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Morphine-induced antinociception in the rat: Supra-additive interactions with imidazoline I2 receptor ligands
European Journal of Pharmacology 669 (2011) 59–65
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Morphine-induced antinociception in the rat: Supra-additive interactions with imidazoline I2 receptor ligands Jun-Xu Li a,⁎, Yanan Zhang b, Jerrold C. Winter a a b
Department of Pharmacology and Toxicology, University at Buffalo, NY 14214, USA Research Triangle Institute, Research Triangle Park, NC 27709, USA
a r t i c l e
i n f o
Article history: Received 11 May 2011 Received in revised form 4 July 2011 Accepted 30 July 2011 Available online 16 August 2011 Keywords: Imidazoline I2 receptor Morphine Antinociception Writhing test
a b s t r a c t 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 (N 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 doseaddition 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. © 2011 Elsevier B.V. All rights reserved.
1. 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 ⁎ Corresponding author at: Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, University at Buffalo, 102 Farber Hall, Buffalo, NY 14214-3000, USA. Tel.: + 1 716 829 2482; fax: + 1 716 829 2801. E-mail address: [email protected] (J.-X. Li). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.07.041
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
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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. 2. Materials and methods 2.1. 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 Figs. 2–4 and a second group of 8 rats contributed to the data of Figs. 5–6. Animals were maintained and experiments were conducted in accordance with the Institutional Animal Care and Use Committee, University at Buffalo, and with the 1996 Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences).
Fig. 2. Antinociceptive effects of morphine, 2-BFI, BU224 and agmatine in a hypertonic saline induced writhing test in rats. Ordinate: percentage of antinociception, expressed as the percentage of rats that did not demonstrate writhing response (n = 9). Abscissa: dose of drugs in milligram per kilogram.
2.2. Behavioral test A hypertonic saline solution-induced writhing test was used in the current study as described previously with minor modification (Fukawa et al., 1980). Preliminary study showed that an injection of 5% saline (intraperitoneally, i.p.) at a volume of 2 ml/kg elicited a reliable writhing response in more than 80% of the rats and the writhing response was stable during repeated injections with an inter-injection period of 20 min. This dosing paradigm was used throughout the study and no more than 4 saline injections (4 cycles) were administered in a given test session. Tests were separated by at least 3 days to minimize the potential drug interactions between test sessions and to decrease the stress due to repeated testing. All the studies were conducted in a quiet behavioral test room next to the animal colony room, with similar lighting, environmental temperature and humidity. Each rat was weighed and put into a clear cage, which served as an observation arena, for a 30 min habituation period. The observation arena was identical to the home cage except that corn cob bedding was used to facilitate observation, whereas wood chip bedding was used for home cages. For each cycle, rats were injected with 5% saline and then immediately put into the observation cage and the number of writhing response was recorded for up to 5 min. All drugs were administered immediately prior to the injection of 5% saline solution. Dose–effect relationships were determined using a cumulative dosing procedure with 0.5 log unit increments. For antagonism studies, an antagonist was administered 10 min before the start of the session. 2.3. Drugs Morphine sulfate, agmatine sulfate, idazoxan hydrochloride ((±)-2[1,4-benzodioxan-2-yl]-2-imidazoline hydrochloride) and yohimbine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Fig. 1. Chemical structures of agmatine and three imidazoline compounds (2-BFI, BU224 and idazoxan).
Fig. 3. Antinociceptive effects of morphine in the presence and absence of different doses of agmatine treatment (n = 9). See Fig. 2 for other details.
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Fig. 4. Composite additive curves and isobolographical plots of morphine + 2-BFI, morphine + BU224 and 2-BFI + BU224. Left three panels: percentage of antinociception plotted as a function of log dose (milligram per kilogram). The dashed lines indicated the expected composite additive curves and the solid lines indicated the observed composite additive curves for the drug mixture. Right panel: the ED50 values (± 95% CL) of one drug plotted as a function of the ED50 values (± 95% CL) of the second drug in the pair of drug interaction studies. The diagonal solid line indicated the line of additivity and the diagonal dashed lines indicated the 95% CL from the line of additivity.
2-BFI hydrochloride (2-(2-benzofuranyl)-2-imidazoline hydrochloride) and BU224 hydrochloride (2-(4, 5-dihydroimidazol-2-yl) quinoline hydrochloride) were synthesized according to the reported procedures (Ishihara and Togo, 2007). All drugs were dissolved in sterile water
and administered i.p. Doses are expressed as milligrams of the form indicated above per kilogram of body weight. Injection volumes were 1 ml/kg. 2.4. Data analysis
Fig. 5. Antinociceptive effects of 2-BFI (left) and BU224 (right) in the presence and absence of yohimbine or idazoxan (n = 8). See Fig. 2 for other details.
The criterion for a writhing response was as defined by Collier et al. (1968): a wave of constriction and elongation passing caudally along the abdominal wall, accompanied by a twisting of the trunk and followed by extension of the hind limbs. The number of rats meeting this criterion was recorded. The absence (inhibition) of this response in 5 min was calculated as percentage of antinociception according to: 100 × (nonresponders/group size) (Raffa et al., 2000). Quantal dose–response curves were generated where possible and the ED50 values and 95% confidence limit (CL) estimates were determined using Pharm Tools Pro version 1.1 for Windows (The McCary Group Inc., Elkins Park, PA, USA) based on Litchfield and Wilcoxon (1949) method. Interactions between drugs that alone produced antinociceptive effects (morphine, 2-BFI and BU224) were assessed using a fixed ratio dose addition analysis. For this analysis, two drugs were combined in a fixed proportion and administered using a cumulative dosing procedure. Dose–response curves for drugs administered alone and
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Fig. 6. Antinociceptive effects of morphine (left), 2-BFI (center) and BU224 (right) in the presence or absence of 100 mg/kg agmatine or 100 mg/kg agmatine + 3.2 mg/kg yohimbine (n = 8). See Fig. 2 for other details. Note that the 100 mg/kg agmatine dose–response curve (left panel) was replotted from Fig. 3 for comparison purpose.
