Introduction

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N-Methyl-D-aspartate (NMDA) receptor antagonists are a chemically and functionally diverse class of compounds that when repeatedly coadministered with morphine, can attenuate the development of tolerance to the antinociceptive effects of morphine (Trujillo and Akil, 1991; Allen and Dykstra, 2000a). In addition, NMDA receptor antagonists can attenuate the development of tolerance to the antinociceptive effects of a variety of other opioid receptor agonists, including the µ-opioid receptor agonists etorphine and dezocine (Allen and Dykstra, 2000b), the -opioid receptor agonist U50,488 (Bhargava and Thorat, 1994), and the -opioid receptor (DOR) agonists DPDPE and deltorphin II (Bhargava and Zhao, 1996a; Zhao and Bhargava, 1996). These findings suggest that endogenous glutamatergic activation of the NMDA receptor may represent a general mechanism whereby tolerance develops to the antinociceptive effects of opioids.

Some investigations emphasize that attenuation of morphine tolerance by NMDA receptor antagonists occurs under experimental conditions and/or with doses of NMDA receptor antagonists that: 1) do not produce antinociceptive effects alone or 2) do not acutely potentiate the antinociceptive effects of morphine (Trujillo and Akil, 1991; Allen and Dykstra, 2000a). Nevertheless, there are data indicating that NMDA receptor antagonists produce antinociception under a variety of experimental conditions (Sadove et al., 1971; France et al., 1989) and relieve pain in some clinical situations (Nelson et al., 1997). NMDA receptor antagonists also increase the magnitude and duration of antinociceptive effectiveness of low doses of morphine under a variety of conditions (Caruso, 2000; Allen and Dykstra, 2001). For example, the noncompetitive NMDA receptor antagonist dizocilpine (MK-801), the competitive NMDA receptor antagonist LY235959, and the glycine-site antagonist (+)-HA-966 each potentiated the antinociceptive effects of low doses of morphine when tested using a squirrel monkey titration procedure (Allen and Dykstra, 2001).

Although there is considerable information available regarding the effect of NMDA receptor antagonists on the antinociceptive effect of morphine and other µ-opioid agonists, less is known about the interaction of NMDA receptor antagonists with opioids that differ in their receptor selectivity. DOR agonists produce antinociceptive effects in a variety of experimental pain assays (Bilsky et al., 1995; Negus et al., 1998; Fraser et al., 2000). Unlike µ-opioid agonists, -opioid agonists do not adversely affect respiration or gastrointestinal function (Porreca et al., 1984; Negus et al., 1994). However, tolerance to the antinociceptive effects of some DOR agonists can develop following repeated treatment (Bhargava and Zhao, 1996a; Zhao and Bhargava, 1996). In addition, some DOR agonists produce proconvulsant effects at higher doses (Comer et al., 1993; Dykstra et al., 1993), and this effect may severely limit their therapeutic potential. Because the tolerance-attenuating and anticonvulsant effects associated with NMDA receptor antagonists may improve the safety profile of DOR agonists as antinociceptive agents, knowledge about their acute interaction is an important goal.

In the present study, we determined whether the noncompetitive NMDA receptor antagonist, dextromethorphan, would potentiate the antinociceptive effects of low doses of the DOR agonist, SNC80 [(+)-4[R)--((2S,5R)-4-allyl-2,5,-dimethyl-l-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide]. SNC80 is a systemically active DOR agonist with 495-fold selectivity for /µ receptors (Calderon et al., 1994; Bilsky et al., 1995). The antinociceptive effects of dextromethorphan and SNC80, alone and in combination, were measured using the squirrel monkey titration procedure. The squirrel monkey titration procedure is an animal model that simultaneously provides a measure of antinociception and response rate (Dykstra, 1985). Because termination of shock (the nociceptive stimulus) requires an operant response (FR 5), responding on the lever [response rate (RR)] is recorded along with the antinociceptive measure [median shock level (MSL)]. Data show that several NMDA receptor antagonists potentiate the effects of morphine in this procedure (Allen and Dykstra, 2001). In addition to testing the effects of dextromethorphan in combination with SNC80, the effects of dextromethorphan in combination with morphine were also examined to extend previous findings indicating that the NMDA receptor antagonists, dizocilpine, LY235959, and (+)-HA-966, potentiate the antinociceptive effects of morphine in squirrel monkeys (Allen and Dykstra, 2001).

