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Pentylindole/Pentylindazole Synthetic Cannabinoids and Their 5-Fluoro Analogs Produce Different Primary Metabolites: Metabolite Profiling for AB-PINACA and 5F-AB-PINACA

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Abstract

Whereas non-fluoropentylindole/indazole synthetic cannabinoids appear to be metabolized preferably at the pentyl chain though without clear preference for one specific position, their 5-fluoro analogs’ major metabolites usually are 5-hydroxypentyl and pentanoic acid metabolites. We determined metabolic stability and metabolites of N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA) and 5-fluoro-AB-PINACA (5F-AB-PINACA), two new synthetic cannabinoids, and investigated if results were similar. In silico prediction was performed with MetaSite (Molecular Discovery). For metabolic stability, 1 μmol/L of each compound was incubated with human liver microsomes for up to 1 h, and for metabolite profiling, 10 μmol/L was incubated with pooled human hepatocytes for up to 3 h. Also, authentic urine specimens from AB-PINACA cases were hydrolyzed and extracted. All samples were analyzed by liquid chromatography high-resolution mass spectrometry on a TripleTOF 5600+ (AB SCIEX) with gradient elution (0.1% formic acid in water and acetonitrile). High-resolution full-scan mass spectrometry (MS) and information-dependent acquisition MS/MS data were analyzed with MetabolitePilot (AB SCIEX) using different data processing algorithms. Both drugs had intermediate clearance. We identified 23 AB-PINACA metabolites, generated by carboxamide hydrolysis, hydroxylation, ketone formation, carboxylation, epoxide formation with subsequent hydrolysis, or reaction combinations. We identified 18 5F-AB-PINACA metabolites, generated by the same biotransformations and oxidative defluorination producing 5-hydroxypentyl and pentanoic acid metabolites shared with AB-PINACA. Authentic urine specimens documented presence of these metabolites. AB-PINACA and 5F-AB-PINACA produced suggested metabolite patterns. AB-PINACA was predominantly hydrolyzed to AB-PINACA carboxylic acid, carbonyl-AB-PINACA, and hydroxypentyl AB-PINACA, likely in 4-position. The most intense 5F-AB-PINACA metabolites were AB-PINACA pentanoic acid and 5-hydroxypentyl-AB-PINACA.

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Abbreviations

AB-PINACA:

N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide

CB:

Cannabinoid

CL:

Clearance

cps:

Counts per second

CYP:

Cytochrome P450

ER:

Extraction ratio

ESI:

Electrospray ionization

FDA:

Food and Drug Administration

HLM:

Human liver microsomes

HRMS:

High-resolution mass spectrometry

IDA:

Information-dependent acquisition

LC-MS:

Liquid chromatography-mass spectrometry

MDF:

Mass defect filter

MS:

Mass spectrometry

MW:

Molecular weight

NADPH:

Nicotinamide adenine dinucleotide phosphate reduced form

NPS:

Novel psychoactive substances

Q:

Qualifier

Q-TOF:

Quadrupole/time of flight

T:

Target

TOF:

Time of flight

REFERENCES

  1. European Monitoring Centre for Drugs and Drug Addiction. Perspectives on drugs: synthetic cannabinoids in Europe. 2014.

  2. American Association of Poison Control Centers. Synthetic marijuana data, updated January 31, 2015. https://aapcc.s3.amazonaws.com/files/library/Syn_Marijuana_Web_Data_through_1.2015.pdf

  3. Gurney SMR, Scott KS, Kacinko SL, Presley BC, Logan BK. Pharmacology, toxicology, and adverse effects of synthetic cannabinoid drugs. Forensic Sci Rev. 2013;26:53–77.

    Google Scholar 

  4. ElSohly MA, Gul W, Wanas AS, Radwan MM. Synthetic cannabinoids: analysis and metabolites. Life Sci. 2014;97(1):78–90.

    Article  CAS  PubMed  Google Scholar 

  5. Chimalakonda KC, Seely KA, Bratton SM, Brents LK, Moran CL, Endres GW, et al. Cytochrome P450-mediated oxidative metabolism of abused synthetic cannabinoids found in K2/Spice: identification of novel cannabinoid receptor ligands. Drug Metab Dispos. 2012;40(11):2174–84.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. De Brabanter N, Esposito S, Geldof L, Lootens L, Meuleman P, Leroux-Roels G, et al. In vitro and in vivo metabolisms of 1-pentyl-3-(4-methyl-1-naphthoyl)indole (JWH-122). Forensic Toxicol. 2013;31(2):212–22.

    Article  Google Scholar 

  7. De Brabanter N, Esposito S, Tudela E, Lootens L, Meuleman P, Leroux-Roels G, et al. In vivo and in vitro metabolism of the synthetic cannabinoid JWH-200. Rapid Commun Mass Spectrom. 2013;27(18):2115–26.

