For more than a decade, new psychoactive substances (NPS) have attracted the interest of healthcare professionals, toxicologists, law enforcement, policymakers, people who are using these drugs, and other stakeholders interested in the psychoactive drugs field. NPS – sometimes also called designer drugs – are typically not controlled by the United Nations’ drug conventions, but thought to pose a potential public health threat comparable to that posed by substances listed therein. Psychoactive drugs – including medicines, dietary supplements, NPS, and controlled substances – have to be seen as commodities, which generates demand from various user groups and communities, though the exposure to such substances is not always intentional, which can lead to dramatic consequences. Exciting opportunities arise from the systematic investigation of NPS, which often begins with studying their analytical features during the development of synthetic routes. On the street level, unambiguous identifications can be a challenge, for example when having to consider the existence of isomers in the absence of reference material. Studying their effects in vitro and in vivo also adds to the understanding of their pharmacokinetic and pharmacodynamic properties. From a chemical and pharmacological perspective, the link between controlled drugs and medicines has long been understood and this extends to the NPS phenomenon as well. Indeed, an appreciable number of NPS originated from drug discovery research aimed to develop new medicines several decades ago. Typically, some overlap exists between drugs that belong to a particular pharmacological or chemical group though some differences can sometimes also be observed, which can be surprising or even unexpected. This presentation will provide some insights into these endeavours using (2- aminopropyl)benzo[b]thiophene analogues (APBTs) as a representative example.

Amongst the new psychoactive substances (NPS), synthetic cannabinoid receptor agonists (SCRAs) constitute the largest class of compounds, while synthetic opioids are undoubtedly the most deadly ones. Both drug classes have in common that they are composed of highly active compounds (often acting at low- or sub-ng/ml level). These NPS classes grow continuously, with new structures containing minor or major structural modifications, to evade legislation and/or detection. This poses a real challenge in terms of detection and monitoring. Hence, currently available assays are struggling to detect these compounds. Immuno-assays only recognize compounds that have substantial structural overlap and have no value at all for detecting structurally unrelated molecules. Also chromatographic assays struggle to detect these newly emerging compounds: LC-MS/MS is restricted by its targeted nature (requiring prior structural knowledge) and also LC-HRMS, either with data-dependent or data-independent acquisition, may struggle to detect unknown compounds (or their metabolites) at very low concentrations. These limitations hold true for both SCRAs and synthetic opioids, for which a wide variety of structures has recently emerged, in part driven by generic legislations banning existing structures. Morover, the pharmacological activity of many newly emerging NPS remains unclear, which complicates prioritization efforts, which ideally focus on those substances that may pose most harm. In this presentation we will present an alternative strategy, independent of antibodies or mass spectrometric detection, as an initial screening tool to test biological matrices for the presence of compounds with cannabinoid or opioid activity. The developed strategy is based upon biological activity and may be used as a first-line screening tool to identify (synthetic) cannabinoid or opioidpositive biological samples, as well as for activity profiling of new SCRAs (acting at the cannabinoid receptors CB1 or CB2), synthetic opioids (acting at the mu opioid receptor) and compounds with psychedelic activity (acting at the serotonin 2A receptor). All above-mentioned receptors are G protein-coupled receptors (GPCRs). Activation of these receptors results in recruitment of signaling molecules, such as G proteins or ?-arrestin 2. Here, we modified HEK293T cells in such a way that they express 1) a GPCR fused to one part of a nanoluciferase molecule, and 2) ?-arrestin 2 or a G protein, fused to the other part of nanoluciferase (NanoBiT® Technology). Addition to the developed cell systems of pure compound or a biological extract will result in receptor activation and, hence, recruitment of the signaling molecule to the GPCR. This brings both parts of the nanoluciferase into close proximity, resulting in a so-called functional complementation, which, after application of a substrate, can easily be monitored via luminescence. The developed assays were used to characterize a wide variety of new synthetic opioids (e.g. belonging to the ‘nitazene‘ class), SCRAs and their metabolites (also including more recent SCRAs evading the recent ‘China ban‘), as well as a wide variety of psychedelics. Moreover, these assays were also successfully used to screen extracts from biofluids (serum, plasma, urine, oral fluid) for opioid or cannabinoid activity, with excellent sensitivity and specificity. In conclusion, these bioassays do not only allow to gain better insight into the potential activity of NPS (helping to steer prioritization efforts), they also offer the opportunity to serve as an alternative and universal first-line screening tool for SCRAs and synthetic opioids in biological matrices.

New psychoactive substances (NPS) are analogues to traditional substances in all major drug groups; amphetamines, cannabis, cocaine, opioids, and benzodiazepines. The diversity and changing drug panorama challenge the analytical toxicologist and comprehensive methods are costly and need advanced instrumentation. High resolution mass spectrometry (e.g. LC-Q-TOF) seems to have analytical advantages that make it an alternative both for quick, broad screening as well as for confirmatory analyses. In post mortem toxicology, NPS from all drug groups have been determined to contribute to the cause of death but NPS from the opioid group have dominated because of high potency and adverse effects related to respiratory depression. However, the interpretation is complicated by the lack of reference data such as concentrations in the living. Indeed, even basic pharmacological data can be missing when an NPS is introduced on the market. Such basic data includes receptor binding and activation studies and animal studies. In this lecture, I will describe current methods for screening and confirmation of NPS in biological matrices, give an overview of current findings, and describe how in vitro methods can be used to investigate NPS effects.