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Structure-metabolism relationships of valine and tert-leucine-derived synthetic cannabinoid receptor agonists: a systematic comparison of the in vitro phase I metabolism using pooled human liver microsomes and high-resolution mass spectrometry

  • Florian Franz
  • Hanna Jechle
  • Maurice Wilde
  • Verena Angerer
  • Laura M. Huppertz
  • Mitchell Longworth
  • Michael Kassiou
  • Manfred Jung
  • Volker AuwärterEmail author
Original Article

Abstract

Purpose

Synthetic cannabinoid receptor agonists, commonly referred to as ‘synthetic cannabinoids’ (SCs), gained popularity as recreational drugs due to their cannabis-like effects. The subclass of valine or tert-leucine-derived SCs has dominated the ‘designer drug’ market in recent years and has been associated with several severe intoxication cases. Most SCs are highly lipophilic compounds and are extensively metabolized prior to renal excretion. Hence, for drug detection in urine samples, the major metabolites of new compounds have to be identified first. The aim of this study was to elucidate structure-metabolism relationships (SMRs) of valine and tert-leucine-derived SCs enabling in-depth understanding of their phase I biotransformation and facilitating the prediction of suitable analytical targets for urine analysis.

Methods

After incubation of 32 different valine/tert-leucine-derived SCs with pooled human liver microsomes (pHLM), the phase I metabolite profile of each compound was characterized using liquid chromatographyquadrupole time-of-flight mass spectrometry. By comparing chemical-structural analogs with the relative abundances of their metabolites, SMRs were studied.

Results

The terminal functionality (amide vs. methyl ester), the amino acid side chain (valine vs. tert-leucine), the core ring system (indole vs. indazole), and the N-alkyl side chain (cyclohexyl methyl vs. pentyl vs. 5-fluoropentyl vs. 4-fluorobenzyl) showed distinct differences of metabolic dehalogenation, dehydrogenation, formation of dihydrodiols, hydrolysis, hydroxylation, and N-dealkylation.

Conclusions

The presented pHLM approach proved to be an effective tool for systematic investigation of SMRs. The information gained from this work may be useful for predicting potential SC metabolites for urine analysis.

Keywords

ICA INACA Metabolite New psychoactive substance 

Notes

Acknowledgements

The authors would like to thank colleagues from different institutions for providing synthetic cannabinoid standards: Dr. Sonja Klemenc (Slovenian National Forensic Laboratory, Ljubljana, Slovenia) and all participants of the ‘RESPONSE’ project providing ADB-CHMICA as well as AMB-CHMICA; Dr. Előd Hidvégi and his group (Hungarian National Institute of Forensic Sciences, Budapest, Hungary) providing MDMB-FUBICA. This study was financially supported by the European Commission [Grants JUST/2011/DPIP/AG/3597 and JUST/2013/ISEC/DRUGS/AG/6421] and the Deutsche Forschungsgemeinschaft [Grant INST 380/92-1 FUGG].

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.

Supplementary material

11419_2018_462_MOESM1_ESM.pdf (1.6 mb)
Supplementary material 1 (PDF 1653 kb)

