The Predictive Value of Plasma Bioactive Lipids on Craving in Human Volunteers With Alcohol Use Disorder.

Background: Alcohol use disorder (AUD) is a chronic relapsing disorder characterized by alcohol seeking and consumption despite negative consequences. Despite the availability of multiple treatments, patients continue to exhibit high relapse rates. Thus, biomarkers that can identify patients at risk for heightened craving are urgently needed. Mounting preclinical and clinical evidence implicates perturbations in bioactive lipid signaling in the neurobiology of craving in AUD. We hypothesize that these lipids are potential biomarkers for predicting alcohol craving in patients with AUD. Methods: This study used archival deidentified clinical data and corresponding plasma specimens from 157 participants in 3 clinical studies of AUD. We evaluated plasma levels of 8 lipid species as predictors of craving in response to in vivo alcohol and affective cues during abstinence. Results: Participants were 109 men and 48 women who met DSM-5 criteria for severe AUD. We found that plasma levels of 12- and 15-HETE, 12/15-lipoxygenase-produced proinflammatory lipids, and palmitoylethanolamide, an anti-inflammatory fatty acid amide hydrolase-regulated lipid metabolite, were differentially correlated with alcohol craving during abstinence, predicting higher craving independent of demographics, alcohol use history, and multiple therapeutic treatments. Conclusions: Our findings highlight the promise of these lipid metabolites as biomarkers of heightened alcohol craving. The results open a novel opportunity for further research and clinical evaluation of these biomarkers to optimize existing treatments and develop new therapeutics for AUD.

Alcohol use disorder (AUD) is a chronic relapsing disorder characterized by the compulsion to seek out and consume alcohol regardless of negative consequences. Despite the availability of multiple treatment options, patients continue to exhibit high relapse rates due in part to craving during abstinence. The current study evaluated the relationship between plasma lipids and alcohol craving during abstinence to determine whether we could predict vulnerable patients independent of treatment approach and history of alcohol use.

Effect of chronic vapor nicotine exposure on affective and cognitive behavior in male mice.

Nicotine use is a leading cause of preventable deaths worldwide, and most of those who attempt to quit will relapse. While electronic cigarettes and other electronic nicotine delivery systems (ENDS) were presented as a safer alternative to traditional cigarettes and promoted as devices to help traditional tobacco smokers reduce or quit smoking, they have instead contributed to increasing nicotine use among youths. Despite this, ENDS also represent a useful tool to create novel preclinical animal models of nicotine exposure that more accurately represent human nicotine use. In this study, we validated a chronic, intermittent, ENDS-based passive vapor exposure model in mice, and then measured changes in multiple behaviors related to nicotine abstinence. First, we performed a behavioral dose curve to investigate the effects of different nicotine inter-vape intervals on various measures including body weight, locomotor activity, and pain hypersensitivity. Next, we performed a pharmacokinetic study to measure plasma levels of nicotine and cotinine following chronic exposure for each inter-vape interval. Finally, we utilized a behavior test battery at a single dosing regimen that produces blood levels equivalent to human smokers in order to characterize the effects of chronic nicotine, vehicle, or passive airflow and identified nicotine-induced impairments in cognitive behavior.

Effect of chronic vapor nicotine exposure on affective and cognitive behavior in male mice.

Nicotine use is a leading cause of preventable deaths worldwide, and most of those who attempt to quit will relapse. While electronic cigarettes and other electronic nicotine delivery systems (ENDS) were presented as a safer alternative to traditional cigarettes and promoted as devices to help traditional tobacco smokers reduce or quit smoking, they have instead contributed to increasing nicotine use among youths. Despite this, ENDS also represent a useful tool to create novel preclinical animal models of nicotine exposure that more accurately represent human nicotine use. In this study, we validated a chronic, intermittent, ENDS-based passive vapor exposure model in mice, and then measured changes in multiple behaviors related to nicotine abstinence. First, we performed a behavioral dose curve to investigate the effects of different nicotine inter-vape intervals on various measures including body weight, locomotor activity, and pain hypersensitivity. Next, we performed a pharmacokinetic study to measure plasma levels of nicotine and cotinine following chronic exposure for each inter-vape interval. Finally, we utilized a behavior test battery at a single dosing regimen that produces blood levels equivalent to human smokers in order to characterize the effects of chronic nicotine, vehicle, or passive airflow and identified nicotine-induced impairments in cognitive behavior.

