Psychopharmacology (1998) 140:20–28
© Springer-Verlag 1998
O R I G I N A L I N V E S T I G AT I O N
Drake Morgan · Mitchell J. Picker
The µ opioid irreversible antagonist beta-funaltrexamine differentiates the discriminative stimulus effects of opioids with high and low efficacy at the µ opioid receptor Received: 19 August 1997 / Final version: 28 March 1998
Abstract The purpose of the present study was to determine the relative intrinsic efficacy of various opioids using the irreversible µ opioid antagonist beta-funaltrexamine (βFNA). To this end, pigeons were trained to discriminate 3.0 (n=6) or 1.8 (n=1) mg/kg morphine from distilled water in a two-key, food-reinforced, drug discrimination procedure. The µ opioids fentanyl, l-methadone, buprenorphine, butorphanol, nalorphine, nalbuphine and levallorphan, as well as the δ opioid BW373U86, substituted completely for the morphine stimulus. The stimulus effects of morphine were antagonized (i.e., produced a significant increase in the ED50 value) by a 10 mg/kg but not a 5 mg/kg dose of βFNA. Antagonist effects of βFNA were observed following a 2-h pretreatment, but not following 26-, 50-, 74-, 98- or 146-h pretreatments. The stimulus effects produced by fentanyl, l-methadone and buprenorphine were not antagonized by doses of βFNA as high as 20, 10 and 10 mg/kg, respectively. The lowest dose of βFNA required to antagonize the stimulus effects of butorphanol was 10 mg/kg, whereas the effects of nalorphine, nalbuphine and levallorphan were antagonized by a dose of βFNA as low as 5 mg/kg. The δ opioid BW373U86 substituted for the morphine stimulus, and this effect was not antagonized by 10 mg/kg βFNA. The pkB values for naloxone (1.0 mg/kg) against the stimulus effects of fentanyl (6.70) and morphine (6.52) were considerably higher than that for BW373U86 (4.60), indicating further that the morphine-like stimulus effects produced by BW373U86 were not mediated by activity at the µ opioid receptor. These findings indicate that the This manuscript partially fulfills the requirements for the Doctor of Philosophy Degree of D. Morgan from the University of North Carolina at Chapel Hill D. Morgan1 · M.J. Picker (✉) Department of Psychology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3270, USA Present address: 1 Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA
strategy of irreversible antagonism can be used effectively to differentiate opioids with varying degrees of intrinsic efficacy at the µ opioid receptor in a pigeon drug discrimination procedure. In particular, the ranking of these drugs by relative intrinsic efficacy at the µ opioid receptor is: l-methadone=fentanyl≥buprenorphine≥morphine≥ butorphanol>nalorphine=nalbuphine=levallorphan. Additionally, the short-acting effect of βFNA in the pigeon suggests that the recovery of µ opioid receptor function varies across species. Key words Drug discrimination · Opioids · Pigeons · Irreversible antagonists · Intrinsic efficacy
Introduction The concept of intrinsic efficacy, which essentially reflects the ability of a drug to activate a receptor once bound to it, encompasses three common assumptions regarding receptor pharmacology: (1) a given drug effect is proportional to receptor occupancy, such that as occupation increases, the magnitude of the effect increases, (2) drugs can typically produce their maximal effects by occupying only a proportion of the available receptor population, and (3) the maximal effects produced by a drug can vary across assays (Mjanger and Yaksh 1991; Kenakin 1993). In drug discrimination procedures, the relative intrinsic efficacy of opioids has been determined by examining their patterns of substitution and antagonism (Shannon and Holtzman 1977; France and Woods 1985; Picker et al. 1992, 1993; Young et al. 1992). For example, in animals trained to discriminate either a low or a high dose of the µ opioids morphine or fentanyl from saline, opioids with high efficacy at the µ opioid receptor substitute completely for the stimulus effects of both training doses. In contrast, opioids with lower efficacy produce complete substitution for the training drug in the low training dose group, but fail to substitute for, and in fact, antagonize the effects of the training drug in the high training dose group.
