Activity-based detection of synthetic cannabinoid receptor agonists in plant materials.

BACKGROUND: Since late 2019, fortification of 'regular' cannabis plant material with synthetic cannabinoid receptor agonists (SCRAs) has become a notable phenomenon on the drug market. As many SCRAs pose a higher health risk than genuine cannabis, recognizing SCRA-adulterated cannabis is important from a harm reduction perspective. However, this is not always an easy task as adulterated cannabis may only be distinguished from genuine cannabis by dedicated, often expensive and time-consuming analytical techniques. In addition, the dynamic nature of the SCRA market renders identification of fortified samples a challenging task. Therefore, we established and applied an in vitro cannabinoid receptor 1 (CB1) activity-based procedure to screen plant material for the presence of SCRAs. METHODS: The assay principle relies on the functional complementation of a split-nanoluciferase following recruitment of ß-arrestin 2 to activated CB1. A straightforward sample preparation, encompassing methanolic extraction and dilution, was optimized for plant matrices, including cannabis, spiked with 5 µg/mg of the SCRA CP55,940. RESULTS: The bioassay successfully detected all samples of a set (n = 24) of analytically confirmed authentic Spice products, additionally providing relevant information on the 'strength' of a preparation and whether different samples may have originated from separate batches or possibly the same production batch. Finally, the methodology was applied to assess the occurrence of SCRA adulteration in a large set (n = 252) of herbal materials collected at an international dance festival. This did not reveal any positives, i.e. there were no samples that yielded a relevant CB1 activation. CONCLUSION: In summary, we established SCRA screening of herbal materials as a new application for the activity-based CB1 bioassay. The simplicity of the sample preparation, the rapid results and the universal character of the bioassay render it an effective and future-proof tool for evaluating herbal materials for the presence of SCRAs, which is relevant in the context of harm reduction.

In vitro activation of the CB1 receptor by the semi-synthetic cannabinoids hexahydrocannabinol (HHC), hexahydrocannabinol acetate (HHC-O) and hexahydrocannabiphorol (HHC-P).

Semi-synthetic cannabinoids (SSCs) including hexahydrocannabinol (HHC) are emerging on the drug market and sold openly as purportedly legal replacements for cannabis and Δ9-THC. By the beginning of 2024, 24 European countries had identified HHC, often sold openly in edibles (foods/candy), vapes and low-THC cannabis flowers and resins. The SSC market is developing rapidly, with HHC acetate (HHC-O), hexahydrocannabiphorol (HHC-P) and others recently identified. These developments may mark the first major change in the market for 'legal' replacements to cannabis since 'Spice' containing synthetic cannabinoids, such as JWH-018, emerged in 2008. Currently, there are some data available on the pharmacology of SSCs, which is crucial for understanding their effects, evaluating health risks and informing public health responses. This study focused on characterizing the in vitro activation of the human CB1 receptor by the (R)- and (S)-epimers of HHC, HHC-P and HHC-O. Using recombinant CHO-K1 cells expressing the human CB1 receptor, the potency (EC50) and efficacy were determined. It was established that (9R)-HHC and (9R)-HHC-P activated the CB1 receptor as partial agonists and with five and two times lower potency compared to JWH-018, respectively, while the (S)-epimers exhibited even lower potency. The (R)-epimer of HHC-O activate the CB1 receptor to even lesser extent and the (S)-epimer showed no activation. For HHC and HHC-P, all epimers exhibited similar level of efficacy. This available evidence suggests cannabimimetic effects of the tested SSC with the exception for the acetates that likely function as pro-drugs in vivo.

In vitro cannabinoid receptor activity, metabolism, and detection in seized samples of CH-PIATA, a new indole-3-acetamide synthetic cannabinoid.

