Abstract
The effects of SNC80 and other structurally related δ-opioid receptor agonists were assessed under conditions of chemically induced hypersensitivity to thermal stimuli in four rhesus monkeys. The shaved tail of each monkey was exposed to warm water (38, 42, 46, and 50°C), and the tail-withdrawal latency from each temperature was recorded. The effects of drugs on the temperature that produced a 10-s tail-withdrawal latency (the T10 value) were examined. Capsaicin (0.01–0.32 mg) injected into the tail of monkeys dose dependently decreased the T10, indicating that capsaicin increased sensitivity to thermal stimuli. A dose of 0.1 mg of capsaicin decreased theT10 from 48.0 to 42.1°C (a −5.9°C change) 15 min after injection. SNC80 (1.0–10.0 mg/kg s.c.) dose dependently blocked the capsaicin-induced decrease in theT10, and 10.0 mg/kg SNC80 fully blocked the effects of capsaicin. The δ-selective antagonist naltrindole (0.1–1.0 mg/kg) dose dependently antagonized the effects of SNC80, whereas a μ-selective dose of the opioid antagonist quadazocine (0.1 mg/kg) did not. Two other δ-selective agonists, SNC162 (1.0–10.0 mg/kg) and SNC243A (1.0–10.0 mg/kg), also dose dependently blocked capsaicin-induced thermal hypersensitivity. In contrast, neither SNC67 (10.0 mg/kg), which is the (−)-enantiomer of SNC80, nor the nonsteroidal anti-inflammatory drug (NSAID) ketorolac (1.0–10.0 mg/kg) modified the effects of capsaicin. SNC80 was also effective in reversing thermal hypersensitivity induced by prostaglandin E2 (0.0158 mg) and Freund's complete adjuvant (10% concentration). These findings suggest that δ-agonists have antinociceptive effects in primates under conditions of chemically induced thermal hypersensitivity and might be effective under a broader range of conditions than clinically available NSAIDs.
Opioids act at three main types of receptors, the μ-, κ-, and δ-opioid receptors (Kieffer, 1995). Although the analgesic activity of μ- and κ-opioid agonists is well established, the potential utility of δ-agonists as analgesics has been examined less extensively, in part because the only selective δ-agonists available for study until recently were peptidic compounds with poor bioavailability. However, both spinal and supraspinal administration of peptidic δ-agonists produce thermal antinociception in mice and rats (e.g., Heyman et al., 1987; Stewart and Hammond, 1993). Moreover, the simultaneous administration of δ-agonists into both spinal and supraspinal sites produces synergistic effects under some conditions (Hurley et al., 1999; Kovelowski et al., 1999). These findings parallel the synergistic antinociceptive effects of spinal and supraspinal administration of μ-agonists and suggest that δ-agonists may be reasonable candidates for further evaluation as potential analgesics.
(±)-BW373U86 was developed as the first selective and nonpeptidic δ-opioid agonist (Chang et al., 1993). As one consequence of its enhanced bioavailability relative to the peptidic agonists, the effects of (±)-BW373U86 could be readily examined in nonhuman primates as well as in rodents. However, (±)-BW373U86 was relatively ineffective in standard assays of antinociception in mice, squirrel monkeys, and rhesus monkeys (Dykstra et al., 1993; Negus et al., 1993b;Wild et al., 1993; see for review, Negus and Picker, 1996). The isomers of (±)-BW373U86 were subsequently resolved (Calderon et al., 1994,1997), and some derivatives of (+)-BW373U86 displayed improved pharmacologic profiles relative to the parent compound. SNC80, theO-methyl derivative of (+)-BW373U86, was reported to be more than 800-fold selective for δ- versus μ-receptors, whereas (±)-BW373U86 was only between 7- and 50-fold selective for δ-receptors (Calderon et al., 1994, 1997). In addition, SNC80 produced more robust antinociceptive effects than (±)-BW373U86 in both mice and rhesus monkeys (Bilsky et al., 1995; Negus et al., 1998). For example, in rhesus monkeys, SNC80 produced dose-dependent antinociception against low intensity thermal stimuli, whereas (±)-BW373U86 was ineffective (Negus et al., 1998). SNC80-induced antinociception was surmountably antagonized by the δ-selective antagonist naltrindole, indicating that these effects were δ-receptor mediated. In addition, the effects of SNC80 were also blocked by pretreatment with (±)-BW373U86, suggesting that SNC80 and (±)-BW373U86 acted at the same receptor type in monkeys, and that SNC80 had higher efficacy at these receptors. Taken together, these findings suggest that SNC80 may have greater potential than the parent compound as an analgesic agent.
