3-CMC, 4-CMC, and 4-BMC Human Metabolic Profiling: New Major Pathways to Document Consumption of Methcathinone Analogues?

Synthetic cathinones represent one of the largest and most abused new psychoactive substance classes, and have been involved in numerous intoxications and fatalities worldwide. Methcathinone analogues like 3-methylmethcathinone (3-MMC), 3-chloromethcathinone (3-CMC), and 4-CMC currently constitute most of synthetic cathinone seizures in Europe. Documenting their consumption in clinical/forensic casework is therefore essential to tackle this trend. Targeting metabolite markers is a go-to to document consumption in analytical toxicology, and metabolite profiling is crucial to support investigations. We sought to identify 3-CMC, 4-CMC, and 4-bromomethcathinone (4-BMC) human metabolites. The substances were incubated with human hepatocytes; incubates were screened by liquid chromatography-high-resolution tandem mass spectrometry and data were mined with Compound Discoverer (Themo Scientific). 3-CMC-positive blood, urine, and oral fluid and 4-CMC-positive urine and saliva from clinical/forensic casework were analyzed. Analyses were supported by metabolite predictions with GLORYx freeware. Twelve, ten, and ten metabolites were identified for 3-CMC, 4-CMC, and 4-BMC, respectively, with similar transformations occurring for the three cathinones. Major reactions included ketoreduction and N-demethylation. Surprisingly, predominant metabolites were produced by combination of N-demethylation and ω-carboxylation (main metabolite in 3-CMC-positive urine), and combination of ß-ketoreduction, oxidative deamination, and O-glucuronidation (main metabolite in 4-CMC-positive urine). These latter metabolites were detected in negative-ionization mode only and their non-conjugated form was not detected after glucuronide hydrolysis; this metabolic pathway was never reported for any methcathinone analogue susceptible to undergo the same transformations. These results support the need for comprehensive screening strategies in metabolite identification studies, to avoid overlooking significant metabolites and major markers of consumption.

Influences of hair dyeing on the distribution shapes of zolpidem and methoxyphenamine in hair.

In order to investigate the influences of hair dyeing on the distribution shapes of drugs in hair, different hair dyeing processes ("semi-permanent coloring without bleaching" and "permanent coloring with bleaching") were performed in vitro on black hair specimens collected from two subjects (Asians) who took a single dose of zolpidem (ZP, 10 mg of ZP tartrate) or methoxyphenamine (MOP, 50 mg of MOP hydrochloride). Under the following three different dyeing conditions, (1) semi-permanent coloring, (2) permanent coloring (once), (3) permanent coloring (twice), drug distributions in single hair specimens were investigated using a 2-mm segmental analysis by liquid chromatography-tandem mass spectrometry. Distribution shapes of drugs changed significantly only under the permanent coloring (twice) condition, resulting in reduced peak concentration and extended distribution width. There was, however, no significant difference in the amounts of drugs in hair between non-treated and dyed specimens, suggesting the drugs hardly leaked out of hair or were only slightly degraded during dyeing. In addition, while assuming contact with aqueous environment such as daily hair washing after dyeing, dyed hair specimens were individually immersed in ultrapure water for 20 hours, then the outflow of drugs in ultrapure water as well as the distribution shapes of drugs remaining in hair were determined. The drug outflow after permanent coloring (once and twice) was significantly larger than those after semi-permanent coloring, and the outflow ratios, [outflow]/([outflow] + [amount remaining in hair]), ranged over 9.8-24% (n = 3) for ZP and 68-71% (n = 3) for MOP after permanent coloring (once), and 54-72% (n = 3) for ZP and 86-91% (n = 3) for MOP after permanent coloring (twice). The distribution shapes of drugs after 20 h of immersion tended to flatten as outflow ratios increased, resulting in no change in the shapes after semi-permanent coloring, and complete collapse of their shapes after permanent coloring (twice). Thus, the present results indicated that hair dyeing involving bleaching and subsequent contact with aqueous environment after dyeing could significantly influence distribution shapes of drugs in hair.

