Acute DOB and PMA Administration Impairs Motor and Sensorimotor Responses in Mice and Causes Hallucinogenic Effects in Adult Zebrafish

The drastic increase in hallucinogenic compounds in illicit drug markets of new psychoactive substances (NPS) is a worldwide threat. Among these, 2, 5-dimetoxy-4-bromo-amphetamine (DOB) and paramethoxyamphetamine (PMA; marketed as "ecstasy") are frequently purchased on the dark web and consumed for recreational purposes during rave/dance parties. In fact, these two substances seem to induce the same effects as MDMA, which could be due to their structural similarities. According to users, DOB and PMA share the same euphoric effects: increasing of the mental state, increasing sociability and empathy. Users also experienced loss of memory, temporal distortion, and paranoia following the repetition of the same thought. The aim of this study was to investigate the effect of the acute systemic administration of DOB and PMA (0.01-30 mg/kg; i.p.) on motor, sensorimotor (visual, acoustic, and tactile), and startle/PPI responses in CD-1 male mice. Moreover, the pro-psychedelic effect of DOB (0.075-2 mg/kg) and PMA (0.0005-0.5 mg/kg) was investigated by using zebrafish as a model. DOB and PMA administration affected spontaneous locomotion and impaired behaviors and startle/PPI responses in mice. In addition, the two compounds promoted hallucinatory states in zebrafish by reducing the hallucinatory score and swimming activity in hallucinogen-like states

Potential of the zebrafish model for the forensic toxicology screening of NPS: A comparative study of the effects of APINAC and methiopropamine on the behavior of zebrafish larvae and mice

Abstract The increased diffusion of the so-called novel psychoactive substances (NPS) and their continuous change in structure andconceivably activity has led to the need of a rapid screening method to detect their biological effects as early as possible after their appearance in the market. This problem is very felt in forensic pathology and toxicology, so the preclinical study is fundamental in the approach to clinical and autopsy cases of difficult interpretation intoxication. Zebrafish is a high-throughput suitable model to rapidly hypothesize potential aversive or beneficial effects of novel molecules. In the present study, we measured and compared the behavioral responses to two novel neuroactive drugs, namely APINAC, a new cannabimimetic drug, and methiopropamine (MPA), a methamphetamine-like compound, on zebrafish larvae (ZL) and adult mice. By using an innovative statistical approach (general additive models), it was found that the spontaneous locomotor activity was impaired by the two drugs in both species: the disruption extent varied in a dose-dependent and time-dependent manner. Sensorimotor function was also altered: i) the visual object response was reduced in mice treated with APINAC, whereas it was not after exposure to MPA; ii) the visual placing responses were reduced after treatment with both NPS in mice. Furthermore, the visual motor response detected in ZL showed a reduction after treatment with APINAC during light-dark and dark-light transition. The same pattern was found in the MPA exposed groups only at the dark-light transition, while at the transition from light to dark, the individuals showed an increased response. In conclusion, the present study highlighted the impairment of spontaneous motor and sensorimotor behavior induced by MPA and APINAC administration in both species, thus confirming the usefulness of ZL as a model for a rapid behavioural-based drug screening

Neurochemical and Behavioral Profiling in Male and Female Rats of the Psychedelic Agent 25I-NBOMe

4-Iodo-2,5-dimethoxy-N-(2-methoxybenzyl)phenethylamine (25I-NBOMe), commonly called "N-Bomb," is a synthetic phenethylamine with psychedelic and entactogenic effects; it was available on the Internet both as a legal alternative to lysergic acid diethylamide (LSD) and as a surrogate of 3,4-methylenedioxy-methamphetamine (MDMA), but now it has been scheduled among controlled substances. 25I-NBOMe acts as full agonist on serotonergic 5-HT2A receptors. Users are often unaware of ingesting fake LSD, and several cases of intoxication and fatalities have been reported. In humans, overdoses of "N-Bomb" can cause tachycardia, hypertension, seizures, and agitation. Preclinical studies have not yet widely investigated the rewarding properties and behavioral effects of this compound in both sexes. Therefore, by in vivo microdialysis, we evaluated the effects of 25I-NBOMe on dopaminergic (DA) and serotonergic (5-HT) transmissions in the nucleus accumbens (NAc) shell and core, and the medial prefrontal cortex (mPFC) of male and female rats. Moreover, we investigated the effect of 25I-NBOMe on sensorimotor modifications as well as body temperature, nociception, and startle/prepulse inhibition (PPI). We showed that administration of 25I-NBOMe affects DA transmission in the NAc shell in both sexes, although showing different patterns; moreover, this compound causes impaired visual responses in both sexes, whereas core temperature is heavily affected in females, and the highest dose tested exerts an analgesic effect prominent in male rats. Indeed, this drug is able to impair the startle amplitude with the same extent in both sexes and inhibits the PPI in male and female rats. Our study fills the gap of knowledge on the behavioral effects of 25I-NBOMe and the risks associated with its ingestion; it focuses the attention on sex differences that might be useful to understand the trend of consumption as well as to recognize and treat intoxication and overdose symptoms

