Assessing the Pharmacological Properties of Novel Psychoactive Substances (NPS) Identified Online: In Silico Studies on Designer Benzodiazepines and Novel Synthetic Opioids

Background By 2022, a total of 1,127 of Novel Psychoactive Substances (NPS) have been identified worldwide and officially reported by the United Nations Office on Drugs and Crime (UNODC) and the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). An analysis of the surface web via the use of a web crawler, NPSfinder®, indicated that the number of NPS could be almost four times higher than that known to both the UNODC and EMCDDA. This is of particular concern, especially if one considers the public health risks and harms associated with NPS use/abuse and the paucity of data related to their pharmacological/toxicity profiles. In particular, in the last few years two NPS classes, i.e. novel synthetic opioids (NSOs) and designer benzodiazepines (DBZDs) were associated with serious side-effects and life-threatening scenarios (i.e., fatalities and overdoses). Gaps in knowledge Hence, with online NPS numbers exceeding those reported by official sources, there is a strong need to address the gap in knowledge concerning the discrepancies between the online and the evidence based NPS market(s); as well as the gap in knowledge concerning lack of pharmacological profiles for most of the newly-identified NPS. Objectives This programme of research aimed to: use data available from NPSfinder®, the UNODC and EMCDDA to assess the current general NPS scenarios, and in particular for DBZDs and NSOs; use in silico computational techniques to predict the biological activity of the emerging NPS; use the predicted values to infer possible health threats associated with the consumption of these substances, underscoring which of the NPS identified online could indeed represent a serious threat to public health; assess the potential of in silico methodologies as preliminary risk assessment tools; and subsequently inform relevant stakeholders about the risks associated with these new NPS. Methods The NPSfinder® web crawler was used to identify NPS which are available/discussed online. A comparison with UNODC and EMCDDA databases was then carried out to assess the extent of the total NPS scenario, and the numbers of the NSOs and DBZDs classes. To appreciate and predict the biological activities of NSOs and DBZDs, in silico models (e.g., quantitative structure-activity relationship (QSAR), Molecular Docking (MD) and pharmacophore mapping) were used as reliable, time- and cost- effective alternatives to the classical approaches such as in vivo, in vitro or preclinical studies. Results and Discussion A total of 4,231 NPS were identified on the surface web, almost four times the numbers reported by both UNDOC and EMCDDA databases (circa 1,127). These results suggest how the online content analysis should be considered as an important source for the assessment of the NPS scenario. The same discrepancy in the total NPS numbers was observed for each NPS class and a total of 115 DBZDs and 371 NSOs were identified compared to 33 and 123 reported by the UNODC respectively. To assess pharmacological profiles of these NSOs and DBZDs identified online, specific QSAR models were developed in MOE® and Forge™. For the prediction of biological activities of DBZDs, the γ-aminobutyric acid A receptor (GABA-AR) was used; the mu opioid receptor (MOR) was used for the NSOs. In addition, for the DBZDs, a set of new potential ligands resulting from “scaffold hopping” exercises conducted with MOE® was also evaluated. The generated QSAR models returned good performance statistics confirming their strong reliability in predicting the biological activity of an unknown or a newly-identified molecule. The DBZDs predicted to be the most active were flubrotizolam, clonazolam, pynazolam and, fluclotizolam, consistently with reported literature and/or drug discussion forums. In particular with flubrotizolam and fluclotizolam, it was found they were discussed on drug fora but not previously identified either by the UNODC or EMCDDA (flubrotizolam only). This suggests the possible presence on the market of very potent NPS which are still unknown to international agencies, potentially representing a serious threat to public health. Worrisome results were also obtained for the class of NSOs, with the identification of new and potent analogues of carfentanyl (10,000 more potent than morphine), i.e., 2-methyl carfentanyl, n-methyl-carfentanyl and butyryl-carfentanyl. Moreover, the scaffold hopping exercise conducted for the DBZDs class, strongly suggested that structural replacement of the pendant phenyl moiety could increase biological activity and highlighted the existence of a still unexplored chemical space for this NPS class. The results obtained with QSAR analysis were supported by molecular docking exercises, which gave an indication of the binding affinity of these NPS towards their respective receptors. Moreover, the binding affinity of a set of DBZDs was assessed for the MOR, in an attempt to assess a possible multi-receptor activity of these molecules. Conclusions The online identification of a great number of NPS, including very potent central nervous system depressants, represents a serious challenge, in particular if one considers that DBZDs and NSOs are usually consumed either together or in combination with stimulants for recreational purposes and self-medication. The high numbers of available molecules, their patterns of use and the paucity of pharmacological data could lead to worrisome outcomes, including the synergy of each NPS class side-effects, which could (and are) increasing the likelihood of respiratory depression, coma, and deaths. To retrieve an extensive picture of the current NPS drug scenario, the online analysis has proven very useful, if not fundamental. Its ability to identify novel mentioned NPS, in a timely manner, makes it a very important tool for a range of activities, including informing law-enforcement and public health stakeholders, supporting the European and United Nations Early Warning Systems impacting and influencing law-making and guiding monitoring/surveillance. Moreover, in silico methodologies, proven as reliable tools for a fast prediction of biological activity, could be used in describing the activity/toxicity profile of novel NPS, aiming at supporting both law enforcement in scheduling process and public health stakeholders in drafting treatment/management educational packages. Finally, the combination of online and in silico analysis could support and improve the risk assessment procedures currently in place for NPS