Metaphedrone (3-Methylmethcathinone): Pharmacological, Clinical, and Toxicological Profile.

Introduction: Synthetic cathinones are a group of novel psychoactive substances used as an alternative to classical recreational drugs. As a result of legal prohibitions on older generations of these compounds, new formulations appeared on the drug market. One of them is metaphedrone (3-methylmethcathinone, 3-MMC), a structural isomer of 4-methylmethcathinone and a psychostimulant drug. Metaphedrone became popular in a large number of countries in a short period of time. The aim: The collection, analysis, and review of relevant research on the subject of metaphedrone in order to present information about the pharmacological, clinical, and toxicological profile of this compound. An assessment of the significance and role of metaphedrone in consumption patterns of novel psychoactive substances among recreational drug users. Methodology: By using search engines like Google Scholar and PubMed, the relevant literature on metaphedrone was looked for and analyzed. The search was not limited to a specific period of time. All information regarding the compound of interest was analyzed and presented. Key results and discussion: All novel psychoactive substances are abused due to their pronounced stimulatory, hallucinogenic, dissociative, and euphoric and/or relaxing characteristics. Users of 3-methylmethcathinone usually opt for this substance for recreational purposes and/or sexual stimulation. Metaphedrone has the potential to cause a psychological dependence to the users. It was determined in relevant studies that most users are from 17 to 50 years of age. Older users usually administer metaphedrone intravenously, while younger ones usually choose snorting and oral ingestion of the drug. In Serbia, metaphedrone is a legally controlled substance. The pharmacodynamic properties make metaphedrone similar to classical recreational drugs. The method of administration, mainly repeated administration in a single session, could be explained using the pharmacokinetic profile of the drug. The most reported symptoms of intoxication were those of a sympathomimetic nature, such as tachycardia, chest pain, hypertension, diaphoresis, and agitation. Most intoxications and fatal outcomes occurred to users who combined several psychoactive substances. The correlation between measured blood concentrations of the drug and outcomes of intoxication was not found. The mechanisms of metaphedrone's toxicity are not fully understood. Conclusions: There is an increasing trend of abuse of metaphedrone among recreational drugs users. Future studies should focus on pharmacological and toxicological effects of metaphedrone on animals and humans.

The psychomotor, reinforcing, and discriminative stimulus effects of synthetic cathinone mexedrone in male mice and rats.

The chronic use of the novel synthetic cathinone mexedrone, like other psychoactive drugs, can be considered addictive, with a high potential for abuse and the ability to cause psychological dependence in certain users. However, little is known about the neurobehavioral effects of mexedrone in association with its potential for abuse. We investigated the abuse potential for mexedrone abuse through multiple behavioral tests. In addition, serotonin transporter (SERT) levels were measured in the synaptosome of the dorsal striatum, and serotonin (5-HT) levels were measured in the dorsal striatum of acute mexedreone (50 mg/kg)-treated mice. To clarify the neuropharmacological mechanisms underlying the locomotor response of mexedrone, the 5-HT2A receptor antagonist M100907 (0.5 or 1.0 mg/kg) was administered prior to the acute injection of mexedrone in the locomotor activity experiment in mice. Mexedrone (10-50 mg/kg) produced a significant place preference in mice and mexedrone (0.1-0.5 mg/kg/infusion) maintained self-administration behavior in rats in a dose-dependent manner. In the drug discrimination experiment, mexedrone (5.6-32 mg/kg) was fully substituted for the discriminative stimulus effects of cocaine in rats. Mexedrone increased locomotor activity, and these effects were reversed by pretreatment with M100907. Acute mexedrone significantly increased c-Fos expression in the dorsal striatum and decreased SERT levels in the synaptosome of the dorsal striatum of mice, resulting in an elevation of 5-HT levels. Taken together, our results provide the possibility that mexedrone has abuse potential, which might be mediated, at least in part, by the activation of the serotonergic system in the dorsal striatum.

