Tail-withdrawal Procedure Research Articles

The Effects of Eating a Traditional High Fat/High Carbohydrate or a Ketogenic Diet on Sensitivity of Female Rats to Morphine.

Patients diagnosed with obesity are prescribed opioid medications at a higher rate than the general population; however, it is not known if eating a high fat diet might impact individual sensitivity to these medications. To explore the hypothesis that eating a high fat diet increases sensitivity of rats to the effects of morphine, 24 female Sprague-Dawley rats (n = 8/diet) ate either a standard (low fat) laboratory chow (17% kcal from fat), a high fat/low carbohydrate (ketogenic) chow (90.5% kcal from fat), or a traditional high fat/high carbohydrate chow (60% kcal from fat). Morphine-induced antinociception was assessed using a warm water tail withdrawal procedure, during which latency (in seconds) for rats to remove their tail from warm water baths was recorded following saline or morphine (0.32-56 mg/kg, i.p.) injections. Morphine was administered acutely and chronically (involving 18 days of twice-daily injections, increasing in 1/4 log dose increments every 3 days: 3.2-56 mg/kg, i.p., to induce dependence and assess tolerance). The adverse effects of morphine (i.e., tolerance, withdrawal, and changes in body temperature) were assessed throughout the study. Acute morphine induced comparable antinociception in rats eating different diets, and all rats developed tolerance following chronic morphine exposure. Observable withdrawal signs and body temperature were also comparable among rats eating different diets; however, withdrawal-induced weight loss was less severe for rats eating ketogenic chow. These results suggest that dietary manipulation might modulate the severity of withdrawal-related weight loss in ways that could be relevant for patients.

Behavioral effects of benzylideneoxymorphone (BOM), a low efficacy µ opioid receptor agonist and a δ opioid receptor antagonist.

Opioids remain the drugs of choice for treating moderate to severe pain, although adverse effects often limit use. Drugs acting concomitantly as agonists at μ opioid receptors and antagonists at δ opioid receptors produce antinociceptive effects with a reduced profile of adverse effects; one such drug, benzylideneoxymorphone (BOM), might further limit adverse effects because it appears to have lower pharmacological efficacy than other μ opioid receptor agonists. The current study compared the acute behavioral effects of BOM with the effects of other μ opioid receptor agonists. Discriminative stimulus and rate-decreasing effects were studied in 1 group of 7 rats discriminating 3.2 mg/kg morphine while responding under a fixed-ratio 10 schedule of food presentation. Antinociceptive effects were determined in a second group of 8 rats using a warm water tail withdrawal procedure. Reinforcing effects were evaluated in a third group of 12 rats with a history of remifentanil self-administration. BOM produced morphine-lever responding and both discriminative stimulus and rate-decreasing effects were antagonized by naltrexone. BOM did not markedly increase tail-withdrawal latencies from water maintained at 50 °C and did not substantially attenuate the antinociceptive effects of morphine. BOM was not self-administered and did not change remifentanil self-administration. Some effects of BOM (e.g., discriminative stimulus effects) appear to be mediated by μ opioid receptors; however, BOM is not self-administered by rats, suggesting that it might have limited abuse liability and a reduced profile of adverse effects compared with currently prescribed opioids.

Opioid-enhancing antinociceptive effects of delta-9-tetrahydrocannabinol and amitriptyline in rhesus macaques.

Cannabinoids can enhance the antinociceptive effects of opioids in a synergistic manner, potentially reducing the analgesic dosage of opioids and improving pain therapy. This strategy has also been used as a rationale to combine certain antidepressants and opioids. In this experiment, opioid-induced thermal antinociception was assessed in rhesus macaques using a warm-water tail-withdrawal procedure with 3 water temperatures (40, 50, and 55 °C). In general, the acute antinociceptive effects of intramuscular (i.m.) cumulative doses of heroin were studied alone or in combination with i.m. (-)-trans-delta-9-tetrahydrocannabinol (THC), cannabinol (CBN), or the tricyclic antidepressant amitriptyline. A nonantinociceptive dose of THC (1 mg/kg) shifted the ED50 for the heroin dose-effect curve 3.6-fold leftward at 50 °C and 1.9-fold leftward at 55 °C compared with heroin alone. When the cannabinoid type-1 receptor (CB1R) antagonist, rimonabant, was administered prior to the most effective THC-heroin combination, rimonabant blocked the THC enhancement of heroin antinociception. When CBN (1-3.2 mg/kg) was administered prior to heroin, or 1 mg/kg of CBN was administered prior to a combination of 0.32 mg/kg of THC and heroin, no shifts were evident in the heroin dose-effect curves at either temperature. However, similar to THC, amitriptyline (0.32-1 mg/kg) administered prior to heroin significantly shifted the heroin dose-effect curve leftward. Heroin produced both dose- and temperature-dependent thermal antinociception in nonhuman primates and THC produced opioid-enhancing effects in a CB1R-dependent manner. These effects of THC were not shared by cannabinol, but were quantitatively similar to that of amitriptyline. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Methocinnamox Produces Sustained Antagonism of the Antinociceptive Effects of Morphine and Persistent Decreases in [3H]DAMGO Binding to μ Opioid Receptors in Rat Cortex

