Neuropharmacology and AnalgesiaCP47,497-C8 and JWH073, commonly found in ‘Spice’ herbal blends, are potent and efficacious CB1 cannabinoid receptor agonists
Introduction
Cannabis sativa (cannabis, marijuana or hashish) is a widely used plant preparation with well-known psychoactive effects (Ameri et al., 1999, Costa, 2007, Howlett, 2002, Howlett et al., 2002, Pertwee, 2008). ‘Spice’ is an herbal blend that is used recreationally for its cannabis-like effects and promoted as an alternative to marijuana (Auwarter et al., 2009, Hudson et al., 2010, Lindigkeit et al., 2009, Uchiyama et al., 2010, Vardakou et al., 2010, Zimmermann et al., 2009). Its use as a recreational drug has resulted in numerous analyses of its chemical constituents (Auwarter et al., 2009, Lindigkeit et al., 2009, Uchiyama et al., 2010). These have led to changes in its legal status (Griffiths et al., 2010, Lindigkeit et al., 2009, McLachlan, 2009, Vardakou et al., 2010), although this has not been without debate (Hammersley, 2010). Recent reports confirm similar physiological responses from ‘Spice’ use and cannabis use (Muller et al., 2010, Zimmermann et al., 2009). Mass spectrometry analyses of different ‘Spice’ preparations reveal that these products contain diverse synthetic cannabinoid additives (Auwarter et al., 2009, Hudson et al., 2010, Lindigkeit et al., 2009, Uchiyama et al., 2010, Vardakou et al., 2010). The cannabinoid JWH018 (Fig. 1A) was common among many of the different products first analyzed (Auwarter et al., 2009). These products also contain CP47,497-C8 (Fig. 1B), a variant of CP47,497 (extending the dimethylheptyl sidechain to a dimethyloctyl one) (Melvin et al., 1993). A third compound, JWH073 (Fig. 1C), the butyl homolog of JWH018, has appeared in more recently tested samples, replacing JWH018 in some cases (Lindigkeit et al., 2009). Interestingly Δ9-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa, was not reported present in the samples of ‘Spice’ that were analyzed, suggesting that the psychoactive effects were likely due to these synthetic cannabinoid additives (Auwarter et al., 2009).
The CB1 cannabinoid receptor is the receptor primarily responsible for mediating the psychoactive effects of THC (Huestis et al., 2001, Monory et al., 2007). CB1 is abundantly expressed in the central nervous system, particularly in areas linked to behaviors affected by THC (Mackie, 2005). In neurons, CB1 is primarily found on axon terminals where it modulates neurotransmission (Chevaleyre et al., 2006, Katona et al., 2006, Mackie, 2005). CB1 activation reduces cellular excitability and the probability of neurotransmitter release (Shen et al., 1996). This ability to inhibit neurotransmission allows both exogenous (such as THC) and endogenous cannabinoid agonists to modulate neuronal communication, and may underlie the psychoactivity of THC. Prolonged activation of CB1 receptors results in desensitization of the receptor and its internalization (Hsieh et al., 1999, Jin et al., 1999, Roche et al., 1999). These processes are thought to be involved in tolerance to drugs of abuse such as THC (Wu et al., 2008).
We previously characterized JWH018 as a potent and efficacious CB1 receptor agonist based on its ability to inhibit neurotransmission and promote CB1 receptor internalization (Atwood et al., 2010). These data indicate that JWH018 may promote the cannabis-like effects of ‘Spice.’ However CP47,497-C8 and JWH073 are also present in many samples of ‘Spice.’ Like JWH018 these compounds only had limited characterization (Aung et al., 2000, Compton et al., 1992, Griffin et al., 1998, Melvin et al., 1984, Melvin et al., 1993, Wiley et al., 1998). We sought to further characterize these two ‘Spice’ additives using assays that are relevant to the physiological effects of drugs of abuse: modulation of neurotransmission and promotion of receptor internalization.
Section snippets
Materials
Drugs and reagents were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Sigma-Aldrich (St Louis, MO, USA). JWH018 was generously provided by John Huffman and was synthesized as described in Huffman et al. (1994). Rimonabant (SR141716) was obtained from the National Institute of Drug Abuse drug supply. Chrm1-EGFP mice were obtained from the Mutant Mouse Regional Resource Center (University of California, Davis, CA, USA). DSMO was used as a solvent for JWH018 and rimonabant. Ethanol was
CP47,497-C8 and JWH073 Inhibit Neurotransmission by Activating CB1 Receptors
As seen in Fig. 3A, bath application of CP47,497-C8 reduced EPSC size. This suppression of EPSCs was concentration dependent with an IC50 of 15.4 nM (2.0 to 118.7 nM) and a maximal suppression at 1 μM to 51.6 ± 8.0% of baseline (Fig. 3C). Furthermore, as seen in Fig. 3A and D, the effect of CP47,497-C8 was reversed by rimonabant, a CB1 receptor antagonist. JWH073 also decreased the magnitude of EPSCs (Fig. 3B) in a concentration-dependent manner (Fig. 3C). The effect of JWH073 tended to be less
Discussion
Following the original identification of synthetic cannabinoid additives in different preparations of ‘Spice’ herbal blends (Auwarter et al., 2009) we extended the characterization of JWH018 to demonstrate that it is indeed a potent and efficacious CB1 receptor agonist (Atwood et al., 2010). The original report along with several more recent ones confirmed that in addition to JWH018, ‘Spice’ also can contain CP47,497-C8 and, more recently, JWH073 (Auwarter et al., 2009, Hudson et al., 2010,
Acknowledgements
Supported by DA11322, DA21696, DA024122, and DA009158, the Gill Center, and the Indiana METACyt Initiative of Indiana University, through a major grant from the Lilly Endowment, Inc.
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