0022-3565/93/2651-0218$03.OO/O
THE
JOURNAL
Copyright
OF PHARMACOLOGY
AND
C 1993 by The American
EXPERIMENTAL
Society
Vol. 265, No. 1
THERAPEUTiCS
for Pharmacology
and Experimental
Printed
Therapeutics
Cannabinoid Structure-Activity
Relationships:
Receptor Binding and in Vivo Activities1
Correlation
DAVID
A. COMPTON,
M. ROSS JOHNSON
LAWRENCE
Department
KENNER
and BILLY
C. RICE, BRIAN
R. MARTIN
R. DE
COSTA,
RAJ
K. RAZDAN,
in U.S.A.
of
S. MELVIN,
Laboratory
Bethesda,
Research
of Pharmacology
and Toxicology,
Medical College of Virginia, Virginia Commonwealth
University,
Richmond,
Wrginia (D.R.C., B.R.M.);
of Medicinal
Chemistry,
Nationallnstitute
of Diabetes and DigestWe
and Kidney Diseases, National Institutes of Health,
Maryland
(K.C.R., B.R.D.); Organix, Inc., Woburn, Massachusetts
(R.K.R.); Pfizer Inc., Groton, Connecticut
(L.S.M.) and Glaxo Inc.,
Triangle Park, North Carolina (M.R.J.)
Accepted
for
November
publication
23, 1992
ABSTRACT
structure-activity
exists
for cannabinoid
drugs,
it is uncertain
actions this receptor
mediates.
This
relationship investigation
was initiated to deter-
indicated
the importance
of side chain structure
to high-affinity
binding, with the most potent analogs (Kl < 10 nM) possessing
either a dimethylheptyl
side-chain,
a similarly
complex
branched
mine which effects might correspond
to binding affinity for the
cannabinoid
receptor,
as well as to explore the binding requirements
of this site. The ability of nearly 60 cannabinoids
to
displace
[3HJCP-55,940
{(-)-3-[2-hydroxy-4-(1
,1 -dimethylheptyl)phenylj-4-[3-hydroxy
propyl]
cyclohexan-1
-oI’ was determined before establishing
correlations
between
receptor
affinity
and in vivo pharmacological
potency.
Analysis of [3H]CP-55,940
binding indicated
a Hill coefficient
of 0.97, a B,
of 499 pM (3.3
pmol/mg
of protein)
and an apparent
Kd of 924 pM. Closer
inspection
indicated
the binding assay exhibited
“zone B” charactenstics,
and use of correction
equations
indicated
a true Kd
for CP-55,940
of 675 pM. The structure-activity
relationship
side chain or a halogen substituent
at the 5’ position.
Comparative analysis of Km values to in viva potency
in a mouse
model
indicated
a high degree
of correlation between
parameters
for
the depression
of spontaneous
locomotor
activity (r = 0.91) and
for the production of antinociception
(r = 0.90), hypothermia
(r
=
0.89) and catalepsy
(r = 0.85). Similarly high correlations
were
demonstrated
between
binding affinity and in viva potency
in
both the rat drug discrimination
model (r = 0.81) and for psychotomimetic
activity
in humans
(r = 0.88). Thus,
these studies
appear to indicate
that the requirements
for activation
of the
cannabinoid
receptor
are similar across different
species,
and
In the time since its isolation
from plant
material
(Gaoni
and Mechoulam,
1964), 9-THC
has come to be recognized
as
the principle
active component
of marihuana.
Despite
the fact
that pharmacological
evidence
such as SAR suggested
the existence
of a receptor
through
which 9-THC
and related
cannabimimetic
drugs
might
be producing
their effects
(Binder
and Franke,
1982), the first attempt
to show the existence
of
such a site by use of radioligand
binding
techniques
failed to
provide
convincing
evidence
that a receptor
existed
in brain
tissue,
although
modest
information
was obtained
for binding
to cultured
cells (Harris
et at., 1978).
The
first
successful
identification
of a cannabinoid
binding
site in brain tissue was
accomplished
by use of a water-soluble
THC analog,
although
binding
affinity
at this site proved
not to be well correlated
with the known
behavioral
effects
of multiple
cannabinoids
(Nye et at., 1985, 1989). Subsequently,
the 5’-trimethylammonium derivative
used as the radioligand
was found to be devoid
of the typical
pharmacological
profile
expected
of a cannabimimetic
(Compton
and Martin,
1990).
Despite
the lack of
evidence
for a binding
site, a correlation
of the analgesic
prop-
Received
1
This
03672
and
for publication
work
was supported
by
a Pharmaceutical
July
8, 1992
by National
Institutes
Manufacturers
on Drug
Association
Abuse
Foundation
Grant
DA
Starter
that receptor
erties
effects
of a series
ability
to
However,
ugation
assay
as the
radioligand
was
nonclassical
adenylyl
cyclase
of a cannabinoid
1988).
site
to mediate
shown
it was
using
the
many of the known
of cannabinoids.
of potent
inhibit
formulation
Unlike
Grant.
binding is sufficient
pharmacological
cannabinoids
receptor
not
until
the
extremely
that
a specific
to exist
in
the 5’-trimethylammonium
model
saturable
brain
tissue
CP-55,940
cannabinoid
radioligand,
their
to the
et at.,
of a centrif-
analgesic
(Devane
led
(Howlett
development
potent
to
in vitro,
activity
binding
et at., 1988).
CP-55,940
has
THC, tetrahydrocannabinol;
SAR, structure-activity
relationship; CBD, cannabidiol; DMHP, dimethylheptyl
pyran; TMA, trimethylammonium; DMH, dimethylheptyl;
BSA, bovine serum albumin; PEI, polyethylenimine;
Ki, dissociation
constant determined from inhibition of
radioligand binding; SA, spontaneous activity; TF, tail-flick; AT, rectal temperature; RI, ring immobility; HHC, hexahydrocannabinol;
CP-56,667, (+)-
ABBREVIATIONS:
CP-55,940
or (+)-3-[2-hydroxy-4-(1
tyl)phenylj-4-[3-hydroxy
218
,1 -dimethylheptyl)phenyl]-4-[3-hydroxypropyljcyclohexan-1-ol;
propyljcyclohexan-1
-ol
CP-55,940,
(-)-3-[2-hydroxy-4-(1
,1-dimethylhep-
Downloaded from jpet.aspetjournals.org by on May 2, 2009
Although
a receptor
which
pharmacological
219
OH
Bacyclc Analog
CannabinoidSAR
1993
Materials
and Methods
TABLE 1
Structures
Male
Sprague-Dawley
Dominion
Laboratories
light/dark
cycle,
rats
(150-200
g)
obtained
from
(Dublin,
VA) were maintained
on a 14:10-hr
and water
ad libitum.
Drugs
and chemicals.
[3H]CP-55,940
was
prepared
by tnitium
reduction,
in the presence of a Pd catalyst, of an analog possessing
a
double bond located
between
the C2 and C3 carbons of the dimethylheptyl side chain (the R substituent
depicted in table 1). 8-THC,
.
THC, ‘-THC,
11-OH-8-THC,
11-OH-9-THC,
CBD, 1’,2’-DMHP
(6.-1Os),
5’-OH-9-THC
and 11-OH-5’-TMA-8-THC
were obtained
from
the National
Institutes
on Drug Abuse. 1-O-Carbamoyl-9-THC
was provided
by the National
Cancer Institute;
nabilone by Eli Lilly
and Company
(Indianapolis,
IN); 5’-Bn-8-THC,
5’-I-8-THC,
5’-F38-THC,
5’-F-8-THC,
2-I-5-THC
and 11-F-8-THC
by Dr. Alexandros Makriyannis
(University
of Connecticut,
Stoma,
CT); 0,2-propamo-8-THC,
9a-OH-HHC,
93-OH-HHC
and 0,10-methano-8-THC
by Dr. Patricia H. Reggio
(Kennesaw
State College, Marietta,
GA) and
(+)- and (-)-11-0H-8-THC-DMH
(HU-210
and HU-211,
respectively) by Dr. Rafael Mechoulam
(Hebrew University,
Jerusalem,
Israel).
