Abstract
This study was performed to assess the interactions that occur betweendelta-and mu opioid receptors by studying effects of the systemically active nonpeptide deltaagonist BW373U86 and the mu agonist fentanyl in mice. Concentrations of the compounds were varied, and analgesic responses were determined by 55°C hot-plate assays. BW373U86 produced hot-plate antinociceptive activity along with convulsive side effects. These effects could be antagonized by the selective deltaantagonist naltrindole. Fentanyl produced hot-plate antinociceptive activity with Straub tail and hyperactivity as side effects. When BW373U86 and fentanyl were coadministered, BW373U86 convulsive activity was attenuated by fentanyl in a dose-dependent manner and the fentanyl-induced Straub tail effect was antagonized by BW373U86, also in a dose-dependent manner. Hot-plate analgesic activity was additive between the two compounds. The delta antagonist naltrindole partially antagonized the ability of BW373U86 to block the fentanyl-induced Straub tail effect. The mu antagonist β- funaltrexamine antagonized the fentanyl-induced blockade of the convulsive effects of BW373U86. These data suggest that complex inhibitory interactions take place between mu anddelta receptors in mice. Future studies are clearly needed to study the neuromodulatory effects of mu anddelta receptors. The widespread use of muagonists in the clinic indicates that a large number of patients exist who could greatly benefit from the conjunctive use ofdelta pharmaceuticals.
Accumulating evidence suggests that interactions occur between mu- and delta opioid receptors (Lee and Smith, 1980; Rothman and Westfall, 1982;Vaught et al., 1982). There also are considerable data indicating that the antinociceptive effects of certain muagonists can be increased or decreased by delta agonists. The first investigators to report such action (Vaught and Takemori, 1979) showed that an intracerebroventricular injection of [Leu5]enkephalin potentiates the antinociceptive action of morphine. Later, studies showed that the antinociceptive action of morphine can be antagonized by [Met5]enkephalin (Leeet al., 1980; Vaught et al., 1982). The agonistic and antagonistic effects of [Leu5] and [Met5]enkephalins apparently occur through the actions ofdelta receptors, because the effects can be blocked by selective delta antagonists (Cotton et al., 1984;Heyman et al., 1989a, 1989b; Jiang et al., 1990b;Portoghese et al., 1988b). All of these results indicate that antinociception can be modulated by an interaction betweendelta and mu receptors. However, only a few studies have investigated the interactive effects of deltareceptor activation on other mu opioid effects; these studies include modulation of endotoxic shock in rats (D’Amato and Holaday, 1984; Holaday et al., 1986; Holaday and D’Amato, 1983) and modulation of mu-mediated changes in urinary bladder motility (Sheldon et al., 1989).
BW373U86 [(±)-4-(α-R∗)-α-(2S∗,5R∗)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide] is a novel nonpeptide delta agonist that can cross the blood-brain barrier. In vitro evidence shows that BW373U86 has a higher affinity for delta opioid receptors (Ki = 1.8 nM) than for muopioid receptors (Ki = 15 nM) in rat brain (Chang et al., 1993). BW373U86 is ∼700 times more potent at delta receptors of mouse vas deferens than atmu receptors of guinea pig ileum (Chang et al., 1993). In vivo pharmacological studies indicate that BW373U86 primarily acts at delta receptors to produce a variety of effects in different species (Comer et al., 1993a, 1993b; Dykstra et al., 1993; Lee et al., 1993; Negus et al., 1993). The mild analgesic effects of BW373U86 are primarily mediated by delta opioid receptors at the spinal level (Wild et al., 1993). In rats, increased locomotor activity and inhibition of acoustic startle reflex are seen after s.c. or intraperitoneal administration of BW373U86, but no antinociceptive activity occurs in the hot-plate, tail-withdrawal, tail-flick or tail-pinch assays (Chang et al., 1993).In vivo characteristics of BW373U86 in mice include antinociceptive activity for the acetic acid-induced writhing and hot-plate assays, along with increased locomotor activity and convulsions (Comer et al., 1993a; Wild et al., 1993). We now report that data obtained with BW373U86 indicate thatmu and delta receptors interact, resulting in a modulation of opioid side effects as well as antinociception in mice.
Methods
Animals.
