Cytotoxicity of new psychoactive substances and other drugs of abuse studied in human HepG2 cells using an adopted high content screening assay.

New psychoactive substances (NPS) are still an emerging issue in clinical and forensic toxicology. Information about their cytotoxic potential is limited or even unavailable before distribution and thus their intake can be of high risk for consumers. The aim of the presented study was to develop a strategy to identify cytotoxic potential of NPS based on a high content screening assay (HCSA) using HepG2 cell line and four fluorescent dyes, namely Hoechst33342, TMRM, CAL-520, and TOTO-3. The HCSA was optimized to work without an automated analyzer by using the model compounds fluvastatin, paracetamol, propranolol, and simvastatin. The following parameters were monitored: stained nuclei as a measure for cell count as well as nuclear size and nuclear intensity (all Hoechst33342), mitochondrial membrane potential (TMRM), cytosolic calcium level (CAL-520), and plasma membrane integrity (TOTO-3). The present study showed strong cytotoxic potential for the NPS 5F-PB-22 and MDAI, moderate effects for MDMA, MDPV, methylone, cathinone, 4-MEC, and mephedrone, and no toxic effects for methamphetamine. To assess the metabolic suitability of HepG2 cells under the chosen conditions, cell culture supernatants were analyzed by liquid chromatography-high resolution-tandem mass spectrometry. Metabolites were merely detected for lipophilic drugs such as 5F-PB-22 and MDPV and in addition with a much lower abundance in comparison to the parent compound but the study only allowed a qualitative look for metabolites and the used liver cell line might not ideal when considering metabolism.

Inhibition and stimulation of the human breast cancer resistance protein as in vitro predictor of drug-drug interactions of drugs of abuse.

Transporter-mediated drug-drug interactions (DDI) may induce adverse clinical events. As drugs of abuse (DOA) are marketed without preclinical safety studies, only very limited information about interplay with membrane transporters are available. Therefore, 13 DOA of various classes were tested for their in vitro affinity to the human breast cancer resistance protein (hBCRP), an important efflux transporter. As adenosine 5'-triphosphate (ATP) hydrolysis is crucial for hBCRP activity, adenosine 5'-diphosphate (ADP) formation was measured and used as in vitro marker for hBCRP ATPase activity. ADP quantification was performed by hydrophilic interaction liquid chromatography coupled to high-resolution tandem mass spectrometry and its amount in test compound incubations was compared to that in reference incubations using the hBCRP substrate sulfasalazine or the hBCRP inhibitor orthovanadate. If DOA caused stimulation or inhibition, further investigations such as Michaelis-Menten kinetic modeling or IC50 value determination were conducted. Among the tested DOA, seven compounds showed statistically significant hBCRP ATPase stimulation. The entactogen 3,4-BDB and the plant alkaloid mitragynine were identified as strongest stimulators. Their affinity to the hBCRP ATPase was lower than that of sulfasalazine but comparable to that of rosuvastatin, another hBCRP model substrate. Five DOA showed statistically significant hBCRP ATPase inhibition. Determination of IC50 values identified the synthetic cannabinoid receptor agonists JWH-200 and WIN 55,212-2 as the strongest inhibitors comparable to orthovanadate. The present study clearly demonstrated that tested DOA show in part high affinities to the hBCRP within the range of model substrates or inhibitors. Thus, there is a risk of hBCRP-mediated DDI, which needs to be considered in clinical settings.

An easy and fast adenosine 5'-diphosphate quantification procedure based on hydrophilic interaction liquid chromatography-high resolution tandem mass spectrometry for determination of the in vitro adenosine 5'-triphosphatase activity of the human breast cancer resistance protein ABCG2.

