Activity-based detection of consumption of synthetic cannabinoids in authentic urine samples using a stable cannabinoid reporter system

Synthetic cannabinoids (SCs) continue to be the largest group of new psychoactive substances (NPS) monitored by the European Monitoring Center of Drugs and Drugs of Abuse (EMCDDA). The identification and subsequent prohibition of single SCs has driven clandestine chemists to produce analogues of increasing structural diversity, intended to evade legislation. That structural diversity, combined with the mostly unknown metabolic profiles of these new SCs, poses a big challenge for the conventional targeted analytical assays, as it is difficult to screen for "unknown" compounds. Therefore, an alternative screening method, not directly based on the structure but on the activity of the SC, may offer a solution for this problem. We generated stable CB1 and CB2 receptor activation assays based on functional complementation of a split NanoLuc luciferase and used these to test an expanded set of recent SCs (UR-144, XLR-11, and their thermal degradation products; AB-CHMINACA and ADB-CHMINACA) and their major phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at the cannabinoid receptors. These active metabolites may prolong the parent compound's psychotropic and physiological effects and may contribute to the toxicity profile. Utility of the generated stable cell systems as a first -line screening tool for SCs in urine was also demonstrated using a relatively large set of authentic urine samples. Our data indicate that the stable CB reporter assays detect CB receptor, activation by extracts of urine in which SCs (or their metabolites) are present at low- or subnanomolar (ng/mL) level. Hence, the developed assays do not only allow activity profiling of SCs and their metabolites, it may also serve as a screening tool, complementing targeted and untargeted analytical assays and preceding analytical (mass spectrometry based) confirmation

Activity-based detection of consumption of synthetic cannabinoids in authentic urine samples using a stable cannabinoid reporter system

Synthetic cannabinoids (SCs) continue to be the largest group of new psychoactive substances (NPS) monitored by the European Monitoring Center of Drugs and Drugs of Abuse (EMCDDA). The identification and subsequent prohibition of single SCs has driven clandestine chemists to produce analogues of increasing structural diversity, intended to evade legislation. That structural diversity, combined with the mostly unknown metabolic profiles of these new SCs, poses a big challenge for the conventional targeted analytical assays, as it is difficult to screen for "unknown" compounds. Therefore, an alternative screening method, not directly based on the structure but on the activity of the SC, may offer a solution for this problem. We generated stable CB1 and CB2 receptor activation assays based on functional complementation of a split NanoLuc luciferase and used these to test an expanded set of recent SCs (UR-144, XLR-11, and their thermal degradation products; AB-CHMINACA and ADB-CHMINACA) and their major phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at the cannabinoid receptors. These active metabolites may prolong the parent compound's psychotropic and physiological effects and may contribute to the toxicity profile. Utility of the generated stable cell systems as a first -line screening tool for SCs in urine was also demonstrated using a relatively large set of authentic urine samples. Our data indicate that the stable CB reporter assays detect CB receptor, activation by extracts of urine in which SCs (or their metabolites) are present at low- or subnanomolar (ng/mL) level. Hence, the developed assays do not only allow activity profiling of SCs and their metabolites, it may also serve as a screening tool, complementing targeted and untargeted analytical assays and preceding analytical (mass spectrometry based) confirmation

Heroin Adulterated with the Novel Synthetic Cannabinoid, 5F-MDMB-PINACA: A Case Series

Introduction: Heroin can be adulterated with various substances that may or may not have pharmacological effects. Here we report a case series of 8 patients who presented to the emergency department after overdose with intravenous heroin preparation adulterated with the synthetic cannabinoid methyl 2-(1-(5-fluoropentyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (5F-MDMB-PINACA).Case Series: Except for one patient, all of them presented with a typical initial opioid toxidrome consisting of central nervous system and respiratory depression along with pinpoint pupils. Naloxone was given to them, triggering severe agitation and combative behavior along with overlapping features of anticholinergic and sympathomimetic toxidrome. All patients required multiple doses of benzodiazepines. Three were successfully treated with physostigmine.Discussion: 5F-MDMB-PINACA is a synthetic cannabinoid that was added to heroin in samples obtained from patients reported in this case series. Patients demonstrated significant agitation after receiving naloxone for opioid toxidrome, presumably because of the removal of the depressant effect of opioids, which unmasked the excitatory effects of the synthetic cannabinoids. Three patients required physostigmine along with the benzodiazepines for control of their agitation, urine retention and abnormal vitals, suggesting the possibility of an anticholinergic toxidrome to have developed in these patients.Conclusion: Heroin contaminated with 5F-MDMB-PINACA exhibits variable severities of anticholinergic effects, some on presentation and others only after opiate antagonism

