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For centuries, ‘khat sessions’ have played a key role in the social and cultural traditions among several communities around Saudi Arabia and most East African countries. The identification of cathinone as the main psychoactive compound of khat leaves, exhibiting amphetamine-like pharmacological properties, resulted in the synthesis of several derivatives structurally similar to this so-called natural amphetamine. Synthetic cathinones were primarily developed for therapeutic purposes, but promptly started being misused and extensively abused for their euphoric effects. In the mid-2000’s, synthetic cathinones emerged in the recreational drug markets as legal alternatives (‘legal highs’) to amphetamine, ‘ecstasyʼ, or cocaine. Currently, they are sold as ‘bath salts’ or ‘plant foodʼ, under ambiguous labels lacking information about their true contents. Cathinone derivatives are conveniently available online or at ‘smartshops’ and are much more affordable than the traditional illicit drugs. Despite the scarcity of scientific data on these ‘legal highs’, synthetic cathinones use became an increasingly popular practice worldwide. Additionally, criminalization of these derivatives is often useless since for each specific substance that gets legally controlled, one or more structurally modified analogs are introduced into the legal market. Chemically, these substances are structurally related to amphetamine. For this reason, cathinone derivatives share with this drug both central nervous system stimulating and sympathomimetic features. Reports of intoxication and deaths related to the use of ‘bath salts’ have been frequently described over the last years, and several attempts to apply a legislative control on synthetic cathinones have been made. However, further research on their pharmacological and toxicological properties is fully required in order to access the actual potential harm of synthetic cathinones to general public health. The present work provides a review on khat and synthetic cathinones, concerning their historical background, prevalence, patterns of use, legal status, chemistry, pharmacokinetics, pharmacodynamics, and their physiological and toxicological effects on animals and humans.

25I-NBOMe, a new psychoactive substance, is a potent 5-HT2A receptor agonist with strong hallucinogenic potential. Recently, it was involved in several fatal and non-fatal intoxication cases. The aim of the present work was to study its phase I and II metabolism and its detectability in urine screening approaches. After application of 25I-NBOMe to male Wistar rats, urine was collected over 24 h. The phase I and II metabolites were identified by LC-HR-MS/MS in urine after suitable workup. For the detectability studies, standard urine screening approaches (SUSA) by GC-MS, LC-MS(n), and LC-HR-MS/MS were applied to rat and also to authentic human urine samples submitted for toxicological analysis. Finally, an initial CYP activity screening was performed to identify CYP isoenzymes involved in the major metabolic steps. 25I-NBOMe was mainly metabolized by O-demethylation, O,O-bis-demethylation, hydroxylation, and combinations of these reactions as well as by glucuronidation and sulfation of the main phase I metabolites. All in all, 68 metabolites could be identified. Intake of 25I-NBOMe was detectable mainly via its metabolites by both LC-MS approaches, but not by the GC-MS SUSA. Initial CYP activity screening revealed the involvement of CYP1A2 and CYP3A4 in hydroxylation and CYP2C9 and CYP2C19 in O-demethylation. The presented study demonstrated that 25I-NBOMe was extensively metabolized and could be detected only by the LC-MS screening approaches. Since CYP2C9 and CYP3A4 are involved in initial metabolic steps, drug-drug interactions might occur in certain constellations.

Compounds derived from known drugs are usually brought on the new psychoactive substance (NPS) market without any previous toxicological risk assessment. The European Union Drugs Agency issued an EU early notification for 3,4-methylenedioxyphenmetrazine (MDPM) in 2024. It is structurally related to the stimulants amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), and phenmetrazine and expected to have similar effects. So far, no scientific reports are available describing its toxicokinetic and analytical profile. This study aimed to provide such data to allow a thorough risk assessment and to ease its analytical detectability in forensic and clinical toxicology and doping control. Data reported include the in vitro plasma protein binding, the in vitro half-life and in vitro metabolism of MDPM by human liver microsomes and S9 fraction (pHLS9) and by HepaRG cells. A monooxygenase mapping and the in vitro cytochrome P450 inhibition of MDPM was elucidated. Results showed that HepaRG cells and pHLS9 formed the same MDPM metabolites via demethylenation and O -methylation and that MDPM has a low plasma protein binding and is a low-turnover drug. Monooxygenase mapping revealed that the demethylenation was exclusively CYP2D6-mediated. MDPM showed strong inhibition of CYP2D6 and moderate inhibition of CYP1A2 and CYP3A4. Polymorphisms or the simultaneous intake of substances that are also CYP2D6 substrates can have a considerable impact on the toxicity of MDPM. Based on in vitro data, the demethylenyl-methyl metabolite of MDPM and the parent compound are recommended as analytical urine screening targets.

