Introduction

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States of nociception, such as allodynia and hyperalgesia, to thermal and mechanical stimuli often occur after tissue injury, inflammation and nerve lesions (e.g., Levine and Taiwo, 1994). Allodynia is a condition in which pain is produced by a stimulus that is normally innocuous, whereas hyperalgesia is increased pain reaction induced by a stimulus that is normally noxious (Willis, 1992). In clinics, many nociceptive conditions such as postoperative pain, cancer and arthritis are associated with inflammation. The development of experimental inflammatory pain models is valuable for investigating the mechanisms of allodynia and hyperalgesia and for evaluating potential antinociceptive agents. Traditionally, most clinically used opioids are predominantly mu opioid agonists (Twycross, 1994). In addition to relieving pain, mu agonists induce a wide variety of other biological effects, and some centrally mediated side effects can be clinically undesirable, such as sedation and respiratory depression. Thus, the existence of peripheral opioid receptors becomes more important for developing local analgesics or peripherally selective opioid agonists. To date, several rodent and clinical studies demonstrate that locally administered opioid agonists can produce pronounced antinociceptive effects by interacting with peripheral opioid receptors in inflamed tissue (Stein et al., 1989, 1991; Barber and Gottschlich, 1992; Kinnman et al., 1997).

Endogenous and exogenous compounds, e.g., formalin, bradykinin, prostaglandin E₂ and Freund's adjuvant, have been used to induce nociception after local application (Stein et al., 1989; Dray and Dickenson, 1991; Negus et al., 1993b). Capsaicin, the pungent ingredient in hot chili peppers, is another compound that has been investigated in both human and rodent behavioral and neurophysiological studies. Topical or intradermal administration of capsaicin to human skin produces burning pain, a flare and a hyperalgesia that is characterized by a lowered pain threshold to heat and a tenderness to innocuous mechanical stimulation (Simone et al., 1987, 1989). These temporary sensory symptoms of the capsaicin model in humans resemble those of patients with neuropathic pain. Several studies have demonstrated that nociception induced by capsaicin is mediated in part by activating a subset of sensory neurons including polymodal nociceptors and thermoceptors (Dray and Dickenson, 1991; LaMotte et al., 1992; Kim et al., 1995; Kinnman and Levine, 1995). In addition, capsaicin stimulates the release of glutamate and neuropeptides such as calcitonin gene-related peptide and substance P from the peripheral and central terminals of sensory neurons (Maggi, 1993; Winter et al., 1995). On the other hand, rodent studies have reported that morphine inhibited microvasodilation in capsaicin-induced peripheral nerve trunk inflammation, presumably through an action on local opioid receptors (Zochodne and Ho, 1993; Schaafsma et al., 1997). Clinical studies also have provided evidence that local or systemic administration of mu opioid agonists attenuate capsaicin-induced allodynia and hyperalgesia in humans (Park et al., 1995; Eisenach et al., 1997; Kinnman et al., 1997). Taken together, these findings support the feasibility of pharmacological studies with an intradermal capsaicin model.

This study was initiated to develop a procedure for thermal allodynia in rhesus monkeys by administering capsaicin to induce local transient nociception. Then, the antinociceptive effect of both fentanyl, a synthetic lipophilic mu agonist, and DAMGO, a selective mu peptide, were compared after local and systemic administration. In addition, antagonist studies were performed to further investigate the role of peripheral mu opioid receptors in this procedure. The aim of this study was to provide a useful experimental pain model and to evaluate the hypothesis that local administration of mu opioid agonists can diminish capsaicin-induced nociception in primates.

	Methods

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Subjects

Six adult male and female rhesus monkeys (Macaca mulatta) with body weights ranging between 7.7 and 12.1 kg (mean weight, 9.6 kg) were used. They were housed individually with free access to water and were fed approximately 25 to 30 biscuits (Purina Monkey Chow) and fresh fruit daily. All monkeys had previous experience in the tail-withdrawal procedure and did not have exposure to opioids for 2 months before the present study.

