Alexander M. Zvonok

Activation of CB2 cannabinoid receptors by AM1241 inhibits experimental neuropathic pain: pain inhibition by receptors not present in the CNS.

We designed AM1241, a selective CB2 cannabinoid receptor agonist, and used it to test the hypothesis that CB2 receptor activation would reverse the sensory hypersensitivity observed in neuropathic pain states. AM1241 exhibits high affinity and selectivity for CB2 receptors. It also exhibits high potency in vivo. AM1241 dose-dependently reversed tactile and thermal hypersensitivity produced by ligation of the L5 and L6 spinal nerves in rats. These effects were selectively antagonized by a CB2 but not by a CB1 receptor antagonist, suggesting that they were produced by actions of AM1241 at CB2 receptors. AM1241 was also active in blocking spinal nerve ligation-induced tactile and thermal hypersensitivity in mice lacking CB1 receptors (CB1-/- mice), confirming that AM1241 reverses sensory hypersensitivity independent of actions at CB1 receptors. These findings demonstrate a mechanism leading to the inhibition of pain, one that targets receptors localized exclusively outside the CNS. Further, they suggest the potential use of CB2 receptor-selective agonists for treatment of human neuropathic pain, a condition currently without consistently effective therapies. CB2 receptor-selective agonist medications are predicted to be without the CNS side effects that limit the effectiveness of currently available medications.

Activation of peripheral cannabinoid CB1 and CB2 receptors suppresses the maintenance of inflammatory nociception: a comparative analysis

Effects of locally administered agonists and antagonists for cannabinoid CB1 and CB2 receptors on mechanical and thermal hypersensitivity were compared after the establishment of chronic inflammation.

Selective Activation of Cannabinoid CB2 Receptors Suppresses Neuropathic Nociception Induced by Treatment with the Chemotherapeutic Agent Paclitaxel in Rats

Activation of cannabinoid CB2 receptors suppresses neuropathic pain induced by traumatic nerve injury. The present studies were conducted to evaluate the efficacy of cannabinoid CB2 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 CB2 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 CB2 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 CB1 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 CB2. Administration of either the CB1 or CB2 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 CB2 receptors may be important therapeutic targets for the treatment of chemotherapy-evoked neuropathy.

Identification of a potent and highly efficacious, yet slowly desensitizing CB1 cannabinoid receptor agonist

The relationship of agonist efficacy to the rate of G protein‐coupled receptor signaling desensitization is controversial. Expressing inwardly rectifying potassium channels (GIRKs) in Xenopus oocytes, we have devised a signaling assay that clearly identifies CB1 cannabinoid receptor agonists with low intrinsic efficacy. In this assay, the synthetic CB1 agonists, AM411, AM782, AM1902, AM2233 and WIN55,212‐2 and the endogenous cannabinoid, 2‐arachidonoyl ester, were full agonists. The synthetic CB1 agonist AM356 (methanandamide), the endogenous cannabinoids, anandamide and 2‐arachidonoyl ether, and the phytocannabinoid, Δ9THC, were partial agonists. The rate of desensitization of CB1 was independent of agonist efficacy. WIN55,212‐2, AM782, AM1902, AM2233, and 2‐arachidonoyl glycerol ester all desensitized quickly, with desensitization rates varying from 14% min−1 to 10% min−1. AM356, AM411, anandamide, and Δ9THC all desensitized considerably slower, at a rate of 5% min−1. Despite high potency and efficacy, AM411 desensitized as slowly as anandamide and Δ9THC. CB1 agonist efficacy and rate of desensitization are not necessarily related.

Self-medication of a cannabinoid CB2 agonist in an animal model of neuropathic pain

