The adverse effects of mu opioid agonists have spurred a renewed interest in using kappa opioid receptor (KOR) agonists as analgesics. KOR agonists also have potential for development as diuretics for the treatment of edema and hypertension. Here, we evaluated the discriminative stimulus, antinociceptive, and diuretic effects of the kappa agonist (±)-trans-U-50488 and its stereoisomers (-)-(1S,2S)-U-50488 or (+)-(1R,2R)-U-50488) alone and in combination with the cannabinoid agonist (-)-CP 55,940. To establish (±)-U-50488 as a discriminative stimulus, rats (n = 12) were trained to discriminate intraperitoneal (i.p.) administration of 5.6 mg/kg of (±)-trans-U-50488 from saline under a fixed-ratio 20 (FR-20) schedule of food reinforcement. Then, antinociception was assessed using two procedures: warm water tail withdrawal and von Frey paw withdrawal. Diuretic effects were assessed in separate rats (n = 6/group). Doses of (±)-U-50488 and (-)-U-50488 that served as discriminative stimuli produced significant increases in urine output, but at lower doses than those that produced antinociception. In contrast, (+)-U-50488 alone had no discriminative stimulus or diuretic effects at the doses tested, but did produce antinociception in the von Frey assay. When three cannabinoids and morphine were tested in the (±)-U-50488 discrimination procedure to determine the similarity of these drugs' discriminative stimulus effects to those for (±)-U-50488, the rank order similarity was (-)-CP 55,940 > (-)-trans-THC > (+)-WIN 55,212-2 ≥ morphine. (-)-CP 55,940 alone (0.056 mg/kg) partially substituted for the discriminative stimulus effects of (±)-U-50488 and produced significant diuretic and antinociceptive effects. (-)-CP 55,940 in combination with (±)-U-50488 also produced a two-fold leftward shift in the discriminative stimulus curve for (±)-U-50488, and near-additive antinociception with (±)-U-50488 and (+)-U-50488. Further, the diuretic effect of (-)-CP 55,940 was enhanced by a dose of (+)-U50488, which itself did not alter urine output. These data together indicate that a combination of cannabinoid and kappa opioid agonists can enhance diuresis, but may have limited potential for serving as opioid-sparing pharmacotherapeutics for treatment of pain.

Increased abuse of synthetic cannabinoids (SCBs) continues to be a public health concern. Preclinical studies have identified SCBs as being THC‐like; however, occurrences of emesis, hallucinations, and seizures have been reported with their usage. These studies examined the premise that the hallucinogenic effects of AM8936, a highly potent synthetic CB1 agonist, resemble the discriminative stimulus effects of another hallucinogen, the 5HT2A agonist (R)(−)2,5‐dimethoxy‐4‐iodoamphetamine (DOI). Male and female Sprague‐Dawley rats (n=6/group) were trained to discriminate either 0.18 mg/kg AM8936 or 0.56 mg/kg DOI from saline under a shock avoidance schedule of reinforcement. In both DOI and AM8936 trained subjects, their respective training dose produced full (>80%) substitution. In the DOI trained subjects, AM8936 (0.032 – 0.18 mg/kg), delta‐9‐tetrahydrocannabinol (Δ9‐THC) (1 – 10 mg/kg), and JWH‐018 (0.1 – 3.2 mg/kg) produced dose‐dependent increases in DOI‐lever responding, but only JWH‐018 and AM8936 produced full substitution. In the AM8936 trained subjects, DOI (0.56 – 3.2 m/k), Δ9‐THC (1 – 10 mg/kg), and JWH‐018 (0.1 – 10 mg/kg) produced dose‐dependent increases in AM8936‐lever responding, but only JWH‐018 produced full (>80%) substitution. Doses of DOI that produced AM8936‐appropriate responding (0.56 – 3.2 mg/kg) were higher than those that resulted in DOI‐appropriate responding (0.1 – 0.8 mg/kg). In addition, the mu‐opioid receptor agonist morphine (1 – 10 mg/kg) (negative control) engendered less than 20% DOI‐lever responding or AM8936‐lever responding up to doses that produced behavioral disruption. These studies indicate that there is bi‐directional overlap in the discriminative stimulus effects of SCBs and a 5‐HT2A agonist, suggesting similarities in their subjective effects.Support or Funding InformationAcknowledgementsFunded by NIH/NIDA: DA043700