The studies cited above assessed the effects of peptidic and nonpeptidic δ-agonists in assays that used noxious thermal or electrical stimuli. However, assays that model the clinical conditions of allodynia and hyperalgesia may provide a greater degree of sensitivity to the potential analgesic effects of drugs. Allodynia is defined as a pain-like response to a normally innocuous stimulus, and hyperalgesia is defined as an exaggerated response to a normally noxious stimulus (Willis, 1992). Allodynia and hyperalgesia associated with inflammation are components of many pain states encountered clinically, such as postoperative pain and arthritis, and models of allodynia and hyperalgesia may be useful for identifying potential therapeutic compounds (Negus et al., 1993b, 1995a). In some models of inflammatory allodynia and hyperalgesia, chemical agents are used to produce hypersensitivity to thermal or mechanical stimuli, and drug effects on chemically induced thermal or mechanical hypersensitivity can then be evaluated. Both peptidic and nonpeptidic δ-agonists are effective in at least some of these models (Stein et al., 1989; Stewart and Hammond, 1994; Butelman et al., 1995; Fraser et al., 2000). For example, peptidic δ-agonists blocked mechanical hypersensitivity produced by intraplantar administration of Freund's complete adjuvant (FCA) in mice (Stein et al., 1989). More recently, we compared the antinociceptive effects of (±)-BW373U86, morphine, and the nonsteroidal anti-inflammatory drug ketorolac in rhesus monkeys (Butelman et al., 1995). All three drugs blocked thermal hypersensitivity produced by the inflammatory mediator bradykinin. However, only morphine blocked thermal hypersensitivity by another inflammatory mediator, prostaglandin E2(PGE2). Thus, even in models of allodynia and hyperalgesia, (±)-BW373U86 may produce antinociceptive effects under only a limited range of conditions.
Because SNC80 has greater selectivity and may have greater efficacy than the parent compound (±)-BW373U86 at δ-opioid receptors, the purpose of the present study was to further evaluate the potential analgesic effects of SNC80 and related piperazinyl benzamides in a model of thermal allodynia and hyperalgesia in rhesus monkeys. Initial studies assessed the ability of these δ-agonists to block thermal hypersensitivity produced by capsaicin. Capsaicin binds to vanilloid receptors located on primary afferent nociceptors (Szallasi and Blumberg, 1999), and previous studies demonstrated that capsaicin-induced thermal hypersensitivity in rhesus monkeys was blocked by both μ- and κ-opioid agonists (Ko et al., 1998, 1999). The stereoselectivity of the antinociceptive effects of SNC80 was assessed using SNC67, the (−)-enantiomer of SNC80. In addition, the role of δ-opioid receptors in mediating the effects of SNC80 was examined in antagonism studies using the δ-selective antagonist naltrindole and the μ-selective antagonist quadazocine. Finally, the generality of the effects of SNC80 against other chemical irritants was examined using PGE2 and FCA.
Materials and Methods
Subjects.
Four male rhesus monkeys (Macaca mulatta) had free access to water and were maintained on a daily diet of fresh fruit and vegetables, multiple vitamins, and 10 to 15 Lab Diet Jumbo Monkey biscuits (PMI Feeds, Inc., St. Louis, MO). A 12-h light/12-h dark cycle was in effect (lights on from 7:00 AM to 7:00 PM).
Animal maintenance and research were conducted in accordance with the guidelines provided by the National Institutes of Health Committee on Laboratory Animal Resources. The laboratory facility was licensed by the United States Department of Agriculture, and research protocols were approved by the McLean Hospital Institutional Animal Care and Use Committee. A consulting veterinarian periodically monitored the health of the monkeys. Monkeys had visual, auditory, and olfactory contact with other monkeys throughout the study. Daily access to rubber toys and puzzle feeders provided additional environmental enrichment.
Procedure.
Monkeys were seated in primate chairs, and the lower 10 cm of the shaved tail of each monkey was exposed to warm water (38, 42, 46, and 50°C). The latency (seconds) for monkeys to remove their tails from warm water was used as a measure of nociception. If monkeys failed to remove their tails within 20 s, the experimenter removed the tail and assigned a latency of 20 s to that measurement. Each experimental session began by determining baseline tail-withdrawal latencies at each temperature. Temperature presentations were separated by approximately 1 min.
Chemically Induced Thermal Hypersensitivity.
Thermal hypersensitivity was produced by the subcutaneous administration of capsaicin (0.01–0.1 mg), PGE2 (0.0158 mg), or FCA (1–10% concentration) into the terminal 1 to 3 cm of the tail. All chemical irritants were injected in a volume of 0.1 ml. To determine the effects of capsaicin, tail-withdrawal latencies were assessed at 15, 30, 45, and 60 min after an injection of capsaicin (0.01–0.1 mg). We have previously reported that a dose of 0.0158 mg of PGE2 injected into the tail produces thermal hypersensitivity (Negus et al., 1995a). To determine the effects of PGE2 in the current experiment, a dose of 0.0158 mg was injected and tail-withdrawal latencies were determined 15, 30, 45, 60, 90, and 120 min later. To determine the effects of FCA (1–10% concentration), tail-withdrawal latencies were assessed 0.25, 0.5, 1, 2, 4, 24, and 48 h after an injection of FCA. Vehicle controls for each chemical irritant were also assessed. A minimum of 1 week separated tests with capsaicin and PGE2, and a minimum of 2 weeks separated tests with FCA.