Interactions with the human breast cancer resistance protein (hBCRP) significantly influence the pharmacokinetic properties of a drug and can even lead to drug-drug interactions. As efflux pump from the ABC superfamily, hBCRP utilized energy gained by adenosine 5'-triphosphate (ATP) hydrolysis for the transmembrane movement of its substrates, while adenosine 5'-diphosphate (ADP) and inorganic phosphate were released. The ADP liberation can be used to detect interactions with the hBCRP ATPase. An ADP quantification method based on hydrophilic interaction liquid chromatography (HILIC) coupled to high resolution tandem mass spectrometry (HR-MS/MS) was developed and successfully validated in accordance to the criteria of the guideline on bioanalytical method validation by the European Medicines Agency. ATP and adenosine 5'-monophosphate were qualitatively included to prevent interferences. Furthermore, a setup consisting of six sample sets was evolved that allowed detection of hBCRP substrate or inhibitor properties of the test compound. The hBCRP substrate sulfasalazine and the hBCRP inhibitor orthovanadate were used as controls. To prove the applicability of the procedure, the effect of amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir on the hBCRP ATPase activity was tested. Nelfinavir, ritonavir, and saquinavir were identified as hBCRP ATPase inhibitors and none of the five HIV protease inhibitors turned out to be an hBCRP substrate. These findings were in line with a pervious publication.

Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.

Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans.

Studies on the metabolism and toxicological detection of the new psychoactive designer drug 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) in human and rat urine using GC-MS, LC-MS(n), and LC-HR-MS/MS.

25I-NBOMe, a new psychoactive substance, is a potent 5-HT2A receptor agonist with strong hallucinogenic potential. Recently, it was involved in several fatal and non-fatal intoxication cases. The aim of the present work was to study its phase I and II metabolism and its detectability in urine screening approaches. After application of 25I-NBOMe to male Wistar rats, urine was collected over 24 h. The phase I and II metabolites were identified by LC-HR-MS/MS in urine after suitable workup. For the detectability studies, standard urine screening approaches (SUSA) by GC-MS, LC-MS(n), and LC-HR-MS/MS were applied to rat and also to authentic human urine samples submitted for toxicological analysis. Finally, an initial CYP activity screening was performed to identify CYP isoenzymes involved in the major metabolic steps. 25I-NBOMe was mainly metabolized by O-demethylation, O,O-bis-demethylation, hydroxylation, and combinations of these reactions as well as by glucuronidation and sulfation of the main phase I metabolites. All in all, 68 metabolites could be identified. Intake of 25I-NBOMe was detectable mainly via its metabolites by both LC-MS approaches, but not by the GC-MS SUSA. Initial CYP activity screening revealed the involvement of CYP1A2 and CYP3A4 in hydroxylation and CYP2C9 and CYP2C19 in O-demethylation. The presented study demonstrated that 25I-NBOMe was extensively metabolized and could be detected only by the LC-MS screening approaches. Since CYP2C9 and CYP3A4 are involved in initial metabolic steps, drug-drug interactions might occur in certain constellations.

P-glycoprotein interactions of novel psychoactive substances - stimulation of ATP consumption and transport across Caco-2 monolayers.

In contrast to drugs for therapeutic use, there are only few data available concerning interactions between P-glycoprotein (P-gp) and drugs of abuse (DOA). In this work, interactions between structurally diverse DOA and P-gp were investigated using different strategies. First, the effect on the P-gp ATPase activity was studied by monitoring of ATP consumption after addition to recombinant, human P-gp. Second, DOA showing an increased ATP consumption were further characterized regarding their transport across filter grown Caco-2- monolayers. Analyses were performed by luminescence and liquid chromatography-mass spectrometry, respectively. Among the nine DOA initially screened, benzedrone, diclofensine, glaucine, JWH-200, MDBC, WIN-55,212-2 showed an increase of ATP consumption in the ATPase stimulation assay. In Caco-2 transport studies, Glaucine, JWH-200, mitragynine, WIN-55,212-2 could moreover be identified as non-transported substrates, but inhibitors of P-gp activity. Thus, drug-drug or drug-food interactions should be very likely for these compounds.

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 liquid-chromatography high-resolution tandem mass spectrometry approach for quantification of nine cytochrome P450 (CYP) model substrate metabolites in an in vitro CYP inhibition cocktail.