The illicit synthetic cathinone mephedrone (4‐methylmethcathinone) often termed a “bath salt” produces discriminative stimulus effects that are similar, but not identical, to those for central nervous system stimulants such as cocaine and methamphetamine. This may be due to its capacity to bind atypically at both dopamine and serotonin transporters compared to those drugs. The purpose of the present study was to further elucidate the mechanism of action underlying mephedrone’s discriminative stimulus effects by administering it with the dopamine type‐1 receptor antagonist SCH‐23390 (n=11) or the catecholamine synthesis inhibitor alpha‐methyl‐tyrosine (alpha‐MT; n=8). Male Long‐Evans rats were trained to discriminate intraperitoneal injections of mephedrone (3.2 mg/kg) from saline under a fixed‐ratio 20 (FR‐20) schedule of food presentation. Mephedrone dose‐effect curves (0.32 – 10 mg/kg) were then established by administering increasing cumulative doses of mephedrone on test days, and comparing those effects with multiple (4–5) injections of saline. The interaction of SCH‐23390 (0.032 – 0.1 mg/kg) and alpha‐MT (200 mg/kg) with mephedrone was examined by administering an acute injection of these drugs prior to increasing cumulative doses of mephedrone. As shown previously by our laboratory, saline administration produced almost exclusively saline‐lever responding while increasing cumulative doses of mephedrone dose‐dependently increased mephedrone‐lever responding. When SCH‐23390 preceded cumulative doses of mephedrone, neither dose alone produced mephedrone‐lever responding and only the larger of the two doses tested (0.1 mg/kg) shifted the mephedrone dose‐effect curve rightward; however, this shift was still less than 2‐fold and produced rate‐decreasing effects when administered alone. Acute administration of alpha‐MT alone also did not produce mephedronelever responding nor decrease rate, and in combination did not produce a marked rightward shift in the mephedrone dose‐effect curve. Taken together, these data indicate that neither SCH‐23390 nor alpha‐MT effectively antagonize the discriminative stimulus effects of mephedrone, and the alpha‐MT data in particular suggest that mephedrone’s discriminative stimulus effects do not depend on newly synthesized catecholamines.

Kappa (κ) opioid receptor agonists can produce prominent analgesic and diuretic effects when administered alone and these effects can be enhanced by the co‐administration of cannabinoids such as CP 55,940. The purpose of the present experiment was to determine if cannabinoids can also enhance the aversive effects of kappa agonists, because these effects substantially reduce their clinical utility as analgesics and aquaretics. To study the aversive effects of kappa agonists and their interaction with cannabinoids, nine male Long‐Evans rats were trained to respond for food under a fixed‐ratio (FR) schedule of food presentation and then implanted with intravenous (i.v.) catheters. Following recovery, the subjects were transitioned to a multiple FR, FR schedule. More specifically, in the presence of a single white stimulus light, food was presented under a fixed‐ratio 30 (FR‐30) schedule (non‐infusion component), whereas presence of both this white stimulus light and a green stimulus light, an i.v. infusion was presented on the first response of each FR and food was presented on the thirtieth FR response (infusion component). When responding was consistent in both components, varying infusion doses of U‐50488 (0.032 – 0.18 mg/kg) replaced the baseline saline infusion until one of three criterion was met: 3 days in which the variability of responding did not vary by ± 20% of the mean, 3 days of responding at less than 0.3 responses per sec, or a maximum of 8 days. After the criterion was met for each dose substitution, the subjects returned to the baseline in which saline served as the infusion and responding was similar between components. To study the interaction of CP 55,940 with the effects of U‐50488, single acute injections of a dose of CP 55,940 (0.056 mg/kg) were administered prior to sessions in which a dose of U‐50488 was available in the infusion component. Each dose‐infusion combination was administered until subjects met the criterion. These effects were then compared to sessions in which one of two doses of naltrexone (1, 3.2 mg/kg) was administered prior the same U‐50488 infusion doses. When infusions of U‐50488 replaced saline, low infusion doses (e.g., 0.056 mg/kg/infusion) selectively suppressed responding in the infusion component, while higher infusion doses (e.g., 0.18 mg/kg/infusion) decreased responding in both components as the total dose of U‐50488 for the session increased. Administration of CP 55,940 did not produce a marked enhancement of the suppressive effects of U‐50488 at the dose tested, and surprisingly, neither dose of naltrexone antagonized its suppressive effects. Together, these data demonstrate the aversive effects of U‐50488 alone, the absence of a marked enhancement by at least one cannabinoid agonist, and the surprising absence of antagonism by the opioid receptor antagonist naltrexone.

