Analgesic effects of mGlu1 and mGlu5 receptor antagonists in the rat formalin test

Neuropharmacology 51 (2006) 623e630 www.elsevier.com/locate/neuropharm

Analgesic effects of mGlu1 and mGlu5 receptor antagonists in the rat formalin test N. Sevostianova, W. Danysz* Merz Pharmaceuticals GmbH, Eckenheimer Landstrasse 100, 60318 Frankfurt/Main, Germany Received 15 December 2005; received in revised form 18 April 2006; accepted 4 May 2006

Abstract mGlu1 and mGlu5 receptors have been implicated in pain associated with inflammation. In the present study, the formalin test was used to measure sustained pain with components of tissue injury. The aims of the present study were to assess: (i) the role of mGlu1 and mGlu5 receptors in inflammatory pain using selective antagonist EMQMCM, 1.25e5 mg/kg, as the mGlu1 receptor antagonist, and MPEP or MTEP, 2.5e10 mg/kg, as mGlu5 receptor antagonist; (ii) the possible interaction between mGlu1 and mGlu5 receptor antagonists and morphine; and (iii) whether tolerance develops to the analgesic effects of these antagonists after prolonged treatment. EMQMCM, MTEP and MPEP significantly reduced the manifestation of both phases of formalin response. However, all these mGlu receptor antagonists did not affect the withdrawal latencies in a model of acute pain (Hargreaves test), which has a different underlying mechanism. In the present study, the suppressive effect on formalin-induced pain behaviour was much stronger when mGlu1 and mGlu5 receptor antagonists were co-injected compared to administration of a single antagonist, but this effect was not seen when mGlu receptor antagonist was co-administered with morphine. This is in contrast to the pronounced inhibitory effects after co-treatment with morphine and the uncompetitive NMDA receptor antagonist memantine. The present study also provides the first direct in vivo evidence that prolonged administration of MTEP (5 mg/kg) over 7 days leads to the development of tolerance to its antinociceptive effects. Such tolerance was not observed when EMQMCM (5 mg/kg) was administered in the same manner. In conclusion, these results provide additional arguments for the role of group I mGlu receptors in pain with inflammatory conditions. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Pain; mGlu receptors; Formalin test; mGlu1; mGlu5; Morphine; Tolerance

1. Introduction The great body of evidence over the last several decades indicates that the excitatory amino acid glutamate plays a pivotal role in nociceptive processing. Glutamate acts at several types of receptors, including ionotropic (cation-specific ion channels divided into three groups: N-methyl-D-aspartate (NMDA), aamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate receptors) and metabotropic receptors (coupled to G-proteins that modulate the intracellular second messengers). The vast number of studies in animals and in humans

* Corresponding author. Tel.: þ49 69 150 3564; fax: þ49 69 596 2150. E-mail address: [email protected] (W. Danysz). 0028-3908/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2006.05.004

demonstrated the ability of NMDA receptor antagonists to attenuate central sensitization and hyperalgesia (Price et al., 1994; Mao, 1999). In addition to ionotropic receptors, the involvement of metabotropic glutamate receptors (mGlu) in pain has been shown in behavioural studies. Metabotropic glutamate receptors are divided into three groups with growing evidence that group I mGlu receptors are implicated in nociceptive transmission and central sensitization. Group I mGlu receptors, comprising mGlu1 and mGlu5 receptors, are coupled to phospholipase C, and their activation leads to inositol 1,4,5-trisphosphate (IP3) production with subsequent release of Ca2þ from intracellular stores (Crawford et al., 2000). Intrathecal administration of group I mGlu receptor agonists induces spontaneous nociceptive behaviours and allodynia (Fisher and Coderre, 1998; Dolan

