Introduction

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Moxonidine belongs to the imidazoline class of compounds and acts centrally. The spinal antinociceptive actions of several adrenergics/imidazolines have been demonstrated in multiple species. Spinally administered clonidine and dexmedetomidine both produce antinociception in rat (Reddy et al., 1980), sheep (Eisenach and Dewan, 1990), mouse (Roerig et al., 1992; Stone et al., 1997), frog (Stevens and Brenner, 1996), and humans (Mendez et al., 1990). The spinal antinociceptive action of moxonidine, however, has not been previously reported.

Spinal administration of clonidine to humans for the treatment of pain has been used in clinical studies since the mid-1980s. Epidural administration of clonidine has since been approved for the treatment of severe cancer pain (Eisenach, 1996). Although clonidine is efficacious for the treatment of both hypertension and pain, treatment with clonidine is frequently accompanied by adverse side effects that include, but are not limited to, sedation and dry mouth (Davies et al., 1977; Wing et al., 1977; Thananopavarn et al., 1982; Eisenach et al., 1989a,b, 1995), rebound withdrawal symptoms (Hokfelt et al., 1970; Reid et al., 1977; Weber, 1980), hypotension (Eisenach et al., 1989a; Mendez et al., 1990), and tolerance (Meyer et al., 1977; Yaksh and Reddy, 1981; Takano and Yaksh, 1993). These reported limitations of the use of clonidine drive the continued search for improved ₂-adrenergic receptor (AR) analgesics and, in particular, the exploration of ₂AR subtype-selective ligands that may invoke analgesia without also invoking undesirable side effects (Codd et al., 1995).

Moxonidine, a selective imidazoline₁ (I₁)/₂ agonist, was developed (Armah and Stenzel, 1981) to be an effective antihypertensive agent acting centrally, predominantly on imidazoline receptors, with an improved side effect profile over clonidine (Plänitz, 1984). Moxonidine has been tested in clinical trials for antihypertensive efficacy since the early 1980s. These studies demonstrate that moxonidine reduces hypertension in patients with mild to moderate hypertension with an efficacy comparable to that of clonidine (Plänitz, 1984, 1986), the angiotensin-converting enzyme inhibitors (Ollivier and Christen, 1994) captopril (Kraft and Vetter, 1994) and enalapril (Küppers et al., 1997), nifedipine, and atenolol; all these studies suggest that moxonidine is well tolerated. Furthermore, several studies (Plänitz, 1984, 1986) report that, at low but effective doses, moxonidine does not invoke the side effects of sedation or dry mouth. At least one study reports that some patients experienced dry mouth and sedation after moxonidine administration (Plänitz, 1984, 1986); however, the incidence of these moxonidine-induced side effects was significantly below (6 of 20, 30%) that reported from patients taking clonidine (17 of 20, 85%). Complementarily, several reports indicate that rebound withdrawal symptoms are not observed after the cessation of moxonidine treatment (Kraft and Vetter, 1994; Webster and Koch, 1996; Ziegler et al., 1996). The hypotensive effect of clonidine (Mendez et al., 1990) may contraindicate its use as an analgesic in some cases. The development of an adrenergic analgesic with reduced hypotensive effects in normotensive subjects would, therefore, represent an advance over the use of clonidine (Tamsen and Gordh, 1984). A comparison of the hemodynamic and behavioral effects induced by orally administered moxonidine (200 µg) and clonidine (200 µg) in normal subjects showed that although both drugs reduced blood pressure, the hypotensive effect of clonidine was significantly greater than that of moxonidine. The side effect reporting was greater in the clonidine-treated group of normal subjects than in the group that received moxonidine (Macphee et al., 1992). This study in normotensives could be predictive of two different outcomes. First, moxonidine may be more efficacious in hypertensive than in normotensive patients possibly due to differential vascular sensitivity and/or sympathetic nervous system activity (Macphee et al., 1992). Second, moxonidine may require greater doses than clonidine to produce a hypotensive effect in normotensive subjects; those greater doses may reveal a side effect incidence comparable to that of effective doses of clonidine. If the former proves to be true, this could mean that moxonidine produces analgesia in normotensive patients with reduced risk of hypotensive side effects.

