Characterization and validation of a spontaneous acute and protracted oxycodone withdrawal model in male and female mice.

Opioid use disorder (OUD) is a serious health problem that may lead to physical dependence, in addition to affective disorders. Preclinical models are essential for studying the neurobiology of and developing pharmacotherapies to treat these problems. Historically, chronic morphine injections have most often been used to produce opioid-dependent animals, and withdrawal signs indicative of dependence were precipitated by administering an opioid antagonist. In the present studies, we have developed and validated a model of dependence on oxycodone (a widely prescribed opioid) during spontaneous withdrawal in male and female C57BL/6J mice. Dependence was induced by chronically administering oxycodone through osmotic minipumps at different doses for 7 days. Somatic withdrawal signs were measured after 3, 6, 24, and 48 h following minipump removal. Additionally, sensitivity to mechanical, thermal, and cold stimuli, along with anxiety-like behavior, were also measured. Our results indicated that spontaneous withdrawal following discontinuation of oxycodone produced an increase in total withdrawal signs after 60 and 120 mg/kg/day regimens of oxycodone administration. These signs were reversed by the administration of clinically approved medications for OUD. In general, both female and male mice showed similar profiles of somatic signs of spontaneous withdrawal. Spontaneous withdrawal also resulted in mechanical and cold hypersensitivity lasting for 24 and 14 days, respectively, and produced anxiety-like behaviors after 2 and 3 weeks following oxycodone removal. These results help validate a new model of oxycodone dependence, including the temporally distinct emergence of somatic, hyperalgesic, and anxiety-like behaviors, potentially useful for mechanistic and translational studies of opioid dependence.

Enantiomeric contributions to methamphetamine's bidirectional effects on basal and fentanyl-depressed respiration in mice.

RATIONALE: Fentanyl remains the primary cause of fatal overdoses, and its co-use with methamphetamine (METH) is a growing concern. We previously demonstrated that racemic METH can either enhance or mitigate opioid-induced respiratory depression (OIRD) dependent upon whether a low or high dose is administered. The optical isomers of METH, dextromethamphetamine (d-METH) and levomethamphetamine (l-METH), differ substantially in their selectivity and potency to activate various monoamine (MA) receptors, and these pharmacological differences may underlie the bidirectional effects of the racemate. Since it is unknown which of METH's MA receptor mechanisms mediate these respiratory effects, examination of METH's pharmacologically distinct enantiomers may provide insight into treatment targets for OIRD. METHODS: The two optical isomers of METH, d-METH and l-METH, were tested in adult male mice to determine their effects on basal and fentanyl-depressed respiratory frequency, tidal volume, and minute ventilation (MVb; i.e., respiratory frequency x tidal volume) using whole-body plethysmography. RESULTS: When tested at dose ranges of 1.0-10 mg/kg, d-METH elevated MVb and l-METH decreased basal MVb. A dose of 30 mg/kg l-METH increased basal MVb. Under fentanyl-depressed conditions, the bidirectional effects of racemic METH were observed with d-METH treatment while l-METH significantly exacerbated OIRD at 1.0 and 3.0 mg/kg. CONCLUSIONS: d-METH and l-METH differentially contribute to the bidirectional respiratory modulation observed by the racemate, with d-METH exhibiting predominantly stimulatory effects and l-METH exhibiting primarily depressant effects depending on dose.

Theophylline reverses oxycodone's but not fentanyl's respiratory depression in mice while caffeine is ineffective against both opioids.

