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Duloxetine, known by its brand name, CymbaltaTM, is a selective serotonin and norepinephrine reuptake inhibitor used to treat major depressive disorders. Determination of patient compliance for duloxetine is typically determined through medication possession ratio (MPR) or plasma concentrations. The purpose of this paper was to characterize normal urinary duloxetine concentrations in patients prescribed duloxetine to monitor patient adherence. Patient data collected from routine screens for duloxetine concentrations in urine were included in this study. Inclusion criteria consisted of patients who were prescribed duloxetine and (i) tested positive for duloxetine, (ii) tested negative for illicit substances and (iii) included creatinine, age and duloxetine dose administered. Of the 5,592 patient urines screened, 2,004 of the results fit into the inclusion criteria. Positive urine concentrations of duloxetine ranged from 50 to 2,722 ng/mL. Duloxetine urine concentrations were normalized to creatinine and dose further characterized by sex, age, body mass index (BMI) and dose in milligrams. Sample distribution included urines collected from 1,487 females and 517 males. The age range of the specimen donors was between 15 and 90 years old with an average age of 52. BMI levels ranged from 13.9 (underweight) to 88.1 (obese), with the average BMI being 33.5. The most common dose of duloxetine prescribed was a daily, oral dose of 60 mg. Analysis of the normalized, transformed creatinine concentrations showed that there was a significant statistical difference (P

Psychedelics, also known as classical hallucinogens, affect processes related to perception, cognition and sensory processing mostly via the serotonin 5-HT(2A) receptor (5-HT(2A)R). This class of psychoactive substances, which includes lysergic acid diethylamide (LSD), psilocybin, mescaline and the substituted amphetamine 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), is receiving renewed attention for their potential therapeutic properties as it relates to psychiatric conditions such as depression and substance use disorders. Current studies focused on the potentially clinical effects of psychedelics on human subjects tend to exclude sex as a biological variable. Much of the understanding of psychedelic pharmacology is derived from rodent models, but most of this preclinical research has only focused on male mice. Here we tested the effects of DOI on head-twitch behavior (HTR) – a mouse behavioral proxy of human psychedelic potential – in male and female mice. DOI elicited more HTR in female as compared to male C57BL/6J mice, a sex-specific exacerbated behavior that was not observed in 129S6/SvEv animals. Volinanserin (or M100907) – a 5-HT(2A)R antagonist – fully prevented DOI-induced HTR in male and female C57BL/6J mice. Accumulation of inositol monophosphate (IP(1)) in the frontal cortex upon DOI administration showed no sex-related effect in C57BL/6J mice. However, the pharmacokinetic properties of DOI differed among sexes – brain and plasma concentrations of DOI were lower 30 and 60 min after drug administration in female as compared to male C57BL/6J mice. Together, these results suggest strain-dependent and sex-related differences in the behavioral and pharmacokinetic profiles of the 5-HT(2A)R agonist DOI in C57BL/6J mice, and support the importance of studying sex as a biological variable in preclinical psychedelic research.

Background and purpose Paclitaxel, a widely used anti-cancer drug, is frequently associated with prolonged and severe peripheral neuropathies (PIPN), associated with neuroinflammation. Currently, PIPN effective treatments are lacking. Peroxisome Proliferator-Activated Receptor-α (PPAR-⍺) can modulate inflammatory responses. Thus, the use of PPAR-⍺ agonists, such as fibrates (fenofibrate and choline-fenofibrate), currently used in dyslipidemia treatment, could represent an interesting therapeutic approach in PIPN. Experimental approach Our studies tested the efficacy of fenofibrate (150 mg/kg, daily, i.p.) and choline fenofibrate (60 mg/kg daily, p.o.) in reversing and preventing the development of PIPN (paclitaxel: 8 mg/kg, i.p., every other day for 4 days) in male and female C57BL/6J mice. Mechanical and cold hypersensitivity, conditioned place preference, sensory nerve action potential (SNAP), as well as the expression of PPAR-⍺, TNF-⍺, IL-1β and IL-6 mRNA were evaluated. Key results While fenofibrate treatment partially reversed and prevented the development of mechanical hypersensitivity, this was completely reversed and prevented by choline-fenofibrate. Both fibrates were able to completely reverse and prevent cold hypersensitivity induced by paclitaxel. The reduction of SNAP amplitude induced by paclitaxel was also reversed by both fenofibrate and choline-fenofibrate. Our results indicate that suppression of paclitaxel-induced hypersensitivity by fibrates involves the regulation of PPAR-⍺ expression and decrease neuroinflammation in DRG. Finally, the co-treatment of Paclitaxel and fenofibric acid (fibrates active metabolite) was tested on different cancer cell lines, no decrease in the antitumoral effect of paclitaxel was observed. Conclusions and implications Taken together, our results show for the first time the therapeutic potential (prevention and reversal) of fibrates in PIPN and opens to a potential pharmacological repurposing of these drugs.

Opioids are among the most effective analgesics and the mainstay of pain management. However, concerns about safety and abuse liability have challenged their widespread use by the medical community. Opioid-sparing therapies include drugs that in combination with opioids have the ability to enhance analgesia while decreasing opioid requirement as well as their side effects. Sex differences in antinociceptive responses to opioids have received increasing attention in recent years. However, the molecular mechanisms underlying sex differences related to opioid-sparing adjuncts remain largely unexplored. Using warm water tail-withdrawal as a mouse model of acute thermal nociception, our data suggest that adjunctive administration of the serotonin 5-HT(2A) receptor (5-HT(2A)R) antagonist volinanserin dose-dependently enhanced potency of the opioid analgesic oxycodone in male, but not female, mice. This antinociceptivelike response induced by oxycodone was also augmented in 5-HT(2A)R knockout (5-HT(2A)R(−/−)) male, but not female mice; an effect that was reversed by Cre-loxP-mediated selective expression of 5-HT(2A)R in dorsal root ganglion (DRG) neurons of 5-HT(2A)R(−/−) littermates. Pharmacological inhibition with volinanserin or genetic deletion in 5-HT(2A)R(−/−) animals potentiated the ability of oxycodone to reduce DRG excitability in male mice. Adjunctive volinanserin did not affect oxycodone-induced conditioned place preference (CPP), whereas it reduced oxycodone-induced locomotor sensitization in male and female mice. Together, these results suggest that adjunctive volinanserin augments opioid-induced antinociception, but not abuse-related behavior, through a sex-specific signaling crosstalk mechanism that requires 5-HT(2A)R expression in mouse DRG neurons. Ultimately, our results may pave the way for the clinical evaluation of volinanserin as a potential sex-specific opioid adjuvant.

BACKGROUND. The phenomenon of alcohol analgesia and tolerance can facilitate misuse and lead to the development of alcohol use disorder (AUD). Numerous alcohol-induced behaviors are genetically influenced; however, it is unknown if alcohol analgesia has a genetic contribution. Rodent studies have shown that alcohol responses differ vastly between two widely studied inbred strains of mice, C57BL/6J (B6) and DBA/2J (D2). Here, we used B6 and D2 mice as an initial behavioral genetic analysis of acute alcohol-induced antinociception. METHODS. The antinociceptive effect of orally-administered alcohol was characterized using the hot plate test in B6 and D2 mice of both sexes. Using the opioid receptor antagonist naloxone, the involvement of the opioid system was assessed. Locomotor activity and blood alcohol concentrations were also measured. Ovariectomized mice were used to evaluate the influence of ovarian sex hormones on alcohol-induced antinociception. RESULTS. Alcohol induced an antinociceptive effect in B6 and D2 male mice in a time- and dose-dependent manner. In addition, D2 male mice were more sensitive to the antinociceptive effect of alcohol than B6 male mice. However, locomotion is not impeded by the tested doses of alcohol in B6 mice. Female D2 and B6 mice failed to show significant antinociceptive effects in alcohol dose-response studies. In addition, alcohol-induced antinociception was still not evident in ovariectomized female mice. Male mice of both strains developed tolerance to this effect after repeated administration of alcohol. Strain differences were found in blood alcohol concentration. Finally, no difference was found in the blockade of alcohol antinociception by 2 mg/kg naloxone. CONCLUSION. Our results indicate that the antinociceptive effects of alcohol in the hot plate test are influenced by strain and sex. These findings support further genetic analysis of alcohol-induced antinociception to identify operative mechanisms and better assess the contribution of this phenotype to AUD.

