Note: Descriptions are shown in the official language in which they were submitted.
CA 02517313 2005-08-26
Methods and Therapies for Potentiating Therapeutic
Activities of a Cannabinoid Receptor Agonist via
Administration of a Cannabinoid Receptor Antagonist
Field of the Invention
A combination therapy is provided for potentiating a
therapeutic activity of a cannabinoid receptor agonist by
co-administration with a cannabinoid receptor antagonist at
a concentration effective to potentiate, but not antagonize,
the therapeutic activity of the cannabinoid receptor
agonist. The present invention thus relates to compositions
and methods for potentiating therapeutic activities of
cannabinoid receptor agonists, including but not limited to,
analgesia, inhibition of nausea or vomiting, treatment of
glaucoma, control of muscle spasticity in movement
disorders, inhibition of neurodegeneration, inhibition of
anxiety, treatment of hypertension, inhibition of
inflammation, treatment of Alzheimer's disease, treatment of
gastrointestinal disorders such as diarrhea, and preventing
or reducing atherosclerosis, and effectively inhibiting the
development of chronic as well as acute tolerance to the
therapeutic actions of the cannabinoid receptor agonists via
ultra low dose cannabinoid receptor antagonist therapy.
Methods for reversing cannabinoid receptor agonist tolerance
and/or restoring therapeutic potency of a cannabinoid
receptor agonist via administration of an ultra low dose of
a cannabinoid receptor antagonist to a subject receiving
cannabinoid receptor agonist therapy are also provided
Background of the Invention
Cannabinoids, like marijuana and similar synthetic
compounds, produce a range of behavioral effects (e. g.,
catalepsy, hypothermia, analgesia, disruption of psychomoter
behavior, short term memory impairment, intoxication,
stimulation of appetite, and anti-emetic effects), although
CA 02517313 2005-08-26
2
tolerance to these effects develops with repeated use
(Dewey, W.L., Pharmacol. Rev. 1986 38: 151-178; also see
Iversen L. Brain 2003 126:1252-1270).
Control of pain via administration of the endogenous
cannabinoids anadamide and palmitoylethanolamine is
described in U.S. Patents 6,348,498 and 6,656,972.
Cannabinoid receptor agonists are also used and/or are
being investigated for use in inhibition of nausea and/or
vomiting (e. g. Nabilone, Cambridge Laboratories); inhibition
of neurodegeneration (Shen, M. and Thayer, S.A. Molecular
Pharmacology 1998 54:459-462); inhibition of
anxiety (Kathuria, S, et al., Nature Medicine 2003 9:76-
81); treatment of gastrointestinal disorders such as
diarrhea (Izzo et al. Naunyn Schmiedebergs Arch Pharmacol
1999 359(1):65-70); treatment of acute inflammation related
to, for example, trauma, chronic inflammation related to
autoimmune diseases, such as Rheumatoid arthritis, Chrohn's
disease, ulcerative colitis (Federico M. et. al. Clin.
Invest. 2004 113:1202-1209) and pulmonary inflammation
(Berdyshev et al. Life Sci. 1998 63(8):PL125-9); glaucoma
(Jarvinen et al., Pharmacology & Therapeutics 2002 95 (2):
203-220); treatment of hypertension (U. S. Patent 6,903,137);
treatment of movement disorders/diseases such as Parkinson's
Disease; treatment of prevention of atherosclerosis
(Steffens et al. Nature 2005 434:782-786); and treatment of
Alzheimer's disease (Ramirez et al., The Journal of
Neuroscience. 2005 25(8) 1904-1913).
Pharmacologically, cannabinoids bind to G-protein
coupled cannabinoid CB1 receptors, located primarily in
neural membranes (Matsuda et al. Nature 1990 346:561-4) and
to CB2 receptors found primarily in cells of the immune
system, for example macrophages in the marginal zone of
spleen, and peripheral neurons (Munro et al. Nature 1993
CA 02517313 2005-08-26
3
365:61-5; Stander et al. J Dermatol Sci. 2005 38:177-88).
2-Arachidonylglycerol, an endogenous ligand for CB1 and CB2
receptors has been suggested to induce the migration of
several types of leukocytes such as macrophage/monocytes
through a CB2-receptor dependent mechanism thereby
stimulating inflammatory reactions and immune response
(Kishimoto et al. J. Biol. Chem. 2003 278(27):24469-24475).
