| 1. |
- Andersson, Maria, et al.
(author)
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Metabolic profiling of new synthetic cannabinoids AMB and 5F-AMB by human hepatocyte and liver microsome incubations and high-resolution mass spectrometry
- 2016
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In: Rapid Communications in Mass Spectrometry. - : WILEY-BLACKWELL. - 0951-4198 .- 1097-0231. ; 30:8, s. 1067-1078
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Journal article (peer-reviewed)abstract
- RationaleAMB (methyl (1-pentyl-1H-indazole-3-carbonyl)-L-valinate)) and its fluoro analog 5F-AMB (methyl (1-(5-fluoropentyl)-1H-indazole-3-carbonyl)-L-valinate) are two new synthetic cannabinoids that are structural analogs of AB-PINACA and 5F-AB-PINACA, respectively. 5F-AMB is scheduled as an illicit drug in China, Germany, Singapore and Japan, and no metabolism data are currently available for either drug. The aim of the present work was to investigate the metabolism of AMB and 5F-AMB and propose appropriate markers to identify their intake in clinical or forensic cases. MethodsAMB and 5F-AMB were incubated in human hepatocytes (10 mol/L) to generate phase I and II metabolites, which were identified with a TripleTOF 5600(+) high-resolution mass spectrometer. AMB and 5F-AMB metabolic stability studies also were performed with human liver microsomes (HLM) to evaluate metabolic clearances, and to adequately design the human hepatocyte experiment. ResultsAMB and 5F-AMB were quickly metabolized in HLM with a 1.1 0.1 and 1.0 +/- 0.2min T-1/2, respectively. The predominant metabolic pathway for AMB and 5F-AMB in hepatocytes was ester hydrolysis, and further oxidation and/or glucuronidation. In total, 19 metabolites were identified for AMB and 17 for 5F-AMB. We describe metabolites to differentiate AMB from 5F-AMB, and metabolites that are common to both analytes due to oxidative defluorination of 5F-AMB. ConclusionsFor the first time, AMB and 5F-AMB metabolism profiles were characterized, providing valuable data for identifying these two novel psychoactive substances. The difficulties of differentiating AMB and 5F-AMB from AB-PINACA/5F-AB-PINACA metabolites also were examined. These data improve the interpretation of urinary markers after AMB and 5F-AMB intake. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA
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| 2. |
- Carlier, Jeremy, et al.
(author)
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In Vitro Metabolite Profiling of ADB-FUBINACA, A New Synthetic Cannabinoid
- 2017
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In: Current Neuropharmacology. - : BENTHAM SCIENCE PUBL LTD. - 1570-159X .- 1875-6190. ; 15:5, s. 682-691
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Journal article (peer-reviewed)abstract
- Background: Metabolite profiling of novel psychoactive substances (NPS) is critical for documenting drug consumption. N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1-Hindazole-3-carboxamide (ADB-FUBINACA) is an emerging synthetic cannabinoid whose toxicological and metabolic data are currently unavailable. Methods: We aimed to determine optimal markers for identifying ADB-FUBINACA intake. Metabolic stability was evaluated with human liver microsome incubations. Metabolites were identified after 1 and 3 h incubation with pooled human hepatocytes, liquid chromatography-high resolution mass spectrometry in positive-ion mode (5600(+) TripleTOF (R), Sciex) and several data mining approaches (MetabolitePilot (TM), Sciex). Results: Metabolite separation was achieved on an Ultra Biphenyl column (Restek (R)); full-scan TOF-MS and information-dependent acquisition MS/MS data were acquired. ADB-FUBINACA microsomal half-life was 39.7 min, with a predicted hepatic clearance of 9.0 mL/min/kg and a 0.5 extraction ratio (intermediate-clearance drug). Twenty-three metabolites were identified. Major metabolic pathways were alkyl and indazole hydroxylation, terminal amide hydrolysis, subsequent glucuronide conjugations, and dehydrogenation. Conclusion: We recommend ADB-FUBINACA hydroxyalkyl, hydroxydehydroalkyl and hydroxylindazole metabolites as ADB-FUBINACA intake markers. N-dealkylated metabolites are not specific ADB-FUBINACA metabolites and should not be used as definitive markers of consumption. This is the first ADB-FUBINACA in vitro metabolism study; in vivo experiments enabling pharmacokinetic and pharmacodynamics studies or urine from authentic clinical/forensic cases are needed to confirm our results.