RATIONALE: The opioid epidemic remains a pressing public health crisis in the United States. Most of these overdose deaths are a result of lethal respiratory depression. In recent years the increasing incidence of opioid-involved overdose deaths has been driven by fentanyl, which is more resistant to adequate reversal by naloxone (NARCAN ®) than semi-synthetic or classical morphinan predecessors like oxycodone and heroin. For this and other reasons (e.g., precipitating withdrawal) non-opioidergic pharmacotherapies to reverse opioid-depressed respiration are needed. Methylxanthines are a class of stimulant drugs including caffeine and theophylline which exert their effects primarily via adenosine receptor antagonism. Evidence suggests methylxanthines can stimulate respiration by enhancing neural activity in respiratory nuclei in the pons and medulla independent of opioid receptors. This study aimed to determine whether caffeine and theophylline can stimulate respiration in mice when depressed by fentanyl and oxycodone. METHODS: Whole-body plethysmography was used to characterize fentanyl and oxycodone's effects on respiration and their reversal by naloxone in male Swiss Webster mice. Next, caffeine and theophylline were tested for their effects on basal respiration. Finally, each methylxanthine was evaluated for its ability to reverse similar levels of respiratory depression induced by fentanyl or oxycodone. RESULTS AND CONCLUSIONS: Oxycodone and fentanyl dose-dependently reduced respiratory minute volume (ml/min; MVb) that was reversible by naloxone. Caffeine and theophylline each significantly increased basal MVb. Theophylline, but not caffeine, completely reversed oxycodone-depressed respiration. In contrast, neither methylxanthine elevated fentanyl-depressed respiration at the doses tested. Despite their limited efficacy for reversing opioid-depressed respiration when administered alone, the methylxanthines safety, duration, and mechanism of action supports further evaluation in combination with naloxone to augment its reversal of opioid-depressed respiration.

Amphetamines modulate fentanyl-depressed respiration in a bidirectional manner.

BACKGROUND: The opioid epidemic remains one of the most pressing public health crises facing the United States. Fentanyl and related synthetic opioid agonists have largely driven the rising rates of associated overdose deaths, in part, because of their surreptitious use as substitutes for other opioids and as adulterants in psychostimulants. Deaths involving opioids typically result from lethal respiratory depression, and it is currently unknown how co-use of psychostimulants with opioids affects respiratory toxicity. Considering psychostimulant overdoses have increased over 3-fold since 2013, and half of those co-involved opioids, this is a cardinal question. METHODS: Naloxone, d-amphetamine (AMPH), and (±)-methamphetamine (METH) were evaluated for their effects on basal and fentanyl-depressed respiration. Minute volume (MVb) was measured in awake, freely moving mice via whole-body plethysmography to quantify fentanyl-induced respiratory depression and its modulation by dose ranges of each test drug. RESULTS: Naloxone immediately reversed respiratory depression induced by fentanyl only at the highest dose tested (10 mg/kg). Both AMPH and METH exhibited bidirectional effects on MVb under basal conditions, producing significant (p ≤ 0.05) depressions then elevations of respiration as dose increased. Under depressed conditions the bidirectional effects of AMPH and METH on respiration were exaggerated, exacerbating and then reversing fentanyl-induced depression as dose increased. CONCLUSIONS: These results indicate that co-use of amphetamines with fentanyl may worsen respiratory depression, but conversely, monoaminergic components of the amphetamines may possibly be exploited to mitigate fentanyl overdose.

Structurally diverse fentanyl analogs yield differential locomotor activities in mice.

Synthetic narcotics have been implicated as the single greatest contributor to increases in opioid-related fatalities in recent years. This study evaluated the effects of nine fentanyl-related substances that have emerged in the recreational drug marketplace, and for which there are no existing or only limited in vivo data. Adult male Swiss Webster mice were administered fentanyl-related substances and their effects on locomotion as compared to MOR agonist standards were recorded. In locomotor activity tests, morphine (100, 180 mg/kg), buprenorphine (1, 10 mg/kg), fentanyl (1, 10 mg/kg), cyclopropylfentanyl (1, 10 mg/kg), cyclopentylfentanyl (10 mg/kg), (±)-cis-3-methylbutyrylfentanyl (0.1, 1, 10 mg/kg), ortho-methylacetylfentanyl (10 mg/kg), para-chloroisobutyrylfentanyl (100 mg/kg), ocfentanil (1, 10 mg/kg), and ortho-fluoroacrylfentanyl (0.1, 1, 10 mg/kg) elicited significant (p ≤ 0.05) dose-dependent increases in locomotion. However, 2,2,3,3-tetramethylcyclopropylfentanyl did not have any effects on locomotion, even when tested up to 100 mg/kg, and 4'-methylacetylfentanyl (10, 100 mg/kg) significantly decreased locomotion. The rank order of efficacy for stimulating locomotion (maximum effect as a % of fentanyl's maximum effect) for fentanyl-related substances relative to MOR agonist standards was cyclopropylfentanyl (108.84 ± 20.21) > fentanyl (100 ± 15.3) > ocfentanil (79.27 ± 16.92) > morphine (75.9 ± 14.5) > (±)-cis-3-methylbutyrylfentanyl (68.04 ± 10.08) > ortho-fluoroacrylfentanyl (63.56 ± 19.88) > cyclopentylfentanyl (56.46 ± 8.54) > para-chloroisobutyrylfentanyl (22.44 ± 8.51) > buprenorphine (11.26 ± 2.30) > ortho-methylacetylfentanyl (9.45 ± 2.92) > 2,2,3,3-tetramethylcyclopropylfentanyl (6.75 ± 1.43) > 4'-methylacetylfentanyl (3.47 ± 0.43). These findings extend in vivo results from previous reports documenting additional fentanyl related-related substances that stimulate locomotion similar to known abused opioids while also identifying some anomalies.

