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Antihyperalgesic effects of the muscarinic receptor ligand vedaclidine in models involving central sensitization in rats
Pain 93 (2001) 221±227
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Antihyperalgesic effects of the muscarinic receptor ligand vedaclidine in models involving central sensitization in rats Harlan E. Shannon*, Carrie K. Jones, Dominic L. Li, Steven C. Peters, Rosa M.A. Simmons, Smriti Iyengar Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA Received 17 July 2000; received in revised form 7 February 2001; accepted 30 March 2001
Abstract It is well established that muscarinic cholinergic agonists produce antinociceptive effects in a number of acute pain models. However, relatively little is known about the effects of muscarinic receptor agonists in models which involve central sensitization in pain pathways. The purpose of the present studies was to evaluate the effects of vedaclidine, a muscarinic receptor mixed agonist/antagonist across receptor subtypes, in models involving central sensitization. Vedaclidine (0.3±10 mg/kg s.c.) produced dose-related antihyperalgesic effects in the formalin test as well as a dose-related reversal of capsaicin-induced mechanical hyperalgesia in rats. In the carrageenan test, vedaclidine (0.1±30 mg/kg) produced a dose-related reversal of both mechanical and thermal hyperalgesia that were antagonized by the muscarinic receptor antagonist scopolamine. In addition, the antihyperalgesic effects of vedaclidine in the carrageenan test were synergistic with the antihyperalgesic effects of the non-steroidal antiin¯ammatory drug ketoprofen, as demonstrated by isobolographic analysis. The present studies demonstrate that vedaclidine produces antihyperalgesic effects in models involving central sensitization, suggesting that vedaclidine, and potentially other muscarinic receptor agonists, may have clinical utility in the management of pain states involving central sensitization, such as neuropathic and in¯ammatory pain states. q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Vedaclidine; Muscarinic cholinergic; Antinociception; Rats
1. Introduction Vedaclidine has high af®nity for muscarinic cholinergic, but not other, receptors and is an agonist at M2 and M4, but an antagonist at M1, M3 and M5, muscarinic receptor subtypes (Shannon et al., 1997). Vedaclidine is ef®cacious in producing acute antinociception in the grid shock, hot plate, tail¯ick and writhing tests in mice and rats (Swedberg et al., 1997). In these acute antinociceptive tests, vedaclidine was approximately equief®cacious to, and approximately 3-fold more potent than, morphine (Swedberg et al., 1997). The antinociceptive effects of vedaclidine were antagonized by the selective muscarinic receptor antagonist scopolamine but not by the opioid receptor antagonist naltrexone (Swedberg et al., 1997). Thus, vedaclidine may be a clinically useful analgesic and an alternative to opioids. The question arises as to whether vedaclidine, in addition * Corresponding author. Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285 USA. Tel.: 1317-276-4360; fax: 11-317276-5546. E-mail address: [email protected] (H.E. Shannon).
to being ef®cacious in models of acute nociception, is ef®cacious in models involving central sensitization in pain pathways. Central sensitization is characterized by altered responsiveness of dorsal horn neurons, expansion of receptive ®elds and plasticity of neuronal connections (e.g. Woolf, 1983; Devor and Wall, 1978; Woolf et al., 1994). The intradermal or subcutaneous injection of capsaicin, which selectively activates primary afferent C-®bers (Baumann et al., 1991), produces sensitization of spinothalamic tract neurons (Simone et al., 1991; Dougherty and Willis, 1992), and produces thermal hyperalgesia and mechanical allodynia in animals (Gilchrist et al., 1996) as well as humans (LaMotte et al., 1991; TorebjoÈrk et al., 1992). Similarly, the subcutaneous injection of formalin produces a biphasic response: an immediate and intense increase in the spontaneous activity followed by a quiescent phase and then a more prolonged increase in cell ®ring of both primary afferents (Heapy et al., 1987; Puig and Sorkin, 1995) as well as dorsal horn neurons (Dickenson and Sullivan, 1987a,b). Behaviorally, the injection of formalin elicits a temporally biphasic increase in licking, biting, ¯inching
0304-3959/01/$20.00 q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(01)00319-0
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and other nocifensive behaviors (e.g. Dubuisson and Dennis, 1977). The initial phase is short lived and likely re¯ects an acute nociceptive state, while the second phase is more extended and re¯ects a state of central sensitization (Dubuisson and Dennis, 1977; Wheeler-Aceto et al., 1990; Coderre et al., 1993). In¯ammation, such as that produced by carrageenan, can produce a state of spinal cord sensitization in which a noxious stimulus produces an exaggerated response (hyperalgesia) and a normally non-noxious stimulus can produce a nocifensive response (allodynia) to both thermal and mechanical stimuli (e.g. Hargreaves et al., 1988; Traub, 1997). Unlike an injection of formalin, however, carrageenan does not increase spontaneous activity of C-®ber afferents, but rather increases the magnitude of C-®ber evoked responses (Stanfa et al., 1992) and decreases the occurrence and magnitude of inhibitory input to spinal cord neurons (Traub, 1997). The purpose of the present study was to determine the antinociceptive effects of vedaclidine in models involving central sensitization. Accordingly, dose-response curves were determined for vedaclidine in the formalin, capsaicin and carrageenan models. In addition, dose-response curves were determined for vedaclidine administered alone and in the presence of the muscarinic cholinergic receptor antagonist scopolamine in the carrageenan model. Further, in order to determine whether the concomitant administration of vedaclidine and a non-steroidal antiin¯ammatory drug (NSAID) was additive or synergistic, dose-response curves were determined in the carrageenan model for vedaclidine and the representative NSAID ketoprofen administered alone and at several ®xed dose-ratios, and the data analyzed isobolographically (Tallarida et al., 1989). 2. Materials and methods 2.1. Animals Adult male Sprague±Dawley rats from Charles River (Portage, MI) weighing 200±250 g were used for the formalin and rotarod (p.o.) experiments or from Harlan Sprague± Dawley (Indianapolis, IN) and weighing 80±100 g for the carrageenan experiments or weighing 250±300 g for the capsaicin and rotarod (s.c.) experiments were used. Animals were group housed with food and water freely available in a temperature and humidity controlled colony room. No animal was used more than once. Lights were turned on between 06:00 and 18:00 h. All experimental protocols were approved by the Eli Lilly Institutional Animal Care and Use Committee. 2.2. Rotarod test The ability of vedaclidine to induce sedation/ataxia was examined using an accelerating rotarod. All rats were given an initial three training trials to maintain posture on a rotarod which accelerated to 17 rev./min in 5 s and main-
