Toxicokinetics of lefetamine and derived diphenylethylamine designer drugs-Contribution of human cytochrome P450 isozymes to their main phase I metabolic steps.

Lefetamine was first marketed in the 1940s as an opioid analgesic. Since withdrawal symptoms were observed during treatment, it became a controlled substance. Its N-ethyl and N-iso-propyl derivatives appeared on the illicit drug market in 2008. Metabolism studies for lefetamine and these derivatives showed that N-dealkylation was the initial step for all three substances in rats. The involvement of the ten most important human cytochrome P450 (CYP) isozymes in this N-dealkylation should be studied now. Studies showed the involvement of CYP1A2, CYP2B6, CYP2C19, and CYP3A4 in N-dealkylation of all three compounds and additionally CYP2D6 for lefetamine and NEDPA. All kinetic profiles followed classic Michaelis-Menten kinetics. Using the relative activity factor approach, the following net clearances were calculated: for lefetamine 8% by CYP1A2, 72% by CYP2B6, 2% by CYP2C19, 1% by CYP2D6, and 17% by CYP3A4; for NEDPA 27% by CYP1A2, 30% by CYP2B6, 23% by CYP2C19, 4% by CYP2D6, and 17% by CYP3A4; for NPDPA 18% by CYP1A2, 24% by CYP2B6, 28% by CYP2C19, and 30% by CYP3A4. In addition, calculated data were compared to chemical inhibition studies in human liver microsomes. Due to the involvement of at least four enzymes in the initial metabolic steps, the risk of CYP-related drug-drug or drug-food interactions should be low.

Lefetamine, a controlled drug and pharmaceutical lead of new designer drugs: synthesis, metabolism, and detectability in urine and human liver preparations using GC-MS, LC-MS(n), and LC-high resolution-MS/MS.

Lefetamine (N,N-dimethyl-1,2-diphenylethylamine, L-SPA) was marketed as an opioid analgesic in Japan and Italy. After being widely abused, it became a controlled substance. It seems to be a pharmaceutical lead for designer drugs because N-ethyl-1,2-diphenylethylamine (NEDPA) and N-iso-propyl-1,2-diphenylethylamine (NPDPA) were confiscated by the German police. In contrast to these derivatives, metabolism and detectability of lefetamine were not studied yet. Therefore, phase I and II metabolism should be elucidated and correlated to the derivatives. Also the detectability using the authors' standard urine screening approaches (SUSA) needed to be checked. As lefetamine was commercially unavailable, it had to be synthesized first. For metabolism studies, a high dose of lefetamine was administered to rats and the urine samples worked up in different ways. Separation and analysis were achieved by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS). In accordance with NEPDA and NPDPA, the following metabolic steps could be proposed: N-oxidation, N-dealkylation, mono- and bis-hydroxylation of the benzene ring, and hydroxylation of the phenyl ring only after N-dealkylation. The di-hydroxy metabolites were conjugated by methylation of one hydroxy group, and hydroxy metabolites by glucuronidation or sulfation. All initial metabolites could also be detected in human liver preparations. After a therapeutic lefetamine dose, the bis-nor, bis-nor-hydroxy, nor-hydroxy, nor-di-hydroxy metabolites could be detected using the authors' GC-MS SUSA and the nor-hydroxy-glucuronide by the LC-MS(n) SUSA. Thus, an intake of lefetamine should be detectable in human urine assuming similar pharmacokinetics.

GC-MS, LC-MS(n), LC-high resolution-MS(n), and NMR studies on the metabolism and toxicological detection of mesembrine and mesembrenone, the main alkaloids of the legal high "Kanna" isolated from Sceletium tortuosum.

Mesembrine and mesembrenone are the main alkaloids of Sceletium tortuosum, a plant species that was used for sedation and analgesia by the KhoiSan, previously known as Hottentots, a tribe in South Africa. After fermentation, the obtained preparation called "Kanna" or "Kougoed" was used by chewing, smoking, or sniffing. Today, Kanna gains popularity by drug users as legal high. For monitoring such consumption, metabolism studies are mandatory because the metabolites are mostly the analytical targets, especially in urine. Therefore, the metabolism of both alkaloids was investigated in rat urine and pooled human liver preparations after several sample work-up procedures. As both alkaloids were not commercially available, they were isolated from plant material by Soxhlet extraction, and their identity confirmed by NMR. The metabolites were identified using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography coupled to linear ion trap high resolution mass spectrometry (LC-HR-MS(n)). Both alkaloids were O- and N-demethylated, dihydrated, and/or hydroxylated at different positions. The phenolic metabolites were partly excreted as glucuronides and/or sulfates. Most of the phase I metabolites identified in rat urine could be detected also in the human liver preparations. After a common user's low dose application of mesembrine, mainly the O- and N demethyl-dihydro, hydroxy, and bis-demethyl-dihydro metabolites, and in case of mesembrenone only the N-demethyl and the N-demethyl-dihydro metabolite could be detected in rat urine using the authors' standard urine screening approaches (SUSA) by GC-MS or LC-MS(n). Thus, it should be possible to monitor a consumption of mesembrine and/or mesembrenone assuming similar pharmacokinetics in humans.

