The psychoactive aminoalkylbenzofuran derivatives, 5-APB and 6-APB, mimic the effects of 3,4-methylenedioxyamphetamine (MDA) on monoamine transmission in male rats.

RATIONALE: The nonmedical use of new psychoactive substances (NPS) is a worldwide public health concern. The so-called "benzofury" compounds, 5-(2-aminopropyl)benzofuran (5-APB) and 6-(2-aminopropyl)benzofuran (6-APB), are NPS with stimulant-like properties in human users. These substances are known to interact with monoamine transporters and 5-HT receptors in transfected cells, but less is known about their effects in animal models. METHODS: Here, we used in vitro monoamine transporter assays in rat brain synaptosomes to characterize the effects of 5-APB and 6-APB, together with their N-methyl derivatives 5-MAPB and 6-MAPB, in comparison with 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA). In vivo neurochemical and behavioral effects of 5-APB (0.3 and 1.0 mg/kg, i.v.) and 6-APB (0.3 and 1.0 mg/kg, i.v.) were assessed in comparison with MDA (1.0 and 3.0 mg/kg, i.v.) using microdialysis sampling in the nucleus accumbens of conscious male rats. RESULTS: All four benzofuran derivatives were substrate-type releasers at dopamine transporters (DAT), norepinephrine transporters (NET), and serotonin transporters (SERT) with nanomolar potencies, similar to the profile of effects produced by MDA and MDMA. However, the benzofurans were at least threefold more potent than MDA and MDMA at evoking transporter-mediated release. Like MDA, both benzofurans induced dose-related elevations in extracellular dopamine and serotonin in the brain, but benzofurans were more potent than MDA. The benzofuran derivatives also induced profound behavioral activation characterized by forward locomotion which lasted for at least 2 h post-injection. CONCLUSIONS: Overall, benzofurans are more potent than MDA in vitro and in vivo, producing sustained stimulant-like effects in rats. These data suggest that benzofuran-type compounds may have abuse liability and could pose risks for adverse effects, especially if used in conjunction with abused drugs or medications which enhance monoamine transmission in the brain.

The synthetic cathinones, butylone and pentylone, are stimulants that act as dopamine transporter blockers but 5-HT transporter substrates.

RATIONALE: Synthetic cathinones continue to emerge in recreational drug markets worldwide. 1-(1,3-Benzodioxol-5-yl)-2-(methylamino)butan-1-one (butylone) and 1-(1,3-benzodioxol-5-yl)-2-(methylamino)pentan-1-one (pentylone) are derivatives of the cathinone compound, 1-(1,3-benzodioxol-5-yl)-2-(methylamino)propan-1-one (methylone), that are being detected in drug products and human casework. OBJECTIVES: The purpose of the present study was to examine the neuropharmacology of butylone and pentylone using in vitro and in vivo methods. METHODS: In vitro uptake and release assays were carried out in rat brain synaptosomes and in cells expressing human dopamine transporters (DAT) and 5-HT transporters (SERT). In vivo microdialysis was performed in the nucleus accumbens of conscious rats to assess drug-induced changes in neurochemistry. RESULTS: Butylone and pentylone were efficacious uptake blockers at DAT and SERT, though pentylone was more DAT-selective. Both drugs acted as transporter substrates that evoked release of [3H]5-HT at SERT, while neither evoked release at DAT. Consistent with the release data, butylone and pentylone induced substrate-associated inward currents at SERT but not DAT. Administration of butylone or pentylone to rats (1 and 3 mg/kg, i.v.) increased extracellular monoamines and motor activity, but pentylone had weaker effects on 5-HT and stronger effects on motor stimulation. CONCLUSIONS: Our data demonstrate that increasing the α-carbon chain length of methylone creates "hybrid" transporter compounds which act as DAT blockers but SERT substrates. Nevertheless, butylone and pentylone elevate extracellular dopamine and stimulate motor activity, suggesting both drugs possess significant risk for abuse.

Tunability of Biodegradable Poly(amine- co-ester) Polymers for Customized Nucleic Acid Delivery and Other Biomedical Applications.