The rapidly evolving synthetic cannabinoid receptor agonist (SCRA) market poses significant challenges for forensic scientists. Since the enactment of a generic ban in China, a variety of new compounds have emerged capable of evading the legislation by carrying new structural features. One recent example of a SCRA with new linker and head moieties is CH-PIATA (CH-PIACA, CHX-PIATA, CHX-PIACA). CH-PIATA bears an additional methylene spacer in the linker moiety between the indole core and the traditional carbonyl component of the linker. This study describes detections in 2022 of this new SCRA in the United States, Belgium, and Scottish prisons. CH-PIATA was detected once in a seized powder by Belgian customs and 12 times in Scottish prisons in infused papers or resin. The metabolites of CH-PIATA were investigated via in vitro human liver microsome (HLM) incubations and eight metabolites were identified, dominated by oxidative biotransformations. A blood sample from the United States was confirmed to contain a mixture of SCRAs including CH-PIATA via presence of the parent and at least five of the metabolites identified from HLM incubations. Furthermore, this paper evaluates the intrinsic in vitro cannabinoid 1 and 2 (CB1 and CB2) receptor activation potential of CH-PIATA reference material and the powder seized by Belgian customs by means of ß-arrestin 2 recruitment assays. Both the reference and the seized powder showed a weak activity at both CB receptors with signs of antagonism found. Based on these results, the expected harm potential of this newly emerging substance remains limited.

Activation of the cannabinoid type 2 (CB2) receptor improves cardiac contractile performance in fish, Brycon amazonicus.

In addition to their well-known classical effects, cannabinoid CB1 and CB2 receptors have also been involvement in both deleterious and protective actions on the heart under various pathological conditions. While the potential therapeutic applications of the endocannabinoid system in the context of cardiovascular function are indeed a viable prospect, significant debate exists within the literature regarding whether CB1, CB2, or a combination of both receptors exert a favorable influence on cardiac function. Hence, the aim of this study was to investigate the effects of CB1 + CB2 or CB2 agonists on cardiac excitation-contraction (E-C) coupling, utilizing fish (Brycon amazonicus) as an experimental model. The CB2 agonist elicited marked positive inotropic and lusitropic responses in isolated ventricular myocardium, induced cyclic adenosine 3',5'-monophosphate (cAMP) production, and upregulated critical Ca2+ handling proteins, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). Our current study demonstrated, for the first time, that CB2 receptor activation-induced effects improved the efficiency of Ca2+ cycling, excitation-contraction coupling (E-C coupling), and cardiac performance in under physiological conditions. Hence, CB2 receptors could be considered a potential therapeutic target for modulating cardiac contractile dysfunctions.

Log D7.4 and plasma protein binding of synthetic cannabinoid receptor agonists and a comparison of experimental and predicted lipophilicity.

The emergence of new synthetic cannabinoid receptor agonists (SCRAs) onto the illicit drugs market continues to cause harm, and the overall availability of physicochemical and pharmacokinetic data for new psychoactive substances is lacking. The lipophilicity of 23 SCRAs and the plasma protein binding (PPB) of 11 SCRAs was determined. Lipophilicity was determined using a validated chromatographic hydrophobicity index (CHI) log D method; tested SCRAs showed moderate to high lipophilicity, with experimental log D7.4 ranging from 2.48 (AB-FUBINACA) to 4.95 (4F-ABUTINACA). These results were also compared to in silico predictions generated using seven commercially available software packages and online tools (Canvas; ChemDraw; Gastroplus; MoKa; PreADMET; SwissADME; and XlogP). Licenced, dedicated software packages provided more accurate lipophilicity predictions than those which were free or had prediction as a secondary function; however, the latter still provided competitive estimates in most cases. PPB of tested SCRAs, as determined by equilibrium dialysis, was in the upper range of the lipophilicity scale, ranging from 90.8% (ADB-BUTINACA) to 99.9% (BZO-HEXOXIZID). The high PPB of these drugs may contribute to reduced rate of clearance and extended durations of pharmacological effects compared to lesser-bound SCRAs. The presented data improve understanding of the behaviour of these drugs in the body. Ultimately, similar data and predictions may be used in the prediction of the structure and properties of drugs yet to emerge on the illicit market.