Fentanyl-related substances elicit antinociception and hyperlocomotion in mice via opioid receptors.

Synthetic opioids have been implicated as the single greatest contributor to rising drug-related fatalities in recent years. This study evaluated mu-opioid receptor (MOR) mediated effects of seven fentanyl-related substances that have emerged in the recreational drug marketplace, and for which there are no existing or only limited in vivo data. Adult male Swiss Webster mice were administered fentanyl-related substances and their effects on nociception and locomotion as compared to MOR agonist standards were observed. In locomotor activity tests, morphine (100, 180 mg/kg), fentanyl (1, 10 mg/kg), beta-methylfentanyl (10 mg/kg), para-methoxyfentanyl (10 mg/kg), fentanyl carbamate (100 mg/kg), and 3-furanylfentanyl (10 mg/kg), elicited significant (p ≤ 0.05) dose-dependent increases in locomotion. However, para-methylfentanyl and beta'-phenylfentanyl did not produce significant effects on locomotion at doses up to 100 mg/kg and phenylfentanyl (100 mg/kg) significantly decreased locomotion. In warm-water tail-withdrawal tests, all substances produced significant dose-dependent increases in antinociception with increasing ED50 values (95% CI) of fentanyl [0.08 mg/kg (0.04-0.16)] > para-methoxyfentanyl [0.43 mg/kg (0.23-0.77)] > 3-furanylfentanyl [0.51 mg/kg (0.36-0.74)] > beta-methylfentanyl [0.74 mg/kg (0.64-0.85)] > para-methylfentanyl [1.92 mg/kg (1.48-2.45)] > fentanyl carbamate [5.59 mg/kg (4.11-7.54)] > morphine [7.82 mg/kg (5.42-11.0)] > beta'-phenylfentanyl [19.4 mg/kg (11.0-34.4)] > phenylfentanyl [55.2 mg/kg (33.5-93.0)]. Naltrexone (1 mg/kg) increased ED50 values several fold with decreasing magnitudes of para-methylfentanyl (63.1×) > para-methoxyfentanyl (22.5×) > beta'-phenylfentanyl (21.0×) > 3-furanylfentanyl (20.6×) > beta-methylfentanyl (19.2×) > phenylfentanyl (5.23×) > fentanyl (3.95×) > fentanyl carbamate (2.21×) > morphine (1.48×). These findings expand upon in vivo results from previous studies and establish that the effects of these fentanyl related-related substances are at least in part mediated by the MOR.

Validation and characterization of oxycodone physical dependence in C57BL/6J mice.

Opioid use disorder is a growing concern in the United States. Mice were used to investigate the mechanisms involving opioid physical dependence and for evaluating medications for treating opioid use disorders. While there are many preclinical reports describing protocols for inducing physical dependence upon morphine, there are fewer preclinical reports describing more contemporary abused prescription opiates. The goal of this study was to characterize and validate a mouse model of oxycodone dependence. Male C57BL/6J mice were injected with saline or increasing doses of oxycodone (9-33 mg/kg) twice daily for 8 days. On the 9th day, mice were challenged with 1 mg/kg naloxone and observed for somatic signs. Mice were pretreated with oxycodone (17, 33, or 75 mg/kg) prior to withdrawal to determine if it could attenuate somatic withdrawal signs. Additional mouse groups were pretreated with 1 mg/kg clonidine. Lastly, we measured somatic signs for 6, 24, and 48 h post-withdrawal during spontaneous and precipitated withdrawal. Pretreating with oxycodone or clonidine dose-dependently prevented the emergence of withdrawal signs. Mice chronically treated with oxycodone exhibited more withdrawal signs than vehicle at 24 h after the final injection during spontaneous withdrawal. In contrast, mice that received repeated naloxone challenges showed peak withdrawal signs at 6 h, and withdrawal signs were significantly greater at all time points compared to vehicle. Reversal of withdrawal effects by positive controls, and establishing spontaneous and precipitated withdrawal paradigms, serve as validation of this model and provide a means to examine novel therapeutics to treat opioid withdrawal.

