Introduction

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Anticonvulsant drugs normally used for the treatment of epilepsy are effective in the management of certain pain states such as trigeminal neuralgia (Loeser, 1994) and central/poststroke pain (Leijon and Boivie, 1989). Newer anticonvulsants such as gabapentin have found a use in the alleviation of neuropathic pain (Rosner et al., 1996).

Remacemide [(±)-2-amino-N-(1-methyl-1,2-diphenylethyl)-acetamide hydrochloride] is a novel anticonvulsant (Palmer et al., 1993) and neuroprotective (Palmer et al., 1995) drug with a high therapeutic index that is presently in phase III clinical trials. A major and active metabolite of remacemide is AR-R12495AA [(±)-1-methyl-1,2-diphenylethylamine-monohydrochloride], which is formed by the des-glycination of remacemide hydrochloride (Heyn et al., 1994) and, in animal models of epilepsy, is more potent than remacemide itself (Palmer et al., 1993). Remacemide and AR-R12495AA affect glutamate-mediated excitatory synaptic transmission via N-methyl-D-aspartate (NMDA) receptors, and this is considered to contribute to their anticonvulsant and neuroprotective activities. Remacemide and AR-R12495AA displace binding of the NMDA receptor-ion channel blocker [³H]MK-801 from rat brain homogenates (Palmer et al., 1992). Remacemide and AR-R12495AA are effective against NMDA-induced seizures (Stagnitto et al., 1990; Garske et al., 1991; Palmer et al., 1992), and voltage-clamp studies in hippocampal neurons have shown that both compounds selectively inhibit NMDA-evoked currents (Subramaniam et al., 1996), with AR-R12495AA possessing higher potency.

The capability of remacemide and AR-R12495AA to affect the NMDA receptor-ion channel complex is of interest in the context of nociception. The NMDA receptor is believed to play an important role in the generation and maintenance of spinal central sensitization and hyperalgesia (Woolf and Thompson, 1991; King and Thompson, 1995). Electrophysiological studies in the spinal cord revealed a long latency excitatory postsynaptic potential resulting from nociceptive afferent or noxious cutaneous stimulation that is partially dependent on NMDA receptor activation (Thompson et al., 1990; Woodley and Kendig, 1991; King et al., 1992). In the spinal cord, these nociceptive afferent-evoked potentials are recorded extracellularly as dorsal root-evoked ventral root potentials (DR-VRPs) of several seconds' duration (Siarey et al., 1992; Thompson et al., 1992; Nagy and Woolf, 1996). Moreover, a cumulative ventral root depolarization (CVRD) can be produced by the temporal summation of DR-VRPs after high-intensity low-frequency stimulation (Thompson et al., 1992; Nagy and Woolf, 1996). CVRD is proposed as a correlate of wind-up, a prolonged increase in the excitability of spinal neurons induced by nociceptive afferent stimulation (Woolf and Thompson, 1991). The amplitudes of the CVRD and wind-up are dependent on NMDA receptors (King et al., 1992; Thompson et al., 1992). These NMDA-related neuroplastic phenomena are believed to underpin central sensitization, a contributory factor in postinjury pain hypersensitivity (Woolf and Thompson, 1991). In support of this, mechanical hyperalgesia induced by algogens such as carrageenan or Freund's complete adjuvant (FCA) is attenuated by NMDA antagonists such as MK-801 (Ren et al., 1992; Ren and Dubner, 1993).

In this electrophysiological investigation, the actions of the anticonvulsant remacemide and its more potent metabolite AR-R12495AA on single-shock DR-VRPs and the summated CVRD were determined in the isolated hemisected neonatal rat spinal cord. MK-801, an established noncompetitive NMDA antagonist, was included for comparison. In animal studies, it has been shown that due to its shorter half-life (0.3 h in rat; Palmer et al., 1993), dosing with remacemide results in a progressive rise in the level of the more stable metabolite AR-R12495AA. Thus, in a parallel behavioral study, AR-R12495AA was tested against carrageenan- and FCA-induced hyperalgesia to determine whether this compound may be effective in a rat model of inflammatory pain. Preliminary data have been presented in abstract form (Asghar et al., 1997, 1998a,b).

