New Psychoactive Substances Intoxications and Fatalities during the COVID-19 Epidemic

Simple Summary We presented a comprehensive, systematic literature review of all published New Psychoactive Substances (NPS)-related intoxications and fatalities during the COVID-19 pandemic (from January 2020 to March 2022). Public implications, such as isolation and social distancing, may have reduced consumption of some drugs. These stressful conditions brought an increase in the use of other drugs, with the illicit market and related misuse of drugs moving to different drugs of abuse, such as NPS. More than 200 cases were reported in Europe, UK, USA and Japan during the pandemic period, with synthetic opioid, synthetic cannabinoids and synthetic cathinones the most representative NPS classes. Importantly, the combined consumption of several NPS classes comprised 30% of all cases. Considering that the pandemic may have reduced the capabilities of forensic toxicology laboratories to report comprehensive information, the data could have led to an underestimation. Abstract In January 2020, the World Health Organization (WHO) issued a Public Health Emergency of International Concern, declaring the COVID-19 outbreak a pandemic in March 2020. Stringent measures decreased consumption of some drugs, moving the illicit market to alternative substances, such as New Psychoactive Substances (NPS). A systematic literature search was performed, using scientific databases such as PubMed, Scopus, Web of Science and institutional and government websites, to identify reported intoxications and fatalities from NPS during the COVID-19 pandemic. The search terms were: COVID-19, SARS-CoV-2, severe acute respiratory syndrome coronavirus 2, coronavirus disease 2019, intox*, fatal*, new psychoactive substance, novel psychoactive substance, smart drugs, new psychoactive substance, novel synthetic opioid, synthetic opioid, synthetic cathinone, bath salts, legal highs, nitazene, bath salt, legal high, synthetic cannabinoid, phenethylamine, phencyclidine, piperazine, novel benzodiazepine, benzodiazepine analogue, designer benzodiazepines, tryptamine and psychostimulant. From January 2020 to March 2022, 215 NPS exposures were reported in Europe, UK, Japan and USA. Single NPS class intoxications accounted for 25, while mixed NPS class intoxications represented only 3 cases. A total of 130 NPS single class fatalities and 56 fatalities involving mixed NPS classes were published during the pandemic. Synthetic opioids were the NPS class most abused, followed by synthetic cathinones and synthetic cannabinoids. Notably, designer benzodiazepines were frequently found in combination with fentalogues. Considering the stress to communities and healthcare systems generated by the pandemic, NPS-related information may be underestimated. However, we could not define the exact impacts of COVID-19 on processing of toxicological data, autopsy and death investigations.


Introduction
The World Health Organization (WHO) declared a Public Health Emergency of International Concern in January 2020 and characterized the outbreak as a pandemic in March 2020.
In response to the COVID-19-pandemic, lockdowns were required to prevent the spread of the disease, resulting in drastic reductions in social contact in public settings and in most individuals' private and professional lives. Such restrictions also led to modifications in the synthesis of chemical precursors, manufacturing and distribution [1,2]. In April 2020, the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) released an alert to the EU Early Warning System Network, focusing on the potential impact of the pandemic on drug markets [1,3].
As reported in the Global Drug Survey [4,5], stressful conditions produced by pandemic restrictions brought an increase in the use of some drugs. According to this survey [4], increases in benzodiazepine use during the pandemic could have arisen from the need to manage anxiety and depression; however, benzodiazepines are highly addictive, which could have led to a serious public health problem. Stringent measures, including isolation and social distancing, resulted in lower consumption of some drugs [1], such as stimulants and hallucinogens.
The illicit market and related misuse of drugs moved to alternative substances [6], such as New Psychoactive Substances (NPS). NPS comprise a heterogenous group of substances [7], such as prescription drugs or research chemicals, which are not controlled under the 1961 Single Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substance, synthetized to mimic the psychoactive effects of commonly abused drugs. NPS consumption [8][9][10][11][12][13][14][15] can cause several adverse effects, such as acute psychosis, bradypnea, angina pectoris, migraine, headaches and life-threatening cardiovascular problems ranging from mild tachycardia to arrhythmias, myocardial infarction and even death.
Considering the many reported cardiovascular complications [16][17][18][19][20] for NPS and commonly abused drugs, and the high life-threatening risk every time an NPS enters into the market, further studies on the side effects of drug consumption, including cardiotoxicity, are required. The most recent literature on NPS did not include comprehensive systematic reviews [13][14][15] on NPS intoxications and fatalities. While synthetic opioids, synthetic cannabinoid and synthetic cathinones represented the most abused NPS classes during the COVID-19 pandemic, co-consumption of different NPS classes raised particular concern.
The pandemic overloaded the healthcare system, reducing the capability of forensic toxicology laboratories and emergency departments to detect and report NPS-related information. As a consequence, the data could be underestimated [1]. To address this concern, we aimed to identify all the intoxications and fatal cases reported, during the pandemic, caused by single or mixed NPS classes, focusing on which NPS class caused the most intoxications and/or fatalities as well as the most frequent combination of them.
Herein, we presented a comprehensive, systematic review of all published NPS-related intoxication instances and fatalities throughout the COVID-19 pandemic (from January 2020 to March 2022).

