Comprehensive evaluation of the pharmacological and toxicological effects of γ-valerolactone as compared to γ-hydroxybutyric acid: Insights from in vivo and in silico models.

Γ-valerolactone (GVL), marketed online as "Tranquilli-G" and "excellent Valium", is used as a legal substitute for γ-hydroxybutyric acid (GHB); however, until now, GVL has only been connected to one Drug-Facilitated Sexual Assault (DFSA) case. Moreover, the pharmaco-toxicological effects of GVL are poorly studied. The aim of this study was to investigate the 1) in vivo effects of gavage administration of GVL (100-3000 mg/kg) on neurological (myoclonia, convulsions), sensorimotor (visual, acoustic, and overall tactile) responses, righting reflex, thermoregulation, motor activity (bar, drag, and accelerod test) and cardiorespiratory changes (heart rate, breath rate, oxygen saturation, and pulse distension) in CD-1 male mice and the 2) in silico ADMET profile of GVL in comparison to GHB and the open active form γ-hydroxyvaleric acid (GHV). The present study demonstrates that GVL inhibits, in a dose-dependent manner, sensorimotor and motor responses and induces cardiorespiratory depression (at a dose of 3000 mg/kg) in mice. The determination of the ED50 in sensorimotor and motor responses revealed that GVL is about 4-5 times less potent than GHB. In silico prediction of ADMET profiles revealed toxicokinetic similarities between GHB and GHV, and differences with GVL. These results suggest that GVL could be used as a substitute for GHB and should be added to forensic toxicology screenings.

Synthetic cannabinoid JWH-073 alters both acute behavior and in vivo/vitro electrophysiological responses in mice.

JWH-073 is a synthetic cannabinoid (SCB) that is illegally marketed within an "herbal blend", causing psychoactive effects more intense than those produced by Cannabis. Users report that JWH-073 causes less harmful effects than other SCBs, misrepresenting it as a "safe JWH-018 alternative", which in turn prompts its recreational use. The present study is aimed to investigate the in vivo pharmacological activity on physiological and neurobehavioral parameters in male CD-1 mice after acute 1 mg/kg JWH-073 administration. To this aim we investigate its effect on sensorimotor (visual, acoustic, and tactile), motor (spontaneous motor activity and catalepsy), and memory functions (novel object recognition; NOR) in mice coupling behavioral and EEG data. Moreover, to clarify how memory function is affected by JWH-073, we performed in vitro electrophysiological studies in hippocampal preparations using a Long-Term Potentiation (LTP) stimulation paradigm. We demonstrated that acute administration of JWH-073 transiently decreased motor activity for up to 25 min and visual sensorimotor responses for up to 105 min, with the highest effects at 25 min (~48 and ~38%, respectively), while the memory function was altered up to 24 h (~33%) in treated-mice as compared to the vehicle. EEG in the somatosensory cortex showed a maximal decrease of α (~23%) and γ (~26%) bands at 15 min, ß (~26%) band at 25 min, a maximal increase of θ (~14%) band at 25 min and δ (~35%) band at 2 h, and a significant decrease of θ (~18%), α (~26%), and ß (~10%) bands during 24 h. On the other hand, EEG in the hippocampus showed a significant decrease of all bands from 10 min to 2 h, with the maximal effect at 30 min for θ (~34%) and γ (~26%) bands and 2 h for α (~36%), ß (~29%), and δ (~15%) bands. Notably, the δ band significant increase both at 5 min (~12%) and 24 h (~19%). Moreover, in vitro results support cognitive function impairment (~60% of decrease) by interfering with hippocampal synaptic transmission and LTP generation. Our results suggest that JWH-073 deeply alters brain electrical responsiveness with minor behavioral symptoms. Thus, it poses a subtle threat to consumers who mistakenly consider it safer than other SCBs.

Effect of -NBOMe Compounds on Sensorimotor, Motor, and Prepulse Inhibition Responses in Mice in Comparison With the 2C Analogs and Lysergic Acid Diethylamide: From Preclinical Evidence to Forensic Implication in Driving Under the Influence of Drugs.

