Molecular Insights and Clinical Outcomes of Drugs of Abuse Adulteration: New Trends and New Psychoactive Substances

Adulteration is a well-known practice of drug manufacturers at different stages of drug production. The intentional addition of active ingredients to adulterate the primary drug may enhance or mask pharmacological effects or may produce more potent drugs to increase the number of available doses and the dealer\u27s profit. Adulterants found in different drugs change over time in response to different factors. A systematic literature search in PubMed and Scopus databases and official international organizations\u27 websites according to PRISMA guidelines was performed. A total of 724 studies were initially screened, with 145 articles from PubMed and 462 from Scopus excluded according to the criteria described in the Method Section. The remaining 117 records were further assessed for eligibility to exclude articles without sufficient data. Finally, 79 studies were classified as non-biological (n = 35) or biological (n = 35 case reports; n = 9 case series) according to the samples investigated. Although the seized samples analyses revealed the presence of well-established adulterants such as levamisole for cocaine or paracetamol/acetaminophen for heroin, the reported data disclosed new adulteration practices, such as the use of NPS as cutting agents for classic drugs of abuse and other NPS. For example, heroin adulterated with synthetic cannabinoids or cocaine adulterated with fentanyl/fentalogues raised particular concern. Notably, adulterants play a role in some adverse effects commonly associated with the primary drug, such as levamisole-adulterated cocaine that may induce vasculitis via an autoimmune process. It is essential to constantly monitor adulterants due to their changing availability that may threaten drug consumers\u27 health

Methylone and MDMA Pharmacokinetics Following Controlled Administration in Humans

The aim of this study is to define, for the first time, human methylone and HMMC plasma pharmacokinetics following controlled administration of 50–200 mg methylone to 12 male volunteers. A new LC-MS/MS method was validated to quantify methylone, MDMA, and their metabolites in plasma. The study was a randomized, cross-over, double-blinded and placebo-controlled study, with a total of 468 plasma samples collected. First, 10 µL of MDMA-d5, MDA-d5 and methylone-d3 internal standards were added to 100 µL of plasma. Two mL of chloroform and ethyl acetate 9:1 (v/v) were then added, mixed well and centrifuged. The supernatant was fortified with 0.1 mL acidified methanol and evaporated under nitrogen. Samples were reconstituted with a mobile phase and injected into the LC-MS/MS instrument. The method was fully validated according to OSAC guidelines (USA). Methylone plasma concentrations increased in a dose-proportional manner, as demonstrated by the increasing maximum concentration (Cmax) and area under the curve of concentrations (AUC). Methylone Cmax values were reported as 153, 304, 355 and 604 ng/mL, AUC0–24 values were reported as 1042.8, 2441.2, 3524.4 and 5067.9 h·ng/mL and T1/2 values as 5.8, 6.4, 6.9 and 6.4 h following the 50, 100, 150 and 200 mg doses, respectively. Methylone exhibited rapid kinetics with a Tmax of 1.5 h for the 50 mg dose and 2 h approximately after all the other doses. HMMC exhibited faster kinetics compared to methylone, with a Cmax value that was 10–14-fold lower and an AUC0–24 value that was 21–29-fold lower. Methylone pharmacokinetics was linear across 50–200 mg oral doses in humans, unlike the previously described non-linear oral MDMA pharmacokinetics. An LC-MS/MS method for the quantification of methylone, MDMA and their metabolites in human plasma was achieved. Methylone exhibited linear pharmacokinetics in humans with oral doses of 50–200 mg

3F-α-pyrrolydinovalerophenone (3F-α-PVP) in vitro human metabolism: Multiple in silico predictions to assist in LC-HRMS/MS analysis and targeted/untargeted data mining

