Microsoft powerpoint - poster_mainz2005_klein

CANNABIDIOL IS A NEGATIVE ALLOSTERIC MODULATOR AT THE µ OPIOID (MOP)
RECEPTOR
M. Kathmann, K. Flau, E. Schlicker
Department of Pharmacology and Toxicology, University of Bonn, Reuterstr. 2b, 53113 Bonn, [email protected] Introduction
∆9-Tetrahydrocannabinol (THC) is the main source of the pharmacological effects caused by the consumption of cannabis, both the marijuana-like action and the medicinal benefits of the plant. However, the non-psychotropic cannabidiol (CBD), several cannabinoid analogues and newly discovered modulators of the endogenous cannabinoid system are also promising candidates for clinical research and therapeutic uses. Cannabinoids exert many effects through activation of G-protein-coupled cannabinoid receptors in the brain and ecific l/mg
D = 0.68 ± 0.02 nM
peripheral tissues. To date, two types of cannabinoid receptors, CB Bmax = 203 ± 7 fmol/mg prot.
identified. Additionally, there is evidence for non-receptor-dependent mechanisms. Cannabidiol (CBD) occurs at high concentration in the cannabis plant but, unlike ∆9- tetrahydrocannabinol (∆9-THC), is devoid of a psychotropic effect, but little is known so far 75 100 125 150 175
[3H]-DAMGO (nM)
In the present study, the possibility that CBD possesses an allosteric effect at opioid Fig. 1 Saturation of specific 3H-DAMGO binding to rat cerebral cortex membranes. Specific binding was defined as that inhibited by
receptors (Vaysee et al. 1987) has been further examined, using the MOP receptor ligand naloxone 10 µM. Scatchard transformation of the saturation data is presented in the right panel. Mean ± SEM from 4 experiments in triplicate. Error bars are not shown when they are smaller than the symbols.
time (min)
3H-DAMGO. For the sake of comparison the CB1 agonist ∆9-THC and the selective CB1 antagonist rimonabant (previous name SR141716) were also included in this study.
Fig. 5 Effects of naloxone 10 µM alone and in the presence of 30 µM ∆9-THC (THC), 30 µM cannabidiol (CBD) and 10 µM
rimonabant (SR) on 3H-DAMGO dissociation in rat cerebral cortex membranes. Mean ± SEM from 4 experiments in triplicate. Error bars are not shown when they are smaller than the symbols.
Conclusions
Cerebral cortex membranes from male Wistar rats were homogenized (Potter-Elvehjem) in 25 volumes of ice-cold Tris-HCl-EDTA buffer (TE-buffer: Tris 50 mM; EDTA 5 mM; pH 7.5) containing 10.27 % sucrose and centrifuged at 1000 x g for 10 min (4°C). The supernatant was centrifuged at 35000 x g for 10 min (4°C) and the pellet was washed twice with TE-buffer. Finally the pellet was 1. The binding of 3H-DAMGO to rat cerebral cortex was displaced by cannabidiol, resuspended in TRIS-buffer (Tris 50 mM; pH 7.4) and frozen at -80°C. In saturation binding experiments seven concentrations of the radioligand 3H-DAMGO (0.1 nM to 10 nM) were used in a final volume of 0.5 ml TRIS-buffer containing 10 µM AEBSF (serine proteinase inhibitor). Incubation was performed at 25°C and lasted for 60 min. 15 30 45 60 75 90 105 120
The opioid receptor antagonist naloxone (10 µM) was used to determine the non-specific binding.
90 105 120
2. The dissociation of 3H-DAMGO induced by naloxone 10 µM was not affected time (min)
time (min)
by rimonabant 10 µM but increased by cannabidiol and ∆9-THC by a factor of Displacement experiments with 3H-DAMGO (0.5 nM) were performed in TRIS-buffer in a final volume of 0.5 ml containing various concentrations of the drugs under study. Naloxone (10 µM) was taken to determine the non-specific binding and AEBSF (10 µM) was 12 and 2, respectively; the respective pEC Fig. 2 Association (left panel) and dissociation (right panel) kinetics of 3H-DAMGO binding to rat cerebral cortex membranes. Mean ±
used to inhibit proteinase. Incubation was performed at 25°C and lasted for 120 min.
SEM from 4 experiments in triplicate. Error bars are not shown when they are smaller than the symbols.
Kinetic experiments. To study 3H-DAMGO association kinetics, assays were prepared in a larger volume of TRIS-buffer allowing 3. The present study adds a further piece of evidence that cannabidiol is a withdrawal of several aliquots of 0.5 ml at adequate time intervals over a period of al least 120 min. Naloxone (10 µM) was taken to negative allosteric modulator at the MOP receptor. This property is shared by determine the non-specific binding and AEBSF (10 µM) was used to inhibit proteinase. 3H-DAMGO dissociation kinetics: Radioligand and membranes were incubated for 45 min at 25 °C and then naloxone (10 µM) or a combination of naloxone and the drugs under study was added. Assays were prepared a larger volume of TRIS-buffer allowing withdrawal of several aliquots of 0.5 ml at adequate time intervals over a period of at least additional 120 min. Data analysis. Experimental data from the individual experiments were analyzed by computer-aided, nonlinear regression analysis using Prism software (Vers. 3.0, Graph Pad®, San Diego, USA). Effects of the allosteric agents on 3H-DAMGO equilibrium binding were analyzed according to the ternary model of allosteric interaction as formulated by Ehlert (1988).
References
Ehlert FJ (1988) Estimation of the affinities of allosteric ligands using radioligand binding and pharmacological null
methods. Mol Pharmacol 33:187-194
Vaysee PJJ, Gardner EL, Zukin RS (1987) Modulation of rat brain opioid receptors by cannabinoids. J Pharm Exp Ther
241:534-539
Drugs [log M]
Drugs (log M)
Fig. 3 Effects on 3H-DAMGO equilibrium binding to rat
Fig. 4 Increase in 3H-DAMGO dissociation kinetics in rat cerebral
cerebral cortex membranes of ∆9-THC (THC), cannabidiol cortex membranes by ∆9-THC (THC) and cannabidiol (CBD). (CBD) and rimonabant (SR). Curve fitting was based on the rimonabant
Mean ± SEM from 4 experiments in triplicate. Error bars are not cannabidiol
ternary complex model of allosteric interactions. Mean ± shown when they are smaller than the symbols.
SEM from 4 experiments in triplicate. Error bars are not shown when they are smaller than the symbols.

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