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α2-adrenergic receptor agonist moxonidine in comparison with clonidine in the formalin test in rats
Pain 85 (2000) 161±167 www.elsevier.nl/locate/pain
Effects of the I1 imidazoline/a 2-adrenergic receptor agonist moxonidine in comparison with clonidine in the formalin test in rats Harlan E. Shannon*, Elizabeth A. Lutz Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285-0510, USA Received 15 June 1999; received in revised form 24 September 1999; accepted 29 October 1999
Abstract Moxonidine is a mixed I1 imidazoline/a 2-adrenergic receptor agonist structurally related to clonidine. In the present studies, moxonidine, like clonidine, produced a dose- and time-related inhibition of formalin-induced behaviors after subcutaneous administration. Moxonidine was equief®cacious to both clonidine and morphine in inhibiting formalin-induced behaviors. The order of potencies of these analgesics was clonidine . moxonidine morphine. The I1 imidazoline preferring antagonist efaroxan produced a dose-dependent antagonism of both moxonidine (5.0 mg/kg) and clonidine (0.5 mg/kg). In addition, the a 2-adrenergic receptor antagonist yohimbine produced a dose-related antagonism of moxonidine, but only partially antagonized clonidine. Prazosin failed to block the effects of either moxonidine or clonidine, indicating a lack of involvement of a 1 as well as a 2B and a 2C receptors. The present results suggest that a 2-adrenergic receptors play an important role in mediating the effects of moxonidine in producing antinociception in the formalin test. Further, the present results demonstrate that the mechanism of action of moxonidine and clonidine differ in that clonidine, but not moxonidine, produces an antinociceptive effect through a yohimbine-insensitive mechanism in the formalin test. q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Moxonidine analgesia; Clonidine analgesia; I1 imidazoline receptor; a 2-Adrenergic receptor; Formalin hyperalgesia
1. Introduction Moxonidine is a mixed I1 imidazoline and a 2-adrenergic receptor agonist with at least a 40-fold selectivity for I1 over a 2 receptors and is related structurally to the imidazoline clonidine, which is approximately fourfold selective for I1 over a 2 receptors (Ferry et al., 1988; Ernsberger et al., 1993). Alpha2-adrenergic receptor agonists of the imidazoline class, including clonidine and dexmedetomidine, have been demonstrated to produce antinociception in a variety of species including mice (e.g. Roerig et al., 1992; Millan et al., 1994; Stone et al., 1997), rats (e.g. Paalzow, 1974; Reddy et al., 1980; Solomon et al., 1989; Takano and Yaksh, 1992; Yaksh et al., 1995) and monkeys (Yaksh and Reddy, 1981; Wang et al., 1985). In addition, clonidine has been demonstrated to produce antinociception after epidural administration in humans (Mendez et al., 1990; Eisenach et al., 1989, 1995) and has been approved by the FDA for epidural administration for the treatment of severe cancer pain (Eisenach, 1996). However, the therapeutic use * Corresponding author. Tel.: 11-317-276-4360; fax: 11-317-2765546. E-mail address: [email protected] (H.E. Shannon)
of clonidine is limited by undesirable side-effects including sedation and dry mouth (e.g. Davies et al., 1977; Eisenach et al., 1989, 1995; Wing et al., 1977), rebound hypertension (Reid et al., 1977; Weber, 1980), and, hypotension in normotensive patients (Eisenach et al., 1989; Mendez et al., 1990). Moxonidine, while approximately equipotent to clonidine in reducing hypertension in humans, has been demonstrated to produce signi®cantly less sedation and dry mouth than clonidine (PlaÈnitz, 1984, 1986; Armah et al., 1988; Kirch et al., 1990), does not produce rebound hypertension (Kraft and Vetter, 1994; Ziegler et al., 1996), and is less likely to produce hypotension in normotensive patients (PlaÈnitz, 1984; MacPhee et al., 1992). It has been suggested that the improved side-effect pro®le of moxonidine over clonidine may be due to the greater selectivity of moxonidine for I1 imidazoline receptors relative to a 2 receptors (e.g. Ernsberger et al., 1993; see also Codd et al., 1995). If moxonidine, like clonidine, also produces analgesia in humans, then moxonidine might represent an analgesic with an improved side-effect pro®le relative to clonidine. Recently, Fairbanks and Wilcox (1999) demonstrated that moxonidine produces antinociception in mice in the tail-¯ick test and the substance P nociceptive test after
0304-3959/00/$20.00 q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(99)00260-2
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intrathecal administration. The antinociceptive effects of both moxonidine and clonidine were blocked by the mixed I1/a 2 antagonist efaroxan as well as by the selective a 2 antagonist SK&F 86466, indicating the involvement of a 2 receptors in mediating the antinociceptive effects of both moxonidine and clonidine after intrathecal administration in mice (Fairbanks and Wilcox, 1999). However, in D79N-a 2a mice which lack functional a 2a receptors, clonidine failed to produce antinociception whereas the potency of moxonidine in producing antinociception was comparable in D79N-a 2a and wild-type mice, indicating that different a 2 receptor subtypes are likely involved in mediating the antinociceptive effects of moxonidine and clonidine (Fairbanks and Wilcox, 1999). In addition, Fairbanks et al. (2000) demonstrated that moxonidine synergized with morphine and deltorphin II, but not DAMGO, to inhibit substance Pinduced behavior after intrathecal administration in mice. Thus, moxonidine may represent a therapeutically useful analgesic with a unique mechanism of action and an improved side-effect pro®le relative to clonidine. The major purpose of the present studies was to determine if moxonidine produces analgesia in the formalin test in rats. Clonidine previously has been shown to produce analgesia in the formalin test in rats and mice after intraplantar (Hong and Abbott, 1996), systemic (Dennis et al., 1980; Tasker and Melzack, 1989) or intrathecal (Przesmycki et al., 1997, 1998; Kanui et al., 1993) administration, and to reduce formalin-induced paw edema after systemic administration (Kulkarni et al., 1986). Accordingly, dose± and time±response curves were determined for both moxonidine and clonidine in the formalin test. A dose±response curve also was determined for morphine for purposes of comparison. A second purpose of the present studies was to determine the potential relative contribution of imidazoline and alpha adrenergic receptors to the antinociceptive effects of moxonidine and clonidine in the formalin test. Therefore, dose±response curves were determined for the I1/a 2 receptor antagonist efaroxan, the a 2 receptor antagonist yohimbine, and the a 1 receptor antagonist prazosin administered alone or with a constant dose of moxonidine or clonidine. The results of the present studies demonstrate that moxonidine, like clonidine, produces analgesia in the formalin test in rats but that the mechanism of action of the two drugs differs.