tained that speed for 40 s (Omnitech Electronics Inc., Columbus, OH). On the following day, a 40 s trial was conducted 1 and 2 h after oral administration, or 0.5, 1 or 2 h after s.c. administration, of vehicle or a dose of vedaclidine. Animals not falling off the rotarod were given a maximum score of 40 s. 2.3. Formalin test The formalin test was conducted using Plexiglas cubicles measuring 25 £ 25 £ 20 cm (length £ width £ height) as described in detail by Simmons et al. (1998) and modi®ed from Shibata et al. (1989) and Wheeler-Aceto et al. (1990). Brie¯y, a mirror placed at the back of the cubicle allowed the unhindered observation of the formalin injected paw. Rats were acclimatized in the cubicles for at least 1 h before the experiment. All testing was conducted between 08:00 and 14:00 h. Vehicle or a dose of drug was administered 30 min before the formalin injection. Formalin (50 ml of a 5% solution in saline) was injected subcutaneously into the dorsal lateral surface of the right hind paw. Observation started immediately after the formalin injection. The number of seconds each rat spent licking the formalininjected hind paw was recorded in 5 min bins for 50 min after the formalin injection. 2.4. Capsaicin test Rats were injected subcutaneously with capsaicin (30 mg in 25 ml) into the plantar surface of the right hind paw at time zero. Ten minutes after the injection of capsaicin, tactile allodynia was evaluated with a calibrated series of von Frey ®laments using the up and down method of Chaplan et al. (1994). Brie¯y, the rats were placed in clear plastic cages (17:5 £ 15 £ 15 cm) ®tted with wire mesh ¯ooring and allowed to acclimate for approximately 5 min. Each ®lament was applied to the mid-plantar surface of each hind paw in a perpendicular fashion and depressed slowly (4±6 s) until bending occurred. In uninjected animals, von Frey ®laments above 15 g lifted the paw without bending and were therefore not used; the maximum withdrawal threshold was thus taken to be 15 g. Vehicle or a dose of drug was injected subcutaneously 20 min before the injection of capsaicin. 2.5. Carrageenan test Rats were injected subcutaneously with l -carrageenan (100 ml of 15 mg/ml solution) into the plantar surface of the right hind paw at time zero. Withdrawal latencies to a nociceptive thermal stimulus were assessed using a modi®cation of the methods of Hargreaves et al. (1988). The device used (Plantar Test, Ugo Basile) consisted of a glass surface on which the rats were placed individually in Plexiglas cubicles (17 £ 13 £ 14 cm). The radiant heat nociceptive stimulus could be delivered to each hind paw separately. A timer was actuated with the stimulus source,
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and the timer and stimulus were stopped when a brisk withdrawal response was detected by photodiode motion sensors. The intensity of the stimulus was adjusted to produce a baseline latency of approximately 15 s. Tactile allodynia was evaluated with a calibrated series of von Frey ®laments as described above. Vehicle or a dose of drug was injected subcutaneously 90 min after the injection of carrageenan, and withdrawal latencies to mechanical and thermal stimuli determined approximately 30 and 40 min later, respectively. In experiments to determine if the concomitant administration of vedaclidine and an NSAID were additive or potentially synergistic in reversing thermal hyperalgesia in the carrageenan test, dose-response curves were determined for vedaclidine and the representative NSAID ketoprofen administered alone and at several ®xed dose-ratios using the thermal stimulus only. 2.6. Drugs Vedaclidine ((1)-S-3-(4-butylthio-1,2,5-thiadiazol-3yl)-1-azabicyclo[2.2.2]octane; butylthio[2.2.2]; LY297802/ NNC11-1053) tartrate was synthesized at Lilly Research Laboratories. Scopolamine hydrobromide, ketoprofen, l carrageenan and capsaicin were from Sigma Chemical Co. (St. Louis, MO). Vedaclidine, scopolamine and carrageenan were dissolved in distilled water. Ketoprofen was prepared as a suspension in 6% acacia. Capsaicin was dissolved in 10% Tween-80 and 90% saline by heating to 708C for 15 min. Drugs were administered either s.c. or orally by gavage in a volume of 1.0 ml/kg.
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Table 1 Lack of effects of vedaclidine on motor performance on the rotarod a Dose
Time on rotarod (s)
mg/kg p.o.
1h
2h
mg/kg s.c.
0.5 h
1h
2h
0 1.0 3.0 10
40 ^ 0 40 ^ 0 37 ^ 3 40 ^ 0
40 ^ 0 40 ^ 0 36 ^ 4 40 ^ 0
0 1.0 3.0 10
40 ^ 0 40 ^ 0 38 ^ 2 34 ^ 2
40 ^ 0 40 ^ 0 38 ^ 2 30 ^ 4
40 ^ 0 40 ^ 0 37 ^ 3 35 ^ 3
a Rats were administered the indicated dose of vedaclidine orally or subcutaneously and tested on the rotarod at the indicated times after administration. Each value represents the mean ^ SEM of 5±6 (p.o.) or 10±11 (s.c.) rats.
line, then the mixture is considered to be additive. If the mixture ED50 lies below the theoretical additive ED50 line, and the con®dence intervals do not overlap that line, then the mixture is considered to be synergistic. ED50 values were determined using a four-parameter logistic equation (De Lean et al., 1978). 3. Results 3.1. Rotarod test Vedaclidine had no signi®cant effect on motor performance as measured on the rotarod up to 2 h after either p.o. or s.c. administration of 1.0±10 mg/kg (Table 1).
2.7. Statistics
3.2. Formalin test
Scoring in the formalin test was carried out according to Coderre et al. (1993) and Abbott et al. (1995). The sum of time spent licking in seconds from minutes 0±5 was considered the early phase while the late phase was taken as the sum of seconds spent licking from 15 to 40 min. In the tactile stimulation test, a withdrawal threshold was calculated as described in Chaplan et al. (1994). In the thermal test, a difference in withdrawal latency between the two hindpaws was calculated as the withdrawal latency of the ipsilateral paw minus the withdrawal latency of the contralateral paw. Data are presented as means ^ SEM. Data were analyzed by one-way analysis of variance and treatment groups were compared to appropriate control groups using a Dunnett's t-test. The interaction between vedaclidine and ketoprofen in the carrageenan test was examined by isobolographic analysis (e.g. Tallarida et al., 1989). Brie¯y, in the isobolograms, the vedaclidine ED50 is plotted on the abscissa and the ketoprofen ED50 on the ordinate. A theoretical line of additive interaction is drawn by connecting the ED50 for vedaclidine alone with that of ketoprofen alone. For each drug combination, an ED50 along with con®dence intervals is calculated for each mixture. If the mixture ED50 lies on the theoretical additive ED50 line, or the con®dence intervals overlap that
When vedaclidine was administered orally 30 min before formalin, vedaclidine produced a dose-dependent antihyperalgesic effect in the formalin test in the rat (Fig. 1). Vedaclidine (0.3±10 mg/kg p.o.) was ef®cacious in reducing formalin-induced behaviors in both the early and the late
Fig. 1. Dose-related inhibition of early as well as late phase formalininduced paw licking behavior by vedaclidine after oral administration in rats. Vehicle or a dose of vedaclidine was administered 30 min before the intraplantar administration of formalin. Each bar represents the mean ^ SEM of one observation in each of 6±11 rats. *P , 0:05 vs. vehicle alone.
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3.3. Capsaicin test Paw withdrawal threshold to a mechanical stimulus was reduced to approximately 3.0 g when determined approximately 30 min after the administration of vehicle and 10 min after the administration of capsaicin (Fig. 2; point above Veh). Thus, capsaicin produced mechanical hyperalgesia in the present studies. Vedaclidine (0.3±10 mg/kg s.c.), administered 20 min before capsaicin, produced a doserelated reversal of capsaicin-induced mechanical allodynia which was signi®cant after doses of 3.0 and 10 mg/kg. Morphine (10 mg/kg s.c.) also produced a complete reversal of capsaicin-induced mechanical allodynia (Fig. 2, point above 10 M). 3.4. Carrageenan test
Fig. 2. Dose-related reversal of capsaicin-induced decreases in tactile threshold by vedaclidine in rats. Vehicle or a dose of vedaclidine was administered s.c. 20 min before capsaicin, and mechanical withdrawal threshold was determined 10 min after the intraplantar administration of capsaicin. The dashed line represents the maximum withdrawal threshold in rats not injected with capsaicin. Each point represents the mean ^ SEM of one observation in each of 12 rats. Veh, vehicle; 10 M, 10 mg/kg morphine. *P , 0:05 vs. vehicle alone.
phase of the test. A dose of 10 mg/kg was required to signi®cantly reduce paw licking time in phase I, while both 3.0 and 10 mg/kg signi®cantly reduced paw licking time in phase II.
