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

Alessia Ligresti, Maria Grazia Cascio, Gareth Pryce, Sanjitha Kulasegram, Irina Beletskaya, Luciano De Petrocellis, Bijali Saha, Anu Mahadevan, Cristina Visintin, Jenny L. Wiley, David Baker, Billy R. Martin, Raj K. Razdan, Vincenzo Di Marzo*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

51 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)83-91
Number of pages9
JournalBritish Journal of Pharmacology
Issue number1
Publication statusPublished - 29 Jan 2009

Bibliographical note

Acknowledgement: The authors acknowledge the Italian Ministry of University and Research (within the FIRB program to VDM), the Multiple Sclerosis Society of Great Britain and Northern Ireland (to DB), the Brain Research Trust (to DB), and the National Institute for Drug Abuse (Grants DA-09789 and DA-08904) for funding this work.


  • 2-arachidonoylglycerol
  • Cannabinoid
  • Endocannabinoid
  • Membrane transport
  • Multiple sclerosis
  • Spasticity


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