-THC-DMH,
8fl-0H-9”-THC,
1-0-methyl-9”-THC,
8-(Nmorpholino)-amino-9”-THC,
3-(norpentyl)-3-propyl-9”-THC,
1O-(6-aminohexyl)-8-THC,
1-0-methyl-8-THC,
1-0-(2-morpholinoethyl)-8-THC,
1-0-methyl-9-THC,
1-0-biphenylmethy1-8-THC,
1O-biphenylmethy1-9”-THC,
1-0-(4-phthalimidobutyl)-8-THC,
1-0methyl-3-norpentyl-3-propyl-9”-THC,
1-O-(4-aminobutyl)-8-THC,
and
received
9-nor-9-carboxy-8-THC
carboxy--THC
hydroxy
and
acid,
acid
and
food
1-0-(3-aminopropyl)-8-THC,
abnormal-CBD
(where
the
Cl
analogs
Bcyclic
Analogs
Ito XIV and XVI
T
XV
R,
Mog
__________________
____________
A,
Ill
IV
V
VI (CP-47,497)
VII
A2
H
H
-C(CH3)CH3
-C(CH3)CH2CH3
H
H
H
-C(CH3)(CH2)CH3
H
H
-C(CH3),(CH2)SCH3
-C(CH3)(CH2MCH3
H
H
H
-C(CH3)S(CH2),CH3
-C(CH3)(CH2)4CH3
-CH3
-C(CH3MCH2)TCH3
-C(CH3)(CH,)CH3
-C(CH3)(CH2)CH3
-C(CH3)(CH2)SCH3
XII
-CH3
-C(CH3)(CH2)SCH3
XIII
-(CH2)OH
-(CH2)OH
-(CH2),OH
-(CH2)OH
VIII
IX
X
Xl (Ci-aOH)
XIV (CP-55,940r
XV (CP-56,667r
XVI
H
-C(CH3MCH2)SCH3
-C(CH3MCH2)SCH3
-C(CH3)(CH2)SCH3
Previously reported as (-)-AC
and (+)-ACisomers
(Little et al., 1988). The
numbering
system
of these nondassical
cannaheeds
is proded
for comparison
to that given in Figure 2.
sb
those
described
by
et at.
Devane
(1988).
After
decapitation
and
removal
of the
until
material
was
grinding
system
the
brain,
the
cortices
of
homogenized
cortex
five
with
(Fisher
was
rats
a Kontes
Scientific,
dissected
were combined.
The
Potten-Elvehjem
glass-Teflon
cortical
NJ). The homogenate
Springfield,
was centrifuged
at 1600 X g for 10 mm, the supernatant
saved
and
combined
with the two subsequent
supernatants
obtained
from washing
(and 1600 x g centrifugation)
of the P1 pellet.
The combined
supennatant
fractions
were
centrifuged
at 39,000
in 50 ml of buffer
x g for
15 mm.
The
P2 pellet
Tnis. HC1, 2 mM Tnis.
EDTA, 5 mM MgCI2, pH 7.0), incubated
for 10 mm at 37”C, then
centrifuged
at 23,000
x g for 10 mm. The P2 membrane
was resuspended
was
resuspended
in 50 ml
of buffer
A, incubated
A (50 mM
again
except
at
30#{176}C
for
40 mm,
x g for 15 mm. The final wash-treated
was resuspended
in assay
buffer B (50 mM Tris HCI,
EDTA, 3 mM MgC12, pH 7.4) to a protein concentration
centrifuged
at
11,000
1 mM
.
2 mg/ml.
mately
The
membrane
equal aliquots
and quickly
methylbutane
(Sigma
-80”C
for no more
an aliquot
of frozen
determined
by the
frozen
Chemical
than
membrane
was
solution
Co.,
2 weeks.
method
preparation
in a bath
St.
Louis,
Before
(1976)
using
into
four
of dry ice and 2-
MO),
thawed
Tris.
of approxi-
divided
performing
was rapidly
ofBradford
then
P2 pellet
then
stored
a binding
and protein
Coomassie
at
assay,
values
brilliant
blue dye (Bio-Rad,
Richmond,
CA) and BSA standards
(fatty
acid free,
Co.) prepared
in assay buffer.
Binding
assay. The methods for nadioligand
binding were essentially
those
described
by Devane
et at. (1988) with the exception
that
bound and free drug were separated
by filtration
rather
than centrifuSigma
Chemical
gation.
Binding
was initiated
phenolic
analog (for displacement
studies)
and a sufficient
quantity
of
(50 mM Tris.HC1,
1 mM Tris.EDTA,
3 mM MgCl2, 5 mg/ml
bring the total incubation
volume
to 1 ml. The concentration
CP-55,940
in displacement
studies was 400 pM, whereas that
ration studies varied from 25 to 2500 pM. Similar
results were
preliminary
centrations
saturation
varied
from
[3HJCP-55,940
of 150 zg of P2 membrane
to test tubes
with
containing
by the addition
9-non-9-
C3 pentyl side chain are transposed)
were synthesized
at
Organix,
Inc. (Woburn, MA).
Analogs
I through
XV (depicted
in table 1), which includes
CP55,940 and its (+) isomer
CP-56,667
as well as t,d-nantradol
were
synthesized
at Central Research,
Pfizer Inc.
Membrane
preparation.
The methods for tissue preparation
were
the
free using visual
landmarks
following
reflection
of cortical
material
from the midline.
The cortex was immersed
in 30 ml of ice-cold
centrifugation
solution
(320 mM sucrose,
2 mM Tnis. EDTA,
5 mM MgCl2). The process
was
rapid
repeated
Animals.
of bicyclic
studies
25 to 5000
(79 Ci/mmol),
in which
the
pM. Nonspecific
a cannabinoid
[3H]CP-55,940
binding
buffer C
BSA) to
of [3H]
in satuobtained
con-
was deter-
Downloaded from jpet.aspetjournals.org by on May 2, 2009
been shown
to possess
a profile
of pharmacological
activity
similar
to that of 9-THC
(Little
et aL, 1988).
Localization
of
the [3HJCP-55,940
binding
site using autoradiographic
techniques
revealed
significant
similarities
among
rat, monkey
and
human
brain tissue,
whereas
limited
SAR suggests
some correlation
between
in vitro displacement
potency
and in vivo
behavioral
potency
(Herkenham
et at., 1990). These
studies
contributed
in part to the evaluation
of a previously
cloned
rat
brain protein,
which was subsequently
expressed
in various
cell
lines as a fully functional
cannabinoid
receptor
capable
of
inhibiting
adenylyl
cyclase
activity
(Matsuda
et aL, 1990),
although
no radioligand
binding
data were presented
in these cell
lines. Since then the human
cannabinoid
receptor
has also been
cloned
(Gerard
et at., 1991).
Although
it is now well established
that a receptor
exists for
the cannabinoids,
it is unclear
whether
this receptor
is responsible for all of the centrally
mediated
actions
of the cannabinoids.
This
SAR
investigation
was initiated
to determine
whether
affinity
for this receptor
correlates
with any or all of
the pharmacological
effects of the cannabinoids.
The ability
of
nearly
60 cannabinoids
to displace
[3HJCP-55,940
from its
binding
site in a membrane
preparation
was determined
via a
filtration
assay in order to evaluate
the structural
requirements
for receptor
recognition.
Additionally,
all of these compounds
have been evaluated
previously
in one or more models
for in
vivo potency.
Thus, binding
data were also determined
in order
to establish
whether
a significant
correlation
existed
between
the affinities
of these drugs
for the binding
site and their in
vivo pharmacological
activities.
220
Compton
mined
by the addition
cannabinoid
et al.
of 1 iM
analogs
mg/ml
ethanolic
centration
Vol. 265
were
stock
of no more
unlabeled
CP-55,940.
by suspension
prepared
without
evaporation
CP-55,940
and all
in buffer C from a 1
of the ethanol
(final
0.4%).
