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, 6 (7), e21917

Phase I Hydroxylated Metabolites of the K2 Synthetic Cannabinoid JWH-018 Retain in Vitro and in Vivo Cannabinoid 1 Receptor Affinity and Activity


Phase I Hydroxylated Metabolites of the K2 Synthetic Cannabinoid JWH-018 Retain in Vitro and in Vivo Cannabinoid 1 Receptor Affinity and Activity

Lisa K Brents et al. PLoS One.


Background: K2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ(9)-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2's high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R).

Methods/principal findings: JWH-018, five potential monohydroxylated metabolites (M1-M5), and one carboxy metabolite (M6) were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [(3)H]CP-55,940, and then for CB1R intrinsic efficacy using an [(35)S]GTPγS binding assay. JWH-018 and M1-M5 bound CB1Rs with high affinity, exhibiting K(i) values that were lower than or equivalent to Δ(9)-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ(9)-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ(9)-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.

Conclusions/significance: Unlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ(9)-THC, may contribute to the greater prevalence of adverse effects observed with JWH-018-containing products relative to cannabis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Structures of JWH-018 and six JWH-018 hydroxylated products.
A. JWH-018 [(1-pentyl-1H-indol-3-yl)-1-naphthalenyl-methanone] B. M1 [(4-hydroxy-1-pentyl-1H-indol-3-yl)(naphthalen-1-yl)methanone] C. M2 [(5-hydroxy-1-pentyl-1H-indol-3-yl)(naphthalen-1-yl)methanone] D. M3 [(6-hydroxy-1-pentyl-1H-indol-3-yl)(naphthalen-1-yl)methanone] E. M4 [(7-hydroxy-1-pentyl-1H-indol-3-yl)naphthalen-1-yl)methanone] F. M5 [(1-(5-hydroxypentyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone] G. M6 [5-(3-(1-naphthoyl)-1H-indol-1-yl)pentanoic acid].
Figure 2
Figure 2. JWH-018 and M1–M5 bind CB1R with equal or greater affinity than Δ9-THC.
JWH-018 and M1, M2, M3, M4, M5, but not M6, completely displaced the radiolabeled cannabinoid [3H]CP-55,940 from CB1Rs (data not shown). Affinities for CB1Rs of JWH-018 and M1–M5 were equivalent to or up to 10-fold greater than that of Δ9-THC (*P<0.05, **P<0.01, ***P<0.001 relative to Δ9-THC, one way ANOVA with Dunnett's Multiple Comparison Test, n = 3–4).
Figure 3
Figure 3. JWH-018 and M1–M5 activate CB1R.
A. Ten µM concentrations of JWH-018, M1, M2, M3, and M5 activated brain GPCRs greater than 10 µM Δ9-THC. Activation by JWH-018, M2, M3 and M5 did not differ from the full CB1R agonist CP-55,940. Values designated with different letters above the error bars are significantly different (P<0.05, one way ANOVA with Tukey's Multiple Comparison post-hoc Test, n = 3–10). B. JWH-018 and M1 stimulated G-proteins more potently and efficaciously than Δ9-THC, n = 3–4. C. GPCR activation by an estimated ED90 concentration (100 nM) of metabolites was blocked by co-incubation with 1 µM of the selective neutral CB1R antagonist O-2050 (**P<0.01, ***P<0.001 vs drug alone, Student's t-test, n = 3–7).
Figure 4
Figure 4. JWH-018 and M1 decreased mouse locomotor activity in a CB1R-dependent manner, similar to Δ9-THC.
A. Intraperitoneal (i.p.) administration of 3 mg/kg JWH-018, 10 mg/kg JWH-018 M1, and 30 mg/kg Δ9-THC decreased locomotor activity relative to vehicle controls over a 10 h time course, beginning 60 min after injection. B. Area under the curve data generated from the 10 h time-course shows 3 mg/kg JWH-018, 10 mg/kg JWH-018 M1, and 30 mg/kg Δ9-THC significantly decrease locomotor activity relative to vehicle controls (*P<0.05 vs. vehicle controls, Kruskal-Wallis one-way ANOVA with Tukey HSD test, n = 5). Co-administration of each cannabinoid with the CB1R-preferring antagonist/inverse agonist AM251 (10 mg/kg) restored locomotor activity to vehicle control levels.
Figure 5
Figure 5. JWH-018 and M1 decreased mouse core body temperature in a CB1R-dependent manner similar to Δ9-THC.
A. Mice administered 3 mg/kg JWH-018 and 10 mg/kg M1 (i.p.) exhibited greater depressions in core body temperature than 30 mg/kg Δ9-THC, but also recovered more quickly over a 10 h time course, resulting in B. equivalent area under the curve values, which were significantly lower than vehicle controls (*P<0.005 vs. vehicle controls, one-way ANOVA with Tukey HSD test, n = 5). Core body temperature was restored to vehicle control levels by coadministration of cannabinoids with the CB1R-preferring antagonist/inverse agonist AM251 (10 mg/kg).

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