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Many new psychoactive substances (NPS) are stimulants, and information about their potency and abuse potential is often lacking. To start addressing this need, a method measuring the inhibition of the dopamine, serotonin, and norepinephrine transporters (DAT, SERT, and NET) by stimulant drugs was developed. The use of a proprietary fluorescent dye mixture and three cell lines (CHO‐K1, HEK 293, and MDCK), each expressing a single transporter, allowed for a semiautomated, one‐pot determination of inhibition in a 384‐well format. The method was validated using well characterized stimulants, including cocaine, amphetamine, 3,4‐methylenedioxymethamphetamine (MDMA), α‐PVP, and fluoxetine and performed similarly to other methods. Seven synthetic cathinones all showed highest potency for DAT inhibition, followed by NET and SERT. The rank potency for DAT inhibition IC50 (nM) was MPHP (4.53) > 4Cl‐α‐PVP (8.05) > 3F‐α‐PVP (12.7) > α‐PiHP (13.4) > N‐ethylpentylone (16.9) > N‐ethylhexedrone (44.5) > 4‐methylpentedrone (261). All but 4‐methylpentedrone were more potent than amphetamine (257) and cocaine (111). The DAT/SERT inhibition ratio for the cathinones was in the range from 5.02 for 4‐methylpentedrone to >3730 for α‐PiHP, compared to 1.64 for cocaine and >4030 for α‐PVP. All seven substances had inhibition profiles similar to those of potent stimulants with high abuse potential. [ABSTRACT FROM AUTHOR]

Acetylbenzylfentanyl, benzoylbenzylfentanyl, 3‐fluoro‐methoxyacetylfentanyl, and 3‐phenylpropanoylfentanyl are fentanyl analogs that have been reported to the European Monitoring Centre for Drugs and Drug Addiction in recent years. The aim of this study was to identify metabolic pathways and potential biomarker metabolites of these fentanyl analogs. The compounds were incubated (5 μM) with cryopreserved hepatocytes for up to 5 h in vitro. Metabolites were analyzed with liquid chromatography–quadrupole time of flight–high‐resolution mass spectrometry (LC‐QTOF‐HRMS). The experiments showed that acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3‐phenylpropanoylfentanyl were mainly metabolized through N‐dealkylation (forming nor‐metabolites) and 3‐fluoro‐methoxyacetylfentanyl mainly through demethylation. Other observed metabolites were formed by mono‐/dihydroxylation, dihydrodiol formation, demethylation, dehydrogenation, amide hydrolysis, and/or glucuronidation. The experiments showed that a large number of metabolites of 3‐phenylpropanoylfentanyl were formed. The exact position of hydroxy groups in formed monohydroxy metabolites could not be established solely based upon recorded MSMS spectra of hepatocyte samples. Therefore, potential monohydroxy metabolites of 3‐phenylpropanoylfentanyl, with the hydroxy group in different positions, were synthesized and analyzed together with the hepatocyte samples. This approach could reveal that the β position of the phenylpropanoyl moiety was highly favored; β‐OH‐phenylpropanoylfentanyl was the most abundant metabolite after the nor‐metabolite. Both metabolites have the potential to serve as biomarkers for 3‐phenylpropanoylfentanyl. The nor‐metabolites of acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3‐fluoro‐methoxyacetylfentanyl do also seem to be suitable biomarker metabolites, as do the demethylated metabolite of 3‐fluoro‐methoxyacetylfentanyl. Identified metabolic pathways and formed metabolites were in agreement with findings in previous studies of similar fentanyl analogs. [ABSTRACT FROM AUTHOR]

4′Cl‐cumyl‐PINACA (SGT‐157), 4′F‐cumyl‐5F‐PINACA (4F‐cumyl‐5F‐PINACA, SGT‐65) and 4′F‐cumyl‐5F‐PICA (4F‐cumyl‐5F‐PICA, SGT‐64) are a series of new halogenated cumyl synthetic cannabinoid receptor agonists (SCRAs). Due to rapid metabolism, monitoring and screening for SCRAs in biological matrices requires identification of their metabolites. It is an essential tool for estimating their spread and fluctuations in the global illicit market. The purpose of this study was to identify human biotransformations of 4′Cl‐cumyl‐PINACA, 4′F‐cumyl‐5F‐PINACA and 4′F‐cumyl‐5F‐PICA in vitro and characterize for the first time the metabolic pathways of halogenated cumyl SCRAs. 4′Cl‐cumyl‐PINACA, 4′F‐cumyl‐5F‐PINACA and 4′F‐cumyl‐5F‐PICA were incubated with human hepatocytes in duplicates for 0, 1, 3 and 5 h. The supernatants were analysed in data‐dependent acquisition on a UHPLC‐QToF‐MS, and the potential metabolites were tentatively identified. A total of 11 metabolites were detected for 4′Cl‐cumyl‐PINACA, 21 for 4′F‐cumyl‐5F‐PINACA and 10 for 4′F‐cumyl‐5F‐PICA. The main biotransformations were oxidative defluorination, followed by hydroxylation with dehydrogenation, N‐dealkylation, dihydrodiol formation and glucuronidation. Hydroxylations were most common at the tail moieties with higher abundancy for indole than indazole compounds. N‐dealkylations were more common for fluorinated tail chain compounds than the non‐fluorinated 4′Cl‐cumyl‐PINACA. In conclusion, many metabolites retained halogen groups at the cumyl moieties which, in various combinations, may be suitable as analytical biomarkers. [ABSTRACT FROM AUTHOR]