In rats administered vehicle 90 min after carrageenan and tested approximately 25 min later, paw withdrawal latencies to a noxious thermal stimulus were, on average, approximately 9.0 s shorter ipsilaterally than contralaterally (Fig. 3, left panel). Thus, carrageenan produced thermal hyperalgesia in the present studies. Vedaclidine (s.c.) produced a dose-related reversal of carrageenan-induced thermal hyperalgesia that was signi®cant after doses of 0.3 mg/kg and higher. When the dose-response curve for vedaclidine was redetermined in the presence of the selective muscarinic receptor antagonist scopolamine (1.0 mg/kg s.c.), the dose-response curve for vedaclidine was shifted to the right approximately 10-fold. In the presence of scopolamine, doses of 3.0 mg/kg of vedaclidine and higher signi®cantly reversed the thermal hyperalgesia. Scopolamine (1.0 mg/kg) administered alone produced no appreciable effects
Fig. 3. Dose-related reversal of carrageenan-induced decreases in paw withdrawal latencies to a thermal stimulus (left panel) and in paw withdrawal thresholds to a mechanical stimulus (right panel) by vedaclidine, and antagonism by scopolamine, in rats. Vehicle or a dose of vedaclidine alone or concomitantly with a dose of scopolamine was administered s.c. 90 min after carrageenan, and approximately 30 min later withdrawal thresholds to non-noxious mechanical stimulation and withdrawal latencies to noxious thermal stimulation were determined. Each point represents the mean ^ SEM of one observation in each of 12 rats. V 1 V, vehicle plus vehicle; V 1 S, vehicle plus 1.0 mg/kg scopolamine *P , 0:05 vs. vehicle plus vehicle.
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on withdrawal latencies to noxious thermal stimulation (Fig. 3, left panel, point above V 1 S). In the same rats, the paw withdrawal threshold to nonnoxious mechanical stimulation was reduced to less than approximately 2.5 g when determined 120 min after carrageenan, and 30 min after vehicle, administration (Fig. 3, right panel). Thus, carrageenan produced mechanical allodynia. Vedaclidine s.c. produced a dose-related reversal of mechanical allodynia that was signi®cant after doses of 3.0± 30 mg/kg. In the presence of scopolamine (1.0 mg/kg), the dose-response curve for vedaclidine was shifted modestly to the right. However, even in the presence of scopolamine, doses of 3.0±30 mg/kg of vedaclidine signi®cantly reversed the mechanical allodynia. Scopolamine (1.0 mg/kg) administered alone produced no appreciable effects on withdrawal thresholds to non-noxious mechanical stimulation (Fig. 3, right panel, point above V 1 S). Higher doses of scopolamine produced hyperactivity which precluded testing in the present experiments. 3.5. Isobolographic analysis of vedaclidine-ketoprofen interactions The data for the concomitant administration of vedaclidine s.c. and the NSAID ketoprofen p.o. in ®xed dose-ratios in the carrageenan test using the thermal stimulus are represented as an isobologram in Fig. 4. In Fig. 4, the doses of vedaclidine contributing to the ED50 values are shown along the abscissa, with the ED50 for vedaclidine alone (0.45 ^ 0.01 mg/kg s.c.) lying on the abscissa, and, the doses of ketoprofen contributing to the ED50 values are
Fig. 4. Isobolograms for the ED50 values for vedaclidine s.c. plotted against the ED50 for ketoprofen p.o. using the thermal stimulus in the carrageenan model. The solid line represents the line of additivity constructed by joining the ED50 values for each drug alone. The isobol points were determined from the ED50 values of vedaclidine and ketoprofen given in ®xed doseratios of 1:5, 1:10 and 1:30. The SEMs are resolved into the vedaclidine (horizontal) and ketoprofen (vertical) components.
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shown on the ordinate, with the ED50 for ketoprofen alone (18.6 ^ 2.4 mg/kg p.o.) lying on the ordinate. The straight line connecting the ED50 values for vedaclidine and ketoprofen is the theoretical line of additivity. The ED50 values for the dose combinations are plotted as points on the isobologram, where the coordinates are the amount of vedaclidine in the combined ED50 and the amount of ketoprofen in the combined ED50. When data for the dose combinations of vedaclidine:ketoprofen of 1:5, 1:10 and 1:30 were plotted isobolographically, all of the data points were below the theoretical line of additivity and none of the SEMs overlapped the line. Thus, vedaclidine and ketoprofen were synergistic (i.e. more than additive) in producing analgesia in the carrageenan test. 4. Discussion The major ®nding of the present studies was that the muscarinic cholinergic receptor ligand vedaclidine, with mixed agonist and antagonist actions across muscarinic cholinergic receptor subtypes (Shannon et al., 1997), produced antinociceptive and antihyperalgesic effects in models involving central sensitization in rats. Vedaclidine produced dose-related reductions in formalin-, capsaicin-, and carrageenan-induced nocifensive behaviors. Vedaclidine has previously been shown to produce antinociception in acute nociceptive models, including grid shock, tail-¯ick, hot plate and writhing models (Swedberg et al., 1997). The present ®ndings are the ®rst demonstration that vedaclidine is ef®cacious in models involving central sensitization of pain pathways. We have previously reported (Swedberg et al., 1997) that vedaclidine does not produce salivation at doses up to 130 times the antinociceptive ED50 and produces tremor only at doses 54 times the antinociceptive ED50. In addition, consistent with the present lack of effects on the rotarod at anti-hyperalgesic/allodynic doses, vedaclidine was without effect on the rotarod at antinociceptive doses (Swedberg et al., 1997). In contrast, the ratios of the sideeffect-to-ef®cacy ED50 values for the non-selective muscarinic agonist oxotremorine were 2±12 (Swedberg et al., 1997). Thus, vedaclidine produces antinociception as well as antihyperalgesia/allodynia in models of both acute and chronic pain with an improved side-effect pro®le relative to nonselective muscarinic agonists, and therefore may be a clinically useful analgesic and an alternative to opioids. Vedaclidine produced dose-related reductions in formalin-induced behaviors. The subcutaneous injection of formalin produces biphasic behavioral as well as electrophysiologic effects (e.g. Dubuisson and Dennis, 1977; Puig and Sorkin, 1995; Dickenson and Sullivan, 1987a,b), with the early phase likely re¯ecting an acute nociceptive state and the late phase a state of central sensitization (e.g. Dubuisson and Dennis, 1977; Wheeler-Aceto et al., 1990; Coderre et al., 1993). The effectiveness of vedaclidine in reducing formalin-induced behaviors in the early phase is
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consistent with the antinociceptive effects of vedaclidine in acute antinociceptive models (Swedberg et al., 1997). The demonstration that vedaclidine also reduces formalininduced behaviors in the late phase indicates that vedaclidine also is ef®cacious in reducing central sensitization in pain pathways. The present ®ndings are consistent with previous reports that the non-selective muscarinic receptor agonist oxotremorine reduced formalin-induced behaviors after subcutaneous or intrathecal administration in rats (Capone et al., 1999; Machelska et al., 1999; Przewlocka et al., 1999). In¯ammation, such as that produced by carrageenan, produces central sensitization accompanied by increases in the magnitude of C-®ber evoked responses and decreases in descending inhibition without increases in spontaneous ®ring of C-®ber afferents (Stanfa et al., 1992; Traub, 1997). In the present studies, vedaclidine produced a doserelated reversal of the thermal and mechanical hyperalgesia produced by carrageenan. Thus, vedaclidine was able to reverse previously established central sensitization, suggesting that it may have a direct action on central sensitization in pain pathways. One possible explanation for the present ®ndings is that vedaclidine may have enhanced descending inhibition. Iwamoto (1991) has previously demonstrated that muscarinic receptor agonists