RATIONALE: Tianeptine is a mu-opioid receptor (MOR) agonist with increasing reports of abuse in human populations. Preclinical data regarding the abuse potential and other opioid-like adverse effects of tianeptine at supratherapeutic doses are sparse. OBJECTIVES: The present study evaluated tianeptine in a rat model of abuse potential assessment and in mouse models of motor, gastrointestinal, and respiratory adverse effects. METHODS: Abuse potential was assessed in adult male Sprague-Dawley rats using an intracranial self-stimulation (ICSS) procedure to determine effects of acute and repeated tianeptine on responding for electrical brain stimulation. Male ICR mice were used to determine the effects of tianeptine in assays of locomotor behavior and gastrointestinal motility. Male Swiss-Webster mice were monitored for respiratory changes using whole-body plethysmography. RESULTS: In rats, acute tianeptine produced weak and delayed evidence for abuse-related ICSS facilitation at an intermediate dose (10 mg/kg, IP) and pronounced, naltrexone-preventable ICSS depression at a higher dose (32 mg/kg, IP). Repeated 7-day tianeptine (10 and 32 mg/kg/day, IP) produced no increase in abuse-related ICSS facilitation, only modest tolerance to ICSS depression, and no evidence of physical dependence. In mice, tianeptine produced dose-dependent, naltrexone-preventable locomotor activation. Tianeptine (100 mg/kg, SC) also significantly inhibited gastrointestinal motility and produced naloxone-reversible respiratory depression. CONCLUSIONS: Tianeptine presents as a MOR agonist with resistance to tolerance and dependence in our ICSS assay in rats, and it has lower abuse potential by this metric than many commonly abused opioids. Nonetheless, tianeptine produces MOR agonist-like acute adverse effects that include motor impairment, constipation, and respiratory depression.

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.

BACKGROUND: Complex interactions between environmental and genetic factors influence the risk for developing alcohol use disorder (AUD) in humans. To date, studies of the impact of environment on AUD risk have primarily focused on psychological characteristics or on the effects of developmental exposure to ethanol. We recently observed that modifying levels of the long-chain ω-3 (LC ω-3) fatty acid, eicosapentaenoic acid (EPA), alters acute physiological responses to ethanol in C. elegans. Because mammals derive ω-3 fatty acids from their diet, here we asked if manipulating dietary levels of LC ω-3 fatty acids can affect ethanol-responsive behaviors in mice. METHODS: We used two well-characterized inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2), that differ in their responses to ethanol. Age-matched young adult male mice were maintained on isocaloric diets that differed only by being enriched or depleted in LC ω-3 fatty acids. Animals were subsequently tested for acute ethanol sensitivity (locomotor activation and sedation), voluntary consumption, and metabolism. Fat deposition was also determined. RESULTS: We found that dietary levels of LC ω-3s altered ethanol sensitivity and consumption in a genotype-specific manner. Both B6 and D2 animals fed high LC ω-3 diets demonstrated lower ethanol-induced locomotor stimulation than those fed low LC ω-3 diets. Ethanol sedation and ethanol metabolism were greater in D2, but not B6 mice on the high LC ω-3 diet. Conversely, LC ω-3 dietary manipulation altered ethanol consumption in B6, but not in D2 mice. B6 mice on a high LC ω-3 diet consumed more ethanol in a 2-bottle choice intermittent access model than B6 mice on a low LC ω-3 diet. CONCLUSIONS: Because ethanol sensitivity is predictive of risk for developing AUD in humans, our data indicate that dietary LC ω-3 levels should be evaluated for their impact on AUD risk in humans. Further, these studies indicate that genetic background can interact with fatty acids in the diet to significantly alter ethanol-responsive behaviors.

Although paclitaxel effectively treats various cancers, its debilitating peripheral neuropathic pain side effects often persist long after treatment has ended. Therefore, a compelling need exists for the identification of novel pharmacologic strategies to mitigate this condition. As inhibitors of monoacylglycerol lipase (MAGL), the primary hydrolytic enzyme of the endogenous cannabinoid, 2-arachidonyolglycerol, produces antinociceptive effects in numerous rodent models of pain, we investigated whether inhibitors of this enzyme (i.e., JZL184 and MJN110) would reverse paclitaxel-induced mechanical allodynia in mice. These drugs dose dependently reversed allodynia with respective ED(50) values (95% confidence limit) of 8.4 (5.2–13.6) and 1.8 (1.0–3.3) mg/kg. Complementary genetic and pharmacologic approaches revealed that the antiallodynic effects of each drug require both cannabinoid receptors, CB(1) and CB(2). MJN110 reduced paclitaxel-mediated increased expression of monocyte chemoattractant protein-1 (MCP-1, CCL2) and phospho-p38 MAPK in dorsal root ganglia as well as MCP-1 in spinal dorsal horn. Whereas the antinociceptive effects of high dose JZL184 (40 mg/kg) underwent tolerance following 6 days of repeated dosing, repeated administration of a threshold dose (i.e., 4 mg/kg) completely reversed paclitaxel-induced allodynia. In addition, we found that the administration of MJN110 to control mice lacked intrinsic rewarding effects in the conditioned place preference (CPP) paradigm. However, it produced a CPP in paclitaxel-treated animals, suggesting a reduced paclitaxel-induced aversive state. Importantly, JZL184 did not alter the antiproliferative and apoptotic effects of paclitaxel in A549 and H460 non-small cell lung cancer cells. Taken together, these data indicate that MAGL inhibitors reverse paclitaxel-induced neuropathic pain without interfering with chemotherapeutic efficacy.

The renal medulla, considered critical for the regulation of salt and water balance and long-term blood pressure control, is enriched in anandamide and two of its major metabolizing enzymes, cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH). Infusion of anandamide (15, 30, and 60 nmol·min−1·kg−1) into the renal medulla of C57BL/6J mice stimulated diuresis and salt excretion in a COX-2- but not COX-1-dependent manner. To determine whether endogenous endocannabinoids in the renal medulla can elicit similar effects, the effects of intramedullary isopropyl dodecyl fluorophosphate (IDFP), which inhibits the two major endocannabinoid hydrolases, were studied. IDFP treatment increased the urine formation rate and sodium excretion in a COX-2- but not COX-1-dependent manner. Neither anandamide nor IDFP affected the glomerular filtration rate. Neither systemic (0.625 mg·kg−1·30 min−1 iv) nor intramedullary (15 nmol·min−1·kg−1·30 min−1) IDFP pretreatment before intramedullary anandamide (15–30 nmol·min−1·kg−1) strictly blocked effects of anandamide, suggesting that hydrolysis of anandamide was not necessary for its diuretic effect. Intramedullary IDFP had no effect on renal blood flow but stimulated renal medullary blood flow. The effects of IDFP on urine flow rate and medullary blood flow were FAAH-dependent as demonstrated using FAAH knockout mice. Analysis of mouse urinary PGE2 concentrations by HPLC-electrospray ionization tandem mass spectrometry showed that IDFP treatment decreased urinary PGE2. These data are consistent with a role of FAAH and endogenous anandamide acting through a COX-2-dependent metabolite to regulate diuresis and salt excretion in the mouse kidney.

This study compared the development of tolerance to two orally bioavailable prescription opioids, oxycodone and hydrocodone, to that of morphine, and the reversal of this tolerance by ethanol. Oxycodone (s.c.) was significantly more potent in the mouse tail-withdrawal assay than either morphine or hydrocodone. Oxycodone was also significantly more potent in this assay than hydrocodone when administered orally. Tolerance was seen following chronic subcutaneous administration of each of the three drugs and by the chronic administration of oral oxycodone, but not following the chronic oral administration of hydrocodone. Ethanol (1 g/kg i.p.) significantly reversed the tolerance to the subcutaneous administration of each of the three opioids that developed when given 30 minutes prior to challenge doses. It took twice as much ethanol, when given orally, to reverse the tolerance to oxycodone. We investigated whether the observed tolerance to oxycodone and its reversal by ethanol were due to biodispositional changes or reflected a true neuronal tolerance. As expected, a relationship between brain oxycodone concentrations and activity in the tail-immersion test existed following administration of acute oral oxycodone. Following chronic treatment, brain oxycodone concentrations were significantly lower than acute concentrations. Oral ethanol (2 g/kg) reversed the tolerance to chronic oxycodone, but did not alter brain concentrations of either acute or chronic oxycodone. These studies show that there is a metabolic component of tolerance to oxycodone; however, the reversal of that tolerance by ethanol is not due to an alteration of the biodisposition of oxycodone, but rather is neuronal in nature.

