Analgesic Interaction Between Morphine with Fentanyl In Experimental Murine Pain

Opioids are among the most effective pain relievers available, however multimodal antinociception between opioids, has not been extensively studied in diverse animal pain models.In this study the pharmacological interaction of morphine with fentanyl was evaluated in different murine pain models by means of isobolographic analysis. In control animals, morphine and fentanyl produced a dose-related antinociceptive action in the murine assays and the rank of potency was: formalin hind paw phase I > formalin phase II > tail ick. Coadministration of morphine with fentanyl, in a xed relation 1:1 of their ED 50 , produces a dose response in all tests and the isobologram resulted in synergism. Fentanyl was more effective than morphine which could be explained according the suggestion that opioids could be acting through other targets, with different binding capacity thru the regulation or activation of non-opioid receptors. Co-administration of morphine with fentanyl induces synergism in all murine trials, conrming the antinociceptive capacity of both opioids which would constitute a promisory idea to multimodal treatment of pain.


Introduction
In the therapy of pain, opioids are among the most effective pain relievers available. These drugs have demonstrated analgesic e cacy in different animal models of pain, including models of tonic pain: acetic acid and formalin writhing test and phasic pain tests: tail movement and hot plate. Opioids comprise a variety of drugs both natural (morphine, codeine) and synthetic (fentanyl, methadone, heroin), all of which are powerful and effective pain relievers, but with high potential for dependency and abuse.
Opioid receptors are coupled to G proteins and divided into subtypes: MOR (µ1, µ2, µ3); DOR (δ1, δ2, δ3); and KOR (κ1, κ2, κ3).They are transmembrane domains, three extracellular and three intracellular loops with N-terminal extracellular amino acid terminations and intracellular C-terminal carboxyl. Opioid receptors are found in both the central and peripheral nervous systems and activation of these receptors by ligands induces signi cant molecular changes inside the cell, such as an inhibition of adenylate cyclase activity, activation of potassium channels and reductions of calcium conductance. In this context, G protein receptor kinases (GRKs) are important modulators of opioid receptor function. Recent data indicate that other signalling pathways also may be involved in morphine activity. Among these are phospholipase C, mitogen-activated kinases (MAP kinases) or β-arrestin. There are endogenous natural ligands for opioid receptors such as β-endorphins, encephalin, dynorphins, and nociceptin/orphanin FQ.
The different opioids, due to their frequent use, deserve to be highlighted: (A) Natural opioid morphine with powerful analgesic effects, used in clinical medicine for almost two hundred years and acts by activating MOR-type receptors in the CNS and PNS. (B) Synthetic opioid fentanyl with pain action similar to morphine, but 50 to 200 times more potent. Like morphine, fentanyl is an agonist of MOR-like receptors in the CNS and PNS. Fentanyl is effective in both induction and maintenance of anesthesia (Pathan and Williams 2012;Corder et al. 2018).
The multimodal analgesia, a method of combining analgesic drugs, is an approach should be used when treating pain reactive to the action of a single analgesic, which leads, for example in the case of opioids, to a potential reduction in undesirable effects such as nausea, vomiting, respiratory depression and also provide relief of pain using different routes of cellular action. When two drugs are taken together if the resulting response can be greater than the sum of the individual response, the interaction between the drugs is synergistic ( Tallarida 2001).
In the present study, the pharmacological co-interaction of morphine with fentanyl was evaluated in different murine pain models by means of isobolographic analysis.

Animals
Male CF-1 mice (25-30g) housed on a 12 h light-dark cycle at 22±1 °C with free access to food and water ad libitum, were used. All procedures with animals from the central nursery were approved by the Animal Care and Use Committee of the Faculty of Medicine. (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 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) tail-ick test (TF) as described previously ( Miranda et al. 2001). A radiant heat, automatic tail ick (Ugo Basile, Comerio, Italy) was used to measure response latencies. Baseline was obtained before protocol and then test latency after administration of drugs. A cut-off time of 8 sec was set to avoid damage of mice tissue. TF latencies controls were 2.65 ± 0.12 (n=12) and converted to % MPE.
(B) the formalin hind paw (FHP) test described previously was used (Miranda et al. 2001). To perform the test 20 µL of 2 % formalin solution was injected into the dorsal surface of the right hind paw. The intensity of pain was assessed as the time, in sec, by the licking or biting of the injected paw. The test show 2 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 values were, phase I: 133.05 ± 7.04 (n =12) and phase II: 157.83 ± 9.10 (n=12). Licking time was converted to % MPE.

Experimental design
The antinociceptive potency of morphine and fentanyl, a dose-response curve from 0.1, 0.3, 1 and 3 mg/kg, i.p. for morphine and 0.03, 0.06, 0.12 and 0.24 mg/kg i.p. for fentanyl was obtained in the WT, TF, FHP and HP tests using at least 6 animals for each at least 4 doses, as can be seen in gure 1. Then the DE 50 , dose that induce 50% of MPE, was calculated from lineal regression of dose-response curves of morphine and fentanyl.

Isobolographic analysis
The interaction morphine with fentanyl ws evaluated by isobolography ( Miranda et al., 2001( Miranda et al., , 2007( Miranda et al., , 2012. The isobolograms were constructed connecting ED 50 of fentanyl in the abscissa with ED 50 of morphine in the ordinate to obtain the additive line. For each combination, the ED 50 was obtained and

Statistical analysis
Results are presented as means ± SEM or 95 % con dence limits (95 % CL). 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) considered statistically signi cant. Statistical analyses were carried out using the program Pharm Tools Pro, version 1.27, Mc Cary Group Inc., PA, USA.

