New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis

Article abstract of DOI:10.1038/sj.bjp.0706418

1. We previously reported that the compound O-2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re-examined the activity of O-2093 in vivo and synthesized four structural analogs (O-2247, O-2248, O-3246, an

Full text of DOI:10.1038/sj.bjp.0706418

British Journal of Pharmacology (2006) 147, 83–91
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New potent and selective inhibitors of anandamide reuptake with
antispastic activity in a mouse model of multiple sclerosis
1
2
3
4
1Alessia Ligresti, Maria Grazia Cascio, Gareth Pryce, Sanjitha Kulasegram, Irina Beletskaya,
5Luciano De Petrocellis, Bijali Saha, Anu Mahadevan, Cristina Visintin, Jenny L. Wiley,
3
3
2
4
2David Baker, Billy R. Martin, Raj K. Razdan & *,1Vincenzo Di Marzo
4
3
2
1Endocannabinoid Research Group, Institute of Biomolecular Chemistry, C.N.R. Pozzuoli (Napoli), Italy; Institute of
Neurology, University College, London; Organix Inc., Woburn, MA, U.S.A.; Department of Pharmacology & Toxicology,
3
4
5
Medical College of Virginia, Virginia Commonwealth University Richmond, VA, U.S.A. and Institute of Cybernetics, C.N.R.
Pozzuoli (Napoli), Italy
1 We previously reported that the compound O-2093 is a selective inhibitor of the reuptake of
the endocannabinoid anandamide (AEA). We have now re-examined the activity of O-2093 in vivo
and synthesized four structural analogs (O-2247, O-2248, O-3246, and O-3262), whose activity was
assessed in: (a)binding assays carried out with membranes from cells overexpressing the human CB
1
and CB2 receptors; (b)assays of transient receptor potential of the vanilloid type-1 (TRPV1)channel
functional activity (measurement of [Ca2 þ ]i); (c) [14C]AEA cellular uptake and hydrolysis assays
in rat basophilic leukaemia (RBL-2H3)cells; (d)the mouse ‘tetrad’ tests (analgesia on a hot plate,
immobility on a ‘ring’, rectal hypothermia and hypolocomotion in an open field); and (e) the limb
spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE)mice, a model of
multiple sclerosis (MS).
2
O-2093, either synthesized by us or commercially available, was inactive in the ‘tetrad’ up to a
20 mg kgÀ1 dose (i.v.). Like O-2093, the other four compounds exhibited low affinity in CB1 (Ki from
1.3 to 410 mM)and CB binding assays (1.3oKio 8 mM), low potency and efficacy in a TRPV1
2
functional assay (EC50410 mM), very low potency as fatty acid Àa1mide hydrolase (FAAH) inhibitors
(IC50425 mM)and were inactive in the ‘tetrad’ up to a 30 mg kg
3
dose (i.v.).
While O-2247 and O-2248 were poor inhibitors of [14C]AEA cellular uptake (IC50440 mM), O-3246
and O-3262 were quite potent in this assay. O-3246, which exhibits only a very subtle structural
difference with O-2093, is the most potent inhibitor of AEA uptake reported in vitro under our
experimental conditions (IC50 ¼ 1.4 mM)and is 12-fold more potent than O-2093.
4
When injected intravenously O-3246 and O-3262, again like O-2093 and unlike O-2247 and
O-2248, significantly inhibited limb spasticity in mice with CREAE.
5
These data confirm the potential utility of selective AEA uptake inhibitors as anti-spasticity drugs
in MS and, given the very subtle chemical differences between potent and weak inhibitors of uptake,
support further the existence of a specific mechanism for this process.
British Journal of Pharmacology (2006) 147, 83–91. doi:10.1038/sj.bjp.0706418;
published online 14 November 2005
Keywords: Cannabinoid; endocannabinoid; 2-arachidonoylglycerol; membrane transport; spasticity; multiple sclerosis
Abbreviations: AEA, arachidonoylethanolamide (anandamide); 2-AG, 2-arachidonoylglycerol; BSA, bovine serum albumin;
CREAE, chronic relapsing experimental allergic encephalomyelitis; DMAP, 4-dimethyl-amino-pyridine; DMEM,
.
