Tackling new psychoactive substances through metabolomics: UHPLC-HRMS study on natural and synthetic opioids in male and female murine models.

Novel psychoactive substances (NPS) represent a broad class of drugs new to the illicit market that often allow passing drug-screening tests. They are characterized by a variety of structures, rapid transience on the drug scene and mostly unknown metabolic profiles, thus creating an ever-changing scenario with evolving analytical targets. The present study aims at developing an indirect screening strategy for NPS monitoring, and specifically for new synthetic opioids (NSOs), based on assessing changes in endogenous urinary metabolite levels as a consequence of the systemic response following their intake. The experimental design involved in-vivo mice models: 16 animals of both sex received a single administration of morphine or fentanyl. Urine was collected before and after administration at different time points; the samples were then analysed with an untargeted metabolomics LC-HRMS workflow. According to our results, the intake of opioids resulted in an elevated energy demand, that was more pronounced on male animals, as evidenced by the increase in medium and long chain acylcarnitines levels. It was also shown that opioid administration disrupted the pathways related to catecholamines biosynthesis. The observed alterations were common to both morphine and fentanyl: this evidence indicate that they are not related to the chemical structure of the drug, but rather on the drug class. The proposed strategy may reinforce existing NPS screening approaches, by identifying indirect markers of drug assumption.

5-HT2A receptors are involved in the pharmaco-toxicological effects of the synthetic cannabinoids JWH-018 and 5F-PB22: In vivo studies in mice.

Over the last years, Synthetic Cannabinoids (SCs) have been among the largest and most frequently seized groups of Novel Psychoactive Substances (NPS). These substances have been frequently detected in biological samples from patients involved in several intoxication and death cases. Their serious adverse effects have been related to their action as potent agonist of cannabinoid CB1 receptors. However, evidence concerning the potential interaction between SCs and serotoninergic mechanisms has emerged. Therefore, this study aims to evaluate the involvement of 5-HT2A receptors in the effects induced by acute systemic administration of 1-pentyl-3-(1-naphthoyl)indole (JWH-018; 1 mg/kg) and quinolin-8-yl 1-pentyfluoro-1H-indole-3-8-carboxylate (5F-PB22; 1 mg/kg). Sensorimotor (visual, acoustic, and tactile) responses, pain threshold (acute mechanical and thermal nociception), core temperature, breath rate and motor performance (stepping activity) have been assessed in CD-1 male mice. The present results pointed out that both substances deeply alter sensorimotor responses, nociceptive threshold, core temperature, breath rate and motor activity in mice. Noteworthy, pretreatment with the selective 5-HT2A receptors antagonist MDL100907 (0.1 mg/kg) at least partially prevented sensorimotor disruption, antinociception and hypothermic effects. Conversely, the respiratory and motor impairment was not prevented. Thus, it states the relevance of serotoninergic 5-HT2A mechanisms on pharmaco-toxicological effects induced by SCs.

Pharmaco-toxicological effects of the novel tryptamine hallucinogen 5-MeO-MiPT on motor, sensorimotor, physiological, and cardiorespiratory parameters in mice-from a human poisoning case to the preclinical evidence.