Effects of δ-Opioid Agonists under Conditions of Thermal Hypersensitivity Produced by Capsaicin.
Initial studies examined the antinociceptive effects of δ-agonists and other drugs under conditions of capsaicin-induced thermal hypersensitivity. Based on observations of the potency and duration of capsaicin (seeResults), the antinociceptive effect of a single dose of drug was assessed 15 min after administration of 0.1 mg of capsaicin. Three series of experiments were conducted.
In experiment 1, the potency and time course of SNC80 were assessed. To examine the potency of SNC80, a single dose of SNC80 (1.0, 3.2, or 10.0 mg/kg) was administered 15 min before capsaicin. To examine the time course of SNC80, a single dose of 10.0 mg/kg SNC80 was administered 4, 15, or 60 min before capsaicin.
In experiment 2, the effects of other opioid and nonopioid drugs on capsaicin-induced thermal hypersensitivity were examined. The three classes of drugs studied were as follows: 1) piperazinyl benzamides structurally similar to SNC80 (SNC67, SNC86, SNC162, and SNC243A), 2) the μ-agonist morphine, and 3) the nonsteroidal anti-inflammatory drug (NSAID) ketorolac. As with SNC80, other piperazinyl benzamides were administered 15 min before capsaicin. In addition, a dose of 3.2 mg/kg SNC86 was administered 15 min before a dose of 10.0 mg/kg SNC80 to determine whether it would modify the effects of SNC80. Morphine was administered 30 min before capsaicin [the time of its peak effect (Negus et al., 1995a)], and ketorolac was administered 60 min before capsaicin [the time of its peak effect (Negus et al., 1995a)].
In experiment 3, the δ-opioid selectivity of the antinociceptive effects of SNC80 was evaluated by pretreating monkeys with the opioid antagonists naltrindole or quadazocine. During antagonism studies, a single dose of naltrindole (0.01, 0.1, or 1.0 mg/kg) or quadazocine (0.1 mg/kg) was administered 30 min before the SNC80. SNC80 was administered 15 min before capsaicin. For comparison, a dose of 1.0 mg/kg naltrindole or 0.1 mg/kg quadazocine was administered 30 min before a dose of 0.32 mg/kg morphine. Morphine was administered 30 min before capsaicin. We have reported previously that naltrindole at doses of up to 1.0 mg/kg acts as a selective δ-antagonist in monkeys, whereas a dose of 0.1 mg/kg quadazocine selectively antagonizes the behavioral effects of μ-agonists and not δ-agonists in monkeys (Negus et al., 1993a, 1998; Brandt et al., 1999).
Effects of SNC80 under Conditions of Thermal Hypersensitivity Produced by PGE2 or FCA.
Capsaicin has a short duration of action that necessitated the administration of drugs before the induction of thermal hypersensitivity. As a result, these experiments evaluated the ability of SNC80 to prevent the effects of capsaicin. However, analgesic drugs used clinically are often administered after an allodynic state has been established. Accordingly, two additional series of experiments were conducted to examine the ability of SNC80 to reverse thermal hypersensitivity when SCN80 was administered after treatment with longer-acting chemical irritants. The first study evaluated the ability of SNC80 to reverse PGE2-induced thermal hypersensitivity. Tail-withdrawal latencies were assessed 15, 30, 45, 60, 90, and 120 min after an injection of 0.0158 mg of PGE2 in the tail. Based on results from a previous study (Negus et al., 1995a) and observations of the duration of PGE2-induced thermal hypersensitivity in the current study (see Results), the ability of SNC80 to reverse the effects of PGE2 was assessed using a cumulative dosing procedure. Three sequential doses of SNC80 were administered in a single session, and each dose increased the total cumulative dose by 0.5 log units. Two overlapping SNC80 dose-effect curves (0.1–1.0 and 0.32–3.2 mg/kg) were determined. Cumulative doses of SNC80 were administered at 15, 30, and 45 min after PGE2, and tail-withdrawal latencies were determined 10 to 15 min after each injection.