enhance descending inhibition in producing acute antinociception. Further experiments are needed to determine what effects vedaclidine may have on descending inhibitory pathways, and whether such effects might in¯uence central sensitization. The subcutaneous injection of capsaicin directly stimulates C-®bers and thus, like the injection of formalin, increases the ®ring of primary afferent neurons (Puig and Sorkin, 1995; Baumann et al., 1991) and produces central sensitization (Simone et al., 1991; Dougherty and Willis, 1992). Pretreatment with vedaclidine produced a doserelated reversal of the mechanical hyperalgesic effects produced by capsaicin. To the best of our knowledge, the present ®ndings that vedaclidine prevented the hyperalgesia produced by capsaicin are the ®rst report that a muscarinic receptor agonist can reduce the hyperalgesia produced by direct stimulation of C-®bers by capsaicin. Further research is needed to determine whether the muscarinic receptors involved in preventing capsaicin-induced hyperalgesia are located peripherally, spinally, or supraspinally. Antagonism of the effects of vedaclidine by the muscarinic cholinergic receptor antagonist scopolamine was investigated in the carrageenan test. Scopolamine (1.0 mg/kg) produced a parallel shift to the right in the vedaclidine dose-response curve when the thermal stimulus was used indicating that vedaclidine produced anti-thermal hyperalgesia by agonist actions at muscarinic cholinergic receptors. These ®ndings corroborate and extend our previous ®ndings that scopolamine antagonizes the antinociceptive effects of vedaclidine in the writhing, tail-¯ick, hot plate and grid shock tests in the mouse (Swedberg et al., 1997). In contrast, scopolamine had little effect on the vedaclidine dose-
response curve using the mechanical stimulus in the carrageenan test. However, approximately 10-fold higher doses of vedaclidine (.3.0 mg/kg) were required to reverse mechanical allodynia than to reverse thermal allodynia (.0.3 mg/kg). Thus, it is perhaps not surprising that higher doses of scopolamine would be needed to antagonize the higher doses of vedaclidine needed to reverse mechanical allodynia. Higher doses of scopolamine produced hyperactivity which, however, precluded testing in the carrageenan test. The anti-thermal allodynia effects of vedaclidine in the carrageenan-induced in¯ammatory model were synergistic with the non-steroidal antiin¯ammatory drug ketoprofen as determined by isobolographic analysis (e.g. Tallarida et al., 1989). When administered alone, the ED50 values for vedaclidine and ketoprofen were approximately 0.45 and 18.5 mg/kg, respectively. However, when administered in a ®xed dose-ratio of 1:10, the ED50 values for vedaclidine and ketoprofen were reduced to approximately 0.15 and 2.0 mg/kg, respectively. These latter doses of vedaclidine and ketoprofen were inactive when administered alone. Thus, vedaclidine and ketoprofen administered in combination were ef®cacious at doses which when administered alone were inactive. It may therefore be possible to administer vedaclidine and ketoprofen, or another NSAID, in combination to provide pain relief clinically at doses which produce fewer or greatly reduced side-effects. The present studies have demonstrated that vedaclidine produces antihyperalgesic/allodynic effects in three models involving central sensitization by agonist actions at muscarinic receptors. Moreover, the antihyperalgesic/allodynic effects of vedaclidine cannot be due to antagonist activity at M1, M3 or M5 receptors because the non-selective muscarinic receptor antagonist scopolamine was without effect when administered alone. Relatively little is known about the potential mechanisms and/or site of action of muscarinic agonists in reducing central sensitization. Muscarinic cholinergic receptors are abundant throughout pain pathways from dorsal root ganglia to somatosensory cortex and therefore could modulate processing of sensory information at several levels. In particular, muscarinic receptors are localized on the super®cial laminae of the dorsal horn in rats where nociceptive Ad and C ®bers terminate (Gillberg and Askmark, 1991; HoÈglund and Baghdoyan, 1997). Thus, muscarinic agonists may enhance descending inhibition and/or act directly on dorsal horn neurons to reduce central sensitization. Alternatively, muscarinic receptor agonists may act more indirectly, for example by modulating nitric oxide pathways. In this regard, Przewlocka et al. (1999) reported that intrathecal oxotremorine reduced formalin-induced hyperalgesia and also reduced the number of formalin-induced nitric oxide synthase-containing neurons in the spinal cord. However, thalamic and cortical sites of action may also be important. Further studies are needed to better understand the mechanism and site of action of muscarinic receptor agonists in
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HoÈglund AU, Baghdoyan HA. M2, M3 and M4 but not M1 muscarinic receptor subtypes are present in rat spinal cord. J Pharmacol Exp Ther 1997;281:470±477. Iwamoto ET. Characterization of the antinociception induced by nicotine in the pedunculopontine tegmental nucleus and the nucleus raphe magnus. J Pharmacol Exp Ther 1991;257:120±133. LaMotte RH, Shain CN, Simone DA, Tsai E-FP. Neurogenic hyperalgesia: psychophysical studies of underlying mechanisms. J Neurophysiol 1991;66:190±211. Machelska H, Pavone F, Capone F, Przewlocka B. Antinociception after both peripheral and intrathecal injection of oxotremorine is modulated by spinal nitric oxide. Eur Neuropsychopharmacol 1999;9:213±217. Przewlocka B, Mika J, Capone F, Machelska H, Pavone F. Intrathecal oxotremorine affects formalin-induced behavior and spinal nitric oxide synthase immunoreactivity in rats. Pharmacol Biochem Behav 1999;62:531±536. Puig S, Sorkin LS. Formalin-evoked activity in identi®ed primary afferent ®bers: systemic lidocaine suppresses phase-2 activity. Pain 1995;64:345±355. Shannon HE, Sheardown MJ, Bymaster FP, Calligaro DO, DeLapp NW, Gidda J, Mitch CH, Sawyer BD, Stengel PW, Ward JS, Olesen PH, Suzdak Pd, Sauerberg P, Swedberg MDB. Pharmacology of butylthio[2.2.2] (LY297802/NNC11-1053): a novel analgesic with mixed muscarinic receptor agonist and antagonist activity. J Pharmacol Exp Ther 1997;281:884±894. Shibata M, Ohkubo T, Takahashi H, Inoki R. Modi®ed formalin test: characteristic biphasic pain response. Pain 1989;38:347±352. Simmons RMA, Li DL, Hoo KY, Deverill M, Ornstein PL, Iyengar SI. Kainate GluR5 receptor subtype mediates the nociceptive response to formalin in the rat. Neuropharmacology 1998;37:25±36. Simone DA, Sorkin LS, Oh U, Chung JM, Owens C, LaMotte RH, Willis WD. Neurogenic hyperalgesia: central neural correlates in responses of spinothalamic tract neurons. J Neurophysiol 1991;66:228±246. Stanfa LC, Sullivan AF, Dickenson AH. Alterations in neuronal excitability and the potency of spinal mu, delta and kappa opioids after carrageenan-induced in¯ammation. Pain 1992;50:345±354. Swedberg MDB, Sheardown MJ, Sauerberg P, Olesen PH, Suzdak PD, Hansen KT, Bymaster FP, Ward JS, Mitch CH, Calligaro DO, DeLapp NW, Shannon HE. Butylthio[2.2.2] ((1)-3(R)-[4-(butylthio)-1,2,5-thiadiazol-3-yl]-1-azabicyclo[2.2.2]octane): an antinociceptive orally active muscarinic agonist in mouse and rat. J Pharmacol Exp Ther 1997;281:876±883. Tallarida RJ, Porreca F, Cowan A. Statistical analysis of drug-drug and sitesite interactions with isobolograms. Life Sci 1989;45:947±996. TorebjoÈrk HE, Lundberg LER, LaMotte RH. Central changes in processing of mechanoreceptive input in capsaicin-induced secondary hyperalgesia in humans. J Physiol 1992;448:765±780. Traub RJ. Spinal modulation of the induction of central sensitization. Br Res 1997;778:34±42. Wheeler-Aceto H, Porreca F, Cowan A. The rat paw formalin test: comparison of noxious agents. Pain 1990;40:229±238. Woolf CJ. Evidence for a central component of post-injury hypersensitivity. Nature 1983;306:686±688. Woolf CJ, Shortland P, Sivilotti LG. Sensitization of high mechanothreshold super®cial dorsal horn and ¯exor motor neurones following chemosensitive primary afferent activation. Pain 1994;58:141±155.
