Enantioseparation and Determination of Mephedrone and Its Metabolites by Capillary Electrophoresis Using Cyclodextrins as Chiral Selectors.

Mephedrone, a psychoactive compound derived from cathinone, is widely used as a designer drug. The determination of mephedrone and its metabolites is important for understanding its possible use in medicine. In this work, a method of capillary electrophoresis for the chiral separation of mephedrone and its metabolites was developed. Carboxymethylated ß-cyclodextrin was selected as the most effective chiral selector from seven tested cyclodextrin derivates. Based on the simplex method, the optimal composition of the background electrolyte was determined: at pH 2.75 and 7.5 mmol·L-1 carboxymethylated ß-cyclodextrin the highest total resolution of a mixture of analytes was achieved. For mephedrone and its metabolites, calibration curves were constructed in a calibration range from 0.2 to 5 mmol·L-1; limits of detection, limits of quantification, precision, and repeatability were calculated, and according to Mandel's fitting test, the linear calibration ranges were determined.

Behavioural, Pharmacokinetic, Metabolic, and Hyperthermic Profile of 3,4-Methylenedioxypyrovalerone (MDPV) in the Wistar Rat.

3,4-methylenedioxypyrovalerone (MDPV) is a potent pyrovalerone cathinone that is substituted for amphetamines by recreational users. We report a comprehensive and detailed description of the effects of subcutaneous MDPV (1-4 mg/kg) on pharmacokinetics, biodistribution and metabolism, acute effects on thermoregulation under isolated and aggregated conditions, locomotion (open field) and sensory gating (prepulse inhibition, PPI). All studies used male Wistar rats. Pharmacokinetics after single dose of 2 mg/kg MDPV was measured over 6 h in serum, brain and lungs. The biotransformation study recorded 24 h urinary levels of MDPV and its metabolites after 4 mg/kg. The effect of 2 mg/kg and 4 mg/kg on body temperature (°C) was measured over 12 h in group- vs. individually-housed rats. In the open field, locomotion (cm) and its spatial distribution were assessed. In PPI, acoustic startle response (ASR), habituation, and PPI were measured (AVG amplitudes). In behavioural experiments, 1, 2, or 4 mg/kg MDPV was administered 15 or 60 min prior to testing. Thermoregulation and behavioural data were analysed using factorial analysis of variance (ANOVA). Peak concentrations of MDPV in sera, lung and brain tissue were reached in under 30 min. While negligible levels of metabolites were detected in tissues, the major metabolites in urine were demethylenyl-MDPV and demethylenyl-methyl-MDPV at levels three-four times higher than the parent drug. We also established a MDPV brain/serum ratio ~2 lasting for ~120 min, consistent with our behavioural observations of locomotor activation and disrupted spatial distribution of behaviour as well as moderate increases in body temperature (exacerbated in group-housed animals). Finally, 4 mg/kg induced stereotypy in the open field and transiently disrupted PPI. Our findings, along with previous research suggest that MDPV is rapidly absorbed, readily crosses the blood-brain barrier and is excreted primarily as metabolites. MDPV acts as a typical stimulant with modest hyperthermic and psychomimetic properties, consistent with a primarily dopaminergic mechanism of action. Since no specific signs of acute toxicity were observed, even at the highest doses used, clinical care and harm-reduction guidance should be in line with that available for other stimulants and cathinones.

Identification of three new phase II metabolites of a designer drug methylone formed in rats by N-demethylation followed by conjugation with dicarboxylic acids.