MAM-2201 acute administration impairs motor, sensorimotor, prepulse inhibition, and memory functions in mice: a comparison with its analogue AM-2201

Rationale1-[(5-fluoropentyl)-1H-indol-3-yl](4-methyl-1-naphthalenyl) methanone (MAM-2201) is a potent synthetic cannabinoid receptor agonist illegally marketed in "spice" products and as "synthacaine" for its psychoactive effects. It is a naphthoyl-indole derivative which differs from its analogue 1-[(5-Fluoropentyl)-1H-indol-3-yl](1-naphthylenyl) methanone (AM-2201) by the presence of a methyl substituent on carbon 4 (C-4) of the naphthoyl moiety. Multiple cases of intoxication and impaired driving have been linked to AM-2201 and MAM-2201 consumption.ObjectivesThis study aims to investigate the in vitro (murine and human cannabinoid receptors) and in vivo (CD-1 male mice) pharmacodynamic activity of MAM-2201 and compare its effects with those induced by its desmethylated analogue, AM-2201.ResultsIn vitro competition binding studies confirmed that MAM-2201 and AM-2201 possess nanomolar affinity for both CD-1 murine and human CB1 and CB2 receptors, with preference for the CB1 receptor. In agreement with the in vitro binding data, in vivo studies showed that MAM-2201 induces visual, acoustic, and tactile impairments that were fully prevented by pretreatment with CB1 receptor antagonist/partial agonist AM-251, indicating a CB1 receptor mediated mechanism of action. Administration of MAM-2201 also altered locomotor activity and PPI responses of mice, pointing out its detrimental effect on motor and sensory gating functions and confirming its potential use liability. MAM-2201 and AM-2201 also caused deficits in short- and long-term working memory.ConclusionThese findings point to the potential public health burden that these synthetic cannabinoids may pose, with particular emphasis on impaired driving and workplace performance

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

Pharmaco-toxicological effects of the novel tryptamine hallucinogen 5-MeO-MiPT on motor, sensorimotor, physiological, and cardiorespiratory parameters in mice—from a human poisoning case to the preclinical evidence

Rationale: The 5-methoxy-N-methyl-N-isopropyltryptamine (5-MeO-MiPT, known online as “Moxy”) is a new psychedelic tryptamine first identified on Italian national territory in 2014. Its hallucinogen effects are broadly well-known; however, only few information is available regarding its pharmaco-toxicological effects. Objectives: Following the seizure of this new psychoactive substances by the Arm of Carabinieri and the occurrence of a human intoxication case, in the current study we had the aim to characterize the in vivo acute effects of systemic administration of 5-MeO-MiPT (0.01–30 mg/kg i.p.) on sensorimotor (visual, acoustic, and overall tactile) responses, thermoregulation, and stimulated motor activity (drag and accelerod test) in CD-1 male mice. We also evaluated variation on sensory gating (PPI, prepulse inhibition; 0.01–10 mg/kg i.p.) and on cardiorespiratory parameters (MouseOx and BP-2000; 30 mg/kg i.p.). Lastly, we investigated the in silico ADMET (absorption, distribution, metabolism, excretion, toxicity) profile of 5-MeO-MiPT compared to 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) and N,N-dimethyltryptamine (DMT). Results: This study demonstrates that 5-MeO-MiPT dose-dependently inhibits sensorimotor and PPI responses and, at high doses, induces impairment of the stimulated motor activity and cardiorespiratory changes in mice. In silico prediction shows that the 5-MeO-MiPT toxicokinetic profile shares similarities with 5-MeO-DIPT and DMT and highlights a cytochrome risk associated with this compound. Conclusions: Consumption of 5-MeO-MiPT can affect the ability to perform activities and pose a risk to human health status, as the correspondence between the effects induced in mice and the symptoms occurred in the intoxication case suggests. However, our findings suggest that 5-MeO-MiPT should not be excluded from research in the psychiatric therapy field