&NA; Drug self‐administration methods were used to test the hypothesis that rats would self‐medicate with a cannabinoid CB2 agonist to attenuate a neuropathic pain state. Self‐medication of the CB2 agonist (R,S)‐AM1241, but not vehicle, attenuated mechanical hypersensitivity produced by spared nerve injury. Switching rats from (R,S)‐AM1241 to vehicle self‐administration also decreased lever responding in an extinction paradigm. (R,S)‐AM1241 self‐administration did not alter paw withdrawal thresholds in sham‐operated or naive animals. The percentage of active lever responding was similar in naive groups self‐administering vehicle or (R,S)‐AM1241. The CB2 antagonist SR144528 blocked both antiallodynic effects of (R,S)‐AM1241 self‐medication and the percentage of active lever responding in neuropathic (but not naive) rats. Neuropathic and sham groups exhibited similar percentages of active lever responding for (R,S)‐AM1241 on a fixed ratio 1 (FR1) schedule. However, neuropathic animals worked harder than shams to obtain (R,S)‐AM1241 when the schedule of reinforcement was increased (to FR6). (R,S)‐AM1241 self‐medication on FR1, FR3, or FR6 schedules attenuated nerve injury‐induced mechanical allodynia. (R,S)‐AM1241 (900 μg intravenously) failed to produce motor ataxia observed after administration of the mixed CB1/CB2 agonist WIN55,212‐2 (0.5 mg/kg intravenously). Our results suggest that cannabinoid CB2 agonists may be exploited to treat neuropathic pain with limited drug abuse liability and central nervous system side effects. These studies validate the use of drug self‐administration methods for identifying nonpsychotropic analgesics possessing limited abuse potential. These methods offer potential to elucidate novel analgesics that suppress spontaneous neuropathic pain that is not measured by traditional assessments of evoked pain. Rats self‐administer a cannabinoid CB2 agonist to attenuate neuropathic pain. This approach may identify analgesics that suppress spontaneous neuropathic pain with limited drug abuse liability.

The cannabinoid CB2 receptor agonist AM1241 enhances neurogenesis in GFAP/Gp120 transgenic mice displaying deficits in neurogenesis

HIV‐1 glycoprotein Gp120 induces apoptosis in rodent and human neurons in vitro and in vivo. HIV‐1/Gp120 is involved in the pathogenesis of HIV‐associated dementia (HAD) and inhibits proliferation of adult neural progenitor cells (NPCs) in glial fibrillary acidic protein (GFAP)/Gp120 transgenic (Tg) mice. As cannabinoids exert neuroprotective effects in several model systems, we examined the protective effects of the CB2 receptor agonist AM1241 on Gp120‐mediated insults on neurogenesis.

Immunofluorescent spectral analysis reveals the intrathecal cannabinoid agonist, AM1241, produces spinal anti-inflammatory cytokine responses in neuropathic rats exhibiting relief from allodynia.

During pathological pain, the actions of the endocannabinoid system, including the cannabinoid 2 receptor (CB2R), leads to effective anti‐allodynia and modifies a variety of spinal microglial and astrocyte responses. Here, following spinal administration of the CB2R compound, AM1241, we examined immunoreactive alterations in markers for activated p38 mitogen‐activated protein kinase, interleukin‐1β (IL‐1β), the anti‐inflammatory cytokine, interleukin‐10 (IL‐10) as well as degradative endocannabinoid enzymes, and markers for altered glial responses in neuropathic rats. In these studies, the dorsal horn of the spinal cord and dorsal root ganglia were examined. AM1241 produced profound anti‐allodynia with corresponding immunoreactive levels of p38 mitogen‐activated kinase, IL‐1β, IL‐10, the endocannabinoid enzyme monoacylglycerol lipase, and astrocyte activation markers that were similar to nonneuropathic controls. In contrast, spinal AM1241 did not suppress the increased microglial responses observed in neuropathic rats. The differences in fluorescent markers were determined within discrete anatomical regions by applying spectral analysis methods, which virtually eliminated nonspecific signal during the quantification of specific immunofluorescent intensity. These data reveal expression profiles that support the actions of intrathecal AM1241 control pathological pain through anti‐inflammatory mechanisms by modulating critical glial factors, and additionally decrease expression levels of endocannabinoid degradative enzymes.

Pharmacological characterization of AM1710, a putative cannabinoid CB2 agonist from the cannabilactone class: antinociception without central nervous system side-effects.

