Identification of cytochrome P450 enzymes involved in the metabolism of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone.

The involvement of human hepatic cytochrome P450 (P450) isoenzymes in the metabolism of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) to 4'-(hydroxymethyl)-alpha-pyrrolidinobutyrophenone (HO-MPBP) was studied using insect cell microsomes with cDNA-expressed human P450s and human liver microsomes (HLM). Incubation samples were analyzed by liquid chromatography-mass spectrometry. Only CYP2D6, CYP2C19, and CYP1A2 were capable of catalyzing MPBP 4'-hydroxylation. According to the relative activity factor approach, these enzymes accounted for 54, 30, and 16% of net clearance. At 1 microM MPBP, the chemical inhibitors quinidine (CYP2D6), fluconazole (CYP2C19), and alpha-naphthoflavone (CYP1A2) reduced metabolite formation in pooled HLM by 83, 53, and 47%, respectively, and at 50 microM MPBP by 41, 47, and 45%, respectively. In experiments with HLM from CYP2D6 and CYP2C19 poor metabolizers, HO-MPBP formation was found to be 78 and 79% lower in comparison with pooled HLM, respectively. From these data, it can be concluded that polymorphically expressed CYP2D6 is mainly responsible for MPBP hydroxylation.

Studies on the toxicological detection of the designer drug 4-bromo-2,5-dimethoxy-beta-phenethylamine (2C-B) in rat urine using gas chromatography-mass spectrometry.

The phenethylamine-derived designer drug 4-bromo-2,5-dimethoxy-beta-phenethylamine (2C-B) is known to be extensively metabolized in various species including humans. In rat urine, 2C-B was found to be excreted mainly via its metabolites. In the current study, the toxicological detection of these metabolites in the authors' systematic toxicological analysis (STA) procedure was examined. The STA procedure using full-scan GC-MS allowed proving an intake of a common drug abusers' dose of 2C-B by detection of the O-demethyl deaminohydroxy and two isomers of the O-demethyl metabolites in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-B in human urine.

Identification of monoamine oxidase and cytochrome P450 isoenzymes involved in the deamination of phenethylamine-derived designer drugs (2C-series).

In recent years, several compounds of the phenethylamine-type (2C-series) have entered the illicit drug market as designer drugs. In former studies, the qualitative metabolism of frequently abused 2Cs (2C-B, 2C-I, 2C-D, 2C-E, 2C-T-2, 2C-T-7) was studied using a rat model. Major phase I metabolic steps were deamination and O-demethylation. Deamination to the corresponding aldehyde was the reaction, which was observed for all studied compounds. Such reactions could in principal be catalyzed by two enzyme systems: monoamine oxidase (MAO) and cytochrome P450 (CYP). The aim of this study was to determine the human MAO and CYP isoenzymes involved in this major metabolic step and to measure the Michaelis-Menten kinetics of the deamination reactions. For these studies, cDNA-expressed CYPs and MAOs were used. The formation of the aldehyde metabolite was measured using GC-MS after extraction. For all compounds studied, MAO-A and MAO-B were the major enzymes involved in the deamination. For 2C-D, 2C-E, 2C-T-2 and 2C-T-7, CYP2D6 was also involved, but only to a very small extent. Because of the isoenzymes involved, the 2Cs are likely to be susceptible for drug-drug interactions with MAO inhibitors.

Studies on the metabolism and toxicological detection of the designer drug 2,5-dimethoxy-4-methyl-beta- phenethylamine (2C-D) in rat urine using gas chromatographic/mass spectrometric techniques.

The phenethylamine-derived designer drug 2,5-dimethoxy-4-methyl-beta-phenethylamine (2C-D) was found to be metabolized in rats by O-demethylation at position 2 or 5 followed by N-acetylation or by deamination with oxidation to the corresponding acids or reduction to the corresponding alcohol. Furthermore, 2C-D was hydroxylated at the methyl group or deaminated followed by reduction to the corresponding alcohol or by oxidation to the corresponding acid. Most of the metabolites were excreted in conjugated form. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS allowed the detection of an intake of a dose of 2C-D in rat urine that corresponds to a common drug user's dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-D in human urine.

New designer drug 4-iodo-2,5-dimethoxy-beta-phenethylamine (2C-I): studies on its metabolism and toxicological detection in rat urine using gas chromatographic/mass spectrometric and capillary electrophoretic/mass spectrometric techniques.