	Materials and Methods

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Animals. Four adult male squirrel monkeys (Saimiri sciureus) weighing between 0.70 and 0.95 kg were housed in pairs in a colony room with a 12-h light/dark cycle. All monkeys had continuous access to water, were maintained on a high-protein monkey diet, and were given fresh fruit and nuts daily. All of the monkeys had previous experience with the titration procedure and had received various opioid compounds but had not received drugs for at least 30 days before the start of the present experiment.

Apparatus. During experimental sessions, each monkey sat in a Plexiglas chair and was held in place by a waist support with its tail secured by a small stock (see Dykstra, 1985). The tail was coated with EKG Sol, a noncorrosive electrode paste (Graphics Control Medical Products Division, Buffalo, NY), to provide a low-resistance electrical contact. Electric shock (110 V a.c., 60 Hz) was delivered through two hinged brass plates that rested on a shaved portion of the tail.

Behavioral Procedure. A shock titration procedure nearly identical to that described by Dykstra (1985) was used. In each session, periods during which an FR 5 schedule of shock titration was in effect alternated with periods of blackout. Each FR 5 titration period began with the illumination of the houselight and presentation of 0.01-mA shock. Shock intensity increased from 0.01 to 2.0 mA in 30 increments. Completion of the FR 5 requirement at a given shock intensity initiated a time-out during which shock was off and the houselight remained illuminated. After the 15-s time-out, the shock resumed at the next lower intensity. If a monkey failed to complete the FR 5 during 15 s at a given shock intensity, the intensity increased by one increment and the response requirement was reset to 5. The FR 5 titration periods usually lasted 15 min. An FR 5 period terminated automatically, however, if the shock intensity rose to the peak intensity of 2.0 mA and the FR 5 requirement was not completed during any of five consecutive 15-s periods. During the blackouts that separated the FR 5 titration periods, the chamber was dark, no shock was delivered, and lever presses had no programmed consequences. Blackouts lasted 20 min. Each session began with an FR 5 titration period and ended after completion of five FR 5 periods.

Pharmacological Procedure. Time-effect curves for morphine, SNC80, dextromethorphan, and all drug combinations were obtained by administering a dose or dose combination to monkeys 20 min before the first FR 5 period. On completion of the first and all subsequent FR 5 periods (at the onset of each blackout), vehicle (sterile water) was injected.

Data Analysis. Two dependent variables were analyzed extensively in this study: MSL (milliamperes) and RR during shock (responses per second). Response rate during time-out, a third dependent variable, was only analyzed for data from vehicle control injections.

Vehicle and Morphine Control Data. For each monkey, data from the seven most recent vehicle control tests were averaged, and the average for a monkey was used in the statistical analysis. For morphine/dextromethorphan and SNC80/dextromethorphan combinations, the effects of the opioid were assessed before and after each series of dose combinations. Thus, there were two opioid-alone curves for each monkey in each of these experiments. Since there was little variability in the effectiveness of morphine for a given monkey, these morphine curves were averaged, and the average for a monkey represented that monkey in the statistical analysis. The results of the statistical analysis did not differ, however, when only one morphine-alone curve was used in place of the averaged morphine curve (data not shown).

Contrasts. When a post hoc analysis of a factor in the repeated-measures ANOVA was conducted, the analysis made the following comparisons: for multiple doses of a drug tested alone, all doses versus vehicle control; and for dose combinations, all combinations versus the effect of morphine or SNC80 alone. All data were analyzed using SAS for Windows version 8e (Cary, NC).