The second study evaluated the ability of SNC80 to reverse FCA-induced thermal hypersensitivity. Tail-withdrawal latencies were assessed 0.25, 0.5, 1, 2, 4, 24, and 48 h after the injection of a concentration of 1 and 10% FCA into the tail. Higher concentrations of FCA were not administered to preclude potential tissue damage. Based on results for the duration of FCA-induced hypersensitivity in the current study (seeResults), the ability of SNC80 to reverse the effects of FCA was assessed using a cumulative dosing procedure. For this study, a single SNC80 dose-effect curve was determined 24 h after the administration of 10% FCA. This time was chosen to examine the antinociceptive effects of SNC80 after a prolonged period of thermal hypersensitivity. Cumulative doses of SNC80 (0.32–3.2 mg/kg) were administered every 15 min, and tail-withdrawal latencies were determined 10 to 15 min after each injection.
Data Analyses.
Temperature-effect curves were generated for each experimental condition for individual monkeys. AT10 value was determined from each temperature-effect curve, and the T10value was defined as the temperature that produced a tail-withdrawal latency of 10 s, which is one-half the maximal tail-withdrawal latency of 20 s (see Negus et al., 1993b). TheT10 was determined by interpolation from a line drawn between the point above and the point below 10 s on the temperature-effect curve. IndividualT10 values were averaged to provide a mean (±1 S.E.M.). For the purposes of the present study, thermal hypersensitivity was operationally defined as a leftward shift in the temperature-effect curve and a decrease in theT10 value. Antinociception was defined as a blockade or reversal of chemically induced thermal hypersensitivity. Antinociception was quantified as the percentage maximum possible effect (% MPE) according to the following equation:
Statistical analysis of pretreatment tests was done using one-way ANOVA for repeated measures. Significant main effects were analyzed further by subsequent paired comparisons using the Student-Newman-Keuls method. The criterion for significance was p < 0.05.
Drugs.
Naltrindole HCl, SNC67, SNC80, SNC86, SNC162, and SNC243A were synthesized by K. C. Rice and colleagues (National Institutes of Health, Bethesda, MD). Quadazocine methanesulfonate was generously supplied by Sanofi Pharmaceuticals (Malvern, PA). Morphine sulfate was supplied by the National Institute on Drug Abuse (NIDA, Bethesda, MD). Prostaglandin E2 and Freund's complete adjuvant were purchased from Sigma Chemical Co. (St. Louis, MO). Capsaicin was purchased from Research Biochemicals International (Natick, MA), and ketorolac tromethamine was purchased as an injectable solution from Abbott Laboratories (North Chicago, IL). The free-base forms of SNC67, SNC80, SNC162, and SNC243A were dissolved in 3% lactic acid and sterile water to a final concentration of 50 mg/ml, and dilutions were made with sterile water. The HCl salt form of SNC86 was dissolved in sterile water. Capsaicin was dissolved in 10% EtOH, 10% Emulphor, and 80% sterile water. FCA was diluted in sesame oil (Sigma Chemical Co.). All other compounds were dissolved or diluted in sterile water. All drugs were administered subcutaneously. Signs of tissue damage were not observed after injections with any of the test compounds. Doses were based on free-base or salt forms described above.
Results
Baseline Thermal Nociception and Capsaicin-Induced Thermal Hypersensitivity.
Under baseline conditions, maximal tail-withdrawal latencies (i.e., 20 s) were typically obtained with temperatures of 38, 42, and 46°C. When the water temperature was increased to 50°C, tail-withdrawal latencies for individual monkeys were between 0.75 and 3 s. The baselineT10 value was 48.2 ± 0.04°C.
Capsaicin produced a dose- and time-dependent thermal hypersensitivity manifested as a leftward shift in the temperature-effect curve and a decrease in the T10 value. Maximal decreases in tail-withdrawal latencies for all doses of capsaicin occurred 15 min after administration, and latencies returned to baseline by 60 min after injection (data not shown). Figure1 shows theT10 value 15 min after administration of vehicle or doses of capsaicin. TheT10 (±1 S.E.M.) under capsaicin vehicle conditions was 48.0 ± 0.3°C (point above “V”). Doses of 0.01 and 0.032 mg of capsaicin only slightly decreased theT10. A larger dose of 0.1 mg of capsaicin decreased the T10 to 42.9 ± 1.0°C (a −5.1°C change from vehicle control). A redetermination of the thermal hypersensitivity produced by 0.1 mg of capsaicin midway through these studies in each monkey was similar to the first determination (T10 = 41.3 ± 0.8°C). Therefore, the first and second determination were combined (T10 = 42.1 ± 0.7°C) in Fig. 1 and used for analysis of % MPE in other figures. A larger dose of 0.32 mg of capsaicin did not further decrease theT10. Based on these results, the antinociceptive effects of drugs were studied 15 min after a dose of 0.1 mg of capsaicin.
Effects of Piperazinyl Benzamide δ-Agonists under Conditions of Capsaicin-Induced Thermal Hypersensitivity.