2. Materials and methods 2.1. Animals Male Sprague±Dawley rats (Harlan Sprague±Dawley, Indianapolis, IN) weighing 180±220 g were used. Rats were housed up to eight per cage in a large colony room, and provided food and water ad libitum with a 12:12 h light/ dark cycle. Each animal was used only once. All procedures
were approved by the Eli Lilly Institutional Animal Care and Use Committee. 2.2. Formalin test The methods used were a modi®cation of the automated method of Jett and Michelson (1996). All testing took place in commercially available startle behavior chambers (Model SR-Lab, San Diego Instruments, San Diego, CA) which detected movements of the rats by means of an accelerometer. At the beginning of an experiment, the rats were injected with vehicle or a dose of drug and individually placed in the restraint cylinders (i.d. 8.5 cm; length 16 cm). Thirty minutes later, the rats were removed from the cylinders and administered formalin (50 ml of a 5% solution in saline) subcutaneously into the plantar surface of the right hindpaw and immediately placed back into the restraining cylinders. The magnitude of the movements were monitored continuously for 60 min in 1-s bins. The number of `agitation' events, de®ned as the number of 1-s bins with a change in dynamic force that exceeded an empirically determined threshold value (20 arbitrary load units, which was determined in pilot experiments to be greater than that produced by animals quietly snif®ng and breathing) were totaled in 5min intervals. As previously described by Jett and Michelson (1996), the formalin-induced movements detected by the system included licking and ¯inching the affected paw as well as hopping and turning. 2.3. Drugs Moxonidine free base (Lilly Research Laboratories, Indianapolis, IN) was dissolved in distilled water with a drop of 1 N HCl. Clonidine HCl, efaroxan HCl, prazosin HCl (Sigma Chemical Co., St. Louis, MO), yohimbine HCl and morphine sulfate (RBI, Natick, MA) were dissolved in deionized water. Doses refer to the form of the drug listed. All drugs were administered subcutaneously in a volume of 1.0 ml/kg. 2.4. Statistics In time-course experiments, the number of `agitation' events were totaled in 5-min intervals and data were analyzed by repeated measures ANOVA. For constructing dose±response curves, the total events during minutes 0±5 after formalin administration were considered to be phase I, and the total number of events during minutes 15±45 were considered to be phase II. Treatment groups were compared to appropriate control groups using ANOVA and Dunnett's t-test. Data are expressed as mean ^ SEM. 3. Results In animals administered vehicle 30 min before formalin, there was an initial peak in the number of events during the ®rst 5-min block after formalin, followed by a decrease in
H.E. Shannon, E.A. Lutz / Pain 85 (2000) 161±167
the number of events in the second 5-min block, and subsequently an increase again in blocks 3 through 9 (Fig. 1, open circles). Thus, our automated method produced results which are highly comparable to the automated method of Jett and Michelson (1996) as well as qualitatively similar to manual methods of counting, scoring or timing components of formalin-induced behavior such as licking or ¯inching (e.g. Dubuisson and Dennis, 1977; Wheeler-Aceto et al., 1990). When moxonidine was administered 30 min before formalin, moxonidine produced a dose- and time-dependent analgesic effect in the formalin test in the rat (Fig. 1). Moxonidine (1.0±10 mg/kg s.c.) was ef®cacious in reducing formalin-induced behaviors in both phase I (the ®rst 5-min block), and phase II (5-min blocks 3 through 9). A dose± response curve was constructed by summing all of the events in the respective phases and is presented in Fig. 2. Like moxonidine, clonidine and morphine also produced dose-related decreases in formalin-induced behaviors in both phase I and phase II of the formalin test (Fig. 2). Clonidine was the most potent in both phases while moxonidine and morphine were approximately equipotent in each phase. In order to determine the potential role of imidazoline and a -adrenergic receptors in the mechanism of action of moxonidine and clonidine, constant doses of either moxonidine (5.0 mg/kg) or clonidine (0.5 mg/kg) were administered with varying doses of either the mixed imidazoline I1 and a 2-adrenergic antagonist efaroxan, the selective a 2-adrenergic receptor antagonist yohimbine, or the selective a 1-adrenergic receptor antagonist prazosin. Neither efaroxan nor yohimbine administered alone produced statistically significant effects on formalin-induced behavior (Fig. 3, left
163
panel). Prazosin produced a modest, but signi®cant reduction in formalin-induced behavior after doses of 0.3 and 10 mg/kg but had no effect after doses of 0.1 and 1.0 mg/kg (Fig. 3, left panel). Both efaroxan (0.1±1.0 mg/kg) and yohimbine (0.1±1.0 mg/kg) produced dose-related reversals of the analgesic effects of 5.0 mg/kg of moxonidine (Fig. 3, center panel), and these two antagonists were equief®cacious and approximately equipotent to each other in reversing moxonidine. Prazosin (0.3±10 mg/kg) had no signi®cant effect on moxonidine-induced analgesia (Fig. 3, center panel). In contrast, while efaroxan produced a complete and dose-related antagonism of the effects of clonidine, the reversal of clonidine by yohimbine was substantially smaller in magnitude than that for efaroxan (Fig. 3, right panel). Prazosin also did not reverse the effects of clonidine (Fig. 3, right panel).