Methylnaltrexone crosses the blood-brain barrier and attenuates centrally-mediated behavioral effects of morphine and oxycodone in mice.

BACKGROUND: Antagonism of peripheral opioid receptors by methylnaltrexone (MNTX) was recently proposed as a potential mechanism to attenuate the development of opioid analgesic tolerance based on experiments conducted in mice. However, reports indicate that MNTX is demethylated to naltrexone (NTX) in mice, and NTX may subsequently cross the blood-brain barrier to antagonize centrally-mediated opioid effects. The goal of this study was to determine whether MNTX alters centrally-mediated behaviors elicited by the opioid analgesics, morphine and oxycodone, and to quantify concentrations of MNTX and NTX in blood and brain following their administration in mice. METHODS: Combinations of MNTX and morphine were tested under acute and chronic conditions in thermal nociceptive assays. Effects of MNTX and NTX pretreatment were assessed in an oxycodone discrimination operant procedure. Blood and brain concentrations of these antagonists were quantified after their administration using liquid chromatography-mass spectrometry. RESULTS: MNTX dose-dependently attenuated acute and chronic morphine antinociception. MNTX and NTX dose-dependently antagonized the discriminative stimulus effects of oxycodone. MNTX and NTX were detected in both blood and brain after administration of MNTX, confirming its demethylation and demonstrating that MNTX itself can cross the blood-brain barrier. CONCLUSIONS: These results provide converging behavioral and analytical evidence that MNTX administration in mice attenuates centrally-mediated effects produced by opioid analgesics and results in functional concentrations of MNTX and NTX in blood and brain. Collectively, these findings indicate that MNTX cannot be administered systemically in mice for making inferences that its effects are peripherally restricted.

Opioid-like antinociceptive and locomotor effects of emerging fentanyl-related substances.

The emergence of several fentanyl-related substances in the recreational drug marketplace has resulted in a surge of opioid overdose deaths in the United States. Many of these substances have never been examined in living organisms under controlled conditions. In the present study, seven fentanyl-related substances were tested in adult male Swiss Webster mice for their effects on locomotion and antinociception and compared to those of fentanyl and morphine. In locomotor activity tests, fentanyl (1, 10 mg/kg), morphine (100, 180 mg/kg), isobutyrylfentanyl (10 mg/kg), crotonylfentanyl (10 mg/kg), para-fluorobutyrylfentanyl (10, 100 mg/kg), para-methoxybutyrylfentanyl (10 mg/kg), thiophenefentanyl (100 mg/kg), and benzodioxolefentanyl (0.1 mg/kg) produced significant (p ≤ 0.05) dose-dependent increases in locomotion. Valerylfentanyl, however, was without effects on locomotion up to 100 mg/kg. In warm-water tail-withdrawal tests, all substances produced significant (p ≤ 0.05) dose-dependent increases in antinociception with increasing ED50 values (CI) of isobutyrylfentanyl [0.0768 mg/kg (0.044-0.128)] > fentanyl [0.0800 mg/kg (0.0403-0.164)] > para-methoxybutyrylfentanyl [0.106 mg/kg (0.0516-0.195)] > crotonylfentanyl [0.226 mg/kg (0.176-0.292)] > para-fluorobutyrylfentanyl [0.908 mg/kg (0.459-1.58)] > thiophenefentanyl [4.66 mg/kg (3.65-5.95)] > valerylfentanyl [6.43 mg/kg (3.91-10.5)] > morphine [7.82 mg/kg (5.42-11.0)] > benzodioxolefentanyl [46.3 mg/kg (25.8-83.4)]. Naltrexone (1 mg/kg) increased antinociceptive ED50 values several fold in decreasing magnitudes of isobutyrylfentanyl (233x) > para-methoxybutyrylfentanyl (37.7x) > thiophenefentanyl (34.6x) > valerylfentanyl (11.9x) > para-fluorobutyrylfentanyl (10.9x) > benzodioxolefentanyl (8.42x) > crotonylfentanyl (6.27x) > fentanyl (3.95x) > morphine (1.48x). These findings establish that locomotor and antinociceptive effects of several fentanyl-related substances are similar to those of morphine and fentanyl and are mediated by opioid receptors.