Figure2 shows that SNC80 produced a dose-dependent (left panel) and time-dependent (right panel) blockade of capsaicin-induced thermal hypersensitivity. Doses of 1.0 and 3.2 mg/kg SNC80 partially blocked the effects of capsaicin. A higher dose of 10.0 mg/kg SNC80 fully blocked the effects of capsaicin, and the ED50 value for SNC80 is shown in Table1. Importantly, although SNC80 increased tail-withdrawal latencies at the intermediate temperature of 46°C, higher temperatures of water (e.g., 50°C) elicited tail-withdrawal latencies near control values demonstrating that monkeys could make the tail-withdrawal response. The antinociceptive effects of SNC80 had a rapid onset and a short duration of action. The effects of SNC80 were near maximal after 4 min, peaked at 15 min, and had nearly dissipated after 60 min.
Figure 3 (left panel) shows the effects of other piperazinyl benzamides on capsaicin-induced thermal hypersensitivity. SNC243A and SNC162 had potencies similar to SNC80 for blocking the effects of capsaicin (Table 1). A dose of 10.0 mg/kg of either SNC243A or SNC162 completely blocked the effects of capsaicin (Fig. 3, left panel). SNC86, the (+)-enantiomer of (±)-BW373U86, only slightly increased the % MPE at the highest dose of 10.0 mg/kg. When administered 15 min before SNC80, a dose of 3.2 mg/kg SNC86 decreased the antinociceptive effects of 10.0 mg/kg SNC80 to 53.6 ± 29.1 (data not shown). SNC67, the (−)-enantiomer of SNC80, did not produce antinociception at a dose of 10.0 mg/kg. Larger doses of SNC67 have been reported to produce convulsions in rhesus monkeys (Brandt et al., 1999) and were not tested.
For comparison with the effects of the δ-agonists, the effects of the μ-agonist morphine and the NSAID ketorolac were also examined (Fig.3, right panel). Morphine dose dependently and completely blocked thermal hypersensitivity produced by capsaicin. A dose of 0.32 mg/kg morphine fully blocked the effects of capsaicin. Morphine was 32-fold more potent than SNC80 (Table 1). In contrast, ketorolac at doses up to 10.0 mg/kg did not block thermal hypersensitivity produced by capsaicin.
Figure 4 shows the effects of 10 mg/kg SNC80 (left panel) or 0.32 mg/kg morphine (right panel) alone and in combination with the δ-selective antagonist naltrindole or the μ-selective opioid antagonist quadazocine. Naltrindole (0.01–1.0 mg/kg) dose dependently antagonized the antinociceptive effects of SNC80 (left panel), and a dose of 1.0 mg/kg naltrindole significantly antagonized the effects of 10.0 mg/kg SNC80. In contrast, 1.0 mg/kg naltrindole did not antagonize the antinociceptive effects of morphine. A μ-selective dose of quadazocine (0.1 mg/kg) antagonized the antinociceptive effects of morphine but not of SNC80.
Effects of SNC80 under Conditions of PGE2- and FCA-Induced Thermal Hypersensitivity.
Figure5 compares the effects of PGE2 and FCA. PGE2 produced a time-dependent thermal hypersensitivity with a longer duration of action than capsaicin. Under baseline conditions, theT10 was 48.2 ± 0.02°C. Maximal thermal hypersensitivity of 0.0158 mg of PGE2occurred 0.5, 0.75, and 1 h after injection, andT10 values (±S.E.M.) at these times were 44.3 (±0.06), 44.3 (±0.02), and 44.2 (±0.02)°C, respectively (approximately a −4°C change from baseline; Fig. 5, left panel). The effects of PGE2 began to dissipate at 1.5 h and were similar to control values at 2 h. FCA also produced a time-dependent thermal hypersensitivity with a long duration of action. However, unlike capsaicin and PGE2, which produced thermal hypersensitivity in all monkeys, concentrations of FCA up to 10% produced thermal hypersensitivity in only two of the three monkeys. Therefore, the third monkey unresponsive to FCA was excluded from the final study. Figure 5 (right panel) shows the magnitude and duration of the effects of FCA. A concentration of 1% FCA had peak effects between 0.5 and 2 h. T10values at these times where between 44.2 and 44.6°C (approximately a −4°C change from baseline). A higher concentration of 10% FCA decreased the T10 to values similar to the lower FCA concentration; however, the duration of effect was substantially longer with 10% FCA and lasted for more than 24 h. Subsequent redetermination of tail-withdrawal latencies (T10 = 48.1 ± 0.03°C) were similar to original determinations.