The opioid crisis continues in the US despite the availability of pharmacotherapies that are effective in some patients. A novel μ opioid receptor antagonist, methocinnamox (MCAM), has a long duration of action in blocking the effects of opioids, which could provide a substantial advantage over currently available medications, particularly for overdose. MCAM has been shown to attenuate the behavioral effects of μ, but not k, opioid receptor agonists, and this antagonism, which can last for several weeks, is thought to be due to pseudoirreversible binding of MCAM to μ opioid receptors. In the current study, a warm water tail withdrawal procedure was used to examine sustained antagonism by MCAM in one group of 8 rats. This study showed that 10 mg/kg MCAM attenuated the antinociceptive effects of morphine (1.78–17.8 mg/kg) 5 and 13 days after MCAM administration with antagonism beginning to wane 21 days after MCAM. In separate groups of 8 rats, that same dose of MCAM decreased [3H]DAMGO binding to μ opioid receptors in rat cortical homogenates measured 1, 5 and 21 days after MCAM injection, indicating that MCAM occupied μ opioid receptors at all time points tested. For example, maximal binding (Bmax) was 28.51 ± 3.33 and 30.96 ± 1.73 fmol/mg protein 5 and 21 days, respectively, after vehicle administration, and 16.03 ± 4.15 and 12.50 ± 1.27 fmol/mg protein 5 and 21 days, respectively, after administration of 10 mg/kg MCAM. Binding affinity (KD) was not markedly changed after MCAM administration. Thus, MCAM produces a sustained decrease in [3H]DAMGO binding to μ opioid receptors in rat cortex that parallels antagonism of the antinociceptive effects of μ opioid receptor agonists. This finding supports the notion that MCAM blocks the behavioral effects of μ opioid receptor agonists by binding pseudoirreversibly to these receptors. The unique binding profile of MCAM to μ opioid receptors could increase the usefulness of MCAM as a treatment for opioid abuse and overdose.Support or Funding InformationSupported by NIH (R01DA005018 and R01048417) and the Welch Foundation (AQ‐0039).

Effectiveness and selectivity of a heroin conjugate vaccine to attenuate heroin, 6-acetylmorphine, and morphine antinociception in rats: Comparison with naltrexone

BackgroundOne emerging strategy to address the opioid crisis includes opioid-targeted immunopharmacotherapies. This study compared effectiveness of a heroin-tetanus toxoid (TT) conjugate vaccine to antagonize heroin, 6-acetylmorphine (6-AM), morphine, and fentanyl antinociception in rats. MethodsAdult male and female Sprague Dawley rats received three doses of active or control vaccine at weeks 0, 2, and 4. Vaccine pharmacological selectivity was assessed by comparing opioid dose-effect curves in 50 °C warm-water tail-withdrawal procedure before and after active or control heroin-TT vaccine. Route of heroin administration [subcutaneous (SC) vs. intravenous [IV)] was also examined as a determinant of vaccine effectiveness. Continuous naltrexone treatment (0.0032-0.032 mg/kg/h) effects on heroin, 6-AM, and morphine antinociceptive potency were also determined as a benchmark for minimal vaccine effectiveness. ResultsThe heroin-TT vaccine decreased potency of SC heroin (5-fold), IV heroin (3-fold), and IV 6-AM (3-fold) for several weeks without affecting IV morphine or SC and IV fentanyl potency. The control vaccine did not alter potency of any opioid. Naltrexone dose-dependently decreased antinociceptive potency of SC heroin, and treatment with 0.01 mg/kg/h naltrexone produced similar, approximate 8-fold decreases in potencies of SC and IV heroin, IV 6-AM, and IV morphine. The combination of naltrexone and active vaccine was more effective than naltrexone alone to antagonize SC heroin but not IV heroin. ConclusionsThe heroin-TT vaccine formulation examined is less effective, but more selective, than chronic naltrexone to attenuate heroin antinociception in rats. Furthermore, these results provide an empirical framework for future preclinical opioid vaccine research to benchmark effectiveness against naltrexone.

Behavioral Effects of Opioid Full and Partial Agonists During Chronic Buprenorphine Treatment.

Buprenorphine, a partial agonist at the μ-opioid receptor, is commonly prescribed for the management of opioid addiction. Notwithstanding buprenorphine's clinical popularity, the relationship between its effectiveness in attenuating relapse-related behavior and its opioid efficacy is poorly understood. Furthermore, changes in the antinociceptive potency or effectiveness of opioid drugs that might occur during buprenorphine treatment have not been characterized. Here, we address these questions by assessing the ability of daily buprenorphine treatment to protect against the reinstatement of drug-seeking behavior by six opioids differing in efficacy (methadone, heroin, oxycodone, buprenorphine, butorphanol, nalbuphine) and, in separate experiments, by determining how such treatment may modify their antinociceptive effects. In one set of experiments, squirrel monkeys were trained to respond under concurrent schedules (choice) of food or intravenous oxycodone presentations. The priming strength of different opioids during sessions in which saline, rather than oxycodone, was available for intravenous self-administration was determined before and during chronic buprenorphine treatment (0.1 or 0.32 mg/kg per day). In other subjects, antinociceptive effects of the different opioids were assessed using cumulative dosing procedures in a modified warm-water tail withdrawal procedure before and during buprenorphine treatment. Results show that, notwithstanding some tolerance, full agonists retain high efficacy in producing priming and antinociceptive effects. In contrast, both the priming strength and antinociceptive effectiveness of partial agonists were decreased. These results suggest that the utility of buprenorphine in the management of opioid addiction, and how it alters the analgesic effects of opioids, can vary depending on the efficacy of the abused or prescribed opioid. SIGNIFICANCE STATEMENT: Our findings indicate that the pharmacological efficacy of abused opioids may predict the ability of buprenorphine to attenuate their relapse-related priming and analgesia-related antinociceptive effects. This information can help inform physicians as to the effectiveness and limitations of buprenorphine as a pharmacotherapy for opioid addiction.

