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Research ArticleBehavioral Pharmacology

Enhancement of Opioid Antinociception by Nicotinic Ligands

Fernando B. de Moura and Jack Bergman
Journal of Pharmacology and Experimental Therapeutics April 2021, 377 (1) 100-107; DOI: https://doi.org/10.1124/jpet.120.000423

Abstract

Nicotine has previously been shown to augment the antinociceptive effects of μ-opioid agonists in squirrel monkeys without producing a concomitant increase in behavioral disruption. The present studies were conducted to extend these findings by determining the ability of the nicotinic acetylcholine receptor (nAChR) agonist epibatidine and partial α4β2 nAChR agonist varenicline to selectively augment the antinociceptive effects of the μ-opioid receptor (MOR) full agonist fentanyl, the MOR partial agonist nalbuphine, and the κ-opioid receptor (KOR) agonist U69,593 in male squirrel monkeys. Results indicate that both nAChR ligands selectively increased the antinociceptive effects of nalbuphine and that epibatidine increased the antinociceptive effects of U69,593 without altering effects on operant behavior. However, neither epibatidine nor varenicline enhanced the antinociceptive effects of fentanyl, perhaps due to its high efficacy. The enhancement of nalbuphine’s antinociceptive effects by epibatidine, but not varenicline, could be antagonized by either mecamylamine or dihydro-β-erythroidine, consistent with α4β2 mediation of epibatidine’s effects but suggesting the involvement of non-nAChR mechanisms in the effects of varenicline. The present results support previous findings showing that an nAChR agonist can serve as an adjuvant for MOR antinociception and, based on results with U69,593, further indicate that the adjuvant effects of nAChR drugs may also apply to antinociception produced by KOR. Our findings support the further evaluation of nAChR agonists as adjuvants of opioid pharmacotherapy for pain management and point out the need for further investigation into the mechanisms by which they produce opioid-adjuvant effects.

SIGNIFICANCE STATEMENT Nicotine has been shown to augment the antinociceptive effects of μ-opioid receptor analgesics without exacerbating their effects on operant performance. The present study demonstrates that the nicotinic acetylcholine receptor (nAChR) agonist epibatidine and partial α4β2 nAChR agonist varenicline can also augment the antinociceptive effects of nalbuphine, as well as those of a κ-opioid receptor agonist, without concomitantly exacerbating their behaviorally disruptive effects. These findings support the view that nAChR agonists and partial agonists may have potential as adjuvant therapies for opioid-based analgesics.

Introduction

Opioids are among the most commonly prescribed medications for the management of pain; however, their use is limited by adverse effects (e.g., respiratory depression, behavioral inhibition, addiction) (Schug et al., 1992; Paulozzi and Ryan, 2006; Clark and Schumacher, 2017). One approach to this problem has been to identify novel drugs (e.g., the development of biased agonists or nonopioid ligands) that may be effective analgesics absent of opioid-like deleterious effects. Although conceptually appealing, this approach has not yet been fruitful. An alternative strategy involves the adjuvant use of selected nonopioids to increase the analgesic potency of prescription opioids [for review, see Li (2019)]. According to this strategy, combination of the adjuvant and opioid drugs should permit the use of lower doses of opioids for effective pain relief and thereby reduce the intensity of adverse effects. A wide range of nonopioid drugs (e.g., NMDA antagonists, cannabinoid agonists, imidazoline I2 agonists) have been investigated preclinically to evaluate this “opioid-sparing” approach (Maguire and France, 2014, 2018; Siemian et al., 2016; Li, 2017), which has enjoyed some clinical success. For example, α2 adrenergic agonists, such as clonidine, have been shown to reduce the need for postoperative morphine, suggesting that these drugs may effectively function as adjuvants for μ-opioid receptor (MOR)-mediated analgesia (Blaudszun et al., 2012; Engelman and Marsala, 2013).

Recently, using an assay combining nociception (warm water tail withdrawal) and operant food-maintained behavior in squirrel monkeys (Withey et al., 2018a), we found that the nicotinic acetylcholine receptor (nAChR) agonist nicotine increased the antinociceptive potency of MOR agonists at doses below those that exacerbated their disruptive effects on operant behavior (de Moura et al., 2019). These effects of nicotine were nAChR subtype–selective (i.e., not evident with muscarinic ligands) and inversely related to efficacy at MOR (i.e., nicotine was more effective in enhancing the antinociceptive effects of the low-efficacy agonist nalbuphine than the high-efficacy agonist fentanyl). These findings were of particular interest because nicotinic ligands have previously been reported to produce antinociception in their own right (Wewers et al., 1999; Zarrindast et al., 1999; Berrendero et al., 2002; Kyte et al., 2018), providing a functional framework for the idea that an nAChR agonist may have utility as opioid adjuvants.

