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Forensic Toxicology

, Volume 35, Issue 1, pp 20–32 | Cite as

In vitro and in vivo human metabolism of a new synthetic cannabinoid NM-2201 (CBL-2201)

  • Xingxing Diao
  • Jeremy Carlier
  • Mingshe Zhu
  • Shaokun Pang
  • Robert Kronstrand
  • Karl B. Scheidweiler
  • Marilyn A. Huestis
Original Article

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 µM NM-2201 was incubated in human liver microsomes (HLMs) for up to 1 h; for metabolite profiling, 10 µM of NM-2201 was incubated in human hepatocytes for 3 h. Two authentic urine specimens from NM-2201-positive cases were subjected to β-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 β-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.

Keywords

NM-2201 CBL-2201 Synthetic cannabinoid In vitro human hepatocyte metabolism High-resolution mass spectrometry Authentic human urine specimen 

Notes

Acknowledgments

This research is supported by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health. NM-2201 was generously donated by the US Drug Enforcement Administration. We also appreciate help from Dr. Ariane Wohlfarth in performing the HLMs metabolic stability assays.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. 1.
    Auwärter V, Dresen S, Weinmann W, Müller M, Pütz M, Ferreiros N (2009) ‘Spice’ and other herbal blends: harmless incense or cannabinoid designer drugs? J Mass Spectrom 44:832–837CrossRefPubMedGoogle Scholar
  2. 2.
    Namera A, Kawamura M, Nakamoto A, Saito T, Nagao M (2015) Comprehensive review of the detection methods for synthetic cannabinoids and cathinones. Forensic Toxicol 33:175–194CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Pertwee RG (2006) Cannabinoid pharmacology: the first 66 years. Br J Pharmacol 147(Suppl 1):S163–S171PubMedPubMedCentralGoogle Scholar
  4. 4.
    Huffman JW, Dai D, Martin BR, Compton DR (1994) Design, synthesis and pharmacology of cannabimimetic indoles. Bioorg Med Chem Lett 4:563–566CrossRefGoogle Scholar
  5. 5.
    Cooper ZD (2016) Adverse effects of synthetic cannabinoids: management of acute toxicity and withdrawal. Curr Psychiatry Rep 18:52CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Scheidweiler KB, Jarvis MJ, Huestis MA (2015) Nontargeted SWATH acquisition for identifying 47 synthetic cannabinoid metabolites in human urine by liquid chromatography-high-resolution tandem mass spectrometry. Anal Bioanal Chem 407:883–897CrossRefPubMedGoogle Scholar
  7. 7.
    Hermanns-Clausen M, Kneisel S, Szabo B, Auwärter V (2013) Acute toxicity due to the confirmed consumption of synthetic cannabinoids: clinical and laboratory findings. Addiction 108:534–544CrossRefPubMedGoogle Scholar
  8. 8.
    Seely KA, Lapoint J, Moran JH, Fattore L (2012) Spice drugs are more than harmless herbal blends: a review of the pharmacology and toxicology of synthetic cannabinoids. Prog Neuropsychopharmacol Biol Psychiatry 39:234–243CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Forrester MB, Kleinschmidt K, Schwarz E, Young A (2012) Synthetic cannabinoid and marijuana exposures reported to poison centers. Hum Exp Toxicol 31:1006–1011CrossRefPubMedGoogle Scholar
  10. 10.
    Young AC, Schwarz E, Medina G, Obafemi A, Feng SY, Kane C, Kleinschmidt K (2012) Cardiotoxicity associated with the synthetic cannabinoid, K9, with laboratory confirmation. Am J Emerg Med 30(1320):e1325–e1327Google Scholar
