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Vol. 289, Issue 3, 1427-1433, June 1999
Department of Pharmacology and Toxicology (C.J.H., S.M., M.J.G., R.D., W.B.C.), Medical College of Wisconsin, Milwaukee, Wisconsin; and Department of Biomedical Sciences (R.A.R., L.A.S., V.M., R.G.P.), University of Aberdeen, Aberdeen, Scotland
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Two subtypes of the cannabinoid receptor (CB1 and CB2) are expressed in
mammalian tissues. Although selective antagonists are available for
each of the subtypes, most of the available cannabinoid agonists bind
to both CB1 and CB2 with similar affinities. We have synthesized two
analogs of N-arachidonylethanolamine (AEA), arachidonylcyclopropylamide (ACPA) and
arachidonyl-2-chloroethylamide (ACEA), that bind to the CB1 receptor
with very high affinity (KI values of
2.2 ± 0.4 nM and 1.4 ± 0.3 nM, respectively) and to the CB2
receptor with low affinity (KI values of
0.7 ± 0.01 µM and 3.1 ± 1.0 µM, respectively). Both
ACPA and ACEA have the characteristics of agonists at the CB1 receptor;
both inhibit forskolin-induced accumulation of cAMP in Chinese hamster
ovary cells expressing the human CB1 receptor, and both analogs
increase the binding of [35S]GTP
S to cerebellar
membranes and inhibit electrically evoked contractions of the mouse vas
deferens. ACPA and ACEA produce hypothermia in mice, and this effect is
inhibited by coadministration of the CB1 receptor antagonist SR141716A.
Therefore, ACPA and ACEA are high-affinity agonists of the CB1 receptor
but do not bind the CB2 receptor, suggesting that structural analogs of
AEA can be designed with considerable selectivity for the CB1 receptor over the CB2 receptor.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Two
cannabinoid receptors, CB1 (Matsuda et al., 1990
) and CB2 (Munro et
al., 1993), have been identified. These two receptors form a distinct
class within the family of G protein-coupled receptors (Matsuda 1997).
They share 40 to 50% amino acid sequence homology and recognize many
of the same ligands. In fact, most high-affinity agonists of the CB1
receptor bind to the CB2 receptor in the same concentration range
(Munro et al., 1993; Felder et al., 1995). Selective antagonists of the
two receptors have been identified: SR141716A, with at least 100-fold
selectivity for CB1 over CB2 (Rinaldi-Carmona et al., 1994), and
SR144528, a selective antagonist of the CB2 receptor (Rinaldi-Carmona
et al., 1998). Several agonists have been identified with selectivity
for the CB2 receptor over the CB1 receptor. These include the naturally
occurring cannabinoid, cannabinol (Munro et al., 1993; Felder et al.,
1995), and several deoxy derivatives of
8-tetrahydrocannabinol (Huffman et al., 1996,
1998). The aminoalkylindole cannabimimetic Win 55212-2 binds to CB1
and CB2 receptors with similar affinity in membranes from tissues in
which the receptors are expressed endogenously (Kuster et al., 1993;
Slipetz et al., 1995) but has higher affinity for the cloned, expressed
CB2 receptor than the CB1 receptor (Felder et al., 1995).
N-Arachidonylethanolamine (AEA or anandamide) is a
brain-derived compound that binds and activates both the CB1 (Devane et al., 1992) and CB2 receptors (Felder et al., 1995; Slipetz et al.,
1995). Several laboratories have synthesized and evaluated structural
analogs of AEA for their ability to bind the CB1 receptor (see Hillard
and Campbell, 1997, for review). To summarize, these studies have shown
that analogs with modifications in the amide head group, particularly
changes in which the head group becomes more hydrophobic, bind to the
CB1 receptor with greater affinity than the parent compound. Less is
known about the affinities of AEA analogs for the CB2 receptor;
however, two anandamide analogs, arachidonyl-2-fluoroethylamide
(Showalter et al., 1996) and (R)-methanandamide (Khanolkar
et al., 1996) have been shown to bind with higher affinity to the CB1
than the CB2 receptor. These results suggest that modifications to the
amide portion of AEA that enhance binding to CB1 reduce binding to CB2.