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Antihyperalgesic effects of the muscarinic receptor ligand vedaclidine in models involving central sensitization in rats Harlan E. Shannon*, Carrie K. Jones, Dominic L. Li, Steven C. Peters, Rosa M.A. Simmons, Smriti Iyengar Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA Received 17 July 2000; received in revised form 7 February 2001; accepted 30 March 2001
Abstract It is well established that muscarinic cholinergic agonists produce antinociceptive effects in a number of acute pain models. However, relatively little is known about the effects of muscarinic receptor agonists in models which involve central sensitization in pain pathways. The purpose of the present studies was to evaluate the effects of vedaclidine, a muscarinic receptor mixed agonist/antagonist across receptor subtypes, in models involving central sensitization. Vedaclidine (0.3±10 mg/kg s.c.) produced dose-related antihyperalgesic effects in the formalin test as well as a dose-related reversal of capsaicin-induced mechanical hyperalgesia in rats. In the carrageenan test, vedaclidine (0.1±30 mg/kg) produced a dose-related reversal of both mechanical and thermal hyperalgesia that were antagonized by the muscarinic receptor antagonist scopolamine. In addition, the antihyperalgesic effects of vedaclidine in the carrageenan test were synergistic with the antihyperalgesic effects of the non-steroidal antiin¯ammatory drug ketoprofen, as demonstrated by isobolographic analysis. The present studies demonstrate that vedaclidine produces antihyperalgesic effects in models involving central sensitization, suggesting that vedaclidine, and potentially other muscarinic receptor agonists, may have clinical utility in the management of pain states involving central sensitization, such as neuropathic and in¯ammatory pain states. q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Vedaclidine; Muscarinic cholinergic; Antinociception; Rats
1. Introduction Vedaclidine has high af®nity for muscarinic cholinergic, but not other, receptors and is an agonist at M2 and M4, but an antagonist at M1, M3 and M5, muscarinic receptor subtypes (Shannon et al., 1997). Vedaclidine is ef®cacious in producing acute antinociception in the grid shock, hot plate, tail¯ick and writhing tests in mice and rats (Swedberg et al., 1997). In these acute antinociceptive tests, vedaclidine was approximately equief®cacious to, and approximately 3-fold more potent than, morphine (Swedberg et al., 1997). The antinociceptive effects of vedaclidine were antagonized by the selective muscarinic receptor antagonist scopolamine but not by the opioid receptor antagonist naltrexone (Swedberg et al., 1997). Thus, vedaclidine may be a clinically useful analgesic and an alternative to opioids. The question arises as to whether vedaclidine, in addition * Corresponding author. Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285 USA. Tel.: 1317-276-4360; fax: 11-317276-5546. E-mail address: [email protected] (H.E. Shannon).
to being ef®cacious in models of acute nociception, is ef®cacious in models involving central sensitization in pain pathways. Central sensitization is characterized by altered responsiveness of dorsal horn neurons, expansion of receptive ®elds and plasticity of neuronal connections (e.g. Woolf, 1983; Devor and Wall, 1978; Woolf et al., 1994). The intradermal or subcutaneous injection of capsaicin, which selectively activates primary afferent C-®bers (Baumann et al., 1991), produces sensitization of spinothalamic tract neurons (Simone et al., 1991; Dougherty and Willis, 1992), and produces thermal hyperalgesia and mechanical allodynia in animals (Gilchrist et al., 1996) as well as humans (LaMotte et al., 1991; TorebjoÈrk et al., 1992). Similarly, the subcutaneous injection of formalin produces a biphasic response: an immediate and intense increase in the spontaneous activity followed by a quiescent phase and then a more prolonged increase in cell ®ring of both primary afferents (Heapy et al., 1987; Puig and Sorkin, 1995) as well as dorsal horn neurons (Dickenson and Sullivan, 1987a,b). Behaviorally, the injection of formalin elicits a temporally biphasic increase in licking, biting, ¯inching
0304-3959/01/$20.00 q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(01)00319-0
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and other nocifensive behaviors (e.g. Dubuisson and Dennis, 1977). The initial phase is short lived and likely re¯ects an acute nociceptive state, while the second phase is more extended and re¯ects a state of central sensitization (Dubuisson and Dennis, 1977; Wheeler-Aceto et al., 1990; Coderre et al., 1993). In¯ammation, such as that produced by carrageenan, can produce a state of spinal cord sensitization in which a noxious stimulus produces an exaggerated response (hyperalgesia) and a normally non-noxious stimulus can produce a nocifensive response (allodynia) to both thermal and mechanical stimuli (e.g. Hargreaves et al., 1988; Traub, 1997). Unlike an injection of formalin, however, carrageenan does not increase spontaneous activity of C-®ber afferents, but rather increases the magnitude of C-®ber evoked responses (Stanfa et al., 1992) and decreases the occurrence and magnitude of inhibitory input to spinal cord neurons (Traub, 1997). The purpose of the present study was to determine the antinociceptive effects of vedaclidine in models involving central sensitization. Accordingly, dose-response curves were determined for vedaclidine in the formalin, capsaicin and carrageenan models. In addition, dose-response curves were determined for vedaclidine administered alone and in the presence of the muscarinic cholinergic receptor antagonist scopolamine in the carrageenan model. Further, in order to determine whether the concomitant administration of vedaclidine and a non-steroidal antiin¯ammatory drug (NSAID) was additive or synergistic, dose-response curves were determined in the carrageenan model for vedaclidine and the representative NSAID ketoprofen administered alone and at several ®xed dose-ratios, and the data analyzed isobolographically (Tallarida et al., 1989). 2. Materials and methods 2.1. Animals Adult male Sprague±Dawley rats from Charles River (Portage, MI) weighing 200±250 g were used for the formalin and rotarod (p.o.) experiments or from Harlan Sprague± Dawley (Indianapolis, IN) and weighing 80±100 g for the carrageenan experiments or weighing 250±300 g for the capsaicin and rotarod (s.c.) experiments were used. Animals were group housed with food and water freely available in a temperature and humidity controlled colony room. No animal was used more than once. Lights were turned on between 06:00 and 18:00 h. All experimental protocols were approved by the Eli Lilly Institutional Animal Care and Use Committee. 2.2. Rotarod test The ability of vedaclidine to induce sedation/ataxia was examined using an accelerating rotarod. All rats were given an initial three training trials to maintain posture on a rotarod which accelerated to 17 rev./min in 5 s and main-