  11. 11.
    Ustundag MF, Ozhan Ibis E, Yucel A, Ozcan H (2015) Synthetic cannabis-induced mania. Case Rep Psychiatry 2015:310930. doi: 10.1155/2015/310930 PubMedPubMedCentralGoogle Scholar
  12. 12.
    Wohlfarth A, Gandhi AS, Pang S, Zhu M, Scheidweiler KB, Huestis MA (2014) Metabolism of synthetic cannabinoids PB-22 and its 5-fluoro analog, 5F-PB-22, by human hepatocyte incubation and high-resolution mass spectrometry. Anal Bioanal Chem 406:1763–1780CrossRefPubMedGoogle Scholar
  13. 13.
    Uchiyama N, Asakawa K, Kikura-Hanajiri R, Tsutsumi T, Hakamatsuka T (2015) A new pyrazole-carboxamide type synthetic cannabinoid AB-CHFUPYCA [N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(cyclohexylmethyl)-3-(4-fluorophenyl)-1H-pyrazole-5-carboxamide] identified in illegal products. Forensic Toxicol 33:367–373CrossRefGoogle Scholar
  14. 14.
    Diao X, Scheidweiler KB, Wohlfarth A, Zhu M, Pang S, Huestis MA (2016) Strategies to distinguish new synthetic cannabinoid FUBIMINA (BIM-2201) intake from its isomer THJ-2201: metabolism of FUBIMINA in human hepatocytes. Forensic Toxicol. doi: 10.1007/s11419-016-0312-2 PubMedCentralGoogle Scholar
  15. 15.
    Holm NB, Nielsen LM, Linnet K (2015) CYP3A4 mediates oxidative metabolism of the synthetic cannabinoid AKB-48. AAPS J 17:1237–1245CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chimalakonda KC, Seely KA, Bratton SM, Brents LK, Moran CL, Endres GW, James LP, Hollenberg PF, Prather PL, Radominska-Pandya A, Moran JH (2012) Cytochrome P450-mediated oxidative metabolism of abused synthetic cannabinoids found in K2/Spice: identification of novel cannabinoid receptor ligands. Drug Metab Dispos 40:2174–2184CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Shevyrin V, Melkozerov V, Nevero A, Eltsov O, Baranovsky A, Shafran Y (2014) Synthetic cannabinoids as designer drugs: new representatives of indol-3-carboxylates series and indazole-3-carboxylates as novel group of cannabinoids. Identification and analytical data. Forensic Sci Int 244:263–275CrossRefPubMedGoogle Scholar
  18. 18.
    Kondrasenko AA, Goncharov EV, Dugaev KP, Rubaylo AI (2015) CBL-2201. Report on a new designer drug: napht-1-yl 1-(5-fluoropentyl)-1H-indole-3-carboxylate. Forensic Sci Int 257:209–213CrossRefPubMedGoogle Scholar
  19. 19.
    Castaneto MS, Wohlfarth A, Pang S, Zhu M, Scheidweiler KB, Kronstrand R, Huestis MA (2015) Identification of AB-FUBINACA metabolites in human hepatocytes and urine using high-resolution mass spectrometry. Forensic Toxicol 33:295–310CrossRefGoogle Scholar
  20. 20.
    Wohlfarth A, Castaneto MS, Zhu M, Pang S, Scheidweiler KB, Kronstrand R, Huestis MA (2015) Pentylindole/pentylindazole synthetic cannabinoids and their 5-fluoro analogs produce different primary metabolites: metabolite profiling for AB-PINACA and 5F-AB-PINACA. AAPS J 17:660–677CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
  22. 22.
    Sobolevsky T, Prasolov I, Rodchenkov G (2012) Detection of urinary metabolites of AM-2201 and UR-144, two novel synthetic cannabinoids. Drug Test Anal 4:745–753CrossRefPubMedGoogle Scholar
  23. 23.
