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Concentrations of delta9-tetrahydrocannabinol and 11-nor-9-carboxytetrahydrocannabinol in blood and urine after passive exposure to Cannabis smoke in a coffee shop.

Cannabinoid concentrations in blood and urine after passive exposure to cannabis smoke under real-life conditions were investigated in this study. Eight healthy volunteers were exposed to cannabis smoke for 3 h in a well-attended coffee shop in Maastricht, Netherlands. An initial blood and urine sample was taken from each volunteer before exposure. Blood samples were taken 1.5, 3.5, 6, and 14 h after start of initial exposure, and urine samples were taken after 3.5, 6, 14, 36, 60, and 84 h. The samples were subjected to immunoassay screening for cannabinoids and analyzed using gas chromatography-mass spectrometry (GC-MS) for Delta(9)-tetrahydrocannabinol (THC), 11-nor-hydroxy-Delta(9)-tetrahydrocannabinol (THC-OH), and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH). It could be demonstrated that all volunteers absorbed THC. However, the detected concentrations were rather small. None of the urine samples produced immunoassay results above the cutoff concentration of 25 ngmL. THC-COOH concentrations up to 5.0 and 7.8 ngmL before and after hydrolysis, respectively, were found in the quantitative GC-MS analysis of urine. THC could be detected in trace amounts close to the detection limit of the used method in the first two blood samples after initial exposure (1.5 and 3.5 h). In the 6 h blood samples, THC was not detectable anymore. THC-COOH could be detected after 1.5 h and was still found in 3 out of 8 blood samples after 14 h in concentrations between 0.5 and 1.0 ngmL.

Young adult Ecstasy users who forego necessary medical care a fairly common occurrence with important health implications.

This study examines the practice of foregoing necessary medical care in a population of young adult Ecstasy users. The objectives are to (1) investigate how the failure to receive needed medical care is related to drug-related outcomes, and (2) identify factors that are associated with receiving versus foregoing needed medical care. Face-to-face, computer-assisted, structured interviews were conducted with 283 active young adult Ecstasy users in Atlanta, Georgia between August 2002 and October 2007. Study participants were recruited using a targeted sampling approach. Results indicated that almost one-third of the young adult Ecstasy users interviewed did not receive the medical care that they needed during the preceding year. Foregoing such care was associated with a variety of adverse drug-related outcomes, including experiencing a greater number of negative effects from using Ecstasy, experiencing a larger number of drug dependency symptoms, a greater likelihood of ever having binged on Ecstasy, and a greater likelihood of being classified as a high end polydrug abuser. Several factors were found to be associated with a greater tendency not to receive the medical care they needed, including race (not being African American), educational attainment (having completed at least high school), self-identification as belonging to the lowest socioeconomic status grouping, low self-esteem, and having experienced sexual abuse during ones formative years.

Sex differences in the effects of marijuana on simulated driving performance.

In the United States, one in six teenagers has driven under the influence of marijuana. Driving under the influence of marijuana and alcohol is equally prevalent, despite the fact that marijuana use is less common than alcohol use. Much of the research examining the effects of marijuana on driving performance was conducted in the 1970s and led to equivocal findings. During that time, few studies included women and driving simulators were rudimentary. Further, the potency of marijuana commonly used recreationally has increased. This study examined sex differences in the acute effects of marijuana on driving performance using a realistic, validated driving simulator. Eighty-five subjects (n 50 males, 35 females) participated in this between-subjects, double-blind, placebo controlled study. In addition to an uneventful, baseline segment of driving, participants were challenged with collision avoidance and distracted driving scenarios. Under the influence of marijuana, participants decreased their speed and failed to show expected practice effects during a distracted drive. No differences were found during the baseline driving segment or collision avoidance scenarios. No differences attributable to sex were observed. This study enhances the current literature by identifying distracted driving and the integration of prior experience as particularly problematic under the influence of marijuana.

The results of an experimental indoor hydroponic Cannabis growing study, using the Screen of Green (ScrOG) method-Yield, tetrahydrocannabinol (THC) and DNA analysis.

The results of an indoor hydroponic Cannabis growth study are presented. It is intended that this work will be of assistance to those with an interest in determining an estimation of yield and value of Cannabis crops. Three cycles of six plants were grown over a period of 1 year in order to ascertain the potential yield of female flowering head material from such an operation. The cultivation methods used were selected to replicate typical indoor hydroponic Cannabis growing operations, such as are commonly encountered by the New Zealand Police. The plants were also tested to ascertain the percentage of the psychoactive chemical Δ-9 tetrahydrocannabinol (THC) present in the flowering head material, and were genetically profiled by STR analysis. Phenotypic observations are related to the data collected. The inexperience of the growers was evidenced by different problems encountered in each of the three cycles, each of which would be expected to negatively impact the yield and THC data obtained. These data are therefore considered to be conservative. The most successful cycle yielded an average of 881g (31.1oz) of dry, groomed female flowering head per plant, and over the whole study the 18 plants yielded a total of 12,360g (436.0oz), or an average of 687g (24.2oz) of dry head per plant. THC data shows significant intra-plant variation and also demonstrates inter-varietal variation. THC values for individual plants ranged from 4.3 to 25.2%. The findings of this study and a separate ESR research project illustrate that the potency of Cannabis grown in New Zealand has dramatically increased in recent years. DNA analysis distinguished distinct groups in general agreement with the phenotypic variation observed. One plant however, exhibiting a unique triallelic pattern at two of the five loci tested, while remaining phenotypically indistinguishable from three other plants within the same grow.

Sexually dimorphic alterations in locomotion and reversal learning after adolescent tetrahydrocannabinol exposure in the rat.

Research suggests that use and abuse of marijuana can be especially harmful if it occurs during adolescence, a period of vast developmental changes throughout the brain. We examined the effects of 2mgkg (9)-tetrahydrocannabinol (THC) administered daily via intra-peritoneal injections during juvenileearly adolescence (postnatal day 22-40) or late adolescence (postnatal day 41-60) on locomotor activity, development of tolerance, and acquisitionretention of spatial avoidance in adulthood. THC caused locomotor depression in both male and female animals dosed during early adolescence but only in female animals dosed during late adolescence. Evidence of reverse tolerance to THC was seen in early adolescent animals only. In the active place avoidance test (APA), male and female animals administered THC during early adolescence made more errors on the reversal trial requiring flexibility in learning, but in animals dosed during late adolescence there were no significant sex or treatment differences. The results of the locomotor activity study indicate that females may be more sensitive to the effects of THC than males, while results of both locomotor activity and APA studies suggest that early adolescents appear to be more vulnerable to these effects than late adolescentsyoung adults.

18-Methoxycoronaridine, a potential anti-obesity agent, does not produce a conditioned taste aversion in rats.

18-Methoxycoronaridine (18-MC), a selective antagonist of alpha3beta4 nicotinic receptors, has been shown to reduce the self-administration of several drugs of abuse. Recently, this agent has also been shown to attenuate sucrose reward, decrease sucrose intake and prevent the development of sucrose-induced obesity in rats. The present experiments were designed to determine whether the latter effect was due to an 18-MC-induced conditioned taste aversion to sucrose. Both 18-MC (20mg kg, i.p.) and control agent, lithium chloride (100mgkg, i.p.), reduced sucrose intake 24h after association with sucrose however, only lithium chloride reduced sucrose intake 72h later. Consistent with previous data, 18-MC appears to have proactive effect for 24h and it does not induce a conditioned taste aversion.

Significant decreases in frontal and temporal 11C-raclopride binding after THC challenge.

Delta9-tetrahydrocannabinol (THC) increases prefrontal cortical dopamine release in animals, but this is yet to be examined in humans. In man, striatal dopamine release can be indexed using 11C-raclopride positron emission tomography (PET), and recent reports suggest that cortical 11C-raclopride binding may also be sensitive to dopaminergic challenges. Using an existing dataset we examined whether THC alters 11C-raclopride binding potential (BP(ND)) in cortical regions. Thirteen healthy volunteers underwent two 11C-raclopride PET scans following either oral 10 mg THC or placebo. Significant areas of decreased cortical 11C-raclopride BP(ND) were identified using whole brain voxel-wise analysis and quantified using a region of interest (ROI) ratio analysis. Effect of blood flow on binding was estimated using a simplified reference tissue model analysis. Results were compared to 11C-raclopride test-retest reliability in the ROIs identified using a separate cohort of volunteers. Voxel-wise analysis identified three significant clusters of decreased 11C-raclopride BP(ND) after THC in the right middle frontal gyrus, left superior frontal gyrus and left superior temporal gyrus. Decreases in 11C-raclopride BPND following THC were greater than test-retest variability in these ROIs. R1, an estimate of blood flow, significantly decreased in the left superior frontal gyrus in the THC condition but was unchanged in the other ROIs. Decreased frontal binding significantly correlated to catechol-o-methyl transferase (COMT) val108 status. We have demonstrated for the first time significant decreases in bilateral frontopolar cortical and left superior temporal gyrus 11C-raclopride binding after THC. The interpretation of these findings in relation to prefrontal dopamine release is discussed.

Disposition of smoked cannabis with high Δ(9)-tetrahydrocannabinol content a kinetic model.

No model exists to describe the disposition and kinetics of inhaled cannabis containing a high THC dose. We aimed to develop a kinetic model providing estimates of the THC serum concentrations after smoking cannabis cigarettes containing high THC doses (up to 69mg THC). Twenty-four male non-daily cannabis users smoked cannabis cigarettes containing 29.3mg, 49.1mg, and 69.4mg THC. Blood samples were collected over a period of 0-8h and serum THC concentrations were measured. A two-compartment open model was fitted on the individual observed data. Large inter-individual variability was observed in the pharmacokinetic parameters. The median pharmacokinetic parameters generated by the model were Cmax175ngmL, Tmax14min, and AUC0-8h8150ng×minmL for the 69.4mg THC dose. Median model results show an almost linear dose response relation for CmaxDose2.8×10(-6)mL and AUC0-8hDose136×10(-6)minmL. However, for increasing dose level, there was a clear decreasing trend CmaxDose3.4, 2.6 and 2.5×10(-6)mL and AUC0-8hDose157, 133 and 117×10(-6)minmL for the 29.3, 49.1 and 69.4mg dose, respectively. Within the restriction of 8h of observation, the apparent terminal half life of THC was 150min. The model offers insight into the pharmacokinetics of THC in recreational cannabis users smoking cannabis containing high doses of THC mixed with tobacco. The model is an objective method for providing serum THC concentrations up to 8h after smoking cannabis with a high THC content (up to 23%).

Estimation of illicit drugs consumption by wastewater analysis in Paris area (France).

Illicit drugs consumption is actually an important public health concern that needs to be well defined to be managed. A new method, expressed as sewage epidemiology has been proposed by Daughton and developed by Zuccato. This method involves estimating the consumption from the measurement of drug residues in sewage. Several studies have been carried out, leading to an assessment of drugs consumption in some European countries. This work, carried out in Paris area (France) brings new data to this assessment and allows a comparison of cocaine and MDMA consumptions with European estimations. Four wastewater treatment plants (WWTPs) have been retained for the study, taking into account biological treatment, volume capacity, geographic location and social environment. Cocaine and its major metabolite benzoylecgonine (BZE), amphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and buprenorphine were measured in raw water and WWTP effluent using HPLC-MSMS after SPE extraction. Amphetamine was rarely detected. Cocaine and BZE were quantified at levels from 5 to 282 ng L(-1) and 15 to 849 ng L(-1), respectively. MDMA and buprenorphine concentrations remained under 20 ng L(-1). Cocaine consumption was estimated from cocaine or BZE concentrations measured in raw water and the results showed significant difference in drug taking during week or weekend. The estimated doses observed in this study are lower than those reported for others countries, especially Spain and Italy. MDMA consumption was estimated at lower levels than cocaine.

Effect of (-)-Delta(9)-tetrahydrocannabinoid on the hepatic redox state of mice.

(-)-Delta(9)-Tetrahydrocannabinol (Delta(9)-THC), a psychoactive component of marijuana, has been reported to induce oxidative damage in vivo and in vitro. In this study, we administered Delta(9)-THC to healthy C57BL6J mice aged 15 weeks in order to determine its effect on hepatic redox state. Mice were divided into 3 groups Delta(9)-THC (N 10), treated with 10 mgkg body weight Delta(9)-THC daily VCtrl (N 10), treated with vehicle 1118, cremophor EL (polyoxyl 35 castor oil)ethanolsaline Ctrl (N 10), treated with saline. Animals were injected ip twice a day with 5 mgkg body weight for 10 days. Lipid peroxidation, protein carbonylation and DNA oxidation were used as biomarkers of oxidative stress. The endogenous antioxidant defenses analyzed were glutathione (GSH) levels as well as enzyme activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and glutathione peroxidase (GPx) in liver homogenates. The levels of mRNA of the cannabinoid receptors CB1 and CB2 were also monitored. Treatment with Delta(9)-THC did not produce significant changes in oxidative stress markers or in mRNA levels of CB1 and CB2 receptors in the liver of mice, but attenuated the increase in the selenium-dependent GPx activity (Delta(9)-THC 8% VCtrl 23% increase) and the GSHoxidized GSH ratio (Delta(9)-THC 61% VCtrl 96% increase), caused by treatment with the vehicle. Delta(9)-THC administration did not show any harmful effects on lipid peroxidation, protein carboxylation or DNA oxidation in the healthy liver of mice but attenuated unexpected effects produced by the vehicle containing ethanolcremophor EL.

Cerebral vasculopathy after 4-bromo-2,5-dimethoxyphenethylamine ingestion.

4-bromo-2,5-dimethoxyphenethylamine (2C-B) is a designer-drug variant of 3,4-methylenedioxymethamphetamine (ecstasy) whose recreational use has increased significantly over the last 10 years. Neurologic consequences of 2C-B usage are currently unknown. A 43-year-old woman experienced severe headaches within 48 hours of taking liquid 2C-B, after which time she developed progressive encephalopathy and quadraparesis, which did not improve over several months. MRA and cerebral angiogram imaging demonstrated profound vascular abnormalities of large, medium, and small-caliber vessels with subsequent watershed infarction. Brain biopsy and cerebrospinal fluid studies ruled out an inflammatory process. This case demonstrates an idiosyncratic and devastating neurologic response to 2C-B, a recreational drug whose popularity has increased with widespread availability of online guides for its synthesis.

Crystallographic characterization of the first reported crystalline form of the potent hallucinogen (R)-2-amino-1-(8-bromobenzo1,2-b5,4-bdifuran-4-yl)propane or bromodragonfly the 11 anhydrous proton-transfer compound with 3,5-dinitrosalicylic acid.

The 11 proton-transfer compound of the potent substituted amphetamine hallucinogen (R)-2-amino-1-(8-bromobenzo1,2-b5,4-bdifuran-4-yl)propane (common trivial name bromodragonfly) with 3,5-dinitrosalicylic acid, namely 1-(8-bromobenzo1,2-b5,4-bdifuran-4-yl)propan-2-aminium 2-carboxy-4,6-dinitrophenolate, C(13)H(13)BrNO(2)().C(7)H(3)N(2)O(7)(-), forms hydrogen-bonded cation-anion chain substructures comprising undulating head-to-tail anion chains formed through C(8) carboxyl-nitro O-H...O associations and incorporating the aminium groups of the cations. The intrachain cation-anion hydrogen-bonding associations feature proximal cyclic R(3)(3)(8) interactions involving both an N()-H...O(phenolate) and the carboxyl-nitro O-H...O associations and aromatic pi-pi ring interactions minimum ring centroid separation 3.566 (2) A. A lateral hydrogen-bonding interaction between the third aminium H atom and a carboxyl O-atom acceptor links the chain substructures, giving a two-dimensional sheet structure. This determination represents the first of any form of this compound and is in the (R) absolute configuration. The atypical crystal stability is attributed both to the hydrogen-bonded chain substructures provided by the anions, which accommodate the aminium proton-donor groups of the cations and give crosslinking, and to the presence of the cation-anion aromatic ring pi-pi interactions.

Interpretation and utility of drug of abuse immunoassays lessons from laboratory drug testing surveys.

To assist with patient diagnosis and management, physicians from pain services, drug treatment programs, and the emergency department frequently request that urine be tested for drugs of abuse. However, urine immunoassays for drugs of abuse have limitations. To use data from the College of American Pathologists Proficiency Testing Surveys to determine and summarize the characteristics, performance, and limitations of urine immunoassays for drugs of abuse. Six years of urine drug testing proficiency surveys were reviewed. Lysergic acid diethylamide and methaqualone are infrequently prescribed or abused and, therefore, testing may be unnecessary. However, implementation of more specific testing for methylenedioxymethamphetamine and oxycodone may be warranted. Each drug of abuse immunoassay exhibits a different cross-reactivity profile. Depending on the cross-reactivity profile, patients with clinically insignificant concentrations of drugs may have false-positive results, and patients with clinically significant concentrations of drugs may have false-negative results. Laboratory directors should be aware of the characteristics of their laboratories assays and should communicate these characteristics to physicians so that qualitative results can be interpreted more accurately. Furthermore, manufacturers claims should be interpreted with caution and should be verified in each organizations patient population, if possible.