Oxycodone-like discriminative stimulus effects of fentanyl-related emerging drugs of abuse in mice.

BACKGROUND: Fentanyl and its structurally related compounds have emerged as the most significant contributors to opioid overdose fatalities in recent years. While there is abundant information about the pharmacological effects of fentanyl, far less is known of its more recently abused analogs. The objective of this study was to determine whether fentanyl and several fentanyl-related substances would engender oxycodone-like responding in a mouse model of oxycodone discrimination. Oxycodone was selected as the training drug due to its high selectivity for mu opioid receptors. Compounds that elicited oxycodone-like responding in this procedure would likely evoke overlapping subjective experiences. METHODS: Adult male C57BL/6 mice were trained to discriminate 1.3 mg/kg oxycodone from vehicle in a food-reinforced, two-lever choice procedure. Generalization tests were conducted with fentanyl and the following fentanyl-related compounds: ocfentanil, 3-furanyl fentanyl, crotonylfentanyl, and valerylfentanyl. RESULTS: Fentanyl and each of its analogs completely generalized to the 1.3 mg/kg oxycodone discriminative stimulus and naltrexone pretreatment significantly decreased oxycodone-like responding for each compound. Rank order potency for engendering oxycodone-appropriate responding was ocfentanil > fentanyl > 3-furanyl fentanyl ≈ crotonylfentanyl > oxycodone > valerylfentanyl. Drug doses that evoked full substitution also significantly suppressed response rates compared to vehicle. CONCLUSIONS: These results indicate that the discriminative stimulus, and by extension, the interoceptive and subjective effects of the tested fentanyl analogs, overlap with those of oxycodone. These observations consequentially support the prediction that they would also engender the likelihood for abuse similar to oxycodone. This article is part of the Special Issue entitled 'Opioid Neuropharmacology: Advances in treating pain and opioid addiction'.

Nociceptin/orphanin FQ receptors modulate the discriminative stimulus effects of oxycodone in C57BL/6 mice.

BACKGROUND: Nociceptin/orphanin FQ (NOP) receptor ligands have shown efficacy as putative analgesics and can modulate the abuse-related effects of opioids, suggesting therapeutic applications. The discriminative stimulus effects of a drug are related to their subjective effects, a predictor of abuse potential. To determine whether activation of NOP receptors could alter the subjective effects of an abused opioid analgesic, a novel oxycodone discrimination was established in mice, characterized with positive and negative controls, and its expression evaluated with a NOP receptor agonist. METHODS: Adult male C57BL/6 mice were trained to discriminate 1.3 mg/kg oxycodone from vehicle in a two-lever operant procedure. The discrimination was characterized with naloxone challenge, and generalization tests with the µ-opioid receptor agonists, heroin and morphine, and the κ-opioid receptor selective agonist, U50488. Subsequently, effects of the NOP agonist Ro64-6198 were evaluated with and without oxycodone. RESULTS: Oxycodone generalization occurred in a dose-dependent manner and was reversed by naloxone pretreatment. Heroin and morphine, but not U50488, substituted for oxycodone. Co-treatment of 1 mg/kg Ro64-6198 with the oxycodone training dose reduced % oxycodone lever responding (%OLR) and restored response rates to vehicle control levels. J-113397, a NOP antagonist, reversed these effects. Co-administration of 1 mg/kg Ro64-6198 with a range of oxycodone doses resulted in rightward dose-effect curve shifts in %OLR and response rates compared to oxycodone alone. CONCLUSIONS: These results provide additional evidence that NOP receptor activation can modulate the subjective effects of opioid analgesics and represent the first characterization of oxycodone's discriminative stimulus effects in mice.

Effects of Acute and Repeated Administration of Oxycodone and Naloxone-Precipitated Withdrawal on Intracranial Self-Stimulation in Rats.