    Andersson M, Diao X, Wohlfarth A, Scheidweiler KB, Huestis MA (2016) Metabolic profiling of new synthetic cannabinoids AMB and 5F-AMB by human hepatocyte and liver microsome incubations and high-resolution mass spectrometry. Rapid Commun Mass Spectrom 30:1067–1078CrossRefPubMedGoogle Scholar
  24. 24.
    Diao X, Pang X, Xie C, Guo Z, Zhong D, Chen X (2014) Bioactivation of 3-n-butylphthalide via sulfation of its major metabolite 3-hydroxy-NBP: mediated mainly by sulfotransferase 1A1. Drug Metab Dispos 42:774–781CrossRefPubMedGoogle Scholar
  25. 25.
    Soars MG, McGinnity DF, Grime K, Riley RJ (2007) The pivotal role of hepatocytes in drug discovery. Chem-Biol Interact 168:2–15CrossRefPubMedGoogle Scholar
  26. 26.
    Castaneto MS, Wohlfarth A, Pang SK, Zhu MS, Scheidweiler KB, Kronstrand R, Huestis MA (2015) Identification of AB-FUBINACA metabolites in human hepatocytes and urine using high-resolution mass spectrometry. Forensic Toxicol 33:295–310CrossRefGoogle Scholar
  27. 27.
    Diao X, Scheidweiler KB, Wohlfarth A, Pang S, Kronstrand R, Huestis MA (2016) In vitro and in vivo human metabolism of synthetic cannabinoids FDU-PB-22 and FUB-PB-22. AAPS J 18:455–464CrossRefPubMedGoogle Scholar
  28. 28.
    Wang P, Zhao Y, Zhu Y, Sun J, Yerke A, Sang S, Yu Z (2016) Metabolism of dictamnine in liver microsomes from mouse, rat, dog, monkey, and human. J Pharm Biomed Anal 119:166–174CrossRefPubMedGoogle Scholar
  29. 29.
    Ellefsen KN, Wohlfarth A, Swortwood MJ, Diao X, Concheiro M, Huestis MA (2016) 4-Methoxy-α-PVP: in silico prediction, metabolic stability, and metabolite identification by human hepatocyte incubation and high-resolution mass spectrometry. Forensic Toxicol 34:61–75CrossRefPubMedGoogle Scholar
  30. 30.
    Baranczewski P, Stanczak A, Sundberg K, Svensson R, Wallin A, Jansson J, Garberg P, Postlind H (2006) Introduction to in vitro estimation of metabolic stability and drug interactions of new chemical entities in drug discovery and development. Pharmacol Rep 58:453–472PubMedGoogle Scholar
  31. 31.
    Swortwood MJ, Carlier J, Ellefsen KN, Wohlfarth A, Diao X, Concheiro-Guisan M, Kronstrand R, Huestis MA (2016) In vitro, in vivo and in silico metabolic profiling of α-pyrrolidinopentiothiophenone, a novel thiophene stimulant. Bioanalysis 8:65–82CrossRefPubMedGoogle Scholar
  32. 32.
    Wang P, Chen H, Sang S (2016) Trapping methylglyoxal by genistein and its metabolites in mice. Chem Res Toxicol 29:406–414CrossRefPubMedGoogle Scholar
  33. 33.
    McNaney CA, Drexler DM, Hnatyshyn SY, Zvyaga TA, Knipe JO, Belcastro JV, Sanders M (2008) An automated liquid chromatography-mass spectrometry process to determine metabolic stability half-life and intrinsic clearance of drug candidates by substrate depletion. Assay Drug Dev Technol 6:121–129CrossRefPubMedGoogle Scholar
  34. 34.
    Diao X-X, Zhong K, Li X-L, Zhong D-F, Chen X-Y (2015) Isomer-selective distribution of 3-n-butylphthalide (NBP) hydroxylated metabolites, 3-hydroxy-NBP and 10-hydroxy-NBP, across the rat blood-brain barrier. Acta Pharmacol Sin 36:1520–1527CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Vikingsson S, Josefsson M, Green H (2015) Identification of AKB-48 and 5F-AKB-48 metabolites in authentic human urine samples using human liver microsomes and time of flight mass spectrometry. J Anal Toxicol 39:426–435CrossRefPubMedGoogle Scholar
  36. 36.