In the last decade, the market for new psychoactive substances has been enriched by numerous psychedelic phenethylamines, which mimic the psychoactive effect of lysergic acid diethylamide (LSD). In particular, the -NBOMe series, which are more potent than their 2C compounds analogs, are considered worthy substitutes for LSD by users. The purpose of this study was to assess the effects of 25H-NBOMe and its halogenated derivatives (25I-NBOMe and 25B-NBOMe) in comparison to their 2C compounds analogs and LSD on the sensorimotor (visual, acoustic, and overall tactile), reaction time, spontaneous (total distance traveled) and stimulated (drag, accelerod test) motor activity, grip strength test, and prepulse inhibition (PPI) responses in mice. Systemic administration of -NBOMe, 2C compounds analogs, and LSD (0.001-10 mg/kg) differently impaired the sensorimotor, reaction time, motor, and PPI responses in mice. In particular, halogenated (25I and 25B)-NBOMe derivatives appear to be more effective than the entire class of 2C compounds analogs in altering visual and acoustic responses, affecting reaction time, and motor and sensory gating in PPI test. In fact, the specific rank order of compounds potency for nearly all of the experiments showed that (25I and 25B)-NBOMe were more potent than 2C compounds analogs and LSD. -NBOMe and 2C compounds analogs impaired not only the reception of incoming sensory stimuli (visual and acoustic), but their correct brain processing (PPI) in an equal and sometimes stronger way than LSD. This sensory impairment directly affected the spontaneous motor response and reaction time of mice, with no change in performance in stimulated motor activity tests. These aspects should be carefully considered to better understand the potential danger that psychedelic phenethylamines, in particular -NBOMe, may pose to public health, with particular reference to decreased performance in driving and hazardous works that require special sensorimotor skills.

In vitro and in vivo pharmaco-dynamic study of the novel fentanyl derivatives: Acrylfentanyl, Ocfentanyl and Furanylfentanyl.

Fentanyl derivatives (FENS) belongs to the class of Novel Synthetic Opioids that emerged in the illegal drug market of New Psychoactive Substances (NPS). These substances have been implicated in many cases of intoxication and death with overdose worldwide. Therefore, the aim of this study is to investigate the pharmaco-dynamic profiles of three fentanyl (FENT) analogues: Acrylfentanyl (ACRYLF), Ocfentanyl (OCF) and Furanylfentanyl (FUF). In vitro, we measured FENS opioid receptor efficacy, potency, and selectivity in calcium mobilization studies performed in cells coexpressing opioid receptors and chimeric G proteins and their capability to promote the interaction of the mu receptor with G protein and ß-arrestin 2 in bioluminescence resonance energy transfer (BRET) studies. In vivo, we investigated the acute effects of the systemic administration of ACRYLF, OCF and FUF (0.01-15 mg/kg i.p.) on mechanical and thermal analgesia, motor impairment, grip strength and cardiorespiratory changes in CD-1 male mice. Opioid receptor specificity was investigated in vivo using naloxone (NLX; 6 mg/kg i.p) pre-treatment. In vitro, the three FENS were able to activate the mu opioid receptor in a concentration dependent manner with following rank order potency: FUF > FENT=OCF > ACRYLF. All compounds were able to elicit maximal effects similar to that of dermorphin, with the exception of FUF which displayed lower maximal effects thus behaving as a partial agonist. In the BRET G-protein assay, all compounds behaved as partial agonists for the ß-arrestin 2 pathway in comparison with dermorphin, whereas FUF did not promote ß-arrestin 2 recruitment, behaving as an antagonist. In vivo, all the compounds increased mechanical and thermal analgesia with following rank order potency ACRYLF = FENT > FUF > OCF and impaired motor and cardiorespiratory parameters. Among the substances tested, FUF showed lower potency for cardiorespiratory and motor effects. These findings reveal the risks associated with the use of FENS and the importance of studying the pharmaco-dynamic properties of these drugs to better understand possible therapeutic interventions in the case of toxicity.

