Over the past decade, new psychoactive substances (NPS) have emerged in the illegal drug market and have continued to attract attention from the international community. Among these, amphetamine-like NPS, classified as stimulants, constitute a significant proportion. However, the pharmacological characteristics and mechanisms underlying addiction to amphetamine-like NPS remain poorly understood. Given that circadian rhythms are linked to the brain stimulation effects of methamphetamine (METH) and amphetamine, we investigated the effects of METH, 1-(4-methoxyphenyl)-N-methylpropan-2-amine (PMMA), and 1-(benzofuran-5-yl)-N-ethylpropan-2-amine (5-EAPB) on intracranial self-stimulation (ICSS) in wild-type (WT) or Period circadian regulator 2 knockout mice. Amphetamine-like drugs increase intracellular Ca2+ levels to provoke dopamine release, so we examined the impact of Per2 knockdown on intracellular Ca2+ levels in PC12 cells to elucidate a potential mechanism underlying NPS-induced ICSS enhancement. Our ICSS results showed that METH and PMMA significantly increased brain stimulation in Per2 knockout mice compared to WT mice. Similarly, METH and PMMA induced higher Ca2+ fluorescence intensity in Per2 knockdown PC12 cells than in control cells. In contrast, 5-EAPB did not produce significant changes in either ICSS or Ca2+ signaling. These findings suggest that Per2 plays a crucial role in the brain stimulation effects of amphetamine-like drugs through the regulation of intracellular Ca2+.

Recently, hexahydrocannabinol (HHC) was posed under strict control in Europe due to the increasing HHC-containing material seizures. The lack of analytical methods in clinical laboratories to detect HHC and its metabolites in biological matrices may result in related intoxication underreporting. We developed and validated a comprehensive GC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC, 9αOH-HHC, 9βOH-HHC, 8(R)OH-9(R)-HHC, 8(S)OH-9(S)HHC, 11OH-9(R)HHC, 11OH-9(S)HHC, 11nor-carboxy-9(R)-HHC, and 11nor-carboxy-9(S)-HHC in whole blood, urine, and oral fluid. A novel QuEChERS extraction protocol was optimized selecting the best extraction conditions suitable for all the three matrices. Urine and blood were incubated with β-glucuronidase at 60 °C for 2 h. QuEChERS extraction was developed assessing different ratios of Na2SO4:NaCl (4:1, 2:1, 1:1, w/w) to be added to 200 µL of any matrix added with acetonitrile. The chromatographic separation was achieved on a 7890B GC with an HP-5ms column, (30 m, 0.25 mm × 0.25 µm) in 12.50 min. The analytes were detected with a triple-quadrupole mass spectrometer in the MRM mode. The method was fully validated following OSAC guidelines. The method showed good validation parameters in all the matrices. The method was applied to ten real samples of whole blood (n = 4), urine (n = 3), and oral fluid (n = 3). 9(R)-HHC was the prevalent epimer in all the samples (9(R)/9(S) = 2.26). As reported, hydroxylated metabolites are proposed as urinary biomarkers, while carboxylated metabolites are hematic biomarkers. Furthermore, 8(R)OH-9(R)HHC was confirmed as the most abundant metabolite in all urine samples.

OBJECTIVE: New psychoactive substances such as N-ethylpentylone (NEP) are continuously emerging in the illicit drug market, and knowledge of their effects and risks, which may vary between sexes, is scarce. Our present study compares some key effects of NEP in male and female mice.
METHODS: Psychostimulant, rewarding and reinforcing effects were investigated by tracking locomotor activity, conditioned place preference (CPP) paradigm and through a self-administration (SA) procedure, respectively, in CD1 mice. Moreover, the expression of early genes (C-fos, Arc, Csnk1e, Pdyn, Pp1r1b and Bdnf in addiction-related brain areas) was assessed by qPCR. Finally, serum and brain levels of NEP were determined by UHPLC-MS/MS.
RESULTS: NEP-treated males experimented locomotor sensitisation and showed higher and longer increases in locomotion as well as higher hyperthermia after repeated administration than females. Moreover, while preference score in the CPP was similar in both sexes, extinction occurred later, and reinstatement was more easily established for males. Female mice self-administered more NEP than males at a higher dose. Differences in early gene expression (Arc, Bdnf, Csnk1e and Ppp1r1b) were found, but the serum and brain NEP levels did not differ between sexes.
CONCLUSIONS: Our results suggest that male mice are more sensitive to NEP psychostimulant and rewarding effects. These differences may be attributed to different early gene expression but not to pharmacokinetic factors. Moreover, males appear to be more vulnerable to the hyperthermic effects of NEP, while females might be more prone to NEP abuse.