Synthetic cathinones (SCs) constitute a heterogenous class of new psychoactive substances (NPS), structurally related to cathinone. SCs represent the widest NPS class, second to synthetic cannabinoids, accounting for approximately 160 different analogues with substitution at the phenyl group, the amine group, or the alkyl chain. In 2020, α-pyrrolidonophenone analogues were the most trafficked SCs, and were involved in many fatalities and intoxication cases. In particular, 3F-α-pyrrolidinovalerophenone (3F-α-PVP) was the cause of the highest number of SC-related fatal intoxications in Sweden in 2018. Minor structural modifications are used to avoid legal controls and analytical detection, but may also induce different toxicological profile. Therefore, the identification of specific markers of consumption is essential to discriminate SCs in clinical and forensic toxicology. In this study, we assessed 3F-α-PVP metabolic profile. 3F-α-PVP was incubated with 10-donor-pooled human hepatocytes, LC-HRMS/MS analysis, and software-assisted data mining. This well-established workflow was completed by in silico metabolite predictions using three different freeware. Ten metabolites were identified after 3 h incubation, including hydrogenated, hydroxylated, oxidated, and N-dealkylated metabolites. A total of 51 phase I and II metabolites were predicted, among which 7 were detected in the incubations. We suggest 3F-α-PVP N-butanoic acid, 3F-α-PVP pentanol, and 3F-α-PVP 2-ketopyrrolidinyl-pentanol as specific biomarkers of 3F-α-PVP consumption. This is the first time that an N-ethanoic acid is detected in the metabolic pathway of a pyrrolidine SC, demonstrating the importance of a dual targeted/untargeted data mining strategy

In silico and in vitro human metabolism of IOX2, a performance-enhancing doping agent

IOX2 is a potent inhibitor of prolyl hydroxylase 2, a key enzyme in the regulation of hypoxia-inducible factor (HIF) and oxygen homeostasis. As such, it can be used to enhance athletic performance and is currently banned by the World Anti-Doping Agency (WADA). Detection of metabolites is critical to demonstrate drug use in doping. However, there is currently little data on IOX2 human metabolism. Our aim was to identify relevant biomarkers of IOX2 use in humans. For this purpose, IOX2 was incubated with 10-donor-pooled human hepatocytes for 3 h, incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS), and LC-HRMS/MS data were screened with Compound Discoverer (Thermo Scientific) for a comprehensive identification of IOX2 metabolites. Additionally, IOX2 human metabolites were predicted with GLORYx open-access software (University of Hamburg, Germany) to assist in the LC-HRMS/MS analysis and data mining. Thirteen metabolites were identified, oxidation at the quinolinyl group, O-glucuronidation, and combinations being predominant biotransformations. The results were consistent with previous animal studies and a single case of oral microdose administration. We suggest hydroxyquinolinyl-IOX2 as major biomarker of IOX2 use in biological samples, glucuronide hydrolysis being critical to increase IOX2 and hydroxyquinolinyl-IOX2 detectability in urine

Molecular insights and clinical outcomes of drugs of abuse adulteration: new trends and new psychoactive substances.

Adulteration is a well-known practice of drug manufacturers at different stages of drug production. The intentional addition of active ingredients to adulterate the primary drug may enhance or mask pharmacological effects or may produce more potent drugs to increase the number of available doses and the dealer's profit. Adulterants found in different drugs change over time in response to different factors. A systematic literature search in PubMed and Scopus databases and official international organizations' websites according to PRISMA guidelines was performed. A total of 724 studies were initially screened, with 145 articles from PubMed and 462 from Scopus excluded according to the criteria described in the Method Section. The remaining 117 records were further assessed for eligibility to exclude articles without sufficient data. Finally, 79 studies were classified as "non-biological" ( = 35) or "biological" ( = 35 case reports; = 9 case series) according to the samples investigated. Although the seized samples analyses revealed the presence of well-established adulterants such as levamisole for cocaine or paracetamol/acetaminophen for heroin, the reported data disclosed new adulteration practices, such as the use of NPS as cutting agents for classic drugs of abuse and other NPS. For example, heroin adulterated with synthetic cannabinoids or cocaine adulterated with fentanyl/fentalogues raised particular concern. Notably, adulterants play a role in some adverse effects commonly associated with the primary drug, such as levamisole-adulterated cocaine that may induce vasculitis via an autoimmune process. It is essential to constantly monitor adulterants due to their changing availability that may threaten drug consumers' health

Human metabolism and basic pharmacokinetic evaluation of AP-238: A recently emerged acylpiperazine opioid