Detection in seized samples, analytical characterization, and in vitro metabolism of the newly emerged 5-bromo-indazole-3-carboxamide synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS) and new structural scaffolds have emerged on the recreational drug market since the enactment of Chinese SCRA analog controls in 2021. This study reports the first SCRAs to be detected with a bromide at the 5 position (5'Br) on the phenyl ring of the indazole core and without a tail moiety. ADB-5'Br-INACA (ADMB-5'Br-INACA) and MDMB-5'Br-INACA were detected in seized samples from Scottish prisons, Belgian customs, and US forensic casework. The brominated analog with a tail moiety, ADB-5'Br-BUTINACA (ADMB-5'Br-BUTINACA), was also detected in Scottish prisons and US forensic casework. The metabolites of these compounds and the predicted compound MDMB-5'Br-BUTINACA were identified through incubation with primary human hepatocytes to aid in their toxicological identification. The bromide on the indazole remains intact on metabolites, allowing these compounds to be easily distinguished in toxicological samples from their non-brominated analogs. Glucuronidation was more common for tail-less analogs than their butyl tail-containing counterparts. Forensic toxicologists are advised to update their analytical methods with the characteristic ions for these compounds, as well as their anticipated urinary markers: amide hydrolysis and monoOH at tert-butyl metabolites (after ß-glucuronidase treatment) for ADB-5'Br-INACA; monoOH at tert-butyl and amide hydrolysis metabolites for ADB-5'Br-BUTINACA; and ester hydrolysis metabolites with additional metabolites for MDMB-5'Br-INACA and MDMB-5'Br-BUTINACA. Toxicologists should remain vigilant to the emergence of new SCRAs with halogenation of the indazole core and tail-less analogs, which have already started to emerge.

New synthetic cannabinoids and the potential for cardiac arrhythmia risk.

Synthetic cannabinoid receptor agonists (SCRAs) have been associated with QT interval prolongation. Limited preclinical information on SCRA effects on cardiac electrogenesis results from the rapid emergence of new compounds and restricted research availability. We used two machine-learning-based tools to evaluate seven novel SCRAs' interaction potential with the hERG potassium channel, an important drug antitarget. Five SCRAs were predicted to have the ability to block the hERG channel by both prediction tools; ADB-FUBIATA was predicted to be a strong hERG blocker. ADB-5Br-INACA and ADB-4en-PINACA showed varied predictions. These findings highlight potentially proarrhythmic hERG block by novel SCRAs, necessitating detailed safety evaluations.

In vitro cannabinoid activity profiling of generic ban-evading brominated synthetic cannabinoid receptor agonists and their analogs.

Following the enactment of a generic ban in China in 2021, the synthetic cannabinoid market has been evolving, now encompassing even wider structural diversity. Compounds carrying a brominated core such as ADB-5'Br-BUTINACA (ADMB-B-5Br-INACA) and tail-less analogs, such as ADB-5'Br-INACA (ADMB-5Br-INACA), MDMB-5'Br-INACA, and ADB-INACA (ADMB-INACA), have been detected since late 2021. This study investigated the cannabinoid receptor (CB) activation potential of synthesized (S)-enantiomers of these substances, as well as of two predicted analogs MDMB-5'Br-BUTINACA (MDMB-B-5Br-INACA) and ADB-5'F-BUTINACA (ADMB-B-5F-INACA), using CB1 and CB2 ß-arrestin 2 recruitment assays and a CB1 intracellular calcium release assay. Surprisingly, the tail-less (S)-ADB-5'Br-INACA and (S)-MDMB-5'Br-INACA retained CB activity, albeit with a decreased potency compared to their tailed counterparts (S)-ADB-5'Br-BUTINACA and (S)-MDMB-5'Br-BUTINACA, respectively, which were potent and efficacious CB1 agonists. Also, at CB2 , tail-less analogs showed a lower potency but increased efficacy. Removing the bromine substitution ((S)-ADB-INACA) resulted in a reduced activity at CB1 ; however, this effect was less prominent at CB2 . Looking at tailed analogs, replacing the bromine with a fluorine substitution ((S)-ADB-5'F-BUTINACA) resulted in an increased potency and efficacy at both receptors. Furthermore, as ADB-5'Br-INACA and MDMB-5'Br-INACA have been frequently detected together in Scottish prisons, this study also evaluated the CB1 receptor activation potential of different mixtures of their respective reference standards, showing no unexpected cannabimimetic effect of combining both substances. Lastly, two powders seized by Belgian Customs and confirmed to contain ADB-5'Br-INACA and MDMB-5'Br-INACA, respectively, were assessed for CB activity. Based on the comparison with their reference standards, varying degrees of purity were suspected.

