The kappa-opioid receptor agonist, triazole 1.1, reduces oxycodone self-administration and enhances oxycodone-induced thermal antinociception in male rats

Abstract

Rationale

Triazole 1.1 is a novel kappa-opioid receptor (KOR) agonist reported to produce antinociception without KOR-typical adverse effects. When combined with the mu-opioid receptor (MOR) agonist, oxycodone, triazole 1.1 blocks oxycodone-induced pruritis without producing sedation-like effects in nonhuman primates. However, it is unknown if triazole 1.1 can reduce the abuse-related effects or enhance the antinociceptive effects of oxycodone similarly to other KOR agonists.

Objectives

The aim of the present study was to quantitatively compare the behavioral effects of triazole 1.1 to the KOR agonists, U50,488h and nalfurafine, on oxycodone self-administration and oxycodone-induced thermal antinociception when administered as mixtures with oxycodone.

Methods

In the self-administration study, male Sprague-Dawley (SD) rats (n=6) self-administered intravenous (i.v.) oxycodone alone (0.056 mg/kg/inj) or combined with U50,488h (0.032–0.32 mg/kg/inj), nalfurafine (0.00032–0.0032 mg/kg/inj), or triazole 1.1 (0.32–1.8 mg/kg/inj) under a progressive-ratio schedule of reinforcement. In a hot plate assay, male SD rats (n=6) received i.v. injections of oxycodone (1.0–5.6 mg/kg), U50,488h (1.0–18.0 mg/kg), nalfurafine (0.01–1.0 mg/kg), or triazole 1.1 (3.2–32.0 mg/kg) alone or in combinations of fixed proportion with oxycodone based on the relative potencies of the single drugs. Each study concluded with administration of the KOR antagonist nor-BNI and some degree of retesting of the previous conditions to verify that the behavioral effects were mediated by KOR activation.

Results

All KOR agonists reduced oxycodone self-administration in a dose-dependent manner. Moreover, all single drugs and drug combinations produced dose-dependent, fully efficacious thermal antinociception. All KOR agonist:oxycodone combinations produced either additive or super-additive thermal antinociception. Finally, each KOR agonist was blocked in effect by nor-BNI in both behavioral measures.

Conclusion

This study demonstrates that triazole 1.1 reduces oxycodone’s reinforcing effects and enhances oxycodone-induced antinociception to degrees that are comparable to typical KOR agonists. Given triazole 1.1’s mild adverse-effect profile, developing MOR-KOR agonist combinations from the triazole 1.1 series may render new pain therapeutics with reduced abuse liability.

Introduction

Mu-opioid receptor (MOR) agonists (e.g., oxycodone) are highly efficacious for the treatment of pain but are limited by side effects (e.g., abuse liability; Chan et al. 2017). Kappa-opioid receptor (KOR) agonists are a class of drugs that are being investigated as potential therapeutics to reduce the side effects of MOR agonists (Townsend et al. 2017; Zamarripa et al. 2020a, b). Like MOR agonists, KOR agonists produce antinociceptive effects in several animal models of nociception (for review, see Jones et al. 2016). Additionally, KOR agonists produce additive antinociceptive effects, and super-additive effects in some cases, when combined with MOR agonists (Ko and Husbands 2009; Miaskowski et al. 1992; Minervini et al. 2018; Negus et al. 2008; Townsend et al. 2017). KOR agonists also reduce pruritus in rodents, nonhuman primates, and humans, including MOR-induced pruritus (Beck et al. 2019; Huskinson et al. 2020; Inan et al. 2019; Kamimura et al. 2017; Ko and Husbands 2009). Moreover, KOR agonists have been shown to attenuate the abuse-related effects of drugs of abuse (e.g., of MOR agonists; Bolanos et al. 1996; Freeman et al. 2014; Glick et al. 1995; Kaski et al. 2019; Negus et al. 2008). However, KOR agonists also produce untoward effects (e.g., dysphoria and psychotomimesis) in humans, which has largely precluded their use as therapeutics (MacLean et al. 2013; Ranganathan et al. 2012).

Recently, a series of KOR agonists have been developed that exhibit full efficacy at the KOR but appear to lack many of the KOR-typical adverse effects (e.g., sedation; for review see Mores et al. 2019). One proposed explanation for the atypical behavioral profiles of these drugs is “biased agonism,” typically defined for opioids (at both the MOR and KOR) as a greater selectivity for activating the G-protein pathway relative to β-arrestin-2 recruitment (Mores et al. 2019; Rankovic et al. 2016; Urban et al. 2007). Following on these observations, some have suggested that the activation of the G-protein signaling pathway is the principal mediator of antinociception while the recruitment of β-arrestin-2 may underlie adverse effects such as dysphoria and sedation (Bedini et al. 2020; Brust et al. 2016). For example, the atypical KOR agonist, nalfurafine, which has been reported to be moderately G-protein biased (4 to 7 times more biased relative to U50,488h; Cao et al., 2020; Schattauer et al. 2017), is clinically approved for the treatment of intractable pruritus due in part to the fact that it does not produce dysphoric or psychotomimetic effects in humans (Kumagai et al. 2010, 2012). Similar to typical KOR agonists, nalfurafine produces thermal antinociception (Bolanos et al. 1996; Townsend et al. 2017) and reduces the abuse-related effects of MOR agonists (Endoh et al. 2000; Kaski et al. 2019; Townsend et al. 2017; Townsend 2021; Zamarripa et al. 2020a, b). When administered alone, nalfurafine reportedly does not produce KOR-typical aversion in rodents in conditioned place aversion (Kaski et al. 2019; Liu et al. 2019) or KOR-typical anhedonia in intracranial self-stimulation (ICSS; Liu et al. 2019) at doses that produce inflammatory antinociception. These findings suggest that KOR agonists that favor the G-protein signaling cascade will be more therapeutically selective than typical KOR agonists like U50,488h, a benchmark drug often used to represent an “unbiased” KOR agonist (Brust et al. 2016; Dunn et al. 2018; Schattauer et al. 2017). However, nalfurafine does produce KOR-typical disruption of motor coordination and operant behavior in rodents (Kaski et al. 2019; Lazenka et al. 2017; Liu et al. 2019; Townsend 2021) and KOR-typical suppression of species-typical behavior in rhesus monkeys (Huskinson et al. 2020) at doses that produce antinociception and antipruritic effects, respectively. Together, these findings suggest that the clinical utility of atypical KOR agonists with at least a moderate preference for the G-protein signaling pathway will still be limited by some degree of KOR-typical side effects (e.g., sedation).