Technical report on N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(cyclohexylmethyl)-1H-indazole-3- carboxamide (ADB-CHMINACA). Annex 1 to the Risk Assessment Report on N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1- (cyclohexylmethyl)-1H-indazole-3-carboxamide (ADB-CHMINACA).

In accordance with Article 6 of Council Decision 2005/387/JHA on the information exchange, risk assessment and control of new psychoactive substances

An index of fatal toxicity for new psychoactive substances

The final, definitive version of this paper has been published in Journal of Psychopharmacology, February 2018, published by SAGE Publishing, All rights reserved.An index of fatal toxicity for new psychoactive substances has been developed based solely on information provided on death certificates. An updated index of fatal toxicity (T f), as first described in 2010, was calculated based on the ratio of deaths to prevalence and seizures for the original five substances (amphetamine, cannabis, cocaine/crack, heroin and 3,4-methylenedioxymethylamphetamine) *. These correlated well with the 2010 index. Deaths were then examined for cases both where the substance was and was not found in association with other substances. This ratio (sole to all mentions; S/A) was then calculated for deaths in the period 1993 to 2016. This new measure of fatal toxicity, expressed by S/A, was well-correlated with the index L n (T f) of the original reference compounds. The calculation of S/A was then extended to a group of new psychoactive substances where insufficient prevalence or seizure data were available to directly determine a value of T f by interpolation of a graph of T f versus S/A. Benzodiazepine analogues had particularly low values of S/A and hence T f. By contrast, γ-hydroxybutyrate/γ-butyrolactone, α-methyltryptamine, synthetic cannabinoid receptor agonists and benzofurans had a higher fatal toxicity.Peer reviewedFinal Accepted Versio

Recent trends in analytical methods to determine new psychoactive substances in hair

New Psychoactive Substances (NPS) belong to several chemical classes, including phenethylamines, piperazines, synthetic cathinones and synthetic cannabinoids. Development and validation of analytical methods for the determination of NPS both in traditional and alternative matrices is of crucial importance to study drug metabolism and to associate consumption to clinical outcomes and eventual intoxication symptoms. Among different biological matrices, hair is the one with the widest time window to investigate drug-related history and demonstrate past intake. The aim of this paper was to overview the trends of the rapidly evolving analytical methods for the determination of NPS in hair and the usefulness of these methods when applied to real cases. A number of rapid and sensitive methods for the determination of NPS in hair matrix has been recently published, most of them using liquid chromatography coupled to mass spectrometry. Hair digestion and subsequent solid phase extraction or liquid-liquid extraction were described as well as extraction in organic solvents. For most of the methods limits of quantification at picogram per milligram hair were obtained. The measured concentrations for most of the NPS in real samples were in the range of picograms of drug per milligram of hair. Interpretation of the results and lack of cut-off values for the discrimination between chronic consumption and occasional use or external contamination are still challenging. Methods for the determination of NPS in hair are continually emerging to include as many NPS as possible due to the great demand for their detection

Synthetic Cannabinoids : psychopharmacology, clinical aspects, and psy-chotic onset