Materials and Methods
In accordance with Prisma guidelines [21], a literature search was performed to identify reported intoxications and fatalities from NPS during the COVID-19 pandemic, specifically from January 2020 to March 2022. PubMed, Scopus, Web of Science and institutional and government websites were searched by two scientists for the following terms, alone or in combination: ("COVID-19" OR "SARS-CoV-2" OR "Severe acute respiratory syndrome coronavirus 2" OR "Coronavirus disease 2019" OR intox* or fatal*) AND ("New psychoactive substance" OR "Novel psychoactive substance" OR "smart drugs" OR "Novel psychoactive substances" OR "New psychoactive substances" OR "Synthetic opioid" OR "Novel synthetic opioids" OR "Novel synthetic opioid" OR "Synthetic opioids" OR "Synthetic cathinone" OR "bath salts" OR "legal highs" OR "nitazene" OR "Synthetic cathinones" OR "bath salt" OR "legal high" OR "synthetic cannabinoid" OR "synthetic cannabinoids" OR phenethylamine OR phencyclidine OR piperazine OR "novel benzodiazepine" OR "Benzodiazepine analogue" OR "designer benzodiazepines" OR tryptamine OR Psychostimulant).
Relevant articles cited in the references of these selected publications were also considered. Articles were screened according to the following criteria: 1.
English, Italian or French language. 2.
Studies involving human exposure to NPS, alone or in combination with other drugs, from January 2020 to March 2022. 3.
Exposure confirmation through qualitative or quantitative toxicology analyses of human biological matrices.
All the data were extracted, curated and analyzed without software aid. Articles were manually screened by three of the authors. Moreover, a second check was performed to avoid researcher bias. Figure 1 shows the Prisma Flowchart of the literature search.

Results
The results of the literature search are shown in Table 1. Out of 5207 records identified, a total of 63 papers met the inclusion criteria (after duplicate removal). In Table 1, all cases reported were clustered by year. Whenever available, each case included a descriptive analysis of individual characteristics (e. g., age, gender), the class of new psychoactive substances (NPS) and relative concentrations in different biological matrices (iliac and femoral blood were reported as peripheral blood), concomitant exposure(s), date and country where the report originated.

Synthetic Opioids Related Intoxications and Deaths
Synthetic opioids [71] are the third largest class of NPS worldwide, increasing from 14 different substances in 2009 to 87 in 2020, of which 22 were reported for the first time. Synthetic opioids have a high abuse liability due to their binding affinity to µ-opioid receptors. Furthermore, their high drug lipophilicity facilitates crossing through the bloodbrain barrier.
During the last decade, the most-abused synthetic opioids were fentanyl and its analogues. Many fentanyl analogues were scheduled starting in 2019 [72], leading to the development of benzimidazoles, an emerging synthetic opioid class that entered the black market as a cheaper alternative to heroin.

Fentanyl and Analogues
Synthetic opioids [77,78] induce euphoria, sedation and respiratory depression, with potencies 100 to 10,000 times greater than morphine, producing primarily fatal cases.
Despite fentanyl-related substances' placement in the FDA's Schedule I in 2018, during the COVID-19 pandemic, 64 deaths involving fentanyl were reported. Fentanyl and its analogues [79] continue to have a critical worldwide impact on drug intoxications and fatalities. Additionally, 90% of seized synthetic opioids were fentanyl.
The most frequent fentalogues reported with fentanyl were p-FF and acetyl fentanyl (and their metabolites), representing 43% of total cases (n = 43).

Other Synthetic Opioids
Brorphine [25,28,40], structurally similar to fentanyl, is a substituted piperidine benzimidazolone. Brorphine was reported in 9 deaths during the COVID-19 pandemic, with in vitro receptor activation assays showing a similar potency and efficacy to fentanyl.

