Cannabinoid CB2 agonists produce antinociception without central nervous system (CNS) side-effects. (5 mg/kg i.p.) produced a longer period of antinociceptive action than DMXAA (ASA404) the aminoalkylindole CB2 agonist (pharmacological profile associated with cannabilactone administration and determine whether compounds of this class show limited CNS side-effects. It remains unknown whether DMXAA (ASA404) systemic administration of cannabilactones such as AM1710 lack cardinal indicators of CB1 receptor activation (e.g. hypothermia hypoactivity motor ataxia) or exhibit an unfavorable CNS profile. This examination is important for validating the therapeutic potential of the cannabilactones for the treatment of pain. The present studies were conducted to evaluate the antinociceptive properties of the cannabilactone AM1710 (Fig 1) (Ki: CB1 vs. CB2: 360 nM vs. 6.7 nM) (Khanolkar cells and purified using the procedure disclosed by Patricelli and colleagues (1998). Recombinant hexa-histidine-tagged human MGL (hMGL) was expressed in cells and purified as reported by Zvonok and colleagues (2008a; 2008b). A high-throughput fluorometric screening assay for rFAAH inhibition using the fluorescent substrate arachidonoyl 7-amino-4-methylcoumarin amide (AAMCA) was performed as previously reported (Ramarao 0.05 was considered statistically significant. 3 Results 3.1 Results of in vitro screen for target selectivity AM1710 demonstrated 17-fold selectivity for mCB2 (Ki = 17+/?10 nM) compared to rCB1 (Ki = 282 +/?91 nM; data are the average +/? standard deviation of five individual experiments run in triplicate). An screen was also used to assess the target selectivity of AM1710 for CB2 receptors. The Novascreen failed to identify off-target activity of AM1710 at 62 different targets including neurotransmitter-related G-protein coupled receptors steroids ion channels second messenger-related prostaglandins growth factors/hormones brain/gut peptides and enzymes (Supplementary File). In the NovaScreen AM1710 did not inhibit [3H]CP55 940 binding to hCB1 at 100 nM but exhibited 50% inhibition of binding at10 0 CD263 nM. In a fluorescence assay AM1710 in concentrations up to 100 μM also failed to inhibit activity of fatty-acid amide hydrolase and monoacylglycerol lipase enzymes implicated in endocannabinoid deactivation (data not shown). 3.2 Brain DMXAA (ASA404) Barrier Penetration of AM1710 An screen was used to determine the ability of AM1710 to cross the blood brain barrier using intravenously administered doses of 1 1 mg/kg. The amount of AM1710 found in the unperfused brain tissue was 0.066%/g of the injected dose while plasma contained 0.000086%/mL (Table DMXAA (ASA404) 1). AM1710 has a low DMXAA (ASA404) brain penetration expected compared to other cannabilactones screened in this class (B/P ratio range = 0.03-1.3 mL/g; unpublished results). Table 1 Brain barrier penetration of AM1710 (1 mg/kg i.v.) 3.3 Behavioral Results 3.3 General Results Thermal paw withdrawal latencies and mechanical paw withdrawal thresholds did not differ between right or left paws for any group. Therefore withdrawal thresholds in all studies are offered as the mean of duplicate measurements averaged across paws. Baseline responses (i.e. thermal paw withdrawal latencies or mechanical withdrawal thresholds) were also comparable between groups prior to administration of drug or vehicle. Baseline paw withdrawal latencies did not differ between groups in any study; therefore baselines in the log dose response plot (Fig 2) were averaged across all doses of the same drug for statistical analyses. Moreover paw withdrawal latencies and thresholds did not differ based upon the order of thermal and mechanical screening at baseline; therefore the two vehicle groups are combined for all those studies offered. Physique 2 (a) Log dose response for AM1710-induced antinociception in the plantar test. (b) Time course of antinociceptive effects observed following administration of AM1710 (5 mg/kg i.p.) in DMXAA (ASA404) comparison with (< 0.05 planned comparison t-test; Table 1). However this same dose did not alter post-injection thresholds relative to the vehicle condition. Moreover antagonist pre-treatment did not alter paw withdrawal thresholds relative to baseline (Table 2). Paw withdrawal thresholds were not altered by (< 0.001; < 0.05 for each comparison). All doses of AM1710.