	Materials and Methods

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Tissue Preparation and Drug Application. After the induction of fatal anesthesia with urethane (2 g · kg¹ i.p.), a dorsal laminectomy was performed, and spinal cords were removed rapidly from Wistar rats aged 10 to 14 days. The cord with dorsal and ventral segmental roots was submerged in ice-cold artificial cerebrospinal fluid (aCSF) and hemisected. The hemicord was placed into a Perspex chamber (volume 1.5 ml), secured onto a Sylgard-coated base, and superfused (10 ml · min¹) at room temperature (21-22°C) with oxygenated (95% O₂, 5% CO₂) aCSF. The composition of aCSF was 128 mM NaCl, 1.9 mM KCl, 1.2 mM KH₂PO₄, 1.3 mM MgSO₄, 2.4 mM CaCl₂, 26 mM NaHCO₃, and 10 mM glucose, pH 7.4.

Electrophysiological Protocols. For extracellular monitoring of DR-VRPs, selected segmental lumbar roots (L3-6) were drawn into separate aCSF-filled glass suction electrodes. The dorsal root was stimulated using constant-current single shocks from an isolated stimulator (Neurolog NL800; Digitimer Ltd., Welwyn Garden City, UK) at A- and C-afferent suprathreshold intensity (200 µA, 200 µs), as determined by sciatic nerve-dorsal root compound action potential analysis (Thompson et al., 1990). The single-shock DR-VRP was recorded from the ventral root using a DC amplifier (Axoclamp 2A; Applied Biosystems, Foster City, CA). Unfiltered waveforms were captured on videotape, and Spike 2 software (Cambridge Electronic Design, Cambridge, UK), which sampled at 3 to 5 kHz, was used for online and offline data analysis of DR-VRP parameters.

Behavioral Protocols and Algogen-Induced Peripheral Inflammation. An acute peripheral inflammation was induced by footpad injection of carrageenan (150 µl, 0.5% in normal sterile saline) in adult Wistar rats (150-180 g) under brief halothane anesthesia. In control animals, this dose of carrageenan induces a rapidly developing mechanical hyperalgesia that reaches a peak at approximately 240 min postinjection (Fig. 5A). The development of mechanical hyperalgesia and paw swelling was followed for a period of 5 h. Nociceptive hind paw withdrawal pressure thresholds were measured using an analgesymeter (Ugo Basile) that operates by gradually applying an increasing calibrated mechanical pressure (g) to the rat hind paw until there is a nociceptive limb withdrawal response. Paw swelling was assessed indirectly by measuring the dorsal-plantar thickness of each hind paw using calibrated vernier calipers (to 0.1 mm). A difference score for hind paw nociceptive mechanical thresholds and for hind paw thickness was calculated by subtracting the contralateral uninjected paw value from that measured in the ipsilateral injected paw. Three baseline readings were taken before injecting AR-R12495AA (dissolved in sterile saline 0.9% w/v, 75 mg · kg¹ i.p.) 30 min before the injection of carrageenan. The effects of AR-R12495AA were compared against those for rats injected i.p. with the drug vehicle (sterile saline 0.9% w/v).

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	Results

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Effects of Remacemide, AR-R12495AA and MK-801 on DR-VRP and CVRD. Single-shock stimulation of lumbar dorsal roots at A- and C-fiber strength produced a DR-VRP. The DR-VRP consisted of a short-latency (<10 ms), presumed monosynaptic, synchronized fast peak (mean amplitude of 3.2 ± 0.3 mV, n = 40) followed by a slow phase that consisted of smaller amplitude asynchronous potentials decaying over several seconds (Figs. 1 and 2). The slow DR-VRP phase had a total duration of 9.4 ± 0.6 s and a t_1/2 decay of 0.30 ± 0.03 s (n = 40).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Reduction in the slow DR-VRP by remacemide and AR-R12495AA. The control slow DR-VRP total duration (left traces) is reduced by (A) 200 µM remacemide and (B) 200 µM AR-R12495AA (right traces). Note the reduction in decay time of the slow DR-VRP caused by remacemide and AR-R12495AA. At this time scale, the stimulus artifacts are merged with the fast peaks of the DR-VRP (arrowheads). C, concentration-response curves for the percentage of depression in slow DR-VRP duration induced by remacemide (), AR-R12495AA (), and MK-801 (). Each data point is mean ± S.E. (n = 6-7).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effects of remacemide and AR-R12495AA on the extracellular DR-VRP elicited by single stimulation of a dorsal root at 200 µA, 200 µs. A, DR-VRPs under control conditions (left trace) and after 100 µM AR-R12495AA (right trace). AR-R12495AA depressed the DR-VRP slow peak t1/2 decay time by 40% and the fast peak by <10%. Stimulus artifact is indicated by arrowheads below records. Concentration-response curves are for the reductions of fast peak amplitude (B) and slow peak t1/2 decay time (C) induced by remacemide (), AR-R12495AA (), and MK-801 () on DR-VRP parameters. Each data point is mean ± S.E. (n = 6 or 7).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   CVRDs elicited by repetitive stimulation (2 Hz) of dorsal roots at 200 µA, 200 µs, for 20s (left traces) are attenuated by (A) 200 µM remacemide and (B) 200 µM AR-R12495AA (right traces). Arrows indicate where CVRD amplitudes were measured. C, concentration-response curves for the reduction in CVRD amplitude induced by remacemide (), AR-R12495AA (), and MK-801 (). Each data point is mean ± S.E. (n = 6 or 7).