New Synthetic Opioids (Benzimidazoles and AP-Series Drugs)
Among all synthetic opioids previously reported [80,81], a new wave of designer opioids raised concern worldwide, entering the market in 2019. Benzimidazoles, structurally unrelated to fentanyl, are synthetic analogues of etonitazene, discovered for the first time in Switzerland by a pharmaceutical company [80]. Recently, benzimidazoles were shown to have a considerable degree of affinity and a higher potency than that of fentanyl for the µ-opioid receptor.
The rapid placement of new substances into controlled drug schedules is one important action to prevent such substances from spreading out of control. Unfortunately, scheduling still remains a lengthy and time-consuming process. Most isotonitazene cases in the USA were reported before isotonitazene was scheduled in June 2020.
After fentanyl-related substances [60] were placed under control, the cinnamoylpiperazine subclass emerged onto the market. 2-Methyl AP-237 and AP-238 were recently reported in several cases (n = 7). This class of compounds [71], due to its piperazine ring, has highly potent analgesic effects. To date, few compounds in this class have been reported, but minimal structural modification could generate even more potent compounds that could easily lead to overdoses and deaths.
Currently, the mechanisms by which NPS affect cardiac function have not yet been elucidated. NPS inhibit monoamine transporters [11,82], potentially giving rise to indirect cardiovascular effects. However, NPS [11] may also have direct effects on cardiomyocyte function, irrespective of circulating norepinephrine levels.

Synthetic Cannabinoids
According to the European Drug Report [7], synthetic cannabinoids are the NPS class with the highest number of substances monitored by the EU Early Warning System in 2021. They mimic the effects of the natural constituent delta9-tetrahydrocannabinol (THC), which is the principal compound producing cannabispsychoactive effects. First synthetized for research purposes, synthetic cannabinoids are sold on the black market. Synthetic cannabinoids [83] are agonists at the CB1 and CB2 cannabinoid receptors, but also ion channels and PPAR receptors, where they can produce severe adverse effects, including disinhibition, euphoria, neurological disorders, agitation, irritability, paranoia, psychiatric conditions and death. There were 19 intoxications and 30 deaths published during the COVID-19 pandemic involving synthetic cannabinoid intake.

F-MDMB-BICA
4F-MDMB-BICA was the most commonly abused synthetic cannabinoid in 2020. Consumption of 4F-MDMB-BICA [37] was associated with a high number of intoxications and deaths in 2020 in the UK and Hungary (n = 5 and n = 21, respectively). No further cases were described after the end of 2020.

MDMB-4en-PINACA
MDMB-4en-PINACA [34] is a CB1 receptor agonist, with a potency three times higher than its analogue JWH-018. In 2020, There were 12 intoxications and 7 fatalities reported [34,37], 6 of which were in combination with other synthetic cannabinoids. While it has been available in the market since 2017, MDMB-4en-PINACA seizures increased during 2020 [61] after two similar analogues, 4F-MDMB-BINACA and 5F-MBMB-PICA, were placed under international control. This example confirmed the importance of rapid detection and scheduling, but also demonstrated that, as soon as a substance is controlled, a new analogue appears on the market.

Synthetic Cathinones
Synthetic cathinones [7] are the second-largest NPS class, based on the EU Early warning system (EWS). As of this writing, 162 synthetic cathinones are monitored by the EU EWS. They have a similar structure to the natural compound cathinone, principally found in Catha edulis, and amphetamines. Synthetic cannabinoids are b-keto analogues of a corresponding phenethylamine. During the COVID-19 pandemic, 42 cases {Formatting Citation} were reported with synthetic cannabinoids, alone or in combination with other NPS classes.

3-CMC
3-CMC has psychostimulant effects similar to those of methcathinone and 4chloromethcathinone (4-CMC), which are Schedule I and II of the 1971 United Nation Single Convention [84] on Psychotropic Substances, respectively.
As reported in the European Drug Report [7], 3-CMC accounted for almost a quarter of the total drug seizures. This increase in 3-CMC seizures could have been related to the recent control of 4-CMC under the international drug system.
In Europe, between 2020 and 2021, a total of 9 fatalities (6 in Poland and 3 in Sweden) were reported in the risk assessment published by the EMCDDA. In some cases, 3-CMC [31] was the only substance identified, but in other cases, multiple stimulants and depressants were also observed.

3-MMC
3-MMC is a synthetic cathinone related to methcathinone and 4-methylmethcathinone (mephedrone, 4-MMC). 3-MMC was reported for the first time in Sweden in September 2012 [32]. The distinction between the two positional isomers requires hyphenated analytical techniques. 3-MMC [32] was reported in 6 fatalities and 2 intoxications. No specific data were available regarding these cases.