in combination were analyzed for parallelism with an F-test (Pharm Tools Pro version 1.1 for Windows). For drug mixtures, the dose– response curves were determined and the individual ED50 values of the two drugs in the mixture were calculated based upon the shared dose–response curves. The sum of the ED50 values of both drugs in the mixture was defined as Zmix. The experimentally determined (observed) ED50 values (Zmix) were compared with the predicted additive ED50 values (Zadd) and were considered significantly different when the 95% CL did not overlap. Zadd and Zmix values were calculated using Pharm Tools Pro version 1.1 for Windows (The McCary Group Inc., Elkins Park, PA, USA) based on the procedures described previously (Stevenson et al., 2005; Tallarida, 2000). Three fixed ratios (1:3, 1:1 and 3:1) were used in the current study. The slopes and the differences of the experimentally determined composite additive curves and the predicted composite additive curves were compared using the F-test. If the calculated F values are greater than the tabular F values, then the two curves are considered significantly different. Conversely, if the calculated F values are smaller than the tabular F values, then the two curves are considered not significantly different. A significant leftward shift of the experimentally determined composite additive curve indicates that the drug mixture produces the effect in a manner that is greater than additivity (supraadditivity). Isobolograms were constructed to examine whether the effects of drug mixtures were additive, supra-additive, or infraadditive (Li et al., 2011b). An isobologram plots equieffective doses (e.g., ED50) of one drug in the presence of different doses of a second drug. If the effects of the two drugs are additive, then the ED50 values (± 95% CL) for the drug combination should overlap with the diagonal line between the ED50 values (±95% CL) for the two drugs alone (line of additivity). If the ED50 values (±95% CL) fall below the limits of the line of additivity, then the effects of the two drugs are considered to be supra-additive (i.e., in the presence of one drug, smaller than predicted doses of a second drug are needed to produce the same effect). If the ED50 values (±95% CL) fall above the limits of the line of additivity, then the effects of the two drugs are considered to be infra-additive (i.e., in the presence of one drug, larger than predicted doses of a second drug are needed to produce the same effect). 3. 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 for ED50 values). The rank order of potency for antinociception was morphine N2-BFI ≈ BU224. Agmatine did not show reliable antinociceptive effects up to a cumulative dose of 100 mg/kg. Because agmatine alone did not decrease the writhing response, it was administered as a pretreatment to determine whether agmatine modified the antinociceptive effects of morphine. In the presence of 10, 32 and 100 mg/kg agmatine, the dose–effect curves for morphine were shifted leftward in a dose-dependent manner (Fig. 3), decreasing the ED50 values from 0.56 [(0.39, 0.69), 95% CL] mg/kg to 0.81 (0.63, 0.99) mg/kg, 0.20 (0.15, 0.25) mg/kg and 0.12 (0.09, 0.15) mg/kg, respectively. Thus, 32 mg/kg and 100 mg/kg agmatine shifted the morphine dose–effect curve 2.8-fold and 4.7-fold leftward, respectively. Fixed ratio dose-addition analyses revealed a supra-additive interaction between morphine and 2-BFI or BU224. For the three fixed ratios studied (1:3, 1:1 and 3:1), the experimentally determined dose–effect curves were all significantly different from the expected composite additive curves (top and central left panels, Fig. 4; calculated F values ≥6.506, tabular F values ≤5.790), although the slopes were not significantly different (calculated F values ≤5.823, tabular F values ≥ 5.990). Thus, when the two drugs were studied in combination, the potencies (Zmix) were increased by 2.43- to 6.75-fold as compared to the expected potencies (Zadd) (Table 2), and this increase was statistically significant, as the 95% CLs did not overlap between Zadd and Zmix. Isobolographical analysis further supports the conclusion of supra-additivity (right panel, Fig. 4). For all the fixed ratios of the drug mixture, the experimentally determined ED50 values fell below the lines of additivity (±95% CL), suggesting a supra-additive interaction. The interaction between 2-BFI and BU224 was additive (bottom panels, Fig. 4). For all the three fixed ratios studied (1:3, 1:1 and 3:1), the slopes of the experimentally determined dose–response
Table 1 ED50 values (mg/kg, 95% CL) and relative potency (compared with BU224) for morphine, 2-BFI and BU224 in inhibiting writhing response (antinociception) in rats. Compound
ED50 value (95% CL)
Relative potency
BU224 2-BFI Morphine
3.51 (2.72, 4.30) 3.13 (2.42, 3.84) 0.56 (0.39, 0.68)
1 1.12 6.28
J.-X. Li et al. / European Journal of Pharmacology 669 (2011) 59–65 Table 2 Observed (experimentally determined) ED50 values (Zmix) and expected additive ED50 values (Zadd), and the ratio of expected/observed ED50 values for drug mixtures for antinociception in rats. Mixture Relative dose (proportion) Morphine:2-BFI 1.86:1 (1:3) 5.59:1 (1:1) 16.78:1 (3:1) Morphine:BU224 2.09:1 (1:3) 6.28:1 (1:1) 18.84:1 (3:1) 2-BFI:BU224 0.37:1 (1:3) 1.12:1 (1:1) 3.37:1 (3:1) a
Zadd (95% CL)
Zmix (95% CL)
Ratio Zadd/Zmix
2.48 (2.08, 2.89) 1.84 (1.545, 2.14) 1.20 (1.01, 1.39)
a
0.50 (0.33, 0.67) 0.27 (0.18, 0.36)a 0.49 (0.36, 0.63)a
4.98 6.75 2.43
2.77 (2.33, 3.22) 2.03 (1.71, 2.36) 1.30 (1.09, 1.50)
0.58 (0.39, 0.76)a 0.51 (0.35, 0.68)a 0.34 (0.27, 0.42)a
4.80 3.96 3.79
3.41 (2.87, 3.96) 3.32 (2.79, 3.85) 3.22 (2.70, 3.74)
2.11 (1.50, 2.72)a 2.64 (2.05, 3.22) 2.77 (2.13, 3.41)
1.62 1.26 1.16
The 95% CL of Zmix did not overlap with the 95% CL of Zadd.