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and Nolan, 2000; Lorrain et al., 2002), whereas mGlu receptor antagonists produce antihyperalgesic effects in animal models of persistent pain (Fisher and Coderre, 1996; Walker et al., 2001a). Several studies showed that group I mGlu receptors have a modulatory role upon NMDA-induced responses via PKC-dependent mechanisms (Pisani et al., 1997; Pisani et al., 2001). Moreover, the abundant expression of mGlu1 and mGlu5 receptors on peripheral sensory neurons and superficially in dorsal horn of the spinal cord, where primary afferent fibres terminate, indicate the involvement of group I mGlu receptors in processes of nociceptive transmission and plasticity (Valerio et al., 1997; Neugebauer, 2001). It has recently been suggested that mGlu receptors contribute to pain states associated with inflammation. Results from electrophysiological and behavioural studies in rats are consistent with the involvement of group I mGlu receptors in the spinal processing of sustained nociceptive input with an inflammatory component evoked by formalin (Fisher and Coderre, 1996) and carrageenan or mustard oil applications (Young et al., 1997). However, although blockade of group I mGlu receptors reverses thermal hyperalgesia in models with inflammatory or nerve injuries, it does not attenuate mechanical hyperalgesia, which also constitutes the neuropathic pain (Walker et al., 2001a; Hudson et al., 2002). In the present study, the formalin test was used to measure sustained pain with tissue injury components. Formalin-induced behaviour is characterised by two phases, where the first phase reflects the acute pain state and the second phase is attributed to spinal cord hyperexcitability and is referred to as the ‘‘tonic’’ pain phase (Coderre and Yashpal, 1994; McCall et al., 1996; Martindale et al., 2001). The purpose of the present study was to assess the role of mGlu1 and mGlu5 receptors in nociception evoked by injection of formalin using selective antagonists. Up to now, the majority of previous behavioural studies have been performed using non-selective mGlu1 receptor ligands, which have poor bioavailability and penetration to the brain (Spooren et al., 2003). Due to the lack of selective ligands, much less is known about the role of mGlu1 than mGlu5 receptors in nociception. EMQMCM (JNJ16567083, 3-ethyl-2-methyl-quinolin-6-yl)(4-methoxy-cyclohexyl)-methanone methanesulfone) is of special interest, as it is one of the first selective noncompetitive and potent mGlu1 receptor antagonist that penetrates the bloodebrain barrier. In the present study, the mGlu1 receptor antagonist EMQMCM (Lesage et al., 2002) and two mGlu5 receptor antagonists, MPEP (2-methyl-6-(phenylethynyl)pyridine (Varney et al., 1999)) and MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (Busse et al., 2004)), were tested either alone or in combination. Additionally, the Hargreaves test was employed to assess whether antagonists of group I mGlu receptors reduce acute pain with a different underlying mechanism than in the first phase of the formalin response. A considerable number of studies have demonstrated the enhancement of analgesic action after application of opiates in combination with NMDA receptor antagonists. Since group I mGlu receptors are also involved in synaptic plasticity and in synaptic pathways of pain transmission, one can assume that co-administration of group I mGlu receptor antagonists

with an opioid receptor agonist will result in enhancement of analgesic action. In the present study, therefore, the possible interaction between mGlu1 and mGlu5 receptor antagonists and morphine was verified in the formalin test. In addition, since the treatment of chronic pain requires long-term administration of drugs, another aim was to investigate whether tolerance develops to the analgesic effects of EMQMCM and MTEP after prolonged treatment. 2. Materials and methods 2.1. Subjects Adult experimentally naive male Sprague-Dawley rats (200e300 g; Janvier, France) were housed in groups of four with food and water available ad libitum and alternating 12 h/12 h dayenight cycle (lights on at 07:00) for at least 6 days before the experiments were started. Colony room temperature and humidity were maintained at 20
1  C and 60
3%, respectively. All experiments were conducted during the light period of a dayenight cycle. The study was approved by the Ethical Committee, Regierungspraesidium Darmstadt, Hessen and performed in accordance with the recommendations and policies of the U.S. National Institutes of Health Guidelines for the Use of Animals. Each animal was used only once.