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Thus, in the high-dose group, these drugs bind to but are unable to activate enough receptors to produce effects on their own, and thus competitively block the effects of higher efficacy opioids (e.g., Shannon and Holtzman 1979; Colpaert and Janssen 1984; Koek and Woods 1989; Young et al. 1992; Picker et al. 1993). Similarly, in morphine-dependent pigeons trained to discriminate between a high training dose of morphine, naltrexone and saline, higher efficacy opioids substitute for the morphine stimulus, whereas lower efficacy opioids substitute for the naltrexone stimulus. Moreover, only relatively high efficacy opioids reverse the naltrexone-appropriate responding evoked by morphine abstinence (France and Woods 1990). Since chronic administration of a drug appears functionally to decrease the population of receptors through which the drug interacts, this technique can also be useful in a drug discrimination procedure to rank opioids on the basis of their relative intrinsic efficacy. Following chronic administration of morphine, for example, the dose-effect curves of low efficacy opioids are shifted to the right and/or downward to a greater extent than the dose-effect curves of higher efficacy opioids (Young et al. 1991; Paronis and Holtzman 1994). Irreversible antagonists also can be used in vivo to rank order drugs on the basis of their relative efficacies, and this approach has proven useful in assays of antinociception (e.g. Zimmerman et al. 1987; Adams et al. 1990; Mjanger and Yaksh 1991; Zernig et al. 1994, 1995), self-administration (e.g. Zernig et al. 1997) and schedule-controlled responding (e.g. Butelman et al. 1996; Pitts et al. 1996). Irreversible antagonists bind to the receptor in such a way as to make them non-functional, effectively limiting the number of receptors with which a drug can interact. In these situations, drugs first decrease in potency and then effectiveness, with the changes in potency and effectiveness being greater for lower than higher efficacy compounds (see Kenakin 1993). Numerous studies have demonstrated that βFNA is an irreversible antagonist at the µ opioid receptor (see Takemori and Portoghese 1985; Martin et al. 1993). βFNA covalently labels µ opioid receptors in several species, including bovine, guinea pig, rat and mouse striatal membranes (Liu-Chen et al. 1993). Furthermore, this binding could be inhibited by naloxone or µ opioids, but not by δ or κ opioids, suggesting high specificity (LiuChen and Philips 1987). The purpose of the present study was to determine the relative intrinsic efficacy of selected opioids that are active at the µ opioid receptor using βFNA. To this end, the dose-effect curves for the opioids fentanyl, l-methadone, morphine, buprenorphine, butorphanol, nalbuphine, nalorphine and levallorphan were determined in the absence and then presence of one or more doses of βFNA in pigeons trained to discriminate morphine from saline. A relatively low dose of morphine was used to establish the discrimination to insure that these opioids produce complete substitution (Young et al. 1992).
Materials and methods Subjects Seven experimentally naive, female, White Carneau pigeons maintained at approximately 80–85% of their free-feeding body weights were used. Each pigeon was singly housed in a climate controlled colony on a 12-h light/dark cycle, with free access to grit and water. Apparatus Seven operant conditioning chambers were used. Each chamber contained two operative, 2.5 cm diameter, illuminated response keys which were 23 cm from the bottom of the chamber. Three seconds access to grain was available through an aperture that centered below the keys approximately 8 cm from the floor, and was illuminated by a 7-W bulb when the hopper was raised. The chambers also contained a white light for ambient illumination, an exhaust fan for ventilation, and white noise to mask extraneous sounds. Data were collected with a microcomputer using software and interfacing supplied by MED Associates Inc. (Georgia, Vt., USA). Drug discrimination training After key-pecking was established, food delivery became contingent upon a single response (fixed ratio 1; FR1). The ratio size required for food delivery was increased over several sessions until an FR20 was in effect. At