Antinociceptive and Discriminative Stimulus Effects of Six Novel Psychoactive Opioid Substances in Male Rats.

Compounds with novel or fentanyl-like structures continue to appear on the illicit drug market and have been responsible for fatalities, yet there are limited preclinical pharmacological data available to evaluate the risk of these compounds to public health. The purpose of the present study was to examine acetyl fentanyl, butyryl fentanyl, 3,4-dichloro-N-[[1-(dimethylamino)cyclohexyl]methyl]benzamide (AH-7921), 1-cyclohexyl-4-(1,2-diphenylethyl)piperazine (MT-45), 4-chloro-N-[1-(2-phenylethyl)-2-piperidinylidene]-benzenesulfonamide (W-15), and 4-chloro-N-[1-[2-(4-nitrophenyl)ethyl]-2-piperidinylidene]-benzenesulfonamide (W-18) for their relative potency to reference opioids and their susceptibility to naltrexone antagonism using the 55oC warm-water, tail-withdrawal assay of antinociception and a morphine drug discrimination assay in male, Sprague-Dawley rats. In the antinociception assay, groups of 8 rats per drug were placed into restraining tubes, their tails were immersed into 40o or 55oC water, and the latency for tail withdrawal was measured with a cutoff time of 15 seconds. In the drug discrimination assay, rats (n = 11) were trained to discriminate between 3.2 mg/kg morphine and saline, subcutaneously, in a two-choice, drug discrimination procedure under a fixed ratio-5 schedule of sucrose pellet delivery. Morphine, fentanyl, and four of the synthetic opioids dose dependently produced antinociception and fully substituted for morphine in the drug discrimination assay with the following rank order of potency: fentanyl > butyryl fentanyl > acetyl fentanyl > AH-7921 > MT45 > morphine. All drugs that produced antinociception or morphine-like discriminative stimulus effects were blocked by naltrexone. W-15 and W-18 did not show antinociceptive or morphine-like discriminative stimulus effects at the doses tested supporting a lack of opioid activity for these two compounds. These findings suggest that butyryl fentanyl, acetyl fentanyl, AH-7941, and MT-45 have abuse liability like other opioid agonists. SIGNIFICANCE STATEMENT: As novel psychoactive substances appear on the illicit drug market, preclinical pharmacological testing is required to assist law enforcement, medical professionals, and legal regulators with decisions about potential public health risks. In this study, four synthetic opioids, acetyl fentanyl, butyryl fentanyl, AH-7921, and MT-45 produced effects similar to fentanyl and morphine and were blocked by naltrexone. These data suggest the four synthetic opioids possess similar abuse liability risks as typical opioid agonists.

Potential learning and memory disruptors and enhancers in a simple, 1-day operant task in mice.

The objective of this study was to develop a rapid, 1-day learning and memory assay in mice that is sensitive to the effects of compounds that could impair or enhance acquisition and retrieval. Swiss-Webster, male mice were placed in experimental chambers for a 1-h acquisition session with an intermittent, audible tone. If a nose-poke response occurred during the tone, an Ensure water solution was presented. After 1 h, the mice returned to the chambers for 2 h. Drugs were injected before or after sessions to determine the effects on acquisition and/or retrieval. Mice injected with saline learned a nose-poke response as measured by decreased latencies to earn 10 reinforcers, increased reinforced response rates, and decreased nonreinforced response rates. Scopolamine and acetazolamide impaired retrieval of the nose-poke response, whereas ketamine only modestly impaired retrieval. Doses of 8-OH-DPAT or the novel carbonic anhydrase activator, MAI27, either had no effect or impaired some measures of responding. Neither 8-OH-DPAT nor MAI27 were able to prevent the modest impairments produced by ketamine. The simple, 1-day operant task is a rapid assay that can be used as an initial screen to test the effects of learning and memory disruptors and potentially enhancers.

Distinct interactions of cannabidiol and morphine in three nociceptive behavioral models in mice.