Understanding methiopropamine, a new psychoactive substance: an in-depth review on its chemistry, pharmacology and implications to human health.

Methiopropamine or 1-(thiophen-2-yl)-2-methylaminopropane (MPA) is a thiophene ring-based structural analogue of methamphetamine, first synthesized in 1942 but become popular when it started to be available for purchase on websites selling 'legal highs' since 2010. While it is legally controlled in many countries, it remains readily accessible and frequently encountered in recreational settings. The growing prevalence of MPA use results in new therapeutic challenges. Relatively few studies have focused on its pharmacodynamics and pharmacokinetics, making it important to better understand its potential risks and harmful effects in humans in terms of its toxicity. This review provides a comprehensive profiling of MPA toxicological properties, including its chemical properties, analytical methods, prevalence, patterns of use, and legal status. Additionally, it discusses the drug's effects on the central nervous system, its potential for addiction, and its adverse physical and mental health effects. Improving the understanding of safety aspects of MPA and how it imposes health threats for public health will guide the development of therapeutic approach of its intoxication and guide the authorities in deciding its legal status.

Cardiotoxicity After Synthetic Cathinone Use; Two Cases, A Case Series and Scoping Review.

The cardiotoxic effects of synthetic cathinones remain largely unknown. In this study, we present two cases, a case series and a scoping review, to explore synthetic cathinone associated cardiotoxicity. Case 1 involved a 28-year-old male with non-ST-elevation myocardial infarction after ingesting a substance containing 4-methylmethcathinone (4-MMC), 3-methylmethcathinon (3-MMC), and methcathinone. Case 2 involved a 49-year-old male with ventricular fibrillation after 4-methylmethcathinone ingestion, who was diagnosed with severe three-vessel disease. A retrospective analysis was performed on self-reported synthetic cathinone poisonings reported to the Dutch Poisons Information Centre from 2012 to 2022. A total of 222 mono-intoxications with cardiotoxicity were included, mostly involving 3-methylmethcathinon (63%). Often tachycardia, hypertension, palpitations, and chest pain were reported. A comprehensive literature search was performed on PubMed to identify the studies reporting cardiac arrest, myocardial infarction, cardiac inflammation, cardiomyopathy, and life-threatening arrhythmias following synthetic cathinone use. A total of 30 articles reporting 40 cases were included. The reported complications included cardiac arrest (n = 28), ventricular tachycardia (n = 4), supraventricular tachycardia (n = 1), ST-elevation myocardial infarction (n = 2), non-ST-elevation myocardial infarction (n = 2), cardiomyopathy (n = 1), and myocarditis (n = 2). A total of ten different associated synthetic cathinones were identified. Cardiac arrest, myocardial infarction, and ventricular arrhythmias have been reported following the use of synthetic cathinones, underscoring the importance of obtaining a detailed recreational drug use history from patients presenting with syncope, chest pain, or palpitations.

Health characteristics associated with chemsex among men who have sex with men: Results from a cross-sectional clinic survey in Norway.

