Romain Pelletier

Les nouvelles substances psychoactives (NPS) représentent un défi croissant pour la toxicologie clinique, médico-légale et les politiques de santé publique. Leur propagation rapide, leur diversité chimique et leur faible niveau de caractérisation analytique compliquent leur identification et leur interprétation toxicologique. Le métabolisme de ces substances, souvent inconnu, constitue un verrou important à la caractérisation de biomarqueurs fiables de consommation. Cette thèse propose une approche transversale visant à identifier des métabolites pertinents de plusieurs NPS à travers une combinaison de modèles expérimentaux (in vivo, in vitro, ex vivo), de prédictions in silico, et de traitement bio-informatique de données par réseau moléculaire. Des études portant sur l’eutylone, le 25E-NBOH ou encore l’AP-237 illustrent l’intérêt de croiser les données issues de matrices humaines réelles, de lignées cellulaires et de modèles alternatifs. L’utilisation de la spectrométrie de masse haute résolution (LC-HRMS), couplée à des outils bio-informatiques comme le réseau moléculaire, a permis d’identifier de nouveaux métabolites comme marqueurs de consommation. Ce travail souligne la complémentarité des approches et propose un arbre décisionnel pour guider les futures investigations métaboliques sur les NPS émergents. Ces résultats apportent des éléments nouveaux pour la communauté scientifique, et complètent les outils de détection en contexte clinique ou médico-légal. Ils ouvrent la voie à des stratégies plus réactives, robustes et anticipatrices pour faire face à l’évolution rapide des substances de synthèse sur le marché illicite.

New psychoactive substances (NPS) pose an increasing challenge for clinical and forensic toxicology due to the initial lack of analytical and metabolic data. This study evaluates the performance of four in silico prediction tools (GLORYx, BioTransformer 3.0, SyGMa, and MetaTrans) in predicting the metabolism of seven NPS from five major chemical families (cathinones, synthetic cannabinoids, synthetic opioids, designer benzodiazepines, and dissociative anesthetics). The predicted metabolites were compared to those reported in the literature. The results revealed that SyGMa was the most exhaustive tool, predicting 437 metabolites, whereas MetaTrans predicted the fewest (61). GLORYx uniquely identified glutathione conjugation, while BioTransformer was particularly effective in predicting phase I reactions. However, no single tool provided complete predictions. Combining the four tools enabled the identification of several key biomarkers consistent with experimental data, such as m/z 238.1443 for eutylone and m/z 381.1926 for etonitazepipne. These findings highlight the need for integrated approaches to optimize metabolite prediction. Future advancements in artificial intelligence-based models could reduce false positives and enhance the accuracy of predictions, thus reinforcing the role of in silico tools in toxicological investigations.

Background: Needleless connectors (NCs) can be disinfected using antiseptic barrier caps (ABCs) to reduce the risk of catheter-related bloodstream infections. However, recent evidence suggests that isopropanol can leak from the ABC into the NC, posing concern about their safe use. We sought to determine in vitro which ABC and NC parameters influence the leakage of isopropanol through the infusion circuit.Methods: We assessed 13 NCs and 4 ABCs available in the European market. In vitro circuits consisting of an isopropanol cap, a NC, and an 11-cm catheter line were created. The circuits were left in place for 1 to 7 days at room temperature to assess the kinetics of isopropanol leakage. Isopropanol content in ABC and in circuit flushing solutions (5 mL NaCl 0.9%) after exposure to the cap were measured using gas chromatography with a flame ionization detector.Results: The leakage of isopropanol from the cap to the NC was dependent on the NC, but not the cap. The NC mechanism did not predict the leakage of isopropanol. The Q-Syte NC exhibited the most isopropanol leakage (7.01±1.03 mg and 28.32±2.62 mg at 24 hours and 7 days, respectively), whereas the Caresite NC had the lowest isopropanol leakage at 7 days (1.69±0.01 mg).Conclusion: The use of isopropanol ABCs can cause isopropanol leakage into the catheter circuit according to NC parameters. Caution should be exercised when using these devices, especially in the pediatric and neonatal population.

N-Benzylphenethylamine derivatives are 5-HT2A receptor agonists with hallucinogenic properties, including NBOMe (N-(2-methoxybenzyl)-2-(3,4,5-trimethoxyphenyl)ethan-1-amine) and NBOH (2-(((2,5-dimethoxyphenethyl)amino)methyl)phenol). We reported here the case of a 23-year-old man who presented a serotoninergic syndrome and a loss of consciousness following the consumption of a powder labelled as 25I-NBOH. Toxicological analyses of biological samples were carried out using a liquid chromatography high-resolution mass spectrometry. Two new psychoactive substances were identified and confirmed with certified reference materials: 25E-NBOH (2-(((4-ethyl-2,5-dimethoxyphenethyl)amino)methyl)phenol) and MDPHP (1-(benzo[d][1,3]dioxol-5-yl)-2-(pyrrolidin-1-yl)hexan-1-one). Pharmaceuticals administered to the patient during his medical care were found in plasma and urine. 25E-NBOH and MDPHP concentrations were respectively at 2.3 ng/mL and 3.4 ng/mL in plasma, and 25.7 ng/mL and 30.5 ng/mL in urine. 25I-NBOH (2-(((4-iodo-2,5-dimethoxyphenethyl)amino)methyl)phenol) was specifically searched in both samples and was not detected. These results are discussed along with a literature review on human cases of exposure to N-benzylphenethylamine derivatives. Using molecular networking approach, we propose the first 25E-NBOH metabolism study using authentic biological samples (plasma and urine). We described seven metabolites (M1 to M7), including two phase I (m/z 330.172; m/z 288.160) and five phase II metabolites (m/z 464.191, m/z 478.207, m/z 492.223, m/z 508.218; m/z 396.156). The M6 (m/z 492.223) was the most intense ion detected in plasma and urine and could be proposed as a relevant 25E-NBOH consumption marker. Overall, we described an original case of 25E-NBOH poisoning and identified metabolites that could potentially be used as consumption markers to detect 25E-NBOH intoxications with a higher confidence level and probably a longer detection window.

