Repeated administration of phytocannabinoid Δ9-THC or synthetic cannabinoids JWH-018 and JWH-073 induces tolerance to hypothermia but not locomotor suppression in mice, and reduces CB1 receptor expression and function in a brain region-specific manner

These studies probed the relationship between intrinsic efficacy and tolerance / cross-tolerance between Δ9-THC and synthetic cannabinoid drugs of abuse (SCBs) by examining in vivo effects and cellular changes concomitant with their repeated administration in mice. Dose-effect relationships for hypothermic effects were determined in order to confirm that SCBs JWH-018 and JWH-073 are higher efficacy agonists than Δ9-THC in mice. Separate groups of mice were treated with saline, sub-maximal hypothermic doses of JWH-018 or JWH-073 (3.0 mg/kg or 10.0 mg/kg, respectively) or a maximally hypothermic dose of 30.0 mg/kg Δ9-THC once per day for 5 consecutive days while core temperature and locomotor activity were monitored via biotelemetry. Repeated administration of all drugs resulted in tolerance to hypothermic effects, but not locomotor effects, and this tolerance was still evident 14 days after the last drug administration. Further studies treated mice with 30.0 mg/kg Δ9-THC once per day for 4 days, then tested with SCBs on day 5. Mice with a Δ9-THC history were cross-tolerant to both SCBs, and this cross-tolerance also persisted 14 days after testing. Select brain regions from chronically treated mice were examined for changes in CB1 receptor expression and function. Expression and function of hypothalamic CB1Rs were reduced in mice receiving chronic drugs, but cortical CB1R expression and function were not altered. Collectively, these data demonstrate that repeated Δ9-THC, JWH-018 and JWH-073 can induce long-lasting tolerance to some in vivo effects, which is likely mediated by region-specific downregulation and desensitization of CB1Rs

Monohydroxylated metabolites of the K2 synthetic cannabinoid JWH-073 retain intermediate to high cannabinoid 1 receptor (CB1R) affinity and exhibit neutral antagonist to partial agonist activity

K2 and several similar purported “incense products” spiked with synthetic cannabinoids are abused as cannabis substitutes. We hypothesized that metabolism of JWH-073, a prevalent cannabinoid found in K2, contributes to toxicity associated with K2 use. Competition receptor binding studies and G-protein activation assays, both performed by employing mouse brain homogenates, were used to determine the affinity and intrinsic activity, respectively, of potential monohydroxylated (M1, M3–M5) and monocarboxylated (M6) metabolites at cannabinoid 1 receptors (CB1Rs). Surprisingly, M1, M4 and M5 retain nanomolar affinity for CB1Rs, while M3 displays micromolar affinity and M6 does not bind to CB1Rs. JWH-073 displays equivalent efficacy to that of the CB1R full agonist CP-55,940, while M1, M3, and M5 act as CB1R partial agonists, and M4 shows little or no intrinsic activity. Further in vitro investigation by Schild analysis revealed that M4 acts as a competitive neutral CB1R antagonist (Kb~40nM). In agreement with in vitro studies, M4 also demonstrates CB1R antagonism in vivo by blunting cannabinoid-induced hypothermia in mice. Interestingly, M4 does not block agonist-mediated responses of other measures in the cannabinoid tetrad (e.g., locomotor suppression, catalepsy or analgesia). Finally, also as predicted by in vitro results, M1 exhibits agonist activity in vivo by inducing significant hypothermia and suppression of locomotor activity in mice. In conclusion, the present study indicates that further work examining the physiological effects of synthetic cannabinoid metabolism is warranted. Such a complex mix of metabolically produced CB1R ligands may contribute to the adverse effect profile of JWH-073-containing products

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

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).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.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

JWH-018 and M1 decreased mouse locomotor activity in a CB1R-dependent manner, similar to Δ⁹-THC.

<p><b>A.</b> 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>B.</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 (*<i>P</i><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.</p

Structures of JWH-018 and six JWH-018 hydroxylated products.

<p><b>A. JWH-018</b> [(1-pentyl-1H-indol-3-yl)-1-naphthalenyl-methanone] <b>B. M1</b> [(4-hydroxy-1-pentyl-1H-indol-3-yl)(naphthalen-1-yl)methanone] <b>C. M2</b> [(5-hydroxy-1-pentyl-1H-indol-3-yl)(naphthalen-1-yl)methanone] <b>D. M3</b> [(6-hydroxy-1-pentyl-1H-indol-3-yl)(naphthalen-1-yl)methanone] <b>E. M4</b> [(7-hydroxy-1-pentyl-1H-indol-3-yl)naphthalen-1-yl)methanone] <b>F. M5</b> [(1-(5-hydroxypentyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone] <b>G. M6</b> [5-(3-(1-naphthoyl)-1H-indol-1-yl)pentanoic acid].</p

JWH-018 and M1 decreased mouse core body temperature in a CB1R-dependent manner similar to Δ⁹-THC.

<p><b>A.</b> 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>B.</b> equivalent area under the curve values, which were significantly lower than vehicle controls (*<i>P</i><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).</p

JWH-018 and M1–M5 activate CB1R.

<p><b>A.</b> 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 <i>post-hoc</i> Test, n = 3–10). <b>B.</b> JWH-018 and M1 stimulated G-proteins more potently and efficaciously than Δ⁹-THC, n = 3–4. <b>C.</b> 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 (**<i>P</i><0.01, ***<i>P</i><0.001 vs drug alone, Student's <i>t</i>-test, n = 3–7).</p