New groups of synthetic “designer drugs” have increased in popularity over the past several years. These products mimic the euphoric effects of other well-known illicit drugs but are advertised as “legal” highs and are sold over the internet, at raves and night clubs, and in head shops. The 2C series drugs are ring-substituted phenethylamines that belong to a group of designer agents similar in structure to 3,4-methylenedioxy- N -methylamphetamine (MDMA, Ecstasy). Understanding the pharmacology and toxicology of these agents is essential in order to provide the best medical care for these patients. This review focuses on the pharmacology, pharmacokinetics, clinical effects, and treatment of 2C drug intoxication based on available published literature. Multiple names under which 2C drugs are sold were identified and tabulated. Common features identified in patients intoxicated with 2Cs included hallucinations, agitation, aggression, violence, dysphoria, hypertension, tachycardia, seizures, and hyperthermia. Patients may exhibit sympathomimetic symptoms or symptoms consistent with serotonin toxicity, but an excited delirium presentation seems to be consistent amongst deaths attributed to 2C drugs; at least five deaths have been reported in the literature in patients intoxicated with 2C drugs. 2C drugs are a group of designer intoxicants, many of which are marketed as legal, but may carry risks that consumers are unaware of. These drugs may be characterized by either serotonergic toxicity or a sympathomimetic toxidrome, but a presentation consistent with excited delirium is consistent amongst the reported 2C-related deaths. Treatment of 2C intoxication is primarily supportive, but immediate action is required in the context of excited delirium, hyperthermia, and seizure activity.

Designer drugs like 2C-I and 25I-NBOMe have emerged as potent psychoactive substances, with several reports linking their consumption to severe poisoning and fatalities. However, there is limited information on their toxicity, particularly in in vivo models. In this manuscript, we evaluate the survival, developmental, and reproductive impact of these designer drugs on the model organism Caenorhabditis elegans (C. elegans). For this purpose, adult worms synchronized at the L1 stage were exposed to growing concentrations of 2C-I and 25I-NBOMe. The animal survival rate and the putative effects of the drugs on C. elegans development and reproductive behavior were assessed after 24 h of exposure. A concentration-dependent decrease in animal survival was observed. 25I-NBOMe was approximately six times more toxic than 2C-I (LC50 values—1.368 mM for 2C-I and 0.236 mM for 25I-NBOMe). Furthermore, sublethal concentrations of both drugs delayed animal development and reduced the total progeny but not its survival. Overall, these findings underscore the developmental and reproductive risks associated with exposure to 2C-I and 25I-NBOMe, even at sublethal concentrations.

Synthetic cathinones have emerged in recreational drug markets as legal alternatives for classical amphetamines. Though currently banned in several countries, 3,4-methylenedioxypyrovalerone (MDPV) is one of the most commonly abused cathinone derivatives worldwide. We have recently reported the potential of MDPV to induce hepatocellular damage, but the underlying mechanisms responsible for such toxicity remain to be elucidated. Similar to amphetamines, a prominent toxic effect of acute intoxications by MDPV is hyperthermia. Therefore, the present in vitro study aimed to provide insights into cellular mechanisms involved in MDPV-induced hepatotoxicity and also evaluate the contribution of hyperthermia to the observed toxic effects. Primary cultures of rat hepatocytes were exposed to 0.2–1.6 mM MDPV for 48 h, at 37 or 40.5 °C, simulating the rise in body temperature that follows MDPV intake. Cell viability was measured through the MTT reduction and LDH leakage assays. Oxidative stress endpoints and cell death pathways were evaluated, namely the production of reactive oxygen and nitrogen species (ROS and RNS), intracellular levels of reduced (GSH) and oxidized (GSSG) glutathione, adenosine triphosphate (ATP) and free calcium (Ca 2+ ), as well as the activities of caspases 3, 8 and 9, and nuclear morphological changes with Hoechst 33342/PI double staining. At 37 °C, MDPV induced a concentration-dependent loss of cell viability that was accompanied by GSH depletion, as one of the first signs of toxicity, observed already at low concentrations of MDPV, with negligible changes on GSSG levels, followed by accumulation of ROS and RNS, depletion of ATP contents and increases in intracellular Ca 2+ concentrations. Additionally, activation of caspases 3, 8, and 9 and apoptotic nuclear morphological changes were found in primary rat hepatocytes exposed to MDPV, indicating that this cathinone derivative activates both intrinsic and extrinsic apoptotic death pathways. The cytotoxic potential of MDPV and all the studied endpoints were markedly aggravated under hyperthermic conditions (40.5 °C). In conclusion, these data suggest that MDPV toxicity in primary rat hepatocytes is mediated by oxidative stress, subsequent to GSH depletion and increased ROS and RNS accumulation, mitochondrial dysfunction, and impairment of Ca 2+ homeostasis. Furthermore, the rise in body temperature subsequent to MDPV abuse greatly exacerbates its hepatotoxic potential.