The recent emergence of new synthetic opioids (NSOs) compounds in the illicit market is increasingly related to fatal cases. Identification and medical care of NSO intoxication cases are challenging, particularly due to high frequency of new products and extensive metabolism. As the study of NSO metabolism is crucial for the identification of these drugs in cases of intoxication, we aimed to investigate the metabolism of the piperazine NSO AP-237 (= bucinnazine). Two complementary approaches (in silico and in vitro) were used to identify putative AP-237 metabolites which could be used as consumption markers. In silico metabolism studies were realized by combining four open access softwares (MetaTrans, SyGMa, Glory X, Biotransformer 3.0). In vitro experiments were performed by incubating AP-237 (20 µM) in differentiated HepaRG cells during 0 h, 8 h, 24 h or 48 h. Cell supernatant were extracted and analyzed by liquid chromatography coupled to high-resolution mass spectrometry and data were reprocessed using three strategies (MetGem, GNPS or Compound Discoverer®). A total of 28 phase I and six phase II metabolites was predicted in silico. Molecular networking identified seven putative phase I metabolites (m/z 203.154, m/z 247.180, m/z 271.180, two m/z 289.191 isomers, m/z 305.186, m/z 329.222), including four previously unknown metabolites. Overall, this cross-disciplinary approach with molecular networking on data acquired in vitro and in silico prediction enabled to propose relevant candidate as AP-237 consumption markers that could be added to mass spectrometry libraries to help diagnose intoxication.

Eutylone is a cathinone-derived synthetic amphetamine scheduled by the World Health Organization and European Monitoring Centre for Drugs and Drug Addiction since 2022 due to its growing consumption. We report here an eutylone intoxication involving a 38-year-old man and a 29-year-old woman in a chemsex context. A bag containing a white crystalline powder labelled as a research product was found in their vehicle. Nuclear magnetic resonance and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analyses identified the powder as eutylone and confirmed purity superior to 99%. LC-HRMS data analysis using molecular networking allowed to propose new eutylone metabolites in blood samples in a graphical manner. We described 16 phase I (e.g. hydroxylated or demethylated) and phase II metabolites (glucuroconjugates and sulfoconjugates). The same metabolites were found both in male and female blood samples. Toxicological analyses measured eutylone concentration in blood samples at 1374 ng/mL and 1536 ng/mL for the man and the woman, respectively. A keto-reduced metabolite (m/z 238.144) was synthesized to permit its quantification at 67 ng/mL and 54 ng/mL in male and female blood samples, respectively. Overall, the identification of these metabolites will increase the knowledge of potential drug consumption markers and allow to implement mass spectrometry databases to better monitor future drug abuse or consumption.

Since the 2000s, an increasing number of new psychoactive substances (NPS) have appeared on the drug market. Arylcyclohexylamine (ACH) compounds such as ketamine, phencyclidine and eticyclidine derivatives are of particular concern, given their rapidly increasing use and the absence of detailed toxicity data. First used mainly for their pharmacological properties in anesthesia, their recreational use is increasing. ACH derivatives have an antagonistic activity against the N-methyl-D-aspartate receptor, which leads to dissociative effects (dissociation of body and mind). Synthetic ketamine derivatives produced in Asia are now arriving in Europe, where most are not listed as narcotics and are, thus, legal. These structural derivatives have pharmacokinetic and pharmacodynamic properties that are sometimes very different from ketamine. Here, we describe the pharmacology, epidemiology, chemistry and metabolism of ACH derivatives, and we review the case reports on intoxication.

Synthetic cathinones constitute a family of new psychoactive substances, the consumption of which is increasingly worldwide. A lack of metabolic knowledge limits the detection of these compounds in cases of intoxication. Here, we used an innovative cross-disciplinary approach to study the metabolism of the newly emerging cathinone chloro-alpha-pyrrolidinovalerophenone (4-Cl-PVP). Three complementary approaches (in silico, in vitro, and in vivo) were used to identify putative 4-Cl-PVP metabolites that could be used as additional consumption markers. The in silico approach used predictive software packages. Molecular networking was used as an innovative bioinformatics approach for re-processing high-resolution tandem mass spectrometry data acquired with both in vitro and in vivo samples. In vitro experiments were performed by incubating 4-Cl-PVP (20 µM) for four different durations with a metabolically competent human hepatic cell model (differentiated HepaRG cells). In vivo samples (blood and urine) were obtained from a patient known to have consumed 4-Cl-PVP. The in silico software predicted 17 putative metabolites, and molecular networking identified 10 metabolites in vitro. On admission to the intensive care unit, the patient’s plasma and urine 4-Cl-PVP concentrations were, respectively, 34.4 and 1018.6 µg/L. An in vivo analysis identified the presence of five additional glucuronoconjugated 4-Cl-PVP derivatives in the urine. Our combination of a cross-disciplinary approach with molecular networking enabled the detection of 15 4-Cl-PVP metabolites, 10 of them had not previously been reported in the literature. Two metabolites appeared to be particular relevant candidate as 4-Cl-PVP consumption markers in cases of intoxication: hydroxy-4-Cl-PVP (m/z 282.1254) and dihydroxy-4-Cl-PVP (m/z 298.1204).