COVID-19 in rheumatic diseases in Italy: first results from the Italian registry of the Italian Society for Rheumatology (CONTROL-19)

OBJECTIVES: Italy was one of the first countries significantly affected by the coronavirus disease 2019 (COVID-19) epidemic. The Italian Society for Rheumatology promptly launched a retrospective and anonymised data collection to monitor COVID-19 in patients with rheumatic and musculoskeletal diseases (RMDs), the CONTROL-19 surveillance database, which is part of the COVID-19 Global Rheumatology Alliance. METHODS: CONTROL-19 includes patients with RMDs and proven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) updated until May 3rd 2020. In this analysis, only molecular diagnoses were included. The data collection covered demographic data, medical history (general and RMD-related), treatments and COVID-19 related features, treatments, and outcome. In this paper, we report the first descriptive data from the CONTROL-19 registry. RESULTS: The population of the first 232 patients (36% males) consisted mainly of elderly patients (mean age 62.2 years), who used corticosteroids (51.7%), and suffered from multi-morbidity (median comorbidities 2). Rheumatoid arthritis was the most frequent disease (34.1%), followed by spondyloarthritis (26.3%), connective tissue disease (21.1%) and vasculitis (11.2%). Most cases had an active disease (69.4%). Clinical presentation of COVID-19 was typical, with systemic symptoms (fever and asthenia) and respiratory symptoms. The overall outcome was severe, with high frequencies of hospitalisation (69.8%), respiratory support oxygen (55.7%), non-invasive ventilation (20.9%) or mechanical ventilation (7.5%), and 19% of deaths. Male patients typically manifested a worse prognosis. Immunomodulatory treatments were not significantly associated with an increased risk of intensive care unit admission/mechanical ventilation/death. CONCLUSIONS: Although the report mainly includes the most severe cases, its temporal and spatial trend supports the validity of the national surveillance system. More complete data are being acquired in order to both test the hypothesis that RMD patients may have a different outcome from that of the general population and determine the safety of immunomodulatory treatments

Variazioni delle risposte neuro-comportamentali e della trasmissione degli endocannabinoidi nei topi dopo la somministrazione ripetuta di JWH-018: sviluppo di un modello in vivo di demenza e correlazione cliniche.