Incidence of prescription opioid abuse and overdose, often led by oxycodone, continues to increase, producing twice as many overdose deaths as heroin. Surprisingly, preclinical reports relevant to oxycodone's abuse-related effects are relatively sparse considering its history and patient usage. The goal of this study was to characterize dose- and time-dependent effects of acute and repeated oxycodone administration in a frequency-rate intracranial self-stimulation (ICSS) procedure, an assay often predictive of drug-related reinforcing effects, in male Sprague-Dawley rats. We hypothesized that oxycodone would produce a biphasic profile of rate-increasing and rate-decreasing effects maintained by ICSS similar to µ-opioid receptor agonists. Oxycodone (0.03, 0.3, 1, and 3 mg/kg, s.c.) produced dose- and time-dependent alterations on ICSS, with the predicted biphasic profile of rate-increasing effects at lower stimulation frequencies followed by rate-decreasing effects at higher frequencies. Peak effects were observed between 30 and 60 minutes, which were reversed by naloxone pretreatment (30 minutes). Tolerance to rate-decreasing effects was observed over a 5-day period when rats were treated with 1 mg/kg oxycodone twice a day. Subsequently, the dosing regimen was increased to 3 mg/kg twice a day over 10 days, although further marked tolerance did not develop. When then challenged with 10 mg/kg naloxone, a significant suppression below baseline levels of ICSS-maintained responding occurred indicative of dependence that recovered to baseline within 5 hours. The results of this study provide the first report of acute and chronic effects of oxycodone on responding maintained by ICSS presentation and the use of ICSS-maintained responding to characterize its tolerance and dependence effects.

Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice.

A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184).

Endocannabinoid contribution to Δ9-tetrahydrocannabinol discrimination in rodents.

The mechanism through which marijuana produces its psychoactive effects is Δ(9)-tetrahydrocannabinol (THC)-induced activation of cannabinoid CB1 receptors. These receptors are normally activated by endogenous lipids, including anandamide and 2-arachidonoyl glycerol (2-AG). A logical "first step" in determination of the role of these endocannabinoids in THC׳s psychoactive effects is to investigate the degree to which pharmacologically induced increases in anandamide and/or 2-AG concentrations through exogenous administration and/or systemic administration of inhibitors of their metabolism, fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), respectively, share THC׳s discriminative stimulus effects. To this end, adult male mice and rats were trained to discriminate THC (5.6 and 3mg/kg, respectively). In Experiment 1, exogenous administration of anandamide or 2-AG did not substitute for THC in mice nor was substitution enhanced by co-administration of the FAAH or MAGL inhibitors, URB597 and N-arachidonyl maleimide (NAM), respectively. Significant decreases in responding may have prevented assessment of adequate endocannabinoid doses. In mice trained at higher baseline response rates (Experiment 2), the FAAH inhibitor PF3845 (10mg/kg) enhanced anandamide substitution for THC without producing effects of its own. The MAGL inhibitor JZL184 increased brain levels of 2-AG in vitro and in vivo, increased THC-like responding without co-administration of 2-AG. In rats, neither URB597 nor JZL184 engendered significant THC-appropriate responding, but co-administration of these two enzyme inhibitors approached full substitution. The present results highlight the complex interplay between anandamide and 2-AG and suggest that endogenous increases of both endocannabinoids are most effective in elicitation of THC-like discriminative stimulus effects.

The endogenous cannabinoid anandamide shares discriminative stimulus effects with ∆(9)-tetrahydrocannabinol in fatty acid amide hydrolase knockout mice.

The endogenous cannabinoid system has been noted for its therapeutic potential, as well as the psychoactivity of cannabinoids such as Δ9-tetrahydrocannabinol (THC). However, less is known about the psychoactivity of anandamide (AEA), an endocannabinoid ligand. Thus, the goals of this study were to establish AEA as a discriminative stimulus in transgenic mice lacking fatty acid amide hydrolase (i.e., FAAH -/- mice unable to rapidly metabolize AEA), evaluate whether THC or oleamide, a fatty acid amide, produced AEA-like responding, and assess for CB(1) mediation of AEA's discriminative stimulus. Mice readily discriminated between 6mg/kg AEA and vehicle in a two-lever drug discrimination task. AEA dose-dependently generalized to itself. THC elicited full AEA-like responding, whereas oleamide failed to substitute. The CB(1) antagonist rimonabant attenuated AEA- and THC-induced AEA-appropriate responding, demonstrating CB(1) mediation of AEA's discriminative stimulus. These findings suggest that, in the absence of FAAH, AEA produces intoxication comparable to THC, and consequently to marijuana.