BACKGROUND: Chemsex typically involves drugs such as GHB/GBL, crystal meth and mephedrone, and is increasingly common among MSM. The behaviour has been found to be associated with sexually transmitted infections (STIs) and mental health problems. We aimed to assess the extent of chemsex engagement and associations with different aspects of health, among MSM attending a free specialist walk-in clinic for STIs in Oslo, Norway. METHODS: Anonymous cross-sectional survey data was collected from June to October 2016. Differences in STI health (chlamydia, gonorrhoea, syphilis, HIV diagnoses), mental health (depression/anxiety) and internalised homonegativity between MSM using and not using GHB/GBL, crystal meth, mephedrone, cocaine or ketamine with sex in the last year were assessed descriptively and in a multivariate logistic regression model. The predictors were number of self-reported chlamydia, gonorrhoea or syphilis diagnoses, HIV diagnosis, depression/anxiety, and degree of internalised homonegativity. We adjusted for age, education level and having lived abroad. RESULTS: Of the 518 MSM respondents, 17% reported sexualised use of either GHB/GBL, crystal meth, mephedrone, cocaine or ketamine in the last year (chemsex). We found significant positive associations between chemsex and self-reported HIV diagnoses (adjusted odds ratio [aOR] = 3.26, 95%CI = 1.37-7.76), number of reported chlamydia, gonorrhoea or syphilis diagnoses in the last year (aOR = 1.63, 95%CI = 1.18-2.12), having lived more than one year abroad (aOR = 2.10, 95%CI = 1.20-3.65), but no significant association with depression/anxiety (aOR = 1.02, 95%CI = 0.53-1.93), nor internalised homonegativity (aOR = 0.62, 95%CI = 0.33-1.19). CONCLUSION: Chemsex engagement in Norway is relatively low compared to findings from STI clinics in other European countries, and GHB/GBL and cocaine the two most commonly used drugs with sex. Chemsex was more common among MSM having lived more than one year abroad, reporting HIV diagnoses and a higher number of either chlamydia, gonorrhoea or syphilis diagnoses in the last year. Health care providers need to be made aware of chemsex as a behavioural phenomenon among MSM, and special care should be afforded to MSM living with HIV and being diagnosed with STIs.

Methylone pre-exposure differentially impacts the aversive effects of MDPV and MDMA in male and female Sprague-Dawley rats: Implications for abuse vulnerability.

BACKGROUND: Polydrug use is well documented in synthetic cathinone users, although the consequences of such use are not well characterized. In pre-clinical research, a pre-exposure to a drug has been reported to attenuate the aversive effects of other drugs which has implications for their abuse potential. The goal of the present study was to investigate the impact of pre-exposure to the synthetic cathinone methylone on the aversive effects of MDPV and MDMA. METHOD: Male and female Sprague-Dawley rats were exposed to 10 mg/kg of methylone every 4th day (for a total of five injections) prior to taste avoidance training with 1.8 mg/kg of MDPV or 1 mg/kg of MDMA. RESULTS: MDPV and MDMA induced taste avoidance in males and females (all p's < 0.05). In males, methylone pre-exposure attenuated the avoidance induced by MDPV and MDMA (all p's < 0.05) with the attenuation greater with MDPV. In females, methylone pre-exposure attenuated avoidance induced by MDPV (all p's < 0.05), but it had no effect on those induced by MDMA (all p's > 0.05). CONCLUSIONS: The effects of exposure to methylone on taste avoidance induced by MDPV and MDMA were drug- (MDPV > MDMA) and sex- (MDMA only in males) dependent. The attenuating effects of methylone pre-exposure on MDPV and MDMA were discussed in terms of their shared neurochemical action. These findings suggest that a history of methylone use may reduce the aversive effects of MDPV and MDMA which may have implications for polydrug use involving the synthetic cathinones.

Binge-like mephedrone treatment induces memory impairment concomitant with brain kynurenic acid reduction in mice.

While mephedrone (4-methylmethcathinone), a synthetic cathinone derivative, is widely abused by adolescents and young adults, the knowledge about its long-term effects on memory processes is limited. Kynurenic acid (KYNA) is a neuroactive metabolite of the kynurenine pathway of tryptophan degradation. KYNA is considered an important endogenous modulator influencing physiological and pathological processes, including learning and memory processes. The aim of this study was to determine whether (A) binge-like mephedrone administration (10.0 and 30.0 mg/kg, intraperitoneally, in 4 doses separated by 2 h) induces memory impairments, assessed 2, 8 and 15 days after mephedrone cessation in the passive avoidance test in mice, and whether (B) KYNA is involved in these memory processes. To clarify the role of KYNA in the mephedrone effects, its production in the murine brain in vivo, and in cortical slices in vitro, as well as the activities of kynurenine aminotransferases (KATs) I and II were assessed. Furthermore, cell line experiments were conducted to investigate the effects of mephedrone on normal human brain cells. Our results showed memory impairments 8 and 15 days after binge-like mephedrone administration. At the same time, reduction in the KYNA level in the murine brain was noted. In vitro studies showed no effect of mephedrone on the production of KYNA in cortical slices or on the activity of the KAT I and II enzymes. Finally, exposure of normal cells to mephedrone in vitro resulted in a modest reduction of cell viability and proliferation.