BackgroundThe current opioid crisis has reinvigorated preclinical research in the evaluation of non-opioid candidate treatments for opioid use disorder (OUD). Emerging evidence suggests 5-HT2C receptor agonists may attenuate the abuse-related effects of opioids. This study evaluated effectiveness of 7-day treatment with the clinically available 5-HT2C agonist lorcaserin on heroin-vs.-food choice in rhesus monkeys. Lorcaserin effects were compared to effects produced by saline substitution and by 7-day treatment with the opioid antagonist naltrexone.MethodsAdult male (1) and female (6) rhesus monkeys were trained to respond under a concurrent schedule of food delivery (1g pellets, fixed-ratio 100 schedule) and intravenous heroin injections (0-0.032 mg/kg/injection, fixed-ratio 10 schedule) during daily 2h sessions. Heroin choice dose-effect functions were determined daily before and following 7-day saline substitution or 7-day continuous treatment with naltrexone (0.0032-0.032 mg/kg/h, IV) or lorcaserin (0.032-0.32 mg/kg/h, IV).ResultsUnder baseline conditions, increasing heroin doses maintained a dose-dependent increase in heroin choice. Both saline substitution and 7-day naltrexone treatment significantly attenuated heroin choice and produced a reciprocal increase in food choice. Continuous lorcaserin treatment significantly increased heroin choice.ConclusionsIn contrast to saline substitution and naltrexone, lorcaserin treatment was ineffective to reduce heroin-vs.-food choice. These preclinical results do not support the therapeutic potential of lorcaserin as a candidate OUD treatment.

Drug self-administration procedures are the gold standard for laboratory research to study mechanisms of drug use disorders and evaluate candidate medications. However, preclinical-to-clinical translation has been hampered by a lack of coordination. To address this limitation, we previously developed homologous intravenous (IV) cocaine choice self-administration procedures in rhesus monkeys and humans, and then demonstrated their functional equivalence. The present studies sought to determine the sensitivity of these procedures to d-amphetamine maintenance. Three (N = 3) rhesus monkeys with histories of cocaine self-administration and 16 (N = 16) humans with cocaine use disorder completed the studies. Monkeys were maintained on IV d-amphetamine (0, 0.019, 0.037 and 0.074 mg/kg/h), and then completed 7 sessions during each condition in which they completed 9 choice trials to receive 0.14 mg/kg/injection IV cocaine (corresponding to 10 mg/70 kg in humans) or 10 food pellets under independent, concurrent progressive-ratio schedules. Humans were maintained on oral extended release d-amphetamine (0, 30 and 60 mg/day, corresponding to the lowest 3 doses in monkeys) and participated in 12 sessions in which they chose money ($6.00) or IV cocaine (0, 3, 10 and 30 mg/70 kg). Blood samples were taken to compare d-amphetamine plasma levels across species. In monkeys and humans, d-amphetamine reduced the number of cocaine choices and produced comparable blood levels at equivalent daily doses. d-Amphetamine had similar efficacy, though lower potency, at reducing choice for an equivalent cocaine dose in monkeys relative to humans. These coordinated studies support the utility of these procedures as a translational model for cocaine use disorder. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

In the present study, we examined whether LDK1258, which produces strong CB(1) receptor allosteric effects in in vitro assays, would elicit in vivo effects consistent with allosteric activity. In initial studies, LDK1258 reduced food consumption and elicited delayed antinociceptive effects in the chronic constrictive injury of the sciatic nerve (CCI) model of neuropathic pain, which unexpectedly emerged 4 h post-injection. UPLC-MS/MS analysis quantified significant levels of LDK1258 in both blood and brain tissue at 30 min post-administration that remained stable up to 4 h. The observation that LDK1258 also produced respective antinociceptive and anorectic effects in rimonabant-treated wild type mice and CB(1) (−/−) mice suggests an off-target mechanism of action. Likewise, LDK1258 produced a partial array of common cannabimimetic effects in the tetrad assay, which were not CB(1) receptor mediated. Additionally, LDK1258 did not substitute for the CB(1) receptor orthosteric agonists CP55,940 or anandamide in the drug discrimination paradigm. In other in vivo assays sensitive to CB(1) receptor allosteric modulators, LDK1258 failed to shift the dose-response curves of either CP55,940 or anandamide in producing thermal antinociception, catalepsy, or hypothermia, and did not alter the generalization curve of either drug in the drug discrimination assay. Thus, this battery of tests yielded results demonstrating that LDK1258 produces antinociceptive effects in the CCI model of neuropathic pain, anorectic effects, and other in vivo pharmacological effects in a manner inconsistent with CB(1) receptor allosterism. More generally, this study offers a straightforward screening assay to determine whether newly synthesized CB(1) receptor allosteric modulators translate to the whole animal.

One proposed factor contributing to the increased opioid overdose deaths is the increased frequency of synthetic opioids, including fentanyl and fentanyl analogs. A treatment strategy currently under development to address the ongoing opioid crisis is immunopharmacotherapies or opioid-targeted vaccines. The present study determined the effectiveness and selectivity of a fentanyl-tetanus toxoid conjugate vaccine to alter the behavioral effects of fentanyl and a structurally dissimilar mu-opioid agonist oxycodone in male rhesus monkeys (n=3-4). Fentanyl and oxycodone produced dose-dependent suppression of behavior in an assay of schedule-controlled responding and antinociception in an assay of thermal nociception (50°C). Acute naltrexone (0.032 mg/kg) produced an approximate 10-fold potency shift for fentanyl to decrease operant responding. The fentanyl vaccine was administered at weeks 0, 2, 4, 9, 19, and 44 and fentanyl or oxycodone potencies in both behavioral assays were redetermined over the course of 49 weeks. The vaccine significantly and selectively shifted fentanyl potency at least 10-fold in both assays at several time points over the entire experimental period. Mid-point titer levels were significantly correlated with fentanyl antinociceptive potency shifts. Antibody affinity for fentanyl as measured by a competitive binding assay increased over time to around 3-4 nM. The fentanyl vaccine also significantly increased fentanyl plasma levels approximately 6-fold consistent with the hypothesis that the vaccine sequesters fentanyl in the blood. Overall, these results support the continued development and evaluation of this fentanyl vaccine to address the ongoing opioid crisis.HighlightsVaccine blunted fentanyl rate-suppression potency ∼ 10-foldVaccine blunted fentanyl antinociceptive potency ∼25-foldFentanyl vaccine was as effective as acute 0.032 mg/kg naltrexoneVaccine was selective for fentanyl and not oxycodoneAntibody immune response ∼ 3 nM affinity for fentanyl

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

Models to assess the addictive-like properties of nicotine in mice are limited. Therefore, we aimed to characterize and validate an addiction index by using an oral nicotine free-choice paradigm in mice. Adult C57BL/6J, DBA/2J, or genetically modified mice carrying deletions for nicotinic acetylcholine receptor (nAChR) subunits, (n = 8–10/sex/group) were given a choice of water or nicotine (10–960 μg/ml) solution using a two-bottle free-choice (2BC) paradigm. In general, oral nicotine intake and preference were higher in female mice compared to males. Absence of nicotine led to withdrawal, and intermittent access resulted in an escalation in consumption and greater nicotine withdrawal than continuous exposure. Additionally, oral nicotine consumption increased nucleus accumbens tyrosine hydroxylase levels. While β2 and α6 KO mice showed a significant decrease in nicotine intake, deletion of α5 nAChRs increased nicotine consumption at high concentrations. Deletion of the α7 subunit altered the observed sex difference in nicotine consumption, with females consuming less than males. The α4β2 partial agonist varenicline decreased oral nicotine consumption. Although addition of quinine to the nicotine solution lowered nicotine intake, mice primed with nicotine did not lower their intake after quinine addition. Nicotine deprivation followed by re-exposure showed increased nicotine consumption, and DBA/2J mice consumed less nicotine compared to C57BL/6J. We validated the mouse 2BC paradigm to study nicotine's addictive-like properties including nicotine intake, preference, withdrawal, and escalation of nicotine consumption during binge drinking or after reinstatement of a deprivation period.