Knowledge about the cytochrome P450 (CYP) inhibition potential of new drug candidates is important for drug development because of its risk of interactions. For novel psychoactive substances (NPS), corresponding data are not available. For developing a general drug inhibition cocktail assay, a liquid-chromatography high-resolution tandem mass spectrometry multi-analyte approach was developed and validated for quantifying low concentrations of O-diethyl phenacetin for CYP 1A2, 7-hydroxy coumarin for CYP 2A6, 4-hydroxy bupropion for CYP 2B6, N-diethyl amodiaquine for CYP 2C8, 4-hydroxy diclofenac for CYP 2C9, 5-hydroxy omeprazole for CYP 2C19, O-dimethyl dextromethorphan for CYP 2D6, 6-hydroxy chlorzoxazone for CYP 2E1, and 6-beta-hydroxy testosterone for CYP 3A in the incubation mixture in the presence of substrates and inhibitors. The tested matrix effects ranged from 63 to 141 % and the recoveries from 95 to 110 %. Time-saving one-point calibration allowed sufficient quantification, although some of the validation results for 7-hydroxy coumarin, 4-hydroxy bupropion, 4-hydroxy diclofenac, and 6-beta-hydroxy testosterone were outside the acceptance criteria (AC) but without influence of the IC50 calculation. Validation showed also that the approach was sensitive and selective using mass spectral multiplexing. In conclusion, the presented assay was suitable for the quantification of the model substrate metabolites and could be used for the development of a CYP inhibition assay for testing most CYPs and a wide range of drugs of abuse.

Methylenedioxy designer drugs: mass spectrometric characterization of their glutathione conjugates by means of liquid chromatography-high-resolution mass spectrometry/mass spectrometry and studies on their glutathionyl transferase inhibition potency.

Methylenedioxy designer drugs of abuse such as 3,4-methylenedioxymethamphetamine (MDMA) can be selectively toxic to serotonergic neurons and glutathione (GSH) adducts have been implicated in its neurotoxicity. The catecholic demethylenyl metabolites of MDMA, 3,4-dihydroxymethamphetamine and 3,4-dihydroxyamphetamine, are metabolically oxidized to the corresponding ortho-quinones, which are highly reactive intermediates. These intermediates can then be conjugated with GSH preventing cellular damage. Furthermore, glutathionyl transferase (GST) activity was described to be irreversibly inhibited by the catechols dopamine, α-methyldopa and their GSH conjugates. Therefore, the aims of the present work were the detection and characterization of GSH conjugates of ten methylenedioxy drugs of abuse and their phase I metabolites as well as to assess their inhibition potency on GST activity. The substrates were incubated using human placental GST with or without preincubation by cytochrome P450 enzymes preparations. GST inhibition was tested using chlorodinitrobenzene GSH conjugation as marker reaction. GSH conjugates were analyzed and characterized using LC-high-resolution-MS/MS. For confirmation of postulated fragmentation patterns, formation of GSH conjugates of selected deuterated analogs (deuterated analogue approach, DAA) of the investigated drugs was explored. For the methylenedioxy amphetamines the following steps could be identified: conjugation of the parent compounds at position 2, 5, 6, of the demethylenyl metabolites at position 2 and 5, and of the further deaminated demethylenyl metabolites at position 2. For the ß-keto-phenylalkylamine and pyrrolidinophenone, conjugation of the demethylenyl metabolites and of the deaminated demethylenyl metabolites at position 2 could be identified. The DAA allowed the differentiation of the 2 and 5/6 isomers by confirmation of the postulated mass spectral fragments. Finally, the tested drugs and phase I metabolites showed no inhibition potency on GST activity.

Michaelis-Menten kinetic analysis of drugs of abuse to estimate their affinity to human P-glycoprotein.

The pharmacokinetics of various important drugs are known to be significantly influenced by the human ABC transporter P-glycoprotein (P-gp), which may lead to clinically relevant drug-drug interactions. In contrast to therapeutic drugs, emerging drugs of abuse (DOA) are sold and consumed without any safety pharmacology testing. Only some studies on their metabolism were published, but none about their affinity to the transporter systems. Therefore, 47 DOAs from various classes were tested for their P-gp affinity using human P-gp (hP-gp) to predict possible drug-drug interactions. DOAs were initially screened for general hP-gp affinity and further characterized by modeling classic Michaelis-Menten kinetics and assessing their K(m) and V(max) values. Among the tested drugs, 12 showed a stimulation of ATPase activity. The most intensive stimulating DOAs were further investigated and compared with the known P-gp model substrates sertraline and verapamil. ATPase stimulation kinetics could be modeled for the entactogen 3,4-methylenedioxy-α-ethylphenethylamine (3,4-BDB), the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI), the abused alkaloid glaucine, the opioid-like drugs N-iso-propyl-1,2-diphenylethylamine (NPDPA), and N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA), with K(m) and V(max) values within the same range as for verapamil or sertraline. As a consequence interactions with other drugs being P-gp substrates might be considered to be very likely and further studies should be encouraged.