Warm Water Tail-Withdrawal Assay

Apparatus and procedure. Antinociception was measured with a procedure that was described previously (Dykstra and Woods, 1986). The subjects were seated in restraint chairs and the lower part of the shaved tail (approximately 15 cm) was immersed in warm water maintained at temperatures of 42, 46 and 50°C. Tail-withdrawal latencies were recorded manually by a computerized timer. A maximum cutoff latency (20 sec) was recorded if the subjects failed to remove their tails by this time. A single dosing procedure was used in all test sessions. Each experimental session began with control determinations at each temperature. Subsequent tail-withdrawal latencies were determined at 5, 15, 30, 45 and 60 min after injection. The subjects were tested one to two times at three temperatures in a varying order, with approximately 1- to 2-min intervals between tests. Experimental sessions were conducted one to two times a week, with at least 3 days between sessions.

Experimental design. Nociceptive effects of capsaicin. Time course of the nociceptive effects of capsaicin (0.01-0.32 mg/tail) was determined twice in each subject. Capsaicin was injected subcutaneously (s.c.) into the terminal 1 to 4 cm of the tail, in a constant 0.1 ml volume. In the time-course study only, monkeys were exposed to four temperatures, 38, 42, 46 and 50°C, during the test session.

Data Analysis

Except for the time-course study, the 15-min time point was used for analysis because this was the time of peak effects of both capsaicin and mu agonists. Individual tail-withdrawal latencies were converted to %MPE by the following formula: %MPE = [(test latency  control latency)/(cutoff latency  control latency)] × 100. Individual control latencies were averaged from two determinations after application of 0.1 mg of capsaicin in the tail in 46°C water. Mean ED₅₀ values were obtained after log transformation of individual ED₅₀ values, which were calculated by least-squares regression with the portion of the dose-effect curves spanning the 50% MPE; 95% confidence limits (95% C.L.) also were determined. Mean ID₅₀ values of antagonists were determined in the same manner by defining the dose that inhibited the 50% MPE of local fentanyl (0.1 mg). Comparison of relative potencies of each compound administered locally or systemically was performed by converting the mg/kg units to total mg units based on individual monkey's body weight (i.e., 0.1 mg/kg corresponds to 1 mg, assuming an approximate monkey weight of 10 kg). In addition, dose ratios (D.R.) were calculated by dividing mean ED₅₀ values in the presence of the antagonist by the base-line ED₅₀ values. A significant difference between dose-effect curves was defined as a lack of overlap in the 95% C.L. of the ED₅₀ values. In the time-course study, a significant reduction in tail-withdrawal latency was determined by use of the Newman-Keuls test (P < .01).

Apparent pK_B values were determined for individual antagonist doses by use of a modified equation (Negus et al., 1993a): pK_B = log [B/(D.R.  1)], where B equals the antagonist dose in moles per kilogram. Mean pK_B values ± 95% C.L. also were calculated from individual pK_B values for quadazocine. Apparent pK_B values were considered to be significantly different when their 95% C.L. values did not overlap.

Drugs

Fentanyl hydrochloride (National Institute on Drug Abuse, Bethesda, MD), DAMGO acetate salt (Sigma, St. Louis, MO), quadazocine methanesulfonate (Sanofi, Malvern, PA), QNTX (MRZ-2663-BR; Dr. H. Merz, Boehringer Ingelheim KG, Germany), nor-binaltorphimine (Dr. H.I. Mosberg, Dept. of Medicinal Chemistry, University of Michigan) and naltrindole hydrochloride (Dr. K.C. Rice, NIH-NIDDK, Bethesda, MD) were dissolved in sterile water. For systemic administration, all compounds were administered s.c. in the back at a volume of 0.1 ml/kg. Capsaicin (Sigma, St. Louis, MO) was dissolved in a solution of Tween 80/ethanol/saline in a ratio of 1:1:8. For local coadministration, all compounds were mixed in capsaicin solution and injected in 0.1 ml volume in the tail.