Cannabinoid and opioid agonists can display overlapping behavioral effects and the combination of these agonists is known to produce enhanced antinociception in several rodent models of acute and chronic pain. The present study investigated the antinociceptive effects of the nonpsychoactive cannabinoid, cannabidiol (CBD) and the µ-opioid agonist morphine, both alone and in combination, using three behavioral models in mice, to test the hypothesis that combinations of morphine and CBD would produce synergistic effects. The effects of morphine, CBD, and morphine/CBD combinations were assessed in the following assays: (a) acetic acid-stimulated stretching; (b) acetic acid-decreased operant responding for palatable food; and (c) hot plate thermal nociception. Morphine alone produced antinociceptive effects in all three models of acute nociception, whereas CBD alone produced antinociception only in the acetic acid-stimulated stretching assay. The nature of the interactions between morphine and CBD combinations were assessed quantitatively based on the principle of dose equivalence. Combinations of CBD and morphine produced synergistic effects in reversing acetic acid-stimulated stretching behavior, but subadditive effects in the hot plate thermal nociceptive assay and the acetic acid-decreased operant responding for palatable food assay. These results suggest that distinct mechanisms of action underlie the interactions between CBD and morphine in the three different behavioral assays and that the choice of appropriate combination therapies for the treatment of acute pain conditions may depend on the underlying pain type and stimulus modality.

Pharmacokinetic application of a bio-analytical LC-MS method developed for 5-fluorouracil and methotrexate in mouse plasma, brain and urine.

In the past we have reported significant cognitive deficits in mice receiving 5-fluorouracil in combination with low-dose methotrexate. To explain such interactions, a pharmacokinetic study was designed. A sensitive bio-analytical method was therefore developed and validated for 5-fluorouracil and methotrexate in mouse plasma, brain and urine with liquid chromatography coupled to a single quadrupole mass spectrometer. Chromatographic separation was accomplished by Agilent® Zorbax® SB-C18 column, with isocratic elution (5 mM ammonium acetate and methanol, 70:30, %v/v) at a flow rate of 300 µL/min. The limit of quantitation for both drugs was 15.6 ng/mL (plasma and brain) and 78.1 ng/mL (urine), with interday and intraday precision and accuracy ≤15% and a total run time of 6 min. This bio-analytical method was used for the pharmacokinetic characterization of 5-fluorouracil and methotrexate in mouse plasma, brain and urine over a period of 24 h. This method allowed characterization of the brain concentrations of 5-fluorouracil over a period of 24 h.

Beta-lactam antibiotic decreases acquisition of and motivation to respond for cocaine, but not sweet food, in C57Bl/6 mice.

No medication is approved to treat cocaine addiction, but mounting evidence suggests that glutamate-directed approaches may reduce cocaine dependence and relapse. We tested the hypotheses that the glutamate transporter subtype 1 activator, ceftriaxone, disrupts acquisition of cocaine self-administration, motivation to self-administer cocaine, and conditioned place preference in mice. Repeated ceftriaxone (200 mg/kg) reduced the ability of mice to acquire cocaine and the motivation to self-administer cocaine after successful acquisition without affecting acquisition of or motivation for sweet food. Repeated ceftriaxone had no effect on cocaine-conditioned place preference. These results suggest that a ß-lactam antibiotic reduces the direct reinforcing strength of cocaine without producing nonspecific deficits in conditioned learning processes.

Acquisition session length modulates consolidation effects produced by 5-HT2C ligands in a mouse autoshaping-operant procedure.

Although the neurotransmitter, 5-hydroxytryptamine (serotonin, 5-HT), has been implicated as a mediator of learning and memory, the specific role of 5-HT receptors in rodents requires further delineation. In this study, 5-HT2C receptor ligands of varying relative intrinsic efficacies were tested in a mouse learning and memory model called autoshaping-operant. On day 1, mice were placed in experimental chambers and presented with a tone on a variable interval schedule. The tone remained on for 6 s or until a nose-poke response occurred to produce a dipper with Ensure solution. Mice were then injected with saline, MK212 (full agonist), m-chlorophenylpiperazine (partial agonist), mianserin, and SB206 553 (inverse agonists), and methysergide and (+)-2-bromo lysergic acid diethylamide (+)-hydrogen tartrate (neutral antagonists). Each compound was injected after either 1 or 2-h acquisition sessions on day 1 to investigate the role of acquisition session length on consolidation. Day 1 injection of the 5-HT2C inverse agonist mianserin produced greater retrieval impairments of the autoshaped operant response on day 2 than any other agent tested. Furthermore, decreasing the length of the acquisition session to 1h significantly increased the difficulty of the autoshaping task further modulating the consolidation effects produced by the 5-HT2C ligands tested.

