Worsening of the Toxic Effects of (±) Cis -4,4′-DMAR Following Its Co-Administration with (±) Trans -4,4′-DMAR: Neuro-Behavioural, Physiological, Immunohistochemical and Metabolic Studies in Mice

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/4.0/).4,4’-Dimethylaminorex (4,4’-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±)cis (0.1–60 mg/kg) and (±)trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±)cis at 1, 10 or 60 mg/kg + (±)trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4′-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±)cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±)trans stereoisomer was ineffective whilst the stereoisomers’ co-administration resulted in a worsening of the toxic (±)cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±)trans stereoisomer reduced the metabolism of the (±)cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.Peer reviewedFinal Published versio

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 databas

Evaluation of the excretion profile and acute behavioral effects of 2-methiopropamine in the animal model.

This communication presents the effects induced by 2-methiopropamine, a structural analogue of methamphetamine, on: sensorimotor responses, body temperature, thermal and mechanical pain, motor activity and neurological changes in mice. The analytical aspect was also considered by carrying out a series of in vitro and in vivo studies to characterize the metabolic profile of 2- methiopropamine and to select the most appropriate diagnostic marker(s) of intake. Systemic administration of 2-methiopropamine in a dose-dependent way inhibits visual placing response, facilitates acoustic response, pinna and vibrissae reflexes, alters the core temperature, causes potent mechanical analgesia and impairs motor activity. Finally, 2-methiopropamine induces tail elevation, restlessness, piloerection and head twitch. Metabolism studies confirm data reported by other Authors, showing that 2-methiopropamine, similarly to amphetamine-like substances, is extensively oxidized to form nor-methiopropamine, hydroxy-methiopropamine, oxo- methiopropamine and hydroxy nor-methiopropamine. The experimental evidences obtained in this study demonstrate that 2-methiopropamine impairs sensorimotor responses and induces motor activity and neurological alterations, suggesting its possible hazard for human health and the need of further studies. At the same time, the results of the metabolism studies confirmed the similarities between amphetamine and 2-methiopropamine

In vitro characterization of the metabolic pathways of four new synthetic cannabinoids

Synthetic cannabinoids are a class of novel psychoactive substances originally developed to explore the function and structure of cannabinoid receptors. At present, they have found great popularity into the world of “recreational” drug use, being among the most widely diffused of the new psychoactive substances. Indeed more than a hundred different varieties of synthetic cannabinoids have been synthetized so far, making their detection in herbal products and in biological fluids a demanding challenge for forensic laboratories. Here the phase I and phase II biochemical reactions involved in the biotransformation pathways of four new cannabinoids (CUMYL-THPINACA, ADB-CHMICA, APP-FUBINACA, APP-CHMINACA) were characterized by LC–MS-based techniques and in vitro models to identify the most appropriate marker(s) of intake. Our data show that the compounds studied are extensively metabolized mainly by CYP3A4 and CYP3A5 isoforms to more than 10 oxidized metabolites, being the mono-hydroxylation the most abundant biotransformation pathways detected. Concerning the phase II metabolism our data suggest that for all the compounds under investigation, most of the phase I metabolites characterized once formed undergo glucuronidation

EVALUATION OF THE METABOLIC PROFILE OF 2-METHIOPROPAMINE BY LC-MS/MS IN MICE AND IN HUMAN LIVER MICROSOMES

Introduction The aim of the present study was to investigate the phase I and phase II metabolism of 2- methiopropamine, a structural analogue of methamphetamine [1-3], after acute administration in mice, in order to select the most appropriate marker(s) of intake, also defining the excretion windows in urine. In vitro metabolism studies were carried out in order to enzymatically synthetize the metabolites of MPA and to set up the most appropriate sample pre-treatment procedures for LC-QqQ analysis. Methods A dose of 10 mg/kg was selected for the in vivo metabolism studies of MPA and urine samples were collected every 3 hours in the range of 0-9 hours after the injection. The In vitro studies were carried out with HLM (human liver microsomes). Samples from both studies were analysed, after extraction with tertbutyl-methyl ether at pH 9, using an Agilent 1200 HPLC equipped with a SUPELCO C18 column coupled to an API4000 triple quadrupole (Sciex) whit ESI source, operating in positive-ion mode. Results To reproduce the in vivo metabolism, the in vitro metabolism protocol was optimized considering different conditions: substrate concentration 20 µM, proteins concentration 0.5 mg/mL, phosphate buffer 0.1 M at pH 7.4, and an incubation time of 4 h at 37°C. Three main metabolites were identified: Nor-MPA, HydroxyMPA, and Nor-hydroxy-MPA. To optimize sample pre-treatment different extraction solvents (tertbutylmethyl ether, diethyl ether and ethylacetate) and pH values (7, 9 and 12) were evaluated. The best recoveries (higher than 70%) for the principal metabolites were obtained at pH 9. In vivo metabolism studies shown the formation of three principal phase I metabolites identified as: Nor-MPA, Hydroxy-MPA, and Oxo-MPA. 2-MPA and its metabolites show a maximum of excretion in the first 3 h from administration to mice showing an increasing conversion into phase II metabolites both glucorono and sulfo-conjugates for MPA, Nor-MPA, Hydroxy-MPA; Nor-Hydroxy-MPA was detected only as a phase II metabolite. Conclusions The excretion profile of 2-MPA after acute administration was evaluated identifying its principal metabolites and their excretion windows in mice. MPA and Nor-MPA are the best markers of intake in the first 0-24 hours after the administration of MPA. The present method was qualitative validated for MPA and Nor-MPA according to WADA technical document[4-5] defining: recovery, LOD, selectivity and repeatability. Novel Aspect There are few studies about MPA metabolism [3]. This is the first study identifying MPA excretion profile including phase I and phase II, also selecting the best markers of intake