Gene therapy promises to treat diseases that arise from genetic abnormalities by correcting the underlying cause of the disease rather than treating the associated symptoms. Successful transfer of nucleic acids into cells requires efficient delivery vehicles that protect the cargo and can penetrate the appropriate cellular barriers before releasing their contents. Many viral vectors and synthetic polycationic vectors for nucleic acid delivery do not translate well from in vitro to in vivo applications due to their instability and toxicity. We synthesized and characterized a library of biocompatible low charge density polymers from a family of poly(amine- co-ester) (PACE) terpolymers produced via enzyme catalyzed polymerization. PACE polymers are highly customizable; we found that the terpolymer composition can be optimized to produce efficient transfection of various nucleic acids-including DNA plasmids, mRNA, and siRNA-in specific cell types with low toxicity. Our findings suggest that the unique tunability of PACEs offers new tools for gene therapy and other biomedical applications.

Evaluation of drug incorporation into hair segments and nails by enantiomeric analysis following controlled single MDMA intakes.

Incorporation rates of the enantiomers of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolite 3,4-methylenedioxyamphetamine (MDA) into hair and nails were investigated after controlled administration. Fifteen subjects without MDMA use received two doses of 125 mg of MDMA. Hair, nail scrapings, and nail clippings were collected 9-77 days after the last administration (median 20 days). Hair samples were analyzed in segments of 1- to 2-cm length. After chiral derivatization with N-(2,4-dinitro-5-fluorophenyl)-L-valinamide, MDMA and MDA diastereomers were analyzed by liquid chromatography-tandem mass spectrometry. Highest concentrations in hair segments corresponded to the time of MDMA intake. They ranged from 101 to 3200 pg/mg and 71 to 860 pg/mg for R- and S-MDMA, and from 3.2 to 116 pg/mg and 4.4 to 108 pg/mg for R- and S-MDA, respectively. MDMA and MDA concentrations in nail scrapings and clippings were significantly lower than in hair samples. There was no significant difference between enantiomeric ratios of R/S-MDMA and R/S-MDA in hair and nail samples (medians 2.2-2.4 for MDMA and 0.85-0.95 for MDA). Metabolite ratios of MDA to MDMA were in the same range in hair and nail samples (medians 0.044-0.055). Our study demonstrates that administration of two representative doses of MDMA was detected in the hair segments corresponding to the time of intake based on average hair growth rates. MDMA was detected in all nail samples regardless of time passed after intake. Comparable R/S ratios in hair and nail samples may indicate that incorporation mechanisms into both matrices are comparable.

Chiral Plasma Pharmacokinetics of 3,4-Methylenedioxymethamphetamine and its Phase I and II Metabolites following Controlled Administration to Humans.

Generally, pharmacokinetic studies on 3,4-methylenedioxymethamphetamine (MDMA) in blood have been performed after conjugate cleavage, without taking into account that phase II metabolites represent distinct chemical entities with their own effects and stereoselective pharmacokinetics. The aim of the present study was to stereoselectively investigate the pharmacokinetics of intact glucuronide and sulfate metabolites of MDMA in blood plasma after a controlled single MDMA dose. Plasma samples from 16 healthy participants receiving 125 mg of MDMA orally in a controlled study were analyzed using liquid chromatography-tandem mass spectroscopy after chiral derivatization. Pharmacokinetic parameters of R- and S-stereoisomers were determined. Sulfates of 3,4-dihydroxymethamphetamine (DHMA), and sulfate and glucuronide of 4-hydroxy-3-methoxymethamphetamine (HMMA) were identified, whereas free phase I metabolites were not detected. Stereoselective differences in Cmax and AUC24 were observed with the following preferences: R>S for MDMA and DHMA 4-sulfate; S>R for 3,4-methylenedioxyamphetamine (MDA), DHMA 3-sulfate, and HMMA glucuronide; and no preference in Cmax for HMMA sulfate. R/S ratios were >1 for all analytes after 24 hours, independent of the initial chiral preference. These are the first data on chiral pharmacokinetics of MDMA phase II metabolites in human plasma in vivo after controlled administration. The main human MDMA metabolites were shown to be sulfate and glucuronide conjugates.

Quantitative Silylation Speciations of Primary Phenylalkyl Amines, Including Amphetamine and 3,4-Methylenedioxyamphetamine Prior to Their Analysis by GC/MS.