La diffusione della Demenza senile è strettamente correlata alle dinamiche demografiche mondiali. I dati epidemiologici mostrano che circa un milione di italiani sono affetti da vari tipologie di Demenza (DD), confermando così la grande rilevanza clinica e sociale di questo argomento. In termini terapeutici, i recenti riconosciuti effetti benefici dell’uso di cannabis nel rallentamento dello sviluppo di molti disturbi neurodegenerativi, sia in modello animale che umano, suggeriscono che il sistema endocannabinoide (ECS) potrebbe in qualche modo essere coinvolto nella fisiopatologia della DD. Tuttavia, gli effetti negativi acuti e cronici indotti dall'assunzione di cannabinoidi sulle prestazioni neurocognitive (per esempio per quanto concerne l'apprendimento, l'attenzione e la memoria di lavoro; deficit nelle abilità verbali, il riconoscimento visivo, e l’attività di memoria prospettica a breve e lungo termine) sono ancora da chiarire. Inoltre, in un contesto d’emergenza mondiale di vendita illegale di sostanze stupefacenti come quello odierno, in cui cannabis e cannabinoidi sintetici (SCBS) rappresentano due tra le principali sostanze d’abuso maggiormente ricercate tra gli utilizzatori abituali in tutta Europa, risulta evidente la necessità di un’imminente individuazione delle loro potenzialità tossicologiche a lungo termine. Pertanto, soffermandosi proprio sul potenziale d’abuso dei SCBS e le possibili conseguenze del loro utilizzo sul ECS, nel presente progetto di Dottorato di Ricarca, si è verificato se il loro utilizzo sub-cronico, anche se per un breve periodo nell’arco della vita (da giovani-adulti), possa rappresentare una concausa nella fisiopatogenesi della demenza senile e se la disregolazione del ECS sia specifica nei vari sottotipi della patologia. In particolare, sono stati avviati una serie di studi pilota preclinici che hanno permesso di individuare se, alla dose efficace testata, vi sia correlazione tra i sintomi manifestati in vivo ed eventuale disregolazione del sistema ECS a seguito della somministrazione protratta di una specifica sostanza. A tal fine, un gruppo di topi (di età compresa tra i 3 e i 4 mesi) è stato trattato con JWH-018 (6 mg/Kg, i.p.) per sette giorni consecutivi (per un totale di 7 iniezioni) seguito da un periodo di riposo (wash-out). Gli animali sono stati poi sacrificati per la raccolta e quantificazione in campioni di sangue e tessuti, dei livelli dei due endocannabinoidi principali: l’acido arachidonoiletanolammide (AEA) e il 2-arachidonoilglicerolo (2-AG). I test eseguiti, dopo periodi di wash-out definiti - primo studio (i): 3 giorni; secondo studio (ii): 15 giorni; terzo studio (iii): 53 giorni – hanno permesso di ottenere i seguenti risultati: (i) alterazione a breve termine delle risposte sensoriali, sensori-motorie, fisiologiche e motorie; incrementata analgesia e acinesia; sospetto sviluppo di tolleranza; (ii) variazione del gating sensoriale; transitoria facilitazione dell'attività motoria spontanea con insorgenza di atteggiamenti tipicamente stereotipici (movimenti su piastra in senso orario) e ansiogeni; ridotta densità recettoriale CB1 nell’ippocampo; alterazione dei livelli di AEA e 2-AG, e dei loro enzimi degradanti (FAAH e MAGL) nello striato; (iii) variazione a lungo termine del gating sensoriale; comportamento depressivo-simile; danneggiamento della memoria di lavoro; ridotta socievolezza; aumentata concentrazione plasmatica di 2-AG. Successivamente agli studi preclinici, ha seguito la valutazione clinica di campioni di pazienti affetti da differenti tipologie di demenza (Alzheimer, mista e Mild Cognitive Impairment o MCI). Questi, sono stati confrontati con campioni di soggetti con funzioni cognitive nella norma.The prevalence of Dementia Diseases (DD) is closely related to the world demographic dynamics. Epidemiological data shows that about one million Italians are affected by various type of DD, thus confirming the great clinical and social relevance of this topic. In therapeutic terms, the remarkable beneficial effects of medical cannabis use in limiting the development of many neurodegenerative disorders both in animal and human models, suggest that the endocannabinoid system (ECS) may underline the umbrella of DD physiopathology. Despite this, the negative effects induced by acute and chronic cannabinoids intake on neurocognitive performance (i.e., learning, attention and working memory; deficits in verbal skills, visual recognition, a delay in visual recall, and short- and long-range prospective memory tasks) is still to be elucidated. In addition, in a current contest of emergency of illegal drugs world’s market, with cannabis and synthetic cannabinoids (SCBS) that represent two of the most sought and sold abuse substances among regular users in Europe, the need for an early identification of their long-term toxicological potential is evident. Therefore, focusing on the SCBS potential abuse and the possible consequences of their use on the ECS, the present PhD project investigates whether their sub-chronic use, even if for a short period of life (as young/adults), may be a factor in the pathogenesis of DD and if ECS dysregulation may be specific to various DD subtypes. In particular, a series of pre-clinical pilot studies have analysed the potential correlation between in vivo symptoms and possible dysregulation of the ECS following prolonged administration of a specific substance at the effective dose tested. For this purpose, a group of mice (aged between 3/4 months) have been treated with JWH-018 (6 mg/kg, i.p.) for seven consecutive days (for a total of 7 injections) followed by a wash-out period. Animals have been then sacrificed for the collection of tissue and blood samples, in which the main two endocannabinoid have been quantified: the N-arachidonoylethanolamine (AEA) and the 2-Arachidonoylglycerol (2-AG). The tests performed, after defined wash-out periods - first study (i): 3 days; second study (ii): 15 days; third study (iii): 53 days - yielded the following results: (i) short-term alteration of sensory, sensory-motor, physiological and motor responses; increased analgesia and acinesia; suspected development of tolerance; (ii) variation of sensory gating; transient facilitation of spontaneous motor activity with onset of characteristically stereotypical attitudes (circular movement of the mouse on themselves) and anxiogenic; reduced CB1 receptor density in the hippocampus; alteration of AEA and 2-AG levels, and their degrading enzymes (FAAH and MAGL) in the striatum; (iii) long-term variation of sensory gating; depressive-like behavior; impairment of working memory; reduced sociability; increased plasmatic concentration of 2-AG. Lastly, a clinical trial was conducted using blood samples of patients with different types of dementia (Alzheimer’s, mixed and Mild Cognitive Impairment - MCI) and subjects with normal cognitive functions. From the results obtained, a trend of increasing AEA concentrations (expressed as the area below the curve) is shown and varies according to the specific DD form taken into account, confirming what has been obtained in preclinical studies. In conclusion, the results of this PhD project show that repeated treatment with JWH-018, also as a result of prolonged wash-out periods, causes cognitive impairment, depressive-like behaviour and social avoidance, potentially related to the behavioural and psychological symptoms of the MCI that typically precedes the development of specific DD

