Over the years, researchers investigated the possible options of reduction of the risk of tolerance and physical dependence related to the chronic application of opioids. Interestingly, development of tolerance to the analgesic action of morphine (attributed to the central nervous system, CNS) was accompanied by the tolerance to the inhibitory effect of this drug in the GI tract. However, tolerance occurred in the upper GI tract, but not in the colon [5]. In this study, we validated two in vitro methods that can be applied in the fast screening of drug ability to induce tolerance and physical dependence in the GI tract. Furthermore, as the results of animal studies showed that there are numerous factors that affect development of tolerance and physical dependence to the analgesic effect of opioids (i.e. activation of CB receptors), we evidenced that simultaneous activation of OR and CB receptors with PR-38, mixed agonist, abolished the development of tolerance and physical dependence in the mouse ileum.

Tolerance to opioids could be evoked either in vivo (repeated administration of drug) or in vitro (incubation of tissue with opioid). Ross et al. [5] reported that longitudinal preparations of the ileum from morphine-tolerant mice (animals with implanted pellet containing 75 mg of morphine) became resistant to stimulation with morphine in vitro (in organ baths): the inhibitory action of morphine on EFS-stimulated contractility in the ileum isolated from morphine pelleted mice was significantly weaker than that observed in the intestine from placebo-implanted or non-implanted mice. In earlier studies on the ileum of guinea pigs (GP), it was shown that the incubation or repeated exposure to morphine in vitro led to the development of tolerance to the effect of morphine on neurogenic contractions in the longitudinal muscles to the same extent as in vivo treatment with opioid [4, 6]. As both, in vivo and in vitro exposure to opioid could induce tolerance, in our study we adjusted an in vitro method of the assessment of tolerance development in the murine GI tract. Our results were in agreement with Rezvani et al. [6], as they found that 60 min incubation of GP ileum preparation with an OR agonist was enough to evoke acute tolerance. We assessed that 60 min time period of incubation with opioid led to the development of tolerance in the mouse ileum, as the inhibitory action of morphine on EFS-stimulated contractions was diminished in the preparations pre-incubated with Tyrode solution containing morphine nor Tyrode solution alone for the same time period.

Taking into consideration the beneficial effect of co-activation of OR and CB1 receptors (i.e. synergistic analgesic activity), we evaluated whether a cannabinoid component of mixed OR/CB ligand (PR-38, derivative of salvinorin A, SA) affected the tolerance forming potency of this compound. In our previous studies we found that SA activated KOP, CB1, CB2 receptors in the GI tract and thus attenuated colitis, modulated neurogenic ion transport in the colon and inhibited the GI transit [13]. However, SA could not be applied in clinics due to the psychoactive activity in the CNS. PR-38 shared some features with SA (agonistic activity at MOP, KOP and CB1 receptors), but it was deprived of the action in the CNS [14]. Using a newly validated method we assessed the potency of PR-38 to develop tolerance in the GI tract. We observed that inhibitory action of PR-38 in tissue incubated for 60 min in Tyrode solution containing PR-38 was not weaker than the effect evoked in the preparation incubated with Tyrode solution alone. In fact, we observed that at higher concentrations of this compound, incubation with PR-38 increased the sensitivity to further activation with the inhibitory action of this drug. We determined that simultaneous activation of OR and CB receptors with PR-38 attenuated the tolerance development in the mouse GI tract, what was in agreement with numerous reports on the impact of CB receptors activation on the analgesic effect of opioid. For example, according to Cichewicz et al. [16], an oral co-treatment with morphine (p.o. twice daily for 7 days: 200 mg/kg for 2 days; 300 mg/kg for latter days) and CB1 agonist, Δ9-tetrahydrocannabinol (Δ9-THC, 20 mg/kg, 15 min prior to morphine, twice daily) did not induce development of tolerance to antinociceptive effect of morphine. Interestingly, Basilico et al. [17] in the study on GP ileal myenteric plexus-longitudinal muscle preparations, reported the opposite on the role of interactions between OR and CB receptors in the tolerance development. In contrast to our study, they used two separate agonists: morphine (as OR agonist) and WIN 55,212–2 (as CB receptors agonist). First, it was assessed that after 5 h time period of incubation either with MOP agonist or CB receptor agonist, the tissues became less sensitive to the further inhibitory action of respective ligands, what indicated a development of tolerance in both cases. Second, it was reported by that 5 h incubation with WIN 55,212–2 (5 × 10⁻⁸ M) decreased twofold maximum inhibitory effect of morphine when compared to the tissue incubated without CB agonist. The same results were obtained in experiments on the tissue that became tolerant to morphine (5 h incubation with opioid, 10⁻⁷ M) and then acutely exposed to WIN 55,212-2 (8 × 10⁻⁷ M). It was determined that co-activation of OR and CB receptors augmented the tolerance development to opioid in the GI tract of GP.

