Craig McKenzie

An emerging new trend reveals that potent synthetic cannabinoid receptor agonists (SCRAs) that were prevalent prior to the Chinese class-wide ban in July 2021 have remained on the market or re-emerged. This is likely because of a new production route where unscheduled tail-less precursors are converted into the desired controlled SCRA via a one-step synthesis. This study reports the discovery of a clandestine production site in Switzerland for this synthesis. Controlled recreations of the synthesis of ADB-BUTINACA (also known as ADB-BINACA or ADMB-BUTINACA) and MDMB-4en-PINACA based on a recipe recovered from the Swiss production site were undertaken. The recreations yielded oils or waxy products with chromatographic purities ranging from 39.3 to 93.4% with the precursors (ADB-INACA or MDMB-INACA) remaining in all products. Mixtures of a precursor (e.g., MDMB-INACA, ADB-INACA, AB-INACA, MDMB-ICA, ADB-IATA, MDMB-5’Me-INACA) with corresponding final SCRA(s) (e.g., MDMB-4en-PINACA, 4F-MDMB-BUTINACA, MDMB-BUTINACA, MDMB-FUBINACA, ADB-BUTINACA, ADB-4en-PINACA, AB-CHMINACA, 4F-MDMB-BUTICA, ADB-FUBIATA) were found in 28 forensic casework samples in the US received between February 2023 and February 2024 and 87 samples seized from the Scottish prisons between February 2023 and July 2024. In vitro CB₁ receptor activity of the precursors, synthesized products, and mixtures of precursors and final SCRAs were examined using β-arrestin 2 recruitment and intracellular calcium release assays. The precursors were much less potent than the final SCRAs and the concentration of the final SCRA dictated the activity of mixtures. This study raises awareness of the emergence of a new production method for SCRAs as observed in multiple countries.

The rapid structural evolution and extensive metabolism of synthetic cannabinoid receptor agonists (SCRAs) following consumption makes their detection in biological samples challenging, especially in urine. In vitro metabolite identification studies are an essential tool for identifying analytical targets to confirm SCRA consumption in clinical and forensic toxicology casework. Systematic studies on structurally related SCRAs allow structure–metabolism relationships (SMRs) to be determined, helping to predict the metabolites of emerging and future compounds. In this study, a series of amino acid-derived 4-pentenyl SCRAs and the OXIZID 4-pentenyl SCRA BZO-4en-POXIZID were incubated at 5 µM with pooled human hepatocytes for up to 3 h. Metabolites were identified using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC–QTOF-MS) and SMRs investigated using the proportion that each type of biotransformation contributed to the total abundance of metabolites for each parent compound. Metabolites were mainly produced via terminal amide/ester hydrolysis, dihydrodiol formation on the tail, hydroxylation and N-dealkylation, but also by ketone formation, dehydrogenation, glucuronidation and combinations thereof. Methyl valinate (MMB) and ethyl valinate (EMB) SCRAs underwent extensive ester hydrolysis (90.9–96.0%), which was less dominant for methyl tert-leucinate (MDMB) SCRAs (47.6–60.7%), with dihydrodiol formation (24.3–27.7%) and hydroxylation (6.2–17.3%) becoming more prominent. For AB-4en-PICA and tert-leucinamide (ADB) SCRAs, hydroxylation was the major metabolic pathway (47.9–58.4%), while tail dihydrodiol formation was most prominent for BZO-4en-POXIZID (61.5%). Clear SMRs were identified for 4-pentenyl SCRAs and can be used to recommend biomarkers of consumption and aid prediction of metabolites of emerging and future SCRAs.

Synthetic cannabinoid receptor agonists (SCRAs) are a group of new psychoactive substances (NPS) that bind to and activate the cannabinoid 1 and 2 (CB₁ and CB₂) receptors. Following the introduction of SCRA analog controls in China in July 2021, new SCRAs with a bromine at the 5 position on the indazole core emerged on the recreational drug market. In this study, the in vitro CB₁ receptor activity of 19 different SCRAs with a halogenated indazole core was systematically examined using a AequoScreen^® CB₁ assay alongside four available non-halogenated analogs, ADB-BUTINACA, MDMB-BUTINACA, MDMB-4en-PINACA, and MDMB-INACA, and the newly emerged MDMB-5′Me-INACA (MDMB-5-methyl-INACA). Of SCRAs with a substitution at the 5 position on the indazole core, analogs with a fluorine had the lowest EC₅₀ values. For SCRAs with a methyl ester head moiety (MMB or MDMB), chlorinated analogs had the next lowest EC₅₀ values followed by brominated analogs; however, the opposite relationship was observed for SCRAs with an amide head moiety (AB or ADB). All halogenated compounds had similar potency to their non-halogenated analog, except the brominated tert-leucine methyl ester SCRAs, which had significantly reduced potency. Of the different head moieties, tert-leucine methyl ester SCRAs were the most potent, followed by tert-leucinamide, valinamide, and valine methyl ester. The potencies of the SCRAs with pentyl (ADB-5′Br-PINACA/ADMB-5′Br-PINACA/ADB-P-5′Br-INACA), pent-4-enyl (ADB-4en-5′Br-PINACA/ADMB-4en-5′Br-PINACA/ADB-4en-P-5′Br-INACA and MDMB-4en-5′Br-PINACA/MDMB-4en-P-5′Br-INACA), and butyl tails (ADB-5′Br-BUTINACA/ADMB-5′Br-BUTINACA/ADB-B-5′Br-INACA) were not significantly different, whereas fluorobenzyl (ADB-5′Br-FUBINACA), tail-less (ADB-5′Br-INACA), and decyl tail (ADB-5′Br-DECINACA/ADMB-5′Br-DECINACA/ADB-D-5′Br-INACA) analogs were significantly less potent.