CB1 receptor agonists produce dose-dependant analgesic
effects, and reduce the neurotransmission in pain related
pathways (Meng et al. Nature 1998 395:381-3). Furthermore,
activation of the CB1 receptor activates inhibitory Gi
proteins which then down-regulate adenylyl cyclase
production of CAMP (Howlett, A.C., Mol. Pharmacol. 1985
27:429-36; Howlett, A.C. and Fleming, R.M. MoI. Pharmacol.
1984 26:532-8; Howlett et al. Mol. Pharmacol. 1986 29:307-
13). Previous studies suggested that Gi protein activation
by CB1 receptors is necessary for cannabinoid-induced
analgesia (Raffa et al. Neurosci Lett 1999 263:29-32).
More recent evidence, however, is indicative of the
behavioral pharmacology of cannabinoids being more complex.
For example, cannabinoid CB1 receptor agonists have been
reported to have dose dependent biphasic effects on
behavior. In particular, low doses of the endocannabinoid
anandamide (10 ~g/kg) were demonstrated to produce increased
locomotion, rearing, defecation, and nociception, and
decreased catalepsy: effects that are opposite to higher
doses (10 mg/kg) (Sulcova et al. Pharmacol. Biochem. Behav.
1998 59:347-52). Furthermore, the CB1 receptor has been
suggested to couple to both the inhibitory Gi-protein, and
the stimulatory Gs-protein(Glass, M. and Felder, C.C., J
Neurosci 1997 17:5327-33; Calandra et al. Eur J Pharmacol.
1999 374:445-55). The ability of the CB1 receptor population
CA 02517313 2005-08-26
4
to couple to both stimulatory and inhibitory G-proteins may
explain cannabinoid-induced biphasic effects on behavior.
Cannabinoid CB1 receptor antagonists are being
developed as a new class of therapeutic agents for drug
addiction (see Le Foll, B, and Goldberg, S.R., J. Pharmacol.
and Exp. Therapeutics 2005 312 (3) :875-883 for review) .
Further, it has been suggested that cannabinoid receptor
antagonists may alter opioid peptide release, thus
facilitating a reduction in alcohol consumption (Manzanares
et al. Alcohol and Alcoholism 2005 40(1):25-34). A synergy
between low dose treatment with opioids and cannabinoid
receptor antagonists at reducing the motivation to consume
alcohol in rats has been disclosed (Gallate et al.
Psychopharmacology 2004 173:210-216).
The opioid and cannabinoid systems have also been
disclosed to be involved in the control of appetite with the
cannabinoid receptor antagonist SR 141716 attenuating
overfeeding induced by morphine administered systemically or
intracranially into the paraventricular nuclear of the
hypothalamus but not food intake induced by administration
of morphine intracranially into the nucleus accumbens (Verty
et al. Psychopharmacology 2003 168:314-323). Peripheral but
not central administration of the cannabinoid receptor
agonist WIN55,212-2 was reported to promote hyperphagia in
partially satiated rats while peripheral, but not central
administration of SR 141716 reduced food intake in rats
(Gomez et al. J. Neurosci. 2002 22(21):9612-9617). The
selective CB1 cannabinoid receptor antagonist SR 141716
(also referred to as ACOMPLIA or RIMONABANT) is currently
under development by Sanofi-Aventis for treatment of obesity
(see drugdevelopment-technology with the extension
.com/projects/rimonabant/ or sanofi-synthelabo with the
extension .us/live/us/en, both of the world wide web).
CA 02517313 2005-08-26
U.S. Patent 6,825,209 discloses amide analogs of SR
141716 with increased CB1 receptor selectivity which are
suggested to be useful in treatment of CB1 receptor related
disorders such as obesity, schizophrenia, memory dysfunction
5 and marijuana abuse.
U.S. Patent 5,547,524 discloses aryl-benzo[b]thiophene
and benzo[b]furan compounds which are antagonists of the CB-
1 receptor in the mammalian central nervous system. These
compounds are suggested to be useful in treating a variety
of disorders associated with cannabinoid stimulation
including depression, cognitive dysfunction, loss of memory
and poor alertness and sensory perception.
Published U.S. Patent Application US2004/0209861
discloses the combination of a CB1 receptor antagonist and a
compound which activates dopaminergic neurotransmission in
the brain in the treatment of Parkinson~s disease.
Published U.S. Patent Application US2005/0014786
discloses tetrahydroquinoline containing compounds which are
cannabinoid-1 receptor modulators and include selective
agonists, partial agonists, inverse agonists, antagonists or
partial antagonists of the cannabinoid receptor. Preferred
are compounds possessing activity as antagonists or inverse
agonists of the CB-1 receptor taught to be useful in
metabolic disorders or psychiatric disorders.