	Results

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Effects of Dextromethorphan Alone on MSL and RR. The effects of dextromethorphan alone on MSL and RR were assessed in four monkeys, and these data are presented in Table 1. Dextromethorphan produced small increases in MSL relative to vehicle control injections. The results of a repeated-measures ANOVA of these data indicated no main effect of dextromethorphan dose [F(3,9) = 2.60, P = 0.12] or time [F(4,12) = 1.02, P = 0.44] but indicated a significant interaction effect [F(12,36) = 2.21, P = 0.03). This small increase in MSL was greatest during the first component, with individual MSLs of 0.06, 0.15, 0.15, and 0.40 mA (mean = 0.19 ± 0.07) for monkeys tested with the highest dose of dextromethorphan (10 mg/kg). In contrast to the effects of dextromethorphan on MSL, dextromethorphan did not meaningfully alter RR across the range of doses tested in this study. The results from the repeated-measures ANOVA using RR as the dependent variable revealed no main effect of dose [F(3,9) = 1.46, P = 0.29] or time [F(4,12) = 1.73, P = 0.21], and revealed a significant interaction [F(12,36) = 2.00, P = 0.05].

Effects of Morphine Alone and in Combination with Dextromethorphan on MSL and RR. Fig. 1 (left, top panel) shows the time course of the antinociceptive effect of morphine alone when tested in four monkeys. Mean (±S.E.) MSL values for the four monkeys to which morphine was administered are presented in Table 1. When administered alone, morphine increased MSL in a manner that was both dose- and time-dependent. The results from the repeated-measures ANOVA of the MSL values revealed a significant main effect of both morphine dose (F4,8 = 71.56, P = 0.0001) and time [F(4,8) = 8.35, P = 0.006] and a significant dose × time interaction [F(16,32) = 3.38, P = 0.002]. For one monkey, the experimental session auto-terminated (see Materials and Methods) during components 2 and 3. The session was discontinued at this point, and naltrexone was administered to this monkey. Because the data for this monkey were not complete, the repeated-measures ANOVA includes only data from three monkeys.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Effect of morphine alone (left panels) and in combination with dextromethorphan (DXM) (center and right panels) on MSL (top panels) and response rate (bottom panels). Ordinate: MSL in milliamperes (top panels) or response rate in responses per second (bottom panels). Abscissa: time after drug administration during which MSL or rate was calculated.

Effects of SNC80 Alone and in Combination with Dextromethorphan on MSL and RR. The effects of SNC80 alone (1.0, 3.0, and 10 mg/kg) on MSL and RR are presented in Fig. 2 (left panels). SNC80 produced small increases in MSL that were dose-related and of short duration. Mean (±S.E.) MSL values for the four monkeys to which SNC80 was administered are presented in Table 1. The highest mean MSL produced by SNC80 was 0.38 (0.15) mA, and this occurred in the first component of an experimental session (25-35 min postinjection) after the administration of 3.0 mg/kg SNC80 (Table 1, Fig. 2). The results from the two-way repeated-measures ANOVA for MSL values reveal a main effect of dose [F(3,9 = 8.40, P = 0.006] and a significant dose × time interaction [F(12,36) = 2.17, P = 0.04] but no main effect of time [F(4,12) = 2.31, P = 0.12]. This same analysis for RR revealed no main effect of dose [F(3,9) = 1.18, P = 0.37] or time [F(4,12 = 2.34, P = 0.11], but a significant dose × time interaction [F(12,36) = 2.32, P = 0.03].

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Effect of SNC80 alone (left panels) and in combination with dextromethorphan (DXM) (center and right panels) on MSL (top panels) and response rate (bottom panels). Ordinate: MSL in milliamperes (top panels) or response rate in responses per second (bottom panels). Abscissa: time after drug administration during which MSL or response rate was calculated.