2.2. Drugs Two percent formaldehyde was made from 1 part of formalin (w36.6%; formalin, Fluka, Taufkirchen, Germany) and 17.3 parts of saline. Morphine sulphate (opioid receptor agonist; Sigma, Deisenhofen, Germany, 3 mg/kg) and memantine (HCl, 1-amino-3,5-dimethyladamantane, uncompetitive NMDA receptor antagonist, Merz Pharmaceuticals, Frankfurt/Main, Germany, 5 mg/kg) were dissolved in physiological saline. EMQMCM (JNJ16567083, (3-ethyl2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfone, 1.25e5 mg/kg), MPEP (2-methyl-6-(phenylethynyl)pyridine, 2.5e10 mg/kg) and MTEP ((3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine, 2.5e10 mg/kg) were synthesised by Merz Pharmaceuticals and dissolved in 10% water solution of Tween 80. Morphine was administered s.c.; glutamate receptor antagonists were injected i.p. The injection volume of morphine and memantine was 1 ml/kg, all antagonists of mGlu receptors were administered in a volume of 2 ml/kg. For interaction studies, mode and time of administration were the same as for single injection experiments, using appropriate vehicles, i.e. each animal had two injections (see Fig. 2e4 legends for description). In experiments aimed to investigate tolerance, either EMQMCM or MTEP (5 mg/kg each) was given once daily for 7 days, and 24 h after the last injection the challenging dose of either EMQMCM or MTEP (5 mg/kg each) was administered 30 min before formalin (see Fig. 5 legend for description).

2.3. Formalin test Rats were placed individually in an open Plexiglas chamber (bowl-like cage 40  35 cm) with a mirror angled at 45  positioned behind to allow an unobstructed view of the paws by the observer. The animals were habituated to the observation bowl for 30 min prior to the experimental sessions. Formalin (50 ml) was injected s.c. into the plantar surface of the rat hind paw using a 27gauge needle. After injection, rats were immediately returned to the observation bowl and the formalin-induced behaviours were recorded for a period of 60 min. All tested substances were injected 30 min before the injection of formalin. The duration of licking and biting of the injected paw was scored using a custom-made software program and quantified every 6 min for the 60-min observation period. The 6-min interval was chosen based on an earlier report on the time course of the first (0e6 min) and second (12e60 min) phases of the formalin-induced facial grooming (Eisenberg et al., 1996).

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2.5. Data analysis The duration of licking/biting the injected paw was analysed by analysis of variance (ANOVA, one or two way depending on the study design) using SigmaStat software (Ver 3.0, SPSS, Chicago, IL, USA). The statistic was made separately to each phase, first phase (0e6 min) and second phase (12e 60 min), and data are expressed as mean
standard error mean (S.E.M.). The HolmeSidak’s post hoc method was used to determine the difference between groups. Data from the Hargreaves test were analysed by one-way ANOVA.

3. Results Injection of formalin into the rats’ hind paws induced the typical biphasic behaviours of licking/biting and flinching/shaking as observed between 0e6 min (first phase) and 12e60 min (second phase), with nearly no responses recorded between 6 and 12 min. 3.1. Effects of antagonists of group I mGlu receptors in the formalin test All antagonists of group I mGlu receptors applied systemically (i.p.) 30 min before the formalin injection produced dose-dependent antinociceptive effects in the first (Fig. 1A)

MPEP

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In animals administered either EMQMCM (Fig. 3A) or MTEP (Fig. 3B) together with morphine, no difference in the duration of licking/biting behaviour was detected as compared to that in rats treated with mGlu receptor antagonist only. Inhibition of the first phase was observed in all treated groups as compared to animals injected with vehicle. Suppression of the second phase was observed in groups injected with EMQMCM (2.5 mg/kg) and MTEP (5 mg/kg) alone, and also in the group administered a single injection of the threshold dose of morphine (3 mg/kg) along with EMQMCM (Fig. 3A). After combined treatment with EMQMCM and morphine or MTEP with morphine, there was a significant (apparently negative) interaction as shown by ANOVA analysis (see Fig. 3 legend for details).

phase II 700 EMQMCM

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3.3. Effects of co-administration of mGlu1 or mGlu5 receptor antagonists with morphine in the formalin test

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The results from experiment assessing the effect of the combination of mGlu1 receptor antagonist EMQMCM with the mGlu5 receptor antagonist MTEP (both at inactive doses of 1.25 mg/kg and 2.5 mg/kg, respectively) are presented in Fig. 2. Two-way ANOVA revealed a significant effect of this treatment in the first and second phases of formalin response. Moreover, ANOVA analysis also indicated significant interaction between these treatments (see Fig. 2 legend for details) suggesting hyperadditive (synergistic) effect.