this time the training dose of morphine (3.0 mg/kg for six pigeons, 1.8 mg/kg for one pigeon) or distilled water was administered before each session. For four pigeons, food delivery was contingent upon responding on the right key after drug administration and on the left key after distilled water administration. The contingencies were reversed for the other three pigeons. During these initial training sessions, the pretreatment time and session length were 15 min and the ratio size was increased to 30 (i.e., FR30). After discriminative control was established, a multiple trial training procedure was initiated. These sessions were one to five components in length, with each component consisting of a 10-min time-out followed by a 6-min response period. At the beginning of the time-out period, either distilled water or the training dose of morphine was administered. After the timeout period, the house light and key lights were illuminated and responding on the injection-appropriate key was reinforced. These sessions consisted of zero to four distilled water components followed by zero to two morphine components. During sessions in which the second to last component was preceded by administration of the training dose of morphine, either distilled water administration or a sham injection preceded the last component, and at the end of this trial the session was terminated. Training sessions were typically conducted 5 days per week. Drug discrimination testing Once stimulus control was established, testing began. Test sessions were conducted only if the percentage of injection-appropriate responses before the first reinforcer was greater than 80% on the preceding 2 training days. During test sessions, the completion of the FR30 on either key resulted in food delivery. Using a cumulative dosing procedure, increasing doses of the test drug were administered at the beginning of each time-out such that the total dose administered increased by 0.25 or 0.5 log units. In tests with naloxone, the dose of naloxone was administered immediately preceding the first test drug dose. In tests with βFNA (except for the time-course studies), the dose of βFNA was administered 2 h before the first component of the test session. After βFNA administration, dose-effect curves of all drugs were determined 2 h, 50 h, and 146 h later. With morphine and nalbuphine, additional tests were conducted (“time-course tests”) in which 10 mg/kg βFNA was administered 26 h before determination of the first dose-effect
22 Table 1 ED50 values (±95% CL) and dose ratios for the dose-effect curves of various opioids before and after βFNA (2 and 50 h pretreatment) administration
a
Dose Ratio = (ED50 after βFNA) / (ED50 before βFNA) b Significant rightward shift (i.e. non-overlapping confidence limits) c Could not be determined
2 h pretreatment ED50 value Morphine alone +5.0 βFNA +10 βFNA Fentanyl alone +10 βFNA +20 βFNA l-Methadone alone +10 βFNA Buprenorphine alone +5.0 βFNA +10 βFNA Butorphanol alone +5.0 βFNA +10 βFNA Nalorphine alone +2.5 βFNA +5.0 βFNA +10 βFNA Nalbuphine alone +2.5 βFNA +5.0 βFNA +10 βFNA +20 βFNA Levallorphan alone +2.5 βFNA +5.0 βFNA +10 βFNA BW373U86 alone +10 βFNA
50 h pretreatment Dose ratioa
0.22 (0.13–0.40) 0.25 (0.095–0.63) 1.1 1.0 (0.48–2.09) 4.5b 0.0056 (0.0030–0.011) 0.0032 (0.0014–0.0074) 0.6 0.0035 (0.0018–0.0065) 0.6 0.038 (0.023–0.060) 0.037 (0.020–0.071) 1.0 0.011 (0.0039–0.030) 0.027 (0.010–0.076) 2.5 0.055 (0.017–0.18) 5.0 0.013 (0.0088–0.019) 0.084 (0.015–0.47) 6.5 0.13 (0.026–0.68) 10.0b 0.11 (0.08–0.16) 0.073 (0.044–0.12) 0.7 0.36 (0.17–0.79) 3.3b 0.84 (0.24–3.02) 7.6b 0.08 (0.05–0.14) 0.12 (0.07–0.20) 1.5 0.64 (0.15–2.80) 7.8b 1.39 (0.55–3.53) 17.0b –c 0.122 (0.06–0.239) 0.095 (0.05–0.17) 0.8 0.797 (0.25–2.54) 6.5b 2.68 (0.56–12.9) 22.0b 0.11 (0.058–0.205) 0.13 (0.049–0.352) 1.2
ED50 value 0.22 (0.13–0.40) 0.22 (0.10–0.46) 0.27 (0.06–1.28) 0.0056 (0.0030–0.011) 0.0048 (0.0024–0.0096) 0.0041 (0.0021–0.0082) 0.038 (0.023–0.060) 0.065 (0.044–0.097) 0.012 (0.0053–0.026) 0.011 (0.0077–0.017) 0.020 (0.013–0.030) 0.013 (0.0088–0.019) 0.010 (0.0049–0.019) 0.025 (0.0089–0.072) 0.11 (0.08–0.16) 0.27 (0.11–0.69) 0.14 (0.07–0.29) 0.10 (0.07–0.15) 0.08 (0.05–0.14) 0.19 (0.10–0.35) 0.12 (0.08–0.24) 0.13 (0.07–0.23) 0.24 (0.11–0.51) 0.122 (0.06–0.239) 0.077 (0.05–0.11) 0.253 (0.08–0.81) 0.072 (0.04–0.15) 0.11 (0.058–0.21) 0.12 (0.08–0.17)