In vivo Dependence Liability Assessment of Novel Fentanyl Analogues: Science to Guide drug Policy

The emergence of fentanyl analogues as drugs of abuse raises concern regarding their dependence liability. Following prolonged opioid administration, physical dependence leads to increased use, which facilitates the development of tolerance and the dangers of overdose. In animal models, opioid dependence can be inferred by assessing signs of withdrawal, with vertical jumping being the most sensitive and easily quantified in the mouse. In this study, we aimed to characterize the dependence liability of fentanyl, morphine, and six fentanyl analogues by assessing antinociception and withdrawal signs elicited by each compound in the mouse. First, to determine antinociceptive effects, we used a warm‐water tail withdrawal procedure using both 50°C and 55°C water. Latencies until mice removed their tails from the water were recorded, and fully effective doses were determined using a 10 second cut‐off to prevent tissue damage. Dose effect‐curves were generated to determine the potency (ED50) of each compound for eliciting antinociception. All analogues were less potent than fentanyl but more potent than morphine. Additionally, all test compounds were fully effective in both 50°C and 55°C water. Next, to assess withdrawal, we treated animals with 3 injections of morphine, fentanyl or the various fentanyl analogues spaced 12 hours apart. Following the first two injections on day 1, we precipitated withdrawal on day 2, 3 hours after the final injection of agonist, using the nonselective opioid antagonist naloxone. Withdrawal sessions lasted 20 minutes and the number of vertical jumps were recorded for each session. Antagonist dose‐effect curves for withdrawal jumps were generated first using an agonist dose one‐log unit higher than the fully effective antinociceptive dose and precipitating withdrawal with 3, 10, and 30 mg/kg of naloxone. Next, we used 30 mg/kg of naloxone to precipitate withdrawal using increasing unit doses of each test compoewund since maximal jumping was elicited using 30 mg/kg of naloxone. Agonist dose‐effect curves were generated starting with the lowest fully effective antinociceptive dose and increasing in half‐log units. Our results indicate that all of these fentanyl derivatives are capable of eliciting dependence and offer valuable insight into their abuse potential.Support or Funding InformationThese studies supported in part by DEA/NCTR (224‐16‐0505R),) and the University of Arkansas for Medical ScienVA (D‐15‐OD‐002cesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pharmacological Characterization of Mitragynine, the Primary Constituent in Kratom (Mitragyna Speciosa)

Kratom (Mitragyna Speciosa) and associated commercial products are to self‐treat pain and opioid dependence. The Drug Enforcement Agency and Food and Drug Administration continue to monitor this unregulated product. Mitragynine is the primary alkaloid constituent of kratom. In vitro radioligand binding assays demonstrate that mitragynine has affinity for mu‐opioid receptors (MORs), and lesser affinity for alpha2‐adrenergic receptors. The present study characterized the pharmacological effects of mitragynine using various behavioral and physiological measurements in rats. In rats trained to discriminate morphine (3.2 mg/kg, i.p.) from vehicle (20% Tween 80 in saline) under a standard two‐lever operant conditioning procedure using a food reinforcer, morphine (0.32–3.2 mg/kg) and the MOR agonist fentanyl (0.0032–0.178 mg/kg) produced dose‐dependent increases in morphine‐lever responding. Mitragynine produced a maximum of 70% morphine‐lever responding up to a dose (56 mg/kg) that markedly decreased response rate. In rats trained to discriminate mitragynine (32 mg/kg, i.p.) from vehicle (20% Tween 80 in saline), mitragynine (5.6–56 mg/kg) dose‐dependently increased mitragynine‐lever responding. Morphine (up to 32 mg/kg) and fentanyl (up to 0.178 mg/kg) produced a maximum of 75% mitragynine‐lever responding. The opioid antagonist naltrexone (0.032 mg/kg) significantly antagonized both training drugs. The alpha2‐adrenergic receptor agonist clonidine (0.0178–0.178 mg/kg) produced a maximum of 38% and 58% druglever responding in morphine‐ and mitragynine‐trained rats, respectively. The alpha2‐adrenergic receptor antagonist yohimbine (1–10 mg/kg, i.p.) produced a maximum of 21% and 48% druglever responding in the respective discriminations. Yohimbine (3.2 mg/kg) significantly antagonized the discriminative stimulus effects of the training doses of morphine and mitragynine. In a warm water (50°C) tail‐withdrawal procedure, morphine (10–56 mg/kg) dosedependently decreased tail‐withdrawal latency up to 100% maximal possible effect. The maximum possible effect of mitragynine (17.8–56 mg/kg) was 40% up to the largest dose that could be safely studied. When measuring rectal temperature, clonidine (0.01–0.1 mg/kg) and the alpha2‐adrenergic receptor agonist lofexidine (0.056–0.56 mg/kg) produced dose‐dependent hypothermia; the hypothermic effects of both clonidine and lofexidine were antagonized by yohimbine (1 mg/kg). Mitragynine did not significantly alter rectal temperature up to a dose of 56 mg/kg. Consistent with in vitro and ex vivo assays, the current results suggests that mitragynine functions as a low efficacy MOR agonist with discriminative stimulus effects that are overlapping yet distinct from those of morphine. These data suggest that alpha2‐adrenergic receptors represent a component of mitragynine's pharmacological mechanism of action in vivo.Support or Funding InformationSupported by NIDA DA25267 and DA48353.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Methocinnamox: Sustained Antagonism of the Antinociceptive Effects of Morphine and Not Spiradoline in Rats