Carbenoxolone is a derivative of glycyrrhetinic acid found in the root of Glycyrrhiza glabra, colloquially known as licorice. It has been used as a treatment for peptic and oral ulcers. In recent years, carbenoxolone has been utilized in basic research for its ability to block gap junctional communication. Better understanding the distribution of carbenoxolone after systemic administration can lead to a better understanding of its potential sites of action. Presented is an ultra high-performance liquid chromatography tandem mass spectrometer (UHPLC-MS/MS) method for the identification and quantification of carbenoxolone in mouse blood and brain tissue. Twenty mice were injected intraperitoneally with 25 mg/kg carbenoxolone and brain tissue and blood were collected for analysis. Blood concentrations (mean ± SD) at 15, 30, 60 and 120 min were determined to be (n = 5) 5394 ± 778, 2636 ± 836, 1564 ± 541 and 846 ± 252 ng/mL, respectively. Brain concentrations (mean ± SD) at 15, 30, 60 and 120 mins were determined to be (n = 5) 171 ± 62, 102 ± 35, 55 ± 10 and 27 ± 9 ng/g, respectively. The analysis of these specimens at the four different time points resulted in blood and brain half-lives in mice of ~43 and 41 min, respectively. The UHPLC-MS/MS method was determined to be sensitive and robust for quantification of carbenoxolone.

'NBOMe' (dimethoxyphenyl-N-[(2-methoxyphenyl)methyl]ethanamine) derivatives are a new class of designer hallucinogenic drugs widely available on the Internet. Currently, 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) is the most popular abused derivative in the USA. There are little published data on the absorption, metabolism and elimination of 25I-NBOMe, or any of the other NBOMe derivatives. Therefore, there are no definitive metabolite biomarkers. We present the identification of fifteen 25I-NBOMe metabolites in phase I and II mouse hepatic microsomal preparations, and analysis of two human urine samples from 25I-NBOMe-intoxicated patients to test the utility of these metabolites as biomarkers of 25I-NBOMe use. The synthesis of two major urinary metabolites, 2-iodo-4-methoxy-5-[2-[(2-methoxyphenyl) methylamino]ethyl]phenol (2-O-desmethyl-5-I-NBOMe, M5) and 5-iodo-4-methoxy-2-[2-[(2-methoxyphenyl)methylamino]ethyl]phenol (5-O-desmethyl-2-I-NBOMe), is also presented. Seven phase II glucuronidated metabolites of the O-desmethyl or the hydroxylated phase I metabolites were identified. One human urine sample contained 25I-NBOMe as well as all 15 metabolites identified in mouse hepatic microsomal preparations. Another human urine sample contained no parent 25I-NBOMe, but was found to contain three O-desmethyl metabolites. We recommend β-glucuronidase enzymatic hydrolysis of urine prior to 25I-NBOMe screening and the use of M5 as the primary biomarker in drug testing.

Podocytopathy and associated nephrotic syndrome (NS) have been reported in a knockout mouse strain (Asah1fl/fl/PodoCre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy of these mice remains unknown. The present study tested whether exosome release from podocytes is enhanced due to Asah1 gene knockout, which may serve as a pathogenic mechanism switching on podocytopathy and associated NS in Asah1fl/fl/PodoCre mice. We first demonstrated the remarkable elevation of urinary exosome excretion in Asah1fl/fl/PodoCre mice compared with WT/WT mice, which was accompanied by significant Annexin-II (an exosome marker) accumulation in glomeruli of Asah1fl/fl/PodoCre mice, as detected by immunohistochemistry. In cell studies, we also confirmed that Asah1 gene knockout enhanced exosome release in the primary cultures of podocyte isolated from Asah1fl/fl/PodoCre mice compared to WT/WT mice. In the podocytes from Asah1fl/fl/PodoCre mice, the interactions of lysosome and multivesicular body (MVB) were demonstrated to be decreased in comparison with those from their control littermates, suggesting reduced MVB degradation that may lead to increase in exosome release. Given the critical role of transient receptor potential mucolipin 1 (TRPML1) channel in Ca2+-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca2+ release through TRPML1 channels is inhibited in the podocytes of Asah1fl/fl/PodoCre mice. By GCaMP3 Ca2+ imaging, it was found that lysosomal Ca2+ release through TRPML1 channels was substantially suppressed in podocytes with Asah1 gene deletion. As an Ac product, sphingosine was found to rescue TRPML1 channel activity and thereby recover lysosome-MVB interaction and reduce exosome release of podocytes from Asah1fl/fl/PodoCre mice. Combination of N, N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, and sphingosine significantly inhibited urinary exosome excretion of Asah1fl/fl/PodoCre mice. Moreover, rescue of Aash1 gene expression in podocytes of Asah1fl/fl/PodoCre mice showed normal ceramide metabolism and exosome secretion. Based on these results, we conclude that the normal expression of Ac importantly contributes to the control of TRPML1 channel activity, lysosome-MVB interaction, and consequent exosome release from podocytes. Asah1 gene defect inhibits TRPML1 channel activity and thereby enhances exosome release, which may contribute to the development of podocytopathy and associated NS.

The NBOMes (N-benzyl-oxy-methyl derivatives of known 2C phenylethylamines) are a new and growing class of potent synthetic stimulants. Case reports provide the bulk of available safety and clinical data for clinicians. We report two cases of NBOMe intoxication with 25C-NBOMe (the first lab-confirmed US case) and 25B-NBOMe, respectively, both confirmed via triple quadrapole mass spectrometry.Case 1: A 16-year-old girl had a generalized seizure after reported use of 25I-NBOMe. She presented with altered mental status, lower extremity rigidity, and elevated CPK (6042 U/L). Despite treatment with benzodiazepines, her lower extremity rigidity persisted and CPK peaked at 47,906 U/L. She was discharged on hospital day 8. Serum and urine specimens confirmed presence of 25C-NBOMe. Case 2: A 15-year-old boy developed bizarre behavior after reported use of 25I-NBOMe. In the ED, he had two generalized seizures and persistent muscle rigidity. CPK peaked at 429 U/L. Seizures were managed with benzodiazepines, and he was discharged within 24 h. Serum specimens revealed 25B-NBOMe.NBOMes are amphetamine derivatives and highly potent 5-HT(2A) receptor agonists. Clinical manifestations are a product of enhanced central sympathetic and serotonergic tone. We report two cases of NBOMe intoxication in patients who believed they used 25I-NBOME, while lab confirmation proved otherwise. Whether unique clinical manifestations are specific to the NBOMe variant, dose, route of administration, or other factors is unknown. Laboratory confirmation may play a role in identifying unexpected NBOMe variants, while contributing to the epidemiologic data on these novel substances.

A new class of synthetic hallucinogens called NBOMe has emerged, and reports of adverse effects are beginning to appear. We report on a case of a suicide attempt after LSD ingestion which was analytically determined to be 25I-NBOMe instead. Clinicians need to have a high index of suspicion for possible NBOMe ingestion in patients reporting the recent use of LSD or other hallucinogens.

A series of in vivo and in vitro assays were conducted to characterize the pharmacological effects of the first generation abused synthetic cannabinoid CP47,497, a racemic bicyclic cannabinoid that is similar in structure to the potent, high-efficacy synthetic cannabinoid CP55,940. CP47,497 was less efficacious than CP55,940 in activating G-proteins and dose-dependently produced common CB1 receptor-dependent pharmacological effects (i.e. catalepsy, hypothermia, antinociception, and hypolocomotion). CP47,497 also substituted for Δ9-tetrahydrocannabinol (THC) in the mouse drug discrimination, indicating that both drugs elicited a similar interceptive stimulus. The pharmacological effects of CP47,497 underwent tolerance following repeated administration and showed cross-tolerance following repeated THC administration, further suggesting a common cannabimimetic mechanism of action. Finally, the CB1 receptor antagonist rimonabant precipitated similar magnitudes of somatic withdrawal responses in mice treated repeatedly with THC or CP47,497. Taken together, these data verify the acute cannabimimetic effects of CP47,497, and indicate tolerance and dependence following repeated administration. The assays used here provide a straightforward approach to characterize the emerging next generation of abused synthetic cannabinoids.

Serious clinical liabilities associated with the prescription of opiates for pain control include constipation, respiratory depression, pruritus, tolerance, abuse, and addiction. A recognized strategy to circumvent these side effects is to combine opioids with other antinociceptive agents. The combination of opiates with the primary active constituent of cannabis (Δ(9)-tetrahydrocannabinol) produces enhanced antinociceptive actions, suggesting that cannabinoid receptor agonists can be opioid sparing. Here, we tested whether elevating the endogenous cannabinoid 2-arachidonoylglycerol through the inhibition of its primary hydrolytic enzyme monoacylglycerol lipase (MAGL), will produce opioid-sparing effects in the mouse chronic constriction injury (CCI) of the sciatic nerve model of neuropathic pain. The dose-response relationships of i.p. administration of morphine and the selective MAGL inhibitor 2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate (MJN110) were tested alone and in combination at equieffective doses for reversal of CCI-induced mechanical allodynia and thermal hyperalgesia. The respective ED50 doses (95% confidence interval) of morphine and MJN110 were 2.4 (1.9-3.0) mg/kg and 0.43 (0.23-0.79) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. Acute antinociceptive effects of the combination of morphine and MJN110 required μ-opioid, CB1, and CB2 receptors. This combination did not reduce gastric motility or produce subjective cannabimimetic effects in the drug discrimination assay. Importantly, combinations of MJN110 and morphine given repeatedly (i.e., twice a day for 6 days) continued to produce antiallodynic effects with no evidence of tolerance. Taken together, these findings suggest that MAGL inhibition produces opiate-sparing events with diminished tolerance, constipation, and cannabimimetic side effects.

