2C-B-Fly-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C. elegans: Confirmation with Synthesized Analytical Standards.

Compounds from the N-benzylphenethylamine (NBPEA) class of novel psychoactive substances are being increasingly utilized in neurobiological and clinical research, as diagnostic tools, or for recreational purposes. To understand the pharmacology, safety, or potential toxicity of these substances, elucidating their metabolic fate is therefore of the utmost interest. Several studies on NBPEA metabolism have emerged, but scarce information about substances with a tetrahydrobenzodifuran ("Fly") moiety is available. Here, we investigated the metabolism of 2-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b']difuran-4-yl)-N-(2-methoxybenzyl)ethan-1-amine (2C-B-Fly-NBOMe) in three different systems: isolated human liver microsomes, Cunninghamella elegans mycelium, and in rats in vivo. Phase I and II metabolites of 2C-B-Fly-NBOMe were first detected in an untargeted screening and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hypothesized metabolites were then synthesized as reference standards; knowledge of their fragmentation patterns was utilized for confirmation or tentative identification of isomers. Altogether, thirty-five phase I and nine phase II 2C-B-Fly-NBOMe metabolites were detected. Major detected metabolic pathways were mono- and poly-hydroxylation, O-demethylation, oxidative debromination, and to a lesser extent also N-demethoxybenzylation, followed by glucuronidation and/or N-acetylation. Differences were observed for the three used media. The highest number of metabolites and at highest concentration were found in human liver microsomes. In vivo metabolites detected from rat urine included two poly-hydroxylated metabolites found only in this media. Mycelium matrix contained several dehydrogenated, N-oxygenated, and dibrominated metabolites.

mTOR inhibitor improves autistic-like behaviors related to Tsc2 haploinsufficiency but not following developmental status epilepticus.

BACKGROUND: Tuberous sclerosis complex (TSC), a multi-system genetic disorder often associated with autism spectrum disorder (ASD), is caused by mutations of TSC1 or TSC2, which lead to constitutive overactivation of mammalian target of rapamycin (mTOR). In several Tsc1+/- and Tsc2+/- animal models, cognitive and social behavior deficits were reversed by mTOR inhibitors. However, phase II studies have not shown amelioration of ASD and cognitive deficits in individuals with TSC during mTOR inhibitor therapy. We asked here if developmental epilepsy, common in the majority of individuals with TSC but absent in most animal models, could explain the discrepancy. METHODS: At postnatal day P12, developmental status epilepticus (DSE) was induced in male Tsc2+/- (Eker) and wild-type rats, establishing four experimental groups including controls. In adult animals (n = 36), the behavior was assessed in the paradigms of social interaction test, elevated plus-maze, light-dark test, Y-maze, and novel object recognition. The testing was carried out before medication (T1), during a 2-week treatment with the mTOR inhibitor everolimus (T2) and after an 8-week washing-out (T3). Electroencephalographic (EEG) activity was recorded in a separate set of animals (n = 18). RESULTS: Both Tsc2+/- mutation and DSE caused social behavior deficits and epileptiform EEG abnormalities (T1). Everolimus led to a persistent improvement of the social deficit induced by Tsc2+/-, while deficits related to DSE did not respond to everolimus (T2, T3). CONCLUSIONS: These findings may contribute to an explanation why ASD symptoms in individuals with TSC, where comorbid early-onset epilepsy is common, were not reliably ameliorated by mTOR inhibitors in clinical studies.

25CN-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C.elegans-Structure Determination and Synthesis of the Most Abundant Metabolites.

