Mechanisms of Endocannabinoid Inactivation:

Functional Consequences

Daniele PiomelliUniversity of California, Irvine

SfN, November 7, 2003

CBR

R

E

T

E

Endocannabinoid signaling

SynapseT

E

FAAH

AT

Anandamide2-AG

O

NH

OH

O

NH

OHArachidonic

acid

O

OH

Ethanolamine/Glycerol

Endocannabinoid deactivation

MGL

Design of endocannabinoid transport inhibitors

RClN

H

O

>100

OR

OCH3NH

>100

OR

CH3NH

>100

OR

CNNH

>100

IC50(M)

OH

OR

NH

21.3 ± 3.4

2.2 ± 0.2O

OHNH

R

OOH

NH

Anandamide

O

OHNH

AM404Beltramo et al., Science, 1997

Piomelli et al., PNAS, 1999

AM404 inhibits anandamide transport in FAAH-deficient mice

***

**

0 105

Time (min)-/-

3000

0

6000

0 105

***

***

Time (min)

Ana

ndam

ide

Tran

spor

t+/+

Fegley et al., submitted

*55

0

110+/+

** *** **

-/-

Anandamide (100 M)

AM404(10 M)

Ana

ndam

ide

Tran

spor

t

**

0-4oC

***

+/+ -/- +/+ -/- +/+ -/-

37oC

AM404 inhibits anandamide transport in FAAH-deficient mice

Time (min)

/ /2

1.5

1

0.5

0

0.5

30 50 70 90

∆ Te

mpe

ratu

re (˚

C)

AM404 protects anandamide from inactivationin FAAH-deficient mice

AEA 2 mg

AEA 2 mg + AM404

AEA 5 mg

25

50

75

0

100

AM404AM1172 (M)

0.1 1 10

**

** **

Mouse cortical neurons

Ana

ndam

ide

Tran

spor

t

O

OHNH

AM404

OH

AM1172

NHO

AM1172: a transport inhibitor not hydrolyzed by FAAH

Endocannabinoid transport inhibition increases brain levels of anandamide

++

***

**

*

- + +- + +-

-

100

0

AEA

(pm

ol/g

)

AmphAM404

50

Giuffrida et al., submitted

C P

N A

A 1.0 CTX

RACL+QUINRACL

300

100

00 60 120 180 300240

AEA

out

flow

(% b

asel

ine)

Time (min)

** ***

*

QUIN

900

300

500

700

100

0

Ana

ndam

ide

outfl

ow(%

bas

elin

e) 1100

0 60 120 180 300240

Time (min)

Dopamine D2-receptor activation of anandamide release

Giuffrida et al., Nat. Neurosci., 1999

AmphAmph+ AM404Amph+AM404+Ri

50 10000

100

200

Time (min)

# of

cag

e cr

ossi

ngs

Inhibition of endocannabinoid transportreduces amphetamine hyperactivity

Giuffrida et al., submitted

AnandamideAnandamide

Dopamine

D2R D1R

CB1R

Psychomotor activity

+

-AM404

Inactivation

Regulation of dopamine transmission byendocannabinoid 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

ET

E

Endocannabinoid signaling

Synapse

TE

c-C6H11

O NH2

0.004

p-C6H10FO >100

R1R IC50(M)

CH3 >100

c-C6H11 0.32

n-C4H9O 0.39

CH3 18.6

c-C6H11 0.063

R1R IC50(M)

R R1

N

O

HO

Design of carbamate inhibitors of FAAH

URB597

O NH2

URB532

O

**** ** ** **

V

1500

500

0FA

AH

act

ivity

(pm

ol/m

in/m

g pr

ot)

1.25 2 4 6Time (h)

1000

Dose (log [mg kg-1])-2

100

50

0FAA

H a

ctiv

ity (%

of c

ontr

ol)

1-1 0

URB532URB597

ID50= 0.60 mg/kg

ID50= 0.15 mg/kg

Inhibition of brain FAAH activity in vivo

Kathuria et al., Nature Medicine, 2003

**

1000

2000

3000

V 597 V 597

50

0

25

Ana

ndam

ide

(pm

ol/g

)

02-

AG

(pm

ol/g

)

Anandamide 2-AG

Effect on brain endocannabinoid levels

Negative

Negative

Negative

Weak

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

* * **

URB532

120Vo

caliz

tion

(% b

asel

ine)

0

80

40

-- 2

-Ri

52

5-

10-

1-

**

120

0

80

40

-- 2

-URB597Ri

.12

.1-

.05-

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 FegleyJin FuSilvana GaetaniAndrea GiuffridaSatish KathuriaJesse Lo VermeFariba 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