After incubation
at 30#{176}C
for 1 hr, binding was terminated
of 2 ml of ice-cold buffer D (50 mM Tnis . HC1, 1 mg/mi
vacuum
filtration
con-
than
through
manifold
(Millipore,
with
2 ml of ice-cold
pretreated
Bedford,
MA).
buffer D, and
filters
by addition
BSA) and
in a 12-well
sampling
Reaction
vessels were washed
the filters washed
twice with
once
4 ml
of ice-cold buffer D. Filters were placed into 20-mi plastic scintillation
vials (Packard,
Downer’s Grove, IL) with 1 ml of distilled
water and
10 ml of Budget-Solve
for 1 hr, the radioactivity
(RPI Corp., Mount
Prospect,
IL). After shaking
present
was determined
by liquid scintillation
spectrometry.
Assay conditions
the combined
were performed
data
were performed
in triplicate,
of three
to six independent
in siliconized
test tubes,
and results
experiments.
which
represent
All assays
were prepared
by
air
drying
(12 hr) the inverted
borosilicate
tubes after two rinses with a
0.1% solution
of AquaSil
(Pierce,
Rockford,
IL). The GF/C glass-fiber
filters
(2.4 cm, Baxter,
McGaw
Park, IL) were pretreated
in a 0.1%
solution
of
Data
pH 7.4 PEI (Sigma Chemical Co.) for at least 6 hr.
The Bmu and Kd values
obtained
from
Scatchard
analysis.
analysis
(Scatchard,
1951;
cation
of
Rosenthal,
1967)
values
were
via
determined
of binding analysis programs
for the Macintosh
Milltown,
NJ). The software
represents
a modifi-
the original
descriptions
of LIGAND
(Munson
and
Rodbard,
1980) and Equilibrium
Binding
Data Analysis
(McPherson,
1983).
Displacement
ICo values were originally
determined
by unweighted
least-squares
linear regression
of log concentration-percent
displacement data and then converted
to Kz values
using
reported
methods
(Chengand
Prusoff, 1973). Statistical
evaluation
ofparallelism
between
displacement
isotherms
was performed
using
ALLFIT
(De Lean
et aL,
1987),
a program
sigmoidal
curves.
for
the
Statistical
simultaneous
analysis
product-moment
coefficients
on the Macintosh
computer
using
Inc., Agoura
Hills,
(Brainpower,
for
curve
fitting
of a family
and generation
the correlation
the StatView
of
of the Pearson
studies was performed
512+ statistical
package
CA).
Results
Assay
characteristics.
sented
a modification
cubation
conditions
Because
this
of the centrifugation
were
evaluated
before
filtration
assay
procedure,
initiation
of
repreall in-
The temperature
dependence
otherwise
standard
conditions
55,940.
Total
tively,
binding
ofthat
at 25 and
determined
of binding
using
35#{176}C
was
tremendously
and
incubation
temperature
of 30”C
was
Treatment
of filters
PEI
reduced
did washing
was
greater
filters
than
with
with
10%
two
of
the
was
4-ml
total
variable.
then
to that
nately,
binding
at 50 g
of this phenomenon
impact
on
saturation
the
and
Kd value
for
binding,
from
150
of protein
(>10%
Scatchard
g
analysis.
saturation
of protein.
proved
of total
displacement
as
binding
a value
were reduced
to 50 zg
than 10% of total radio-
derived
using
the
assays.
nonspecific
maximum
found
Thus,
rinses
of buffer.
Total
radioactivity
added,
considered
added,
binding
in subsequent
when protein
concentrations
binding
maintained
at less
similar
respec-
at 40#{176}C
total
used
However,
and total
sis was
[3HJCP-
71%,
highly
normally
activity
determined
pM
66 and
at 30#{176}C,
whereas
decreased
was
400
analyUnfortu-
highly variable.
The
radioactivity
bound)
analysis
is addressed
under
“Discussion.”
Scatchard
ducted
over
analysis.
a period
Independent
of 1 year have
results.
example
of
An
independent
experiment
curve
as an
shown
the
Scatchard
is shown
inset.
saturation
resulted
analysis
in figure
Computer
analyses
conin reproducible
1 with
analysis
of
a
single
the saturation
of saturation
data
(n = 5, simultaneous
analysis
with LIGAND)
indicated
a Kd of
924 ± 140 pM and a B,,,55 of 499 ± 60 pM (3.3 ± 0.6 pmol/mg
protein)
and a Hill coefficient
of 0.97 ± 0.01.
Because
cannabinoids
bind to albumin
(Garrett
and Hunt,
1974;
Haque
and
Poddar,
1984),
BSA
can
be included
in buffers
to act
as the cannabinoid
carrier
while in an aqueous
medium.
To evaluate
the potential
interference
of BSA binding
on
receptor
binding,
the quantity
of BSA present
in the assay
buffer
(buffer
C) was
reduced
from
ml in order
to determine
whether
Scatchard
analysis.
At a concentration
5 mg/ml
there
to 2.5 and
was
an
of 2.5 mg/ml
1.0 mgI
effect
on
of BSA
the EBDA/LIGAND
analysis
(n = 2) indicated
a Kd of 1160 ±
260 pM, a Bmax of 705 ± 117 pM and a Hill coefficient
of 0.99
± 0.01. At a concentration
of 1.0 mg/ml
of BSA
the EBDA/
LIGAND
analysis
(n = 2) indicated
a Kd of 932 ± 102 pM, a
satura-
The optimal
incubation
conditions
were found to
be similar
to those previously
described
(Devane
et al., 1988).
Thus,
the results
for characterization
are not shown.
Unlike
the previously
published
method,
nonspecific
binding
was defined as the binding
observed
in the presence
of 1 MM CP55,940. Specific
binding
averaged
90% in the displacement
studies
where
radioligand
concentration
was 400 pM. The
protein
dependency
of the assay
was determined
using
the
tion
analysis.
standard
assay
conditions
for
all preparations
and
incubations
with [3HJCP-55,940
having
been arbitrarily
held at 400 pM.
Similar
to the previously
published
assay,
binding
was found
to be linear from 20 to 50 g of protein
per tube, but there was
a high degree of variability
in the binding
at these low protein
levels.
However,
variability
in response
was minimized
at a
higher protein
range, and binding
was linear (r = 0.95) from 20
to 200
ofprotein.
There was a suggestion
of a deviation
from
linearity
above
175 tg of protein,
so subsequent
assays
were
conducted
using 150 tg of protein
per tube. The time course of
binding
was evaluated
under standard
conditions
using 400 pM
[3H]CP-55,940.
Binding
increased
from
10 to 50 mm, and
maximal
binding
was maintained
through
3 hr suggesting
no
degradation
of radioligand
or receptor
during this period. Thus,
subsequent
assays
were conducted
with an incubation
time of
I
0
100
200
300
Bound
400
500
600
(fmol)
1. Scatchard
analysis of [3H]CP-55,940
receptor binding. This
saturation
experiment
(inset) and resulting Scatchard
analysis is representative of the binding obtained with rat cortical tissue. In this particular
experiment the Kd was found to be 799 pM and the B
equal to 4.0
Fig.
pmol/mg
of protein.
Downloaded from jpet.aspetjournals.org by on May 2, 2009
the KELL package
computer
(Biosoft,
1 hr.
under
Cannabinold
1993
Bmax of 661
displacement
± 49 pM
and
assays
were
a Hill
coefficient
performed
using
of 0.98
± 0.01.
the 5 mg/ml
Thus,
concen-
tration
of BSA.
Drug
displacement.
A primary
focus of this investigation
was the determination
of the SAR for binding
to the cannabinoid receptor
by evaluating
analogs
with as much structural
diversity
and widely
varying
potencies
as possible.
A series of
16 nonclassical
bicyclic
cannabinoids
were evaluated
in displacement
studies,
and as these analogs
were previously
identified by roman
numerals
(Compton
et at., 1992),
the same
designation
was used in these
studies.
The structure
of CP55,940 and related
bicyclic
cannabinoids
are shown
in table 1.
Naturally
occurring
analogs
chosen
for displacement
studies
included
9-THC
and CBD (fig. 2). Many
analogs
evaluated
possessed
a side
chain
other
than
the
typical
straight
chain
well
as
as the
to
ing correlation
1 zM.
