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Mechanisms of Endocannabinoid Inactivation: Functional Consequences

Explore the deactivation mechanisms of endocannabinoids, their impact on signaling and synapses, and the design of inhibitors. Learn how AM404 and other compounds affect endocannabinoid transport and levels in the brain.

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Mechanisms of Endocannabinoid Inactivation: Functional Consequences

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

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

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

  4. O IC50(mM) R N H CH3 >100 O O N H N H OH OH O >100 Anandamide R O >100 N H Cl OH N H OH >100 O R AM404 21.3 ± 3.4 N H O O R R 2.2 ± 0.2 R N H OCH3 N H CN Design of endocannabinoid transport inhibitors Beltramo et al., Science, 1997 Piomelli et al., PNAS, 1999

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

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

  7. 0.5 AEA 2 mg 0 0.5 AEA 5 mg ∆ Temperature (˚C) AEA 2 mg + AM404 1 1.5 2 / / 30 70 90 50 Time (min) AM404 protects anandamide from inactivation in FAAH-deficient mice

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

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

  10. ** 1100 * * * 900 Anandamide outflow (% baseline) 700 * CTX A 1 . 0 500 300 100 C P QUIN 0 0 60 120 180 240 300 N A Time (min) 300 AEA outflow (% baseline) 100 RACL RACL+QUIN 0 0 60 120 180 240 300 Time (min) Dopamine D2-receptor activation of anandamide release Giuffrida et al., Nat. Neurosci., 1999

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

  12. Regulation of dopamine transmission by endocannabinoid transport inhibition Anandamide AM404 Dopamine D2R D1R + Anandamide Psychomotor activity - Inactivation CB1R 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. T T CBR R Endocannabinoid signaling E E Synapse E

  15. H O N R1 R O R R1 IC50(mM) R R1 IC50(mM) CH3 >100 c-C6H11 0.063 O NH2 CH3 18.6 O NH2 c-C6H11 0.004 c-C6H11 0.32 URB597 n-C4H9 0.39 p-C6H10F >100 O O O URB532 Design of carbamate inhibitors of FAAH

  16. 100 1500 1000 FAAH activity (% of control) FAAH activity (pmol/min/mg prot) 50 500 URB532 ** ** URB597 ** ** ** 0 0 V .25 1 2 4 6 -2 -1 0 1 Dose (log [mg kg-1]) Time (h) Inhibition of brain FAAH activity in vivo ID50= 0.15 mg/kg ID50= 0.60 mg/kg Kathuria et al., Nature Medicine, 2003

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

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

  19. 120 120 80 80 * * ** Vocaliztion (% baseline) ** 40 40 0 0 URB532 - 1 5 10 5 - URB597 - .05 .1 .1 - Ri - - - - 2 2 Ri - - - 2 2 Anxiolytic-like effects of FAAH inhibitors: Isolation-induced vocalizations Kathuria et al., Nature Medicine, 2003

  20. 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

  21. 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

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