The opioid epidemic continues in the US. Currently available pharmacotherapies for opioid abuse and overdose include mu opioid receptor agonists (methadone and buprenorphine) and antagonists (naltrexone and naloxone), and while each treatment can be effective, they have limitations. A novel mu opioid receptor antagonist, methocinnamox (MCAM), has a long duration of action in blocking the abuse‐related and toxic effects of opioids, and its antagonist effects are not surmounted by subsequent administration of mu opioid receptor agonists. Consequently, MCAM might overcome the limitations of other antagonists, although other problems could emerge with long‐term blockade of mu opioid receptors (e.g. prescription opioids would be ineffective for treating pain). The current study measured antinociception using a warm water tail withdrawal procedure in rats and examined the persistence, surmountability, and selectivity of antagonism by MCAM. In 1 group of 8 rats, morphine dose‐effect curves were determined 1 day after MCAM administration and periodically thereafter, and in a different group of 8 rats, a dose‐effect curve for the kappa opioid receptor agonist spiradoline was determined 1 day after MCAM administration with morphine dose‐effect curves determined periodically thereafter. Morphine (1.78–17.8 mg/kg) and spiradoline (1–10 mg/kg) dose‐dependently increased the latency for rats to remove their tails from water maintained at 50°C. MCAM (1–10 mg/kg) attenuated the antinociceptive effects of morphine in a dose‐ and time‐dependent manner, with 10 mg/kg MCAM producing a 30‐fold rightward shift in the morphine dose‐effect curve 1 day after it was administered. For at least 37 days after administration of the largest dose of MCAM, doses of 178–560 mg/kg of morphine were needed to produce a maximal antinociceptive effect. In contrast, the spiradoline dose‐effect curve was not changed 1 day after administration of 10 mg/kg MCAM, although in the same rats the antinociceptive effects of morphine were attenuated 4 days later (i.e. 5 days after MCAM administration). Thus, MCAM produces a sustained and selective antagonism of the antinociceptive effects of mu opioid receptor agonists. Under these conditions, antagonism by MCAM was surmounted by very large doses of morphine, although it remains to be determined whether the antinociceptive effects of those doses of morphine are mediated by mu opioid receptors. Nevertheless, the selectivity of MCAM in antagonizing the effects of mu opioid receptor agonists suggests that pain could be treated through mechanisms other than mu opioid receptors in patients that are taking MCAM for opioid abuse or overdose.Support or Funding InformationSupported by NIH (R01DA005018; R01DA07315) and the Welch Foundation (AQ‐0039).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

U50,488 Enantiomers Differentially Contribute to its Analgesic, Diuretic, and Discriminative Stimulus Effects