More recently, triazole 1.1, a KOR agonist exhibiting high G-protein bias, was introduced (28 times more biased relative to U50,488h; Brust et al. 2016; Zhou et al. 2013). Triazole 1.1 is arguably the most behaviorally atypical of the next-generation KOR agonists for a number of reasons. First, triazole 1.1 produces KOR-mediated antinociception and antipruritic effects in rodents at doses that do not affect motor performance (Brust et al. 2016). Second, antinociceptive doses of triazole 1.1 do not alter dopamine levels in the nucleus accumbens in mice (Brust et al. 2016), a neurochemical consequence that is thought to contribute to dysphoria and aversion (Crowley and Kash 2015; Shippenberg et al. 2001). However, what sets triazole 1.1 apart the most is the fact that it is the only KOR agonist that has been demonstrated to reverse reductions in behavior caused by a nociceptive stimulus (Brust et al., 2016). This is significant because KOR agonists, which are known to produce sedative effects, have historically failed to reverse pain-depressed behavior (Lazenka et al. 2018; Legakis et al. 2020; Leitl et al. 2014; Negus et al. 2010, 2011, 2015), presumably due to concomitant sedation that precludes the ability of these compounds to exhibit therapeutic effects through measures that operationalize antinociception through increases in behavioral rate. Recently, we reported that triazole 1.1, when combined with oxycodone, attenuated oxycodone-induced itch in rhesus monkeys at doses that did not produce other typical KOR-mediated effects (e.g., sedation) that were observed with the typical KOR agonists, salvinorin A and U50,488h (Huskinson et al. 2020). While this finding suggests that triazole 1.1 reduces MOR agonist-induced itch, it has yet to be determined if triazole 1.1 can reduce the reinforcing effects of MOR agonists and enhance MOR agonist-induced antinociception as has been reported with other KOR agonists (Kaski et al. 2019; Ko and Husbands 2009; Minervini et al. 2018; Negus et al. 2008; Townsend et al. 2017; Zamarripa et al. 2020a, b).

The current study investigated the ability of triazole 1.1 to reduce the reinforcing effects of oxycodone and enhance oxycodone-induced thermal antinociception and compared its effects to the KOR agonists U50,488h and nalfurafine in male Sprague-Dawley rats. Dose-response assessments for contingently administered U50,488h, nalfurafine, and triazole 1.1 as reducers of oxycodone self-administration were determined using a progressive-ratio (PR) schedule of reinforcement. Additionally, dose-response assessments for thermal antinociception for oxycodone, U50,488h, nalfurafine, and triazole 1.1 were determined using the hot plate assay. Each KOR agonist was then combined with oxycodone in mixtures of fixed proportions to determine if triazole 1.1 could enhance MOR-mediated thermal antinociception in a manner comparable to U50,488h and nalfurafine.

Method

Subjects

Male Sprague-Dawley rats (Envigo Laboratories, New Jersey, USA) at approximately 65 days of age and weighing 275–299 g on arrival were acclimated to their housing chambers for at least 1 week prior to experiments. Subjects were pair housed with ad libitum access to food and water and were maintained on a reversed 12-h light/dark cycle (lights off at 0700) with experiments occurring during the dark phase. Experiments were conducted in compliance with the National Research Council’s Guide for Care and Use of Laboratory Animals (2011) and approved by the University of Mississippi Medical Center’s Institutional Animal Care and Use Committee.

Surgery

All subjects received intravenous (i.v.) catheters implanted into their right jugular veins as described in Zamarripa et al. (2018). Briefly, catheters were guided subcutaneously to a vascular access port (Instech Laboratories, Plymouth Meeting, PA, USA) that was placed behind the shoulder blades. Subjects received carprofen (5.0 mg/kg; subcutaneous [s.c.]) once pre-operatively and twice post-operatively (24 and 48 h following surgery) for analgesic support. For the 7-day post-surgical period, subjects were treated with the antibiotic, enrofloxacin (5.0 mg/kg; s.c.), to prevent infection, and catheters were infused daily with 0.1 ml of heparinized saline (30 U/ml; i.v.) to facilitate patency. Subjects were allowed to recover for 7 days prior to self-administration sessions. Once testing began, catheters were flushed daily with 0.1 ml of heparinized saline after each self-administration session. Every 14 days, patency was verified by infusions of Brevital (5.0 mg/kg; i.v.; Zamarripa et al. 2018), followed by 0.1 ml of heparinized saline. Catheters were considered patent if ataxia was apparent within 3 sec. If catheters were considered non-functional, a new catheter was implanted into the left jugular vein, and the subject continued in the experiment. Postoperative care after implantation of a second catheter was identical to that described above.

Apparatus

Operant chambers

Six operant test chambers (Med-Associates, St. Albans, VT, USA) were used for the self-administration procedure. Self-administration sessions occurred daily and began at approximately 0800. Operant chambers were equipped with two retractable levers and corresponding white stimulus lights mounted above each lever. Drug injections were delivered at a rate of 0.018 ml/s from 10-ml plastic syringes that were seated in infusion pumps outside of the operant box (Med-Associates, St. Albans, VT, USA). Infusion pumps were connected to the implanted ports using a single, polyethylene tube that was contained in a spring-arm leash (Instech Laboratories). A PC equipped with Med-Associates software (Med-PC for Windows; St. Albans, VT, USA) controlled experimental conditions and recorded data.

Hot plate apparatus

A hot plate analgesiometer (Omnitech Electronics, Columbus, OH, USA) with a plastic enclosure (11″ L, 11″ W, 8″ H) was used to test the thermal antinociceptive effects of the drugs and drug combinations. Thermal antinociception sessions occurred no less than 72 h apart and began at approximately 1630. The hot plate was maintained at 52.5 °C during each experimental session. Latencies were recorded by a blinded observer with a pedal-operated digital timer.