Dose ratio 1.0 1.2 0.9 0.7 1.7 0.9 1.7 0.8 1.9 2.5 1.3 0.9 2.4 1.5 1.6 3.0 0.6 2.1 0.6 1.1
curve, with the dose-effect curve then redetermined after 74 and 98 h. During these testing periods, training sessions were not held on the intervening days. Generally, at least 2 weeks intervened between βFNA administrations. To ensure that the dose-effect curves did not change as a function of repeated testing, multiple dose-effect curves were obtained throughout the experiment for fentanyl (n=2 determinations), morphine (n=3), and nalbuphine (n=2). In these cases, mean data were determined for an individual animal, and these means were averaged to produce the group dose-effect curve displayed. Data analysis The percentage of responses on the drug-appropriate key before delivery of the first reinforcer was calculated for each drug and drug dose. This measure was displayed as a function of dose of the drug. ED50 values and 95% confidence limits were determined using loglinear interpolation from at least 3 points on the ascending limb of the dose-effect curve. Data from individual animals were used to determine ED50 values. Dose ratios were calculated by dividing a drug’s ED50 following administration of βFNA by its ED50 in the absence of βFNA. Rightward shifts in the dose-effect curve were considered significant if there was no overlap in the 95% confidence limits of the ED50 values. Tests were also conducted on the slope of the dose-effect curve determined for a drug alone and in combination with βFNA (procedure 11; Tallarida and Murray 1987). With the exceptions of 10 mg/kg βFNA in combination with butorphanol and nalorphine, βFNA failed to alter the slopes of the dose-effect functions. In tests with naloxone, pkB values were determined using the following formula: pkB=–log [B/(DR–1)], where B is the molecular weight of the antagonist and DR is the dose ratio (i.e., ED50 after naloxone/ED50 before naloxone).
Fig. 1 Effects of morphine (n=7) alone and when tested at various time points after pretreatment with 5 and 10 mg/kg βFNA. Data are shown from both the 2-, 50- and 146-h tests and from the timecourse tests (see Materials and methods). Data are presented as the percent drug-appropriate responding before delivery of the first reinforcer as a function of drug dose. Data were excluded from the analysis if the pigeon failed to complete at least 30 responses on either key. ● Alone, ● 2 h, ■ 26 h, ▼ 50 h, ▲ 146 h
23 Drugs The following drugs were used: morphine sulfate, buprenorphine hydrochloride, nalorphine hydrochloride, l-methadone hydrochloride, beta-funaltrexamine (all provided by the National Institute on Drug Abuse); fentanyl citrate (Janssen Pharmaceuticals, Beerse, Belgium): butorphanol tartrate (supplied by Bristol-Meyers, Wallingford, Conn., USA); nalbuphine hydrochloride (Research Biochemical Inc., Natick, Mass., USA); levallorphan tartrate (supplied by Hoffmann-La Roche, Inc., Nutley, N.J., USA); naloxone hydrochloride (Sigma Chemical Co., St Louis, Mo., USA); and BW373U86 (supplied by Burroughs Wellcome Co., Research Triangle Park, N.C., USA). Doses for all drugs are expressed in terms of the salts. All drugs were dissolved in distilled water and administered intramuscularly (IM) in an injection volume of 0.5–1.0 ml/kg. In several cases, the drugs were dissolved in distilled water and a small amount of lactic acid.
Results
and 10 mg/kg βFNA. When administered alone, morphine produced dose-dependent increases in drug-appropriate responding with complete substitution (≥80% drug-appropriate responding) obtained at doses of 1.0 and 3.0 mg/kg. Following pretreatments at 2, 50 and 146 h, a dose of 5 mg/kg βFNA failed to alter the morphine dose-effect curve. As shown in Table 1, this dose of βFNA did not significantly alter morphine’s ED50. The stimulus effects of morphine were antagonized by a 2-h pretreatment of 10 mg/kg βFNA, as indicated by a 4.5-fold rightward shift in the dose-effect curve and a significant increase in the ED50 value. By 26 h after βFNA administration and at all other time points examined, the dose-effect curve of morphine remained at control levels. When administered alone and in combination with βFNA, morphine did not markedly alter rates of responding (data not shown).