The present studies were conducted to explore the ability of nAChR agonists to augment the antinociceptive effects of opioids. Using assay conditions previously employed to study nicotine (Withey et al., 2018a; de Moura et al., 2019), the effects of epibatidine and varenicline alone and in combination with the MOR agonists fentanyl or nalbuphine, or the κ-opioid receptor (KOR) agonist U69,593, were investigated in squirrel monkeys. Epibatidine, a high-efficacy agonist that selectively activates the α4β2 nAChR, has previously been evaluated as a candidate analgesic in its own right, whereas varenicline, a relatively nonselective agonist with low efficacy at α4β2 nAChRs, is currently marketed for smoking cessation (Grady et al., 2010(Nirogi et al., 2013)(Rollema et al., 2007)). These ligands, as well as the MOR agonists fentanyl and nalbuphine, were selected for study primarily to evaluate the role of α4β2 nAChR and MOR efficacy in their expected adjuvant effects. The inclusion of U69,593 in these studies permitted an initial evaluation of whether nicotinic agonists might also enhance KOR-mediated antinociception.

Materials and Methods

Subjects.

Adult male squirrel monkeys (n = 4; Saimiri sciureus) were housed in stainless steel cages in a climate-controlled vivarium under a 12-hour light/dark cycle at the McLean Hospital Animal Care Facility (licensed by the US Department of Agriculture). Subjects had unrestricted access to water and were fed a daily allotment of high-protein primate chow (Purina Monkey Chow, St. Louis, MO) supplemented with multivitamins and fruit, per veterinary recommendation. All procedures and protocols were approved by the Institutional Animal Care and Use Committee at McLean Hospital, and housing was compliant with guidelines provided by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animals Resources, Commission on Life Sciences, National Research Council (2011). All subjects previously served in similar studies with nAChR and MOR agonists (de Moura et al., 2019).

Apparatus.

Experimental sessions were conducted 5 days a week, between 9 AM and 1 PM. During experimental sessions, subjects were placed in customized Plexiglas chairs as previously described (Withey et al., 2018a; de Moura et al., 2019). Briefly, the seated subject faced two levers, each 8 cm below a stimulus light that could be illuminated during the session. The subjects also faced and had easy access to a customized Plexiglas receptacle between the levers into which fluid could be delivered for consumption. During sessions, a syringe pump (model PHM-100-10; Med Associates, Inc., Georgia, VT) outside the chamber could be operated to deliver sweetened condensed milk via Tygon tubing into the receptacle’s reservoir (0.15 ml in 0.84 seconds). The rear portion of the chair was designed so that the tail of the subject could hang freely, permitting its end to be immersed in a water-filled container; only the distal 3 to 4 cm of the tail was immersed in water during experiments, allowing the subject to remove its tail from the water at any time without assistance. All experimental events were programmed and recorded through a commercially available interface (MED-PC; Med Associates Inc., St. Albans, VT).

Behavioral Procedures.

The procedures in this study have been previously described (Withey et al., 2018a; de Moura et al., 2019). Briefly, the daily session was composed of four 15-minute components, each divided into a 10-minute long time-out, followed by a 5-minute test period. The test period was initiated by the illumination of red stimulus light above one lever. The completion of 10 responses (FR10) on that lever within 20 seconds (limited hold 20 seconds) terminated the red lights and triggered the delivery of 30% milk in water (v/v) into the reservoir. A 30-second short time-out followed the completion of the FR requirement or the expiration of the limited hold, after which the red stimulus lights were reilluminated and the FR10 schedule was again in effect. During the 30-second time-out, the distal portion of the subject’s tail was immersed in 35, 50, 52, or 55°C water, and the latency to withdraw the tail from the water was recorded. The trial was terminated by withdrawal of the tail from water by the subject or, if 10 seconds elapsed without tail withdrawal, removal of the heated water by the experimenter. The 10-second cutoff served to avoid the possibility of tissue damage. These session parameters permitted at least six tail-withdrawal measurements within each component. The order of water temperatures within and across components was randomized, with the proviso that determinations in each test component occurred no more than once at 50, 52, or 55°C and at least three times at 35°C. The water temperature of 35°C was selected to control for conditioned responses to water because previous data have shown that it does not induce a tail-withdrawal response under nondrug conditions (Withey et al., 2018a).