Procedures

Oxycodone self-administration training

Subjects (n=6) were trained to self-administer oxycodone (0.056 mg/kg/injection) on a fixed-ratio 1 (FR1) schedule of reinforcement. The training dose was selected based on previous work showing it to be reliable for acquisition of self-administration and to be a maximally reinforcing dose in rats under a progressive ratio (PR) schedule of reinforcement (Townsend et al. 2017; Zamarripa et al. 2020b). The maximum number of injections for this phase was set at 20, with the session running 2 h or until 20 injections occurred. The response ratio was increased to FR5 once a subject met one of the following criteria: (a) 3 consecutive days of 15 or more injections, (b) 2 consecutive days of 20 injections, or (c) after 14 days of self-administration at FR1 without reaching either of the previous two criteria. Additionally, at least 80% of total session responses had to occur on the active lever. Subsequently, self-administration under the FR5 schedule of reinforcement was maintained until one of the following criteria were met: (a) 3 consecutive days of 15 or more injections, (b) 2 consecutive days of 20 injections. Failure to meet either of these criteria resulted in removal from the study. Notably, all subjects that met the criteria under the FR5 schedule did so by self-administering the total 20 injections of 0.056 mg/kg/injections oxycodone for two consecutive sessions. As such, the less stringent 3-day criterion with fewer injections was not applied.

Once subjects completed training under the FR schedule, subjects self-administered the training dose of oxycodone on a PR schedule of reinforcement. Session time increased to a fixed 4-h time with no limit on injections earned. The response-cost series was derived from a logit equation previously described by Thomsen et al (2005): 19 × [1 + log (step/7–0.3 × step))], rounded to the nearest integer up to step 23, after which the ratio was increased by 12 for all subsequent ratios (e.g., 76, 88, 100), resulting in the following response-cost values: 3, 9, 13, 16, 18, 20, 22, 24, 25, 27, 28, 29, 31, 32, 34, 35, 37, 39, 41, 44, 47, 52, 64, 76, 88, and so forth. Training was considered complete when the following stability criteria were met: (a) injections earned across 3 days were within 20% of the 3-session mean and (b) no upward or downward trends.

Once self-administration was stable under the PR schedule, the oxycodone syringe was substituted with a saline syringe, and responding was extinguished. Training under this condition for each subject was considered complete once the following criteria were met: (a) saline injections earned were at least half the mean number of oxycodone injections, (b) injections earned across 3 days were within 20% of the 3-session mean, and (c) there were no upward or downward trends.

Effects of contingently administered KOR agonists on oxycodone self-administration

Once training was completed, each subject self-administered the training dose of oxycodone alone (0.056 mg/kg/injection) or combined with a range of doses of U50,488h (0.032–0.32 mg/kg/injection), nalfurafine (0.00032–0.0032 mg/kg/injection), or triazole 1.1 (0.32–1.8 mg/kg/injection) under the PR schedule described above. Dose ranges for each KOR agonist included a dose that did not affect oxycodone self-administration up to a dose that reduced oxycodone self-administration to control levels (i.e., comparable to saline level responding). All drug conditions and doses were administered in a counterbalanced fashion such that no subject received the same order of conditions as another subject. Stability criteria were (a) injections earned across 3 days had to be within 20% of the 3-session mean and (b) no upward or downward trends. A saline test condition was included within the counterbalanced sequence as a non-reinforcer control.

After each completed test of a drug combination condition and previous to the subsequent test, the training dose of oxycodone (0.056 mg/kg/injection) was offered for one session on the PR schedule to monitor the stability of responding for the training dose over the time period required to test all drug conditions, and this “return to baseline” session was followed by a series of saline sessions sufficient to reduce responding to within 20% of each subject’s pre-test saline injection baseline. Notably, the mean oxycodone injections for each of these sessions were within 20% of the pre-test baseline injection mean for all subjects, and all subjects decreased responding to within 20% of their pre-test saline levels within 3 sessions of saline availability. As such, all test conditions were conducted within a context of comparable baseline performance.

Following completion of the dose-response determinations for each of the KOR agonists, all subjects received an injection of the long-acting KOR antagonist, nor-binaltorphimine (nor-BNI; 15 mg/kg, s.c.; Flax et at. 2015), at least 24 h prior to self-administration tests to determine if the reduction in oxycodone self-administration was mediated by activity at the KOR. Nor-BNI injections were repeated every 10 days to maintain receptor occupancy (Melief et al. 2011). Testing conditions were comprised of self-administration sessions identical to the testing design described above with the exception that the conditions were constrained to oxycodone alone (0.056 mg/kg/injection) or that same dose of oxycodone combined with the dose of each KOR agonist that produced the greatest reduction in oxycodone self-administration (i.e., the highest doses tested for each KOR agonist): U50,488h (0.32 mg/kg/injection), nalfurafine (0.0032 mg/kg/injection), and triazole 1.1 (1.8 mg/kg/injection). All drug conditions were administered in an irregular order across subjects, and stability criteria and between-test baseline conditions were identical to those described above.

Thermal antinociception

Thermal antinociception was evaluated in a separate cohort of rats than those that were used in the self-administration study. The study began with a total of 8 subjects, with 6 of the subjects completing all of the single-drug and drug combination conditions described below. All rats were implanted with i.v. catheters as described above, and all test drugs were administered i.v. On day 1 of the study, each subject was placed on the unheated apparatus for an acclimation period of 60 s before being removed from the hot plate and placed in their home cage. Following this acclimation exposure period, the surface of the apparatus was heated to 52.5 °C. Each subject received an injection of saline, followed by a 15-min period before being placed on the hot plate for a test of nociception. This injection test series was repeated for a total of 5 serial tests to acclimate the subjects to the cumulative-dosing procedure (described below). Notably, all subjects were removed from the hot plate immediately following a nociceptive response, which was defined as a paw lift or lick, a shake, or vocalization.

All test sessions started with a saline injection followed 15 min later by placement on the hot plate heated to 52.5 °C to serve as a within-session control response. Subjects were removed from the hot plate immediately after emitting a nociceptive response, and latency (in seconds) to emit the response was recorded. Following the saline control test, subjects received increasing doses oxycodone (0.32–5.6 mg/kg), U50,488h (1.0–18.0 mg/kg), nalfurafine (0.01–1.0 mg/kg), or triazole 1.1 (3.2–32.0 mg/kg). All doses of a particular drug were tested within a single session using a cumulative-dosing approach that rendered a dose range in half-log intervals that encompassed a full range effect (≤20 to ≥80% maximum possible effect). If 60 s passed without a nociceptive response, subjects were removed from the hot plate by the experimenter. Tests of nociception occurred 15 min after each injection and were followed immediately after by the next injection in the test series. Oxycodone, U50,488h, nalfurafine, and triazole 1.1 were tested in all subjects on different days in a counterbalanced order, and all tests occurred at least 72 h apart. An experimenter blinded to the test conditions scored and recorded response latencies during the session, while another experimenter administered injections.