1. Methylone (3,4-methylenedioxy-N-methylcathinone, MDMC), which appeared on the illicit drug market in 2004, is a frequently abused synthetic cathinone derivative. Known metabolic pathways of MDMC include N-demethylation to normethylone (3,4-methylenedioxycathinone, MDC), aliphatic chain hydroxylation and oxidative demethylenation followed by monomethylation and conjugation with glucuronic acid and/or sulphate. 2. Three new phase II metabolites, amidic conjugates of MDC with succinic, glutaric and adipic acid, were identified in the urine of rats dosed subcutaneously with MDMC.HCl (20 mg/kg body weight) by LC-ESI-HRMS using synthetic reference standards to support identification. 3. The main portion of administered MDMC was excreted unchanged. Normethylone, was a major urinary metabolite, of which a minor part was conjugated with dicarboxylic acids. 4. Previously identified ring-opened metabolites 4-hydroxy-3-methoxymethcathinone (4-OH-3-MeO-MC), 3-hydroxy-4-methoxymeth-cathinone (3-OH-4-MeO-MC) and 3,4-dihydroxymethcathinone (3,4-di-OH-MC) mostly in conjugated form with glucuronic and/or sulphuric acids were also detected. 5. Also, ring-opened metabolites derived from MDC, namely, 4-hydroxy-3-methoxycathinone (4-OH-3-MeO-C), 3-hydroxy-4-methoxycathinone (3-OH-4-MeO-C) and 3,4-dihydroxycathinone (3,4-di-OH-C) were identified for the first time in vivo.

Study on the metabolism of 5,6-methylenedioxy-2-aminoindane (MDAI) in rats: identification of urinary metabolites.

1. 5,6-Methylenedioxy-2-aminoindane (MDAI) is a member of aminoindane drug family with serotoninergic effect, which appeared on illicit drug market as a substitute for banned stimulating and entactogenic drugs. 2. Metabolism of MDAI, which has been hitherto unexplored, was studied in rats dosed with a subcutaneous dose of 20 mg MDAI.HCl/kg body weight. The urine of rats was collected within 24 h after dosing for analyses by HPLC-ESI-HRMS and GC/MS. 3. The main metabolic pathways proceeding in parallel were found to be oxidative demethylenation followed by O-methylation and N-acetylation. These pathways gave rise to five metabolites, namely, 5,6-dihydroxy-2-aminoindane, 5-hydroxy-6-methoxy-2-aminoindane, N-acetyl-5,6-methylenedioxy-2-aminoindane, N-acetyl-5,6-dihydroxy-2-aminoindane and N-acetyl-5-hydroxy-6-methoxy-2-aminoindane, which were found predominantly in the form of corresponding glucuronides and sulphates. However, the main portion of administered MDAI was excreted unchanged. 4. Minor metabolites formed primarily by hydroxylation at various sites include cis- and trans-1-hydroxy-5,6-methylenedioxy-2-aminoindane, 5,6-methylenedioxyindan-2-ol and 4-hydroxy-5,6-methylenedioxy-2-aminoindane. 5. Identification of all metabolites except for glucuronides, sulphates and tentatively identified 4-hydroxy-5,6-methylenedioxy-2-aminoindane was supported by synthesised reference standards.

Emerging toxicity of 5,6-methylenedioxy-2-aminoindane (MDAI): Pharmacokinetics, behaviour, thermoregulation and LD50 in rats.

MDAI (5,6-Methylenedioxy-2-aminoindane) has a reputation as a non-neurotoxic ecstasy replacement amongst recreational users, however the drug has been implicated in some severe and lethal intoxications. Due to this, and the fact that the drug is almost unexplored scientifically we investigated a broad range of effects of acute MDAI administration: pharmacokinetics (in sera, brain, liver and lung); behaviour (open field; prepulse inhibition, PPI); acute effects on thermoregulation (in group-/individually-housed rats); and systemic toxicity (median lethal dose, LD50) in Wistar rats. Pharmacokinetics of MDAI was rapid, maximum median concentration in serum and brain was attained 30min and almost returned to zero 6h after subcutaneous (sc.) administration of 10mg/kg MDAI; brain/serum ratio was ~4. MDAI particularly accumulated in lung tissue. In the open field, MDAI (5, 10, 20 and 40mg/kg sc.) increased exploratory activity, induced signs of behavioural serotonin syndrome and reduced locomotor habituation, although by 60min some effects had diminished. All doses of MDAI significantly disrupted PPI and the effect was present during the onset of its action as well as 60min after treatment. Unexpectedly, 40mg/kg MDAI killed 90% of animals in the first behavioural test, hence LD50 tests were conducted which yielded 28.33mg/kg sc. and 35mg/kg intravenous but was not established up to 40mg/kg after gastric administration. Disseminated intravascular coagulopathy (DIC) with brain oedema was concluded as a direct cause of death in sc. treated animals. Finally, MDAI (10, 20mg/kg sc.) caused hyperthermia and perspiration in group-housed rats. In conclusion, the drug had fast pharmacokinetics and accumulated in lipohilic tissues. Behavioural findings were consistent with mild, transient stimulation with anxiolysis and disruption of sensorimotor processing. Together with hyperthermia, the drug had a similar profile to related entactogens, especially 3,4-metyhlenedioxymethamphetamine (MDMA, ecstasy) and paramethoxymethamphetamine (PMMA). Surprisingly subcutaneous MDAI appears to be more lethal than previously thought and its serotonergic toxicity is likely exacerbated by group housing conditions. MDAI therefore poses greater risks to physical and mental health than recognised hitherto.