	Discussion

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In this study, dextromethorphan alone did not produce antinociceptive effects across the range of doses tested. This result is in agreement with the animal literature that examines the antinociceptive efficacy of dextromethorphan in models of acute or phasic pain. In general, dextromethorphan lacks antinociceptive efficacy in rat and mouse tail-withdrawal, tail-flick, and hot-plate procedures. However, France et al. (1989) have shown that dextrorphan and other NMDA receptor antagonists increase rhesus monkey tail-withdrawal latencies from 50 and 55°C water.

Studies with human experimental pain models also tend to show that dextromethorphan is not effective at reducing the pain associated with an acute nociceptive thermal or electrical stimulus (Price et al., 1994; Ilkjaer et al., 1997) or ischemic pain (Plesan et al., 2000). However, dextromethorphan is effective at reducing postinjury hyperalgesia (Ilkjaer et al., 1997) and the temporal summation of second- or late-phase, C-fiber pain (Price et al., 1994). Similarly, it has been demonstrated that dextromethorphan has antinociceptive efficacy in the formalin test, an animal model that measures sensitized responses to noxious stimuli (Elliot et al., 1995).

This distinction between the effectiveness of dextromethorphan on acute pain versus sensitized responses to pain is also consistent with the results from clinical investigations with dextromethorphan. In general, dextromethorphan appears ineffective as an analgesic when administered to patients postoperatively for the management of pain associated with knee surgery (Wadhwa et al., 2001) or for the pain associated with facial neuralgias (Gilron et al., 2000), cancer pain (Mercandante et al., 1998), or neuropathic pain (McQuay et al., 1994). However, high doses of oral dextromethorphan were effective at reducing pain associated with diabetic neuropathy (Nelson et al., 1997). In contrast, single doses of dextromethorphan administered preoperatively significantly reduce the severity of postoperative pain and opioid consumption associated with various surgical procedures (e.g., Helmy and Bali, 2001). Thus, dextromethorphan appears to minimize the development of a painful condition (e.g., postincisional pain) more reliably than it reduces either 1) the severity of acutely painful stimuli or 2) the severity of pain associated with a pre-existing condition.

A second major finding from this study is that dextromethorphan potentiates the antinociceptive effects of morphine in a dose-dependent manner. Antinociceptive effects of dextromethorphan/morphine combinations were near-maximal in this procedure, even though neither the morphine nor dextromethorphan doses used in the combination experiments increased MSL when administered alone. Importantly, these dose combinations did not adversely alter the rate at which monkeys responded on the lever to terminate the nociceptive stimulus. Thus, this potentiating effect of dextromethorphan on low-dose morphine antinociception cannot be accounted for by the potential motor-impairing effects of these drugs. Furthermore, these results extend our previous findings that show that a noncompetitive antagonist (dizocilpine), a competitive antagonist (LY235959), and a glycine-site antagonist [(+)-HA-966] potentiate the effects of morphine in this procedure (Allen and Dykstra, 2001).

Potentiation of low-dose morphine antinociception has also been demonstrated in rat tail-flick assays (Manning et al., 1996) as well as in a rat model of peripheral neuropathy (Kauppila et al., 1998). Similarly, dextromethorphan appears to potentiate the analgesic effects of morphine in humans after oral and orthopedic surgery (Caruso, 2000). The combination of morphine with dextromethorphan appears to be an effective analgesic with a favorable side effect profile (Caruso, 2000) and may represent an improvement over the management of pain with morphine alone.

In the present study, the DOR agonist SNC80 produced small but statistically significant increases in MSL when administered alone. Previous research from this laboratory shows that BW373U86, the parent compound from which SNC80 was originally synthesized (Calderon et al., 1994), produced half-maximal increases in MSL in this procedure (Dykstra et al., 1993). However, doses of BW373U86 that increased MSL often produced tremors and/or convulsions. Also in contrast to the present findings, SNC80 has antinociceptive efficacy in a variety of procedures, including the rhesus monkey tail-withdrawal assay (Negus et al., 1998), the rat paw-pressure assay (Fraser et al., 2000), and the mouse warm water tail-flick and hot-plate assays (Bilsky et al., 1995). It is possible that doses sufficient to produce larger increases in MSL were not tested in this study. No dose of SNC80 tested suppressed response rate, and thus, it is possible that higher doses may further increase MSL. It was noted, however, that two of the four monkeys in this study exhibited proconvulsant behavior immediately after administration of 10 mg/kg SNC80, and for this reason higher doses were not tested. To our knowledge, this effect has not been quantified in squirrel monkeys by any investigator.