To quantify potency for each drug, ED₅₀ values were calculated from the dose-response functions for each subject using nonlinear regression, and these values were averaged across subjects to determine ED₅₀ values for each drug (GraphPad Prism 8.0). Subjects were then tested with oxycodone combined with each KOR agonist in fixed proportions to determine if the combinations produced additive or interactive (i.e., sub- or super-additive) antinociceptive effects. Because it has been reported that drug interactions can be ratio dependent, three ratios were tested for each combination (Tallarida 2000). Specifically, combinations were tested in 1:1, 3:1, and 1:3 proportions of oxycodone:KOR agonist based on the relative ED₅₀ values of the single drugs (see Table 1 for actual dose proportions). Notably, the hot plate study began with 8 subjects, and the averaged ED₅₀ values used to determine the 1:1, 3:1, and 1:3 mixture proportions for testing were based on these values. However, two of the subjects were removed from the study during the mixture tests due to catheter failure and were therefore excluded from the analyses and figures. Because the removal of the two subjects resulted in modest alterations of the averaged ED₅₀ values, the original designation of the proportions of 1:1, 3:1, and 1:3 no longer served as accurate descriptions of the mixture ratios for the six subjects used in the analysis. For this reason, the mixture ratios are described in terms of their absolute dose ratios, with oxycodone fixed at a normalized value of 1 (see Table 1). For all drug combination conditions, tests occurred in a counterbalanced order across the oxycodone-KOR agonist combinations and across the ratios for each combination, and all tests occurred at least 72 h apart.

Table 1.

Predicted additive ED₅₀ (Z_add) and experimentally determined ED₅₀ (Z_mix) values for oxycodone combined with each KOR agonist from the measures of thermal nociception. Also listed are predicted additive ED₂₅ and ED₇₅ (Z_add) and experimentally determined ED₂₅ and ED₇₅ (Z_mix) values for oxycodone and nalfurafine combinations. CI, confidence interval

Combination ratios	Zadd (95% CI)	Zmix (95% CI)	Relationship
ED50 oxycodone:U50,488h
1:1.7	4.0 (3.03–4.97)	4.11 (2.09–6.14)	Additive
1:5	6.15 (4.70–7.62)	7.11 (3.96–10.27)	Additive
1:15	8.29 (6.29–10.3)	16.65 (2.97–30.32)	Additive
ED25 oxycodone:nalfurafine
1:0.02	0.84 (0.55–1.27)	0.40 (0.23–0.67)	Additive
1:0.06	0.57 (0.37–0.85)	0.36 (0.32–0.44)	Additive
1:0.2	0.31 (0.19–0.50)	0.42 (0.17–0.88)	Additive
ED50 oxycodone:nalfurafine
1:0.02	1.44 (0.93–1.96)	0.99 (0.49–1.50)	Additive
1:0.06	1.03 (0.69–1.42)	0.51 (0.39–0.61)	Super-additive*
1:0.2	0.61 (0.33–0.90)	0.52 (0.22–0.82)	Additive
ED75 oxycodone:nalfurafine
1:0.02	2.16 (1.45–3.36)	1.68 (1.12–2.94)	Additive
1:0.06	1.57 (1.03–2.80)	0.63 (0.24–0.78)	Super-additive*
1:0.2	0.99 (0.60–2.24)	2.78 (1.55–6.79)	Additive
ED50 oxycodone:triazole 1.1
1:4	5.66 (3.93–7.39)	5.9 (3.82–7.98)	Additive
1:12	9.4 (6.511–2.42)	14.19 (8.31–20.07)	Additive
1:36	13.27 (9.07–17.47)	5.13 (3.49–6.77)	Super-additive*

^*Significant difference between Z_add and Z_mix (non-overlapping 95% confidence intervals)

Following completion of all drug combination tests, each subject completed an oxycodone redetermination test that was procedurally identical to the original test to determine if oxycodone’s antinociceptive potency or other performance characteristics were affected by the repeated-testing arrangement required for the completion of the full range of drug combination tests in each subject. Subsequently, to verify that the effects of each KOR agonist were in fact KOR-dependent, each subject received an injection of nor-BNI (15 mg/kg, s.c.; Flax et al. 2015) 24 h prior to tests of antinociception, and the individual KOR agonists and oxycodone alone were retested in a counterbalanced order using the cumulative-dosing method described above. During this testing phase, nor-BNI injections were repeated every 10 days to maintain nor-BNI occupation of the KOR (Melief et al. 2011). Due to solubility constraints with triazole 1.1, the highest dose in the dose-response range for each KOR agonist was only increased by one ¼ logarithmic unit over the original dose series (the upper limit of our ability to increase triazole 1.1 dose).

Data analysis

Self-administration

The average number of injections earned per session for saline alone, a discrete dose of oxycodone alone (0.056 mg/kg/injection), or a discrete dose of oxycodone (0.056 mg/kg/injection) combined with each of three doses of U50,488h (0.032–0.32 mg/kg/injection), nalfurafine (0.00032–0.0032 mg/kg/injection), or triazole 1.1 (0.32–1.8 mg/kg/injection) were analyzed using a one-way repeated measures analysis of variance (ANOVA) with the within-subject factor of test condition (i.e., saline alone, oxycodone alone, and oxycodone combined with three doses each of U50,488h, nalfurafine, and triazole 1.1). Bonferroni’s multiple comparisons were used to make planned comparisons according to the following series: (a) saline compared to oxycodone alone or oxycodone combined with three doses each of U50,488h, nalfurafine, or triazole 1.1, and (b) oxycodone alone compared to oxycodone combined with three doses each of U50,488h, nalfurafine, or triazole 1.1. The relative potencies of U50,488h, nalfurafine, and triazole 1.1 to reduce oxycodone self-administration were determined by using nonlinear regression to quantify ED₅₀ values for each subject. For the regression analyses, the top constraint for each KOR agonist was set to the mean number of oxycodone injections for a single subject and the bottom constraint was set to that subject’s saline value. Individual-subject ED₅₀ values were then averaged to determine the overall ED₅₀ value and 95% confidence interval for each KOR agonist (GraphPad Prism 8.0). ED₅₀ values for the KOR agonists were considered to be significantly different if the 95% confidence intervals did not overlap.