Cases of fatal para methoxy amphetamine (PMA) poisoning in the material of the Forensic Medicine Department, Medical University Of Białystok, Poland.

The issue of sudden deaths due to acute para methoxy amphetamine (PMA) poisoning is presented in the report. The analysis included three cases autopsied at the Forensic Medicine Department in Białystok at the beginning of 2009. The toxicological analysis of samples of blood and urine did not confirm the presence of MDMA, also known as ecstasy, but it revealed the presence of para methoxy amphetamine (PMA). During post-mortem examinations, the cause of the death was not established in either case. Based on the above investigations it may be said that the common cause of death was acute para methoxy amphetamine (PMA) poisoning.

Elevated brain monoamine oxidase A binding in the early postpartum period.

The early postpartum period is a time of high risk for a major depressive episode (or postpartum depression), with a prevalence of 13%. During this time, there is a heightened vulnerability for low mood because postpartum blues is common. Severe postpartum blues can herald the onset of postpartum depression. The neurobiological mechanisms to explain postpartum blues and the high risk for the onset of postpartum depression in the first few weeks after delivery are unclear. Estrogen levels drop 100- to 1000-fold during the first 3 to 4 days postpartum, and changes in estrogen levels have an inverse relationship with monoamine oxidase A (MAO-A) density. However, MAO-A levels have never been measured in the early postpartum period. To determine whether brain MAO-A binding is elevated in the early postpartum period. Case-control study. Tertiary care academic psychiatric hospital in Toronto, Ontario, Canada. Fifteen healthy women who were 4 to 6 days postpartum and 15 healthy women who had not recently been postpartum underwent carbon 11-labeled harmine positron emission tomography scanning. All women were nonsmoking and medication free. MAO-A total distribution volume, an index of MAO-A density, was measured in prefrontal cortex, anterior cingulate cortex, anterior temporal cortex, thalamus, dorsal putamen, hippocampus, and midbrain. MAO-A total distribution volume was significantly elevated (mean, 43%) throughout all analyzed brain regions during the early postpartum period. Elevated MAO-A levels in the early postpartum period can be interpreted as a marker of a monoamine-lowering process that contributes to the mood change of postpartum blues. Rather than a purely psychosocial model, we propose a neurobiological model of estrogen decline, followed by elevated MAO-A binding, low mood, and subsequently a period of high risk for major depressive episodes. Our model has important implications for preventing postpartum depression and for developing therapeutic strategies that target or compensate for elevated MAO-A levels during postpartum blues.

Cannabinoid administration increases 5HT1A receptor binding and mRNA expression in the hippocampus of adult but not adolescent rats.

The endocannabinoid and serotonin systems share a high level of overlap in terms of the physiological processes that they regulate, however, little is known about their functional interactions particularly during adolescence, a vulnerable period for both the development of psychosis and for initiation to substance use. In the present study, the effects of cannabinoid treatment on serotonin 5HT1A receptor density and mRNA expression were investigated in two age groups Adolescent (postnatal day 35) and adult (postnatal day 70) rats were injected with the synthetic cannabinoid HU210 (25, 50 or 100 microgkg) or vehicle for 1, 4 or 14 days and sacrificed 24 h after the last injection. 5HT1A receptor density was measured in different brain regions using (3)H8-OH-DPAT quantitative autoradiography whereas mRNA expression was measured in adjacent brain sections. Higher levels of both serotonin 5HT1A receptor binding and mRNA expression were observed in limbic regions in adolescent control animals compared to adults. 5HT1A receptor density was increased by 23% in the CA1 region of the hippocampus of adult rats treated with 100 microgkg HU210 for 4 days compared to vehicle treated controls. The same treatment increased mRNA expression by 27% and by 14% in the CA1 region and dentate gyrus of the hippocampus respectively. 5HT1A receptor density was increased by 22% in the CA1 of adult animals treated with 50 microg HU210, by 26% in the dentate gurus of adult rats treated with 100 microg for 14 days. By contrast, 5HT1A receptor density or mRNA expression was not affected in the brain of adolescent animals in any of the brain regions examined. These results suggest that cannabinoid treatment has differential effects on serotonin-related neurochemistry in adolescent compared to adult rats. The effects in the adult brain may compromise hippocampal function and could account for the cognitive deficits seen in habitual heavy cannabis users.

Caffeine promotes dopamine D1 receptor-mediated body temperature, heart rate and behavioural responses to MDMA (ecstasy).

Caffeine exacerbates the acute toxicity of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) in rats characterised by hyperthermia, tachycardia and lethality. Depletion of central catecholamine stores and dopamine D(1) receptor blockade have been reported to attenuate the ability of caffeine to exacerbate MDMA-induced hyperthermia. Here, we investigate whether dopamine D(1) and D(2) receptors mediate the effects of caffeine on MDMA-induced changes in body temperature, heart rate and locomotor activity. All parameters were recorded continuously in individually housed rats using bioradiotelemetry from 1 h prior to 4 h following caffeine (10 mgkg, s.c.) andor MDMA (10 mgkg, s.c.) administration. Co-administration of caffeine with MDMA provoked a switch from MDMA-induced hypothermia and bradycardia to hyperthermia and tachycardia without influencing MDMA-induced hyperlocomotion. Pre-treatment with a specific dopamine D(15) antagonist SCH 23390 (1 mgkg) enhanced MDMA-induced hypothermia and blocked the ability of caffeine to provoke a switch from MDMA-induced hypothermia to hyperthermia. Furthermore, SCH 23390 blocked MDMA-induced hyperactivity and the ability of caffeine to promote a tachycardic response to MDMA. By contrast, pre-treatment with the selective D(2) antagonist, sulpiride (100 mgkg) blocked MDMA-induced hypothermia, failed to influence the ability of caffeine to promote tachycardia whilst enhancing MDMA-induced hyperactivity. Our results highlight the importance of dopamine D(1) and D(2) receptors in shaping the behavioural and physiological response to MDMA and suggest that the ability of caffeine to provoke MDMA-induced toxicity is associated with the promotion of dopamine D(1) over D(2) receptor-related responses.

Modulation of prepulse inhibition and stereotypies in rodents no evidence for antipsychotic-like properties of histamine H3-receptor inverse agonists.

H(3)-receptor inverse agonists raise a great interest as innovative therapeutics in several central disorders. Whereas their procognitive properties are well established, their antipsychotic-like properties are still debated. We further explored the effect of maximal doses (3-10 mgkg) of ciproxifan, BF2.649, and ABT-239, three selective H(3)-receptor inverse agonists, on deficits of prepulse inhibition (PPI) induced by apomorphine, MK-801, and phencyclidine (PCP). Their effect was also investigated on stereotypies induced by apomorphine and methamphetamine. Ciproxifan, BF2.649, and ABT-239 did not reverse the PPI impairment produced by apomorphine (0.5 mgkg, subcutaneous) in rats. Ciproxifan and BF2.649 did not reverse the impairment induced in mice by MK-801 (0.3 mgkg). Ciproxifan and BF2.649 also failed to reverse the disruption induced in mice by PCP (5-10 mgkg). Low to moderate doses of haloperidol (0.1-0.4 mgkg, intraperitoneal), alone or co-administered with BF2.649, did not reverse MK-801-induced PPI disruption. A high dose (1 mgkg) of haloperidol partially reversed the MK-801-induced deficit and BF2.649 tended to increase this effect, although nonsignificantly. Whereas stereotypies induced in mice by apomorphine and methamphetamine were totally suppressed by haloperidol, the decrease induced by ciproxifan was partial against apomorphine and very low, if any, against methamphetamine. Their total absence of effect in several validated animal models of the disease does not support antipsychotic properties of H(3)-receptor inverse agonists. However, their positive effects previously reported in behavioral tasks addressing learning, attention, and memory maintain the interest of H(3)-receptor inverse agonists for the treatment of cognitive symptoms of schizophrenia as adjunctive medications.

Elevated BDNF protein level in cortex but not in hippocampus of MDMA-treated Dark Agouti rats a potential link to the long-term recovery of serotonergic axons.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a widely used recreational drug known to cause selective long-term serotonergic damage. In this study, we examined the pattern of BDNF protein expression 1 day, 3, 8, 12 and 24 weeks after a single 15mgkg i.p. dose of MDMA to adolescent Dark Agouti rats. In parallel, we measured either tryptophan-hydroxylase immunoreactive (TpH IR) axon density, or (3)H-paroxetine-binding in parietal cortex and hippocampus, two brain areas known to have different recovery capacity after MDMA, to test whether BDNF-levels were associated with the long-term recovery of serotonergic fibers after a neurotoxic dose of MDMA. Both TpH IR axon density and (3)H-paroxetine-binding were significantly decreased 3 weeks after the treatment in both brain areas but while normalization in both parameters was found in parietal cortex 24 weeks after treatment, significant decreases remained evident in the hippocampus. In the parietal cortex, a significant reduction in BDNF protein levels was found in the acute phase after treatment (1 day), which was followed by a robust increase 8 weeks later and a return to control levels by 12 weeks. In contrast, no significant alteration of BDNF protein level was found in the hippocampus at any time points. This absence of any significant increase in BDNF protein levels in the hippocampus, and the persistence in this region of decreases in TpH IR axon density and (3)H-paroxetine-binding, raises the possibility that BDNF has an important role in the long-term recovery of serotonergic axons after MDMA treatment.

A randomized, double-blinded, crossover pilot study assessing the effect of nabilone on spasticity in persons with spinal cord injury.

To determine whether nabilone, a synthetic cannabinoid, alleviates spasticity in people with spinal cord injury (SCI). A double-blind, placebo-controlled crossover study. Outpatient rehabilitation clinics. We recruited volunteers (N12) with SCI and spasticity. One subject, a paraplegic man, dropped out of the study because of an unrelated cause. Eleven subjects completed the study all subjects were men with an average age of 42.36 years 6 of them were persons with tetraplegia, and 5 were persons with paraplegia. The subjects received either nabilone or placebo during the first 4-week period (0.5mg once a day with option to increase to 0.5mg twice a day), and then outcome measures were assessed. After a 2-week washout, subjects were crossed over to the opposite arm. The primary outcome was the Ashworth Scale for spasticity in the most involved muscle group, in either the upper or lower extremities, chosen by the subject and clinician. The secondary outcomes included the sum of the Ashworth Scale in 8 muscle groups of each side of the body measured by the clinician Spasm Frequency Scale and visual analog scale, reported by the subject Wartenberg Pendulum Test, in order to quantify severity of spasticity and the Clinicians and Subjects Global Impression of Change. One subject dropped out during the placebo arm because of an unrelated urinary stricture, and 11 subjects completed the study. There was a significant decrease on active treatment for the Ashworth in the most involved muscle (mean difference - SD, .909-.85 P.003), as well as the total Ashworth score (P.001). There was no significant difference in other measures. Side effects were mild and tolerable. Nabilone may be beneficial to reduce spasticity in people with SCI. We recommend a larger trial with a more prolonged treatment period and an option to slowly increase the dosage further.

Cannabidiol attenuates the appetitive effects of Delta 9-tetrahydrocannabinol in humans smoking their chosen cannabis.

Worldwide cannabis dependence is increasing, as is the concentration of Delta(9)-tetrahydrocannabinol (THC) in street cannabis. At the same time, the concentration of the second most abundant cannabinoid in street cannabis, cannabidiol (CBD), is decreasing. These two cannabinoids have opposing effects both pharmacologically and behaviorally when administered in the laboratory. No research has yet examined how the ratio of these constituents impacts on the appetitivereinforcing effects of cannabis in humans. A total of 94 cannabis users were tested 7 days apart, once while non-intoxicated and once while acutely under the influence of their own chosen smoked cannabis on dependence-related measures. Using an unprecedented methodology, a sample of cannabis (as well as saliva) was collected from each user and analyzed for levels of cannabinoids. On the basis of CBD THC ratios in the cannabis, individuals from the top and bottom tertiles were directly compared on indices of the reinforcing effects of drugs, explicit liking, and implicit attentional bias to drug stimuli. When intoxicated, smokers of high CBD THC strains showed reduced attentional bias to drug and food stimuli compared with smokers of low CBD THC. Those smoking higher CBD THC strains also showed lower self-rated liking of cannabis stimuli on both test days. Our findings suggest that CBD has potential as a treatment for cannabis dependence. The acute modulation of the incentive salience of drug cues by CBD may possibly generalize to a treatment for other addictive disorders.

1,2-ethane bis-1-amino-4-benzamidine is active against several brain insult and seizure challenges through anti-NMDA mechanisms targeting the 3H-TCP binding site and antioxidant action.

Five bis-benzamidines were screened towards murine magnesium deficiency-dependent audiogenic seizures, unravelling two compounds with efficacious doses 50 (ED(50)) less than 10mgkg. They were also screened against maximal electroshock and subcutaneous pentylenetetrazole-induced seizures, and explored for superoxide -scavenging activity. 1,2-Ethane bis-1-amino-4-benzamidine (EBAB) was selected and evaluated in 6 Hz seizure test (ED(50)49 mgkg) and at 4 microgkg in focal cerebral ibotenate poisoning in pups (sizes of both white and grey matter wounds were halved). EBAB was further tested on NMDA-induced seizures in mice (ED(50)6 mgkg) and on (3)H-TC -binding to a rodent cerebral preparation (IC(50)1.4 microM). Taken as a whole, present data emphasise the suitability of bis-benzamidines as templates for designing brain protective compounds.

Relationship between color and pigment production in two stone biofilm-forming cyanobacteria (Nostoc sp. PCC 9104 and Nostoc sp. PCC 9025).

Previous studies have provided evidence that color measurements enable on site quantification of superficial biofilms, thereby avoiding the need for sampling. In the present study, the efficiency of color measurements to evaluate to what extent pigment production is affected by environmental parameters such as light intensity, combined nitrogen and nutrient availability, was tested with two cyanobacteria, Nostoc sp. strains PCC 9104 and PCC 9025, which form biofilms on stone. Both strains were acclimated, in aerated batch cultures for 2 weeks, to three different culture media BG-11, BG-11(0), and BG-11(0)10 at either high or low light intensity. The content of chlorophyll a, carotenoids, and phycocyanins was measured throughout the experiment, together with variations in the color of the cyanobacteria, which were represented in the CIELAB color space. The results confirmed that the CIELAB color parameters are correlated with pigment content in such a way that variations in the latter are reflected as variations in color.

Beyond acid suppression new pharmacologic approaches for treatment of GERD.

Proton pump inhibitors are highly successful in treating gastroesophageal reflux disease, but a significant proportion of patients have persistent symptoms from weakly or nonacidic reflux. Transient lower esophageal sphincter relaxation (TLESR) represents the dominant mechanism of gastroesophageal reflux and has therefore become the most intensely investigated therapeutic target. The triggering of TLESR involve the vagal pathways and the gamma-aminobutyric type B (GABA(B)) and metabotropic glutamate type 5 (mGluR5) receptors. Baclofen is a GABA(B) receptor agonist that is effective in inhibiting TLESR and reducing the number of reflux episodes, but is associated with significant central nervous system (CNS) side effects. The newer GABA(B) agonists, such as AZD9343 and AZD3355, and mGluR5 antagonists, such as 2-methyl-6-(phenylethynyl)-pyridine (MPEP), have been shown in small, randomized, controlled trials to have comparable efficacy to baclofen, but possibly a more favorable CNS side effect profile. Cannibinoid agonists, such as Delta(9)-THC, have also been demonstrated to reduce TLESRs and reflux events respectively. Macrolide antibiotics (eg, erythromycin) show early promise in a select group of patients with possible reflux associated post-lung transplant problems.

Methylenedioxymethamphetamine inhibits mitochondrial complex I activity in mice a possible mechanism underlying neurotoxicity.