Evidence-based successful example of a structure-based approach for the prediction of designer fentanyl-like molecules

© 2024 The Author(s). Published by Elsevier Ltd on behalf of International Society for the Study of Emerging Drugs. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/In 2019, we published three innovative quantitative structure-activity relationship models (QSAR) for predicting the affinity of mu-opioid receptor (µOR) ligands. The three different models were then combined to produce a consensus model used to explore the chemical landscape of 3000 virtual fentanyl-like structures, also generated by us by a theoretical scaffold-hopping approach to explore potential novel active substances and predict their activity. Interestingly, five years have passed, and some of the virtual predicted compounds have been identified/reported to e.g. the EU Early Warning System or the United Nations Office on Drugs and Crime, thus confirming our warning hypothesis that new emerging drugs from our screen would find way to the market.Peer reviewe

Recent Changes in Drug Abuse Scenario: The Novel Psychoactive Substances (NPS) Phenomenon

copyright 2019 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.Final Published versio

The Psychonauts’ Benzodiazepines; Quantitative Structure-Activity Relationship (QSAR) Analysis and Docking Prediction of Their Biological Activity

© 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/).Designer benzodiazepines (DBZDs) represent a serious health concern and are increasingly re-ported in polydrug consumption-related fatalities. When new DBZDs are identified, very limited information is available on their pharmacodynamics. Here, computational models (e.g., quantita-tive structure-activity relationship/QSAR and Molecular Docking) were used to analyse DBZDs identified online by an automated web crawler (NPSfinder®) and to predict their possible activi-ty/affinity on the gamma-aminobutyric acid receptors (GABA-ARs). The computational software MOE was used to calculate 2D QSAR models, perform docking studies on crystallised GABA-A receptors (6HUO, 6HUP) and generate pharmacophore queries from the docking conformational results. 101 DBZDs were identified online by NPSfinder®. The validated QSAR model predicted high biological activity values for 41% of these DBDZs. These predictions were supported by the docking studies (good binding affinity) and the pharmacophore modelling confirmed the im-portance of the presence and location of hydrophobic and polar functions identified by QSAR. This study confirms once again the importance of web-based analysis in the assessment of drug scenarios (DBZDs), and how computational models could be used to acquire fast and reliable in-formation on biological activity for index novel DBZDs, as preliminary data for further investiga-tions.Peer reviewe

Semaglutide as a Possible Calmodulin Binder: Ligand-Based Computational Analyses and Relevance to Its Associated Reward and Appetitive Behaviour Actions

© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Semaglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, has gained considerable attention as a therapeutic agent for type 2 diabetes mellitus and obesity. Despite its clinical success, the precise mechanisms underlying its pharmacological effects remain incompletely understood. In this study, we employed ligand-based drug design strategies to investigate potential off-target interactions of semaglutide. Through a comprehensive in silico screening of semaglutide’s structural properties against a diverse panel of proteins, we have identified calmodulin (CaM) as a putative novel target of semaglutide. Molecular docking simulations revealed a strong interaction between semaglutide and CaM, characterized by favourable binding energies and a stable binding pose. Further molecular dynamics simulations confirmed the stability of the semaglutide–CaM complex, emphasizing the potential for a physiologically relevant interaction. In conclusion, our ligand-based drug design approach has uncovered calmodulin as a potential novel target of semaglutide. This discovery sheds light on the complex pharmacological profile of semaglutide and offers a promising direction for further research into the development of innovative therapeutic strategies for metabolic disorders. The CaM, and especially so the CaMKII, system is central in the experience of both drug- and natural-related reward. It is here hypothesized that, due to semaglutide binding, the reward pathway-based calmodulin system may be activated, and/or differently regulated. This may result in the positive semaglutide action on appetitive behaviour. Further studies are required to confirm these findings.Peer reviewe