To our knowledge, the present experiments comprise the first report of an antinociceptive action of moxonidine. We tested moxonidine for potential antinociceptive action in the tail-flick thermal nociceptive test and the SP nociceptive behavioral test in ICR mice, mixed C57BL/6 × 129/Sv (WT) mice, and C57BL/6 × 129/Sv mice with a mutation (D79N) in the _2a receptor subtype that renders that receptor dysfunctional (MacMillan et al., 1996) (not a null mutation). These experiments revealed that moxonidine produces antinociception in both strains of mice and in the D79N-_2a mutant line in both tests of antinociception. We also tested the ability of the ₂AR-selective antagonist SK&F 86466 to inhibit moxonidine-induced antinociception in both strains of mice and in the line of D79N-_2a mutated mice. Moxonidine is reported to have a 30-fold lower affinity for ₂ARs compared with that of clonidine (Armah et al., 1988; Ferry et al., 1988) and is thought to preferentially activate the putative I₁ receptor (Ernsberger et al., 1993). Therefore, we also used an antagonist strategy to attempt to identify the relative I₁/₂AR contributions to moxonidine-mediated antinociception. Finally, we tested for potential involvement of descending noradrenergic mechanisms in the spinal action of moxonidine using the selective noradrenergic neurotoxin 6-hydroxydopamine (6-OHDA) in an effort to destroy descending noradrenergic nerve terminals.

	Materials and Methods

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Animals. Experimental subjects were 20- to 25-g male ICR mice (Harlan, Madison, WI) or 15- to 20-g male and female mice (gender-matched) with a mixed C57BL/6 × 129/Sv genetic background (designated WT for wild type). We also used male and female mice with this same background (C57BL/6 × 129/Sv) with a "hit and run" gene-targeted mutation (D79N) that renders the _2aAR dysfunctional (MacMillan et al., 1996; Stone et al., 1997). These experiments were approved by the Institutional Animal Care and Use Committee. Subjects were housed in groups of 5 to 10 in 25 × 48 × 15-cm plastic cages in a temperature- and humidity-controlled environment. Subjects were maintained on a 12-h light/dark cycle and had free access to food and water. In the experiment using the noradrenergic neurotoxin 6-OHDA, the mice that were tested 3 days after treatment (saline or 6-OHDA) were randomized and retested 9 days later on day 14 after treatment. In all other experiments, each animal was used only once.

Chemicals. Moxonidine [4-chloro-5-(2-imidazolin-2-ylamino)-6-methoxy-2-methylpyrimidine] chloride was a generous gift of Solvay Pharma GmbH (Hannover, Germany). Norepinephrine (NE) was purchased from Sigma Chemical (St. Louis, MO) and was prepared fresh for each experiment in acidified saline. Smith Kline & French (King of Prussia, PA) donated the SK&F 86466 [6-chloro-2,3,4,5-tetrahydro-3-methyl-1-H-3-benzazepine]. Zeneca (Wilmington, DE) donated the dexmedetomidine [(+)-(S)-4-[1-(2,3-dimethylphenyl) ethyl]1H-imidazole]. Efaroxan [2-(2-ethyl-2,3-dihydrobenzofuranyl)-2-imidazoline] hydrochloride was purchased from Research Biochemicals International (Natick, MA). SP was purchased from Sigma Chemical Co. (St. Louis, MO). Clonidine HCl (2-[2,6-dichloroaniline]-2-imidazoline) was from Boehringer-Ingelheim Ltd. Moxonidine was dissolved in 1% acetic acid and diluted with acidified saline (pH 3.2-4.0, 0.01 N acetic acid). NE and SP were dissolved in acidified saline. All other drugs were dissolved in 0.9% saline. All drugs were administered intrathecally in a 5-µl volume in conscious mice according to the method of Hylden and Wilcox (1980) as modified by Wigdor and Wilcox (1987).

Thermal Nociception. Thermal nociceptive responsiveness was determined using the warm water (52.5°C) immersion tail-flick test. The latency to the first rapid tail-flick represented the behavioral endpoint (Janssen et al., 1963). Baseline measurements of tail-flick latencies were collected on all mice before testing. The mean baseline tail-flick latency of the ICR mice was 3.6 s (S.D. = 0.69 s, n = 31); ICR mice that failed to respond within 5 s to baseline tests were excluded from analysis. The mean baseline tail-flick latency of the WT and D79N-_2a mutant mice was 6.4 s (S.D. = 2.6 s, n = 250). The baseline tail-flick latency did not differ (unpaired t test: p > .05) between the WT and D79N-_2a mice (WT: mean = 6.5 s, S.D. = 2.5 s, S.E.M. = 0.27 s, n = 133 mice; D79N-_2a mice: mean = 6.2 s, S.D. = 2.7 s, S.E.M. = 0.24 s, n = 85 mice). WT and D79N-_2a mice that failed to respond within 11.5 s (a value of more than 2 S.D. from the mean) were eliminated from analysis (5.5%) (Horan et al., 1991). The percent of maximum possible antinociceptive effect (% MPE) of injected (drug or saline control) was determined according to the following formula:

In all three lines of mice, a maximum score of 100% was assigned to those animals not responding before a 12-s (ICR) or 15-s (WT/D79N-_2a) cutoff to avoid tissue injury. In each case in which the animal did not respond before the cutoff, the tail was examined for loss of motor control. Only animals capable of movement in their tails were included in analysis.