Morphine
Fentanyl and l-methadone
Figure 1 shows the effects of morphine alone and when tested at various time points after pretreatment with 5.0
Figure 2 shows the effects of fentanyl and l-methadone alone and following a 2-h pretreatment with selected
Fig. 2 Effects of fentanyl (n=7), l-methadone (n=5), buprenorphine (n=5), butorphanol (n=6), nalorphine (n=6) and levallorphan (n=5) alone and in combination with βFNA administered 2 h before the session on the percentage of morphine-appropriate responding. Data are presented as the percent drug-appropriate responding before delivery of the first reinforcer as a function of drug dose. Data were excluded from the analysis if the pigeon failed to complete at least 30 responses on either key. ● Alone, ◆ +2.5 βFNA, ■ +5.0 βFNA, ● +10 βFNA, ▲ +20 βFNA
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Fig. 4 Effects of BW373U86 (n=5) alone and in combination with 10.0 mg/kg βFNA when administered 2 h before the session, and in combination with 1.0 mg/kg naloxone on the percentage of morphine-appropriate responding. Data are presented as the percent drug-appropriate responding before delivery of the first reinforcer as a function of drug dose. Data were excluded from the analysis if the pigeon failed to complete at least 30 responses on either key. ● Alone, ● +10 βFNA, ▼ +1.0 NLX
buprenorphine dose-effect curve to the right in a dosedependent manner, based on changes in ED50 values, these shifts were not statistically significant (Table 1). The lack of statistical significance was partially due to large degrees of individual differences in sensitivity to βFNA’s effects. That is, some animals were not affected by βFNA, whereas others failed to respond on the drugappropriate key when tested with the highest doses of buprenorphine. When administered alone or in combination with βFNA, buprenorphine failed to significantly alter response rates (data not shown). Fig. 3 Effects of nalbuphine (n=7) alone and when tested at various time points after pretreatment with 2.5, 5.0 and 10 mg/kg βFNA on the percentage of morphine-appropriate responding. Data are presented as the percent drug-appropriate responding before delivery of the first reinforcer as a function of drug dose. Data were excluded from the analysis if the pigeon failed to complete at least 30 responses on either key. ● Alone, ● 2 h, ■ 26 h, ▼ 50 h, ▲ 146 h
doses of βFNA. When administered alone, fentanyl and l-methadone produced dose-dependent increases in drugappropriate responding with complete substitution obtained at the highest doses tested. The dose-effect curves for both fentanyl and l-methadone were not altered by doses as high as 20 and 10 mg/kg βFNA, respectively (see also Table 1). Fentanyl and l-methadone, alone or in combination with βFNA, did not markedly alter rates of responding (data not shown). Buprenorphine Figure 2 also shows the effects of buprenorphine alone and in combination with βFNA, administered 2 h before determination of the dose-effect curve. When administered alone, buprenorphine substituted completely for the morphine stimulus. Although βFNA appeared to shift the
Butorphanol, nalorphine and levallorphan Figure 2 shows the effects of butorphanol, nalorphine and levallorphan alone and following a 2-h pretreatment with selected doses of βFNA. Butorphanol produced dose-dependent increases in drug-appropriate responding with complete substitution obtained at the highest dose tested. The lowest dose of βFNA tested, 5 mg/kg, did not significantly alter butorphanol’s ED 50 (Table 1). The 10 mg/kg dose of βFNA produced a 10-fold increase in ED50 values, and decreased the slope of the dose-effect curve. Even at the highest dose of butorphanol tested (0.3 mg/kg), complete substitution for the morphine stimulus was not obtained. Nalorphine produced dose-dependent increases in drug-appropriate responding, with complete substitution at 0.3 mg/kg. The lowest dose of βFNA tested (2.5 mg/kg) did not antagonize the effects of nalorphine, whereas the 5 and 10 mg/kg doses decreased nalorphine’s potency such that there was a 3.3- and 7.6-fold increase in ED50 values, respectively (Table 1). When combined with 10 mg/kg βFNA, the slope of nalorphine’s dose-effect curve decreased, such that 3.0 mg/kg nalorphine failed to substitute completely.