Figure 6 compares the effects of SNC80 on thermal hypersensitivity produced by PGE2 and FCA. Cumulative doses of SNC80 administered during the times of peak effect after PGE2 (i.e., 0.5–1 h) dose dependently reversed the effects of PGE2, and 3.2 mg/kg SNC80 fully reversed PGE2-induced thermal hypersensitivity (Fig. 6). SNC80 was 5-fold more potent in reversing the effects of PGE2 than in blocking the effects of capsaicin (Table 1). Cumulative doses of SNC80 were also administered 24 h after 10% FCA. SNC80 dose dependently reversed the effects of FCA, and a dose of 3.2 mg/kg SNC80 fully reversed FCA-induced thermal hypersensitivity in both monkeys. The ED50 for SNC80 to reverse the effects of FCA was similar to the ED50 of SNC80 to reverse the effects of PGE2 (Table 1).
Discussion
The present series of studies was conducted to assess the effects of SNC80 and other related δ-agonists under conditions of thermal hypersensitivity in rhesus monkeys. δ-Agonists produced dose-dependent, stereoselective, and naltrindole-reversible antinociception against three different chemical irritants. Moreover, SNC80 was able both to prevent thermal hypersensitivity when it was administered before the chemical irritant (capsaicin) and to reverse thermal hypersensitivity when it was administered after the chemical irritant (PGE2 and FCA). Finally, δ-agonists produced antinociception as effectively as morphine and more effectively than the clinically available NSAID ketorolac. These findings suggest that δ-agonists may be useful clinically for the management of some types of inflammatory pain, such as postoperative pain or arthritis.
Thermal Hypersensitivity Produced by Chemical Irritants.
The thermal hypersensitivity produced by capsaicin and PGE2 observed in the present study replicates and confirms earlier findings in rhesus monkeys (Negus et al., 1993b,1995a; Ko et al., 1998, 1999). Capsaicin is a plant-derived compound that acts as an agonist at vanilloid receptors located on primary afferent nociceptors (Szallasi and Blumberg, 1999). In contrast, PGE2 is a metabolite of the arachidonic acid cascade that is produced endogenously under conditions of inflammation or tissue damage, and it is thought to produce thermal hypersensitivity by binding to endoperoxide receptors that are also located on primary afferent nociceptors (Halushka et al., 1989; Taiwo et al., 1989). Like capsaicin and PGE2, FCA also produced thermal hypersensitivity in monkeys. FCA is a heat-killed macrobacteria that produces thermal hypersensitivity by provoking an inflammatory response at the site of injection. Taken together, these findings indicate that thermal hypersensitivity can be produced in monkeys by chemical irritants with differing mechanisms of action. Moreover, thermal hypersensitivity induced by these irritants provides a model for the assessment of drugs that may be useful for the treatment of inflammatory pain.
Effects of δ-Agonists on Capsaicin-Induced Thermal Hypersensitivity.
SNC80, SNC162, and SNC243A dose dependently blocked the effects of capsaicin. The antinociceptive effects of SNC80 were stereoselective, because SNC67, the (−)-enantiomer of SNC80, did not block thermal hypersensitivity produced by capsaicin. In addition, the antinociceptive effects of SNC80 were dose dependently antagonized by δ-selective doses of naltrindole and not by a μ-selective dose of quadazocine. These findings suggest that the antinociceptive effects of SNC80 were mediated by δ-opioid receptors and extend our previous studies on the δ-mediated behavioral effects of SNC80 in rhesus monkeys (Negus et al., 1998; Brandt et al., 1999). These results also agree with the finding that peptidic δ-agonists attenuate chemically induced thermal hypersensitivity in rodents (e.g., Stein et al., 1989;Stewart and Hammond, 1994; Zhou et al., 1998; Fraser et al., 2000).
In time course studies, 10.0 mg/kg SNC80 produced antinociception with a rapid onset of action, but these effects lasted for less than 1 h. Previous studies have examined the time course of other behavioral effects produced by SNC80 in rhesus monkeys (Negus et al., 1998; Brandt et al., 1999). In agreement with the present findings, SNC80 also produced discriminative stimulus effects and decreases in rates of schedule-controlled behavior with a rapid onset of action. However, these behavioral effects of SNC80 were still present after 1 h and took several hours to dissipate completely. Moreover, these other behavioral effects were produced by lower doses of SNC80. The reason for this difference in the potency and time course of SNC80 in producing different behavioral effects is not known. One possibility is that blockade of capsaicin-induced thermal hypersensitivity requires higher levels of δ-receptor stimulation than the other behavioral measures, and as a result, antinociception can be achieved only with high doses of high efficacy δ-agonists.