In the past 15 years, close to 1000 of new psychoactive substances (NPS) have been reported in Europe and globally. At the time of identification, data on safety, toxicity and carcinogenic potential of many NPS are not available or very limited. To work more efficiently, a strategy and collaboration between the Public Health Agency of Sweden (PHAS) and the National Board of Forensic Medicine was established involving in vitro receptor activity assays to demonstrate neurological activity of NPS. This report summarizes the first results on the synthetic cannabinoid receptor agonists (SCRAs), and subsequent actions taken by PHAS. A total of 18 potential SCRAs were selected by PHAS for in vitro pharmacological characterization. 17 compounds could be acquired and investigated for their activity on the human cannabinoid-1 (CB1) receptors expressed together with the AequoScreen system in CHO-K1 cells. Dose-response curves were established using eight different concentrations in triplicates at three occasions with JWH-018 as reference. For the MDMB-4en-PINACA, MMB-022, ACHMINACA, ADB-BUTINACA, 5F-CUMYL-PeGACLONE, 5C-AKB48, NM-2201, 5F-CUMYL-PINACA, JWH-022, 5Cl-AB-PINACA, MPhP-2201, 5F-AKB57 the half maximal effective concentration values ranged from 2.2 nM (5F-CUMYL-PINACA) to 171 nM (MMB-022). EG-018 and 3,5-AB-CHMFUPPYCA were none-active. The results contributed to 14 of these compounds being scheduled as narcotics in Sweden. In conclusion, many of the emerging SCRAs are potent activators of the CB1 receptor in vitro , although some lack activity or are partial agonists. The new strategy proved useful when data on psychoactive effects of the SCRAs under investigation were not available or limited. • Synthetic cannabinoids receptor agonists (SCRAs) continue to emerge in Europe. • Pharmacological data is often missing, which impedes the risk assessment of SCRAs. • We report the cannabinoid-1 (CB1) receptor activity of 17 SCRAs & it's impact on the Swedish legislation. [ABSTRACT FROM AUTHOR]

Synthetic cannabinoids are a group of psychoactive drugs presently widespread among drug users in Europe. Analytical methods to measure these compounds in urine are in demand as urine is a preferred matrix for drug testing. For most synthetic cannabinoids, the parent compounds are rarely detected in urine. Therefore urinary metabolites are needed as markers of drug intake. AB-FUBINACA was one of the top three synthetic cannabinoids most frequently found in seizures and toxicological drug screening in Sweden (2013-2014). Drug abuse is also reported from several other countries such as the USA and Japan. In this study, 28 authentic case samples were used to identify urinary markers of AB-FUBINACA intake using liquid chromatography quadrupole tandem time of flight mass spectrometry and human liver microsomes. Three metabolites suitable as markers of drug intake were identified and at least two of them were detected in all but one case. In total, 15 urinary metabolites of AB-FUBINACA were reported, including hydrolxylations on the indazole ring and the amino-oxobutane moiety, dealkylations and hydrolysis of the primary amide. No modifications on the fluorobenzyl side-chain were observed. The parent compound was detected in 54% of the case samples. Also, after three hours of incubation with human liver microsomes, 77% of the signal from the parent compound remained. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Flualprazolam is a novel designer benzodiazepine, structurally related to alprazolam, flubromazolam and triazolam. In the last couple of years, it has been frequently detected in seizures and in forensic cases in Sweden and Finland. However, there is a lack of published blood concentrations for the drug, which presents difficulties when assessing its relevance for the cause of death. A quantitative method for the determination of flualprazolam in post-mortem blood was developed and validated, and subsequently used to analyse samples from 33 deaths previously screened as testing positive for flualprazolam in Sweden and Finland. Most of the cases in the study were accidental deaths (61 %) or suicides (18 %). The median (range) flualprazolam concentration was 18.0 (3.0-68) ng/g. The majority of the deceased were male (82 %) and the median age was 30 years. The median age in the Swedish cases was significantly higher (35 years) than in the Finnish cases (23 years) (p< 0.05). Poly-drug use and particularly the concomitant use of flualprazolam and opioids were very common in the study population. Most of the cases that were positive for flualprazolam were fatal poisonings by a drug (N=23), and in 13 cases, flualprazolam was implicated in the cause of death. Combining the resources of two countries in which all post-mortem toxicology is centralised provided a more comprehensive insight into the toxicology of flualprazolam. Research on novel psychoactive substances, such as flualprazolam, is required in order to be able to provide scientific evidence on the risks of these new substances for drug administration and potential users. [ABSTRACT FROM AUTHOR]