Ethanol enhanced MDPV- and cocaine-induced aggressive behavior in mice: Forensic implications.

BACKGROUND: Reports concerning the causal link between aggressive behavior and use and abuse of different substances (i.e., alcohol, MDPV) can be found in the literature. Nonetheless, the topic concerning the effects of acute ethanol administration on MDPV and cocaine induced aggressive behavior has yet to be thoroughly investigated. The aim of this study was to investigate such synergistic effects. MATERIALS AND METHODS: A total of 360 male mice were employed in the study. Ethanol was diluted with saline solution and administered 10 min before MDPV or cocaine injection via oral gavage needles. Similarly, MDPV and cocaine were dissolved in saline solution and administered by intraperitoneal injection. Different associations of specific drug doses were then tested. To investigate the acute effects of MDPV and cocaine and their interaction with ethanol on aggression in mice, a resident-intruder test was used. RESULTS: Ethanol alone was ineffective at dosages of 0.05 g/kg and 0.25 g/kg but increased the aggressiveness of the mice at 0.125 g/kg. Similarly, the injection of both cocaine alone and MDPV alone did not significantly increase the aggressiveness of the mice; conversely, the combination of ethanol and cocaine and ethanol and MDPV enhanced aggression at specific ethanol dosages (0.05 g/kg and 0.125 g/kg). CONCLUSION: This study demonstrated that acute ethanol administration enhances MDPV- and cocaine-induced aggressive behavior in mice. This aggressive response is particularly enhanced when MDVP and cocaine are coupled with specific ethanol dosages, proving that psychostimulant drugs may act synergistically under certain conditions.

Worsening of the Toxic Effects of (±)Cis-4,4'-DMAR Following Its Co-Administration with (±)Trans-4,4'-DMAR: Neuro-Behavioural, Physiological, Immunohistochemical and Metabolic Studies in Mice.

4,4'-Dimethylaminorex (4,4'-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±)cis (0.1-60 mg/kg) and (±)trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±)cis at 1, 10 or 60 mg/kg + (±)trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4'-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±)cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±)trans stereoisomer was ineffective whilst the stereoisomers' co-administration resulted in a worsening of the toxic (±)cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±)trans stereoisomer reduced the metabolism of the (±)cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.

Comparison of N-methyl-2-pyrrolidone (NMP) and the "date rape" drug GHB: behavioral toxicology in the mouse model.

N-methyl-2-pyrrolidone (NMP) and γ-hydroxybutyrate acid (GHB) are synthetic solvents detected in the recreational drug market. GHB has sedative/hypnotic properties and is used for criminal purposes to compromise reaction ability and commit drug-facilitated sexual assaults and other crimes. NMP is a strong solubilizing solvent that has been used alone or mixed with GHB in case of abuse and robberies. The aim of this experimental study is to compare the acute pharmaco-toxicological effects of NMP and GHB on neurological signs (myoclonia, convulsions), sensorimotor (visual, acoustic, and overall tactile) responses, righting reflex, thermoregulation, and motor activity (bar, drag, and accelerod test) in CD-1 male mice. Moreover, since cardiorespiratory depression is one of the main adverse effects related to GHB intake, we investigated the effect of NMP and GHB on cardiorespiratory changes (heart rate, breath rate, oxygen saturation, and pulse distension) in mice. The present study demonstrates that NMP inhibited sensorimotor and motor responses and induced cardiorespiratory depression, with a lower potency and efficacy compared to GHB. These results suggest that NMP can hardly be used alone as a substance to perpetrate sexual assault or robberies.

Low-normal doses of methiopropamine induce aggressive behaviour in mice.