RATIONALE: The 5-methoxy-N-methyl-N-isopropyltryptamine (5-MeO-MiPT, known online as "Moxy") is a new psychedelic tryptamine first identified on Italian national territory in 2014. Its hallucinogen effects are broadly well-known; however, only few information is available regarding its pharmaco-toxicological effects. OBJECTIVES: Following the seizure of this new psychoactive substances by the Arm of Carabinieri and the occurrence of a human intoxication case, in the current study we had the aim to characterize the in vivo acute effects of systemic administration of 5-MeO-MiPT (0.01-30 mg/kg i.p.) on sensorimotor (visual, acoustic, and overall tactile) responses, thermoregulation, and stimulated motor activity (drag and accelerod test) in CD-1 male mice. We also evaluated variation on sensory gating (PPI, prepulse inhibition; 0.01-10 mg/kg i.p.) and on cardiorespiratory parameters (MouseOx and BP-2000; 30 mg/kg i.p.). Lastly, we investigated the in silico ADMET (absorption, distribution, metabolism, excretion, toxicity) profile of 5-MeO-MiPT compared to 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) and N,N-dimethyltryptamine (DMT). RESULTS: This study demonstrates that 5-MeO-MiPT dose-dependently inhibits sensorimotor and PPI responses and, at high doses, induces impairment of the stimulated motor activity and cardiorespiratory changes in mice. In silico prediction shows that the 5-MeO-MiPT toxicokinetic profile shares similarities with 5-MeO-DIPT and DMT and highlights a cytochrome risk associated with this compound. CONCLUSIONS: Consumption of 5-MeO-MiPT can affect the ability to perform activities and pose a risk to human health status, as the correspondence between the effects induced in mice and the symptoms occurred in the intoxication case suggests. However, our findings suggest that 5-MeO-MiPT should not be excluded from research in the psychiatric therapy field.

Sex-specific behavioural, metabolic, and immunohistochemical changes after repeated administration of the synthetic cannabinoid AKB48 in mice.

BACKGROUND AND PURPOSE: AKB48 is a synthetic cannabinoid illegally sold for its psychoactive cannabis-like effects that have been associated with acute intoxication and whose effects are poorly known. EXPERIMENTAL APPROACH: Using a behavioural, neurochemical, and immunohistochemical approach, we investigated the pharmaco-toxicological effects, pharmacokinetics, and neuroplasticity at cannabinoid CB1 receptors in the cerebellum and cortex induced by repeated AKB48 administration in male and female mice. KEY RESULTS: The effects of AKB48 varied significantly depending on sex and treatment duration. The first injection impaired sensorimotor responses and reduced body temperature, analgesia, and breath rate to a greater extent in females than in males; the second injection induced stronger effects in males while the third injection of AKB48 induced weaker responses in both sexes, suggesting emergence of tolerance. The CB1 receptor antagonist NESS-0327 prevented the effects induced by repeated AKB48, confirming a CB1 receptor-mediated action. Blood AKB48 levels were higher in females than in males and repeated administration caused a progressive rise of AKB48 levels in both sexes, suggesting an inhibitory effect on cytochrome activity. Finally, immunohistochemical analysis revealed higher expression of CB1 receptors in the cerebellum and cortex of females, and a rapid CB1 receptor down-regulation in cerebellar and cortical areas following repeated AKB48 injections, with neuroadaptation occurring generally more rapidly in females than in males. CONCLUSION AND IMPLICATIONS: We have shown for the first time that AKB48 effects significantly vary with prolonged use and that sex affects the pharmacodynamic/pharmacokinetic responses to repeated administration, suggesting a sex-tailored approach in managing AKB48-induced intoxication.

Effect of Repeated Administration of ɣ-Valerolactone (GVL) and GHB in the Mouse: Neuroadaptive Changes of the GHB and GABAergic System.