SP Nociceptive Test. Nociceptive responsiveness was also tested in the SP nociceptive test. The SP assay is a sensitive indicator of milder analgesics (Hylden and Wilcox, 1981). A constant dose of SP (10-20 ng) was injected intrathecally to produce approximately 40 to 60 behaviors (scratches and bites directed to the hindlimbs) in the first minute after injection. The dose of SP required to produce this number of behaviors was confirmed with each new experiment. Coadministration of opioid or adrenergic analgesics dose-dependently inhibits those behaviors (Hylden and Wilcox, 1983). To test the ability of moxonidine, dexmedetomidine, clonidine, and NE to inhibit SP-induced behavior, the drugs were coadministered with SP and inhibition was expressed as a percent of the mean response of the control group (determined with each new experiment) according to the following equation:
In some experiments, antagonists were coadministered with the moxonidine-SP combinations. In the case of the SK&F 86466 compound and efaroxan, dose-antagonism curves were determined for their respective abilities to antagonize the inhibition of SP behavior by an 80% effective dose of moxonidine. The ED₈₀ value was calculated from the regression line of the dose-inhibition curve. This measure was expressed by the equation:
Each dose selected from the calculated ED₈₀ value was tested experimentally on the day of the percent antagonism experiment. In the event that the experimental dose did not produce an exact 80% inhibition, the equation was adjusted to reflect exact percent inhibition that the antagonist was acting against (e.g., 70 or 90 for 70% or 90%, respectively).

Antagonism of Moxonidine

Dose-response curves to moxonidine and dexmedetomidine were generated in ICR mice in the SP nociceptive test. ED₈₀ doses were calculated according to the method of Tallarida and Murray (1987). ICR, WT, and D79N-_2a mutant mice were coadministered with either SK&F 86466 (0.01-10 nmol i.t.) or efaroxan (0.001-10 nmol i.t.) and an ED₈₀ dose of either moxonidine (ICR: efaroxan, 0.2 nmol; SK&F 86466, 0.3 nmol; WT: 1.5 nmol; D79N: 8.6 nmol i.t.) or dexmedetomidine (ICR: efaroxan, 0.2 nmol; SK&F 86466, 0.8 nmol) in the SP nociceptive test. Inhibition curves for SK&F 86466 or efaroxan were generated and the ID₅₀ values were calculated according to the method of Tallarida and Murray (1987).

6-OHDA Treatment. ICR mice were injected intrathecally with the noradrenergic neurotoxin 6-OHDA (5 µg i.t.) (Fig. 6). This dose of 6-OHDA was selected because it has been demonstrated to be effective when administered intrathecally in mice; Fasmer and colleagues (1986) demonstrated that uptake of ³H-NE was dramatically reduced in mouse spinal cord 14 days after the i.t. injection of 5 µg of 6-OHDA. At 3 and 14 days after treatment, saline-treated mice and 6-OHDA-treated mice were injected with different doses of moxonidine or NE. Dose-inhibition curves were collected and ID₅₀ values calculated for each drug in each treatment group. The 14-day time point represents the same mice used for the 3-day time point. Mice from the first group were randomized for the subsequent treatment (NE or moxonidine) to reduce potential confound from previous drug exposure. The time points of 3 and 14 days treatment were selected to be comparable to the testing days used by Fasmer et al. (1986); this group observed differences in the responses to nociceptive stimuli of 6-OHDA-treated and control mice on day 3 but not on day 14 after treatment.

Statistical Analysis. Data describing antinociception are expressed as % MPE or percent inhibition with S.E.M. In experiments in which full dose-response curves were generated, a minimum of three doses was used for each drug or combination of drugs. Statistical comparisons of potencies are based on the confidence limits of the ED₅₀ values. A shift in a dose-response curve is considered significant when the calculated ED₅₀ value of one curve falls outside the confidence limits of the ED₅₀ value of the curve to which it is being compared. The ED₅₀ values and confidence limits were calculated according to the method of Tallarida and Murray (1987).