Dulbecco’s modified Eagle’s medium; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl; FAAH, fatty
acid amide hydrolase; HEK, human embryonic kidney; MPE, maximum possible effect; MS, multiple sclerosis;
NMR, nuclear magnetic resonance; RBL-2H3, rat basophilic leukaemia; TLC, thin layer chromatography;
TRPV1, transient receptor potential of the vanilloid type-1
Introduction
Endocannabinoids are defined as endogenous agonists of the
G-protein-coupled receptors for Cannabis psychoactive prin-
ciple D9-tetrahydrocannabinol. To date, two such receptors
have been cloned, the CB1 and CB2 cannabinoid receptors
(Howlett, 2002, for a review), and, together with endocanna-
binoids, they have been implicated in several physiological and
pathological conditions in mammals (Di Marzo et al., 2004a;
Howlett et al., 2004, for reviews). Like all endogenous
mediators, the levels of the two major endocannabinoids,
arachidonoylethanolamide (anandamide)(AEA)(Devane
et al., 1992)and 2-arachidonoylglycerol (2-AG)(Mechoulam
et al., 1995; Sugiura et al., 1995)are regulated by specific
biosynthetic and degradative reactions catalyzed by enzymes
that have been recently characterized and cloned (Di Marzo
et al., 2004a). AEA and 2-AG are produced ‘on demand’
*Author for correspondence at: Istituto di Chimica Biomolecolare-
Endocannabinoid Research Group, Consiglio Nazionale delle Ri-
cerche, Via Campi Flegrei 34, Comprensorio Olivetti, Fabbr 70,
Pozzuoli (Napoli)80078, Italy; E-mail: vdimarzo@icmib.na.cnr.it

84
A. Ligresti et al
Potent and selective inhibitors of anandamide
following cell stimulation, immediately released to act upon
cannabinoid receptors and ultimately inactivated via cellular
reuptake and intracellular hydrolysis. The possible existence of
a plasma membrane protein mediating both endocannabinoid
release after de novo biosynthesis, and endocannabinoid
reuptake following activation of CB1 and CB2 cannabinoid
receptors has been investigated and is still controversial (Di
Marzo et al., 1994; Glaser et al., 2003; Fegley et al., 2004;
Ligresti et al., 2004; Oddi et al., 2005; and Hillard & Jarrahian,
2003; McFarland & Barker, 2004 for reviews). Nevertheless,
many substances have been developed that are capable of
selectively inhibiting the cellular reuptake of AEA (Lopez-
Rodriguez et al., 2003; Ortar et al., 2003)without interfering
with other proteins nor with the major enzyme involved in the
degradation of this compound, fatty acid amide hydrolase
(FAAH)(Cravatt et al., 1996). In some cases, the capability of
a compound to inhibit AEA reuptake is dramatically impaired
by introducing very subtle changes in its chemical structure,
such as inversion of the stereochemistry of a chiral center
(Piomelli et al., 1999; Di Marzo et al., 2004b; Ligresti et al.,
2004), thus supporting the existence of a specific protein to
facilitate this process. Among the potential therapeutic effects
shared by ‘direct’ agonists of CB1 cannabinoid receptors and
inhibitors of endocannabinoid reuptake (which by elevating
endocannabinoid levels, act as ‘indirect’ agonists of these
receptors)(Di Marzo et al., 2004a), one of the most promising
is the capability of inhibiting limb spasticity in mice with
chronic relapsing experimental allergic encephalomyelitis
(CREAE), a model of multiple sclerosis (MS) (Baker et al.,
2000; 2001; de Lago et al., 2004).
the putative AEA membrane transporter. Through the
chemical modification of O-2093, we have systematically
increased property to inhibit AEA cellular uptake, thus
obtaining the most potent inhibitor of AEA reuptake thus
far reported under our experimental conditions. Finally, we
confirm that potent inhibitors of AEA transport across the
cell membrane are also very efficacious inhibitors of spasticity
in the CREAE model of MS, with no apparent cannabimi-
metic side effects.
Methods
Synthesis of compounds
The synthesis of analogs of O-2093 (MW ¼ 616.70)was carried
out in the same manner as we reported for the synthesis of O-
2093 (Di Marzo et al., 2002). For the synthesis of O-2247 and
O-2248 (Figure 1, MW ¼ 619.15 and 605.12, respectively), the
appropriate secondary amines were synthesized from 4-
chlorobenzaldehyde by reductive alkylation with 2,4-dichlor-
obenzylamine or 2,4-dichloroaniline, respectively (triacetoxy-
borohydride/acetic acid/CH2CL2)in 56–62% yield (Abdel-
Magid et al., 1996). Condensation of these secondary amines
with the acid chloride of arachidonic acid formed O-2247 and
O-2248. O-3246 and O-3262 (Figure 1, MW ¼ 616.70 and
547.81.12, respectively)were similarly synthesized from the
corresponding secondary amines. In the case of O-3262, the (1-
.
(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl)EDCI/4-
dimethyl-amino-pyridine (DMAP)procedure was used for
condensation with arachidonic acid rather than the acid
chloride procedure. All compounds were characterized on
the basis of their [1H] nuclear magnetic resonance spectra
(run on a Jeol Eclipse 300 MHz)and elemental analyses
(Figure 1). O-2093 was also purchased from Tocris, Bristol,
U.K. After purification, the compounds were 495% pure on
the basis of nuclear magnetic resonance (NMR), thin layer
chromatography (TLC), mass spectrometric and elemental
analyses. They were stable if kept under nitrogen and in the
freezer (À201C).