Recreational use of illicit methiopropamine (MPA) is a public health concern because it produces neurochemical effects comparable with those induced by methamphetamine (METH). The present study investigated the effects of MPA on the expression of an aggressive behaviour. Eighty CD-1 male mice, after receiving intraperitoneal injection of saline, MPA (0.01-10 mg/kg), METH (0.01-10 mg/kg), or AMPH (0.01-10 mg/kg), once a week over a 5-week period, underwent the resident-intruder test and spontaneous locomotor activity measurement. Results showed that all psychostimulants induce aggressive behaviour even at low doses, with a dose-dependent increase and a time-dependent sensitisation. MPA potency was similar to METH and superior to AMPH. Therefore, MPA-induced aggressive behaviour may appear even at MPA dosages free of cardiovascular or other behavioural adverse effects and could become a non-intentional side effect that users experience after increasing and repeating MPA consumption.

Magnetic Resonance Spectroscopy in Adolescent Cannabis Users: Metabolites in the Anterior Cingulate Cortex Reflects Individual Differences in Personality Traits and can Affect Rehabilitation Compliance.

INTRODUCTION: The anterior cingulate cortex (ACC) has shown to play a role in impulsivity, fear, and anxiety. Considering, its high glutamate receptor density, it was chosen as a region of interest to investigate the role of glutamate transmission in drug dependance. We investigated the correlations between personality trait scores and glutamate-to-glutamine (Glx) ratio concentrations in the ACC in order to evaluate if (1) personality traits may increase the probability of drug use and (2) drug use can modify cerebral metabolic pattern contributing to addictive behaviors. MATERIALS AND METHODS: Glx ratio concentrations in the ACC region were measured with high-resolution multivoxel proton magnetic resonance spectroscopy (1H-MRS). Personality traits were evaluated utilizing Cloninger's TCI-revised test. Bivariate correlations between personality scores of 28 teens cannabis users (males, mean age = 18.54 ± 2.80) were evaluated. RESULTS: In the ACC, we observed negative correlation between GG concentrations (r = -0.44, P = 0.05) and co-operativeness values (CO), choline (cho), and novelty seeking (NS) values (r = -0,45, P = 0.05). Low levels of glutamate and high levels of cho in the ACC were closely related to the CO and NS personality traits. CONCLUSIONS: Metabolic and personality patterns seems to be related to the risk of substance predisposition in adolescents. Our data contribute a possible support to the "top-down" control of the ACC on brain metabolism, due to the particular cerebral metabolic pattern found in "drug-using" adolescents.

Novel halogenated synthetic cannabinoids impair sensorimotor functions in mice.

JWH-018-Cl, JWH-018-Br and AM-2201 (JWH-018 halogenated-derivatives; JWH-018-R compounds) are synthetic cannabinoid agonists illegally marketed as "Spice", "K2", "herbal blend" and research chemicals for their cannabis-like psychoactive effects. In rodents, JWH-018 and its halogenated derivatives reproduce the typical effects of Δ9-tetrahydrocannabinol (Δ9-THC), i.e. hypothermia, analgesia, hypolocomotion and akinesia. Yet, the effects of JWH-018-R compounds on sensorimotor functions are still unknown. This study was designed to investigate the effect of an acute intraperitoneal (i.p.) administration of JWH-018-R compounds (0.01-6 mg/kg) on sensorimotor functions in mice and to compare them to those caused by the reference compound JWH-018 and Δ9-THC. A well validated battery of behavioral tests was used to investigate the effects of these synthetic cannabinoids on the visual, auditory and tactile responses in mice, while the pre-pulse inhibition (PPI) test was used to investigate their effect on sensorimotor gating. The effect of the synthetic cannabinoids on spontaneous locomotion was also measured by a video tracking analysis to assess potential cannabinoid-induced motor impairment. Results showed that, similarly to JWH-018, systemic administration of JWH-018-R compounds inhibits sensorimotor and PPI responses at lower doses (0.01-0.1 mg/kg) and reduced spontaneous locomotion at intermediate/high doses (1-6 mg/kg). All effects were prevented by the administration of the selective cannabinoid CB1 receptor antagonist/inverse agonist AM-251 thus confirming a CB1 receptor-mediated action. Finding that lower doses of JWH-018-R compounds selectively impair sensorimotor and PPI responses without affecting locomotion should be carefully considered to better understand the potential danger that halogenated-derivatives of JWH-018 may pose to public health, with particular reference to decreased performance in driving and hazardous works.