We have used this principle to design two analogs, arachidonylcyclopropylamide and arachidonyl-2-chloroethylamide, which
would be predicted to be high-affinity CB1 ligands due to the
hydrophobic character of the amide substitution. As predicted, these
analogs bind to the CB1 receptor with very high affinity but have low
affinity for the CB2 receptor, exhibiting selectivity ratios of 325 and
2200, respectively. We report in this article that both compounds are
agonists of the CB1 receptor and, therefore, represent the first
high-affinity cannabinoid agonists that exhibit significant selectivity
for CB1 over CB2 receptors.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Compound Syntheses.
Arachidonylcyclopropylamide (ACPA; Fig.
1) was synthesized from arachidonic acid
(0.033 mmol) in anhydrous tetrahydrofuran (200 µl) and was stirred
with 5 µl of triethylamine (0.036 mmol) and isobutyl chloroformate
(0.033 mmol) at 0°C for 30 min. Cyclopropylamine (5 µl, 0.072 mmol)
was added, and the reaction mixture was stirred at 0°C for 3 h.
The reaction mixture was diluted with water and ether, and the ether
extract was washed successively with water, 5% sodium bicarbonate
solution, and water. After evaporation of the solvent, the product was
purified by silica gel chromatography using 40% ethyl acetate in
hexane as eluent; the yield was 88%. NMR was used to confirm the
structural identity: 1H NMR (300 MHz,
CDCl3) 
0.49 (s, 2 H),0.77 (d,
J = 6.3 Hz, 2 H), 0.90 (t, J = 6.5 Hz, 3 H), 1.20 to 1.1.40 (m, 6 H), 1.65 to 1.80 (m, 2 H), 2.00 to 2.20 (m, 6 H), 2.71 (s, 1 H), 2.75 to 2.85 (m, 6 H), 5.30 to 5.45 (m, 8 H), 5.61 (broad s,
1 H); 13C NMR (75 MHz,
CDCl3) 6.88 (2 C), 14.28, 22.78, 25.87, 25.82 (4 C), 26.84, 27.41, 29.51, 31.71, 36.11, 127.70, 128.04, 128.35 (2 C),
128.78, 128.93, 129.93, 130.70, 174.44.
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0.9 (t, J = 6.5 Hz, 3 H), 1.20 to 1.40 (m, 6 H), 1.70 to 1.80 (m, 2 H), 2.0 to 2.20 (m, 4 H), 2.23 (t, J = 7.3 Hz, 2 H),
2.70 to 2.90 (m, 6 H), 3.50 to 3.70 (m, 4 H), 5.32 to 5.50 (m, 8 H),
5.82 (broad s, 1 H); 13C NMR (75 MHz,
CDCl3) 14.29, 22.78, 25.59, 25.83 (3 C),
26.81, 27.42, 29.52, 31.72, 36.13, 41.32, 44.43, 127.72, 128.02, 128.32, 128.44, 128.80, 129.08, 129.18, 130.71, 173.23.
Radioligand Binding Methods.
The affinities of the compounds
for the CB1 receptor were determined using rat cerebellar membranes and
[3H]CP55940 as described previously
(Hillard et al., 1995).
80°C for no more than
1 month. Spleen membranes (50 µg of protein) were incubated with
[3H]CP55940 (0.5-1 nM) for 1 h at 30°C
in a final volume of 0.2 ml of TME buffer containing 0.1% fatty acid
free BSA. Nonspecific binding was defined as
[3H]CP55940 bound in the presence of 5 µM Win
55212-2. Bound and free radioligand were separated by filtration
(Hillard et al., 1995).