curves were not significantly different from those of the expected composite additive curves (calculated F values ≤ 2.621, tabular F values ≥5.990), and the curves were also not significantly different (calculated F values ≤2.978, tabular F values ≥5.14). Isobolographical analysis suggested an additive interaction (right panel, Fig. 4), as evidenced by the fact that the experimentally determined ED50 values overlapped with the line of additivity (±95% CL). The antinociceptive potencies of morphine, 2-BFI and BU224 between the two groups of animals were not significantly different (compare Fig. 2 and Fig. 6). The ED50 values for morphine were 0.56 (0.39, 0.69) mg/kg and 0.63 (0.46, 0.81) mg/kg, respectively. The ED50 values for 2-BFI were 3.13 (2.42, 3.84) and 3.18 (2.23, 4.13) mg/kg, respectively. The ED50 values for BU224 were 3.51 (2.72, 4.30) and 3.18 (2.23, 4.13) mg/kg, respectively. Thus, the within-group comparisons were conducted using respective dose–effect curves as references. The antinociceptive effects of 2-BFI and BU224 were attenuated by pretreatment with the non-selective I2 receptor antagonist/α2 adrenoceptor antagonist idazoxan, but not by the selective α2 adrenoceptor antagonist yohimbine. In the presence of 1 mg/kg idazoxan, the dose–effect curve of 2-BFI was shifted 4.4-fold rightward, increasing ED50 values from 3.18 (2.23, 4.13) to 14.15 (8.95, 19.34) mg/kg (Fig. 5). A dose of 3.2 mg/kg yohimbine did not markedly alter the effect of 2-BFI in that the ED50 value (5.06 [3.67, 6.45] mg/kg) was not significantly different from that without yohimbine treatment. Similar results were observed for BU224. Idazoxan (1 mg/kg) shifted the dose–effect curve of BU224 3.9-fold rightward, increasing ED50 values from 3.18 (2.23, 4.13) to 12.28 (6.56, 18.01) mg/kg, and comparison of the two curves approached statistical significance (calculated F values = 17.231, tabular F values = 19.000). A dose of 3.2 mg/kg yohimbine did not change the dose–effect curve of BU224 (ED50 = 3.18 [2.23, 4.13] mg/kg). Although pretreatment with 100 mg/kg agmatine markedly shifted the morphine dose–effect curve leftward (Fig. 3), a dose of 3.2 mg/kg yohimbine pretreatment prevented this enhancement. Thus, in the presence of 3.2 mg/kg yohimbine and 100 mg/kg agmatine, the ED50 value was 0.55 (0.28, 0.83) mg/kg, which was not significantly different from that without drug treatment (0.63 [0.46, 0.81] mg/kg) (left panel, Fig. 6). Pretreatment with 100 mg/kg agmatine also shifted the dose–effect curves of 2-BFI and BU224 5.5and 8.7-fold leftward, respectively (Fig. 6). The ED50 values of 2-BFI and BU224 were decreased from 3.18 (2.23, 4.13) mg/kg to 0.58 (0.29, 0.86) mg/kg, and from 3.18 (2.23, 4.13) mg/kg to 0.37 (0.26, 0.48) mg/ kg, respectively, by 100 mg/kg agmatine. The enhancement of the antinociceptive effects of 2-BFI and BU224 by agmatine was prevented by co-administration of 3.2 mg/kg yohimbine (center
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and right panels, Fig. 6). In the presence of 3.2 mg/kg yohimbine and 100 mg/kg agmatine, the ED50 values of 2-BFI and BU224 were 2.26 (1.43, 3.09) mg/kg and 2.00 (1.44, 2.55), respectively. 4. 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 produce marked antinociceptive effect under this condition, but enhanced the antinociceptive effect of morphine, 2-BFI and BU224, and pharmacological antagonism studies revealed that the effect was primarily mediated by α2 adrenoceptors. This represents the first study demonstrating that selective I2 receptor ligands have substantial antinociceptive effects and produce antinociceptive synergy with morphine, which is in accordance with the idea that I2 receptors could be a target for developing new analgesics (Li and Zhang, 2011). When administered intraperitoneally, many irritant chemicals elicit a characteristic writhing response in rodents (Le Bars et al., 2001). Writhing induced by hypertonic saline in rats as a procedure to evaluate analgesic agents was first described in 1980 (Fukawa et al., 1980). Compared with the widely used acetic acid-induced writhing test, this procedure is more sensitive and specific to analgesics from diverse pharmacological classes (Fukawa et al., 1980). Unlike the acetic acid-induced writhing test, which produces robust writhing responses which persist for minutes, hypertonic saline produces reliable but very brief writhing responses in rats. More importantly, repeated treatment with hypertonic saline does not cause tissue damage and does not demonstrate tolerance to the stimulus (Fukawa et al., 1980), which is especially suitable for studying repeated drug treatment and a within-group design. For instance, writhing usually occurred within 10 s after 5% saline injection, and ceased within 60 s in the current study. In a group of 9 rats, 5% saline produced an average of 2.7 (range = [0, 5]) writhing responses for each rat and the total responses did not markedly change within a session of 4 treatments. Thus, a quantal data analysis was employed in the current study. Imidazoline receptors recognize compounds with an imidazoline ring, a concept first proposed by Bousquet et al. (1984). Imidazoline I2 receptors are receptors 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). The cationic polyamine, agmatine, was proposed as the endogenous imidazoline receptor ligand in 1994 (Li et al., 1994) and the physiological and pharmacological functions of agmatine have been extensively studied. One of the most widely recognized roles of agmatine is pain modulation (Li and Zhang, 2011). However, agmatine non-selectively binds to imidazoline I1, I2 receptors, and α2 adrenoceptors, and also has low affinity for NMDA receptors and nicotinic receptors (Halaris and Plietz, 2007; Li et al., 1994; Loring, 1990; Regunathan and Reis, 1996). Agmatine also modulates nitric oxidase synthase activity (Berkels et al., 2004). Agmatine shows antinociceptive activity and enhances the antinociceptive effects of morphine in several animal models of acute, inflammatory and neuropathic pain. Its effect is thought to be mediated by α2 adrenoceptors, imidazoline I2 receptors, serotonergic 5-HT2 receptors, or modulation of nitric oxygen synthesis, depending on the experimental conditions (Li and Zhang, 2011). The current study showed that the effect of agmatine for enhancing morphine, 2-BFI and BU224 antinociception was mediated by α2 adrenoceptors, as the enhancement was completely prevented by the selective α2 adrenoceptor antagonist, yohimbine. Interestingly, the enhancement of agmatine is not limited to opioids induced antinociception. For
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example, agmatine also enhances the antinociceptive effects of drugs acting at cannabinoid CB1 receptors (Aggarwal et al., 2009). It is surprising that agmatine alone did not inhibit the writhing response in the present study, as it has been shown to be effective in the same assay (Li et al., 1999). The reason for this discrepancy is unclear; however, several important differences exist between the two studies. Previous study used both male and female Wistar rats while the current study used male Sprague–Dawley rats. Agmatine was administered subcutaneously in the previous study and i.p. in the current study. It has been shown that the route of drug administration plays an important role in the effects of agmatine (Nguyen et al., 2003). Thus, these methodological differences may contribute to the observed discrepancy. Although several studies employing the non-selective α2 adrenoceptor antagonist/imidazoline I2 receptor antagonist idazoxan have suggested that the antinociceptive effects of agmatine are mediated by imidazoline I2 receptors under some conditions, consensus is dampened by the non-selective nature of both agmatine and idazoxan. The application of highly selective imidazoline I2 receptor ligands could greatly improve our confidence of the role of I2 receptors in pain modulation. Although