Single Exposure to the Cathinones MDPV and α-PVP Alters Molecular Markers of Neuroplasticity in the Adult Mouse Brain

Synthetic cathinones have gained popularity among young drug users and are widely used in the clandestine market. While the cathinone-induced behavioral profile has been extensively investigated, information on their neuroplastic effects is still rather fragmentary. Accordingly, we have exposed male mice to a single injection of MDPV and α-PVP and sacrificed the animals at different time points (i.e., 30 min, 2 h, and 24 h) to have a rapid readout of the effect of these psychostimulants on neuroplasticity in the frontal lobe and hippocampus, two reward-related brain regions. We found that a single, low dose of MDPV or α-PVP is sufficient to alter the expression of neuroplastic markers in the adult mouse brain. In particular, we found increased expression of the transcription factor Npas4, increased ratio between the vesicular GABA transporter and the vesicular glutamate transporter together with changes in the expression of the neurotrophin Bdnf, confirming the widespread impact of these cathinones on brain plasticity. To sum up, exposure to low dose of cathinones can impair cortical and hippocampal homeostasis, suggesting that abuse of these cathinones at much higher doses, as it occurs in humans, could have an even more profound impact on neuroplasticity

Urinary excretion and effects on visual placing response in mice of gamma-valero-lactone, an alternative to gamma‐hydroxy-butyrate for drug-facilitated sexual assault

Γ-Valero-lactone (GVL) is a freely marketed organic solvent and food additive. GVL is also an underrated legal substitute for γ-hydroxybutyric acid (GHB), and it is sold under the trade name "Tranquili-G". GVL is usually not included in forensic drug testing, even though it has been reported in drug-facilitated sexual assaults (DFSA). To date, few studies verified the effectiveness of GVL as a hypnotic/narcotic substance, and no data are available on its urinary excretion profile. This work aims to fill this knowledge gap and lead to the introduction of GVL in forensic drug testing, especially when DFSA is suspected. To monitor the timeframe of GVL activity in vivo, we have assessed the effects on sensorimotor responses in visual placing tests carried out on CD-1 mice at a dose of 400 mg/kg of GVL, administered by gastric gavage. In parallel, samples of the in vitro and in vivo metabolism studies were analyzed using a rapid and cost-effective analytical procedure based on gas chromatography coupled to mass spectrometry. Our data confirmed that GVL impairs visual placing response in mice in the first 4 h after the intake, and they also show that GVL is a less potent substitute of GHB. The urinary excretion profile of GVL is consistent with the results of the behavioral study, with a maximum of excretion in the first 5 h after the intake. GVL is only detectable in the first 0–8 h after the intake. Our data confirmed the same rapid urinary excretion rate of GHB in urine and the related forensic implications, with the risk of possible false-negative results, especially when DFSA is suspected