N-Benzylphenethylamines are novel psychedelic substances increasingly used for research, diagnostic, or recreational purposes. To date, only a few metabolism studies have been conducted for N-2-methoxybenzylated compounds (NBOMes). Thus, the available 2,5-dimethoxy-4-(2-((2-methoxybenzyl)amino)ethyl)benzonitrile (25CN-NBOMe) metabolism data are limited. Herein, we investigated the metabolic profile of 25CN-NBOMe in vivo in rats and in vitro in Cunninghamella elegans (C. elegans) mycelium and human liver microsomes. Phase I and phase II metabolites were first detected in an untargeted screening, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of the most abundant metabolites by comparison with in-house synthesized reference materials. The major metabolic pathways described within this study (mono- and bis-O-demethylation, hydroxylation at different positions, and combinations thereof, followed by the glucuronidation, sulfation, and/or N-acetylation of primary metabolites) generally correspond to the results of previously reported metabolism of several other NBOMes. The cyano functional group was either hydrolyzed to the respective amide or carboxylic acid or remained untouched. Differences between species should be taken into account in studies of the metabolism of novel substances.

Two immunoassays for the detection of 2C-B and related hallucinogenic phenethylamines.

INTRODUCTION: The use of new psychoactive substances as drugs of abuse has dramatically increased over the last years. Hallucinogenic phenethylamines gained particular popularity as they have both stimulating and psychedelic effects. Although generally perceived as safe, these illicit drugs pose a serious health risk; they have been linked to cases of severe poisoning or even deaths. Therefore, simple, cost-effective and reliable methods are needed for rapid determination of abused hallucinogens. METHODS: For this purpose, two haptens derived from 2C-H were designed, synthesized and subsequently attached to a carrier protein. Polyclonal antibodies obtained from a rabbit immunized with one of the prepared immunogens were used for the development of two immunoassays. RESULTS: In this study, a lateral flow immunoassay (LFIA) and an enzyme linked immunosorbent assay (ELISA) for the detection of 2C-B and related hallucinogenic phenethylamines in urine were developed. The presented LFIA is primarily suitable for on-site monitoring as it is simple and can provide a visual evidence of 2C-B presence within a few minutes. Its reasonable sensitivity (LODLFIA = 15 ±â€¯7 ng mL-1) allows detection of the drug presence in urine after acute exposure. For greater accuracy, highly sensitive ELISA (LODELISA = 6 ±â€¯3 pg mL-1) is proposed for toxicological quantitative analyses of positive samples captured by the LFIA. DISCUSSION: The comparison of the ELISA with the well-established UHPLC-MS-MS method shows excellent agreement of results, which confirms good potential of the ELISA to be used for routine analyses of 2C-B and related hallucinogenic phenethylamines of both main sub-families.

Lateral flow immunoassay and enzyme linked immunosorbent assay as effective immunomethods for the detection of synthetic cannabinoid JWH-200 based on the newly synthesized hapten.

In recent years, the use of synthetic cannabinoids (SCs) as drugs of abuse has greatly increased. SCs are associated with a risk of severe poisoning or even death. Therefore, more rapid, cost effective and reliable methods are needed, especially for the screening of drivers after traffic accidents and for detailed toxicological analysis in forensic laboratories. In this study, we developed a lateral flow immunoassay (LFIA) and an enzyme linked immunosorbent assay (ELISA) for the detection of JWH-200 in oral fluids. For this purpose a new hapten was prepared using a ten-step synthetic route. The developed immuno methods are based on antibodies obtained from rabbit immunized with synthesized hapten conjugated to carrier protein. The proposed methods are highly sensitive (LODLFIA = 0.08 ±â€¯0.04 ng mL-1; LODELISA = 0.04 ±â€¯0.02 ng mL-1). They were applied to the quantification of JHW-200 in spiked oral fluids. The recoveries ranged from 82 to 134% for both methods. The results correlated excellently with results obtained using UHPLC-MS/MS (R2LFIA = 0.99; R2ELISA = 0.99). Our developed methods could be an important tool for analyses of JWH-200 in human oral fluids. The one-step LFIA is particularly suitable for roadside and on-site monitoring due to the rapid qualitative results it delivers, while the ELISA is especially useful for laboratory quantitative analyses of positive samples captured by LFIA.