Anesth Analg 2002;94:1542-1546
© 2002 International Anesthesia Research Society
TECHNOLOGY, COMPUTING, AND SIMULATION
The Role of Spinal Opioid Receptors in Antinociceptive Effects Produced by Intrathecal Administration of Hydromorphone and Buprenorphine in the Rat
Gopi A. Tejwani, PhD, and
Anil K. Rattan, PhD
Department of Pharmacology, Ohio State University, College of Medicine and Public Health, Columbus
Address correspondence and reprint requests to Gopi A. Tejwani, PhD, Department of Pharmacology, Ohio State University, College of Medicine and Public Health, 5197 Graves Hall, 333 W 10th Ave., Columbus, OH 43210-1239. Address e-mail to Tejwani.1{at}osu.edu
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Abstract
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The intrathecal administration of morphine has been the standard therapy to control long-term intractable pain. Recently, a panel of pain therapy experts suggested that because of the lack of efficacy or because of the side effects produced by morphine in some patients, other drugs, such as hydromorphone and buprenorphine, should be investigated for their analgesic properties. We designed this study to compare the efficacy of intrathecal hydromorphone and buprenorphine to suppress thermal nociception in male Sprague-Dawley rats. An additional objective was to understand whether hydromorphone and buprenorphine bind and act as agonists to µ-, 
-, and 
-spinal opioid receptors. Intrathecally-administered hydromorphone and buprenorphine produced a dose- and time-dependent increase in the tail-flick response latency in rats. The 50% effective dose value for the antinociceptive effect of buprenorphine and hydromorphone were 4 and 69.5 nmol/L, respectively. Both drugs act as agonists to µ-opioid receptors, as determined by their ability to displace [3H]-DAMGO from the spinal opioid receptors and by the ability of an opioid receptor antagonist, naloxone, to reverse their antinociceptive effects. Buprenorphine also has an agonistic effect on the -opioid receptors. For the first time, we report that intrathecal buprenorphine is approximately 17 times more effective than hydromorphone in inhibiting thermal pain, and buprenorphine produces its antinociceptive effect by acting as an agonist at both µ- and -spinal opioid receptors. Naloxone administered intrathecally was effective in preventing the antinociceptive effects of subsequent intrathecal injections of buprenorphine.
IMPLICATIONS:Hydromorphone and buprenorphine are two important drugs used for pain relief. We observed that intrathecal buprenorphine is 17 times more potent than hydromorphone to inhibit pain in rats. Both drugs exert their effects through specific spinal opioid receptors.
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Introduction
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Intrathecally-applied drugs such as opioids are important clinically to produce pain relief as well as to understand the mechanism of antinociception involving drug-spinal receptor interactions (1,2). In addition to morphine (3), two other opioids, hydromorphone (3) and buprenorphine (4,5), have been used intrathecally to produce pain relief in humans. Hydromorphone, a semi-synthetic derivative of morphine, is considered to be a first-line opioid analgesic for postoperative pain management and for the treatment of certain forms of cancer pain (6). Hydromorphone is a µ-opioid receptor agonist and is approximately 5 to 7 times as potent as morphine (6,7). Like hydromorphone, buprenorphine is also semi-synthetic but an oripavine alkaloid (thebaine) derivative. Buprenorphine also has been used intrathecally for pain relief in humans and has a systemic potency of 20 to 70 times that of morphine (4, 8). Unlike hydromorphone, buprenorphine is a mixed agonist–antagonist drug, exhibiting partial agonistic activity towards µ-opioid receptors and antagonistic action at -opioid receptors (9). However, some studies have reported that buprenorphine may have agonistic activity at -opioid receptors and could be a poor choice for intrathecal use because buprenorphine-induced analgesia exhibits a lack of naloxone reversibility (10). We designed the present study to explore the relative efficacy of hydromorphone and buprenorphine in producing antinociception after the intrathecal administration in rats. Naloxone and selective antagonists for µ-, -, and -opioid receptors were used to reverse the antinociception produced by hydromorphone and buprenorphine. We also determined the binding of hydromorphone and buprenorphine to µ-, -, and -opioid receptors in the spinal cord.
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Methods
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This study received previous approval from the Ohio State University Institutional Laboratory Animal Care and Use Committee. Male Sprague-Dawley rats weighing 250–300 g were anesthetized with ketamine and xylazine and placed into a stereotaxic apparatus. Briefly, a polyethylene tube (PE 10 catheter, 0.75 mm in diameter, 12-cm long) was inserted through a slit in the cisternal membrane approximately 8.5 cm down the spinal subarachnoid space to the rostral aspect of the lumbar enlargement according to the method described earlier (1,2,11). The catheter was fixed to the skull by cranioplastic cement, and the rats were allowed to recover for at least a week.
