Neuropharmacology and Analgesia
Morphine-induced antinociception in the rat: Supra-additive interactions with imidazoline I2 receptor ligands

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Abstract

Pain remains a significant clinical challenge and currently available analgesics are not adequate to meet clinical needs. Emerging evidence suggests the role of imidazoline I2 receptors in pain modulation primarily from studies of the non-selective imidazoline receptor ligand, agmatine. However, little is known of the generality of the effect to selective I2 receptor ligands. This study examined the antinociceptive effects of two selective I2 receptor ligands 2-BFI and BU224 (> 2000-fold selectivity for I2 receptors over α2 adrenoceptors) in a hypertonic (5%) saline-induced writhing test and analyzed their interaction with morphine using a dose-addition analysis. Morphine, 2-BFI and BU224 but not agmatine produced a dose-dependent antinociceptive effect. Both composite additive curve analyses and isobolographical plots revealed a supra-additive interaction between morphine and 2-BFI or BU224, whereas the interaction between 2-BFI and BU224 was additive. The antinociceptive effect of 2-BFI and BU224 was attenuated by the I2 receptor antagonist/α2 adrenoceptor antagonist idazoxan but not by the selective α2 adrenoceptor antagonist yohimbine, suggesting an I2 receptor-mediated mechanism. Agmatine enhanced the antinociceptive effect of morphine, 2-BFI and BU224 and the enhancement was prevented by yohimbine, suggesting that the effect was mediated by α2 adrenoceptors. Taken together, these data represent the first report that selective I2 receptor ligands have substantial antinociceptive activity and produce antinociceptive synergy with opioids in a rat model of acute pain. These data suggest that drugs acting on imidazoline I2 receptors may be useful either alone or in combination with opioids for the treatment of pain.

Introduction

Pain remains a major health problem that markedly reduces quality of life of a large segment of the population and imparts high health costs and economic loss to society. Opioids are the drugs of choice for many pain conditions. However, the unwanted effects related to repeated opioid use including pruritus, constipation and physical dependence limit adequate dosing in the clinic (Annemans, 2011). New analgesics that retain the therapeutic effects but circumvent some of the unwanted effects are in great clinical demand.

One strategy for improved treatment of pain is to combine one opioid with another pharmacologically unrelated drug in the hope that the drug mixture increases the analgesic efficacy while not altering or perhaps diminishing adverse effects of the opioid. However, the practice of this scientifically valid drug development strategy has achieved only modest success thus far (Smith, 2008). For example, opioids in combination with acetaminophen are widely used for pain management. However, the unwanted effects of the drug mixture are similar to those of opioids alone and non-medical use is common (Zacny et al., 2003). This underscores the need to identify new drug targets for analgesic development.

Imidazoline receptors are a group of receptors that recognize compounds with an imidazoline ring, a concept first proposed by Bousquet et al. (1984). Later studies established that the α2 adrenoceptor agonist and imidazoline compound clonidine primarily exerts its hypotensive activity by acting on imidazoline receptors (Head and Mayorov, 2006) and the receptors that have high binding affinity with 3H-para-aminoclonidine and 3Hidazoxan are termed imidazoline I1 receptors (Regunathan and Reis, 1996). Two selective I1 receptor ligands, moxonidine and rilmenidine, are currently used for treating hypertension (Sica, 2007). Imidazoline I2 receptors are binding sites that bind 3H-idazoxan and 3H-2-BFI with high affinity and 3H-para-aminoclonidine and 3H-clonidine with much lower affinity (Regunathan and Reis, 1996). Imidazoline I2 receptors might be implicated in several psychiatric disorders including depression, opioid addiction and neurodegenerative diseases as the density of I2 receptors is significantly different in patients who suffer from those disorders as compared to control (Garcia-Sevilla et al., 1999). However, the possible functional relationship between I2 receptors and these disorders remains to be elucidated (Garcia-Sevilla et al., 1999). Autoradiographical studies indicate that I2 receptors are widely distributed in the central nervous systems, with high bindings to the area postrema, interpeduncular nucleus, arcuate nucleus, mammillary peduncle, ependyma and pineal gland (Lione et al., 1998). Emerging evidence indicates that the cationic polyamine, agmatine, possesses antinociceptive and analgesic activity both in animals and in man (Li and Zhang, 2011). Agmatine is a non-selective low-affinity imidazoline I1 and I2 receptor ligand but also has affinity for α2 adrenoceptors, NMDA receptors, and nicotinic receptors, and also inhibits nitric oxide production (Berkels et al., 2004, Loring, 1990). The mechanisms of the antinociceptive effects of agmatine primarily involve I2 receptors and α2 adrenoceptors (Li et al., 1999, Roerig, 2003). Although the antinociceptive effects of α2 adrenoceptor agonists are well established, there are only limited data concerning the antinociceptive effects of I2 receptor ligands, and few studies employ selective I2 receptor ligands (Gentili et al., 2006, Sanchez-Blazquez et al., 2000).

Consistent with the effects of agmatine, selective I2 receptor ligands enhance the antinociceptive effects of morphine and attenuate the development of tolerance to morphine antinociception for pain following thermal stimulation (Boronat et al., 1998, Sanchez-Blazquez et al., 2000). However, previous studies only employed one procedure (radiant tail flick) and a single dose is typically used. Thus, it is unclear of the extent to which these findings relate to other models of pain and the nature of the interaction between I2 receptors and opioid receptors. This study investigated the antinociceptive effects of agmatine, morphine and two selective I2 receptor ligands 2-BFI and BU224 (Fig. 1) using a hypertonic saline-induced writhing test in the rat. Furthermore, potential receptor mechanisms were explored using pharmacological antagonists and the application of quantitative pharmacological analysis.

Section snippets

Subjects

Two groups of adult male Sprague–Dawley rats (Harlan, Indianapolis, IN) were housed individually under a 12/12-h light/dark cycle beginning at 6:00 a.m. (experiments were conducted during the light period) with free access to standard rodent chow and water in the home cage. One group of 9 rats contributed to the data shown in Fig. 2, Fig. 3, Fig. 4 and a second group of 8 rats contributed to the data of Fig. 5, Fig. 6. Animals were maintained and experiments were conducted in accordance with

Results

The hypertonic (5%) saline-induced writhing response was stable within sessions. The writhing response for both groups of rats was examined at the beginning and the completion of the study and the percentage of rats demonstrating writhing response for four consecutive 20-min cycles were (mean ± S.E.M.): 89% ± 7.9%, 94% ± 3.4%, 92% ± 5.3% and 88% ± 0.4%.

Morphine and the selective imidazoline I2 receptor ligands, 2-BFI and BU224, produced antinociceptive effects in a dose-related manner (Fig. 2; see Table 1

Discussion

The primary finding of the current study is that the selective imidazoline I2 receptor ligands, 2-BFI and BU224, alone had antinociceptive effects and in combination with morphine produced antinociceptive effects in a supra-additive manner in a writhing test. Pharmacological antagonism studies indicate that the effects of 2-BFI and BU224 were mediated by I2 receptors but not by α2 adrenoceptors. In contrast, the non-selective I2 receptor ligand/α2 adrenoceptor agonist agmatine alone did not

Acknowledgments

The authors thank Wonjin Shin for her expert technical assistance. None of the authors has any conflict of interest with this work.

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