PII S0024-3205(98)00238-0

PHARMACOLGY LETTERS
Accelerated Communication

COMPLEX PHARMACOLOGY OF NATURAL CANNABINOIDS : EVIDENCE FOR PARTIAL AGONIST ACTIVITY OF ⁹-TETRAHYDROCANNABINOL AND ANTAGONIST ACTIVITY OF CANNABIDIOL ON RAT BRAIN CANNABINOID RECEPTORS

Francois Petitet*, Bernadette Jeantaud, Michel Reibaud, Assunta Imperato and Marie-Christine Dubroeucq

Rhône-Poulenc Rorer S.A., C.R.V.A., B.P. 14, 13 Quai Jules Guesde, 94403 Vitry-sur-Seine Cedex, France.

(Submitted December 4, 1997; acepted January 7, 1998; received in final form April 8, 1998)

Key words: cannabinoid, D ⁹-tetrahydrocannabinol, cannabinol, cannabidiol, [³⁵S]GTP- g -S binding.

Introduction

The psychoactive compound from Cannabis sativa, the marijuana plant, ⁹-tetrahydrocannabinol ( ⁹-THC) was isolated in 1964 by Mechoulam's group (1). This compound binds to cannabinoid CB₁ as well CB₂ receptors with affinities around 40 nM. ⁹-THC has been regarded as being a cannabinoid agonist inhibiting cAMP production (2) and inhibiting Ca²⁺ channels in NG108-15 (3). In vivo ⁹-THC induced in mice the classical behavioral ½tetrad©: hypomotility, catalepsy, analgesia and hypothermia. These effects could be antagonized by the newly developed CB₁ selective antagonist SR141716A (4,5) underlining their central origin, since CB₁ receptors are mainly expressed in the brain. Recently however, ⁹-THC was postulated to be a partial agonist on CB₂ receptors (6,7) and on CB₁ receptors (8,9).

Cannabinol and cannabidiol are nonpsychoactive compounds found in Cannabis sativa. Cannabinol is a weak cannabinoid receptor ligand but interestingly is able to discriminate pharmacologically between CB₁ and CB₂ receptors (10). Cannabidiol has a very low affinity (in the micromolar range) for CB₁ as well for CB₂ receptors and was found to be anticonvulsant in animal models of epilepsy and in humans with epilepsy (11). In addition, cannabidiol has been shown to have anti-spasmodic (12)and anxiolytic properties (13). The antagonistic properties of cannabidiol were postulated a long time ago by Karniol and Carlini (14) considering that in some cases, cannabidiol could reverse the behavioral effects induced by ⁹-THC.

Recently, a method was developed to examine receptor activation of G proteins in brain slices or membranes by stimulating the binding of the hydrolysis-resistant GTP analog [³⁵S]GTP- g -S with agonists (15,16). Recent reports shows that cannabinoid agonists are able to stimulate [³⁵S]GTP- g -S binding on slices (8,17) or membranes (18,19). We describe in this study using [³⁵S]GTP- g -S binding on rat cerebellar membranes, evidence for a partial agonist activity of ⁹-THC and antagonist activity of cannabidiol on brain CB₁ receptors.

Methods

Materials

SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride) and CP55940 ((cis)-3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-(trans)-4-(3-hydroxypropyl)cyclohexanol) were synthesized in the chemistry department of Rhône-Poulenc Rorer, France. [³⁵S]GTP- g -S (specific activity : 1250 Ci/mmol) was obtained from N.E.N. GTP- g -S, and guanosine 5'-diphosphate (GDP) were from Sigma as well as ⁹-tetrahydrocannabinol ( ⁹-THC), cannabinol, cannabidiol and fatty acid free bovine serum albumin (BSA).

Preparation of rat cerebellar membranes

Rat cerebellar membranes were prepared as previously described by Kuster et al. (20). Male Sprague Dawley rats (200 - 300 g) (Iffa Credo, France) were killed by decapitation and their cerebella rapidly removed. Tissue was homogenized (1 : 100 w/v) in ice-cold buffer consisting of : HEPES 20 mM, MgCl₂ 1 mM, pH 7.4 and centrifuged at 48,000 x g for 20 min at 4—C. The pellet was resuspended in the same amount of ice-cold buffer and centrifuged for a second time as above. Finally, the membranes were resuspended in the buffer (1 : 120 w/v) and stored at -80—C until use.

Binding of [³⁵S]GTP- g -S to rat cerebellar membranes

Rat cerebellar membranes (15 ´g per tube) prepared as above were incubated with [³⁵S]GTP- g -S (0.2 nM) in a final volume of 500 ´l 20 mM HEPES buffer (pH 7.4) containing 1 mM MgCl₂, 1 mM EGTA, 0.1 mM GDP and 0.5 mg/ml fatty acid free BSA with or without the compound to be tested. After a 90 min incubation at 30—C the medium was filtered through Whatman GF/B filters pretreated for 3 - 4 h with a solution of 5 mg/ml fatty acid free BSA. After a wash with 12 ml of cold buffer, membrane-bound radioactivity was counted by liquid scintillometry using ReadySolv Ó (Beckman). All assays were run in triplicate.

Results

Effects of CP55940, D ⁹-THC cannabinol and cannabidiol on [³⁵S]GTP- g -S binding to rat cerebellar membranes.