Animal models.

As clinical studies reveal that chemotherapeutic agents may impair several different cognitive domains in humans, the development of preclinical animal models is critical to assess the degree of chemotherapy-induced learning and memory deficits and to understand the underlying neural mechanisms. In this chapter, the effects of various cancer chemotherapeutic agents in rodents on sensory processing, conditioned taste aversion, conditioned emotional response, passive avoidance, spatial learning, cued memory, discrimination learning, delayed-matching-to-sample, novel-object recognition, electrophysiological recordings and autoshaping is reviewed. It appears at first glance that the effects of the cancer chemotherapy agents in these many different models are inconsistent. However, a literature is emerging that reveals subtle or unique changes in sensory processing, acquisition, consolidation and retrieval that are dose- and time-dependent. As more studies examine cancer chemotherapeutic agents alone and in combination during repeated treatment regimens, the animal models will become more predictive tools for the assessment of these impairments and the underlying neural mechanisms. The eventual goal is to collect enough data to enable physicians to make informed choices about therapeutic regimens for their patients and discover new avenues of alternative or complementary therapies that reduce or eliminate chemotherapy-induced cognitive deficits.

Sex and cannabinoid CB1 genotype differentiate palatable food and cocaine self-administration behaviors in mice.

Both cannabinoid CB1 receptor knockout and antagonism produce well-established attenuation of palatable food and drug self-administration behavior. Although cannabinoid drugs have received attention as pharmacotherapeutics for various disorders, including obesity and addiction, it is unclear whether these agents produce equivalent behavioral effects in females and males. In this study, acquisition of 32% corn oil or 10% Ensure self-administration, and maintenance of corn oil, Ensure, or 0.56 mg/kg/infusion cocaine self-administration under both fixed ratio (FR)-1 and progressive ratio (PR) schedule of reinforcement, was compared in male and female wild type (WT) and CB1 knockout (KO) mice. Furthermore, the effect of pretreatment with the CB1 antagonist SR141716 (0.3-3.0) on Ensure self-administration in male and female WT and CB1 KO mice was assessed. CB1 genotype and sex significantly interacted to produce an attenuation of acquisition and maintenance of Ensure self-administration and PR self-administration for both Ensure and cocaine in male CB1 KO mice. In contrast, male CB1 KO mice showed no deficit in acquisition and maintenance of FR-1 responding or in PR responding maintained by corn oil. Sex differences also arose within genotypes for responding maintained under all three reinforcers. Lastly, pretreatment with SR141716 attenuated Ensure self-administration in WT and CB1 KO mice but was approximately five-fold more potent in WT mice than in CB1 KOs. The present data add to a small but growing literature suggesting that the cannabinoid system may be differentially sensitive in its modulation of appetitive behavior in males versus females.

Effects of dopaminergic and serotonergic compounds in rats trained to discriminate a high and a low training dose of the synthetic cathinone mephedrone.