Previous research from this laboratory demonstrated that male outbred rats (Long-Evans) can be trained to prefer ethanol (10% v/v) over water during 30-min home-cage sessions and that higher ethanol concentrations (18-32% v/v) can serve as a reinforcer under various operant schedules. Further, we have shown that two neurosteroids, dehydroepiandrosterone (DHEA) and pregnanolone, can readily decrease ethanol self-administration in males. The present study used the same procedures in an attempt to systematically replicate the previous findings in female outbred rats. Rats were first trained to self-administer ethanol in the home cage using a saccharin-fading procedure. Subsequently, a two-bottle preference test was initiated by substituting different ethanol concentrations after subjects reliably consumed 10% ethanol alone. Water was always available during this phase. Next, subjects were transitioned to a fixed-ratio 10 (FR-10) schedule of reinforcement with 0.1 mL of ethanol (18% v/v) serving as the reinforcer so that a concentration-effect curve could be established. Upon completion, subjects were transitioned to an FR-10 FR-20 multiple schedule of ethanol (32% v/v) and food reinforcement to determine whether noncontingent ethanol, DHEA, and pregnanolone could selectively decrease ethanol intake. Not surprisingly, female subjects preferentially consumed ethanol over water at concentrations of 3.2-18% (v/v) during the home-cage procedure, and significantly increased the mean dose of ethanol consumed and blood ethanol concentration (BEC). Similarly, increasing concentrations under an FR-10 schedule significantly increased the dose of ethanol presented and BEC compared to control (water). Finally, under the multiple schedule, noncontingent injections of ethanol (0.32-1.8 g/kg), DHEA (10 -100 mg/kg), and pregnanolone (1.8-32 mg/kg) dose-dependently decreased food- and ethanol maintained responding and the dose of ethanol presented. BEC was significantly decreased by the neurosteroids, but increased by ethanol due to its noncontingent administration. Together, these data replicate only a subset of the data previously obtained in males, suggesting there are sex differences particularly with respect to the effects of DHEA and pregnanolone. (C) 2018 Elsevier Inc. All rights reserved.

Currently, more than half of the drugs in clinical use are chiral compounds. In many instances the enantiomers of chiral drugs demonstrate pronounced differences in biological activity, toxicology, pharmacokinetics, and metabolism. Administration of racemic U‐50,488, a highly selective chiral κ‐opioid receptor agonist, produces a marked sodium and potassium sparing diuresis (i.e. aquaresis), dysphoria, and analgesia in conscious rats. Though U‐50,488 enantiomers have been the subject of various behavioral and analgesic studies, there is a distinct lack of data pertaining to isomers and kappa induced aquaresis. Therefore, the aim of the present study was to pharmacologically characterize the renal excretory effects produced by the isolated (−)‐(1S,2S)‐U‐50,488 versus (+)‐(1R,2R)‐U50,488 enantiomers. Methods Male Sprague‐Dawley rats with an indwelling intracerebroventricular (ICV) cannula were surgically implanted with an arterial, venous, and bladder catheter and continuously infused intravenously (i.v.) with iso‐saline. After stabilization, mean arterial pressure (MAP), heart rate (HR), and urine flow rate (V) were measured in conscious rats for 20‐min before (control) and 90‐min after (experimental) ICV bolus injection (1 ug) of (−)‐(1S,2S)‐U‐50,488, (+)‐(1R,2R)‐U50,488, racemic (+/−)‐U‐50,488, or vehicle (n=6/group). Urine samples were analyzed for urinary sodium excretion (UNaV) and urinary potassium excretion (UKV). Results ICV injection of racemic U‐50,488 (1 ug) produced a significant diuretic (control (C), 56±6, 30‐min, 152±7 ul/min), antinatriuretic (C, 8.7±2.2, 30 min, 3.7±2 ueq/min), and antikaluretic (C, 1.1±0.5; 0.4±0.4 ueq/min) response. At the same dose, both the (−)‐(1S,2S)‐U‐50,488 and (+)‐(1R,2R)‐U50,488 isomers produced a significant sodium and potassium sparing diuresis without changing MAP or HR. At 1 ug, (−)‐(1S,2S)‐U‐50,488 significantly increased peak urine flow rate (C, 58±6 ul/min; 20 min 196±29 ul/min) and decreased urinary sodium (C, 8.9±2.3 μeq/min; 30 min 3.9±0.8 μeq/min) and potassium (C, 0.8±0.2 μeq/min, 30 min, 0.2±0.1 μeq/min) excretion. In contrast, 1 ug of (+)‐(1R,2R)‐U50,488 increased urine flow rate to a lessor magnitude (C, 57±6 ul/min; 10 min 119±26 ul/min) while decreasing urinary sodium excretion (C, 8.0±0.7 μeq/min, 30 min 4.3±1.5 μeq/min) but not urinary potassium excretion. ICV pretreatment (1ug, 10 min) with the kappa antagonist nor‐BNI abolished the kappa‐mediated diuresis by both enantiomers and racemic U‐50,488.ConclusionsThese results indicate (−)‐(1S,2S)‐U‐50,488 is the predominant isomer responsible for producing the centrally mediated aquaresis observed with racemic U‐50,488. Blockade of the aquaresis by ICV nor‐BNI indicates that both enantiomers mediate their renal excretory effects through activation of central kappa receptors. Further renal excretory studies are needed to fully elucidate the pharmacological variances between these two compounds and determine whether a selective entaniomer of U‐50,488 may be used therapeutically as a water diuretic.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.