A novel, quantitative trimethylsilylation approach derivatizing 11 primary phenylalkyl amines (PPAAs), including amphetamine (A) and 3,4-methylenedioxyamphetamine (MDA), was noted. Triggering the fully derivatized ditrimethylsilyl (diTMS) species with the N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) reagent, a new principle was recognized followed by GC/MS. In the course of method optimization, the complementary impact of solvents (acetonitrile, ACN; ethyl acetate, ETAC; pyridine, PYR) and catalysts (trimethylchlorosilane, TMCS; trimethyliodosilane, TMIS) was studied: the role of solvent and catalyst proved to be equally crucial. Optimum, proportional, huge responses were obtained with the MSTFA/PYR = 2/1-9/1 (v/v) reagent applying catalysts; A and MDA needed the TMIS, while the rest of PPAAs provided the diTMS products also with TMCS. Similar to derivatives generated with hexamethyldisilazane and perfluorocarboxylic acid (HMDS and PFCA) ( Molnár et al. Anal. Chem. 2015 , 87 , 848 - 852 ), the fully silylated PPAAs offer several advantages. Both of our methods save time and cost by allowing for direct injection of analytes into the column; this is in stark contrast with the requirement to evaporate acid anhydrides by nitrogen prior to their injection. Efficiences of the novel catalyzed trimethylsilylation (MSTFA) and our recently introduced (now, for A and MDA extended) acylation principle were contrasted. Catalyzed trimethylsilylation led to diTMS derivatives resulting in on average a 1.7 times larger response compared to the corresponding acylated species. Catalyzed trimethylsilylation of PPAAs, A, and MDA were characterized with retention, mass fragmentation, and analytical performance properties (R(2), LOQ values). The practical utility of ditrimethylsilyation was shown by analyzing A in urine and mescaline (MSC) in cactus samples.

Molecular basis of the selective binding of MDMA enantiomers to the alpha4beta2 nicotinic receptor subtype: synthesis, pharmacological evaluation and mechanistic studies.

The α4ß2 nicotinic acetylcholine receptor (nAChR) is a molecular target of 3,4-methylenedioxymethamphetamine (MDMA), a synthetic drug also known as ecstasy, and it modulates the MDMA-mediated reinforcing properties. However, the enantioselective preference of the α4ß2 nAChR subtype still remains unknown. Since the two enantiomers exhibit different pharmacological profiles and stereoselective metabolism, the aim of this study is to assess a possible difference in the interaction of the MDMA enantiomers with this nAChR subtype. To this end, we report a novel simple, yet highly efficient enantioselective synthesis of the MDMA enantiomers, in which the key step is the diastereoselective reduction of imides derived from optically pure tert-butylsulfinamide. The enantioselective binding to the receptor is examined using [(3)H]epibatidine in a radioligand assay. Even though the two enantiomers induced a concentration-dependent binding displacement, (S)-MDMA has an inhibition constant 13-fold higher than (R)-MDMA, which shows a Hill's coefficient not significantly different from unity, implying a competitive interaction. Furthermore, when NGF-differentiated PC12 cells were pretreated with the compounds, a significant increase in binding of [(3)H]epibatidine was found for (R)-MDMA, indicating up-regulation of heteromeric nAChR in the cell surface. Finally, docking and molecular dynamics studies have been used to identify the binding mode of the two enantiomers, which provides a structural basis to justify the differences in affinity from the differential interactions played by the substituents at the stereogenic centre of MDMA. The results provide a basis to explore the distinct psychostimulant profiles of the MDMA enantiomers mediated by the α4ß2 nAChR subtype.

5-Iodo-2-aminoindan (5-IAI): chemistry, pharmacology, and toxicology of a research chemical producing MDMA-like effects.

In 2010, an internet snapshot of EMCDDA anticipated the presence of 5-iodo-2-aminoindan (5-IAI) within the recreational drug market. In 2011, this compound, a psychoactive derivative of 2-aminoindane, was identified in recreational products sold in the United Kingdom. 5-IAI is a rigid analogue of p-iodoamphetamine producing MDMA-like effects. The aim of this paper is to summarize the clinical, pharmacological, and toxicological information about this new potential drug of abuse.

Screening of methylenedioxyamphetamine- and piperazine-derived designer drugs in urine by LC-MS/MS using neutral loss and precursor ion scan.