RATIONALE: The underlying pharmacological mechanisms of mephedrone, especially as related to interactions with different neurotransmitter systems, are a critical area of study as mephedrone continues to be abused. OBJECTIVE: Direct-acting 5-HT2A/2C receptor agonists and antagonists and D1-3 receptor antagonists were examined in two groups of rats trained to discriminate mephedrone. A high dose of mephedrone was trained to extend previous results with traditional monoamine transporter inhibitors and substrate releasers. A very low dose of mephedrone was trained to preferentially capture serotonergic activity and to minimize the influence of rate-decreasing effects on substitution patterns. Selective 5-HT2A/2C and D1-3 receptor antagonists were examined in both groups. METHODS: Male Sprague-Dawley rats were trained to discriminate either a low dose of 0.5 mg/kg mephedrone (N = 24) or a high dose of 3.2 mg/kg mephedrone (N = 11) from saline. RESULTS: In the low training-dose group, mephedrone, MDMA, methamphetamine, d-amphetamine, cocaine, and enantiomers of mephedrone substituted for mephedrone; mCPP partially substituted overall for mephedrone; and DOI, WAY163909, and morphine failed to substitute for mephedrone. In the high training-dose group, only mephedrone and MDMA substituted for mephedrone. Sulpiride produced a small antagonism of the low training dose of mephedrone while SCH23390, SB242084, and ketanserin altered response rates. CONCLUSIONS: A lower training dose of mephedrone produces a discriminative stimulus fully mimicked by MDMA, methamphetamine, cocaine, and d-amphetamine, whereas a higher training dose of mephedrone requires a discriminative stimulus that was only mimicked by MDMA. Dopaminergic or serotoninergic antagonists failed to produce significant blockade of mephedrone at either training dose.

Effects of a Cannabinoid1 receptor antagonist and Serotonin2C receptor agonist alone and in combination on motivation for palatable food: a dose-addition analysis study in mice.

The cannabinoid and serotonin systems modulate feeding behavior in humans and laboratory animals. The present study assessed whether a cannabinoid (CB)(1) receptor antagonist and a serotonin (5-HT)(2C) receptor agonist alone and in combination attenuate motivation for the liquid nutritional drink Ensure as measured by a progressive ratio (PR) schedule of reinforcement in male C57BL/6 mice. Pretreatment (15 min i.p.) with either the CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716) (SR; Rimonabant or Acomplia) or the 5-HT(2C) receptor agonist m-chlorophenylpiperazine (mCPP) dose-dependently decreased the maximum ratio completed under the PR schedule (break point) in mice. ED(25) values for SR and mCPP to decrease break point were determined, and the relative potency of each drug alone was quantified. Fixed dose-ratio pairs of SR/mCPP based on their relative potency were then administered. Dose-addition analysis comparing the experimentally determined potency for SR/mCPP combinations with their predicted additive potency revealed that SR/mCPP combinations in 1:1 and 2:1 ratios based on relative potency produced significant synergistic attenuation of break point for Ensure. The ED(25) values for decreasing break point were consistently lower than ED(25) values for decreasing response rate, and synergistic effects of SR/mCPP combinations on break point were seen independent of synergistic effects on response rate. These results indicate that cannabinoid CB(1) and serotonin 5-HT(2C) receptors are involved in motivated feeding behavior in mice and that these compounds can synergistically modulate motivation for palatable food with the synergy dependent upon the ratio of SR/mCPP in the combination.

Effects of chemotherapeutic agents 5-fluorouracil and methotrexate alone and combined in a mouse model of learning and memory.

RATIONALE: The concern that adjuvant cancer chemotherapy agents cause cognitive impairment in a significant number of patients has been expressed by patients and healthcare providers, but clinical studies have yielded conflicting results to date. OBJECTIVE: We directly tested two commonly used chemotherapeutic agents in a mouse model of learning and memory. MATERIALS AND METHODS: In the present study, mice were conditioned to respond for a liquid reinforcer (Ensure solution) in the presence of an audible tone on day 1 as a measure of acquisition and were then required to perform the same response on day 2 as a measure of retrieval and retention. Methotrexate and 5-fluorouracil were administered prior to the day 1 session. RESULTS: Methotrexate (1.0-32 mg/kg) alone failed to alter mean latency acquisition, retrieval, or reinforced response rates. Similar to scopolamine, a known amnesic in this assay, 5-fluorouracil (3-75 mg/kg) failed to alter response rates or acquisition latency on day 1 but significantly altered latency to retrieve a previously learned response on day 2. In combination, 3.2 mg/kg methotrexate plus 75 mg/kg 5-fluorouracil significantly increased day 1 and day 2 acquisition and retrieval latencies without altering response rates or motivation to respond as measured by progressive ratio responding. CONCLUSION: Taken together, these data demonstrate that 5-fluorouracil causes increased latencies for retrieval of previously learned behavioral responses and that combination of chemotherapeutic agents may produce greater delays than either agent alone, including when neither agent alone does so.