The market of sport supplement in the digital era: A netnographic analysis of perceived risks, side-effects and other safety issues

© 2021 The Authors. Published by Elsevier Ltd on behalf of International Society for the Study of Emerging Drugs. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ )Background: The market of sport supplements is expanding worldwide. Such phenomenon is often supported by captivating marketing strategies and social media advertising providing unscientifically founded claims, thus raising safety concerns. The aim of our study is to provide a comprehensive analysis of the online market, patterns of use, perceived risks and other safety issues on supplement use as reported in online fitness communities. Methods: A mixed method approach was employed. An automatized web-based monitoring tool (Brand24®) was used to track the most popular supplements and related discussions according to the number of interactions between users and shares; the number and category of websites; the social media reach; and the most popular hashtags. Results were assessed through a netnographic qualitative analysis of online fitness fora, to identify motivations of intake, self-reported side effects andthe overall safety perception reliability of supplements information online. Results: A social media reach of over four million individuals, inclusive of 18595 posts, emerged from our search. The most cited supplements were “Whey Protein”, “Branched Chain Amino-Acid”, “Creatine”, “Multivitamin supplements” and “Nitric Oxide boosters”. Supplements were mainly taken for muscle gain (23%), increase energy (17%), and weight loss (8%). Although the web narrative on supplementation was overall positive, a wide range of side effects were reported by 19% of fitness fora users. These included acne (9%), water retention (9%), stomach pain (9%), rashes (7%), erectile dysfunctions (7%) and weight gain (5%). Concerns about contamination (47%), counterfeit content (17%) and the presence of hidden ingredients (11%) were also recorded. Conclusions: In a poorly regulated context, where unsolicited social media posts have replaced the typical advice provided by professionals, efforts should be made to ensure the reliability of the provided information to avoid the insurgence of unwanted adverse effects and safeguard public health.Peer reviewe

GLP-1 Receptor Agonists and Related Mental Health Issues; Insights from a Range of Social Media Platforms Using a Mixed-Methods Approach

© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/The emergence of glucagon-like peptide-1 receptor agonists (GLP-1 RAs; semaglutide and others) now promises effective, non-invasive treatment of obesity for individuals with and without diabetes. Social media platforms’ users started promoting semaglutide/Ozempic as a weight-loss treatment, and the associated increase in demand has contributed to an ongoing worldwide shortage of the drug associated with levels of non-prescribed semaglutide intake. Furthermore, recent reports emphasized some GLP-1 RA-associated risks of triggering depression and suicidal thoughts. Consistent with the above, we aimed to assess the possible impact of GLP-1 RAs on mental health as being perceived and discussed in popular open platforms with the help of a mixed-methods approach. Reddit posts yielded 12,136 comments, YouTube videos 14,515, and TikTok videos 17,059, respectively. Out of these posts/entries, most represented matches related to sleep-related issues, including insomnia (n = 620 matches); anxiety (n = 353); depression (n = 204); and mental health issues in general (n = 165). After the initiation of GLP-1 RAs, losing weight was associated with either a marked improvement or, in some cases, a deterioration, in mood; increase/decrease in anxiety/insomnia; and better control of a range of addictive behaviors. The challenges of accessing these medications were a hot topic as well. To the best of our knowledge, this is the first study documenting if and how GLP-1 RAs are perceived as affecting mood, mental health, and behaviors. Establishing a clear cause-and-effect link between metabolic diseases, depression and medications is difficult because of their possible reciprocal relationship, shared underlying mechanisms and individual differences. Further research is needed to better understand the safety profile of these molecules and their putative impact on behavioral and non-behavioral addictions.Peer reviewe

Illicit COVID-19 products online: A mixed-method approach for identifying and preventing online health risks

© 2023 Catalani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Aims: The COVID-19 pandemic triggered a demand for vaccines, cures, and the need of related documentation for travel, work and other purposes. Our project aimed to identify the illicit availability of such products across the Dark Web Markets (DWMs). Methods: A retrospective search for COVID-19 related products was carried out across 118 DWMs since the start of the pandemic (March 2020-October 2021). Data on vendors as well as advertised goods such as asking price, marketplace, listed date were collected and further validated through additional searches on the open web to verify the information relating to specific marketplaces. Both quantitative and qualitative methods were used for data analysis. Results: Forty-two listings of unlicenced COVID-19 cures and vaccination certificates were identified across 8 marketplaces sold by 25 vendors with significant variation in prices. The listings were found to be geographically specific and followed the progression of the pandemic in terms of availability. Correlations between vendor portfolios of COVID-19 products and variety of goods of other illicit nature such as illegal weaponry, medication/drugs of abuse also emerged from our analysis. Conclusion: This study is one of the first attempts to identify the availability of unlicenced COVID-19 products on DWMs. The easy accessibility to vaccines, fake test certificates and hypothetical/illegal cures poses serious health risks to (potential) buyers due to the uncontrolled nature of such products. It also exposes buyers to an unwanted contact with vendors selling a variety of other dangerous illicit goods. Further monitoring and regulatory responses should be implemented to protect the health and safety of citizens especially at times of global crisis.Peer reviewe