Ki
values
could
not
be
728
obtained
pM
to more
for
16 of
the compounds,
all of which failed to displace
at least 50% at
10 M
drug.
The displacement
curves
for a representative
sample
of drugs
are shown
in figure
3. Statistical
analysis
(ALLFIT;
P < .05) indicated
that
the displacement
curves
of
most analogs
were parallel
to that of 9-THC
and CP-55,940.
This suggested
competitive
inhibition
of radioligand
binding
(rather
than noncompetitive
inhibition)
is an indication
that
receptor
recognition
occurs in a similar
fashion
for most of the
cannabinoid
drugs,
suggesting
binding
at a single site on the
receptor.
However,
not all displacement
curves could be shown
to be parallel
to that of 9-THC,
which
is described
in more
detail later (see under “Discussion”).
Correlation
studies.
The compounds
showing
a measurable
affinity
for the cannabinoid
receptor
were compared
to their
abilities
to produce
pharmacological
effects
in the mouse
(fig.
were
previously
to
has
other
relatively
long
been
weak
lation
analysis
in the
mouse
dose
analogs
were
not
because
these
compounds
that
were
greater
than
evaluated)
and
the human.
The potency
be
devoid
of
the
data
were
not
RT
behavioral
in any
corre-
ED50
values
possessed
mg/kg
100
available
solid
line
(the
in either
that
(two-tailed)
The
dashed
in each
against
the
potencies
mean
is a noticeable
plot
highest
the
rat
or
determined
mouse
represents
were
statistically
on the
when
ED
obtained
the
based
line
was obtained
previously
in
(Martin
the
linear
The correlation
coefficients
were 0.91, 0.90, 0.89 and
All correlations
level
was
procedures
data set.
RI evaluations
and
tively.
.01
of 29 analogs
included
in
et aL,
1987; Little et at., 1988, 1989; Compton
et aL, 1989, 1990, 1992;
Compton
and Martin,
1990; Reggio
et al., 1990, 1991; Charalambous
et aL, 1991). Not all compounds
were active
in all four
evaluations
which
included
measures
of SA, antinociception
via a TF assay,
hypothermia
via RT evaluation
and a RI or
catalepsy
measure.
The log ED
(tmol/kg)
value was plotted
against
the corresponding
log Ki (nM)
value for each analog
multiple
values.
than
from
values
were
conduct-
recognized
CBD
greater
ranged
potency
values
before
cause
of that
obtained
Human
kilogram
per
psychoactive
properties
found with 9-THC
and demonstrated
to have
a profile of pharmacological
activity
different
from that
of \9-THC
(Compton
et aL, 1990),
this compound
was not
included
in any of the correlation
analyses.
Similarly,
seven
4). The
values
studies.
(fig. 5). All ED
(as with ED
values)
the compound.
Be-
(fig.
Ki
micromoles
(=1.0),
and
more potent
sion
2. The
human
of
expressed
relative
to 9-THC
the smaller
the number,
the
than
4 tM.
The maximum
observed
displacement
averaged
approximately
90% for all compounds
with Ki values
of less
than 1 tiM, and varied from a 64 to 86% (for CBD and (+)-11OH-z8-THC-DMH,
respectively)
for compounds
with Ki values
table
rat and
units
in each
plot
value
the
for each
obtained
between
from
procedures.
the
at the
P <
product-moment
depicts
the KI value
in all four mouse
divergence
significant
Pearson
regres-
for SA, TF,
0.85, respec-
mean
linear
regression
drug
was plotted
averaging
the
Although
behavioral
there
potency
and the actual
antinociception
potency
of drugs
in the locomotor
(SA) and
(TF) paradigms,
it is also obvious
that there is
little
in the
difference
paradigms.
model,
the
cumbersome,
yet
potency.
Besides
establishing
and
general
temperature
(RT)
and
It is clear that for general
discussions
single mean ED
value (not given)
still
pharmacological
a relatively
accurate,
correlations
indicator
receptor
in
mouse
the
whether
and potency
(RI)
of the mouse
is a much less
between
activity
was of great
interest
to determine
existed
between
receptor
affinity
immobility
of drug
affinity
model,
correlations
in the rat
it
also
drug
11-OH-Et8-THC-DMH
Fig. 2. Structures of cannabinoids.
9-THC is numbered
according
to the dibenzopyran
nomenclature,
and related
analogs given in this report (including
8-THC derivatives)
have been named similarly. In contrast, CBD is typically
numbered according
to the monoterpinoid
nomenclature.
Also shown is the extremely potent analog 11-OH-5-THC-
DMH, as well as the exocyclic
THC.
cannabinoid
analog
Downloaded from jpet.aspetjournals.org by on May 2, 2009
pentyl observed
for 9-THC.
A common
side chain modification
that often results
in a more potent
cannabinoid
is the substitution
of a DMH side chain (Razdan,
1986; Little et al., 1989).
One example
of such an analog
chosen
for evaluation
in these
studies
was 11-OH-8-THC-DMH
(fig. 2). Other
analogs
included were derivatives
ofthe exocyclic
cannabinoid
9”-THC
(fig. 2).
The calculated
KI values
obtained
from experimentally
determined
IC
values
for 59 different
compounds
are listed in
4)
converted
221
SAR
222
Compton
et aI.
Vol. 265
TABLE 2
K, values
of cannabinoid
analogs
Analog
K,
±S.E.
Analog
K,
nM
±S.E.
nM
(-)-1 1-OH-8-THC-DMH
CP-55,940
[(-)-AC,
XIV]
0.728
0.924
0.1 13
0.140
8-THC
IX
126
163
16
35
I-Nantradol
XVI
1 .06
1 .55
0.24
0.85
9a-OH-HHC
911-THC
171
236
8
55
1.56
1 .59
0.08
0.24
X
IV
381
735
28
14
VII
4.73
1 .34
8-OH-911-THC
820
85
XI
5’-Br-8-THC
6.15
7.63
1 .99
1 .41
1-O-methyI-911-THC
8-(N-morpholino)-amino-i9’1-THC
827
869
71
71
XII
7.70
2.05
3-(NorpentyI)-3-propyl-911-THC
5’-I-8-THC
7.77
2.40
1-O-(6-Aminohexyl)-i8-THC
0.35
1’,2’-DMHP
8-THC-DMH
(6a-1Oa)
9.54
(+)-11-OH-8-THC-DMH
1990
1430
0.9
6.6
3.3
0.8
1 .7
10.2
2.3
1 1-OH-5’-TMA-8-THC
1 -O-MethyI-t8-THC
1 -O-(2-MorphoIinoethyl)-8-THC
d-Nantradol
III
CBD
1-O-Methyl-9-THC
21 10
2170
3040
3100
3760
4350
460
110
460
550
70
61.7
87.6
89.0
1 07
1 24
126
5.0
6.4
15.5
28
11
9
O,2-Propano-z8-THC
I
II
XIII
1-O-biphenylmethyI-8-THC
1-O-biphenylmethyl-19’1-THC
5’-Fr-THC
Nabilone
VIII
1 1-OH--THC
9-THC
1 1-OH-8-THC
5’-F--THC
>10,000
>10,000
1-O-Methy3-(norpentri)-
3-PropyI-9’1-THC
1-O-(4-Aminobutyl)--THC
9-Nor-9-carboxy-8-THC
390
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
(acid)
>10,000
1-O-(3-AminopropyI)-6-THC
9-Nor-9-Carboxy-9-THC
(acid)
O,10-Methano-9-THC
1-O-Carbamoyl-9-THC
Abnormal-CBD
(abn-CBD)
>10,000
>10,000
>10,000
>10,000
>10,000
panel of figure 5. The correlation
coefficient
was 0.81, which
was statistically
significant
at the P < .01 level (two-tailed).
Despite
the potential
for species differences,
and because
of the
similarity
between
the cloned
rat (Matsuda
et at., 1990) and
human
cannabinoid
receptors
(Gerard
et at., 1991), a correlation
100
80
analysis
60
was
data on the
dan, 1986).