3,4-methylenedioxymethamphetamine (MDMA) causes a persistent loss of dopaminergic cell bodies in the substantia nigra of mice. Current evidence indicates that such neurotoxicity is due to oxidative stress but the source of free radicals remains unknown. Inhibition of mitochondrial electron transport chain complexes by MDMA was assessed as a possible source. Activities of mitochondrial complexes after MDMA were evaluated spectrophotometrically. In situ visualization of superoxide production in the striatum was assessed by ethidium fluorescence and striatal dopamine levels were determined by HPLC as an index of dopaminergic toxicity. 3,4-methylenedioxymethamphetamine decreased mitochondrial complex I activity in the striatum of mice, an effect accompanied by an increased production of superoxide radicals and the inhibition of endogenous aconitase. alpha-Lipoic acid prevented superoxide generation and long-term toxicity independent of any effect on complex I inhibition. These effects of alpha-lipoic acid were also associated with a significant increase of striatal glutathione levels. The relevance of glutathione was supported by reducing striatal glutathione content with L-buthionine-(S,R)-sulfoximine, which exacerbated MDMA-induced dopamine deficits, effects suppressed by alpha-lipoic acid. The nitric oxide synthase inhibitor, N(G)-nitro-L-arginine, partially prevented MDMA-induced dopamine depletions, an effect reversed by L-arginine but not D-arginine. Finally, a direct relationship between mitochondrial complex I inhibition and long-term dopamine depletions was found in animals treated with MDMA in combination with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Inhibition of mitochondrial complex I following MDMA could be the source of free radicals responsible for oxidative stress and the consequent neurotoxicity of this drug in mice.

MDMA toxicity and pathological consequences a review about experimental data and autopsy findings.

Studies conducted in humans or in animals explored the presence, nature and potential causes of 3,4-methylenedioxymethamphetamine (MDMA) toxicity. According to literature, there are four principal types of such serious toxicity hepatic, cardiovascular, cerebral and hyperpyrexic. The molecular mechanisms involved in the genesis of these toxic effects are not yet fully clarified, but the oxidative stress, excitotoxicity, and mitochondrial dysfunction appear to be causal events that converge to mediate MDMA-induced toxicity. Studies conducted on animals demonstrated that the acute administration of MDMA elicits cardiovascular responses that are similar to those elicited by d-amphetamine, and that these responses appear to involve catecholaminergic and non-catecholaminergic-dependent mechanisms. Although there is undeniable evidence of MDMA-induced cardiac toxicity, the mechanism responsible remains to be clarified. While many reports both in humans and in animals have demonstrated MDMA-induced liver damage, the underlying mechanism accounting for hepatic toxicity is poorly understood. Various mechanisms may contribute to MDMA-induced liver toxicity, including the metabolism of MDMA, the increased efflux of neurotransmitters, the oxidation of biogenic amines, and hyperthermia. The molecular mechanisms involved in the genesis of these toxic effects are not yet fully clarified, but the oxidative stress, excitotoxicity, and mitochondrial dysfunction appear to be causal events that converge to mediate MDMA-induced neurotoxicity, as measured by loss of various markers of dopaminergic and serotonergic terminals. The evidence is overwhelming that MDMA produces acute and long-lasting toxic anatomic effects in animals and humans. Anatomical and functional MDMA consequences must be better understood.

Mice in ecstasy advanced animal models in the study of MDMA.

The party drug 3,4-methylenedioxymethamphetamine -better known as MDMA or ecstasy- has numerous effects on the human body, characterized by a rush of energy, euphoria and empathy. However, also a multitude of toxicneurotoxic effects have been ascribed to MDMA, based upon case reports and studies in animals. Given the intrinsic difficulties associated with controlled studies in human beings, most of our insights into the biology of MDMA have been gained through animal studies. The vast majority of these studies utilizes a pharmacological approach to elucidate the mechanisms by which MDMA exerts its effects. Advances in genetics during the last decade have led to the development of several mouse models (transgenic or knockout) that have greatly contributed to our understanding of MDMA biology. This review provides an overview of these genetically modified animal models, in the light of some characteristic effects of MDMA, e.g. hyperlocomotion, neurotoxicity, hyperthermia, behaviour or rewarding. Without a shadow of a doubt, the next decade will bring many more advanced animal models, such as mice with site-specific deletion or rescue of genes and more genetically modified rat models. These models will further improve our knowledge on the pharmacology and toxicity of MDMA and, possibly, may assist in developing therapies coping with potential damage in abusers of MDMA and other drugs, as well as in patients suffering from specific neuronal pathologies.

Mechanisms of MDMA (ecstasy)-induced oxidative stress, mitochondrial dysfunction, and organ damage.

Despite numerous reports about the acute and sub-chronic toxicities caused by MDMA (3,4-methylenedioxymethamphetamine, ecstasy), the underlying mechanism of organ damage is poorly understood. The aim of this review is to present an update of the mechanistic studies on MDMA-mediated organ damage partly caused by increased oxidativenitrosative stress. Because of the extensive reviews on MDMA-mediated oxidative stress and tissue damage, we specifically focus on the mechanisms and consequences of oxidative-modifications of mitochondrial proteins, leading to mitochondrial dysfunction. We briefly describe a method to systematically identify oxidatively-modified mitochondrial proteins in control and MDMA-exposed rats by using biotin-N-maleimide (biotin-NM) as a sensitive probe for oxidized proteins. We also describe various applications and advantages of this Cys-targeted proteomics method and alternative approaches to overcome potential limitations of this method in studying oxidized proteins from MDMA-exposed tissues. Finally we discuss the mechanism of synergistic drug-interaction between MDMA and other abused substances including alcohol (ethanol) as well as application of this redox-based proteomics method in translational studies for developing effective preventive and therapeutic agents against MDMA-induced organ damage.

Causes and effects of cellular oxidative stress as a result of MDMA abuse.

3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) is a substituted amphetamine with potent central nervous stimulant effects. Increasing evidence suggests that one way of MDMA-induced toxicity involves the production of reactive oxygen and reactive nitrogen species and a subsequent production of oxidativenitrosative stress. The free radicals can originate from several molecular pathways (oxidative deamination of monoamine, metabolic pathways, cathecolamines autoxidation, and hyperthermia) and their harmful effect causing potential biological damage such as lipoperoxidation and cellular death. The role of oxidative stress in mediating MDMA toxicity is illustrated by decreases in the activity of the endogenous enzymatic and non enzymatic antioxidants observed in cells in vitro and in animals model. This review examines the available evidence for the involvement of oxidative stress in the mechanisms of MDMA-induced cellular damage with the aim to contribute to the understanding of the cellular and molecular mechanisms involved in MDMA toxicity.

Post-mortem (re)distribution of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) human and animal data.

In this paper, the distribution and redistribution of the amphetamine derivative, 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is brought into focus. Animal experimental data were compared with internationally reported MDMA-related human fatalities in general, these turned out to be parallel with each other. Due to its inherent properties (e.g. significant volume of distribution), MDMA is liable to postmortem redistribution. Indeed, very high concentrations have been found in cardiac blood and tissues located centrally in the body (blood-rich organs such as lungs and liver in particular). This confirms that post-mortem redistribution due to diffusion from higher to lower concentration can easily take place, mainly at longer post-mortem intervals and when putrefaction occurs. Therefore, we can conclude that for post-mortem quantitation of amphetamine and derivatives, and MDMA in particular, peripheral blood sampling (e.g. femoral vein) remains compulsory. However, if the latter is impossible, MDMA quantification in a few alternative matrices such as vitreous humour and iliopsoas muscle may provide additional information to come to a reliable conclusion. Furthermore, it should be stressed that--at present--it is impossible to estimate the individual susceptibility to the various possible adverse effects of MDMA, which implies that it is impossible to provide a safe or therapeutic blood MDMA level. Therefore, in current forensic practice, the post-mortem pathological and toxicological findings should form an entity in order to draw a well-grounded conclusion.

Neurotoxicity of ecstasy (MDMA) an overview.

Ecstasy (MDMA) is a powerful hallucinogenic drug which has raised concern worldwide because of its high abuse liability. A plethora of studies have demonstrated that MDMA has the potential to induce neurotoxicity both in human and laboratory animals. Although research on MDMA has been carried out by many different laboratories, the mechanism underlying MDMA induced toxicity has not been fully elucidated. MDMA has the ability to reduce serotonin levels in terminals of axons in the cortex of rats and mice. Recently we have shown that it also has the potential to produce degenerate neurons in discrete areas of the brain such as insular and parietal cortex, thalamus, tenia tecta and bed nucleus of stria terminalis (BST). Acute effects of MDMA can result in a constellation of changes including arrthymias, hypertension, hyperthermia, serotonin (5-HT) syndrome, liver problems, seizures and also long lasting neurocognitive impairments including mood disturbances. In human MDMA abusers, there is evidence for reduction of serotonergic biochemical markers. Several factors may contribute to the MDMA-induced neurotoxicity, especially hyperthermia. Other factors potentially influencing MDMA toxicity include monoamine oxidase metabolism of dopamine and serotonin, nitric oxide generation, glutamate excitotoxicity, serotonin 2A receptor agonism and the formation of MDMA neurotoxic metabolites. In this review we will cover the following topics pharmacological mechanisms, metabolic pathways and acute effects in laboratory animals, as well as in humans, with special attention on the mechanism and pathology of MDMA induced neurotoxicity.

The cardiovascular and cardiac actions of ecstasy and its metabolites.

The recreational use of 3, 4 methylenedioxymethamphetamine (ecstasy or MDMA) has increased dramatically over the past thirty years due to its ability to increase stamina and produce feelings of emotional closeness and wellbeing. In spite of the popular perception that MDMA is a safe drug, there is a large literature documenting that the drug can produce significant neurotoxicity, especially in serotonergic and catecholaminergic systems. There are also experimental and clinical data which document that MDMA can alter cardiovascular function and produce cardiac toxicity, including rhythm disturbances, infarction and sudden death. This manuscript will review the literature documenting the cardiovascular responses elicited by MDMA in humans and experimental animals and will examine the underlying mechanisms mediating these responses. We will also review the available clinical, autopsy and experimental data linking MDMA with cardiac toxicity. Most available data indicate that oxidative stress plays an important role in the cardiotoxic actions of MDMA. Moreover, new data indicates that redox active metabolites of MDMA may play especially important roles in MDMA induced toxicity.

Mechanisms underlying the hepatotoxic effects of ecstasy.

3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is a worldwide illegally used amphetamine-derived designer drug known to be hepatotoxic to humans. Jaundice, hepatomegaly, centrilobular necrosis, hepatitis and fibrosis represent some of the adverse effects caused by MDMA in the liver. Although there is irrefutable evidence of MDMA-induced hepatocellular damage, the mechanisms responsible for that toxicity remain to be thoroughly clarified. One well thought-of mechanism imply MDMA metabolism in the liver into reactive metabolites as responsible for the MDMA-elicited hepatotoxicity. However, other factors, including MDMA-induced hyperthermia, the increase in neurotransmitters efflux, the oxidation of biogenic amines, polydrug abuse pattern, and environmental features accompanying illicit MDMA use, may increase the risk for liver complications. Liver damage patterns of MDMA in animals and humans and current research on the mechanisms underlying the hepatotoxic effects of MDMA will be highlighted in this review.

MDMA administration and heat shock proteins response foreseeing a molecular link.

Molecular and cellular mechanisms of MDMA-induced toxicity have been extensively studied in a number of experimental models. Nevertheless, only few studies investigated the involvement of HSPs (molecular chaperones) in MDMA organs toxicity. In the present minireview we highlight this subject analysing the results of these studies conducted especially on brain tissue. Despite of it seems obvious that HSPs overexpression is a protective reaction against MDMA treatment, the molecular mechanisms for exerting their action are far to be undiscovered. At the same time, we need of comprehensive studies concerning the whole range of Hspschaperones expressions in all organs after acute and chronic administration of MDMA.

Myocardial expression of TNF-alpha, IL-1beta, IL-6, IL-8, IL-10 and MCP-1 after a single MDMA dose administered in a rat model.

Indirect effects of 3,4-methylenedioxy-N-methylamphetamine (MDMA) and metabolites on the cardiac cells are well-known, the mechanism(s) underlying direct MDMA-induced cardiotoxicity remaining to be clarified. To better understand the immuno-inflammatory phenomena accompanying the cardiac alterations during MDMA administration, we conducted a study in an in vivo animal model to evaluate the cellular morphological alterations related to the biological response between MDMA administration and inflammatory cytokines (tumor necrosis factor-alpha, IL-1beta, IL-6, 8, 10, and monocyte chemotactic protein-1). A total of 25 male rats were used. The effects were evaluated at 6, 16 and 24 hours after a single dose MDMA administered (20 mgkg i.p.). We found high levels of the cardioinhibitory cytokines in rat heart after 3 and 6 hs from MDMA administration. Strongest reaction was observed at 24 hs for TNF-alpha, IL-1beta, IL-6, 8, 10 and for MCP-1. Furthermore, we still determined the presence of MDMA and MDA in the plasma of rats treated with MDMA intra-peritoneal single injection it was present as early at 6 hs and still present 24 hs after treatment. Western blot analysis in cardiac samples demonstrated the IL-1beta and IL-6 reactions in rats died spontaneously at fourth hour. The rise of the selective cardioinhibitory cytokines may be interpreted as the adaptive response of jeopardized myocardium to the cardiac dysfunction resulting from MDMA injection.

Metabolism and toxicologic analysis of tryptamine-derived drugs of abuse.

5-Methoxy-N,N-dialkyltryptamines are tryptamine derivatives that possess strong hallucinogenic effects. Because of their escalating popularity and potent physiological effects, an increasing number of acute poisoning cases have been reported in various countries. For their metabolism in humans, only a few studies have been reported. Thus, based on previous studies, the authors forecasted and synthesized authentic standards of their expected metabolites, which retained the structural characteristics of the parent drugs. Using these authentic standards, several urine specimens from abusers and rats were analyzed by gas chromatography mass spectrometry, high-performance liquid chromatography mass spectrometry, and high-performance liquid chromatography tandem mass spectrometry. The present study reveals that four metabolic pathways of great quantitative significance for 5-Methoxy-N,N-dialkyltryptamines to four characteristic metabolites, which retain structural characteristics identifiable with the parent compound, exist in humans and rats. The finding in the present study will be of great importance in the study on the metabolism of other psychotomimetic tryptamine-derived drugs of abuse and in forensic toxicologic analyses.

Evidence of increased activation underlying cognitive control in ecstasy and cannabis users.

Evidence suggests that users of ecstasy (3,4-methylenedioxymethamphetamine) have behavioural and cognitive deficits and show increased impulsivity. Impulse control impairments have been shown to be common to a number of addictive behaviours and may constitute a risk factor for drug abuse and dependence. The aim of this study was to investigate brain activation during response inhibition and performance monitoring in current recreational drug users who predominantly used ecstasy. Twenty drug users (ten female) and twenty healthy controls were scanned during performance of a response-inhibition GONOGO task using functional magnetic resonance imaging. No performance deficits were evident. However, the drug user group revealed elevated frontal and parietal BOLD response during successful inhibitions, and temporal, frontal, and cingulate hyperactivity during commission errors. In addition, the users showed reduced deactivation in the default-mode network during task performance. Whether contributing to or arising from drug use, these results reveal dysregulation in brain regions subserving cognitive control and default-mode processes in current recreational drug users mirroring effects previously observed for harder drugs of abuse.

Assessment of auditory sensory processing in a neurodevelopmental animal model of schizophrenia--gating of auditory-evoked potentials and prepulse inhibition.

The use of translational approaches to validate animal models is needed for the development of treatments that can effectively alleviate cognitive impairments associated with schizophrenia, which are unsuccessfully treated by the current available therapies. Deficits in pre-attentive stages of sensory information processing seen in schizophrenia patients, can be assessed by highly homologues methods in both humans and rodents, evident by the prepulse inhibition (PPI) of the auditory startle response and the P50 (termed P1 here) suppression paradigms. Treatment with the NMDA receptor antagonist PCP on postnatal days 7, 9, and 11 reliably induce cognitive impairments resembling those presented by schizophrenia patients. Here we evaluate the potential of early postnatal PCP (20mgkg) treatment in Lister Hooded rats to induce post-pubertal deficits in PPI and changes, such as reduced gating, in the P1 suppression paradigm in the EEG. The results indicate that early postnatal PCP treatment to rats leads to a reduction in PPI of the acoustic startle response. Furthermore, treated animals were assessed in the P1 suppression paradigm and produced significant changes in auditory-evoked potentials (AEP), specifically by an increased P1 amplitude and reduced P2 (P200 in humans) gating. However, the treatment neither disrupted normal P1 gating nor reduced N1 (N100 in humans) amplitude, representing two phenomena that are usually found to be disturbed in schizophrenia. In conclusion, the current findings confirm measures of early information processing to show high resemblance between rodents and humans, and indicate that early postnatal PCP-treated rats show deficits in pre-attentional processing, which are distinct from those observed in schizophrenia patients.

Cannabis with high δ9-THC contents affects perception and visual selective attention acutely an event-related potential study.