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| 3. |
- Diao, Xingxing, et al.
(author)
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In vitro and in vivo human metabolism of a new synthetic cannabinoid NM-2201 (CBL-2201)
- 2017
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In: Forensic Toxicology. - : SPRINGER. - 1860-8965 .- 1860-8973. ; 35:1, s. 20-32
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Journal article (peer-reviewed)abstract
- In 2014, NM-2201 (CBL-2201), a novel synthetic cannabinoid (SC), was detected by scientists at Russian and US laboratories. It has been already added to the list of scheduled drugs in Japan, Sweden and Germany. Unfortunately, no human metabolism data are currently available, which makes it challenging to confirm its intake, especially given that all SCs investigated thus far have been found to be extensively metabolized. The present study aims to recommend appropriate marker metabolites by investigating NM-2201 metabolism in human hepatocytes, and to confirm the results in authentic human urine specimens. For the metabolic stability assay, 1 A mu M NM-2201 was incubated in human liver microsomes (HLMs) for up to 1 h; for metabolite profiling, 10 A mu M of NM-2201 was incubated in human hepatocytes for 3 h. Two authentic urine specimens from NM-2201-positive cases were subjected to beta-glucuronidase hydrolysis prior to analysis. The identification of metabolites in hepatocyte samples and urine specimens was achieved with high-resolution mass spectrometry via information-dependent acquisition. NM-2201 was quickly metabolized in HLMs, with an 8.0-min half-life. In human hepatocyte incubation samples, a total of 13 NM-2201 metabolites were identified, generated mainly from ester hydrolysis and further hydroxylation, oxidative defluorination and subsequent glucuronidation. M13 (5-fluoro PB-22 3-carboxyindole) was found to be the major metabolite. In the urine specimens, the parent drug NM-2201 was not detected; M13 was the predominant metabolite after beta-glucuronidase hydrolysis. Therefore, based on the results of our study, we recommend M13 as a suitable urinary marker metabolite for confirming NM-2201 and/or 5F-PB-22 intake.
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| 4. |
- Diao, Xingxing, et al.
(author)
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In Vitro and In Vivo Human Metabolism of Synthetic Cannabinoids FDU-PB-22 and FUB-PB-22
- 2016
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In: AAPS Journal. - : SPRINGER. - 1550-7416. ; 18:2, s. 455-464
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Journal article (peer-reviewed)abstract
- In 2014, FDU-PB-22 and FUB-PB-22, two novel synthetic cannabinoids, were detected in herbal blends in Japan, Russia, and Germany and were quickly added to their scheduled drugs list. Unfortunately, no human metabolism data are currently available, making it challenging to confirm their intake. The present study aims to identify appropriate analytical markers by investigating FDU-PB-22 and FUB-PB-22 metabolism in human hepatocytes and confirm the results in authentic urine specimens. For metabolic stability, 1 mu M FDU-PB-22 and FUB-PB-22 was incubated with human liver microsomes for up to 1 h; for metabolite profiling, 10 mu M was incubated with human hepatocytes for 3 h. Two authentic urine specimens from FDU-PB-22 and FUB-PB-22 positive cases were analyzed after beta-glucuronidase hydrolysis. Metabolite identification in hepatocyte samples and urine specimens was accomplished by high-resolution mass spectrometry using information-dependent acquisition. Both FDU-PB-22 and FUB-PB-22 were rapidly metabolized in HLM with half-lives of 12.4 and 11.5 min, respectively. In human hepatocyte samples, we identified seven metabolites for both compounds, generated by ester hydrolysis and further hydroxylation and/or glucuronidation. After ester hydrolysis, FDU-PB-22 and FUB-PB-22 yielded the samemetabolite M7, fluorobenzylindole-3-carboxylic acid (FBI-COOH). M7 and M6 (hydroxylated FBI-COOH) were the major metabolites. In authentic urine specimens after beta-glucuronidase hydrolysis, M6 and M7 also were the predominant metabolites. Based on our study, we recommend M6 (hydroxylated FBI-COOH) and M7 (FBI-COOH) as suitable urinary markers for documenting FDU-PB-22 and/or FUB-PB-22 intake.