4. Discussion The major ®nding of the present studies was that moxonidine, which is an I1 imidazoline/a 2-adrenergic receptor agonist (e.g. Ernsberger et al., 1993), produced a robust antinociceptive effect in the formalin test in rats. Moreover, moxonidine was equief®cacious to both clonidine and morphine in reducing formalin-induced behaviors. Furthermore, moxonidine was approximately equipotent to morphine in the formalin test but approximately tenfold less potent than clonidine. The present ®ndings corroborate and extend the previous ®ndings that moxonidine produces antinociception in the mouse tail-¯ick and substance P nociceptive tests after intrathecal administration in mice (Fairbanks and Wilcox, 1999; Fairbanks et al., 2000). In humans,
Fig. 1. Dose- and time-related effects of moxonidine in reducing nocifensive behaviors produced by the intraplantar injection of formalin in rats. Each point represents the mean ^ SEM of eight rats. Abscissa: consecutive 5-min blocks; ordinate: number of events.
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Fig. 2. Dose±response curves for moxonidine, clonidine and morphine in the formalin test in rats. Each point represents the total number of events during phase I or phase II of the 45-min time course after formalin was injected (see Fig. 1), and is the mean ^ SEM of 7±8 rats. Points above V represent the effects of vehicle. Abscissa: dose of drug in mg/kg; ordinate: total number of events during the ®rst 5 min (phase I, left panel) or the ®nal 35 min (phase II, right panel). *P , 0:05 vs. Vehicle, Dunnett's t-test.
moxonidine appears to have an improved side-effect pro®le relative to clonidine (see Section 1). If moxonidine also produces analgesia in humans, then it has the potential to be as ef®cacious an analgesic as clonidine but with an improved side-effect pro®le. Several lines of evidence suggest that I1 imidazoline receptors play at least a role in the analgesic effects of moxonidine. Moxonidine has approximately a 40-fold higher af®nity for I1 imidazoline receptors (2.3 ^ 0.5 nM) than for a 2-adrenergic receptors (75 ^ 8 nM) whereas clonidine is only approximately fourfold selective for I1 (1.0 ^ 0.3) over a 2 (3.8 ^ 1.0) receptors (Ernsberger et al., 1993). Based upon these in vitro binding af®nities, moxonidine might be expected to act preferentially at I1 receptors in vivo, and to be approximately two- to threefold less potent than clonidine. In the present studies, moxonidine was approximately tenfold less potent than clonidine in vivo. The tenfold difference in potency observed in vivo, while not identical, is closer to the difference in potency in vitro at I1 compared with the difference in potency in vitro at a 2 receptors between moxonidine and clonidine, suggesting that I1 receptors may be involved in mediating the analgesic effects of moxonidine in the formalin test. In addition, the analgesic effects of moxonidine were antagonized by the I1 receptor preferring antagonist efaroxan (Haxhiu et al., 1994). However, differences in potency in vivo need not necessarily re¯ect potencies in vitro due to differences in drug metabolism and distribution, and, the degree of receptor reserve (e.g. Takano and Yaksh, 1993; Kenakin, 1997). Further, the a 2 receptor antagonist yohimbine, which does
not bind to imidazoline receptors (Piletz et al., 1996), was equipotent and equief®cacious to efaroxan in antagonizing the analgesic effects of moxonidine, indicating that a 2 receptors play an important role in meditating the analgesic effects of moxonidine. Thus, the present data suggest that the antinociceptive effects of moxonidine in the formalin test are mediated primarily by a 2 rather than I1 receptors. In contrast to the results with moxonidine, the effects of clonidine in the formalin test, while antagonized completely by efaroxan, were only partially antagonized by yohimbine. Thus, the present results suggest that I1 receptors play a greater role than a 2 receptors in mediating the analgesic effects of clonidine in the formalin test. The present ®ndings corroborate and extend those of previous investigators. Dennis et al. (1980) found that yohimbine only partially antagonized clonidine in the formalin test; however, yohimbine administered alone at doses of 1.0 and 4.0 mg/kg i.p. produced statistically signi®cant analgesia. Similarly, Tasker and Melzack (1989) found that a single dose of yohimbine (2.0 mg/kg) only partially, and nonsigni®cantly, reversed the effects of clonidine in the formalin test. Although yohimbine is .1000-fold selective for a 2 receptors over I1 receptors, it has an af®nity for serotonin1A receptors which is only approximately twofold lower than its af®nity for a 2 receptors (Winter and Rabin, 1992). Thus, it is possible that yohimbine might have produced effects through serotonin1A receptors which masked any antagonism of clonidine by yohimbine at a 2 receptors. However, in the present studies, we did not observe a statistically significant effect of yohimbine when administered alone over the
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Fig. 3. Antagonism of moxonidine and clonidine by yohimbine and efaroxan during phase II of the formalin test in rats. (Left) Lack of effects of the alphaadrenergic receptor antagonists efaroxan, yohimbine and prazosin administered alone in the formalin test. (Middle) Antagonism of moxonidine (5.0 mg/kg) by the mixed imidazoline and a 2-adrenergic receptor antagonist efaroxan and the selective a 2-adrenergic antagonist yohimbine but not by the a 1-adrenergic antagonist prazosin in the formalin test in rats. (Right) Antagonism of clonidine (0.5 mg/kg) by the mixed imidazoline and a 2-adrenergic receptor antagonist efaroxan, partial reversal by the selective a 2-adrenergic receptor antagonist yohimbine and lack of antagonism by the selective a 1-adrenergic receptor antagonist prazosin in the formalin test in rats. Each point represents the total number of events during the 35-min of phase II after formalin was injected, and is the mean of 4±8 rats. Points above Veh represent the effects of vehicle alone, points above V 1 V represent the effects of vehicle 1 vehicle, points above V 1 Mox represent the effects of vehicle 1 5:0 mg/kg moxonidine, and points above V 1 Cl represent the effects of vehicle 1 0:5 mg/kg clonidine. Abscissa: dose of antagonist in mg/kg; ordinate: total number of events during the 35 min of phase II. *P , 0:05 vs. vehicle or vehicle 1 vehicle, Dunnett's t-test. # P , 0:05 vs. vehicle 1 moxonidine or vehicle 1 clonidine, Dunnett's t-test.