Hydromorphone efficacy and treatment protocol impact on tolerance and mu-opioid receptor regulation.

This study examined the antinociceptive (analgesic) efficacy of hydromorphone and hydromorphone-induced tolerance and regulation of mu-opioid receptor density. Initially s.c. hydromorphone's time of peak analgesic (tail-flick) effect (45 min) and ED50 using standard and cumulative dosing protocols (0.22 mg/kg, 0.37 mg/kg, respectively) were determined. The apparent analgesic efficacy (tau) of hydromorphone was then estimated using the operational model of agonism and the irreversible mu-opioid receptor antagonist clocinnamox. Mice were injected with clocinnamox (0.32-25.6 mg/kg, i.p.) and 24 h later, the analgesic potency of hydromorphone was determined. The tau value for hydromorphone was 35, which suggested that hydromorphone is a lower analgesic efficacy opioid agonist. To examine hydromorphone-induced tolerance, mice were continuously infused s.c. with hydromorphone (2.1-31.5 mg/kg/day) for 7 days and then morphine cumulative dose response studies were performed. Other groups of mice were injected with hydromorphone (2.2-22 mg/kg/day) once, or intermittently every 24 h for 7 days. Twenty-four hours after the last injection, mice were tested using morphine cumulative dosing studies. There was more tolerance with infusion treatments compared to intermittent treatment. When compared to higher analgesic efficacy opioids, hydromorphone infusions induced substantially more tolerance. Finally, the effect of chronic infusion (31.5 mg/kg/day) and 7 day intermittent (22 mg/kg/day) hydromorphone treatment on spinal cord mu-opioid receptor density was determined. Hydromorphone did not produce any change in mu-opioid receptor density following either treatment. These results support suggestions that analgesic efficacy is correlated with tolerance magnitude and regulation of mu-opioid receptors when opioid agonists are continuously administered. Taken together, these studies indicate that analgesic efficacy and treatment protocol are important in determining tolerance and regulation of mu-opioid receptors.

The analgesic efficacy of fentanyl: relationship to tolerance and mu-opioid receptor regulation.

This study determined if fentanyl analgesic efficacy predicts the magnitude of tolerance and mu-opioid receptor regulation. To estimate efficacy, mice were injected i.p. with saline or clocinnamox (CCAM), an irreversible mu-opioid receptor antagonist, (0.32-25.6 mg/kg) and 24 h later fentanyl cumulative dose-response studies were conducted. CCAM dose dependently shifted the fentanyl dose-response function to the right. The apparent efficacy (tau) of fentanyl, based on the operational model of agonism, was estimated as 58, indicating that fentanyl is a high analgesic efficacy agonist. Next, mice were infused with fentanyl (1, 2 or 4 mg/kg/day) for 7 days. Controls were implanted with placebo pellets. At the end of 7 days, morphine cumulative dose-response studies or mu-opioid receptor saturation binding studies were conducted. Fentanyl infusions dose dependently decreased morphine potency with the highest fentanyl dose reducing morphine potency by approximately 6 fold. Chronic infusion with fentanyl (4 mg/kg/day) significantly reduced mu-opioid receptor density by 28% without altering affinity, whereas lower infusion doses had no effect. Taken together, the present results strengthen the proposal that opioid analgesic efficacy predicts mu-opioid receptor regulation and the magnitude of tolerance.

A Prospective Evaluation of Drug Discrimination in Pharmacology.