The findings with SNC86 in the present study are consistent with this hypothesis. SNC86 is the active (+)-enantiomer of (±)-BW373U86, and like SNC80, SNC86 and (±)-BW373U86 decrease rates of schedule-controlled behavior and substitute for cocaine in monkeys trained to discriminate cocaine from saline (Negus et al., 1993a, 1998;Brandt et al., 1999). However, SNC86 and (±)-BW373U86 were less effective than SNC80 in producing several other behavioral effects in rhesus monkeys. For example, SNC86 and (±)-BW373U86 were less effective than SNC80 in producing thermal antinociception (Negus et al., 1998), antinociception against PGE2 or capsaicin [cf. Butelman et al. (1995) and present study], and cocaine-like discriminative stimulus effects in monkeys trained to discriminate cocaine from saline (Negus et al., 1995b, 1998). Similarly, (±)-BW373U86 was less effective than SNC80 in producing antinociception in mice (cf. Wild et al., 1993; Bilsky et al., 1995) and in enhancing the discriminative stimulus effects of cocaine in squirrel monkeys (cf. Spealman and Bergman, 1994; Rowlett and Spealman, 1998). Finally, pretreatment with (±)-BW373U86 attenuated the antinociceptive effects of SNC80 (Negus et al., 1998), and in the present study, pretreatment with SNC86 attenuated the antinociceptive effects of SNC80 against capsaicin-induced thermal hypersensitivity. Taken together, these results are consistent with the view that SNC80 may have higher efficacy than SNC86 or (±)-BW373U86 at δ-opioid receptors. These findings also suggest that SNC86 has sufficient efficacy to produce some SNC80-like effects, such as suppression of operant response rates and SNC80-like discriminative stimulus effects (Brandt et al., 1999), but that it does not have sufficient efficacy to block capsaicin-induced thermal hypersensitivity.
Previous studies found that μ-, κ-, and δ-agonists can reverse chemically induced hypersensitivity in rats (Levine and Taiwo, 1989;Stein et al., 1989; Zhou et al., 1998; Fraser et al., 2000) and μ- and κ-agonists can reverse chemically induced thermal hypersensitivity in monkeys (Ko et al., 1998, 1999) by acting at peripheral opioid receptors. The present study did not determine whether SNC80 and related δ-agonists produced antinociception by acting at central or peripheral δ-receptors. However, as noted above, the potency of SNC80 for producing antinociception in the present study was similar to or lower than its potency for producing other behavioral effects that are presumably mediated by central δ-opioid receptors (e.g., rate suppression, discriminative stimulus effects, and antinociceptive effects against noxious thermal stimuli). Although these findings do not exclude a role for peripheral receptors in mediating δ-agonist-induced antinociception, they do suggest that central receptors are activated by antinociceptive doses of SNC80, and these central receptors may contribute to the antinociceptive effects of δ-agonists. In support of this conclusion, intracerebroventricular administration of SNC80 reverses thermal hypersensitivity associated with the administration of FCA into the plantar surface of the hindpaw in rats (Fraser et al., 2000).
Effects of SNC80 on PGE2- and FCA-Induced Thermal Hypersensitivity.
Capsaicin had a short duration of action that necessitated the administration of drugs before the induction of thermal hypersensitivity. However, analgesic agents used clinically are typically administered after a state of inflammatory pain has been established. Accordingly, we also conducted studies with longer-acting chemical irritants to determine the degree to which SNC80 could reverse chemically induced thermal hypersensitivity. SNC80 produced a dose-dependent and complete reversal of the effects of both PGE2 and FCA. Moreover, SNC80 was more potent in reversing the effects of PGE2 and FCA than it was in blocking the effects of capsaicin. Taken together, these findings indicate that δ-agonists produce antinociceptive effects under a wide range of conditions in rhesus monkeys.
There are several possible explanations for the difference in the potency of SNC80 across chemical irritants. First, PGE2 and FCA produced smaller decreases in theT10 value than capsaicin, and these weaker effects may have been more sensitive to the antinociceptive effects of SNC80. Second, PGE2 and FCA produce thermal hypersensitivity by different mechanisms of action than capsaicin (see above), and these different mechanisms of action may be more sensitive to modulation by δ-receptor activation. Finally, recent anatomical evidence suggests that δ-receptors are predominately localized intracellularly on the membranes of large dense-core vesicles in the terminals of small dorsal root ganglia neurons. These receptors may be externalized onto the plasmalemma under conditions of chemical stimulation or inflammatory pain that would be expected to stimulate these neurons (Zhang et al., 1998). These findings raise the possibility that prolonged thermal hypersensitivity, in contrast to the short thermal hypersensitivity induced by capsaicin, may increase the density of active δ-opioid receptors and the potency of δ-opioid agonists. Consistent with this last view, we and others have demonstrated that δ-agonists produce antinociceptive effects under conditions of chemically induced thermal hypersensitivity (Butelman et al., 1995; current study) but only weak antinociceptive effects in the absence of inflammation or chemical stimulation (Dykstra et al., 1993; Negus et al., 1998; M. R. Brandt, unpublished observation). Moreover, a recent study has demonstrated that the potency of the δ-agonist [d-Ala2,Glu4]deltorphin is progressively enhanced 4 h, 4 days, and 2 weeks after the intraplantar administration of FCA into the hindpaw of rats (Hurley and Hammond, 2000). Together, these findings suggest that the therapeutic potential of δ-agonists may be most evident under conditions of allodynia and hyperalgesia.