	Results

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Control tail-withdrawal latencies. The monkeys used in this study showed a consistent profile in tail-withdrawal responses. They kept their tails in 38, 42 and 46°C water for 20 sec (cutoff latency) and removed their tails from 50°C water rapidly (typically 1-3 sec). The thermal pain thresholds in monkeys used in this study were similar to other studies in primates. For example, it has been reported that monkeys frequently escaped the 51°C stimuli, but almost never the 43 and 47°C temperatures; human subjects have described 43°C as slightly warm, 47°C as distinctly warm but not painful, and 51°C as a clearly painful stimulus (Kupers et al., 1997).

Nociceptive effects of capsaicin. When capsaicin (0.01-0.32 mg) was injected into the tail, it produced a transient nociception (thermal allodynia), which was indicated as reduced tail-withdrawal latencies, both in a dose- and temperature-dependent manner (fig. 1; upper panel). In particular, both 0.1 and 0.32 mg of capsaicin caused rapid tail-withdrawal latencies of approximately 2 sec in 46°C water, 15 min after injection (fig. 1; lower panel). However, all doses of capsaicin caused a general nociception, which was not temperature-dependent, within the first 5 min after administration (data only shown in 46°C water). For our purposes, a standard dose of 0.1 mg capsaicin was chosen to induce allodynia in 46°C water for further studies.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Thermal allodynia caused by capsaicin locally applied in the tail. (Upper panel) temperature-effect curves measured at 15 min after injection. (Lower panel) time course of capsaicin-induced thermal allodynia in 46°C water. All points represent the mean ± S.E.M. (n = 6). Asterisks represent significant difference (P < .01) from vehicle.

Antinociceptive effects of mu agonists. Figure 2 compares the antinociceptive effects of both fentanyl and DAMGO after local and systemic administration. Local administration of fentanyl (0.0032-0.1 mg) dose-dependently inhibited capsaicin (0.1 mg)-induced thermal allodynia in 46°C water (fig. 2, upper left panel). However, the high dose of fentanyl (0.1 mg), when applied in the back, was not effective against capsaicin, and it was not locally effective against a noxious stimulus, 50°C water, in the absence of capsaicin. After systemic administration, fentanyl (0.01-0.056 mg/kg) also dose-dependently inhibited capsaicin-induced allodynia (fig. 2, lower left panel). Comparing the potency of both administration routes, local administration of fentanyl was approximately 15-fold more potent than systemic injection (table 1).

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Antinociceptive effects of fentanyl (left panels) and DAMGO (right panels) administered locally (hashed bars, in the tail) or systemically (filled bars, in the back) against 46°C water in the presence of 0.1 mg capsaicin or against 50°C water in the absence of capsaicin. Each value represents the mean ± S.E.M. (n = 3-6). Abscissae (all panels): agonist doses in mg or mg/kg (s.c.). Ordinates (all panels): percent of maximum possible effect (%MPE). Each data point was obtained 15 min after injection. See "Methods" for other details.

Antagonism of mu agonist-induced antinociception. Local administration of quadazocine (0.001-0.032 mg) antagonized the local antinociceptive effects of fentanyl (0.1 mg) against capsaicin in a dose-dependent manner (fig. 3). When the locally effective dose of quadazocine (0.032 mg) was applied in the back, it did not antagonize local fentanyl. This locally effective dose of quadazocine also significantly antagonized local DAMGO (0.032 mg) (data not shown). Although systemic administration of quadazocine (0.0032-0.1 mg/kg) dose-dependently antagonized local fentanyl, the relative antagonist potency of quadazocine was approximately 80-fold higher after local versus systemic injections (table 1).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Antagonist effects of quadazocine administered locally (hashed bars, in the tail) and systemically (filled bars, in the back) against local fentanyl in 46°C water in the presence of capsaicin. CTRL represents the effects of coadministration of 0.1 mg of capsaicin and 0.1 mg of fentanyl in the tail. Other details are as in figure 2.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Antagonist effects of quaternary naltrexone administered locally (hashed bars, in the tail) and systemically (filled bars, in the back) against local fentanyl in 46°C water in the presence of capsaicin. CTRL represents the effects of coadministration of 0.1 mg of capsaicin and 0.1 mg of fentanyl in the tail. Other details are as in figure 2.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Quadazocine potency in antagonizing systemic fentanyl against either 50°C water without capsaicin or 46°C water in the presence of capsaicin. Open symbols represent dose-effect curves determined alone. Closed symbols represent dose-effect curves obtained 30 min after quadazocine (0.1 mg/kg) pretreatment. All points represent the mean ± S.E.M. (n = 3). Abscissae (all panels): fentanyl dose in mg/kg. Ordinates (all panels): %MPE. Each data point was obtained 15 min after fentanyl injection in a single dosing procedure.