Kappa (κ) opioid receptor agonists can produce prominent analgesic and diuretic effects. However, these agonists also produce dysphoria, which markedly reduces their clinical utility. The purpose of the present experiment was to determine if the optical isomers of the κ agonist U50,488 ((±)‐trans‐3,4‐dichloro‐N‐methyl‐N‐[2‐(1‐pyrrolidinyl)‐cyclohexyl]benzeneacetamide) might be differentially effective in producing these κ‐mediated effects. In addition, the effects of specific cannabinoids were tested alone and in combination with the U50,488 racemate on several measures of κ activity. To establish some of U50,488's subjective effects as a discriminate stimulus, a group (n=12) of rats were trained to discriminate 5.6 mg/kg from saline under a fixed‐ratio 20 (FR‐20) schedule of food reinforcement. Then, following sessions in which the enantiomers or the cannabinoids were administered, antinociception was assessed in these rats using both a warm‐water tail‐withdrawal procedure and a paw‐withdrawal (Von Frey) procedure. In separate groups of rats (n=6/group), U50,488 and its enantiomers, (−)‐(1S,2S)‐U50,488 and (+)‐(1R,2R)‐U50,488, were administered intracerebroventricularly (ICV) to determine their capacity for producing an aquaresis. With respect to the discrimination procedure, both (−)‐(1S,2S)‐U50,488 and delta‐9‐tetrahydrocannabinol (Δ9‐THC) produced dose‐dependent increases in U50,488‐lever responding, but only (−)‐(1S,2S)‐U50,488 produced full (>80%) substitution. In contrast, both (+)‐(1R,2R)‐U50,488 and the mu opioid receptor agonist morphine (negative control) engendered less than 20% U50,488‐lever responding up to doses that significantly decreased overall response rate. Doses of U50,488 and (−)‐(1S,2S)‐U50,488 that produced U50,488‐appropriate responding also produced antinociception by dose‐dependently increasing both tail‐ and paw‐withdrawal latencies, whereas (+)‐(1R,2R)‐U50,488 was inactive in both assays. Finally, a synergistic interaction between the cannabinoids and κ opioids occurred when a dose of 0.056 mg/kg of CP‐55,940, a non‐selective CB1/CB2 receptor agonist, was administered prior to ineffective doses of U50,448. This dose of CP‐55,940 produced a small effect alone and potentiated the effects of low doses of U50,488 in the paw‐withdrawal assay (i.e., there was an upward shift in the dose‐effect curve). While all of the κ agonists tested produced an aquaresis, as measured by an increase in urine output, an equivalent dose of each produced a rank order of (−)‐(1S,2S)‐U50,488 > U50,488 > (+)‐(1R,2R)‐U50,488. These increases in urine output were also blocked by a pretreatment with the κ antagonist norbinaltorphimine (nor‐BNI), indicating these effects were mediated by κ opioid receptors. Taken together, these data indicate that the (−)‐(1S,2S)‐U50,488 enantiomer produces discriminative, antinociceptive, and diuretic effects that are most similar to those of the racemate, and provide evidence for a synergistic interaction between κ opioids and cannabinoid receptors. Further studies are needed in order to reevaluate the clinical potential of κ opioid receptor agonists as therapeutic targets as either analgesics or diuretics.Support or Funding InformationNIH P30GM106392 (DRK)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effectiveness comparisons of G-protein biased and unbiased mu opioid receptor ligands in warm water tail-withdrawal and drug discrimination in male and female rats

One emerging strategy to address the opioid crisis is the development of mu opioid receptor (MOR) ligands that preferentially signal the G-protein vs. β-arrestin pathway. The present study compared the relative potency and effectiveness of two G-protein biased (GPB)-MOR ligands TRV130 and SR-14968 to five unbiased MOR ligands (NAQ, nalbuphine, buprenorphine, morphine, and methadone) on therapeutic-related (e.g. antinociception) and abuse-related (e.g. discriminative stimulus effects) endpoints. Male and female rats were tested in a warm water tail-withdrawal procedure (50 °C) or trained to discriminate fentanyl (0.04 mg/kg, SC) from saline in a two-lever food-reinforced discrimination procedure. TRV130 and SR-14968 were approximately two-fold more potent to produce fentanyl stimulus effects vs. antinociception. Morphine, fentanyl, and methadone were significantly more potent in the fentanyl discrimination vs. tail withdrawal procedure. In addition, maximum antinociceptive and discriminative stimulus effects of fixed-proportion fentanyl/naltrexone mixtures (1:0.018, 1:0.054, 1:0.18, 1:0.3, and 1:0.54) were used to quantify 1) the relative in vivo efficacy of the two GPB-MOR agonists and five unbiased MOR ligands, and 2) potential species differences in MOR ligand effects between rats and monkeys. The efficacy-effect function generated from the fentanyl/naltrexone mixtures stratified the five unbiased ligands consistent with agonist-stimulated GTPγS binding (NAQ = nalbuphine < buprenorphine < morphine < methadone). However, TRV130 and SR-14968 produced greater antinociception and less fentanyl-like stimulus effects than was predicted. Furthermore, there was a significant positive correlation between rat and monkey antinociceptive effects. Overall, these results demonstrate GPB-MOR agonists produce undesirable abuse-related effects, albeit with slightly better potency and efficacy ratios compared to unbiased agonists.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Concurrent Assessment of the Antinociceptive and Behaviorally Disruptive Effects of Opioids in Squirrel Monkeys

Although the clinical application of opioids for pain management is often hindered by undesired behavioral impairment, preclinical assays of antinociception typically do not provide information regarding the behaviorally disruptive effects of opioids that may accompany their antinociceptive effects. To address this, we modified a warm water tail withdrawal procedure to determine concurrently the effects of opioids on tail withdrawal latency (antinociception) and indices of food-maintained operant behavior (rates of responding and reinforcement density) in squirrel monkeys. Six opioid agonists were tested, and all produced dose-dependent antinociception and impairment of operant behavior. The ratio of median effective dose (ED50) values for both measures (behavioral impairment:antinociception) was used as a quantitative measure of therapeutic index. Nalbuphine had the highest ED50 ratio (4.88), reflecting antinociception with minimal behavioral disruption. Oxycodone, heroin, buprenorphine, and methadone all produced similar ED50 ratios (.82–1.14), whereas butorphanol yielded a significantly lower ED50 ratio (.17) reflecting behavioral disruption at doses producing only minimal antinociception. The antinociceptive and behaviorally disruptive effects of oxycodone and buprenorphine were further characterized using Schild analysis to calculate apparent pA2 values for antagonism of the 2 drugs by naltrexone. These analyses suggest that µ-receptor mechanisms likely mediate the antinociceptive as well as behaviorally disruptive effects of oxycodone (pA2 values: 8.13 and 8.57) and buprenorphine (pA2 values: 8.6 and 7.9). PerspectiveThis article presents an assay that allows for the concurrent assessment of the antinociceptive and behaviorally disruptive effects of opioids. Our results show that the tail withdrawal assay in squirrel monkeys can provide a useful index of the behavioral selectivity with which opioids produce antinociception.