dose range of 0.1±1.0 mg/kg, although there was a nonsigni®cant effect of yohimbine at 1.0 mg/kg. Furthermore, yohimbine completely antagonized moxonidine at doses which only partially blocked clonidine. Therefore, any effects of yohimbine at serotonin1A, or any other, receptors are highly unlikely to have prevented yohimbine from antagonizing clonidine. Thus, the majority of the evidence demonstrates that clonidine produces analgesia in the formalin test through an efaroxan-sensitive but relatively yohimbine-insensitive mechanism. We interpret these ®ndings to indicate that I1 imidazoline receptors may play a greater role than a 2 receptors in mediating the analgesic effects of clonidine in the formalin test. The ®ndings that yohimbine does not completely antagonize clonidine in the formalin test are in contrast to results in other nociceptive tests. For example, Tasker and Melzack (1989) found that although yohimbine (2.0 mg/kg) nonsigni®cantly affected clonidine in the formalin test, this same dose of yohimbine completely antagonized the effects of clonidine in the tail-¯ick test. Similarly, the intrathecal administration of yohimbine antagonized the analgesic effects of intrathecally administered clonidine in the hot-plate (Takano and Yaksh, 1992), tail-¯ick (Monroe
et al., 1995), and thermal paw-withdrawal (Buerkle and Yaksh, 1998) tests. Further, yohimbine antagonized the anti-allodynic effects of clonidine in rats with L5 and L6 nerve ligation (Yaksh et al., 1995). Thus, the effects of clonidine in the formalin test appear to be mediated by mechanisms different from those which mediate its effects in other nociceptive tests. Prazosin did not antagonize either moxonidine or clonidine in the formalin test in the present studies. Prazosin is selective for a 1 over a 2 receptors, and its lack of antagonist activity demonstrates a lack of involvement of a 1 receptors in mediating the effects of moxonidine and clonidine in the formalin test. However, it is now appreciated that prazosin has preferential, and appreciable, af®nity for the a 2B and a 2C over the a 2A subtypes of a 2 receptors (Blaxall et al., 1991; Link et al., 1992; UhleÂn et al., 1992). Thus, the lack of antagonism by prazosin suggests that a 2B and a 2C, as well as a 1, adrenergic receptors play little if any role in mediating the effects of moxonidine and clonidine in the formalin test. However, the effects of selective a 2B and a 2C antagonists, when they become available, on moxonidine antinociception, as well as the effects of moxonidine in a 2B and a 2C receptor knockout mice, are needed to more completely
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determine the relative role of a 2B and a 2C receptors in mediating moxonidine-induced antinociception. Several studies using genetically altered mice recently have demonstrated that the a 2A-adrenergic receptor subtype is the primary mediator of a 2-adrenergic analgesia. Lakhlani et al. (1997) demonstrated that in a mouse line expressing a point mutation (D79N) in the a 2A-adrenergic receptor, the a 2A agonist dexmedetomidine did not produce analgesia in the hot plate test. In the same mouse line, Stone et al. (1997) found that the intrathecal administration of the nonselective a 2 agonist UK 14304 had no analgesic effect in the tail-¯ick test or the substance-P nociceptive test. Further, Hunter et al. (1997) reported that dexmedetomidine did not produce analgesia in the tail immersion test in D79N mutant mice, but did produce analgesia in a 2B and a 2C receptor knockout mice. Moreover, Hunter et al. (1997) also demonstrated that the sedative and hypothermic effects of dexmedetomidine were abolished in the D79N mice but not the a 2B and a 2C receptor knockout mice. The ®ndings with D79N mutant mice have led to the conclusion that selective a 2A-adrenergic receptor agonists are unlikely to provide analgesia with an acceptable therapeutic pro®le (Hunter et al., 1997). However, the demonstration that moxonidine produces antinociception (Fairbanks and Wilcox, 1999; present report) by a 2 receptors other than the a 2a subtype (Fairbanks and Wilcox, 1999), suggests that a 2 agonists selective for other subtypes, perhaps like moxonidine, may provide analgesia with an acceptable therapeutic pro®le. Further experiments are needed to determine the relative role of a 2 receptor subtypes in mediating moxonidine- and clonidine-induced antinociception in other animal models.
