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.

The metabolism of 3‐chloromethcathinone (3‐CMC) was studied after controlled administration in a murine model using the dried blood spot (DBS) technique for the sampling, storage and purification of blood samples. Liquid chromatography–high‐resolution mass spectrometry (LC‐HRMS) was used for the identification of metabolites and investigation of their fragmentation pattern. Subsequently, a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for their identification and 3‐CMC quantification in routine workload. The main metabolites identified were two stereoisomers of dihydro‐CMC, N‐demethyl‐CMC, and dihydro‐N‐demethyl‐CMC. The stability of 3‐CMC and of its metabolites deposited on DBS was evaluated by replicate analyses after 30, 50, and 90 days, demonstrating a decrease in concentration. It was more pronounced for 3‐CMC, with −67% and −82% percentage deviation from the initial concentrations, and for N‐demethyl 3‐CMC (decrease comprised between −48% and −88%) than for the di‐hydro metabolites, ranging from −5% to −37%. Regardless, all of them were detectable till 90 days after deposition as DBS. The possibility of identifying 3‐CMC and its metabolites with high sensitivity is an invaluable tool for the diagnosis of exposure to the substance, also in low doses or after some hours, and for various applications in clinical and forensic toxicology, such as driving under the influence, drug‐facilitated crimes, and addiction to intoxications. DBS demonstrated to be a reliable technique for the sampling, storage, and purification of the blood specimen for 3‐CMC and metabolite detection.

To date, the exposure to Synthetic Cannabinoids (SCs) has been linked to unanticipated psychiatric symptoms such as agitation, psychosis, and aggressive behavior. In line with this, preclinical studies have shown that acute and long-term exposure to these compounds can result in psychostimulant effects that may be related to CB₁-mediated and dopamine-dependent mechanisms. This study focuses on the progressive effects induced by repeated injection of 1-pentyl-3-(1-naphthoyl)indole JWH-018 (6 mg/kg, i.p.) on the locomotor activity and aggressive behavior in adult male ICR-CD1® mice. Thus, the interaction with the cannabinoid CB₁ receptor-preferring antagonist/inverse agonist AM-251 (6 mg/kg, i.p.), the dopamine D_1/5 receptor antagonist SCH23390 (0.1 mg/kg, i.p.), and the dopamine D_2/3 receptor antagonist haloperidol (0.05 mg/kg, i.p.) have been evaluated. Expression and distribution of D₁ and D₂ receptors and tyrosine hydroxylase (TH) have been also investigated by immunohistochemistry on brain and cerebellar samples to explore potential neuroplastic events. The repeated treatment with JWH-018 lead to the exacerbation of unanticipated psychomotor agitation, progressively increasing spontaneous locomotion and aggressiveness. Pre-treatment with AM-251 prevents the effects induced by the SC first, third and seventh injection. SCH23390 and haloperidol significantly attenuate and fully prevent the effects induced by JWH-018 seventh injection when pre-administered, respectively, alone and in combination. Behavioral changes observed in JWH-018-treated mice are accompanied by alterations in cortical, hippocampal, striatal and cerebellar D₁, D₂ and TH gene expression levels. The present results demonstrated that repeated treatment with high dosage of JWH-018 induces psycho-stimulants effects via both CB₁ receptor-mediated and dopamine-dependent mechanisms.

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-HT_2A 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-HT_2A 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-HT_2A mechanisms on pharmaco-toxicological effects induced by SCs.

Alpha-Pyrrolidinohexanophenone (α-PHP) is a synthetic pyrovalerone derivative with structural characteristics and stimulant effects on humans comparable to α-PVP and MDPV. Since its pharmaco-toxicological effects have been poorly investigated, the aim of this study was to evaluate the acute effects of α-PHP (0.1-30mg/kg; i.p.) on behavioral responses in CD-1 male and female mice. Sex-related differences in pharmacokinetic profile of α-PHP (30mg/kg; i.p.) in mice were evaluated by analyzing i) the urine concentration of α-PHP and its metabolites at different time points, and ii) α-PHP levels in plasma, brain, and kidneys at 35min after the injection. Clinical data related to α-PHP intoxications, recorded by the Pavia Poison Control Centre (PCC) are also described. The present study shows that female mice were more sensitive to the effects of α-PHP on visual object, tactile, mobility time, and hypothermia, but males showed a deeper effect on visual placing. Both sexes developed analgesia to the mechanical stimulation, but only males showed a slight increase in enduring the thermal stimulation. Male mice showed higher plasma levels of α-PHP and a different elimination of α-PHP and metabolites than females. Case reports highlighted severe toxidromes characterized by Central Nervous System alterations (psychomotor agitation, tremors/fasciculations, hallucinations), cardiovascular toxicity signs (tachycardia, tachypnoea, thoracic pain) and other peripheral symptoms (hyperthermia, rhabdomyolysis). Our findings highlight the importance of the in vivo investigation of the effects and pharmacokinetic differences in male and female mice, to make contribution to the translational toxicological and forensic sex-related value.