Summary of the Invention
An object of the present invention is to provide a
composition comprising a cannabinoid receptor agonist, at a
concentration effective to produce a desired therapeutic
effect, and a cannabinoid receptor antagonist, at a
concentration effective to potentiate, but not antagonize,
the therapeutic effect of the cannabinoid receptor agonist.
CA 02517313 2005-08-26
6
These compositions provide useful therapeutic agents for
pain, nausea or vomiting, glaucoma, a movement disorder,
neurodegeneration, anxiety, acute inflammation, chronic
inflammation, pulmonary inflammation, Alzheimer~s disease,
gastrointestinal disorders such as diarrhea, hypertension
and atherosclerosis.
Another object of the present invention is to provide a
method for potentiating a therapeutic effect of a
cannabinoid receptor agonist in a subject which comprises
administering to the subject, in combination with a
cannabinoid receptor agonist, a cannabinoid receptor
antagonist at a concentration effective to potentiate, but
not antagonize the therapeutic effect of the cannabinoid
receptor agonist.
Another object of the present invention is to provide a
method for inhibiting development of acute tolerance to a
therapeutic action of a cannabinoid receptor agonist in a
subject which comprises administering to the subject, in
combination with a cannabinoid receptor agonist, a
cannabinoid receptor antagonist at a concentration effective
to potentiate, but not antagonize, the therapeutic effect of
the cannabinoid receptor agonist.
Another object of the present invention is to provide a
method for inhibiting development of chronic tolerance to a
therapeutic action of a cannabinoid receptor agonist in a
subject which comprises administering to the subject, in
combination with a cannabinoid receptor agonist, a
cannabinoid receptor antagonist at a concentration effective
to potentiate, but not antagonize, the therapeutic effect of
the cannabinoid receptor agonist.
Another object of the present invention is to provide a
method for reversing tolerance to a therapeutic action of a
cannabinoid receptor agonist and/or restoring therapeutic
CA 02517313 2005-08-26
7
potency of a cannabinoid receptor agonist in a subject which
comprises administering a cannabinoid receptor antagonist to
a subject receiving a cannabinoid receptor agonist, said
cannabinoid receptor antagonist being administered at a
concentration effective to potentiate, but not antagonize
the therapeutic effect of the cannabinoid receptor agonist.
Another object of the present invention is to provide a
method for treating a subject suffering from a condition
treatable with a cannabinoid receptor agonist comprising
administering to the subject a cannabinoid receptor agonist
at a concentration effective to produce a therapeutic effect
and a cannabinoid receptor antagonist at a concentration
effective to potentiate, but not antagonize, the therapeutic
effect of the cannabinoid receptor agonist. This method is
useful in treating subjects suffering from conditions
including, but not limited to, pain, nausea or vomiting,
glaucoma, a movement disorder, neurodegeneration, anxiety,
acute inflammation, chronic inflammation, pulmonary
inflammation, Alzheimer's disease, gastrointestinal
disorders such as diarrhea, hypertension and
atherosclerosis.
Brief Description of the Figures
Figure lA and 1B are line graphs illustrating the
antinociceptive properties of the cannabinoid receptor
agonist WIN 55 212-2 (WIN) in the rat tail flick test being
enhanced by ultra-low doses of the cannabinoid CB1 receptor
antagonist SR 141716 (SR) following a single injection of
this combination therapy. Group mean (+/- SEM)
antinociception is represented as percent mean possible
effect (MPE) using the tail-flick test. In Figure lA the
cannabinoid receptor agonist WIN was administered at 0.0625
mg/kg and the cannabinoid CB1 receptor antagonist SR was
CA 02517313 2005-08-26
8
administered at the ultra low dose of 0.55 ng/kg and 0.055
ng/kg. In Figure 1B, the cannabinoid receptor agonist WIN
was administered at 0.09375 mg/kg and the cannabinoid CB1
receptor antagonist SR was administered at the ultra low
dose of 0.83 ng/kg and 0.083 ng/kg. Vehicle data are re-
plotted from Figure lA in Figure 1B. Animals receiving
vehicle alone are depicted by filled circles. Animals
receiving WIN alone are depicted by open circles. Animals
receiving WIN plus SR at 0.55 ng/kg (Figure lA) or 0.83
ng/kg (Figure 1B) are depicted by filled triangles. Animals
receiving WIN plus SR at 0.055 ng/kg (Figure IA) or 0.083
ng/kg (Figure 1B) are depicted by open triangles. Animals
receiving SR alone at 0.55 ng/kg (Figure lA) or 0.83 ng/mg
(Figure 1B) are depicted by filled squares. Each group had
8 animals. All injections were administered intravenously.