	Discussion

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The present study was designed to develop a model of capsaicin-induced thermal allodynia in rhesus monkeys and to investigate the role of peripheral mu opioid receptors in this model. Capsaicin caused allodynia in the warm water tail-withdrawal assay. Local administration of fentanyl and DAMGO significantly diminished capsaicin-induced allodynia in a dose-dependent manner. This antinociception was antagonized by a small dose of either quadazocine or quaternary naltrexone applied locally. These results support the hypothesis that activation of peripheral mu opioid receptors can relieve nociception caused by capsaicin, which is thought to be mediated by stimulating primary afferent C- and A-fibers (Holzer, 1991; Winter et al., 1995).

Nociceptive effects of capsaicin. It has been suggested that burning pain induced by capsaicin is mediated through the activation of a heat-gated ion channel (Caterina et al., 1997). Exposure of nociceptor terminals to capsaicin leads to excitation of the neuron and local release of inflammatory mediators (Szolcsányi, 1993; Winter et al., 1995). In human and rodent studies, capsaicin has been used widely to evoke nociceptive responses for investigation of antinociceptive agents (Park et al., 1995; Gilchrist et al., 1996; Eisenach et al., 1997). In the present study, s.c. administration of capsaicin (0.1 mg) into the tail produced thermal allodynia in 46°C water. This quick-onset and short-lasting nociceptive effect was mediated locally in the tail, because the same amount of capsaicin, s.c. applied in the back, had no effect on the tail-withdrawal latency (data not shown).

Antinociceptive effects of mu agonists. The present study provides the first behavioral demonstration that local administration of selective mu opioid agonists, fentanyl and DAMGO, dose-dependently inhibited capsaicin-induced thermal allodynia in non-human primates. However, the locally effective doses of both mu agonists, when applied in the back, did not inhibit capsaicin-induced allodynia. This indicates that the site of fentanyl- and DAMGO-induced antinociception against capsaicin is located in the tail. It has been suggested that stimulation of local opioid receptors is likely to inhibit the sensory afferent barrage and vasodilatation by reducing the release of vasoactive substances from capsaicin-sensitive neurons (Barthó et al., 1990, 1992; Zochodne and Ho, 1993; Schaafsma et al., 1997).

Antagonism of mu agonist-induced antinociception. Local administration of both quadazocine and QNTX dose-dependently antagonized local inhibition of fentanyl against capsaicin-induced allodynia. However, the locally effective dose of both antagonists, when applied in the back, did not antagonize local fentanyl. This observation confirms the local agonist study, which indicates that the site of action of locally applied mu opioids is in the tail. Similarly, a greater relative potency of both antagonists following local versus systemic routes was observed. In particular, local QNTX was at least 300-fold more potent than systemic QNTX, which has been studied up to a dose of 3.2 mg/kg. Quaternary naltrexone is an N-methylated derivative of naltrexone. Although it is an effective opioid antagonist in vitro, systemic QNTX is ineffective in precipitating withdrawal in morphine-dependent rhesus monkeys at doses up to 8000 times larger than the effective dose of naltrexone (Valentino et al., 1983). Such large differential potency could be explained by the fact that most quaternary compounds have poor distribution after systemic administration (Brown and Goldberg, 1985).