Effects of AR-R12495AA on Algogen-Induced Mechanical Hyperalgesia. In adult Wistar rats (n = 6), hind paw footpad injection of carrageenan (150 µl, 0.5% in normal sterile saline) induced a mechanical hyperalgesia that was measured as a progressive reduction in the gram pressure (g) required to elicit the ipsilateral paw withdrawal threshold. This nociceptive threshold reduction was manifest at 60 min postinjection and reached its peak at approximately 240 min postinjection (Fig. 4A). Ipsilateral paw swelling, an index of inflammation, developed rapidly after carrageenan and was maximum at approximately 180 min after carrageenan (Fig. 4B). AR-R12495AA (i.p. 75 mg · kg¹ 30 min before carrageenan) strongly attenuated the mechanical hyperalgesia at all of the measured 60- to 300-min time points (Fig. 4A) and significantly reduced the amount of paw swelling that followed carrageenan (Fig. 4B).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effects of AR-R12495AA on carrageenan- and FCA-induced mechanical hyperalgesia and paw swelling. Intradermal carrageenan reduced the gram pressure (g) threshold for paw withdrawal over a period of 60 to 300 min (A) and elicited paw swelling (B). These effects were attenuated by pretreatment with AR-R12495AA (single dose, 75 mg · kg1 i.p.) (A and B). The basal values for paw withdrawal thresholds and paw thickness before any treatment (30-min time point) were 63 ± 5 g and 5.2 ± 0.1 mm, respectively. The FCA-induced reduction in the paw withdrawal threshold (C) and increased paw thickness (D) were attenuated by AR-R12495AA (30 mg · kg1 i.p. daily). Basal values for paw withdrawal thresholds and paw thickness before any treatment (0-min time point) were 71 ± 1 g and 5.7 ± 0.2 mm, respectively. Data are mean ± S.E. (n = 6, *P  .05, unpaired t test, significantly different from vehicle-treated rats).

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	Discussion

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Remacemide and its des-glycinated metabolite AR-R12495AA significantly depressed primary afferent-mediated synaptic potentials elicited by single-shock stimuli (DR-VRP) and the cumulative depolarization generated by low-frequency repetitive stimuli (CVRD). The most pronounced drug actions were on the slow DR-VRP total duration and t_1/2 decay. The slow DR-VRP and CVRD are strongly dependent on activation of high-threshold afferents and NMDA receptors (Thompson et al., 1990; King et al., 1992; Thompson et al., 1992; Farkas and Ono, 1995). The actions of remacemide and AR-R12495AA on the slow components of the DR-VRP and on CVRD amplitude would be consistent, therefore, with previously published data on NMDA receptor-ion channel modulation by these drugs. For example, in the cortical wedge preparation, AR-R12495AA attenuated NMDA-induced depolarization (Hu and Davies, 1995a), and in whole-cell voltage-clamp experiments in hippocampal neurons, remacemide and AR-R12495AA (Subramaniam et al., 1996) blocked NMDA-evoked currents. Furthermore, studies in dissociated cultured spinal neurons demonstrated that remacemide and AR-R12495AA reduced NMDA-induced depolarizations (Wamil et al., 1996).