This document is the Accepted Manuscript version of the following article: Giovanni Martinotti, Rita Santacroce, Duccio Papanti, Yasmine Elgharably, Mariya Prilutskaya, Ornella Corazza, ‘Synthetic Cannabinoids: Psychopharmacology, Clinical Aspects, and Psychotic Onset’, CNS & Neurological Disorders – Drug Targets, Vol. 16, 2017. Under embargo. Embargo end date: 13 April 2018. The published manuscript is available at EurekaSelect via: https://doi.org/10.2174/1871527316666170413101839. Published by Bentham Science.Synthetic Cannabinoids (SC) are the widest and most diffused class of Novel Psychoactive Substances. SC are chemically heterogeneous and structurally dissimilar from delta-9-tetrahydrocannabinol, being full agonists of the endocannabinoid system receptors CB1 and CB2. Desired effects include euphoria, talkativeness, feelings of joy and laughter, relaxation. With respect to cannabis, SC intake may also be associated with quicker arise of the effects, shorter duration of action, and larger levels of hangover. SC are more psychoactive than cannabis: symptoms may include a wide range of clinically relevant posi-tive, negative and cognitive psychopathological symptoms that mimic symptoms of schizophrenia. The risk of two widespread symptoms of SC intoxication, namely agitation and cardiotoxicity, exceeds this of traditional cannabis of 3.8 and 9.2 times respectively. A number of deaths have been related to SC ingestion, either on their own or in combination with other recreational drugs. Prompt and reliable in-formation available for health professionals, more specific analytic techniques, and designed preventive strategies are all required to face this unprecedented challenge.Peer reviewedFinal Accepted Versio

AB-PINACA. Critical Review Report

AB-PINACA (N-[(2S)-1-amino-3-methyl-1-oxobutan-2-yl]-1-pentyl-1H-indazole-3-carboxamide), originally developed by Pfizer Inc. as a synthetic cannabinoid receptor agonist (SCRA), has been encountered outside medical settings as a synthetic constituent and found in herbal smoking mixtures that are sold under a variety of brand names. It is common for retailers to purchase bulk quantities of the synthetic substance and add the synthetic material to plant matter that is then distributed onto the market. However, AB-PINACA is also available in powdered form as a “research chemical”. The identification of AB-PINACA was reported first in 2012 by Japan and more forensic detections began to emerge in other countries in 2013. Data from law enforcement suggest that AB-PINACA was one of the most prevalent substances in the United States of America in 2014, which dropped again in the following years. A small number of in vitro and in vivo studies are currently available but the data indicate that AB-PINACA binds to and activates human CB1 and CB2 receptors at low nanomolar concentrations, and that it induces a number of biological responses also triggered by the naturally occurring phytocannabinoid Δ9-THC. In tetrad assays (locomotor suppression, antinociception, hypothermia, and catalepsy), for example, AB- PINACA was shown to be 2- to 14-fold more potent than Δ9-THC and the effects were rimonabant-reversible. In some in vitro assays, AB-PINACA acted as a full agonist with slight selectivity for the hCB2 receptor. AB-PINACA also fully substituted for Δ9-THC in the drug discrimination paradigm and was 1.5-fold more potent than the training drug, which suggests that AB-PINACA may have abuse liability similar to Δ9-THC and/or other internationally controlled synthetic cannabinoid receptor agonists. Reports indicate an increasing trend for SCRAs being implicated in mini epidemics that have been associated with severe adverse drug effects including deaths. Reported adverse drug reactions associated with a range of SCRAs frequently include gastrointestinal (e.g. nausea/hyperemesis), neurological (e.g. hallucination, agitation, anxiety, paranoia, confusion, delusions, catatonia, lethargy, psychosis (including susceptible individuals)), cardiovascular (e.g. tachycardia, hypertension) and renal (e.g. acute kidney failure) features. The total number of cases reported in the scientific literature that make a causal link with AB-PINACA is very small. Intoxications and deaths associated with AB-PINACA have been reported but very few details are available. ‘Driving Under the Influence’ cases linked to AB-PINACA intoxication and ‘Drug Recognition Expert’ exams revealed significant impairment. Although not specific to just AB-PINACA, there are indications that socially vulnerable and stigmatized drug users for example found in homeless and prison populations, are increasingly associated with problematic use of SCRA products. Heavy use of SCRAs has been associated with problematic withdrawal symptoms even though further research is needed to investigate the underlying mechanisms. Epidemiological data, such as prevalence of use, abuse and dependence information specifically related to AB-PINACA could not be identified

Technical report on 1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide (CUMYL-4CN-BINACA). Annex 1 to the Risk Assessment Report on 1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3- carboxamide (CUMYL-4CN-BINACA).

In accordance with Article 6 of Council Decision 2005/387/JHA on the information exchange, risk assessment and control of new psychoactive substances