Synthetic Cannabinoid Metabolites Research Articles

Separation and Characterization of Synthetic Cannabinoid Metabolite Isomers Using SLIM High-Resolution Ion Mobility-Tandem Mass Spectrometry (HRIM-MS/MS).

Synthetic cannabinoids, a subclass of new psychoactive substances (NPS), are laboratory-made substances that are chemically similar to those found naturally in the cannabis plant. Many of these substances are illicitly manufactured and have been associated with severe health problems, prompting a need to develop analytical methods capable of characterizing both known and previously undetected compounds. This work focuses on a novel Structures for Lossless Ion Manipulations (SLIM) IM-MS approach to the differentiation and structural characterization of synthetic cannabinoid metabolites, specifically MDA-19/BUTINACA, JWH-018, and JWH-250 isomer groups. These different compound classes are structurally very similar, differing only in the position of one or a few functional groups; this yielded similarity in measured collision cross section (CCS) values. However, the high resolution of SLIM IM provided adequate separation of many of these isomers, such as sodiated JWH-250 metabolites N-4-OH, N-5-OH, and 5-OH, which displayed CCS of 187.5, 182.5, and 202.3 Å2, respectively. In challenging cases where baseline separation was precluded due to nearly identical CCS, such as for JWH-018 isomers, simple derivatization by dansyl chloride selectively reacted with the 6-OH compound to provide differentiation of all isomers using a combination of CCS and m/z. Finally, the opportunity to use this method for structural elucidation of unknowns was demonstrated by using SLIM IM mobility-aligned MS/MS fragmentation. Different MDA-19/BUTINACA isomers were first mobility separated and could then be individually activated, yielding unique fragments for both targeted identification and structural determination. Overall, the described SLIM IM-MS/MS workflow provides significant potential as a rapid screening tool for the characterization of emerging NPS such as synthetic cannabinoids and their metabolites.

Synthesis and structure determination of a synthetic cannabinoid CUMYL-THPINACA metabolite with differentiation between the ortho-, meta-, and para-hydroxyl positions of the cumyl moiety.

Synthetic cannabinoids, a type of new psychoactive substances, are likely to be rapidly metabolized; thus, the detection of their metabolites, rather than the parent compound, is a common method used to prove drug consumption. Although the analysis of metabolites is generally performed by mass spectrometry, it is limited to structural estimation because of few commercially available standards. In particular, distinguishing between positional isomers is difficult. Synthetic cannabinoids with a cumyl moiety can be hydroxylated at the cumyl moiety during metabolism, but it remains unclear whether the hydroxylation occurs at the ortho, meta, or para position. This study determined the structures of a metabolite formed by mono-hydroxylation at the cumyl moiety of the synthetic cannabinoid CUMYL-THPINACA, used as a model compound. Chemical synthesis was performed to create possible metabolites with one hydroxyl group at the ortho, meta, or para positions of the cumyl moiety. Using the synthesized metabolites and liquid chromatography-quadrupole time-of-flight mass spectrometry, the metabolite detected in the microsomal reaction of CUMYL-THPINACA was identified as a compound mono-hydroxylated at the para position based on retention time and product ion spectra. Moreover, the rapid metabolism of CUMYL-THPINACA was demonstrated with an in vitro half-life of 4.9 min and the identified metabolite could be detected for a relatively long time in vitro. The synthesized metabolite may be utilized as a good reference standard for proof of CUMYL-THPINACA consumption. These findings have potential applications in the synthesis of metabolites of other synthetic cannabinoids bearing a cumyl moiety.

Study of the Metabolic Profiles of "Indazole-3-Carboxamide" and "Isatin Acyl Hydrazone" (OXIZID) Synthetic Cannabinoids in a Human Liver Microsome Model Using UHPLC-QE Orbitrap MS.

Unregulated core structures, "isatin acyl hydrazones" (OXIZIDs), have quietly appeared on the market since China legislated to ban seven general core scaffolds of synthetic cannabinoids (SCs). The fast evolution of SCs presents clinical and forensic toxicologists with challenges. Due to extensive metabolism, the parent compounds are barely detectable in urine. Therefore, studies on the metabolism of SCs are essential to facilitate their detection in biological matrices. The aim of the present study was to elucidate the metabolism of two cores, "indazole-3-carboxamide" (e.g., ADB-BUTINACA) and "isatin acyl hydrazone" (e.g., BZO-HEXOXIZID). The in vitro phase I and phase II metabolism of these six SCs was investigated by incubating 10 mg/mL pooled human liver microsomes with co-substrates for 3 h at 37 °C, and then analyzing the reaction mixture using ultrahigh-performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry. In total, 9 to 34 metabolites were detected for each SC, and the major biotransformations were hydroxylation, dihydrodiol formation (MDMB-4en-PINACA and BZO-4en-POXIZID), oxidative defluorination (5-fluoro BZO-POXIZID), hydrogenation, hydrolysis, dehydrogenation, oxidate transformation to ketone and carboxylate, N-dealkylation, and glucuronidation. Comparing our results with previous studies, the parent drugs and SC metabolites formed via hydrogenation, carboxylation, ketone formation, and oxidative defluorination were identified as suitable biomarkers.