An additional mechanism of drug action that cannot be excluded on the basis of these data is through ion channel modulation. Data from hippocampal neurons (Norris and King, 1997) and dissociated spinal neurons (Wamil et al., 1996) showed a remacemide- and AR-R12495AA-induced limitation of firing frequency, an indirect indication of sodium channel blockade. The concentration of remacemide and AR-R12495AA required to block sodium channels is similar to that required for block of NMDA-evoked responses (Wamil et al., 1996). In the hemisected spinal cord preparation, sodium channels have been demonstrated to play an important role in the slow phase of the DR-VRP and in CVRD amplitude (Nagy and Woolf, 1996). Thus, part of the attenuation in the slow potentials by remacemide and AR-R12495AA observed in the current investigation could be attributable to sodium channel block. A direct voltage-clamp study of ionic current modulation by remacemide and its analogs will be required to clarify the precise mechanism of action for modulation of primary afferent-mediated synaptic potentials.

Although the principal actions of remacemide and AR-R12495AA were against the long latency components of the DR-VRP, at concentrations of >100 µM, there was a significant reduction of the DR-VRP fast peak. Given that the DR-VRP fast peak is most effectively blocked by non-NMDA receptor antagonists (Thompson et al., 1992), it is possible these higher drug concentrations could modulate non-NMDA receptor-mediated events. However, against this is the fact that in the cortical wedge preparation, -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA)-induced depolarizations were unaffected by AR-R12495AA (Hu and Davies, 1995a). In cultured hippocampal neurons, currents evoked by kainate or AMPA were insensitive to either drug (Subramaniam et al., 1996). A role for NMDA receptors in fast monosynaptic spinal neurotransmission has been identified (King et al., 1992), and so some of the attenuation by remacemide and AR-R12495AA could be ascribed to this. An alternative explanation is that the reduction in fast peak amplitude reflects sodium channel modulation rather than excitatory amino acid receptor antagonism. In this study, the well established noncompetitive NMDA antagonist MK-801, which also limits sustained repetitive firing and modulates sodium channels (Rothman, 1988; Wamil and McLean, 1992), effectively reduced the DR-VRP fast peak amplitude with an IC₅₀ value lower than that for AR-R12495AA or remacemide. Also relevant is the fact that in mouse cortical slices, MK-801 and AR-R12495AA inhibit veratridine-stimulated release of glutamate and aspartate (Srinivasan et al., 1995).

The IC₅₀ values for remacemide, AR-R12495AA, and MK-801 against DR-VRP parameters and CVRD calculated in the present investigation are higher than those obtained in previous studies. Thus, in cultured hippocampal neurons, the (S)-(+)- and (R)-()-des-glycine analogs of remacemide tested against 10 µM NMDA had IC₅₀ values of 0.7 and 4 µM, respectively, whereas (R)-(+)-and (S)-()-remacemide had IC₅₀ values of 67 and 75 µM (Subramaniam et al., 1996). In cultured spinal cord neurons (Wamil and McLean, 1992; Wamil et al., 1996) remacemide, AR-R12495AA and MK-801 limited sustained repetitive firing and antagonized NMDA (10 µM) depolarizations with IC₅₀ values of 7.9, 1.2, and 0.01 to 0.1 µM, respectively. However, a range of IC₅₀ values have been reported in different models under a variety of experimental conditions. In the hippocampal slice, limitation of sustained repetitive firing by remacemide and AR-R12495AA had IC₅₀ values of 66 and 60 µM (Norris and King, 1997). In cortical wedges prepared from epilepsy-prone DBA/2 mice (Hu and Davies, 1995b), AR-R12495AA reduced spontaneous depolarizations, afterpotentials, and NMDA (10 µM) depolarizations with IC₅₀ values of 102, 50, and 43 µM, respectively. Apart from the different experimental protocols and methodologies used, there are several other explanations for variability in cited potencies. For example, complete drug penetration of lipophilic compounds such as remacemide, AR-R12495AA, and MK-801 will be much harder to achieve in hemisected cord compared with cultured neurons. Another possible explanation is that our experiments in the spinal cord were performed at room temperature, whereas those with cultured spinal neurons (Wamil and McLean, 1992; Wamil et al., 1996) were performed at 37°C. In the rat hemicord preparation, DR-VRPs and associated slow components are optimal at 20-22°C and are not seen above 25°C (Bagust, 1993).