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| 5. |
- Diao, Xingxing, et al.
(author)
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Strategies to distinguish new synthetic cannabinoid FUBIMINA (BIM-2201) intake from its isomer THJ-2201: metabolism of FUBIMINA in human hepatocytes
- 2016
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In: Forensic Toxicology. - : SPRINGER. - 1860-8965 .- 1860-8973. ; 34:2, s. 256-267
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Journal article (peer-reviewed)abstract
- Since 2013, a new drugs-of-abuse trend attempts to bypass drug legislation by marketing isomers of scheduled synthetic cannabinoids (SCs), e.g., FUBIMINA (BIM-2201) and THJ-2201. It is much more challenging to confirm a specific isomers intake and distinguish it from its structural analog because the isomers and their major metabolites usually have identical molecular weights and display the same product ions. Here, we investigated isomers FUBIMINA and THJ-2201 and propose strategies to distinguish their consumption. THJ-2201 was scheduled in the US, Japan, and Europe; however, FUBIMINA is easily available on the Internet. We previously investigated THJ-2201 metabolism in human hepatocytes, but human FUBIMINA metabolism is unknown. We aim to characterize FUBIMINA metabolism in human hepatocytes, recommend optimal metabolites to confirm its consumption, and propose strategies to distinguish between intakes of FUBIMINA and THJ-2201. FUBIMINA (10 mu M) was incubated in human hepatocytes for 3 h, and metabolites were characterized with high-resolution mass spectrometry (HR-MS). We identified 35 metabolites generated by oxidative defluorination, further carboxylation, hydroxylation, dihydrodiol formation, glucuronidation, and their combinations. We recommend 5-OH-BIM-018 (M34), BIM-018 pentanoic acid (M33), and BIM-018 pentanoic acid dihydrodiol (M7) as FUBIMINA specific metabolites. THJ-2201 produced specific metabolite markers 5-OH-THJ-018 (F26), THJ-018 pentanoic acid (F25), and hydroxylated THJ-2201 (F13). Optimized chromatographic conditions to achieve different retention times and careful selection of specific product ion spectra enabled differentiation of isomeric metabolites, in this case FUBIMINA from THJ-2201. Our HR-MS approach should be applicable for differentiating future isomeric SCs, which is especially important when different isomers have different legal status.
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| 6. |
- Swortwood, Madeleine J., et al.
(author)
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First metabolic profile of PV8, a novel synthetic cathinone, in human hepatocytes and urine by high-resolution mass spectrometry
- 2016
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In: Analytical and Bioanalytical Chemistry. - : SPRINGER HEIDELBERG. - 1618-2642 .- 1618-2650. ; 408:18, s. 4845-4856
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Journal article (peer-reviewed)abstract
- Novel psychoactive substances (NPS) are ever changing on the drug market, making it difficult for toxicology laboratory methods to stay current with so many new drugs. Recently, PV8, a synthetic pyrrolidinophenone, was detected in seized products in Japan (2013), The Netherlands (2014), and Germany (2014). There are no controlled PV8 administration studies, and no pharmacodynamic and pharmacokinetic data. The objective was to determine PV8s metabolic stability in human liver microsome (HLM) incubation and its metabolism following human hepatocyte incubation and high-resolution mass spectrometry (HRMS) with a Thermo Scientific Q-Exactive. Data were acquired with a full-scan data-dependent mass spectrometry method. Scans were thoroughly data mined with different data processing algorithms and analyzed in WebMetaBase. PV8 exhibited a relatively short 28.8 min half-life, with an intrinsic 24.2 mu L/min/mg microsomal clearance. This compound is predicted to be an intermediate clearance drug with an estimated human 22.7 mL/min/kg hepatic clearance. Metabolic pathways identified in vitro included: hydroxylation, ketone reduction, carboxylation, N-dealkylation, iminium formation, dehydrogenation, N-oxidation, and carbonylation. The top three in vitro metabolic pathways were di-hydroxylation amp;gt; ketone reduction amp;gt; gamma-lactam formation. Authentic urine specimen analyses revealed the top three metabolic pathways were aliphatic hydroxylation amp;gt; ketone reduction + aliphatic hydroxylation amp;gt; aliphatic carboxylation, although the most prominent peak was parent PV8. These data provide useful urinary metabolite targets (aliphatic hydroxylation, aliphatic hydroxylation + ketone reduction, aliphatic carboxylation, and di-hydroxylation) for forensic and clinical testing, and focus reference standard companies synthetic efforts to provide commercially available standards needed for PV8 biological specimen testing.
