Pharmacological evaluation of the natural constituent of Cannabis sativa, cannabichromene and its modulation by Δ⁹-tetrahydrocannabinol

Abstract

In contrast to the numerous reports on the pharmacological effects of Δ⁹-tetrahydrocannabinol (THC), the pharmacological activity of another substituent of Cannabis sativa, cannabichromene (CBC) remains comparatively unknown. In the present study, we investigated whether CBC elicits cannabinoid activity in the tetrad assay, which consists of the following four endpoints: hypomotility, antinociception, catalepsy, and hypothermia. Because cannabinoids are well documented to possess anti-inflammatory properties, we examined CBC, THC, and combination of both phytocannabinoids in the lipopolysaccharide (LPS) paw edema assay. CBC elicited activity in the tetrad that was not blocked by the CB₁ receptor antagonist, rimonabant. Moreover, a behaviorally inactive dose of THC augmented the effects of CBC in the tetrad that was associated with an increase in THC brain concentrations. Both CBC and THC elicited dose-dependent anti-inflammatory effects in the LPS-induced paw edema model. The CB₂ receptor, SR144528 blocked the anti-edematous actions of THC, but not those produced by CBC. Isobolographic analysis revealed that the anti-edematous effects of these cannabinoids in combination were additive. Although CBC produced pharmacological effects, unlike THC, its underlying mechanism of action did not involve CB₁ or CB₂ receptors. In addition, there was evidence of a possible pharmacokinetic component in which CBC dose-dependently increased THC brain levels following an i.v. injection of 0.3 mg/kg THC. In conclusion, CBC produced a subset of behavioral activity in the tetrad assay and reduced LPS-induced paw edema through a noncannabinoid receptor mechanism of action. These effects were augmented when CBC and THC were co-administered.

Introduction

While Δ⁹-tetrahydrocannabinol (THC) is recognized as the primary psychoactive constituent in Cannabis sativa (Gaoni and Mechoulam, 1964), the issue of whether other constituents in this plant contribute to its pharmacological effects, have pharmacological effects of their own, or modulate the effects of THC remains of interest from a drug abuse perspective as well as for the development of cannabinoid-based medications. One particular class of constituents in marijuana, the phytocannabinoids (of which THC is the prototypical type), has received considerable attention for their contribution to the pharmacological effects of marijuana. Of the approximately 70 identified phytocannabinoids in marijuana, many are present in trace amounts, and some of these compounds can be quantitatively abundant in the plant (McPartland and Russo, 2001, ElSohly and Slade, 2005). For example, the respective percentages of THC and cannabidiol (CBD), a non-psychoactive phytocannabinoid, from confiscated cannabis preparations are 8.8% and 0.4% (Mehmedic et al., 2010). CBD is documented to possess anxiolytic-like, antinociceptive, anti-psychotic-like, anti-inflammatory, and anti-oxidant effects (McPartland and Russo, 2001, Mechoulam et al., 2007).

The pharmacological actions of another phytocannabinoid, cannabichromene (CBC) (Mechoulam et al., 1968), present at relatively high concentrations in certain strains of marijuana remain to be extensively investigated. Turner et al. (1980) and Holley et al. (1975) noted that “drug type” marijuana primarily contained higher concentrations of CBC than CBD, while “fiber type” marijuana contained higher concentrations of CBD than CBC. This classification of “drug type” or “fiber type” marijuana is related to some strains of marijuana that have more potent psychotropic effects compared to other strains that cultivated as hemp for their fiber. While CBC is the second most abundant cannabinoid in some strains of marijuana growing in the United States (Brown and Harvey, 1990), it represents 0.3% of the constituents from confiscated cannabis preparations (Mehmedic et al., 2010).

Despite the presence of CBC in certain marijuana strains, relatively few studies have investigated the pharmacological effects of this compound. CBC was shown to have analgesic properties and potentiated the analgesic effects of THC in the mouse tail-flick assay (Davis and Hatoum, 1983). It also was found to prolong hexabarbital-induced hypnosis (Hatoum et al., 1981). In addition, it potentiated bradycardia caused by THC, though it did not induce bradycardia on its own (O’Neil et al., 1979). CBC was reported to induce sedation and ataxia in canines (Gaoni and Mechoulam, 1966); however, subsequent studies failed to replicate these effects (Gaoni and Mechoulam, 1971). Of relevance to the present study, CBC and its analogs have been reported to have anti-inflammatory properties in vivo and in vitro (Turner and ElSohly, 1981, Wirth et al., 1980a, Wirth et al., 1980b).

THC, other naturally occurring psychoactive cannabinoids, and synthetic cannabinoids produce their pharmacological effects predominantly through CB₁ and CB₂ receptors, with the former receptor largely responsible for behavioral and metabolic actions of these drugs, and latter receptor generally associated with the modulation of immune responses. This class of drugs produces a myriad of effects, including locomotor suppression, catalepsy, antinociception, and hypothermia, which collectively are known as the tetrad assay and is used to screen cannabinoid activity (Fride, 2006, Martin et al., 1991). The pharmacological effects of cannabinoids in each of these four in vivo tests yield a high positive correlation with their affinity for the CB₁ receptor (Compton et al., 1993). Importantly, CB₁ receptor binding affinity also positively correlates with drug discrimination studies in rats and psychoactivity in humans. Accordingly, the tetrad assay has had great utility because of its high predictive value in identifying compounds that possess marijuana-like effects. However, there are currently no published reports that evaluated CBC in the tetrad.

Phytocannabinoids, such as THC, have been effective anti-inflammatory agents in a variety of inflammatory models and there is substantial in vivo and in vitro evidence that both cannabinoid receptors (CB₁ and CB₂) are involved in anti-inflammatory processes (Zurier, 2003). Although both cannabinoid receptors are found on various populations of immune cells, CB₂ receptors are far more abundant than CB₁ receptors (Croxford and Yamamura, 2005). Many studies have shown that stimulation of CB₂ receptors inhibits pro-inflammatory cytokine release triggered by inflammatory agents, such as lipopolysaccharide (LPS), a constituent of the outer membrane of the cell wall of most gram negative bacteria (Puffenbarger et al., 2000). Intraplantar administration of LPS elicits paw edema that has been utilized to investigate the anti-inflammatory effects of various classes of anti-inflammatory compounds including steroidal and non-steroidal anti-inflammatory drugs (Kanaan et al., 1997), as well as cannabinoid receptor agonists (Naidu et al., 2010). However, there are presently no published reports on the possible anti-inflammatory effects of THC or CBC in this LPS-induced inflammatory paw model.

There were five objectives of the present study. The first goal was to determine whether CBC produces cannabinoid activity in the tetrad (locomotor suppression, catalepsy, antinociception, and hypothermia). Second, we evaluated whether this phytocannabinoid would elicit anti-edematous effects in the LPS-induced paw edema model. The third aim was to determine whether CBC produces its pharmacological effects in the aforementioned assays through CB₁ or CB₂ receptor mechanisms of action, using the respective receptor antagonists, rimonabant and SR144528. The fourth goal was to investigate the ability of CBC to modulate the pharmacological effects of THC in the tetrad and the LPS-induced paw edema model. The final goal was to examine whether co-administration of THC and CBC alters blood and brain levels of each phytocannabinoid.