Rapid Simultaneous Determination of 11 Synthetic Cannabinoids in Urine by Liquid Chromatography-Triple Quadrupole Mass Spectrometry

Synthetic cannabinoids are a series of synthetic substances that mimic the effects of natural cannabinoids and produce a much stronger toxicity than natural cannabinoids, and they have become the most abused family of new psychoactive substances. A solid-phase extraction–liquid chromatography–triple quadrupole/linear ion trap mass spectrometry method has been developed to determine 11 synthetic cannabinoids in rat urine. Oasis HLB cartridge was selected to simultaneously extract synthetic cannabinoids for pretreatment. The effects of the loading solution and elution reagent volume on the recovery were investigated. The optimized acetonitrile proportion and elution reagent volume were determined by both high recovery and low solvent consumption. The results showed that the linear coefficients of determination of 11 types of synthetic cannabinoids ranged from 0.993 to 0.999, the limit of quantitation ranged from 0.01 to 0.1 ng/mL, and the spiked recoveries ranged from 69.90% to 118.39%. The research presented here provides a validated liquid chromatography tandem mass spectrometry method to accurately identify and quantitate synthetic cannabinoid metabolites in urine samples.

Advances in urinary biomarker research of synthetic cannabinoids.

New psychoactive substances (NPS) are chemical compounds designed to mimic the action of existing illicit recreational drugs. Synthetic cannabinoids (SCs) are a subclass of NPS which bind to the cannabinoid receptors, CB1 and CB2, and mimic the action of cannabis. SCs have dominated recent NPS seizure reports worldwide. While urine is the most common matrix for drug-of-abuse testing, SCs undergo extensive Phase I and Phase II metabolism, resulting in almost undetectable parent compounds in urine samples. Therefore, the major urinary metabolites of SCs are usually investigated as surrogate biomarkers to identify their consumption. Since seized urine samples after consuming novel SCs may be unavailable in a timely manner, human hepatocytes, human liver microsomes and human transporter overexpressed cell lines are physiologically-relevant in vitro systems for performing metabolite identification, metabolic stability, reaction phenotyping and transporter experiments to establish the disposition of SC and its metabolites. Coupling these in vitro experiments with in vivo verification using limited authentic urine samples, such a two-pronged approach has proven to be effective in establishing urinary metabolites as biomarkers for rapidly emerging SCs.

Human phase-I metabolism and prevalence of two synthetic cannabinoids bearing an ethyl ester moiety: 5F-EDMB-PICA and EDMB-PINACA.

Around 2017, with the appearance of 5F-EDMB-PINACA, synthetic cannabinoids (SCs) carrying an ethyl ester moiety at the linked group started spreading on the market of new psychoactive substances (NPS). In 2020 and 2021, the indole analog of 5F-EDMB-PINACA (5F-EDMB-PICA) and the non-fluorinated analog of this compound (EDMB-PINACA) were analytically characterized. Here, we present suitable urinary markers to prove the consumption of these two ethyl analogs. Ten authentic urine samples for each compound were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qToF-MS). Anticipated phase-I metabolites detected in urine samples were confirmed in vitro by applying a pooled human liver microsomes (pHLM) assay. Prevalence data were obtained from urines collected for abstinence control and submitted to a screening method for SC metabolites. Ten phase-I metabolites of 5F-EDMB-PICA and 18 of EDMB-PINACA were detected by LC-qToF-MS analysis of authentic urine specimens. The main in-vivo metabolites were built by ester hydrolysis, often coupled to further metabolic processes. Investigation of phase-I biotransformation led to the identification of ester hydrolysis, monohydroxylation, and defluorination products as the most suitable urinary biomarkers for 5F-EDMB-PICA. Metabolites formed by ester hydrolysis coupled to ketone formation and by monohydroxylation are suggested for the detection of EDMB-PINACA. From October 1, 2020 to February 1, 2022, among positive urine samples, 5.4% and 10.1% tested positive 5F-EDMB-PICA and EDMB-PINACA, respectively. Due to common metabolites shared among structurally related SCs, the unequivocal detection of their consumption remains challenging for forensic laboratories and requires sensitive methods to monitor multiple metabolites, ideally including highly specific species.