Structural and pharmacological analysis of O-2050, a putative neutral cannabinoid CB(1) receptor antagonist.

Rimonabant, the prototypic antagonist of cannabinoid CB(1) receptors, has been reported to have inverse agonist properties at higher concentrations, which may complicate its use as a tool for mechanistic evaluation of cannabinoid pharmacology. Consequently, recent synthesis efforts have concentrated on discovery of a neutral antagonist using a variety of structural templates. The purpose of this study was to evaluate the pharmacological properties of the putative neutral cannabinoid CB(1) receptor antagonist O-2050, a sulfonamide side chain analog of Δ(8)-tetrahydrocannabinol. O-2050 and related sulfonamide cannabinoids exhibited good affinity for both cannabinoid CB(1) and CB(2) receptors. While the other sulfonamide analogs produced cannabinoid agonist effects in vivo (e.g., activity suppression, antinociception, and hypothermia), O-2050 stimulated activity and was inactive in the other two tests. O-2050 also decreased food intake in mice, an effect that was reminiscent of that produced by rimonabant. Unlike rimonabant, however, O-2050 did not block the effects of cannabinoid agonists in vivo, even when administered i.c.v. In contrast, O-2050 antagonized the in vitro effects of cannabinoid agonists in [(35)S]GTPγS and mouse vas deferens assays without having activity on its own in either assay. Further evaluation revealed that O-2050 fully and dose-dependently substituted for Δ(9)-tetrahydrocannabinol in a mouse drug discrimination procedure (a cannabinoid agonist effect) and that it inhibited forskolin-stimulated cyclic AMP signaling with a maximum efficacy of approximately half that of the full agonist CP55,940 [(-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol]. Together, these results suggest that O-2050 is not a viable candidate for classification as a neutral cannabinoid CB(1) receptor antagonist.

Kappa opioid mediation of cannabinoid effects of the potent hallucinogen, salvinorin A, in rodents.

RATIONALE: Salvinorin A, the primary psychoactive derivative of the hallucinogenic herb Salvia divinorum, is a potent and highly selective kappa-opioid receptor (KOR) agonist. Several recent studies, however, have suggested endocannabinoid system mediation of some of its effects. OBJECTIVES: This study represents a systematic examination of this hypothesis. METHODS: Salvinorin A was isolated from S. divinorum and was evaluated in a battery of in vitro and in vivo procedures designed to detect cannabinoid activity, including CB(1) receptor radioligand and [(35)S]GTPgammaS binding, calcium flux assay, in vivo cannabinoid screening tests, and drug discrimination. RESULTS: Salvinorin A did not bind to nor activate CB(1) receptors. In vivo salvinorin A produced pronounced hypolocomotion and antinociception (and to a lesser extent, hypothermia). These effects were blocked by the selective KOR antagonist, JDTic, but not by the CB(1) receptor antagonist rimonabant. Interestingly, however, rimonabant attenuated KOR activation stimulated by U69,593 in a [(35)S]GTPgammaS assay. Salvinorin A did not substitute for Delta(9)-tetrahydrocannabinol (THC) in mice trained to discriminate THC. CONCLUSIONS: These findings suggest that similarities in the pharmacological effects of salvinorin A and those of cannabinoids are mediated by its activation of KOR rather than by any direct action of salvinorin A on the endocannabinoid system. Further, the results suggest that rimonabant reversal of salvinorin A effects in previous studies may be explained in part by rimonabant attenuation of KOR activation.

Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo.

Delta(9)-tetrahydrocannabinol (THC), the psychoactive component of marijuana, and other direct cannabinoid receptor (CB1) agonists produce a number of neurobehavioral effects in mammals that range from the beneficial (analgesia) to the untoward (abuse potential). Why, however, this full spectrum of activities is not observed upon pharmacological inhibition or genetic deletion of either fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), enzymes that regulate the two major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), respectively, has remained unclear. Here, we describe a selective and efficacious dual FAAH/MAGL inhibitor, JZL195, and show that this agent exhibits broad activity in the tetrad test for CB1 agonism, causing analgesia, hypomotilty, and catalepsy. Comparison of JZL195 to specific FAAH and MAGL inhibitors identified behavioral processes that were regulated by a single endocannabinoid pathway (e.g., hypomotility by the 2-AG/MAGL pathway) and, interestingly, those where disruption of both FAAH and MAGL produced additive effects that were reversed by a CB1 antagonist. Falling into this latter category was drug discrimination behavior, where dual FAAH/MAGL blockade, but not disruption of either FAAH or MAGL alone, produced THC-like responses that were reversed by a CB1 antagonist. These data indicate that AEA and 2-AG signaling pathways interact to regulate specific behavioral processes in vivo, including those relevant to drug abuse, thus providing a potential mechanistic basis for the distinct pharmacological profiles of direct CB1 agonists and inhibitors of individual endocannabinoid degradative enzymes.