    Article  PubMed  Google Scholar 

  8. Gandhi A, Zhu M, Pang S, Wohlfarth A, Scheidweiler K, Liu H-f, et al. First characterization of AKB-48 metabolism, a novel synthetic cannabinoid, using human hepatocytes and high-resolution mass spectrometry. AAPS J. 2013;15(4):1091–9.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Gandhi AS, Wohlfarth A, Zhu M, Pang S, Castaneto M, Scheidweiler KB, et al. High-resolution mass spectrometric metabolite profiling of a novel synthetic designer drug, N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135), using cryopreserved human hepatocytes and assessment of metabolic stability with human liver microsomes. Drug Test Anal. 2014. doi:10.1002/dta.1662.

    Google Scholar 

  10. Gandhi AS, Zhu M, Pang S, Wohlfarth A, Scheidweiler KB, Huestis MA. Metabolite profiling of RCS-4, a novel synthetic cannabinoid designer drug, using human hepatocyte metabolism and time of flight mass spectrometry. Bioanalysis. 2014;6(11):1471–85.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Grigoryev A, Kavanagh P, Melnik A. The detection of the urinary metabolites of 1-[(5-fluoropentyl)-1H-indol-3-yl]-(2-iodophenyl)methanone (AM-694), a high affinity cannabimimetic, by gas chromatography-mass spectrometry. Drug Test Anal. 2013;5(2):110–5.

    Article  CAS  PubMed  Google Scholar 

  12. Grigoryev A, Kavanagh P, Melnik A. The detection of the urinary metabolites of 3-[(adamantan-1-yl)carbonyl]-1-pentylindole (AB-001), a novel cannabimimetic, by gas chromatography-mass spectrometry. Drug Test Anal. 2012;4(6):519–24.

    Article  CAS  PubMed  Google Scholar 

  13. Grigoryev A, Melnik A, Savchuk S, Simonov A, Rozhanets V. Gas and liquid chromatography-mass spectrometry studies on the metabolism of the synthetic phenylacetylindole cannabimimetic JWH-250, the psychoactive component of smoking mixtures. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879(25):2519–26.

    Article  CAS  PubMed  Google Scholar 

  14. Grigoryev A, Savchuk S, Melnik A, Moskaleva N, Dzhurko J, Ershov M, et al. Chromatography-mass spectrometry studies on the metabolism of synthetic cannabinoids JWH-018 and JWH-073, psychoactive components of smoking mixtures. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879(15–16):1126–36.

    Article  CAS  PubMed  Google Scholar 

  15. Hutter M, Broecker S, Kneisel S, Auwärter V. Identification of the major urinary metabolites in man of seven synthetic cannabinoids of the aminoalkylindole type present as adulterants in ‘herbal mixtures’ using LC-MS/MS techniques. J Mass Spectrom. 2012;47(1):54–65.

    Article  CAS  PubMed  Google Scholar 

  16. Hutter M, Moosmann B, Kneisel S, Auwärter V. Characteristics of the designer drug and synthetic cannabinoid receptor agonist AM-2201 regarding its chemistry and metabolism. J Mass Spectrom. 2013;48(7):885–94.

    Article  CAS  PubMed  Google Scholar 

  17. Kavanagh P, Grigoryev A, Melnik A, Simonov A. The identification of the urinary metabolites of 3-(4-methoxybenzoyl)-1-pentylindole (RCS-4), a novel cannabimimetic, by gas chromatography-mass spectrometry. J Anal Toxicol. 2012;36(5):303–11.

    Article  CAS  PubMed  Google Scholar 

  18. Sobolevsky T, Prasolov I, Rodchenkov G. Detection of urinary metabolites of AM-2201 and UR-144, two novel synthetic cannabinoids. Drug Test Anal. 2012;4(10):745–53.

    Article  CAS  PubMed  Google Scholar 

  19. Wohlfarth A, Gandhi A, Pang S, Zhu M, Scheidweiler K, Huestis M. 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. 2014;406(6):1763–80.

    Article  CAS  PubMed  Google Scholar 

  20. Wohlfarth A, Pang S, Zhu M, Gandhi AS, Scheidweiler KB, Huestis MA. Metabolism of RCS-8, a synthetic cannabinoid with cyclohexyl structure, in human hepatocytes by high-resolution MS. Bioanalysis. 2014;6(9):1187–200.

    Article  CAS  PubMed  Google Scholar 

  21. Wohlfarth A, Pang S, Zhu M, Gandhi AS, Scheidweiler KB, Liu H-F, et al. First metabolic profile of XLR-11, a novel synthetic cannabinoid, obtained by using human hepatocytes and high-resolution mass spectrometry. Clin Chem. 2013;59(11):1638–48.

    Article  CAS  PubMed  Google Scholar 

  22. Zhang Q, Ma P, Cole R, Wang G. Identification of in vitro metabolites of JWH-015, an aminoalkylindole agonist for the peripheral cannabinoid receptor (CB2) by HPLC-MS/MS. Anal Bioanal Chem. 2006;386(5):1345–55.

    Article  CAS  PubMed  Google Scholar 

  23. Makriyannis A, Deng H. Cannabimimetic indole derivatives, patent WO/2001/028557. 2001.

  24. Uchiyama N, Matsuda S, Wakana D, Kikura-Hanajiri R, Goda Y. 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. 2013;31(1):93–100.