The symbol ~~°~~ is representative of a significant difference
from vehicle. The symbol ~~*~~ is representative of a
significant difference from WIN 0.0625 mg/kg. The symbol ~~
is representative of a significant difference from WIN
0.09375 mg/kg
Figure 2 is a line graph illustrating that daily
administrations of ultra-low doses of the cannabinoid CB1
receptor antagonist SR 141716 (SR) with the cannabinoid
receptor agonist WIN 55 212-2 (WIN) prevented the
development of tolerance to an analgesic action of the
cannabinoid receptor agonist in the rat tail-flick test.
Group mean (+/- SEM) antinociception is represented as
percent mean possible effect (MPE). Each group had 8
animals. All injections were administered intravenously.
Animals receiving vehicle alone are depicted by filled
circles. Animals receiving WIN at 0.125 mg/kg alone are
depicted by open circles. Animals receiving WIN plus SR at
1.1 ng/kg are depicted by filled triangles. Animals
CA 02517313 2005-08-26
9
receiving WIN plus SR at 0.11 ng/kg are depicted by open
triangles. Animals receiving SR alone at 1.1 ng/kg are
depicted by filled squares. The symbol "°" is representative
of a significant difference from vehicle. The symbol "*" is
representative of a significant difference from WIN 0.125
mg/kg.
Detailed Description of the Invention
It has now been found that administration of ultra low
doses of a cannabinoid receptor antagonist potentiates
regional cannabinoid receptor agonist analgesia and inhibits
the development of tolerance to cannabinoid receptor
agonists. It is expected that administration of an ultra
low dose of cannabinoid receptor antagonist will also
reverse tolerance to a cannabinoid receptor agonist in a
subject tolerant to cannabinoid receptor agonist therapy
and/or restore potency of a cannabinoid receptor agonist in
such subjects. The present invention provides new
combination therapies for potentiating a therapeutic
activity of a cannabinoid receptor agonist and inhibiting
development of chronic and/or acute tolerance to a
cannabinoid receptor agonist involving co-administration of
a cannabinoid receptor agonist with a cannabinoid receptor
antagonist. An aspect of the present invention thus relates
to compositions comprising a cannabinoid receptor agonist
and an ultra low dose of a cannabinoid receptor antagonist.
Another aspect of the present invention relates to methods
for potentiating a therapeutic action of a cannabinoid
receptor agonist and/or effectively inhibiting the
development of acute as well as chronic tolerance to a
therapeutic action of a cannabinoid receptor agonist by co-
administering the cannabinoid receptor agonist with an ultra
low dose of a cannabinoid receptor antagonist. Another
CA 02517313 2005-08-26
aspect of the present invention relates to methods for
reversing tolerance to an therapeutic action of a
cannabinoid receptor agonist and/or restoring therapeutic
potency of a cannabinoid receptor agonist by administering
5 an ultra low dose of a cannabinoid receptor antagonist to a
subject already receiving a cannabinoid receptor agonist.
The new combination therapies of the present invention are
expected to be useful in optimizing the use of cannabinoid
drugs in various applications including but not limited to:
10 pain management, e.g. management of acute post-surgical
pain, obstetrical pain, acute and chronic inflammatory pain,
pain associated with conditions such as multiple sclerosis
and cancer, pain associated with trauma, pain associated
with migraines, neuropathic pain, central pain and
management of chronic pain syndrome of a non-malignant
origin such as chronic back pain; inhibition of nausea
and/or vomiting; glaucoma; and inhibiting spasticity and
controlling movement in movement disorders such as
Parkinson's disease.
Cannabinoid receptor antagonist useful in the
combination therapies and methods of the present invention
include, but are in no way limited to, antagonists of
cannabinoid 1 (CB1) receptors, antagonists of cannabinoid 2
(CB2)receptors, and antagonists of CB1 and CB2 receptors.
In a preferred embodiment, the cannabinoid receptor
antagonist is a CB1 receptor antagonists. Examples of
cannabinoid receptor antagonists useful in the present
invention include, but are in no way limited to, SR 141716,
AM-251 (Torvis Cookson, Bristol, UK), AM281 (Torvis Cookson,
Bristol, UK) LY320135 (Eli Lilly, Inc. Indiana), and SR
144528 (Rinaldi-Carmona et al. J Pharmacol Exp Ther 1998
284:644-650). Exemplary cannabinoid receptor antagonists
useful in the present invention are also set forth in U.S.