CB1 activity of three classes of synthetic cannabinoids and their metabolites – The tail needs to know what the head is doing

Synthetic cannabinoids (SCs) are amongst the most prevalent of the new psychoactive substances. They encompass various structural classes with a wide range of CB1 activity. Most SCs are extensively bio-transformed in vivo and in vitro. The available data on the CB1 activity of SC metabolites are limited but such knowledge is needed to evaluate SC toxicology, duration of effects and to develop bioassays for screening purposes. To evaluate any structure-activity relationships, we determined the human CB1 receptor efficacy and potency of three SC classes and their metabolites. An activity assay incorporating AequoScreen recombinant CHO-K1 cells expressing the human CB1 receptor was used to study SCs and their metabolites. Dose-response curves were prepared in triplicate in three independent experiments and GraphPad Prism was used to calculate substance efficacy and potency. Eight concentrations, 29 nM–60 μM, were used for each drug and JWH-018 was used as a reference. The three SC classes investigated were (i) JWH-018, AM2201, THJ-018 and THJ-2201 and their corresponding 4-OH-pentyl and 5OH-pentyl metabolites; (ii) MMB-4en-PICA (MMB022) and MDMB-4en-PINACA and their corresponding ester-hydrolysis and dihydrodiol metabolites; and (iii) 5F-MDMB-PINACA, 5F-MDMB-PICA, 4F-MDMB-BINACA, and 4F-MDMB-BICA and their oxidative-defluorination and ester-hydrolysis metabolites. Parent SC potency values ranged from 7-200 nM. The 4-OH-pentyl metabolites of JWH-018, AM2201, THJ-018 and THJ-2201 had similar potency to their respective parent SC. The 5-OH-pentyl-JWH-018 and 5-OH-pentyl-THJ-018 metabolites were 5–10 times less potent than their parent SCs. No CB1 activity was observed for MMB-4en-PICA ester-hydrolysis and dihydrodiol metabolites, possibly due to the lower activity of the parent and a subsequent large loss of activity, whilst the dihydrodiol and ester-hydrolysis MDMB-4en-PINACA metabolites were 53 times and 130 times less potent respectively than MDMB-4en-PINACA. 5F-MDMB-PINACA and 5F-MDMB-PICA oxidative-defluorinated metabolites were 7 and 27 times less potent than the parent SCs. The 4OH-butyl metabolites of 4F-MDMB-BINACA, and 4F-MDMB-BICA were 6 and 11 times less potent than the parent drug, respectively, and their ester-hydrolysis metabolites were 21-47 times less potent. Where hydroxylations occur at the non-terminal (4) position of the pentyl tail of JWH-018 and THJ -2201 there is no loss of CB1 activity. The CB1 potency of the JWH-018 and THJ -2201 5OH-pentyl metabolites is reduced but in the same potency range as those reported for other parent SCs. Dihydrodiol metabolites of 4en-compounds show a large reduction in CB1 activity (> 53 times) compared to the parent, as do their ester hydrolysis metabolites (> 130-times). The oxidative defluorinated metabolites of the 4F-MDMB and 5F-MDMB compounds retain some CB1 activity (7–27 times reduction) but a more significant loss of activity is observed when ester hydrolysis occurs in the head group (21–417 times reduction). The head and core moieties of SCs are most influential in determining CB1 potency. The tail, when fulfilling certain steric requirements, acts as an anchor point in the CB1 receptor, influencing the overall position and rigidity of the SC in the CB1 receptor. From a urinary bioassay perspective, where metabolites can be 10 times more abundant than parent SCs, the activity of the metabolites might be important for SC detection. We have established a structure-activity relationship for three SC classes and their metabolites. The relative change in CB1 activity caused by a biotransformation on the tail of the SC depends on the head structure present and biotransformation of the head group leads to a greater reduction in potency than single hydroxylations on the tail. So, “the tail of the SC metabolite needs to know what the head is doing” when activating the CB1 receptor. The study is part of Eurostars-2 Joint Programme NPS-REFORM.