We have previously shown that the chemical modification of
arvanil, a nonselective, albeit relatively potent, AEA uptake
inhibitor with high potency against spasticity in CREAE mice
(Melck et al., 1999; Brooks et al., 2002), yields compounds that
still selectively inhibit the uptake process without exhibiting
two other properties typical of the starting compound, that is
activation of both CB1 cannabinoid receptors and, particu-
larly, the transient receptor potential vanilloid type 1 (TRPV1)
channel (Di Marzo et al., 2001; 2002). One of these
compounds, O-2093 (Figure 1)served as the lead compound
for the present study. Apart from inhibiting AEA uptake,
O-2093 was inactive at CB1 and TRPV1 receptors and did
not significantly inhibit FAAH. In the present study, we
have designed and synthesized four structural analogs of
O-2093 (Figure 1)with the aim of increasing its efficacy at
Assay of AEA cellular reuptake
The effect of compounds on the uptake of [14C]AEA by rat
basophilic leukemia (RBL-2H3)cells was studied by using
O
R
N
O-2093, R=
O-3262, R=
O-3246, R=
R
OH
OH
Cl
OH
Cl
O
Cl
O-2247, n=1
O-2248, n=0
N
(CH2)n
Cl
Cl
Figure 1 Chemical structures of O-2093 and its four novel analogs.
British Journal of Pharmacology vol 147 (1)

A. Ligresti et al
Potent and selective inhibitors of anandamide
85
2.4 mM (10,000 c.p.m.)of [ 14C]AEA as described previously
(Bisogno et al., 1997). Cells were incubated with [14C]AEA for
5 min at 371C, in the presence or absence of varying
concentrations of the inhibitors. Residual [14C]AEA in the
incubation medium after extraction with CHCl3/CH3OH 2 : 1
(by volume), determined by scintillation counting of the
lyophilized organic phase, was used as a measure of the
AEA that was taken up by cells (De Petrocellis et al.,
2000). Previous studies (Bisogno et al., 1997)had shown that
after a 5-min incubation the amount of [14C]AEA that
disappeared from the medium of RBL-2H3 cells is found
mostly (490%)as unmetabolized [ 14C]AEA in the cell extract.
Nonspecific binding of [14C]AEA to cells and plastic dishes was
determined in the presence of 100 mM AEA and was never
higher than 30%. Data are expressed as the concentration
exerting 50% inhibition of AEA uptake (IC50)calculated
by GraphPads.
Assay of binding activity at CB1 and CB2 receptors
For the CB1 receptor, [3H]CP-55,940 (KD ¼ 690 pM)binding to
P2 membranes was conducted as described previously, except
whole rat brain (rather than cortex only)was used. Displace-
ment curves were generated by incubating drugs with 1 nM of
[3H]CP-55,940. The assays were performed in triplicate, and
the results represent the combined data from three individual
experiments. CB2 receptor binding was carried out in Chinese
hamster ovary (CHO)cells that were maintained in Dulbecco’s
modified Eagle’s medium (DMEM)with 10% fetal clone II
(HyClone, Logan UT, U.S.A.)and 5% CO at 371C in a
2
Forma incubator. Cell lines were created by transfection of
CB2pcDNA3 into CHO cells by the Lipofectamine reagent
(Life Technologies, Gaithersburg, MD, U.S.A.). The human
CB2 cDNA was provided by Dr Sean Munro (MRC, Cam-
bridge, U.K.). Stable transformants were selected in growth
medium containing geneticin (1 mg mlÀ1, reagent, Life Tech-
nologies, Gaithersburg, MD, U.S.A.). Cells were harvested in
phosphate-buffered saline containing 1 mM EDTA and cen-
trifuged at 500 Â g. The cell pellet was homogenized in 10 ml of
solution A (50 mM Tris-HCl, 320 mM sucrose, 2 mM EDTA,
5 mM MgCl2, pH 7.4). The homogenate was centrifuged at
1600 Â g (10 min), the supernatant saved, and the pellet washed
three times in solution A with subsequent centrifugation. The
combined supernatants were centrifuged at 100,000 Â g
(60 min). The (P2 membrane)pellet was resuspended in 3 ml
of buffer B (50 mM Tris-HCl, 1 mM EDTA, 3 mM MgCl2, pH
Assay of FAAH
The effect of compounds on the enzymatic hydrolysis of AEA
was studied as described previously (Bisogno et al., 1997),
using membranes prepared from RBL-2H3 cells, incubated
with the test compounds and [14C]AEA (2.4 mM)in 50 m M Tris-
HCl, pH 9, for 30 min at 371C. [14C]Ethanolamine produced
from [14C]AEA hydrolysis was measured by scintillation
counting of the aqueous phase after extraction of the
incubation mixture with two volumes of CHCl3/CH3OH 2 : 1
(by volume). Data are expressed as the concentration exerting
50% inhibition of AEA hydrolysis (IC50), calculated by
GraphPad.