NAPE-PLD regulates specific baseline affective behaviors but is dispensable for inflammatory hyperalgesia.

N-acyl-ethanolamine (NAEs) serve as key endogenous lipid mediators as revealed by manipulation of fatty acid amide hydrolase (FAAH), the primary enzyme responsible for metabolizing NAEs. Preclinical studies focused on FAAH or NAE receptors indicate an important role for NAE signaling in nociception and affective behaviors. However, there is limited information on the role of NAE biosynthesis in these same behavioral paradigms. Biosynthesis of NAEs has been attributed largely to the enzyme N-acylphosphatidylethanolamine Phospholipase D (NAPE-PLD), one of three pathways capable of producing these bioactive lipids in the brain. In this report, we demonstrate that Nape-pld knockout (KO) mice displayed reduced sucrose preference and consumption, but other baseline anxiety-like or depression-like behaviors were unaltered. Additionally, we observed sex-dependent responses in thermal nociception and other baseline measures in wildtype (WT) mice that were absent in Nape-pld KO mice. In the Complete Freund's Adjuvant (CFA) model of inflammatory arthritis, WT mice exhibited sex-dependent changes in paw edema that were lost in Nape-pld KO mice. However, there was no effect of Nape-pld deletion on arthritic pain-like behaviors (grip force deficit and tactile allodynia) in either sex, indicating that while NAPE-PLD may alter local inflammation, it does not contribute to pain-like behaviors associated with inflammatory arthritis. Collectively, these findings indicate that chronic and systemic NAPE-PLD inactivation will likely be well-tolerated, warranting further pharmacological evaluation of this target in other disease indications.

THC and CBD: Villain versus Hero? Insights into Adolescent Exposure.

Cannabis is the most used drug of abuse worldwide. It is well established that the most abundant phytocannabinoids in this plant are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). These two compounds have remarkably similar chemical structures yet vastly different effects in the brain. By binding to the same receptors, THC is psychoactive, while CBD has anxiolytic and antipsychotic properties. Lately, a variety of hemp-based products, including CBD and THC, have become widely available in the food and health industry, and medical and recreational use of cannabis has been legalized in many states/countries. As a result, people, including youths, are consuming CBD because it is considered "safe". An extensive literature exists evaluating the harmful effects of THC in both adults and adolescents, but little is known about the long-term effects of CBD exposure, especially in adolescence. The aim of this review is to collect preclinical and clinical evidence about the effects of cannabidiol.

Locus coeruleus activation during environmental novelty gates cocaine-induced long-term hyperactivity of dopamine neurons.

A key feature of the brain is the ability to handle novelty. Anything that is new will stimulate curiosity and trigger exploration. Novelty preference has been proposed to predict increased sensitivity to cocaine. Different brain circuits are activated by novelty, but three specific brain regions are critical for exploring a novel environment: the noradrenergic neurons originating from the locus coeruleus (LC), the dopaminergic neurons from the ventral tegmental area (VTA), and the hippocampus. However, how exploring a novel environment can interfere with the reward system and control cocaine impact on VTA dopamine neuron plasticity is unclear. Here, we first investigated the effects of exposure to a novel environment on the tonic electrophysiological properties of VTA dopamine neurons. Then, we explored how exposure to a novel environment controls cocaine-evoked plasticity in dopamine neurons. Our findings indicate that LC controls VTA dopamine neurons under physiological conditions but also after cocaine.