Study on the metabolic process of synthetic cannabinoids 4F-MDMB-BINACA and 4F-MDMB-BICA in human liver microsome and zebrafish model via UHPLC-QE Orbitrap MS

In order to address the increasing abuse of synthetic cannabinoids, on July 1, 2021, China listed the whole category of synthetic cannabinoids in the Supplementary Catalog for the Control of Non-medicinal Narcotic Drugs and Psychotropic Substances. Because synthetic cannabinoids metabolize rapidly, techniques are urgently needed to identify the phase I metabolites of new synthetic cannabinoids, as well as the symbol metabolites, which can be used for detection in real cases. In this study, we used pooled human liver microsome (pHLM) and zebrafish combined with ultra-high-performance liquid chromatography (UHPLC) Q Exactive Orbitrap MS to identify the phase Imetabolites of two new synthetic cannabinoids 4F-MDMB-BICA and 4F-MDMB-BINACA in vitro and in vivo, respectively. We studied the toxicokinetics of 4F-MDMB-BICA and 4F-MDMB-BINACA by sampling from a pHLM incubation system at different time points to study the change in metabolites over time. We detected a total of 14 metabolites of 4F-MDMB-BINACA and 16 metabolites of 4F-MDMB-BICA in this study. Metabolites of 4F-MDMB-BICA were detected in vitro for the first time. One metabolite of 4F-MDMB-BINACA, M05, was discovered for the first time. Based on the toxicokinetics results, we recommend three metabolites (M03, M11, M12) of 4F-MDMB-BINACA and three metabolites (M10, M12, M14) of 4F-MDMB-BICA as their symbol metabolites. The results showed that these two structurally similar synthetic cannabinoids 4F-MDMB-BINACA and 4F-MDMB-BICA had similar metabolic processes, as well as similar structures of their main symbol metabolites.

Long-term stability of 24 synthetic cannabinoid metabolites spiked into whole blood and urine for up to 168days, and the comparable study for the 6 metabolites in non-spiked real case specimens stored for 1-5years.

The aim of this study is to investigate the stabilities of the 24 synthetic cannabinoid metabolites (SCMs) in blood and urine at various temperatures from - 30 to 37 ℃ stored for 1-168days. In addition, experiments of stabilities at lower temperatures and for much longer duration have been performed as described below. The quantification was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The blank blood and urine spiked with SCMs and non-spiked real case (authentic) specimens were incubated at 37 ℃ up to 56days and at 22, 4 or - 30 ℃ up to 168days. The non-spiked authentic blood and urine specimens were also stored at - 30 or - 80 ℃ for 1, 3 or 5years to investigate stabilities during very long time frames. All the 24 SCMs were much more stable in urine than in blood at 37, 22 or 4 ℃. All 24 SCMs spiked into blood or urine were stable at - 30 ℃ for up to 168days. The 6 SCMs in the authentic specimens exhibited long stabilities at - 30 or - 80 ℃ for 3-5years. Some tendencies were observed according to the relation between the structures of SCMs and their stabilities. The long-term stabilities of 24 SCMs in spiked samples and those of 6 SCMs in the authentic specimens were examined using LC-MS/MS. SCMs were largely very stable and usable several years after storage at - 30 or - 80 ℃.

Simultaneous screening of 239 synthetic cannabinoids and metabolites in blood and urine samples using liquid chromatography–high resolution mass spectrometry

Synthetic cannabinoids (SCs) are new psychoactive substances that function as endocannabinoid CB1 and CB2 receptor agonists. Abuse of SCs can lead to symptoms such as confusion, dizziness, and even death. At present, Synthetic cannabinoids constitute one of the largest groups of new psychoactive substances and become popular recreational drugs of abuse for their psychoactive properties. The continuous transformation of SCs also leads to an endless emergence of new types. An efficient, high-throughput screening method is therefore very important for their identification. This paper describes a liquid chromatography–high resolution mass spectrometry (LC-HRMS) method for simultaneously screening 179 SCs and 80 SC metabolites in blood and urine. Simple acetonitrile was used to precipitate the blood and urine proteins, and the supernatants obtained after centrifugation were analyzed. The LC-HRMS run time was 20 min. The mass spectrometer used an ESI source with a scanning range of m/z 100–1000. LC-HRMS provided accurate mass, retention time, and fragment ions for qualitative analysis. The method validation results showed that the limits of detection (LODs) for over 80% compounds were 5 ng/mL in blood and urine samples. At low concentrations (50 ng/mL), 229 compounds (95.8%) in the blood showed recoveries of more than 50%, and 232 compounds (97.1%) had matrix effects greater than 80%. In the urine, 219 compounds (91.6%) had recoveries above 50%, and the matrix effects of 234 compounds (97.9%) were greater than 80%. This method was successfully applied to actual forensic cases.