Route of administration influences substitution patterns in rats trained to discriminate methadone vs. vehicle.

Replacement therapy with the synthetic mu-opioid agonist methadone is an efficacious treatment for opioid abuse. While much is known about methadone's pharmacology, its discriminative stimulus properties remain largely unexplored. The present study sought to establish methadone discrimination in rats. Moreover, some research suggests that route of administration alters the discriminative stimulus of methadone. Thus, the present study also compared intraperitoneal (i.p.) and subcutaneous (s.c.) routes of administration. Male Sprague-Dawley rats were trained to discriminate 3.0mg/kg methadone (i.p.) from vehicle in a two-lever discrimination procedure. Generalization tests were conducted with a variety of compounds administered i.p. and s.c. Methadone fully substituted for itself, yielding ED(50)s of 1.5mg/kg (i.p.) and 0.2mg/kg (s.c.). Naltrexone (i.p.), an opioid antagonist produced a dose-dependent reduction in methadone-appropriate responding. The methadone stereoisomers fully substituted for methadone when given s.c.; however, when administered i.p., (+) and (-) methadone produced partial and no substitution, respectively. Heroin fully generalized to methadone regardless of administration route, while morphine fully substituted when given s.c., but not i.p. The kappa-agonist U50-488 failed to generalize to methadone with either route of administration. These results demonstrated that methadone's discriminative stimulus is mediated through mu-opioid receptor activity and is similar to that of commonly abused opioids (heroin, morphine). Additionally, route of administration produced differential results for many of the drugs tested, suggesting decreased drug bioavailability following i.p. administration due to hepatic first pass metabolism. Taken together, these results suggest that methadone's shared subjective effects with abused opioids, as well as its unique metabolic properties contribute to its efficacy in opioid maintenance therapy.

Further characterization of the discriminative stimulus properties of the atypical antipsychotic drug clozapine in C57BL/6 mice: role of 5-HT(2A) serotonergic and alpha (1) adrenergic antagonism.

RATIONALE: The discriminative stimulus properties of the atypical antipsychotic drug (APD) clozapine (CLZ) have recently been studied in C57BL/6 mice, a common background strain for genetic alterations. However, further evaluation is needed to fully characterize CLZ's discriminative cue in this strain of mice. OBJECTIVES: The objectives of the study were to confirm the previous findings using a shorter pretreatment time and to further characterize the receptor mechanisms mediating the discriminative stimulus properties of CLZ by testing APDs, selective ligands, and N-desmethylclozapine (CLZ's major metabolite) in C57BL/6 mice. MATERIALS AND METHODS: C57BL/6 male mice were trained to discriminate 2.5 mg/kg CLZ (s.c.) from vehicle in a two-lever drug discrimination task. RESULTS: Generalization testing with CLZ yielded an ED(50) = 1.19 mg/kg. Substitution testing with APDs showed that the atypical APDs quetiapine, sertindole, zotepine, iloperidone, and melperone fully substituted for CLZ (> or =80% CLZ-appropriate responding), but aripiprazole did not. The typical APDs chlorpromazine and thioridazine substituted for CLZ (fluphenazine and perphenazine did not). The serotonin (5-HT) (2A) antagonist M100907 and the alpha(1)-adrenoceptor antagonist prazosin fully substituted for CLZ. The H(1) histaminergic antagonist pyrilamine, dopamine agonist amphetamine, and the selective serotonin reuptake inhibitor fluoxetine did not substitute for CLZ. While N-desmethylclozapine did not substitute for CLZ when tested alone, N-desmethylclozapine plus a low dose of CLZ combined in an additive manner produced full substitution. CONCLUSIONS: CLZ's discriminative cue in C57BL/6 mice is a "compound" cue mediated in part by antagonism of 5-HT(2A) and alpha(1) receptors.