3-Methylmethcathinone (3-MMC) Poisonings: Acute Clinical Toxicity and Time Trend Between 2013 and 2021 in the Netherlands.

STUDY OBJECTIVE: The synthetic cathinone 3-methylmethcathinone (3-MMC, or metaphedrone) has recently gained popularity. We studied the numbers of 3-MMC poisonings over time and the clinical effects following poisonings with 3-MMC. METHODS: We performed a retrospective study on the numbers of self-reported 3-MMC poisonings to the Dutch Poisons Information Center (DPIC) from 2013 to June 2021. For poisonings reporting 3-MMC only, the symptoms were extracted and the Poisoning Severity Score (PSS) was determined. From 2016 to June 2019, a prospective cohort study on poisonings reporting only 3-MMC was performed, in which details on the clinical courses were collected through telephone interviews. RESULTS: From 2013 to June 2021, the DPIC was consulted on 184 3-MMC poisonings. The number of poisonings increased from 1 in 2013 to 70 in the first half of 2021. In 84 poisonings with only 3-MMC (46%), sympathomimetic symptoms were commonly reported, including tachycardia (n=29, 35%), hypertension (n=17, 20%), and agitation (n=16, 19%). The initial PSS was usually minor (n=37, 44%) to moderate (n=39, 46%). Five patients (6%) experienced severe effects, including 3 patients experienced severe hypertension (systolic blood pressure >180 mmHg; n=3) and nonfatal cardiac arrest (n=1). Sympathomimetic symptoms (n=8) were also reported in the prospective cohort study. The percentage of moderate poisonings increased (n=6, 75%), and 1 (13%) severe poisoning was observed. Analytical confirmation of 3-MMC exposure was performed in 2 cases. CONCLUSION: The number of 3-MMC poisonings reported to the DPIC has increased over time. Most poisonings with 3-MMC resulted in moderate toxicity and involved sympathomimetic effects, while severe effects were observed in 5 cases.

Determination of famprofazone, amphetamine and methamphetamine in liver samples using enzymatic cell dispersion and SPE-LC-ESI-MS.

The use of small amounts of sample presents advantages in chromatographic analyses that have made this a current trend following the development of increasingly sensitive analytical techniques. Biological sample preparation methods, especially for rigid or semi-rigid matrices, are also under constant development, focusing on a more efficient extraction and in obtaining cleaner residues for analysis. In this context, the aim of this study was to present a validated a liquid chromatography-mass spectrometry (LC-MS) method for the quantification of famprofazone and its metabolites, methamphetamine and amphetamine in liver, using enzymatic cell dispersion promoted by collagenase, followed by protein precipitation and solid phase extraction (SPE) for sample extraction, concentration and clean-up. Potentially relevant variables for enzymatic cell dispersion concerning efficiency, such as enzyme concentration, temperature, buffering, agitation, and mechanical effect of stainless-steel spheres were assessed. Recovery evaluations were performed during the optimization of each step to ensure minimal loss of analytes. Linearity, the limit of detection (LOD) and limit of quantification (LOQ), stability, carryover, matrix effect, precision and bias were evaluated using fortified blank samples. An authentic sample was obtained from a controlled daily oral administration of 200 mg famprofazone to pigs for five days. The procedure was optimized for 500 mg of liver tissue, obtaining 99.9 ± 9.3% of digested collagen and 90.2 ± 1.7% of dispersed cells, without the tissue losses that usually ensue during crushing or grinding processes. Precision (CV%) was ≤ 10% and bias was ≤ 13% for all analytes. The LOQ was 5 ng/g for all analytes. The mean famprofazone concentration was 9.3 ± 0.53 ng/g, and mean metabolite concentrations were 16.7 ± 1.67 and 24.3 ± 1.36 ng/g for amphetamine and methamphetamine, respectively.