The development of tolerance is usually accompanied by the occurrence of physical dependence. Thus, we attempted to adjust in vitro WR method that would enable to evaluate the potency of opioids to develop physical dependence in a fast and simple way. In experiments with morphine we measured that addition of naloxone into the organ bath induced a significant increase in the amplitude of the intestinal contractions, which we interpreted as a sign of acute physical dependence–withdrawal response (WR). In the past, Collier et al. [4] adjusted the method based on the assessment of WR in the GP ileal preparations. They used numerous set ups which varied in the concentration of opioid (normorphine 0.01–1 pM), time of incubation (2–24 h) and the temperature of incubation (5, 22 or 37 ℃). It was observed that in the ileum incubated with normorphine (2–24 h), the exposition to naloxone (0.03 pM) at 22 ℃ produced significant WR, while there was no WR response in the control tissue that was incubated without OR agonist. The WR was also induced in the tissue incubated with opioid after washout and exchange of the incubation buffer. Collier et al. [4] concluded that a single, short-term exposition to opioid was not enough to induce physical dependence, but Valeri et al. [12] found that even short (few minutes) exposition to opioid evoked physical dependence. A significant naloxone-induced WR was noted in the GP ileum incubated with morphine (10^–7 M) for 5 min, what was similar to the results obtained in our study in which the exposition to morphine lasted 10 min.

To evaluate how the co-activation of OR and CB receptors with PR-38 affected the development of physical dependence, we examined the presence of naloxone-induced WR in the tissue incubated with this mixed agonist. The only one report on the involvement of CB receptors in the opioid-related physical dependence in the GI tract was obtained by Basilico et al. [17]. It was observed, that in GP ileal myenteric plexus preparations that became tolerant to morphine (5 h incubation, 10⁻⁷ M) the exposition to naloxone (10⁻⁵ M) resulted in strong WR. However, in the tissue samples that rendered tolerant to opioid, the addition of WIN 55,212-2 (5 × 10⁻⁸ M) 15 min before challenging with naloxone significantly abolished the WR. Similarly, in our study there was no naloxone-induced WR in the tissue incubated with PR-38. Both, our results and the experiments performed by Basilico et al., indicated that simultaneous activation of OR and CB receptors diminished the development of physical dependence. Noteworthy, we determined the role of cannabinoid component of PR-38 in the physical dependence development in the setups with CB antagonist, AM-251. In particular, strong WR was noted after the addition of naloxone in the tissue that was exposed to AM-251 prior to the incubation with PR-38. Therefore, we confirmed that the simultaneous activation of OR and CB receptors with mixed agonist resulted in reduction of physical dependence.

CB and OR exhibit several common features, such as: their neuroanatomical distribution, functional over-lapping (pain, reward process, anxiety, immune, or the action in the GI system), cellular co-localization and similarities in signal transduction. There are multiple possible mechanisms of cross-talk between MOP and CB receptors, as they both belong to the G protein—coupled receptors family and transduce signals via activation of Gi/o proteins, what is followed by inhibition of the adenylyl cyclase activity, activation of Ca2 + channel, neurotransmitter release and stimulation of the MAP kinase cascade [18, 19]. The interactions between OR and CB receptors could be a consequence of the ability of OR and CB receptors to form functional heterodimers [20]. Moreover, there is a possible contribution of allosteric modulation: agonist-occupied CB receptors act as an allosteric modulator of the partner opioid receptor and conversely [21]. For instance, it was reported that cannabidiol is a negative allosteric modulator of MOP: cannabidiol abolished the inhibitory action of DAMGO in the electrically induced twitch response test in the mouse vas deferens [22]. Interestingly, SA (parent compound to PR-38), besides being partial agonist of MOP, acts as a negative allosteric modulator of MOP [23]. Allosteric modulation of OR through activation of CB1 can potentially lead to the decreased potency of development of tolerance and physical dependence [21]. The beneficial effect of PR-38 on the tolerance and physical dependence development could result from the allosteric modulation of opioid receptors, but it needs to be determined if PR-38 is an allosteric modulator of OR in further research.