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3.2. Effects of co-administration of mGlu1 and mGlu5 receptor antagonists in the formalin test

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The hind paw thermal nociceptive threshold was assessed with a heat thermal stimulator, manufactured by Dr. G. Ozaki of the University of California (San Diego, USA). Four rats from different groups were placed in the clear plastic chambers (10  26  10 cm) on a glass floor and allowed to acclimatize to their environment for 20 min before testing. The radiant heat was positioned manually directly beneath the heel portion of the plantar surface of hind paw. To assess the nociceptive response to thermal stimuli, the baseline levels for the right and left hind paw were determined for each animal. The rats were then injected with drugs/vehicle and immediately returned to the chambers. The tests commenced 30 min later by activation of the stimulus, which initiated a timing circuit. The time interval between the application of the light and the hind paw withdrawal response was defined as the paw withdrawal latency. In the absence of a response, the stimulation was automatically terminated at 40 s to avoid tissue injury, and that time was assigned as the response latency. Baseline response latencies averaged approximately 7e8 s.

and second phases of formalin response (Fig. 1B). Significant analgesic effects of the mGlu1 receptor antagonist EMQMCM were observed starting from the dose of 2.5 mg/kg both for the first and second phases. Similarly, the mGlu5 receptor antagonists starting at the dose of 5 mg/kg had significant inhibitory effects on both phases (see Fig. 1 legend for details).

0 2.5 5 10

2.4. Hargreaves test

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Dose (mg/kg)

Fig. 1. Effects of antagonists of group I mGlu receptors on the nociceptive response induced by formalin. EMQMCM (1.25e5 mg/kg), MTEP (2.5e10 mg/kg), MPEP (2.5e10 mg/kg) or vehicle (10% Tween) was given i.p. 30 min prior to the injection of formalin. Data are presented as mean
S.E.M. duration (in seconds) of the paw licking and biting cumulatively for the first phase (panel A, one-way ANOVA: F (3,32) ¼ 7.3, P < 0.001; F (4,42) ¼ 6.2, P < 0.001; F (3,29) ¼ 3.9, P ¼ 0.02 for EMQMCM, MTEP and MPEP, respectively) and for the second phase (panel B, one-way ANOVA: F (3,32) ¼ 4.8, P ¼ 0.007; F (4,42) ¼ 10.1, P < 0.001; F (3,29) ¼ 3.3, P ¼ 0.03 for EMQMCM, MTEP and MPEP, respectively). Asterisks denote significant differences from groups treated with vehicle, P < 0.05, HolmeSidak test. N ¼ 8e9.

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626

phase I

uncompetitive NMDA receptor antagonist memantine (5 mg/ kg) together with morphine (3 mg/kg) resulted in an enhancement of analgesic action, thus serving as a positive control for the interaction between antagonists of group I mGlu receptors and morphine. Two-way analysis of variance demonstrated the significant effects of memantine alone in both phases of formalin-induced pain behaviour. The HolmeSidak multiple comparison test indicated the significant inhibitory effects of morphine alone (3 mg/kg) in the first phase. There was no statistically significant interaction between morphine and memantine. However, co-administration of the substances led to a more pronounced attenuation of both phases of formalininduced pain response, indicating an additive effect (see Fig. 4 legend for details).

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MTEP (mg/kg) Fig. 2. Effects of co-administration of mGlu1 and mGlu5 receptor antagonists on the nociceptive response induced by formalin. Rats were treated with EMQMCM (1.25 mg/kg), MTEP (2.5 mg/kg) or vehicle (10% Tween) alone or in combination 30 min before the injection of formalin. Two-way ANOVA revealed significant effects of EMQMCM (first phase: F (1,28) ¼ 15.1, P < 0.001; second phase F (1,28) ¼ 21.0, P < 0.001), MTEP (first phase: F (1,28) ¼ 18.2, P < 0.001; second phase: F (1,28) ¼ 12.8, P ¼ 0.001) and their interaction (first phase: F (1,28) ¼ 4.7, P ¼ 0.04; second phase: F (1,28) ¼ 5.2, P ¼ 0.03). Data are presented as mean
S.E.M. duration (in seconds) of the paw licking and biting cumulatively for the first and second phases of formalin-induced response. Asterisks and hash signs denote significant differences between the group treated with vehicle and the group treated with a single injection of one of the substances. P < 0.05, HolmeSidak test. N ¼ 8.