Background Recent years have seen a rise in the diversity and use of synthetic cannabinoids. The present study evaluated the behavioral effects of the third-generation indazole-3-carboxamide-type synthetic cannabinoid, AB-FUBINACA. Methods Adult male and female C57BL/6J mice were treated with AB-FUBINACA (0−3 mg/kg, i.p.) and tested repeatedly in the tetrad battery measuring catalepsy, antinociception, hypothermia, and locomotor activity. Mice treated with AB-FUBINACA (≥2 mg/kg, i.p.) displayed classic cannabinoid effects in the tetrad that were blocked by the CB1 receptor selective antagonist rimonabant. To address tolerance and withdrawal effects, a second group of mice was injected with AB-FUBINACA (3 mg/kg, s.c.) or vehicle consisting of 5% ethanol, 5% Kolliphor EL, and 90 % saline every 12 h and tested daily in modified tetrad over the course of 5 days. On the 6th day, withdrawal was precipitated using rimonabant (3 mg/kg, s.c.), and somatic signs of withdrawal (i.e., head twitches and paw tremors) were quantified. Results Although mice did not develop tolerance to AB-FUBINACA or cross-tolerance to Δ9-tetrahydrocannabinol (THC; 50 mg/kg, i.p.), somatic precipitated withdrawal signs were observed. Repeated tetrad testing up to 48 h post injection indicated that AB-FUBINACA effects are relatively short-lived, as compared with THC. Brain levels of AB-FUBINACA, as quantified by UHPLC-MS/MS, were undetectable 4 h post injection. Conclusions These data indicate that the cannabinoid effects of AB-FUBINACA are relatively short-lived, yet sufficient to induce dependence in mice.

Opioid-sparing adjuncts are treatments that aim to reduce the overall dose of opioids needed to achieve analgesia, hence decreasing the burden of side effects through alternative mechanisms of action. Lorcaserin is a serotonin 5-HT2C receptor (5-HT2CR) agonist that has recently been reported to reduce abuse-related effects of the opioid analgesic oxycodone. The goal of our studies was to evaluate the effects of adjunctive lorcaserin on opioid-induced analgesic-like behavior using the tail-flick reflex (TFR) test as a mouse model of acute thermal nociception. We show that whereas subcutaneous (s.c.) administration of lorcaserin alone was inactive on the TFR test, adjunctive lorcaserin (s.c.) significantly increased the potency of oxycodone as an antinociceptive drug. This effect was prevented by the 5-HT2CR antagonist SB242084. A similar lorcaserin (s.c.)-induced adjunctive phenotype was observed upon administration of the opioid analgesics morphine and fentanyl. Remarkably, we also show that, opposite to the effects observed via s.c. administration, intrathecal (i.t.) administration of lorcaserin alone induced antinociceptive TFR behavior, an effect that was not prevented by the opioid receptor antagonist naloxone. This route of administration (i.t.) also led to a significant augmentation of oxycodone-induced antinociception. Lorcaserin (s.c.) did not alter the brain or blood concentrations of oxycodone, which suggests that its adjunctive effects on opioid-induced antinociception do not depend upon changes in opioid metabolism. Together, these data indicate that lorcaserin-mediated activation of the 5-HT2CR may represent a new pharmacological approach to augment opioid-induced antinociception. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

Head-twitch behavior (HTR) is the behavioral signature of psychedelic drugs upon stimulation of the serotonin 5-HT2A receptor (5-HT2AR) in rodents. Following the previous report of a semi-automated detection of HTR based on the dynamics of mouse’s head movement, here we present a system for the identification of individual HTR events in a fully automated fashion. The validity of this fully automated HTR detection system was tested with the psychedelic drug DOI in 5-HT2AR-KO mice, and via evaluation of potential sources of false-positive and false-negative HTR events. The increased throughput in data processing achieved via automation afforded the possibility of conducting otherwise time consuming HTR time-course studies. To further assess the versatility of our system, we also explored the pharmacological interactions between 5-HT2AR and the metabotropic glutamate receptor 2 (mGluR2). Our data demonstrate the potentiation effect of the mGluR2/3 antagonist LY341495 on DOI-induced HTR, as well as the HTR-blocking effect of the mGluR2/3 agonist and antipsychotic drug in development LY404039. This fully automated system can contribute to speed up our understanding of 5-HT2AR’s pharmacology and its characteristic behavioral outputs in rodents.

The kidneys contribute to the control of body fluid and electrolytes and the long-term regulation of blood pressure through various systems, including the endocannabinoid system. Previously, we showed that inhibition of the two major endocannabinoid-hydrolyzing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, in the renal medulla increased the rate of urine excretion (UV) and salt excretion without affecting mean arterial pressure (MAP). The present study evaluated the effects of a selective FAAH inhibitor, N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidine carboxamide (PF-3845) on MAP and renal functions. Infusion of PF-3845 into the renal medulla of C57BL/6J mice reduced MAP during the posttreatment phases and increased UV at 15 and 30 nmol/min per gram kidney weight (g kwt), relative to the pretreatment control phase. Intravenous PF-3845 administration reduced MAP at the 7.5, 15, and 30 doses and increased UV at the 15 and 30 nmol⋅min−1⋅g−1 kwt doses. PF-3845 treatment elevated sodium and potassium urinary excretion and medullary blood flow. Homozygous FAAH knockout mice were refractory to intramedullary PF-3845-induced changes in MAP, but UV was increased. Both MAP and UV responses to intramedullary PF-3845 in C57BL/6J mice were diminished by pretreatment with the cannabinoid type 1 receptor-selective antagonist, rimonabant (3 mg/kg, ip) but not the cyclooxygenase 2-selective inhibitor, celecoxib (15 mg/kg, iv). Liquid chromatography-tandem mass spectrometry analyses showed increased anandamide in kidney tissue and 2-arachidonoyl glycerol in plasma after intramedullary PF-3845. These data suggest that inhibition of FAAH in the renal medulla leads to both a diuretic and blood pressure-lowering response mediated by elevated anandamide in kidney tissue or 2-arachidonoyl glycerol in plasma.

One complicating factor in cocaine addiction may be concurrent exposure and potential dependence on nicotine. The aim of the present study was to determine the effects of continuous nicotine treatment and subsequent termination on cocaine versus food choice in rhesus monkeys (Macaca mulatta). For comparison, we also determined effects of the nicotinic receptor antagonist mecamylamine on cocaine versus food choice during continuous saline and nicotine treatment. Rhesus monkeys (N = 3) responded under a concurrent schedule of food pellet (1 g) and intravenous cocaine (0-0.1 mg/kg/injection) availability. Saline and ascending nicotine doses (0.1-1.0 mg/kg/hr, intravenous) were continuously infused for 7-day treatment periods and separated by 24-hr saline treatment periods. Acute effects of mecamylamine (0.32-1.8 mg/kg, intramuscular, 15 min pretreatment) were determined during continuous saline and 0.32-mg/kg/hr nicotine treatments. During saline treatment, cocaine maintained a dose-dependent increase in cocaine choice. Nicotine treatment did not alter cocaine versus food choice. In contrast, preference of 0.032 mg/kg/injection cocaine was attenuated 24 hr following termination of 0.32-mg/kg/hr nicotine treatment, despite no somatic abstinence signs being observed. Acute mecamylamine enhanced cocaine choice during saline treatment and mainly suppressed rates of behavior during nicotine treatment. Overall, continuous nicotine exposure, up to 1 mg/kg/hr, does not enhance cocaine choice and does not produce nicotine dependence, as demonstrated by the lack of abstinence signs.

