New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis.
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) [(14)C]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). 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 microM) and CB(2) binding assays (1.3<K(i)< 8 microM), low potency and efficacy in a TRPV1 functional assay (EC(50)>10 microM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC(50)>25 microM) and were inactive in the 'tetrad' up to a 30 mg kg(-1) dose (i.v.). While O-2247 and O-2248 were poor inhibitors of [(14)C]AEA cellular uptake (IC(50)>40 microM), 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 microM) and is 12-fold more potent than O-2093. 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. 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.
Ligresti
Alessia
A
Endocannabinoid Research Group, Institute of Biomolecular Chemistry, C.N.R. Pozzuoli (Napoli), Italy.
Cascio
Maria Grazia
MG
Pryce
Gareth
G
Kulasegram
Sanjitha
S
Beletskaya
Irina
I
De Petrocellis
Luciano
L
Saha
Bijali
B
Mahadevan
Anu
A
Visintin
Cristina
C
Wiley
Jenny L
JL
Baker
David
D
Martin
Billy R
BR
Razdan
Raj K
RK
Di Marzo
Vincenzo
V
D000818:Animals; D001095:Arachidonic Acids / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002460:Cell Line; D004195:Disease Models, Animal; D063388:Endocannabinoids; D051379:Mice; D009103:Multiple Sclerosis / Q000188:drug therapy* / Q000378:metabolism; D009128:Muscle Spasticity / Q000188:drug therapy*; D009466:Neuromuscular Blocking Agents / Q000494:pharmacology*; D053284:Polyunsaturated Alkamides / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats
D001095:Arachidonic Acids; D063388:Endocannabinoids; D009466:Neuromuscular Blocking Agents; D053284:Polyunsaturated Alkamides; C078814:anandamide