Abstract
The NSO_MAP project investigates the pharmaco-toxicological and forensic risks of Nitazenes, a potent class of New Synthetic Opioids (NSO) emerging on illicit markets. Combining in vivo (mouse and zebrafish larvae) and in vitro (human cells) studies with advanced analytical techniques, the project characterizes their toxic effects, metabolism, genotoxicity, and identifies key biomarkers. Nitazene exposure will be evaluated across neurological, cardiovascular, and gender-specific responses. Mass spectrometry (LC-HRMS) will support metabolite identification in biological fluids and tissues, flow cytometry will support genotoxicity evaluation. The outcomes will aid forensic toxicology, emergency response, and legislative control, improving national and European strategies against NSO spread. Results will be shared with public agencies and scientific communities to enhance monitoring and prevention.
Results achieved
The NSO_MAP project has generated one of the most comprehensive toxicological characterizations currently available for Nitazene Synthetic Opioids, a rapidly expanding class of new psychoactive substances (NPS) that has become an important public health concern due to its extremely high potency and its association with severe intoxications and fatal overdoses. Nitazenes are synthetic opioids acting as highly potent μ-opioid receptor agonists. Over the last few years, numerous new derivatives have appeared on the illicit drug market, often replacing or contaminating heroin and fentanyl preparations. Their extraordinary potency, together with the limited toxicological information available, has created major challenges for clinicians, forensic laboratories and public health authorities. Within this context, the NSO_MAP project aimed to provide an integrated toxicological framework capable of improving the understanding of both the acute and long-term health risks associated with these emerging compounds. To achieve this objective, the project combined complementary experimental approaches, including in vitro studies on human cell models, developmental toxicity studies using zebrafish embryos and larvae, in vivo pharmacotoxicological investigations in murine models, metabolite characterization and computational pharmacokinetic modelling. The integration of these different methodologies allowed the project to investigate Nitazene toxicity from multiple biological perspectives while promoting the reduction of animal experimentation through the implementation of alternative models and in silico approaches. One of the most relevant achievements of the project was the establishment of a comparative pharmacotoxicological profile of several Nitazene derivatives. Experimental studies demonstrated that these substances possess an extremely high potency, producing profound respiratory depression and marked cardiovascular impairment at very low doses. Respiratory depression emerged as the major life-threatening effect, confirming the exceptional risk associated with exposure to these compounds. The project also demonstrated that impairment of sensorimotor functions occurs at doses close to those producing analgesic effects, highlighting the narrow safety margin that characterizes this class of synthetic opioids. These findings provide valuable information for the interpretation of intoxication cases and for improving emergency clinical management. Another important result concerns the characterization of Nitazene metabolism. The project demonstrated that some metabolites are not simply inactive degradation products but may retain, or even increase, the toxicological activity of the parent compound. In particular, functional validation of N-desethyl-isotonitazene showed respiratory and cardiac toxicity comparable to or greater than that of isotonitazene itself. These findings indicate that metabolic transformation can substantially amplify toxicological burden and suggest that forensic and clinical toxicology should systematically consider active metabolites when evaluating poisoning cases. The project also provided important information on the potential long-term biological effects of Nitazenes. Through internationally validated in vitro assays performed according to OECD Guideline 487, several Nitazene derivatives were shown to induce chromosomal damage, while others did not exhibit detectable genotoxic activity. Moreover, metabolic activation increased the genotoxic potential of specific compounds, demonstrating that genotoxic risk is compound-specific and may depend on biotransformation processes. These results reveal that different Nitazene derivatives cannot be considered toxicologically equivalent and underline the need for individual risk assessment of newly emerging analogues. A further innovative aspect of the project was the use of zebrafish embryos and larvae as an alternative vertebrate model for developmental toxicology. These studies identified developmental cardiotoxicity, morphological alterations and behavioural changes consistent with central nervous system depression, confirming the sensitivity of this model for the early screening of highly potent synthetic opioids. The successful implementation of this approach represents an important contribution toward the adoption of experimental strategies consistent with the principles of Replacement, Reduction and Refinement (3Rs) in biomedical research. During the course of the project, the research plan was successfully adapted to the rapidly evolving landscape of new psychoactive substances. As new Nitazene derivatives emerged and were identified by the European Union Drugs Agency (EUDA) and national early warning systems as substances of increasing concern, the experimental activities were strategically updated to include these compounds. At the same time, computational ADMET modelling was integrated with experimental data to predict pharmacokinetic behaviour, blood-brain barrier penetration and metabolic pathways, allowing the project to maintain a comprehensive pharmacological characterization while optimizing available resources and minimizing additional animal experimentation. Overall, NSO_MAP has produced an integrated dataset that combines mechanistic cellular studies, developmental toxicology, in vivo functional pharmacology and computational modelling. This multidisciplinary approach has generated new scientific knowledge on the mechanisms underlying Nitazene toxicity and has established a scalable framework that can be applied to the evaluation of newly emerging synthetic opioids in the future. Beyond its scientific achievements, the project has contributed to strengthening national expertise in the field of emerging synthetic opioids through the active involvement of early-career researchers, PhD candidates and Master's students, who received advanced training in modern toxicological methodologies including flow cytometry, metabolite analysis by high-resolution mass spectrometry, behavioural pharmacology, respiratory physiology and computational toxicology. The project has also generated scientific publications, conference presentations and academic theses, ensuring broad dissemination of the results and promoting knowledge transfer to the scientific community, forensic laboratories and public health authorities. The knowledge generated by NSO_MAP provides valuable support for forensic toxicology, clinical toxicology and public health surveillance. The identification of highly toxic Nitazene derivatives, the demonstration of metabolite-driven toxicity, the characterization of compound-specific genotoxicity and the development of predictive toxicological models contribute to improving risk assessment, facilitating early warning activities and supporting more effective management of intoxications caused by these emerging synthetic opioids. The methodological framework established by the project also represents a solid basis for future investigations on newly emerging psychoactive substances, reinforcing preparedness against evolving drug-related public health threats. This project was supported by the European Union – NextGenerationEU under the PRIN 2022 programme. The results presented here reflect the scientific activities carried out within the NSO_MAP project and contribute to advancing knowledge on the toxicological characterization of emerging Nitazene synthetic opioids.Dettagli del progetto
Responsabile scientifico: Monia Lenzi
Strutture Unibo coinvolte:
Dipartimento di Farmacia e Biotecnologie
Coordinatore:
Università degli Studi di Ferrara - Amministrazione Centrale(Italy)
Contributo totale Unibo: Euro (EUR) 99.043,00
Durata del progetto in mesi: 24
Data di inizio
16/10/2023
Data di fine:
28/02/2026
