Insights into the metabolism of CH-PIATA-A novel synthetic cannabinoid featuring an acetamide linker.

The recent change from the popular carboxamide to an acetamide (ATA) linker scaffold in synthetic cannabinoid receptor agonists (SCRAs) can be interpreted as an attempt to circumvent legal regulations, setting new analytical challenges. Metabolites of N-cyclohexyl-2-(1-pentyl-1 H-indol-3-yl)acetamide: CH-PIATA, the second ATA type SCRA detected in the EU, were investigated in urine and serum samples by LC-HRMS-MS and LC-MS-MS. Two different in vitro models, a pHLM assay and HepG2-cells, as well as an in silico prediction by GLORYx freeware assisted in metabolite formation/identification. CH-PIATA was extensively metabolized, leading to metabolites formed primarily by mono- and dihydroxylation. For urine and serum specimens, monohydroxylation at the indole core or the methylene spacer of the acetamide linker (M1.8), carboxylic acid formation at the N-pentyl side chain (M3.1) and degradation of the latter leading to a tentatively identified N-propionic acid metabolite (M5.1) are suggested as reliable markers for substance intake. The N-propionic acid metabolite could not be confirmed in the in vitro assays as it includes multiple consecutive metabolic reactions. Furthermore, CH-PIATA could be detected as parent substance in blood samples, but not in urine. Both in vitro assays and the in silico tool proved suitable for predicting metabolites of CH-PIATA. Considering effort and costs, pHLM incubations seem to be more effective for metabolite prediction in forensic toxicology than HepG2 cells. The highlighted Phase I metabolites serve as reliable urinary targets for confirming CH-PIATA use. The in silico approach is advantageous when reference material is unavailable.

ADB-HEXINACA-a novel synthetic cannabinoid with a hexyl substituent: phase I metabolism in authentic urine samples, a case report and prevalence on the German market.

Synthetic cannabinoid receptor agonists (SCRAs) are one of the largest groups of new psychoactive substances (NPS). Yet, another novel analog started spreading on the NPS market around 2021. Soon after, the substance could be analytically characterized in herbal material as ADB-HEXINACA, an SCRA containing a hexyl-substituted tail on the indazole core. Here, we present suitable urinary markers to prove the consumption of this analog, a case report of acute polydrug intoxication and data on its prevalence in Germany. Anticipated phase I metabolites were detected in 12 authentic urine samples that were collected for abstinence control and analyzed by ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer (UPLC-qToF-MS). The results of in vivo samples were confirmed by analysis of in vitro incubates with pooled human liver microsomes (pHLMs). Forensic samples were used to assess the prevalence of ADB-HEXINACA. Thirty-two phase I metabolites were detected in the authentic urine samples. The main metabolites resulted from amide hydrolysis in combination with either monohydroxylation or ketone formation at the hexyl moiety (M15 and M26), the monitoring of which is recommended as a proof of consumption. ADB-HEXINACA was detected in 3.5% of SCRA positive urine samples collected for abstinence control in Freiburg up to December 2022 and in 5.5% of the SCRA positive blood/serum samples. The hexyl substituent of ADB-HEXINACA allows for the detection of specific urinary biomarkers suggested as analytical targets to confirm its prior intake. ADB-HEXINACA had a rather low prevalence in Germany, alternating months of higher prevalence with periods of total absence.

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.