Rats were randomized to various treatment groups. Hydromorphone hydrochloride (obtained from Wyeth Labs, Madison, NJ) and buprenorphine hydrochloride (obtained from Norwich Eaton Pharmaceutical Inc, Norwich, NY) were used for the intrathecal administration using a 10-µL Hamilton syringe (Hamilton Co, Reno, NV). The total volume injected was 20 µL (5 µL of saline + 10 µL of drug + 5 µL of saline) and was kept constant in all experiments. The control rats received only the vehicle. A different group of six rats was used for every dose of each drug injected intrathecally in a volume of 10 µL as follows: (a) hydromorphone alone (4, 20, and 40 or 60 µg, or 12.4, 62.2, and 124.3 or 186.5 nmol/L, respectively) and (b) buprenorphine alone (0.3, 1.5, and 3 or 4.5 µg, or 0.6, 30, and 6 or 9 nmol/L, respectively).
After the last injection of the drug, the rats were anesthetized with ketamine and xylazine, and 20 µL of 3% ferric chloride and 1% potassium ferrocyanide in 10% formalin solution was injected into the catheter to aid in the evaluation of catheter placement, as indicated by a Prussian blue color. The rats were dissected, and the location of the catheter tip was verified by staining caudal region. Data from the rats with malpositioned catheters were excluded from the study, as described earlier (1).
To determine the involvement of the opioid system in mediating the antinociceptive effect of hydromorphone and buprenorphine in some rats, an intrathecal dose of 100 µg/10 µL of naloxone or 100 µg/10 µL of a selective -opioid receptor antagonist, 1-cyclopenthyl-5-(1,2,3,4,5,6-hexahydroxy-3,6,11-trimethyl-2-methano-3-benzazocin)-3-pentatone methane sulfonate (WIN 44,441–3), obtained from the Sterling Winthrop Research Institute, Rensselaer, NY, was administered 15 min before the administration of these drugs (12).
The rats were tested for their nociceptive responses using the tail-flick (TFL) test, as described earlier (1,2,11,13,14). TFL response latencies are presented as mean ± SEM. Comparisons between predrug and postdrug treatment were performed with a one-way analysis of variance followed by the Newman-Keuls t-test. In analysis of the relationship between the effects of drugs on TFL, latencies were converted to a percentage of maximal possible effect (MPE), where
equation
P values <0.05 were considered to be statistically significant.
To determine whether hydromorphone and buprenorphine had any effect on the binding of the specific opioid ligands to various subtypes of spinal cord opioid receptors, spinal cords from normal rats not injected with any drugs were used for the binding studies of radioactive ligands. The details of the binding assays for µ (DAMGO), (DSLET), and (ethylketocyclazocine [EKC]) are given in our earlier reports (1,2,11,13–15).
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Results
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Intrathecally-administered hydromorphone (4–60 µg; Fig. 1A) and buprenorphine (0.30–4.5 µg; Fig. 1B) produced a dose- and time-dependent increase in the TFL response latency in rats. The peak antinociceptive effect was observed 10–15 min after the administration of the larger doses (40–60 µg) and 20–30 min after the administration of the smaller doses (4–20 µg) of hydromorphone (Fig. 1A). At smaller doses (4–20 µg), the antinociceptive effect of hydromorphone lasted approximately 1 h compared with more than 6 h of antinociceptive effect observed at the largest dose (60 µg). In contrast to the hydromorphone effect, the antinociceptive effect of intrathecal buprenorphine developed slowly (30–60 min after the administration of the drug at larger doses [3–4.5 µg] and 2 h after the administration of the drug at a smaller dose [1.5 µg]). However, similar to hydromorphone, the antinociceptive effect of buprenorphine lasted up to 6 h at the largest dose used (Fig. 1B). On an equimolar basis, buprenorphine was more potent than hydromorphone in producing an antinociceptive effect. The effective dose (50% effective dose value) for an antinociceptive effect of buprenorphine and hydromorphone were 4 nmol/L (2 µg) and 69.5 nmol/L (22.4 µg), respectively (Fig. 2A).
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Figure 1. Dose- and time-dependent antinociception produced by the intrathecal administration of hydromorphone (A) and buprenorphine (B) in the rat. Each point represents the percentage maximum possible effect (%MPE) as a mean of tail-flick latency (TFL). *P < 0.05 (compared with rats injected with saline solution at their respective times).