Cannabis intake has been reported to affect cognitive functions such as selective attention. This study addressed the effects of exposure to cannabis with up to 69.4mg Delta(9)-tetrahydrocannabinol (THC) on Event-Related Potentials (ERPs) recorded during a visual selective attention task. Twenty-four participants smoked cannabis cigarettes with four doses of THC on four test days in a randomized, double blind, placebo-controlled, crossover study. Two hours after THC exposure the participants performed a visual selective attention task and concomitant ERPs were recorded. Accuracy decreased linearly and reaction times increased linearly with THC dose. However, performance measures and most of the ERP components related specifically to selective attention did not show significant dose effects. Only in relatively light cannabis users the Occipital Selection Negativity decreased linearly with dose. Furthermore, ERP components reflecting perceptual processing, as well as the P300 component, decreased in amplitude after THC exposure. Only the former effect showed a linear dose-response relation. The decrements in performance and ERP amplitudes induced by exposure to cannabis with high THC content resulted from a non-selective decrease in attentional or processing resources. Performance requiring attentional resources, such as vehicle control, may be compromised several hours after smoking cannabis cigarettes containing high doses of THC, as presently available in Europe and Northern America.

Delta9-tetrahydrocannabinol is a full agonist at CB1 receptors on GABA neuron axon terminals in the hippocampus.

Marijuana impairs learning and memory through actions of its psychoactive constituent, delta-9-tetrahydrocannabinol (Delta(9)-THC), in the hippocampus, through activation of cannabinoid CB1 receptors (CB1R). CB1Rs are found on glutamate and GABA neuron axon terminals in the hippocampus where they control neurotransmitter release. Previous studies suggest that Delta(9)-THC is a partial agonist of CB1Rs on glutamate axon terminals in the hippocampus, whereas its effects on GABA terminals have not been described. Using whole-cell electrophysiology in brain slices from C57BL6J mice, we examined Delta(9)-THC effects on synaptic GABA IPSCs and postsynaptic GABA currents elicited by laser-induced photo-uncaging (photolysis) of alpha-carboxy-2-nitrobenzyl (CNB) caged GABA. Despite robust inhibition of synaptic IPSCs in wildtype mice by the full synthetic agonist WIN55,212-2, using a Tween-80 and DMSO vehicle, Delta(9)-THC had no effects on IPSCs in this, or in a low concentration of another vehicle, randomly-methylated beta-cyclodextrin (RAMEB, 0.023%). However, IPSCs were inhibited by Delta(9)-THC in 0.1% RAMEB, but not in neurons from CB1R knockout mice. Whereas Delta(9)-THC did not affect photolysis-evoked GABA currents, these responses were prolonged by a GABA uptake inhibitor. Concentration-response curves revealed that the maximal effects of Delta(9)-THC and WIN55,212-2 were similar, indicating that Delta(9)-THC is a full agonist at CB1Rs on GABA axon terminals. These results suggest that Delta(9)-THC inhibits GABA release, but does not directly alter GABA(A) receptors or GABA uptake in the hippocampus. Furthermore, full agonist effects of Delta(9)-THC on IPSCs likely result from a much higher expression of CB1Rs on GABA versus glutamate axon terminals in the hippocampus.

Plaque deposition dependent decrease in 5-HT2A serotonin receptor in AbetaPPswePS1dE9 amyloid overexpressing mice.

Intrahippocampal injections of aggregated amyloid-beta (Abeta)1-42 in rats result in memory impairment and in reduction of hippocampal 5-HT2A receptor levels. In order to investigate how changes in 5-HT2A levels and functionality relate to the progressive accumulation of Abeta protein, we studied 5-HT2A receptor regulation in double transgenic AbetaPPswePS1dE9 mice which display excess production of Abeta and age-dependent increase in amyloid plaques. Three different age-groups, 4-month-old, 8- month-old, and 11-month-old were included in the study. 3H-MDL100907, 3H-escitalopram, and 11C-PIB autoradiography was performed for measuring 5-HT2A receptor, serotonin transporter (SERT), and Abeta plaque levels in medial prefrontal cortex (mPFC), prefrontal cortex (PFC), frontoparietal cortex (FPC), dorsal and ventral hippocampus, and somatosensory cortex. To investigate 5-HT2A receptor functionality, animals were treated with the 5-HT2A receptor agonist DOI and head-twitch response (HTR) subsequently recorded. Expression level of the immediate early gene c-fos was measured by in situ hybridization. We found that the age-related increase in Abeta plaque burden was accompanied by a significant decrease in 5-HT2A receptor binding in mPFC in the 11-month-old group. The changes in 5-HT2A receptor binding correlated negatively with 11C-PIB binding and were not accompanied by decreases in SERT binding. Correspondingly, 11-month-old transgenic mice showed diminished DOI-induced HTR and reduced increase in expression of c-fos mRNA in mPFC and FPC. These observations point towards a direct association between Abeta accumulation and changes in 5-HT2A receptor expression that is independent of upstream changes in the serotonergic system.

Selective electrochemical discrimination between dopamine and phenethylamine-derived psychotropic drugs using electrodes modified with an acyclic receptor containing two terminal 3-alkoxy-5-nitroindazole rings.

Electrochemical discrimination between dopamine and psychotropic drugs which have in common a skeletal structure of phenethylamine, can be obtained using acyclic receptors L(1) and L(2), containing two terminal 3-alkoxy-5-nitroindazole rings. Upon attachment to graphite electrodes, L(1) and L(2) exhibit a well-defined, essentially reversible solid state electrochemistry in contact with aqueous media, based on electrolyte-assisted reduction processes involving successive cation and anion insertionbinding. As a result, a distinctive, essentially Nernstian electrochemical response is obtained for phenethylammonium ions of methamphetamine (METH), p-methoxyamphetamine (PMA), amphetamine (AMPH), mescaline (MES), homoveratrylamine (HOM), phenethylamine (PEA) and dopamine (DA) in aqueous media.

Detection of Delta9-tetrahydrocannabinol and amphetamine-type stimulants in oral fluid using the Rapid Stat point-of-collection drug-testing device.

The Rapid Stat assay, a point-of-collection drug-testing device for detection of amphetamines, cannabinoids, cocaine, opiates, methadone, and benzodiazepines in oral fluid, was evaluated for cannabis and amphetamine-type stimulants. The Rapid Stat tests (n 134) were applied by police officers in routine traffic checks. Oral fluid and blood samples were analyzed using gas chromatography-mass spectrometry (GC-MS) for Delta(9)-tetrahydrocannabinol, amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyethylamphetamine, and methylenedioxyamphetamine. The comparison of GC-MS analysis of oral fluid with the Rapid Stat results for cannabis showed a sensitivity of 85%, a specificity of 87%, and a total confirmation rate of 87%. When compared with serum, the sensitivity of the cannabis assay decreased to 71%, the specificity to 60%, and the total confirmation rate to 66%. These findings were possibly caused by an incorrect reading of the THC test results. Comparison of the Rapid Stat amphetamine assay with GC-MS in oral fluid showed a sensitivity of 94%, a specificity of 97%, and a total confirmation rate of 97%. Compared with serum, a sensitivity of 100%, a specificity of 90%, and a total confirmation rate of 92% was found. The amphetamine assay must, therefore, be regarded as satisfactory.

The abuse potential of the synthetic cannabinoid nabilone.

Nabilone is a synthetic cannabinoid prescription drug approved in Canada since 1981 to treat chemotherapy-induced nausea and vomiting. In recent years, off-label use of nabilone for chronic pain management has increased, and physicians have begun to express concerns about nabilone becoming a drug of abuse. This study evaluates the evidence for abuse of nabilone, which is currently ill-defined. Scientific literature, popular press and internet databases were searched extensively for evidence of nabilone abuse. Focused interviews with medical professionals and law enforcement agencies across Canada were also conducted. The scientific literature and popular press reviews found very little reference to nabilone abuse. Nabilone is perceived to produce more undesirable side effects, to have a longer onset of action and to be more expensive than smoked cannabis. The internet review revealed rare and isolated instances of recreational use of nabilone. The database review yielded little evidence of nabilone abuse, although nabilone seizures and thefts have occurred in Canada in the past few years, especially in Ontario. Most law enforcement officers reported no instances of nabilone abuse or diversion, and the drug has no known street value. Medical professionals reported that nabilone is not perceived to be a matter of concern with respect to its abuse potential. Reports of nabilone abuse are extremely rare. However, follow-up of patients using nabilone for therapeutic purposes is prudent and should include assessment of tolerance and dependence. Prospective studies are also needed to definitively address the issue of nabilone abuse.

Testing for cannabis in the work-place a review of the evidence.

Urinalysis testing in the work-place has been adopted widely by employers in the United States to deter employee drug use and promote drug-free work-places. In other countries, such as Canada, testing is focused more narrowly on identifying employees whose drug use puts the safety of others at risk. We review 20 years of published literature on questions relevant to the objectives of work-place drug testing (WPDT), with a special emphasis on cannabis, the most commonly detected drug. We conclude (i) that the acute effects of smoking cannabis impair performance for a period of about 4 hours (ii) long-term heavy use of cannabis can impair cognitive ability, but it is not clear that heavy cannabis users represent a meaningful job safety risk unless using before work or on the job (iii) urine tests have poor validity and low sensitivity to detect employees who represent a safety risk (iv) drug testing is related to reductions in the prevalence of cannabis positive tests among employees, but this might not translate into fewer cannabis users and (v) urinalysis has not been shown to have a meaningful impact on job injuryaccident rates. Urinalysis testing is not recommended as a diagnostic tool to identify employees who represent a job safety risk from cannabis use. Blood testing for active tetrahydrocannabinol (THC) can be considered by employers who wish to identify employees whose performance may be impaired by their cannabis use.

Inhibition of monoamine oxidase activity by cannabinoids.

Brain monoamines are involved in many of the same processes affected by neuropsychiatric disorders and psychotropic drugs, including cannabinoids. This study investigated in vitro effects of cannabinoids on the activity of monoamine oxidase (MAO), the enzyme responsible for metabolism of monoamine neurotransmitters and affecting brain development and function. The effects of the phytocannabinoid Delta(9)-tetrahydrocannabinol (THC), the endocannabinoid anandamide (N-arachidonoylethanolamide AEA), and the synthetic cannabinoid receptor agonist WIN 55,212-2 (WIN) on the activity of MAO were measured in a crude mitochondrial fraction isolated from pig brain cortex. Monoamine oxidase activity was inhibited by the cannabinoids however, higher half maximal inhibitory concentrations (IC(50)) of cannabinoids were required compared to the known MAO inhibitor iproniazid. The IC(50) was 24.7 micromoll for THC, 751 micromoll for AEA, and 17.9 micromoll for WIN when serotonin was used as substrate (MAO-A), and 22.6 micromoll for THC, 1,668 micromoll for AEA, and 21.2 micromoll for WIN when phenylethylamine was used as substrate (MAO-B). The inhibition of MAOs by THC was noncompetitive. N-Arachidonoylethanolamide was a competitive inhibitor of MAO-A and a noncompetitive inhibitor of MAO-B. WIN was a noncompetitive inhibitor of MAO-A and an uncompetitive inhibitor of MAO-B. Monoamine oxidase activity is affected by cannabinoids at relatively high drug concentrations, and this effect is inhibitory. Decrease of MAO activity may play a role in some effects of cannabinoids on serotonergic, noradrenergic, and dopaminergic neurotransmission.

Antinociceptive activity of Delta9-tetrahydrocannabinol non-ionic microemulsions.

Delta(9)-Tetrahydrocannabinol (Delta(9)-THC), the major psychoactive constituent of Cannabis sativa L., has been widely studied for its potential pharmaceutical application in the treatment of various diseases and disturbs. This sparingly soluble terpeno-phenolic compound is not easy to handle and to be formulated in pharmaceutical preparations. The aim of this work was to develop a stable aqueous Delta(9)-THC formulation acceptable for different ways of administration, and to evaluate the therapeutic properties of the new Delta(9)-THC based preparation for pain treatment. Due to the thermodynamic stability and advantages of microemulsion based systems, the study was focused on the identification of aqueous microemulsion based systems containing Delta(9)-THC. Oil in water Delta(9)-THC microemulsions were individuated through phase diagrams construction, using the non-ionic surfactant Solutol HS15, being this surfactant acceptable for parenteral administration in human. A selected microemulsion samples containing 0.2 wt% of Delta(9)-THC, stable up to 52 degrees C, was successfully assayed on animal models of pain. Significant antinociceptive activity has been detected by both intraperitoneal and intragastric administration of the new Delta(9)-THC pharmaceutical preparation. The effect has been highlighted in shorter time if compared to a preparation of the same active principle based on previously reported conventional preparation.

Functional expression of 5-HT2A receptor in osteoblastic MC3T3-E1 cells.

In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT(2) receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT(2A) and 5-HT(2C) receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT(2A) receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT(2A) receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERKMAPK pathways in MC3T3-E1 cells.

Antibacterial and antifungal activities of (beta)-carboline alkaloids of Peganum harmala (L) seeds and their combination effects.

The β-carboline alkaloids of Peganum harmala L were extracted through a bioassay-guided fractionation and their antimicrobial activities were investigated. Results revealed significant differences (P>0.05) between compounds depending on the microorganism tested and the application method. When examined individually, harmine was the most effective against Proteus vulgaris, Bacillus subtilis and Candida albicans where inhibition zones ranged between 21.2 and 24.7 mm. Potentiality of the alkaloids was increased when applied as binary mixtures suggesting a kind of synergistic interaction with inhibition zones reaching 31.5mm with the total alkaloidal extract. We recommended the use of such compounds as new antimicrobial biorationals.

Neurocognitive impairments in MDMA and other drug users MDMA alone may not be a cognitive risk factor.

MDMA (3,4-methylenedioxymethamphetamine Ecstasy) is an amphetamine derivative with mild hallucinogenic and stimulant qualities. MDMA leads to serotonin (5-hydroxytryptamine 5-HT) neurotoxicity and has been linked to cognitive impairments. It remains unclear whether these impairments are due to MDMA versus other drug use. Neurocognitive functioning was measured in a sample of abstinent polydrug users (n 52) with a range of MDMA use and healthy nondrug controls (n 29). Participants completed a comprehensive neuropsychological battery and self-report measures of drug use. Polydrug users performed worse than controls on spatial span and spatial working memory (ps < .05). Among polydrug users, lifetime marijuana use significantly predicted verbal learning and memory performance (p < .01), while MDMA use was not predictive of cognitive impairment. This study and our previous report (Hanson, Luciana, Sullwold, 2008) suggest that moderate MDMA use does not lead to persistent impairments above and beyond that associated with generally heavy drug use, but polydrug use may lead to dose-related temporal and frontoparietal dysfunction. Marijuana use may be particularly problematic. Cause-effect relations are unclear.

The administration of psilocybin to healthy, hallucinogen-experienced volunteers in a mock-functional magnetic resonance imaging environment a preliminary investigation of tolerability.

This study sought to assess the tolerability of intravenously administered psilocybin in healthy, hallucinogen-experienced volunteers in a mock-magnetic resonance imaging environment as a preliminary stage to a controlled investigation using functional magnetic resonance imaging to explore the effects of psilocybin on cerebral blood flow and activity. The present pilot study demonstrated that up to 2 mg of psilocybin delivered as a slow intravenous injection produces short-lived but typical drug effects that are psychologically and physiologically well tolerated. With appropriate care, this study supports the viability of functional magnetic resonance imaging work with psilocybin.

Serotonergic dystrophy induced by excess serotonin.

Administration of certain serotonin-releasing amphetamine derivatives (fenfluramine andor 3,4-methylenedioxymethamphetamine, MDMA, ecstasy) results in dystrophic serotonergic morphology in the mammalian brain. In addition to drug administration, dystrophic serotonergic neurites are also associated with neurodegenerative disorders. We demonstrate here that endogenously elevated serotonin in the Drosophila CNS induces aberrant enlarged varicosities, or spheroids, that are morphologically similar to dystrophic mammalian serotonergic fibers. In Drosophila these spheroids are specific to serotonergic neurons, distinct from typical varicosities, and form only after prolonged increases in cytoplasmic serotonin. Our results also suggest that serotonin levels during early development determine later sensitivity of spheroid formation to manipulations of the serotonin transporter (SERT). Elevated serotonin also interacts with canonical protein aggregation and autophagic pathways to form spheroids. The data presented here support a model in which excess cytoplasmic neurotransmitter triggers a cell-specific pathway inducing aberrant morphology in fly serotonergic neurons that may be shared in certain mammalian pathologies.

Abnormal mGlu 5 receptorendocannabinoid coupling in mice lacking FMRP and BC1 RNA.