3.4. Effects of co-administration of NMDA receptor antagonist memantine with morphine in formalin test A considerable number of studies have demonstrated that blockade of NMDA receptors by antagonists enhances opiate analgesia. In the current study, the administration of the

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saline morphine 3 mg/kg

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Repetitive treatment with group I mGlu receptor antagonists was used to verify whether tolerance develops to the analgesic effects. EMQMCM (Fig. 5A), MTEP (Fig. 5B) or vehicle was administered once per day at a dose of 5 mg/kg for 7 days, and the formalin test was performed on the 8th day of the experiment with preceded challenging dose (5 mg/kg) of respective antagonist. EMQMCM (5 mg/kg) produced inhibition of nociceptive behaviours after one week pre-exposure to EMQMCM or vehicle (Fig. 5A). Statistical analysis showed significant antinociceptive effects of EMQMCM at dose 5 mg/kg when it was applied acutely, 30 min before the injection of formalin (P < 0.001). There was no significant interaction between acute and chronic treatment (see Fig. 5 legend for details). MTEP (5 mg/kg) injected 30 min prior to the test, produced significant attenuation of formalin-induced licking/biting

Duration of licking/biting (s)

Duration of licking/biting (s)

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3.5. Effects of group I mGlu receptor antagonists after repetitive administration in the formalin test

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Fig. 3. Effects after co-administration of mGlu1 or mGlu5 receptor antagonist with morphine on the nociceptive response induced by formalin. Panel A: rats were treated s.c. with morphine (3 mg/kg, two-way ANOVA: F (1,31) ¼ 29.5, P < 0.001 and F (1,31) ¼ 11.4, P ¼ 0.02 for the first and second phases, respectively), saline and EMQMCM (2.5 mg/kg, two-way ANOVA: F (1,31) ¼ 38.2, P < 0.001 and F (1,31) ¼ 37.9, P < 0.001 for the first and second phases, respectively) or its vehicle (10% Tween) 30 min before the injection of formalin. Two-way ANOVA also revealed the negative interaction for the second phase (F (1,31) ¼ 7.8, P ¼ 0.009). Panel B: rats were treated s.c. with morphine (3 mg/kg, F (1,23) ¼ 9.2, P ¼ 0.006 for the first phase), saline and MTEP (5 mg/kg, P < 0.05 according to post hoc comparison for the first and second phases, however, two-way ANOVA showed the lack of effects of MTEP on both phases) or its vehicle (10% Tween) 30 min before the formalin test. Two-way ANOVA also revealed the negative interaction: F (1,23) ¼ 4.8, P ¼ 0.038 and F (1,23) ¼ 4.5, P ¼ 0.046 for the first and second phases, respectively. Data are presented as mean
S.E.M. duration (in seconds) of the paw licking and biting cumulatively for the first and second phases of formalin-induced response. Asterisks denote significant differences from groups treated with vehicles, P < 0.05, HolmeSidak test. N ¼ 8e9.

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phase I

3.6. Effects of antagonists of group I mGlu receptors in Hargreaves test

phase II

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Neither the mGlu1 receptor antagonist (EMQMCM) nor the mGlu5 receptor antagonist (MTEP or MPEP) affected the paw withdrawal latency measured in response to thermal stimulus using the Hargreaves test (Fig. 6).