New insights into methoxetamine mechanisms of action: Focus on serotonergic 5-HT2 receptors in pharmacological and behavioral effects in the rat.

Methoxetamine (MXE) is a dissociative substance of the arylcyclohexylamine class that has been present on the designer drug market as a ketamine-substitute since 2010. We have previously shown that MXE (i) possesses ketamine-like discriminative and positive rewarding effects in rats, (ii) affects brain processing involved in cognition and emotional responses, (iii) causes long-lasting behavioral abnormalities and neurotoxicity in rats and (iv) induces neurological, sensorimotor and cardiorespiratory alterations in mice. To shed light on the mechanisms through which MXE exerts its effects, we conducted a multidisciplinary study to evaluate the various neurotransmitter systems presumably involved in its actions on the brain. In vivo microdialysis study first showed that a single administration of MXE (0.25 and 0.5 mg/kg, i.v.) is able to significantly alter serotonin levels in the rat medial prefrontal cortex (mPFC) and nucleus accumbens. Then, we observed that blockade of the serotonin 5-HT2 receptors through two selective antagonists, ketanserin (0.1 mg/kg, i.p.) and MDL 100907 (0.03 mg/kg, i.p.), at doses not affecting animals behavior per se, attenuated the facilitatory motor effect and the inhibition on visual sensory responses induced by MXE (3 mg/kg, i.p.) and ketamine (3 mg/kg, i.p.), and prevented MXE-induced reduction of the prepulse inhibition in rats, pointing to the 5-HT2 receptors as a key target for the recently described MXE-induced sensorimotor effects. Finally, in-vitro electrophysiological studies revealed that the GABAergic and glutamatergic systems are also likely involved in the mechanisms through which MXE exerts its central effects since MXE inhibits, in a concentration-dependent manner, NMDA-mediated field postsynaptic potentials and GABA-mediated spontaneous currents. Conversely, MXE failed to alter both the AMPA component of field potentials and presynaptic glutamate release, and seems not to interfere with the endocannabinoid-mediated effects on mPFC GABAergic synapses. Altogether, our results support the notion of MXE as a NMDA receptor antagonist and shed further lights into the central mechanisms of action of this ketamine-substitute by pointing to serotonin 5-HT2 receptors as crucial players in the expression of its sensorimotor altering effects and to the NMDA and GABA receptors as potential further important targets of action.

Human Neuronal Cell Lines as An In Vitro Toxicological Tool for the Evaluation of Novel Psychoactive Substances.

Novel psychoactive substances (NPS) are synthetic substances belonging to diverse groups, designed to mimic the effects of scheduled drugs, resulting in altered toxicity and potency. Up to now, information available on the pharmacology and toxicology of these new substances is very limited, posing a considerable challenge for prevention and treatment. The present in vitro study investigated the possible mechanisms of toxicity of two emerging NPS (i) 4'-methyl-alpha-pyrrolidinoexanophenone (3,4-MDPHP), a synthetic cathinone, and (ii) 2-chloro-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA), a phenethylamine. In addition, to apply our model to the class of synthetic opioids, we evaluated the toxicity of fentanyl, as a reference compound for this group of frequently abused substances. To this aim, the in vitro toxic effects of these three compounds were evaluated in dopaminergic-differentiated SH-SY5Y cells. Following 24 h of exposure, all compounds induced a loss of viability, and oxidative stress in a concentration-dependent manner. 2-Cl-4,5-MDMA activates apoptotic processes, while 3,4-MDPHP elicits cell death by necrosis. Fentanyl triggers cell death through both mechanisms. Increased expression levels of pro-apoptotic Bax and caspase 3 activity were observed following 2-Cl-4,5-MDMA and fentanyl, but not 3,4-MDPHP exposure, confirming the different modes of cell death.

Repeated exposure to JWH-018 induces adaptive changes in the mesolimbic and mesocortical dopaminergic pathways, glial cells alterations, and behavioural correlates.