Transcriptional silencing of the gene encoding the fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS). FMRP acts as a translational repressor at central synapses, and molecular and synaptic plasticity studies have shown that the absence of this protein alters metabotropic glutamate 5 receptors (mGlu5Rs)-mediated signaling. In the striatum of mice lacking FMRP, we found enhanced activity of diacylglycerol lipase (DAGL), the enzyme limiting 2-arachidonoylglicerol (2-AG) synthesis, associated with altered sensitivity of GABA synapses to the mobilization of this endocannabinoid by mGlu5R stimulation with DHPG. Mice lacking another repressor of synaptic protein synthesis, BC1 RNA, also showed potentiated mGlu5R-driven 2-AG responses, indicating that both FMRP and BC1 RNA act as physiological constraints of mGlu5Rendocannabinoid coupling at central synapses. The effects of FMRP ablation on DAGL activity and on DHPG-mediated inhibition of GABA synapses were enhanced by simultaneous genetic inactivation of FMRP and BC1 RNA. In double FMRP and BC1 RNA lacking mice, striatal levels of 2-AG were also enhanced compared with control animals and to single mutants. Our data indicate for the first time that mGlu5R-driven endocannabinoid signaling in the striatum is under the control of both FMRP and BC1 RNA. The abnormal mGlu5R2-AG coupling found in FMRP-KO mice emphasizes the involvement of mGlu5Rs in the synaptic defects of FXS, and identifies the modulation of the endocannabinoid system as a novel target for the treatment of this severe neuropsychiatric disorder.

RIM1alpha and interacting proteins involved in presynaptic plasticity mediate prepulse inhibition and additional behaviors linked to schizophrenia.

Several presynaptic proteins involved in neurotransmitter release in the CNS have been implicated in schizophrenia in human clinical genetic studies, in postmortem studies, and in studies of putative animal models of schizophrenia. The presynaptic protein RIM1alpha mediates presynaptic plasticity and cognitive function. We now demonstrate that mice deficient in RIM1alpha exhibit abnormalities in multiple schizophrenia-relevant behavioral tasks including prepulse inhibition, response to psychotomimetic drugs, and social interaction. These schizophrenia-relevant behavioral findings are relatively selective to RIM1alpha-deficient mice, as mice bearing mutations in the RIM1alpha binding partners Rab3A or synaptotagmin 1 only show decreased prepulse inhibition. In addition to RIM1alphas involvement in multiple behavioral abnormalities, these data suggest that alterations in presynaptic forms of short-term plasticity are linked to alterations in prepulse inhibition, a measure of sensorimotor gating.

Direct analysis of Salvia divinorum leaves for salvinorin A by thin layer chromatography and desorption electrospray ionization multi-stage tandem mass spectrometry.

Salvia divinorum is widely cultivated in the US, Mexico, Central and South America and Europe and is consumed for its ability to produce hallucinogenic effects similar to those of other scheduled hallucinogenic drugs, such as LSD. Salvinorin A (SA), a kappa opiod receptor agonist and psychoactive constituent, is found primarily in the leaves and to a lesser extent in the stems of the plant. Herein, the analysis of intact S. divinorum leaves for SA and of acetone extracts separated using thin layer chromatography (TLC) is demonstrated using desorption electrospray ionization (DESI) mass spectrometry. The detection of SA using DESI in the positive ion mode is characterized by several ions associated with the compound - MH(), MNH(4)(), MNa(), 2MNH(4)(), and 2MNa(). Confirmation of the identity of these ions is provided through exact mass measurements using a time-of-flight (ToF) mass spectrometer. The presence of SA in the leaves was confirmed by multi-stage tandem mass spectrometry (MS(n)) of the MH() ion using a linear ion trap mass spectrometer. Direct analysis of the leaves revealed several species of salvinorin in addition to SA as confirmed by MS(n), including salvinorin B, C, DE, and divinatorin B. Further, the results from DESI imaging of a TLC separation of a commercial leaf extract and an acetone extract of S. divinorum leaves were in concordance with the TLCDESI-MS results of an authentic salvinorin A standard. The present study provides an example of both the direct analysis of intact plant materials for screening illicit substances and the coupling of TLC and DESI-MS as a simple method for the examination of natural products.

An investigation of factors associated with depressive symptoms among a sample of regular ecstasy consumers.

Methylenedioxymethamphetamine affects the central serotonergic system, and there is some evidence for an association between ecstasy use (drugs sold as methylenedioxymethamphetamine) and depression. The aim of the present study was to investigate the incidence of self-reported depression and associated help-seeking among a sample of regular ecstasy users. A further aim was to examine the correlates of depressive symptomatology in this population. 100 regular ecstasy consumers (at least monthly use) were interviewed as part of the Ecstasy and Related Drug Reporting System in Tasmania, Australia. Participants were also administered epidemiological measures of depression (Center for Epidemiological Studies Depression Scale) and psychological distress (Kessler Psychological Distress Scale). One quarter (23%) of participants self-reported recent experience of depression, a rate notably greater than the general population. However, only one third of these participants had attended a health professional for this issue. A range of drug use factors (e.g. frequency and quantity of ecstasy use, frequent cannabis or methamphetamine use, intravenous drug use, polydrug use, binge drug use, harmful alcohol use, and elevated psychological dependence scores for ecstasy and methamphetamine) were associated with high levels of depressive symptomatology. These findings are consistent with an association between depressive symptomatology and ecstasy and other drug use. Harm reduction strategies which target drug use factors such as those identified in this study may also aid in the reduction of the experience of depression. Considering the low levels of help-seeking among this population, improving awareness and access to information and treatment for depression may also be important.

Differential roles of phase I and phase II enzymes in 3,4-methylendioxymethamphetamine-induced cytotoxicity.

Metabolism plays an important role in the toxic effects caused by 3,4-methylenedioxymethamphetamine (MDMA). Most research has focused on the involvement of CYP2D6 enzyme in MDMA bioactivation, and less is known about the contribution of other cytochrome P450 (P450) and phase II metabolism. In this study, we researched the differential roles of phase I P450 enzymes CYP1A2, CYP3A4, and CYP2D6 and phase II enzymes glutathione S-transferase (GST) and catechol-O-methyltransferase (COMT) on the toxic potential of MDMA. MDMA acts as inhibitor of its own metabolism with a relative potency of inhibition of CYP2D>CYP3A>> CYP1A in rat liver microsomes and in human liver immortalized human liver epithelial cells (THLE) cells transfected with individual CYP1A2, CYP3A4, or CYP2D6. Cytotoxicity measurements by 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in THLE cells showed that the inhibition of phase I enzymes CYP1A2 by alpha-naphthoflavone and CYP3A4 by troleandomycin does not affect MDMA-induced cytotoxicity. MDMA metabolism by CYP2D6 significantly increased cytotoxicity, which was counteracted by CYP2D6 inhibition by quinidine. Inhibition of COMT by 2-fluoro-3,4-dihydroxy-5-nitrobenzophenone (Ro-41-0960) and GST by buthionine sulfoximine showed that COMT is mainly involved in detoxification of CYP2D6-formed MDMA metabolites, whereas glutathione (GSH) is mainly involved in detoxification of CYP3A4-formed MDMA metabolites. Liquid chromatographytandem mass spectrometry analyses of MDMA-metabolites in the THLE cell culture media confirmed formation of the specific MDMA metabolites and corroborated the observed cytotoxicity. Our data suggest that CYP2D6 as well as CYP3A4 play an important role in MDMA bioactivation. In addition, further studies are needed to address the differential roles of CYP3A4 and GSHGST in MDMA bioactivation and detoxification.

Impulsivity and executive functions in polysubstance-using rave attenders.

Rave parties are characterized by high levels of drug use and polysubstance-using patterns that may be especially harmful for psychological and neuropsychological functioning. The aim of this study was to conduct a comprehensive assessment of different aspects of impulsivity and executive functions in a sample of polysubstance-using rave attenders. We collected data from two groups rave attenders (RvA, n 25) and drug-free healthy comparison individuals (HCI, n 27). RvA were regular users of cannabis, cocaine, methampethamine, hallucinogens, and alcohol. The assessment protocol included a drug-taking interview, the UPPS-P Impulsive Behavior Scale, the delay-discounting questionnaire and a set of neuropsychological tests taxing different aspects of executive functions response speed, working memory, reasoning, response inhibition and switching, self-regulation, decision making, and emotion perception. For impulsivity measures, RvA had significantly elevated scores on lack of perseverance and positive and negative urgency, but did not differ from controls on lack of premeditation or sensation seeking. For neuropsychological functioning, RvA had significantly poorer performance on indices of analogical reasoning, processing speed, working memory, inhibitionswitching errors, and decision making, but performed similar to controls on indices of self-regulation, reversal learning, and emotion processing. Peak and binge alcohol and drug use were positively correlated with positive urgency, and negatively correlated with performance on executive indices. Rave attenders have selective alterations of impulsive personality and executive functions. These findings can contribute to delineate the neuropsychological profiles that distinguish recreational polysubstance use from substance dependence.

CRF receptor 1 regulates anxiety behavior via sensitization of 5-HT2 receptor signaling.

Stress and anxiety disorders are risk factors for depression and these behaviors are modulated by corticotrophin-releasing factor receptor 1 (CRFR1) and serotonin receptor (5-HT(2)R). However, the potential behavioral and cellular interaction between these two receptors is unclear. We found that pre-administration of corticotrophin-releasing factor (CRF) into the prefrontal cortex of mice enhanced 5-HT(2)R-mediated anxiety behaviors in response to 2,5-dimethoxy-4-iodoamphetamine. In both heterologous cell cultures and mouse cortical neurons, activation of CRFR1 also enhanced 5-HT(2) receptor-mediated inositol phosphate formation. CRFR1-mediated increases in 5-HT(2)R signaling were dependent on receptor internalization and receptor recycling via rapid recycling endosomes, resulting in increased expression of 5-HT(2)R on the cell surface. Sensitization of 5-HT(2)R signaling by CRFR1 required intact PDZ domain-binding motifs at the end of the C-terminal tails of both receptor types. These data suggest a mechanism by which CRF, a peptide known to be released by stress, enhances anxiety-related behavior via sensitization of 5-HT(2)R signaling.

Chronic treatment with fluoxetine decreases cerebral metabolic responses to the 5-HT1A agonist 8-hydroxy-2(di-N-propylamino)tetralin and increases those to the 5-HT2A2C agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane and to the dopaminergic agonist apomorphine.

Fluoxetine is a selective serotonin (5-HT) reuptake inhibitor that, when given chronically, alters different neurotransmitter systems. To assess functional changes occurring in the 5-HT and dopaminergic systems, we investigated the effects of 5-HT(1A) agonist 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT), of the 5-HT(2A2C) agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and of the dopamine D(12) agonist apomorphine (APO) on behavior and on regional cerebral metabolic rates for glucose (rCMRglc) in rats pretreated for 3weeks with saline or fluoxetine (8mgkgday). Behavioral effects were assessed for 8-OH-DPAT by scoring the 5-HT syndrome, for DOI by counting head shakes and for APO with an activity monitor. rCMRglc were measured with quantitative autoradiographic (14)C2-deoxyglucose technique in 60 brain regions at 10min after acute administration of 8-OH-DPAT 1mgkg, at 30min after DOI 5mgkg or at 10min after APO 1mgkg. Chronic fluoxetine did not alter the 5-HT syndrome by 8-OH-DPAT, decreased head shakes by DOI and enhanced hyperlocomotion by APO. 8-OH-DPAT produced rCMRglc increases in sensorimotor regions that were unaffected by fluoxetine pretreatment and diffuse metabolic decrements that were attenuated by fluoxetine in limbic and raphe areas (17% and 4% mean decreases, respectively, in saline control and fluoxetine-pretreated rats). DOI produced widespread rCMRglc declines that were intensified by fluoxetine (14% and 20% decreases, in control and fluoxetine rats). APO caused rCMRglc increases in 22 brain regions that were potentiated by fluoxetine in dopaminergic motor areas (10% and 25% increases, in control and fluoxetine rats). In conclusion, fluoxetine enhances 5-HT neurotransmission by blunting responsivity of 5-HT(1A) autoreceptors and increasing that of 5-HT(2A2C) postsynaptic receptors and enhances dopaminergic D(12) receptor neurotransmission.

Risky car following in abstinent users of MDMA.

Ecstasy (MDMA) use raises concerns because of its association with risky driving. We evaluated driving performance and risk taking in abstinent recreational MDMA users in a simulated car following task that required continuous attention and vigilance. Drivers were asked to follow two car lengths behind a lead vehicle (LV). Three sinusoids generated unpredictable LV velocity changes. Drivers could mitigate risk by following further behind the erratic LV. From vehicle trajectory data we performed a Fourier analysis to derive measures of coherence, gain, and delay. These measures and headway distance were compared between the different groups. All MDMA drivers met coherence criteria indicating cooperation in the car following task. They matched periodic changes in LV velocity similar to controls (abstinent THC users, abstinent alcohol users, and non-drug users), militating against worse vigilance. While all participants traveled approximately 55 mph (89 kph), the MDMA drivers followed 64 m closer to the LV and demonstrated 1.04 s shorter delays to LV velocity changes than other driver groups. The simulated car following task safely discriminated between driving behavior in abstinent MDMA users and controls. Abstinent MDMA users do not perform worse than controls, but may assume extra risk. The control theory framework used in this study revealed behaviors that might not otherwise be evident.

The effects of cannabis and alcohol on simulated arterial driving Influences of driving experience and task demand.

This study compared the effects of three doses of cannabis and alcohol (placebo, low and high doses), both alone and in combination, on the driving performance of young, novice drivers and more experienced drivers. Alcohol was administered as ethanol (95%) mixed with orange juice in doses of approximately 0, 0.4 and 0.6gkg. Cannabis was administered by inhalation of smoke from pre-rolled cannabis cigarettes (supplied by the National Institute of Drug Abuse, USA). Active cigarettes contained 19 mg delta-9-THC. Using a counterbalanced design, the simulated driving performance of 25 experienced and 22 inexperienced drivers was tested under the nine different drug conditions in an arterial driving environment during which workload was varied through the drive characteristics as well as through the inclusion of a secondary task. High levels of cannabis generally induced greater impairment than lower levels, while alcohol at the doses used had few effects and did not produce synergistic effects when combined with cannabis. Both cannabis and alcohol were associated with increases in speed and lateral position variability, high dose cannabis was associated with decreased mean speed, increased mean and variability in headways, and longer reaction time, while in contrast alcohol was associated with a slight increase in mean speed. Given the limitations of the study, it is of great interest to further explore the qualitative impairments in driving performance associated with cannabis and alcohol separately and how these impairments may manifest in terms of crash characteristics.

Effects of a beta-blocker on the cardiovascular response to MDMA (Ecstasy).

MDMA (3,4-methylenedioxymethamphetamine, Ecstasy) produces tachycardia and hypertension and is rarely associated with cardiovascular and cerebrovascular complications. In clinical practice, beta-blockers are often withheld in patients with stimulant intoxication because they may increase hypertension and coronary artery vasospasm due to loss of beta(2)-mediated vasodilation and unopposed alpha-receptor activation. However, it is unknown whether beta-blockers affect the cardiovascular response to MDMA. The effects of the non-selective beta-blocker pindolol (20 mg) on the cardiovascular effects of MDMA (1.6 mgkg) were investigated in a double-blind placebo-controlled crossover study in 16 healthy subjects. Pindolol prevented MDMA-induced increases in heart rate. Peak values (mean-SD) for heart rate were 84-13 beatsmin after MDMA vs 69-7 beatsmin after pindolol-MDMA. In contrast, pindolol pretreatment had no effect on increases in mean arterial blood pressure (MAP) after MDMA. Peak MAP values were 115-11 mm Hg after MDMA vs 114-11 mm Hg after pindolol-MDMA. Pindolol did not change adverse effects of MDMA. The results of this study indicate that beta-blockers may prevent increases in heart rate but not hypertensive and adverse effects of MDMA.

Rimonabant-induced Delta9-tetrahydrocannabinol withdrawal in rhesus monkeys discriminative stimulus effects and other withdrawal signs.

Marijuana-dependent individuals report using marijuana to alleviate withdrawal, suggesting that pharmacotherapy of marijuana withdrawal could promote abstinence. To identify potential pharmacotherapies for marijuana withdrawal, this study first characterized rimonabant-induced Delta(9)-tetrahydrocannabinol (Delta(9)-THC) withdrawal in rhesus monkeys by using drug discrimination and directly observable signs. Second, drugs were examined for their capacity to modify cannabinoid withdrawal. Monkeys receiving chronic Delta(9)-THC (1 mgkg12 h s.c.) discriminated the cannabinoid antagonist rimonabant (1 mgkg i.v.) under a fixed ratio schedule of stimulus-shock termination. The discriminative stimulus effects of rimonabant were dose-dependent (ED(50) 0.25 mgkg) and accompanied by head shaking. In the absence of chronic Delta(9)-THC treatment (i.e., in nondependent monkeys), a larger dose (3.2 mgkg) of rimonabant produced head shaking and tachycardia. Temporary discontinuation of Delta(9)-THC treatment resulted in increased responding on the rimonabant lever, head shaking, and activity during the dark cycle. The rimonabant discriminative stimulus was attenuated fully by Delta(9)-THC (at doses larger than mgkg12 h) and the cannabinoid agonist CP 55940 5-(1,1-dimethylheptyl)-2-5-hydroxy-2-(3-hydroxypropyl)cyclohexylphenol, and partially by the cannabinoid agonist WIN 55212-2 (R)-()-2, 3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo1,2,3-de-1,4-benzoxazin-6-yl-1-naphthalenylmethanone mesylate and the alpha(2)-adrenergic agonist clonidine. In contrast, a benzodiazepine (diazepam) and monoamine agonist (cocaine) did not attenuate the rimonabant discriminative stimulus. Head shaking was attenuated by all test compounds. These results show that the discriminative stimulus effects of rimonabant in Delta(9)-THC-treated monkeys are a more pharmacologically selective measure of cannabinoid withdrawal than rimonabant-induced head shaking. These results suggest that cannabinoid and noncannabinoid (alpha(2)-adrenergic) agonists are potentially useful therapeutics for marijuana dependence inasmuch as they attenuate the subjective experience of Delta(9)-THC withdrawal.