Male CD-1 mice (Charles River Laboratories, Raleigh, NC) weighing 24 to 34 g on injection were housed in groups of 15 in a temperature-controlled room (22±1°C) on a 12-hr dark/light cycle with food and water available ad libitum.
Analgesic testing.
At the time of testing, 10 animals were placed in a clean cage and transported to the laboratory. The mice were given 1 hr to adapt to the new environment. Analgesic responses were determined using a hot plate with timer (Ugo Basile, Varese, Italy) and a temperature of 55±0.5°C as the nociceptive stimulus. Each mouse was removed from the hot plate when either a jumping escape response occurred, hind paws were licked or a maximal cutoff time of 50 sec was reached. Two initial preinjection analgesia readings were collected as control measures. The first was used as a habituation procedure and was disregarded. The second, taken 5 to 10 min later, was used as the pre-injection control base-line response time. Drugs were administered by an s.c. route of injection at the back of neck. Hot-plate measures were performed at 10-, 30- and 60-min time intervals after drug treatment. Thereafter, measurements were taken in 30-min intervals until drug action had terminated.
Convulsion scoring.
Groups of 10 mice were injected s.c. with test drugs, and the percentage of animals that showed a seizure response was scored. To achieve a positive seizure score, subjects must have exhibited convulsive activity followed by a period of catalepsy. Convulsive activity was characterized by clonic movements that encompassed the entire body. The postictal period of catalepsy was characterized by a loss of locomotor activity for a 5- to 15-min period and a loss of the righting reflex. Normal behavior patterns were typically resumed after the cataleptic period.
Straub tail measures.
Straub tail effect values were recorded by visual observation with the use of a protractor. Straub tail scores were recorded using the horizontal as 0 degrees and measuring the increase of tail curvature in 15-degree increments up to 90 degrees.
Drugs.
Drugs used in the study were BW373U86 (Burroughs Wellcome, Research Triangle Park, NC), fentanyl citrate (Sigma Chemical, St. Louis, MO), midazolam (Hoffmann-LaRoche, Nutley, NJ), β-FNA hydrochloride (Research Biochemicals, Natick, MA) and NTI (synthesized according to Portoghese et al., 1988a). BW373U86 and the delta antagonist NTI (Portoghese et al., 1988b) were dissolved in a 0.9% NaCl solution to concentrations used in the text. β-FNA and fentanyl were dissolved using water to the required concentrations. Some dimethylsulfoxide (15% of the total volume) was used to enhance the solubility of midazolam. Fentanyl and BW373U86 were often coadministered. They were each dissolved in a stock solution, and then the required volume of each was pipetted to a separate vial to reach final concentrations. To ensure that receptors were saturated, the opioid antagonists and midazolam were administered at a set time before the individual agonist or mixed agonists were injected. Midazolam was injected 10 min and NTI was injected 20 min before mu and delta agonist administration. β-FNA was injected once every 24 hr over a 3-day period (total of three injections). Mu and deltaagonists were administered 24 hr after the last β-FNA injection.
Data analysis.
Statistical analyses were performed on all data using an analysis of variance, the Student’s t test or a χ2 test, depending on the nature of the data. Differences were considered to be significant at a P < .05 level. ED50 values were calculated with regression equations plotted on a probit scale. The following equation was used to determine the MPE score:
Results
Hot-plate antinociceptive response.
Although thedelta opioid agonist BW373U86 was inactive in the hot-plate assay with rats (Chang et al., 1993), in mice the compound induced a time- and dose-dependent antinociceptive response in the hot-plate assay (figs. 1 and 2). As in the rat, BW373U86 appeared (by visual observation) to substantially increase locomotor activity. In mice, hyperactivity persisted for ∼30 min, or during the period before the occurrence of a convulsion. The antinociceptive response of BW373U86 (F = 23.65,df = 4/2, P < .0001) peaked at 10 min after the injection, and the duration of action was short, lasting ∼45 to 60 min at the highest dose tested (fig. 1). The antinociceptive effects of BW373U86 were produced with a 0.5 mg/kg dose and reached a nearly maximal effect at an s.c. dose of 10 mg/kg (fig. 2). The ED50 values for the production of analgesic and seizure effects by BW373U86 were 2 and 5 mg/kg, respectively.