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 β‐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.

Synthetic cannabinoid receptor agonists (SCRAs) remain a major public health concern, with their use implicated in intoxications and drug-related deaths worldwide. Increasing our systematic understanding of SCRA metabolism supports clinical and forensic toxicology casework, facilitating the timely identification of analytical targets for toxicological screening procedures and confirmatory analysis. This is particularly important as new SCRAs continue to emerge on the illicit drug market. In this work, the metabolism of ADB-HEXINACA (ADB-HINACA, N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-hexyl-1H-indazole-3-carboxamide), which has increased in prevalence in the United Kingdom and other jurisdictions, was investigated using in vitro techniques. The (S)-enantiomer of ADB-HEXINACA was incubated with pooled human hepatocytes over 3 hours to identify unique and abundant metabolites using liquid chromatography–quadrupole time-of-flight mass spectrometry. In total, 16 metabolites were identified, resulting from mono-hydroxylation, di-hydroxylation, ketone formation (mono-hydroxylation then dehydrogenation), carboxylic acid formation, terminal amide hydrolysis, dihydrodiol formation, glucuronidation and combinations thereof. The majority of metabolism took place on the hexyl tail, forming ketone and mono-hydroxylated products. The major metabolite was the 5-oxo-hexyl product (M9), while the most significant mono-hydroxylation product was the 4-hydroxy-hexyl product (M8), both of which were confirmed by comparison to in-house synthesized reference standards. The 5-hydroxy-hexyl (M6) and 6-hydroxy-hexyl (M7) metabolites were not chromatographically resolved, and the 5-hydroxy-hexyl product was the second largest mono-hydroxylated metabolite. The structures of the terminal amide hydrolysis products without (M16, third largest metabolite) and with the 5-positioned ketone (M13) were also confirmed by comparison to synthesized reference standards, along with the 4-oxo-hexyl metabolite (M11). The 5-oxo-hexyl and 4-hydroxy-hexyl metabolites are suggested as biomarkers for ADB-HEXINACA consumption.

Drug use within prisons is increasingly complex and unpredictable. Benzodiazepines are currently one of the most common drugs detected in individuals leaving Scottish prisons; however, understanding illicit benzodiazepine use within prisons and assessing the potential harm to individuals is challenging due to the lack of available analytical data on the substances circulating. Increasingly, materials, such as paper and clothing, infused with novel benzodiazepines have been identified as a smuggling route into Scottish prisons. Methods were developed for the qualitative and quantitative analysis of benzodiazepines using gas chromatography–mass spectrometry (GC–MS) and applied to 495 seized samples from 11 Scottish prisons, including papers, cards, blotters, powders, tablets, and clothing. Evolution in the benzodiazepines being detected was demonstrated, with etizolam being the most prevalent throughout 2020/2021 following which flubromazepam and bromazolam detections increased. Additionally, significant changes in the smuggling methods and drug formats detected occurred over time following policy changes within prisons. These data represent the first reported widescale etizolam quantitation data and demonstrate high levels of variability across all sample types, most notably within tablets (0.34–2.33 mg per tablet). Additionally, concentration mapping of a whole seized card sample revealed the total concentration of drug present (312.5 mg) and demonstrated variability across the surface of the card (1.16–1.87 mg/cm²). These data highlight the challenges of consistent dosing for individuals and the high risks of unintentional overdose. Increased understanding of the challenge of such drug smuggling and benzodiazepine use will aid in the development of strategies to reduce supply and mitigate harm.