Major Metabolites of the Synthetic Cannabinoid 5F‐ADB Retain High Affinity and Full Efficacy at CB1 Receptors; Potential Mechanism Contributing to Enhanced Toxicity?

Synthetic cannabinoids (SCs) are a large group of abused psychoactive compounds that elicit numerous toxic effects not observed with marijuana, including death. SCs are abused worldwide and metabolites of these drugs are often detected as a means of drug identification. SC metabolites resulting from Phase I metabolism have been shown to retain affinity and activity at cannabinoid receptors. However, whether these active compounds contribute to toxicity of SCs is unknown. 5-Fluoro-ADB (5F-ADB), also known as 5-Fluoro-MDMB-PINACA, is a commonly abused third-generation synthetic cannabinoid agonist based on an indazole-structure. Significantly, over 40 recent fatalities have been attributed to 5F-ADB overdose. A potential mechanism underlying 5F-ADB-induced mortality is drug-related dysrhythmia resulting in sudden death. Analysis of postmortem central and peripheral blood samples have revealed abnormally high concentrations of an ester-hydrolysis Phase I metabolite of 5F-ADB, identified as metabolite 7 (5F-ADB-M7). We propose that high concentrations of potent and efficacious active metabolites, such as 5F-ADB-M7 and others, accumulate with the parent drug 5F-ADB and produce additive and/or synergistic effects that possibly contribute to fatal intoxication following overdose. As a first step to test this hypothesis, in vitro studies were conducted to compare the affinity and activity of tetrahydrocannabinol (THC; the primary psychoactive component in marijuana), 5F-ADB, 5F-ADB-M7 and a second major metabolite, 5F-ADB-M2 at cannabinoid type-1 (CB1) receptors. Competition receptor binding studies, using mouse brain membranes, showed that all four cannabinoids bound with high affinity to CB1 receptors with a rank order of affinity (Ki values) of 5F-ADB > 5F-ADB-M2 > THC > 5F-ADB-M7. Furthermore, functional experiments examining modulation of G-protein activity demonstrated that 5F-ADB and both Phase I metabolites acted as full agonists at CB1 receptors, relative to partial agonist effects produced by THC. Subsequently, in vivo studies were conducted utilizing the cannabinoid tetrad in mice to compare the effects of THC with those of 5F-ADB-M2. Both 5F-ADB-M2 and THC produced CB1-mediated dose-dependent hypothermia, analgesia, and locomotor suppression, within 30 minutes of injection; however, catalepsy was not elicited by the metabolite. Consistent with higher CB1 affinity determined from in vitro studies, 5F-ADB-M2 produced tetrad effects with higher potency compared to THC. Future studies are planned to examine in vivo effects of 5F-ADB-M7 compared to THC. If 5F-ADB metabolites exhibit in vivo activity similar to that predicted from the in vitro studies presented here, this suggests that the enhanced, often fatal, effects of 5F-ADB may be due to an accumulation of the parent and active, high affinity metabolites acting at CB1 receptors.

Involuntary 5F-ADB-related intoxication following e-cigarette use

The detection of synthetic cannabinoid (SC) intoxication cases is challenging, even more when the involved SC identification is requested in a forensic context. This situation can be complicated by new modes of SC consumption, non-specific symptomatology, and analytical pitfalls. To illustrate these issues, we report the case of a 16-year-old man who presented symptoms evocating of a seizure disorder in the minutes following the use of a friend's e-cigarette. At admission in the emergency department, his electroencephalogram was interpreted as coherent with a recent seizure episode. 5F-ADB, a third generation SC, was detected in the e-liquid and in an early collected (H2 after the e-cigarette use) serum sample (0.50µg/L), but not in urine samples (H18 and H38). One 5F-ADB metabolite, O-desmethyl-5F-ADB (M5), was detectable in urine up to at least 38h after intoxication. Neither 5F-ADB nor its metabolites could be detected in victim's hair sampled 3months after the intoxication. Although leading to a non-specific symptomatology, acute SC intoxication should be considered when the case history is related to e-cigarette or e-liquid use. Early biological samples are recommended, even if analytical screening can be positive for SC metabolites in urine sampled until 2days after exposure. Accordingly, data from the literature and the present case underscore the relevance of adding both main 5F-ADB metabolites (M5 and 5-OH-pentyl-ADB) to mass spectrum databases used for toxicological screening in order to reduce the risk of false-negative results in intoxication cases involving 5F-ADB.