	Results

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Moxonidine-Induced Antinociception

Moxonidine Produces Antinociception in ICR Mice in Tail-Flick Test and SP Nociceptive Test. We determined dose-response curves (Fig. 1) for the effects of moxonidine in the tail-flick test [0.1, 0.3, and 1 nmol, i.t; ED₅₀ = 0.5 nmol (0.3-0.7)] and in the SP nociceptive behavioral test in [(0.01, 0.03, 0.1, and 0.3 nmol, i.t; ED₅₀ = 0.04 nmol (0.03-0.07)]. The lower ED₅₀ value of moxonidine in the SP test versus the tail-flick test is consistent with comparisons made between the tests for other antinociceptive compounds. We have observed that both opioid and adrenergic agonists produce antinociception in the SP nociceptive test with greater potency compared with their respective potencies in the tail-flick assay (Hylden and Wilcox, 1983). Furthermore, the ED₅₀ values of moxonidine in both tests are comparable to those observed with spinally administered morphine or clonidine (see Discussion).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Intrathecally administered moxonidine produces spinal antinociception. A, tail-flick test: dose-response curves to moxonidine were generated in ICR mice in the warm water immersion tail-flick assay [ED50 = 0.5 nmol (0.3-0.7) ]. B, SP test: dose-response curves to moxonidine were generated in ICR mice in the SP nociceptive behavioral assay [ED50 = 0.04 nmol (0.03-0.07) ].

Antagonism of Moxonidine-Induced Antinociception

SKF-86466 and Efaroxan Dose-Dependently Antagonize Moxonidine-Induced Antinociception in SP Nociceptive Test. We compared the abilities of the ₂AR-selective antagonist SK&F 86466 (Hieble et al., 1986) and the I₁/₂AR-selective mixed antagonist efaroxan (Haxhiu et al., 1994) to antagonize the effects of moxonidine and dexmedetomidine in the SP test in ICR mice.

SK&F 86466 and Efaroxan Antagonism of Dexmedetomidine in ICR Mice. As a positive control, we antagonized dexmedetomidine antinociception with both SK&F 86466 and efaroxan. On each experimental day, we generated a dose-response curve to dexmedetomidine in ICR mice and the calculated the ED₈₀ value from the regression line. For the SK&F 86466 antagonism experiment, the ED₅₀ value of dexmedetomidine was calculated to be 0.0013 nmol and the ED₈₀ dose was calculated to be 0.8 nmol. This constant dose of dexmedetomidine was administered with varying doses of SK&F 86466 (0.1, 0.3, 1, 3, 6, and 8 nmol i.t.) to generate a dose-inhibition curve (Fig. 2A). A high dose of SK&F 86466 (8 nmol i.t.) alone did not affect the number of SP-elicited behaviors (data not shown). Similarly, we observed that a single dose of the nonimidazoline ₂AR-selective antagonist yohimbine (2.5 nmol) effectively antagonized high efficacy doses of moxonidine (3 nmol, data not shown) in the tail-flick test. For the efaroxan antagonism experiment, the ED₅₀ value of dexmedetomidine was calculated to be 0.003 nmol (0.001-0.1) and the ED₈₀ dose was calculated to be 0.2 nmol. This constant dose of dexmedetomidine was administered with varying doses of efaroxan (0.01, 0.1, 0.3, and 1 nmol i.t.) to generate a dose-inhibition curve (Fig. 2A). A high dose of efaroxan (1 nmol i.t.) did not affect the number of SP-elicited behaviors (data not shown). The ID₅₀ value of SK&F 86466 antagonism of dexmedetomidine was 1.5 nmol (0.87-2.6), which is 15-fold greater than the ID₅₀ value of efaroxan (0.1 nmol, 0.028-0.38) antagonism of dexmedetomidine.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   SK&F 86466 and efaroxan antagonize dexmedetomidine- and moxonidine-induced inhibition of SP-elicited behavior in ICR mice. A, dose-response curves to dexmedetomidine [SKF experiment: ED50 = 0.0013 nmol (0.0002-0.01); EFA experiment: ED50 = 0.003 nmol (0.001-0.01); ED80 = 0.2 nmol] were generated to determine the appropriate ED80 doses (data not shown). Dose-antagonism curves (shown) for the ability of SK&F 86466 [ID50 = 1.5 nmol (0.87-2.6), ] and efaroxan [ID50 = 0.1 nmol (0.028-0.38), ] to inhibit the effect of the ED80 dose of dexmedetomidine were generated. B, dose-response curves to moxonidine [SKF experiment: ED50 = 0.04 nmol (0.03-0.07); ED80 = 0.3 nmol; EFA experiment: ED50 = 0.06 nmol (0.04-0.08); ED80 = 0.2 nmol)] were generated to determine the appropriate ED80 doses (data not shown). Dose-antagonism curves (shown) were generated for the ability of SK&F 86466 [ID50 = 0.15 nmol (0.079-0.29), ; efaroxan: ID50 = 0.05 nmol (0.014-0.18 ] to inhibit the effect of the ED80 doses of moxonidine.