Studies are described on the metabolism and the toxicological analysis of the phenethylamine-derived designer drug 4-iodo-2,5-dimethoxy-beta-phenethylamine (2C-I) in rat urine using gas chromatographic/mass spectrometric (GC/MS) techniques, and for a particular question, using capillary electrophoretic/mass spectrometric (CE/MS) techniques. The identified metabolites indicated that 2C-I was metabolized on the one hand by O-demethylation in position 2 and 5, respectively, followed either by N-acetylation or by deamination with subsequent oxidation to the corresponding acid or reduction to the corresponding alcohol, respectively. The latter metabolite was hydroxylated in beta-position and further oxidized to the corresponding oxo metabolite. On the other hand, 2C-I was metabolized by deamination with subsequent oxidation to the corresponding acid or reduction to the corresponding alcohol, respectively. 2C-I and most of its metabolites were partially excreted in conjugated form. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of an intake of a dose of 2C-I in rat urine that corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-I in human urine.

Toxicokinetics of drugs of abuse: current knowledge of the isoenzymes involved in the human metabolism of tetrahydrocannabinol, cocaine, heroin, morphine, and codeine.

This review summarizes the major metabolic pathways of the drugs of abuse, tetrahydrocannabinol, cocaine, heroin, morphine, and codeine, in humans including the involvement of isoenzymes. This knowledge may be important for predicting their possible interactions with other xenobiotics, understanding pharmaco-/toxicokinetic and pharmacogenetic variations, toxicological risk assessment, developing suitable toxicological analysis procedures, and finally for understanding certain pitfalls in drug testing. The detection times of these drugs and/or their metabolites in biological samples are summarized and the implications of the presented data on the possible interactions of drugs of abuse with other xenobiotics, ie, inhibition or induction of individual polymorphic and nonpolymorphic isoenzymes, discussed.

Studies on the metabolism and toxicological detection of the designer drug 4-ethyl-2,5-dimethoxy-beta-phenethylamine (2C-E) in rat urine using gas chromatographic-mass spectrometric techniques.

The phenethylamine-derived designer drug 4-ethyl-2,5-dimethoxy-beta-phenethylamine (2C-E) was found to be mainly metabolized in rats by O-demethylation, N-acetylation, hydroxylation of the ethyl side chain at C2' or at C1' followed by oxidation at C1' to the corresponding ketone, by deamination followed by reduction to the corresponding alcohols or by oxidation to the corresponding acids, and finally combinations of these steps. Most of the metabolites were excreted in conjugated form. The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS allowed the detection of an intake of a dose of 2C-E in rat urine that corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-E in human urine.

New designer drug 2,5-dimethoxy-4-ethylthio-beta-phenethylamine (2C-T-2): studies on its metabolism and toxicological detection in rat urine using gas chromatography/mass spectrometry.

Studies are described on the metabolism and the toxicological analysis of the phenethylamine-derived designer drug 2,5-dimethoxy-4-ethylthio-beta-phenethylamine (2C-T-2) in rat urine using gas chromatography/mass spectrometry (GC/MS) after enzymatic cleavage of conjugates, liquid-liquid extraction and derivatization. The structures of 14 metabolites were assigned tentatively by detailed interpretation of their mass spectra. Identification of these metabolites indicated that 2C-T-2 was metabolized by sulfoxidation followed by N-acetylation and either hydroxylation of the S-ethyl side chain or demethylation of one methoxy group, O-demethylation of the parent compound followed by N-acetylation and sulfoxidation, deamination followed by reduction to the corresponding alcohol followed by partial glucuronidation and/or sulfation or by oxidation to the corresponding acid followed either by partial glucuronidation or by degradation to the corresponding benzoic acid derivative followed by partial glucuronidation. Furthermore, 2C-T-2 was metabolized by N-acetylation of the parent compound followed either by O-demethylation and sulfoxidation or by S-dealkylation, S-methylation and sulfoxidation. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction microwave-assisted acetylation allowed the detection of an intake of a dose of 2C-T-2 in rat urine, which corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-T-2 in human urine.

Screening for and validated quantification of phenethylamine-type designer drugs and mescaline in human blood plasma by gas chromatography/mass spectrometry.