Drug Testing Procedures.

Dose-response functions for antinociception and decreases in operant response rates produced by the MOR agonists fentanyl, nalbuphine, and the KOR agonist U69,593 (La Regina et al., 1988) were determined in all subjects using cumulative dosing procedures, i.e., by administering graded increasing intramuscular doses at the beginning of successive components (i.e., at the onset of the 10-minute time-out). For example, if the starting dose was 0.32 μg/kg fentanyl, prior to second component, an intramuscular injection of 0.68 μg/kg was administered, 2.2 μg/kg was administered prior to the third component, and 6.8 μg/kg was administered before the fourth and final component, so that a dose-response function including the doses of 0.32, 1, 3.2, and 10 μg/kg fentanyl would be measured through the experimental session. In other experiments, the effects of each opioid were also determined after intramuscular injections of the nAChR agonists epibatidine (0.56 μg/kg) or varenicline (0.032 mg/kg). These doses of the nAChR agonists were selected on the basis of previous data (Withey et al., 2018b) and dose-ranging studies showing that these doses were the highest that did not completely abolish food-maintained responding. In the present experiments, saline or nAChR agonists were administered immediately prior to the session to permit evaluation of their effects in the first component, and the first dose of each opioid was administered in the second component. Subsequent studies to evaluate antagonism of nicotinic modulation of opioid effects were conducted by administering either the nonselective nAChR antagonist mecamylamine or the selective α4β2 nAChR antagonist dihydro-β-erythroidine (DhβE) (Grady et al., 2010; de Moura and McMahon, 2017) 5 minutes prior to the administration of the nAChR agonists or saline vehicle. The doses of mecamylamine and DHβE were selected on the basis of previous studies in squirrel monkeys (Withey et al., 2018b). Drug testing occurred no more than once per week throughout the present studies.

Data Analyses.

Latency to withdraw the tail from water was expressed as the elapsed time from immersion of the tail in water to its removal by the subject or experimenter; operant response rate was measured as responses per second and expressed as a percentage of the subject’s overall rate of responding during control sessions. The effects of successive injections of saline on both tail-withdrawal latency and response rates across components were determined at least monthly and provided baseline values (control sessions). Overall baseline values for tail-withdrawal latency and response rate in the present study are expressed as the means of control values across subjects (±S.E.M). Results from drug experiments are similarly calculated for individual subjects and expressed as the mean of data across subjects (±S.E.M).

Effects of nAChR Agonists Alone or Combined with nAChR Antagonists.

A repeated measures one-way ANOVA was used to compare response rates after administration of saline and varenicline (0.032 mg/kg) or epibatidine (0.56 μg/kg), alone or in the presence of mecamylamine (0.1 mg/kg) or DHβE (0.1 mg/kg). Linear regression was used to fit lines to the linear portion of dose-response functions in individual subjects (GraphPad Prism version 5.0 for Windows; GraphPad Software, San Diego, CA). For response rate, the linear portion of the curve was defined to include not more than one dose that produced <20% of control response rate and not more than one dose that produced >80% of control response rate. For tail-withdrawal latency, the linear portion of the curve was defined to include not more than one dose that produced a <2-second latency and not more than one dose that produced a >8-second latency.

Effects of Opioids Alone or After Treatment with nAChR Agonists or nAChR Agonist-Antagonist Combinations.

Dose-response data for opioids alone or after treatment with nAChR agonists or nAChR agonist-antagonist combinations were fitted to straight lines and were considered significant if the slope differed significantly from 0. The slopes and intercepts of the dose-response functions for an opioid alone or after each type of treatment were compared using an F-ratio test (GraphPad Prism), with statistical significance set at P < 0.05. ED50 values for each type of treatment were determined using linear interpolation. If the dose-response function for an nAChR agonist in combination with an opioid had a slope >0 but the group average latency was not at least 5 seconds, the ED50 value was estimated using linear regression, provided that tail-withdrawal latency in at least half the subjects was >5. Linear regression was used to determine a common slope to calculate potency ratios with corresponding 95% confidence limits (CL) per Tallarida (2000). If the 95% confidence limits of a potency ratio did not include 1, the ED50 values were considered significantly different.