Pharmacological insights emerging from the characterization of a large collection of synthetic cannabinoid receptor agonists designer drugs.

Synthetic cannabinoid receptor agonists (SCRAs) constitute the largest and most defiant group of abuse designer drugs. These new psychoactive substances (NPS), developed as unregulated alternatives to cannabis, have potent cannabimimetic effects and their use is usually associated with episodes of psychosis, seizures, dependence, organ toxicity and death. Due to their ever-changing structure, very limited or nil structural, pharmacological, and toxicological information is available to the scientific community and the law enforcement offices. Here we report the synthesis and pharmacological evaluation (binding and functional) of the largest and most diverse collection of enantiopure SCRAs published to date. Our results revealed novel SCRAs that could be (or may currently be) used as illegal psychoactive substances. We also report, for the first time, the cannabimimetic data of 32 novel SCRAs containing an (R) configuration at the stereogenic center. The systematic pharmacological profiling of the library enabled the identification of emerging Structure-Activity Relationship (SAR) and Structure-Selectivity Relationship (SSR) trends, the detection of ligands exhibiting incipient cannabinoid receptor type 2 (CB2R) subtype selectivity and highlights the significant neurotoxicity of representative SCRAs on mouse primary neuronal cells. Several of the new emerging SCRAs are currently expected to have a rather limited potential for harm, as the evaluation of their pharmacological profiles revealed lower potencies and/or efficacies. Conceived as a resource to foster collaborative investigation of the physiological effects of SCRAs, the library obtained can contribute to addressing the challenge posed by recreational designer drugs.

Novel Cannabinoid Receptor 2 (CB2) Low Lipophilicity Agonists Produce Distinct cAMP and Arrestin Signalling Kinetics without Bias.

Cannabinoid Receptor 2 (CB2) is a promising target for treating inflammatory diseases. We designed derivatives of 3-carbamoyl-2-pyridone and 1,8-naphthyridin-2(1H)-one-3-carboxamide CB2-selective agonists with reduced lipophilicity. The new compounds were measured for their affinity (radioligand binding) and ability to elicit cyclic adenosine monophosphate (cAMP) signalling and ß-arrestin-2 translocation with temporal resolution (BRET-based biosensors). For the 3-carbamoyl-2-pyridone derivatives, we found that modifying the previously reported compound UOSS77 (also known as S-777469) by appending a PEG2-alcohol via a 3-carbomylcyclohexyl carboxamide (UOSS75) lowered lipophilicity, and preserved binding affinity and signalling profile. The 1,8-naphthyridin-2(1H)-one-3-carboxamide UOMM18, containing a cis configuration at the 3-carboxamide cyclohexyl and with an alcohol on the 4-position of the cyclohexyl, had lower lipophilicity but similar CB2 affinity and biological activity to previously reported compounds of this class. Relative to CP55,940, the new compounds acted as partial agonists and did not exhibit signalling bias. Interestingly, while all compounds shared similar temporal trajectories for maximal efficacy, differing temporal trajectories for potency were observed. Consequently, when applied at sub-maximal concentrations, CP55,940 tended to elicit sustained (cAMP) or increasing (arrestin) responses, whereas responses to the new compounds tended to be transient (cAMP) or sustained (arrestin). In future studies, the compounds characterised here may be useful in elucidating the consequences of differential temporal signalling profiles on CB2-mediated physiological responses.

"Spice"-related deaths in and around Munich, Germany: A retrospective look at the role of synthetic cannabinoid receptor agonists in our post-mortem cases over a seven-year period (2014-2020).

