Of the different classes of NPS, the growing group of new synthetic opioids (NSOs) is of particular concern owing to the high risk of overdose. Prior to 2019, the majority of emerging NSOs were analogues of fentanyl; in recent years, this balance has shifted towards the appearance of non-fentanyl-related NSOs, shaping what could be considered the current “post-fentanyl analogue era”. Insight into the biological effects of emerging NSOs such as nitazenes is crucial to inform stakeholders of the potential harm that is linked with the presence of these substances on recreational drug markets. The first part of this presentation will focus on the pharmacological characterization of recent and emerging non-fentanyl NSOs by means of in vitro and in vivo techniques. At the in vitro level, the focus lies with binding to and activation of the µ-opioid receptor (MOR), the primary molecular target for clinically applied and abused opioids. In vivo, the effects of various NSOs in rodents can be evaluated by means of behavioural assays (including evaluation of antinociception, locomotor activity, and body temperature changes). The second part of this presentation will zoom in on the use of an activity-based assay to detect the presence of NSOs in biological samples. This non-targeted technique has the potential to become a complementary tool to mass spectrometry-based methods to aid in the detection of NPS, specifically NSOs. Taken together, approaching the NSO problem from a pharmacological perspective may help to navigate this complex landscape in terms of risk assessment, prioritization, and detection.

Due to their psychoactive effects, synthetic cathinones (SCs) were confirmed as the second most prevalent class of NPS in 2024, underscoring their widespread availability and use. When the first generation synthetic cathinones (methylone,mephedrone, MDPV) became illegal, a second and third generationemerged and a lack of information exists on their molecular structure and metabolic pathway, making their identification in biological samples.challenging. This is critical in the case of cathinones that undergo extensive metabolic degradation, for which the identification of metabolites in urine constitutes the only possible way of attesting their consumption,
In this concern, our study group investigated the kinetics and metabolism of most popular last-generation cathinones in consumers.
The main metabolic pathways of the latest generation synthetic cathinones N-ethylhexedrone (NEH), N-Ethylpentedrone (NEP) and 4-Chloromethcathinone (4-CMC, clephedrone) in urine suggested hydroxy-NEP, hydroxy-NEH and hydroxy-4-CMC as target analytes in the pharmacotoxicological analyses, so as to increase detection time after parent compound intake.
Disposition of α-pyrrolidinovalerophenone (α-PVP) in urine and oral fluid showed a later time peak than non-pyrrolidine SCs and N-butanoic acid metabolite as the most abundant metabolite whereas β-OH-α-PVP was a minor metabolite.
Between 2022 and 2024, Italy reported 106 cases of intoxication associated with SCs consumption. MDPHP was the most frequently involved cathinone, either consumed alone or in combination with other substances. Two fatalities exclusively linked to MDPHP consumption were also reported between 2023 and 2024.
These findings highlight the evolving landscape of SCs distribution and consumption, their kintetics and metabolism emphasizing the need for continued monitoringdevoted to public health interventions.

The rapid emergence and structural diversification of New Psychoactive Substances (NPS), particularly synthetic cannabinoids (SCs) and psychedelics, pose significant challenges for forensic detection and clinical toxicology in Singapore and worldwide. Understanding their metabolism and pharmacokinetics (PK) is crucial for identifying reliable biomarkers and rationalizing toxicities. This presentation highlights our recent research, in collaboration with Health Sciences Authority (HSA), Singapore, elucidating NPS disposition.
A two-pronged approach using in vitro liver subcellular systems, recombinant enzymes, and transporter assays, corroborated with authentic urine analysis, was employed. Key findings include: OAT3-mediated renal transport contributing to the urinary abundance of ester hydrolysis metabolites (EHMs) of 5F-MDMB-PINACA/4F-MDMB-BINACA; identification of stable hydroxylated (M6, M11, M14) and oxidative deaminated (M15) urinary biomarkers for ADB-BUTINACA; and parent/mono-hydroxylated metabolites serving as biomarkers for OXIZID SCs. Additionally, CES-mediated transesterification with alcohol was observed for indazole-3-carboxamide SCs, yielding potential co-abuse biomarkers. For psilocin, we identified 4-HIA as the intermediate metabolite and established MAO-A as the enzyme initiating this conversion, while also determining its significant extrahepatic clearance.
Collectively, these studies provide critical metabolic pathways, enzyme contributions, PK insights, and validated urinary biomarkers, enhancing our ability to detect NPS use and assess associated risks.