index of 0.13; according to Tallarida [28], an interaction index of less than one indicates supra-additive synergism. This finding agrees with data from Guindon et al. [17], who reported a synergistic effect when combining IBU and anandamide, an endogenous cannabinoid, using local administration. In the present study, IBU was administered systemically to involve the peripheral and central mechanisms. In addition to the inhibition of prostaglandin synthesis at the peripheral level, IBU can also stop synthesis in the CNS. It has also been reported that when administered at the same site, IBU can inhibit (nitric oxide) NO synthesis [29] and decrease the nociceptive effects generated by glutamate and substance P [30], contributing to its analgesic activity. In our study, WIN was administered via the intraplantar route to activate only local cannabinoid receptors, suggesting that different mechanisms probably contribute to the observed supra-additive synergism (Figure 5).

The degree of involvement of the cannabinoid receptors observed in the supra-additive synergism for pain inhibition was explored in this study, and differences were found with previous reports such as the results of Guindon et al. [17], who reported that the CB₁ receptor participated more strongly in the synergism observed with IBU and anandamide. In this respect, we observed that the AM281 antagonist reduced the effect of the combination at 45%, while the AM630 antagonist produced a reduction of 76%. Thus, the CB₂ receptor showed stronger participation in the synergism of our COMB with these experimental conditions. Other results agree that the CB₂ receptor agonists administered via the intraplantar route have similar efficacy to morphine by the same route [21].

In addition, it is known that the CB₂ receptor is expressed in leukocytes, neutrophils, monocytes, and T cells, among other cells involved in inflammation [12]). It has also been reported to play an essential role in controlling the acute inflammatory response [31]. This effect has been explained in some experimental models because the activation of this receptor can reduce neutrophil recruitment [32] and decrease IL-6 secretion [24], resulting in anti-inflammatory effects. Thus, the activation of CB₂ receptors could inhibit the release of inflammatory and pain mediators in the late phase of the formalin test. Together with the inhibition of prostaglandin synthesis induced by ibuprofen, this information supports the synergistic response of COMB in our study (Figure 5).

With regard to the CB₁ receptors that are expressed in nociceptors, Agarwal et al. [14] reported that these receptors play a significant role in analgesia since these receptors induce endocannabinoid release both peripherally and centrally. Moreover, small doses of synthetic cannabinoid agonists reduce inflammatory and neuropathic pain. In our study, low doses of WIN administered in the hind paw produced supra-additive synergism in which the CB₁ receptor showed weaker participation than CB₂.

The effect of COMB was avoided completely when the antagonists of the CB₁ and CB₂ receptors were tested together, since animals presented a higher number of nociceptive behaviors than the control group. This result demonstrates the synergistic role of both cannabinoid receptors in the antinociceptive effect of the combination. It is known that the activation of the CB₁ and CB₂ cannabinoid receptors by WIN inhibits the activity of TRPV1 receptors [33,34], which are involved in several types of pain including those associated with inflammatory pain induced by chemicals such as formalin. Therefore, the synergism obtained with the COMB treatment might be in part due to the activation of the CB₂ receptors in the inflammatory cells by WIN, which leads to the inhibition of inflammatory mediators such as interleukins and cytokines so that these mediators will no longer desensitize the nociceptor. On the other hand, WIN also activates the CB₁ receptor located on nociceptors, contributing to the inhibition of the nociceptive signal (Figure 5). In addition, endocannabinoids are released under stress stimuli such as inflammation and pain [35]. Calignano et al. [36] reported that the levels of the endocannabinoids anandamide and palmitoylethanolamide in rat paw skin could activate the CB₁ and CB₂ receptors, respectively, attenuating nociceptive behaviors. Therefore, when both the CB₁ and CB₂ receptor antagonists were co-administered, the nociceptive effect was higher than in the control group. These mechanisms, added to the peripheral inhibition of prostaglandins and the central mechanisms activated by IBU, induced the synergism observed in our experimental design.

There are studies that have demonstrated the participation of the cannabinoid system in analgesia [14,21] as well as other studies that have found synergism or addition with combinations of NSAIDs and cannabinoids [1,37]. However, there is a need for studies to determine the dosage, safety, and administration conditions. Our study demonstrated that synergism occurred with the coadministration of IBU plus WIN when administered by different routes. The administration of IBU by the systemic route activates peripheral and central mechanisms, whereas the administration of WIN by the local route activates only peripheral mechanisms, avoiding adverse effects in the CNS.

The limitation of this study is that no adverse effects were analyzed. Further studies are needed to determine if this combination might be associated with possible adverse effects. Nevertheless, our results provide evidence of the potential benefit of this combination for pain therapy, and suggest that a formulation integrating a non-selective cannabinoid with a NSAID such as IBU might be useful for pain relief.