7.4)to yield
a protein concentration of approximately
1 mg mlÀ1. The tissue preparation was divided into equal
aliquots, frozen on dry ice, and stored at À701C. Binding was
initiated by the addition of 40–50 mg membrane protein to
silanized tubes containing [3H]CP-55,940 (102.9 Ci mmolÀ1
)
Assay of functional activity at TRPV1 receptors
and a sufficient volume of buffer C (50 mM Tris-HCl, 1 mM
EDTA, 3 mM MgCl2, and 5 mg mlÀ1 fatty acid free bovine
serum albumin (BSA), pH 7.4) to bring the total volume to
0.5 ml. The addition of 1 mM unlabeled CP-55,940 was used to
assess nonspecific binding. Following incubation (301C for
1 h), binding was terminated by the addition of 2 ml of ice-cold
buffer D (50 mM Tris-HCl, pH 7.4, plus 1 mg mlÀ1 BSA)and
rapid vacuum filtration through Whatman GF/C filters
(pretreated with polyethyleneimine [0.1%] for at least 2 h).
Tubes were rinsed with 2 ml of ice-cold buffer D, which was
also filtered, and the filters subsequently rinsed twice with 4 ml
of ice-cold buffer D. Before radioactivity was quantitated by
liquid scintillation spectrometry, filters were shaken for 1 h in
5 ml of scintillation fluid.
Overexpression of human TRPV1 cDNA into human
embryonic kidney (HEK)293 cells was carried out as described
previously (Hayes et al., 2000). Cells were grown as mono-
layers in minimum essential medium supplemented with
nonessential amino acids, 10% fetal calf serum, and 0.2 mM
glutamine and maintained under 95% O2/5% CO2 at 371C.
The effect of the substances on [Ca2 þ ]i was determined by
using Fluo-3 (Molecular Probes, Eugene, OR, U.S.A.), a
selective intracellular fluorescent probe for Ca2 þ (De Petro-
cellis et al., 2000; Smart et al., 2000). Cells were transferred
into six-well dishes coated with poly-L-lysine (Sigma, Milan,
Italy)1 day prior to experiments and grown in the culture
medium mentioned above. On the day of the experiment, the
cells (50–60,000 per well)were loaded for 2 h at 25 1C with 4 mM
Fluo-3 methylester in dimethyl sulfoxide containing 0.04%
Pluoronic. After loading, cells were washed with Tyrode’s
solution, pH ¼ 7.4, trypsinized, resuspended in Tyrode’s
solution, and transferred to the cuvette of the fluorescence
detector (PerkinElmer LS50B)under continuous stirring.
Experiments were carried out by measuring cell fluorescence
at 251C (lEX ¼ 488 nm, lEM ¼ 540 nm)before and after the
addition of the test compounds at various concentrations. The
efficacy of the effect was determined by comparing it to the
analogous effect observed with 4 mM ionomycin. In some cases,
the experiments were repeated in the presence of the TRPV1
antagonist capsazepine, at a concentration (20 nM)sufficient to
block the effect of 100 nM capsaicin.
Pharmacological effects in mice
Cannabinoids were dissolved in a mixture of ethanol (5%),
Emulphor (North American Chemicals, Cranbury, NJ,
U.S.A.)(5%,) and saline (90%)for i.v. administration. The
analogs were administered to male ICR mice by tail-vein
injection and evaluated for their ability to produce hypomo-
tility, hypothermia, antinociception and ring immobility. This
route of administration limits pharmacokinetic problems
associated with first pass metabolism and lipophilicity of
compounds. Prior to testing in any of the tetrad procedures,
mice were acclimated to the experimental setting (ambient
temperature 22–241C)for at least 1 h. Subsequently, mice were
injected with one of the compounds or with O-3262 or vehicle
British Journal of Pharmacology vol 147 (1)

86
A. Ligresti et al
Potent and selective inhibitors of anandamide
followed 5 min later by an injection of AEA (potentiation
tests). Each mouse was tested in two procedures (either
spontaneous activity and tail flick or rectal temperature and
ring immobility). Tail-flick latency or rectal temperature was
measured at 4 min after the last injection. At 1 min after
measurement of antinociception or rectal temperature, mice
were placed in individual activity chambers where spontaneous
activity was measured for 10 min or they were placed on the
ring immobility apparatus for 5 min. Preinjection control
values were determined for rectal temperature and tail-flick
latency (in s). Activity was measured as total number of
interruptions of 16 photocell beams per chamber during the
10-min test and expressed as % inhibition of activity of the
vehicle group. Maximum latency of 10 s was used for the tail
flick test. Antinociception was calculated as percent of
maximum possible effect (%MPE), as in formula below.
Control latencies typically ranged from 1.5 to 4.0 s. Rectal
temperature was measured with a telethermometer (Yellow
Springs Instrument Co., Yellow Springs, OH, U.S.A.)and a
thermistor probe (model YSI 400; Markson LabSales Inc.,
Hillsboro, OR, U.S.A.)inserted at a depth of 2 mm and was
expressed as the difference between control temperature
(before injection)and temperature following drug administra-
tion. During placement on the ring immobility apparatus, the
total amount of time (in s)that the mouse remained motionless
was measured. This value was divided by 300 s and multiplied
by 100 to obtain a percent immobility rating. The criterion for
ring immobility was the absence of all voluntary movement,
including snout and whisker movement. Given their poor CB1
and CB2 cannabinoid receptor binding affinities, the four
analogs of O-2093 were evaluated at a single dose of
30 mg kgÀ1 (n ¼ 5–6 mice per test). O-2093 was evaluated over
fore, the forces from individual limbs were assessed, pairwise,
using Analysis of Variance tests (Baker et al., 2001).