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4. Discussion

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Memantine (mg/kg) Fig. 4. Effects after co-administration of the NMDA receptor antagonist memantine with morphine on nociceptive behaviour induced by formalin. Rats were treated s.c. with saline or morphine (3 mg/kg, two-way ANOVA: F (1,29) ¼ 10.4, P ¼ 0.003 and F (1,29) ¼ 11.2, P ¼ 0.002 for the first and second phases, respectively), memantine (5 mg/kg, two-way ANOVA: F (1,29) ¼ 8.6, P ¼ 0.006 and F (1,29) ¼ 25.0, P < 0.001 for the first and second phases, respectively) or vehicles (saline). ANOVA revealed a lack of interaction (F (1,29) ¼ 0.01, P ¼ 0.916 and F (1,29) ¼ 2.14, P ¼ 0.154 for the first and second phases, respectively). Data are presented as mean
S.E.M. duration (in seconds) of the paw licking and biting cumulatively for the first and second phases of formalin-induced response. Asterisks and hash signs denote significant differences from group treated with vehicle and with a single injection of one of the substances, respectively. P < 0.05, HolmeSidak test. N ¼ 8e9.

behaviour in rats injected for 7 days with vehicle, but not in rats injected with MTEP (Fig. 5B). Two-way ANOVA confirmed significant effects of acute and chronic treatment with MTEP in the first phase. However, ANOVA revealed significant (apparently negative) interaction between repetitive and acute treatment with MTEP (see Fig. 5 legend for details).

Duration of licking/biting (s)

A

phase I

All group I mGlu receptor antagonists tested here significantly reduced the manifestation of both phases of formalinevoked pain behaviour. It is well known that injection of formalin into the hind paw causes the release of glutamate from peripheral terminals of primary afferent neurons at the site of inflammation (Carlton et al., 1995; Lawand et al., 1997). Since group I mGlu receptors are widely expressed at both central (Shigemoto et al., 1993; Valerio et al., 1997) and peripheral terminals (Bhave et al., 2001), it is likely that the observed antinociceptive effects of EMQMCM, MTEP and MPEP in the formalin test result from blockade of neurotransmission through presynaptically located mGlu1 and mGlu5 receptors. These results are consistent with previous studies where local (Dogrul et al., 2000) and systemic administration of mGlu1 and mGlu5 receptor antagonists significantly reduced inflammatory hyperalgesia (Bhave et al., 2001; Walker et al., 2001b). In the present study, all tested group I mGlu receptor antagonists affected the first and second phases of the formalin response to a similar extent. The inhibitory effects in the first phase could either indicate acute analgesic action or unspecific motor impairment by mGlu receptor antagonists. Modulation of the withdrawal latencies by EMQMCM, MTEP and MPEP was therefore assessed in the Hargreaves test, a model of acute pain. None of these agents produced a significant

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Fig. 5. Effects of repetitive administration with group I mGlu receptor antagonists on acute inhibition of formalin nociception by these antagonists. Rats were treated for 7 days with EMQMCM (5 mg/kg, panel A), MTEP (5 mg/kg, panel B) or vehicle (10% Tween). On the 8th day EMQMCM or MTEP at the dose of 5 mg/kg was administered 30 min before the injection of formalin. Two-way ANOVA confirmed significant effects of acute but not chronic treatment with EMQMCM (F (1,29) ¼ 28.2, P < 0.001 and F (1,29) ¼ 36.6, P < 0.001 for the first and second phases, respectively), and no significant interaction between these treatments. In case of MTEP there was a significant effect of both acute (F (1,29) ¼ 9.7, P ¼ 0.004 and F (1,29) ¼ 8.1, P ¼ 0.008 for the first and second phases, respectively) and chronic treatment (F (1,29) ¼ 4.8, P ¼ 0.036 only for the first phase). Moreover, two-way ANOVA revealed significant interaction between treatments (F (1,29) ¼ 6.3, P ¼ 0.018 and F (1,29) ¼ 5.3, P ¼ 0.028 for the first and second phases, respectively). Data are presented as mean
S.E.M. duration (in seconds) of the paw licking and biting cumulatively for the first and second phases of formalin-induced response. Asterisks denote significant differences from groups treated with vehicle. P < 0.05, HolmeSidak test. N ¼ 7e10.