25 Table 2 ED50 values (±95% CL) for the dose-effect curves of various opioids before and after naloxone administration
ED50 value (±95% CL)
Dose ratioa
pkB (±SEM)b
Fentanyl alone +1 Naloxone
0.0064 (0.0041–0.010) 0.11 (0.038–0.30)
15.7
6.70 (±0.29)
Morphine alone +1 Naloxone
0.20 (0.12–0.32) 2.69 (0.91–7.92)
13.6
6.52 (±0.29)
BW373U86 alone +1 Naloxone
0.11 (0.058–0.20) 0.30 (0.20–0.46)
2.7
4.60 (±1.26)
a
Dose ratio=(ED50 after naloxone)/(ED50 before naloxone) b pkB value=–log [B/(DR–1)], where B is the molecular weight of the antagonist and DR is the dose ratio
Like the other opioids tested, levallorphan substituted completely for the morphine stimulus. Whereas a 2.5 mg/kg dose of βFNA failed to alter the levallorphan dose-effect curve, doses of 5.0 and 10 mg/kg βFNA shifted the levallorphan dose-effect curve 6.5 and 22fold to the right, respectively (Table 1). When administered alone and in combination with βFNA, butorphanol, nalorphine and levallorphan did not produce marked decreases in rates of responding (data not shown). Nalbuphine Figure 3 shows the effects of nalbuphine administered alone and after several doses of βFNA given at pretreatment times ranging from 2 to 146 h. When administered alone, nalbuphine substituted completely for the morphine stimulus. Although the nalbuphine dose-effect curve was not altered by the 2.5 mg/kg dose of βFNA, the 5.0 and 10 mg/kg βFNA doses shifted the nalbuphine dose-effect curve 7.8 and 17-fold to the right, respectively. When a dose of 20 mg/kg βFNA was administered 2 h prior to nalbuphine, the dose-effect curve was shifted to the right. An ED50 value could not be determined, but the magnitude of this rightward shift appeared comparable to that obtained following administration of 10 mg/kg βFNA. Twenty-six hours after administration of 10 mg/kg βFNA, and by 50 h after administration of the other doses of βFNA, the nalbuphine dose-effect curve returned to control levels. Nalbuphine did not produce marked effects on rates of responding when administered alone or in combination with βFNA (data not shown). BW373U86 As shown in Fig. 4, BW373U86 substituted completely for the morphine stimulus, and the BW373U86 dose-effect curve was not altered by a 10 mg/kg dose of βFNA. Figure 4 also shows that a 1.0 mg/kg dose of naloxone produced a 2.7-fold parallel, rightward shift in the BW373U86 dose-effect curve (Table 2), yielding a pkB value for naloxone of 4.6 (±1.26, SEM). This rightward shift was considerably smaller than the 14- and 16-fold parallel, rightward shifts obtained when 1.0 mg/kg naloxone was combined with morphine and fentanyl, respectively (Table 2). Against the stimulus effects of mor-
phine and fentanyl, the pkB values for naloxone were 6.52 (±0.29) and 6.7 (±0.29), respectively. BW373U86 alone or in combination with βFNA did not produce marked effects on rates of responding. When combined with naloxone, BW373U86 produced a dose-dependent decrease in response rate, with the highest doses markedly decreasing rates of responding (data not shown).
Discussion In the present study, l-methadone, fentanyl, morphine, buprenorphine, butorphanol, nalorphine, nalbuphine and levallorphan substituted completely for the morphine stimulus, a finding in accord with numerous studies demonstrating that these drugs produce µ opioid-like stimulus effects (e.g., Shannon and Holtzman 1977; Young et al. 1992; Picker et al. 1993; Morgan and Picker 1996). Although these opioids produced similar substitution patterns, their relative intrinsic efficacy at the µ opioid receptor could be differentiated on the basis of their sensitivity to antagonism by the irreversible antagonist βFNA. In particular, the stimulus effects of nalbuphine, nalorphine and levallorphan were antagonized by a dose of βFNA as low as 5 mg/kg, whereas the stimulus effects of morphine and butorphanol were antagonized by a dose as low as 10 mg/kg. In contrast to these findings, βFNA failed to antagonize the stimulus effects of fentanyl and l-methadone even when tested up to doses as high as 20 and 10 mg/kg, respectively. The stimulus effects of buprenorphine were not significantly antagonized by 5 or 10 mg/kg βFNA, a possible consequence of large individual differences across animals in sensitivity to βFNA. These findings suggest that the ranking of these drugs by relative intrinsic efficacy at the µ opioid receptor is: l-methadone=fentanyl≥buprenorphine≥morphine≥butorphanol>nalorphine=nalbuphine=levallorphan. This rank order closely parallels those obtained in various in vitro studies (e.g. Emmerson et al. 1996) and many in vivo studies (see below) using other methods to determine intrinsic efficacy, suggesting that the in vivo use of βFNA is a useful tool for determining relative intrinsic efficacy. The finding that βFNA antagonized the stimulus effects of morphine is consistent with those reported previously in subjects trained to discriminate morphine from saline (France and Woods 1987; Suzuki et al. 1995; Holtzman 1997). Of particular interest was that in pigeons