several selective imidazoline I2 receptor ligands are currently available (Gentili et al., 2006; Hudson et al., 2003), few studies have examined the antinociceptive effects of these compounds. Two studies reported that several selective I2 receptor ligands alone showed no antinociceptive effects in a radiant tail flick test in mice, but all enhanced the effects of morphine (Gentili et al., 2006; SanchezBlazquez et al., 2000), which provides the first line of evidence that selective I2 receptor ligands can increase opioid antinociception. This study found that two highly selective I2 receptor ligands, 2-BFI and BU224, produced dose-dependent antinociception in a rat writhing test. This effect was not due to general behavioral suppression, since sedative agents such as chlorpromazine and pentobarbital are inactive in this assay (Fukawa et al., 1980) and, up to a dose of 10 mg/kg, both 2-BFI and BU224 did not alter the locomotor activity in rats (unpublished observation). It has been shown that 2-BFI and BU224 (14 mg/kg, i.p.) increase the rotational behavior in unilateral nigrostriatal dopamine pathway lesioned rats (Macinnes and Duty, 2004), and our observed lack of effect of 2-BFI and BU224 on the locomotor activity could be due to the lower dose used (10 mg/kg) and different neurobiological mechanisms of the two behavioral endpoints. The effects were primarily mediated by I2 receptors but not by α2 adrenoceptors, as both ligands have N2000-fold selectivity for I2 receptors over α2 adrenoceptors in vitro as indicated by radioligand binding studies using 3H-clonidine and 3H-2-BFI as radioligands (Hudson et al., 1999). In addition, the antinociceptive effects were markedly attenuated by the non-selective α2 adrenoceptor antagonist/I2 receptor ligand idazoxan, but not by the selective α2 adrenoceptor antagonist yohimbine. It should be noted that yohimbine also has high affinity for 5-HT1A receptors (Winter and Rabin, 1992), although it is unlikely that 5-HT1A receptor plays a prominent role under this condition, as activation of 5-HT1A receptors does not produce antinociception in models of acute pain (Millan, 1994). BU224 reportedly does not have antinociceptive effects in a tail flick test but prevents the effect of 2-BFI to enhance morphine antinociception (Sanchez-Blazquez et al., 2000). In fact, BU224 is regularly used as an antagonist of imidazoline I2 receptors (Jou et al., 2004; Su et al., 2009), although BU224 behaves like 2-BFI under some conditions (Macinnes and Duty, 2004). Taken together, this may indicate that BU224 is an I2 receptor ligand with lower positive efficacy than 2-BFI, and its pharmacological activity depends on the efficacy requirement of the bioassay (Li and Zhang, 2011). In the present study, 2-BFI and BU224 produced antinociceptive effects with similar potency. This may suggest that the efficacy demand of the writhing test is lower than the tail flick test, and that the positive efficacy of BU224 at I2 receptors is adequate for exerting antinociception under this condition.
Dose-addition analysis is a powerful approach to analyze the nature of drug interactions (e.g., additive, supra-additive or infraadditive) in a quantitative manner when two drugs produce similar effects (Tallarida, 2000). When interactions occur, the effect may depend upon the proportions of the drugs in the mixture. For instance, the interaction between a serotonin 5-HT1A receptor agonist and a 5HT2 receptor agonist for decreasing food-maintained responding is additive when only a small proportion of the 5-HT1A receptor agonist is available in the drug mixture but the interaction is clearly infra-additive when the proportion of 5-HT1A receptor agonist is increased (Li et al., 2011a). In the current study, when two drugs with different mechanisms of action (morphine and 2-BFI or BU224) were studied in combination, the interaction for antinociception was supraadditive across a broad range of drug proportions, revealing clear antinociceptive synergy between morphine and I2 receptor ligands. On the other hand, when two drugs with the same mechanism of action (2-BFI and BU224) were studied in combination, only additive interaction was observed. This is not surprising in that when two drugs with similar mechanisms of action are given in combination, they would be expected to appear as a single drug. One interesting finding of the current study was that agmatine markedly enhanced the antinociceptive effects of 2-BFI and BU224 by activating α2 adrenoceptors. These receptors are critically involved in pain modulation and agonists such as clonidine are effective for treating some intractable clinical pain conditions (Fairbanks et al., 2009; Newsome et al., 2008). Future studies exploring the interaction between I2 receptor ligands and α2 adrenoceptor agonists for antinociception are warranted. Although I2 receptor ligands such as agmatine and 2-BFI enhanced the antinociceptive effects of morphine, suggesting the therapeutic potential of these drug combinations, the enthusiasm could be dampened if these drugs also increase the unwanted effects of opioids. Current available data indicate that agmatine does not increase morphine induced intestinal motility inhibition (Kolesnikov et al., 1996), blocks morphine induced hyperthermia (Rawls et al., 2007), attenuates the development of tolerance to morphine antinociception, blocks chronic morphine treatment induced physical withdrawal symptoms and decreases abuse-related effects of opioids (Li and Zhang, 2011; Wu et al., 2008). The effects of selective I2 receptor ligands such as 2-BFI and BU224 on the unwanted effects of morphine are less clear, although limited data indicate that 2-BFI markedly attenuates precipitation induced withdrawal symptoms in morphine dependent animals (Hudson et al., 1999). Thus, I2 receptor ligands seem to increase the therapeutic (antinociceptive) effects but decrease the unwanted (e.g., tolerance, constipation, and physical dependence) effects of opioids, which further enforces the notion of using I2 receptor ligands and opioids as combination therapies for pain (Li and Zhang, 2011). In summary, this study used quantitative pharmacological analysis to examine the interaction between morphine and selective imidazoline I2 receptor ligands in a writhing test in rats. Selective I2 receptor ligands, 2-BFI and BU224, produced antinociceptive effects and antagonism studies confirmed that the antinociceptive effects were primarily mediated by I2 receptors. Both 2-BFI and BU224 enhanced the antinociceptive effects of morphine and dose-addition analysis revealed a supra-additive interaction. Agmatine enhanced the antinociceptive effects of morphine, 2-BFI and BU224, and the effect was mediated by α2 adrenoceptors. Combined with the finding that selective I2 receptor ligands block the development of tolerance to morphine antinociception (Boronat et al., 1998), these data suggest that selective I2 receptor ligands could be used as a monotherapy or combined with opioids as an adjunct for treating some pain conditions. 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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Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Morphine-induced antinociception in the rat: Supra-additive interactions with imidazoline I2 receptor ligands Jun-Xu Li a,⁎, Yanan Zhang b, Jerrold C. Winter a a b
Department of Pharmacology and Toxicology, University at Buffalo, NY 14214, USA Research Triangle Institute, Research Triangle Park, NC 27709, USA
a r t i c l e
i n f o
Article history: Received 11 May 2011 Received in revised form 4 July 2011 Accepted 30 July 2011 Available online 16 August 2011 Keywords: Imidazoline I2 receptor Morphine Antinociception Writhing test
a b s t r a c t 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 (N 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 doseaddition 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. © 2011 Elsevier B.V. All rights reserved.