11-OH-8-THC,
correlation
vs. human
be statistically
.40
.
20
performed
on the
analogs
in this
study
for
which
behavioral
potency
in humans
was available
(RazThese analogs
were l-nantradol,
DMHP,
nabilone,
11-OH-z9-THC,
8-THC
and 9-THC.
The
coefficient
of the regression
of the log values of KI
potency
data
(fig. 5) was 0.88,
which also proved
to
significant
at the P < .01 level (two-tailed).
0
-10
-8
-6
Discussion
-4
Log Drug Concentration
Fig. 3. Displacement
of bound [3H]CP-55,940
by various
cannabinoid
analogs.
discrimination
model
as well as in previously
reported
psychoactivity
values
in humans
A correlation
analysis
was performed
using analogs
for which drug discrimination
data in the
rat model was available
(Razdan,
1986; Balster
and Prescott,
1992).
These
analogs
were 1-nantradol,
z8-THC-DMH,
CP47497
THC
Ki
(or
and
vs. rat
VI),
nabilone,
9-THC.
drug
11-OH-8-THC,
The linear
discrimination
11-OH-9-THC,
regression
of the
potency
is shown
log values
of
in the upper
The results
presented
in this manuscript
behavioral
potency
of cannabinoids
in the
dicted
by establishing
the affinity
of the
receptor
labeled
by [3HJCP-55,940.
The
demonstrating
this correlation
of in vitro
in vivo agonist
potency
was linear regression
data, as previously
used for both dopamine
Seeman,
1980)
and
opiate
receptors
(Loh
clearly indicate
that
mouse
can be precannabinoid
for the
method
chosen
for
receptor
affinity
to
analysis
of log-log
(Seeman
et at., 1978;
et aL, 1978),
rather
than
via nongraphic
statistical
correlations
which
generally
only establish
simple
rank ordering
(Stahl
et aL, 1977;
Furchgott, 1978). Drugs with Ki values
in the 0.8 to 5.0 iM range
were either
inactive
at i.v. doses
up to 100 mg/kg,
showed
Downloaded from jpet.aspetjournals.org by on May 2, 2009
5’-OH-9-THC
2-I--THC
1 1-F-’-THC
93-OH-HHC
V
1-O-(4-PhthalimicIobutyI)--THC
35
150
19.9
22.3
28.5
38.4
40.7
54.9
57.0
VI
CP-56,667; (+)-AC, XV
968
1730
223
CannabinoidSAR
1993
3
*2
2
C
.!
i
I-
0
-1
-1
a
-2
-2
3
3
2
C
a
C#{176}
a
E
-1
-2
0
-1
3
0
1
3
2
4
Log K
2
from previously
other
behavioral
2
effects
of
cannabinoids
generated
tissue
are
0
1
2
3
4
Log
Fig. 4. Linear correlations
dependent
These
of log EDse values
(mol/kg)
2. The potency
for cannabinoids
of these analogs
data
and the log Kt values given in table
administered
i.v. in the mouse on four pharmacological
measures have
been reported previously (see under “Results”), and include reduction of
SA, antinociception
TF procedure, hypothermia as measured via AT and
immobility
(or catalepsy) in the RI measure. The dashed line represents
the linear regression that would be obtained if the mean EDse value of
the four measures
was plotted instead of that shown for each individual
behavior.
highly
effect,
not
necessarily
apply
here. For
receptor
to behavioral
example,
affinity
effects
it should
is predictive
not
of
are
model
that
neural
some
paths
been
with
such
dicative
ofthe
in man
pounds
vitro
potency
suggests
properties,
the
not
model
here,
receptor
evaluated
cortex).
affinity
for the
the whole spec-
effects
described
that
development
to design
only
one
action
here.
of a
has been
drugs
which
pharmacological
act
or antiemesis.
a drug
generally
been
been
activity
possesses
in humans.
has
which would
and Prescott,
cannabinoid
studies
that
correlated
of the
with
produce
fact
of psychoactivity
presented
note
behaviors
outside
of pharmacological
whether
has
anxiolytic,
highly
of pharmacological
model
discrimination
widely
has
cannabimi-
considered
to
However,
considered
be
the
in-
produce
cannabimimetic
1992).
Based
upon
this
study
affinity
actions
the comprovides
evidence
that
in
is predictive
of in vivo
in the rat drug discrimination
model.
that
those
properties
of cannabinoids
crimination
tor labeled
A strong
binding
it
predictive
(Balster
drug
or sedation
mouse
were
lying
It may be difficult
while
producing
to determine
of drugs
with
selectivity
as analgesia
the
used
metic
consistent
to attain.
receptor
Although
rat-drug
minimal
activity
or only showed
activity
in a limited
number
of evaluations.
Drugs with 1<1 values in the 1 to 100 pM range
proved
potent
in the production
of hypoactivity,
hypothermia,
antinociception
and catalepsy.
Similarly,
drugs with Ki values
in the 100 to 800 nM range also generally
proved
moderately
potent in the production
of all four mouse measures,
although
there were minor
exceptions.
Although
it would seem highly
probable
that these
four distinct
pharmacological
effects
are
mediated
by different
neuronal
pathways/mechanisms,
the potencies
of cannabinoids
across all four results were very similar.
This point
was demonstrated
by the fact that the mean
or
average
potency
of the cannabinoids
could be correlated
with
receptor
affinity
just as well as potency
in any individual
behavioral
evaluation.
However,
it must be cautioned
that this
not reported
that cannabinoid
upon
cannabinoid
difficult
via this
may
rat brain
it is likely
as
the fact that 1(1 values
were
tissue and by using only cortical
These
findings
suggest
that any
cannabinoid
receptor
will apparently
trum
of cannabinoid
pharmacological
-2
-1
by using
(although
such
actions
until appropriate
it is interesting
to
behavioral
results
in the mouse
with
receptor
affinity,
despite
0
of cannabinoids
be concluded
results (see under “Results”).
anticonvulsant
and antiemetic
have been conducted.
Lastly,
3
generalization
published
This
correlation
by which
dis-
is possible
are produced
via activation
of the recepby [3H]CP-55,940.
positive
correlation
was also obtained
between
affinity
and
psychoactivity
in humans.
This
suggests
that
in vitro cannabinoid
receptor
affinity
is predictive
of
psychoactivity
in humans.
Although
this conclusion
is based
upon
a limited
number
of compounds,
it seems
likely
that
potency
at the cannabinoid
receptor
in one species is indicative
of potency
in another
species,
and activation
of this receptor
is
responsible
for the production
of psychoactivity
in humans
as
well as the behavioral
effects
in rodents.
These
correlations
Downloaded from jpet.aspetjournals.org by on May 2, 2009
Fig. 5. Linear correlation of log KI values to the log EDse values (mol/
kg) for cannabinoids
in the rat drug discrimination procedure, and log Ki
values to the relative potencies of analogs to 9-THC. The scale chosen
is that used in figure 3. In vivo data for rats and humans were obtained
Ri
224
Comptonetal.
VoL 265
suggests
that either
rodent
model
may be used to determine
whether
novel cannabinoid
compounds
might possess
the psychoactive
properties
for which 9-THC
is abused
by humans.
This study represents
the first comprehensive
report
on the
SAR of cannabinoid
receptor
binding.
These
data support
the
SAR of cannabinoids
as described
by in vivo pharmacological
results.
Affinity
for the cannabinoid
receptor
is greatly
increased
by altering
the side chain.
The structures
of 8-THC
and 9-THC
include
a pentyl
side chain
at the C3 position.
However,
increasing
the side chain
to a seven-carbon
atom
length
and
branching
the
side
chain
by
adding
two
methyl
is shortened
to
a propyl
side
chain.
Thus,
these
data
specify
the requirements
for cannabinoid
side chain
length
and/or
branching
for receptor
affinity.
Affinity
for the cannabinoid
receptor
is increased
by halogenation
of the 5’ end of the side chain. Either
bromination
or
iodination
produced
analogs
in the 8-THC
series
with KI
values
of approximately
8 nM. Both
the 5’-fluoro
and 5’trifluoro
analogs
were somewhat
less potent.