Application of Receptor Theory to the Design and Use of Fixed-Proportion Mu-Opioid Agonist and Antagonist Mixtures in Rhesus Monkeys.

Receptor theory predicts that fixed-proportion mixtures of a competitive, reversible agonist (e.g., fentanyl) and antagonist (e.g., naltrexone) at a common receptor [e.g., mu-opioid receptors (MORs)] will result in antagonist proportion-dependent decreases in apparent efficacy of the agonist/antagonist mixtures and downward shifts in mixture dose-effect functions. The present study tested this hypothesis by evaluating behavioral effects of fixed-proportion fentanyl/naltrexone mixtures in a warm-water tail-withdrawal procedure in rhesus monkeys (n = 4). Fentanyl (0.001-0.056 mg/kg) alone, naltrexone (0.032-1.0 mg/kg, i.m.) alone, and fixed-proportion mixtures of fentanyl/naltrexone (1:0.025, 1:0.074, and 1:0.22) were administered in a cumulative-dosing procedure, and the proportions were based on published fentanyl and naltrexone Kd values at MOR in monkey brain. Fentanyl alone produced dose-dependent antinociception at both 50 and 54°C thermal intensities. Up to the largest dose tested, naltrexone alone did not alter nociception. Consistent with receptor theory predictions, naltrexone produced a proportion-dependent decrease in the effectiveness of fentanyl/naltrexone mixtures to produce antinociception. The maximum effects of fentanyl, naltrexone, and each mixture were also used to generate an efficacy-effect scale for antinociception at each temperature, and this scale was evaluated for its utility in quantifying 1) efficacy requirements for antinociception at 50 and 54°C and 2) relative efficacy of six MOR agonists that vary in their efficacies to produce agonist-stimuated GTPγS binding in vitro (from lowest to highest efficacy: 17-cyclopropylmethyl-3,14β-dihyroxy-4,5α-epoxy-6α-[(3'-isoquinolyl)acetamindo]morphine, nalbuphine, buprenorphine, oxycodone, morphine, and methadone). These results suggest that fixed-proportion agonist/antagonist mixtures may offer a useful strategy to manipulate apparent drug efficacy for basic research or therapeutic purposes.

Antinociceptive effects of mixtures of mu opioid receptor agonists and cannabinoid receptor agonists in rats: Impact of drug and fixed-dose ratio

Pain is a significant clinical problem, and there is a need for effective pharmacotherapies with fewer adverse effects than currently available drugs (e.g., mu opioid receptor agonists). Cannabinoid receptor agonists enhance the antinociceptive effects of mu opioid receptor agonists, but it remains unclear which drugs and in what proportion will yield the most effective and safest treatments. The antinociceptive effects of the mu opioid receptor agonists etorphine and morphine alone and in combination with the cannabinoid receptor agonists Δ9-THC and CP55940 were studied in male Sprague-Dawley rats (n = 16) using a warm water tail withdrawal procedure. The ratio of opioid to cannabinoid (3:1, 1:1, and 1:3) varied for each mixture. Drugs administered alone or as pairwise mixtures of an opioid and a cannabinoid dose-dependently increased tail withdrawal latency. Mixtures with morphine produced supra-additive (CP55940) and additive (Δ9-THC) effects, whereas mixtures with etorphine and either cannabinoid were sub-additive. The interactions were not different among ratios for a particular mixture. The nature of the interaction between opioids and cannabinoids with regard to antinociceptive effects varies with the particular drugs in the mixture, which can have implications for designing combination therapies for pain.

Effects of acute and repeated treatment with the biased mu opioid receptor agonist TRV130 (oliceridine) on measures of antinociception, gastrointestinal function, and abuse liability in rodents.