Results
Effect of O-2093 and its analogs on [14C]AEA uptake
by RBL-2H3 cells
When coincubated with [14C]AEA and intact RBL-2H3 cells,
where an active transport of AEA across the cell membrane
has been described (Bisogno et al., 1997), O-2093 inhibited the
cellular uptake of the radiolabeled compound with an
IC50 ¼ 17.372.0 mM (Figure 2), very similar to that previously
reported by us (11.571.3 mM, Di Marzo et al., 2002). On the
other hand, O-3246, an isomer of O-2093, was more than 10-
fold more potent, exhibiting an IC50 ¼ 1.470.2 mM and a 100%
inhibition of uptake at 5 mM (Figure 2). Intriguingly, the
analog with no chlorine atoms on both aromatic moieties, that
is O-3262, exhibited an IC50 of 2.870.3 mM (Figure 2)that was
intermediate between O-2093 and O-3246. Finally, the two
analogs with chlorine atoms and no hydroxy group on both
aromatic moieties, that is O-2247 and O-2248, only exhibited
very weak activity against [14C]AEA uptake (IC50 440 mM,
Table 1).
Effect of O-2093 and its analogs on [14C]AEA hydrolysis
by RBL-2H3 cells
As reported previously (Di Marzo et al., 2002), O-2093 was
inactive (IC50450 mM)on [ 14C]AEA hydrolysis, which in this
study was tested using membranes from RBL-2H3 cells. All
four new O-2093 analogs synthesized for this study were also
inactive (IC50450 mM), except for O-2248, which exhibited
some weak activity (IC50 ¼ 27.973.4 mM)(Table 1).
a dose range of 3–20 mg kgÀ1
.
ꢀ
ꢁ
ðtest latency À control latencyÞ
10s À control latency
%MPE ¼
Â100
Effect of O-2093 and its analogs on human
TRPV1-mediated enhancement of [Ca2 þ
]
i
Assay of limb spasticity in CREAE mice
As reported previously (Di Marzo et al., 2002), O-2093 was
only very weakly active at enhancing [Ca2 þ ]i in HEK cells
Spasticity was induced in ABH mice following the induction of
experimental allergic encephalomyelitis (EAE)using syngenic
spinal cord homogenate in Freund’s adjuvant on day 0 and 7.
Mice exhibited relapsing-remitting episodes of paralysis and
spasticity developed typically after 2–3 relapses, at about 80–
100 days postinduction as described previously (Baker et al.,
2000). Spasticity was assessed by measuring the force required
for hindlimb flexion against a strain gauge prior to and
following the administration of compound (Baker et al., 2000).
These were injected intravenously in the same vehicle as above,
in order to limit pharmacokinetic problems associated with
first pass metabolism and lipophilicity of cannabinoids, and
observed when using other administration routes that may
interfere with speed and level of efficacy, as has been reported
previously (Baker et al., 2000). Spasticity, resulting from
accumulating neurological deficit is associated with limb
stiffness and was measured during remission from active
paralytic attacks, where limbs lack functional movement and
exhibit weak resistance to flexion (Baker et al., 2000). There is
significant variation between the degree of spasticity between
individual limbs and animals, which makes direct comparison
between different groups difficult (Baker et al., 2000). There-
O-2093
100
O-3246
O-3262
O-2247
O-2248
50
0
-7
-6
-5
-4
logM
Figure 2 Effect of O-2093 and its four novel analogs on [14C]AEA
uptake by RBL-2H3 cells. Data are means7s.e. of three different
experiments.
British Journal of Pharmacology vol 147 (1)

A. Ligresti et al
Potent and selective inhibitors of anandamide
87
Table 1 Summary of the in vitro activity of the O-2093 and its four novel analogs
Compound
[14C]AEA uptake by RBL- [14C]AEA hydrolysis by
rCB1 (Ki, mM)
hCB2 (Ki, mM) [Ca2+]i in HEK cells expressing
hTRPV1 (max. effect at 10 mM,
as % of the effect of 4 mM
2H3 cells (IC50, mM)
RBL-2H3 cell membranes
(IC50, mM)
ionomycin)
O-2093
O-3246
O-3262
O-2247
O-2248
OMDM-2
17.372.0
1.470.2
2.870.3
43.374.1
450
450
450
450
450
27.973.4
450
1.2970.11
2.6970.19
2.0270.02
410
410
5.1070.30
2.3870.51
2.1870.38
1.3170.12
8.0470.80
5.1270.21
4.9571.40
27.172.6*
18.271.4*
24.472.2*
16.771.8
8.171.1
4.170.8
31.073.2*
Data are reported as IC50 (mM)for the uptake and hydrolysis assays, Ki (mM)for the binding assays, and as maximal effect (i.e. as % of the
effect on calcium observed with 4 mM ionomycin), for the [Ca2+]i assay, and are means7s.e.m. of 3 experiments. Asterisk denotes
compounds that were tested in this assay also in the presence of 20 nM of the TRPV1 antagonist capsazepine, with no change in the effect
observed. The previously reported (Ortar et al., 2003; de Lago et al., 2004)inhibitor of AEA uptake, OMDM-2, was used as reference
compound.