The utility of in vitro metabolism studies coupled to liquid chromatography-tandem mass spectrometry for the selection of diagnostic markers of novel psychoactive substances: the case of 4,4’-DMAR

para-Methyl-4-methylaminorex (4,4’-DMAR) is a synthetic, substituted oxazoline derivative and analogue of the scheduled aminorex and 4-methylaminorex (4-MAR), both of which originally designed and synthesized as appetite suppressants. The psychostimulant properties of these substances are due to the interaction with dopamine transporter (DAT), serotonin transporter (SERT) and norepinephrine transporter (NET). Chemically, because of the two chiral centers in the oxazoline ring, 4,4’-DMAR exist as two different racemic (±) –cis and (±) –trans mixture or four distinct enantiomers; recent evidence suggests that only the cis isomers show a biological activity; furthermore, if cis and trans isomers are administered in combination, the effects of the cis isomer are enhanced. A recently validated method was used to detect the main metabolites in rat plasma and brain tissues and it allowed to identify four metabolites, due to hydroxylation, oxidation, hydrolysis and deamination. (Lucchetti, et al., 2017) Here we describe the in vitro metabolism of cis-4,4’-DMAR, investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on samples obtained after incubation with pooled human liver microsomes (HLM) and recombinant isoforms of cytochrome P450 and uridine diphospho glucuronosyl-transferase, with the aim of identifying the most suitable diagnostic marker(s) of intake, to be targeted in human urine in case of voluntary assumption. Following incubation with HLM, no metabolite was detected for the trans isomer, while six metabolites were found for cis-4,4’-DMAR as a result of mono-hydroxylation, di-hydroxylation and oxidation of the hydroxylated derivatives. The main cytochrome P450 isoforms involved in metabolism of this substance seems to be cytochrome P450 2D6 (CYP2D6), which is a polymorphic one. The chromatograms also shows the presence of a small amount of trans-4,4’-DMAR, probably due to the incubation with HLM or the exposure to heat. The same analysis was performed on the racemic mixture and it shows that this association leads to a lower amount of metabolites. This result suggests that the trans isomer could exert an inhibitory effect on the cis metabolism

Metabolic profile and pharmaco-toxicological effects of MPA in mouse model

Methiopropamine (MPA) is a thiophene ring-based structural analog of methamphetamine that is categorized as a novel psychoactive substance. MPA acts primary as a norepinephrine-dopamine reuptake inhibitor and, secondary, as a serotonin reuptake inhibitor. In human, MPA induces stimulation, alertness and increase of focus and energy. However, important side-effects were reported after MPA administration such as tachycardia, anxiety, panic attacks, perspiration, headache and difficulty in breathing. To date, few data are available on the pharmaco-toxicological effects of MPA and on its metabolism in vivo. To this purpose we investigated the acute in vivo effects induced by MPA on visual, acoustic and tactile sensorimotor responses, body temperature, pain threshold, motor activity, cardiovascular and cardiorespiratory changes in CD-1 male mice. In vitro (from human liver microsomes preparation) and in vivo (from CD-1 male mice urine) metabolism studies were also conducted in order to characterize the phase I metabolic profile of MPA, with the aim to select both the best marker(s) of intake and the responsible of the potential behavioural effects. Systemic administration of MPA (0.01-30 mg/kg) impaired visual placing response, facilitated acoustic and tactile response, induced hypothermia, increased mechanical and thermal analgesia stimulated locomotor activity and induced motor stereotypies in mice. MPA strongly affected cardiovascular and respiratory parameters. It induced tachycardia, increased diastolic and systolic blood pressure, caused vasoconstriction. Moreover, MPA increased breath rate but it reduced SpO2 saturation. The acute administration of MPA at 10 and 30 mg/kg caused the death (~30% and ~40%, respectively) of mice. Metabolism studies show that MPA is exensively oxidated to form mainly nor-MPA, hydroxy-MPA, oxo-MPA. The best markers of intake and the compounds detected in huge amount during the behavioural tests are the MPA itself and its hydroxylated and demethylated metabolites. The experimental evidence obtained in this study demonstrates for the first time that MPA impairs sensorimotor responses, has psychostimulant effect, causes cardiovascular and respiratory alterations, thus suggesting its possible hazard for human health. This research has been funded by the Drug Policies Department, Presidency of the Council of Ministers, Italy (project: “Effects of NPS: development of a multicentric research for the information enhancement of the Early Warning System” to MM) and FIRB 2012 to FDG