The mu agonist fentanyl was also observed to produce analgesia (ED50 = 50 μg/kg), and the coadministration of BW373U86 and fentanyl produced additive antinociceptive effects (fig.3). Results do not demonstrate a clear potentiation of analgesic activity (note the similarity of slopes between doses in fig.3). Most importantly, analgesic effects of fentanyl were not decreased after BW373U86 administration, and thus the delta andmu agonists do not have antagonistic effects in a hot-plate paradigm. The BW373U86-induced hot-plate antinociceptive response was antagonized by the delta antagonist NTI (F = 27.8, df = 3/7, P < .0003), which did not affect fentanyl analgesia (F = 0.55, df = 2/6, P = NS; fig. 4A). Analgesia induced by 10 mg/kg BW373U86 s.c. was effectively blocked by a 0.5 mg/kg dose of NTI, suggesting that the hot-plate antinociceptive activity of BW373U86 was mediated by delta receptors. Antagonism of deltareceptors does not, however, play a significant role in modulating the analgesic effects produced by fentanyl (fig. 4A). The anticonvulsant midazolam did not have analgesic effects and did not affect the analgesia produced by BW373U86 (fig. 4B).
Fentanyl attenuates the convulsive response of BW373U86.
Convulsions produced by BW373U86 in mice are brief, nonreoccurring incidents that are typically followed by a period of catalepsy (Comeret al., 1993a). In the present study, the minimal dose needed to produce convulsions in at least 1 mouse in a group of 10 was 1 mg/kg s.c. Convulsive activity occurred in 80% of the animals with a dose of 10 mg/kg s.c. (fig. 5). The convulsive activity of BW373U86 is about 2.5-fold less potent than the antinociceptive activity (fig. 2). The administration of the deltaantagonist NTI antagonized the convulsive effects of BW373U86 in a dose-dependent manner (fig. 5; χ2 = 30.9,df = 3, P < .0001). A dose of 1 mg/kg of NTI completely eliminated convulsions induced by 10 mg/kg BW373U86. As observed by Comer et al. (1993a), BW373U86-induced convulsive activity was completely blocked by the anticonvulsant midazolam at a dose of 3.2 mg/kg s.c. (fig. 5; χ2 = 36.7,df = 1, P < .0001).
The data from figure 2 do not reveal whether 373U86-induced increases in hot-plate latencies represent true antinociception or are, in part, a reflection of animals being behaviorally compromised by some degree of convulsive activity. Most importantly, the data from figures 4B and5 indicate that the antinociceptive effect of BW373U86 is not a consequence of convulsive effects. When BW373U86 seizure activity is blocked by midazolam (fig. 5), the hot-plate antinociceptive response of BW373U86 still occurs (see fig. 4B).
Fentanyl did not have convulsive activity at doses tested up to 500 μg/kg s.c. Administration of the mu agonist fentanyl did, in a dose-dependent manner, reduce the percentage of mice that had BW373U86-induced seizures (McNemar’s χ2 = 193.3,df = 6, P < .0001) but did not completely eliminate all convulsions (fig. 6). These results lead to the conclusion that mu receptors can modulate BW373U86-induced convulsive activity. To confirm this, a study was performed using β-FNA, a specific and irreversible muopioid antagonist (Jiang et al., 1990a, 1990b; Takemori and Portoghese, 1985; Ward et al., 1982) to test the hypothesis that mu receptors can modulate BW373U86-induced convulsive activity.
Mice were injected (s.c.) a total of three times with 10 mg/kg β-FNA (once a day over a 3-day period). The antinociceptive effect of fentanyl was significantly reduced from maximum levels to ∼50% of the MPE after β-FNA pretreatment (t = 4.36,df = 2, P < .05; fig 7A). The antinociceptive effect of BW373U86 was not affected by β-FNA pretreatment (t = 0.67, df = 5, NS). These data confirm that β-FNA treatment under the present protocol produced selective antagonistic effects at mu-, but notdelta-, opioid receptors.