1. 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 ⁎ Corresponding author at: Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, University at Buffalo, 102 Farber Hall, Buffalo, NY 14214-3000, USA. Tel.: + 1 716 829 2482; fax: + 1 716 829 2801. E-mail address: [email protected] (J.-X. Li). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.07.041
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
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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. 2. Materials and methods 2.1. 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 Figs. 2–4 and a second group of 8 rats contributed to the data of Figs. 5–6. Animals were maintained and experiments were conducted in accordance with the Institutional Animal Care and Use Committee, University at Buffalo, and with the 1996 Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences).
Fig. 2. Antinociceptive effects of morphine, 2-BFI, BU224 and agmatine in a hypertonic saline induced writhing test in rats. Ordinate: percentage of antinociception, expressed as the percentage of rats that did not demonstrate writhing response (n = 9). Abscissa: dose of drugs in milligram per kilogram.
2.2. Behavioral test A hypertonic saline solution-induced writhing test was used in the current study as described previously with minor modification (Fukawa et al., 1980). Preliminary study showed that an injection of 5% saline (intraperitoneally, i.p.) at a volume of 2 ml/kg elicited a reliable writhing response in more than 80% of the rats and the writhing response was stable during repeated injections with an inter-injection period of 20 min. This dosing paradigm was used throughout the study and no more than 4 saline injections (4 cycles) were administered in a given test session. Tests were separated by at least 3 days to minimize the potential drug interactions between test sessions and to decrease the stress due to repeated testing. All the studies were conducted in a quiet behavioral test room next to the animal colony room, with similar lighting, environmental temperature and humidity. Each rat was weighed and put into a clear cage, which served as an observation arena, for a 30 min habituation period. The observation arena was identical to the home cage except that corn cob bedding was used to facilitate observation, whereas wood chip bedding was used for home cages. For each cycle, rats were injected with 5% saline and then immediately put into the observation cage and the number of writhing response was recorded for up to 5 min. All drugs were administered immediately prior to the injection of 5% saline solution. Dose–effect relationships were determined using a cumulative dosing procedure with 0.5 log unit increments. For antagonism studies, an antagonist was administered 10 min before the start of the session. 2.3. Drugs Morphine sulfate, agmatine sulfate, idazoxan hydrochloride ((±)-2[1,4-benzodioxan-2-yl]-2-imidazoline hydrochloride) and yohimbine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Fig. 1. Chemical structures of agmatine and three imidazoline compounds (2-BFI, BU224 and idazoxan).
Fig. 3. Antinociceptive effects of morphine in the presence and absence of different doses of agmatine treatment (n = 9). See Fig. 2 for other details.
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Fig. 4. Composite additive curves and isobolographical plots of morphine + 2-BFI, morphine + BU224 and 2-BFI + BU224. Left three panels: percentage of antinociception plotted as a function of log dose (milligram per kilogram). The dashed lines indicated the expected composite additive curves and the solid lines indicated the observed composite additive curves for the drug mixture. Right panel: the ED50 values (± 95% CL) of one drug plotted as a function of the ED50 values (± 95% CL) of the second drug in the pair of drug interaction studies. The diagonal solid line indicated the line of additivity and the diagonal dashed lines indicated the 95% CL from the line of additivity.
2-BFI hydrochloride (2-(2-benzofuranyl)-2-imidazoline hydrochloride) and BU224 hydrochloride (2-(4, 5-dihydroimidazol-2-yl) quinoline hydrochloride) were synthesized according to the reported procedures (Ishihara and Togo, 2007). All drugs were dissolved in sterile water
and administered i.p. Doses are expressed as milligrams of the form indicated above per kilogram of body weight. Injection volumes were 1 ml/kg. 2.4. Data analysis
Fig. 5. Antinociceptive effects of 2-BFI (left) and BU224 (right) in the presence and absence of yohimbine or idazoxan (n = 8). See Fig. 2 for other details.
The criterion for a writhing response was as defined by Collier et al. (1968): a wave of constriction and elongation passing caudally along the abdominal wall, accompanied by a twisting of the trunk and followed by extension of the hind limbs. The number of rats meeting this criterion was recorded. The absence (inhibition) of this response in 5 min was calculated as percentage of antinociception according to: 100 × (nonresponders/group size) (Raffa et al., 2000). Quantal dose–response curves were generated where possible and the ED50 values and 95% confidence limit (CL) estimates were determined using Pharm Tools Pro version 1.1 for Windows (The McCary Group Inc., Elkins Park, PA, USA) based on Litchfield and Wilcoxon (1949) method. Interactions between drugs that alone produced antinociceptive effects (morphine, 2-BFI and BU224) were assessed using a fixed ratio dose addition analysis. For this analysis, two drugs were combined in a fixed proportion and administered using a cumulative dosing procedure. Dose–response curves for drugs administered alone and
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Fig. 6. Antinociceptive effects of morphine (left), 2-BFI (center) and BU224 (right) in the presence or absence of 100 mg/kg agmatine or 100 mg/kg agmatine + 3.2 mg/kg yohimbine (n = 8). See Fig. 2 for other details. Note that the 100 mg/kg agmatine dose–response curve (left panel) was replotted from Fig. 3 for comparison purpose.