SK&F 86466 and Efaroxan Antagonism of Moxonidine in ICR Mice in SP Test. On each experimental day, we generated a dose-response curve to moxonidine in ICR mice and calculated the ED₈₀ value from the regression line. For the SK&F 86466 antagonism experiment, the ED₅₀ value of moxonidine was 0.04 nmol (0.03-0.07) and the ED₈₀ dose was calculated to be 0.3 nmol. This constant dose of moxonidine was administered with varying doses of SK&F 86466 (0.01, 0.1, and 1 nmol i.t.) to generate a dose-antagonism curve (Fig. 2B). Similarly, we observed that a single dose of the yohimbine (0.3 nmol) effectively antagonized high-efficacy doses of moxonidine (0.3 nmol, data not shown) in the SP test. For the efaroxan antagonism experiment, the ED₅₀ value of moxonidine was 0.06 nmol (0.04-0.08) and the ED₈₀ dose was calculated to be 0.2 nmol. This constant dose of moxonidine was administered with varying doses of efaroxan (0.001, 0.01, 0.1, and 1 nmol i.t.) to generate a dose-inhibition curve (Fig. 2B). The ID₅₀ value of efaroxan antagonism of moxonidine was 0.05 nmol (0.014-0.18), which did not differ significantly from the ID₅₀ value of SK&F 86466 (0.15 nmol, 0.079-0.29) antagonism of moxonidine.

Adrenergic Receptor Subtype Participation in Moxonidine-Induced Antinociception

Moxonidine Produces Antinociception in WT Mice and D79N-_2a Mutant Mice in Tail-Flick and SP Nociceptive Tests. The ability of SK&F 86466 to antagonize moxonidine in ICR mice strongly suggested the participation of an ₂AR in moxonidine-induced antinociception. Other studies suggest that the _2aAR subtype is the primary mediator of ₂AR agonist-mediated antinociception (Stone et al., 1997). To examine the participation of the _2aAR subtype in moxonidine-mediated antinociception, we tested moxonidine for its ability to produce antinociception in mice with a mutation (D79N) in the _2aAR. This mutation results in a functional disruption of the _2aAR and an 80% knock-down in receptor binding in membrane preparations from brain (MacMillan et al., 1996). To begin, we tested for moxonidine-induced antinociception in the D79N-_2aAR mutant mouse and WT counterpart line and compared potency to that of clonidine. Moxonidine dose-dependently (0.01, 0.03, 0.06, 0.1, and 3 nmol i.t.) produced potent antinociception in WT mice in the tail-flick test [Fig. 3A, ED₅₀ = 0.17 nmol (0.09-0.32)]. Clonidine also dose-dependently [1, 10, 60, and 100 nmol i.t. ED₅₀ = 28 nmol (11-72)] produced potent antinociception in WT mice in the tail-flick test (Fig. 3B). We also tested both moxonidine and clonidine for the inhibition of SP-induced behavior. Moxonidine [0.1, 0.3, and 1 nmol i.t. ED₅₀ = 0.4 (0.3-0.5)] (Fig. 4A), clonidine [0.1, 1, and 10 nmol i.t. ED₅₀ = 2.7 (1.3-5.4)] (Fig. 4B), and NE (0.001, 0.03, 0.1, 0.3, 1 pmol i.t. ED₅₀ = 0.087 pmol (0.016-0.48), Fig. 4C) all dose-dependently inhibited the SP-induced nociceptive behavior. We then evaluated moxonidine antinociception in D79N-_2aAR mice in the tail-flick test [0.01, 0.1, 1, and 3 nmol, ED₅₀ = 0.32 (0.074-1.6)] and in the SP nociceptive test [0.1, 1, and 10 nmol, ED₅₀ = 1.1 (0.7-1.7)]. In both tests, moxonidine potency was approximately 2-fold lower in the _2a mutant mice than in their WT counterparts. In contrast, clonidine did not produce antinociception in the D79N-_2aAR mice in either the tail-flick (Fig. 3B) or the SP test (Fig. 4B) at doses up to 100 (SP test) or 300 (tail-flick test) nmol. In contrast to both moxonidine and clonidine, the nonselective catecholamine ₂AR agonist NE produced antinociception in the D79N-_2aAR mice [0.3, 100, and 300 pmol i.t. ED₅₀ = 68 pmol (8.1-564), Fig. 4C] but with 780-fold decreased potency relative to their WT counterparts.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Moxonidine produces spinal antinociception in the tail-flick test in WT and D79N-2a mutant mice. A, tail-flick test: moxonidine. Dose-response curves to moxonidine were generated in WT [ED50: 0.17 nmol (0.09-0.32), ] and D79N-2a [ED50: 0.32 nmol (0.074-1.6), ] mice. B, tail-flick test: clonidine. Varying doses of clonidine were administered intrathecally to WT [ED50: 28 nmol (11-72), ] and D79N-2a (ED50 not calculable, ) mice.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Moxonidine produces spinal antinociception in the SP test in WT and D79N-2a mutant mice. A, SP test: moxonidine. Dose-response curves to moxonidine were generated in WT [ED50 = 0.4 (0.3-0.5), ] and D79N-2a [ED50 = 1.1 (0.7-1.7), ] mice. B, SP test: clonidine. Varying doses of clonidine were administered intrathecally to WT [ED50 = 2.7 (1.3-5.4), ] and D79N-2a (ED50 not calculable, ) mice. C, SP test: norepinephrine. Varying doses of NE were administered intrathecally to WT [ED50 = 0.087 pmol (0.016-0.48), ] and D79N-2a [ED50 = 68 pmol (8.1-564)] mice.