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).

Acute and repeated administration of MDPV increases aggressive behavior in mice: forensic implications.

MDPV is a synthetic cathinone illegally marketed and consumed for its psychostimulant effects, which are similar to those produced by cocaine, amphetamines, and MDMA. Clinical reports indicate that MDPV produces euphoria, increases alertness, and at high doses causes agitation, psychosis, tachycardia and hypertension, hallucinations, delirium, hyperthermia, rhabdomyolysis, and even death. In rodents, MDPV reproduces the typical physiological effects of psychostimulant drugs, demonstrating greater potency than cocaine. Nevertheless, its role in aggressive behavior has been reported but not yet experimentally confirmed. Therefore, the aim of this study was to evaluate the effects of acute and repeated MDPV (0.01-10 mg/kg i.p.) administration on aggressive behavior in mice and to compare them with those of cocaine (0.01-10 mg/kg i.p.) administration. To this purpose, the resident-intruder test in isolated mice and the spontaneous and stimulated aggressiveness tests for group-housed mice were employed. The present study shows for the first time that MDPV enhances aggressive behavior and locomotion in mice with greater potency and efficacy than cocaine treatment. Moreover, the aggressive and locomotor responses are enhanced after repeated administration, indicating that a sensitization mechanism comes into play. These results, although from preclinical investigation, are suggestive that human MDPV intake could be a problem for public health and the criminal justice system. Thus, investigation by police officers and medical staff is needed to prevent interpersonal violence induced by the consumption of synthetic cathinones.

Psychostimulant Effect of the Synthetic Cannabinoid JWH-018 and AKB48: Behavioral, Neurochemical, and Dopamine Transporter Scan Imaging Studies in Mice.

JWH-018 and AKB48 are two synthetic cannabinoids (SCBs) belonging to different structural classes and illegally marketed as incense, herbal preparations, or chemical supply for theirs psychoactive cannabis-like effects. Clinical reports from emergency room reported psychomotor agitation as one of the most frequent effects in people assuming SCBs. This study aimed to investigate the psychostimulant properties of JWH-018 and AKB48 in male CD-1 mice and to compare their behavioral and biochemical effects with those caused by cocaine and amphetamine. In vivo studies showed that JWH-018 and AKB48, as cocaine and amphetamine, facilitated spontaneous locomotion in mice. These effects were prevented by CB1 receptor blockade and dopamine (DA) D1/5 and D2/3 receptors inhibition. SPECT-CT studies on dopamine transporter (DAT) revealed that, as cocaine and amphetamine, JWH-018 and AKB48 decreased the [123I]-FP-CIT binding in the mouse striatum. Conversely, in vitro competition binding studies revealed that, unlike cocaine and amphetamine, JWH-018 and AKB48 did not bind to mouse or human DAT. Moreover, microdialysis studies showed that the systemic administration of JWH-018, AKB48, cocaine, and amphetamine stimulated DA release in the nucleus accumbens (NAc) shell of freely moving mice. Finally, unlike amphetamine and cocaine, JWH-018 and AKB48 did not induce any changes on spontaneous [3H]-DA efflux from murine striatal synaptosomes. The present results suggest that SCBs facilitate striatal DA release possibly with different mechanisms than cocaine and amphetamine. Furthermore, they demonstrate, for the first time, that JWH-018 and AKB48 induce a psychostimulant effect in mice possibly by increasing NAc DA release. These data, according to clinical reports, outline the potential psychostimulant action of SCBs highlighting their possible danger to human health.

Alcohol, caffeine, and nicotine consumption in adolescents: hair analysis versus self-report.

