Selective activation of cannabinoid CB2 receptors suppresses neuropathic nociception induced by treatment with the chemotherapeutic agent paclitaxel in rats.

Activation of cannabinoid CB(2) receptors suppresses neuropathic pain induced by traumatic nerve injury. The present studies were conducted to evaluate the efficacy of cannabinoid CB(2) receptor activation in suppressing painful peripheral neuropathy evoked by chemotherapeutic treatment with the antitumor agent paclitaxel. Rats received paclitaxel (2 mg/kg i.p./day) on 4 alternate days to induce mechanical hypersensitivity (mechanical allodynia). Mechanical allodynia was defined as a lowering of the threshold for paw withdrawal to stimulation of the plantar hind paw surface with an electronic von Frey stimulator. Mechanical allodynia developed in paclitaxel-treated animals relative to groups receiving the Cremophor EL/ethanol/saline vehicle at the same times. Two structurally distinct cannabinoid CB(2) agonists, the aminoalkylindole (R,S)-AM1241 [(R,S)-(2-iodo-5-nitrophenyl)-[1-((1-methyl-piperidin-2-yl)methyl)-1H-indol-3-yl]-methanone] and the cannabilactone AM1714 (1,9-dihydroxy-3-(1',1'-dimethylheptyl)-6H-benzo[c]chromene-6-one), produced a dose-related suppression of established paclitaxel-evoked mechanical allodynia after systemic administration. Pretreatment with the CB(2) antagonist SR144528 [5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-N-(1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl)-1H-pyrazole-3-carboxamide], but not the CB(1) antagonist SR141716 [5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide], blocked the antiallodynic effects of both (R,S)-AM1241 and AM1714. Moreover, (R)-AM1241, but not (S)-AM1241, suppressed paclitaxel-evoked mechanical allodynia relative to either vehicle treatment or preinjection thresholds, consistent with mediation by CB(2). Administration of either the CB(1) or CB(2) antagonist alone failed to alter paclitaxel-evoked mechanical allodynia. Moreover, (R,S)-AM1241 did not alter paw withdrawal thresholds in rats that received the Cremophor EL vehicle in lieu of paclitaxel, whereas AM1714 induced a modest antinociceptive effect. Our data suggest that cannabinoid CB(2) receptors may be important therapeutic targets for the treatment of chemotherapy-evoked neuropathy.

Cannabilactones: a novel class of CB2 selective agonists with peripheral analgesic activity.

The identification of the CB2 cannabinoid receptor has provided a novel target for the development of therapeutically useful cannabinergic molecules. We have synthesized benzo[ c]chromen-6-one analogs possessing high affinity and selectivity for this receptor. These novel compounds are structurally related to cannabinol (6,6,9-trimethyl-3-pentyl-6 H-benzo[ c]chromen-1-ol), a natural constituent of cannabis with modest CB2 selectivity. Key pharmacophoric features of the new selective agonists include a 3-(1',1'-dimethylheptyl) side chain and a 6-oxo group on the cannabinoid tricyclic structure that characterizes this class of compounds as "cannabilactones." Our results suggest that the six-membered lactone pharmacophore is critical for CB2 receptor selectivity. Optimal receptor subtype selectivity of 490-fold and subnanomolar affinity for the CB2 receptor is exhibited by a 9-hydroxyl analog 5 (AM1714), while the 9-methoxy analog 4b (AM1710) had a 54-fold CB2 selectivity. X-ray crystallography and molecular modeling show the cannabilactones to have a planar ring conformation. In vitro testing revealed that the novel compounds are CB2 agonists, while in vivo testing of cannabilactones 4b and 5 found them to possess potent peripheral analgesic activity.

(-)-7'-Isothiocyanato-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM841), a high-affinity electrophilic ligand, interacts covalently with a cysteine in helix six and activates the CB1 cannabinoid receptor.

The CB1 cannabinoid receptor has been shown to play important physiological roles in the central nervous system, as well as peripherally, and is a target for development of therapeutic medications. To gain insight on the ligand binding site(s) and structural features of activation, we designed and synthesized (-)-7'-isothiocyanato-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM841), a classical cannabinoid affinity label that incorporates an isothiocyanate substituent as an electrophilic reactive group capable of interacting irreversibly with a suitably located and properly oriented nucleophilic amino acid residue at or near the binding site. To obtain evidence for the site of covalent attachment of AM841, C6.47, identified in part by interactive ligand docking, was mutated to serine, alanine, and leucine to reduce or eliminate the nucleophilic character. Wild-type (WT) and mutant CB1 receptors were evaluated for their abilities to recognize a series of cannabinergic ligands. Each bound comparably to WT, excluding C6.47L, which displayed a reduced affinity for 3H-labeled (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940), AM841, 11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM4056), and (-)-7'-bromo-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM4043) and an improvement in affinity for (-)-trans-delta9-tetrahydrocannabinol (delta9-THC). The affinity of 3H-labeled [2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](naphthyl)methanone (WIN55212-2) was unchanged across all mutants. It is noteworthy that AM841 was shown to bind irreversibly to WT CB1 but exhibited no covalent attachment with the mutants and behaved as an agonist suggesting irreversible attachment to C6.47 maintains CB1 in its active state. The evidence presented identifies C6.47 as the site of covalent bond formation with AM841 and combined with the binding data fully supports the molecular modeling. These studies present the first report of tandem applications of affinity labeling, site-directed mutagenesis, and interactive ligand docking for CB1.