As investigators, we use many methodologies to answer both practical and theoretical questions in our field. Occasionally, we must stop and collect the latest findings or trends and then look forward to where our ideas, findings, and hypotheses may take us. Similar to volumes that were published in previous years on drug discrimination (Glennon and Young, Drug discrimination applications to medicinal chemistry and drug studies. Wiley, Hoboken, 2011; Ho et al., Drug discrimination and state dependent learning. Academic Press, New York, 1978), this collection in Current Topics in Behavioral Neurosciences serves as a current analysis of the continued value of the drug discrimination procedure to the fields of pharmacology, neuroscience, and psychology and as a stepping stone to where drug discrimination methodology can be applied next, in both a practical and theoretical sense. This final chapter represents one investigator's perspective on the utility and possibilities for a methodology that she fell in love with over 30 years ago.

Effect of cannabinoid CB1 receptor antagonist SR141716A and CB1 receptor knockout on cue-induced reinstatement of Ensure and corn-oil seeking in mice.

The cannabinoid CB1 receptor antagonist SR141716A decreases cue-induced reinstatement of sucrose and drug seeking in rats. Reinstatement behavior is not well characterized in C57Bl/6 mice, including CB1 receptor knockout mice generated on a C57Bl/6 background. In the present study, male C57Bl/6, CB1 knockout (CB1 KO), and wild-type littermate (WT) mice were trained to respond for the sweet reinforcer Ensure or corn oil. Responding was maintained on a fixed ratio 1 (FR1) schedule of reinforcement for 10 days, and then extinguished by the removal of the reinforcer and associated cues. Subsequently, the effect of either pretreatment with SR141716A or CB1 receptor knockout on cue-induced reinstatement of Ensure or corn-oil seeking was assessed. Both 1.0 and 3.0 mg/kg SR141716A decreased reinstatement of Ensure seeking in C57Bl/6 mice. A tenfold higher dose of SR141716A (10.0 mg/kg) was required to attenuate reinstatement behavior in C57Bl/6 mice responding for corn oil, suggesting that CB1 receptors may be selectively involved in the neurobiology underlying reinstatement of responding for some food reinforcers but not others. Whereas CB1 receptor antagonism selectively attenuated reinstatement of responding for Ensure, genetic deletion of the CB1 receptor produced only a trend in decreasing reinstatement of Ensure seeking, and did not attenuate reinstatement of corn-oil seeking. Baseline differences in levels of operant responding were also observed in WT vs CB1 KO mice maintained by Ensure and corn oil. This and other possible reasons for the observed discrepancy between pharmacological blockade vs genetic invalidation of the CB1 receptor on reinstatement of Ensure seeking are discussed.

In vivo Schild regression analyses using nonselective 5-HT2C receptor antagonists in a rat operant behavioral assay.

RATIONALE: Although competitive antagonism experiments are critical tools in the classification of potential pharmacotherapies, no studies have quantitatively compared the potencies of 5-HT(2C) receptor antagonists using the Schild regression analysis in vivo. OBJECTIVES: To evaluate the behavioral effects of 5-HT(2C) receptor agonists and antagonists, a series of nonselective 5-HT(2C) receptor antagonists, the 5-HT(2A/2C) receptor antagonist ketanserin, the 5-HT(2B) receptor antagonist SB 204,741, the 5-HT(2B/2C) receptor antagonist SB 200,646, and the peripherally acting 5-HT(2C) receptor antagonist RS102,221 were evaluated for their capacity to competitively antagonize the agonists MK212, mCPP, or BW723C86 in rats. MATERIALS AND METHODS: Male Sprague-Dawley rats (N = 28) were trained to respond under a fixed ratio 10 schedule of food reinforcement. A multiple-trial, cumulative-dosing procedure was used to evaluate the capacity of the compounds to suppress response rates. RESULTS: MK212, mCPP, and the 5-HT(2B) receptor agonist BW723C86 dose-dependently decreased response rates. Only metergoline, mianserin, and methysergide produced a dose-dependent antagonism of the rate-decreasing effects of both mCPP and MK212. Apparent pA(2) analysis indicated that metergoline, mianserin, and methysergide were approximately equipotent as antagonists overall. Metergoline and mianserin failed to block the rate-decreasing effects of BW723C86. Ketanserin, SB 200,646, SB 204,741, and RS102,221 failed to block either mCPP or MK212, suggesting that 5-HT(2A), 5-HT(2B), or peripheral 5-HT(2C) receptors do not play a primary role in the rate-decreasing effects of these two agonists. CONCLUSIONS: Taken together, these antagonism profiles suggest that the agonists MK212 and mCPP produce their rate-decreasing effects through a combination of 5-HT receptors with the 5-HT(2C) receptor playing a prominent but not exclusive role.