in combination were analyzed for parallelism with an F-test (Pharm Tools Pro version 1.1 for Windows). For drug mixtures, the dose– response curves were determined and the individual ED50 values of the two drugs in the mixture were calculated based upon the shared dose–response curves. The sum of the ED50 values of both drugs in the mixture was defined as Zmix. The experimentally determined (observed) ED50 values (Zmix) were compared with the predicted additive ED50 values (Zadd) and were considered significantly different when the 95% CL did not overlap. Zadd and Zmix values were calculated using Pharm Tools Pro version 1.1 for Windows (The McCary Group Inc., Elkins Park, PA, USA) based on the procedures described previously (Stevenson et al., 2005; Tallarida, 2000). Three fixed ratios (1:3, 1:1 and 3:1) were used in the current study. The slopes and the differences of the experimentally determined composite additive curves and the predicted composite additive curves were compared using the F-test. If the calculated F values are greater than the tabular F values, then the two curves are considered significantly different. Conversely, if the calculated F values are smaller than the tabular F values, then the two curves are considered not significantly different. A significant leftward shift of the experimentally determined composite additive curve indicates that the drug mixture produces the effect in a manner that is greater than additivity (supraadditivity). Isobolograms were constructed to examine whether the effects of drug mixtures were additive, supra-additive, or infraadditive (Li et al., 2011b). An isobologram plots equieffective doses (e.g., ED50) of one drug in the presence of different doses of a second drug. If the effects of the two drugs are additive, then the ED50 values (± 95% CL) for the drug combination should overlap with the diagonal line between the ED50 values (±95% CL) for the two drugs alone (line of additivity). If the ED50 values (±95% CL) fall below the limits of the line of additivity, then the effects of the two drugs are considered to be supra-additive (i.e., in the presence of one drug, smaller than predicted doses of a second drug are needed to produce the same effect). If the ED50 values (±95% CL) fall above the limits of the line of additivity, then the effects of the two drugs are considered to be infra-additive (i.e., in the presence of one drug, larger than predicted doses of a second drug are needed to produce the same effect). 3. 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 for ED50 values). The rank order of potency for antinociception was morphine N2-BFI ≈ BU224. Agmatine did not show reliable antinociceptive effects up to a cumulative dose of 100 mg/kg. Because agmatine alone did not decrease the writhing response, it was administered as a pretreatment to determine whether agmatine modified the antinociceptive effects of morphine. In the presence of 10, 32 and 100 mg/kg agmatine, the dose–effect curves for morphine were shifted leftward in a dose-dependent manner (Fig. 3), decreasing the ED50 values from 0.56 [(0.39, 0.69), 95% CL] mg/kg to 0.81 (0.63, 0.99) mg/kg, 0.20 (0.15, 0.25) mg/kg and 0.12 (0.09, 0.15) mg/kg, respectively. Thus, 32 mg/kg and 100 mg/kg agmatine shifted the morphine dose–effect curve 2.8-fold and 4.7-fold leftward, respectively. Fixed ratio dose-addition analyses revealed a supra-additive interaction between morphine and 2-BFI or BU224. For the three fixed ratios studied (1:3, 1:1 and 3:1), the experimentally determined dose–effect curves were all significantly different from the expected composite additive curves (top and central left panels, Fig. 4; calculated F values ≥6.506, tabular F values ≤5.790), although the slopes were not significantly different (calculated F values ≤5.823, tabular F values ≥ 5.990). Thus, when the two drugs were studied in combination, the potencies (Zmix) were increased by 2.43- to 6.75-fold as compared to the expected potencies (Zadd) (Table 2), and this increase was statistically significant, as the 95% CLs did not overlap between Zadd and Zmix. Isobolographical analysis further supports the conclusion of supra-additivity (right panel, Fig. 4). For all the fixed ratios of the drug mixture, the experimentally determined ED50 values fell below the lines of additivity (±95% CL), suggesting a supra-additive interaction. The interaction between 2-BFI and BU224 was additive (bottom panels, Fig. 4). For all the three fixed ratios studied (1:3, 1:1 and 3:1), the slopes of the experimentally determined dose–response
Table 1 ED50 values (mg/kg, 95% CL) and relative potency (compared with BU224) for morphine, 2-BFI and BU224 in inhibiting writhing response (antinociception) in rats. Compound
ED50 value (95% CL)
Relative potency
BU224 2-BFI Morphine
3.51 (2.72, 4.30) 3.13 (2.42, 3.84) 0.56 (0.39, 0.68)
1 1.12 6.28
J.-X. Li et al. / European Journal of Pharmacology 669 (2011) 59–65 Table 2 Observed (experimentally determined) ED50 values (Zmix) and expected additive ED50 values (Zadd), and the ratio of expected/observed ED50 values for drug mixtures for antinociception in rats. Mixture Relative dose (proportion) Morphine:2-BFI 1.86:1 (1:3) 5.59:1 (1:1) 16.78:1 (3:1) Morphine:BU224 2.09:1 (1:3) 6.28:1 (1:1) 18.84:1 (3:1) 2-BFI:BU224 0.37:1 (1:3) 1.12:1 (1:1) 3.37:1 (3:1) a
Zadd (95% CL)
Zmix (95% CL)
Ratio Zadd/Zmix
2.48 (2.08, 2.89) 1.84 (1.545, 2.14) 1.20 (1.01, 1.39)
a
0.50 (0.33, 0.67) 0.27 (0.18, 0.36)a 0.49 (0.36, 0.63)a
4.98 6.75 2.43
2.77 (2.33, 3.22) 2.03 (1.71, 2.36) 1.30 (1.09, 1.50)
0.58 (0.39, 0.76)a 0.51 (0.35, 0.68)a 0.34 (0.27, 0.42)a
4.80 3.96 3.79
3.41 (2.87, 3.96) 3.32 (2.79, 3.85) 3.22 (2.70, 3.74)
2.11 (1.50, 2.72)a 2.64 (2.05, 3.22) 2.77 (2.13, 3.41)
1.62 1.26 1.16
The 95% CL of Zmix did not overlap with the 95% CL of Zadd.
