Neuropharmacology and Analgesia
CP47,497-C8 and JWH073, commonly found in ‘Spice’ herbal blends, are potent and efficacious CB1 cannabinoid receptor agonists

https://doi.org/10.1016/j.ejphar.2011.01.066Get rights and content

Abstract

‘Spice’ is an herbal blend that has been reported to produce cannabis-like effects when smoked and is marketed as an alternative to marijuana. Synthetic additives have been identified in numerous ‘Spice’ preparations from different sources. Common among many of the preparations were the compounds JWH018 and a dimethyloctyl variant of CP47,497 (CP47,497-C8) and, more recently JWH073. The synaptic effects of each of these compounds were uncharacterized. We previously reported that JWH018 is a potent and efficacious CB1 cannabinoid receptor agonist. In this study we have examined the abilities of CP47,497-C8 and JWH073 to inhibit neurotransmission in cultured autaptic hippocampal neurons. Each inhibited EPSCs with an efficacy and potency similar to JWH018. We also analyzed these compounds' effects on promoting internalization of CB1 receptors in HEK293 cells stably expressing CB1 receptors. Similar to our neurotransmission data, CP47,497-C8 internalized CB1 in a fashion indistinguishable from JWH018. However, JWH073 was less potent and produced slower internalization than JWH018 and CP47,497-C8. It appears that ‘Spice’ contains a number of cannabinoid receptor agonists that activate CB1 receptors to inhibit synaptic transmission with similar potencies and efficacies. It is highly probable that the cannabis-like effects of ‘Spice’ are due to the presence of these and analogous synthetic additives acting on CB1 receptors.

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.

References (40)

  • I. Vardakou et al.

    Spice drugs as a new trend: mode of action, identification and legislation

    Toxicol. Lett.

    (2010)
  • A. Ameri et al.

    Effects of the endogeneous cannabinoid, anandamide, on neuronal activity in rat hippocampal slices

    Br. J. Pharmacol.

    (1999)
  • B.K. Atwood et al.

    JWH018, a common constituent of ‘Spice’ herbal blends, is a potent and efficacious cannabinoid CB(1) receptor agonist

    Br. J. Pharmacol.

    (2010)
  • V. Auwarter et al.

    ‘Spice’ and other herbal blends: harmless incense or cannabinoid designer drugs?

    J. Mass Spectrom.

    (2009)
  • J.M. Bekkers et al.

    Excitatory and inhibitory autaptic currents in isolated hippocampal neurons maintained in cell culture

    Proc. Natl Acad. Sci. USA

    (1991)
  • V. Chevaleyre et al.

    Endocannabinoid-mediated synaptic plasticity in the CNS

    Annu. Rev. Neurosci.

    (2006)
  • D.R. Compton et al.

    Pharmacological profile of a series of bicyclic cannabinoid analogs: classification as cannabimimetic agents

    J. Pharmacol. Exp. Ther.

    (1992)
  • B. Costa

    On the pharmacological properties of delta9-tetrahydrocannabinol (THC)

    Chem. Biodivers.

    (2007)
  • E.J. Furshpan et al.

    Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for cholinergic, adrenergic, and dual-function neurons

    Proc. Natl Acad. Sci. USA

    (1976)
  • G. Griffin et al.

    Evaluation of cannabinoid receptor agonists and antagonists using the guanosine-5′-O-(3-[35S]thio)-triphosphate binding assay in rat cerebellar membranes

    J. Pharmacol. Exp. Ther.

    (1998)
  • Cited by (95)

    • Cannabimimetic effects of abused indazole-carboxamide synthetic cannabinoid receptor agonists AB-PINACA, 5F-AB-PINACA and 5F-ADB-PINACA in mice: Tolerance, dependence and withdrawal

      2022, Drug and Alcohol Dependence
      Citation Excerpt :

      SCRAs exert their effects via binding to, and activation of, G protein-coupled cannabinoid type-1 (CB1R) and type-2 (CB2R) receptors. Like the phytocannabinoid ∆9-tetrahydrocannabinol (THC), the psychoactive effects of SCRAs are mediated by interactions with CB1Rs (Atwood et al., 2011); however, SCRAs bind with higher affinity (Wiley et al., 1998) and activate CB1Rs with greater efficacy than THC (Compton et al., 1992; Huffman and Padgett, 2005). Despite a decreasing trend in SCRA use in the USA, the United Nations Office on Drugs and Crime Early Warning Advisory on New Psychoactive Substances reported that as of December 2019, SCRAs still account for about one-third of all new drugs appearing on the global market (UNODC, 2020).

    • Review of the Endocannabinoid System

      2021, Biological Psychiatry: Cognitive Neuroscience and Neuroimaging
      Citation Excerpt :

      This likely varies across synapses, as sometimes THC mimics 2-AG effects (124). On the other hand, spice compounds are high-efficacy agonists, fully and indiscriminately activating CB1 receptors and countering AEA signaling (AEA is a low-efficacy agonist) (125,126). The ECS is present from the earliest stage of pregnancy, in the preimplantation embryo and uterus (127), in the placenta (128), and in the developing fetal brain (129).

    • Regional trends in suspected synthetic cannabinoid exposure from January 2016 to September 2019 in the United States

      2020, Drug and Alcohol Dependence
      Citation Excerpt :

      Although SCs can have similar pharmacological actions to certain phytocannabinoids, such as tetrahydrocannabinol (THC), there are important differences. THC only partially or weakly binds to and activates the cannabinoid receptor (CB1) (Atwood et al., 2011). However, some SCs fully bind and activate CB1, resulting in stronger psychoactive effects and longer duration of action than cannabis (Atwood et al., 2010).

    View all citing articles on Scopus
    View full text