Classical agonists at the cannabinoid receptor increase the amount of [³⁵S]GTP- g -S binding. The most potent agonist is CP55940 which increases [³⁵S]GTP- g -S binding by about 80 % with an EC₅₀ of 53 nM. ⁹-THC is less potent (EC₅₀ = 216 nM) but the maximal effect obtained at high concentration (10 ´M) represents 54 % of stimulation. Cannabinol is less potent than CP55940 (EC₅₀ = 187 nM) and the maximal [³⁵S]GTP- g -S binding stimulation observed at 10 ´M is only 24 %. Cannabidiol is devoid of agonist activity even at high concentration (10 ´M). Fig. 1.

Fig. 1: Comparison of the stimulation of [³⁵S]GTP- g -S binding to rat cerebellar membranes by (filled circle) CP55940, (filled square) ⁹-THC, (open square) cannabinol and (inverted filled triangle) cannabidiol. Assays were performed as described in Methods for 90 min at 30—C. Values represent percentage of specific binding and are given as means ˜ s.e.m. from three to eight separate experiments performed in triplicate.

Antagonist activity of SR141716A, cannabidiol and D ⁹-THC on CP55940-induced stimulation of [³⁵S]GTP- g -S binding to rat cerebellar membranes.

The effect of cannabidiol (10 ´M) was evaluated on CP55940-induced stimulation of [³⁵S]GTP- g -S binding. Cannabidiol is able to shift the concentration-response curve to the right. The EC₅₀ for CP55940 in the presence of 10 ´M cannabidiol being around 500 nM. Surprisingly, given the concentrations of CP55940 (0.1 to 3 nM) [³⁵S]GTP- g -S binding was 18 - 22 % lower than the control situation. Fig. 2.

Fig. 2: CP55940 potently stimulates [³⁵S]GTP- g -S binding on rat cerebellar membranes (87 % of stimulation and EC₅₀ = 53 nM) (filled circle). In the presence of 10 ´M of cannabidiol (o ) the dose response curve of CP55940 is shifted to the right.

The inhibitory effects of SR141716A, ⁹-THC and cannabidiol were compared using a concentration of CP55940 giving a maximal [³⁵S]GTP- g -S binding stimulation (1 ´M). The IC₅₀ for SR141716A is 11.70 ˜ 1.20 nM; > 10 000 nM for ⁹-THC and 3350 ˜ 580 nM for cannabidiol. Fig. 3. But at 10 ´M ⁹-THC induced a significant antagonism, the effect of CP55940 being inhibited by approximately 30 %. Fig. 3. In the same conditions cannabinol (even at 10 ´M) did not antagonize the effect of CP55940.

Fig. 3: The maximal effect of CP55940 was obtained at 1 ´M (filled circle) and could be reversed by SR141716A (filled diamond), cannabidiol (open circle). (left panel) or ⁹-THC (open square) (right panel).

The effects of D ⁹-THC and cannabinol are not additive.

Cannabinol increases [³⁵S]GTP- g -S binding by 24 % at 1 ´M and ⁹-THC, at the same concentration, by 46 %. The combination of these two agonists (both at 1 ´M) gives a stimulation of [³⁵S]GTP- g -S binding of 53 ˜ 6 % which was not significantly different from ⁹-THC-induced stimulation. Fig. 4.

Fig. 4: ⁹-THC stimulates [³⁵S]GTP- g -S binding on rat cerebellar membranes by 46 % at 1 ´M (first column) and cannabinol by 24 % (second column). No additive effect was shown by the combination of ⁹-THC and cannabinol (both at 1 ´M) (third column).

Discussion

The marijuana plant may contain different proportions of ⁹-THC, cannabinol and cannabidiol and it is clear that the ratios of ⁹-THC, cannabinol and cannabidiol are important when interpreting the pharmacological or recreational effects of marijuana use. We and others have previously shown that the binding of the stable GTP analog [³⁵S]GTP- g -S on a rat cerebellar homogenate model provides a good functional test for cannabinoid receptors (18,19). In this test ⁹-THC behaves as a partial agonist giving only half of the maximal effect obtained with the full agonist CP55940. Moreover at high concentrations ⁹-THC exhibits antagonist properties. Comparable methodological approaches led to similar results recently published (8,9). Cannabinol appears as a weak agonist giving only 24% of the maximal effect even at high concentrations. Unlike ⁹-THC cannabinol is not an antagonist at high doses. Furthermore the effect of ⁹-THC and cannabinol are not additive. The intrinsic activity of cannabinol seems to be very small. Cannabinol, in man, exhibits either a very weak cannabinoid activity or partly potentiates the ⁹-THC effect (for review see 21).

We have also demonstrated that cannabidiol is a weak antagonist acting in the micromolar range. The evidence for antagonistic properties of cannabidiol in vivo are controversial: we have not been able to block CP55940-induced hypothermia in mice (unpublished results). Nonetheless, some evidence exists in the litterature: cannabidiol has been reported to reduce the psychological effects of ⁹-THC in humans (22) and animals (23) as well as the ⁹-THC-induced catatonia in mice and hypothermia in rabbits (14).

In conclusion, by using a functional assay in vitro, we provide here evidence that natural cannabinoids have a complex pharmacology. ⁹-THC acts as a partial agonist and, at high concentrations, exhibits antagonistic properties while cannabinol has a very weak intrinsic activity. Moreover, cannabidiol was shown to be a weak antagonist with micromolar potency.

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* Corresponding author: François Petitet, Neurochemistry Dpt, Rhône-Poulenc Rorer S.A., C.R.V.A., B.P. 14, 13 Quai Jules Guesde, 94403 Vitry-sur-Seine Cedex, France. Tel.: 33 1 55 71 34 78, Fax: 33 1 55 71 36 53, e-mail: Francois.PETITET@RP-RORER.FR