TRV130 (oliceridine; N-[(3-methoxythiophen-2-yl)methyl]-2-[(9 R)-9-pyridin-2-yl-6-oxaspiro[4.5]decan-9-yl]ethanamine) is a novel mu opioid receptor (MOR) agonist that preferentially activates G-protein versus β-arrestin signaling pathways coupled to MORs. Prevailing evidence suggests that TRV130 and other G-protein-biased MOR agonists may produce therapeutic analgesic effects with reduced adverse effects compared to existing MOR agonists. This study compared the effects of acute and repeated TRV130 administration on measures of antinociception, gastrointestinal function, and abuse liability in rodents. We hypothesized that TRV130 would produce robust and sustained antinociception and abuse-related effects during repeated treatment, but that tolerance would develop to gastrointestinal inhibition. Antinociception was assessed using a warm-water tail-withdrawal procedure in mice. Gastrointestinal function was assessed in mice using an in vivo measure of fecal output and in vitro assays of colonic propulsion and of colon and ileum circular muscle contraction. Abuse liability was assessed in rats using an intracranial self-stimulation (ICSS) procedure. (+)-TRV130 was administered with acute and repeated dosing regimens, and (-)-TRV130 was also examined in the ICSS procedure to assess stereoselectivity. Acute (+)-TRV130 treatment produced robust antinociception, complete inhibition of gastrointestinal function, and weak abuse-related effects. Repeated (+)-TRV130 treatment failed to produce tolerance to antinociception or gastrointestinal inhibition, and abuse-related effects were enhanced by repeated treatment. Effects of acute and repeated (+)-TRV130 in these procedures resemble effects of morphine, with the exception that TRV130 antinociception was more resistant to tolerance. (-)-TRV130 was inactive. These results suggest that TRV130 retains undesirable constipating and abuse-related effects during repeated treatment despite its bias for G-protein signaling.

Controlling for Individual Differences in Temperature Sensitivity Reduces Potency Differences Among Subjects in a Warm Water Tail Withdrawal Procedure in Rhesus Monkeys

Warm water tail withdrawal is a widely used procedure for assessing the antinociceptive effects of drugs, including opioids. Traditionally, this assay uses the same predetermined temperature(s) to measure tail withdrawal latencies for all subjects in a group; however, under some conditions, the potency of drugs for increasing tail withdrawal latencies varies markedly among subjects. That variance might result from individual differences in sensitivity to drug effects and/or individual differences in sensitivity to the thermal stimuli. This study tested the hypothesis that variance in the antinociceptive potency of opioids is due, in part, to baseline differences in sensitivity to thermal stimuli among individual subjects. Four rhesus monkeys were tested using a warm water tail withdrawal assay under two conditions. In the first condition, the antinociceptive effects of morphine (0.56–5.6 mg/kg, s.c.) and fentanyl (0.0056–0.056 mg/kg, s.c.) were assessed with a single water temperature, 54°C. Both morphine and fentanyl dose dependently increased tail withdrawal latencies; however, for both drugs, there was a greater than 3‐fold difference in dose that produced a full effect across subjects. In the second condition, temperature‐effect curves were first determined for individual subjects by varying the water temperature from 40 to 54°C. Tail withdrawal latency decreased in all subjects in a temperature‐dependent manner; however, the lowest temperature that significantly decreased tail withdrawal latency varied depending on the subject. The temperatures that reduced tail withdrawal latency to less than four seconds ranged from 48ºC to 52ºC. When the antinociceptive effects of morphine and fentanyl were redetermined using these temperatures that were functionally equivalent among monkeys, rather than the same temperature (54°C), the previous between‐subject differences in potency were reduced such that effective doses of either morphine or fentanyl were the same among subjects. These results demonstrate that differences among individual subjects in the antinociceptive potency of opioids can be minimized by using functionally equivalent temperatures in a warm water tail withdrawal assay to accommodate for differences in sensitivity to a thermal stimulus. Manipulations that alter drug sensitivity or effectiveness, such as administering drugs in combination, can be assessed in a more quantifiably rigorous manner by controlling for individual variability in temperature sensitivity.Support or Funding InformationSupported by USPHS Grants R01DA05018, T32DA03111, and K05DA017918.

Effects of the imidazoline I2 receptor agonist 2-BFI on the development of tolerance to and behavioural/physical dependence on morphine in rats.

This study examined the effects of imidazoline I2 receptor agonists on the development of tolerance to and physical dependence on repeated morphine treatment in rats. Two groups of rats (n=9 per group) were trained to lever press for sucrose (10%) presentation under a fixed-ratio 10 schedule. The rate-suppressing effects of the opioid receptor ligands morphine and naltrexone and the I2 receptor agonist 2-BFI were examined weekly in rats treated with either daily morphine (20mg·kg(-1) , s.c.), alone or in combination with 2-BFI (10mg·kg(-1) ) for 3weeks. Changes in body weight were measured following naltrexone tests in both groups of rats. In separate experiments, the antinociceptive effects of morphine were assessed using a warm-water tail-withdrawal procedure in rats before and after daily treatments (7days) with morphine (32mg·kg(-1) , i.p.) alone or in combination with various doses of the I2 receptor agonists 2-BFI, BU224 and CR4056. Daily treatment for 3 weeks, with morphine in combination with 2-BFI produced significantly less tolerance to the rate-suppressing effects of morphine and produced a decreased sensitivity to the rate-suppressing effects of naltrexone as well as decreased naltrexone-induced weight loss, compared with morphine-alone group. Repeated treatment for 7days with morphine produced antinociceptive tolerance, which was attenuated by co-administration with 2-BFI, BU224 or CR4056. Imidazoline I2 receptor agonists attenuated the development of tolerance to and physical dependence on morphine, further supporting the therapeutic potential of combining I2 receptor agonists and opioids for pain treatment.