BACKGROUND: Gamma-hydroxybutyric acid (GHB) at low dosages has anxiolytic effects and promotes REM sleep and low-wave deep sleep. In the U.S., the legal form of GHB is prescribed to adults suffering from narcolepsy-associated cataplexy; the sodium salt of GHB is reserved for alcohol-addiction treatment. GHB is also a molecule of abuse and recreational use, it is a controlled substance in several countries, so gamma-valerolactone (GVL) has frequently been used as a legal substitute for it. GHB's abuse profile is most likely attributable to its anxiolytic, hypnotic, and euphoric properties, as well as its widespread availability and inexpensive/low cost on the illicit market. METHODS: Our study is focused on evaluating the potential effects on the mouse brain after repeated/prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) through behavioral study and immunohistochemical analysis using the markers tetraspanin 17 (TSPAN17), aldehyde dehydrogenase 5 (ALDH5A1), Gamma-aminobutyric acid type A receptor (GABA-A), and Gamma-aminobutyric acid type B receptor (GABA-B). RESULTS: Our findings revealed that prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) can have effects on a component of the mouse brain, the intensity of which can be assessed using immunohistochemistry. The findings revealed that long-term GHB administration causes a significant plastic alteration of the GHB signaling system, with downregulation of the putative binding site (TSPAN17) and overexpression of ALDH5A1, especially in hippocampal neurons. Our findings further revealed that GABA-A and GABA-B receptors are downregulated in these brain locations, resulting in a greater decrease in GABA-B expression. CONCLUSIONS: The goal of this study, from the point of view of forensic pathology, is to provide a new methodological strategy for better understanding the properties of this controversial substance, which could help us better grasp the unknown mechanism underlying its abuse profile.

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.

Genotoxicity Evaluation of The Novel Psychoactive Substance MTTA.

MTTA, also known as mephtetramine, is a stimulant novel psychoactive substance characterized by a simil-cathinonic structure. To date, little has been studied on its pharmaco-toxicological profile, and its genotoxic potential has never been assessed. In order to fill this gap, the aim of the present work was to evaluate its genotoxicity on TK6 cells in terms of its ability to induce structural and numerical chromosomal aberrations by means of a cytofluorimetric protocol of the "In Vitro Mammalian Cell Micronucleus (MN) test". To consider the in vitro effects of both the parental compound and the related metabolites, TK6 cells were treated with MTTA in the absence or presence of an exogenous metabolic activation system (S9 mix) for a short-term time (3 h) followed by a recovery period (23 h). No statistically significant increase in the MNi frequency was detected. Specifically, in the presence of S9 mix, only a slight increasing trend was observable at all tested concentrations, whereas, without S9 mix, at 75 µM, almost a doubling of the negative control was reached. For the purposes of comprehensive evaluation, a long-term treatment (26 h) was also included. In this case, a statistically significant enhancement in the MNi frequency was observed at 50 µM.

MAM-2201 acute administration impairs motor, sensorimotor, prepulse inhibition, and memory functions in mice: a comparison with its analogue AM-2201.

RATIONALE: 1-[(5-fluoropentyl)-1H-indol-3-yl](4-methyl-1-naphthalenyl) methanone (MAM-2201) is a potent synthetic cannabinoid receptor agonist illegally marketed in "spice" products and as "synthacaine" for its psychoactive effects. It is a naphthoyl-indole derivative which differs from its analogue 1-[(5-Fluoropentyl)-1H-indol-3-yl](1-naphthylenyl) methanone (AM-2201) by the presence of a methyl substituent on carbon 4 (C-4) of the naphthoyl moiety. Multiple cases of intoxication and impaired driving have been linked to AM-2201 and MAM-2201 consumption. OBJECTIVES: This study aims to investigate the in vitro (murine and human cannabinoid receptors) and in vivo (CD-1 male mice) pharmacodynamic activity of MAM-2201 and compare its effects with those induced by its desmethylated analogue, AM-2201. RESULTS: In vitro competition binding studies confirmed that MAM-2201 and AM-2201 possess nanomolar affinity for both CD-1 murine and human CB1 and CB2 receptors, with preference for the CB1 receptor. In agreement with the in vitro binding data, in vivo studies showed that MAM-2201 induces visual, acoustic, and tactile impairments that were fully prevented by pretreatment with CB1 receptor antagonist/partial agonist AM-251, indicating a CB1 receptor mediated mechanism of action. Administration of MAM-2201 also altered locomotor activity and PPI responses of mice, pointing out its detrimental effect on motor and sensory gating functions and confirming its potential use liability. MAM-2201 and AM-2201 also caused deficits in short- and long-term working memory. CONCLUSION: These findings point to the potential public health burden that these synthetic cannabinoids may pose, with particular emphasis on impaired driving and workplace performance.