Synthetic cannabinoid receptor agonists (SCRAs, "Spice") are a diverse group of recreational drugs, with their structural and pharmacological variability still evolving. Forensic toxicologists often rely on previous reports to assess their role in intoxication cases. This work provides detailed information on the "Spice"-related fatalities around Munich, Germany, from 2014 to 2020. All cases underwent an autopsy. Pharmaceutical and illicit drugs were detected and quantified in post-mortem peripheral blood or liver by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on circumstantial evidence, only those cases for which a prior consumption was suspected underwent additional analyses for SCRAs and other new psychoactive substances in post-mortem blood, liver or antemortem specimens. Drug concentrations, pathological findings at autopsy and case histories were considered to assess and rank the SCRAs' involvement in each death. Concentration ranges for the individual substances in blood were defined and their distribution patterns over the investigated period were determined and correlated with their legal status and local police seizures. We identified 41 different SCRAs among 98 fatalities. 91.8% were male, at a median age of 36 years. SCRAs played a causative role in 51%, contributory role in 26%, and an insignificant role in 23% of cases. In correlation with local police seizures and legal status, 5F-ADB was the most prevalent in our cases, followed by 5F-MDMB-PICA and AB-CHMINACA. Cumyl-CBMICA and 5F-MDMB-P7AICA were among the least frequently detected SCRAs. "Spice"-related fatalities and SCRAs' causative role have significantly decreased among our cases since the German New Psychoactive Substances Act.

Cannabinoid Receptor 1 Agonist ACEA and Cannabinoid Receptor 2 Agonist GW833972A Attenuates Cell-Mediated Immunity by Different Biological Mechanisms.

Cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) are components in the endocannabinoid system that play significant roles in regulating immune responses. There are many agonists for the cannabinoid receptors; however, their effects on T cell regulation have not been elucidated. In the present study, we determined the effects of the CB1 selective agonist ACEA and the CB2 selective agonist GW833972A on T cell responses. It was found that both agonists impaired anti-CD3 monoclonal antibody induced T cell proliferation. However, ACEA and GW833972A agonists down-regulated the expression of activation markers on CD4+ and CD8+ T cells and co-stimulatory molecules on B cells and monocytes in different manners. Moreover, only GW833972A suppressed the cytotoxic activities of CD8+ T cells without interfering in the cytotoxic activities of CD4+ T cells and NK cells. In addition, the CB2 agonist, but not CB1 agonist, caused the reduction of Th1 cytokine production. Our results demonstrated that the CB1 agonist ACEA and CB2 agonist GW833972A attenuated cell-mediated immunity in different mechanisms. These agonists may be able to be used as therapeutic agents for inducing T cell hypofunction in inflammatory and autoimmune diseases.

Efficacy and safety of olorinab, a full agonist of the cannabinoid receptor 2, for the treatment of abdominal pain in patients with irritable bowel syndrome: Results from a phase 2b randomized placebo-controlled trial (CAPTIVATE).

BACKGROUND: Olorinab is a highly selective, peripherally acting, full agonist of cannabinoid receptor 2. This study assessed the efficacy and safety of olorinab to treat abdominal pain in patients with irritable bowel syndrome with diarrhea (IBS-D) and constipation (IBS-C). METHODS: CAPTIVATE was a phase 2b, randomized, double-blind, placebo-controlled, parallel-group trial. Eligible participants aged 18-70 years with IBS-C and IBS-D diagnosed per Rome IV received olorinab 10 mg, 25 mg, or 50 mg three times daily (TID) or placebo TID for 12 weeks. The primary endpoint was the change in patient-reported average abdominal pain score (AAPS) from baseline to Week 12. KEY RESULTS: A total of 273 participants were randomized to receive olorinab 10 mg (n = 67), olorinab 25 mg (n = 67), olorinab 50 mg (n = 69), or placebo (n = 70). Although a treatment response was observed across all groups, the weekly change in average AAPS from baseline to Week 12 was not significantly different between placebo and any olorinab dose. In a prespecified subgroup analysis of participants with a baseline AAPS ≥6.5, olorinab 50 mg (n = 35) significantly improved AAPS compared with placebo (n = 30) (p = 0.014). Adverse event rates were comparable between olorinab and placebo and there were no reported serious adverse events or deaths. CONCLUSION AND INFERENCES: Although olorinab was well-tolerated and improved weekly AAPS, the primary endpoint was not met. However, in participants with moderate-to-severe pain at baseline (AAPS ≥6.5), olorinab 50 mg significantly improved weekly AAPS compared with placebo. CLINICALTRIALS: gov: NCT04043455.