SKF-86466 and Efaroxan Dose-Dependently Antagonize Moxonidine-Induced Antinociception in SP Nociceptive Test. The ability of moxonidine to produce antinociception in both WT and D79N-_2a mice suggested the participation of some ₂AR, although perhaps not _2aAR. However, moxonidine is also thought to act at the imidazoline receptor, which could explain its effectiveness in the D79N-_2a mutant mice. To address this issue, we tested the ability of the ₂AR-selective antagonist SK&F 86466 as well as the mixed I₁/₂AR-selective antagonist efaroxan to antagonize the effects of moxonidine and dexmedetomidine WT and D79N-_2a mice in the SP test.

WT Mice. ED₈₀ doses of moxonidine were estimated from previous dose-response curves and confirmed by administering that dose (1.46 nmol) against SP. This constant dose of moxonidine was administered with varying doses of SK&F 86466 (0.1, 0.2, 0.5, and 1 nmol i.t.) to generate a dose-inhibition curve [ID₅₀ = 0.4 nmol (0.3-0.5)] (Fig. 5A). Similarly, we observed that a single dose of the yohimbine (0.3 nmol) effectively antagonized high-efficacy doses of moxonidine in WT mice (0.1 nmol, data not shown). Efaroxan (0.1, 0.3, and 1 nmol i.t.) antagonized this same ED₈₀ dose of moxonidine with an ID₅₀ value of 0.24 nmol (0.18-0.33) (Fig. 5B); this value does not differ significantly from the ID₅₀ value of SK&F 86466. 

View larger version (24K): [in this window] [in a new window]   Fig. 5.   SK&F 86466 and efaroxan antagonize moxonidine-induced inhibition of SP-elicited behavior in WT and D79N-2a mutant mice. A, WT mice: dose-antagonism curves were generated to SK&F 86466 [ID50 = 0.4 nmol (0.3-0.5), ] and efaroxan [ID50 = 0.24 (0.18-0.33), ] against a constant ED80 dose of moxonidine (1.46 nmol) B, D79N-2a mutant mice: dose-antagonism curves were generated to SK&F 86466 [ID50 = 1 nmol (0.68-1.5 nmol i.t.) ] and efaroxan [ID50 = 4.6 nmol (3.1-6.8), ] against a constant ED80 dose of moxonidine (8.56 nmol for SK&F 86466 and 10 nmol for efaroxan) (two different experimental days). Efaroxan (10 nmol i.t.) did not produce a behavioral effect when administered alone to D79N-2a mice, nor did it affect SP-elicited behavior (data not shown).

D79N-_2a Mutant Mice. ED₈₀ doses of moxonidine were estimated from previous dose-response curves (Fig. 2B) and confirmed by administering that dose against SP. This constant dose of moxonidine (8.6 nmol i.t.) was administered with varying doses of SK&F 86466 (0.3, 1, and 3 nmol i.t.). SK&F 86466 dose-dependently antagonized moxonidine with an ID₅₀ value of 1 nmol (0.68-1.5) (Fig. 5A). Similarly, we observed that a single dose of yohimbine (2.5 nmol) effectively antagonized high-efficacy doses of moxonidine (6 nmol) in D79N-_2a mutant mice (data not shown). Efaroxan dose-dependently (1, 3, and 10 nmol i.t.) antagonized an ED₈₀ dose of moxonidine (10 nmol i.t.) with an ID₅₀ value 4.6 nmol (3.1-6.8); the ID₅₀ value of efaroxan is significantly (4.5-fold) greater than the ID₅₀ value of SK&F 86466.