CA 02517313 2005-08-26
11
Patents 6,825,209,5,547,524, and 6,916,838 and published
U.S. Patent Application 2005/0014786.
The cannabinoid receptor antagonist is included in the
compositions and administered in the methods of the present
invention at an ultra low dose. By ultra low dose, as used
herein, it is meant a concentration of cannabinoid receptor
antagonist that potentiates, but does not antagonize, a
therapeutic effect of a cannabinoid receptor agonist. Thus,
in one embodiment, by the term "ultra low dose" it is meant
a concentration of the cannabinoid receptor antagonist lower
than that established by those skill in the art to
significantly block or inhibit cannabinoid receptor
activity. For example, in one embodiment, by ultra low dose
of cannabinoid receptor antagonist it is meant a
concentration ineffective at G;~o-coupled CB1 receptor
blockade. For example, ultra low doses of SR 141716
demonstrated herein to potentiate the analgesic activity of
the cannabinoid receptor agonist WIN 55 212-2 are
approximately 300,000-fold lower than an optimal dose of SR
141716 producing a blockade of the Giro-coupled CB1
receptors. Ultra low doses useful in the present invention
for other cannabinoid CB1 receptor antagonists can be
determined routinely by those skilled in the art in
accordance with their known effective concentrations as Gi~o-
coupled CB1 receptor blockers and the methodologies
described herein for SR 141716. In general, in this
embodiment, however, by "ultra low" it is meant a dose of
cannabinoid receptor antagonist of at least 1,000- to
100,000,000-fold lower than a dose of cannabinoid receptor
antagonist producing a blockade of Giro-coupled CB1
receptors. As will be understood by the skilled artisan
upon reading this disclosure, however, other means for
measuring cannabinoid receptor antagonism can be used.
CA 02517313 2005-08-26
12
In an alternative embodiment, by "ultra low dose" it is
meant a concentration of cannabinoid receptor antagonist
which is significantly less than the concentration of
cannabinoid receptor agonist to be administered. Thus, in
this embodiment, the ultra low dose of cannabinoid receptor
antagonist is expressed as a ratio with respect to the dose
of cannabinoid receptor agonist administered or to be
administered. In this embodiment a preferred ratio for an
ultra low dose is a ratio of 1:10,000, 1:100,000 or
1:1,000,000 or any raion in between of cannabinoid receptor
antagonist to cannabinoid receptor agonist.
Examples of cannabinoid receptor agonists useful in the
combination therapies and methods of the present invention
include, but are in no way limited to endocannabinoid, (-)-
trans-delta-9-tetrahydrocannabinol (delta-9-THC), CP-55,940,
arachidonylethanolamide (anandamide), WIN-55,212-2, HU-210,
HU-243, arachidonyl-2-chloroethylamide,
arachidonylcyclopropylamide, O-1812, 2-arachidonoyl
glycerol, dronabinol (marinol), sativex, cannabidiol,
cannabinol, cannabichromene, cannabigerol and
phytocannabinoids. The concentration of cannabinoid
receptor agonist included in the compositions of the present
invention and used in the methodologies described herein is
a concentration effective to produce a therapeutic effect.
Such concentrations are well known to the skilled artisan
and can typically range between 0.02 mg to 100 mg depending
upon the cannabinoid receptor agonist selected, the route of
administration, the frequency of the administration, and the
condition being treated. As demonstrated herein, however,
co-administration of a cannabinoid receptor agonist with an
ultra low dose of a cannabinoid receptor antagonist
potentiates the therapeutic effect of analgesia of the
cannabinoid receptor agonist.
CA 02517313 2005-08-26
13
For purposes of the present invention, by "therapeutic
effect" or "therapeutic activity" or "therapeutic action" it
is meant a desired pharmacological activity of a cannabinoid
receptor agonist useful in the inhibition, prevention or
treatment of pain, nausea or vomiting, glaucoma, a movement
disorder, neurodegeneration, anxiety, acute inflammation,
chronic inflammation, pulmonary inflammation, Alzheimer's
disease, gastrointestinal disorders such as diarrhea,
hypertension or atherosclerosis.