References

  1. 1.
    Di Marzo V, Bifulco M, De Petrocellis L (2004) The endocannabinoid system and its therapeutic exploitation. Nat Rev Drug Discov 3:771–784.  https://doi.org/10.1038/nrd1495 CrossRefGoogle Scholar
  2. 2.
    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 44:832–837.  https://doi.org/10.1002/jms.1558 CrossRefGoogle Scholar
  3. 3.
    Uchiyama N, Kikura-Hanajiri R, Kawahara N, Haishima Y, Goda Y (2009) Identification of a cannabinoid analog as a new type of designer drug in a herbal product. Chem Pharm Bull 57:439–441.  https://doi.org/10.1248/cpb.57.439 CrossRefGoogle Scholar
  4. 4.
    EMCDDA (2017) Perspectives on drugs: synthetic cannabinoids in Europe. Publications Office of the European Union, Luxembourg. http://www.emcdda.europa.eu/system/files/publications/2753/POD_Synthetic%20cannabinoids_0.pdf. Accessed 27 Sep 2018
  5. 5.
    European database on new drugs (EDND) european monitoring centre for drugs and drug addiction (EMCDDA). https://ednd.emcdda.europa.eu/html.cfm/index6555EN.html. Accessed 22 Jun 2018
  6. 6.
    Aldlgan AA, Torrance HJ (2016) Bioanalytical methods for the determination of synthetic cannabinoids and metabolites in biological specimens. TrAC Trends Anal Chem 80:444–457.  https://doi.org/10.1016/j.trac.2016.03.025 CrossRefGoogle Scholar
  7. 7.
    Uchiyama N, Matsuda S, Wakana D, Kikura-Hanajiri R, Goda Y (2013) New cannabimimetic indazole derivatives, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA) and N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA) identified as designer drugs in illegal products. Forensic Toxicol 31:93–100.  https://doi.org/10.1007/s11419-012-0171-4 CrossRefGoogle Scholar
  8. 8.
    Buchler IP, Hayes MJ, Hegde SG, Hockerman SL, Jones DE, Kortum SW, Rico JG, Tenbrink RE, Wu KK (2009) Indazole derivatives. Patent WO2009106980A2Google Scholar
  9. 9.
    Buchler IP, Hayes MJ, Hegde SG, Hockerman SL, Jones DE, Kortum SW, Rico JG, TenBrink RE, Wu KK (2011) Indazole derivatives. Patent US2011/0028447A1Google Scholar
  10. 10.
    Banister SD, Moir M, Stuart J, Kevin RC, Wood KE, Longworth M, Wilkinson SM, Beinat C, Buchanan AS, Glass M, Connor M, McGregor IS, Kassiou M (2015) Pharmacology of indole and indazole synthetic cannabinoid designer drugs AB-FUBINACA, ADB-FUBINACA, AB-PINACA, ADB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, ADBICA, and 5F-ADBICA. ACS Chem Neurosci 6:1546–1559.  https://doi.org/10.1021/acschemneuro.5b00112 CrossRefGoogle Scholar
  11. 11.
    Banister SD, Longworth M, Kevin R, Sachdev S, Santiago M, Stuart J, Mack JBC, Glass M, McGregor IS, Connor M, Kassiou M (2016) Pharmacology of valinate and tert-leucinate synthetic cannabinoids 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and their analogues. ACS Chem Neurosci 7:1241–1254.  https://doi.org/10.1021/acschemneuro.6b00137 CrossRefGoogle Scholar
  12. 12.
    Longworth M, Banister SD, Mack JBC, Glass M, Connor M, Kassiou M (2016) The 2-alkyl-2H-indazole regioisomers of synthetic cannabinoids AB-CHMINACA, AB-FUBINACA, AB-PINACA, and 5F-AB-PINACA are possible manufacturing impurities with cannabimimetic activities. Forensic Toxicol 34:286–303.  https://doi.org/10.1007/s11419-016-0316-y CrossRefGoogle Scholar
  13. 13.