Drugs.

All doses were calculated in terms of the drug’s base weight and were administered intramuscularly. Drugs were fentanyl citrate (Sigma-Aldrich, St. Louis, MO), nalbuphine hydrochloride hydrate (Sigma-Aldrich), (±)-epibatidine dihydrochloride hydrate (Sigma-Aldrich), varenicline tartrate (Sigma-Aldrich), dihydro-β-erythroidine (Tocris Biosciences, Minneapolis, MN), and mecamylamine hydrochloride (Sigma-Aldrich).

Results

Control Response Rate and Tail-Withdrawal Latencies.

Control rates of operant responding (i.e., after saline administration) were 2.81 (±0.82) responses per second. Tail-withdrawal latencies (means ± S.E.M.) after the administration of saline averaged 1.3 (±0.19), 1.2 (±0.14), and 1.1 (±0.08) seconds at 50, 52, and 55°C, respectively (Fig. 1).

Fig. 1.
Fig. 1.

Rate-decreasing effects and tail-withdrawal latencies at 50, 52, and 55°C for fentanyl alone (open circles) or in combination with varenicline (top panels, gray circles) or epibatidine (bottom panels, gray circles) (n = 4). Left panel: ordinate, response rate normalized to baseline rate of responding. Right panels: ordinate, tail-withdrawal latency in seconds. Error bars depict ±S.E.M. Sec=seconds.

Effects of Opioids Alone.

Dose-response data for the effects of opioids on operant behavior and tail-withdrawal latency are shown in Fig. 1⇓–3; ED50 values are summarized in Table 1. The high-efficacy MOR agonist fentanyl dose-dependently decreased rates of responding while concomitantly increasing tail-withdrawal latencies at all warmed water temperatures (Fig. 1, left panels; Table 1). In contrast, doses up to 3.2 mg/kg of the low-efficacy MOR agonist nalbuphine did not appreciably alter rates of responding and increased tail-withdrawal latency at only two of the three water temperatures (50 and 52°C) (Fig 2,3; Table 1). Nalbuphine was without antinociceptive effect when the water temperature was 55°C. The KOR agonist U69,593, like fentanyl, dose-dependently decreased the rate of operant responding and increased tail-withdrawal latencies at all water temperatures (Fig 4).

Fig. 2.
Fig. 2.

Rate-decreasing effects and tail-withdrawal latencies at 50, 52, and 55°C for nalbuphine alone (open circles) or in combination with varenicline (top panels, gray circles), varenicline + 0.1 mg/kg mecamylamine (top panels, filled circles), epibatidine (bottom panels, gray circles), or epibatidine + 0.1 mg/kg mecamylamine (bottom panels, filled circles) (n = 4). Left panel: ordinate, response rate normalized to baseline rate of responding. Right panels: ordinate, tail-withdrawal latency in seconds. Error bars depict ±S.E.M. Sec=seconds

Fig. 3.
Fig. 3.

Rate-decreasing effects and tail-withdrawal latencies at 50, 52, and 55°C for nalbuphine alone (open circles) or in combination with varenicline (top panels, gray circles), varenicline + 0.1 mg/kg DHβE (top panels, filled squares), epibatidine (bottom panels, gray circles), or epibatidine + 0.1 mg/kg DHβE (bottom panels, filled squares) (n = 4). Left panel: ordinate, response rate normalized to baseline rate of responding. Right panels: ordinate, tail-withdrawal latency in seconds. Error bars depict ±S.E.M. Sec=seconds.

View this table:
TABLE 1

ED50 values and potency ratios of fentanyl, nalbuphine, and U69,593 alone and in combination with various doses of epibatidine or varenicline

ED50 values in mg/kg with corresponding 95% confidence limits for fentanyl, nalbuphine, and U69,593 alone and in the presence of various doses of epibatidine or varenicline in 50, 52, and 55°C warm water. Potency ratios are calculated when available with corresponding 95% confidence limits. Increases in antinociceptive potency are considered statistically significant if the 95% confidence limits of the potency ratios of drug alone or in the presence of nicotine does not include 1. PR=Potency Ratio; Var=varenicline; Epi=epibatidine; NA=not available.