tained that speed for 40 s (Omnitech Electronics Inc., Columbus, OH). On the following day, a 40 s trial was conducted 1 and 2 h after oral administration, or 0.5, 1 or 2 h after s.c. administration, of vehicle or a dose of vedaclidine. Animals not falling off the rotarod were given a maximum score of 40 s. 2.3. Formalin test The formalin test was conducted using Plexiglas cubicles measuring 25 £ 25 £ 20 cm (length £ width £ height) as described in detail by Simmons et al. (1998) and modi®ed from Shibata et al. (1989) and Wheeler-Aceto et al. (1990). Brie¯y, a mirror placed at the back of the cubicle allowed the unhindered observation of the formalin injected paw. Rats were acclimatized in the cubicles for at least 1 h before the experiment. All testing was conducted between 08:00 and 14:00 h. Vehicle or a dose of drug was administered 30 min before the formalin injection. Formalin (50 ml of a 5% solution in saline) was injected subcutaneously into the dorsal lateral surface of the right hind paw. Observation started immediately after the formalin injection. The number of seconds each rat spent licking the formalininjected hind paw was recorded in 5 min bins for 50 min after the formalin injection. 2.4. Capsaicin test Rats were injected subcutaneously with capsaicin (30 mg in 25 ml) into the plantar surface of the right hind paw at time zero. Ten minutes after the injection of capsaicin, tactile allodynia was evaluated with a calibrated series of von Frey ®laments using the up and down method of Chaplan et al. (1994). Brie¯y, the rats were placed in clear plastic cages (17:5 £ 15 £ 15 cm) ®tted with wire mesh ¯ooring and allowed to acclimate for approximately 5 min. Each ®lament was applied to the mid-plantar surface of each hind paw in a perpendicular fashion and depressed slowly (4±6 s) until bending occurred. In uninjected animals, von Frey ®laments above 15 g lifted the paw without bending and were therefore not used; the maximum withdrawal threshold was thus taken to be 15 g. Vehicle or a dose of drug was injected subcutaneously 20 min before the injection of capsaicin. 2.5. Carrageenan test Rats were injected subcutaneously with l -carrageenan (100 ml of 15 mg/ml solution) into the plantar surface of the right hind paw at time zero. Withdrawal latencies to a nociceptive thermal stimulus were assessed using a modi®cation of the methods of Hargreaves et al. (1988). The device used (Plantar Test, Ugo Basile) consisted of a glass surface on which the rats were placed individually in Plexiglas cubicles (17 £ 13 £ 14 cm). The radiant heat nociceptive stimulus could be delivered to each hind paw separately. A timer was actuated with the stimulus source,
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and the timer and stimulus were stopped when a brisk withdrawal response was detected by photodiode motion sensors. The intensity of the stimulus was adjusted to produce a baseline latency of approximately 15 s. Tactile allodynia was evaluated with a calibrated series of von Frey ®laments as described above. Vehicle or a dose of drug was injected subcutaneously 90 min after the injection of carrageenan, and withdrawal latencies to mechanical and thermal stimuli determined approximately 30 and 40 min later, respectively. In experiments to determine if the concomitant administration of vedaclidine and an NSAID were additive or potentially synergistic in reversing thermal hyperalgesia in the carrageenan test, dose-response curves were determined for vedaclidine and the representative NSAID ketoprofen administered alone and at several ®xed dose-ratios using the thermal stimulus only. 2.6. Drugs Vedaclidine ((1)-S-3-(4-butylthio-1,2,5-thiadiazol-3yl)-1-azabicyclo[2.2.2]octane; butylthio[2.2.2]; LY297802/ NNC11-1053) tartrate was synthesized at Lilly Research Laboratories. Scopolamine hydrobromide, ketoprofen, l carrageenan and capsaicin were from Sigma Chemical Co. (St. Louis, MO). Vedaclidine, scopolamine and carrageenan were dissolved in distilled water. Ketoprofen was prepared as a suspension in 6% acacia. Capsaicin was dissolved in 10% Tween-80 and 90% saline by heating to 708C for 15 min. Drugs were administered either s.c. or orally by gavage in a volume of 1.0 ml/kg.
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Table 1 Lack of effects of vedaclidine on motor performance on the rotarod a Dose
Time on rotarod (s)
mg/kg p.o.
1h
2h
mg/kg s.c.
0.5 h
1h
2h
0 1.0 3.0 10
40 ^ 0 40 ^ 0 37 ^ 3 40 ^ 0
40 ^ 0 40 ^ 0 36 ^ 4 40 ^ 0
0 1.0 3.0 10
40 ^ 0 40 ^ 0 38 ^ 2 34 ^ 2
40 ^ 0 40 ^ 0 38 ^ 2 30 ^ 4
40 ^ 0 40 ^ 0 37 ^ 3 35 ^ 3
a Rats were administered the indicated dose of vedaclidine orally or subcutaneously and tested on the rotarod at the indicated times after administration. Each value represents the mean ^ SEM of 5±6 (p.o.) or 10±11 (s.c.) rats.
line, then the mixture is considered to be additive. If the mixture ED50 lies below the theoretical additive ED50 line, and the con®dence intervals do not overlap that line, then the mixture is considered to be synergistic. ED50 values were determined using a four-parameter logistic equation (De Lean et al., 1978). 3. Results 3.1. Rotarod test Vedaclidine had no signi®cant effect on motor performance as measured on the rotarod up to 2 h after either p.o. or s.c. administration of 1.0±10 mg/kg (Table 1).