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.

Mephedrone (4‐methylmethcathinone) is one of the major constituents of “bath salts.” It is a drug with discriminative effects similar to CNS stimulants and it can produce auditory and visual hallucinations, as well as problematic cardiovascular effects. This study compared the discriminative stimulus effects of mephedrone (0.32–10 mg/kg) with other prototypical drugs with CNS effects, such as ketamine (1.8–18 mg/kg), bupropion (5.6–56 mg/kg), and 3,4‐methylenedioxyamphetamine (MDA) (0.32–5.6 mg/kg). In addition, mephedrone was administered in combination with cocaine (5.6–32 mg/kg), which inhibits the reuptake of catecholamines and serotonin. Rats (n=6) were trained to discriminate an intraperitoneal injection of 3.2 mg/kg mephedrone from saline under a fixed‐ratio 20 schedule. Following training, increasing cumulative doses of mephedrone produced dose‐dependent increases in mephedrone‐lever responding, with full substitution considered to be greater than 80% responding and partial substitution to be greater than 50% responding. During substitution tests, ketamine never produced more than 17% drug‐lever responding up to doses that significantly decreased overall response rate. In addition, bupropion and MDA produced 36% and 77% drug‐lever responding, respectively, without significantly decreasing response rate. When 3.2 mg/kg of cocaine was administered prior to increasing doses of mephedrone, there was no substitution for cocaine alone; however, this dose produced a rightward shift in the dose‐effect curve for mephedrone‐lever responding. Unlike this dose, 5.6 mg/kg of cocaine alone did not substitute for mephedrone or shift the mephedrone dose‐effect curve. When the dose of cocaine was increased to 10 mg/kg, there was still no mephedrone substitution, but this dose increased the mephedrone‐lever responding of the low doses of mephedrone. Finally, 18 and 32 mg/kg of cocaine produced 38.1% and 77.7% substitution, respectively, and dose‐dependently increased the substitution of the low doses of mephedrone (0.32 – 1.8 mg/kg). This data suggest that mephedrone may be similar to cocaine in having a higher affinity for the serotonin transorter (SERT) than the dopamine transporter (DAT), but more efficacy at DAT than SERT. Certainly, due to the capacity of cocaine to only enhance the substitution of the low mephedrone doses, the discriminative stimulus effects of the training dose of mephedrone are mediated predominately by dopamine rather than serotonin.Support or Funding InformationThis research was supported by the Louisiana Board of Regents Fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abuse of cathinones has been a worldwide health concern for some time. Their chemical structures and wide variation in pharmacodynamic effects have led to clinical and preclinical effects that can be both similar to and different from other psychoactive substances such as methylenedioxymethamphetamine (MDMA), methamphetamine, and cocaine.