Early warning systems detect new psychoactive substances (NPS), while dedicated monitoring programs and routine drug and toxicology testing identify fluctuations in prevalence. We report the increasing prevalence of the synthetic cannabinoid receptor agonist (SCRA) ADB‐BUTINACA (N‐[1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl]‐1‐butyl‐1H‐indazole‐3‐carbox‐amide). ADB‐BUTINACA was first detected in a seizure in Sweden in 2019, and we report its detection in 13 routine Swedish forensic toxicology cases soon after. In January 2021, ADB‐BUTINACA was detected in SCRA‐infused papers seized in Scottish prisons and has rapidly increased in prevalence, being detected in 60.4% of the SCRA‐infused papers tested between January and July 2021. In this work, ADB‐BUTINACA was incubated with human hepatocytes (HHeps), and 21 metabolites were identified in vitro, 14 being detected in authentic case samples. The parent drug and metabolites B9 (mono‐hydroxylation on the n‐butyl tail) and B16 (mono‐hydroxylation on the indazole ring) are recommended biomarkers in blood, while metabolites B4 (dihydrodiol formation on the indazole core), B9, and B16 are suitable biomarkers in urine. ADB‐4en‐PINACA (N‐[1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl]‐1‐[pent‐4‐en‐1‐yl]‐1H‐indazole‐3‐carboxamide) was detected in Scottish prisons in December 2020, but, unlike ADB‐BUTINACA, prevalence has remained low. ADB‐4en‐PINACA was incubated with HHeps, and 11 metabolites were identified. Metabolites E3 (dihydrodiol formed in the tail moiety) and E7 (hydroxylation on the linked/head group) are the most abundant metabolites in vitro and are suggested as urinary biomarkers. The in vitro potencies of ADB‐BUTINACA (EC₅₀, 11.5 nM and ADB‐4en‐PINACA (EC₅₀, 11.6 nM) are similar to that of MDMB‐4en‐PINACA (EC₅₀, 4.3 nM). A third tert‐leucinamide SCRA, ADB‐HEXINACA was also detected in prison samples and warrants further investigation.

In vitro pharmacokinetic studies were conducted on enantiomer pairs of twelve valinate or tert-leucinate indole and indazole-3-carboxamide synthetic cannabinoid receptor agonists (SCRAs) detected on the illicit drug market to investigate their physicochemical parameters and structure-metabolism relationships (SMRs). Experimentally derived Log D_7.4 ranged from 2.81 (AB-FUBINACA) to 4.95 (MDMB-4en-PINACA) and all SCRAs tested were highly protein bound, ranging from 88.9 ± 0.49% ((R)-4F-MDMB-BINACA) to 99.5 ± 0.08% ((S)-MDMB-FUBINACA). Most tested SCRAs were cleared rapidly in vitro in pooled human liver microsomes (pHLM) and pooled cryopreserved human hepatocytes (pHHeps). Intrinsic clearance (CL_int) ranged from 13.7 ± 4.06 ((R)-AB-FUBINACA) to 2944 ± 95.9 mL min⁻¹ kg⁻¹ ((S)-AMB-FUBINACA) in pHLM, and from 110 ± 34.5 ((S)-AB-FUBINACA) to 3216 ± 607 mL min⁻¹ kg⁻¹ ((S)-AMB-FUBINACA) in pHHeps. Predicted Human in vivo hepatic clearance (CL_H) ranged from 0.34 ± 0.09 ((S)-AB-FUBINACA) to 17.79 ± 0.20 mL min⁻¹ kg⁻¹ ((S)-5F-AMB-PINACA) in pHLM and 1.39 ± 0.27 ((S)-MDMB-FUBINACA) to 18.25 ± 0.12 mL min⁻¹ kg⁻¹ ((S)-5F-AMB-PINACA) in pHHeps. Valinate and tert-leucinate indole and indazole-3-carboxamide SCRAs are often rapidly metabolised in vitro but are highly protein bound in vivo and therefore predicted in vivo CL_H is much slower than CL_int. This is likely to give rise to longer detection windows of these substances and their metabolites in urine, possibly as a result of accumulation of parent drug in lipid-rich tissues, with redistribution into the circulatory system and subsequent metabolism.

The synthetic cannabinoid receptor agonist (SCRA) market is transnational, and the availability of individual SCRAs changes regularly in response to national and international legislative controls. This generates a cyclic pattern and near constant evolution of SCRA compounds. This study reports toxicology‐based and/or seized sample‐based prevalence data relating to SCRA use in prisons from Germany, the United Kingdom (UK; Scotland and Wales), and the United States (US), representing 4427 individual test results. The study examines SCRA detections in prisons from July 2018 to September 2020, and where possible, prison‐based data are compared with SCRA prevalence data in the wider population. The relative influence of Chinese, other international, and national drug legislation on the prevalence of individual SCRAs in prisons is also considered. tert‐Leucinate‐ and valinate‐indole‐ and indazole‐3‐carboxamides were the most common SCRA detections, and MDMB‐4en‐PINACA was one of the most commonly detected SCRAs in all jurisdictions by September 2020. However, despite there being a global production and supply market, there were notable regional differences. Analog controls in German and US legislation may have led to increased compound diversity that is not reflected in the UK which has both analog controls and a blanket ban on psychoactive substances. While there were regional differences, SCRA prevalence in prisons closely aligned with the SCRAs detected on the local market, demonstrating that SCRA (and possibly other NPS) monitoring programs in prisons can act as early warning systems for the wider population in that given jurisdiction.