This study describes a method for the screening of methylenedioxyamphetamine- and piperazine-derived compounds in urine by liquid chromatography-tandem mass spectrometry. These substances, characterized by possessing common moieties, are screened using precursor ion and neutral loss scan mode and then quantified in multiple reaction monitoring acquisition mode. Based on the product-ion spectra of different known molecules, chosen as 'model', characteristic neutral losses and product ions were selected: piperazines were detected in precursor ion scan of m/z 44 and neutral loss of 43 and 86 while amphetamines in precursor ion scan of m/z 133, 135 and 163. The applicability of the screening approach was studied in blank urine spiked with selected analytes and processed by solid-phase extraction. Linearity, matrix effect, precision, accuracy, limits of detection and limits of quantification were evaluated both for the screening and the quantification methods. The ability of the screening method to provide semi-quantitative data was demonstrated. This method appears to be a useful tool for the identification of designer drugs derived from piperazines or methylenedioxyamphetamines and can be potentially applied to other drug classes.

New chlorinated amphetamine-type-stimulants disinfection-by-products formed during drinking water treatment.

Previous studies have demonstrated high removal rates of amphetamine-type-stimulants (ATSs) through conventional drinking water treatments; however the behaviour of these compounds through disinfection steps and their transformation into disinfection-by-products (DBPs) is still unknown. In this work, for the first time, the reactivity of some ATSs such as amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) with chlorine has been investigated under simulated and real drinking water treatment conditions in order to evaluate their ability to give rise to transformation products. Two new DBPs from these illicit drugs have been found. A common chlorinated-by-product (3-chlorobenzo)-1,3-dioxole, was identified for both MDA and MDEA while for MDMA, 3-chlorocatechol was found. The presence of these DBPs in water samples collected through drinking water treatment was studied in order to evaluate their formation under real conditions. Both compounds were generated through treatment from raw river water samples containing ATSs at concentration levels ranging from 1 to 15 ng/L for MDA and from 2.3 to 78 ng/L for MDMA. One of them, (3-chlorobenzo)-1,3-dioxole, found after the first chlorination step, was eliminated after ozone and GAC treatment while the MDMA DBP mainly generated after the postchlorination step, showed to be recalcitrant and it was found in final treated waters at concentrations ranging from 0.5 to 5.8 ng/L.

Comparative neuropharmacology of three psychostimulant cathinone derivatives: butylone, mephedrone and methylone.

BACKGROUND AND PURPOSE: Here, we have compared the neurochemical profile of three new cathinones, butylone, mephedrone and methylone, in terms of their potential to inhibit plasmalemmal and vesicular monoamine transporters. Their interaction with 5-HT and dopamine receptors and their psychostimulant effect was also studied. EXPERIMENTAL APPROACH: Locomotor activity was recorded in mice following different doses of cathinones. Monoamine uptake assays were performed in purified rat synaptosomes. Radioligand-binding assays were carried out to assess the affinity of these compounds for monoamine transporters or receptors. KEY RESULTS: Butylone, mephedrone and methylone (5-25 mg·kg(-1) ) caused hyperlocomotion, which was prevented with ketanserin or haloperidol. Methylone was the most potent compound inhibiting both [(3) H]5-HT and [(3) H]dopamine uptake with IC(50) values that correlate with its affinity for dopamine and 5-HT transporter. Mephedrone was found to be the cathinone derivative with highest affinity for vesicular monoamine transporter-2 causing the inhibition of dopamine uptake. The affinity of cathinones for 5-HT(2A) receptors was similar to that of MDMA. CONCLUSIONS AND IMPLICATIONS: Butylone and methylone induced hyperlocomotion through activating 5-HT(2A) receptors and increasing extra-cellular dopamine. They inhibited 5-HT and dopamine uptake by competing with substrate. Methylone was the most potent 5-HT and dopamine uptake inhibitor and its effect partly persisted after withdrawal. Mephedrone-induced hyperlocomotion was dependent on endogenous 5-HT. Vesicular content played a key role in the effect of mephedrone, especially for 5-HT uptake inhibition. The potency of mephedrone in inhibiting noradrenaline uptake suggests a sympathetic effect of this cathinone.

Estimation of community-wide drugs use via stereoselective profiling of sewage.