Synthesis of nine potential synthetic cannabinoid metabolites with a 5F-4OH pentyl side chain from a scalable key intermediate

Synthetic cannabinoid metabolites with a 5F-4OH pentyl side chain are of great interest, and several synthetic methods have been developed for the synthesis of these types of metabolites in the literature, which is either lengthy or inappropriate for upscaling. Herein, a straightforward method has been developed and reported for the synthesis of a key intermediate in four steps with an overall yield of 49% on a multigram scale, and its application in the synthesis of nine potential metabolites of synthetic cannabinoids with a 5F-4OH pentyl side chain.

Determination of the Synthetic Cannabinoids JWH-122, JWH-210, UR-144 in Oral Fluid of Consumers by GC-MS and Quantification of Parent Compounds and Metabolites by UHPLC-MS/MS.

The consumption of synthetic cannabinoids (SCs) has significantly increased in the last decade and the analysis of SCs and their metabolites in human specimens is gaining interest in clinical and forensic toxicology. A pilot study has been carried out using a combination of an initial last generation gas chromatography-mass spectrometry (GC-MS) screening method for the determination of JWH-122, JWH-210, UR-144) in oral fluid (OF) of consumers and an ultra-high performance liquid chromatography high resolution mass spectrometry (UHPLC-HRMS) confirmatory method for the quantification of the parent compounds and their metabolites in the same biological matrix. OF samples were simply liquid-liquid extracted before injecting in both chromatographic systems. The developed methods have been successfully validated and were linear from limit of quantification (LOQ) to 50 ng/mL OF. Recovery of analytes was always higher than 70% and matrix effect always lower than 15% whereas intra-assay and inter-assay precision and accuracy were always better than 16%. After smoking 1 mg JWH-122 or UR-144 and 3 mg JWH-210, maximum concentration of 4.00–3.14 ng/mL JWH-122, 8.10–7.30 ng/mL JWH-210 ng/mL and 7.40 and 6.81 ng/mL UR-144 were measured by GC-MS and UHPLC-HRMS respectively at 20 min after inhalation. Metabolites of JWH 122 and 210 were quantified in OF by UHPLC-HRMS, while that of UR144 was only detectable in traces. Our results provide for the first time information about disposition of these SCs and their metabolites in consumers OF. Last generation GC-MS has proven useful tool to identify and quantify parent SCs whereas UHPLC-HRMS also confirmed the presence of SCs metabolites in the OF of SCs consumers.

Diagnosing intake and rationalizing toxicities associated with 5F-MDMB-PINACA and 4F-MDMB-BINACA abuse.

5F-MDMB-PINACA and 4F-MDMB-BINACA are synthetic cannabinoids (SCs) that elicit cannabinoid psychoactive effects. Defining pharmacokinetic-pharmacodynamic (PK-PD) relationships governing SCs and their metabolites are paramount to investigating their in vivo toxicological outcomes. However, the disposition kinetics and cannabinoid receptor (CB) activities of the primary metabolites of SCs are largely unknown. Additionally, reasons underlying the selection of ester hydrolysis metabolites (EHMs) as urinary biomarkers are often unclear. Here, metabolic reaction phenotyping was performed to identify key metabolizing enzymes of the parent SCs. Hepatic clearances of parent SCs and their EHMs were estimated from microsomal metabolic stability studies. Renal clearances were simulated using a mechanistic kidney model incorporating in vitro permeability and organic anionic transporter 3 (OAT3)-mediated uptake data. Overall clearances were considered in tandem with estimated volumes of distribution for in vivo biological half-lives (t1/2) predictions. Interactions of the compounds with CB1 and CB2 were investigated using a G-protein coupled receptor activation assay. We demonstrated that similar enzymatic isoforms were implicated in the metabolism of 5F-MDMB-PINACA and 4F-MDMB-BINACA. Our in vivo t1/2 determinations verified the rapid elimination of parent SCs and suggest prolonged circulation of their EHMs. The pronounced attenuation of the potencies and efficacies of the metabolites against CB1 and CB2 further suggests how toxic manifestations of SC abuse are likely precipitated by augmented exposure to parent SCs. Notably, basolateral OAT3-mediated uptake of the EHMs substantiates their higher urinary abundance. These novel insights underscore the importance of mechanistic, quantitative and systematic characterization of PK-PD relationships in rationalizing the toxicities of SCs.