For purposes of the present invention, by potentiate,
it is meant that administration of the cannabinoid receptor
antagonist enhances, extends or increases the therapeutic
activity of the cannabinoid receptor agonist and/or results
in a decreased amount of cannabinoid receptor agonist being
required to produce the therapeutic effect. Thus, as will
be understood by the skilled artisan upon reading this
disclosure, the effective concentrations of cannabinoid
receptor agonist included in the combination therapies of
the present invention may be decreased as compared to an
established effective concentration for the cannabinoid
receptor agonist when administered alone. The amount of the
decrease for other cannabinoid receptor agonists can be
determined routinely by the skilled artisan based upon
ratios described herein for WIN 55 212-2 and SR 141716.
This decrease in required cannabinoid receptor agonist
to achieve the same therapeutic benefit will decrease any
unwanted side effects associated with cannabinoid receptor
agonist therapy. Thus, the combination therapies of the
present invention also provide a means for decreasing
unwanted side effects of cannabinoid receptor agonist
therapy.
By "antagonize" as used herein, it is meant an
inhibition or decrease in therapeutic effect or action of a
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14
cannabinoid receptor agonist resulting from addition of a
cannabinoid receptor antagonist which renders the
cannabinoid receptor agonist ineffective therapeutically for
the condition being treated.
S By "tolerance" as used herein, it is meant a loss of
drug potency and is produced by most, if not all currently
used cannabinoid receptor agonists. Chronic or acute
tolerance can be a limiting factor in the clinical
management of cannabinoid receptor agonist drugs as
cannabinoid receptor agonist potency is decreased upon
exposure to the cannabinoid receptor agonist. By "chronic
tolerance" it meant a decrease in potency which can develop
after drug exposure over several or more days. However, loss
of cannabinoid receptor agonist drug potency may also be
seen in pain conditions such as neuropathic pain without
prior cannabinoid receptor agonist drug exposure as
neurobiological mechanisms underlying the genesis of
tolerance and neuropathic pain are similar (Mao et al. Pain
1995 61:353-364) This type of tolerance is referred to
herein as "acute tolerance".
Tolerance has been explained in terms of drug receptor
desensitization or internalization. It has also been
explained on the basis of an adaptive increase in levels of
pain transmitters such as glutamic substance P or CGRP, and
in the case of the opioid system a switch in opioid receptor
coupling from Gai/o to Gas associated G proteins.
Inhibition of tolerance and maintenance of cannabinoid
receptor agonist potency are important therapeutic goals in
cannabinoid receptor agonist therapies which, as
demonstrated herein, are achieved via the combination
therapies of the present invention.
The ability of the combination therapies of the present
invention to potentiate the therapeutic action of analgesia
CA 02517313 2005-08-26
of a cannabinoid receptor agonist and/or inhibit chronic
cannabinoid receptor agonist tolerance upon co-
administration of an ultra low dose of a cannabinoid
receptor antagonist was demonstrated in tests of thermal
5 (rat tail flick) antinociception. In these experiments, the
cannabinoid CB1 receptor antagonist used was SR 141716
(referred to herein as SR). The cannabinoid receptor
agonist was WIN 55 212-2 (referred to herein as WIN). As
will be understood by the skilled artisan upon reading this
10 disclosure, however, the combination of cannabinoid receptor
agonist and cannabinoid receptor antagonist selected for
these experiments as well as the therapeutic action measured
are merely exemplary and are in no way limiting to the scope
of this invention.
15 Figures lA and 1B show the tail-flick data from animals
given single injections of the cannabinoid receptor agonist
WIN alone, the cannabinoid receptor antagonist SR alone, or
a combination of the cannabinoid receptor agonist WIN and
the cannabinoid receptor antagonist SR. In Figure lA
animals administered a cannabinoid receptor agonist received
0.625 mg/kg WIN. Animals receiving a cannabinoid CB1
receptor antagonist were administered either 0.55 ng/kg or
0.055 ng/kg SR. In Figure 1B animals administered a
cannabinoid receptor agonist received 0.09375 mg/kg WIN.
Animals receiving a cannabinoid receptor antagonist were
administered either 0.83 ng/kg or 0.083 ng/kg SR.
Nociceptive testing was performed every 10 minutes following
administration of the therapeutic compounds over a 90 minute
period. When all drug groups were combined, a main effect
of time [F(3.51,221.11)=66.64, P<0.001] was revealed,
whereby tail-flick latency mean possible effects (MPEs)
decreased across time. Furthermore, there was a main drug
CA 02517313 2005-08-26
16
effect [F(8,63)=16.00, P<0.001], and a drug X post-injection
time interaction, [F(28.08,221.11)=7.62, P<0.001].