For both the CB1- and CB2-binding assays, incubations were carried out
in the presence of 150 µM phenylmethylsulfonyl fluoride to inhibit
ligand catabolism by AEA amidohydrolase (Childers et al., 1994; Hillard
et al., 1995). Competing ligands were added to the incubation in 1 µl
of dimethyl sulfoxide (DMSO). In each assay, binding was determined in
triplicate or quadruplicate; IC50 values were
calculated using nonlinear regression to fit the data to a one-site
binding equation (Prism, GraphPad Software, San Diego).
KI values were calculated from the
IC50 values using the formula of Cheng and
Prusoff (1973).
The binding of [35S]GTPS was carried out
using a modification of previously published methods (Wieland and
Jakobs 1994). Cerebellar membranes (5 µg of protein) were incubated
with 0.65 nM [35S]GTPS in TME buffer
containing 0.1% BSA, 10 µM GDP, 150 µM phenylmethylsulfonyl fluoride, and 150 mM NaCl. The incubation was carried out for 30 min at
37°C; bound and free [35S]GTPS were
separated by filtration. Nonspecific binding was defined using 10 µM
Gpp[NH]p. Cannabinoid ligands were added to the incubations in 1 µl
of DMSO; control incubates contained DMSO alone. In each experiment,
the percent increase in [35S]GTPS binding in
response to agonist was calculated using the DMSO-treated membranes as
the control. The EC50 values and maximal agonist-induced increase in [35S]GTPS
binding were determined by fitting the data to a sigmoidal concentration-response curve using nonlinear regression (Prism, GraphPad Software, San Diego, CA).
Adenylyl Cyclase Assays. Cannabinoid receptor inhibition of adenylyl cyclase activity was determined in Chinese hamster ovary (CHO) cells stably transfected with cDNA encoding either the human CB1 or human CB2 receptors. The cells were kindly provided by Drs. G. Disney and A. Green (Glaxo Wellcome Research and Development, Medicines Research Center, Stevenage, England). Cells were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (Ham's F-12) supplemented with 2 mM glutamine, 600 µg/ml geneticin, and 300 µg/ml hygromycin.
Cells were preincubated for 30 min at 37°C with cannabinoid and isobutylmethylxanthine (50 µM) in PBS containing 1 mg/ml BSA and 100 µM phenylmethylsulfonyl fluoride. Forskolin was added (final concentration, 2 µM), and the incubation was continued for 30 min. The reaction was terminated by addition of 0.1 M HCl followed by centrifugation to remove cell debris. The pH of the supernatant was adjusted to 8 to 9 using 1 M NaOH, and cyclic AMP (cAMP) content was measured by radioimmunoassay (Biotrak; Amersham, Arlington Heights, IL). Cannabinoids were dissolved in ethanol as 1-mg/ml stock solutions and diluted to final concentrations in assay buffer. Forskolin and isobutylmethylxanthine were dissolved in DMSO. Effects of the test compounds on forskolin-stimulated cAMP production have been expressed in percentage terms. This was calculated from the equation [100 × (f' b)]/(f b), where
f' is cAMP production in the presence of forskolin and
the test compound, f is cAMP production in the presence of
forskolin alone, and b is basal cAMP production.
Mouse Vas Deferens Experiments.
Vasa deferentia were
obtained from albino MF1 mice weighing 32 to 42 g. Each tissue was
mounted in a 4-ml organ bath at an initial tension of 0.5g
as described previously (Pertwee et al., 1993). The baths contained
Krebs' solution, which was kept at 37°C and bubbled with 95%
O2 and 5% CO2. The
composition of the Krebs' solution was 118.2 mM NaCl, 4.75 mM KCl,
1.19 mM KH2PO4, 25.0 mM
NaHCO3, 11.0 mM glucose, and 2.54 mM
CaCl2·6H2O. Isometric contractions were evoked by stimulation with 0.5-s trains of three pulses of 110% maximal voltage (train frequency, 0.1 Hz; pulse duration, 0.5 ms) through platinum and stainless steel electrodes attached to the upper and lower ends of each bath, respectively. Stimuli were generated by a Grass S48 stimulator, then amplified (Med-Lab channel attenuator; Stag Instruments, Chalgrove, Oxford, UK) and divided to yield separate outputs to four organ baths (Med-Lab StimuSplitter). Contractions were monitored by computer (Apple
Macintosh LC or Quadra 650, Cupertino, CA) using a data recording and
analysis system (MacLab) that was linked via preamplifiers (Macbridge)
to Dynamometer UF1 transducers (Harvard Apparatus, Edenbridge, Kent, UK).