The piperazine analogue para-fluorophenylpiperazine alters timing of the physiological effects of the synthetic cannabinoid receptor agonist AMB-FUBINACA, without changing its discriminative stimulus, signalling effects, or metabolism.

AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA), which has been associated with substantial abuse and health harm since 2016 in many countries including New Zealand. A characteristic of AMB-FUBINACA use in New Zealand has included the observation that forensic samples (from autopsies) and drugs seized by police have often been found to contain para-fluorophenylpiperazine (pFPP), a relatively little-characterised piperazine analogue that has been suggested to act through 5HT1a serotonin receptors. In the current study, we aimed to characterise the interactions of these two agents in rat physiological endpoints using plethysmography and telemetry, and to examine whether pFPP altered the subjective effects of AMB-FUBINACA in mice trained to differentiate a cannabinoid (THC) from vehicle. Though pFPP did not alter the ability of AMB-FUBINACA to substitute for THC, it did appear to abate some of the physiological effects of AMB-FUBINACA in rats by delaying the onset of AMB-FUBINACA-mediated hypothermia and shortening duration of bradycardia. In HEK cells stably expressing the CB1 cannabinoid receptor, 5HT1a, or both CB1 and 5HT1a, cAMP signalling was recorded using a BRET biosensor (CAMYEL) to assess possible direct receptor interactions. Although low potency pFPP agonism at 5HT1a was confirmed, little evidence for signalling interactions was detected in these assays: additive or synergistic effects on potency or efficacy were not detected between pFPP and AMB-FUBINACA-mediated cAMP inhibition. Experiments utilising higher potency, classical 5HT1a ligands (agonist 8OH-DPAT and antagonist WAY100635) also failed to reveal evidence for mutual CB1/5HT1a interactions or cross-antagonism. Finally, the ability of pFPP to alter the metabolism of AMB-FUBINACA in rat and human liver microsomes into its primary carboxylic acid metabolite via carboxylesterase-1 was assessed by HPLC; no inhibition was detected. Overall, the effects we have observed do not suggest that increased harm/toxicity would result from the combination of pFPP and AMB-FUBINACA.

In vitro characterization of the pyrazole-carrying synthetic cannabinoid receptor agonist 5F-3,5-AB-PFUPPYCA and its structural analogs.

The synthetic cannabinoid receptor agonist (SCRA) market is undergoing important changes since the enactment of the 2021 class-wide generic SCRA ban in China, one of the most important source countries for new psychoactive substances (NPS). Recently, various compounds with new structural features, synthesized to bypass this legislation, have entered the recreational drug market. Certain monocyclic pyrazole-carrying "FUPPYCA" SCRAs have been sporadically detected since 2015 without gaining further popularity. However, as evidenced by their recent detection in Scottish prisons, 5F-3,5-AB-PFUPPYCA and 3,5-ADB-4en-PFUPPYCA have re-emerged, potentially triggered by the new legislative ban. The aim of this study was to characterize the in vitro intrinsic CB1 and CB2 receptor activation potential of 5F-3,5-AB-PFUPPYCA and 3,5-ADB-4en-PFUPPYCA, as well as 4 analogs (5F-3,5-ADB-PFUPPYCA, 3,5-AB-CHMFUPPYCA, 5,3-AB-CHMFUPPYCA and 5,3-ADB-4en-PFUPPYCA) using live cell ß-arrestin 2 recruitment assays. Most analogs were essentially inactive at either CB1 or CB2, with only 3,5-AB-CHMFUPPYCA, 5,3-AB-CHMFUPPYCA and 5,3-ADB-4en-PFUPPYCA showing a limited activation potential at CB1. Furthermore, the importance of the position of the tail structure was demonstrated, with 5,3 regioisomers being more active than their 3,5 analogs. Moreover, all compounds exhibited antagonistic behavior at both receptors, which may be associated with their structural resemblance to cannabinoid antagonists and inverse agonists. Although the 3,5 regioisomers of these "FUPPYCA" SCRAs circumvent the Chinese ban, it is unlikely that these SCRAs will pose a major threat to public health, given the lack of pronounced CB receptor activity.