Drugs of abuse screening in urine as part of a metabolite-based LC-MSn screening concept.

Today, immunoassays and several chromatographic methods are in use for drug screening in clinical and forensic toxicology and in doping control. For further proof of the authors' new metabolite-based liquid chromatography-mass spectrometry (LC-MS(n)) screening concept, the detectability of drugs of abuse and their metabolites using this screening approach was studied. As previously reported, the corresponding reference library was built up with MS(2) and MS(3) wideband spectra using a LXQ linear ion trap with electrospray ionization in the positive mode and full scan information-dependent acquisition. In addition to the parent drug spectra recorded in methanolic solution, metabolite spectra were identified after protein precipitation of urine from rats after administration of the corresponding drugs and added to the library. This consists now of data of over 900 parent compounds, including 87 drugs of abuse, and of over 2,300 metabolites and artifacts, among them 436 of drugs of abuse. Recovery, process efficiency, matrix effects, and limits of detection for selected drugs of abuse were determined using spiked human urine, and the resulting data have been acceptable. Using two automatic data evaluation tools (ToxID and SmileMS), the intake of 54 of the studied drugs of abuse could be confirmed in urine samples of drug users after protein precipitation and LC separation. The following drugs classes were covered: stimulants, designer drugs, hallucinogens, (synthetic) cannabinoids, opioids, and selected benzodiazepines. The presented LC-MS(n) method complements the well-established gas chromatography-mass spectroscopy procedure in the authors' laboratory.

Absorption, distribution, metabolism and excretion pharmacogenomics of drugs of abuse.

Pharmacologic and toxic effects of xenobiotics, such as drugs of abuse, depend on the genotype and phenotype of an individual, and conversely on the isoenzymes involved in their metabolism and transport. The current knowledge of such isoenzymes of frequently abused therapeutics such as opioids (oxycodone, hydrocodone, methadone, fentanyl, buprenorphine, tramadol, heroin, morphine and codeine), anesthetics (γ-hydroxybutyric acid, propofol, ketamine and phencyclidine) and cognitive enhancers (methylphenidate and modafinil), and some important plant-derived hallucinogens (lysergide, salvinorin A, psilocybin and psilocin), as well as of nicotine in humans are summarized in this article. The isoenzymes (e.g., cytochrome P450, glucuronyltransferases, esterases and reductases) involved in the metabolism of drugs and some pharmacokinetic data are discussed. The relevance of such data is discussed for predicting possible interactions with other xenobiotics, understanding pharmacokinetic behavior and pharmacogenomic variations, assessing toxic risks, developing suitable toxicological analysis procedures, and finally for interpretating drug testing results.

Whole-cell biotransformation assay for investigation of the human drug metabolizing enzyme CYP3A7.

The cytochrome P450 isoform CYP3A7 (wildtype) is the major form of CYP in human fetal liver. Since it is not exclusively expressed in the fetus but also in a significant number of adults, CYP3A7 has been moving into the focus of investigation on adverse drug reactions and interindividual differences in drug metabolism in the last few years. In addition, CYP3A7 is overexpressed in hepatocellular carcinoma (HCC), where it contributes to the elimination of drugs. We here report the development of a convenient and reliable whole-cell system for testing CYP3A7 activity using recombinant fission yeast. As expected, catalytic properties of wild type CYP3A7.1 and its polymorphic form CYP3A7.2 towards DHEA and testosterone resembled those reported previously. Interestingly, both isoforms of CYP3A7 did not metabolize the anti-cancer drug sorafenib (which is approved for the treatment of HCC), while CYP3A4 produced the N-oxide in our system, as expected. This finding suggests that CYP3A7 activity does not influence the effectiveness of this anti-cancer drug against HCC. Furthermore, CYP3A7-expressing fission yeast cells specifically converted a luciferin-derivate (luciferin-PFBE) to a luminescent product and this activity can conveniently be monitored by spectrometry, which allowed the determination of IC50-values for the broad-range P450 inhibitors econazole and miconazole, respectively. We believe that these new tools for a fast and easy investigation of substrates and inhibitors of human CYP3A7 will contribute to the gain of important insights for drug metabolism, efficacy and safety.