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Dose (mg/kg) Fig. 6. Effects of antagonists of group I mGlu receptors in the Hargreaves test. Tests were performed before (baseline) and 30 min after the drug injections. Data are presented as mean
S.E.M. paw withdrawal latencies (in seconds). N ¼ 6e8.

effect. These data are in line with other studies that showed lack of effects on thermal and mechanical pain thresholds by mGlu receptor antagonists (Dogrul et al., 2000; Walker et al., 2001b). There are differences between the mechanisms of acute pain in response to application of heat stimulus and the cascade of events after administration of a chemical substance, which leads to the development of inflammation (Tegeder et al., 2004). Electron microscopic studies have indicated that both mGlu1 and mGlu5 receptors are expressed on a subset of nociceptive nerve terminals (Bhave et al., 2001) and are up-regulated during inflammation (Coggeshall and Carlton, 1999). Moreover, Hu et al. (2002) hypothesized that activation of peripheral group I mGlu receptors during inflammation increases thermal sensitivity by enhancing vanilloid receptor function. Vanilloid receptor activity also increases within the acidic environment of inflamed tissues. These findings imply that pre-treatment with group I mGlu receptor antagonists might be sufficient to block inflammation-induced changes that develop during the first phase of the formalin response. Another possibility is that the impairment of motor function could explain the apparent inhibitory effects of group I mGlu receptor antagonists in the first phase of formalinevoked pain response. However, our previous studies employing the rotarod test showed the decreased performance only at higher doses than were used in the current study (EMQMCM impaired rotarod performance at 10 mg/kg and MTEP at 20 mg/kg (Pietraszek et al., 2005)). In the present study, the suppressive effect on the expression of formalin-induce pain behaviour was much stronger (synergistic) when mGlu1 and mGlu5 receptor antagonists were co-injected as compared to each agent alone (1.25 mg/ kg and 2.5 mg/kg, respectively, for EMQMCM and MTEP). This observation suggests cross-talk between mGlu1 and mGlu5 receptors in the pathomechanism of inflammatory pain. Morphine is commonly used for the management of pain, although with some limitations related to the development of

tolerance and other side effects (constipation, nausea, and sedation). The enhancement of narcotic analgesic effects by other agents would potentially allow lower narcotic doses to be administered and therefore lower the risk of the above-mentioned side effects. Animal and clinical studies show that combined treatment with opiate and NMDA receptor antagonists can reduce the dose of opiate required for pain relief (Price et al., 2000; Unlugenc et al., 2003). Several lines of evidence indicate that NMDA and group I mGlu receptor antagonists share a variety of in vivo effects (Fundytus and Coderre, 1994; Fundytus et al., 1997). However, in the present study, the administration of morphine together with EMQMCM or MTEP at the lowest doses, which produced suppression of the formalin-evoked response alone, did not result in augmentation of analgesic effects. Moreover, the statistical analysis showed the negative interaction between MTEP and morphine in the second phase of formalin-evoked pain response, suggesting inhibition of MTEP effect by morphine, however, post hoc test failed to show significant difference between group treated with MTEP only versus that treated with a combination of MTEP and morphine. Thus, this observation requires further elucidation. It should be stressed that these results contrast to the enhancement of antinociceptive effects seen in the present study after co-treatment with morphine and the uncompetitive NMDA receptor antagonist memantine. Although neither of the group I mGlu receptor antagonists enhanced the analgesic effects when they were co-administered with morphine, several studies have shown the ability of mGlu receptor antagonists to reduce the severity of abstinence symptoms following withdrawal from morphine (Fundytus and Coderre, 1997), to prevent the development of morphine rewarding effects (Aoki et al., 2004) and to inhibit tolerance to morphine (Kozela et al., 2003; Narita et al., 2005). To our knowledge, this is the first study addressing the influence of mGlu receptor antagonists upon acute opiate analgesia in the formalin test, and further studies are necessary to verify if this phenomenon extends to different types of pain. The present study also provides the first evidence that prolonged administration of mGlu5 receptor antagonists (chronic administration of MTEP for 7 days) leads to the development of tolerance to antinociceptive effects in the formalin test. Such tolerance to the analgesic effect was not observed for mGlu1 receptor antagonist EMQMCM. This is in line with a study by Steckler et al. (2005) showing a lack of tolerance to the anxiolytic effect of mGlu1 antagonist JNJ16259685 (which has the same scaffold as EMQMCM) after treatment for 14 days in the lick suppression test. There are few publications addressing the issue of development of tolerance to pharmacological effects of mGlu5 receptor antagonists. Tolerance did not develop to the anxiolytic- and antidepressant-like effects of MTEP in rats after one week of treatment (Pilc et al., 2002; Klodzinska et al., 2004). Gain of antiparkinsonian-like efficacy of MPEP was seen in the reaction time task in rats after chronic administration, and lack of tolerance to antidyskinetic action was recently reported (Breysse et al., 2002; Dekundy et al., 2003). Other studies, however, showed that tolerance to anxiolytic activity developed very rapidly