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Takano Y, Yaksh TL. Characterization of the pharmacology of intrathecally administered alpha-2 agonists and antagonists in rats. J Pharmacol Exp Ther 1992;261:764±772. Takano Y, Yaksh TL. Chronic spinal infusion of dexmedetomidine, ST-91 and clonidine: spinal alpha2 adrenoceptor subtypes and intrinsic activity. J Pharmacol Exp Ther 1993;264:327±335. Tasker RAR, Melzack R. Different alpha-receptor subtypes are involved in clonidine-produced analgesia in different pain tests. Life Sci 1989;44:9±17. UhleÂn S, Xia Y, Chhajlani V, Felder CC, Wikberg JES. [ 3H]MK912 binding delineates two a 2-adrenergic receptor subtypes in rat CNS one of which is identical with the cloned pA2d a 2-adrenoceptor. Br J Pharmacol 1992;106:986±995. Wang YC, Su CF, Lin MT. The site and the mode of analgesic actions exerted by clonidine in monkeys. Exp Neurol 1985;90:479±488. Weber MA. Discontinuation syndrome following cessation of treatment with clonidine and other antihypertensive agents. J Cardiovasc Pharmacol 1980;2:S73±S89. Wheeler-Aceto H, Porreca F, Cowan A. The rat paw formalin test: comparison of noxious agents. Pain 1990;40:229±238. Wing LM, Reid JL, Davies DS, Neill EA, Tippett P, Dollery CT. Pharmacokinetic and concentration-effect relationships of clonidine in essential hypertension. Eur J Clin Pharmacol 1977;12:463±469. Winter JC, Rabin RA. Yohimbine as a serotonergic agent: evidence from receptor binding and drug discrimination. J Pharmacol Exp Ther 1992;263:682±689. Yaksh TL, Reddy SVR. Studies in the primate on the analgesic effects associated with intrathecal actions of opiates, a -adrenergic agonists and baclofen. Anesthesiology 1981;54:2786±2794. Yaksh TL, Pogrel JW, Lee YW, Chaplan SR. Reversal of nerve ligationinduced allodynia by spinal alpha-2 adrenoceptor agonists. J Pharmacol Exp Ther 1995;272:207±214. Ziegler D, Haxhiu MA, Kaan EC, Papp JG, Ernsberger P. Pharmacology of moxonidine, an I1-imidazoline receptor agonist. J Cardiovasc Pharmacol 1996;27:S26±S37.
Effects of the I1 imidazoline/a 2-adrenergic receptor agonist moxonidine in comparison with clonidine in the formalin test in rats Harlan E. Shannon*, Elizabeth A. Lutz Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285-0510, USA Received 15 June 1999; received in revised form 24 September 1999; accepted 29 October 1999
Abstract Moxonidine is a mixed I1 imidazoline/a 2-adrenergic receptor agonist structurally related to clonidine. In the present studies, moxonidine, like clonidine, produced a dose- and time-related inhibition of formalin-induced behaviors after subcutaneous administration. Moxonidine was equief®cacious to both clonidine and morphine in inhibiting formalin-induced behaviors. The order of potencies of these analgesics was clonidine . moxonidine morphine. The I1 imidazoline preferring antagonist efaroxan produced a dose-dependent antagonism of both moxonidine (5.0 mg/kg) and clonidine (0.5 mg/kg). In addition, the a 2-adrenergic receptor antagonist yohimbine produced a dose-related antagonism of moxonidine, but only partially antagonized clonidine. Prazosin failed to block the effects of either moxonidine or clonidine, indicating a lack of involvement of a 1 as well as a 2B and a 2C receptors. The present results suggest that a 2-adrenergic receptors play an important role in mediating the effects of moxonidine in producing antinociception in the formalin test. Further, the present results demonstrate that the mechanism of action of moxonidine and clonidine differ in that clonidine, but not moxonidine, produces an antinociceptive effect through a yohimbine-insensitive mechanism in the formalin test. q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Moxonidine analgesia; Clonidine analgesia; I1 imidazoline receptor; a 2-Adrenergic receptor; Formalin hyperalgesia
1. Introduction Moxonidine is a mixed I1 imidazoline and a 2-adrenergic receptor agonist with at least a 40-fold selectivity for I1 over a 2 receptors and is related structurally to the imidazoline clonidine, which is approximately fourfold selective for I1 over a 2 receptors (Ferry et al., 1988; Ernsberger et al., 1993). Alpha2-adrenergic receptor agonists of the imidazoline class, including clonidine and dexmedetomidine, have been demonstrated to produce antinociception in a variety of species including mice (e.g. Roerig et al., 1992; Millan et al., 1994; Stone et al., 1997), rats (e.g. Paalzow, 1974; Reddy et al., 1980; Solomon et al., 1989; Takano and Yaksh, 1992; Yaksh et al., 1995) and monkeys (Yaksh and Reddy, 1981; Wang et al., 1985). In addition, clonidine has been demonstrated to produce antinociception after epidural administration in humans (Mendez et al., 1990; Eisenach et al., 1989, 1995) and has been approved by the FDA for epidural administration for the treatment of severe cancer pain (Eisenach, 1996). However, the therapeutic use * Corresponding author. Tel.: 11-317-276-4360; fax: 11-317-2765546. E-mail address: [email protected] (H.E. Shannon)
of clonidine is limited by undesirable side-effects including sedation and dry mouth (e.g. Davies et al., 1977; Eisenach et al., 1989, 1995; Wing et al., 1977), rebound hypertension (Reid et al., 1977; Weber, 1980), and, hypotension in normotensive patients (Eisenach et al., 1989; Mendez et al., 1990). Moxonidine, while approximately equipotent to clonidine in reducing hypertension in humans, has been demonstrated to produce signi®cantly less sedation and dry mouth than clonidine (PlaÈnitz, 1984, 1986; Armah et al., 1988; Kirch et al., 1990), does not produce rebound hypertension (Kraft and Vetter, 1994; Ziegler et al., 1996), and is less likely to produce hypotension in normotensive patients (PlaÈnitz, 1984; MacPhee et al., 1992). It has been suggested that the improved side-effect pro®le of moxonidine over clonidine may be due to the greater selectivity of moxonidine for I1 imidazoline receptors relative to a 2 receptors (e.g. Ernsberger et al., 1993; see also Codd et al., 1995). If moxonidine, like clonidine, also produces analgesia in humans, then moxonidine might represent an analgesic with an improved side-effect pro®le relative to clonidine. Recently, Fairbanks and Wilcox (1999) demonstrated that moxonidine produces antinociception in mice in the tail-¯ick test and the substance P nociceptive test after
0304-3959/00/$20.00 q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(99)00260-2