Pharmacological characterization of 3,4-methylenedioxyamphetamine (MDA) analogs and two amphetamine-based compounds: N,α-DEPEA and DPIA.

3,4-methylenedioxyamphetamine (MDA) is a psychoactive compound chemically related to the entactogen MDMA. MDA shares some of the entactogenic effects of MDMA but also exerts stimulant effects and psychedelic properties at higher doses. Here, we examined the pharmacological properties of MDA analogs and related amphetamine-based compounds detected in street drug samples or in sport supplements. We examined the key pharmacological mechanisms including monoamine uptake inhibition and release using human embryonic kidney 293 cells stably transfected with the respective human transporters. Additionally, we assessed monoamine transporter and receptor binding and activation properties. MDA, its fluorinated analogs, as well as the α-ethyl containing BDB and the dimeric amphetamine DPIA inhibited NET with the greatest potency and preferentially inhibited 5-HT vs. dopamine uptake. The ß­methoxy MDA analog 3C-BOH and the amphetamine-based N,α-DEPEA inhibited NET and preferentially inhibited dopamine vs. 5-HT uptake. The test drugs mediated efflux of at least one monoamine with the exception of DPIA. Most compounds bound to 5-HT2A and 5-HT2C receptors (Ki ≤ 10 µM) and several substances activated the 5-HT2A and 5-HT2B receptor as partial or full agonists. Furthermore, several compounds interacted with adrenergic receptors and the trace amine-associated receptor 1 (TAAR1) in the micromolar range. The pharmacological profiles of some fluorinated and nonfluorinated MDA analogs resemble the profile of MDMA. In contrast, 3C-BOH and N,α-DEPEA displayed more pronounced dopaminergic activity similar to amphetamine. Pharmacokinetics and pharmacodynamics studies are necessary to better establish the risks and therapeutic potential of the tested drugs.

Development of an Electrochemical Sensor Using a Modified Carbon Paste Electrode with Silver Nanoparticles Capped with Saffron for Monitoring Mephedrone.

Mephedrone, also known as 4-methylmethcathinone, is growing into a prominent recreational drug for young people. When it came to detecting mephedrone, limited efforts were made using electrochemical sensors. As a result, this application depicts the fabrication of a new, sensitive, selective, and economical electrochemical sensor capable of detecting mephedrone by using silver nanoparticles capped with saffron produced through electropolymerization to modify carbon paste electrodes (CPEs). Silver nanoparticles (AgNPs) were capped with saffron (AgNPs@Sa) using a green method. AgNPs@Sa were studied using electron scanning microscopy (SEM) and UV-vis spectroscopy. The sensor was evaluated under the optimum condition to determine its analytical features. The results showed that this procedure had a wide linear range, low detection limit and sufficient reproducibility. Furthermore, the sensor posed sufficient stability. Moreover, it was applied in the determination of mephedrone in urine samples, showing the potential applicability of this electrochemical sensor in real sample analysis.

Alteration of Ethanol Reward by Prior Mephedrone Exposure: The Role of Age and Matrix Metalloproteinase-9 (MMP-9).