Overall, the magnitude of antinociception produced by each of the three opioids was inversely related to the water temperature (Table 1). Raising the temperature of water from 50 to 55°C decreased the maximum latency to tail withdrawal from 8.1 (±1.1) to 7.0 (±2.0) seconds after fentanyl, from 7.6 (±1.4) to 1.8 (±0.30) seconds after nalbuphine, and from 10 (±0) to 8.1 (±1.1) seconds after U69,593. Decreases in the magnitude of antinociceptive effects in 55°C water were accompanied by decreases in opioid potency from values recorded in 50°C water, as reflected by >2-fold decreases in potency for fentanyl [2.5 (0.6–11)] and U69,593 [2.2 (0.7–6.4)].

Modification of Opioid Effects by Varenicline and Epibatidine.

Varenicline (0.032 mg/kg) alone or after injection of mecamylamine (0.1 mg/kg) or DHβE (0.1 mg/kg) did not significantly disrupt operant response rate (F3,15 = 4.1, P = 0.11), whereas epibatidine (0.56 μg/kg) significantly decreased rates of responding. The rate-decreasing effects of epibatidine were not significantly antagonized by either mecamylamine or DHβE (F4,37 = 2.5, P = 0.058). When administered prior to saline, neither varenicline nor epibatidine significantly increased tail-withdrawal latencies to <2.5 seconds (Fig 1⇑-3).

Varenicline and epibatidine did not significantly alter the potency with which fentanyl or nalbuphine decreased rates of operant responding (P > 0.05; Fig 1⇑-3). Similarly, neither varenicline (0.032 mg/kg) nor epibatidine (0.56 μg/kg) significantly enhanced the antinociceptive effects of the high-efficacy MOR agonist fentanyl regardless of water temperature (P > 0.05; Fig. 1). In contrast, the antinociceptive effects of the low-efficacy MOR agonist nalbuphine were significantly augmented by both epibatidine and varenicline P > 0.05; Fig 2,3; Table 1). Epibatidine markedly increased the antinociceptive potency of nalbuphine at 50 and 52°C warmed water and increased the tail-withdrawal latency from 55°C water for nalbuphine alone [from 1.8 (±0.30) to 6.5 (±2.2) seconds; Fig 2,3, bottom panels]. The effects of the low-efficacy α4β2 nAChR agonist varenicline on nalbuphine’s antinociceptive effects were of a lesser magnitude than those of the high-efficacy α4β2 nAChR agonist epibatidine, and varenicline approximately doubled the latency to tail-withdrawal from 55°C water compared with nalbuphine alone [to 3.5 (±2.2) seconds; Fig 2,3, top panels].

The nonselective and α4β2-selective nAChR antagonists mecamylamine (0.1 mg/kg) and DHβE (0.1 mg/kg), respectively, were used to examine mechanisms by which epibatidine and varenicline might enhance nalbuphine’s antinociceptive effects. Pretreatment with either nAChR antagonist attenuated the augmentation of the antinociceptive effects of nalbuphine by epibatidine [50°C: (mecamylamine: F2,28 = 4.7, P = 0.018), (DHβE: F2,28 = 5.3, P = 0.011); 52°C: (mecamylamine: F2,28 = 4.1, P = 0.027), (DHβE: F2,28 = 3.6, P = 0.045)] (Fig 2,3, bottom panels). However, neither nAChR antagonist blocked varenicline’s ability to enhance the antinociceptive effects of nalbuphine [50°C: (mecamylamine: F2,19 = 1.5, P = 0.26), (DHβE: F2,19 = 0.69, P = 0.51); 52°C: (mecamylamine: F2,19 = 0.41, P = 0.67), (DHβE: F2,19 = 0.58, P = 0.57)] (Fig 2,3, top panels).

As shown in Fig 4, epibatidine did not significantly alter the rate-decreasing effects of U69,593 (F2,15 = 0.26, P = 0.77) but did enhance its antinociceptive effects. Based on ED50 values, the potency of U69,593 to engender tail-withdrawal latency from water temperatures was increased up to 5.1-fold after epibatidine pretreatment (Fig. 4; Table 1).

Fig. 4.
Fig. 4.