Introduction 2C designer drugs have been in use since the 1970s, but new drugs continue to develop from substitutions to the base phenethylamine structure. This creates new clinical profiles and difficulty with laboratory confirmation. 2-(4-Iodo-2,5-dimethoxyphenyl)- N -[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) is a relatively new 2C drug that is more potent than structural 2C analogs; exposure reports are rare. Testing for 2C drugs is developing; specific testing for new analogs such as 25I-NBOMe is a challenge. These drugs do not reliably trigger a positive result on rapid drug immunoassays. Additionally, most facilities with confirmatory testing capabilities will not identify 25I-NBOMe; methods for detecting 25I-NBOMe in biological samples have not been clearly described nor have optimal metabolic targets for detecting 25I-NBOMe ingestion. Case Report An 18-year-old female presented following use of 25I-NBOMe. She had an isolated brief seizure, tachycardia, hypertension, agitation, and confusion. She improved with intravenously administered fluids and benzodiazepines and was discharged 7 h postingestion. Urine was analyzed using quantitative LC-MS/MS methodology for 25I-NBOMe, 2-(4-chloro-2,5-dimethoxyphenyl)- N -[(2-methoxyphenyl)-methyl]ethanamine (25C-NBOMe), and 2-(2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25H-NBOMe). 25I-NBOMe was found at a concentration of 7.5 ng/mL, and 25H-NBOMe was detected as well. Additional testing was pursued to characterize the metabolism of 25I-NBOMe; the sample was reanalyzed with UPLC–time-of-flight mass spectrometry to identify excreted metabolites. The sample was additionally analyzed for the presence of 2,5-dimethoxy-4-iodophenethylamine (2C-I), 4-bromo-2,5-dimethoxyphenethylamine (2C-B), and 1-(2,5-dimethoxy-4-ethylphenyl)-2-aminoethane (2C-E). Discussion This is a report of a patient presenting following exposure to 25I-NBOMe, a dangerous member of the evolving 2C drug class. The exposure was confirmed in a unique manner that could prove helpful in guiding further patient analysis and laboratory studies.

In vitro cytochrome P450 (CYP) inhibition assays are common approaches for testing the inhibition potential of drugs for predicting potential interactions. In contrast to marketed medicaments, drugs of abuse, particularly the so-called novel psychoactive substances, were not tested before distribution and consumption. Therefore, the inhibition potential of methylenedioxy-derived designer drugs (MDD) of different drug classes such as aminoindanes, amphetamines, benzofurans, cathinones, piperazines, pyrrolidinophenones, and tryptamines should be elucidated. The FDA-preferred test substrates, split in two cocktails, were incubated with pooled human liver microsomes and analysed after protein precipitation using LC-high-resolution-MS/MS. IC 50 values were determined of MDD showing more than 50 % inhibition in the prescreening. Values were calculated by plotting the relative metabolite concentration formed over the logarithm of the inhibitor concentration. All MDD showed inhibition against CYP2D6 activity and most of them in the range of the clinically relevant CYP2D6 inhibitors quinidine and fluoxetine. In addition, the beta -keto compounds showed inhibition of the activity of CYP2B6, 5,6-MD-DALT of CYP1A2 and CYP3A, and MDAI of CYP2A6, all in the range of clinically relevant inhibitors. In summary, all MDD showed inhibition of the activity of CYP2D6, six of CYP1A2, three of CYP2A6, 13 of CYP2B6, two of CYP2C9, six of CYP2C19, one of CYP2E1, and six of CYP3A. These results showed that the CYP inhibition by MDD might be clinically relevant, but further studies are needed for final conclusions.