This paper explores possibilities of applying enantiomeric profiling to solving problems related to estimation of drugs usage in communities via the sewage epidemiology approach: for the identification of whether drug residue results from consumption of illicit drug or metabolism of other drugs, verification of potency of used drugs and monitoring of changing patterns of drugs abuse. Due to the very complex nature of wastewater used in sewage epidemiology, which comes from the whole community rather than one individual, verification of the above is challenging but vital in accurate estimations of drugs abuse as well as providing comprehensive information regarding drug abuse trends. The results of this study indicated that amphetamine in raw wastewater was enriched with R(-)-enantiomer due to its abuse as racemate. Methamphetamine was found to be racemic or to be enriched with S(+)-enantiomer. MDMA was enriched with R(-)-MDMA, which was to be expected as MDMA is abused as racemate. MDA was enriched with S(+)-enantiomer, which suggests that its presence might be associated with MDMA abuse and not intentional MDA use. Out of the four possible isomers of ephedrine only natural 1R,2S(-)-ephedrine and 1S,2S(+)-pseudoephedrine were detected in raw wastewater and their diastereomeric fractions were found to be season dependent with higher contribution from 1S,2S(+)-pseudoephedrine over winter months and an enrichment with 1R,2S(-)-ephedrine during the spring and summer months. These findings were accompanied by a decrease of cumulative concentration of ephedrines throughout the sampling campaign between February and August. This is a very important finding indicating that non-enantioselective measurement of ephedrine concentrations cannot be a reliable indicator of actual potency of ephedrines used.

Simultaneous determination of HFBA-derivatized amphetamines and ketamines in urine by gas chromatography-mass spectrometry.

To facilitate the analysis of targeted drugs under high sample volume testing environment, an extraction, derivatization and gas chromatographic-mass spectrometric analysis method was developed for simultaneously determination of amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), ketamine, and norketamine in urine. This method utilized solid-phase extraction in conjunction with derivatization using heptafluorobutyric anhydride (HFBA) as the derivatization reagent. Using a 1-mL sample, the limits of quantitation achieved for the analysis of AMP, MAMP, MDA, MDMA, MDEA, ketamine, and norketamine were 25, 15, 60, 60, 70, 25, and 30 ng/mL, respectively. Upper limits of quantitation were 8000 ng/mL for all amphetamines and 6000 ng/mL for ketamine and norketamine. Except for dehydronorketamine (DHNK), within-day and between-day precisions (as expressed in CV%) for quality control samples were ≤ 3.1% and ≤ 4.95%, respectively. Except DHNK, the within-day accuracy ranged between 96.0% and 110.7% and the between-day accuracy ranged between 96.9% and 108.7%. A group of 107 urine samples previously determined to contain the target analytes were analyzed by this new approach. Quantitative data produced by both methods agreed well. With this new approach, we were able to use a single analytical protocol to conduct the confirmation test for samples that preliminarily tested positive (by immunoassay) for amphetamines, ketamine, or both.

GC-MS studies on side chain regioisomers related to substituted methylenedioxyphenethylamines: MDEA, MDMMA, and MBDB.

Three regioisomeric 3,4-methylenedioxyphenethylamines having the same molecular weight and major mass spectral fragments of equal mass have been reported as drugs of abuse in forensic studies in recent years. These compounds are 3,4-methylenedioxy-N-ethylamphetamine (MDEA), 3,4-methylenedioxy-N,N-dimethylamphetamine (MDMMA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB). A series of seven additional side chain regioisomers have mass spectra essentially equivalent to the three controlled drug substances, all have molecular weight of 207 and major fragment ions in their electron ionization mass spectra at m/z 72 and 135/136. The trifluoroacetyl, pentafluoropropionyl, and heptafluorobutryl derivatives of the primary and secondary regioisomeric amines were evaluated in GC-MS studies. The mass spectra for these derivatives were significantly individualized, and the resulting unique fragment ions allowed for specific side chain identification. The trifluoroacetyl and heptafluorobutryl derivatives provided more specific fragment ions for molecular individualization among these regioisomeric substances. These perfluoroacyl derivatives showed reasonable resolution on the polar stationary phase Rtx-200.

Role of water in molecular docking simulations of cytochrome P450 2D6.