    Andernach L, Pusch S, Weber C, Schollmeyer D, Münster-Müller S, Pütz M, Opatz T (2016) Absolute configuration of the synthetic cannabinoid MDMB-CHMICA with its chemical characteristics in illegal products. Forensic Toxicol 34:344–352.  https://doi.org/10.1007/s11419-016-0321-1 CrossRefGoogle Scholar
  14. 14.
    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 30:1067–1078.  https://doi.org/10.1002/rcm.7538 CrossRefGoogle Scholar
  15. 15.
    Carlier J, Diao X, Scheidweiler KB, Huestis MA (2017) Distinguishing intake of new synthetic cannabinoids ADB-PINACA and 5F-ADB-PINACA with human hepatocyte metabolites and high-resolution mass spectrometry. Clin Chem 63:1008–1021.  https://doi.org/10.1373/clinchem.2016.267575 CrossRefGoogle Scholar
  16. 16.
    Carlier J, Diao X, Wohlfarth A, Scheidweiler K, Huestis MA (2017) In vitro metabolite profiling of ADB-FUBINACA, a new synthetic cannabinoid. Curr Neuropharmacol 15:682–691.  https://doi.org/10.2174/1570159X15666161108123419 CrossRefGoogle Scholar
  17. 17.
    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 33:295–310.  https://doi.org/10.1007/s11419-015-0275-8 CrossRefGoogle Scholar
  18. 18.
    Erratico C, Negreira N, Norouzizadeh H, Covaci A, Neels H, Maudens K, van Nuijs ALN (2015) In vitro and in vivo human metabolism of the synthetic cannabinoid AB-CHMINACA. Drug Test Anal 7:866–876.  https://doi.org/10.1002/dta.1796 CrossRefGoogle Scholar
  19. 19.
    Franz F, Angerer V, Moosmann B, Auwärter V (2017) Phase I metabolism of the highly potent synthetic cannabinoid MDMB-CHMICA and detection in human urine samples. Drug Test Anal 9:744–753.  https://doi.org/10.1002/dta.2049 CrossRefGoogle Scholar
  20. 20.
    Mogler L, Franz F, Rentsch D, Angerer V, Weinfurtner G, Longworth M, Banister SD, Kassiou M, Moosmann B, Auwärter V (2018) Detection of the recently emerged synthetic cannabinoid 5F-MDMB-PICA in ‘legal high’ products and human urine samples. Drug Test Anal 10:196–205.  https://doi.org/10.1002/dta.2201 CrossRefGoogle Scholar
  21. 21.
    Hasegawa K, Minakata K, Gonmori K, Nozawa H, Yamagishi I, Watanabe K, Suzuki O (2018) Identification and quantification of predominant metabolites of synthetic cannabinoid MAB-CHMINACA in an authentic human urine specimen. Drug Test Anal 10:365–371.  https://doi.org/10.1002/dta.2220 CrossRefGoogle Scholar
  22. 22.
    Kavanagh P, Grigoryev A, Krupina N (2017) Detection of metabolites of two synthetic cannabimimetics, MDMB-FUBINACA and ADB-FUBINACA, in authentic human urine specimens by accurate mass LC–MS: a comparison of intersecting metabolic patterns. Forensic Toxicol 35:284–300.  https://doi.org/10.1007/s11419-017-0356-y CrossRefGoogle Scholar
  23. 23.
    Kusano M, Zaitsu K, Taki K, Hisatsune K, Nakajima J, Moriyasu T, Asano T, Hayashi Y, Tsuchihashi H, Ishii A (2018) Fatal intoxication by 5F-ADB and diphenidine: detection, quantification and investigation of their main metabolic pathways in humans by LC/MS/MS and LC/Q-TOFMS. Drug Test Anal 10:284–293.  https://doi.org/10.1002/dta.2215 CrossRefGoogle Scholar
  24. 24.
    Li J, Liu C, Li T, Hua Z (2018) UPLC-HR-MS/MS-based determination study on the metabolism of four synthetic cannabinoids, ADB-FUBICA, AB-FUBICA, AB-BICA and ADB-BICA, by human liver microsomes. Biomed Chromatogr 32:e4113.  https://doi.org/10.1002/bmc.4113 CrossRefGoogle Scholar
  25. 25.