In vitro characterization of new psychoactive substances at the μ-opioid, CB1, 5HT1A, and 5-HT2A receptors—On-target receptor potency and efficacy, and off-target effects

New psychoactive substances (NPS) appear on the recreational market on a monthly basis, with unclear toxicology, resulting in an increasing number of fatalities. Identification of drug targets and potencies is crucial for understanding and treating intoxications and for scheduling processes. In this study 60 NPS and metabolites belonging to opioids, cannabinoids and serotonergic hallucinogens classes were screened for in vitro activation of the μ-opioid, CB1, 5-HT1A and 5-HT2A receptors using the AequoZen cell system. Fentanyl and NBOMe analogues were chosen for full dose-response characterization of the μ-opioid and 5-HT2A receptors, respectively.Most substances activated their corresponding target receptor. The most potent μ-opioid receptor agonists were 2-fluorofentanyl (EC50 = 1.0 nM), carfentanil (EC50 = 2.7 nM) and acrylfentanyl (EC50 = 2.8 nM) and in total a >1500-fold difference was seen among the tested compounds. Moreover, furanylfentanyl, 4-methoxybutyrylfentanyl and valerylfentanyl acted as partial agonists of the μ-receptor. On the 5-HT2A receptor, bromo-dragonfly showed the highest potency (EC50 = 0.05 nM, 400 times more potent than LSD), followed by most NBOMe compounds with EC50 values ranging from 0.11 nM (for 25N-NBOMe) to 1.3 nM (for 25T4-NBOMe)). Off-target activation of the μ-opioid receptor was identified for piperazines, phenethylamines (in particular NBOMe and 2C compounds) and tryptamines. Moreover, the synthetic cannabinoid metabolite 3-carboxy indole PB-22 activated the 5-HT2A receptor. Bromo-dragonfly was the only compound that activated all four receptors. These results highlight the possible interplay of known and unknown NPS targets and unveil its complexity. Moreover, the detailed, quantitative information presented facilitates our further understanding of NPS toxicology.

Metabolic profiling of synthetic cannabinoid 5F-ADB and identification of metabolites in authentic human blood samples via human liver microsome incubation and ultra-high-performance liquid chromatography/high-resolution mass spectrometry.

Indazole carboxamide synthetic cannabinoids, a prevalent class of recreational drugs, are a major clinical, forensic and public health challenge. One such compound, 5F-ADB, has been implicated in fatalities worldwide. Understanding its metabolism and distribution facilitates the development of laboratory assays to substantiate its consumption. Synthetic cannabinoid metabolites have been extensively studied in urine; studies identifying metabolites in blood are limited and no data on the metabolic stability (half-life, clearance and extraction ratio) of 5F-ADB have been published prior to this report. The in vitro metabolism of 5F-ADB was elucidated via incubation with human liver microsomes for 2 h at 37°C. Samples were collected at multiple time points to determine its metabolic stability. Upon identification of metabolites, authentic forensic human blood samples underwent liquid-liquid extraction and were screened for metabolites. Extracts were analyzed via ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS) operated in positive electrospray ionization mode. Seven metabolites were identified including oxidative defluorination (M1); carboxypentyl (M2); monohydroxylation of the fluoropentyl chain (M3.1/M3.2) and indazole ring system (M4); ester hydrolysis (M5); and ester hydrolysis with oxidative defluorination (M6). The half-life (3.1 min), intrinsic clearance (256.2 mL min-1 kg-1 ), hepatic clearance (18.6 mL min-1 kg-1 ) and extraction ratio (0.93) were determined for the first time. In blood, M1 was present in each sample as the most abundant substance; two samples contained M5; one contained 5F-ADB, M1 and M5. 5F-ADB is rapidly metabolized in HLM. 5F-ADB, M1 and M5 are pharmacologically active at the cannabinoid receptors (CB1 /CB2 ) and M1 and M5 may contribute to a user's impairment profile. The results demonstrate that it is imperative that synthetic cannabinoid assays screen for pharmacologically active metabolites, especially for drugs with short half-lives. The authors propose that M1 and M5 are appropriate markers to include in laboratory blood tests screening for 5F-ADB.

