UC Irvine UC Irvine Previously Published Works Title Modulation of anxiety through blockade of anandamide hydrolysis. Permalink https://escholarship.org/uc/item/9vp7s85t Journal Nature medicine, 9(1) ISSN 1078-8956 Authors Kathuria, Satish Gaetani, Silvana Fegley, Darren et al. Publication Date 2003 DOI 10.1038/nm803 License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California ARTICLES Modulation of anxiety through blockade of anandamide hydrolysis SATISH KATHURIA1, SILVANA GAETANI1, DARREN FEGLEY1, FERNANDO VALIÑO1, ANDREA DURANTI2, ANDREA TONTINI2, MARCO MOR3, GIORGIO TARZIA2, GIOVANNA LA RANA4, ANTONIO CALIGNANO4, ARCANGELA GIUSTINO5, MARIA TATTOLI5, MAURA PALMERY6, VINCENZO CUOMO6 & DANIELE PIOMELLI1 1Department of Pharmacology, University of California, Irvine, California, USA 2Institute of Medicinal Chemistry, University of Urbino, Urbino, Italy 3Pharmaceutical Department, University of Parma, Parma, Italy 4Department of Experimental Pharmacology, University of Naples, Naples, Italy 5Department of Pharmacology and Human Physiology, University of Bari, Bari, Italy 6Department of Pharmacology and General Physiology, University of Rome “La Sapienza”, Rome, Italy Correspondence should be addressed to D.P.; e-mail: [email protected] Published online 2 December 2002; doi:10.1038/nm803 The psychoactive constituent of cannabis, ∆9-tetrahydrocannabinol, produces in humans subjec- tive responses mediated by CB1 cannabinoid receptors, indicating that endogenous cannabi- noids may contribute to the control of emotion. But the variable effects of ∆9-tetrahydrocannabinol obscure the interpretation of these results and limit the therapeutic po- tential of direct cannabinoid agonists. An alternative approach may be to develop drugs that am- plify the effects of endogenous cannabinoids by preventing their inactivation. Here we describe a class of potent, selective and systemically active inhibitors of fatty acid amide hydrolase, the en- zyme responsible for the degradation of the endogenous cannabinoid anandamide. Like clinically used anti-anxiety drugs, in rats the inhibitors exhibit benzodiazepine-like properties in the ele- vated zero-maze test and suppress isolation-induced vocalizations. These effects are accompa- nied by augmented brain levels of anandamide and are prevented by CB1 receptor blockade. Our results indicate that anandamide participates in the modulation of emotional states and point to fatty acid amide hydrolase inhibition as an innovative approach to anti-anxiety therapy. Anandamide, the naturally occurring amide of arachidonic acid the therapeutic potential of FAAH inhibition remains essen- with ethanolamine, meets all key criteria of an endogenous tially unexplored. cannabinoid substance1: it is released on demand by stimulated neurons2,3; it activates cannabinoid receptors with high affin- Lead identification and optimization ity1; and it is rapidly eliminated through a two-step process con- Despite its unusual catalytic mechanism6, FAAH is blocked by a sisting of carrier-mediated transport followed by intracellular variety of serine hydrolase inhibitors, including compounds hydrolysis2,4. Anandamide hydrolysis is catalyzed by the en- with activated carbonyls16. Therefore we examined whether es- zyme fatty acid amide hydrolase (FAAH), a membrane-bound ters of carbamic acid such as the anti-cholinesterase agent car- serine hydrolase5,6 that also cleaves other bioactive fatty acid baryl (compound 1; Table 1) might inhibit FAAH activity in rat ethanolamides such as oleoylethanolamide7 and palmi- brain membranes. Although compound 1 was ineffective, its po- toylethanolamide8. Mutant mice lacking the gene encoding sitional isomer 2 produced a weak inhibition of FAAH (half-max- 9 µ FAAH (Faah) cannot metabolize anandamide and, although imal inhibitory concentration (IC50) = 18.6 ± 0.7 M; mean ± fertile and generally normal, show signs of enhanced anan- s.e.m., n = 3), which was enhanced by replacing the N-methyl damide activity at cannabinoid receptors such as reduced pain substituent with a cyclohexyl group (compound 3; IC50 = 324 ± sensation9. This is suggestive that drugs targeting FAAH may 31 nM). The aryl ester 4, the benzyloxyphenyl group of which heighten the tonic actions of anandamide, while possibly can be regarded as an elongated bioisosteric variant of the naph- avoiding the multiple and often unwanted effects produced by thyl moiety of compound 2, inhibited the activity of FAAH with ∆9 ∆9 -tetrahydrocannabinol ( -THC) and other direct-acting a potency (IC50 = 396 ± 63 nM) equivalent to that of compound cannabinoid agonists10,11. To test this hypothesis, potent, selec- 3. A conformational analysis of compound 4 revealed families of tive and systemically active inhibitors of intracellular FAAH ac- accessible conformers differing mainly in the torsion angle tivity are needed. However, most current inhibitors of this around the O–CH2 bond, with substituents in anti or gauche enzyme lack the target selectivity and biological availability re- conformations (data not shown). As