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
Graphical abstract
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
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