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intrathecal administration. The antinociceptive effects of both moxonidine and clonidine were blocked by the mixed I1/a 2 antagonist efaroxan as well as by the selective a 2 antagonist SK&F 86466, indicating the involvement of a 2 receptors in mediating the antinociceptive effects of both moxonidine and clonidine after intrathecal administration in mice (Fairbanks and Wilcox, 1999). However, in D79N-a 2a mice which lack functional a 2a receptors, clonidine failed to produce antinociception whereas the potency of moxonidine in producing antinociception was comparable in D79N-a 2a and wild-type mice, indicating that different a 2 receptor subtypes are likely involved in mediating the antinociceptive effects of moxonidine and clonidine (Fairbanks and Wilcox, 1999). In addition, Fairbanks et al. (2000) demonstrated that moxonidine synergized with morphine and deltorphin II, but not DAMGO, to inhibit substance Pinduced behavior after intrathecal administration in mice. Thus, moxonidine may represent a therapeutically useful analgesic with a unique mechanism of action and an improved side-effect pro®le relative to clonidine. The major purpose of the present studies was to determine if moxonidine produces analgesia in the formalin test in rats. Clonidine previously has been shown to produce analgesia in the formalin test in rats and mice after intraplantar (Hong and Abbott, 1996), systemic (Dennis et al., 1980; Tasker and Melzack, 1989) or intrathecal (Przesmycki et al., 1997, 1998; Kanui et al., 1993) administration, and to reduce formalin-induced paw edema after systemic administration (Kulkarni et al., 1986). Accordingly, dose± and time±response curves were determined for both moxonidine and clonidine in the formalin test. A dose±response curve also was determined for morphine for purposes of comparison. A second purpose of the present studies was to determine the potential relative contribution of imidazoline and alpha adrenergic receptors to the antinociceptive effects of moxonidine and clonidine in the formalin test. Therefore, dose±response curves were determined for the I1/a 2 receptor antagonist efaroxan, the a 2 receptor antagonist yohimbine, and the a 1 receptor antagonist prazosin administered alone or with a constant dose of moxonidine or clonidine. The results of the present studies demonstrate that moxonidine, like clonidine, produces analgesia in the formalin test in rats but that the mechanism of action of the two drugs differs.
2. Materials and methods 2.1. Animals Male Sprague±Dawley rats (Harlan Sprague±Dawley, Indianapolis, IN) weighing 180±220 g were used. Rats were housed up to eight per cage in a large colony room, and provided food and water ad libitum with a 12:12 h light/ dark cycle. Each animal was used only once. All procedures
were approved by the Eli Lilly Institutional Animal Care and Use Committee. 2.2. Formalin test The methods used were a modi®cation of the automated method of Jett and Michelson (1996). All testing took place in commercially available startle behavior chambers (Model SR-Lab, San Diego Instruments, San Diego, CA) which detected movements of the rats by means of an accelerometer. At the beginning of an experiment, the rats were injected with vehicle or a dose of drug and individually placed in the restraint cylinders (i.d. 8.5 cm; length 16 cm). Thirty minutes later, the rats were removed from the cylinders and administered formalin (50 ml of a 5% solution in saline) subcutaneously into the plantar surface of the right hindpaw and immediately placed back into the restraining cylinders. The magnitude of the movements were monitored continuously for 60 min in 1-s bins. The number of `agitation' events, de®ned as the number of 1-s bins with a change in dynamic force that exceeded an empirically determined threshold value (20 arbitrary load units, which was determined in pilot experiments to be greater than that produced by animals quietly snif®ng and breathing) were totaled in 5min intervals. As previously described by Jett and Michelson (1996), the formalin-induced movements detected by the system included licking and ¯inching the affected paw as well as hopping and turning. 2.3. Drugs Moxonidine free base (Lilly Research Laboratories, Indianapolis, IN) was dissolved in distilled water with a drop of 1 N HCl. Clonidine HCl, efaroxan HCl, prazosin HCl (Sigma Chemical Co., St. Louis, MO), yohimbine HCl and morphine sulfate (RBI, Natick, MA) were dissolved in deionized water. Doses refer to the form of the drug listed. All drugs were administered subcutaneously in a volume of 1.0 ml/kg. 2.4. Statistics In time-course experiments, the number of `agitation' events were totaled in 5-min intervals and data were analyzed by repeated measures ANOVA. For constructing dose±response curves, the total events during minutes 0±5 after formalin administration were considered to be phase I, and the total number of events during minutes 15±45 were considered to be phase II. Treatment groups were compared to appropriate control groups using ANOVA and Dunnett's t-test. Data are expressed as mean ^ SEM. 3. Results In animals administered vehicle 30 min before formalin, there was an initial peak in the number of events during the ®rst 5-min block after formalin, followed by a decrease in
H.E. Shannon, E.A. Lutz / Pain 85 (2000) 161±167
the number of events in the second 5-min block, and subsequently an increase again in blocks 3 through 9 (Fig. 1, open circles). Thus, our automated method produced results which are highly comparable to the automated method of Jett and Michelson (1996) as well as qualitatively similar to manual methods of counting, scoring or timing components of formalin-induced behavior such as licking or ¯inching (e.g. Dubuisson and Dennis, 1977; Wheeler-Aceto et al., 1990). When moxonidine was administered 30 min before formalin, moxonidine produced a dose- and time-dependent analgesic effect in the formalin test in the rat (Fig. 1). Moxonidine (1.0±10 mg/kg s.c.) was ef®cacious in reducing formalin-induced behaviors in both phase I (the ®rst 5-min block), and phase II (5-min blocks 3 through 9). A dose± response curve was constructed by summing all of the events in the respective phases and is presented in Fig. 2. Like moxonidine, clonidine and morphine also produced dose-related decreases in formalin-induced behaviors in both phase I and phase II of the formalin test (Fig. 2). Clonidine was the most potent in both phases while moxonidine and morphine were approximately equipotent in each phase. In order to determine the potential role of imidazoline and a -adrenergic receptors in the mechanism of action of moxonidine and clonidine, constant doses of either moxonidine (5.0 mg/kg) or clonidine (0.5 mg/kg) were administered with varying doses of either the mixed imidazoline I1 and a 2-adrenergic antagonist efaroxan, the selective a 2-adrenergic receptor antagonist yohimbine, or the selective a 1-adrenergic receptor antagonist prazosin. Neither efaroxan nor yohimbine administered alone produced statistically significant effects on formalin-induced behavior (Fig. 3, left
163
panel). Prazosin produced a modest, but signi®cant reduction in formalin-induced behavior after doses of 0.3 and 10 mg/kg but had no effect after doses of 0.1 and 1.0 mg/kg (Fig. 3, left panel). Both efaroxan (0.1±1.0 mg/kg) and yohimbine (0.1±1.0 mg/kg) produced dose-related reversals of the analgesic effects of 5.0 mg/kg of moxonidine (Fig. 3, center panel), and these two antagonists were equief®cacious and approximately equipotent to each other in reversing moxonidine. Prazosin (0.3±10 mg/kg) had no signi®cant effect on moxonidine-induced analgesia (Fig. 3, center panel). In contrast, while efaroxan produced a complete and dose-related antagonism of the effects of clonidine, the reversal of clonidine by yohimbine was substantially smaller in magnitude than that for efaroxan (Fig. 3, right panel). Prazosin also did not reverse the effects of clonidine (Fig. 3, right panel).