New phenylethylamine derivatives are among the most commonly abused new psychoactive substances. They are synthesized and marketed in lieu of classical amphetaminic stimulants, with no previous safety testing. Our study aimed to determine the in vitro hepatotoxicity of two benzofurans [6-(2-aminopropyl)benzofuran (6-APB) and 5-(2-aminopropyl)benzofuran (5-APB)] that have been misused as ‘legal highs’. Cellular viability was assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, following 24-h drug exposure of human hepatoma HepaRG cells (EC50 2.62 mM 5-APB; 6.02 mM 6-APB), HepG2 cells (EC50 3.79 mM 5-APB; 8.18 mM 6-APB) and primary rat hepatocytes (EC50 964 μM 5-APB; 1.94 mM 6-APB). Co-incubation of primary hepatocytes, the most sensitive in vitro model, with CYP450 inhibitors revealed a role of metabolism, in particular by CYP3A4, in the toxic effects of both benzofurans. Also, 6-APB and 5-APB concentration-dependently enhanced oxidative stress (significantly increased reactive species and oxidized glutathione, and decreased reduced glutathione levels) and unsettled mitochondrial homeostasis, with disruption of mitochondrial membrane potential and decline of intracellular ATP. Evaluation of cell death mechanisms showed increased caspase-8, -9, and -3 activation, and nuclear morphological changes consistent with apoptosis; at concentrations higher than 2 mM, however, necrosis prevailed. Concentration-dependent formation of acidic vesicular organelles typical of autophagy was also observed for both drugs. Overall, 5-APB displayed higher hepatotoxicity than its 6-isomer. Our findings provide new insights into the potential hepatotoxicity of these so-called ‘safe drugs’ and highlight the putative risks associated with their use as psychostimulants.Key pointsThe isomer 5-APB is more hepatotoxic than 6-APB.At biologically relevant concentrations, CYP2D6 and 3A4 contribute to the toxification of psychoactive 5-APB and 6-APB. CYP2E1 seems also implicated in 5-APB bioactivation.Both benzofury concentration-dependently increase oxidative species, disturb mitochondrial potential homeostasis and energetic levels.5-APB and 6-APB stimulate autophagy and apoptosis at low in vitro concentrations.

The piperazine derivatives most frequently consumed for recreational purposes are 1-benzylpiperazine, 1-(3,4-methylenedioxybenzyl) piperazine, 1-(3-trifluoromethylphenyl) piperazine and 1-(4-methoxyphenyl) piperazine. Generally, they are consumed as capsules, tablets or pills but also in powder or liquid forms. Currently, the precise mechanism by which piperazine designer drugs induce hepatotoxicity and whether they act by a common pathway is unclear. To answer this question, we performed a gene array study with rat hepatocytes incubated with the four designer drugs. Non-cytotoxic concentrations were chosen that neither induce a decrease in reduced glutathione or ATP depletion. Analysis of the gene array data showed a large overlap of gene expression alterations induced by the four drugs. This ‘piperazine designer drug consensus signature’ included 101 up-regulated and 309 down-regulated probe sets ( p  < 0.05; FDR adjusted). In the up-regulated genes, GO groups of cholesterol biosynthesis represented a dominant overrepresented motif. Key enzymes of cholesterol biosynthesis up-regulated by all four piperazine drugs include sterol C4-methyloxidase, isopentyl-diphosphate-Δ-isomerase, Cyp51A1, squalene epoxidase and farnesyl diphosphate synthase. Additionally, glycoprotein transmembrane nmb, which participates in cell adhesion processes, and fatty acid desaturase 1, an enzyme that regulates unsaturation of fatty acids, were also up-regulated by the four piperazine designer drugs. Regarding the down-regulated probe sets, only one gene was common to all four piperazine derivatives, the betaine-homocysteine- S -methyltransferase 2. Analysis of transcription factor binding sites of the ‘piperazine designer drug consensus signature’ identified the sterol regulatory element binding protein (SREBP-1) as strongly overrepresented in the up-regulated genes. SREBP transcription factors are known to regulate multiple genes of cholesterol metabolism. In conclusion, the present study shows that piperazine designer drugs act by up-regulating key enzymes of cholesterol biosynthesis which is likely to increase the risk of phospholipidosis and steatosis.