When comparing drug groups, there was no difference
between vehicle and 0.0625 mg/kg WIN-treated animals
[t(14)=2.63], but tail flick latencies of vehicle-treated
animals were significantly different from those of animals
injected with 0.09375 WIN [t(14)=4.629, Ps0.005]. The tail-
flick latency MPEs were also compared between WIN alone
groups and the combined WIN and ultra-low dose SR groups.
There was no difference between the 0.0625 mg/kg WIN alone
group and the same dose of WIN mixed with the 0.055 ng/kg SR
[t(14)=3.28], but a slightly higher ultra-low dose of SR
(0.55ng/kg) in combination with 0.0625 mg/kg WIN showed
longer tail-flick latencies compared to the same dose of WIN
alone [t(14)=6.72, Ps0.005]. The combination treatment of
0.09375 mg/kg WIN with both the 0.83 and 0.083 ng/kg ultra-
low dose SR produced longer tail-flick latencies compared to
the same doses of WIN alone [t(14)=9.63, Ps0.005, and
t(14)=8.16, Ps0.005 respectively]. Thus, as demonstrated by
these experiments, ultra-low doses of a cannabinoid receptor
antagonist increase cannabinoid receptor agonist-induced
tail-flick thresholds.
Figure 2 shows the tail-flick data from animals given
repeated daily injections of a cannabinoid receptor agonist
and/or a cannabinoid receptor antagonist for 7 days, and
tested every other day for nociceptive responses. In this
experiment, animals administered the cannabinoid receptor
agonist received 0.125 mg/kg WIN. Animals administered the
cannabinoid receptor antagonist received either 1.1 ng/kg or
0.11 ng/kg SR. A main effect observed in this experiment
was a change in tail-flick latencies across test days
[F(2.98,104.30)=59.36, P<0.001]. Furthermore, there was a
CA 02517313 2005-08-26
17
main effect of drug [F(4,35)=73.05, P<0.001], and a day x
drug group interaction [F(11.92,104.30)=13.92, P<0.001].
When comparing drug groups, 0.125 mg/kg WIN-treated
animals were significantly different from vehicle treated
animals [t(14)=10.44, P<_0.01] whereby the WIN group had
greater tail-flick latencies. Most importantly, animals
injected with 0.125 mg/kg WIN in combination with ultra-low
dose SR at 1.1 and 0.11 ng/kg showed greater tail flick
latency thresholds compared to WIN alone (t(14)=9.77,
P<_0.01; and t(14)=14.35, P<_0.01; respectively]. Thus, as
demonstrated by these experiments, ultra-low doses of a
cannabinoid antagonist blocked the development of tolerance
to the antinociceptive effect of a cannabinoid receptor
agonist.
On the day following the last injection, animals were
sacrificed and tissue samples of the brain and lumbar spinal
cord were collected. Co-immunoprecipitation experiments
were performed on the tissues to determine the G-protein
sub-type coupling profile of activated CB1 receptors.
Results for striatal tissue indicated that WIN-induced
tolerance is associated with a switch in the CB1 receptor G-
protein coupling from the Gai type to the Gas type.
Furthermore, ultra-low dose SR 141716 prevented this
coupling switch. Thus, while not wishing to be bound to any
particular theory, this prevention of the coupling switch by
an ultra-low dose SR 141716 may be responsible for
preventing WIN 55,212-2 induced antinociception.
Accordingly, as shown herein, ultra-low doses of a
cannabinoid receptor antagonist enhanced antinociception
induced by a cannabinoid receptor agonist and increased the
duration of the antinociceptive effect of the cannabinoid
receptor agonist. Furthermore, cannabinoid receptor
agonist-induced tolerance was prevented when an ultra-low
CA 02517313 2005-08-26
18
dose of a cannabinoid antagonist was co-administered.
Although both the SR to WIN dose ratios of 1:100,000 and
1:1,000,000 were effective, the 1:1,000,000 dose ratio
appears slightly (although not significant) better at
preventing WIN-induced tolerance. Thus, as shown herein, a
behavioral effect of a cannabinoid receptor agonist is
enhanced by a cannabinoid receptor antagonist. Further, it
is believed that ultra low doses of a cannabinoid receptor
antagonist will prevent development of acute tolerance to
cannabinoid receptor agonists as well as reverse tolerance
and/or restore the potency of cannabinoid receptor agonists
in animals already tolerant to the analgesic action of the
cannabinoid receptor agonist.