To evaluate effects of nor-BNI on the ability of the KOR agonists to reduce oxycodone self-administration, a one-way repeated measures ANOVA was conducted with the within-subject factor of test condition (i.e., saline alone, 0.056 mg/kg/inj oxycodone alone, and this dose of oxycodone combined with 0.32 mg/kg/inj U50,488h, 0.0032 mg/kg/inj nalfurafine, or 1.8 mg/kg/inj triazole 1.1). Due to the long-acting effects of nor-BNI, all antagonism tests were conducted after completion of the dose-response determinations for the KOR agonists. Therefore, the highest doses of each KOR agonist that were tested prior to the antagonism phase of the study were retested after nor-BNI treatment, and the before and after results were compared. Bonferroni’s multiple comparisons were used to make planned comparisons according to the following: (a) saline to oxycodone alone or combined with the highest dose of each KOR agonist before and after nor-BNI administration (U50,488h 0.32mg/kg/injection, nalfurafine 0.0032 mg/kg/injection, triazole 1.1 1.8 mg/kg/injection), and (b) oxycodone alone to oxycodone combined with the highest dose of each KOR agonist before and after nor-BNI administration.

Thermal antinociception

Hot plate latencies were expressed as % maximum possible effect (%MPE) and were calculated using the following equation:

$$\%\text{MPE}=[(\text{test}\text{latency}-\text{saline}\text{latency}\text{latency})/(60-\text{saline}\text{latency})\times 100]$$

where the test latency was the latency for the rat to emit a nociceptive response after administration of a dose of a drug, and saline latency was the latency to emit a response following the initial injection (which was saline). %MPE for each drug condition was plotted as a function of logarithmic dose. Dose ranges were selected such that the lowest dose corresponded to <image> 20% effect and the highest dose corresponded to <image> 80% effect. To compare relative potencies of oxycodone, U50,488h, nalfurafine, and triazole 1.1 in thermal antinociception, the dose-response data for each single-drug condition were fit with nonlinear regression in each subject to determine ED₅₀ values, and these values were averaged to render mean ED₅₀ values and 95% confidence intervals for the drug conditions as described above. ED₅₀ values between single-drug conditions were considered to be significantly different if the confidence intervals did not overlap. Additionally, all dose-response functions were compared to oxycodone for parallelism (i.e., constant relative potency across doses between two or more functions) as described by Tallarida (2000).

Drug-interaction tests for thermal antinociception between oxycodone combined with either U50,488h, nalfurafine, or triazole 1.1 were conducted with dose-addition analysis, which compares predicted additive ED₅₀ values (Z_add) with experimentally determined ED₅₀ values (Z_mix) of each drug combination. Nonlinear regression was used to determine the ED₅₀ values for oxycodone alone and the corresponding KOR agonist alone for each subject as described above, and Z_add values were calculated for each subject for the designated drug combinations as described by Tallarida (2000) and according to the following:

$${\text{Z}}_{\text{add}}\text{=}\text{fA}\text{+}\text{(1}\text{−}\text{f)B}$$

where A was the ED₅₀ value of oxycodone alone, and B was the ED₅₀ value of the corresponding KOR agonist, and ƒ was a mixture-specific fractional multiplier. The value of ƒ was determined using the equation:

$$\text{f}=\text{MR}÷(\text{MR}+{\text{RP}}_{\text{A}})$$

where MR was the mixture ratio of oxycodone divided by the KOR agonist (in terms of concentration) in a particular mixture, and RP_A was the relative potency ratio (in ED₅₀ terms) of oxycodone divided by the KOR agonist when the drugs were tested alone. Thus, the summing of the two ED₅₀ values derived from the single-drug determinations for a mixture of interest (e.g., oxycodone + triazole 1.1) rendered a Z_add value for each subject, and these values were then averaged across subjects to render the Z_add value and 95% confidence interval for a particular drug combination at a particular ratio.

Z_mix values were calculated by summing the ED₅₀ values for each drug within a drug combination of a particular ratio for each subject, and these values were then averaged across subjects to render Z_mix and a 95% confidence interval for a particular drug mixture of a particular ratio. Mean Z_add and Z_mix values were considered to be significantly different if 95% confidence intervals did not overlap. As stated above, parallelism was tested for each function relative to the dose-response determination for oxycodone. Due to differences in the slopes of the oxycodone and nalfurafine dose-effect functions (data not shown), dose-addition analyses were also performed for the ED₂₅ and ED₇₅ effect levels to determine if the nonlinearity between oxycodone and nalfurafine rendered different results in the interaction tests.

The presentation of additive effects between oxycodone combined with each of the three KOR agonists were displayed graphically using isobolograms (Tallrida 2000). Here, mean ED₅₀ values for oxycodone and each KOR agonist were plotted on the x- and y-axes, respectively. These ED₅₀ values were connected by a line representing the coordinates of predicted additive ED₅₀ values of combinations (i.e., line of additivity). If the ED₅₀ coordinates of a combination fell above the line of additivity, a sub-additive interaction was suggested. Conversely, if the ED₅₀ coordinates of a mixture fell below the line of additivity, a super-additive interaction was suggested (i.e., synergy).

The stability of oxycodone’s antinociceptive potency across the repeated tests was determined by comparing the averaged ED₅₀ values across subjects and confidence intervals for the original oxycodone function to the oxycodone redetermination after all of the mixtures were tested. ED₅₀ values were considered to be significantly different if the confidence intervals did not overlap. In addition, %MPE values averaged across subjects at each dose for the two determinations were analyzed using a two-way repeated measures ANOVA with the within-subject factors of time (oxycodone, oxycodone redetermination) and dose, which allowed for direct comparisons between the functions at each dose.

For the antagonism portion of the study, full antagonism of effect was defined as a reduction of antinociception (i.e., %MPE) to 20% or less as determined with visual inspection of the data.