Drug use is prevalent in prisons with drugs associated with depressant effects found to be more prevalent than stimulants. Synthetic cathinones (SCats; often sold as \"bath salts\", \"ecstasy\", \"molly\", and \"monkey dust\") are the second largest category of new psychoactive substances (NPS) currently monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and are commonly used as substitutes for regulated stimulants, such as amphetamine, cocaine, and MDMA. N,N-dimethylpentylone (also known as dimethylpentylone, dipentylone, and bk-DMBDP) was detected for the first time in the Scottish prisons in seven powder samples seized between January and July 2023. Samples were analyzed using gas chromatography-mass spectrometry (GC-MS), ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QToF-MS), and nuclear magnetic resonance imaging (NMR). Dimethylpentylone was detected alongside other drugs in four samples, including the novel benzodiazepine desalkylgidazepam (bromonordiazepam) and the synthetic cannabinoid receptor agonists (SCRAs) MDMB-INACA and MDMB-4en-PINACA.

Systematic retrospective processing of previously analysed biological samples has been proven to be a valuable tool in the search for new drugs (e.g. new psychoactive substances (NPS)) and for quality assessment in clinical and forensic toxicology. In a previous study, we developed a strategy for retrospective data-analysis using a personalized library of synthetic cannabinoids, designer benzodiazepines and synthetic opioids obtained from the crowdsourced database HighResNPS (https://highresnps.com). In this study, the same strategy was employed for the compounds within the groups of NPS that were not previously included such as synthetic cathinones, phenethylamines, aminoindanes, arylalkylamines, piperazine derivates, piperidines, pyrrolidines, indolalkylamines and arylcyclohexylamines. Synthetic opioids and designer benzodiazepines, which were not part of the previous study, were also included. To enhance the effectiveness of the retrospective analysis, a predicted retention time was included for all entries. Data files from the analysis of 2186 forensic post mortem samples with an Agilent Technologies 6540 ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) performed in the laboratory from January 2014 to December 2021 were retrospectively processed with the up-to-date library. Tentative findings were classified in two groups: The findings where MS/MS data was acquired for library match (category 1) and the less certain findings where such data lacked (category 2). Five compounds of category 1 (three synthetic cathinones and two indolalkylamines) were identified in 12 samples. Only one of the findings, 4-MEAPP (4-methyl-α-ethylaminopentiophenone), was deemed plausible after reviewing case information. As many as 501 presumably positive category 2 findings were detected. Using the predicted retention time as an additional criterion the number was significantly reduced but still too high for a manual review. This work has demonstrated that the strategy developed in the previous study can be applied to other NPS groups. However, it is important to note the limitations such a method may have in detecting compounds at very low concentrations.

The development of lysergic acid diethylamide (LSD) derivatives and analogs continues to inform the design of novel receptor probes and potentially new medicines. On the other hand, a number of newly developed LSD derivatives have also emerged as recreational drugs, leading to reports of their detection in some countries. One position in the ergoline scaffold of LSD that is frequently targeted is the N1-position; numerous N1-alkylcarbonyl LSD derivatives have been reported where the acyl chain is attached to the indole nitrogen, for example, in the form of linear n-alkane substituents, which represent higher homologs of the prototypical 1-acetyl-N,N-diethyllysergamide (1A-LSD, ALD-52). In this study, 1-hexanoyl-LSD (1H-LSD, SYN-L-027), a novel N1-acyl LSD derivative, was characterized analytically using standard techniques, followed by evaluation of its in vivo behavioral effects using the mouse head-twitch response (HTR) assay in C57BL/6J mice. 1H-LSD induced the HTR, with a median effective dose (ED50) of 192.4 μg/kg (equivalent to 387 nmol/kg), making it roughly equipotent to ALD-52 when tested previously under similar conditions. Similar to other N1-acylated analogs, 1H-LSD is anticipated to by hydrolyzed to LSD in vivo and acts as a prodrug. It is currently unknown whether 1H-LSD has appeared as on the research chemical market or is being used recreationally.

UNASSIGNED: Cannabis is the most common recreational drug worldwide and synthetic cannabinoid receptor agonists are currently the largest group of new psychoactive substances. The aim of this study was to compare the clinical features and outcomes of lone acute cannabis toxicity with lone acute synthetic cannabinoid receptor agonist toxicity in a large series of presentations to European emergency departments between 2013-2020.
UNASSIGNED: Self-reported drug exposure, clinical, and outcome data were extracted from the European Drug Emergencies Network Plus which is a surveillance network that records data on drug-related emergency department presentations to 36 centres in 24 European countries. Cannabis exposure was considered the control in all analyses. To compare the lone cannabis and lone synthetic cannabinoid receptor agonist groups, univariate analysis using chi squared testing was used for categorical variables and non-parametric Mann-Whitney U- testing for continuous variables. Statistical significance was defined as a P value of <0.05.
UNASSIGNED: Between 2013-2020 there were 54,314 drug related presentations of which 2,657 were lone cannabis exposures and 503 lone synthetic cannabinoid receptor agonist exposures. Synthetic cannabinoid receptor agonist presentations had statistically significantly higher rates of drowsiness, coma, agitation, seizures and bradycardia at the time of presentation. Cannabis presentations were significantly more likely to have palpitations, chest pain, hypertension, tachycardia, anxiety, vomiting and headache.
UNASSIGNED: Emergency department presentations involving lone synthetic cannabinoid receptor agonist exposures were more likely to have neuropsychiatric features and be admitted to a psychiatric ward, and lone cannabis exposures were more likely to have cardiovascular features. Previous studies have shown variability in the acute toxicity of synthetic cannabinoid receptor agonists compared with cannabis but there is little comparative data available on lone exposures. There is limited direct comparison in the current literature between lone synthetic cannabinoid receptor agonist and lone cannabis exposure, with only two previous poison centre series and two clinical series. Whilst this study is limited by self-report being used to identify the drug(s) involved in the presentations, previous studies have demonstrated that self-report is reliable in emergency department presentations with acute drug toxicity.
UNASSIGNED: This study directly compares presentations with acute drug toxicity related to the lone use of cannabis or synthetic cannabinoid receptor agonists. It supports previous findings of increased neuropsychiatric toxicity from synthetic cannabinoid receptor agonists compared to cannabis and provides further data on cardiovascular toxicity in lone cannabis use.