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| 7. |
- Swortwood, Madeleine J., et al.
(author)
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In vitro, in vivo and in silico metabolic profiling of -pyrrolidinopentiothiophenone, a novel thiophene stimulant
- 2016
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In: Bioanalysis. - : FUTURE SCI LTD. - 1757-6180 .- 1757-6199. ; 8:1, s. 65-82
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Journal article (peer-reviewed)abstract
- Background: Little or no pharmacological or toxicological data are available for novel psychoactive substances when they first emerge, making their identification and interpretation in biological matrices challenging. Materials & methods: A new synthetic cathinone, alpha-pyrrolidinopentiothiophenone (alpha-PVT), was incubated with hepatocytes and samples were analyzed using liquid chromatography coupled to a Q Exactive(TM) Orbitrap mass spectrometer. Authentic urine specimens from suspected -PVT cases were also analyzed. Scans were data mined with Compound Discoverer for identification and structural elucidation of metabolites. Results/conclusion: Seven alpha-PVT metabolites were identified in hepatocyte incubations, and in the authentic urine samples, also with an additional monohydroxylated product and a glucuronide of low intensity. alpha-PVT dihydroxypyrrolidinyl, alpha-PVT 2-ketopyrrolidinyl, alpha-PVT hydroxythiophenyl and alpha-PVT thiophenol had the most intense in vivo signals.
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| 8. |
- Wohlfarth, Ariane, et al.
(author)
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25C-NBOMe and 25I-NBOMe metabolite studies in human hepatocytes, in vivo mouse and human urine with high-resolution mass spectrometry.
- 2017
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In: Drug Testing and Analysis. - : John Wiley & Sons. - 1942-7603 .- 1942-7611. ; 9:5, s. 680-698
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Journal article (peer-reviewed)abstract
- 25C-NBOMe and 25I-NBOMe are potent hallucinogenic drugs that recently emerged as new psychoactive substances. To date, a few metabolism studies were conducted for 25I-NBOMe, whereas 25C-NBOMe metabolism data are scarce. Therefore, we investigated the metabolic profile of these compounds in human hepatocytes, an in vivo mouse model and authentic human urine samples from forensic cases. Cryopreserved human hepatocytes were incubated for 3 h with 10 μM 25C-NBOMe and 25I-NBOMe; samples were analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS) on an Accucore C18 column with a Thermo QExactive; data analysis was performed with Compound Discoverer software (Thermo Scientific). Mice were administered 1.0 mg drug/kg body weight intraperitoneally, urine was collected for 24 h and analyzed (with or without hydrolysis) by LC-HRMS on an Acquity HSS T3 column with an Agilent 6550 QTOF; data were analyzed manually and with WebMetabase software (Molecular Discovery). Human urine samples were analyzed similarly. In vitro and in vivo results matched well. 25C-NBOMe and 25I-NBOMe were predominantly metabolized by O-demethylation, followed by O-di-demethylation and hydroxylation. All methoxy groups could be demethylated; hydroxylation preferably occurred at the NBOMe ring. Phase I metabolites were extensively conjugated in human urine with glucuronic acid and sulfate. Based on these data and a comparison with synthesized reference standards for potential metabolites, specific and abundant 25C-NBOMe urine targets are 5'-desmethyl 25C-NBOMe, 25C-NBOMe and 5-hydroxy 25C-NBOMe, and for 25I-NBOMe 2' and 5'-desmethyl 25I-NBOMe and hydroxy 25I-NBOMe. These data will help clinical and forensic laboratories to develop analytical methods and to interpret results. Copyright © 2016 John Wiley & Sons, Ltd.
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