Elsevier

Biochemical Pharmacology

Volume 83, Issue 7, 1 April 2012, Pages 952-961
Biochemical Pharmacology

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

https://doi.org/10.1016/j.bcp.2012.01.004Get rights and content

Abstract

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  40 nM). 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.

Introduction

K2, Spice and a variety of similar “incense products” (hereafter referred to collectively as “K2”) are currently emerging drugs of abuse with psychotropic effects mimicking those of marijuana [1], [2], [3]. K2 is made by adulterating plant matter with any of several diverse mixtures of synthetic cannabinoids, which are molecules that act in the brain similarly to Δ9-tetrahydrocannabinol (Δ9-THC), the major psychoactive molecule present in marijuana. Many of the most prevalent of these synthetic cannabinoids are structurally distinct relative to Δ9-THC (Fig. 1A and B) and individual K2 products are often quite variable in composition and unpredictable in content. For example, one study details the analytical detection of 11 different synthetic cannabinoids across 40 batches of 16 different incense products in various combinations and proportions from brand to brand and from batch to batch, even within brands [4]. Many K2 components were previously unregulated by legislative authorities in the U.S., and K2 use is undetectable by standard drug urine tests. Reportedly, these properties contribute to the motivation for K2 use by individuals seeking to attain the mood-altering effects of cannabis, while evading detection, prosecution and potential incarceration [5]. Compared to marijuana, K2 use is associated with an apparently higher prevalence of severe adverse effects, such as hypertension, tachycardia, hallucinations, agitation, seizures and panic attacks that often require immediate medical care [6], [7], [8], [9], [10], [11], [12]. The American Association of Poison Control Centers (AAPCC) reported handling 2874 calls in the year 2010 regarding toxicities experienced by individuals after using K2 [13]. Data and reports such as these prompted the United States Drug Enforcement Administration (USDEA) to temporarily classify five common K2 synthetic cannabinoids (JWH-018, JWH-073, JWH-200, CP-47,497, and cannabicyclohexanol) as Schedule I substances on March 1, 2011, until greater understanding regarding the health consequences of their use can be established [14]. Despite this ban, as of November 30, 2011, a reported 6348 calls regarding K2 use have been made to the AAPCC in 2011 alone [15], which is more than double the 2010 report, indicating an apparent persistence of K2 use that results in adverse effects [5], [16]. All of these data are particularly alarming given the recent finding that one in nine high school seniors admitted to using K2 over the past year, making K2 the second most frequently used illicit drug, after marijuana, among high school seniors [17].

Synthetic cannabinoids found in K2, as well as Δ9-THC and other cannabinoids, induce psychotropic effects by binding and activating cannabinoid 1 receptors (CB1Rs) in the CNS [18], [19]. CB1Rs are G-protein coupled receptors (GPCRs) found in highest abundance in the brain, and in lesser amounts in the liver [20], muscle and adipose tissues [21], gastrointestinal tract [22], bone [23], and reproductive system [24]. Most scientific data available regarding K2 to date has focused on determining product composition [4], [25], detecting useful biomarkers for compound detection in urine and serum [26], [27], [28], and reporting commonly observed adverse clinical effects [10], [11]. However, there is a general lack of knowledge concerning K2 metabolism, pharmacology and toxicology.

One synthetic cannabinoid often present in K2 is JWH-073 [25], [29], [30]. JWH-073 is a member of the JWH aminoalkylindole family, which was originally synthesized to study the endocannabinoid system [31]. “Co-abuse” of JWH-073 with JWH-018 (a commonly abused CB1R full agonist that is structurally similar to JWH-073) has been anecdotally reported to reduce JWH-018-induced anxiety, resulting in a more “mellow”, cannabis-like high compared to use of JWH-018 alone [32].

Although little is known concerning the biotransformation of the synthetic cannabinoids present in K2, initial studies have demonstrated that several Phase I monohydroxylated and carboxylated metabolites of both JWH-018 and JWH-073 are the major metabolites excreted in the urine of K2 users [26], [27], [28], [33], [34]. Recently, our laboratory reported that several monohydroxylated JWH-018 metabolites unexpectedly retain high affinity and intrinsic activity at CB1Rs [35], leading us to suggest that these and/or additional active metabolites likely contribute to the mechanism of K2 toxicity. Here, we hypothesize that biotransformation of JWH-073 produces similar metabolites (Fig. 1) possessing high affinity and/or activity at CB1Rs, resulting in complex interactions with other synthetic cannabinoids and their metabolites present in K2. The combined action of all active synthetic cannabinoids formed likely produces an “entourage effect” that contributes to the increased incidence of severe adverse effects observed with K2 relative to marijuana use. Therefore, we first examined the in vitro affinity and activity of one carboxylated and four monohydroxylated derivatives of JWH-073 at CB1Rs. These initial findings led us to further characterize the in vitro and in vivo pharmacology of two molecules, M1 and M4, for potential actions as a CB1R agonist and antagonist, respectively.