Results
Antinociception of morphine and fentanyl.
In control mice, i.p. administration of morphine and fentanyl produced a dose-related antinociceptive action in the FHP and TF assays and the ED 50 with SEM resultant are shown in Table 1 and Fig.1. The order of analgesic ratio for morphine was: phase I of FHP > phase II of FHP > TF. In fentanyl the order was: phase II of FHP > phase I of FHP > TF. Comparison of the potency of NSAIDs was: phase I of FHP > phase II of PHF > TF. Data in Table 1.

Analysis of interaction morphine with fentanyl
The i.p. coadministration of morphine with fentanyl, in relation 1:1 of their ED 50 , produces a dose response in all experimental conditions. The isobologram demonstrated that the opioids combination, in the FHP and TF, resulted in a synergistic interaction of different magnitude, as can be seen in Table 2 and Fig.2 . The degree of potency of the mixture, according the interaction index, revealed the following rank: FHP, phase II > TF > FHP, phase I. These changes were accompanied by similar modi cation of ED 50 values (see Table 2).

Discussion
Opioids are widely used in the treatment of pain. Opioids activate, at the supraspinal level, the MOR receptors of the GABAergic interneurons of the descending pathway of pain modulation and the dorsal horn of the spinal cord. These GABA changes contribute to opioid-induced antinociception. However, antinociception induced by them has not been extensively studied in different animal pain models. The most of the major studies have been conducted with isolated opioids, but have not been used in combination, as suggested by multimodal analgesia. This study demonstrated that morphine and fentanyl induce antinociception in FHP and HP tests, with different antinociceptive potency, with fentanyl had a higher effect than morphine, in all tests, results consistent with the analgesic properties of fentanyl being approximately 100 times more potent than morphine. Furthermore, fentanyl has a different binding capacity, expressed in nmol, from 0.7 to 1.9, compared to morphine from 1.02 to 4 ( Zaveri et al. 2001;Waldhoer et al. 2004).
The present results are in agreement with previous studies, which demonstrate that morphine and fentanyl induce analgesia in different animal pain tests, such as acetic acid writhing, formalin hind paw, hot plate and tail ick assays (Miranda et al., 2001(Miranda et al., , 2007(Miranda et al., , 2012Romero et al. 2010). Co-administration of morphine with fentanyl demonstrated synergism in the FHP and TF assays being more powerful in the FHP-I and less potent in phase II of FHP. These results are in agreement with the general basis of synergism which propose that the effect occurs when two drugs are co-administered with same effect but have a different mechanism of action. In the present work, the interaction could be attributed to various levels of cellular function, among which pain receptors, messengers or mediators can be mentioned.
Opioids are known to interact with speci c receptors (MOR, DOR, KOR, NOP) with different selectivity and pharmacological and biochemical studies have demonstrated the existence of modulatory interactions between opioid and receptors. It has been suggested that morphine antinociception is preferentially mediated through MOR and it is believed that most clinical opioids exert their antinociceptive effects through MOR. Opioids may show different e ciencies probably due to MOR receptor subtypes, since 5 splice variants of the mouse-MOR have been described, so as functional consequences, they could explain the greater e cacy of fentanyl over morphine ( Pasternak 2004;Anathan 2006).
The study shows that fentanyl is a more effective antinociceptive opioid than morphine. This nding is consistent with a previous study that revealed different mechanisms for the antinociception of morphine and fentanyl (Morgan et al. 2020). Furthermore, it could be explained by the suggestion that some opioids could be acting through other mechanisms. Thus, a multi-opioid study using radioligand binding and functional activity assays found novel interactions, including activation of the monoamine transporter. Besides, morphine interacts with α2 adrenergic receptors (α2A, α2B and α2C), on the contrary, fentanyl did not show a nity for α2 adrenoceptors, this effect may have an impact on the pharmacological actions of morphine. Also, it has been reported that various opioids inhibit monoamine transporters (NET and SERT) and interact with 5-HT receptors. Fentanyl has a nity for the 5-HT1A and 5-HT2A receptors, but there are no correlated data available regarding monoamine transporters, for both fentanyl and morphine (Sirohi et al. 2008;Hocker et al. 2009;Keith et al. 2019;Luethi and Liechti 2020).
The preceding antecedents show that there are interactions between opioids that are modulated by other non-opioid receptors in addition to the opioid receptors themselves and that could explain the differences between the antinociceptive activity induced by morphine and that obtained by fentanyl in the present work.

Conclusion
The present study, the e cacy of fentanyl in FHP and TF assays was relatively greater compared to the morphine. The greater effectiveness of fentanyl could lie by a different binding capacity or in the regulation or activation of opioids and non-opioid receptors. Co-administration of morphine with fentanyl induces synergism in all murine trials, con rming the antinociceptive capacity of both opioids, which would constitute a promisory inkling to treatment of pain.

Competing interests
All the authors declare that they have no con icts of interest Funding The study was not funded Tables   TABLE 1: ED 50 values with SEM in mg/kg and analgesic ratio (AR) for the antinociceptive activity of morphine and fentanyl in algesimeter tests of mice.