Beta-keto amphetamines: studies on the metabolism of the designer drug mephedrone and toxicological detection of mephedrone, butylone, and methylone in urine using gas chromatography-mass spectrometry.

In recent years, a new class of designer drugs has appeared on the drugs of abuse market in many countries, namely, the so-called beta-keto (bk) designer drugs such as mephedrone (bk-4-methylmethamphetamine), butylone (bk-MBDB), and methylone (bk-MDMA). The aim of the present study was to identify the metabolites of mephedrone in rat and human urine using GC-MS techniques and to include mephedrone, butylone, and methylone within the authors' systematic toxicological analysis (STA) procedure. Six phase I metabolites of mephedrone were detected in rat urine and seven in human urine suggesting the following metabolic steps: N-demethylation to the primary amine, reduction of the keto moiety to the respective alcohol, and oxidation of the tolyl moiety to the corresponding alcohols and carboxylic acid. The STA procedure allowed the detection of mephedrone, butylone, methylone, and their metabolites in urine of rats treated with doses corresponding to those reported for abuse of amphetamines. Besides macro-based data evaluation, an automated evaluation using the automated mass spectral deconvolution and identification system was performed. Mephedrone and butylone could be detected also in human urine samples submitted for drug testing. Assuming similar kinetics in humans, the described STA procedure should be suitable for proof of an intake of the bk-designer drugs in human urine.

The breast feeding mother and xenon anaesthesia: four case reports. Breast feeding and xenon anaesthesia.

BACKGROUND: Four nursing mothers consented to anaesthesia for urgent surgery only on condition that their ability to breast feed would not be impaired. METHODS: Following induction of general anaesthesia with propofol and remifentanil, 65-69% xenon supplemented with remifentanil was used as an inhalational anaesthetic for maintenance. RESULTS: After finishing surgery the women could be extubated between 2:52 and 7:22 minutes. The women were fully alert just minutes after extubation and spent about 45 minutes in the recovery room before discharge to a regular ward. They resumed regular breast feeding some time later. The propofol concentration in the blood was measured after 0, 30, 90, and 300 minutes and in the milk after 90 and 300 minutes. Just 90 minutes after extubation, the concentration of propofol in the milk was limited (> 3 mg/l) so that pharmacological effects on the babies were excluded after oral intake. Also, no traces of xenon gas were found in the maternal milk at any time. After propofol induction and maintenance of anaesthesia with xenon in combination with a water-soluble short-acting drug like remifentanil, the concentration of propofol in maternal milk is low (> 3 mg/l 90 min after anesthesia) and harmless after oral intake. CONCLUSIONS: These results, as well as the rapid elimination and absence of metabolism of xenon, are of great interest to nursing mothers. General anaesthesia with propofol for induction only, combined with remifentanil and xenon for maintenance, has not yet been described in breast feeding mothers.

Gas chromatography-mass spectrometry detection of a norfluoxetine artifact in hydrolyzed urine samples may falsely indicate tranylcypromine ingestion.

In several cases, fluoxetine, its metabolites, its known artifacts, and supposedly tranylcypromine were detected in urine using the authors' systematic toxicological analysis (STA) procedure based on acid hydrolysis, extraction, and acetylation. As fluoxetine and tranylcypromine are absolutely contraindicated drugs and in none of the cases was tranylcypromine prescribed, the question of whether the detected compound might have been formed by fluoxetine and/or its metabolites arose. Therefore, rat urine taken after dosing with fluoxetine was screened in the same way. In addition, aqueous solutions of fluoxetine, norfluoxetine, tranylcypromine, and a mixture of the latter two drugs were worked-up and analyzed according to the STA and without hydrolysis. In urine specimens obtained from rats dosed with fluoxetine, tranylcypromine was detected as well as in the solution of worked-up norfluoxetine including hydrolysis. Its underlying mass spectrum could be identified by detailed interpretation of the fragmentation patterns as acetylated 3-phenyl-propyl-2-ene-amine. This compound could be postulated as hydrolysis product of norfluoxetine formed by ether cleavage and water elimination. Although this spectrum shows nearly the same fragmentation patterns as that of acetylated tranylcypromine, both compounds could finally be differentiated by their retention indices and by using the positive-ion chemical ionization mode.