Additive antinociceptive effects of mixtures of the κ-opioid receptor agonist spiradoline and the cannabinoid receptor agonist CP55940 in rats

Pain is a significant clinical problem, and there is a need for pharmacotherapies that are more effective with fewer adverse effects than currently available medications. Cannabinoid receptor agonists enhance the antinociceptive effects of μ-opioid receptor agonists; it is unclear whether they impact the effects of agonists acting at other opioid receptors. κ-Opioid receptor agonists have antinociceptive effects, but their clinical use is precluded by adverse effects; however, their therapeutic potential might be realized if antinociceptive effects could be selectively enhanced. In this study, the antinociceptive effects of the cannabinoid receptor agonist CP55940 and the κ-opioid receptor agonist spiradoline, alone and in combination, were studied in rats (n=7) using a warm water tail-withdrawal procedure. When administered alone, CP55940 (0.032-1.0 mg/kg) and spiradoline (1.0-32.0 mg/kg) increased tail-withdrawal latency, and mixtures of CP55940 and spiradoline (ratios of 1 : 3, 1 : 1, and 3 : 1) produced additive effects. It remains to be determined whether this additive interaction between a κ-opioid receptor agonist and a cannabinoid receptor agonist is selective for antinociception and whether it can be generalized to other drugs.

Characterization of the Discriminative Stimulus Effects of a NOP Receptor Agonist Ro 64-6198 in Rhesus Monkeys.

Nociceptin/orphanin FQ receptor (NOP) agonists have been reported to produce antinociceptive effects in rhesus monkeys with comparable efficacy to μ-opioid receptor (MOP) agonists, but without their limiting side effects. There are also known to be species differences between rodents and nonhuman primates (NHPs) in the behavioral effects of NOP agonists. The aims of this study were the following: 1) to determine if the NOP agonist Ro 64-6198 could be trained as a discriminative stimulus; 2) to evaluate its pharmacological selectivity as a discriminative stimulus; and 3) to establish the order of potency with which Ro 64-6198 produces discriminative stimulus effects compared with analgesic effects in NHPs. Two groups of rhesus monkeys were trained to discriminate either fentanyl or Ro 64-6198 from vehicle. Four monkeys were trained in the warm-water tail-withdrawal procedure to measure antinociception. Ro 64-6198 produced discriminative stimulus effects that were blocked by the NOP antagonist J-113397 and not by naltrexone. The discriminative stimulus effects of Ro 64-6198 partially generalized to diazepam, but not to fentanyl, SNC 80, ketocyclazocine, buprenorphine, phencyclidine, or chlorpromazine. Fentanyl produced stimulus effects that were blocked by naltrexone and not by J-113397, and Ro 64-6198 did not produce fentanyl-appropriate responding in fentanyl-trained animals. In measures of antinociception, fentanyl, but not Ro 64-6198, produced dose-dependent increases in tail-withdrawal latency. Together, these results demonstrate that Ro 64-6198 produced stimulus effects in monkeys that are distinct from other opioid receptor agonists, but may be somewhat similar to diazepam. In contrast to previous findings, Ro 64-6198 did not produce antinociception in the majority of animals tested even at doses considerably greater than those that produced discriminative stimulus effects.

Impact of efficacy at the μ-opioid receptor on antinociceptive effects of combinations of μ-opioid receptor agonists and cannabinoid receptor agonists.

Cannabinoid receptor agonists, such as Δ(9)-tetrahydrocannabinol (Δ(9)-THC), enhance the antinociceptive effects of μ-opioid receptor agonists, which suggests that combining cannabinoids with opioids would improve pain treatment. Combinations with lower efficacy agonists might be preferred and could avoid adverse effects associated with large doses; however, it is unclear whether interactions between opioids and cannabinoids vary across drugs with different efficacy. The antinociceptive effects of μ-opioid receptor agonists alone and in combination with cannabinoid receptor agonists were studied in rhesus monkeys (n = 4) using a warm water tail withdrawal procedure. Etorphine, fentanyl, morphine, buprenorphine, nalbuphine, Δ(9)-THC, and CP 55,940 (2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5-(2-methyloctan-2-yl)phenol) each increased tail withdrawal latency. Pretreatment with doses of Δ(9)-THC (1.0 mg/kg) or CP 55,940 (0.032 mg/kg) that were ineffective alone shifted the fentanyl dose-effect curve leftward 20.6- and 52.9-fold, respectively, and the etorphine dose-effect curve leftward 12.4- and 19.6-fold, respectively. Δ(9)-THC and CP 55,940 shifted the morphine dose-effect curve leftward only 3.4- and 7.9-fold, respectively, and the buprenorphine curve only 5.4- and 4.1-fold, respectively. Neither Δ(9)-THC nor CP 55,940 significantly altered the effects of nalbuphine. Cannabinoid receptor agonists increase the antinociceptive potency of higher efficacy opioid receptor agonists more than lower efficacy agonists; however, because much smaller doses of each drug can be administered in combinations while achieving adequate pain relief and that other (e.g., abuse-related) effects of opioids do not appear to be enhanced by cannabinoids, these results provide additional support for combining opioids with cannabinoids to treat pain.