Profiling the vendors of COVID‐19 related product on the Darknet: An observational study

/© 2023 The Author(s). Published by Elsevier Ltd on behalf of International Society for the Study of Emerging Drugs. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)BACKGROUND: In a time of unprecedented global change, the COVID-19 pandemic has led to a surge in demand of COVID-19 vaccines and related certifications. Mainly due to supply shortages, counterfeit vaccines, fake documentation, and alleged cures to illegal portfolios, have been offered on darkweb marketplaces (DWMs) with important public health consequences. We aimed to profile key DWMs and vendors by presenting some in-depth case studies.METHODS: A non-systematic search for COVID-19 products was performed across 118 DWMs. Levels of activity, credibility, content, COVID-19 product listings, privacy protocols were among the features retrieved. Open web fora and other open web sources were also considered for further analysis of both functional and non functional DWMs. Collected data refers to the period between January 2020 and October 2021.RESULTS: A total of 42 relevant listings sold by 24 vendors across eight DWMs were identified. Four of these markets were active and well-established at the time of the study with good levels of credibility. COVID-19 products were listed alongside other marketplace content. Vendors had a trusted profile, communicated in English language and accepted payments in cryptocurrencies (Monero or Bitcoin). Their geographical location included the USA, Asia and Europe. While COVID-19 related goods were mostly available for regional supply, other listings were also shipped worldwide.INTERPRETATION: Findings emerging from this study rise important questions about the health safety of certain DWMs activities and encourage the development of targeted interventions to overcome such new and rapidly expanding public health threats.FUNDING: CovSaf, National Research centre on Privacy, Harm Reduction and Adversarial Influence Online (REPHRAIN), Commonwealth Fund.Peer reviewe

Benzodiazepine Boom: Tracking Etizolam, Pyrazolam, and Flubromazepam from Pre-UK Psychoactive Act 2016 to Present Using Analytical and Social Listening Techniques

© 2024 The Author(s). Licensee MDPI, Basel, Switzerland. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Introduction: The designer benzodiazepine (DBZD) market continues to expand whilst evading regulatory controls. The widespread adoption of social media by pro-drug use communities encourages positive discussions around DBZD use/misuse, driving demand. This research addresses the evolution of three popular DBZDs, etizolam (E), flubromazepam (F), and pyrazolam (P), available on the drug market for over a decade, comparing the quantitative chemical analyses of tablet samples, purchased from the internet prior to the implementation of the Psychoactive Substances Act UK 2016, with the thematic netnographic analyses of social media content. Method: Drug samples were purchased from the internet in early 2016. The characterisation of all drug batches were performed using UHPLC-MS and supported with 1H NMR. In addition, netnographic studies across the platforms X (formerly Twitter) and Reddit, between 2016–2023, were conducted. The latter was supported by both manual and artificial intelligence (AI)-driven thematic analyses, using numerous.ai and ChatGPT, of social media threads and discussions. Results: UHPLC-MS confirmed the expected drug in every sample, showing remarkable inter/intra batch variability across all batches (E = 13.8 ± 0.6 to 24.7 ± 0.9 mg; F = 4.0 ± 0.2 to 23.5 ± 0.8 mg; P = 5.2 ± 0.2 to 11.5 ± 0.4 mg). 1H NMR could not confirm etizolam as a lone compound in any etizolam batch. Thematic analyses showed etizolam dominated social media discussions (59% of all posts), with 24.2% of posts involving sale/purchase and 17.8% detailing new administration trends/poly-drug use scenarios. Artificial intelligence confirmed three of the top five trends identified manually. Conclusions: Purity variability identified across all tested samples emphasises the increased potential health risks associated with DBZD consumption. We propose the global DBZD market is exacerbated by surface web social media discussions, recorded across X and Reddit. Despite the appearance of newer analogues, these three DBZDs remain prevalent and popularised. Reporting themes on harm/effects and new developments in poly-drug use trends, demand for DBZDs continues to grow, despite their potent nature and potential risk to life. It is proposed that greater controls and constant live monitoring of social media user content is warranted to drive active regulation strategies and targeted, effective, harm reduction strategies.Peer reviewe