2.7. Statistics
3.2. Formalin test
Scoring in the formalin test was carried out according to Coderre et al. (1993) and Abbott et al. (1995). The sum of time spent licking in seconds from minutes 0±5 was considered the early phase while the late phase was taken as the sum of seconds spent licking from 15 to 40 min. In the tactile stimulation test, a withdrawal threshold was calculated as described in Chaplan et al. (1994). In the thermal test, a difference in withdrawal latency between the two hindpaws was calculated as the withdrawal latency of the ipsilateral paw minus the withdrawal latency of the contralateral paw. Data are presented as means ^ SEM. Data were analyzed by one-way analysis of variance and treatment groups were compared to appropriate control groups using a Dunnett's t-test. The interaction between vedaclidine and ketoprofen in the carrageenan test was examined by isobolographic analysis (e.g. Tallarida et al., 1989). Brie¯y, in the isobolograms, the vedaclidine ED50 is plotted on the abscissa and the ketoprofen ED50 on the ordinate. A theoretical line of additive interaction is drawn by connecting the ED50 for vedaclidine alone with that of ketoprofen alone. For each drug combination, an ED50 along with con®dence intervals is calculated for each mixture. If the mixture ED50 lies on the theoretical additive ED50 line, or the con®dence intervals overlap that
When vedaclidine was administered orally 30 min before formalin, vedaclidine produced a dose-dependent antihyperalgesic effect in the formalin test in the rat (Fig. 1). Vedaclidine (0.3±10 mg/kg p.o.) was ef®cacious in reducing formalin-induced behaviors in both the early and the late
Fig. 1. Dose-related inhibition of early as well as late phase formalininduced paw licking behavior by vedaclidine after oral administration in rats. Vehicle or a dose of vedaclidine was administered 30 min before the intraplantar administration of formalin. Each bar represents the mean ^ SEM of one observation in each of 6±11 rats. *P , 0:05 vs. vehicle alone.
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3.3. Capsaicin test Paw withdrawal threshold to a mechanical stimulus was reduced to approximately 3.0 g when determined approximately 30 min after the administration of vehicle and 10 min after the administration of capsaicin (Fig. 2; point above Veh). Thus, capsaicin produced mechanical hyperalgesia in the present studies. Vedaclidine (0.3±10 mg/kg s.c.), administered 20 min before capsaicin, produced a doserelated reversal of capsaicin-induced mechanical allodynia which was signi®cant after doses of 3.0 and 10 mg/kg. Morphine (10 mg/kg s.c.) also produced a complete reversal of capsaicin-induced mechanical allodynia (Fig. 2, point above 10 M). 3.4. Carrageenan test
Fig. 2. Dose-related reversal of capsaicin-induced decreases in tactile threshold by vedaclidine in rats. Vehicle or a dose of vedaclidine was administered s.c. 20 min before capsaicin, and mechanical withdrawal threshold was determined 10 min after the intraplantar administration of capsaicin. The dashed line represents the maximum withdrawal threshold in rats not injected with capsaicin. Each point represents the mean ^ SEM of one observation in each of 12 rats. Veh, vehicle; 10 M, 10 mg/kg morphine. *P , 0:05 vs. vehicle alone.
phase of the test. A dose of 10 mg/kg was required to signi®cantly reduce paw licking time in phase I, while both 3.0 and 10 mg/kg signi®cantly reduced paw licking time in phase II.
In rats administered vehicle 90 min after carrageenan and tested approximately 25 min later, paw withdrawal latencies to a noxious thermal stimulus were, on average, approximately 9.0 s shorter ipsilaterally than contralaterally (Fig. 3, left panel). Thus, carrageenan produced thermal hyperalgesia in the present studies. Vedaclidine (s.c.) produced a dose-related reversal of carrageenan-induced thermal hyperalgesia that was signi®cant after doses of 0.3 mg/kg and higher. When the dose-response curve for vedaclidine was redetermined in the presence of the selective muscarinic receptor antagonist scopolamine (1.0 mg/kg s.c.), the dose-response curve for vedaclidine was shifted to the right approximately 10-fold. In the presence of scopolamine, doses of 3.0 mg/kg of vedaclidine and higher signi®cantly reversed the thermal hyperalgesia. Scopolamine (1.0 mg/kg) administered alone produced no appreciable effects
Fig. 3. Dose-related reversal of carrageenan-induced decreases in paw withdrawal latencies to a thermal stimulus (left panel) and in paw withdrawal thresholds to a mechanical stimulus (right panel) by vedaclidine, and antagonism by scopolamine, in rats. Vehicle or a dose of vedaclidine alone or concomitantly with a dose of scopolamine was administered s.c. 90 min after carrageenan, and approximately 30 min later withdrawal thresholds to non-noxious mechanical stimulation and withdrawal latencies to noxious thermal stimulation were determined. Each point represents the mean ^ SEM of one observation in each of 12 rats. V 1 V, vehicle plus vehicle; V 1 S, vehicle plus 1.0 mg/kg scopolamine *P , 0:05 vs. vehicle plus vehicle.
H.E. Shannon et al. / Pain 93 (2001) 221±227
on withdrawal latencies to noxious thermal stimulation (Fig. 3, left panel, point above V 1 S). In the same rats, the paw withdrawal threshold to nonnoxious mechanical stimulation was reduced to less than approximately 2.5 g when determined 120 min after carrageenan, and 30 min after vehicle, administration (Fig. 3, right panel). Thus, carrageenan produced mechanical allodynia. Vedaclidine s.c. produced a dose-related reversal of mechanical allodynia that was signi®cant after doses of 3.0± 30 mg/kg. In the presence of scopolamine (1.0 mg/kg), the dose-response curve for vedaclidine was shifted modestly to the right. However, even in the presence of scopolamine, doses of 3.0±30 mg/kg of vedaclidine signi®cantly reversed the mechanical allodynia. Scopolamine (1.0 mg/kg) administered alone produced no appreciable effects on withdrawal thresholds to non-noxious mechanical stimulation (Fig. 3, right panel, point above V 1 S). Higher doses of scopolamine produced hyperactivity which precluded testing in the present experiments. 3.5. Isobolographic analysis of vedaclidine-ketoprofen interactions The data for the concomitant administration of vedaclidine s.c. and the NSAID ketoprofen p.o. in ®xed dose-ratios in the carrageenan test using the thermal stimulus are represented as an isobologram in Fig. 4. In Fig. 4, the doses of vedaclidine contributing to the ED50 values are shown along the abscissa, with the ED50 for vedaclidine alone (0.45 ^ 0.01 mg/kg s.c.) lying on the abscissa, and, the doses of ketoprofen contributing to the ED50 values are
Fig. 4. Isobolograms for the ED50 values for vedaclidine s.c. plotted against the ED50 for ketoprofen p.o. using the thermal stimulus in the carrageenan model. The solid line represents the line of additivity constructed by joining the ED50 values for each drug alone. The isobol points were determined from the ED50 values of vedaclidine and ketoprofen given in ®xed doseratios of 1:5, 1:10 and 1:30. The SEMs are resolved into the vedaclidine (horizontal) and ketoprofen (vertical) components.