Lysergic acid diethylamide (LSD) analogs have emerged as new psychoactive substances (NPS) since the mid-2010s, and new compounds continue to emerge for recreational use. Since the end of 2023, \"1D-AL-LAD\" appeared on X (formerly Twitter) and other websites. As for the compound \"1D-LSD\" (which also has \"1D\" in the name), several studies show that the ingredient of seized blotter paper printed \"1D-LSD\" was actually 1-(2-thienoyl)-LSD (1T-LSD). However, there are no reports of seizures of 1-(1,2-dimethylcyclobutanecarbonyl)-LSD (1D-LSD). Accordingly, it was considered that all or at least a certain percentage of \"1D-AL-LAD (1-(1,2-dimethylcyclobutanecarbonyl)-6-allyl-nor-LSD)\" is actually 1-(2-thienoyl)-6-allyl-nor-LSD (1T-AL-LAD). This compound is handled by a number of distributors as of April 2024; therefore, it should be characterized in advance if seized. In this study, 1T-AL-LAD was synthesized and characterized using nuclear magnetic resonance spectroscopy, Fourier transform-infrared spectroscopy, liquid chromatography/high-resolution mass spectrometry (LC/HRMS) and gas chromatography/MS (GC/MS). This compound was easily distinguished from previously reported lysergamides. There were some differences in the detectability of 1T-AL-LAD compared with other lysergamides using GC/MS and the fragmentation patterns in LC/HRMS. These differences can be reasonably explained. This information will be of help to determine this substance in seized materials should it emerge on the market.

Substituted phenethylamines including 2C (2,5-dimethoxyphenethylamines) and NBOMe (N-(2-methoxybenzyl)phenethylamines) drugs are potent psychoactive substances with little to no knowledge available on their toxicity. In the present in vitro study, we explored the mechanisms underlying the neurotoxicity of six substituted phenethylamines: 2C-T-2, 2C-T-4, 2C-T-7 and their corresponding NBOMes. These drugs were synthesized and chemically characterized, and their cytotoxicity (0-1000 μM) was evaluated in differentiated SH-SY5Y cells and primary rat cortical cultures, by the NR uptake and MTT reduction assays. In differentiated SH-SY5Y cells, mitochondrial membrane potential, intracellular ATP and calcium levels, reactive oxygen species production, and intracellular total glutathione levels were also evaluated. All the tested drugs exhibited concentration-dependent cytotoxic effects towards differentiated SH-SY5Y cells and primary rat cortical cultures. The NBOMe drugs presented higher cytotoxicity than their counterparts, which correlates with the drug\'s lipophilicity. These cytotoxic effects were associated with mitochondrial dysfunction, evident through mitochondrial membrane depolarization and lowered intracellular ATP levels. Intracellular calcium imbalance was observed for 2C-T-7 and 25T7-NBOMe, implying a disrupted calcium regulation. Although reactive species levels remained unchanged, a reduction in intracellular total GSH content was observed. Overall, these findings contribute to a deeper understanding of these drugs, shedding light on the mechanisms underpinning their neurotoxicity.

With a rising demand of cocaine over the last years, it is likely that unregulated new psychoactive substances with similar effects such as indatraline ((1R,3S)-3-(3,4-dichlorophenyl)-N-methyl-2,3-dihydro-1H-inden-1-amine) and troparil (Methyl (1R,2S,3S,5S)-8-methyl-3-phenyl-8-azabicyclo[3.2.1]octane-2-carboxylate) become popular as well. Both substances share a similar pharmacological profile as cocaine, while their potency is higher, and their duration of action is longer. This study investigated their metabolic fate in rat urine and incubations using pooled human liver S9 fraction (pHLS9). Indatraline formed two phase I and four phase II metabolites, with aromatic hydroxylation and glucuronidation being the main metabolic steps. All metabolites were detected in rat urine, while the parent compound was not detectable. Although low in abundance, indatraline metabolites were well identifiable due to their specific isotopic patterns caused by chlorine. Troparil formed four phase I and three phase II metabolites, with demethylation being the main metabolic step. Hydroxylation of the tropane ring, the phenyl ring, and combinations of these steps, as well as glucuronidation, were found. Phase I metabolites were detectable in rat urine and pHLS9, while phase II metabolites were only detectable in rat urine.