Cytochrome P450-mediated oxidative metabolism of abused synthetic cannabinoids found in K2/Spice: identification of novel cannabinoid receptor ligands

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 (CB(1)) 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 (K(m) = 0.81-7.3 μM) and low to high reaction velocities (V(max) = 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 (f(m) = <0.2). In vitro studies demonstrated that the primary metabolites produced in humans display high affinity and intrinsic activity at the CB(1) receptor, which was attenuated by the CB(1) 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.

Fig. 1.

Structures of JWH-018 and AM2201 and hydroxyl metabolites. JWH-018 (1) and AM2201 (2) are structurally similar, with AM2201 differing by the presence of terminal alkyl fluorine atom. The ring hydroxylated metabolites shown are JWH-018 4-OH (3), JWH-018 5-OH (4), JWH-018 6-OH (5), and JWH-018 7-OH (6) and alkyl side chain metabolites are JWH-018 ω-OH (7), JWH-018 ω-COOH (8), JWH-018 ω-1-OH (9), and AM2201 ω-1-OH (10).

Fig. 2.

LC chromatograms of the hydroxyl and carboxyl metabolites of JWH-018 and AM2201 in human liver microsomes and human urine. Resulting chromatographs produced from incubation of 10 μM JWH-018 (A) and AM2201 (B) with 75 μg of HLM as explained under Materials and Methods and from representative human urine collected after suspected use of JWH-018 (C) or AM2201 (D) are shown. Chromatographs shown were generated after treatment of the urine with β-glucuronidase as described under Materials and Methods. The numbers above the chromatographic peaks correspond to the hydroxylated and carboxylated metabolites of JWH-018 and AM2201 shown in Fig. 1. Concentrations of the respective metabolites in B are as follows: JWH-018 ω-OH (8.53 ng/ml), JWH-018 ω-COOH (4.7 ng/ml), and JWH-018 ω-1-OH (9.6 ng/ml). Concentrations of respective metabolites in D are as follows: JWH-018 ω-OH (19.6 ng/ml), JWH-018 ω-COOH (9.2 ng/ml), and JWH-018 ω-1-OH (1.06 ng/ml). Different color tracings are representative of the SRM experiments (Supplemental Table 1) used to detect analytes 1 to 10 (Fig. 1). Specific LC-MS/MS conditions are provided under Materials and Methods.

Fig. 3.

Oxidation of JWH-018 (A) and AM2201 (B) by human recombinant cytochromes P450. Oxidation of JWH-018 (A) and AM2201 (B) to ω-OH, ω-1-OH, and ω-COOH metabolites. Activity is normalized to hepatic abundance of specific P450s (Rowland-Yeo et al., 2003) and is expressed as picomoles of product per minute per milligram protein.

Fig. 4.

LC chromatograms of the hydroxyl and carboxyl metabolites of JWH-018 and AM2201 produced from human cDNA-expressed recombinant P450s. Resulting chromatographs of hydroxylated and carboxylated metabolites produced from incubation of 10 μM JWH-018 with CYP1A2 (A), CYP2C9 (B), CYP2C19 (C), and CYP2D6 (D) and 10 μM AM2201 with CYP1A2 (E), CYP2C9 (F), CYP2C19 (G), and CYP2D6 (H) as mentioned under Materials and Methods are shown. The numbers above the chromatographic peaks correspond to the hydroxylated and carboxylated metabolites of JWH-018 and AM2201 shown in Fig. 1. Different color tracings are representative of the SRM experiments (Supplemental Table 1) used to detect analytes 1 to 10 (Fig. 1). Specific LC-MS/MS conditions are provided under Materials and Methods.

Fig. 5.

JWH-018 and its ω-OH and ω-1-OH metabolites (A) and AM2201 and its ω-1-OH metabolite (B) bind the CB₁ receptor with nanomolar affinity. JWH-018, AM2201, and ω-OH and ω-1-OH metabolites, but not the ω-COOH metabolite, completely displaced the radiolabeled cannabinoid [³H]CP-55,940 from the CB₁ receptor.

Fig. 6.

A, JWH-018, AM2201, and their ω-OH and ω-1-OH metabolites activate mouse CB₁ receptor; 10 μM JWH-018 and its ω-OH and ω-1-OH metabolites activated mouse CB₁ receptor with greater efficacy than 10 μM Δ⁹-THC. JWH-018, AM2201, and the ω-1-OH metabolites of each displayed full agonist activity comparable to that of CP-55,940. The ω-OH metabolite of JWH-018 displayed efficacy similar to that of the parent drug. No G-protein activity was observed with the ω-COOH metabolite of JWH-018. [³⁵S]GTPγS specific binding is expressed as a percentage of CP-55,940 specific binding. Values designated with different letters above the error bars are significantly different (p < 0.05, one-way ANOVA with Newman-Keuls multiple comparison post hoc test, n = 3). B, G-protein stimulation in mouse brain by JWH-018, AM2201, and their ω-OH and ω-1-OH metabolites is blocked by CB₁ receptor selective neutral antagonist O-2050. G-protein activation by 1 μM CP-55,940 and JWH-018 is blocked to a similar extent by 1 μM O-2050. Significant attenuation of G-protein stimulation by 1 μM AM2201 is blocked by 1 μM O-2050. G-protein stimulation by 1 μM Δ⁹-THC, ω-OH, and ω-1-OH metabolites of JWH-018 and AM2201 is blocked to a similar extent by 1 μM O-2050. [³⁵S]GTPγS specific binding is expressed as a percentage of CP-55,940. Values designated with different letters above the error bars are significantly different (p < 0.05, one-way ANOVA with Newman-Keuls multiple comparison post hoc test, n = 3).