Abstract
Abuse of synthetic cannabinoids (SCs), such as [1-naphthalenyl-(1-pentyl-1H-indol-3-yl]-methanone (JWH-018) and [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone (AM2201), is increasing at an alarming rate. Although very little is known about the metabolism and toxicology of these popular designer drugs, mass spectrometric analysis of human urine specimens after JWH-018 and AM2201 exposure identified monohydroxylated and carboxylated derivatives as major metabolites. The present study extends these initial findings by testing the hypothesis that JWH-018 and its fluorinated counterpart AM2201 are subject to cytochrome P450 (P450)-mediated oxidation, forming potent hydroxylated metabolites that retain significant affinity and activity at the cannabinoid 1 (CB1) receptor. Kinetic analysis using human liver microsomes and recombinant human protein identified CYP2C9 and CYP1A2 as major P450s involved in the oxidation of the JWH-018 and AM2201. In vitro metabolite formation mirrored human urinary metabolic profiles, and each of the primary enzymes exhibited high affinity (Km = 0.81–7.3 μM) and low to high reaction velocities (Vmax = 0.0053–2.7 nmol of product · min−1 · nmol protein−1). The contribution of CYP2C19, 2D6, 2E1, and 3A4 in the hepatic metabolic clearance of these synthetic cannabinoids was minimal (fm = <0.2). In vitro studies demonstrated that the primary metabolites produced in humans display high affinity and intrinsic activity at the CB1 receptor, which was attenuated by the CB1 receptor antagonist (6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (O-2050). Results from the present study provide critical, missing data related to potential toxicological properties of “K2” parent compounds and their human metabolites, including mechanism(s) of action at cannabinoid receptors.
Footnotes
This work was funded by the National Institutes of Health National Institute of General Medical Sciences [Grant GM075893] (to A.R.-P.); Association of Public Health Laboratories [Innovation Award] (to J.H.M.); and the University of Arkansas for Medical Sciences Translational Research Institute (to C.L.), which is funded by the National Center for the Advancement of Translational Science [UL1-TR000039].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
ABBREVIATIONS:
- JWH-018
- [1-naphthalenyl-(1-pentyl-1H-indol-3-yl]-methanone
- AM2201
- [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone
- CB1
- cannabinoid type 1 receptor
- CB2
- cannabinoid type 2 receptor
- P450
- cytochrome P450
- HLM
- human liver microsomes
- CP-55,940
- 5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol
- O-2050
- (6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran
- GTPγS
- guanosine 5′-O-(3-thio)triphosphate
- OR
- oxidoreductase
- MS/MS
- tandem mass spectrometry
- SRM
- specific reaction monitoring
- IDA
- information-dependent acquisition
- EPI
- enhanced product ion
- BSA
- bovine serum albumin
- WIN55,212-2
- (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN-55,212-2)
- ANOVA
- analysis of variance.
- Received June 28, 2012.
- Accepted August 17, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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