curves were not significantly different from those of the expected composite additive curves (calculated F values ≤ 2.621, tabular F values ≥5.990), and the curves were also not significantly different (calculated F values ≤2.978, tabular F values ≥5.14). Isobolographical analysis suggested an additive interaction (right panel, Fig. 4), as evidenced by the fact that the experimentally determined ED50 values overlapped with the line of additivity (±95% CL). The antinociceptive potencies of morphine, 2-BFI and BU224 between the two groups of animals were not significantly different (compare Fig. 2 and Fig. 6). The ED50 values for morphine were 0.56 (0.39, 0.69) mg/kg and 0.63 (0.46, 0.81) mg/kg, respectively. The ED50 values for 2-BFI were 3.13 (2.42, 3.84) and 3.18 (2.23, 4.13) mg/kg, respectively. The ED50 values for BU224 were 3.51 (2.72, 4.30) and 3.18 (2.23, 4.13) mg/kg, respectively. Thus, the within-group comparisons were conducted using respective dose–effect curves as references. The antinociceptive effects of 2-BFI and BU224 were attenuated by pretreatment with the non-selective I2 receptor antagonist/α2 adrenoceptor antagonist idazoxan, but not by the selective α2 adrenoceptor antagonist yohimbine. In the presence of 1 mg/kg idazoxan, the dose–effect curve of 2-BFI was shifted 4.4-fold rightward, increasing ED50 values from 3.18 (2.23, 4.13) to 14.15 (8.95, 19.34) mg/kg (Fig. 5). A dose of 3.2 mg/kg yohimbine did not markedly alter the effect of 2-BFI in that the ED50 value (5.06 [3.67, 6.45] mg/kg) was not significantly different from that without yohimbine treatment. Similar results were observed for BU224. Idazoxan (1 mg/kg) shifted the dose–effect curve of BU224 3.9-fold rightward, increasing ED50 values from 3.18 (2.23, 4.13) to 12.28 (6.56, 18.01) mg/kg, and comparison of the two curves approached statistical significance (calculated F values = 17.231, tabular F values = 19.000). A dose of 3.2 mg/kg yohimbine did not change the dose–effect curve of BU224 (ED50 = 3.18 [2.23, 4.13] mg/kg). Although pretreatment with 100 mg/kg agmatine markedly shifted the morphine dose–effect curve leftward (Fig. 3), a dose of 3.2 mg/kg yohimbine pretreatment prevented this enhancement. Thus, in the presence of 3.2 mg/kg yohimbine and 100 mg/kg agmatine, the ED50 value was 0.55 (0.28, 0.83) mg/kg, which was not significantly different from that without drug treatment (0.63 [0.46, 0.81] mg/kg) (left panel, Fig. 6). Pretreatment with 100 mg/kg agmatine also shifted the dose–effect curves of 2-BFI and BU224 5.5and 8.7-fold leftward, respectively (Fig. 6). The ED50 values of 2-BFI and BU224 were decreased from 3.18 (2.23, 4.13) mg/kg to 0.58 (0.29, 0.86) mg/kg, and from 3.18 (2.23, 4.13) mg/kg to 0.37 (0.26, 0.48) mg/ kg, respectively, by 100 mg/kg agmatine. The enhancement of the antinociceptive effects of 2-BFI and BU224 by agmatine was prevented by co-administration of 3.2 mg/kg yohimbine (center
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and right panels, Fig. 6). In the presence of 3.2 mg/kg yohimbine and 100 mg/kg agmatine, the ED50 values of 2-BFI and BU224 were 2.26 (1.43, 3.09) mg/kg and 2.00 (1.44, 2.55), respectively. 4. 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 produce marked antinociceptive effect under this condition, but enhanced the antinociceptive effect of morphine, 2-BFI and BU224, and pharmacological antagonism studies revealed that the effect was primarily mediated by α2 adrenoceptors. This represents the first study demonstrating that selective I2 receptor ligands have substantial antinociceptive effects and produce antinociceptive synergy with morphine, which is in accordance with the idea that I2 receptors could be a target for developing new analgesics (Li and Zhang, 2011). When administered intraperitoneally, many irritant chemicals elicit a characteristic writhing response in rodents (Le Bars et al., 2001). Writhing induced by hypertonic saline in rats as a procedure to evaluate analgesic agents was first described in 1980 (Fukawa et al., 1980). Compared with the widely used acetic acid-induced writhing test, this procedure is more sensitive and specific to analgesics from diverse pharmacological classes (Fukawa et al., 1980). Unlike the acetic acid-induced writhing test, which produces robust writhing responses which persist for minutes, hypertonic saline produces reliable but very brief writhing responses in rats. More importantly, repeated treatment with hypertonic saline does not cause tissue damage and does not demonstrate tolerance to the stimulus (Fukawa et al., 1980), which is especially suitable for studying repeated drug treatment and a within-group design. For instance, writhing usually occurred within 10 s after 5% saline injection, and ceased within 60 s in the current study. In a group of 9 rats, 5% saline produced an average of 2.7 (range = [0, 5]) writhing responses for each rat and the total responses did not markedly change within a session of 4 treatments. Thus, a quantal data analysis was employed in the current study. Imidazoline receptors recognize compounds with an imidazoline ring, a concept first proposed by Bousquet et al. (1984). Imidazoline I2 receptors are receptors 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). The cationic polyamine, agmatine, was proposed as the endogenous imidazoline receptor ligand in 1994 (Li et al., 1994) and the physiological and pharmacological functions of agmatine have been extensively studied. One of the most widely recognized roles of agmatine is pain modulation (Li and Zhang, 2011). However, agmatine non-selectively binds to imidazoline I1, I2 receptors, and α2 adrenoceptors, and also has low affinity for NMDA receptors and nicotinic receptors (Halaris and Plietz, 2007; Li et al., 1994; Loring, 1990; Regunathan and Reis, 1996). Agmatine also modulates nitric oxidase synthase activity (Berkels et al., 2004). Agmatine shows antinociceptive activity and enhances the antinociceptive effects of morphine in several animal models of acute, inflammatory and neuropathic pain. Its effect is thought to be mediated by α2 adrenoceptors, imidazoline I2 receptors, serotonergic 5-HT2 receptors, or modulation of nitric oxygen synthesis, depending on the experimental conditions (Li and Zhang, 2011). The current study showed that the effect of agmatine for enhancing morphine, 2-BFI and BU224 antinociception was mediated by α2 adrenoceptors, as the enhancement was completely prevented by the selective α2 adrenoceptor antagonist, yohimbine. Interestingly, the enhancement of agmatine is not limited to opioids induced antinociception. For