BACKGROUND: Clinical reliability of self-reported data for alcohol, caffeine, and nicotine consumptions is lacking, particularly in adolescents. OBJECTIVES: To compare a self-report questionnaire and hair analysis to assess the reliability and effectiveness of the self-report. METHODS: A cross-sectional study on 14-15-year-old Italian students (n = 874, 38% males, 62% females) was performed comparing self-reported data to hair analysis. The latter quantified hair concentrations of caffeine, nicotine, cotinine, ethyl glucuronide (EtG), and fatty acid ethyl esters (FAEEs) using mass spectrometry. RESULTS: Concordance between self-report and hair testing ranged from good to poor across substances and levels of use: poor for heavy alcohol intake (EtG: k = 0.36, 20 positive cases by hair analysis, false negative by self-report, 2.3% of total sample; FAEE k = 0.31, 25 positive cases, 2.9% of total sample); fair to poor for active smokers (k = 0.40, 125 positive cases, 14.3% of total sample); and moderate for caffeine (k = 0.57, 56 positive cases, 6.4% of total sample). CONCLUSIONS: Epidemiological studies on alcohol, caffeine, and nicotine consumption in adolescents may benefit from the inclusion of toxicological analysis on hair samples to overcome the under-reporting phenomenon of questionnaires and detect more cases of problematic substance use.

Effect of the novel synthetic cannabinoids AKB48 and 5F-AKB48 on "tetrad", sensorimotor, neurological and neurochemical responses in mice. In vitro and in vivo pharmacological studies.

RATIONALE: AKB48 and its fluorinate derivate 5F-AKB48 are two novel synthetic cannabinoids belonging to a structural class with an indazole core structure. They are marketed as incense, herbal preparations or chemical supply for their psychoactive Cannabis-like effects. OBJECTIVES: The present study was aimed at investigating the in vitro and in vivo pharmacological activity of AKB48 and 5F-AKB48 in male CD-1 mice and comparing their in vivo effects with those caused by the administration of Δ9-THC and JWH-018. RESULTS: In vitro competition binding experiments performed on mouse and human CB1 and CB2 receptors revealed a nanomolar affinity and potency of the AKB48 and 5F-AKB48. In vivo studies showed that AKB48 and 5F-AKB48, induced hypothermia, increased pain threshold to both noxious mechanical and thermal stimuli, caused catalepsy, reduced motor activity, impaired sensorimotor responses (visual, acoustic and tactile), caused seizures, myoclonia, hyperreflexia and promoted aggressiveness in mice. Moreover, microdialysis study in freely moving mice showed that systemic administration of AKB48 and 5F-AKB48 stimulated dopamine release in the nucleus accumbens. Behavioural, neurological and neurochemical effects were fully prevented by the selective CB1 receptor antagonist/inverse agonist AM 251. CONCLUSIONS: For the first time, the present study demonstrates the overall pharmacological effects induced by the administration of AKB48 and 5F-AKB48 in mice and suggests that the fluorination can increase the power and/or effectiveness of SCBs. Furthermore, this study outlines the potential detrimental effects of SCBs on human health.

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.

Population surveys compared with wastewater analysis for monitoring illicit drug consumption in Italy in 2010-2014.

BACKGROUND: Monitoring consumption by population surveys (PS) is an important way to challenge the spread of illicit drugs (ID). To improve the information, we explored a complementary method, particularly wastewater analysis (WWA). METHODS: We estimated the prevalence of use by PS, and the consumption by WWA, of cocaine, opioids, cannabis, methamphetamine and MDMA (ecstasy) from 2010 to 2014 in Italy and compared the results. RESULTS: According to PS, cannabis and cocaine were the ID most used in Italy (last month prevalence 3.0% and 0.43% respectively in 2010) followed by opioids (0.17%) and amphetamines (0.14%). WWA gave similar findings, with cannabis consumption (4.35 g THC/day/1000 inhabitants) exceeding cocaine (0.78 g), heroin (0.092 g), methamphetamine and MDMA (0.103 g). The time trend investigated by PS showed significant decreases for all ID from 2010 to 2012. WWA also indicated a reduction of consumption for methamphetamine (p<0.0001) and heroin (p<0.01). Both methods showed an increase for cannabis in 2014 (p<0.001) with the other ID unchanged. Spatial investigations by WWA showed that cannabis and cocaine were consumed significantly more in central Italy than in the north and south. PS indicated the same but only for cannabis. WWA was helpful to study weekly patterns of consumption, showing increases in cocaine and MDMA at weekends. CONCLUSIONS: PS and WWA were confirmed as complementary methods and when used together improved the information on ID use in Italy. We suggest that the combined use of the two approaches can give better information on ID use in the population.