Quantification of the relative potencies

The ED₅₀ values from self-administration and thermal antinociception (described above) were used to calculate within-drug quotients to relate the relative potencies between the two behavioral endpoints for each KOR agonist using the following equation:

$$={\text{ED}}_{50\text{antinociception}}{\text{/}\text{ED}}_{50}{}_{\text{self-adminstration}}$$

Higher relative values rendered from this calculation indicate a trajectory of increased potency for a KOR agonist to reduce oxycodone self-administration relative to its potency to produce thermal antinociception.

Drugs

Oxycodone hydrochloride was generously provided by the National Institute on Drug Abuse (NIDA) Drug Supply Program (Rockville, MD, USA). Nalfurafine hydrochloride, U50,488h, and nor-BNI were synthesized and provided by Dr. Thomas Prisinzano at the University of Kentucky (Lexington, KY, USA). Triazole 1.1 was synthesized and provided by Dr. Bruce Blough at the Research Triangle Institute (Research Triangle Park, NC, USA). Oxycodone, nalfurafine, and U50,488h were prepared in 0.9% sterile saline. Triazole 1.1 was dissolved in a vehicle of 1:1:8 ethanol:Tween 80:sterile water. Nor-BNI was dissolved in a vehicle of 1% lactic acid in sterile water.

Results

Oxycodone self-administration

All six subjects acquired oxycodone self-administration according to the criteria described above (mean = 8.7 sessions from a range of 7–10 sessions). Following acquisition, all six subjects completed all test conditions. Figure 1A illustrates the average number of oxycodone injections earned per session (± SEM) when administered alone (0.056 mg/kg/inj) or as a combination with various doses of U50,488h, nalfurafine, or triazole 1.1. A repeated measures ANOVA revealed a main effect of test condition (F [2.607, 13.03] = 15.64; p = 0.0002). Specifically, Bonferroni’s multiple comparisons revealed that oxycodone alone or as a mixture with nalfurafine at 0.00032 mg/kg/inj or triazole 1.1 at 0.32 mg/kg/inj were all self-administered at levels significantly greater than saline (all p’s < 0.05). Furthermore, Bonferroni’s multiple comparisons revealed that oxycodone mixed with U50,488h at 0.32 mg/kg/inj, nalfurafine at 0.0032 mg/kg/inj, and triazole 1.1 at 1.8 mg/kg/inj were all self-administered at levels significantly lower than oxycodone alone (all p’s < 0.05). The ED₅₀ (95% confidence intervals) values were 0.08 (0.05–0.11), 0.002 (0.0007–0.012), and 1.04 (0.5–1.7) mg/kg for U50,488h, nalfurafine, and triazole 1.1, respectively. The confidence intervals indicated a significant difference (i.e., the confidence intervals did not overlap) between all three KOR agonists in potency to decrease oxycodone self-administration, resulting in the potency order of: nalfurafine > U50,488h > triazole 1.1.

Fig. 1.

Panel A illustrates the mean (± SEM) number of injections earned per session under a progressive-ratio schedule of reinforcement for i.v. saline (lower shaded gray bar), oxycodone alone (0.056 mg/kg/inj; upper shaded gray bar), or this dose of oxycodone combined with U50,488h (0.032–0.32 mg/kg/inj), nalfurafine (0.00032–0.0032 mg/kg/inj), and triazole 1.1 (0.32–1.8 mg/kg/inj). A single number sign (#) indicates significant differences from saline. A single asterisk (*) indicates significant difference from oxycodone alone. Panel B illustrates the effects of nor-BNI pretreatment (15.0 mg/kg; s.c.) on the mean number of injections (± SEM) earned per session under a progressive-ratio schedule of reinforcement for i.v. saline, oxycodone (0.056 mg/kg/inj), or this dose of oxycodone combined with U50,488h (0.032 mg/kg/inj), nalfurafine (0.0032 mg/kg/inj), and triazole 1.1 (1.8 mg/kg/inj). A single asterisk (*) indicates significant difference from saline.A single en dash (−) denotes absence of a condition or treatment; a single pus sign (+) denotes presence of a certain agonist or treatment

Figure 1B illustrates the average number of injections earned per session (± SEM) of oxycodone (0.056 mg/kg/injection) combined with U50,488h (0.32 mg/kg/injection), nalfurafine (0.0032 mg/kg/injection), or triazole 1.1 (1.8 mg/kg/injection) with and without pretreatment with nor-BNI prior to testing. A repeated measures ANOVA revealed a significant main effect of test condition (F[2.312, 11.56] = 31.89; p < 0.0001). Specifically, oxycodone, either alone or when combined with each of the KOR agonists, was self-administered at levels significantly greater than saline following pretreatment with nor-BNI (all p’s < 0.05), indicating that the ability of the KOR agonists to reduce oxycodone self-administration was blocked by KOR antagonism.

Thermal antinociception

Figure 2A illustrates the thermal antinociceptive effects (± SEM) of oxycodone, U50,488h, nalfurafine, and triazole 1.1 aggregated across subjects in the hot plate test. %MPE values are plotted as a function of dose for each drug. All agonists produced full antinociception (i.e., ≥ 80% MPE) at the highest dose tested for each drug, resulting in ED₅₀ values (95% confidence intervals) of 1.86 (1.22–2.49) mg/kg for oxycodone, 12.7 (7.3–18.1) mg/kg for U50,488h, 0.2 (0.020–0.38) mg/kg for nalfurafine, and 17.1 (11.6–22.5) mg/kg for triazole 1.1. The confidence intervals indicated significant differences (i.e., the confidence intervals did not overlap) in potency between most drugs to produce antinociception with a potency order of nalfurafine > oxycodone > U50,488h = triazole 1.1. Figure 2B illustrates the thermal antinociceptive effects for oxycodone, U50,488h, nalfurafine, and triazole 1.1 following pretreatment with the KOR antagonist, nor-BNI. The thermal antinociceptive effects of U50,488h, nalfurafine, and triazole 1.1, but not oxycodone, were completely blocked (i.e., < 20% MPE) by pretreatment with nor-BNI.

Fig. 2.