Rate-decreasing effects and tail-withdrawal latencies at 50, 52, and 55°C for U69,593 alone (open circles) or in combination with epibatidine (gray circles) (n = 4). Left panel: ordinate, response rate normalized to baseline rate of responding. Right panels: ordinate, tail-withdrawal latency in seconds. Error bars depict ±S.E.M. Sec=seconds

Discussion

The present results show that the high-efficacy MOR agonist fentanyl dose-dependently increased tail-withdrawal latency at all temperatures (50, 52, and 55°C) and consistently decreased rates of operant responding, whereas the low-efficacy MOR agonist nalbuphine increased tail-withdrawal latencies at 50 and 52, but not 55°C, and had no significant rate-decreasing effects. The efficacy-related effects of fentanyl and nalbuphine in the present study are consistent with previously described effects of other MOR agonists on concomitantly measured nociception and operant behavior (Withey et al., 2018a; de Moura et al., 2019). The present results also extend previous findings with nicotine (de Moura et al., 2019), showing enhancement of the MOR partial agonist nalbuphine’s antinociceptive effects by both the full α4β2 agonist epibatidine and the partial α4β2 agonist varenicline.

In contrast to previous findings with nicotine (de Moura et al., 2019), neither epibatidine nor varenicline enhanced the antinociceptive effects of the MOR high-efficacy agonist fentanyl at doses studied here. These findings strengthen the idea that the effectiveness of nAChR ligands in enhancing MOR antinociception may be inversely related to the efficacy of the MOR agonist. However, the relationship between the reported efficacy of nicotinic ligands and their adjuvant effects on opioid antinociception are less certain. On the one hand, the greater enhancement of opioid antinociception by epibatidine than varenicline in the present studies suggests that the magnitude of such adjuvant effects may also be related to the efficacy of the nAChR ligand, presumably at the α4β2 subtype of nAChR. On the other hand, nicotine (de Moura et al., 2019), but not epibatidine (present study), enhanced fentanyl’s antinociceptive effects, even though both nAChR ligands are considered to be full α4β2 agonists.

The inability of epibatidine to enhance the antinociceptive effects of fentanyl in the present study was unexpected but may have been related to the dose of epibatidine tested. In an earlier study (de Moura et al., 2019), the dose of nicotine that enhanced fentanyl’s antinociceptive potency without disrupting operant behavior was 1/4 log unit higher than the dose that increased the antinociceptive potency of nalbuphine whereas, in the present study, the highest dose of epibatidine (0.0056 mg/kg) that did not substantively decrease operant behavior was the same with both fentanyl and nalbuphine. Possibly, a higher dose of epibatidine would have enhanced the antinociceptive effects of fentanyl, albeit with a loss of behavioral selectivity, i.e., increases in antinociception coupled with decreases in the rates of operant responding. Alternatively, the differing effects of nicotine and epibatidine may reflect functionally meaningful differences in their nAChR subtype selectivity. Nicotine is relatively nonselective in activating different subtypes of nAChRs, whereas epibatidine is approximately 10-fold more selective than nicotine in binding to α4β2 sites than to other nAChR subtypes (e.g., α7; Grady et al., 2010). Perhaps actions mediated via other subtypes of nAChR contributed to the adjuvant effects of nicotine in a behaviorally selective manner, i.e., enhancing opioid antinociception without disrupting operant behavior. However, this explanation, although attractive, does not easily reconcile the ability of the α4β2-selective antagonist DHβE, like the nonselective antagonist mecamylamine, to attenuate the adjuvant effects of epibatidine with nalbuphine. In the absence of data with other subtype-selective nAChR antagonists, the ability of nAChR ligands to activate α4β2 nAChRs seems to be the most likely mechanism mediating nAChR enhancement of opioid antinociception.

Like nicotine and epibatidine, the α4β2 nAChR partial agonist varenicline enhanced nalbuphine’s antinociceptive effects without evidence of behavioral disruption. Surprisingly, the adjuvant effects of varenicline were not antagonized by doses of DHβE or mecamylamine that could attenuate the adjuvant effects of nicotine and epibatidine. This lack of antagonism may indicate that the doses of either DHβE or mecamylamine were not sufficient to attenuate the adjuvant effects of varenicline. However, this suggestion is difficult to reconcile with the idea that the nAChR mechanisms that mediate varenicline’s opioid-adjuvant effects are similar to those that mediate the effects of nicotine or epibatidine. An alternative explanation is that non–α4β2-mediated mechanisms contribute more prominently to the opioid-adjuvant effects of varenicline than to those of nicotine or epibatidine. For example, in contrast to the α4β2 subtype selectivity of epibatidine, the affinity of varenicline at the α7 subtype of nAChR is comparable to its affinity at the α4β2 subtype (Grady et al., 2010). Previous reports also have noted varenicline is insensitive to in vivo antagonism by DHβE [(de Moura and McMahon, 2016, 2017, 2019); but see Moerke et al. (2017)]. From this perspective, varenicline may also be less sensitive to mecamylamine’s nonsurmountable actions at nAChRs, thus requiring higher pretreatment doses for antagonism. Because of its high sequence homology to nAChRs (Werner et al., 1994), the 5HT3 receptor has also been investigated as a potential off-target site of varenicline. Indeed, varenicline has been shown to be a potent full agonist at 5HT3 receptors (Lummis et al., 2011). Thus, it is possible that the effects of varenicline on 5HT3 receptors could be producing the opioid antinociceptive enhancing effects, thereby explaining why mecamylamine and DHβE failed to antagonize varenicline. However, this is unlikely inasmuch as inhibition, but not activation, of 5HT3 receptors has been shown to produce analgesia or antinociception (Riering et al., 2004). At this time, it is unclear whether and how non–α4β2 nAChR actions of varenicline may have contributed to the present results.