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Figure 2. (A) Dose-dependent antinociception produced by hydromorphone and buprenorphine after the intrathecal administration of drugs in the rat. Each point represents the percentage maximum possible effect (% MPE) as a mean of tail-flick latency (TFL). Data showing the maximum effect produced, at any time, taken from Figure 1. (B) Effect of opioid receptor antagonists on the antinociception produced by hydromorphone (Hy) and buprenorphine (Bu) in the rat. Naloxone (Na, 100 µg) and -receptor antagonist WIN 44,441–3 (Wi, 100 µg) were injected intrathecally 15 min before the injection of hydromorphone or buprenorphine. TFL latency was measured 30 min after the intrathecal injection of hydromorphone (60 µg, 186.5 nmol/L) or buprenorphine (4.5 µg, 9.0 nmol/L). *P < 0.05 (compared with hydromorphone or buprenorphine injected alone).
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To investigate whether hydromorphone and buprenorphine produce their antinociceptive effects by interacting with specific spinal opioid receptors, we did two different sets of experiments. In one experiment, we determined the antinociceptive effect of these drugs in the presence and the absence of a nonspecific opioid receptor antagonist, such as naloxone, and a specific -opioid receptor antagonist, WIN 44,441–3 (Fig. 2B). Intrathecal injection of 100 µg of naloxone or WIN 44,441–3 had no effect on the basal antinociception (Fig. 2B). Naloxone significantly abolished (P < 0.05) hydromorphone- and buprenorphine-induced antinociception (Fig. 2B). However, WIN 44,441–3 had no effect on the antinociception produced by hydromorphone but significantly abolished (P < 0.05) antinociception produced by buprenorphine. In the second experiment, we assessed the binding of hydromorphone and buprenorphine to the spinal opioid receptors by displacing specific opioid receptor ligands. The ligands used were [3H]-naloxone (binds predominantly to µ- but also to - and -opioid receptors), [3H]-DAMGO (µ-specific), [3H]-DSLET (-specific), and [3H]-EKC (-specific). Both, hydromorphone and buprenorphine decreased the binding of [3H]-naloxone significantly (Fig. 3A). Hydromorphone was much more effective than buprenorphine in displacing [3H]-DAMGO (Fig. 3B), indicating that hydromorphone predominantly binds to µ-opioid receptors in the spinal cord. Hydromorphone decreased the binding of [3H]-DSLET (Fig. 3C), but the effect was not as great as its effect in displacing [3H]-DAMGO (Fig. 3B), indicating that hydromorphone has more affinity for µ- than -opioid receptors in spinal cord. Buprenorphine completely abolished the binding of [3H]-EKC (Fig. 3D), indicating that it predominantly binds to -opioid receptors in the spinal cord.
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Figure 3. Effect of hydromorphone or buprenorphine on the binding of ligands to various subtypes of spinal opioid receptors. The concentration of ligand used was: (A) [3H]-naloxone (0.5 nmol/L), (B) [3H]-DAMGO (1 nmol/L), (C) [3H]-DSLET (1 nmol/L), and (D) [3H]-ethylketocyclazocine (EKC) (2 nmol/L). When [3H]-EKC was used, DAMGO and DADLE (100 nmol/L each) were included in the assay mixture to suppress the binding of this ligand to µ- and -opioid receptors. The details of the assay system are given in a previous report (15).
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Discussion
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Morphine by long-term intraspinal infusion has been used to relieve intractable pain. Recently, an expert panel (16) suggested that because of the lack of efficacy or because of the side effects produced by morphine in some patients, other drugs, such as hydromorphone, should be a high priority for research. Intrathecal hydromorphone has been widely used because it is approximately 5 to 7 times more potent than morphine and has a longer duration of action and fewer side effects (3). We report here that given intrathecally, buprenorphine is approximately 17 times more potent than hydromorphone for suppressing thermal nociception in the rat (Fig. 2A). Both hydromorphone and buprenorphine seem to exert their effects through the spinal opioid receptors.