Panel A: percent of maximum possible effect (%MPE) for thermal antinociception (hot plate) as a function of dose for oxycodone (0.32–5.6 mg/kg; i.v.), U50,488h (1.0–18 mg/kg; i.v.), nalfurafine (0.01–1.0 mg/kg; i.v.), and triazole 1.1 (3.2–32.0 mg/kg; i.v.). Panel B: %MPE for thermal antinociception as a function of dose for oxycodone (0.32–5.6 mg/kg; i.v.), U50,488h (1.0–32 mg/kg; i.v.), nalfurafine (0.01–1.8 mg/kg; i.v.), and triazole 1.1 (3.2–56 mg/kg; i.v.) following pretreatment with the kappa-opioid receptor antagonist, nor-BNI (15.0 mg/kg; s.c.). The gray bars in each panel indicate the saline control condition for each test series, and the width of the bars denote ± SEM. Error bars for the data points also denote ± SEM

Panels A, B, and C of Fig. 3 illustrate the thermal antinociceptive effects (± SEM) of oxycodone combined with U50,488h, nalfurafine, and triazole 1.1, respectively. %MPE values for the drug mixtures are plotted as a function of oxycodone-equivalent dose. All drug combinations, regardless of KOR agonist, produced full antinociception and produced leftward shifts in dose-response functions relative to oxycodone alone. Panels D, E, and F of Fig. 3 illustrate the isobolographic representations of the ED₅₀ values (± 95% confidence intervals) for each of the KOR agonists plotted as coordinates with the ED₅₀ values (± 95% confidence intervals) of oxycodone in each of the drug combinations studied, and Table 1 lists the predicted Zadd ED₅₀ values and the experimentally determined Z_mix ED₅₀ values for the drug combinations. A comparison between the predicted Z_add ED₅₀ values and the experimentally determined Z_mix ED₅₀ values indicated no significant differences (i.e., confidence intervals overlapped) except in three cases. Specifically, the Zmix ED₅₀ values for 1:0.06 oxycodone:nalfurafine and the 1:36 oxycodone:triazole 1.1 combinations were significantly smaller than their respective Z_add ED₅₀ values as determined by the non-overlapping confidence intervals, thus indicating super-additive interactions.

Fig. 3.

Panels A, B, and C illustrate percent maximum possible effect (%MPE) for thermal antinociception (hot plate) as a function of oxycodone dose for oxycodone alone or combined in fixed proportions with A U50,488h, B nalfurafine, and C triazole 1.1. Error bars indicate ± SEM. Panels D, E, and F illustrate isobolographic representations of fixed proportion combinations of oxycodone and D U50,488h, E nalfurafine, and F triazole 1.1. A single asterisk (*) indicates a super-additive interaction at a proportion. Error bars indicate ± 95% confidence intervals.

Due to differences in the slope of the oxycodone and nalfurafine dose-effect functions, additional comparisons were made at the ED₂₅ and ED₇₅ effect levels for nalfurafine (data not illustrated). A comparison between the predicted Z_add ED₇₅ values and the experimentally determined Z_mix ED₇₅ values revealed a significant difference for the 1:0.06 oxycodone:nalfurafine drug combination, indicating a super-additive interaction at this level.

Supplemental Figure 1 illustrates the redetermination of the oxycodone dose-response function, plotted together with the original oxycodone dose-response function. A comparison between the ED₅₀ values indicated no significant difference between the redetermination of oxycodone and the original oxycodone dose-response function (i.e., confidence intervals overlapped). Furthermore, a two-way repeated measures ANOVA revealed a significant main effect of dose (F [3, 30] = 56.38; p < 0.0001) but not time (F [1, 10] = 0.5765; p = 0.4652) nor a dose × time interaction (F [3, 30] = 0.0.1635; p = 0.9201).

Potency relations for reducing self-administration and producing antinociception

Table 2 lists the ED₅₀ values from self-administration and thermal antinociception which were used to calculate a within-drug quotient relating the relative potencies for each KOR agonist to reduce self-administration and produce thermal antinociception. The quotient values were 158.8 for U50,488h, 100 for nalfurafine, and 16.4 for triazole 1.1.

Table 2.

Relative potencies for each KOR agonist expressed as ED₅₀ values (95% confidence intervals) in mg/kg units in self-administration and thermal antinociception. Quotients relate potencies for each KOR agonist in self-administration and thermal antinociception. Higher relative values in the right column indicate a trajectory of increased potency for a KOR agonist to reduce oxycodone self-administration relative to its potency to produce thermal antinociception

	Self-administration	Hot plate	Hot plate/self-administration
U50,488h	0.08 (0.052–0.11)	12.7 (7.3–18.1)	158.8
Nalfurafine	0.002 (0.0007–0.012)	0.2 (0.02–0.38)	100
Triazole 1.1	1.04 (0.5–1.7)	17.1 (11.6–22.5)	16.4

Discussion

The novel findings from the current study were that triazole 1.1 decreased the reinforcing effectiveness and increased the antinociceptive potency of oxycodone in a dose-dependent manner. In the hot plate study, certain ratios of the oxycodone/nalfurafine and the oxycodone/triazole 1.1 combinations produced super-additive antinociception. The KOR antagonist, nor-BNI, reversed all effects of each KOR agonist, indicating that all compounds produced effects through a KOR-dependent mechanism. Ordinal arrangements of potency for the three KOR agonists were comparable for reduction of self-administration and thermal antinociception, though the magnitude of the scalar relation between behavioral endpoints within each KOR agonist markedly differed.