Acute Cardiovascular and Cardiorespiratory Effects of JWH-018 in Awake and Freely Moving Mice: Mechanism of Action and Possible Antidotal Interventions?

JWH-018 is the most known compound among synthetic cannabinoids (SCs) used for their psychoactive effects. SCs-based products are responsible for several intoxications in humans. Cardiac toxicity is among the main side effects observed in emergency departments: SCs intake induces harmful effects such as hypertension, tachycardia, chest pain, arrhythmias, myocardial infarction, breathing impairment, and dyspnea. This study aims to investigate how cardio-respiratory and vascular JWH-018 (6 mg/kg) responses can be modulated by antidotes already in clinical use. The tested antidotes are amiodarone (5 mg/kg), atropine (5 mg/kg), nifedipine (1 mg/kg), and propranolol (2 mg/kg). The detection of heart rate, breath rate, arterial oxygen saturation (SpO2), and pulse distention are provided by a non-invasive apparatus (Mouse Ox Plus) in awake and freely moving CD-1 male mice. Tachyarrhythmia events are also evaluated. Results show that while all tested antidotes reduce tachycardia and tachyarrhythmic events and improve breathing functions, only atropine completely reverts the heart rate and pulse distension. These data may suggest that cardiorespiratory mechanisms of JWH-018-induced tachyarrhythmia involve sympathetic, cholinergic, and ion channel modulation. Current findings also provide valuable impetus to identify potential antidotal intervention to support physicians in the treatment of intoxicated patients in emergency clinical settings.

Ethanol enhances JWH-018-induced impairment of sensorimotor and memory functions in mice: From preclinical evidence to forensic implication in Driving Under the Influence of Drugs.

BACKGROUND: Several new Synthetic Cannabinoids have appeared each year since their introduction into the illicit drug market as recreational drugs. Among these, naphtalen-1-yl-(1-pentylindol-3-yl) methanone (JWH-018) is one of the most detected compounds in biological samples from patients involved in intoxication or death cases. Furthermore, consumption of JWH-018 has been linked to several cases of Driving Under the Influence of Drugs (DUID) suggesting that effects induced by this compound can affect individuals' ability to drive. METHODS: Given the high spread of polydrug consumption and the wide number of alcohol-related traffic accidents, this study aims to investigate the acute effects induced by co-administration of JWH-018 with ethanol on sensorimotor and motor responses, grip strength and memory functions in CD-1 male mice. Acute impairments induced by JWH-018 and ethanol alone have also been investigated, in order to compare their effects with that induced by their concurrent administration. RESULTS: In vivo behavioral experiments revealed a worsening of the cognitive and sensorimotor disruption after the co-administration of JWH-018 with ethanol compared to single compounds. CONCLUSIONS: These animal-based findings suggest a potential increased impairment on psychomotor performances which could be related to driving abilities posed by poly-drug consumption involving SCs and ethanol.

Pharmaco-Toxicological Effects of Atypical Synthetic Cathinone Mephtetramine (MTTA) in Mice: Possible Reasons for Its Brief Appearance over NPSs Scene.

Over the last year, NPSs have been steadily on the rise in the illicit drug market. Among these, synthetic cathinones seem to become increasingly popular among young adults, mainly because of their ability to replicate the effects of traditional psychostimulant drugs, such as cocaine, MDMA and amphetamines. However, scarce data are available about the in vivo pharmaco-toxicology of these new substances. To this end, this study focused on evaluation of effects induced by repeated administration of mephtetramine (MTTA 0.1-30 mg/kg i.p.) in mice. This atypical cathinone highlighted a sensorial (inhibition of visual and acoustic reflexes) and transient physiological parameter (decrease in breath rate and temperature) change in mice. Regarding motor activity, both a dose-dependent increase (accelerod test) and biphasic effect (drag and mobility time test) have been shown. In addition, blood and urine samples have been analysed to enrich the experimental featuring of the present study with reference to evaluation of potential toxicity related to consumption of MTTA. The latter analysis has particularly revealed important changes in blood cells count and blood and urine physicochemical profile after repeated treatment with this atypical cathinone. Moreover, MTTA induced histological changes in heart, kidney and liver samples, emphasizing its potential toxicity.