BACKGROUND AND PURPOSE: Spice/K2 herbal mixtures, containing synthetic cannabinoids such as JWH-018, have been marketed as marijuana surrogates since 2004. JWH-018 has cannabinoid CB1 receptor-dependent reinforcing properties and acutely increases dopaminergic transmission selectively in the NAc shell. Here, we tested the hypothesis that repeated administration of JWH-018 (i) modulates behaviour, (ii) affects dopaminergic transmission and its responsiveness to motivational stimuli, and (iii) is associated with a neuroinflammatory phenotype. EXPERIMENTAL APPROACH: Rats were administered with JWH-018 once a day for 14 consecutive days. We then performed behavioural, electrophysiological, and neurochemical evaluation at multiple time points after drug discontinuation. KEY RESULTS: Repeated JWH-018 exposure (i) induced anxious and aversive behaviours, transitory attentional deficits, and withdrawal signs; (ii) decreased spontaneous activity and number of dopamine neurons in the VTA; and (iii) reduced stimulation of dopaminergic transmission in the NAc shell while potentiating that in the NAc core, in response to acute JWH-018 challenge. Moreover, (iv) we observed a decreased dopamine sensitivity in the NAc shell and core, but not in the mPFC, to a first chocolate exposure; conversely, after a second exposure, dialysate dopamine fully increased in the NAc shell and core but not in the mPFC. Finally, selected dopamine brain areas showed (v) astrogliosis (mPFC, NAc shell and core, VTA), microgliosis (NAc shell and core), and downregulation of CB1 receptors (mPFC, NAc shell and core). CONCLUSION AND IMPLICATIONS: Repeated exposure to JWH-018 may provide a useful model to clarify the detrimental effects of recurring use of Spice/K2 drugs.

Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

Modeling drug exposure in rodents using e-cigarettes and other electronic nicotine delivery systems.

Smoking tobacco products is the leading cause of preventable death worldwide. Coordinated efforts have successfully reduced tobacco cigarette smoking in the United States; however, electronic cigarettes (e-cigarette) and other electronic nicotine delivery systems (ENDS) recently have replaced traditional cigarettes for many users. While the clinical risks associated with long-term ENDS use remain unclear, advancements in preclinical rodent models will enhance our understanding of their overall health effects. This review examines the peripheral and central effects of ENDS-mediated exposure to nicotine and other drugs of abuse in rodents and evaluates current techniques for implementing ENDS in preclinical research.

Pharmacological and Behavioral Effects of the Synthetic Cannabinoid AKB48 in Rats.

AKB48 is a designer drug belonging to the indazole synthetic cannabinoids class, illegally sold as herbal blend, incense, or research chemicals for their psychoactive cannabis-like effects. In the present study, we investigated the in vivo pharmacological and behavioral effects of AKB48 in male rats and measured the pharmacodynamic effects of AKB48 and simultaneously determined its plasma pharmacokinetic. AKB48 at low doses preferentially stimulated dopamine release in the nucleus accumbens shell (0.25 mg/kg) and impaired visual sensorimotor responses (0.3 mg/kg) without affecting acoustic and tactile reflexes, which are reduced only to the highest dose tested (3 mg/kg). Increasing doses (0.5 mg/kg) of AKB48 impaired place preference and induced hypolocomotion in rats. At the highest dose (3 mg/kg), AKB48 induced hypothermia, analgesia, and catalepsy; inhibited the startle/pre-pulse inhibition test; and caused cardiorespiratory changes characterized by bradycardia and mild bradipnea and SpO2 reduction. All behavioral and neurochemical effects were fully prevented by the selective CB1 receptor antagonist/inverse agonist AM251. AKB48 plasma concentrations rose linearly with increasing dose and were correlated with changes in the somatosensory, hypothermic, analgesic, and cataleptic responses in rats. For the first time, this study shows the pharmacological and behavioral effects of AKB48 in rats, correlating them to the plasma levels of the synthetic cannabinoid. Chemical Compound Studied in This Article: AKB48 (PubChem CID: 57404063); AM251 (PubChem CID: 2125).