the latter conformations quired for in vivo studies12–14, whereas newer compounds, more closely resembled the shape of the naphthyl derivative 3, though promising, have not yet been characterized15,16. Thus, we hypothesized that they might be responsible for the interac- 76 NATURE MEDICINE • VOLUME 9 • NUMBER 1 • JANUARY 2003 ARTICLES a b c d e Fig. 1 The FAAH inhibitors URB532 and URB597 block [3H]anandamide [3H]anandamide in neurons, whereas the anandamide transport inhibitor degradation in intact brain neurons. a, Concentration-dependent inhibi- AM404 (AM, 10 µM) reduces it. V, vehicle. d, Release of non-metabo- tion of [3H]anandamide hydrolysis by URB597 (open squares) and lized [3H]anandamide from neurons treated with URB597 (10 nM) dur- URB532 (filled circles) in primary cultures of rat cortical neurons. ing a 15-min incubation in the absence or presence of AM404 (AM, 10 Baseline FAAH activity was 242.6 ± 3.9 cpm well min–1 (n = 4). b, Unlike µM). e, Time course of [3H]anandamide release from neurons treated URB532 (3 µM) or URB597 (10 nM), the inactive analog 8 (10 µM) has with URB597 (10 nM). *P < 0.05; **P < 0.01 versus vehicle-treated neu- no effect on [3H]anandamide degradation. V, vehicle. c, URB532 (3 µM) rons; ANOVA with Tukey’s post-hoc test (n = 4–8). , vehicle; í, treat- and URB597 (10 nM) promote accumulation of non-metabolized ments. Error bars, s.e.m. tion of compound 4 with the active site of FAAH. Testing this distinction is that in the α-keto heterocycle series potency is hypothesis led to the design of the biphenyl derivative 5 (IC5 0 = strongly dependent on the hydrophobicity of the flexible acyl 63 ± 9 nM), which was further optimized by systematic modifi- chain, whereas in the carbamate series potency is modulated by cations of the distal phenyl group, resulting in the potent in- the shape of the rigid aromatic moiety. Accordingly, when we re- hibitor 6 (IC50 = 4.6 ± 1.6 nM; Table 1). The lead optimization placed the biphenyl of compound 5 with a 5-phenylpentyl group, process will be reported elsewhere. representing the most effective acyl chain in the α-keto heterocy- Kinetic analyses and dialysis experiments indicate that com- cle series, the inhibitory activity was lost (compound 7; Table 1). pounds 4 and 6 may inhibit FAAH activity through an irreversible Compounds 4 (URB532) and 6 (URB597) blocked the FAAH- interaction with the enzyme (data not shown), possibly due to a catalyzed hydrolysis of exogenous [3H]anandamide in primary nucleophilic attack of an active serine residue on the carbamate cultures of intact cortical neurons, with IC50 values that paral- group. This mechanism sets the present compounds apart from leled those obtained in membrane preparations (URB532, 214 ± the α-keto heterocycle derivatives described previously16, which 79 nM; URB597, 0.50 ± 0.05 nM; n = 8; Fig. 1a). By contrast, act as competitive FAAH inhibitors. A further indication of such a compound 8, an analog of URB532 that does not inhibit FAAH in membranes (Table 1), had no such effect (Fig. 1b). Moreover, Table 1 Structures of selected carbamate inhibitors of FAAH activity URB532 and URB597 selectively impaired the breakdown of [3H]anandamide without reducing its carrier-mediated uptake, H 3 O N R R causing non-metabolized [ H]anandamide to accumulate in, and O 1 eventually exit from, the neurons. Thus, after a 4-minute incu- RR1 IC50 (nM) bation with [3H]anandamide, the intracellular content of non-metabolized [3H]anandamide was higher in inhibitor- 1CH>100,000 3 treated neurons than in control neurons (Fig. 1c). As expected, the anandamide transport blocker N-(4-hydroxy- 2CH3 18,600 ± 708 phenyl)arachidonamide (AM404) had an opposite effect, sub- 3 4 3 c-C6H11 324 ± 31 stantially reducing [ H]anandamide internalization (Fig. 1c). When neurons treated with URB597 were exposed for 4 minutes 3 4 O n-C4H9 396 ± 63 to [ H]anandamide and then incubated for 15 minutes in an URB532 [3H]anandamide-free solution, 42.6 ± 8.7% of the accumulated 3 5 c-C6H11 63 ± 9 [ H]anandamide was released back into the medium (n = 3; Fig. 1d). This process was linear with time (Fig. 1e) and was not in- O NH 6 2 c-C6H11 4.6 ± 1.6 hibited by AM404 (Fig. 1d), indicating that it occurred through passive diffusion rather than reverse transport. No such time-de- pendent release was observed in control neurons, the medium of URB597 which contained only residual levels of [3H]anandamide carried 7 c-C H >100,000 6 11 over from the pre-incubation period. These studies identify a new class of carbamate inhibitors of FAAH activity, which po- 8 p-FC6H4 >100,000 O tently block anandamide breakdown in intact brain neurons. Values reported are the concentrations required to inhibit FAAH activity by 50% (IC50, nM), and are expressed as the mean ± s.e.m. of at least three independent experi- Target selectivity ments. They were calculated from concentration-response curves, by using non-linear URB532 and URB597 inhibited FAAH, but did not affect the ac- regression analysis as implemented in the Prism 2.0 software package.