9-tetrahydrocannabinol
(9-THC) (Pertwee et al., 1992). Drug additions
were made in volumes of 10 or 31 µl.
Inhibition of electrically evoked contractions has been calculated by
comparing the amplitude of the twitch response after each addition of
an agonist with its amplitude immediately before the first addition of
the agonist. Values with their 95% CLs for the maximal degree of
inhibition of the twitch response produced (Emax) and for agonist concentrations
producing 50% of the Emax (EC50) were calculated using nonlinear regression
analysis (Prism, GraphPad Software).
Determination of Mouse Rectal Temperature. Male ICR mice weighing between 25 and 35 g were used in these studies. Rectal temperatures were determined using a Yellow Springs thermister inserted to a depth of 2.5 cm. All drugs were dissolved in 100% ethanol and administered i.v. or i.p. to the mice in an emulsion of ethanol/emulphor EL-60/saline (1:1:18). Vehicle solutions contained the same amounts of ethanol and emulphor.
Statistics. The binding and adenylyl cyclase data were analyzed for significant differences among mean values using one-way ANOVA followed by Tukey's post hoc test to compare individual means when warranted. The vas deferens data were compared using overlap of the 96% confidence intervals of the mean. The temperature data were analyzed by one-way ANOVA followed by Dunnett's modification of the t test to compare treatment means with a single control.
Materials.
3[H]CP55940 (165 Ci/mmol) and
[35S]GTPS (1200 Ci/mmol) were purchased from
NEN Life Sciences (Boston, MA). Emulphor EL-620 (now called Alkamuls
EL-620) was kindly provided by Rhone-Poulenc (Cranbury, NJ). AEA was
purchased from Cayman Chemical Company (Ann Arbor, MI). Win 55212-2
was purchased from RBI (Natick, MA). SR141716A and SR144528 were kindly
provided by Sanofi Recherche (Montpellier, France); CP55940 was a gift
from Pfizer Central Research (Groton, CT); and
9-THC was obtained from the National Institute
on Drug Abuse (Rockville, MD). All other drugs and chemicals were of
the highest grade possible and were purchased from standard commercial sources.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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ACPA and ACEA Have Higher Affinity for the CB1 Receptor Than the
CB2 Receptor.
The affinities of the arachidonyl amides for the CB1
receptor were determined in rat cerebellar membranes. Previous studies have demonstrated that rat cerebellar membranes are a rich source of
CB1 receptors (Herkenham et al., 1990; Breivogel et al., 1997). CB2
receptor expression has been demonstrated in brain-derived microglial
cells (Kearn and Hillard 1998); however, it is unlikely that these
cells contribute significantly to the total cannabinoid receptor pool
in cerebellar homogenates.
; Childers et al.,
1994) (Table 1 and Fig.
2). The CB2-selective antagonist SR144528
does not inhibit the binding of [3H]CP55940 to
rat cerebellar membranes at a concentration of 100 nM (data not shown),
supporting the argument that essentially all of the cannabinoid
receptors in cerebellar membranes are the CB1 subtype. Both ACPA and
ACEA bind to the CB1 receptor with very high affinity, with
KI values of 2.2 and 1.4 nM,
respectively (Table 1 and Fig. 2). The
KI values for ACPA and ACEA are not significantly different from each other or from the
KI for Win 55212-2, and both are
significantly different from the KI
for AEA (p < .05).
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).