Neurochemical and Behavioral Profiling in Male and Female Rats of the Psychedelic Agent 25I-NBOMe.

4-Iodo-2,5-dimethoxy-N-(2-methoxybenzyl)phenethylamine (25I-NBOMe), commonly called "N-Bomb," is a synthetic phenethylamine with psychedelic and entactogenic effects; it was available on the Internet both as a legal alternative to lysergic acid diethylamide (LSD) and as a surrogate of 3,4-methylenedioxy-methamphetamine (MDMA), but now it has been scheduled among controlled substances. 25I-NBOMe acts as full agonist on serotonergic 5-HT2A receptors. Users are often unaware of ingesting fake LSD, and several cases of intoxication and fatalities have been reported. In humans, overdoses of "N-Bomb" can cause tachycardia, hypertension, seizures, and agitation. Preclinical studies have not yet widely investigated the rewarding properties and behavioral effects of this compound in both sexes. Therefore, by in vivo microdialysis, we evaluated the effects of 25I-NBOMe on dopaminergic (DA) and serotonergic (5-HT) transmissions in the nucleus accumbens (NAc) shell and core, and the medial prefrontal cortex (mPFC) of male and female rats. Moreover, we investigated the effect of 25I-NBOMe on sensorimotor modifications as well as body temperature, nociception, and startle/prepulse inhibition (PPI). We showed that administration of 25I-NBOMe affects DA transmission in the NAc shell in both sexes, although showing different patterns; moreover, this compound causes impaired visual responses in both sexes, whereas core temperature is heavily affected in females, and the highest dose tested exerts an analgesic effect prominent in male rats. Indeed, this drug is able to impair the startle amplitude with the same extent in both sexes and inhibits the PPI in male and female rats. Our study fills the gap of knowledge on the behavioral effects of 25I-NBOMe and the risks associated with its ingestion; it focuses the attention on sex differences that might be useful to understand the trend of consumption as well as to recognize and treat intoxication and overdose symptoms.

Sales and Advertising Channels of New Psychoactive Substances (NPS): Internet, Social Networks, and Smartphone Apps.

In the last decade, the trend of drug consumption has completely changed, and several new psychoactive substances (NPS) have appeared on the drug market as legal alternatives to common drugs of abuse. Designed to reproduce the effects of illegal substances like cannabis, ecstasy, cocaine, or ketamine, NPS are only in part controlled by UN conventions and represent an emerging threat to global public health. The effects of NPS greatly differ from drug to drug and relatively scarce information is available at present about their pharmacology and potential toxic effects. Yet, compared to more traditional drugs, more dangerous short- and long-term effects have been associated with their use, and hospitalizations and fatal intoxications have also been reported after NPS use. In the era of cyberculture, the Internet acts as an ideal platform to promote and market these compounds, leading to a global phenomenon. Hidden by several aliases, these substances are sold across the web, and information about consumption is shared by online communities through drug fora, YouTube channels, social networks, and smartphone applications (apps). This review intends to provide an overview and analysis of social media that contribute to the popularity of NPS especially among young people. The possibility of using the same channels responsible for their growing diffusion to make users aware of the risks associated with NPS use is proposed.

Neuropharmacology of New Psychoactive Substances (NPS): Focus on the Rewarding and Reinforcing Properties of Cannabimimetics and Amphetamine-Like Stimulants.