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trained with a 10 mg/kg dose of morphine, a 10 mg/kg dose of βFNA shifted the morphine dose-effect curve to the right and downward (France and Woods 1987). In contrast, a 3.0 mg/kg dose of morphine was used to established the discrimination in the present study, and the 10 mg/kg dose of βFNA produced only a 4.5-fold rightward shift in the morphine dose-effect curve. One possible consequence of increasing the training dose of a µ opioid is functionally to increase the relative efficacy requirement of the task (e.g. Young et al. 1992; Picker et al. 1993; reviewed in Young 1991). If these changes in training dose were altering the efficacy requirement, the effectiveness of βFNA should also be altered. In particular, increasing the efficacy requirement should increase the effectiveness of βFNA and vice versa. This suggestion is supported by findings in pigeons trained to discriminate dezocine, a µ opioid with lower efficacy than morphine, from saline, where 10 mg/kg βFNA failed to antagonize the stimulus effects of morphine and produced an approximately 10-fold shift in the nalbuphine dose-effect curve (Picker 1997), as opposed to the 4.5and 17-fold shifts observed in the present study. In the present study, the stimulus effects of fentanyl and l-methadone were not antagonized by βFNA, a finding in accord with these drugs having higher efficacy at the µ opioid receptor than morphine as measured in various in vivo (e.g. Brase 1986; Paronis and Holtzman 1992; Duttaroy and Yoburn 1995) and in vitro (e.g. Magnan et al. 1982; Ivarsson and Neil 1989) preparations. Because it has been demonstrated that the stimulus effects produced by fentanyl and l-methadone are mediated by activity at the µ opioid receptor (Shannon and Holtzman 1976; Picker et al. 1993), it appears that βFNA tested failed to alkylate a sufficient number of receptors to produce a change in the drug’s potency. This is in contrast to a recent finding by Holtzman (1997) in which rats were trained to discriminate morphine from saline, and 10 µg βFNA administered intracisternally shifted the fentanyl dose-effect curve 2.3-fold to the right. One possibility is that the efficacy requirement in the present procedure is lower than in the Holtzman study (1997), resulting in βFNA being less effective. This is supported by the finding that nalbuphine and levallorphan do not substitute completely for the morphine training stimulus used in the Holtzman study (e.g., Shannon and Holtzman 1977), suggesting a higher efficacy requirement than the present discrimination. Other possibilities include potential species differences, differences due to the route of drug administration, or length of pretreatment period. Buprenorphine and butorphanol substitute for high and low training doses of fentanyl and morphine (Negus et al. 1990; Picker et al. 1993). However, there is ample evidence from drug discrimination (e.g., Schaefer and Holtzman 1981; France and Woods 1990) and antinociception (Dykstra et al. 1993; Butelman et al. 1995; Walker et al. 1995; Morgan and Picker 1996) studies to suggest that these opioids have a slightly lower degree of intrinsic efficacy than morphine. In the present study, 10 mg/kg but not 5 mg/kg βFNA shifted the butorphanol dose-effect
curve to the right. The degree of antagonism observed with butorphanol (10-fold) was larger than that observed with morphine (4.5-fold) suggesting that butorphanol has slightly lower efficacy than morphine. In contrast to butorphanol, βFNA pretreatment failed to alter buprenorphine’s ED50 values in a statistically significant manner. In a previous study, βFNA shifted the buprenorphine dose-effect curve to the right further than morphine (6.5fold versus 4.9-fold) (Holtzman 1997). The reasons for these differences are not readily apparent, but may be due partially to the considerable inter-animal variability observed in the present study. In the present study, the stimulus effects of these drugs were antagonized by 5 mg/kg βFNA, a dose lower than that required to antagonize the stimulus effects of morphine, butorphanol or buprenorphine, suggesting lower intrinsic efficacy at the µ opioid receptor. This conclusion is consistent with findings from numerous studies which suggest that nalorphine, nalbuphine and levallorphan have lower intrinsic efficacy at the µ opioid receptor than morphine, as well as butorphanol and buprenorphine (France and Woods 1985, 1990; Zimmerman et al. 1987; Dykstra 1990; Young et al. 1992; Picker et al. 1992, 1993; Morgan and Picker 1996). Taken together, these studies suggest that nalorphine, nalbuphine