Abuse of synthetic stimulant compounds resulting in significant toxicity is being increasingly reported by poison centers. Toxicologic assessment is complicated by inconsistent manufacturing processes and limited laboratory testing. We describe a case of self-reported exposure to 25-I (25I-NBOMe), a novel phenethylamine derivative, with subsequent quantification in serum.An 18-year-old male presented to the emergency department (ED) with severe agitation and hallucinations after jumping out of a moving car. He was tachycardiac (150-160 bpm) and hypertensive (150-170 mm Hg systolic and 110 mg Hg diastolic), and required physical restraints and treatment with intravenous lorazepam administration. His symptoms gradually improved and vital signs returned to normal over 48 h, though he continued to have episodes of aggressiveness. An assay was developed by our analytical toxicology laboratory for 25-I, and serum obtained during ED evaluation and treatment was found to contain 0.76 ng/ml of 25-I. Case discussion. For 25I-NBOMe, 25-I is a common abbreviation for 25I-NBOMe, which is a (n-benzyl) phenethylamine in the 2C "family."Initially synthesized for research, cases of self-reported use of 25-I have recently appeared in the literature, some of which contain qualitative urine confirmation. There are no commercially available quantitative assays, and no previous reports have published serum concentrations. 25-I is a potent new synthetic drug with apparent significant behavioral toxicity that can be detected and quantified in serum.

Background Use of synthetic “marijuana” has increased in recent years, produced adverse effects and prompted the temporary DEA ban of five specific cannabinoid analogs, including JWH-018. The objectives of the current study include determining the chemical content of the herbal product, Buzz, assessing its behavioral effects upon inhalation exposure to mice, determining whether CB1 receptors mediate its pharmacological activity, and ascertaining its biodisposition in blood and various organs. Methods Using a nose-only exposure system, mice were exposed to smoke produced from combustion of an herbal incense product, Buzz, which contained 5.4% JWH-018. Cannabimimetic effects following smoke exposure were evaluated using the tetrad procedure, consisting of the following indices: hypomotility, antinociception, catalepsy, and hypothermia. Additionally, blood and tissues were collected for JWH-018 quantification. Results Inhalation exposure to Buzz produced dose-related tetrad effects similar to marijuana as well as dose-related increased levels of JWH-018 in the blood, brain, heart, kidney, liver, lung, and spleen. The behavioral effects were blocked by rimonabant, a CB1 receptor antagonist. Effects produced by Buzz were similar in magnitude and time-course to those produced by marijuana, though equipotent doses of Buzz and marijuana yielded considerably lower brain levels of JWH-018 than THC for the respective materials. Conclusions Inhalation exposure to a product containing JWH-018 penetrates into the brain and other organs and produces CB1 receptor-mediated behavioral pharmacological effects in mice. The increased potency of JWH-018 compared to THC, the variable amount of drug added to various herbal products, and unknown toxicity, undoubtedly contribute to public health risks of synthetic cannabinoids.

The kidneys play an important role in the long-term regulation of blood pressure by control of salt and water balance in the body through various systems including the endocannabinoid system. The endocannabinoid system consists of the two major cannabinoid receptor agonists, anandamide (AEA) and 2-arachidonylglycerol (2-AG), their hydrolyzing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), and the cannabinoid receptors, CB(1) and CB(2). AEA can be converted into 12- and 15(S)-hydroperoxyeicosatetraenoic acid ethanolamides by 12-LOX and 15-LOX, respectively and can form epoxyeicosatrienoic acid-(EET-EAs) (5,6-, 8,9-, 11,12-, 14,15- ) and hydroxyeicosatetraenoic acid- (HETE) ethanolamides. Furthermore, the EET-EAs produce a secondary metabolism by microsomal epoxide hydrolase to form the corresponding dihydroxyeicosatetraenoic acid-EAs (DiHETE-EA). Reference material was not available for DiHETE-EA. These metabolites were synthesized by incubation of the corresponding EET-EAs with mouse liver cytosol containing epoxide hydrolases. Presented is a solid phase extraction and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for the extraction and quantitation of AEA, 2-AG, their metabolites, Oleoylethanolamide (OEA), and Palmitoylethanolamide (PEA), and the in vivo formation of the dihydroxyeicosatetraenoic acid-EAs in kidney after a single intravenous bolus administration of 20 mg/kg of anandamide in C57BL/6J and FAAH KO mice.

While Marijuana continues to be the most widely used illicit drug, abuse of synthetic cannabinoid (SCB) compounds in ‘Spice’ or ‘K2’ herbal incense products has emerged as a significant public health concern in many European countries and in the USA. Several of these SCBs have been declared Schedule I controlled substances but detection and quantification in biological samples remain a challenge. Therefore, we present a liquid chromatography–tandem mass spectrometry method after liquid–liquid extraction for the quantitation of CP-47,497, CP-47,497-C8 and JWH-250 in mouse brain. We report data for linearity, limit of quantification, accuracy/bias, precision, recovery, selectivity, carryover, matrix effects and stability experiments which were developed and fully validated based on Scientific Working Group for Forensic Toxicology guidelines for forensic toxicology method validation. Acceptable coefficients of variation for accuracy/bias, within- and between-run precision and selectivity were determined, with all values within ±15% of the target concentration. Validation experiments revealed degradation of CP-47, 497 and CP-47,497-C8 at different temperatures, and significant ion suppression was produced in brain for all compounds tested. The method was successfully applied to detect and quantify CP-47,497 in brains from mice demonstrating significant cannabimimetic behavioral effects as assessed by the classical tetrad paradigm.

Poklis and Backer published a survey of the concentrations of fentanyl and norfentanyl that could be expected in urine from patients using Duragesic®, a transdermal fentanyl patch. That study employed a relatively small number of patient data points and analysis by Gas Chromatography/Mass Spectrometry. This work examines a larger population of patient positives for fentanyl and norfentanyl to determine whether more than a decade later the original report remains accurate in predicting the range and median levels of fentanyl and norfentanyl concentrations physicians can expect to see from their patients. Additionally, these data were transformed to develop a model that results in a near Gaussian distribution of urine drug test results. This retrospective approach was developed to transform and normalize urine drug testing results to provide a historical picture of expected patient values for this important analgesic. The resulting near Gaussian distribution is dose independent and as such should be of value to physicians in quickly assessing whether their patient is consistent with this historical population in the broad terms of this model. While this comparison alone is not definitive for adherence with a treatment regimen, together with patient interviews, prescription history and other clinical criteria, it can add an idea of expected patient values from urine drug testing.

Although the majority of cannabinoid users smoke marijuana, the preponderance of laboratory animal research is based on administration of Delta9-tetrahydrocannabinol (Delta9-THC) or other cannabinoid agents via injection. The aim of the present study was to evaluate the impact of inhaling marijuana, or ethanol-extracted placebo smoke in the mouse model of cannabinoid activity by assessing inhibition of spontaneous activity, antinociception, catalepsy, and body temperature. In order to determine dosimetry, blood levels of Delta9-THC were obtained following either marijuana exposure or intravenous injection of Delta(9)-THC. Inhalation exposure to marijuana produced dose-related increases in antinociception and catalepsy, with estimated ED50 doses of Delta9-THC of 2.4 and 3.8 mg/kg, respectively. However, hypothermia and locomotor depression occurred in both the placebo- and marijuana-exposed mice. The CB1 receptor antagonist, SR 141716A antagonized the antinociceptive effects of marijuana (AD50 = 0.6 mg/kg), but only slightly decreased marijuana-induced catalepsy, and failed to alter either the hypothermic or locomotor depressive effects. In contrast, SR 141716A antagonized the antinociceptive, cataleptic, and hypothermic effects of intravenously administered Delta9-THC in mice that were exposed to air alone, though all subjects exhibited locomotor depression, possibly related to the restraint. In accordance with reports of others, these data suggest that exposure to smoke alone has pharmacological consequences. Our findings also indicate that marijuana-induced antinociception is mediated through a CB1-receptor mechanism of action and are consistent with the notion that Delta9-THC is mainly responsible for this effect.