Mephedrone, a synthetic cathinone, is widely abused by adolescents and young adults. The aim of this study was to determine: (i) whether prior mephedrone exposure would alter ethanol reward and (ii) whether age and matrix metalloproteinase-9 (MMP-9) are important in this regard. In our research, male Wistar rats at postnatal day 30 (PND30) received mephedrone at the dose of 10 mg/kg, i.p., 3 times a day for 7 days. To clarify the role of MMP-9 in the mephedrone effects, one mephedrone-treated group received minocycline, as an MMP-9 antagonist. Animals were then assigned to conditioned place preference (CPP) procedure at PND38 (adolescent) or at PND69 (adult). After the CPP test (PND48/79), expression of dopamine D1 receptors (D1R), Cav1.2 (a subtype of L-type calcium channels), and MMP-9 was quantified in the rat ventral striatum (vSTR). The influence of mephedrone administration on the N-methyl-D-aspartate glutamate receptors (NMDAR) subunits (GluN1, GluN2A, and GluN2B) was then assessed in the vSTR of adult rats (only). These results indicate that, in contrast with adolescent rats, adult rats with prior mephedrone administration appear to be more sensitive to the ethanol effect in the CPP test under the drug-free state. The mephedrone effect in adult rats was associated with upregulation of D1R, NMDAR/GluN2B, MMP-9, and Cav1.2 signaling. MMP-9 appears to contribute to these changes in proteins expression because minocycline pretreatment blocked mephedrone-evoked sensitivity to ethanol reward. Thus, our results suggest that prior mephedrone exposure differentially alters ethanol reward in adolescent and adult rats.

Excretion of mephedrone and its phase I metabolites in urine after a controlled intranasal administration to healthy human volunteers.

Mephedrone is a stimulant drug structurally related to cathinone. At present, there are no data available on the excretion profile of mephedrone and its metabolites in urine after controlled intranasal administration to human volunteers. In this study, six healthy male volunteers nasally insufflated 100 mg of pure mephedrone hydrochloride (Day 1). Urine was collected at different timepoints on Day 1 and then on Days 2, 3 and 30. Samples were analysed for the presence of mephedrone and its metabolites, namely, dihydro-mephedrone, nor-mephedrone (NOR), hydroxytolyl-mephedrone, 4-carboxy-mephedrone (4-carboxy) and dihydro-nor-mephedrone (DHNM), by a validated liquid chromatography-tandem mass spectrometry method. All analytes were detected in urine, where 4-carboxy (Cmax = 29.8 µg/ml) was the most abundant metabolite followed by NOR (Cmax = 377 ng/ml). DHNM was found at the lowest concentrations (Cmax = 93.1 ng/ml). Analytes exhibited a wide range of detection windows, but only 4-carboxy and DHNM were detectable in all samples on Day 3, extending the detection time of mephedrone use. Moreover, mephedrone had a mean renal clearance of 108 ± 140 ml/min, and 1.3 ± 1.7% of unchanged parent drug was recovered in urine in the first 6 h post administration. It is hoped that this novel information will be useful in future studies involving mephedrone and other stimulant drugs.

In vitro metabolic profile of mexedrone, a mephedrone analog, studied by high- and low-resolution mass spectrometry.

Mexedrone is a synthetic cathinone structurally related to mephedrone, which belongs to the class of N-alkyl cathinone derivatives, whose metabolic profile has not been fully clarified yet. This study considers the in vitro phase I metabolism of mexedrone, to pre-select the most appropriate marker(s) of intake. Mexedrone was incubated in the presence of either human liver microsomes or single recombinant CYP450 isoforms. The metabolic profile was outlined by ultra-high-performance liquid chromatography coupled to both high- and low-resolution mass spectrometry. In detail, the phase I metabolic profile of mexedrone was initially defined by a time-of-flight analyzer, while the chemical structures of the detected metabolites and the potential presence of minor metabolites were subsequently studied by tandem mass spectrometry, using a triple quadrupole analyzer. The main phase I metabolic reactions were hydroxylation and N- and O-dealkylation. The CYP450 isoforms most involved were CYP2C19, responsible for the formation of both hydroxylated and dealkylated metabolites, followed by CYP2D6 and CYP1A2, involved in the hydroxylation reactions only. Finally, a significant fraction of mexedrone unchanged was also detected. Based on this evidence, the most appropriate markers of intake are mexedrone unchanged and the hydroxylated metabolites.