and levallorphan have lower efficacy than morphine, and that the larger shifts in the dose-effect curves after βFNA administration observed in the present study reflect this lower efficacy. One potential application of irreversible antagonists such as βFNA is to quantify the degree of receptor inactivation through the use of various analytical methods (e.g. Furchgott 1966; Black and Leff 1983; Zernig et al. 1997). One requirement of these analyses is that the maximal effect produced by the agonist and the slope of the dose-effect curve decrease (i.e., flattening of the dose-effect curve) after irreversible antagonist administration. In the present study, neither the slopes of the dose-effect curves nor the maximal effects produced were substantially altered (with very few exceptions); therefore, these analyses could not be conducted. Even though the slopes were not altered, the fact that there was a differential degree of antagonism across agonists suggests βFNA is acting as an “irreversible” antagonist, rather than a “competitive” antagonist. A time course analysis of βFNA’s effects against the stimulus effects of morphine and nalbuphine indicated that there was a substantial degree of antagonism 2 h after administration, and when tested at 26 h the dose-effect curves for these drugs were at control levels, regardless of the initial degree of antagonism. In other studies using pigeons, antagonism of the effects of µ opioids was observed at 10 min and at 2.5 h after βFNA administration (France and Woods 1987; Mattox et al. 1994). These pretreatment times differ considerably from the 24 or 48 h (or longer) pretreatment times used in studies with rats, squirrel monkeys and rhesus monkeys (Gmerek and Woods 1985; Zimmerman et al. 1987; Adams et al. 1990; Mjanger and Yaksh 1991; Suzuki et al. 1995; Pitts et al. 1996; Holtzman 1997). For example in rats, the
27
rate-decreasing effects of butorphanol were antagonized by βFNA 24 h through 18 days after administration and failed to return to control levels until 36 days after administration (Pitts et al. 1996). Similarly, βFNA decreased the total number of µ opioid receptors in rats for at least 18 days and antagonized the reinforcing effects of heroin for 10 days (Martin et al. 1995). If the time it takes until the return to control levels reflects the rate at which new µ opioid receptors are generated (Zernig et al. 1994, 1996), it would appear that there exist profound differences across species in the rate at which new µ opioid receptors are generated after alkylation. It is possible that the administration of an opioid agonist 2 h after βFNA may result in a situation where the agonist protects the receptors from alkylation by βFNA (e.g., Sanchez-Blazquez and Garzon 1989; Paronis et al. 1993), thereby lessening βFNA’s effects. The time course studies show that the morphine and nalbuphine dose-effect curves were at control levels 26 h after βFNA pretreatment, with no intervening agonist treatment. This suggests that βFNA has a very short duration of action in the pigeon compared to other species. The δ opioid BW373U86 substituted for the morphine stimulus, and this effect was not reversible by βFNA. The finding that the pkB value for naloxone as an antagonist of the effects of BW373U86 was lower than those for naloxone against the morphine and fentanyl stimulus is more consistent with a δ than a µ opioid-receptor mediated action. Thus, these results extend previous findings demonstrating that δ opioids can produce µ opioid-like stimulus effects through a non-µ and presumably δ opioid mechanism (Negus et al. 1996; Picker et al. 1996; Picker 1997). Taken together, these findings suggest that irreversible antagonists can be used as tools to group or rank drugs by their intrinsic efficacy in a drug discrimination procedure. This ranking was obtained by examining the patterns of antagonism by βFNA, where differential shifts in the dose-effect curves for the various opioids were used to infer differences in intrinsic efficacy. Furthermore, the results from this study are consistent with a number of others examining the effects of these drugs in various species across many different behavioral and physiological procedures. Acknowledgements Animals used in this study were maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of North Carolina, and the “Guide for the Care and use of Laboratory Animals” (Institute of Laboratory Animal Resources, National Academy Press, 1996). This work was supported by US Public Service Grant DA10277 from the National Institute on Drug Abuse. D. M. was supported by Training Grant DA07244 and Predoctoral Fellowship DA05669 from the National Institute on Drug Abuse. The authors would like to thank Chris Mathewson for technical assistance.
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