The only other
drugs
in this study with substituents
at the 5’ position
were 5’OH-9-THC
and 11-OH-5’-TMA-z8-THC.
Although
the 5’OH analog
showed
good affinity,
the trimethylammonium
analog possessed
very weak affinity
for the cannabinoid
receptor
labeled
by [3HJCP-55,940.
The finding
that the trimethylammonium
analog
lacks strong affinity
for the CP-55,940
binding
site is consistent
with the binding
profile
and pharmacological
activity
of the parent
compound
5’-TMA-8-THC
(Nye et aL,
1985;
Compton
and Martin,
1990).
These binding
data also confirm
the enantiospecificity
anticipated based upon in vivo pharmacological
results.
The affinity
ratio between
1,d-nantradol
was greater
than
2900, and that
between
(+)and (-)-11-OH-8-THC-DMH
was greater
than
2700, although
that between
CP-55,940
and CP-56,667
was a
value of only 67. These
findings
are also consistent
with the
observations
that these weak/inactive
stereoisomers
have not
proven
to be antagonists
(Little
et aL, 1988).
The expected
profile
of a cannabinoid
antagonist
would be a
compound
with high receptor
affinity
and no in vivo agonistic
activity.
Such
a compound
would
be expected
to block
the
agonistic
actions
of a cannabinoid
by competitively
binding
to
the receptor.
Besides
the inactive
stereoisomers
just discussed,
several
other
compounds
have previously
been found
to be
these
in in vivo
inactive
analogs
pharmacological
have
been
evaluations.
evaluated
Almost
for antagonist
all of
prop-
erties
and, like the inactive
stereoisomers
mentioned
above,
have not been found to be capable
of blocking
or reducing
the
in vivo effects of 9-THC.
The primary
reason
now appears
to
be due to the fact that these
inactive
analogs
simply
do not
bind to the receptor,
as evidenced
by the fact that most do not
displace
the radioligand
even at 10-tiM concentrations.
However, some inactive
compounds
were found to possess
measurable binding
affinities,
but those drugs had affinities
in the 0.8
to 4.0 M range, and drugs with such weak affinity
would not
necessarily
be expected
to be effective
antagonists
at the doses
normally
used. Interestingly,
the carboxylic
acid metabolite
of
9-THC
has been reported
to antagonize
some effects
of THC (Burstein
et aL, 1987),
but it does not appear
to bind to
the cannabinoid
receptor.
Similarly,
the acid metabolite
of
THC
also does not bind to the cannabinoid
receptor,
which
presumably
would explain
its inability
to antagonize
the effects
of 9-THC
(unpublished
results).
The K,, value of 924 pM obtained
here was somewhat
larger
than
the 133 pM value
obtained
initially
by Devane
et at.
(1988), but very close to the 950 pM value
reported
subsequently
by the same laboratory
using a filtration
assay (Houston et at., 1991).
However,
the Bmax value obtained
in this
investigation
was larger than either the 1.85 or the 0.94 pmol/
mg of protein
values
reported
previously
(Devane
et at., 1988;
Houston
et aL, 1991). However,
use of traditional
Scatchard
methods
or saturation
analysis
of radioligand
binding
data
requires
(among
other
conditions)
that total binding
be less
than
10% of the total radioactivity
added,
otherwise
the approximation
that “free” radioligand
be set equal to “total radioligand
added”
is unacceptable.
This condition
can occur
if
there
is a very high concentration
of receptor
present
under
the assay conditions.
However,
if receptor
concentration
cannot
be assumed
to be negligible,
then classical
conditions
are violated and the traditional
approach
to determining
K,, and Bmaz
are inappropriate.
Specifically,
if the ratio Bmax/Kd
5
greater
than [1/10] then the assay shifts from “zone A” behavior,
where
Scatchard
analysis
is acceptable,
to “zone
B” behavior,
where
alternate
equations
describing
ligand
binding
must
be used
(Goldstein,
1949; Boeynaems
and Dumont,
1980). In this report
the Bmaz/Kd
ratio
equals
499/924
(or 0.54),
thus
corrected
equations
for saturation
and displacement
studies
should
have
been
used.
Interestingly,
although
the previously
described
binding
assay by Devane
et at. (1988)
exhibited
the identical
characteristic
(a Bm.x/Kd
ratio of 92/133
= 0.69),
the autoradiographic
study by Herkenham
et aL (1990) with a 15-nM
K,,
value did not (a Bmax/Kd
ratio of 82/15,000
=
0.006).
Additionally,
although
these binding
data were gathered
under zone
B conditions,
the curvilinear
characteristics
would
not have
been
readily
observed
with experimental
data since Bm.jKd
ratios
1.0
are only slightly
different
from idealized
curves
(Chang
et at., 1975). With use of the proper
binding
formulas,
the Scatchard
representation
of the data is slightly
convex
upward
(Boeynaems
and Dumont,
1980), but linear
graphical
representations
have been developed
(Riggs et at., 1970; Boeynaems
and
Dumont,
1980),
and
the
mathematical
relationship
between
the apparent
dissociation
constant
and receptor
concentration
described
(Chang
et al., 1975). Because
the deviation
from accepted
(or ideal) conditions
is minimal,
the Bm.,, value
can be set equal to that found by computer
analysis
(499 pM).
However,
the true value of the Kd must be calculated
as the
Downloaded from jpet.aspetjournals.org by on May 2, 2009
groups at the first carbon
of the backbone,
will produce
a DMH
derivative
with a greatly
increased
affinity.
In fact, ofthe nearly
60 compounds
evaluated,
there
are 12 compounds
with Ki
values
of less than
10 nM, and eight of these contain
a DMH
substituent.
Two of the other
drugs
also possess
side chains
that are branched,
long carbon backbones.
One is levonantradol
and the other (VII) possesses
a dimethyloctyl
side chain. Only
two of the 12 drugs
possess
the typical
pentyl
side chain.
Interestingly,
both of these cannabinoids
are halogenated
(Br
or I) at the 5’ (terminal)
end of the side chain.
It should
also
be pointed
out that some restrictions
do apply to the approach
of altering
the carbon
length
of the side chain
to increase
receptor
affinity.
Within
the series
of nonclassical
bicyclic
cannabinoids,
increasing
the dimethyl
side chain carbon
backbone
to 9, 10 or 1 1 carbons
as in analogs
VIII, IX and X,
respectively,
diminishes
receptor
affinity.
Similarly,
decreasing
the backbone
length to 6, 5, 4, 3 or 2 carbons,
or removal
of the
side chain as in analogs
V, IV, III, II, I and XIII, respectively,
also reduces
receptor
affinity.
Receptor
affinity
is reduced
from
236 nM for 9”-THC
to 968 nM when the pentyl
side chain
inactive
Cannabinoid
1993
correlation
between
the
ability
of a wide
variety
of cannabinoids
to bind to the receptor
labeled
by [3H]CP-55,940
and their
ability
to produce
in vivo pharmacological
effects.
These
correlations
suggest
a lack of species
differences
in terms
of
receptor
SAR, despite
the fact that the pharmacological
effect(s) measured
between
each species do not necessarily
appear
to be related
to one another.
Additionally,
these correlations
were established
using a set of cannabinoids
incorporating
a
wide degree of structural
diversity,
and this set includes
natural
cannabinoids,
lated)
cannabinoid
metabolites,
dimethylheptyl
(or re-
side
chain
analogs,
nonclassical
bicyclic
cannabinoids,
halogenated
analogs
and other synthetic
analogs
including
stereoisomers.
Thus,
in the process
of establishing
these correlations, data presented
here further
enhance
the body of knowledge concerning
the structural
requirements
for binding
to the
cannabinoid
receptor.
However,
evidence
is presented
suggest-
ing caution
be used in analyzing
radioligand
binding
data from
assays of tissue homogenates,
because
zone B binding
behavior
may be encountered.
This appears
to be due to the fact that
the density
of cannabinoid
receptors
is extremely
high, especially compared
to most other receptor
systems.