Effect of JWH-250, JWH-073 and their interaction on "tetrad", sensorimotor, neurological and neurochemical responses in mice.

JWH-250 and JWH-073 are two synthetic cannabinoid agonists with nanomolar affinity at CB1 and CB2 receptors. They are illegally marketed within "herbal blend" for theirs psychoactive effects greater than those produced by Cannabis. Recently, we analyzed an "herbal" preparation containing a mixture of both JWH-250 and JWH-073. The present study was aimed at investigating the in vitro and in vivo pharmacological activity of JWH-250 and JWH-073 in male CD-1 mice. In vitro competition binding experiments performed on mouse and human CB1 and CB2 receptors revealed a nanomolar affinity and potency of the JWH-250 and JWH-073. In vivo studies showed that JWH-250 and JWH-073, administered separately, induced a marked hypothermia, increased pain threshold to both noxious mechanical and thermal stimuli, caused catalepsy, reduced motor activity, impaired sensorimotor responses (visual, acoustic and tactile), caused seizures, myoclonia, hyperreflexia and promote aggressiveness in mice. Moreover, microdialysis study in freely moving mice showed that systemic administration of JWH-250 and JWH-073 stimulated dopamine release in the nucleus accumbens in a dose-dependent manner. Behavioral, neurological and neurochemical effects were fully prevented by the selective CB1 receptor antagonist/inverse agonist AM 251. Co-administration of ineffective doses of JWH-250 and JWH-073 impaired visual sensorimotor responses, improved mechanical pain threshold and stimulated mesolimbic DA transmission in mice, living unchanged all other behavioral and physiological parameters. For the first time the present study demonstrates the overall pharmacological effects induced by the administration of JWH-250 and JWH-073 in mice and it reveals their potentially synergistic action suggesting that co-administration of different synthetic cannabinoids may potentiate the detrimental effects of individual compounds increasing their dangerousness and abuse potential.

Neurophysiological, psychological and behavioural correlates of rTMS treatment in alcohol dependence.

BACKGROUND: Addiction is associated with dorso-lateral prefrontal cortex (DLPFC) dysfunction and altered brain-oscillations. High frequency repetitive transcranial magnetic stimulation (HFrTMS) over DLPFC reportedly reduces drug craving. Its effects on neuropsychological, behavioural and neurophysiological are unclear. METHODS: We assessed psychological, behavioural and neurophysiological effects of 4 sessions of 10-min adjunctive HFrTMS over the left DLPFC during two weeks during a residential programme for alcohol detoxification. Participants were randomized to active HFrTMS (10 Hz, 100% motor threshold) or sham. Immediately before the first and after the last session, 32-channels EEG was recorded and alcohol craving Visual Analogue Scale, Symptom Check List-90-R, Numeric Stroop task and Go/No-go task administered. Tests were repeated at 1-month follow-up. RESULTS: 17 subjects (mean age 44.7 years, 4 F) were assessed. Active rTMS subjects performed better at Stroop test at end of treatment (p=0.036) and follow up (p=0.004) and at Go-NoGo at end of treatment (p=0.05) and follow up (p=0.015). Depressive symptoms decreased at end of active treatment (p=0.036). Active-TMS showed an overall decrease of fast EEG frequencies after treatment compared to sham (p=0.026). No significant modifications over time or group emerged for craving and number of drinks at follow up. CONCLUSION: 4 HFrTMS sessions over two weeks on the left DLPFC can improve inhibitory control task and selective attention and reduce depressive symptoms. An overall reduction of faster EEG frequencies was observed. Nonetheless, this schedule is ineffective in reducing craving and alcohol intake.