Triazole 1.1 produced a dose-dependent decrease in the reinforcing effects of oxycodone that was comparable to U50,488h and nalfurafine. This finding is in agreement with previous reports that demonstrate that KOR agonists reduce the reinforcing effects of MOR agonists in rodents (Townsend 2021; Townsend et al. 2017; Xi et al. 1998; Zhang and Kreek 2020) and nonhuman primates (Negus et al. 2008; Zamarripa et al. 2020a) and extends these findings to a KOR agonist that exhibits relatively high G-protein signaling bias (Brust et al. 2016). As reviewed above, triazole 1.1 is highly atypical among KOR agonists in terms of its effects on behavior and neurochemistry (Brust et al. 2016). One potential explanation for the KOR-mediated reductions in self-administration is that KOR agonists decrease drug-induced dopamine accumulation within mesocorticolimbic structures related to drug reward (for review see Bruijnzeel 2009). However, unlike U50,488H, triazole 1.1 does not alter brain dopamine levels in the nucleus accumbens or affect locomotor activity in mice at antinociceptive and antipruritic doses (up to 30 mg/kg, s.c.; Brust et al., 2016). Moreover, triazole 1.1 reverses pain-depressed ICSS responding in rats at a dose that does not affect ICSS in the absence of pain (24.0 mg/kg, IP; Brust et al., 2016). In the current report, triazole 1.1, when contingently self-administered with oxycodone, reduced oxycodone self-administration to vehicle levels at a dose of 1.8 mg/kg (i.v.), suggesting that its potency to reduce self-administration is relatively high compared to the measures reviewed above in Brust et al. (2016). Thus, it is possible that triazole 1.1’s ability to reduce oxycodone self-administration occurs independent of dopamine alterations or gross motor impairments. To address the neurochemistry question, a reasonable next step would be to compare the dopamine-altering effects of triazole 1.1 to prototypical KOR agonists in the presence of oxycodone in rats. To address the potential motor-impairment issue, we are currently testing the effectiveness of triazole 1.1 in rhesus monkeys as a deterrent of oxycodone choice in a drug vs. food choice procedure, an approach that renders a measure of reinforcing effect independent of response rate (Banks and Negus, 2017). However, it will be important to conduct similar choice work in rats (e.g., using the methodology of Townsend [2021]) to allow for more meaningful comparisons of KOR agonist and MOR agonist doses across measures of self-administration, antinociception, and other endpoints (e.g., ICSS, in vivo microdialysis).

In the hot plate study, all KOR agonists produced maximally efficacious, KOR-mediated thermal antinociception. These findings agree with previous reports that KOR agonists produce antinociceptive effects (Brust et al. 2016; Kaski et al. 2019; Ko and Husbands, 2009; Leighton et al. 1988; McCurdy et al. 2006; Townsend et al. 2017). Additionally, this is the second report to demonstrate that triazole 1.1 produces thermal antinociception in rodents. Notably, the fully efficacious dose in the current report was 32.0 mg/kg (i.v.), but a similar dose (30.0 mg/kg; s.c.) was not fully efficacious in mice tested in the tail flick assay (Brust et al. 2016). The discrepancies between the two reports may be due to differences including but not limited to approach (hot plate vs. tail flick), species (rat vs. mouse), and/or route of administration (i.v. vs. s.c.).

All oxycodone:KOR agonist mixtures produced at least additive thermal antinociception. The results with both U50,488h and nalfurafine are consistent with previous reports indicating that KOR agonists can enhance MOR-mediated antinociception in rodents (Briggs et al. 2009; Kaski et al. 2019; Minervini et al. 2018; Townsend et al. 2017) and rhesus monkeys (Ko and Husbands 2009; Negus et al. 2008). Nalfurafine produced super-additive effects at the 1:0.06 oxycodone:nalfurafine combination and additive effects at other ratios, which replicates previous findings from our laboratory demonstrating that a similar ratio of oxycodone and nalfurafine produces synergistic antinociception when combined in rats (Townsend et al. 2017). Similar to nalfurafine, combining oxycodone with triazole 1.1 produced additive or super-additive antinociception, indicating that triazole 1.1, while atypical in a number of measures, enhances MOR agonist-induced thermal antinociception as effectively as other KOR agonists.

Although potency orders for each KOR agonist in self-administration and thermal antinociception were the same, the relative potencies between measures within each KOR agonist varied to a high degree. A within-drug quotient was determined for each KOR agonist by dividing the ED₅₀ to produce thermal antinociception by the ED₅₀ to reduce self-administration, and these “windows” indicated that U50,488h was much more potent to reduce oxycodone self-administration than it was to produce antinociception (i.e., a large potency window). The magnitudes of the potency windows for nalfurafine and triazole 1.1 were 63% and 10%, respectively, of the U50,488h window. These results suggest that the doses of triazole 1.1 required to produce antinociception are much closer to the doses required to attenuate the reinforcing effects of MOR agonists like oxycodone. One interpretation from this finding is that the degree of G-protein signaling bias may vary systematically with potency windows relating abuse deterrence to antinociception. Consistent with this notion, nalfurafine, which has been reported to be moderately G-protein biased (Cao et al., 2020; Schattauer et al. 2017), rendered an intermediate potency window among the KOR agonists tested, and triazole 1.1, which is reported to be heavily G-protein biased (Brust et al. 2016), had the smallest potency window. Conclusions about the relation between signaling bias and potency window in light of the current data are premature, however, because the current study did not determine the intracellular signaling profiles for any of the KOR agonists at rat KORs, nor did it evaluate any β-arrestin-2-prefering KOR agonists. Therefore, the only firm conclusion that can be made from the current data is that triazole 1.1 can attenuate the reinforcing effects of oxycodone and augment oxycodone-induced thermal antinociception, but that the relative potencies between the two endpoints for this atypical KOR agonist are much closer than for U50,488h and nalfurafine.

Limitations of the current report must be taken into consideration when interpreting the present findings. First, both behavioral assays were conducted in male rats only. Numerous reports have demonstrated sex differences in behavioral responsivity to both MOR and KOR agonists (Becker and Chartoff 2019; Charoff and Mavrikaki 2015; Lawson et al. 2010; Liu et al. 2013; Negus et al. 2002). While the current study reports positive findings for combinations of oxycodone and triazole 1.1, future studies must address the generality of these findings in female subjects. Second, while the matched routes of administration (i.e., intravenous) between the two behavioral measures facilitated direct comparisons between the three KOR agonists, prescription opioids are most commonly administered orally (da Costa et al. 2014; Leppert et al. 2018). Future work must therefore include tests of oral effects to enhance translation. Third, the current report included only one modality of nociception (thermal). It is therefore unknown if the antinociception results in the current study will generalize to more translational models of nociception (e.g., inflammatory, neuropathic pain). Finally, it should be noted that measures of pain-stimulated behavior like the one used in the current report (i.e., hot plate) can produce false positives with drugs that increase response latencies through gross motor effects (e.g., sedation). Measures of pain-depressed behavior are better suited to separate antinociceptive from motor-impairing effects. As stated above, triazole 1.1 has been reported to reverse pain-depressed behavior in rats, demonstrating that it produces selective antinociception (Brust et al. 2016). However, future work must test the generality of this effect when it is combined with MOR agonists like oxycodone to strengthen conclusions regarding selective antinociception.