Section snippets

Materials

All compounds were stored at −20 °C, thawed and diluted in vehicle for use in subsequent experiments. JWH-073, M1, M3–M6 (Fig. 1) were purchased from Cayman Chemical (Ann Arbor, MI), and diluted to a stock solution with a final concentration of either 10−2 M (for [35S]GTPγS binding assays) or 10−3 M (for competition receptor binding) in 100% ethanol. JWH-018 was synthesized as previously described [36], [37], [38] and validated by [1H] Nuclear Magnetic Resonance (NMR), [13C] NMR, Distortionless

JWH-073, M1, M4, and M5 bind to CB1Rs with intermediate to high affinity

Saturation binding experiments using the radiolabeled, high-affinity cannabinoid agonist [3H]CP-55,940 determined that mouse brain homogenates employed for these experiments contain a CB1R density of 2.44 ± 0.15 pmol/mg protein, to which [3H]CP-55,940 binds with a Kd of 0.37 ± 0.07 nM (n = 3). To determine the affinity (Ki) of JWH-073, M1, and M3–M6 (Fig. 1) for CB1Rs, initial competition receptor binding studies with [3H]CP-55,940 were conducted (Fig. 2, Table 1). Specifically, the ability of

Discussion

This study is the first to report that potential monohydroxylated metabolites of JWH-073 retain physiologically relevant, high (M1), intermediate (M4 and M5) and low (M3) affinity for CB1Rs. M1, M3, and M5 also activate G-proteins in a CB1R-dependent manner with partial agonist activity equivalent to that produced by the major psychoactive constituent in marijuana, Δ9-THC. M1 was further characterized for potential in vivo activity and, similar to JWH-073, induces hypothermia and suppresses

Acknowledgements

The work was supported by the Association of Public Health Laboratories Award (JHM), US PHS Grant RR020146 (WEF), UAMS Pilot Study Award (PLP) and a Pilot Research Award from the University of Arkansas for Medical Sciences Center for Clinical and Translational Research, supported by grant number 1UL1RR029884 from the National Center for Research Resources (JHM, WEF, and PLP). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of

References (72)

  • A. Grigoryev et al.

    Chromatography-mass spectrometry studies on the metabolism of synthetic cannabinoids JWH-018 and JWH-073, psychoactive components of smoking mixtures

    J Chromatogr B Analyt Technol Biomed Life Sci

    (2011)
  • J.W. Huffman et al.

    Structure-activity relationships for 1-alkyl-3-(1-naphthoyl)indoles at the cannabinoid CB(1) and CB(2) receptors: steric and electronic effects of naphthoyl substituents. New highly selective CB(2) receptor agonists

    Bioorg Med Chem

    (2005)
  • H.C. Cheng

    The power issue: determination of KB or Ki from IC50. A closer look at the Cheng-Prusoff equation, the Schild plot and related power equations

    J Pharmacol Toxicol Methods

    (2001)
  • M.M. Aung et al.

    Influence of the N-1 alkyl chain length of cannabimimetic indoles upon CB(1) and CB(2) receptor binding

    Drug Alcohol Depend

    (2000)
  • B.K. Atwood et al.

    CP47,497-C8 and JWH073, commonly found in ‘Spice’ herbal blends, are potent and efficacious CB(1) cannabinoid receptor agonists

    Eur J Pharmacol

    (2011)
  • B.B. Gorzalka et al.

    Putative role of endocannabinoid signaling in the etiology of depression and actions of antidepressants

    Prog Neuropsychopharmacol Biol Psychiatry

    (2011)
  • C. Li et al.

    Role of the endocannabinoid system in food intake, energy homeostasis and regulation of the endocrine pancreas

    Pharmacol Ther

    (2011)
  • B.A. Watkins et al.

    The endocannabinoid signaling system: a marriage of PUFA and musculoskeletal health

    J Nutr Biochem

    (2010)
  • B.B. Gorzalka et al.

    Male-female differences in the effects of cannabinoids on sexual behavior and gonadal hormone function

    Horm Behav

    (2010)
  • D.J. Allsop et al.

    The Cannabis Withdrawal Scale development: patterns and predictors of cannabis withdrawal and distress

    Drug Alcohol Depend

    (2011)
  • C.E. Beyer et al.

    Depression-like phenotype following chronic CB1 receptor antagonism

    Neurobiol Dis

    (2010)
  • F.A. Moreira et al.

    Central side-effects of therapies based on CB1 cannabinoid receptor agonists and antagonists: focus on anxiety and depression

    Best Pract Res Clin Endocrinol Metab

    (2009)
  • J. Bergman et al.

    Some effects of CB1 antagonists with inverse agonist and neutral biochemical properties

    Physiol Behav

    (2008)
  • K.S. Sink et al.

    The CB1 inverse agonist AM251, but not the CB1 antagonist AM4113, enhances retention of contextual fear conditioning in rats

    Pharmacol Biochem Behav

    (2010)
  • G. Kunos et al.

    Should peripheral CB(1) cannabinoid receptors be selectively targeted for therapeutic gain

    Trends Pharmacol Sci

    (2009)
  • V. Auwarter et al.

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

    J Mass Spectrom

    (2009)
  • K.A. Seely et al.

    Marijuana-based drugs: innovative therapeutics or designer drugs of abuse

    Mol Interv

    (2011)
  • S. Hudson et al.

    Use of high-resolution accurate mass spectrometry to detect reported and previously unreported cannabinomimetics in herbal high products

    J Anal Toxicol

    (2010)
  • D. Vearrier et al.

    A teenager with agitation: higher than she should have climbed

    Pediatr Emerg Care

    (2010)
  • H. Muller et al.

    Panic attack after spice abuse in a patient with ADHD

    Pharmacopsychiatry

    (2010)
  • A.C. Young et al.

    Cardiotoxicity associated with the synthetic cannabinoid, K9, with laboratory confirmation

    Am J Emerg Med

    (2011)
  • J. Simmons et al.

    Three cases of spice exposure

    Clin Toxicol (Phila)

    (2011)
  • J. Wehrman

    Fake marijuana spurs more than 4,500 calls to U.S. poison centers

    (2011)
  • M.M. Leonhart

    Schedules of controlled substances: temporary placement of five synthetic cannabinoids into schedule I

    (2010)
  • AAPCC. Synthetic marijuana data, <http://www.aapcc.org/dnn/Portals/0/Synthetic Marijuana Data for Website...
  • X. Hu et al.

    College students and use of K2: an emerging drug of abuse in young persons

    Subst Abuse Treat Prev Policy

    (2011)
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