Characterization of the metabolic profile of novel psychoactive substances by a combination of in vitro and in vivo studies and chromatographic-spectrometric techniques

This PhD work is focused on those Novel Psychoactive Substances (NPS) which could exert effects as doping agent in sport competitions. Analysis were carried out at “Laboratorio Antidoping FMSI” of Rome part of laboratories around the world accredited by WADA to conduct human doping control sample analyses. Different substances were selected thorough the years of study, among those NPS with structures and action similar to other compounds already prohibited in sport competitions. The substances of interest were selected according to University of Ferrara department of morphology, surgery and experimental medicine, section of legal medicine, a collaborative centre of Italian Early Warning System (IEWS) and University Cattolica of Rome. This collaboration led to the establishment of a multicentric collaborative group for the IEWS. The aim was to introduce these selected NPS as recognized doping agents searched by laboratories routine drug test of World Antidoping Agency (WADA), or forensic toxicology laboratories. With this aim specific compounds were selected and their potential effects were evaluated through in vivo behavioural studies employing murine model as a model of human behaviour and metabolism. The substances were administered to mice groups and behavioral studies were carried out at University of Ferrara to establish potential stimulant effects, which configure substances as stimulant compound in-competition. When a substance has showed typical effects as a doping agent, metabolism studies were carried out by our laboratory employing human liver microsomes or CYPs isoform as a model of human oxidative metabolism, and samples were analysed through liquid chromatography mass spectrometry techniques. The selected substances are: Methiopropamine, 4,4’ Dimethylaminorex, Pyrovalerone, Methedrone, γ-valerolactone, ADB-CHMICA, CUMYL-THPINACA. 13 All of these substances show potential stimulant effects known in literature or proved by behavioral studies provided by University of Ferrara. They were selected in accordance with IEWS alert on their abuse in Italy and their potential implication with fatal intoxication. For selected substances (Methiopropamine, GVL, 4,4’ DMAR) tissue damage were also estimated by collaboration with University Cattolica of Rome. For all the substances in vitro metabolism studies were carried out to in order to investigate metabolic pathways reactions and select the most suitable markers of intake. Methiopropamine, 4,4’-dimethylaminorex, and γ-valerolactone are substances of interest in forensic analysis as potential hazardous novel abused recreational drugs with unknown metabolism and /or excretion. The in vivo metabolism of these compounds was therefore studied employing mice as metabolism model. The matrix selected was urine as the elected matrix for doping and toxicological analysis. Collected data from the three unit were linked and proposed to IEWS with the aim to bring up the unknowledge on these substances and to propose the introduction of fundamental data on their toxicology, effects and metabolism on EWS international database