BW373U86-induced seizure activity was again observed to be significantly attenuated by fentanyl administration (fig. 7; χ2 = 21.5, df = 1, P < .0001). Interestingly, the convulsive effect of BW373U86 appeared to be slightly decreased from 80% to 67% after β-FNA treatment, but the effect was not observed to be significant (χ2 = 0.63,df = 1, P = NS; fig. 7B). It is unknown whether the current β-FNA treatment, by itself, would have altered nociceptive or seizure thresholds; however, published reports of studies that used similar methods do not reveal any such effects of β-FNA (Heyman et al., 1989a; Jiang et al., 1991). β-FNA treatment did, however, antagonize the ability of fentanyl to attenuate BW373U86-induced seizure activity (χ2 = 7.3, df = 1, P < .007, BW373U86 + fentanyl group vs. BW373U86 + fentanyl + βFNA group). In β-FNA-pretreated mice that received a 10 mg/kg dose of BW373U86 and a 200 μg/kg fentanyl dose, convulsive activity returned from 20% to nearly 60% compared to the same dose treatment without preinjected β-FNA (fig. 7B). There was no significant difference between the data for the group that received all three treatments and the group that received BW373U86 and β-FNA, suggesting that fentanyl did not attenuate BW373U86 seizure activity in β-FNA-treated mice (χ2 = 0.39, df = 1, P = NS). Overall, results indicate that the ability of fentanyl to block BW373U86-induced seizures is due to mu receptor activity.
BW373U86 inhibits fentanyl-induced Straub tail effect.
Muscle rigidity is one of the side effects of fentanyl that, in mice, results in a Straub tail effect. The degree of tail curvature increases proportionally as the dose of fentanyl is increased (fig.8). The administration of BW373U86 decreased fentanyl-induced Straub tail in a dose-dependent manner (F = 69.4, df = 5/458, P < .0001; fig. 8). NTI was administered to mice in combination with BW373U86 and fentanyl to test the hypothesis that the BW373U86 inhibitory effect on fentanyl-induced muscle rigidity is the result of deltareceptor activation. Neither BW373U86 nor NTI alone induced the Straub tail effect in mice (fig. 9). Results depicted in figure9 illustrate that fentanyl produced ∼30 degrees of Straub tail curvature at a 150 μg/kg dose s.c. BW373U86, at a 10 mg/kg dose, almost completely inhibited the Straub tail effect induced by 150 μg/kg fentanyl (t = 8.3, df = 58, P < .0001). NTI (0.5 mg/kg s.c.) also reduced Straub tail curvature from 30 degrees to ∼18 degrees (t = 3.23,df = 58, P < .002) but did not eliminate the occurrence of Straub tail (fig. 9). A possible explanation for this effect is that NTI does have some affinity for mu receptors and thus can exhibit mu antagonist effects (Portogheseet al., 1988b). When fentanyl, BW373U86 and NTI were coadministered, the degree of curvature was ∼15 degrees, which was significantly different from the 5-degree curvature observed in subjects treated with fentanyl and BW373U86 (t = 4.65,df = 58, P < .0001). Results indicate that NTI administration can, at least partially, antagonize the ability of BW373U86 to block fentanyl-induced Straub tail. The degree of tail curvature was returned to the level produced by NTI alone (fig. 9). Results indicate that the delta agonist BW373U86 can inhibit fentanyl-induced Straub tail through direct effects ondelta receptors.
Discussion
Previous reports (Comer et al., 1993a; Wild et al., 1993) described antinociceptive effects of BW373U86 in acetic acid-induced writhing and tail-flick tests in mice. The present studies confirm the antinociceptive activity of BW373U86 in a 55°C hot-plate test in mice. Analgesic effects of BW373U86 do not appear to be a consequence of seizure activity, because antinociceptive effects are still observed when seizure activity is blocked with the anticonvulsant midizolam (figs. 4 and 5). The hot-plate antinociceptive activity of BW373U86 was antagonized by NTI at doses of <1 mg/kg. In contrast, the irreversible mu antagonist β-FNA, under conditions that significantly reduce the antinociceptive response of the muagonist fentanyl, did not affect the antinociceptive effect of BW373U86. Convulsive activity induced by BW373U86 was also antagonized by NTI in a dose-dependent manner (fig. 5). The data provide evidence that BW373U86 produces antinociceptive and convulsive effects in mice through actions at NTI-selective delta receptors.