Opioid agonist efficacy predicts the magnitude of tolerance and the regulation of mu-opioid receptors and dynamin-2.

It has been proposed that opioid agonist efficacy may play a role in tolerance and the regulation of opioid receptor density. To address this issue, the present studies estimated the in vivo efficacy of three opioid agonists and then examined changes in spinal mu-opioid receptor density following chronic treatment in the mouse. In addition, tolerance and regulation of the trafficking protein dynamin-2 were determined. To evaluate efficacy, the method of irreversible receptor alkylation was employed and the efficacy parameter tau estimated. Mice were injected with the irreversible mu-opioid receptor antagonist clocinnamox (0.32-25.6 mg/kg, i.p), and 24 h later, the analgesic potency of s.c. morphine, oxycodone and etorphine were determined. Clocinnamox dose-dependently antagonized the analgesic effects of morphine, etorphine and oxycodone. The shift to the right of the dose-response curves was greater for morphine and oxycodone compared to etorphine and the highest dose of clocinnamox reduced the maximal effect of morphine and oxycodone, but not etorphine. The order of efficacy calculated from these results was etorphine>morphine>oxycodone. Other mice were infused for 7 days with oxycodone (10-150 mg/kg/day, s.c.) or etorphine (50-250 microg/kg/day, s.c.) and the analgesic potency of s.c. morphine determined. The low efficacy agonist (oxycodone) produced more tolerance than the high efficacy agonist (etorphine) at equi-effective infusion doses. In saturation binding experiments, the low efficacy opioid agonists (morphine, oxycodone) did not regulate the density of spinal mu-opioid receptors, while etorphine produced approximately 40% reduction in mu-opioid receptor density. Furthermore, etorphine increased spinal dynamin-2 abundance, while oxycodone did not produce any significant change in dynamin-2 abundance. Overall, these data indicate that high efficacy agonists produce less tolerance at equi-effective doses. Furthermore, increased efficacy was associated with mu-opioid receptor downregulation and dynamin-2 upregulation. Conversely, lower efficacy agonists produced more tolerance at equi-effective doses, but did not regulate mu-opioid receptor density or dynamin-2 abundance. Taken together, these studies indicate that agonist efficacy plays an important role in tolerance and regulation of receptors and trafficking proteins.

Single and combined effects of Δ9 -tetrahydrocannabinol and cannabidiol in a mouse model of chemotherapy-induced neuropathic pain.

BACKGROUND AND PURPOSE: The non-psychoactive phytocannabinoid cannabidiol (CBD) can affect the pharmacological effects of Δ9 -tetrahydrocannabinol (THC). We tested the possible synergy between CBD and THC in decreasing mechanical sensitivity in a mouse model of paclitaxel-induced neuropathic pain. We also tested the effects of CBD on oxaliplatin- and vincristine-induced mechanical sensitivity. EXPERIMENTAL APPROACH: Paclitaxel-treated mice (8.0 mg·kg-1 i.p., days 1, 3, 5 and 7) were pretreated with CBD (0.625-20.0 mg·kg-1 i.p.), THC (0.625-20.0 mg·kg-1 i.p.) or CBD + THC (0.04 + 0.04-20.0 + 20.0 mg·kg-1 i.p.), and mechanical sensitivity was assessed on days 9, 14 and 21. Oxaliplatin-treated (6.0 mg·kg-1 i.p., day 1) or vincristine-treated mice (0.1 mg·kg-1 i.p. days 1-7) were pretreated with CBD (1.25-10.0 mg·kg-1 i.p.), THC (10.0 mg·kg-1 i.p.) or THC + CBD (0.16 mg·kg-1 THC + 0.16 mg·kg-1 CBD i.p.). KEY RESULTS: Both CBD and THC alone attenuated mechanical allodynia in mice treated with paclitaxel. Very low ineffective doses of CBD and THC were synergistic when given in combination. CBD also attenuated oxaliplatin- but not vincristine-induced mechanical sensitivity, while THC significantly attenuated vincristine- but not oxaliplatin-induced mechanical sensitivity. The low dose combination significantly attenuated oxaliplatin- but not vincristine-induced mechanical sensitivity. CONCLUSIONS AND IMPLICATIONS: CBD may be potent and effective at preventing the development of chemotherapy-induced peripheral neuropathy, and its clinical use may be enhanced by co-administration of low doses of THC. These treatment strategies would increase the therapeutic window of cannabis-based pharmacotherapies.