Cannabinoid CB(2) agonists produce antinociception without central nervous system (CNS) side-effects. This study was designed to characterize the pharmacological and antinociceptive profile of AM1710, a CB(2) agonist from the cannabilactone class of cannabinoids. AM1710 did not exhibit off-target activity at 63 sites evaluated. AM1710 also exhibited limited blood brain barrier penetration. AM1710 was evaluated in tests of antinociception and CNS activity. CNS side-effects were evaluated in a modified tetrad (tail flick, rectal temperature, locomotor activity and rota-rod). Pharmacological specificity was established using CB(1) (SR141716) and CB(2) (SR144528) antagonists. AM1710 (0.1-10mg/kg i.p.) produced antinociception to thermal but not mechanical stimulation of the hindpaw. AM1710 (5mg/kg i.p.) produced a longer duration of antinociceptive action than the aminoalkylindole CB(2) agonist (R,S)-AM1241 (1mg/kg i.p.) at maximally antinociceptive doses. Antinociception produced by the low (0.1mg/kg i.p.) dose of AM1710 was blocked selectively by the CB(2) antagonist SR144528 (6mg/kg i.p.), whereas antinociception produced by the high dose of AM1710 (5mg/kg i.p.) was blocked by either SR144528 (6mg/kg i.p.) or SR141716 (6mg/kg i.p.). AM1710 did not produce hypoactivity, hypothermia, tail flick antinociception, or motor ataxia when evaluated in the tetrad at any dose. In conclusion, AM1710, a CB(2)-preferring cannabilactone, produced antinociception in the absence of CNS side-effects. Thus, any CB(1)-mediated antinociceptive effects of this compound may be attributable to peripheral CB(1) activity. The observed pattern of pharmacological specificity produced by AM1710 is consistent with limited blood brain barrier penetration of this compound and absence of CNS side-effects.

Therapeutic modulation of cannabinoid lipid signaling: metabolic profiling of a novel antinociceptive cannabinoid-2 receptor agonist.

AIMS AM-1241, a novel, racemic cannabinoid-2 receptor (CB2) ligand, is the primary experimental agonist used to characterize the role of CB2-mediated lipid signaling in health and disease, including substance abuse disorders. In vivo pharmacological effects have been used as indirect proxies for AM-1241 biotransformation processes that could modulate CB2 activity. We report the initial pre-clinical characterization of AM-1241 biotransformation and in vivo distribution. MAIN METHODS AM-1241 metabolism was characterized in a variety of predictive in vitro systems (Caco-2 cells; mouse, rat and human microsomes) and in the mouse in vivo. Liquid chromatography and mass spectrometry techniques were used to quantify AM-1241 tissue distribution and metabolic conversion. KEY FINDINGS AM-1241 bound extensively to plasma protein/albumin. A pharmacological AM-1241 dose (25mg/kg, i.v.) was administered to mice for direct determination of its plasma half-life (37 min), following which AM-1241 was quantified in brain, spleen, liver, and kidney. After p.o. administration, AM-1241 was detected in plasma, spleen, and kidney; its oral bioavailability was ~21%. From Caco-2 permeability studies and microsomal-based hepatic clearance estimates, in vivo AM-1241 absorption was moderate. Hepatic microsomal metabolism of AM-1241 in vitro generated hydroxylation and demethylation metabolites. Species-dependent differences were discovered in AM-1241s predicted hepatic clearance. Our data demonstrate that AM-1241 has the following characteristics: a) short plasma half-life; b) limited oral bioavailability; c) extensive plasma/albumin binding; d) metabolic substrate for hepatic hydroxylation and demethylation; e) moderate hepatic clearance. SIGNIFICANCE These results should help inform the design, optimization, and pre-clinical profiling of CB2 ligands as pharmacological tools and medicines.

[INCREMENT]9-Tetrahydrocannabinol discriminative stimulus effects of AM2201 and related aminoalkylindole analogs in rats.

The recent recreational use of synthetic cannabinoid ligands, collectively referred to as ‘Spice’, has raised concerns about their safety and possible differences in their biological effect(s) from marijuana/&Dgr;9-tetrahydrocannabinol (THC). AM2201, a highly efficacious, potent cannabinoid receptor 1 (CB1R) agonist, is a recently detected compound in ‘Spice’ preparations. Furthermore, structural analogs of AM2201 are now being found in ‘Spice’. The present studies were conducted to investigate their &Dgr;9-THC-like effects using drug (&Dgr;9-THC) discrimination in rats. Results show that the tested compounds were potent cannabinergics that generalized to the response to &Dgr;9-THC, with AM2201 being most potent, exhibiting a 14-fold potency difference over &Dgr;9-THC. The other analogs were between 2.5-fold and 4-fold more potent than THC. Surmountable antagonism of AM2201 with the selective CB1R antagonist/inverse agonist rimonabant also established that the discrimination is CB1R dependent. Time-course data reveal that AM2201 likely peaks rapidly with an in-vivo functional half-life of only 60 min. The present data confirm and extend previous observations regarding &Dgr;9-THC-like effects of ‘Spice’ components.