Metabolic profile of mephedrone: Identification of nor-mephedrone conjugates with dicarboxylic acids as a new type of xenobiotic phase II metabolites.

Metabolic profile of mephedrone (4-methylmethcathinone, 4-MMC), a frequently abused recreational drug, was determined in rats in vivo. The urine of rats dosed with a subcutaneous bolus dose of 20mg 4-MMC/kg was analysed by LC/MS. Ten phase I and five phase II metabolites were identified by comparison of their retention times and MS(2) spectra with those of authentic reference standards and/or with the MS(2) spectra of previously identified metabolites. The main metabolic pathway was N-demethylation leading to normephedrone (4-methylcathinone, 4-MC) which was further conjugated with succinic, glutaric and adipic acid. Other phase I metabolic pathways included oxidation of the 4-methyl group, carbonyl reduction leading to dihydro-metabolites and ω-oxidation at the position 3'. Five of the metabolites detected, namely, 4-carboxynormephedrone (4-carboxycathinone, 4-CC), 4-carboxydihydronormephedrone (4-carboxynorephedrine, 4-CNE), hydroxytolyldihydro-normephedrone (4-hydroxymethylnorephedrine, 4-OH-MNE) and conjugates of 4-MC with glutaric and adipic acid, have not been reported as yet. The last two conjugates represent a novel, hitherto unexploited, type of phase II metabolites in mammals together with an analogous succinic acid conjugate of 4-MC identified by Pozo et al. (2015). These conjugates might be potentially of great importance in the metabolism of other psychoactive amines.

Carcinogenic 3-nitrobenzanthrone but not 2-nitrobenzanthrone is metabolised to an unusual mercapturic acid in rats.

3-Nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspect human carcinogen found in diesel exhaust. Its isomer 2-nitrobenzanthrone (2-NBA) has also been found in ambient air. These isomers differ in mutagenicity in Salmonella by 2-3 orders of magnitude. To identify their urinary metabolites and also to assess the assumed differences in their excretion, rats were dosed orally with 2mg/kg b.w. of either 2-NBA or 3-NBA. Their urine was collected for two consecutive days after dosage. Both LC-ESI-MS and GC-MS confirmed formation of the corresponding aminobenzanthrones (ABA). Excretion of these metabolites within the first day after dosing with 2- and 3-ABA amounted to 0.32±0.06 and 0.83±0.40% of the doses, respectively, while the excretion within the second day was by one order of magnitude lower. A novel mercapturic acid metabolite of 3-NBA was identified in urine by LC-ESI-MS as N-acetyl-S-(3-aminobenzanthron-2-yl)cysteine (3-ABA-MA) by comparison with the authentic standard. Its excretion amounted to 0.49±0.15 and 0.02±0.01% of dose within the first and second day after dosing, respectively. In contrast, no mercapturic acid was detected in the urine of rats dosed with 2-NBA. Observed difference in the mercapturic acid formation between 2- and 3-NBA is a new distinctive feature reflecting differences in the critical step of their metabolism, i.e., benzanthronylnitrenium ion formation that is intrinsically associated with biological activities of these two isomers. Moreover, 3-ABA-MA is a promising candidate biomarker of exposure to the carcinogenic 3-NBA.