Neurological Manifestations Associated with Synthetic Cannabinoid Use- A Case Series

Background: Synthetic Cannabinoid (SC) use has emerged as a growing public health threat in the United States. Several unexpected cases, presenting with a constellation of unrelated symptoms, but all having toxicity linked to SC use, have been reported in the last decade (2010-2019). Methods: We report a cluster of several independent cases where patients were admitted having different neurological manifestations. Extensive and expensive work-ups were performed. Upon further inspection, extended toxicology screens were found to be positive for SC metabolites. Results: It is alarming to observe that several reports highlight an increase in the varied and significant morbidity associated after SC use. Various SC compositions have been synthesized and distributed, with new molecules being generated at a staggering rate leading to unexpected manifestations. Conclusion: Young people are the most frequent users owing to its recreational effects, its easy accessibility, lower cost and difficulty in being detected in the urine by routine drug screens. From a hospital quality improvement perspective, efforts to characterize the presence of newly generated SC molecules and establish more accessible in-house screening methods will be a starting step in reducing the associated cost-burden. This will also minimize the unnecessary invasive procedures performed on a specific patient. From a socioeconomic viewpoint, solid and systematic crosstalk with increased recognition and reporting mechanism between the healthcare staff and public health personnel is strongly warranted to support state and federal regulatory efforts in combating this ongoing SC epidemic.

Evaluation of Synthetic Cannabinoid Metabolites in Human Blood in the Absence of Parent Compounds: A Stability Assessment.

Synthetic cannabinoids represent a chemically diverse class of novel psychoactive substances (NPS) responsible for large analytical and interpretative challenges for forensic toxicologists. Between 2016 and 2019, the three most prevalent synthetic cannabinoids in the United States were MMB-FUBINACA (FUB-AMB), 5F-MDMB-PINACA (5F-ADB) and 5F-MDMB-PICA, based on results from seized drug and toxicology testing. In 2018, accurate determination of synthetic cannabinoid positivity was brought into question as it was determined that the metabolites of these drug species were present in the absence of parent compounds in forensically relevant blood samples. During this study, the stability of MMB-FUBINACA, 5F-MDMB-PINACA and 5F-MDMB-PICA was evaluated, as well as the characterization of breakdown products. A liquid-liquid extraction method was assessed for recovery of basic parent compounds and acidic metabolites and deemed fit for use in this study. Analysis was performed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) using a SCIEX TripleTOF® 5600+. All three synthetic cannabinoids were found to be unstable when stored in blood at either room temperature or refrigerated; all analytes were considerably more stable when stored in the freezer. All three synthetic cannabinoids degraded to their respective butanoic acid metabolites: MMB-FUBINACA 3-methylbutanoic acid, 5F-MDMB-PINACA 3,3-dimethylbutanoic acid and 5F-MDMB-PICA 3,3-dimethylbutanoic acid. All three of these metabolites were studied and determined to be stable in blood at all storage conditions. Considering these results, our laboratory continued testing for synthetic cannabinoid metabolites in blood samples and found 83 positives (21%) for only a synthetic cannabinoid metabolite. A case report is presented herein where 5F-MDMB-PINACA 3,3-dimethylbutanoic acid was identified in the absence of 5F-MDMB-PINACA. Forensic toxicologists should be aware of the results of this study as they directly impact analytical consideration for test development and implementation, as well as interpretation of findings.

Metabolism, CB1 cannabinoid receptor binding and in vivo activity of synthetic cannabinoid 5F-AKB48: Implications for toxicity

AKB48 and its fluorinated derivative 5F-AKB48 are synthetic cannabinoids (SCs) which have caused hospitalizations and deaths in human users. Abuse of SCs is dangerous because users may mistake them for natural cannabis, which is generally considered to be unlikely to elicit adverse effects. The present studies were designed to investigate the in vitro oxidative metabolism of 5F-AKB48 by human microsomal fractions from different organs and sexes as well as recombinant human cytochrome P450s (P450s). Mass spectrometry data tentatively provides evidence for the existence of mono-, di-, and trihydroxylated metabolites in a successive metabolism. Experiments utilizing P450s revealed that the most active enzymes (CYP2D6, CYP2J2, CYP3A4, and CYP3A5) effectively produced mono- and dihydroxylated metabolites, while CYP3A4/5 also produced significant amounts of the trihydroxylated metabolite. Moreover, although the affinity and potency of Phase I metabolite 4OH-5F-AKB48 is reduced when compared to that of the parent drug, this metabolite nevertheless retains similar high affinity for CB1 receptors, and greater efficacy for G protein activation, when compared to THC. Finally, 5F-AKB48 produced time- and dose-dependent cannabimimetic effects in mice which were more potent, but shorter acting, than those of Δ9-THC, and were attenuated by prior treatment with the CB1 antagonist rimonabant. Based on our data, we hypothesize that while many cases of toxicity result from genetic mutations, which can lead to a decrease or even absence of activity for Phase I drug-metabolizing enzymes, other P450s could potentially increase their role in the metabolism of these SCs. Because many metabolites of SCs remain biologically active, they could contribute to the deleterious effects of these substances.