Depressive-like effects of the kappa opioid receptor agonist salvinorin A are associated with decreased phasic dopamine release in the nucleus accumbens.

Kappa opioid receptors (KORs) have been implicated in depressive-like states associated with chronic administration of drugs of abuse and stress. Although KOR agonists decrease dopamine in the nucleus accumbens (NAc), KOR modulation of phasic dopamine release in the core and shell subregions of the NAc-which have distinct roles in reward processing-remains poorly understood. Studies were designed to examine whether the time course of effects of KOR activation on phasic dopamine release in the NAc core or shell are similar to effects on motivated behavior. The effect of systemic administration of the KOR agonist salvinorin A (salvA)-at a dose (2.0 mgkg) previously determined to have depressive-like effects-was measured on electrically evoked phasic dopamine release in the NAc core or shell of awake and behaving rats using fast scan cyclic voltammetry. In parallel, the effects of salvA on intracranial self-stimulation (ICSS) and sucrose-reinforced responding were assessed. For comparison, a threshold dose of salvA (0.25 mgkg) was also tested. The active, but not threshold, dose of salvA significantly decreased phasic dopamine release without affecting dopamine reuptake in the NAc core and shell. SalvA increased ICSS thresholds and significantly lowered breakpoint on the progressive ratio schedule, indicating a decrease in motivation. The time course of the KOR-mediated decrease in dopamine in the core was qualitatively similar to the effects on motivated behavior. These data suggest that the effects of KOR activation on motivation are due, in part, to inhibition of phasic dopamine signaling in the NAc core.

Effects of metabotropic glutamate receptor 23 agonism and antagonism on schizophrenia-like cognitive deficits induced by phencyclidine in rats.

Dysregulation of glutamate neurotransmission may play a role in cognitive deficits in schizophrenia. Manipulation of glutamate signaling using drugs acting at metabotropic glutamate receptors has been suggested as a novel approach to treating schizophrenia-related cognitive dysfunction. We examined how the metabotropic glutamate receptor 23 agonist LY379268 and the metabotropic glutamate receptor 23 antagonist LY341495 altered phencyclidine-induced disruptions in performance in the 5-choice serial reaction time task. This test assesses multiple cognitive modalities characteristically impaired in schizophrenia that are disrupted by phencyclidine administration. Acute LY379268 alone did not affect 5-choice serial reaction time task performance, except for nonspecific response suppression at high doses. Acute LY379268 administration exacerbated phencyclidine-induced disruption of attentional performance in this task, while acute LY341495 did not alter 5-choice serial reaction time task performance during phencyclidine exposure. Chronic LY341495 impaired attentional performance in the 5-choice serial reaction time task by itself, but attenuated phencyclidine-induced excessive timeout responding. The mixed effects of metabotropic glutamate receptor 23 agonism and antagonism on cognitive performance under baseline conditions and after disruption with phencyclidine demonstrate that different aspects of cognition may respond differently to a given pharmacological manipulation, indicating that potential antipsychotic or pro-cognitive medications need to be tested for their effects on a range of cognitive modalities. Our findings also suggest that additional mechanisms, besides cortical glutamatergic transmission, may be involved in certain cognitive dysfunctions in schizophrenia.

Discovery and development of endocannabinoid-hydrolyzing enzyme inhibitors.

Fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL) are hydrolytic enzymes which degrade the endogenous cannabinoids (endocannabinoids) N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), respectively. Endocannabinoids are an important class of lipid messenger molecules that are produced on demand in response to elevated intracellular calcium levels. They recognize and activate the cannabinoid CB(1) and CB(2) receptors, the molecular targets for Delta(9)-tetrahydrocannabinol (Delta(9)-THC) in marijuana evoking several beneficial therapeutic effects. However, in vivo the cannabimimetic effects of AEA and 2-AG remain weak owing to their rapid inactivation by FAAH and MGL, respectively. The inactivation of FAAH and MGL by specific enzyme inhibitors increases the levels of AEA and 2-AG, respectively, producing therapeutic effects such as pain relief and depression of anxiety.

Anxiety-like effects of SR141716-precipitated delta9-tetrahydrocannabinol withdrawal in mice in the elevated plus-maze.

Marijuana discontinuation has been recently reported to be anxiogenic in humans, which may predict relapse. Limited animal research has been carried out to model this withdrawal-associated negative affect. The current study sought to investigate the potential anxiety-like effects of cannabinoid withdrawal in mice. Male ICR mice were injected s.c. with delta9-tetrahydrocannabinol (THC) at 10mgkg or vehicle once daily for 10 days. To precipitate withdrawal, the cannabinoid CB1 antagonist SR141716 (0.3, 1.0, or 3.0mgkg) or vehicle was administrated i.p. 4h following the last THC or vehicle treatment. Thirty minutes later, mice were tested on the elevated plus-maze (EPM) for 5min. SR141716 did not significantly change EPM behaviors in vehicle-treated mice. In contrast, SR141716 precipitated a reduction in exploration of the open arms of EPM in mice repeatedly treated with THC vs vehicle. At 3.0mgkg, SR141716 significantly reduced % open arm entries of the total arm entries, % open arm time of total time in arms, and the absolute time spent in open arms. No significant differences in the number of closed or total arm entries were observed, indicating that the behavioral changes were not due to altered motor activity. Collectively, the present results constitute the first evidence that cannabinoid withdrawal produces anxiety-like effects in mice. This animal model may help to identify the mechanisms that contribute to adaptations in the neuronal circuitry of the brain that are expressed as emotional symptoms of cannabinoid withdrawal.

Validation of an enzyme immunoassay for detection and semiquantification of cannabinoids in oral fluid.

Oral fluid (OF) is gaining prominence as an alternative matrix for monitoring drugs of abuse in the workplace, criminal justice, and driving under the influence of drugs programs. It is important to characterize assay performance and limitations of screening techniques for Delta(9)-tetrahydrocannabinol (THC) in OF. We collected OF specimens by use of the Quantisal OF collection device from 13 daily cannabis users after controlled oral cannabinoid administration. All specimens were tested with the Immunalysis SweatOF THC Direct ELISA and confirmed by 2-dimensional GC-MS. The limit of detection was <1 microgL THC equivalent, and the assay demonstrated linearity from 1 to 50 microgL, with semiquantification to 200 microgL. Intraplate imprecision (n 7) ranged from 2.9% to 7.7% CV, and interplate imprecision (n 20) was 3.0%-9.1%. Cross-reactivities at 4 microgL were as follows 11-hydroxy-THC, 198% Delta(8)-tetrahydrocannabinol (Delta(8)-THC), 128% 11-nor-9-carboxy-THC (THCCOOH), 121% THC (target), 98% cannabinol, 87% THCCOOH-glucuronide, 11% THC-glucuronide, 10% and cannabidiol, 2.4%. Of 499 tested OF specimens, 52 confirmed positive (THC 2.0-290 microgL), with 100% diagnostic sensitivity at the proposed Substance Abuse and Mental Health Services Administration screening cutoff of 4 microgL cannabinoids and GC-MS cutoff of 2 microgL THC. Forty-seven specimens screened positive but were not confirmed by 2D-GC-MS, yielding 89.5% diagnostic specificity and 90.6% diagnostic efficiency. Thirty-one of 47 unconfirmed immunoassay positive specimens were from 1 individual and contained >400 ngL THCCOOH, potentially contributing to cross-reactivity. The Immunalysis SweatOF THC Direct ELISA is an effective screening procedure for detecting cannabinoids in OF.

Role of kappa-opioid receptors in the effects of salvinorin A and ketamine on attention in rats.

Disruptions in perception and cognition are characteristic of psychiatric conditions such as schizophrenia. Studies of pharmacological agents that alter perception and cognition in humans might provide a better understanding of the brain substrates of these complex processes. One way to study these states in rodents is with tests that require attention and visual perception for correct performance. We examined the effects of two drugs that cause disruptions in perception and cognition in humans-the kappa-opioid receptor (KOR) agonist salvinorin A (salvA 0.125-4.0 mgkg) and the non-competitive NMDA receptor antagonist ketamine (0.63-20 mgkg)-on behavior in rats using the 5-choice serial reaction time task (5CSRTT), a food-motivated test that quantifies attention. We also compared the binding profiles of salvA and ketamine at KORs and NMDA receptors. SalvA and ketamine produced the same pattern of disruptive effects in the 5CSRTT, characterized by increases in signs often associated with reduced motivation (omission errors) and deficits in processing (elevated latencies to respond correctly). Sessions in which rats were fed before testing suggest that reduced motivation produces a subtly different pattern of behavior. Pretreatment with the KOR antagonist JDTic (10 mgkg) blocked all salvA effects and some ketamine effects. Binding and function studies revealed that ketamine is a full agonist at KORs, although not as potent or selective as salvA. SalvA and ketamine have previously under-appreciated similarities in their behavioral effects and pharmacological profiles. By implication, KORs might be involved in some of the cognitive abnormalities observed in psychiatric disorders such as schizophrenia.

FAAH-- mice display differential tolerance, dependence, and cannabinoid receptor adaptation after delta 9-tetrahydrocannabinol and anandamide administration.

Repeated administration of Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa, induces profound tolerance that correlates with desensitization and downregulation of CB(1) cannabinoid receptors in the CNS. However, the consequences of repeated administration of the endocannabinoid N-arachidonoyl ethanolamine (anandamide, AEA) on cannabinoid receptor regulation are unclear because of its rapid metabolism by fatty acid amide hydrolase (FAAH). FAAH(--) mice dosed subchronically with equi-active maximally effective doses of AEA or THC displayed greater rightward shifts in THC dose-effect curves for antinociception, catalepsy, and hypothermia than in AEA dose-effect curves. Subchronic THC significantly attenuated agonist-stimulated (35)SGTP gamma S binding in brain and spinal cord, and reduced (3)HWIN55,212-2 binding in brain. Interestingly, AEA-treated FAAH(--) mice showed less CB(1) receptor downregulation and desensitization than THC-treated mice. Experiments examining tolerance and cross-tolerance indicated that the behavioral effects of THC, a low efficacy CB(1) receptor agonist, were more sensitive to receptor loss than those of AEA, a higher efficacy agonist, suggesting that the expression of tolerance was more affected by the intrinsic activity of the ligand at testing than during subchronic treatment. In addition, the CB(1) receptor antagonist, rimonabant, precipitated a markedly reduced magnitude of withdrawal in FAAH(--) mice treated subchronically with AEA compared with mice treated repeatedly with THC. The findings that repeated AEA administration produces lesser adaptive changes at the CB(1) receptor and has reduced dependence liability compared with THC suggest that pharmacotherapies targeting endocannabinoid catabolic enzymes are less likely to promote tolerance and dependence than direct acting CB(1) receptor agonists.

Enhancement in sample collection for the detection of MDMA using a novel planar SPME (PSPME) device coupled to ion mobility spectrometry (IMS).

Trace detection of illicit drugs challenges the scientific community to develop improved sensitivity and selectivity in sampling and detection techniques. Ion mobility spectrometry (IMS) is one of the prominent trace detectors for illicit drugs and explosives, mostly due to its portability, high sensitivity and fast analysis. Current sampling methods for IMS rely on wiping suspected surfaces or withdrawing air through filters to collect particulates. These methods depend greatly on the particulates being bound onto surfaces or having sufficient vapour pressure to be airborne. Many of these compounds are not readily available in the headspace due to their low vapour pressure. This research presents a novel SPME device for enhanced air sampling and shows the use of optimized IMS by genetic algorithms to target volatile markers andor odour signatures of illicit substances. The sampling method was based on unique static samplers, planar substrates coated with sol-gel polydimethyl siloxane (PDMS) nanoparticles, also known as planar solid-phase microextraction (PSPME). Due to its surface chemistry, high surface area and capacity, PSPME provides significant increases in sensitivity over conventional fibre SPME. The results show a 50-400 times increase in the detection capacity for piperonal, the odour signature of 3,4-methylenedioxymethamphetamine (MDMA). The PSPME-IMS technique was able to detect 600 ng of piperonal in a 30 s extraction from a quart-sized can containing 5 MDMA tablets, while detection using fibre SPME-IMS was not attainable. In a blind study of six cases suspected to contain varying amounts of MDMA in the tablets, PSPME-IMS successfully detected five positive cases and also produced no false positives or false negatives. One positive case had minimal amounts of MDMA resulting in a false negative response for fibre SPME-IMS.

Quantitation of tetrahydrocannabinol in hair using immunoassay and liquid chromatography with tandem mass spectrometric detection.

A quantitative analytical procedure for the determination of Delta(9)-tetrahydrocannabinol (THC) in hair has been developed and validated using liquid chromatography with tandem mass spectral detection (LC-MSMS). Specimens that were determined as containing cannabinoids following immunoassay testing were quantified using solid-phase extraction followed by liquid chromatographic separation and tandem mass spectral detection in positive electrospray ionization mode. For confirmation, two transitions were monitored and one ratio determined. Samples being reported as positive were required to have both transitions present, the ratio of quantifying transition to qualifying transition being within 20% of that determined from known calibration standards. The limit of quantitation and the limit of detection was 10 pgmg. The percentage recovery of the THC from hair at 20 pgmg was 56% and a matrix effect of the hair showed an ion suppression percentage of -51%. The immunochemical screening method was performed following a rapid aqueous extraction, requiring only 10 mg of hair the confirmatory procedure required 20 mg of hair. The methods were applied to proficiency specimens from the Society of Hair Testing, which had been received in August 2008.

Temporal indication of cannabis use by means of THC glucuronide determination.

According to the regulations of the World Anti-Doping Agency (WADA), the use of cannabinoids is forbidden in competition. In doping controls, the detection of cannabinoid misuse is based on the analysis of the non-psychoactive metabolite 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (carboxy-THC). The determination of values greater than 15 ngmL in urine represents an adverse analytical finding however, no accurate prediction of the time of application is possible as the half-life of carboxy-THC ranges between three and four days. Consequently the detection of carboxy-THC in doping control urine samples collected in competition might also result from cannabis use in out-of-competition periods. The analysis of the glucuronide of the pharmacologically active delta 9-tetrahydrocannabinol (THC-gluc) may represent a complementary indicator for the detection of cannabis misuse in competition.An assay for the determination of THC-gluc in human urine was established. The sample preparation consisted of liquid-liquid extraction of urine specimens, and extracts were analysed by liquid chromatographytandem mass spectrometry (LC-MSMS). Authentic doping-control urine samples as well as specimens obtained from a controlled smoking study were analysed and assay characteristics such as specificity, detection limit (0.1 ngmL), precision (>90%), recovery ( approximately 80%), and extraction efficiency (90%) were determined.

Is anti-doping analysis so far from clinical, legal or forensic targets The added value of close relationships between related disciplines.

There are many areas of common interest between anti-doping laboratories and those working in the clinical, legal and forensic fields. In addition to methodological similarities, there are aspects of the findings in sport drug testing that overlap with other fields in such a way that sport drug testing and clinical, legal or forensic work may benefit from mutual interaction. Three recent examples are presented from the authors experience. Case report 1 concerns the clinical relevance of hCG findings in sport drug testing as potential indicators of the presence of a (testicular) tumour in athletes. Case report 2 refers to difficulties that accredited laboratories can encounter due to differences between national legal systems and the administrative regulation systems of sport authorities. The example involves a network of blood collection for further autologous transfusion. Case report 3 relates to additional forensic-type investigations needed to interpret a situation where intoxication of a whole delegation was responsible for apparent doping cases. Clinical, legal and forensic fields must recognize the added value that some results and developments coming from anti-doping laboratories may have. At the same time anti-doping analysts should be aware of new issues, methodologies and problems appearing in related fields.

High frequency plant regeneration from leaf derived callus of high Δ9-tetrahydrocannabinol yielding Cannabis sativa L.