Table 2 Potentiation of cannabimimetic effects of sub-maximal doses of AEA by the AEA uptake inhibitor, O-3262
(30 mg kgÀ1, i.v.)
Treatment condition
% Inhibition of locomotion
Antinociception (% MPE)
Vehicle+vehicle
Vehicle+O-3262, 30 mg kgÀ1
Vehicle+AEA, 3 mg kgÀ1
Vehicle+AEA, 10 mg kgÀ1
O-3262, 30 mg kgÀ1+AEA, 3 mg kgÀ1
O-3262, 30 mg kgÀ1+AEA, 10 mg kgÀ1
077.8
38710.9
F
4977.0
F
973.0
1975.8
32714.2
F
83711.5**
F
8273.6*
Data are reported as means7s.e.m.. Asterisks indicate significant difference from vehicle+10 mg kgÀ1 AEA (*)or from vehicle+3 mg
kgÀ1 AEA (**)(i.e., doses of AEA that were sub-maximal for the measure)as well as from the vehicle+vehicle condition and the
vehicle+O-3262 condition (Po0.05 as determined by one-way ANOVA with Newman–Kuels post hoc tests).
transfected with the human recombinant TRPV1 (maximal
stimulation at 10 mM was 24.272.0% of the effect of 4 mM
ionomycin). Likewise, all four new O-2093 analogs synthesized
for this study were also very weakly active (maximal
stimulation at 10 mM ranged between 8 and 24% of the effect
of 4 mM ionomycin)and, at least in the case of O-3246 and
O-3262 (i.e. the two most potent compounds on AEA uptake),
it was not sensitive to the TRPV1 antagonist, capsazepine,
20 nM)(Table 1.)
contrast to the lack of effect observed with each compound
alone, potentiation of the effects of a submaximal dose of AEA
by O-3262 (one of the most potent inhibitors of AEA uptake)
was observed for locomotor inhibition and antinociception
(Table 2).
Effect of O-2093 and its analogs on limb spasticity
in CREAE mice
All compounds except O-2247 and O-2248, tested at the dose
of 1 mg kgÀ1 (i.v.), exerted a strong inhibition of the force
required to bend individual spastic limbs to full flexion against
a strain gauge. The effect was maximal (30.3–32.7%)after
30 min from administration (Table 3). As the basal level
of spasticity can vary significantly among individual mice,
and several (10–15)mice are therefore required to obtain
reliable data even for one dose, it was not normally possible to
perform a full dose–response curve for all compounds.
However, a lower dose of O-2093 (0.05 mg kgÀ1 i.v.)remained
active, thus suggesting that the higher doses used may have
been supra-optimal and accounting for a relative lack of
discrimination between O-3246, O-3242 O-2093 in this in vivo
assay (Table 3).
Effect of O-2093 and its analogs in CB1 and CB2 receptor
binding assays and in the mouse ‘tetrad’
Binding affinities for CB1 and CB2 cannabinoid receptors
were notably low for O-2093 and for its four analogs. Affinity
for the CB1 cannabinoid receptor ranged from 1.29 to
410 mM. As a group, affinities for CB2 cannabinoid receptors
were only slightly better, ranging from 1.31 to 8.04 mM.
Consistent with their poor binding affinities at CB1 cannabi-
noid receptors, none of the five compounds showed a complete
cannabimimetic profile in the ‘tetrad’ of in vivo mouse tests up
to a dose of 30 mg kgÀ1 (data not shown). Unlike in our
previous study, O-2093 did not produce antinociceptive or
hypothermic effects in ICR mice, regardless of source of the
drug (in house synthesis or Tocris Ltd., U.K.). Although
O-2093 moderately suppressed spontaneous activity (68%
inhibition), it did so only at the 20 mg kgÀ1 dose. Lack of overt
‘tetrad’ effects were also evident in ABH mice when the
compounds were tested at up to 5 mg kgÀ1 i.v. (not shown). In
Discussion
Previous investigations have shown that inhibitors of endo-
cannabinoid cellular uptake are capable of exerting strong
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88
A. Ligresti et al
Potent and selective inhibitors of anandamide
Table 3 Effect of O-2093 and its four novel analogs on limb spasticity in mice with CREAE
Compound (dose)
n
Time from administration
Mean resistance to flexion force(N)7s.e.