Interestingly, although both SNC80 and dextromethrophan failed to increase MSL greatly, dextromethorphan (10 mg/kg) increased MSL when administered in combination with SNC80. This increase in MSL occurred without a marked suppression of response rate, indicating that monkeys actively titrated the stimulus intensity at higher milliampere values. These behavioral data complement the findings from in vitro and electrophysiological investigations of the interactions between activity at DORs and NMDA receptors. Activation of the NMDA receptor with NMDA attenuates DPDPE inhibition of forskolin-stimulated cAMP production in neuroblastoma × glioma hybrid cells (NG108-15) in a manner that is blocked by the noncompetitive NMDA receptor antagonist, ketamine (Cai et al., 1997). DPDPE also has been shown to inhibit NMDA-evoked responses of nociceptive neurons in the medullary dorsal horn (Wang and Mokha, 1996) and NMDA receptor antagonists can reduce the response of spinothalamic tract neurons to the presentation of noxious thermal, mechanical, chemical, and electrical stimuli (Dougherty et al., 1992).

The only behavioral evidence for an interaction between DOR agonists and NMDA receptor antagonists on nociceptive responding prior to this investigation reveals an attenuation of the acute antinociceptive effects of DOR agonists by NMDA receptor antagonists (Bhargava and Zhao, 1996b; Suzuki et al., 2000). In both of these studies, DOR agonists were administered to mice i.c.v., and the effects of i.c.v. or systemically administered NMDA receptor antagonists on DOR agonist-induced antinociception was measured using hot-plate and tail-flick assays. It is possible that DOR agonists, when administered i.c.v., are less likely to interact with NMDA receptors in a manner that potentiates their antinociceptive effect. Indeed, DORs are abundant in the spinal cord (Honda and Arvidsson, 1995), and given that NMDA receptor antagonists alone can reduce the response of spinothalamic tract neurons to noxious stimuli (Dougherty et al., 1992), it is possible that the spinal cord is the site of action for this drug interaction.

Although not yet formally tested, the effect/side effect profiles of DOR agonists and NMDA receptor antagonists may compliment each other in some ways to produce an attractive analgesic preparation. First, NMDA receptor antagonists attenuate the development of tolerance to the antinociceptive effects of the DOR agonists DPDPE (Zhao and Bhargava, 1996) and deltorphin II (Bhargava and Zhao, 1996a) under some conditions. Second, DOR agonists that exhibit proconvulsant effects do so at doses that are equal to or higher than the doses required for antinociception (Comer et al., 1993; Dykstra et al., 1993). The ability to use low doses of DOR agonists as analgesics when combined with NMDA antagonists might then reduce or eliminate the potential for proconvulsant effects observed with DOR agonists. Finally, NMDA receptor antagonists may act as anticonvulsants on their own to prevent the emergence of convulsions that might otherwise occur with a DOR agonist when administered alone. The anticonvulsant effects of NMDA receptor antagonists generally (Koek and Colpaert, 1990), and dextromethorphan in particular (Leander et al., 1988), have been well established. Importantly, NMDA receptor antagonists have been shown to prevent convulsions elicited by classes of drugs other than NMDA, such as cocaine (Witkin et al., 1999). Although the highest dose of SNC80, when administered alone, appeared to produce proconvulsant effects in two of four monkeys in this study, no proconvulsant activity was observed in any monkey that received SNC80/dextromethorphan combinations even though the antinociceptive efficacy of these combinations was greater than that observed with SNC80 alone. Additional preclinical research is necessary to address these issues.