4. Discussion The major ®nding of the present studies was that moxonidine, which is an I1 imidazoline/a 2-adrenergic receptor agonist (e.g. Ernsberger et al., 1993), produced a robust antinociceptive effect in the formalin test in rats. Moreover, moxonidine was equief®cacious to both clonidine and morphine in reducing formalin-induced behaviors. Furthermore, moxonidine was approximately equipotent to morphine in the formalin test but approximately tenfold less potent than clonidine. The present ®ndings corroborate and extend the previous ®ndings that moxonidine produces antinociception in the mouse tail-¯ick and substance P nociceptive tests after intrathecal administration in mice (Fairbanks and Wilcox, 1999; Fairbanks et al., 2000). In humans,
Fig. 1. Dose- and time-related effects of moxonidine in reducing nocifensive behaviors produced by the intraplantar injection of formalin in rats. Each point represents the mean ^ SEM of eight rats. Abscissa: consecutive 5-min blocks; ordinate: number of events.
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Fig. 2. Dose±response curves for moxonidine, clonidine and morphine in the formalin test in rats. Each point represents the total number of events during phase I or phase II of the 45-min time course after formalin was injected (see Fig. 1), and is the mean ^ SEM of 7±8 rats. Points above V represent the effects of vehicle. Abscissa: dose of drug in mg/kg; ordinate: total number of events during the ®rst 5 min (phase I, left panel) or the ®nal 35 min (phase II, right panel). *P , 0:05 vs. Vehicle, Dunnett's t-test.
moxonidine appears to have an improved side-effect pro®le relative to clonidine (see Section 1). If moxonidine also produces analgesia in humans, then it has the potential to be as ef®cacious an analgesic as clonidine but with an improved side-effect pro®le. Several lines of evidence suggest that I1 imidazoline receptors play at least a role in the analgesic effects of moxonidine. Moxonidine has approximately a 40-fold higher af®nity for I1 imidazoline receptors (2.3 ^ 0.5 nM) than for a 2-adrenergic receptors (75 ^ 8 nM) whereas clonidine is only approximately fourfold selective for I1 (1.0 ^ 0.3) over a 2 (3.8 ^ 1.0) receptors (Ernsberger et al., 1993). Based upon these in vitro binding af®nities, moxonidine might be expected to act preferentially at I1 receptors in vivo, and to be approximately two- to threefold less potent than clonidine. In the present studies, moxonidine was approximately tenfold less potent than clonidine in vivo. The tenfold difference in potency observed in vivo, while not identical, is closer to the difference in potency in vitro at I1 compared with the difference in potency in vitro at a 2 receptors between moxonidine and clonidine, suggesting that I1 receptors may be involved in mediating the analgesic effects of moxonidine in the formalin test. In addition, the analgesic effects of moxonidine were antagonized by the I1 receptor preferring antagonist efaroxan (Haxhiu et al., 1994). However, differences in potency in vivo need not necessarily re¯ect potencies in vitro due to differences in drug metabolism and distribution, and, the degree of receptor reserve (e.g. Takano and Yaksh, 1993; Kenakin, 1997). Further, the a 2 receptor antagonist yohimbine, which does
not bind to imidazoline receptors (Piletz et al., 1996), was equipotent and equief®cacious to efaroxan in antagonizing the analgesic effects of moxonidine, indicating that a 2 receptors play an important role in meditating the analgesic effects of moxonidine. Thus, the present data suggest that the antinociceptive effects of moxonidine in the formalin test are mediated primarily by a 2 rather than I1 receptors. In contrast to the results with moxonidine, the effects of clonidine in the formalin test, while antagonized completely by efaroxan, were only partially antagonized by yohimbine. Thus, the present results suggest that I1 receptors play a greater role than a 2 receptors in mediating the analgesic effects of clonidine in the formalin test. The present ®ndings corroborate and extend those of previous investigators. Dennis et al. (1980) found that yohimbine only partially antagonized clonidine in the formalin test; however, yohimbine administered alone at doses of 1.0 and 4.0 mg/kg i.p. produced statistically signi®cant analgesia. Similarly, Tasker and Melzack (1989) found that a single dose of yohimbine (2.0 mg/kg) only partially, and nonsigni®cantly, reversed the effects of clonidine in the formalin test. Although yohimbine is .1000-fold selective for a 2 receptors over I1 receptors, it has an af®nity for serotonin1A receptors which is only approximately twofold lower than its af®nity for a 2 receptors (Winter and Rabin, 1992). Thus, it is possible that yohimbine might have produced effects through serotonin1A receptors which masked any antagonism of clonidine by yohimbine at a 2 receptors. However, in the present studies, we did not observe a statistically significant effect of yohimbine when administered alone over the
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Fig. 3. Antagonism of moxonidine and clonidine by yohimbine and efaroxan during phase II of the formalin test in rats. (Left) Lack of effects of the alphaadrenergic receptor antagonists efaroxan, yohimbine and prazosin administered alone in the formalin test. (Middle) Antagonism of moxonidine (5.0 mg/kg) by the mixed imidazoline and a 2-adrenergic receptor antagonist efaroxan and the selective a 2-adrenergic antagonist yohimbine but not by the a 1-adrenergic antagonist prazosin in the formalin test in rats. (Right) Antagonism of clonidine (0.5 mg/kg) by the mixed imidazoline and a 2-adrenergic receptor antagonist efaroxan, partial reversal by the selective a 2-adrenergic receptor antagonist yohimbine and lack of antagonism by the selective a 1-adrenergic receptor antagonist prazosin in the formalin test in rats. Each point represents the total number of events during the 35-min of phase II after formalin was injected, and is the mean of 4±8 rats. Points above Veh represent the effects of vehicle alone, points above V 1 V represent the effects of vehicle 1 vehicle, points above V 1 Mox represent the effects of vehicle 1 5:0 mg/kg moxonidine, and points above V 1 Cl represent the effects of vehicle 1 0:5 mg/kg clonidine. Abscissa: dose of antagonist in mg/kg; ordinate: total number of events during the 35 min of phase II. *P , 0:05 vs. vehicle or vehicle 1 vehicle, Dunnett's t-test. # P , 0:05 vs. vehicle 1 moxonidine or vehicle 1 clonidine, Dunnett's t-test.