P value
O–2247 (1 mg kgÀ1 i.v)7
Baseline
0.237
0.258
0.250
0.208
0.261
70.020
0.24970.027
0.22170.018
0.24970.031
+10 min
+30 min
+60 min
NS
NS
NS
O–2248 (1 mg kgÀ1 i.v)10
O–3246 (1 mg kgÀ1 i.v)15
O–3262 (1 mg kgÀ1 i.v)14
Baseline
Baseline
Baseline
Baseline
70.034
0.25270.031
0.27470.033
0.25570.019
+10 min
+30 min
+60 min
NS
NS
NS
70.018
0.24070.017
0.17470.013
0.17370.011
+10 min
+30 min
+60 min
NS
Po0.001
Po0.001
70.011
0.15370.007
0.14070.008
0.15370.009
+10 min
+30 min
+60 min
Po0.001
Po0.001
Po0.001
O–2093 (1 mg kgÀ1 i.v)13
70.033
0.19970.023
0.18270.022
+10 min
+30 min
P ¼ 0.005
Po0.001
O–2093 (0.05 mg kgÀ1 i.v)13
Baseline
0.172
70.048
0.11770.045
0.11170.037
0.09570.033
+10 min
+30 min
+60 min
Po0.001
Po0.001
Po0.001
Following the development of spasticity during the course of experimental allergic encephalomyelitis, the force required to bend
individual spastic limbs to full flexion against a strain gauge was assessed. Compounds were injected at the dose indicated and recordings
following drug administration were compared to baseline levels, using repeated measures Analysis of Variance. NS ¼ non-significant
(P40.05).
activity in each of the four tests of the mouse ‘tetrad’ of
behavioral actions (analgesia on a ‘hot plate’, immobility on a
‘ring’, rectal hypothermia and hypolocomotion in an ‘open
field’ (Martin et al., 1991)) only when they also exert strong
functional activity at cannabinoid CB1 receptors or at vanilloid
TRPV1 channels (Di Marzo et al., 2001; 2002; Wiley &
Martin, 2003; de Lago et al., 2004). One exception appeared to
be O-2093, an endocannabinoid uptake inhibitor previously
developed by us, which exerted weak or no activity at
cannabinoid and TRPV1 receptors and yet exhibited activity
in the mouse ‘tetrad’ (Di Marzo et al., 2002). In the present
study, we modified the chemical structure of O-2093 with the
aim of increasing its potency at the putative AEA membrane
transporter. The first surprising finding of this study, however,
was that O-2093, either obtained from a commercial source or
resynthesized by us, did not produce cannabimimetic activity
in the four behavioral tests carried out in mice, as it had in our
previous study (Di Marzo et al., 2002). This new finding has
now been confirmed several times since the publication of that
original study. Since in all cases: (1)O-2093 was always 495%
pure after its preparation, and stable for up to 4 months when
kept at À201; and (2)the assays were carried out exactly under
the same conditions of administration, it is unlikely that this
discrepancy is due to impurities present in the first batch and
absent in the batches used in the present study, or to
pharmacokinetic factors. A possible explanation is that in
the previous study, where we assessed the activity of eight
structurally similar compounds, all of which were active in the
‘tetrad’ assays, an unfortunate exchange of samples may have
occurred during the preparation of O-2093 aliquots for
storage. Another possibility is that the batch used in the
previous study may have been degraded to active compounds
immediately before its testing. For these reasons, we repeated
in the present study also the in vitro assays originally
performed with O-2093 (Di Marzo et al., 2002). In this case,
we found similar results, except for a slightly lower potency in
the uptake assay. In conclusion, we can confirm here that the
compound is an inhibitor of endocannabinoid uptake with
little or no affinity for cannabinoid CB1 and CB2 receptors, nor
activity at TRPV1 vanilloid receptors and FAAH. Impor-
tantly, the affinity of O-2093 at CB2 receptors had not been
evaluated in our first study with this compound (Di Marzo
et al., 2002).
The most interesting data from this study, however, concern
four O-2093 analogs that we have synthesized here, some of
which show striking pharmacological features. All new
compounds share with O-2093 very weak, if any, activity as
FAAH inhibitors, CB1 and CB2 ligands, TRPV1 agonists, or in
the mouse ‘tetrad’ of tests. However, two of these derivatives,
O-3246 and O-3262, exhibited potent activity as inhibitors of
AEA cellular uptake. Interestingly, in O-3246 the simple shift
of the two chlorine atoms in the two aromatic moieties of O-
2093 from orto to meta to the 4-hydroxy-groups, results in an
12-fold increase in potency in this assay, without causing any
other significant change in the other assays carried out in this
study. Indeed, O-3246, with an IC50 of 1.4 mM, is the most
potent inhibitor of AEA cellular uptake ever found using our
experimental conditions. In fact, it has been demonstrated by
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A. Ligresti et al
Potent and selective inhibitors of anandamide
89
many authors that the potency and efficacy of endocannabi-
noid uptake inhibitors depends very much on the experimental
set-up of the assay (see e.g. Bisogno et al., 2001; Fowler et al.,
2004). Not only the position but also the presence of the
chlorine atoms on the two aromatic moieties of O-2093
seems to be important for activity against the putative
membrane transporter responsible for AEA uptake. In fact,
O-3262, which differs from the previous two analogs by
having no chlorine atoms, also exhibited potency sixfold
higher than O-2093, although twofold lower than O-3246.