In fact, it has
been
pointed
out by others
that the density
of cannabinoid
receptors
is close to values
typically
observed
for receptor
systems
activated
by amino
acid neurotransmitters.
It is also
clear that the use of BSA in aqueous
media does not significantly
alter binding
of cannabinoids
to the receptor,
despite
that
fact that
cannabinoids
bind to serum
proteins,
which
actually
allows
presented
exists
the
here
drugs
to which
that
almost
ligands
may
actions
spectrum
(analgesia,
possess
of in
effects),
finding
vitro
Further
analysis
with structures
such
drugs
binding
Lastly,
at a single
even
and
of cannabinoid
different
from
or DMH
here
a novel
analog
cannabinoids
suggests
selective
the
specific
pharmacological
receptor
action
related
presented
search
may
prove
recog-
such as CBD may
by interacting
at this
or by another
receptor
presented
here which
In time, more selective
SAR
using
that of the bicyclic
55,940
may also prove useful,
as data presented
some
cannabinoids
may
bind to this receptor
different
from that of 9-THC.
Examples
of other
receptor
ligands
might be the aminoalkylindole
2(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](l-napthalenyl)methane
possibly
data
receptor
etc., rather
than the entire
or possess
antagonist
propwill require
continued
comin vivo behavioral
actions.
psychoactivity,
of cannabinoid
parison
bind
cannabinoids
actions
either
recognition
site,
No evidence
is
is likely to occur.
which
solution.
cannabinoid
some
emerge
However,
into
a single
all cannabinoids
nition
site.
However,
produce
pharmacological
receptor
at a different
mechanism
altogether.
would suggest
which
erties.
to be placed
suggests
to either
here.
However,
for a cannabinoid
to be very
ligands
nature
of CPhere indicates
in a fashion
cannabinoid
WIN-55,212or
the
halogenated
presented
data
antagonist
or one
of
difficult.
Acknowledgments
The authors
gratefully
thank
D. Troy Bridgen
and Xin Wei for technical
assistance.
The authors
are also grateful
to Drs. Richard
Rothman
and Allyn
Howlett
for helpful suggestions
regarding
the initial cannabinoid
binding
studies.
References
BALSTER,
in rats
R. L. AND PRESCOVF,
as a model
for cannabis
62, 1992.
M.
AND
F. Hucho,
pp. 151-161,
BINDER,
FRANKE,
W. R.: 9-Tetrahydrocannabinol
intoxication.
Neurosci.
Biobehav.
I.: Is there
Walter
a THC
de Gruyter
discrimination
Rev. 16: 55-
receptor?
In Neuroreceptors,
Press, Berlin,
1982.
ed.
by
J. M. AND DUMONT,
J. E.: Concepts
of zones
A, B and C. In
Outlines
of Receptor
Theory,
ed. by J. M. Boenaems
and J. E. Dumont,
pp.
17-23, Elsevier/North-Holland
Biomedical
Press, Amsterdam,
1980.
BRADFORD,
M. M.: A rapid and sensitive
method for the quantitation
of microgram quantities
of protein
utilizing
the principle
of protein-dye
binding.
Anal.
BOEYNAEMS,
Biochem.
BURSTEIN,
72:
S.,
248-254,
HUNTER,
1976.
S. A., LATHAM,
V. AND RENZULLI,
L.: A major metabolite
of .‘-tetrahythocannabinol
reduces
its cataleptic
effect in mice. Expenientia
43: 402-403,
1987.
CHANG,
K.-J., JACOBS, S. AND CUATRECASAS,
P.: Quantitative
aspects
of hormone-receptor
interactions
of high affinity.
Effect
of receptor
concentration
and measurement
of dissociation
constants
of labeled
and unlabeled
hormones.
Biochim.
Biophys.
Acta 406: 294-303, 1975.
CHARALAMBOUS,
A., LIN, S., MARCINIAK,
G., BANIJAMALI,
A. R., FRIEND,
F. L.,
COMPTON,
D. R., MARTIN,
B. R. AND MAKRIYANNIS,
A.: Pharmacological
evaluation
of halogenated
.8-THC
analogs.
Pharmacol.
Biochem.
Behav.
40:
509-512,
1991.
Y..C. AND PRUSOFF,
W. H.: Relationship
between
the inhibition
constant (K,) and the concentration
of inhibitor
which causes 50 percent inhibition
(I,) on an enzymatic
reaction.
Biochem.
Pharmacol.
22: 3099-3108,
1973.
COMPTON,
D. R., JOHNSON,
M. R., MELVIN,
L. S. AND MARTIN,
B. R.: PharCHENG,
macological
evaluation
ofa series
of bicyclic
cannabinoid
analogs:
Classification
as cannabimimetic
agents.
J. Pharmacol.
Exp. Ther. 260: 201-209, 1992.
COMPTON,
D. R., LITTLE,
P. J., MARTIN,
B. R., GILMAN,
J. W., SAHA, J. K.,
JORAPUR,
V. S., SARD, H. P. AND RAZDAN,
R. K.: Synthesis
and pharmacolog-
ical evaluation
of amino,
azido, and nitrogen
mustard
analogues
of lO-substituted cannabidiol
and 11- or 12-substituted
8-tetrahydrocannabinol.
J. Med.
Chem. 33: 1437-1443,
1990.
COMPTON,
D. R., LIrFLE,
P. J., MARTIN,
B. R., SAHA, J. K., GILMAN,
J., SARD,
H. AND RAZDAN, R. K.: Synthesis
and pharmacological
evaluation
of mercaptoand thioacetylanalogues
of cannabidiol
and 8-tetrahydrocannabinol.
Eur. J.
Med. Chem. 24: 293-298, 1989.
CoMrroN,
D. R. AND MARTIN,
B. R.: Pharmacological
evaluation
ofwater
soluble
cannabinoids
and related
analogs.
Life Sci. 46: 1575-1585,
1990.
DE LEAN, A., MUNSON,
P. J. AND RODBARD,
D.: Simultaneous
analysis of families
of siginoidal
curves:
Application
to bioassay,
radioligand
assay, and physiological dose-response
curves.
Am. J. Physiol.
235: E97-E102,
1987.
DEVANE,
W. A., DYSARZ, F. A., III, JOHNSON,
M. R., MELVIN,
L. S. AND
Downloaded from jpet.aspetjournals.org by on May 2, 2009
K,, (924 pM) minus
one-half
the Bmax (Boeynaems
and Dumont,
1980; Chang
et al., 1975),
which means
the true
K,, is equal to 675 pM. The Ki values
of displacing
drugs were
calculated
in table 2 using the Cheng-Prussoff
formula
(Cheng
and Prusoff,
1973) based
upon a K,, value
of 675 pM and a
corrected
free radioligand
concentration
of 280 pM. No further
corrections
to the Ki values
were necessary
because,
under
conditions
where
the Bmax/Kd
ratio <2.0, the exact displacement
equations
for zone B conditions
(Chang
et al., 1975) revert
to
more traditional
analysis
methods
for competitive
inhibitors.
The competitive
nature
of the binding
of these analogs
was
suggested
by statistical
analysis
(ALLFIT;
P < .05), which
indicated
that the displacement
curves
of almost
all analogs
were parallel
to that of 9-THC
(and CP-55,940).
This suggests
that receptor
recognition
occurs
in a similar
fashion
for most
of the cannabinoid
drugs,
and that
the actual
site on the
receptor
to which
drugs
bind is identical.
However,
not all
displacement
curves
could be shown
to be parallel
to that of
i9-THC.
Due to the limited
number
of data points
that could
be generated
for many analogs
with KI values
greater
than
1
M, the ALLFIT
program
was unable
to fit sigmoid
curves for
subsequent
comparisons.
Therefore,
the nature
of the binding
of these compounds
to the cannabinoid
receptor
is unknown.
This was not the situation
with all compounds
of weak potency.
Analysis
was possible
for the comparison
of 9-THC
with CBD
and all (+)-isomers.
Results
indicated
that displacement
curves
for CBD and XV (or CP-56,667
the isomer
of CP-55,940)
were
not parallel
to that of 9-THC.