[3H]CP55940 exhibits saturable binding to
spleen membranes with a KD of 2.8 ± 0.4 nM and Bmax of 571 ± 21 fmol/mg protein. The CB2-selective antagonist SR144528 (Rinaldi-Carmona
et al., 1998) competes for [3H]CP55940 binding
in spleen with a KI of 4.23 ± 1.0 nM, whereas the CB1-selective antagonist/inverse agonist SR141716A
(Rinaldi-Carmona et al., 1994; Bouaboula et al., 1997) has no effect on
the binding of [3H]CP55940 to spleen membranes
at concentrations up to 1 µM.
As expected, both Win 55212-2 and AEA compete for
[3H]CP55940 binding to CB2 receptors in spleen
(Table 1 and Fig. 3). Win 55212-2 has
significantly higher affinity for the CB2 receptor in spleen than the
CB1 receptor in brain, whereas AEA has greater affinity for the brain
CB1 receptor (p < .05). These results are in very
close agreement with a previous report (Showalter et al., 1996). Both
ACPA and ACEA compete for [3H]CP55940 binding
in spleen, however, with low affinity relative to both AEA and their
affinity for the CB1 receptor (Table 1 and Fig. 3). The
KI value for ACPA is significantly
greater than the KI for AEA
(p < .05).
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ACPA and ACEA Are Agonists of the CB1 Receptor. The CB1 receptor agonist activity of the arachidonyl amides was determined in several ways. In the first set of experiments, inhibition of forskolin-stimulated adenylyl cyclase activity was measured in CHO cells stably expressing either hCB1 or hCB2 receptor. The nonselective cannabinoid agonist CP55940 potently inhibits cAMP accumulation in both cell lines (Table 2). In agreement with the binding data, neither ACEA nor ACPA inhibit forskolin-stimulated adenylyl cyclase activity in cells expressing the CB2 receptor. However, both ACPA and ACEA are very potent inhibitors of cAMP accumulation in cells expressing the CB1 receptor. The IC50 values for ACPA and ACEA are not significantly different from each other and are both significantly less than the IC50 value for AEA (p < .05). All of the ligands investigated produced nearly complete inhibition of cAMP accumulation; the Emax values were not significantly different from each other.
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S binding to cerebellar membranes.
The first step in the activation of intracellular signaling by G
protein-coupled receptors is the induction of an exchange of GDP for
GTP on the guanine nucleotide binding site of the alpha subunit of a
heterotrimeric G protein. The effects of various cannabinoid receptor
agonists on GDP-GTP exchange can be determined from agonist-induced
binding of the nonhydrolyzable GTP analog,
[35S]GTPS (Selley et al., 1996; Breivogel et
al., 1997; Burkey et al., 1997). Both Win 55212-2 and AEA increase the
binding of [35S]GTPS; as has been reported
previously (Selley et al., 1996; Burkey et al., 1997), Win 55212-2 is
both more potent (i.e., has a lower EC50) and
more efficacious than AEA (Table 3). Both
ACPA and ACEA increase the binding of
[35S]GTPS to cerebellar membranes (Table 3
and Fig. 4). The arachidonyl amides are
significantly more potent than AEA (p < .05) but are not significantly more potent than Win 55212-2. ACPA and ACEA are
equiefficacious, and the Emax values
obtained in the presence of these analogs are not significantly
different from the Emax value produced
by either Win 55212-2 or CP55940. The increase in
[35S]GTPS binding to cerebellar membranes
induced by both ACPA and ACEA is inhibited completely by coincubation
of the membranes with the CB1-selective antagonist, SR141716A (Fig. 4).
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). Both ACPA and ACEA potently inhibit
electrically induced contractions (Table 4). There is no significant difference
among the potencies of the three ligands. The
Emax values for both ACPA and ACEA are significantly less than 100%; only ACPA is significantly less efficacious than CP55940.
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ACPA and ACEA Mimic the Effects of AEA on Body Temperature In
Vivo.