Selective and nonselective serotonin antagonists block the aversive stimulus properties of MK212 and m-chlorophenylpiperazine (mCPP) in mice.

Serotonin(2C) (5-HT(2C)) receptors have been implicated to treat mood disorders such as depression and anxiety. In the present study, the capacities of two 5-HT(2C) agonists, MK212 and mCPP, to produce conditioned taste aversions in mice were evaluated. On two training days, Swiss-Webster male mice (19-34g) were trained to associate the flavor of a novel solution with the injection of various doses of MK212 or mCPP. On two alternate training days, mice were trained to associate a different flavored solution with an injection of saline. For testing, both flavored solutions were presented simultaneously and an avoidance of the MK212 or mCPP-paired solution indicated conditioned taste aversion. Robust conditioned taste aversions were observed to solutions paired with 1.0 or 10mg/kg MK212 or mCPP. Acquisition of conditioned taste aversions was blocked by nonselective serotonin antagonists cyproheptadine, bromo-LSD, metergoline, methysergide and mianserin. Selective 5-HT(2B/2C) antagonist SB206,553 blocked both MK212- and mCPP-induced conditioned taste aversion although selective 5-HT(2B/2C) antagonist SB200,646 only blocked mCPP-induced conditioned taste aversion. In a single-bottle procedure, MK212, bromo-LSD, and mianserin failed to alter acquisition rate of a LiCl-induced conditioned taste aversion. Taken together, these data indicate that the serotonin agonists MK212 and mCPP produce conditioned taste aversion and that these effects are mediated predominantly through 5-HT(2C) receptors.

Opioid antagonists differ according to negative intrinsic efficacy in a mouse model of acute dependence.

The purpose of the present study is to compare the capacity of opioid antagonists to elicit withdrawal jumping in mice following two acute pretreatment doses of the opioid agonist morphine. Antagonists that precipitate vigorous withdrawal jumping across both morphine treatment doses are hypothesized to be strong inverse agonists at the mu-opioid receptor, whereas antagonists that elicit withdrawal jumping in mice treated with the high but not the low dose of morphine are hypothesized to be weak inverse agonists. Male, Swiss-Webster mice (15-30 g) were acutely treated with 56 or 180 mg kg(-1) morphine 4 h prior to injection with naloxone, naltrexone, diprenorphine, nalorphine, or naloxonazine. Vertical jumping, paw tremors, and weight loss were recorded. Naloxone, naltrexone, and diprenorphine produced withdrawal jumping after 56 and 180 mg kg(-1)morphine pretreatment. Nalorphine and naloxonazine produced moderate withdrawal jumping after 180 mg kg(-1) morphine pretreatment, but failed to elicit significant withdrawal jumping after 56 mg kg(-1) morphine pretreatment. Nalorphine and naloxonazine blocked the withdrawal jumping produced by naloxone. All antagonists produced paw tremors and weight loss although these effects were generally not dose-dependent. Taken together, these findings reveal a rank order of negative intrinsic efficacy for these opioid antagonists as follows: naloxone=naltrexone> or =diprenorphine>nalorphine=naloxonazine. Furthermore, the observation that nalorphine and naloxonazine blocked the naloxone-induced withdrawal jumping provides additional evidence that nalorphine and naloxonazine are weaker inverse agonists than naloxone.