An efficient in vitro propagation protocol for rapidly producing Cannabis sativa plantlets from young leaf tissue was developed. Using gas chromatography-flame ionization detection (GC-FID), high THC yielding elite female clone of a drug-type CANNABIS variety (MX) was screened and its vegetatively propagated clones were used for micropropagation. Calli were induced from leaf explant on Murashige and Skoog medium supplemented with different concentrations (0.5, 1.0, 1.5, and 2.0 µM) of indole- 3-acetic acid (IAA), indole- 3- butyric acid (IBA), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy-acetic acid (2,4-D) in combination with 1.0 µM of thidiazuron (TDZ) for the production of callus. The optimum callus growth and maintenance was in 0.5 µM NAA plus 1.0 µM TDZ. The two-month-old calli were subcultured to MS media containing different concentrations of cytokinins (BAP, KN, TDZ). The rate of shoot induction and proliferation was highest in 0.5 µM TDZ. Of the various auxins (IAA, IBA, and NAA) tested, regenerated shoots rooted best on half strength MS medium (12 - MS) supplemented with 2.5 µM IBA. The rooted plantlets were successfully established in soil and grown to maturity with no gross variations in morphology and cannabinoids content at a survival rate of 95 % in the indoor growroom.

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

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

Selective potentiation of the metabotropic glutamate receptor subtype 2 blocks phencyclidine-induced hyperlocomotion and brain activation.

Previous preclinical and clinical studies have demonstrated the efficacy of group II metabotropic glutamate receptor (mGluR) agonists as potential antipsychotics. Recent studies utilizing mGluR2-, mGluR3-, and double knockout mice support that the antipsychotic effects of those compounds are mediated by mGluR2. Indeed, biphenyl indanone-A (BINA), an allosteric potentiator of mGluR2, is effective in experimental models of psychosis, blocking phencyclidine (PCP)-induced hyperlocomotion and prepulse inhibition deficits in mice. In this study, we administered the NMDA receptor antagonist PCP (5.6 mgkg i.p.) to rats, an established animal model predictive of schizophrenia. Here, we show that BINA (32 mgkg i.p.) attenuated PCP-induced locomotor activity in rats. Using behaviorally relevant doses of BINA and PCP, we performed pharmacological magnetic resonance imaging (phMRI) to assess the specific brain regions that underlie the psychotomimetic effects of PCP, and examined how BINA modulated the PCP-induced functional changes in vivo. In anesthetized rats, acute administration of PCP produced robust, sustained blood oxygenation level-dependent (BOLD) activation in specific cortical, limbic, thalamic, and striatal regions. Pretreatment with BINA suppressed the amplitude of the BOLD response to PCP in the prefrontal cortex, caudaute-putamen, nucleus accumbens, and mediodorsal thalamus. Our results show key brain structures underlying PCP-induced behaviors in a preclinical model of schizophrenia, and, importantly, its reversal by potentiation of mGluR2 by BINA, revealing specific brain regions functionally involved in its pharmacological action. Finally, our findings bolster the growing body of evidence that mGluR2 is a viable target for the treatment of schizophrenia.

Cannabis constituents modulate δ9-tetrahydrocannabinol-induced hyperphagia in rats.

The hyperphagic effect of Delta9-tetrahydrocannabinol (Delta9THC) in humans and rodents is well known. However, no studies have investigated the importance of Delta9THC composition and any influence other non-Delta9THC cannabinoids present in Cannabis sativa may have. We therefore compared the effects of purified Delta9THC, synthetic Delta9THC (dronabinol), and Delta9THC botanical drug substance (Delta9THC-BDS), a Delta9THC-rich standardized extract comparable in composition to recreationally used cannabis. Adult male rats were orally dosed with purified Delta9THC, synthetic Delta9THC, or Delta9THC-BDS, matched for Delta9THC content (0.34-2.68 mgkg). Prior to dosing, subjects were satiated, and food intake was recorded following Delta9THC administration. Data were then analyzed in terms of hourly intake and meal patterns. All three Delta9THC substances tested induced significant hyperphagic effects at doses >or0.67 mgkg. These effects included increased intake during hour one, a shorter latency to onset of feeding and a greater duration and consumption in the first meal. However, while some differences in vehicle control intakes were observed, there were significant, albeit subtle, differences in pattern of effects between the purified Delta9THC and Delta9THC-BDS. All Delta9THC compounds displayed classical Delta9THC effects on feeding, significantly increasing shortterm intake whilst decreasing latency to the first meal. We propose that the subtle adjustment to the meal patterns seen between the purified Delta9THC and Delta9THC-BDS are due to non-Delta9THC cannabinoids present in Delta9THC-BDS. These compounds and other non-cannabinoids have an emerging and diverse pharmacology and can modulate Delta9THC-induced hyperphagia, making them worth further investigation for their therapeutic potential.

Lack of positive allosteric modulation of mutated alpha(1)S267I glycine receptors by cannabinoids.

Loss of inhibitory synaptic transmission within the dorsal horn of the spinal cord plays a key role in the development of chronic pain following inflammation or nerve injury. Inhibitory postsynaptic transmission in the adult spinal cord involves mainly glycine. Ajulemic acid and HU210 are non-psychotropic, synthetic cannabinoids. Cannabidiol is a non-psychotropic plant constituent of cannabis sativa. There are hints that non-cannabinoid receptor mechanisms of these cannabinoids might be mediated via glycine receptors. In this study, we investigated the impact of the amino acid residue serine at position 267 on the glycine-modulatory effects of ajulemic acid, cannabidiol and HU210. Mutated alpha(1)S267I glycine receptors transiently expressed in HEK293 cells were studied by utilising the whole-cell clamp technique. The mutation of the alpha(1) subunit TM2 serine residue to isoleucine abolished the co-activation and the direct activation of the glycine receptor by the investigated cannabinoids. The nature of the TM2 (267) residue of the glycine alpha(1) subunit is crucial for the glycine-modulatory effect of ajulemic acid, cannabidiol and HU210. An investigation of the impact of such mutations on the in vivo interaction of cannabinoids with glycine receptors should permit a better understanding of the molecular determinants of action of cannabinoids.

Qualitative GC-MS assessment of TCP and TAMORF elimination in rats.

Nerve agents are highly toxic organophosphorus (OP) compounds. They inhibit acetylcholinesterase (AChE), an enzyme that hydrolyses acetycholine (ACh) in the nervous system. Pathophysiological changes caused by OP poisonings are primarily the consequence of surplus ACh on cholinergic receptors and in the central nervous system. Standard treatment of OP poisoning includes combined administration of carbamates, atropine, oximes and anticonvulsants. In order to improve therapy, new compounds have been synthesised and tested. Tenocyclidine (TCP) and its adamantane derivative 1-2-(2-thienyl)-2-adamantyl morpholine (TAMORF) have shown interesting properties against soman poisoning. In this study, we developed a qualitative GC-MS method to measure elimination of TCP and TAMORF through rat urine in order to learn more about the mechanisms through which TCP protects an organism from OP poisoning and to determine the duration of this protective effect. GC-MS showed that six hours after treatment with TCP, rat urine contained only its metabolite 1-thienylcyclohexene, while urine of rats treated with TAMORF contained both TAMORF and its metabolites.

Toxicological methods for tracing drug abuse chromatographic, spectroscopic and biological characterisation of ecstasy derivatives.

Analysis often reveals variability in the composition of ecstasy pills from pure 3,4-methylenedioxymethamphetamine (MDMA) to mixtures of MDMA derivatives, amphetamine, and other unidentified substances. For a comprehensive toxicological analysis one needs to know all steps to MDMA synthesis which may originate impurities. The aim of this study was to synthesise and determine the chemical-physical and in vitro biological properties of a series of MDMA derivatives.3,4-methylendioxyphenyl-2-nitropropene (MDNP) was obtained by condensation of piperonal with an excess of nitroethane in the presence of ammonium acetate. MDNP was then reduced to methylenedioxyamphetamine (MDA) by LiAlH3. All compounds were analysed using HPLC and spectroscopic technique Raman, nuclear magnetic resonance (NMR), or infrared (IR) at all the steps of synthesis. In addition, we assessed the biological potentials of these compounds by measuring in vitro their (i) blood cellwhole blood partition coefficient, (ii) binding to plasmatic proteins (Fbp), and (iii) membrane adsorption. Chemical structure was determined with antibody fluorescence polarisation immunoassay (FPIA). This study showed the presence of solid impurities, particularly of a neurotoxic compound of Al3 in the final products. FPIA identified the aminoethane group close to the substituted benzene ring, but did not detect the two major precursors of MDMA MDNP and piperonal. Raman spectroscopy is an attractive alternative technique to characterise ecstasy pills and it can identify stereoisomeric forms such as cis-MDNP and trans-MDNP, which exhibit signals at 1650 cm-1 and 1300 cm-1, respectively.

The 5-HT 2A serotonin receptor enhances cell viability, affects cell cycle progression and activates MEK-ERK12 and JAK2-STAT3 signalling pathways in human choriocarcinoma cell lines.

Previous results from our group have demonstrated the expression of the 5-HT(2A) receptor and a mitogenic effect of serotonin in human trophoblast. The objectives of the present study were to investigate the role of the 5-HT(2A) receptor in trophoblast cells and to determine the signalling pathways activated by this receptor. We investigated the effect of (-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI), a selective 5-HT(2A) agonist, on cell cycle progression and cell viability in BeWo and JEG-3 cells. We also investigated, by co-immunoprecipitation and western blot analysis, the involvement of the MEK-ERK12 and JAK2-STAT3 signalling pathways following activation of the placental 5-HT(2A) receptor. Our results showed a concentration-dependent increase of cell viability by DOI, which was reversed by ketanserin, a selective 5-HT(2A) receptor antagonist. Furthermore, activation of the 5-HT(2A) receptor by DOI increased cell entry into the G2M and S phase (DNA synthesis) in BeWo and JEG-3 cells, respectively. In addition, stimulation of BeWo and JEG-3 cells by DOI activated both the MEK-ERK12 and the JAK2-STAT3 signalling pathways. This study demonstrated that the 5-HT(2A) receptor increases cell viability and affects cell cycle progression in human trophoblast cell lines as well as activates the MEK-ERK12 and JAK2-STAT3 intracellular signalling pathways, which are related to survival, differentiation, migration and invasion. These findings indicate that serotonin through the activation of the 5-HT(2A) receptor is a key regulator of placentation and may play a role in the pathophysiology of certain pregnancy disorders associated with alterations in placental development, such as preeclampsia, gestational diabetes and preterm birth.

Factor structure of the Chinese version of the University of Rhode Island change assessment in Taiwanese adolescents who abuse MDMA or methamphetamine.

The University of Rhode Island Change Assessment (URICA) is a widely used tool for measuring subjects readiness to change substance-using behaviors. The aim of this study was to examine the factor structure of the Chinese version of the URICA (C-URICA) in Taiwanese adolescents who have used methylenedioxymethamphetamine (MDMA) or methamphetamine (MAMP). Pre-test and post-test data from the C-URICA from 92 adolescents in a juvenile abstinence center who had used MDMA or MAMP were analyzed. We used confirmatory factor analysis (CFA) to examine the adequacy of the factor structure of the C-URICA. CFA indicated that a three-factor structure (pre-contemplation, contemplation action, maintenance) had a better goodness of fit than four-factor (pre-contemplation, contemplation, action, maintenance), and one-factor structures. The results indicated that the URICA may have different factor structures when used in the population different from the original adult population with alcohol drinking.

Adolescent drug dealing and raceethnicity a population-based study of the differential impact of substance use on involvement in drug trade.

Among adolescents, peers are an important source of drug procurement. However, little is known about factors associated with youths involvement in drug trade. The aim of the study is to identify substance use behaviors and contextual factors related to drug dealing among Black and White adolescents. The sample consisted of 13,706 White and Black youths who completed the National Survey on Drug Use and Health. Separate backward logistic regression was used to identify substance use behaviors and contextual factors associated with drug dealing among Black and White youths. Among White youths, drug dealing was associated with use of marijuana, hallucinogens, cocaine, prescription drug misuse, availability of cocaine, and socioeconomic status (SES). Among Black youths, marijuana use and availability of crack and marijuana were associated with drug dealing. For White youths, substance use seems to be more relevant to drug dealing. Consequently, preventing and treating substance abuse may reduce involvement in the illegal distribution of drugs among White youths. More research is needed to identify risk and protective factors for drug dealing among Black adolescents.

Intramolecular transacetylation in salvinorins D and E.

Extraction of fresh Salvia divinorum leaves afforded salvinorins E and D as potential biosynthesis precursors of salvinorin A, a major metabolite and a potent hallucinogen. Attempts at HPLC purification of salvinorin E (2) with acetonitrile as a solvent revealed an equilibrium with its regioisomer, salvinorin D (3), in a 35 ratio. The presence of both compounds was readily observed in the (1)H NMR spectrum. This spontaneous formation of the mixture of isomers occurs via a dynamic intramolecular transacetylation process.

Regulation of subthalamic neuron activity by endocannabinoids.

High levels of anandamide are located in the basal ganglia. The subthalamic nucleus (STN) is considered to be an important modulator of basal ganglia output. The present study aims at characterizing the modulation of the electrical activity of STN neurons by exogenous anandamide or endocannabinoids. Single-unit extracellular recordings in anesthetized rats and patch-clamp techniques in rat brain slices containing the STN were performed. Immunohistochemical assays were used. In vivo, anandamide administration produced two opposite effects (inhibition or stimulation) on STN neuron firing rates, depending of the precise location of the neuron within the nucleus. These effects were enhanced by prior inhibition of fatty acid amide hydrolase with URB597, but not by the inhibitor of carrier-mediated anandamide transport AM404. Rimonabant, a specific CB(1) receptor antagonist, also produced inhibition or stimulation of STN neuron activity when administered alone or after anandamide. These effects seem to be mediated by indirect mechanisms since (1) STN neuron activity is not modified by the cannabinoid agonist Delta(9)-tetrahydrocannabinol (Delta(9)-THC) in vitro (2) no depolarization-induced suppression of inhibition phenomena were observed and (3) CB(1) receptor immunolabeling was not detected in the STN, but was abundant in areas which project efferents to this nucleus. Moreover, chemical lesion of the globus pallidus abolished the stimulatory effect of anandamide and microinfusion of anandamide into the prefrontal cortex led to inhibition of STN neuron activity. The present results show that endocannabinoids exert a tonic control on STN activity via receptors located outside the nucleus. These findings may contribute to enhance our understanding of the role of the endocannabinoid system in motor control.

Functional characteristics of serotonin 5-HT2A and 5-HT2C receptors in the brain and the expression of the 5-HT2A and 5-HT2C receptor genes in aggressive and non-aggressive rats.

The functional activity of serotonin 5-HT(2A) and 5-HT(2C) receptors and the expression of the genes encoding them were studied in Norway rats bred for 60 generations for the presence and absence of high levels of stress-evoked aggression to humans. There were no significant differences in the levels of 5-HT(2A) receptor mRNA in the midbrain, frontal cortex, and hippocampus and the extents of head twitching evoked by the 5-HT(2A) agonist DOI in rats with and without genetically determined high levels of aggression. Administration of the selective 5-HT(2C) agonist MK-212 weakened reflex startle in response to an acoustic signal (the acoustic startle response) in non-aggressive animals but had no significant effects on the response in aggressive animals. Increases in the level of 5-HT(2C) receptor mRNA were seen in the frontal cortex and hippocampus in non-aggressive rats as compared with aggressive animals. Increases in the expression of the 5-HT(2C) receptor gene and the functional state of 5-HT(2C) receptors were seen in the brains of non-aggressive rats, without any changes in the 5-HT(2A) receptor mRNA level or receptor sensitivity this is evidence for the involvement of 5-HT(2C) receptors in the mechanisms inhibiting fear-evoked aggressive behavior.

Drug seeking in response to a priming injection of MDMA in rats relationship to initial sensitivity to self-administered MDMA and dorsal striatal dopamine.

In laboratory animals, exposure to priming injections of 3,4-methylenedioxymethamphetamine (MDMA) produced drug seeking following extinction of MDMA self-administration. This study aimed to evaluate whether the magnitude of drug seeking was related to latency to acquisition of MDMA self-administration and increases in striatal dopamine, as measured by in-vivo microdialysis. Rats were given daily access to MDMA self-administration until they earned a total of 240 infusions (total intake of 165 mgkg MDMA). Twelve of the 20 rats acquired self-administration within the temporal limits of the study and the latency to meet the criterion ranged from 9 d to 37 d. An experimenter-administered injection of MDMA (10.0 mgkg i.p.) produced drug seeking in these rats, and the number of responses was significantly higher than responses produced by rats that failed to meet the criterion or by yoked control rats that received the drug passively. For rats that met the criterion, drug seeking was negatively correlated with the number of days to self-administer the criterion number of MDMA infusions and positively correlated with MDMA-produced dopamine in the dorsal striatum. Importantly, MDMA-produced dopamine overflow was greater for the rats that met the criterion. These findings suggest that drug seeking is influenced by initial sensitivity to the reinforcing effects of MDMA and to drug-produced increases in striatal dopamine.