Behavioral and Pharmacokinetics Studies of N-Methyl-2-Aminoindane (NM2AI) in Mice: An Aminoindane Briefly Used in the Illicit Drug Market.

Drug forums are considered as the main platform sources that have contributed to the increase in NPS popularity, especially for those not yet known to law enforcement and therefore not yet illegal. An example is the new synthetic stimulant NM2AI, which has a very short history of human use and abuse. Little is known regarding this compound, but some information from internet forums and the scientific literature indicates NM2AI as a structural derivate of MDAI, which is known for its entactogenic activity. Indeed, the purpose of this study is to evaluate, for the first time, the in vivo acute effect induced by the intraperitoneal injection of NM2AI (1-10-30-100 mg/kg) in mice. We demonstrate the sensory (by visual placing and object tests) and physiological (core temperature measurement) function variations, nociceptor (by tail pinch test) and strength (grip test) alterations, and sensorimotor (time on rod and mobility) decrease. Moreover, we verify the mild hallucinogenic effect of NM2AI (by startle/prepulse inhibition test). Lastly, we perform a pharmacokinetic study on mice blood samples, highlighting that the main active metabolite of NM2AI is 2-aminoindane (2AI). Taken together, our data confirm the suspected entactogenic activity of NM2AI; however, these in vivo effects appear atypical and less intense with respect to those induced by the classic stimulants, in surprising analogy with what is reported by networked users.

The Old and the New: Cardiovascular and Respiratory Alterations Induced by Acute JWH-018 Administration Compared to Δ9-THC-A Preclinical Study in Mice.

Several new psychoactive substances (NPS) are responsible for intoxication involving the cardiovascular and respiratory systems. Among NPS, synthetic cannabinoids (SCs) provoked side effects in humans characterized by tachycardia, arrhythmias, hypertension, breathing difficulty, apnoea, myocardial infarction, and cardiac arrest. Therefore, the present study investigated the cardio-respiratory (MouseOx Plus; EMKA electrocardiogram (ECG) and plethysmography TUNNEL systems) and vascular (BP-2000 systems) effects induced by 1-naphthalenyl (1-pentyl-1H-indol-3-yl)-methanone (JWH-018; 0.3-3-6 mg/kg) and Δ9-tetrahydrocannabinol (Δ9-THC; 0.3-3-6 mg/kg), administered in awake CD-1 male mice. The results showed that higher doses of JWH-018 (3-6 mg/kg) induced deep and long-lasting bradycardia, alternated with bradyarrhythmia, spaced out by sudden episodes of tachyarrhythmias (6 mg/kg), and characterized by ECG electrical parameters changes, sustained bradypnea, and systolic and transient diastolic hypertension. Otherwise, Δ9-THC provoked delayed bradycardia (minor intensity tachyarrhythmias episodes) and bradypnea, also causing a transient and mild hypertensive effect at the tested dose range. These effects were prevented by both treatment with selective CB1 (AM 251, 6 mg/kg) and CB2 (AM 630, 6 mg/kg) receptor antagonists and with the mixture of the antagonists AM 251 and AM 630, even if in a different manner. Cardio-respiratory and vascular symptoms could be induced by peripheral and central CB1 and CB2 receptors stimulation, which could lead to both sympathetic and parasympathetic systems activation. These findings may represent a starting point for necessary future studies aimed at exploring the proper antidotal therapy to be used in SCs-intoxicated patient management.

Sensorimotor Alterations Induced by Novel Fentanyl Analogs in Mice: Possible Impact on Human Driving Performances.