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(<3 days) after repeated administration of mGlu5 receptor antagonists (Roppe et al., 2004; Busse et al., 2004). The authors indicated that tolerance was not due to changes in mGlu5 receptor number, their sensitivity, changes in receptor occupancy, or pharmacokinetic alterations (unpublished observations of Busse et al. mentioned in the discussion, Busse et al., 2004). Moreover, the phenomenon of tolerance seems not to be specific for a class of scaffolds, but rather a receptor specific phenomenon since agents having different chemical structures produced such decreases in pharmacodynamic responses upon repetitive dosing (Roppe et al., 2004). Recently, de Novellis et al. (2004) reported that the analgesic effect of MPEP was limited to the first 2e3 days after nerve injury in rats with established neuropathic pain. They hypothesized that, over time, persistently high levels of ectopic discharge may cause molecular modifications, including NMDA receptor sensitization, which lead to non-apoptotic excitotoxic neural death and the development of mechanical hyperalgesia that MPEP was unable to reverse. Another example of tolerance to the analgesic effects has been demonstrated for the mGlu2/3 receptor agonist (LY379268) in the formalin, carrageenan and capsaicin tests (Jones et al., 2005). The present data show development of tolerance to the analgesic effect of mGlu5 antagonist and thereby indicate that tolerance development may not be limited only to tests of anxiety, but also extends at least to some types of the pain. The question remains whether such tolerance would also be observed in clinical situations. In conclusion, the present results provide additional evidence for the role of group I metabotropic glutamate receptors in pain with inflammatory conditions. The potential therapeutic use looks encouraging especially for mGlu1 receptor antagonists, since no tolerance develops to antinociceptive activity after repetitive treatment, in contrast to mGlu5 receptor antagonists. References Aoki, T., Narita, M., Shibasaki, M., Suzuki, T., 2004. Metabotropic glutamate receptor 5 localized in the limbic forebrain is critical for the development of morphine-induced rewarding effect in mice. Eur. J. Neurosci. 20, 1633e1638. Bhave, G., Karim, F., Carlton, S.M., Gereau, R.W., 2001. Peripheral group I metabotropic glutamate receptors modulate nociception in mice. Nat. Neurosci. 4, 417e423. Breysse, N., Baunez, C., Spooren, W., Gasparini, F., Amalric, M., 2002. Chronic but not acute treatment with a metabotropic glutamate 5 receptor antagonist reverses the akinetic deficits in a rat model of parkinsonism. J. Neurosci. 22, 5669e5678. Busse, C.S., Brodkin, J., Tattersall, D., Anderson, J.J., Warren, N., Tehrani, L., et al., 2004. The behavioral profile of the potent and selective mGlu5 receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) in rodent models of anxiety. Neuropsychopharmacology 29, 1971e1979. Carlton, S.M., Hargett, G.L., Coggeshall, R.E., 1995. Localization and activation of glutamate receptors in unmyelinated axons of rat glabrous skin. Neurosci. Lett. 197, 25e28. Coderre, T.J., Yashpal, K., 1994. Intracellular messengers contributing to persistent nociception and hyperalgesia induced by L-glutamate and substance P in the rat formalin pain model. Eur. J. Neurosci. 6, 1328e1334. Coggeshall, R.E., Carlton, S.M., 1999. Evidence for an inflammation-induced change in the local glutamatergic regulation of postganglionic sympathetic efferents. Pain 83, 163e168.

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