Methamphetamine is a globally abused drug that is metabolized to amphetamine, which also produces abuse-related behavioral effects. However, the contributing role of methamphetamine metabolism to amphetamine in methamphetamine's abuse-related subjective effects is unknown. This preclinical study was designed to determine 1) the relationship between plasma methamphetamine levels and methamphetamine discriminative stimulus effects and 2) the contribution of the methamphetamine metabolite amphetamine in the discriminative stimulus effects of methamphetamine in rhesus monkeys. Adult male rhesus monkeys (n=3) were trained to discriminate 0.18 mg/kg intramuscular (+)-methamphetamine from saline in a two-key food-reinforced discrimination procedure. Time course of saline, (+)-methamphetamine (0.032-0.32 mg/kg), and (+)-amphetamine (0.032-0.32 mg/kg) discriminative stimulus effects were determined. Parallel pharmacokinetic studies were conducted in the same monkeys to determine plasma methamphetamine and amphetamine levels after methamphetamine administration and amphetamine levels after amphetamine administration for correlation with behavior in the discrimination procedure. Both methamphetamine and amphetamine produced full, ≥90%, methamphetamine-like discriminative stimulus effects. Amphetamine displayed a slightly, but significantly, longer duration of action than methamphetamine in the discrimination procedure. Both methamphetamine and amphetamine behavioral effects were related to methamphetamine and amphetamine plasma levels by a clockwise hysteresis loop indicating acute tolerance had developed to the discriminative stimulus effects. Furthermore, amphetamine levels after methamphetamine administration were absent when methamphetamine stimulus effects were greatest and peaked when methamphetamine discriminative stimulus effects returned to saline-like levels. Overall, these results demonstrate the methamphetamine metabolite amphetamine does not contribute to methamphetamine's abuse-related subjective effects.

Synthetic cathinones are beta-ketone amphetamine analogs that have emerged as a heterogeneous class of abused compounds that function as either monoamine transporter substrates or inhibitors. Pre-clinical drug discrimination procedures are useful for interrogating structure-activity relationships of abuse-related drug effects; however, in vivo structure-activity relationship comparisons between synthetic cathinones with different mechanisms of action are lacking. The aim of the present study was to determine whether the cocaine-like discriminative stimulus effects of the monoamine transporter inhibitor alpha-pyrrolidinovalerophenone (alpha-PVP) and the monoamine transporter substrate methcathinone were differentially sensitive to 3,4-methylenedioxy and 4-methyl substitutions. Male rhesus monkeys (n = 4) were trained to discriminate intramuscular cocaine (0.32 mg/kg) from saline in a two-key food-reinforced discrimination procedure. Potency and timecourse of cocaine-like discriminative stimulus effects were determined for (±)-alpha-PVP, (±)-methcathinone and their 3,4-methylenedioxy or 4-methyl analogs. Alpha-PVP and methcathinone produced dose- and time-dependent cocaine-like effects. A 3,4-methylenedioxy addition to either alpha-PVP or methcathinone (methylone) did not alter the potency or efficacy to produce cocaine-like effects, but did prolong the time course. A 4-methyl addition to alpha-PVP (pyrovalerone) did not alter the potency or efficacy to produce cocaine-like effects, but did prolong the time course. In contrast, addition of a 4-methyl moiety to methcathinone (4MMC; mephedrone) significantly attenuated efficacy to produce cocaine-like effects. Overall, these results suggest different structural requirements for cocaine-like discriminative stimulus effects of monoamine transporter inhibitor and substrate synthetic cathinone analogs. Given that 4MMC is more hydrophobic than MDMC, these results suggest that hydrophobicity may be an important determinant for limiting monoamine transporter substrate abuse-related behavioral effects.

Cannabichromene (CBC) is a phytocannabinoid, the second most abundant cannabinoid quantitatively in marijuana. CBC has been shown to produce antinociception and anti-inflammatory effects in rodents. This method is validated for the measurement of THC and CBC simultaneously after extraction from mouse blood or brain. Whole brain harvested from mice was homogenized 2:1 (v/w) with normal saline. Fifty nanograms of THC-d₃ was added to 0.5 mL of heparinized mouse blood, brain homogenate, and THC and CBC fortified blood or brain calibrators, then equilibrated overnight at 5 °C. Two milliliters of "ice cold" acetonitrile was added drop-wise while the sample was vortex mixed, and then the sample was centrifuged and stored overnight at -30 °C. The cannabinoids were extracted from the acetonitrile layer with 2 mL of 0.2 N NaOH and 4 mL of hexane/ethyl acetate (9:1). The solvent was isolated and evaporated to dryness. Trimethylsilyl derivatives were prepared and then analyzed by gas chromatography-mass spectrometry. Linearity in blood and brain of THC and CBC was 2-10,000 ng/mL (ng/g). THC and CBC recovery ranged from 56 to 78% in blood and brain. Precision was demonstrated at 100 ng/mL and 1000 ng/mL with CVs < 15%. The validated method allows for blood and brain concentrations of cannabinoids to be quantificated and correlated with pharmacological effects produced in mice.

In contrast to the numerous reports on the pharmacological effects of Δ(9)-tetrahydrocannabinol (THC), the pharmacological activity of another substituent of Cannabis sativa, cannabichromene (CBC) remains comparatively unknown. In the present study, we investigated whether CBC elicits cannabinoid activity in the tetrad assay, which consists of the following four endpoints: hypomotility, antinociception, catalepsy, and hypothermia. Because cannabinoids are well documented to possess anti-inflammatory properties, we examined CBC, THC, and combination of both phytocannabinoids in the lipopolysaccharide (LPS) paw edema assay. CBC elicited activity in the tetrad that was not blocked by the CB(1) receptor antagonist, rimonabant. Moreover, a behaviorally inactive dose of THC augmented the effects of CBC in the tetrad that was associated with an increase in THC brain concentrations. Both CBC and THC elicited dose-dependent anti-inflammatory effects in the LPS-induced paw edema model. The CB(2) receptor, SR144528 blocked the anti-edematous actions of THC, but not those produced by CBC. Isobolographic analysis revealed that the anti-edematous effects of these cannabinoids in combination were additive. Although CBC produced pharmacological effects, unlike THC, its underlying mechanism of action did not involve CB(1) or CB(2) receptors. In addition, there was evidence of a possible pharmacokinetic component in which CBC dose-dependently increased THC brain levels following an i.v. injection of 0.3mg/kg THC. In conclusion, CBC produced a subset of behavioral activity in the tetrad assay and reduced LPS-induced paw edema through a noncannabinoid receptor mechanism of action. These effects were augmented when CBC and THC were co-administered.

The present study tested a hypothesis that excess accumulation of sphingolipid, ceramide, its metabolites, or a combination contributes to the development of obesity and associated kidney damage. Liquid chromatography/mass spectrometry analysis demonstrated that C57BL/6J mice on the high-fat diet (HFD) had significantly increased plasma total ceramide levels compared with animals fed a low-fat diet (LFD). Treatment of mice with the acid sphingomyelinase (ASMase) inhibitor amitriptyline significantly attenuated the HFD-induced plasma ceramide levels. Corresponding to increase in plasma ceramide, the HFD significantly increased the body weight gain, plasma leptin concentration, urinary total protein and albumin excretion, glomerular damage index, and adipose tissue ASMase activity compared with the LFD-fed mice. These HFD-induced changes were also significantly attenuated by treatment of mice with amitriptyline. In addition, the decline of plasma glucose concentration after an intraperitoneal injection of insulin (0.15 U/kg b.wt.) was more sustained in mice on the HFD with amitriptyline than on the HFD alone. Intraperitoneal injection of glucose (3 g/kg b.wt.) resulted in a slow increase followed by a rapid decrease in the plasma glucose concentration in LFD and HFD plus amitriptyline-treated mice, but such blood glucose response was not observed in HFD-fed mice. Immunofluorescence analysis demonstrated a decrease in the podocin and an increase in the desmin in the glomeruli of HFD-fed mice compared with the LFD and HFD plus amitriptyline-treated mice. In conclusion, our results reveal a pivotal role for ceramide biosynthesis in obesity, metabolic syndrome, and associated kidney damage.

Direct-acting cannabinoid receptor agonists are well known to reduce hyperalgesic responses and allodynia after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Alternatively, inhibiting fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principal enzymes responsible for the degradation of the respective endogenous cannabinoids, anandamide (AEA) and 2-arachydonylglycerol (2-AG), reduce nociception in a variety of nociceptive assays, with no or minimal behavioral effects. In the present study we tested whether inhibition of these enzymes attenuates mechanical allodynia, and acetone-induced cold allodynia in mice subjected to chronic constriction injury of the sciatic nerve. Acute administration of the irreversible FAAH inhibitor, cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester (URB597), or the reversible FAAH inhibitor, 1-oxo-1-[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL-135), decreased allodynia in both tests. This attenuation was completely blocked by pretreatment with either CB1 or CB2 receptor antagonists, but not by the TRPV1 receptor antagonist, capsazepine, or the opioid receptor antagonist, naltrexone. The novel MAGL inhibitor, 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) also attenuated mechanical and cold allodynia via a CB1, but not a CB2, receptor mechanism of action. Whereas URB597 did not elicit antiallodynic effects in FAAH(-/-) mice, the effects of JZL184 were FAAH-independent. Finally, URB597 increased brain and spinal cord AEA levels, whereas JZL184 increased 2-AG levels in these tissues, but no differences in either endo-cannabinoid were found between nerve-injured and control mice. These data indicate that inhibition of FAAH and MAGL reduces neuropathic pain through distinct receptor mechanisms of action and present viable targets for the development of analgesic therapeutics.