Synthetic cannabinoid receptor agonists are monoamine oxidase-A selective inhibitors.

Synthetic cannabinoid receptor agonists (SCRAs) are one of the fastest growing classes of recreational drugs. Despite their growth in use, their vast chemical diversity and rapidly changing landscape of structures make understanding their effects challenging. In particular, the side effects for SCRA use are extremely diverse, but notably include severe outcomes such as cardiac arrest. These side effects appear at odds with the main putative mode of action, as full agonists of cannabinoid receptors. We have hypothesized that SCRAs may act as MAO inhibitors, owing to their structural similarity to known monoamine oxidase inhibitors (MAOI's) as well as matching clinical outcomes (hypertensive crisis) of 'monoaminergic toxicity' for users of MAOIs and some SCRA use. We have studied the potential for SCRA-mediated inhibition of MAO-A and MAO-B via a range of SCRAs used commonly in the UK, as well as structural analogues to prove the atomistic determinants of inhibition. By combining in silico and experimental kinetic studies we demonstrate that SCRAs are MAO-A-specific inhibitors and their affinity can vary significantly between SCRAs, most notably affected by the nature of the SCRA 'head' group. Our data allow us to posit a putative mechanism of inhibition. Crucially our data demonstrate that SCRA activity is not limited to just cannabinoid receptor agonism and that alternative interactions might account for some of the diversity of the observed side effects and that these effects can be SCRA-specific.

The mystery behind the apprehensions of the selective cannabinoid receptor type-2 agonist BZO-HEXOXIZID (MDA-19) as a drug of abuse.

PURPOSE: MDA-19 or BZO-HEXOXIZID (N'-[(3Z)-1-(1-hexyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-benzohydrazide), in a more recent nomenclature, was first synthesized in 2008 as a selective type-2 cannabinoid receptor (CB2) agonist due to its potential to treat neuropathic pain. In Brazil, this substance was identified in a series of 53 apprehensions between September 2021 and February 2022. Nevertheless, what intrigues toxicologists is that BZO-HEXOXIZID does not exert significant type-1 cannabinoid receptor (CB1) agonism-which is responsible for the well-known psychoactivity of Δ-9-tetrahydrocannabinol. Thus, the objective of this work is to report the first apprehension and identification of BZO-HEXOXIZID in Brazil and to discuss pharmacologically the possible reasons why a CB2 agonist has been incorporated to the illicit market. METHODS: Suspected seized samples were sent to the Laboratory of the Scientific Police of the State of Sao Paulo. After the screening, samples were confirmed for the presence of BZO-HEXOXIZID using chromatography gas-mass spectrometry, Fourier-transform infrared spectroscopy and nuclear magnetic resonance techniques. RESULTS: Of the 53 samples analyzed, 25 contained only BZO-HEXOXIZID and 28 with mixtures, of which 11 with the CB1 agonist ADB-BUTINACA. Other substances were found in association such as cocaine and caffeine. CONCLUSIONS: BZO-HEXOXIZID was detected in a series of seized materials for the first time in Brazil. Nevertheless, there are still unanswered questions regarding the use of this selective CB2 agonist as a drug of abuse.

Analysis of illicit drugs in purchased and seized electronic cigarette liquids from the United Kingdom 2014-2021.

Increasing popularity and known shortfalls in the regulation of electronic cigarettes (ECs) emphasises the urgent need for closer content monitoring and for comprehensible information on their possible health effects. This study investigated components of EC liquids in samples submitted from 2014 to 2021 and discussed the trends driven by legislation changes. Samples originating from prisoners, teenagers and 'test purchases' of commercially available ECs were analysed by gas chromatography-mass spectrometry (GC-MS). For those containing delta-9-tetrahydrocannabinol (THC) and/or cannabidiol (CBD), the content of these components was quantified by liquid chromatography with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) to show variation of these compounds in EC liquids; 112 EC liquids were included in this study. Nicotine was detected in 87 (78%) of the EC liquids analysed. Twenty-two, including samples from before and after introduction of the UK Psychoactive Substances Act (2016), contained one or more synthetic cannabinoid receptor agonist (SCRA). THC was detected in only 11 samples, whereas a single sample was found to contain CBD only. Six samples contained a mixture of THC and CBD. In all cases where information was available, the THC/CBD content was less than that stated on the product label. The data collected showed great variation in EC liquid content. Therefore, it is important that users are educated regarding risks associated with EC use. Additionally, substances now controlled under both the UK Misuse of Drugs Act and Psychoactive Substances Act were present. These substances each carry a potential risk to health, which is possibly exacerbated if multiple compounds are inhaled concomitantly.