High sensitive calixarene-based sensor for detection of dopamine by electrochemical and acoustic methods.

We synthesized 25,26,27,28-tetrakis(11-sulfanylundecyloxy)calix[4]arene (CALIX) sensitive to dopamine and confirmed its structure by (1)H NMR and mass spectrometry. Chemisorption of CALIX molecules or their mixtures with 1-dodecanethiols (DDT) or hexadecanethiols (HDT) resulted in formation of compact low permeable monolayers as revealed by cyclic voltammetry at presence of redox probe [Fe(CN)(6)](3-/4-). These self-assembled monolayers (SAMs) served as sensor for dopamine. Thickness shear mode acoustic method (TSM) has been used for study the interaction of dopamine with calixarene SAM. The admittance spectra of TSM transducer have been measured and used for simultaneous determination of the changes in series resonant frequency, f(S), and motional resistance, R(m), respectively. Addition of dopamine resulted in substantial decrease of f(S) and increase of R(m), which is evidence on increased viscoelastic contribution into the acoustic properties of the sensing layer. Limit of detection (LOD) for dopamine was 50 pM, which is much better in comparison with so far reported lowest LOD for dopamine-sensitive electrochemical sensors (20 nM). The sensor allowed discrimination between dopamine and epinephrine.

Partially O-alkylated thiacalix[4]arenes: synthesis, molecular and crystal structures, conformational behavior.

NMR spectroscopy, X-ray diffraction analysis, and quantum chemical calculations were used for conformational behavior study of partially alkylated thiacalix[4]arenes bearing methyl (1), ethyl (2), or propyl (3) groups at the lower rim. The conformational properties are governed by two basic effects: (i) stabilization by intramolecular hydrogen bonds, and (ii) sterical requirements of the alkoxy groups at the lower rim. While the monosubstituted derivatives 1a and 3a adopt the cone conformation in solution, distally disubstituted compounds 1b, 1'b, 2b, 2'b, 3b, and 3'b exhibit several interesting conformational features. They prefer pinched cone conformation in solution, and, except for 3'b, they form also 1,2-alternate conformation, which is flexible and undergoes rather fast transition between two identical structures. The crystal structures of the compounds 1b, 2b, 2'b, and 3b revealed yet quite rare 1,2-alternate conformation forming molecular channels held together by pi-pi interactions. Different channels-with hexagonal symmetry, 0.26 nm wide-are formed in the crystal structure of the pinched cone conformation of 3b. An uncommon hydrogen bonding pattern was found in dimethoxy and dipropoxy derivatives 1'b and 3'b that adopt distorted cone conformations in crystal. Trialkoxy-substituted compounds 1c and 3c adopt the partial cone conformation in solution. A higher mobility of methyl derivative 1c enables also existence of the cone conformer.

Biotransformation of heterocyclic dinitriles by Rhodococcus erythropolis and fungal nitrilases.

2,6-Pyridinedicarbonitrile (1a) and 2,4-pyridinedicarbonitrile (2a) were hydrated by Rhodococcus erythropolis A4 to 6-cyanopyridine-2-carboxamide (1b; 83% yield) and 2-cyanopyridine-4-carboxamide (2b; 97% yield), respectively, after 10 min. After 118 h, the intermediates 1b or 2b were transformed into 2,6-pyridinedicarboxamide (1c; 35% yield) and 2,6-pyridinedicarboxylic acid (1d; 60% yield) or 2-cyanopyridine-4-carboxylic acid (2c; 64% yield), respectively. The nitrilase from Fusarium solani afforded cyanocarboxylic acids 1e and 2c after 118 h (yields 95 and 62%, respectively). 3,4-Pyridinedicarbonitrile (3a) and 2,3-pyrazinedicarbonitrile (4a) were inferior substrates of nitrile hydratase and nitrilase.