New designer drug alpha-pyrrolidinovalerophenone (PVP): studies on its metabolism and toxicological detection in rat urine using gas chromatographic/mass spectrometric techniques.

The aim of the present study was to identify the metabolites of the new designer drug alpha-pyrrolidinovalerophenone (PVP) in rat urine using GC/MS techniques. Eleven metabolites of PVP could be identified suggesting the following metabolic steps: hydroxylation of the side chain followed by dehydrogenation to the corresponding ketone; hydroxylation of the 2''-position of the pyrrolidine ring followed by dehydrogenation to the corresponding lactam or followed by ring opening to the respective aliphatic aldehyde and further oxidation to the respective carboxylic acid; degradation of the pyrrolidine ring to the corresponding primary amine; and hydroxylation of the phenyl ring, most probably in the 4'-position. The authors' screening procedure for pyrrolidinophenones allowed the detection of PVP metabolites after application of a dose corresponding to a presumed user's dose. In addition, the involvement of nine different human cytochrome P450 (CYP) isoenzymes in the side chain hydroxylation of PVP was investigated and CYP 2B6, 2C19, 2D6, and 3A4 were found to catalyze this reaction.

Human CYP4Z1 catalyzes the in-chain hydroxylation of lauric acid and myristic acid.

Overexpression of human CYP4Z1, a cytochrome P450 enzyme, has been correlated with poor prognosis in human cancer. However, its catalytic properties are not yet known. We expressed this P450 in Schizosaccharomyces pombe and demonstrate by whole-cell biotransformation assays CYP4Z1-dependent in-chain hydroxylation of lauric and myristic acid, which in both cases leads to the formation of four different monohydroxylated products at positions omega-2, omega-3, omega-4, and omega-5, respectively. The CYP4Z1-expressing fission yeast should be a new valuable tool for testing cancer drugs or for the development of new prodrug strategies.

Metabolism and toxicological detection of the designer drug N-(1-phenylcyclohexyl)-3-methoxypropanamine (PCMPA) in rat urine using gas chromatography-mass spectrometry.

Studies on the metabolism and the toxicological detection of the phencyclidine-derived designer drug N-(1-phenylcyclohexyl)-3-methoxypropanamine (PCMPA) in rat urine are described using gas chromatographic-mass spectrometric (GC-MS) techniques. Based on the identified metabolites, the following metabolic pathways could be postulated: N-dealkylation, O-demethylation partially followed by oxidation of the resulting alcohol to the corresponding carboxylic acid, hydroxylation of the cyclohexyl ring at different positions, and aromatic hydroxylation. The formed metabolites were identical to those of the homologue N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA) with exception of the mono hydroxyl metabolites of PCEPA. All PCMPA metabolites were partially excreted in conjugated form. An intake of a common drug users' dose of PCMPA could be detected in rat urine by the authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation. The STA should be suitable for proof of an intake of PCMPA also in human urine assuming similar metabolism.

Metabolism and toxicological detection of the designer drug 4-chloro-2,5-dimethoxyamphetamine in rat urine using gas chromatography-mass spectrometry.

Studies are described on the metabolism and the toxicological analysis of the amphetamine-derived designer drug 4-chloro-2,5-dimethoxyamphetamine (DOC) in rat urine using gas chromatographic-mass spectrometric techniques. The metabolites identified indicated that DOC was metabolized by O-demethylation at position 2 or 5 of the phenyl ring partly followed by glucuronidation and/or sulfation. The authors' systematic toxicological analysis procedure using full-scan gas chromatography-mass spectrometry after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of an intake of a dose of DOC in rat urine that corresponds to a common drug user's dose. Assuming similar metabolism, the STA procedure described should be suitable as proof of an intake of DOC in human urine.