Pharmacology, prevalence in Germany, and analytical data of cyclobutylmethyl- and norbornylmethyl-type synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) are distributed on the drug market to produce THC-like effects while evading routine drug testing and legislation. The cyclobutylmethyl (CBM) and norbornylmethyl (NBM) side chain specifically circumvented the German legislation and led to the emergence of exploratory SCRAs in 2019-2021. The NBM SCRAs were detected post-amendment of the new psychoactive substances act in 2020, which scheduled all CBM SCRAs. All six SCRAs are full agonists at the cannabinoid receptor 1 compared with Δ9 -tetrahydrocannabinol and CP-55,940. The CBM SCRAs showed binding affinities of Ki : 29.4-0.65 nm and potencies of EC50 : 483-40.1 nm (CBMICA << CBMINACA < CBMeGaClone). The norbornyl derivatives exhibited high affinities (Ki : 1.87-0.25 nm), with indazole being the most affine. Functional activity data confirmed that the indazole derivative tends to be the most potent of all three NBM SCRAs (EC50 : 169-1.78 nm). The sterically demanding NBM side chain increased the affinity and activity of almost all core structures. Future studies should be conducted on similarly voluminous side chain moieties. The 'life cycle' of all SCRAs on the drug market was less than a year. Notably, Cumyl-CBMICA was the most prevalent while also having the weakest cannabimimetic properties. Quantification of Cumyl-CBMICA during peak consumption in late 2019 and early 2020 revealed an increase in the concentration on the herbal material, which, together with forum entries and blog posts, corroborates the low in vitro cannabimimetic properties. Seizure prevalence data indicate that almost all SCRAs continue to be identified in 2022, potentially due to remaining stocks.

In vitro metabolic fate of the synthetic cannabinoid receptor agonists (quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate [QMMSB]) and (quinolin-8-yl 4-methyl-3-((propan-2-yl)sulfamoyl)benzoate [QMiPSB]) including isozyme mapping and carboxylesterases activity testing.

The synthetic cannabinoid receptor agonists (SCRAs) (quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate [QMMSB]) and (quinolin-8-yl 4-methyl-3-((propan-2-yl)sulfamoyl)benzoate [QMiPSB], also known as SGT-46) are based on the structure of quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) that has been identified on seized plant material in 2011. In clinical toxicology, knowledge of the metabolic fate is important for their identification in biosamples. Therefore, the aim of this study was the identification of in vitro Phase I and II metabolites of QMMSB and QMiPSB in pooled human liver S9 fraction (pHLS9) incubations for use as screening targets. In addition, the involvement of human monooxygenases and human carboxylesterases (hCES) was examined. Analyses were performed by liquid chromatography coupled with high-resolution tandem mass spectrometry. Ester hydrolysis was found to be an important step in the Phase I metabolism of both SCRAs, with the carboxylic acid product being found only in negative ionization mode. Monohydroxy and N-dealkyl metabolites of the ester hydrolysis products were detected as well as glucuronides. CYP2C8, CYP2C9, CYP3A4, and CYP3A5 were involved in hydroxylation. Whereas enzymatic ester hydrolysis of QMiPSB was mainly catalyzed by hCES1 isoforms, nonenzymatic ester hydrolysis was also observed. The results suggest that ester hydrolysis products of QMMSB and QMiPSB and their glucuronides are suitable targets for toxicological screenings. The additional use of the negative ionization mode is recommended to increase detectability of analytes. Different cytochrome P450 (CYP) isozymes were involved in the metabolism; thus, the probability of drug-drug interactions due to CYP inhibition can be assessed as low.