New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis

1. We previously reported that the compound O-2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re-examined the activity of O-2093 in vivo and synthesized four structural analogs (O-2247, O-2248, O-3246, and O-3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB ₁ and CB ₂ receptors; (b) assays of transient receptor potential of the vanilloid type-1 (TRPV1) channel functional activity (measurement of [Ca ²⁺] _i); (c) [ ¹⁴C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL-2H3) cells; (d) the mouse 'tetrad' tests (analgesia on a hot plate, immobility on a 'ring', rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS). 2. O-2093, either synthesized by us or commercially available, was inactive in the 'tetrad' up to a 20 mg kg ^-1 dose (i.v.). Like O-2093, the other four compounds exhibited low affinity in CB ₁ (K _i from 1.3 to > 10 μM) and CB ₂ binding assays (1.3 < K _i < 8 μM), low potency and efficacy in a TRPV1 functional assay (EC ₅₀ > 10 μM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC ₅₀ > 25 μM) and were inactive in the 'tetrad' up to a 30 mg kg ^-1 dose (i.v.). 3. While O-2247 and O-2248 were poor inhibitors of [ ¹⁴C]AEA cellular uptake (IC ₅₀ > 40 μM), O-3246 and O-3262 were quite potent in this assay. O-3246, which exhibits only a very subtle structural difference with O-2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC ₅₀ = 1.4 μM) and is 12-fold more potent than O-2093. 4. When injected intravenously O-3246 and O-3262, again like O-2093 and unlike O-2247 and O-2248, significantly inhibited limb spasticity in mice with CREAE. 5. These data confirm the potential utility of selective AEA uptake inhibitors as anti-spasticity drugs in MS and, given the very subtle chemical differences between potent and weak inhibitors of uptake, support further the existence of a specific mechanism for this process.