Designer Benzodiazepines
Recently [85], there was a significant increase in the number of new DBZPs. The mechanism of action on the central nervous system is via the GABA A receptor. GABA induces inhibitory effects through the central nervous system, including hypnosis, sedation or amnesia. DBZPs have a high abuse potential. Between 2020 and 2021 [14] clonazolam, diclazepam, etizolam, flualprazolam and flubromazolam were included in Schedule IV of the Convention of Psychotropic Substances of 1971.
Arylcyclohexylamines and phenethylamines have similar mechanisms of action to tryptamines and elicit stimulating effects; hence, they are included in the same NPS subgroup. Only 2 cases were reported for phenethylamine; 2 were also reported for arylcyclohexylamines.

NPS Cardiotoxicity Implications
The exact incidence of toxic myocardial injury and consequent toxic cardiomyopathy is difficult to establish [87] due to the high degree of variability in toxic exposure and to the presence of any underlying cardiac conditions. Toxic damage could therefore trigger a series of reactions in cardiac cells leading to changes in myocardial morphology, biochemistry and physiology. For these reasons, mechanisms behind the development of toxic cardiomyopathy appeared multifactorial and complex. In fact, the effects of drugs on the heart may be aggravated in the presence of comorbidities/cotreatments since they affect ion channel expression and/or activity, mitochondrial function, electro-mechanical coupling and modification of extracellular matrix composition favoring the induction of arrhythmias, contractile dysfunction and, potentially, cardiomyocyte death. Some toxic substances [88] are able to produce direct or indirect toxicity to cardiomyocytes. Direct toxicity, present in several experimental models of toxic cardiomyopathy, can be mediated by reactive oxygens species (ROS) production, which determines lipid peroxidation through membrane interactions and mitochondrial oxidative stress, or by Mitogen-Activated Protein Kinases (MAPKs). The generated ROS may also further induce DNA damage, especially DNA single-strand breaks.
Indirect toxicity [89] can instead be mediated by a neurohormonal response involving excessive catecholamine release or blocking noradrenaline-reuptake mechanism. The major representatives are indirect sympathomimetics and nonselective sympathomimetics, which affect both the vascular system (via noradrenaline actions on α1-adrenergic receptors) and the heart (due to noradrenaline actions on β1-adrenoreceptors).

Conclusions
We undertook a systematic literature review to identify the NPS-related intoxications and fatalities during the COVID-19 pandemic. Although we observed a large diffusion of several NPS from different structural classes, from January 2020 to March 2022, synthetic opioids emerged as the most-abused class of NPSs, causing fatalities when consumed, both alone and in combination with other NPS classes. New designer benzodiazepines were frequently found alongside fentanyl analogues in drug-related deaths displaying respiratory depressant properties. Such combinations may substantiate the assumption that, in times of stress and crisis, vulnerable people and polydrug users prefer drugs that can be used in solitude to escape anxiety, uncertainty and distress-e.g., that generated by the COVID-19 pandemic. Moreover, we observed that many intoxications due to single NPS-exposure were caused by synthetic cannabinoids, followed by synthetic cathinones and synthetic opioids. Considering intoxications observed in polydrug users, a mixture of different NPS classes were observed, including synthetic cathinones, synthetic opioids, arylcyclohexylamines, natural NPS and designer benzodiazepines.
From 2020 to 2022 the number of NPS exposures changed, from 123 cases in 2020 to 71 cases in 2021 and 21 cases up from January-March 2022. Nonetheless, this represented a decrease during those years, though we were unable to clarify whether the cause of this decline was caused by a delay in toxicological processing or a decrease in drug consumption.
The wide range of NPS chemical structures represents the main challenge for their detection in toxicological laboratories. Reference standard availability, method development, the scarcity of knowledge about NPS metabolism and the serious ongoing health emergency could have caused a loss of information about NPS-related intoxications and fatalities. Therefore, hyphenated analytical techniques, validated and updated methods are necessary to ensure reliable results in the identification of analytes. In acknowledgment of this concern, we observed that liquid chromatography mass spectrometry was the most suitable technique used in the cases reported [90,91].
The main limitations of this study were (1) the lack of information regarding concentrations in biological matrices of NPS and their metabolites and (2) the difficulty in identifying which NPS class caused intoxication or death. Comprehension of the analytical data is needed to better understand NPS toxic/lethal blood concentrations, metabolism and postmortem redistribution. Funding: The review paper was partially funded from the following Projects "National Early Warning System on Drugs (SNAP)" and "Implementation of the identification and study of the effects of NPS" funded by the Italian Department of Antidrug policies.