Pharmacological evaluation of the natural constituent of Cannabis sativa, cannabichromene and its modulation by Δ(9)-tetrahydrocannabinol

Abstract

In contrast to the numerous reports on the pharmacological effects of Δ(9)-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 CB(1) 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 CB(2) 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 CB(1) or CB(2) 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 0.3mg/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.

Figure 1

Comparison between the dose-response of intravenously administered CBC alone and in combination with an inactive dose of THC (i.e., 0.3 mg/kg) in the mouse tetrad. When given alone, only 100 mg/kg CBC produced locomotor suppression (A), catalepsy (B), antinociception (C), and hypothermia (D) in mice. The inactive dose of THC (0.3 mg/kg) produced a leftward shift in the dose-response curve of CBC when compared to the dose-response of CBC administered alone for catalepsy (B), antinociception (C), and hypothermia (D), but not for hypomotility (A). The results are presented as mean ± S.E.M., n=6 mice per group. * p < 0.05 for CBC alone vs. vehicle control mice. # p < 0.05 for CBC + THC (0.3 mg/kg) vs. vehicle + vehicle control mice.

Figure 2

The pharmacological effects of CBC for locomotor suppression (A), catalepsy (B), antinociception (C), and hypothermia (D) are not mediated by the CB₁ receptor. Mice were intravenously administered an i.p. injection of vehicle or rimonabant (3 mg/kg) followed 10 min later by an i.v. injection of vehicle or CBC (100 mg/kg). ANOVA revealed a significant main effect of CBC treatment (represented as asterisks) for locomotor suppression (A), catalepsy (B), and hypothermia (D), but not for antinociception (C). Rimonabant (3 mg/kg) failed to block the pharmacological effects of CBC. The results are presented as mean ± S.E.M., n=6 mice per group.

Figure 3

Quantification of blood and brain tissue concentrations of THC and CBC in mice administered a dose range of CBC with and without an inactive dose of THC. Mice were given an i.v. injection of CBC (vehicle, 3, 10, 30, or 100 mg/kg) followed by a second i.v. injection 10 min later of vehicle or THC (0.3 mg/kg). Blood and brain tissue were harvested at 30 min after the first injection. A. THC blood levels. B. THC Brain levels. C. CBC blood levels with and without THC (0.3 mg/kg). D. CBC brain levels with and without THC (0.3 mg/kg). The results are presented as mean ± S.E.M., n=5 or 6 mice per group. *p < 0.05 vs. vehicle + 0.3 mg/kg THC; #p < 0.0001 vs. 100 mg/kg CBC (with or without 0.3 mg/kg THC; $p < 0.0001 vs. 100 mg/kg CBC.

Figure 4

Distinct receptor mechanisms of action mediate the anti-edematous effects of THC and CBC. A. Anti-edematous effects of i.p. administered CBC and THC. Neither drug affected paw thickness in the contralateral paw (data not shown). The results are presented as mean ± S.E.M., n=6 mice per group. Mean pre-injection paw thickness ± SEM values for LPS-injected and saline-injected paws for the mice in the CBC experiment were 1.82 mm ± 0.008 and 1.82 mm ± 0.008, respectively. Mean pre-injection paw thickness ± SEM values for LPS-injected and saline-injected paws for the mice in the THC experiment were 1.99 mm ± 0.013 and 1.99 mm ± 0.012, respectively. *p < 0.05 vs. vehicle control group (THC); #p < 0.05 vs. vehicle control group (CBC). B. The anti-inflammatory effects of THC were blocked by SR144528 (SR2), but not by rimonabant (SR), indicating a CB₂ receptor mediated effect. C. The anti-inflammatory effects of CBC were not blocked by either rimonabant (SR1) or SR144528 (SR2), indicating that neither CB₁ nor CB₂ receptors played a necessary role in CBC’s actions. Collective baseline mean paw thickness values for mice depicted in the experiments in Panels B and C were 1.89 ± 0.008 mm and 1.85 ± 0.010 mm. The results are presented as mean ± S.E.M., n=6 mice per group. *p < 0.05 vs. vehicle/vehicle group; #p < 0.05 vs. vehicle/THC group.

Figure 5

The anti-inflammatory dose-response of CBC and THC administered in combination yield an additive interaction. Dose response curves from CBC alone (A) and THC alone (B) from Figure 4A. In the 1:1 combination study, mice were administered vehicle, 4 mg/kg CBC:0.4 mg/kg THC, 12 mg/kg CBC:1.2 mg/kg THC or 40 mg/kg CBC:4 mg/kg THC. The results are presented as mean ± S.E.M., n=6 mice per group. The respective baseline mean ± S.E.M. paw thickness values for mice in the 1:1 combination study, CBC alone, and THC alone experiments were 1.88 ± 0.008, 1.82 ± 0.008, and 1.99 ± 0.013 mm. *p < 0.05 vs. vehicle control group in the single drug experiments; # p < 0.05 vs. vehicle control group combination drug experiment. C. Isobologram analysis of the anti-inflammatory effects of CBC and THC dosed in an equipotent combination. The ED₅₀ values for CBC and THC were calculated from the dose-response curve of each individual drug. The experimental ED₅₀ value overlapped with the theoretical additive ED₅₀ value indicating an additive relationship between the two drugs.