The cannabinoids produce robust hypothermia in mice (Dewey,
1986). Like the other cannabinoid receptor agonists, AEA produces a
significant hypothermia, although the effect is of shorter duration and
lower maximum than the effect produced by
9-THC (Smith et al., 1994). ACPA, ACEA, and
AEA all reduce rectal body temperature in mice in a dose-related manner
(Fig. 5). All three compounds produced
significant decreases in rectal temperature at a dose of 1 mg/kg. At 1 mg/kg, ACEA significantly reduced rectal temperature for at least 30 min following injection, whereas AEA was only significantly effective
for the first 5 min. Two-way ANOVA of the 1 mg/kg data indicates no
significant differences among the drugs.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Previous studies have demonstrated that AEA binds to both the CB1
and CB2 receptors with moderate affinity (Devane et al., 1992; Felder
et al., 1995). Furthermore, recent studies have suggested that
modifications in the AEA structure that enhance ligand affinity for the
CB1 receptor reduce affinity for the CB2 receptor (Showalter et al.,
1996; Edgemond et al., 1998). In the present study, we report the
synthesis and characterization of two analogs of AEA, ACPA and ACEA.
Both of these arachidonyl amides are high-affinity agonists of the CB1
receptor: 1) These compounds compete for binding with
[3H]CP55940 to cerebellar membranes with
KI values in the low nanomolar range.
2) They inhibit forskolin-stimulated adenylyl cyclase activity in CHO
cells transfected with hCB1 at low nanomolar concentrations. 3) They
inhibit the electrically induced contractions of mouse vasa deferentia
at picomolar concentrations. In addition, both analogs induce the
exchange of GDP for GTP at nanomolar concentrations, and this effect is
blocked by the CB1-selective antagonist SR141716A (Rinaldi-Carmona et
al., 1994). Taken together, these results strongly support the
contention that both ACPA and ACEA are high-affinity agonists of the
CB1 receptor.
In contrast, the analogs bind with low affinity to the CB2 receptor and
do not affect adenylyl cyclase activity in CHO cells transfected with
the CB2 receptor. The calculated potency ratios, based upon the
dissociation constants of the ligands for the two receptors, indicate
that ACPA has greater than 300-fold selectivity for CB1, and ACEA has
greater than 2000-fold selectivity. The only other cannabinoid ligands
with reported selectivity of greater than 25-fold are SR141716A
(Rinaldi-Carmona et al., 1994; Felder et al., 1995; Showalter et al.,
1996), (R)-methanandamide (Khanolkar et al., 1996), and
arachidonyl-(2'-fluoroethyl)amide (Showalter et al., 1996).
As has been shown previously by other investigators (Selley et al.,
1996; Burkey et al., 1997), AEA is significantly less efficacious than
both Win 55212-2 and CP55940 when [35S]GTPS
binding is used to assess efficacy. AEA has also been shown to be a
partial agonist in studies of calcium channel inhibition by the CB1
receptor (Mackie et al., 1993). Neither ACPA nor ACEA behaves as a
partial agonist in the [35S]GTPS assay
(i.e., produce a lower Emax than the
full agonist Win 55212-2). Similarly, ACEA and ACPA produce
Emax values that are not different
from CP55940 in the adenylyl cyclase assay carried out using
transfected CHO cells. However, in the vas deferens assay, ACPA has a
significantly lower Emax value than
CP55940, which indicates that it is not a fully efficacious agonist in this assay. The Emax values of a
partial agonist vary from assay to assay due to differences in the
density of receptors; partial agonists are more apparent in
preparations that are "receptor limited" or have a low number of
spare receptors. In sum, our data suggest an efficacy order of Win
55212-2 
CP55940 ACEA > ACPA > AEA.
As has been shown previously, THC and AEA produce significant decreases
in rectal temperature following i.v. administration in mice (Adams et
al., 1998). This effect is mimicked by ACEA and ACPA. The hypothermia
produced by ACEA and ACPA is blocked by the CB1 receptor antagonist
SR141716A, supporting the involvement of the CB1 receptor in this
effect. Interestingly, the hypothermia produced by AEA was attenuated
by SR141716A but did not reach statistical significance. Similarly,
Adams et al. (1998) reported that SR141716A reversed the hypothermic
effects of 9-THC and CP55940 but not AEA. It
is possible, as the authors of that study point out, that the
conversion of AEA to arachidonic acid and its metabolites at high doses
may account for some noncannabinoid temperature effects.