Although the IC₅₀ values are variable across studies, there is good agreement on the rank potencies of these compounds. Comparing the action of two drugs against the slow DR-VRP and CVRD, AR-R12495AA was moderately (2×) more potent than the parent compound remacemide. In electrophysiological investigations of attenuation of NMDA-mediated responses in cultured spinal or hippocampal neurons, AR-R12495AA had a higher potency relative to that of remacemide (Subramaniam et al., 1996; Wamil et al., 1996). The effect of remacemide increased with greater drug exposure time (Wamil et al., 1996), and this may reflect a slow access of remacemide or the time required for its des-glycination to AR-R12495AA (Heyn et al., 1994). Furthermore, AR-R12495AA is a more potent inhibitor of the sodium channel than is remacemide (Palmer et al., 1995; Wamil et al., 1996). The rank potency order for the three drugs in this study was MK-801 > AR-R12495AA > remacemide. Other studies, such as the NMDA-induced seizure model, have also shown that MK-801 is much more effective than remacemide and AR-R12495AA (Palmer et al., 1993). The greater potency of MK-801 is likely to be due to its higher affinity at the NMDA receptor compared with remacemide and AR-R12495AA (Palmer et al., 1993, 1995; Hudzik et al., 1996) and/or higher affinity at other sites such as sodium channels (Wamil and McLean, 1992).

The ability of remacemide and AR-R12495AA to potentially affect spinal somatosensory pathways through their actions at the NMDA receptor-ion channel complex and/or sodium channels is important in the context of nociception and pain. Other NMDA antagonists, such as MK-801, D-amino-phosphonovalerate and memantine, limit hyperalgesia in behavioral models of localized inflammation or peripheral neuropathy (Ren et al., 1992; Ren and Dubner, 1993; Eisenberg et al., 1995; Lawand et al., 1997). There is evidence that selective sodium channel inhibition attenuates spinal nociceptive responses in a manner not fully accounted for by simple conduction block (Jaffe and Rowe, 1995). In behavioral studies of adult rats subjected to either carrageenan- or FCA-induced inflammation, mechanical hyperalgesia and paw swelling were attenuated by AR-R12495AA. For effectiveness against carrageenan-induced hyperalgesia, a single dose of 75 mg · kg¹ i.p. AR-R12495AA was required. For protection against FCA-induced mechanical hyperalgesia, a daily dosing regimen of 30 mg · kg¹ i.p. AR-R12495AA was used. The finding of a reduction in peripheral edema indicates a putative peripheral action of AR-R12495AA that, in addition to any central action, could also contribute to the attenuation of mechanical hyperalgesia. Glutamate receptors, including the NMDA subtype, are localized on unmyelinated axons of rat glabrous skin (Carlton et al., 1995). Peripheral intra-articular (Lawand et al., 1997) or intraplantar (Jackson et al., 1995) injection of glutamate results in a hyperalgesia that is attenuated by peripherally administered NMDA and non-NMDA receptor antagonists. Interestingly, non-NMDA antagonists, but not NMDA antagonists, have been previously shown to reduce carrageenan/kaolin-induced paw swelling (Sluka et al., 1994).

Because pharmacokinetic data are not yet available for AR-R12495AA i.p., no direct conclusion can be made about final brain concentrations and effective therapeutic levels. In this study, AR-R12495AA at 75 mg · kg¹ (single i.p. dose) and 30 mg · kg¹ (daily i.p. for 19 days) were effective against carrageenan- and FCA-induced hyperalgesia, respectively. As a comparison, in mice, the ED₅₀ value for protection against NMDA-induced seizures was 57.4 mg · kg¹ i.p. for remacemide and 32.4 mg · kg¹ i.p. for AR-R12495AA i.p. (Palmer et al., 1993). In human studies of efficacy in epilepsy, an add-on remacemide dose of 600 mg · day¹ for 28 days effectively reduced the seizure frequency by 33% (Palmer et al., 1993). In view of these electrophysiological and behavioral data, it would be valuable to assess further the anticonvulsants remacemide and AR-R12495AA as potentially useful analgesics.