    Takayama T, Suzuki M, Todoroki K, Inoue K, Min JZ, Kikura-Hanajiri R, Goda Y, Toyo’oka T (2014) UPLC/ESI-MS/MS-based determination of metabolism of several new illicit drugs, ADB-FUBINACA, AB-FUBINACA, AB-PINACA, QUPIC, 5F-QUPIC and α-PVT, by human liver microsome. Biomed Chromatogr 28:831–838.  https://doi.org/10.1002/bmc.3155 CrossRefGoogle Scholar
  26. 26.
    Thomsen R, Nielsen LM, Holm NB, Rasmussen HB, Linnet K, Consortium I (2015) Synthetic cannabimimetic agents metabolized by carboxylesterases. Drug Test Anal 7:565–576.  https://doi.org/10.1002/dta.1731 CrossRefGoogle Scholar
  27. 27.
    Vikingsson S, Gréen H, Brinkhagen L, Mukhtar S, Josefsson M (2016) Identification of AB-FUBINACA metabolites in authentic urine samples suitable as urinary markers of drug intake using liquid chromatography quadrupole tandem time of flight mass spectrometry. Drug Test Anal 8:950–956.  https://doi.org/10.1002/dta.1896 CrossRefGoogle Scholar
  28. 28.
    Wohlfarth A, Castaneto MS, Zhu MS, Pang SK, 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 17:660–677.  https://doi.org/10.1208/s12248-015-9721-0 CrossRefGoogle Scholar
  29. 29.
    Wurita A, Hasegawa K, Minakata K, Gonmori K, Nozawa H, Yamagishi I, Suzuki O, Watanabe K (2016) Identification and quantification of metabolites of AB-CHMINACA in a urine specimen of an abuser. Legal Med 19:113–118.  https://doi.org/10.1016/j.legalmed.2015.07.011 CrossRefGoogle Scholar
  30. 30.
    Carlier J, Diao X, Sempio C, Huestis MA (2017) Identification of new synthetic cannabinoid ADB-CHMINACA (MAB-CHMINACA) metabolites in human hepatocytes. AAPS J 19:568–577.  https://doi.org/10.1208/s12248-016-0037-5 CrossRefGoogle Scholar
  31. 31.
    Mardal M, Dalsgaard PW, Qi B, Mollerup CB, Annaert P, Linnet K (2018) Metabolism of the synthetic cannabinoids AMB-CHMICA and 5C-AKB48 in pooled human hepatocytes and rat hepatocytes analyzed by UHPLC-(IMS)-HR-MSE. J Chromatogr B 1083:189–197.  https://doi.org/10.1016/j.jchromb.2018.03.016 CrossRefGoogle Scholar
  32. 32.
    Grigoryev A, Kavanagh P, Pechnikov A (2016) Human urinary metabolite pattern of a new synthetic cannabimimetic, methyl 2-(1-(cyclohexylmethyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate. Forensic Toxicol 34:316–328.  https://doi.org/10.1007/s11419-016-0319-8 CrossRefGoogle Scholar
  33. 33.
    Ullrich R, Hofrichter M (2007) Enzymatic hydroxylation of aromatic compounds. Cell Mol Life Sci 64:271–293.  https://doi.org/10.1007/s00018-007-6362-1 CrossRefGoogle Scholar

Copyright information

© Japanese Association of Forensic Toxicology 2019

Authors and Affiliations

  • Florian Franz
    • 1
    • 2
  • Hanna Jechle
    • 1
    • 2
  • Maurice Wilde
    • 1
    • 2
    • 3
  • Verena Angerer
    • 1
    • 2
  • Laura M. Huppertz
    • 1
    • 2
  • Mitchell Longworth
    • 4
  • Michael Kassiou
    • 4
  • Manfred Jung
    • 5
  • Volker Auwärter
    • 1
    • 2
    Email author
  1. 1.Institute of Forensic Medicine, Medical CenterUniversity of FreiburgFreiburgGermany
  2. 2.Faculty of MedicineUniversity of FreiburgFreiburgGermany
  3. 3.Hermann Staudinger Graduate SchoolUniversity of FreiburgFreiburgGermany
  4. 4.School of ChemistryThe University of SydneySydneyAustralia
  5. 5.Institute of Pharmaceutical SciencesUniversity of FreiburgFreiburgGermany

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