Advertisement

The AAPS Journal

, Volume 17, Issue 3, pp 660–677 | Cite as

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

  • Ariane Wohlfarth
  • Marisol S. Castaneto
  • Mingshe Zhu
  • Shaokun Pang
  • Karl B. Scheidweiler
  • Robert Kronstrand
  • Marilyn A. Huestis
Research Article

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.

KEY WORDS

5-fluoro-AB-PINACA AB-PINACA in silico prediction metabolism synthetic cannabinoids 

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

Notes

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

Supplementary material

12248_2015_9721_Fig8_ESM.jpg (450 kb)
Supplementary Fig. A

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

12248_2015_9721_MOESM1_ESM.eps (1.9 mb)
High resolution image (EPS 1899 kb)
12248_2015_9721_Fig9_ESM.jpg (425 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)

12248_2015_9721_MOESM2_ESM.eps (1.8 mb)
High resolution image (EPS 1840 kb)
12248_2015_9721_MOESM3_ESM.docx (49 kb)
Supplementary Table 1 (DOCX 48.5 kb)
12248_2015_9721_MOESM4_ESM.docx (49 kb)
Supplementary Table 2 (DOCX 49 kb)
12248_2015_9721_MOESM5_ESM.docx (13 kb)
Supplementary Table 3 (DOCX 13 kb)

REFERENCES

  1. 1.
    European Monitoring Centre for Drugs and Drug Addiction. Perspectives on drugs: synthetic cannabinoids in Europe. 2014.Google Scholar
  2. 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. 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. 4.
    ElSohly MA, Gul W, Wanas AS, Radwan MM. Synthetic cannabinoids: analysis and metabolites. Life Sci. 2014;97(1):78–90.CrossRefPubMedGoogle Scholar
  5. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  6. 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.CrossRefGoogle Scholar
  7. 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.CrossRefPubMedGoogle Scholar
  8. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  9. 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. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  11. 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.CrossRefPubMedGoogle Scholar
  12. 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.CrossRefPubMedGoogle Scholar
  13. 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.CrossRefPubMedGoogle Scholar
  14. 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.CrossRefPubMedGoogle Scholar
  15. 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.CrossRefPubMedGoogle Scholar
  16. 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.CrossRefPubMedGoogle Scholar
  17. 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.CrossRefPubMedGoogle Scholar
  18. 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.CrossRefPubMedGoogle Scholar
  19. 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.CrossRefPubMedGoogle Scholar
  20. 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.CrossRefPubMedGoogle Scholar
  21. 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.CrossRefPubMedGoogle Scholar
  22. 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.CrossRefPubMedGoogle Scholar
  23. 23.
    Makriyannis A, Deng H. Cannabimimetic indole derivatives, patent WO/2001/028557. 2001.Google Scholar
  24. 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.CrossRefGoogle Scholar
  25. 25.
    European Monitoring Centre for Drugs and Drug Addiction. EMCDDA—Europol 2013 annual report on the implementation of Council Decision 2005/387/JHA2014.Google Scholar
  26. 26.
    Pfizer Inc. Indazole derivatives, patent WO/2009/106982. 2009.Google Scholar
  27. 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. 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.CrossRefPubMedGoogle Scholar
  29. 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.PubMedGoogle Scholar
  30. 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.PubMedGoogle Scholar
  31. 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.CrossRefPubMedGoogle Scholar
  32. 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.CrossRefPubMedGoogle Scholar
  33. 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.Google Scholar
  34. 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.CrossRefPubMedGoogle Scholar
  35. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  36. 36.
    Park BK, Kitteringham NR. Effects of fluorine substitution on drug metabolism: pharmacological and toxicological implications. Drug Metab Rev. 1994;26(3):605–43.CrossRefPubMedGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2015

Authors and Affiliations

  • Ariane Wohlfarth
    • 1
  • Marisol S. Castaneto
    • 1
  • Mingshe Zhu
    • 2
  • Shaokun Pang
    • 3
  • Karl B. Scheidweiler
    • 1
  • Robert Kronstrand
    • 4
    • 5
  • Marilyn A. Huestis
    • 1
  1. 1.Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug AbuseNational Institutes of HealthBaltimoreUSA
  2. 2.Department of BiotransformationBristol-Myers Squibb, Research and DevelopmentPrincetonUSA
  3. 3.AB SCIEXRedwood CityUSA
  4. 4.Department of Forensic Genetics and Forensic ToxicologyNational Board of Forensic MedicineLinköpingSweden
  5. 5.Division of Drug ResearchLinköping UniversityLinköpingSweden

Personalised recommendations