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example, agmatine also enhances the antinociceptive effects of drugs acting at cannabinoid CB1 receptors (Aggarwal et al., 2009). It is surprising that agmatine alone did not inhibit the writhing response in the present study, as it has been shown to be effective in the same assay (Li et al., 1999). The reason for this discrepancy is unclear; however, several important differences exist between the two studies. Previous study used both male and female Wistar rats while the current study used male Sprague–Dawley rats. Agmatine was administered subcutaneously in the previous study and i.p. in the current study. It has been shown that the route of drug administration plays an important role in the effects of agmatine (Nguyen et al., 2003). Thus, these methodological differences may contribute to the observed discrepancy. Although several studies employing the non-selective α2 adrenoceptor antagonist/imidazoline I2 receptor antagonist idazoxan have suggested that the antinociceptive effects of agmatine are mediated by imidazoline I2 receptors under some conditions, consensus is dampened by the non-selective nature of both agmatine and idazoxan. The application of highly selective imidazoline I2 receptor ligands could greatly improve our confidence of the role of I2 receptors in pain modulation. Although several selective imidazoline I2 receptor ligands are currently available (Gentili et al., 2006; Hudson et al., 2003), few studies have examined the antinociceptive effects of these compounds. Two studies reported that several selective I2 receptor ligands alone showed no antinociceptive effects in a radiant tail flick test in mice, but all enhanced the effects of morphine (Gentili et al., 2006; SanchezBlazquez et al., 2000), which provides the first line of evidence that selective I2 receptor ligands can increase opioid antinociception. This study found that two highly selective I2 receptor ligands, 2-BFI and BU224, produced dose-dependent antinociception in a rat writhing test. This effect was not due to general behavioral suppression, since sedative agents such as chlorpromazine and pentobarbital are inactive in this assay (Fukawa et al., 1980) and, up to a dose of 10 mg/kg, both 2-BFI and BU224 did not alter the locomotor activity in rats (unpublished observation). It has been shown that 2-BFI and BU224 (14 mg/kg, i.p.) increase the rotational behavior in unilateral nigrostriatal dopamine pathway lesioned rats (Macinnes and Duty, 2004), and our observed lack of effect of 2-BFI and BU224 on the locomotor activity could be due to the lower dose used (10 mg/kg) and different neurobiological mechanisms of the two behavioral endpoints. The effects were primarily mediated by I2 receptors but not by α2 adrenoceptors, as both ligands have N2000-fold selectivity for I2 receptors over α2 adrenoceptors in vitro as indicated by radioligand binding studies using 3H-clonidine and 3H-2-BFI as radioligands (Hudson et al., 1999). In addition, the antinociceptive effects were markedly attenuated by the non-selective α2 adrenoceptor antagonist/I2 receptor ligand idazoxan, but not by the selective α2 adrenoceptor antagonist yohimbine. It should be noted that yohimbine also has high affinity for 5-HT1A receptors (Winter and Rabin, 1992), although it is unlikely that 5-HT1A receptor plays a prominent role under this condition, as activation of 5-HT1A receptors does not produce antinociception in models of acute pain (Millan, 1994). BU224 reportedly does not have antinociceptive effects in a tail flick test but prevents the effect of 2-BFI to enhance morphine antinociception (Sanchez-Blazquez et al., 2000). In fact, BU224 is regularly used as an antagonist of imidazoline I2 receptors (Jou et al., 2004; Su et al., 2009), although BU224 behaves like 2-BFI under some conditions (Macinnes and Duty, 2004). Taken together, this may indicate that BU224 is an I2 receptor ligand with lower positive efficacy than 2-BFI, and its pharmacological activity depends on the efficacy requirement of the bioassay (Li and Zhang, 2011). In the present study, 2-BFI and BU224 produced antinociceptive effects with similar potency. This may suggest that the efficacy demand of the writhing test is lower than the tail flick test, and that the positive efficacy of BU224 at I2 receptors is adequate for exerting antinociception under this condition.
Dose-addition analysis is a powerful approach to analyze the nature of drug interactions (e.g., additive, supra-additive or infraadditive) in a quantitative manner when two drugs produce similar effects (Tallarida, 2000). When interactions occur, the effect may depend upon the proportions of the drugs in the mixture. For instance, the interaction between a serotonin 5-HT1A receptor agonist and a 5HT2 receptor agonist for decreasing food-maintained responding is additive when only a small proportion of the 5-HT1A receptor agonist is available in the drug mixture but the interaction is clearly infra-additive when the proportion of 5-HT1A receptor agonist is increased (Li et al., 2011a). In the current study, when two drugs with different mechanisms of action (morphine and 2-BFI or BU224) were studied in combination, the interaction for antinociception was supraadditive across a broad range of drug proportions, revealing clear antinociceptive synergy between morphine and I2 receptor ligands. On the other hand, when two drugs with the same mechanism of action (2-BFI and BU224) were studied in combination, only additive interaction was observed. This is not surprising in that when two drugs with similar mechanisms of action are given in combination, they would be expected to appear as a single drug. One interesting finding of the current study was that agmatine markedly enhanced the antinociceptive effects of 2-BFI and BU224 by activating α2 adrenoceptors. These receptors are critically involved in pain modulation and agonists such as clonidine are effective for treating some intractable clinical pain conditions (Fairbanks et al., 2009; Newsome et al., 2008). Future studies exploring the interaction between I2 receptor ligands and α2 adrenoceptor agonists for antinociception are warranted. Although I2 receptor ligands such as agmatine and 2-BFI enhanced the antinociceptive effects of morphine, suggesting the therapeutic potential of these drug combinations, the enthusiasm could be dampened if these drugs also increase the unwanted effects of opioids. Current available data indicate that agmatine does not increase morphine induced intestinal motility inhibition (Kolesnikov et al., 1996), blocks morphine induced hyperthermia (Rawls et al., 2007), attenuates the development of tolerance to morphine antinociception, blocks chronic morphine treatment induced physical withdrawal symptoms and decreases abuse-related effects of opioids (Li and Zhang, 2011; Wu et al., 2008). The effects of selective I2 receptor ligands such as 2-BFI and BU224 on the unwanted effects of morphine are less clear, although limited data indicate that 2-BFI markedly attenuates precipitation induced withdrawal symptoms in morphine dependent animals (Hudson et al., 1999). Thus, I2 receptor ligands seem to increase the therapeutic (antinociceptive) effects but decrease the unwanted (e.g., tolerance, constipation, and physical dependence) effects of opioids, which further enforces the notion of using I2 receptor ligands and opioids as combination therapies for pain (Li and Zhang, 2011). In summary, this study used quantitative pharmacological analysis to examine the interaction between morphine and selective imidazoline I2 receptor ligands in a writhing test in rats. Selective I2 receptor ligands, 2-BFI and BU224, produced antinociceptive effects and antagonism studies confirmed that the antinociceptive effects were primarily mediated by I2 receptors. Both 2-BFI and BU224 enhanced the antinociceptive effects of morphine and dose-addition analysis revealed a supra-additive interaction. Agmatine enhanced the antinociceptive effects of morphine, 2-BFI and BU224, and the effect was mediated by α2 adrenoceptors. Combined with the finding that selective I2 receptor ligands block the development of tolerance to morphine antinociception (Boronat et al., 1998), these data suggest that selective I2 receptor ligands could be used as a monotherapy or combined with opioids as an adjunct for treating some pain conditions. 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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