The cannabinoid agonist HU-210: Pseudo-irreversible discriminative stimulus effects in rhesus monkeys

Abstract

Synthetic cannabinoid abuse and case reports of adverse effects have raised concerns about the pharmacologic mechanisms underlying in vivo effects. Here, a synthetic cannabinoid identified in abused products (HU-210) was compared to the effects of Δ⁹-THC and two other synthetic cannabinoid agonists used extensively in pre-clinical studies (CP 55,940 and WIN 55,212-2). One group of monkeys discriminated ∆⁹-THC (0.1 mg/kg i.v.); a separate group received chronic ∆⁹-THC (1 mg/kg/12 h s.c.) and discriminated rimonabant (1 mg/kg i.v.). CP 55,940, HU-210, ∆⁹-THC, and WIN 55,212-2 produced ∆⁹-THC lever responding. HU-210 had a long duration (i.e., 1–2 days), whereas that of the other cannabinoids was 5 h or less. Rimonabant (1 mg/kg) produced surmountable antagonism; single dose-apparent affinity estimates determined in the presence of ∆⁹-THC, CP 55,940, and WIN 55,212-2 did not differ from each other. In contrast, rimonabant (1 mg/kg) produced a smaller rightward shift in the HU-210 dose–effect function. In ∆⁹-THC treated monkeys, the relative potency of CP 55,940, ∆⁹-THC, and WIN 55,212-2 to attenuate the discriminative stimulus effects of rimonabant was the same as that evidenced in the ∆⁹-THC discrimination, whereas HU-210 was unexpectedly more potent in attenuating the effects of rimonabant. In conclusion, the same receptor subtype mediates the discriminative stimulus effects of ∆⁹-THC, CP 55,940 and WIN 55,212-2. The limited effectiveness of rimonabant to either prevent or reverse the effects of HU-210 appears to be due to very slow dissociation or pseudo-irreversible binding of HU-210 at cannabinoid receptors.

Introduction

The goal of this study was to compare the in vivo pharmacology of the synthetic cannabinoid HU-210, recently detected in Spice herbal blends, to Δ⁹-tetrahydrocannabinol (∆⁹-THC) and other synthetic cannabinoids including CP 55,940 and WIN 55,212-2. ∆⁹-THC is the natural product in Cannabis that exerts psychopharmacological effects that are primarily responsible for the widespread use of Cannabis. ∆⁹-THC is an agonist at two G-protein coupled receptor subtypes designated CB₁ and CB₂. CB₁ receptors are abundant in the hippocampus, basal ganglia, cortex, amygdala and cerebellum (Herkenham et al., 1991, Gifford et al., 1999), and are also present in adipose tissue, skeletal muscle and liver (Lindborg et al., 2010, Wu et al., 2011). CB₂ receptors are expressed mainly in the immune system. ∆⁹-THC is a tricyclic terpenoid derivative bearing a benzopyran moiety. Two other CB₁ and CB₂ receptor agonists of synthetic origin include CP 55,940, a bicyclic analog of ∆⁹-THC lacking the pyran ring, and WIN 55,212-2, an aminoalkylindole (Palmer et al., 2002 for review). CP 55,940 and WIN 55,212-2 have been used extensively as reference compounds in pre-clinical studies involving relatively novel cannabinoids.

HU-210 is one synthetic cannabinoid that has been added to non-Cannabis plant material and marketed under a variety of trade names such as Spice or K2 in the United Kingdom, apparently in an attempt to circumvent laws banning Cannabis (Fattore and Fratta, 2011). HU-210 belongs to the same chemical class as ∆⁹-THC and was initially synthesized in the laboratory of Raphael Mechoulam at the Hebrew University. HU-210 is a high-affinity CB₁ and CB₂ receptor agonist (Burkey et al., 1997, Howlett et al., 2002) and was reported to be highly potent in producing effects associated with cannabinoid agonism in rats (12.5–100 µg/kg i.p.) and pigeons (12.5–50 μg/kg, s.c.), including discriminative stimulus effects, decreased locomotor activity, rearing, and grooming, long-lasting hypothermia of a greater magnitude than that produced by Δ⁹-THC, increased vocalization and circling, and sedative effects (Järbe et al., 1989, Ovadia et al., 1995, Ferrari et al., 1999). CB₁ receptors appear to mediate the in vivo effects of HU-210 as evidenced by attenuation of those effects by CB₁ receptor antagonists (e.g., rimonabant; Bosier et al., 2010, Janoyan et al., 2002) as well as in CB₁ receptor knockout mice (Zimmer et al., 1999).

Cannabinoid discrimination procedures in rhesus monkeys have been used previously to demonstrate that synthetic cannabinoids detected in Spice products, including JWH-018 and JWH-073, exert discriminative stimulus effects by acting at the same receptors as those mediating the effects of ∆⁹-THC. In rhesus monkeys discriminating ∆⁹-THC, both JWH-018 and JWH-073 substituted for ∆⁹-THC (Ginsberg et al., 2012). Schild analysis with rimonabant was used to compare the receptor site(s) of action of ∆⁹-THC, JWH-018, and JWH-073. Rimonabant appeared to be a simple, competitive, and reversible antagonist of each agonist, as evidenced by slopes of Schild plots that were not significantly different from unity. The apparent affinity (pA₂) or potency of rimonabant did not differ among agonists, suggesting that the same receptors mediated the discriminative stimulus effects of ∆⁹-THC, JWH-018, and JWH-073. Moreover, each of these agonists attenuated rimonabant-induced ∆⁹-THC withdrawal in a second drug discrimination assay with a relative potency similar to that for producing discriminative stimulus effects in the ∆⁹-THC discrimination assay. Collectively, these drug discrimination data strongly suggest that ∆⁹-THC, JWH-018, and JWH-073 act at the same receptor to produce subjective effects.

Here, the effects of HU-210, Δ⁹-THC, CP 55,940, and WIN 55212-2 were compared in drug discrimination assays in rhesus monkeys that have documented utility for conducting quantitative analysis of drug interactions. One group of monkeys discriminated Δ⁹-THC (0.1 mg/kg i.v.) from vehicle. A second group of monkeys discriminated rimonabant (1 mg/kg i.v.) while receiving 1 mg/kg of Δ⁹-THC s.c. every 12 h. The Δ⁹-THC discrimination assay was used to examine the potency and time course of HU-210, CP 55,940 and WIN 55,212-2, as well as antagonism of their effects by rimonabant. ∆⁹-THC treated monkeys discriminating rimonabant were used to examine the capacity of rimonabant to reverse the effects of ∆⁹-THC, CP 55,940, WIN 55,212-2, and HU-210. CP 55,940 and WIN 55,212-2 were shown to attenuate the rimonabant discriminative stimulus previously (Stewart and McMahon, 2010), although potency was only determined in the presence of a single dose of rimonabant. Here, the potency of agonists was determined from the relationship between agonist dose and magnitude of rightward shift in the rimonabant dose–effect function as described (Ginsburg et al., 2012). The results strongly suggest that HU-210 binds pseudo-irreversibly to CB₁ receptors, defined as a marked decrease in the rate of offset of receptor binding (Kenakin, 2009), which in turn interferes with the binding of rimonabant.