dose range of 0.1±1.0 mg/kg, although there was a nonsigni®cant effect of yohimbine at 1.0 mg/kg. Furthermore, yohimbine completely antagonized moxonidine at doses which only partially blocked clonidine. Therefore, any effects of yohimbine at serotonin1A, or any other, receptors are highly unlikely to have prevented yohimbine from antagonizing clonidine. Thus, the majority of the evidence demonstrates that clonidine produces analgesia in the formalin test through an efaroxan-sensitive but relatively yohimbine-insensitive mechanism. We interpret these ®ndings to indicate that I1 imidazoline receptors may play a greater role than a 2 receptors in mediating the analgesic effects of clonidine in the formalin test. The ®ndings that yohimbine does not completely antagonize clonidine in the formalin test are in contrast to results in other nociceptive tests. For example, Tasker and Melzack (1989) found that although yohimbine (2.0 mg/kg) nonsigni®cantly affected clonidine in the formalin test, this same dose of yohimbine completely antagonized the effects of clonidine in the tail-¯ick test. Similarly, the intrathecal administration of yohimbine antagonized the analgesic effects of intrathecally administered clonidine in the hot-plate (Takano and Yaksh, 1992), tail-¯ick (Monroe
et al., 1995), and thermal paw-withdrawal (Buerkle and Yaksh, 1998) tests. Further, yohimbine antagonized the anti-allodynic effects of clonidine in rats with L5 and L6 nerve ligation (Yaksh et al., 1995). Thus, the effects of clonidine in the formalin test appear to be mediated by mechanisms different from those which mediate its effects in other nociceptive tests. Prazosin did not antagonize either moxonidine or clonidine in the formalin test in the present studies. Prazosin is selective for a 1 over a 2 receptors, and its lack of antagonist activity demonstrates a lack of involvement of a 1 receptors in mediating the effects of moxonidine and clonidine in the formalin test. However, it is now appreciated that prazosin has preferential, and appreciable, af®nity for the a 2B and a 2C over the a 2A subtypes of a 2 receptors (Blaxall et al., 1991; Link et al., 1992; UhleÂn et al., 1992). Thus, the lack of antagonism by prazosin suggests that a 2B and a 2C, as well as a 1, adrenergic receptors play little if any role in mediating the effects of moxonidine and clonidine in the formalin test. However, the effects of selective a 2B and a 2C antagonists, when they become available, on moxonidine antinociception, as well as the effects of moxonidine in a 2B and a 2C receptor knockout mice, are needed to more completely
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determine the relative role of a 2B and a 2C receptors in mediating moxonidine-induced antinociception. Several studies using genetically altered mice recently have demonstrated that the a 2A-adrenergic receptor subtype is the primary mediator of a 2-adrenergic analgesia. Lakhlani et al. (1997) demonstrated that in a mouse line expressing a point mutation (D79N) in the a 2A-adrenergic receptor, the a 2A agonist dexmedetomidine did not produce analgesia in the hot plate test. In the same mouse line, Stone et al. (1997) found that the intrathecal administration of the nonselective a 2 agonist UK 14304 had no analgesic effect in the tail-¯ick test or the substance-P nociceptive test. Further, Hunter et al. (1997) reported that dexmedetomidine did not produce analgesia in the tail immersion test in D79N mutant mice, but did produce analgesia in a 2B and a 2C receptor knockout mice. Moreover, Hunter et al. (1997) also demonstrated that the sedative and hypothermic effects of dexmedetomidine were abolished in the D79N mice but not the a 2B and a 2C receptor knockout mice. The ®ndings with D79N mutant mice have led to the conclusion that selective a 2A-adrenergic receptor agonists are unlikely to provide analgesia with an acceptable therapeutic pro®le (Hunter et al., 1997). However, the demonstration that moxonidine produces antinociception (Fairbanks and Wilcox, 1999; present report) by a 2 receptors other than the a 2a subtype (Fairbanks and Wilcox, 1999), suggests that a 2 agonists selective for other subtypes, perhaps like moxonidine, may provide analgesia with an acceptable therapeutic pro®le. Further experiments are needed to determine the relative role of a 2 receptor subtypes in mediating moxonidine- and clonidine-induced antinociception in other animal models.
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