In recent years, several newer designer drugs of the so-called 2C series such as 2C-D, 2C-E, 2C-P, 2C-B, 2C-I, 2C-T-2, and 2C-T-7 have entered the illicit drug market as recreational drugs. Some fatal intoxications involving 2C-T-7 have been reported. Only scarce data have been published about analyses of these substances in human blood and/or plasma. This paper describes a method for screening and simultaneous quantification of the above-mentioned compounds and their analog mescaline in human blood plasma. The analytes were analyzed by gas chromatography/mass spectrometry in the selected-ion monitoring mode, after mixed-mode solid-phase extraction (HCX) and derivatization with heptafluorobutyric anhydride. The method was fully validated according to international guidelines. Validation data for 2C-T-2 and 2C-T-7 were unacceptable. For all other analytes, the method was linear from 5 to 500 microg/L and the data for accuracy (bias) and precision (coefficient of variation) were within the acceptance limits of +/-15% and <15%, respectively (within +/-20% and <20% near the limit of quantification of 5 microg/L).

New designer drug, 2,5-dimethoxy-4-propylthio-beta-phenethylamine (2C-T-7): studies on its metabolism and toxicological detection in rat urine using gas chromatography/mass spectrometry.

Studies are described on the metabolism and toxicological analysis of the phenethylamine-derived designer drug 2,5-dimethoxy-4-propylthio-beta-phenethylamine (2C-T-7) in rat urine using gas chromatography/mass spectrometry (GC/MS). The identified metabolites indicated that 2C-T-7 was metabolized by hydroxylation of the propyl side chain followed by N-acetylation and sulfoxidation and also by deamination followed by oxidation to the corresponding acid or by reduction to the corresponding alcohol. To a minor extent, 2C-T-7 was also metabolized by S-dealkylation followed by N-acetylation, S-methylation and sulfoxidation. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction microwave-assisted acetylation allowed the detection of an intake of a dose of 2C-T-7 in rat urine that corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-T-7 in human urine.

Studies on the human metabolism and the toxicologic detection of the cough suppressant dropropizine in urine using gas chromatography-mass spectrometry.

Studies are described on the metabolism and the toxicologic analysis of the nonopioid cough suppressant dropropizine [R,S-3-(4-phenyl-1-piperazinyl)1,2-propandiol, DRO] in human urine using gas chromatography-mass spectrometry (GC-MS). The metabolism studies showed that DRO was metabolized in humans mainly by hydroxylation of the aromatic ring, by N-dealkylation of the parent drug and of the hydroxyl-metabolite to the corresponding N-phenylpiperazines, and by degradation of the piperazine moiety. The authors' systematic toxicologic analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction, and microwave-assisted acetylation allowed the unambiguous detection of DRO and its above-mentioned metabolites in human urine up to about 32 hours after intake of a single common therapeutic dose. The target analytes were found to be the parent compound DRO (earlier phase of excretion) and the hydroxylated metabolite para-hydroxy-DRO (later phase of excretion). Both allowed unambiguous detection of an intake of DRO and also differentiation from other phenylpiperazine derivatives.

Identification of human cytochrome P450 2D6 as major enzyme involved in the O-demethylation of the designer drug p-methoxymethamphetamine.

p-Methoxymethamphetamine (PMMA) is a new designer drug, listed in many countries as a controlled substance. Several fatalities have been attributed to the abuse of this designer drug. Previous in vivo studies using Wistar rats had shown that PMMA was metabolized mainly by O-demethylation. The aim of the study presented here was to identify the human hepatic cytochrome P450 (P450) enzymes involved in the biotransformation of PMMA to p-hydroxymethamphetamine. Baculovirus-infected insect cell microsomes, pooled human liver microsomes (pHLMs), and CYP2D6 poor-metabolizer genotype human liver microsomes (PM HLMs) were used for this purpose. Only CYP2D6 catalyzed O-demethylation. The apparent K(m) and V(max) values in baculovirus-infected insect cell microsomes were 4.6 +/- 1.0 microM and 92.0 +/- 3.7 pmol/min/pmol P450, respectively, and 42.0 +/- 4.0 microM and 412.5 +/- 10.8 pmol/min/mg protein in pHLMs. Inhibition studies with 1 microM quinidine showed significant inhibition of the metabolite formation (67.2 +/- 0.6%; p < 0.0001), and comparison of the metabolite formation between pHLMs and PM HLMs revealed significantly lower metabolite formation in the incubations with PM HLMs (87.3 +/- 1.1%; p < 0.0001). According to these studies, CYP2D6 is the major P450 involved in O-demethylation of PMMA.