Diphenidine, a new psychoactive substance: metabolic fate elucidated with rat urine and human liver preparations and detectability in urine using GC-MS, LC-MSn , and LC-HR-MSn.

Diphenidine is a new psychoactive substance (NPS) sold as a 'legal high' since 2013. Case reports from Sweden and Japan demonstrate its current use and the necessity of applying analytical procedures in clinical and forensic toxicology. Therefore, the phase I and II metabolites of diphenidine should be identified and based on these results, the detectability using standard urine screening approaches (SUSAs) be elucidated. Urine samples were collected after administration of diphenidine to rats and analyzed using different sample workup procedures with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-(high resolution)-mass spectrometry (LC-(HR)-MS). With the same approaches incubates of diphenidine with pooled human liver microsomes (pHLM) and cytosol (pHLC) were analyzed. According to the identified metabolites, the following biotransformation steps were proposed in rats: mono- and bis-hydroxylation at different positions, partly followed by dehydrogenation, N,N-bis-dealkylation, and combinations of them followed by glucuronidation and/or methylation of one of the bis-hydroxy-aryl groups. Mono- and bis-hydroxylation followed by dehydrogenation could also be detected in pHLM or pHLC. Cytochrome-P450 (CYP) isozymes CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were all capable of forming the three initial metabolites, namely hydroxy-aryl, hydroxy-piperidine, and bis-hydroxy-piperidine. In incubations with CYP2D6 hydroxy-aryl and hydroxy-piperidine metabolites were detected. After application of a common users' dose, diphenidine metabolites could be detected in rat urine by the authors' GC-MS as well as LC-MSn SUSA. Copyright © 2016 John Wiley & Sons, Ltd.

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.

Biotransformation and detectability of the designer drug 2,5-dimethoxy-4-propylphenethylamine (2C-P) studied in urine by GC-MS, LC-MS(n), and LC-high-resolution-MS(n).

2,5-Dimethoxy-4-propylphenethylamine (2C-P) is a hallucinogenic designer drug of the phenethylamine class, the so-called 2Cs, named according to the ethyl spacer between the nitrogen and the aromatic ring. The aims of the present work were to identify the phases I and II metabolites of 2C-P. In addition, the detectability of 2C-P and its metabolites in urine as proof of an intake in clinical or forensic cases was tested. According to the identified metabolites, the following pathways were proposed: N-acetylation; deamination followed by reduction to the corresponding alcohol and oxidation to carbonic acid; mono- and bis-hydroxylation at different positions; mono- and bis-O-demethylation, followed by glucuronidation, sulfation, or both; and combination of these steps. Proof of an intake of a common user's dose of 2C-P was possible by both standard urine screening approaches, the GC-MS as well as the LC-MS(n) approach.

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.

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.

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.