Exclusion of Autoreceptor-Containing Noradrenergic Terminals. Dose-response curves to moxonidine- and NE-induced inhibition of SP-elicited behavior were collected 3 and 14 days after i.t. treatment with either saline or 6-OHDA (Fasmer et al., 1986). At both time points, the potency of NE was significantly increased in mice treated with 6-OHDA [ED₅₀ = 3 days, 12 pmol (10-13); 14 days, 32 pmol (25-41)] compared with saline-treated controls [ED₅₀ = 3 days, 32 pmol (23-44); 14 days, 72 pmol (40-130)]. This may indicate a supersensitization of postsynaptic ₂ARs after the destruction of presynaptic noradrenergic terminals by the 6-OHDA toxin (Swerdlow and Koob, 1989). In contrast, moxonidine potency did not differ between 6-OHDA [ED₅₀ = 3 days, 140 pmol (100-180); 14 days, 282 pmol (145-551)] compared with saline-treated controls [ED₅₀ = 3 days, 130 pmol (100-170); 14 days, 327 pmol (197-549)] at either time point.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Moxonidine produced potent antinociception in the tail-flick test and the SP nociceptive test in two different strains of mice: ICR and C57BL/6 × 129/Sv (WT). Two ₂AR antagonists [efaroxan (I₁/₂) and SK&F 86466 (₂)] dose-dependently antagonized moxonidine-induced antinociception, confirming the requirement for ₂AR. Moxonidine potency was decreased 2-fold in the D79N-_2a mutant mice compared with their WT counterparts. This stands in sharp contrast to the complete lack of a spinal antinociceptive effect of clonidine in the D79N-_2a mutant mice and substantially decreased potency for NE. Taken together, these data suggest that moxonidine-induced antinociception is governed primarily by an ₂AR subtype, but not likely the _2aAR subtype.

Moxonidine-Induced Antinociception. The ability of ₂-adrenergic agonists to prolong tail-flick latencies is well established (Reddy et al., 1980; Yasuoka and Yaksh, 1983; Milne et al., 1985; Solomon et al., 1989; Ossipov et al., 1990). Spinal administration of moxonidine dose-dependently increased tail-flick latency in two strains of mice: ICR (Fig. 1) and C57BL/6 × 129/Sv (Fig. 3A). Moxonidine and morphine have similar tail-flick ED₅₀ values in both ICR mice (Fairbanks and Wilcox, 1997) and C57BL/6 × 129/Sv (WT) mice (Stone et al., 1997): in ICR mice, moxonidine (0.5 nmol, 0.3-0.7), morphine (1.2 nmol, 0.7-2.4); and in WT mice, moxonidine (0.17 nmol, 0.09-0.32), morphine (0.52 nmol, 0.36-0.74). Morphine remains the gold standard with which other analgesics are compared. The observation that moxonidine prolongs tail-flick latencies with potency comparable to that of morphine illustrates the relevance of moxonidine-induced antinociception. Like morphine, clonidine represents the gold standard for ₂AR-mediated analgesia. Interestingly, we observed that the potency of moxonidine in the tail-flick test is 100-fold greater than that of clonidine [Fairbanks and Wilcox, 1999; ED₅₀ = 49 nmol (29-82) in ICR mice Fig. 1AB] and 164-fold greater than that of clonidine in WT mice (Fig. 3AB). That moxonidine is more potent than clonidine further attests to the relevance of moxonidine-induced antinociception.

Receptor Subtype Participation in Moxonidine-Induced Antinociception. Elucidation of the specific receptors that mediate antinociceptive drug action remains an area of intense investigation. Pharmacological antagonism has proved to be effective in determining the opioid receptor subtypes (µ, , or ) activated by various opioid agonists. Unfortunately, ₂AR antagonists are insufficiently selective between the subtypes _2a, _2b, and _2c to make similar distinctions. Generation of antisense oligonucleotides and mutant mouse lines with absent or disrupted _2aAR, _2bAR, and _2cAR permitted studies to determine the selective function of these subtypes. These studies have concluded that the _2aAR mediates opioid-adrenergic synergy (Stone et al., 1997), epileptogenesis (Janumpalli et al., 1998), and sedation (Mizobe et al., 1996; Hunter et al., 1997; Lakhlani et al., 1997). Moxonidine is an ₂AR agonist; however, both moxonidine and clonidine are also thought to act at a nonadrenergic receptor, the imidazoline receptor, to produce an antihypertensive action (Ziegler et al., 1996). Moxonidine binds preferentially to the putative imidazoline binding site over the ₂ARs: moxonidine was 33-fold selective for I₁ receptors versus ₂ARs (Ernsberger et al., 1993). In comparison, clonidine is much less (3.8-fold) selective for I₁ receptors over ₂ARs. Considering the selectivity of moxonidine for I₁ receptors together with immunohistochemical localization of imidazoline receptors in superficial dorsal horn of the spinal cord (Ruggiero et al., 1998), it is conceivable that imidazoline receptors participate in moxonidine-mediated antinociception in concert with ₂ARs.