The present study tested the hypothesis that homocysteine (Hcys)-induced ceramide production stimulates lipid rafts (LRs) clustering on the membrane of glomerular endothelial cells (GECs) to form redox signalling platforms by aggregation and activation of NADPH oxidase subunits and thereby enhances superoxide (O2*-) production, leading to glomerular endothelial dysfunction and ultimate injury or sclerosis. Using confocal microscopy, we first demonstrated a co-localization of LR clusters with NADPH oxidase subunits, gp91(phox) and p47(phox) in the GECs membrane upon Hcys stimulation. Immunoblot analysis of floated detergent-resistant membrane fractions found that in LR fractions NADPH oxidase subunits gp91(phox) and p47(phox) are enriched and that the activity of this enzyme dramatically increased. We also examined the effect of elevated Hcys on the cell monolayer permeability in GECs. It was found that Hcys significantly increased GEC permeability, which was blocked by inhibition of LR redox signalling platform formation. Finally, we found that Hcys-induced enhancement of GEC permeability is associated with the regulation of microtubule stability through these LR-redox platforms. It is concluded that the early injurious effect of Hcys on the glomerular endothelium is associated with the formation of redox signalling platforms via LR clustering, which may lead to increases in glomerular permeability by disruption of microtubule network in GECs.

We present a traumatic fatality of a 19-year-old man who had ingested blotter paper containing 25I-NBOMe [2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine]. Postmortem specimens were analyzed by high performance liquid chromatography with tandem mass spectrometry (HPLC/MS/MS). Toxicology findings for fluids based upon blood or urine calibrators were as follows: peripheral blood, 405 pg/mL; heart blood, 410 pg/mL; urine, 2.86 ng/mL; and vitreous humor, 99 pg/mL. While findings based upon the method of standard additions were: gastric contents, 7.1 μg total; bile, 10.9 ng/g; brain, 2.54 ng/g and liver, 7.2 ng/g. To our knowledge the presented case is the first postmortem case of 25I-NBOMe intoxication documented by toxicological analysis of tissues and body fluids.

We present a case of 4-bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (25B-NBOMe), an N-benzyl phenethylamines derivative, intoxication and a high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method for detection and quantification of 25B-NBOMe. A 19-year-old male was found unresponsive with generalized grand mal seizure activity. On the second day of hospitalization, a friend admitted that the patient used 'some unknown drug' called 25B. Serum and urine collected were sent to the Virginia Commonwealth University Medical Center Toxicology Laboratory for analysis. An HPLC-MS/MS method for the identification and quantification of 25B-NBOMe using 2-(2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25H-NBOMe) as the internal standard (ISTD) was developed. As this is a novel, single-case presentation, an assay validation was performed prior to testing to ensure the reliability of the analytical results. The serum and urine specimens were determined to contain 180 pg/ml and1900 pg/ml of 25B-NBOMe, respectively.

The CB1 receptor represents a promising target for the treatment of several disorders including pain-related disease states. However, therapeutic applications of Δ(9)-tetrahydrocannabinol and other CB1 orthosteric receptor agonists remain limited because of psychoactive side effects. Positive allosteric modulators (PAMs) offer an alternative approach to enhance CB1 receptor function for therapeutic gain with the promise of reduced side effects. Here we describe the development of the novel synthetic CB1 PAM, 6-methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011), which augments the in vitro and in vivo pharmacological actions of the CB1 orthosteric agonists CP55,940 and N-arachidonoylethanolamine (AEA). ZCZ011 potentiated binding of [(3)H]CP55,940 to the CB1 receptor as well as enhancing AEA-stimulated [(35)S]GTPγS binding in mouse brain membranes and β-arrestin recruitment and ERK phosphorylation in hCB1 cells. In the whole animal, ZCZ011 is brain penetrant, increased the potency of these orthosteric agonists in mouse behavioral assays indicative of cannabimimetic activity, including antinociception, hypothermia, catalepsy, locomotor activity, and in the drug discrimination paradigm. Administration of ZCZ011 alone was devoid of activity in these assays and did not produce a conditioned place preference or aversion, but elicited CB1 receptor-mediated antinociceptive effects in the chronic constriction nerve injury model of neuropathic pain and carrageenan model of inflammatory pain. These data suggest that ZCZ011 acts as a CB1 PAM and provide the first proof of principle that CB1 PAMs offer a promising strategy to treat neuropathic and inflammatory pain with minimal or no cannabimimetic side effects.

Background: Chronic amphetamine treatment decreases cocaine consumption in preclinical and human laboratory studies and in clinical trials. Lisdexamfetamine is an amphetamine prodrug in which L-lysine is conjugated to the terminal nitrogen of d -amphetamine. Prodrugs may be advantageous relative to their active metabolites due to slower onsets and longer durations of action; however, lisdexamfetamine treatment’s efficacy in decreasing cocaine consumption is unknown. Methods: This study compared lisdexamfetamine and d -amphetamine effects in rhesus monkeys using two behavioral procedures: (1) a cocaine discrimination procedure (training dose = 0.32mg/kg cocaine, i.m.); and (2) a cocaine-versus-food choice self-administration procedure. Results: In the cocaine-discrimination procedure, lisdexamfetamine (0.32–3.2mg/kg, i.m.) substituted for cocaine with lower potency, slower onset, and longer duration of action than d -amphetamine (0.032–0.32mg/kg, i.m.). Consistent with the function of lisdexamfetamine as an inactive prodrug for amphetamine, the time course of lisdexamfetamine effects was related to d -amphetamine plasma levels by a counter-clockwise hysteresis loop. In the choice procedure, cocaine (0–0.1mg/kg/injection, i.v.) and food (1g banana-flavored pellets) were concurrently available, and cocaine maintained a dose-dependent increase in cocaine choice under baseline conditions. Treatment for 7 consecutive days with lisdexamfetamine (0.32–3.2mg/kg/day, i.m.) or d -amphetamine (0.032–0.1mg/kg/h, i.v.) produced similar dose-dependent rightward shifts in cocaine dose-effect curves and decreases in preference for 0.032mg/kg/injection cocaine. Conclusions: Lisdexamfetamine has a slower onset and longer duration of action than amphetamine but retains amphetamine’s efficacy to reduce the choice of cocaine in rhesus monkeys. These results support further consideration of lisdexamfetamine as an agonist-based medication candidate for cocaine addiction.

Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca(2+) channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [(35)S]GTPγS (guanylyl-5'-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [(35)S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist-stimulated CB1R signaling and downregulation of CB1Rs. Thus, CRIP1a appears to act as a broad negative regulator of CB1R function.

The disposition of the cannabimimetic naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) in mice following inhalation of the smoke of the herbal incense product (HIP) 'Buzz' is presented. A high-pressure liquid chromatography with electrospray ionization triple quadrupole mass spectrometer (HPLC/MS/MS) method was validated for the analysis of JWH-018 in the specimens using deuterated Δ(9) -tetrahydrocannabinol (d(3) -THC) as the internal standard. JWH-018 was isolated by cold acetonitrile liquid-liquid extraction. Chromatographic separation was performed on a Zorbaz eclipse XDB-C(18) column. The assay was linear from 1 to 1000 ng/mL. Six C57BL6 mice were sacrificed 20 min after exposure to the smoke of 200 mg 'Buzz' containing 5.4% JWH-018. Specimen concentrations of JWH-018 were: blood, 54-166 ng/mL (mean 82 ± 42 ng/mL); brain, 316-708 ng/g (mean 510 ± 166 ng/g); and liver, 1370-3220 ng/mL (mean 1990 ± 752 ng/mL). The mean blood to brain ratio for JWH-018 was 6.8 and ranged from 4.2 to 10.9. After exposure, the responses of the mice were consistent with cannabinoid receptor type 1 activity: body temperatures dropped 7.3 ± 1.1 °C, and catalepsy, hyperreflexia, straub tail and ptosis were observed. The brain concentrations and physiological responses are consistent with the hypothesis that the behavioral effects of 'Buzz' are attributable to JWH-018.