Development and validation of a fast and simple multi-analyte procedure for quantification of 40 drugs relevant to emergency toxicology using GC-MS and one-point calibration.

Diagnosis and prognosis of poisonings should be confirmed by comprehensive screening and reliable quantification of xenobiotics, for example by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS). The turnaround time should be short enough to have an impact on clinical decisions. In emergency toxicology, quantification using full-scan acquisition is preferable because this allows screening and quantification of expected and unexpected drugs in one run. Therefore, a multi-analyte full-scan GC-MS approach was developed and validated with liquid-liquid extraction and one-point calibration for quantification of 40 drugs relevant to emergency toxicology. Validation showed that 36 drugs could be determined quickly, accurately, and reliably in the range of upper therapeutic to toxic concentrations. Daily one-point calibration with calibrators stored for up to four weeks reduced workload and turn-around time to less than 1 h. In summary, the multi-analyte approach with simple liquid-liquid extraction, GC-MS identification, and quantification over fast one-point calibration could successfully be applied to proficiency tests and real case samples.

Lefetamine-derived designer drugs N-ethyl-1,2-diphenylethylamine (NEDPA) and N-iso-propyl-1,2-diphenylethylamine (NPDPA): metabolism and detectability in rat urine using GC-MS, LC-MSn and LC-HR-MS/MS.

N-Ethyl-1,2-diphenylethylamine (NEDPA) and N-iso-propyl-1,2-diphenylethylamine (NPDPA) are two designer drugs, which were confiscated in Germany in 2008. Lefetamine (N,N-dimethyl-1,2-diphenylethylamine, also named L-SPA), the pharmaceutical lead of these designer drugs, is a controlled substance in many countries. The aim of the present work was to study the phase I and phase II metabolism of these drugs in rats and to check for their detectability in urine using the authors' standard urine screening approaches (SUSA). For the elucidation of the metabolism, rat urine samples were worked up with and without enzymatic cleavage, separated and analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS). According to the identified metabolites, the following metabolic pathways for NEDPA and NPDPA could be proposed: N-dealkylation, mono- and bis-hydroxylation of the benzyl ring followed by methylation of one of the two hydroxy groups, combinations of these steps, hydroxylation of the phenyl ring after N-dealkylation, glucuronidation and sulfation of all hydroxylated metabolites. Application of a 0.3 mg/kg BW dose of NEDPA or NPDPA, corresponding to a common lefetamine single dose, could be monitored in rat urine using the authors' GC-MS and LC-MS(n) SUSA. However, only the metabolites could be detected, namely N-deethyl-NEDPA, N-deethyl-hydroxy-NEDPA, hydroxy-NEDPA, and hydroxy-methoxy-NEDPA or N-de-iso-propyl-NPDPA, N-de-iso-propyl-hydroxy-NPDPA, and hydroxy-NPDPA. Assuming similar kinetics, an intake of these drugs should also be detectable in human urine.

The in vivo and in vitro metabolism and the detectability in urine of 3',4'-methylenedioxy-alpha-pyrrolidinobutyrophenone (MDPBP), a new pyrrolidinophenone-type designer drug, studied by GC-MS and LC-MS(n.).

3',4'-Methylenedioxy-alpha-pyrrolidinobutyrophenone (MDPBP), a designer drug of the pyrrolidinophenone-type, was first seized in Germany in 2009. It was also identified in 'legal high' samples investigated in the UK. Therefore, the aim of the presented work was to identify its in vivo and in vitro phase I and II metabolites using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-ion trap mass spectrometry (LC-MS(n) ). Furthermore, detectability of MDPBP in rat and human urine using standard urine screening approaches (SUSA) by GC-MS and LC-MS(n) was studied. The metabolites were isolated either directly or after enzymatic cleavage of conjugates by solid-phase extraction (C18, HCX). The metabolites were then analyzed and structures proposed after GC-MS (phase I) and LC-MS(n) (phase II). Based on these identified metabolites, the following main metabolic steps could be proposed: demethylenation followed by methylation of one hydroxy group, aromatic and side chain hydroxylation, oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Furthermore, in rat urine after a typical user's dose as well as in human urine, mainly the metabolites could be detected using the authors' SUSA by GC-MS and LC-MS(n) . Thus, it should be possible to monitor an application of MDPBP assuming similar toxicokinetics in humans. Finally, CYP2C19 and CYP2D6 could be identified as the isoenzymes mainly responsible for demethylenation.

Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC-MS, LC-MSn, and LC-HR-MSn.

Xylazine is used in veterinary medicine for sedation, anesthesia, and analgesia. It has also been reported to be misused as a horse doping agent, a drug of abuse, a drug for attempted sexual assault, and as source of accidental or intended poisonings. So far, no data concerning human metabolism have been described. Such data are necessary for the development of toxicological detection methods for monitoring drug abuse, as in most cases the metabolites are the analytical targets. Therefore, the metabolism of xylazine was investigated in rat and human urine after several sample workup procedures. The metabolites were identified using gas chromatography (GC)-mass spectrometry (MS) and liquid chromatography (LC) coupled with linear ion trap high-resolution multistage MS (MS(n)). Xylazine was N-dealkylated and S-dealkylated, oxidized, and/or hydroxylated to 12 phase I metabolites. The phenolic metabolites were partly excreted as glucuronides or sulfates. All phase I and phase II metabolites identified in rat urine were also detected in human urine. In rat urine after a low dose as well as in human urine after an overdose, mainly the hydroxy metabolites were detected using the authors' standard urine screening approaches by GC-MS and LC-MS(n). Thus, it should be possible to monitor application of xylazine assuming similar toxicokinetics in humans.

Studies on the in vivo contribution of human cytochrome P450s to the hepatic metabolism of glaucine, a new drug of abuse.

Glaucine ((S)-5,6,6a,7-tetrahydro-1,2,9,10-tetramethoxy-6-methyl-4H-dibenzo [de,g]quinoline), main isoquinoline alkaloid of Glaucium flavum (Papaveraceae), is used as antitussive, but also as recreational drug of abuse. Glaucine was mainly metabolized by O- and N-demethylation to four isomers in rats. So far, only scarce pharmacokinetic data were available. Therefore, the aim of the presented study was to assess the involvement of the ten most important cytochrome P450 (P450) isoforms in the main metabolic steps and determination of their kinetic parameters using the metabolite formation approach. Reference standards of investigated metabolites were synthesized for quantification. In addition, the impact of isomeric standards was tested for calibration and the use of simple peak area ratios on the kinetic constants and resulting contribution of P450 isoforms on estimated hepatic clearance. Kinetic profiles of all metabolite formations followed classic Michaelis-Menten behavior. Km values were between 25 and 140µM, Vmax between 0.10 and 1.92pmol/min/pmol. Using the relative activity factor approach, the hepatic clearance was calculated to be 27 and 73% for 2-O-demethylation by CYP1A2 and CYP3A4, 82, 3, and 15% for 9-O-demethylation by CYP1A2, CYP2C19, and CYP2D6, and finally <1 and 99% for N-demethylation by CYP2D6 and CYP3A4. These data were confirmed by inhibition tests. The calibration mode for determination of the metabolite concentrations had no relevant impact on the estimation of in vivo hepatic clearance of glaucine. As glaucine was metabolized via three initial steps and different P450 isoforms were involved in the hepatic clearance of glaucine, a clinically relevant interaction with single inhibitors should not be expected.

Studies on the metabolism and toxicological detection of glaucine, an isoquinoline alkaloid from Glaucium flavum (Papaveraceae), in rat urine using GC-MS, LC-MS(n) and LC-high-resolution MS(n).

Glaucine ((S)-5,6,6a,7-tetrahydro-1,2,9,10-tetramethoxy-6-methyl-4H-dibenzo [de,g]quinoline) is an isoquinoline alkaloid and main component of Glaucium flavum (Papaveraceae). It was described to be consumed as recreational drug alone or in combination with other drugs. Besides this, glaucine is used as therapeutic drug in Bulgaria and other countries as cough suppressant. Currently, there are no data available concerning metabolism and toxicological analysis of glaucine. To study both, glaucine was orally administered to Wistar rats and urine was collected. For metabolism studies, work-up of urine samples consisted of protein precipitation or enzymatic cleavage followed by solid-phase extraction. Samples were afterwards measured by liquid chromatography (LC) coupled to low or high-resolution mass spectrometry (HR-MS). The phase I and II metabolites were identified by detailed interpretation of the corresponding fragmentations, which were further confirmed by determination of their elemental composition using HR-MS. From these data, the following metabolic pathways could be proposed: O-demethylation at position 2, 9 and 10, N-demethylation, hydroxylation, N-oxidation and combinations of them as well as glucuronidation and/or sulfation of the phenolic metabolites. For monitoring a glaucine intake in case of abuse or poisoning, the O- and N-demethylated metabolites were the main targets for the gas chromatography-MS and LC-MS(n) screening approaches described by the authors. Both allowed confirming an intake of glaucine in rat urine after a dose of 2 mg/kg body mass corresponding to a common abuser's dose.