225
shown on the ordinate, with the ED50 for ketoprofen alone (18.6 ^ 2.4 mg/kg p.o.) lying on the ordinate. The straight line connecting the ED50 values for vedaclidine and ketoprofen is the theoretical line of additivity. The ED50 values for the dose combinations are plotted as points on the isobologram, where the coordinates are the amount of vedaclidine in the combined ED50 and the amount of ketoprofen in the combined ED50. When data for the dose combinations of vedaclidine:ketoprofen of 1:5, 1:10 and 1:30 were plotted isobolographically, all of the data points were below the theoretical line of additivity and none of the SEMs overlapped the line. Thus, vedaclidine and ketoprofen were synergistic (i.e. more than additive) in producing analgesia in the carrageenan test. 4. Discussion The major ®nding of the present studies was that the muscarinic cholinergic receptor ligand vedaclidine, with mixed agonist and antagonist actions across muscarinic cholinergic receptor subtypes (Shannon et al., 1997), produced antinociceptive and antihyperalgesic effects in models involving central sensitization in rats. Vedaclidine produced dose-related reductions in formalin-, capsaicin-, and carrageenan-induced nocifensive behaviors. Vedaclidine has previously been shown to produce antinociception in acute nociceptive models, including grid shock, tail-¯ick, hot plate and writhing models (Swedberg et al., 1997). The present ®ndings are the ®rst demonstration that vedaclidine is ef®cacious in models involving central sensitization of pain pathways. We have previously reported (Swedberg et al., 1997) that vedaclidine does not produce salivation at doses up to 130 times the antinociceptive ED50 and produces tremor only at doses 54 times the antinociceptive ED50. In addition, consistent with the present lack of effects on the rotarod at anti-hyperalgesic/allodynic doses, vedaclidine was without effect on the rotarod at antinociceptive doses (Swedberg et al., 1997). In contrast, the ratios of the sideeffect-to-ef®cacy ED50 values for the non-selective muscarinic agonist oxotremorine were 2±12 (Swedberg et al., 1997). Thus, vedaclidine produces antinociception as well as antihyperalgesia/allodynia in models of both acute and chronic pain with an improved side-effect pro®le relative to nonselective muscarinic agonists, and therefore may be a clinically useful analgesic and an alternative to opioids. Vedaclidine produced dose-related reductions in formalin-induced behaviors. The subcutaneous injection of formalin produces biphasic behavioral as well as electrophysiologic effects (e.g. Dubuisson and Dennis, 1977; Puig and Sorkin, 1995; Dickenson and Sullivan, 1987a,b), with the early phase likely re¯ecting an acute nociceptive state and the late phase a state of central sensitization (e.g. Dubuisson and Dennis, 1977; Wheeler-Aceto et al., 1990; Coderre et al., 1993). The effectiveness of vedaclidine in reducing formalin-induced behaviors in the early phase is
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consistent with the antinociceptive effects of vedaclidine in acute antinociceptive models (Swedberg et al., 1997). The demonstration that vedaclidine also reduces formalininduced behaviors in the late phase indicates that vedaclidine also is ef®cacious in reducing central sensitization in pain pathways. The present ®ndings are consistent with previous reports that the non-selective muscarinic receptor agonist oxotremorine reduced formalin-induced behaviors after subcutaneous or intrathecal administration in rats (Capone et al., 1999; Machelska et al., 1999; Przewlocka et al., 1999). In¯ammation, such as that produced by carrageenan, produces central sensitization accompanied by increases in the magnitude of C-®ber evoked responses and decreases in descending inhibition without increases in spontaneous ®ring of C-®ber afferents (Stanfa et al., 1992; Traub, 1997). In the present studies, vedaclidine produced a doserelated reversal of the thermal and mechanical hyperalgesia produced by carrageenan. Thus, vedaclidine was able to reverse previously established central sensitization, suggesting that it may have a direct action on central sensitization in pain pathways. One possible explanation for the present ®ndings is that vedaclidine may have enhanced descending inhibition. Iwamoto (1991) has previously demonstrated that muscarinic receptor agonists enhance descending inhibition in producing acute antinociception. Further experiments are needed to determine what effects vedaclidine may have on descending inhibitory pathways, and whether such effects might in¯uence central sensitization. The subcutaneous injection of capsaicin directly stimulates C-®bers and thus, like the injection of formalin, increases the ®ring of primary afferent neurons (Puig and Sorkin, 1995; Baumann et al., 1991) and produces central sensitization (Simone et al., 1991; Dougherty and Willis, 1992). Pretreatment with vedaclidine produced a doserelated reversal of the mechanical hyperalgesic effects produced by capsaicin. To the best of our knowledge, the present ®ndings that vedaclidine prevented the hyperalgesia produced by capsaicin are the ®rst report that a muscarinic receptor agonist can reduce the hyperalgesia produced by direct stimulation of C-®bers by capsaicin. Further research is needed to determine whether the muscarinic receptors involved in preventing capsaicin-induced hyperalgesia are located peripherally, spinally, or supraspinally. Antagonism of the effects of vedaclidine by the muscarinic cholinergic receptor antagonist scopolamine was investigated in the carrageenan test. Scopolamine (1.0 mg/kg) produced a parallel shift to the right in the vedaclidine dose-response curve when the thermal stimulus was used indicating that vedaclidine produced anti-thermal hyperalgesia by agonist actions at muscarinic cholinergic receptors. These ®ndings corroborate and extend our previous ®ndings that scopolamine antagonizes the antinociceptive effects of vedaclidine in the writhing, tail-¯ick, hot plate and grid shock tests in the mouse (Swedberg et al., 1997). In contrast, scopolamine had little effect on the vedaclidine dose-
response curve using the mechanical stimulus in the carrageenan test. However, approximately 10-fold higher doses of vedaclidine (.3.0 mg/kg) were required to reverse mechanical allodynia than to reverse thermal allodynia (.0.3 mg/kg). Thus, it is perhaps not surprising that higher doses of scopolamine would be needed to antagonize the higher doses of vedaclidine needed to reverse mechanical allodynia. Higher doses of scopolamine produced hyperactivity which, however, precluded testing in the carrageenan test. The anti-thermal allodynia effects of vedaclidine in the carrageenan-induced in¯ammatory model were synergistic with the non-steroidal antiin¯ammatory drug ketoprofen as determined by isobolographic analysis (e.g. Tallarida et al., 1989). When administered alone, the ED50 values for vedaclidine and ketoprofen were approximately 0.45 and 18.5 mg/kg, respectively. However, when administered in a ®xed dose-ratio of 1:10, the ED50 values for vedaclidine and ketoprofen were reduced to approximately 0.15 and 2.0 mg/kg, respectively. These latter doses of vedaclidine and ketoprofen were inactive when administered alone. Thus, vedaclidine and ketoprofen administered in combination were ef®cacious at doses which when administered alone were inactive. It may therefore be possible to administer vedaclidine and ketoprofen, or another NSAID, in combination to provide pain relief clinically at doses which produce fewer or greatly reduced side-effects. The present studies have demonstrated that vedaclidine produces antihyperalgesic/allodynic effects in three models involving central sensitization by agonist actions at muscarinic receptors. Moreover, the antihyperalgesic/allodynic effects of vedaclidine cannot be due to antagonist activity at M1, M3 or M5 receptors because the non-selective muscarinic receptor antagonist scopolamine was without effect when administered alone. Relatively little is known about the potential mechanisms and/or site of action of muscarinic agonists in reducing central sensitization. Muscarinic cholinergic receptors are abundant throughout pain pathways from dorsal root ganglia to somatosensory cortex and therefore could modulate processing of sensory information at several levels. In particular, muscarinic receptors are localized on the super®cial laminae of the dorsal horn in rats where nociceptive Ad and C ®bers terminate (Gillberg and Askmark, 1991; HoÈglund and Baghdoyan, 1997). Thus, muscarinic agonists may enhance descending inhibition and/or act directly on dorsal horn neurons to reduce central sensitization. Alternatively, muscarinic receptor agonists may act more indirectly, for example by modulating nitric oxide pathways. In this regard, Przewlocka et al. (1999) reported that intrathecal oxotremorine reduced formalin-induced hyperalgesia and also reduced the number of formalin-induced nitric oxide synthase-containing neurons in the spinal cord. However, thalamic and cortical sites of action may also be important. Further studies are needed to better understand the mechanism and site of action of muscarinic receptor agonists in
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