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

2. CBR R E T E Endocannabinoid signaling Synapse T E

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

4. 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 ± 3.4 2.2 ± 0.2 O OH NHNH R O NHNH Anandamide O OH NHNH AM404 Beltramo et al., Science, 1997 Piomelli et al., PNAS, 1999

5. AM404 inhibits anandamide transport in FAAH-deficient mice *** ** 0105 Time (min) -/- 3000 0 6000 0 10 5 *** Time (min) Anandamide Transport +/+ Fegley et al., submitted

6. * 55 0 110 +/+ ** *** ** -/- Anandamide (100  M) AM404 (10  M) Anandamide Transport ** 0-4 o C *** +/+ -/- +/+ -/- +/+ -/- 37 o C AM404 inhibits anandamide transport in FAAH-deficient mice

7. Time (min) / 2 1.5 1 0.5 0 30 50 7090 ∆ Temperature (˚C) AM404 protects anandamide from inactivation in FAAH-deficient mice AEA 2 mg AEA 2 mg + AM404 AEA 5 mg

8. 25 50 75 0 100 AM404 AM1172 (  M) 0.1 1 10 ** Mouse cortical neurons Anandamide Transport O OH NHNH AM404 OH AM1172 NH O AM1172: a transport inhibitor not hydrolyzed by FAAH

9. Endocannabinoid transport inhibition increases brain levels of anandamide ++ *** ** * -++ -++- - 100 0 AEA (pmol/g) Amph AM404 50 Giuffrida et al., submitted

10. RACL+QUIN RACL 300 100 0 060120180300240 AEA outflow (% baseline) Time (min) ** ** * * QUIN 900 300 500 700 100 0 Anandamide outflow (% baseline) 1100 060120180300240 Time (min) Dopamine D 2 -receptor activation of anandamide release Giuffrida et al., Nat. Neurosci., 1999

11. Amph Amph+ AM404 Amph+AM404+Ri 50 100 0 0 200 Time (min) # of cage crossings Inhibition of endocannabinoid transport reduces amphetamine hyperactivity Giuffrida et al., submitted

12. 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

13. 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

14. CBR R E T E Endocannabinoid signaling Synapse T E

15. c-C 6 H 11 O NH 2 0.004 p-C 6 H 10 F O >100 R1R1 R IC 50 (  M) CH 3 >100 c-C 6 H 11 0.32 n-C 4 H 9 O 0.39 CH 3 18.6 c-C 6 H 11 0.063 R1R1 R IC 50 (  M) R R1R1 N O H O Design of carbamate inhibitors of FAAH URB597 O NH 2 URB532 O

16. ** V 1500 500 0 FAAH activity (pmol/min/mg prot) 1.25246 Time (h) 1000 Dose (log [mg kg -1 ]) -2 100 50 0 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

17. ** 1000 2000 3000 V 597V 50 0 25 Anandamide (pmol/g) 0 2-AG (pmol/g) Anandamide2-AG Effect on brain endocannabinoid levels

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

20. Anxiolytic-like effects of FAAH inhibitors: Isolation-induced vocalizations * * ** URB532 120 Vocaliztion (% baseline) 0 80 40 - -2 - Ri 5 2 5 - 10 - 1 - ** 120 0 80 40 - -2 - URB597 Ri.1 2 -.05 - Kathuria et al., Nature Medicine, 2003

21. 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

22. 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