ACEA and ACPA have 35- to 50-fold higher affinity for the CB1 receptor
than AEA, yet produce the same degree of hypothermia in vivo at a dose
of 1 mg/kg. These data suggest that bioavailability plays a significant
role in the in vivo potency of ACEA and ACPA. Indeed, Willoughby et al.
(1997) have demonstrated that only a small fraction of
[3H]AEA administered to mice is found in the
brain. They found that AEA was very rapidly degraded to arachidonic
acid and other more polar metabolites. It is very likely that the same
is true of ACPA and ACEA; both analogs inhibit the hydrolysis of AEA to
arachidonic acid and ethanolamine (data not shown), which suggests they
may be substrates of the amidohydrolase that catabolizes AEA (Deutsch and Chin 1993; Desarnaud et al., 1995; Hillard et al., 1995).
In summary, both ACPA and ACEA are high-affinity agonists of the CB1 receptor. In contrast, they are very poor ligands for the CB2 receptor and, therefore, represent the most selective CB1 agonist currently available. Both analogs produce hypothermia in vivo that is reversed by the CB1-selective antagonist SR141716A. These analogs represent the most selective ligands for the CB1 receptor over the CB2 receptor yet reported and provide interesting lead compounds for further structure activity studies.
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Acknowledgments |
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We are grateful to Sean Tracy and Nicole Tonn for technical assistance.
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Footnotes |
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Accepted for publication February 1, 1999.
Received for publication October 20, 1998.
1 This work was supported by National Institutes of Health Grants DA08098 (to C.J.H.), DA09155 (to W.B.C., C.J.H.), and DA09789 (to R.G.P.); Grants 039538 and 047980 from the Wellcome Trust (to R.G.P., R.A.R.); and by a grant from the European Social Fund (to V.M.). A preliminary report of these findings was made at the 1997 meeting of the International Cannabinoid Research Society.
Send reprint requests to: Cecilia J. Hillard, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI. E-mail: chillard{at}mcw.edu
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Abbreviations |
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AEA, N-arachidonylethanolamine;
CB1, neuronal cannabinoid receptor;
CB2, spleen cannabinoid receptor;
CHO, Chinese hamster ovary;
9-THC, 9-tetrahydrocannabinol;
DMSO, dimethyl sulfoxide;
ACPA, arachidonylcyclopropylamide;
ACEA, arachidonyl-2-chloroethylamide;
TME, Tris, magnesium, and EDTA.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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8-THC derivatives: Selective ligands for the CB2 receptor. 1998 Symposium on the Cannabinoids, La Grande Motte, France, International Cannabinoid Research Society, Burlington, VT.-thio)triphosphate binding by G proteins.
Methods Enzymol
237:
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C. Leterrier, D. Bonnard, D. Carrel, J. Rossier, and Z. Lenkei Constitutive Endocytic Cycle of the CB1 Cannabinoid Receptor J. Biol. Chem., August 20, 2004; 279(34): 36013 - 36021. [Abstract] [Full Text] [PDF]
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A. H. Lichtman, E. G. Hawkins, G. Griffin, and B. F. Cravatt Pharmacological Activity of Fatty Acid Amides Is Regulated, but Not Mediated, by Fatty Acid Amide Hydrolase in Vivo J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 73 - 79. [Abstract] [Full Text] [PDF]
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A. C. Howlett, F. Barth, T. I. Bonner, G. Cabral, P. Casellas, W. A. Devane, C. C. Felder, M. Herkenham, K. Mackie, B. R. Martin, et al. International Union of Pharmacology. XXVII. Classification of Cannabinoid Receptors Pharmacol. Rev., June 1, 2002; 54(2): 161 - 202. [Abstract] [Full Text] [PDF]
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