Operating a vehicle is a complex task that requires multiple cognitive functions and psychomotor skills to cooperate. Driving might be impaired by licit or illicit drugs, including novel psychoactive substances (NPS) and novel synthetic opioids (NSO), the effects of which are still yet to be elucidated in humans. In the present work, a revision of the literature regarding the psychomotor impairing effects of Fentanyl (FENT) and three analogues (Acrylfentanyl, Ocfentanyl and Furanylfentanyl) is presented, as emerged by experimental studies on humans, driving under the influence of a drug (DUID) and intoxication cases. An experimental study on a mouse model evaluated the sensorimotor alterations induced by FENT and the three fentalogs. Acute systemic administration of the four opioids (0.01-15 mg/kg i.p.) dose-dependently decreased the visual object and placing tests, the acoustic and the tactile responses of mice. The preclinical data are in accordance with the data that emerged from the revision of the literature regarding experimental data on humans, driving under the influence of drugs and intoxication cases, suggesting that novel synthetic opioids might affect the psychomotor performances on daily human tasks with a particular focus on driving.

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.

In Vivo Bio-Activation of JWH-175 to JWH-018: Pharmacodynamic and Pharmacokinetic Studies in Mice.

3-(1-Naphthalenylmethyl)-1-pentyl-1H-indole (JWH-175) is a synthetic cannabinoid illegally marketed for its psychoactive cannabis-like effects. This study aimed to investigate and compare in vitro and in vivo pharmacodynamic activity of JWH-175 with that of 1-naphthalenyl (1-pentyl-1H-indol-3-yl)-methanone (JWH-018), as well as evaluate the in vitro (human liver microsomes) and in vivo (urine and plasma of CD-1 male mice) metabolic profile of JWH-175. In vitro binding studies showed that JWH-175 is a cannabinoid receptor agonist less potent than JWH-018 on mouse and human CB1 and CB2 receptors. In agreement with in vitro data, JWH-175 reduced the fESPS in brain hippocampal slices of mice less effectively than JWH-018. Similarly, in vivo behavioral studies showed that JWH-175 impaired sensorimotor responses, reduced breath rate and motor activity, and increased pain threshold to mechanical stimuli less potently than JWH-018. Metabolic studies demonstrated that JWH-175 is rapidly bioactivated to JWH-018 in mice blood, suggesting that in vivo effects of JWH-175 are also due to JWH-018 formation. The pharmaco-toxicological profile of JWH-175 was characterized for the first time, proving its in vivo bio-activation to the more potent agonist JWH-018. Thus, it highlighted the great importance of investigating the in vivo metabolism of synthetic cannabinoids for both clinical toxicology and forensic purposes.

Epigenetic Studies for Evaluation of NPS Toxicity: Focus on Synthetic Cannabinoids and Cathinones.

In the recent decade, numerous new psychoactive substances (NPSs) have been added to the illicit drug market. These are synthetized to mimic the effects of classic drugs of abuse (i.e., cannabis, cocaine, etc.), with the purpose of bypassing substance legislations and increasing the pharmacotoxicological effects. To date, research into the acute pharmacological effects of new NPSs is ongoing and necessary in order to provide an appropriate contribution to public health. In fact, multiple examples of NPS-related acute intoxication and mortality have been recorded in the literature. Accordingly, several in vitro and in vivo studies have investigated the pharmacotoxicological profiles of these compounds, revealing that they can cause adverse effects involving various organ systems (i.e., cardiovascular, respiratory effects) and highlighting their potential increased consumption risks. In this sense, NPSs should be regarded as a complex issue that requires continuous monitoring. Moreover, knowledge of long-term NPS effects is lacking. Because genetic and environmental variables may impact NPS responses, epigenetics may aid in understanding the processes behind the harmful events induced by long-term NPS usage. Taken together, "pharmacoepigenomics" may provide a new field of combined study on genetic differences and epigenetic changes in drug reactions that might be predictive in forensic implications.

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.