Comparative Neuropharmacology and Pharmacokinetics of Methamphetamine and Its Thiophene Analog Methiopropamine in Rodents.

Methiopropamine is a novel psychoactive substance (NPS) that is associated with several cases of clinical toxicity, yet little information is available regarding its neuropharmacological properties. Here, we employed in vitro and in vivo methods to compare the pharmacokinetics and neurobiological effects of methiopropamine and its structural analog methamphetamine. Methiopropamine was rapidly distributed to the blood and brain after injection in C57BL/6 mice, with a pharmacokinetic profile similar to that of methamphetamine. Methiopropamine induced psychomotor activity, but higher doses were needed (Emax 12.5 mg/kg; i.p.) compared to methamphetamine (Emax 3.75 mg/kg; i.p.). A steep increase in locomotor activity was seen after a modest increase in the methiopropamine dose from 10 to 12.5 mg/kg, suggesting that a small increase in dosage may engender unexpectedly strong effects and heighten the risk of unintended overdose in NPS users. In vitro studies revealed that methiopropamine mediates its effects through inhibition of norepinephrine and dopamine uptake into presynaptic nerve terminals (IC50 = 0.47 and 0.74 µM, respectively), while the plasmalemmal serotonin uptake and vesicular uptake are affected only at high concentrations (IC50 > 25 µM). In summary, methiopropamine closely resembles methamphetamine with regard to its pharmacokinetics, pharmacodynamic effects and mechanism of action, with a potency that is approximately five times lower than that of methamphetamine.

4-Fluoromethamphetamine (4-FMA) induces in vitro hepatotoxicity mediated by CYP2E1, CYP2D6, and CYP3A4 metabolism.

4-Fluoromethamphetamine (4-FMA) is an amphetamine-like psychoactive substance with recognized entactogenic and stimulant effects, but hitherto unclear toxicological mechanisms. Taking into consideration that the vast majority of 4-FMA users consume this substance through oral route, the liver is expected to be highly exposed. The aim of this work was to determine the hepatotoxic potential of 4-FMA using in vitro hepatocellular models: primary rat hepatocytes (PRH), human hepatoma cell lines HepaRG and HepG2, and resorting to concentrations ranging from 37 µM to 30 mM, during a 24-h exposure. EC50 values, estimated from the MTT viability assay data, were 2.21 mM, 5.59 mM and 9.57 mM, for each model, respectively. The most sensitive model, PRH, was then co-exposed to 4-FMA and cytochrome P450 (CYP) inhibitors to investigate the influence of metabolism on the toxicity of 4-FMA. Results show that CYP2E1, CYP3A4 and CYP2D6 have major roles in 4-FMA cytotoxicity. Inhibition of CYP2D6 and CYP3A4 led to left-geared shifts in the concentration-response curves of 4-FMA, hinting at a role of these metabolic enzymes for detoxifying 4-FMA, while CYP2E1 inhibition pointed towards a toxifying role of this enzyme in 4-FMA metabolism at physiologically-relevant concentrations. The drug also destabilised mitochondrial membrane potential and decreased ATP levels, increased the production of reactive oxygen and nitrogen species and compromised thiol antioxidant defences. 4-FMA further affected PRH integrity by interfering with the machinery of apoptosis and necrosis, increasing the activity of initiator and effector caspases, and causing loss of cell membrane integrity. Potential for autophagy was also observed. This research contributes to the growing body of evidence regarding the toxicity of new psychoactive substances, in particular regarding their hepatotoxic effects; the apparent influence of metabolism over the resulting cytotoxicity of 4-FMA shows that there is a substantial degree of unpredictability of the consequences for users that could be independent of the dose.