Antinociceptive Effects of Morphine and Ketorolac under Conditions of Capsaicin-Induced Thermal Hypersensitivity.
Results from the current study confirm and extend previous studies indicating that morphine potently prevents thermal hypersensitivity produced by capsaicin and PGE2 in monkeys (Negus et al., 1993b, 1995a). In contrast, the NSAID ketorolac did not prevent thermal hypersensitivity produced by capsaicin, and previous studies found that ketorolac did not reverse thermal hypersensitivity produced by PGE2 (Negus et al., 1995a). The failure of ketorolac to produce antinociceptive effects under these conditions of chemically induced thermal hypersensitivity was probably not a result of inadequate dosing. In the present study, ketorolac was tested up to a dose of 10.0 mg/kg, which is approximately 10 times the recommended analgesic dose for humans (i.e., 60 mg/70 kg). In addition, a dose of 1.0 mg/kg ketorolac effectively blocked thermal hypersensitivity induced by bradykinin in rhesus monkeys (Negus et al., 1995a). Thus, in contrast to δ-agonists, NSAIDS are relatively ineffective in attenuating thermal hypersensitivity induced by agents that act directly on primary nociceptors.
Potential Adverse Effects.
Numerous adverse effects are associated with nonopioid and opioid analgesics. For example, NSAIDs may produce gastric irritation, μ-opioid agonists produce respiratory depression and have abuse potential in humans, and κ-opioid agonists have been shown to produce subjective effects in humans that have been described as “dysphoric” or “psychotomimetic”. In the present study, doses of δ-agonists that produced antinociception under conditions of chemically induced thermal hypersensitivity did not produce signs of overt behavioral toxicity. In particular, although SNC80 may produce convulsions in some strains of mice (e.g., Bilsky et al., 1995), antinociceptive doses of SNC80 did not produce convulsions in the present study, and in previous studies, doses of SNC80 up to 56 mg/kg did not produce convulsions or other signs of severe toxicity in rhesus monkeys (Negus et al., 1998; Negus, 1999). However, in agreement with previous findings, antinociceptive doses of δ-agonists did produce mild sedation, and similar doses of SNC80 and other δ-agonists have been shown to decrease rates of schedule-controlled responding and to produce discriminative stimulus effects (Negus et al., 1998; Brandt et al., 1999). The potential impact of these effects on the clinical utility of SNC80 and related δ-agonists remains to be determined.
Acknowledgment
We thank Beth Moseley, D.V.M. for veterinary assistance.
Footnotes
-
Send reprint requests to: S. Stevens Negus, Ph.D., Harvard Medical School-McLean Hospital, Alcohol and Drug Abuse Research Center, 115 Mill St., Belmont, MA 02178-9106. E-mail:negus{at}mclean.org
-
↵1 Current address: Wyeth-Ayerst Research, Wyeth Neuroscience, CN-8000, Princeton, NJ 08543-8000. E-mail:brandtm{at}war.wyeth.com
-
These studies were supported in part by Grants RO1-DA11460, P50-DA04059, T32-DA0752, and KO5-DA00101 from the National Institute on Drug Abuse (NIDA), National Institutes of Health. We also thank the NIDA for partial support for the Laboratory of Medicinal Chemistry, National Institute of Diabetics, Digestive and Kidney Diseases and National Institute of Health, Bethesda, MD.
- Abbreviations:
- (±)-BW373U86
- (±)-4-[(αR*)-α-[2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide
- SNC80
- (+)-4-[(αR)-α-[2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-methoxybenzyl]-N,N-diethylbenzamide
- SNC67
- (−)-4-[(αS)-α-[2R,5S)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-methoxybenzyl]-N,N-diethylbenzamide
- SNC86
- (+)-4-[(αS)-α-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide
- SNC162
- (+)-4-[(αR)-α-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-benzyl]-N,N-diethylbenzamide
- SNC243A
- (+)-4-[(αR)-α-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-fluorobenzyl]-N,N-diethylbenzamide
- FCA
- Freund's complete adjuvant
- % MPE
- percentage maximum possible effect
- NSAID
- nonsteroidal anti-inflammatory drug
- PGE2
- prostaglandin E2
- T10
- temperature producing a 10-s tail-withdrawal latency
- Received September 5, 2000.
- Accepted November 20, 2000.
- The American Society for Pharmacology and Experimental Therapeutics