Mephedrone (4-methylmethcathinone), a constituent of the recreational substances known as "bath salts", is a synthetic cathinone that can produce auditory and visual hallucinations, as well as problematic cardiovascular effects. This study compared the discriminative stimulus effects of mephedrone (0.32-10 mg/kg) with other prototypical drugs of abuse: cocaine (0.56-32 mg/kg), d-amphetamine (0.18-3.2 mg/kg), ketamine (1.8-18 mg/kg), phencyclidine (PCP, 1-5.6 mg/kg), heroin (1-10 mg/kg), 2,5-dimethoxy-4-iodoamphetamine (R-DOI, 0.1-1 mg/kg), Δ9-tetrahydrocannabinol (Δ9-THC 0.56-5.6 mg/kg), 3,4-methylenedioxyamphetamine (MDA, 0.32-5.6 mg/kg), methylphenidate (1-10 mg/kg), and 3,4-methylenedioxypyrovalerone (MDPV, 0.56-5.6 mg/kg). The discriminative stimulus effects of mephedrone were also assessed after administration of the sigma receptor antagonist rimcazole (0.32-10 mg/kg), the relatively selective norepinephrine transporter (NET) inhibitor desipramine (1.8-18 mg/kg), and the selective serotonin transporter (SERT) inhibitor fluoxetine (1-18 mg/kg). Initially, rats were trained to discriminate an intraperitoneal injection of mephedrone (3.2 mg/kg) from saline under a fixed-ratio 20 schedule of food presentation. Following training, cumulative doses of mephedrone and the other drugs were administered to test for substitution (80% drug-lever responding). Of the drugs tested, including those that were tested in combination with mephedrone (i.e., rimcazole, desipramine, and fluoxetine), only cocaine fully substituted for mephedrone without substantially decreasing response rate. In addition, the three drugs administered in combination with mephedrone shifted the cumulative dose-effect curves leftward (percent drug-lever responding) and down (response rate), although fluoxetine did so in a dose-dependent manner ranging from antagonism to potentiation. In summary, the discriminative stimulus effects of mephedrone were most similar to those for the central nervous system (CNS) stimulant, cocaine, and SERT and DAT activity were necessary for these effects.

Mephedrone (4‐methylmethcathinone) was one of the major constituents of the recreational substances referred to as “bath salts”. It is a synthetic cathinone and psychostimulant that can produce auditory and visual hallucinations, as well as problematic cardiovascular effects. This study compared the discriminative stimulus effects of mephedrone (0.32–10 mg/kg) with other prototypical drugs with central nervous system effects, such as ketamine (1.8–18 mg/kg), methylphenidate (1.0–10 mg/kg), and 3,4‐methylenedioxyamphetamine (MDA) (0.32–5.6 mg/kg). Additionally, a selective serotonin reuptake inhibitor, fluoxetine, was administered alone and in combination with mephedrone in order to assess the involvement of the serotonin transporter (SERT). Rats (n=11) were trained to discriminate an intraperitoneal injection of mephedrone from saline under a fixed‐ratio 20 schedule of food presentation. Following training, increasing cumulative doses of mephedrone (0.32–10 mg/kg) produced dose‐dependent increases in mephedrone‐lever responding, with full substitution considered to be greater than 80% drug‐lever responding and partial substitution greater than 50% drug‐lever responding. During substitution tests, MDA and methylphenidate produced 77% and 56% drug‐lever responding, respectively, without significantly decreasing rate of responding (i.e., a 20% decrease from control). Ketamine never produced more than 17% drug‐lever responding at any dose tested. When cumulative doses of fluoxetine (1.8–18 mg/kg) were administered alone, there was little to no substitution. However, when given as a pretreatment, 3.2 mg/kg fluoxetine in combination with mephedrone produced a small (less than two‐fold) shift to the right in the dose‐effect curve for mephedrone‐lever responding. As the pretreatment dose of fluoxetine increased to 5.6 and 10 mg/kg, the ascending portion of the dose‐effect curve for drug‐lever responding shifted to the left and upward. In addition, the 5.6 and 10 mg/kg doses of fluoxetine both partially substituted when administered alone as a bolus injection prior to mephedrone, something which was not seen when administered in cumulative doses. Taken together, these data indicate that the discriminative stimulus effects of mephedrone partially overlap with stimulants such as methylphenidate and MDA, and are also mediated by SERT due to the capacity of fluoxetine to dose‐dependently shift the ascending portion of the curve for mephedrone's discriminative stimulus effects. These data also suggest that doses in the range of 3.2–10 mg/kg likely involve other neurotransmitters such as dopamine.Support or Funding InformationBoard of Regents Fellowship