Active-site water molecules form an important component in biological systems, facilitating promiscuous binding or an increase in specificity and affinity. Taking water molecules into account in computational approaches to drug design or site-of-metabolism predictions is currently far from straightforward. In this study, the effects of including water molecules in molecular docking simulations of the important metabolic enzyme cytochrome P450 2D6 are investigated. The structure and dynamics of water molecules that are present in the active site simultaneously with a selected substrate are described, and based on this description, water molecules are selected to be included in docking experiments into multiple protein conformations. Apart from the parent substrate, 11 similar and 53 dissimilar substrates are included to investigate the transferability of active-site hydration sites between substrates. The role of water molecules appears to be highly dependent on the protein conformation and the substrate.

Investigations on the human hepatic cytochrome P450 isozymes involved in the metabolism of 3,4-methylenedioxy-amphetamine (MDA) and benzodioxolyl-butanamine (BDB) enantiomers.

3,4-Methylenedioxy-amphetamine (MDA) and benzodioxolyl-butanamine (BDB) are chiral designer drugs distributed on the illicit drug market and they are also N-dealkyl metabolites of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy, Adam), 3,4-methylenedioxyethylamphetamine (MDEA, Eve), and N-methyl-benzodioxolyl-butanamine (MBDB, Eden), respectively. MDA and BDB are mainly metabolized via demethylenation to the corresponding catecholamines. The aim of the present work was to elucidate the contribution of the relevant human P450s in the demethylenation of the MDA and BDB enantiomers. They were incubated using heterologously expressed human P450s and the corresponding metabolites dihydroxyamphetamine and 1,2-dihydroxy-4-[2-amino-butyl]benzene were determined. Highest contributions to the demethylenation as calculated from the enzyme kinetic data were obtained for CYP2D6 (MDA and BDB) and additionally CYP3A4 in the case of BDB at substrate concentrations corresponding to plasma concentrations of recreational users. A preferred transformation of the S-enantiomer could be observed for the CYP2D6- and CYP3A4-catalyzed reactions.

Thermal desorption counter-flow introduction atmospheric pressure chemical ionization for direct mass spectrometry of ecstasy tablets.

A novel approach to the analysis of ecstasy tablets by direct mass spectrometry coupled with thermal desorption (TD) and counter-flow introduction atmospheric pressure chemical ionization (CFI-APCI) is described. Analytes were thermally desorbed with a metal block heater and introduced to a CFI-APCI source with ambient air by a diaphragm pump. Water in the air was sufficient to act as the reactive reagent responsible for the generation of ions in the positive corona discharge. TD-CFI-APCI required neither a nebulizing gas nor solvent flow and the accompanying laborious optimizations. Ions generated were sent in the direction opposite to the air flow by an electric field and introduced into an ion trap mass spectrometer. The major ions corresponding to the protonated molecules ([M + H](+)) were observed with several fragment ions in full scan mass spectrometry (MS) mode. Collision-induced dissociation of protonated molecules gave characteristic product-ion mass spectra and provided identification of the analytes within 5 s. The method required neither sample pretreatment nor a chromatographic separation step. The effectiveness of the combination of TD and CFI-APCI was demonstrated by application to the direct mass spectrometric analysis of ecstasy tablets and legal pharmaceutical products.

4-Hydroxy-3-methoxymethamphetamine glucuronide as a phase II metabolite of 3,4-methylenedioxymethamphetamine: enzyme-assisted synthesis and involvement of human hepatic uridine 5'-diphosphate-glucuronosyltransferase 2B15 in the glucuronidation.

3,4-Methylenedioxymethamphetamine (MDMA), one of the most popular illicit recreational drugs, is metabolized primarily into 4-hydroxy-3-methoxymethamphetamine (HMMA) by drug-metabolizing enzymes. HMMA is further metabolized by phase II enzymes to give the glucuronide or sulfate which is excreted into urine. In the present study, enzyme kinetic studies with various microsomes showed that rat liver microsomes pretreated with Aroclor 1254 were most suitable for the enzyme-assisted synthesis of the glucuronide (HMMA-Gluc). This method selectively produced the beta-anomer of HMMA-Gluc in a very high, isolated yield (71%), and with a purity that was sufficient for use in an analysis of MDMA intake and for enzyme kinetic studies. We also identified, by an LC-MS method, the human uridine 5'-diphosphate-glucuronosyltransferase (UGT) isoforms that catalyze the glucuronidation of HMMA. Among 12 isoforms of human recombinant UGT expressed in insect cells, UGT2B15 was the only isoform that showed adequate enzymatic activity in catalyzing HMMA glucuronidation with K(m) and V(max) values of 3.8 mM and 1.6 nmol/min/mg protein, respectively. The finding that UGT2B15 is capable of HMMA glucuronidation suggests this isoform may have an important in vivo role in human MDMA metabolism.