New psychoactive substances (NPS) are a heterogeneous and rapidly evolving class of molecules available on the global illicit drug market (e.g smart shops, internet, "dark net") as a substitute for controlled substances. The use of NPS, mainly consumed along with other drugs of abuse and/or alcohol, has resulted in a significantly growing number of mortality and emergency admissions for overdoses, as reported by several poison centers from all over the world. The fact that the number of NPS have more than doubled over the last 10 years, is a critical challenge to governments, the scientific community, and civil society [EMCDDA (European Drug Report), 2014; UNODC, 2014b; Trends and developments]. The chemical structure (phenethylamines, piperazines, cathinones, tryptamines, synthetic cannabinoids) of NPS and their pharmacological and clinical effects (hallucinogenic, anesthetic, dissociative, depressant) help classify them into different categories. In the recent past, 50% of newly identified NPS have been classified as synthetic cannabinoids followed by new phenethylamines (17%) (UNODC, 2014b). Besides peripheral toxicological effects, many NPS seem to have addictive properties. Behavioral, neurochemical, and electrophysiological evidence can help in detecting them. This manuscript will review existing literature about the addictive and rewarding properties of the most popular NPS classes: cannabimimetics (JWH, HU, CP series) and amphetamine-like stimulants (amphetamine, methamphetamine, methcathinone, and MDMA analogs). Moreover, the review will include recent data from our lab which links JWH-018, a CB1 and CB2 agonist more potent than Δ(9)-THC, to other cannabinoids with known abuse potential, and to other classes of abused drugs that increase dopamine signaling in the Nucleus Accumbens (NAc) shell. Thus the neurochemical mechanisms that produce the rewarding properties of JWH-018, which most likely contributes to the greater incidence of dependence associated with "Spice" use, will be described (De Luca et al., 2015a). Considering the growing evidence of a widespread use of NPS, this review will be useful to understand the new trends in the field of drug reward and drug addiction by revealing the rewarding properties of NPS, and will be helpful to gather reliable data regarding the abuse potential of these compounds.

Native CB1 receptor affinity, intrinsic activity and accumbens shell dopamine stimulant properties of third generation SPICE/K2 cannabinoids: BB-22, 5F-PB-22, 5F-AKB-48 and STS-135.

In order to investigate the in vivo dopamine (DA) stimulant properties of selected 3rd generation Spice/K2 cannabinoids, BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, their in vitro affinity and agonist potency at native rat and mice CB1 receptors was studied. The compounds bind with high affinity to CB1 receptors in rat cerebral cortex homogenates and stimulate CB1-induced [(35)S]GTPγS binding with high potency and efficacy. BB-22 and 5F-PB-22 showed the lowest Ki of binding to CB1 receptors (0.11 and 0.13 nM), i.e., 30 and 26 times lower respectively than that of JWH-018 (3.38 nM), and a potency (EC50, 2.9 and 3.7 nM, respectively) and efficacy (Emax, 217% and 203%, respectively) as CB1 agonists higher than JWH-018 (EC50, 20.2 nM; Emax, 163%). 5F-AKB-48 and STS-135 had higher Ki for CB1 binding, higher EC50 and lower Emax as CB1 agonists than BB-22 and 5F-PB-22 but still comparatively more favourable than JWH-018. The agonist properties of all the compounds were abolished or drastically reduced by the CB1 antagonist/inverse agonist AM251 (0.1 µM). No activation of G-protein was observed in CB1-KO mice. BB-22 (0.003-0.01 mg/kg i.v.) increased dialysate DA in the accumbens shell but not in the core or in the medial prefrontal cortex, with a bell shaped dose-response curve and an effect at 0.01 mg/kg and a biphasic time-course. Systemic AM251 (1.0 mg/kg i.p.) completely prevented the stimulant effect of BB-22 on dialysate DA in the NAc shell. All the other compounds increased dialysate DA in the NAc shell at doses consistent with their in vitro affinity for CB1 receptors (5F-PB-22, 0.01 mg/kg; 5F-AKB-48, 0.1 mg/kg; STS-135, 0.15 mg/kg i.v.). 3rd generation cannabinoids can be even more potent and super-high CB1 receptor agonists compared to JWH-018. Future research will try to establish if these properties can explain the high toxicity and lethality associated with these compounds.