In Vivo Bio-Activation of JWH-175 to JWH-018: Pharmacodynamic and Pharmacokinetic Studies in Mice

3-(1-Naphthalenylmethyl)-1-pentyl-1H-indole (JWH-175) is a synthetic cannabinoid illegally marketed for its psychoactive cannabis-like effects. This study aimed to investigate and compare in vitro and in vivo pharmacodynamic activity of JWH-175 with that of 1-naphthalenyl (1-pentyl-1H-indol-3-yl)-methanone (JWH-018), as well as evaluate the in vitro (human liver microsomes) and in vivo (urine and plasma of CD-1 male mice) metabolic profile of JWH-175. In vitro binding studies showed that JWH-175 is a cannabinoid receptor agonist less potent than JWH-018 on mouse and human CB1 and CB2 receptors. In agreement with in vitro data, JWH-175 reduced the fESPS in brain hippocampal slices of mice less effectively than JWH-018. Similarly, in vivo behavioral studies showed that JWH-175 impaired sensorimotor responses, reduced breath rate and motor activity, and increased pain threshold to mechanical stimuli less potently than JWH-018. Metabolic studies demonstrated that JWH-175 is rapidly bioactivated to JWH-018 in mice blood, suggesting that in vivo effects of JWH-175 are also due to JWH-018 formation. The pharmaco-toxicological profile of JWH-175 was characterized for the first time, proving its in vivo bio-activation to the more potent agonist JWH-018. Thus, it highlighted the great importance of investigating the in vivo metabolism of synthetic cannabinoids for both clinical toxicology and forensic purposes

Prescription drugs misuse in “clubbers” and disco goers in Ibiza

Funding Information: This study was partly funded by the European Project entitled Analysis, Knowledge dissemination, Justice implementation and Special Testing of Novel Synthetic Opioids—JUST-2017-AG-DRUG. Publisher Copyright: © Copyright © 2020 di Giannantonio, Negri, Schiavone, Vannini, Pettorruso, De-Giorgio, Verrastro, Trabace, Corbo, Gottardo, Camuto, Mazzarino, Barra, De Berardis, Lopez, Del Villar, Schifano and Martinotti. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Prescription drugs misuse and its related risks are considered a worldwide public health issue. Current trends show that the extent of such phenomenon may not be limited to subjects with psychiatric disorders, as it also spreads to dance party and nightclub attendees, who often consume prescription drugs in combination with alcohol and psychoactive substances. This study aims to report the sociodemographic data and the psychiatric and clinical features of a sample of clubbers reporting prescription drugs use. Methods: Patients admitted to the psychiatry ward of the Can Misses Hospital in Ibiza were recruited for the study during a span of four consecutive years (2015-2018). The inclusion criteria were age 18-75 years old and the intake of psychoactive substances or more than five alcohol units during the previous 24 hours. Substances use habits, psychopathological features and use of unprescribed pharmaceuticals were investigated. Urine samples were collected and analysed using Gas Chromatography/Mass Spectrometry. Results: A total of 110 subjects with psychoactive substance intoxication were recruited for the study. Among these, 37 (40%) disclosed the use of prescription drugs without medical supervision. The most common compounds were benzodiazepines (66%), antiepileptic drugs (8%), antidepressants (6%), opioids (6%), antipsychotics (6%), stimulants (6%) and Non-Steroidal Anti Inflammatory Drugs (NSAIDs, 2%).Prescription drugs misuse was negatively associated with the use of psychodysleptics (Two-tailed Fisher’s exact test p=0,018, = -0,262). Conclusions: The use of prescription drugs is also common among clubbers, usually characterised by low propensity to be prescribed benzodiazepines, antipsychotics, or antidepressants. Prescription drugs may be an alternative to classic and novel psychoactive compounds or may be used to tamper and self-medicate the effects determined by the use of substances. Party goers should be adequately informed about possible risks of co-intake of psychoactive substances and prescription drugs to prevent serious medical and psychiatric consequences.Peer reviewe