    Article  CAS  Google Scholar 

  25. European Monitoring Centre for Drugs and Drug Addiction. EMCDDA—Europol 2013 annual report on the implementation of Council Decision 2005/387/JHA2014.

  26. Pfizer Inc. Indazole derivatives, patent WO/2009/106982. 2009.

  27. Drug Enforcement Administration. Schedules of controlled substances: temporary placement of three synthetic cannabinoids into schedule I (AB-CHMINACA, AB-PINACA, THJ-2201). Fed Register. 2014;79(244):75767–71.

    Google Scholar 

  28. Takayama T, Suzuki M, Todoroki K, Inoue K, Min JZ, Kikura-Hanajiri R, et al. 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. 2014;28(6):831–8.

    Article  CAS  PubMed  Google Scholar 

  29. Thomsen R, Nielsen LM, Holm NB, Rasmussen HB, Linnet K, the IC. Synthetic cannabimimetic agents metabolized by carboxylesterases. Drug Test Anal. 2014. doi:10.1002/dta.173.

    PubMed  Google Scholar 

  30. Baranczewski P, Stanczak A, Sundberg K, Svensson R, Wallin A, Jansson J, et al. Introduction to in vitro estimation of metabolic stability and drug interactions of new chemical entities in drug discovery and development. Pharmacol Rep. 2006;58(4):453–72.

    CAS  PubMed  Google Scholar 

  31. McNaney CA, Drexler DM, Hnatyshyn SY, Zvyaga TA, Knipe JO, Belcastro JV, et al. 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. 2008;6(1):121–9.

    Article  CAS  PubMed  Google Scholar 

  32. Lavé T, Dupin S, Schmitt C, Valles B, Ubeaud G, Chou RC, et al. The use of human hepatocytes to select compounds based on their expected hepatic extraction ratios in humans. Pharm Res. 1997;14(2):152–5.

    Article  PubMed  Google Scholar 

  33. Bolze S, Lacombe O, Durand G, Chaimbault P, Massiere F, Gay-Feutry C, et al. Standardization of a LC/MS/MS method for the determination of acyl glucuronides and their isomers. Curr Sep. 2002;20(2):55–9.

    CAS  Google Scholar 

  34. Shipkova M, Armstrong VW, Oellerich M, Wieland E. Acyl glucuronide drug metabolites: toxicological and analytical implications. Ther Drug Monit. 2003;25(1):1–16.

    Article  CAS  PubMed  Google Scholar 

  35. Horng H, Benet LZ. The nonenzymatic reactivity of the acyl-linked metabolites of Mefenamic acid toward amino and thiol functional group bionucleophiles. Drug Metab Dispos. 2013;41(11):1923–33.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Park BK, Kitteringham NR. Effects of fluorine substitution on drug metabolism: pharmacological and toxicological implications. Drug Metab Rev. 1994;26(3):605–43.

    Article  CAS  PubMed  Google Scholar 

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ACKNOWLEDGMENTS

This research was supported by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health. AB-PINACA and 5F-AB-PINACA were generously donated by the Drug Enforcement Administration. Molecular Discovery kindly provided the MetaSite software.

Conflict of Interest

None

Author information

Authors and Affiliations

  1. Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Boulevard, Baltimore, Maryland, 21224, USA

    Ariane Wohlfarth, Marisol S. Castaneto, Karl B. Scheidweiler & Marilyn A. Huestis

  2. Department of Biotransformation, Bristol-Myers Squibb, Research and Development, Princeton, New Jersey, 08543, USA

    Mingshe Zhu

  3. AB SCIEX, Redwood City, California, 94404, USA

    Shaokun Pang

  4. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758, Linköping, Sweden

    Robert Kronstrand

  5. Division of Drug Research, Linköping University, 58185, Linköping, Sweden

    Robert Kronstrand

Authors
  1. Ariane Wohlfarth
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  2. Marisol S. Castaneto
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  3. Mingshe Zhu
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  4. Shaokun Pang
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  5. Karl B. Scheidweiler
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  7. Marilyn A. Huestis
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Corresponding author

Correspondence to Marilyn A. Huestis.

Electronic Supplementary Material

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Supplementary Fig. A

Proposed human hepatic metabolic pathway of AB-PINACA; ambiguous assignments of functional groups are shown as Markush structures (JPEG 449 kb)

High resolution image (EPS 1899 kb)

Supplementary Fig. B

Proposed human hepatic metabolic pathway of 5F-AB-PINACA; ambiguous assignments of functional groups are shown as Markush structures (JPEG 424 kb)

High resolution image (EPS 1840 kb)

Supplementary Table 1

(DOCX 48.5 kb)

Supplementary Table 2

(DOCX 49 kb)

Supplementary Table 3

(DOCX 13 kb)

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Wohlfarth, A., Castaneto, M.S., Zhu, M. et al. 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 (2015). https://doi.org/10.1208/s12248-015-9721-0

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