As a consequence of recently implemented legal restrictions on fentanyl analogs, a new generation of acylpiperazine opioids appeared on the illicit drug market. AP-238 was the latest opioid in this series to be notified by the European Early Warning System in 2020 and was involved in an increasing number of acute intoxications. AP-238 metabolism was investigated to provide useful markers of consumption. For the tentative identification of the main phase I metabolites, a pooled human liver microsome assay was performed. Further, four whole blood and two urine samples collected during post-mortem examinations and samples from a controlled oral self-administration study were screened for anticipated metabolites. In total, 12 AP-238 phase I metabolites were identified through liquid chromatography-quadrupole time-of-flight mass spectrometry in the in vitro assay. All of these were confirmed in vivo, and additionally, 15 phase I and five phase II metabolites were detected in the human urine samples, adding up to a total of 32 metabolites. Most of these metabolites were also detected in the blood samples, although mostly with lower abundances. The main in vivo metabolites were built by hydroxylation combined with further metabolic reactions such as O-methylation or N-deacylation. The controlled oral self-administration allowed us to confirm the usefulness of these metabolites as proof of intake in abstinence control. The detection of metabolites is often crucial to documenting consumption, especially when small traces of the parent drug can be found in real samples. The in vitro assay proved to be suitable for the prediction of valid biomarkers of novel synthetic opioid intake

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

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

Development of enantioselective high-performance liquid chromatography-tandem mass spectrometry method for quantitative determination of methylone and some of its metabolites in oral fluid

In the present study an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the first time for quantitative determination of the recreational drug of abuse methylone and its major metabolites in oral fluid. The simultaneous chemo- and enantioseparation of methylone and its major metabolites was performed on a polysaccharide-based chiral column based on amylose tris(5-chloro-3-methylphenylcarbamate) as chiral selector (Lux i-Amylose-3) with methanol containing 0.4 % (v/ v) aqueous ammonium hydroxide as mobile phase. The time required for enantioselective analysis of methylone and its 2 major metabolites was 15 min.This method was fully validated following the Organization of Scientific Area Committees (OSAC) for Forensic Science guidelines. This method was applied for the enantioselective determination of methylone and its metabolites in oral fluid and enantioselectivity in metabolism and pharmacokinetic of the parent compound and metabolites was observed.While the first enantiomer of methylone was found at higher concentration, both metabolites shown greater concentration for the second enantiomer. The results revealed that MET undergoes an enantioselective biotransformation to its metabolites HMMC and MDC, with S-(-)-MET more rapidly metabolized and eliminated from the body

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

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

Methylone and MDMA Pharmacokinetics Following Controlled Administration in Humans

The aim of this study is to define, for the first time, human methylone and HMMC plasma pharmacokinetics following controlled administration of 50-200 mg methylone to 12 male volunteers. A new LC-MS/MS method was validated to quantify methylone, MDMA, and their metabolites in plasma. The study was a randomized, cross-over, double-blinded and placebo-controlled study, with a total of 468 plasma samples collected. First, 10 µL of MDMA-d5, MDA-d5 and methylone-d3 internal standards were added to 100 µL of plasma. Two mL of chloroform and ethyl acetate 9:1 (v/v) were then added, mixed well and centrifuged. The supernatant was fortified with 0.1 mL acidified methanol and evaporated under nitrogen. Samples were reconstituted with a mobile phase and injected into the LC-MS/MS instrument. The method was fully validated according to OSAC guidelines (USA). Methylone plasma concentrations increased in a dose-proportional manner, as demonstrated by the increasing maximum concentration (Cmax) and area under the curve of concentrations (AUC). Methylone Cmax values were reported as 153, 304, 355 and 604 ng/mL, AUC0-24 values were reported as 1042.8, 2441.2, 3524.4 and 5067.9 h·ng/mL and T1/2 values as 5.8, 6.4, 6.9 and 6.4 h following the 50, 100, 150 and 200 mg doses, respectively. Methylone exhibited rapid kinetics with a Tmax of 1.5 h for the 50 mg dose and 2 h approximately after all the other doses. HMMC exhibited faster kinetics compared to methylone, with a Cmax value that was 10-14-fold lower and an AUC0-24 value that was 21-29-fold lower. Methylone pharmacokinetics was linear across 50-200 mg oral doses in humans, unlike the previously described non-linear oral MDMA pharmacokinetics. An LC-MS/MS method for the quantification of methylone, MDMA and their metabolites in human plasma was achieved. Methylone exhibited linear pharmacokinetics in humans with oral doses of 50-200 m