KOR agonists have well documented antinociceptive properties (Negus et al., 2010; Le Naour et al., 2014(Negus et al., 2008)(Negus et al., 2012)) but, because of unacceptable side effects, have limited promise for the management of pain (Pfeiffer et al., 1986; Wadenberg, 2003; MacLean et al., 2013; Ranganathan et al., 2012). Nevertheless, KOR agonists continue to be investigated as candidate medications for pain management because, unlike most MOR agonists, they do not display significant abuse potential (Ranganathan et al., 2012) and have only limited respiratory depressant effects (Shook et al., 1990). The ability of epibatidine to enhance the antinociceptive potency of the KOR agonist U69,593 without concomitant behavioral disruption suggests that nAChR agonists could play a role in the further development of KOR-based analgesics. However, the present data with U69,593 must be viewed cautiously, as the magnitude of epibatidine’s effect on KOR antinociception was comparatively low—less than its enhancement of the effects of nalbuphine. It may be that the ability of nAChR ligands to modulate KOR antinociception is less pronounced than their ability to mediate MOR antinociception. Alternatively, considering that U69,593 is a high-efficacy KOR agonist (Butelman et al., 2004), it also may be that epibatidine more effectively enhances the antinociceptive effects of low-efficacy than high-efficacy KOR agonists, as appears to be the case for low- versus high-efficacy MOR agonists. Additional studies comparing the ability of nicotinic ligands to modulate antinociception produced by KOR agonists varying in efficacy are necessary to evaluate the viability of this novel strategy for pain management.

In summary, the present results support the hypothesis that nAChR agonists can, under suitable conditions, serve as adjuvants for opioid-based analgesia. Furthermore, our findings show that the magnitude of such effects may be inversely related to opioid agonist efficacy and perhaps directly related to nAChR agonist efficacy, suggesting that full agonists at nAChRs combined with opioid partial agonists, e.g., epibatidine with nalbuphine, may optimize adjuvant enhancement of opioid analgesia. Finally, the adjuvant effects of nAChR agonists appear to be mediated, at least in part, by α4β2 nAChR activation, although the role of other nAChR mechanisms deserves closer scrutiny.

Acknowledgments

The authors would like to thank Dr. Roger D. Spealman for comments on an earlier version of this manuscript and Margaret Branham for her technical assistance.

Authorship Contributions

Participated in research design: de Moura, Bergman.

Conducted experiments: de Moura.

Performed data analysis: de Moura.

Wrote or contributed to the writing of the manuscript: de Moura, Bergman.

Footnotes

    • Received November 17, 2020.
    • Accepted January 11, 2021.

  • This work was supported by National Institutes of Health National Institute on Drug Abuse [Grant R01 DA035857] and the Harvard Medical School Livingston Fellowship.

  • No author has an actual or perceived conflict of interest with the contents of this article.

  • https://doi.org/10.1124/jpet.120.000423.

Abbreviations

CL
confidence limit
DHβE
dihydro-β-erythroidine
KOR
κ-opioid receptor
MOR
μ-opioid receptor
nAChR
nicotinic acetylcholine receptor

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Research ArticleBehavioral Pharmacology

Varenicline and Epibatidine as Opioid Adjuvants

Fernando B. de Moura and Jack Bergman
Journal of Pharmacology and Experimental Therapeutics April 1, 2021, 377 (1) 100-107; DOI: https://doi.org/10.1124/jpet.120.000423
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