There are three major types of opioid receptors (µ, , and ) in large concentration in the substantia gelatinosa of the dorsal horn of the spinal cord, a region that is a major site for early integration of nociceptive input (16,17). In search of new opioids to replace morphine, efforts have been directed to develop compounds that have both antinociceptive as well as opioid antagonistic properties. Such compounds would show agonistic properties towards a particular subtype of opioid receptors while opposing the effect of another subtype of opioid receptors. Buprenorphine has a similar potency and high affinity for both the µ- and -opioid receptor subtypes and acts as a partial agonist at µ receptor and antagonizing -receptors (18). However, some investigators believe that buprenorphine induces analgesia by acting as an agonist at the -receptors (19) specifically through a subtype of -opioid receptor called a 3-opioid receptor (18). The results shown in Figure 2B on the antagonism of buprenorphine-induced antinociception by a -opioid receptor antagonist, WIN 44,441–3 (12), indicate that buprenorphine may act as an agonist at the -opioid receptors. However, it is also possible that buprenorphine produces its antinociceptive action by binding to µ-opioid receptors. Naloxone, an opioid receptor antagonist that has at least 15 to 16 times more affinity for µ-opioid receptors than for - and -opioid receptors (20,21), antagonizes the antinociceptive effects produced by buprenorphine and hydromorphone (Fig. 2B), indicating an agonistic effect of buprenorphine and hydromorphone on the µ receptor. WIN 44,441–3 did not reverse the antinociception produced by hydromorphone (Fig. 2B), indicating a preferential binding of hydromorphone to µ-opioid receptors than to -opioid receptors. However, hydromorphone has more affinity than buprenorphine for µ-opioid receptors (Fig. 3B), and buprenorphine has much more affinity than hydromorphone for -opioid receptors, as indicated by binding studies (Fig. 3D).
Whether buprenorphine acts as an agonist or antagonist at various opioid receptors also may depend on whether these opioid receptors are present in the spinal cord or in the brain. Intrathecally-administered buprenorphine acts as a potent analgesic (Fig. 1B) and as an opioid receptor agonist (Fig. 2B). However, Romero et al. (21), by using [35S]GTP-
-S functional binding assay, have suggested that buprenorphine is a potent µ- and -opioid receptor antagonist. Opioid receptors are coupled to the superfamily of 7 transmembrane G-proteins (GI/GO family) (17). By using an assay that detects the activation of receptors by measuring agonist-stimulated binding of the hydrolysis-resistant GTP analog, [35S]-GTP--S, Romero et al. (21) have reported that buprenorphine did not stimulate [35S]-GTP--S binding in the guinea pig caudate brain region, indicating a lack of agonistic activity. Moreover, buprenorphine antagonized µ-, -, and -stimulated [35S]-GTP--S binding. The µ-antagonist effect of buprenorphine was confirmed in the rat brain using autoradiography (21).
There are contradictory reports on the reversibility of antinociception produced by buprenorphine by naloxone. Schmauss and Yaksh (22) reported the reversal of buprenorphine-induced antinociception by naloxone. Dickenson et al. (10) reported that antinociception produced (as measured by monitoring the inhibition of both C and Aß fiber-evoked response) by 125 µg of buprenorphine could not be reversed by 50 µg of naloxone. In our studies, 100 µg of naloxone easily prevented buprenorphine-induced antinociception (Fig. 2B), suggesting that a larger ratio of naloxone:buprenorphine must be used to reverse the buprenorphine-induced anti-nociception. More recently, Guirimand et al. (23) reported that 0.4 mg/kg IV of naloxone did not reverse the effects produced by intrathecal buprenorphine on a C-fiber reflex in the rat. However, Guirimand et al. (23) injected naloxone after the intrathecal administration of buprenorphine. In the present studies, we administered naloxone before the intrathecal injection of buprenorphine. Buprenorphine is nonionized and a highly lipophilic opioid that passes rapidly via the arachnoid granulations into venous and lymphatic vessels (24). In addition, buprenorphine exhibits a slow dissociation from its receptors (25,26). Therefore, we recommend that to determine the ability of naloxone to antagonize the effects of buprenorphine, naloxone should be administered before the administration of buprenorphine. However, in the clinical setting, previous administration of naloxone is impractical and has no bearing on the potential for naloxone to reverse opioid side effects caused by previously administered intrathecal buprenorphine.
In summary, we report that when given at an equimolar concentration, intrathecal buprenorphine is approximately 17 times more effective than hydromorphone in inhibiting thermal nociception in the rat. Both drugs exert their antinociceptive effects by acting as an agonist at the µ-spinal opioid receptors. Buprenorphine also acts as an agonist at the -spinal opioid receptors. Naloxone prevented the antinociceptive effects produced by hydromorphone and buprenorphine when injected intrathecally before the administration of these drugs.
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Accepted for publication January 24, 2002.
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Abstract
Introduction