Lack of Participation of Autoreceptors on Descending Noradrenergic Terminals in Moxonidine-Induced Antinociception. It is conceivable that SK&F 86466-sensitive moxonidine-induced antinociception could require the participation of autoreceptors on descending noradrenergic terminals. In other words, moxonidine-induced antinociception could be mediated by action at autoreceptors on descending noradrenergic terminals, resulting in the release of NE to act on postsynaptic ₂ARs. In support of the participation of autoreceptors and subsequent NE release, Klimscha et al. (1997) have demonstrated that intrathecally administered clonidine increased the concentration of NE in spinal cord dorsal horn microdiasylate in sheep. Furthermore, Armah and colleagues (Ziegler et al., 1996) found that the hypotensive effects of moxonidine in rabbits were substantially diminished with 6-OHDA pretreatment, implicating the participation of catecholamine stores in the activity of moxonidine. The present experiments revealed that NE potency increased in animals treated with 6-OHDA compared with saline-treated control animals (Fig. 6). This apparent supersensitivity after a catecholaminergic lesion suggests increased affinity of postsynaptic ₂ARs (Post et al., 1987; Swerdlow and Koob, 1989), indicating that the 6-OHDA treatment successfully destroyed descending noradrenergic terminals. If moxonidine-induced antinociception requires postsynaptic activation of ₂ARs by release of NE from descending terminal, the potency of moxonidine should increase in parallel with that of the exogenously applied NE. However, moxonidine potency did not differ between 6-OHDA-treated and saline-treated animals at either 3 or 14 days after treatment (Fig. 6). We interpret this result to indicate that that moxonidine-induced antinociception does not require the release of NE. This is in contrast to supersensitization observed with clonidine-mediated antinociception after 6-OHDA lesioning (Post et al., 1987). These results combined with the observation that (in these murine models of nociception) both clonidine and NE demonstrate selectivity for the _2aAR suggests that the _2aAR receptor is the subtype that becomes supersensitive after 6-OHDA administration. This selectivity could explain why moxonidine-induced antinociception does not display supersensitization after 6-OHDA lesioning. Further studies would be needed to confirm this proposal.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Pretreatment with the noradrenergic neurotoxin 6-OHDA increases norepinephrine potency but not moxonidine potency in ICR mice. ICR mice were injected either with saline or 5 µg of 6-OHDA. Three days later, dose-response curves to NE and to moxonidine were collected in both groups of mice using the SP test. A, 3 days pretreatment: dose-response curves to NE were generated in saline-treated [ED50 = 32 pmol (23-44) , dashed line] and 6-OHDA-treated [ED50 = 12 pmol (10-13), left , solid line] mice; dose-response curves to moxonidine in saline-treated [ED50 = 130 pmol (100-170), , dashed line] and 6-OHDA-treated [ED50 = 140 pmol (100-180), , solid line] mice. B, 14 days pretreatment: dose-response curves to NE were generated in saline-treated [ED50 = 72 pmol (40-130), , dashed line] and 6-OHDA-treated [ED50 = 32 pmol (25-41), leftward , solid line] mice; dose-response curves to moxonidine in saline-treated [ED50 = 327 pmol (197-549), , dashed line] and 6-OHDA-treated [ED50 = 282 pmol (145-551), , solid line] mice.

Conclusion. To our knowledge, this study is the first to report an antinociceptive application for the first of a new generation of antihypertensive compounds, moxonidine. Based on the scientific experience with other adrenergic agonists such as clonidine and dexmedetomidine, we anticipate that the antinociceptive effect of moxonidine will generalize to other species. Unlike antinociception induced by clonidine, dexmedetomidine, UK-14,304, and NE, moxonidine-induced antinociception requires ₂AR activation independent of the _2aAR subtype. Others have suggested that differential receptor selectivities (imidazoline receptor versus ₂AR) account for the improved side effect liabilities of moxonidine versus clonidine when given systemically for the treatment of hypertension. We speculate that our observed differences in ₂AR subtype requirements for the effects of moxonidine and clonidine could also result in differential side effect profiles in spinal administration of moxonidine for the treatment of pain.