A potentiation and modulation of the antinociceptive response of morphine by delta agonist peptides has been previously documented (Heyman et al., 1989a, 1989b; Vaught and Takemori, 1979). Dykstra et al. (1993) also demonstrated that BW373U86 potentiates morphine and l-methadone antinociception in an electric shock titration assay in monkeys. In the present study, we were unable to demonstrate a clear potentiation by BW373U86 of the antinociceptive response of fentanyl in the mouse hot-plate test. The antinociceptive effect of fentanyl and BW373U86 in our hot plate test was purely additive. This finding is not surprising because the potentiating effect of delta agonists has not been reported for highly efficacious mu agonists (i.e., , [d-Ala2,N-MePhe4,Gly-ol5]enkephalin, PL 017 and sufentanil; Heyman et al., 1989b). Fentanyl is known to be a highly effective mu agonist (Jiang et al., 1990b; Porreca et al., 1992; Stevens and Yaksh, 1989), and thus BW373U86 would not be expected to potentiate analgesia induced by fentanyl.
The interactive modulation of mu and deltareceptor effects can be extended to effects of the nonpeptidedelta agonist BW373U86 and the selective muagonist fentanyl. In mice, BW373U86 induces seizure activity via delta receptor activation. The mu opioid agonist fentanyl can antagonize the ability of BW373U86 to produce seizures. Additionally, the selective mu antagonist β-FNA significantly blocked the ability of fentanyl to inhibit seizures produced by BW373U86 (fig. 7). Thus, the ability of BW373U86 to produce seizures can be attenuated by fentanyl through an agonist action atmu receptors.
Fentanyl also induces muscle rigidity, such as Straub tail, throughmu receptor activation. Conversely, fentanyl-induced muscle rigidity can be inhibited by the delta agonist BW373U86. Apparently, delta receptor effects of BW373U86 inhibitmu agonist-induced muscle rigidity, because thedelta antagonist NTI can partially antagonize the ability of BW373U86 to inhibit fentanyl-induced Straub tail (fig. 9). The present data indicate that a delta opioid agonist can attenuate some effects of a mu agonist (i.e., muscle rigidity) and a mu agonist can antagonize some effects of adelta agonist (i.e., seizure activity). Interestingly, the delta agonist BW373U86 and themu agonist fentanyl can selectively antagonize each other’s effects because a combination of the two agonists results in additive analgesic effects.
It is curious that Straub tail was not observed after fentanyl and BW373U86 administration, when Straub tail has been reported to be produced by the delta peptide DPDPE (Murray and Cowan, 1990). We have never observed a Straub tail response in male CD1 mice after the administration of specific nonpeptide delta agonists. As suggested by Murray and Cowan (1990), the Straub tail response seen after DPDPE administration is probably due to mu receptor activation. The question of why delta effects of DPDPE failed to block the mu-induced Straub tail remains unanswered, but critical factors could be the strain of mouse used or pharmacodynamic differences produced by the route of administration (s.c. vs. intracerebroventricular).
A previous report described the existence of a mu anddelta interaction in a rat model of opioid dependence. Leeet al. (1993) demonstrated that BW373U86 does not produce physical dependence alone but can attenuate the development and expression of abstinence precipitated by naloxone in morphine-dependent rats. Our data clearly indicate that a complex in vivointeraction takes place between delta- andmu-mediated effects in mice. It will be interesting to find out whether similar interactions occur in other species and with other opioid effects.
In conclusion, many complex interactions occur between muand delta receptors. These effects contribute to the modulation of antinociception and side effects. In our study, BW373U86 decreased fentanyl-induced muscle rigidity (e.g., Straub tail), whereas fentanyl decreased the convulsions caused by BW373U86. Coadministration of both compounds produced an additive analgesic effect in a mouse hot-plate assay. Further studies are clearly needed to provide a detailed understanding of the intricate interaction that takes place between delta- and mu opioidreceptors.
Footnotes
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Send reprint requests to: Hugh O. Pettit, Ph.D., Delta Pharmaceuticals, Inc., MCB/HLB Complex, Research Triangle Institute, 3040 Cornwallis Rd., Research Triangle Park, NC 27709.
- Abbreviations:
- NTI
- naltrindole
- β-FNA
- β-funaltrexamine
- MPE
- maximal percent effect
- DPDPE
- lsqb]d-Pen2,d-Pen5]enkephalin
- s.c.
- subcutaneous
- Received August 5, 1996.
- Accepted March 18, 1997.
- The American Society for Pharmacology and Experimental Therapeutics