Mechanisms of Endocannabinoid Inactivation: Functional Consequences Daniele Piomelli University of California, Irvine SfN, November 7, 2003

CBR R E T E Endocannabinoid signaling Synapse T E

FAAH AT Anandamide 2-AG O NHNH OH O NHNH Arachidonic acid O OH Ethanolamine/Glycerol Endocannabinoid deactivation MGL

Design of endocannabinoid transport inhibitors R Cl NHNH O >100 O R OCH 3 NHNH >100 O R CH 3 NHNH >100 O R CN NHNH >100 IC 50 (  M) OH O R NHNH 21.3 ± ± 0.2 O OH NHNH R O NHNH Anandamide O OH NHNH AM404 Beltramo et al., Science, 1997 Piomelli et al., PNAS, 1999

AM404 inhibits anandamide transport in FAAH-deficient mice *** ** 0105 Time (min) -/ *** Time (min) Anandamide Transport +/+ Fegley et al., submitted

* /+ ** *** ** -/- Anandamide (100  M) AM404 (10  M) Anandamide Transport ** 0-4 o C *** +/+ -/- +/+ -/- +/+ -/- 37 o C AM404 inhibits anandamide transport in FAAH-deficient mice

Time (min) / ∆ Temperature (˚C) AM404 protects anandamide from inactivation in FAAH-deficient mice AEA 2 mg AEA 2 mg + AM404 AEA 5 mg

AM404 AM1172 (  M) ** Mouse cortical neurons Anandamide Transport O OH NHNH AM404 OH AM1172 NH O AM1172: a transport inhibitor not hydrolyzed by FAAH

Endocannabinoid transport inhibition increases brain levels of anandamide ++ *** ** * AEA (pmol/g) Amph AM Giuffrida et al., submitted

RACL+QUIN RACL AEA outflow (% baseline) Time (min) ** ** * * QUIN Anandamide outflow (% baseline) Time (min) Dopamine D 2 -receptor activation of anandamide release Giuffrida et al., Nat. Neurosci., 1999

Amph Amph+ AM404 Amph+AM404+Ri Time (min) # of cage crossings Inhibition of endocannabinoid transport reduces amphetamine hyperactivity Giuffrida et al., submitted

Anandamide Dopamine D2RD2R D1RD1R CB 1 R Psychomotor activity + - AM404 Inactivation Regulation of dopamine transmission by endocannabinoid transport inhibition Giuffrida et al., Nat. Neurosci., 1999

Conclusions AM404 protects anandamide from deactivation by inhibiting endocannabinoid transport AM404 reduces psychostimulant drug actions by elevating brain endocannabinoid levels Endocannabinoid transport might serve as target for the therapy of psychostimulant dependence or withdrawal

CBR R E T E Endocannabinoid signaling Synapse T E

c-C 6 H 11 O NH p-C 6 H 10 F O >100 R1R1 R IC 50 (  M) CH 3 >100 c-C 6 H n-C 4 H 9 O 0.39 CH c-C 6 H R1R1 R IC 50 (  M) R R1R1 N O H O Design of carbamate inhibitors of FAAH URB597 O NH 2 URB532 O

** V FAAH activity (pmol/min/mg prot) Time (h) 1000 Dose (log [mg kg -1 ]) FAAH activity (% of control) 10 URB532 URB597 ID 50 = 0.60 mg/kg ID 50 = 0.15 mg/kg Inhibition of brain FAAH activity in vivo Kathuria et al., Nature Medicine, 2003

** V 597V Anandamide (pmol/g) 0 2-AG (pmol/g) Anandamide2-AG Effect on brain endocannabinoid levels

Negative Weak Catalepsy Hypothermia Stimulation of feeding Analgesia Hypolocomotion Pharmacological actions of FAAH inhibitors: lack of cannabinoid-like activity

Anxiolytic-like effects of FAAH inhibitors: Isolation-induced vocalizations * * ** URB Vocaliztion (% baseline) Ri ** URB597 Ri Kathuria et al., Nature Medicine, 2003

Conclusions URB597 is a potent, selective and systemically active FAAH inhibitor URB597 does not produce overt cannabinoid-like effects, but has anxiolytic-like properties that are mediated by its ability to elevate brain anandamide levels FAAH may serve as target for the treatment of anxiety and depression

Thanks to: All the members of my lab, but particularly: Darren Fegley Jin Fu Silvana Gaetani Andrea Giuffrida Satish Kathuria Jesse Lo Verme Fariba Oveisi All our collaborators, but particularly: V. Cuomo (Rome, Italy) F. Rodríguez de Fonseca (Malaga, Spain) G. Tarzia, A. Duranti, A. Tontini (Urbino, Italy) M.Mor (Parma, Italy) NIDA for financial support