This indicates
binding
of a
noncompetitive
nature,
and suggests
that the pharmacological
actions
of these two drugs may not be by mechanisms
identical
to that of 9-THC.
Interestingly,
displacement
curves
for (+)11-OH-8-THC-DMH
and dextronantradol
were parallel
to
that of 9-THC,
which suggests
binding
on the receptor
at the
same site as that for 9-THC.
In conclusion,
data presented
here establish
a high degree of
apparent
225
SAR
226
Compton
St al
Vol. 265
and characterization
34: 605-613, 1988.
HowLrrr,
A. C.: Determination
in rat brain. Mol. Phaninacol.
FURCHGOTT,
R.
radioligand-binding
Y.
GA0NI,
active
F.:
Pharmacological
studies.
AND MECHOULAM,
constituent
GARRETT,
E. R.
protein
binding
characterization
J. Am. Chem.
to
1978.
partial
1646-1647,
synthesis
1964.
and
86:
C. A.: Physicochemical
HUNT,
its relation
of receptors:
Soc.
of -tetrahydrocannabinol.
T. I.: Structure
receptor
cloned cDNA.
Fed. Proc. 37: 115-120,
R.: Isolation,
structure,
of hashish.
AND
ofa cannabinoid
of an
properties,
solubility, and
Sci. 63: 1056-1064,
J. Pharm.
1974.
C. M.,
GERARD,
C.,
MOLLEREAU,
cloning of a human
cannabinoid
Biochem. J. 279: 129-134, 1991.
GOLDSTEIN,
A.: The
interactions
G.
VASSART,
receptor
which
is also
in testis.
and
plasma
proteins.
Pharmacol.
Rev.
ofspecific
6:
21-27,
of cannabinoids
with bovine
AND MARTIN,
binding
sites.
Life
1991.
HowLrrr,
sical
CARCHMAN,
cannabinoid
A. C.,
cannabinoid
M. R.,
JOHNSON,
analgetics
inhibit
MELVIN,
adenylate
L. S. AND MILNE,
G. M.: Nonclascyclase:
development
of a can33: 297-302, 1988.
chemical
effects
of 1 1-OH-dimethylheptyl-8-tetrahydrocannabinol.
col. Biochem.
Behav.
32: 661-666,
1989.
H. H., Law,
involvement
P. Y.,
OSTWALD,
of cerebroside
T.,
sulfate
CHO,
in opiate
practical
experiments.
binding
MUNSON,
P. J. AND
for characterization
and
functional
expression
of the
1990.
computer-based
approach
to the analysis
of
Comput.
Programs
Biomed.
17: 107-114,
RODBARD,
D.: LIGAND:
A versatile
of ligand-binding
systems.
Anal.
computerized
approach
107:
Biochem.
220-239,
1980.
Nvz, J. S.,
SELTZMAN,
H. H., Prrr,
cannabinoid
binding
sites
1anmonium--tetrahydrocannabinol.J.
1985.
cannabinoid
SNOWMAN,
binding
in brain
C. G.
AND
S. S.: High-affinity
SNYDER,
membranes
labeled
with [3H)-5’-trimethyPharmacol.
Exp. Ther. 234: 784-791,
A. M.,
site:
VOGLMAIER,
S. AND SNYDER,
Regulation
by ions, ascorbic
acid,
S. H.: High-affinity
and
nucleotides.
T.
M.
Pharma-
P. H.,
B.
cannabinoid
A. D.,
SELTZMAN,
H. H.,
R.: An investigation
W. R. AND
hydroxyl
in
Mol.
H. AND BoURGEoIS,
S.: lac Repressor-operator
interaction.
I. Equilibrium
studies.
J. Mol. Biol. 48: 67-83,
1970.
ROSENTHAL,
H. E.: A graphic method for the determination
and presentation
of
binding
parameters
in a complex
system.
Anal. Biochem.
20: 525-532, 1967.
SCATCHARD,
G.: The attractions
of proteins
for small molecules
and ions. Ann.
RIGGS,
activity.
CoMrroN,
D. R., PREsco’rr,
of the role of the phenolic
PharmacoL
38: 854-862,
1990.
SUZUKI,
N.Y. Aced. Sci. 51: 1951.
SEEMAN,
P.: Brain dopamine
receptors.
Pharmacol.
SEEMAN,
P., TEDESCO,
J. L., LEE, T., CHAU-WONG,
J., WHITAKER,
P. M., MCMANUS,
C., TFrFLER,
W. C. AND BROWN, G. M.: Dopamine receptors
Rev.
32:
229-313,
1980.
M., MULLER,
P., BOWLES,
M., WEINREICH,
P., FRIEND,
in the central
nervous system.
37: 130-136, 1978.
K. D., VAN BEvER, W., JANSSEN,
P. AND SIMoN, E. J.: Receptor
affinity
and pharmacologic
potency
of a series of narcotic
analgesic,
anti-diarrheal
and
neuroleptic
drugs. Eur. J. Pharmacol.
46: 199-205, 1977.
Fed.
Proc.
STAHL,
AND
receptor
1991.
REGGIO,
MARTIN,
WAY,
binding.
E. L.:
Fed.
Possible
Proc.
37:
1978.
MARTIN,
B. R., COMPTON,
P. M.: Pharmacological
nabinoids.
MAT5UDA,
Natl.
L. A.,
Inst.
L0LAIT,
D. R.,
S. J.,
P. J., MARTIN, T. J. AND BEARDSLEY,
of agonist
and antagonist
activity
of canRes. Monogr.
Ser. 79: 108-122,
1987.
LITTLE,
evaluation
Drug Abuse
BROWNSTEIN,
J.
52: 1892-7, 1989.
RAZDAN,
R. K.: Structure-activity
relationships
in cannabinoids.
Pharmacol.
Rev. 38: 75-149, 1986.
REGGIO,
P. H., McGAUGHEY,
G. B., ODEAR, D. F., SELTZMAN,
H. H., C0MPT0N,
D. R. AND MARliN,
B. R.: A national search for the separation
of psychoactivity
and analgesia
in cannabinoids.
Pharmacol.
Biochem.
Behav.
40: 479-486,
M. J.,
YOUNG,
A. C.
AND BONNER,
Send
reprint
cology
and
University,
requests
Toxicology,
Richmond,
to: David R. Compton,
Medical
College
VA 23298-0613.
Ph.D., Department
of Virginia,
Virginia
of Phanma-
Commonwealth
Downloaded from jpet.aspetjournals.org by on May 2, 2009
nabinoid receptor model. Mol. Pharmacol.
P. J., C0MPT0N, D. R., JoHNsoN,
M. R., MELVIN, L. S. AND MARTIN,
B. R.: Pharmacology
and steneoselectivity
of structurally
novel cannabinoids
in mice. J. Pharmacol.
Exp. Ther. 247: 1046-1051, 1988.
LImE,
P. J., COMPTON,
D. R., MECHOULAM,
R. AND MARTIN, B. R.: StereoLITTLE,
LOH,
radioligand
1983.
receptor
561-564,
Neurochem.
B. R.: Evidence for the existence
Sci. 22: 1131-1138, 1978.
HERKENHAM,
M., LYNN, A. B., LITFLE,
M. D., JoHNsoN,
M. R., MELVIN, L. S.,
DECOSTA,
B. R. AND RICE, K. C.: Cannabinoid
receptor
localization
in the
brain. Proc. Natl. Acad. Sci. U.S.A. 87: 1932-1936,
1990.
HOUSTON,
D. B., EVANS, D. M., HowLrrr,
A. C. AND MELVIN, L. S.: [3HJCP55.940 binding
to the cannabinoid
receptor
in brain.
Biotech
Update
(Du Pont)
HARRIs,
R. A.
expressed
346:
G. A.: A
MCPHERSON,
Nvs, J. S.,
of drugs
1: 102-165, 1949.
HAQUE,
S. J. AND PODDAR, M. K.: Interactions
serum albumin. Biosci. Rep. 4: 239-43,
1984.
L. S.,
M.: Molecular
AND PARMENTIER,
of a cannabinoid
Nature