Enantioselectivity in the methylation of the catecholic phase I metabolites of methylenedioxy designer drugs and their capability to inhibit catechol-O-methyltransferase-catalyzed dopamine 3-methylation.

The designer drugs R,S-3,4-methylenedioxy-methamphetamine (MDMA, Ecstasy), R,S-3,4-methylenedioxy-ethylamphetamine (MDEA, Eve), and R,S-N-methyl-benzodioxolyl-butanamine (MBDB, Eden) are chiral compounds, and their in vitro and in vivo metabolism is enantioselective with a preference for the S-enantiomer caused in part by P450-mediated demethylenation. As the elimination of the catecholamine metabolites could also be enantioselective, the aim of the present study was to investigate the O-methylation to the corresponding methoxy derivatives catalyzed by the soluble or membrane-bound form of the catechol-O-methyltransferase (COMT). As all three compounds showed substrate inhibition effects during the incubation, their inhibition potential was quantified using the methylation of dopamine as a marker reaction. For investigation of the catechol-O-methylation catalyzed by the soluble form of the COMT (sCOMT), incubations with human liver cytosol (HLC) were performed. Human liver microsomes (HLM) were used for investigation of the membrane-bound form. For inhibition studies, 3-hydroxytyramine (dopamine) was incubated in HLC. The respective catechols were added at various concentrations to check whether they influence the methylation of 3-hydroxytyramine. Our data showed that the S-enantiomers of all studied catecholamines were preferably O-methylated by both types of COMT. Comparing the resulting kinetics of the HLC and HLM assays, the affinity for all substrates was 10-fold higher for the membrane-bound COMT, whereas the turnover rate was 10-fold higher for the soluble COMT. Uncompetitive inhibition of dopamine methylation could be observed for all tested catechols. In conclusion, elimination of the catecholamine metabolites of MDMA, MDEA, and MBDB was shown to be enantioselective and might therefore contribute to the different pharmacokinetic properties observed for both enantiomers. Furthermore, the catecholic metabolites were identified to be uncompetitive inhibitors of the sCOMT localized in HLC.

Stereoselective differences in the cytochrome P450-dependent dealkylation and demethylenation of N-methyl-benzodioxolyl-butanamine (MBDB, Eden) enantiomers.

In the present study, cytochrome P450 isozymes (P450) involved in the stereoselective metabolism of the designer drug N-methyl-benzodioxolyl-butanamine (MBDB, Eden) were identified for the first time. Demethylenation and N-demethylation of racemic MBDB as well as its single enantiomers were investigated using cDNA-expressed insect cell microsomes. After incubation of MBDB, the two resulting main metabolites 1,2-dihydroxy-4-[2-(methylamino)butyl]benzene (DHMBB) and benzodioxolyl-butanamine (BDB) were separated and quantified by achiral gas chromatography-mass spectrometry after chiral derivatization with S-heptafluorobutyrylprolyl chloride. Dealkylation was mainly catalyzed by CYP2B6 and CYP2C19, demethylenation was additionally catalyzed by CYP1A2, CYP2D6, and CYP3A4. The most abundant isozymes after in vitro-in vivo correlation using the relative activity factor approach are CYP2B6 for N-dealkylation and CYP2D6 for demethylenation the second step being the most relevant. In addition, inhibition studies towards MBDB biotransformation using the CYP2D6 selective inhibitor quinidine confirmed the dominant role of this polymorphic isozyme in total MBDB metabolism. In general, at low substrate concentrations the S-enantiomer is metabolized at a higher rate foremost by CYP2C19. These findings are in line with results previously reported for the corresponding ring substituted amphetamines 3,4-methylenedioxy-methamphetamine and 3,4-methylenedioxy-ethylamphetamine. In conclusion, the main enzyme responsible for MBDB metabolism after in vitro-in vivo correlation is CYP2D6, whereas CYP2C19 is the most enantioselective.