    Lave T, Dupin S, Schmitt C, Valles B, Ubeaud G, Chou RC, Jaeck D, Coassolo P (1997) The use of human hepatocytes to select compounds based on their expected hepatic extraction ratios in humans. Pharmaceut Res 14:152–155CrossRefGoogle Scholar
  37. 37.
    Thomsen R, Nielsen LM, Holm NB, Rasmussen HB, Linnet K (2015) Synthetic cannabimimetic agents metabolized by carboxylesterases. Drug Test Anal 7:565–576CrossRefPubMedGoogle Scholar
  38. 38.
    Michael JP (1999) Quinoline, quinazoline and acridone alkaloids. Nat Prod Rep 16:697–709CrossRefPubMedGoogle Scholar
  39. 39.
    Diao X, Wohlfarth A, Pang S, Scheidweiler KB, Huestis MA (2016) High-resolution mass spectrometry for characterizing the metabolism of synthetic cannabinoid THJ-018 and its 5-fluoro analog THJ-2201 after incubation in human hepatocytes. Clin Chem 62:157–169CrossRefPubMedGoogle Scholar
  40. 40.
    Li XD, Xia SQ, Lv Y, He P, Han J, Wu MC (2004) Conjugation metabolism of acetaminophen and bilirubin in extrahepatic tissues of rats. Life Sci 74:1307–1315CrossRefPubMedGoogle Scholar
  41. 41.
    Gao C, Zhang H, Guo Z, You T, Chen X, Zhong D (2012) Mechanistic studies on the absorption and disposition of scutellarin in humans: selective OATP2B1-mediated hepatic uptake is a likely key determinant for its unique pharmacokinetic characteristics. Drug Metab Dispos 40:2009–2020CrossRefPubMedGoogle Scholar
  42. 42.
    Xie C, Zhou J, Guo Z, Diao X, Gao Z, Zhong D, Jiang H, Zhang L, Chen X (2013) Metabolism and bioactivation of famitinib, a novel inhibitor of receptor tyrosine kinase, in cancer patients. Br J Pharmacol 168:1687–1706CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Gao R, Li L, Xie C, Diao X, Zhong D, Chen X (2012) Metabolism and pharmacokinetics of morinidazole in humans: identification of diastereoisomeric morpholine N +-glucuronides catalyzed by UDP glucuronosyltransferase 1A9. Drug Metab Dispos 40:556–567CrossRefPubMedGoogle Scholar
  44. 44.
    Li Y, Wang K, Jiang Y-Z, Chang X-W, Dai C-F, Zheng J (2014) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits human ovarian cancer cell proliferation. Cell Oncol 37:429–437CrossRefGoogle Scholar
  45. 45.
    Li AP, Gorycki PD, Hengstler JG, Kedderis GL, Koebe HG, Rahmani R, de Sousas G, Silva JM, Skett P (1999) Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel. Chem-Biol Interact 121:117–123CrossRefPubMedGoogle Scholar

Copyright information

© Japanese Association of Forensic Toxicology and Springer Japan (outside the USA) 2016

Authors and Affiliations

  • Xingxing Diao
    • 1
  • Jeremy Carlier
    • 1
  • Mingshe Zhu
    • 2
  • Shaokun Pang
    • 3
  • Robert Kronstrand
    • 4
    • 5
  • Karl B. Scheidweiler
    • 1
  • Marilyn A. Huestis
    • 1
    • 6
  1. 1.Chemistry and Drug Metabolism Section, Clinical Pharmacology and Therapeutics Branch, Intramural Research Program, National Institute on Drug AbuseNational Institutes of HealthBaltimoreUSA
  2. 2.Department of BiotransformationBristol-Myers Squibb, Research and DevelopmentPrincetonUSA
  3. 3.SCIEXRedwood CityUSA
  4. 4.Department of Forensic Genetics and Forensic ToxicologyNational Board of Forensic MedicineLinköpingSweden
  5. 5.Department of Drug ResearchUniversity of LinköpingLinköpingSweden
  6. 6.University of Maryland School of MedicineBaltimoreUSA

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