Therefore, in order for these analogs to inhibit AEA
uptake efficaciously, they must have the chlorine atoms
meta to the 4-hydroxy groups, or not have them at all. On
the other hand, the 4-hydroxy groups on the two aromatic
moieties are necessary for activity since the two O-2093
analogs that lack these groups, and have chlorine atoms
instead, that is O-2247 and O-2248, are significantly less
active than O-2093. This observation is in agreement
with previous structure-activity studies on arvanil (Di Marzo
et al., 2001; 2002), which showed that the 4-hydroxy group
in other analogs of this compound are fundamental for
both their activity as AEA uptake inhibitors and as TRPV1
agonists.
VDM-11, OMDM1 and OMDM2 (de Lago et al., 2004), and
UCM707 (de Lago et al., 2005). Here, we have also evaluated
the effects of O-2093 and its four new analogs on limb
spasticity, and found that only those compounds which
inhibited AEA cellular uptake exerted a strong inhibitory
action on spasticity. We did not observe any real significant
difference in efficacy in this assay between the least and
more potent uptake inhibitors studied here, that is O-2093
and O-3246. All three active compounds could yield similar
effects, with maximal reductions in spasticity maximal after
30 min from administration ranging from 30.3 to 32.7% of
control, and persistent up to 60 min. Although caution is
required when comparing results in different groups of animals
as the level of spasticity can vary significantly between animals
(Baker et al., 2000), this level of inhibition is similar to that
reported previously with cannabinoid receptor agonists
(Baker et al., 2000; Brooks et al., 2002). Indeed, a lower dose
of O-2093 (0.05 mg kgÀ1)still exerts a 34% reduction in
spasticity 30 min after administration, thus indicating that
these three compounds may be active as antispastic drugs
also at lower doses. This suggests that the doses tested here
for the other four compounds (1 mg kgÀ1, i.v.)may have
been already at the plateau of the antispasticity effect of the
active ones. However, although unlikely given their strong
chemical similarities, there may be differences in the pharma-
cokinetic properties of the differing compounds, which
could affect their in vivo efficacy compared to their in vitro
potency and may account for the observation that O-3246
appeared to be slower in reducing spasticity compared to
O-2093 or O-3262. While, based on the present data, we
cannot (and do not intend to)establish a correlation between
the potencies of the five compounds as AEA uptake inhibitors
and their efficacy as antispastic agents, our findings do support
further the use of such inhibitors as templates for the
development of new anti-spastic medicaments to be used in
MS. The recent finding that AEA uptake inhibitors are
effective at decreasing the progress, and not only the signs, of
this disorder in animal models (Mestre et al., 2005; Ortega-
Gutierrez et al., 2005), widens even more their therapeutic
potential in MS.
The finding that, in a complex molecule such as O-2093, the
simple structural change leading to O-3246 causes a dramatic
increase in the potency for the inhibition of AEA uptake can
be considered as further indirect evidence for the presence of
one or more specific proteins facilitating this process, and
AEA transport across the cell membrane in general. Further-
more, the observation that both O-3246 and O-3262 exert
little, if any, inhibition of AEA intracellular hydrolysis, which
in RBL-2H3 cells is catalyzed by FAAH, confirm recent
evidence (Fegley et al., 2004; Ligresti et al., 2004; Ortega-
Gutierrez et al., 2004)that this latter protein is not solely
responsible for AEA cellular uptake, as instead had been
proposed by earlier studies (Glaser et al., 2003). Clearly, by
virtue of its selectivity for AEA uptake and its lack of ‘central’
cannabimimetic activity (as assessed here in the mouse ‘tetrad’
of tests), O-3246 and O-3262 can serve as templates for the
development of a new generation of potent endocannabinoid
uptake inhibitors with several potential therapeutic applica-
tions (see Di Marzo et al., 2004a for review). Importantly,
when coadministered with inactive doses of AEA, O-3262 was
capable of rendering this endocannabinoid active at inhibiting
nociception and locomotor behavior, in agreement with the
action of this compound as an inhibitor of AEA inactivation
also in vivo.
In conclusion, we have reported here the development of
two potent and selective AEA uptake inhibitors and have
demonstrated their activity as potential antispastic drugs in
MS. Moreover, we have provided further strong, albeit still
indirect, evidence for the existence of a specific process
responsible for endocannabinoid transport across the cell
membrane.
Baker et al. (2001)discovered that inhibitors of endocanna-
binoid cellular uptake have the capability of inhibiting limb
spasticity in mice with CREAE, a model of MS. This
observation has been confirmed with six different uptake
inhibitors, that is AM404 and arvanil (which may also act via
TRPV1 receptors (Baker et al., 2001; Brooks et al., 2002)),
The authors acknowledge the Italian Ministry of University and
Research (within the FIRB program to VDM), the Multiple Sclerosis
Society of Great Britain and Northern Ireland (to DB), the Brain
Research Trust (to DB), and the National Institute for Drug Abuse
(Grants DA-09789 and DA-08904)for funding this work.
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a full agonist
(Received July 19, 2005
Accepted August 23, 2005
Published online 14 November 2005)
British Journal of Pharmacology vol 147 (1)