Simultaneous determination of eight sympathomimetic amines in urine by gas chromatographymass spectrometry.

A GC method was developed for the identification and quantitation of eight sympathomimetic amines in urine, i.e., amphetamine, methamphetamine, mephentermine, ephedrine, pseudoephedrine, methylenedioxyamphetamine, methylenedioxymethamphetamine, and methylenedioxyethylamphetamine. Methoxyphenamine was used as the internal standard (IS). The assay is rapid, sensitive, and simple to perform. It involves a liquid-liquid extraction procedure with simultaneous in-solution derivatization of the organic layer with pentafluorobenzoyl chloride (PFB-CI), followed by GCMS analysis. These derivatives and the IS were extracted from 1 mL alkaline urine into hexane before derivatization with PFB-CI. The organic layer was then removed and evaporated to dryness before dissolution with hexane for GCMS analysis. Calibration curves for each analyte showed linearity in the range of 25-5000 ngmL (r2 > or 0.997). Recoveries ranged from 88 to 99%, with the precision of recoveries typically < or 5%. The LOD values ranged from 7 to 28 ngmL, and the LOQ values ranged from 23 to 94 ngmL. At least four ions were available for each analyte for confirmation of identity by MS.

Preparation and characterization of inclusion complexes of a hemisuccinate ester prodrug of delta9-tetrahydrocannabinol with modified beta-cyclodextrins.

Delta(9)-Tetrahydrocannabinol hemisuccinate (THC-HS), an ester prodrug of Delta(9)-tetrahydrocannabinol (THC) has been investigated for its potential to form inclusion complexes with modified synthetic beta-cyclodextrins (CDs). Phase solubility studies were performed to determine the stoichiometric ratio of complexation of THC-HS with random methylated beta-cyclodextrin (RAMEB) and 2-hydroxypropyl beta-cyclodextrin (HPBCD). THC-HSRAMEB and THC-HSHPBCD solid systems were prepared by lyophilization and the lyophilized complexes were characterized by Fourier transform infrared (FT-IR) spectroscopy, proton nuclear magnetic spectroscopy, and molecular modeling techniques. The formation of inclusion complexes of THC-HSRAMEB and THC-HSHPBCD was demonstrated by an A(L) type curve with the slopes less than unity by the phase solubility method. The association constants for THC-HSRAMEB and THC-HSHPBCD were found to be 562.48 and 238.83 M(-1), respectively. The stoichiometry of both of the complexes was found to be 11 as determined from the Jobs plot. This was confirmed by (1)H NMR and FT-IR techniques. The results obtained from the molecular modeling studies were in accordance with the data obtained from nuclear magnetic resonance and FT-IR. The docking studies revealed the most probable mode of binding of THC-HS with RAMEB in which the alkyl chain was submerged in the hydrophobic pocket of the CD molecule and hydrogen bonding interactions were observed between the hemisuccinate ester side chain of THC-HS and the rim hydroxy groups of RAMEB. The solubility of THC-HS was significantly higher in RAMEB compared to HPBCD. Solid dispersions of THC-HS with CDs will be further utilized to develop oral formulations of THC-HS with enhanced bioavailability.

Beta-keto amphetamines studies on the metabolism of the designer drug mephedrone and toxicological detection of mephedrone, butylone, and methylone in urine using gas chromatography-mass spectrometry.

In recent years, a new class of designer drugs has appeared on the drugs of abuse market in many countries, namely, the so-called beta-keto (bk) designer drugs such as mephedrone (bk-4-methylmethamphetamine), butylone (bk-MBDB), and methylone (bk-MDMA). The aim of the present study was to identify the metabolites of mephedrone in rat and human urine using GC-MS techniques and to include mephedrone, butylone, and methylone within the authors systematic toxicological analysis (STA) procedure. Six phase I metabolites of mephedrone were detected in rat urine and seven in human urine suggesting the following metabolic steps N-demethylation to the primary amine, reduction of the keto moiety to the respective alcohol, and oxidation of the tolyl moiety to the corresponding alcohols and carboxylic acid. The STA procedure allowed the detection of mephedrone, butylone, methylone, and their metabolites in urine of rats treated with doses corresponding to those reported for abuse of amphetamines. Besides macro-based data evaluation, an automated evaluation using the automated mass spectral deconvolution and identification system was performed. Mephedrone and butylone could be detected also in human urine samples submitted for drug testing. Assuming similar kinetics in humans, the described STA procedure should be suitable for proof of an intake of the bk-designer drugs in human urine.

Antidepressant-like effect of delta9-tetrahydrocannabinol and other cannabinoids isolated from Cannabis sativa L.

The antidepressant action of cannabis as well as the interaction between antidepressants and the endocannabinoid system has been reported. This study was conducted to assess the antidepressant-like activity of Delta(9)-THC and other cannabinoids. Cannabinoids were initially evaluated in the mouse tetrad assay to determine doses that do not induce hypothermia or catalepsy. The automated mouse forced swim (FST) and tail suspension (TST) tests were used to determine antidepressant action. At doses lacking hypothermic and cataleptic effects (1.25, 2.5, and 5 mgkg, i.p.), both Delta(9)-THC and Delta(8)-THC showed a U-shaped dose response with only Delta(9)-THC showing significant antidepressant-like effects at 2.5 mgkg (p<0.05) in the FST. The cannabinoids cannabigerol (CBG) and cannabinol (CBN) did not produce antidepressant-like actions up to 80 mgkg in the mouse FST, while cannabichromene (CBC) and cannabidiol (CBD) exhibited significant effect at 20 and 200mgkg, respectively (p<0.01). The antidepressant-like action of Delta(9)-THC and CBC was further confirmed in the TST. Delta(9)-THC exhibited the same U-shaped dose response with significant antidepressant-like action at 2.5 mgkg (p<0.05) while CBC resulted in a significant dose-dependent decrease in immobility at 40 and 80 mgkg doses (p<0.01). Results of this study show that Delta(9)-THC and other cannabinoids exert antidepressant-like actions, and thus may contribute to the overall mood-elevating properties of cannabis.

Impairment due to cannabis and ethanol clinical signs and additive effects.

Studies have shown that the impairing effects of Delta-9-tetrahydrocannabinol (THC) are dose-related. Cannabis intake increases the risk of traffic accidents. The purpose of this study was to see how different clinical tests and observations were related to blood THC concentrations and to determine whether the combined influence of THC and ethanol was different from either drug alone. A retrospective cross-sectional forensic database study. Drivers apprehended by the police suspected of driving under the influence of alcohol and other drugs. We investigated 589 cases positive for THC only. In addition, 894 cases with THC and ethanol were included. A comparison was made with 3480 drivers with only ethanol in their blood and 79 drivers who tested negative. Data were analytical results of blood samples and the 27 clinical tests and observations included in the Norwegian clinical test for impairment (CTI). No relationship was found between blood THC concentration and most of the CTI tests. Blood THC concentration was, however, related to conjunctival injection, pupil dilation and reaction to light and to the overall risk of being judged impaired. When THC and ethanol were detected together the risk of being judged impaired was increased markedly. This study demonstrates that cannabis impairs driving ability in a concentration-related manner. The effect is smaller than for ethanol. The effect of ethanol and cannabis taken simultaneously is additive. Conjunctival injection, dilated pupils and slow pupil reaction are among the few signs to reveal THC influence.

Effects of daily chlorpromazine administration on behavioural and physiological parameters in the rat.

Chlorpromazine is a classical neuroleptic drug which produces both therapeutic effects as well as unwanted side effects in human such as sedation, autonomic, endocrine and neurological effects. It is thought that blockade of dopamine D-2 receptors caused by chlorpromazine induces these untoward side effects. Pre-clinical studies on catalepsy has been proposed as an animal model for neuroleptic induced extrapyramidal side effects. The drug also blocks certain stereotypic behaviours in animals induced by dopamine agonists such as apomorphine and amphetamine. These stereotypic behaviours are circling, chewing, rearing, grooming and hyperactivity. Daily administration of chlorpromazine (1, 3 and 10 mgkg, i.p) to rats for 21 days induced catalepsy, tolerance to catalepsy and locomotor sensitization following PCP (10 mgkg, i.p) challenge. These results suggest that daily chlorpromazine treatment induced DANMDA-receptor sensitization to total locomotor activity following PCP challenge. Furthermore, there were no changes in other behavioural parameters assessed. Surprisingly daily chlorpromazine administration in rats also produced no changes in other physiological parameters assessed (body weight, food and water intake).

Rapid development of tolerance to sub-anaesthetic dose of ketamine an oculomotor study in macaque monkeys.

Ketamine, a non-competitive N-methyl-D -aspartic acid antagonist, has been widely used for anaesthetic purposes. At sub-anaesthetic dosage, it induces a dissociative state similar to schizophrenia. The discovery of this effect on dissociative state has led to its use as a pharmacological model of schizophrenia and has also been responsible for its illegal use as a recreational drug. Whereas the former has provided invaluable information, the latter has demonstrated that repeated administration of ketamine induces tolerance. Surprisingly, a review of the relevant literature shows that tolerance to sub-anaesthetic doses of ketamine is largely unreported in neuropharmacological studies. In order to investigate this caveat, we have performed a post hoc analysis of the behavioural effects induced by repeated injections of sub-anaesthetic doses of ketamine observed in five consecutive monkeys performing two oculomotor tasks. Ketamine effects were quantified by the animals performances and latencies in a prosaccade and an antisaccade task, two oculomotor paradigms that are impaired after ketamine administration. Although the result of the initial injections confirmed a clear behavioural effect of ketamine injections in all monkeys, subsequent administrations showed that a tolerance eventually appeared in all monkeys. The profile of this tolerance exhibited however a large inter-subject variability. Psychopharmacological experiments using ketamine as a pharmacological model of psychosis should therefore consider the kinetic and time course of these effects in each individuals and take them into account in the design of experimental protocols.

Reinstatement of extinguished amphetamine self-administration by 3,4-methylenedioxymethamphetamine (MDMA) and its enantiomers in rhesus monkeys.

The effectiveness of MDMA and its enantiomers to reinstate responding previously maintained by drug self-administration has not been thoroughly investigated. The present study was designed to compare the reinstatement effects of amphetamine, the piperazine-analog BZP, SR(-)-MDMA, S()-MDMA, R(-)-MDMA, and fenfluramine on behavior maintained under a second-order schedule of intravenous amphetamine self-administration in rhesus monkeys (n4). Following saline substitution and extinction, a range of doses of amphetamine, BZP, SR(-)-MDMA, S()-MDMA, R(-)-MDMA, and fenfluramine were administered i.v. as non-contingent priming injections in order to characterize their effectiveness to reinstate responding previously maintained by amphetamine self-administration. Priming injections of amphetamine, BZP, SR(-)-MDMA, and S()-MDMA induced significant reinstatement effects. In contrast, neither R(-)-MDMA nor fenfluramine effectively reinstated behavior. Pretreatment with the selective serotonin transporter inhibitor, fluoxetine, attenuated the reinstatement effects of SR(-)-MDMA, S()-MDMA, and BZP but had no significant effect on amphetamine-primed reinstatement. Given the profile of neurochemical effects published previously, these findings suggest that the reinstatement effects of MDMA are mediated primarily by dopamine release however, the attenuation of MDMA-induced reinstatement by fluoxetine supports previous research demonstrating the complex behavioral pharmacology of MDMA-like drugs and that the reinstatement effects of MDMA are at least partially mediated by serotonergic mechanisms.

A double-blind, randomized, placebo-controlled, parallel-group study of Sativex, in subjects with symptoms of spasticity due to multiple sclerosis.

Muscle spasticity is common in multiple sclerosis (MS), occurring in more than 60% of patients. To compare Sativex with placebo in relieving symptoms of spasticity due to MS. A 15-week, multicenter, double-blind, randomized, placebo-controlled, parallel-group study in 337 subjects with MS spasticity not fully relieved with current anti-spasticity therapy. The primary endpoint was a spasticity 0-10 numeric rating scale (NRS). Intention-to-treat (ITT) analysis showed a non-significant improvement in NRS score, in favor of Sativex. The per protocol (PP) population (79% of subjects) change in NRS score and responder analyses (> or 30% improvement from baseline) were both significantly superior for Sativex, compared with placebo -1.3 versus -0.8 points (change from baseline, p0.035) and 36% versus 24% (responders, p0.040). These were supported by the time to response (ITT p0.068 PP p0.025) analyses, carer global impression of change assessment (p0.013) and timed 10-meter walk (p0.042). Among the subjects who achieved a > or 30% response in spasticity with Sativex, 98, 94 and 73% reported improvements of 10, 20 and 30%, respectively, at least once during the first 4 weeks of treatment. Sativex was generally well tolerated, with most adverse events reported being mild-to-moderate in severity. The 0-10 NRS and responder PP analyses demonstrated that Sativex treatment resulted in a significant reduction in treatment-resistant spasticity, in subjects with advanced MS and severe spasticity. The response observed within the first 4 weeks of treatment appears to be a useful aid to prediction of respondernon-responder status.

Effect of the CB1 cannabinoid agonist WIN 55212-2 on the acquisition and reinstatement of MDMA-induced conditioned place preference in mice.

Numerous reports indicate that MDMA users consume other psychoactive drugs, among which cannabis is one of the most common. The aim of the present study was to evaluate, using the conditioned place preference, the effect of the cannabinoid agonist WIN 55,212-2 on the rewarding effects of MDMA in mice. In the first experiment adolescent mice were initially conditioned with 1.25, 2.5 or 5 mgkg of MDMA or 0.1 or 0.5 mgkg of WIN and subsequently with both drugs. Reinstatement of the extinguished preference by priming doses was performed in the groups that showed CPP. In the second experiment, animals were conditioned with 2.5 or 5 mgkg of MDMA and, after extinction, reinstatement of the preference was induced by 0.5 or 0.1 mgkg of WIN. A low dose of WIN 55212-2 (0.1 mgkg) increased the rewarding effects of low doses of MDMA (1.25 mgkg), although a decrease in the preference induced by MDMA (5 and 2.5 mgkg) was observed when the dose of WIN 55212-2 was raised (0.5 mgkg). The CB1 antagonist SR 141716 also increased the rewarding effects of the lowest MDMA dose and did not block the effects of WIN. Animals treated with the highest WIN dose plus a non-neurotoxic dose of MDMA exhibited decreases of striatal DA and serotonin in the cortex. On the other hand, WIN 55212-2-induced CPP was reinstated by priming injections of MDMA, although WIN did not reinstate the MDMA-induced CPP. These results confirm that the cannabinoid system plays a role in the rewarding effects of MDMA and highlights the risks that sporadic drug use can pose in terms of relapse to dependence. Finally, the potential neuroprotective action of cannabinoids is not supported by our data on the contrary, they are evidence of the potential neurotoxic effect of said drugs when administered with MDMA.

Validation of a two-dimensional gas chromatography mass spectrometry method for the simultaneous quantification of cannabidiol, Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC, and 11-nor-9-carboxy-THC in plasma.

A sensitive analytical method for simultaneous quantification of sub-nanogram concentrations of cannabidiol (CBD), Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) in plasma is presented for monitoring cannabinoid pharmacotherapy and illicit cannabis use. Analytes were extracted from 1 mL plasma by solid-phase extraction, derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane, and analyzed by two-dimensional gas chromatography mass spectrometry (2D-GCMS) with cryofocusing. The lower calibration curve was linear from 0.25-25 ngmL for CBD and THC, 0.125-25 ngmL for 11-OH-THC and 0.25-50 ngmL for THCCOOH. A second higher linear range from 5-100 ngmL, achieved through modification of injection parameters, was validated for THC, 11-OH-THC, and THCCOOH and was only implemented if concentrations exceeded the lower curve upper limit of linearity. This procedure prevented laborious re-extraction by allowing the same specimen to be re-injected for quantification on the high calibration curve. Intra- and inter-assay imprecision, determined at four quality control concentrations, were <or7.8% CV. Analytical bias was within -9.2% of target and extraction efficiencies were >or72.9% for all analytes. Analytes were stable when stored at 22 degrees C for 16 h, 4 degrees C for 48 h, after three freeze-thaw cycles at -20 degrees C and when stored on the autosampler for 48 h. This sensitive and specific 2D-GCMS assay provides a new means of simultaneously quantifying CBD, THC and metabolite biomarkers in clinical medicine, forensic toxicology, workplace drug testing, and driving under the influence of drugs programs.