In addition to analgesia, based upon these experiments,
it is expected that the combination therapies of the present
invention will by useful in potentiating other therapeutic
activities of cannabinoid receptor agonists, including but
not limited to, inhibition of nausea or vomiting,
alleviation of symptoms an/or treatment of glaucoma, and
control of muscle spasticity in movement disorders.
As demonstrated herein, cannabinoid receptor agonists
and cannabinoid receptor antagonists can be administered
intravenously. Further, it is expected that these
therapeutic compounds will be effective following other
modes of systemic as well as local administration.
Accordingly, the combination therapies of the invention may
be administered systemically or locally, and by any suitable
route such as oral, transdermal, inhalation, subcutaneous,
intraocular, intravenous, intramuscular, intrathecally,
epidurally or intraperitoneal administration, and the like
(e. g., by injection). Preferably, the cannabinoid receptor
agonist and cannabinoid receptor antagonist are administered
simultaneously via the same route of administration.
CA 02517313 2005-08-26
19
However, it is expected that administration of the compounds
separately, via the same route or different route of
administration, within a time frame during which each
therapeutic compound remains active, will also be effective
therapeutically as well as in alleviating tolerance to the
cannabinoid receptor agonist. Further, it is expected that
administration of a cannabinoid receptor antagonist to a
subject already receiving cannabinoid receptor agonist
treatment will reverse any tolerance to the cannabinoid
receptor agonist and restore therapeutic potency of the
cannabinoid receptor agonist. Thus, treatment with the
cannabinoid receptor agonist and cannabinoid receptor
antagonist in the combination therapy of the present
invention need not begin at the same time. Instead,
administration of the cannabinoid receptor antagonist may
begin several days, weeks, months or more after treatment
with the cannabinoid receptor agonist.
Accordingly, for purposes of the present invention, the
therapeutic compounds, namely the cannabinoid receptor
agonist and the cannabinoid receptor antagonist, can be
administered together in a single pharmaceutically
acceptable vehicle or separately, each in their own
pharmaceutically acceptable vehicle.
As used herein, by the term "therapeutic compound", it
is meant to refer to a cannabinoid receptor agonist and/or a
cannabinoid receptor antagonist.
As used herein "pharmaceutically acceptable vehicle"
includes any and all solvents, excipients, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like which are
compatible with the activity of the therapeutic compound and
are physiologically acceptable to a subject. An example of
the pharmaceutically acceptable vehicle is buffered normal
CA 02517313 2005-08-26
saline (0.15 M NaCl). The use of such media and agents for
pharmaceutically active substances is well known in the art.
Except insofar as any conventional media or agent is
incompatible with the therapeutic compound, use thereof in
5 the compositions suitable for pharmaceutical administration
is contemplated. Supplementary active compounds can also be
incorporated into the compositions.
Carrier or substituent moieties useful in the present
invention may also include moieties which allow the
10 therapeutic compound to be selectively delivered to a target
organ. For example, delivery of the therapeutic compound to
the brain may be enhanced by a carrier moiety using either
active or passive transport (a "targeting moiety").
Illustratively, the carrier molecule may be a redox moiety,
15 as described in, for example, U.S. Patents 4,540,654 and
5,389,623, both to Bodor. These patents disclose drugs
linked to dihydropyridine moieties which can enter the
brain, where they are oxidized to a charged pyridinium
species which is trapped in the brain. Thus drugs linked to
20 these moieties accumulate in the brain. Other carrier
moieties include compounds, such as amino acids or
thyroxine, which can be passively or actively transported in
vivo. Such a carrier moiety can be metabolically removed in
vivo, or can remain intact as part of an active compound.
Structural mimics of amino acids (and other actively
transported moieties) including peptidomimetics, are also
useful in the invention. As used herein, the term
"peptidomimetic" is intended to include peptide analogues
which serve as appropriate substitutes for peptides in
interactions with, for example, receptors and enzymes. The
peptidomimetic must possess not only affinity, but also
efficacy and substrate function. That is, a peptidomimetic
exhibits functions of a peptide, without restriction of
CA 02517313 2005-08-26
21
structure to amino acid constituents. Peptidomimetics,
methods for their preparation and use are described in
Morgan et al. (1989), the contents of which are incorporated
herein by reference. Many targeting moieties are known, and
include, for example, asialoglycoproteins (see e.g., Wu,
U.S. Patent 5,166,320) and other ligands which are
transported into cells via receptor-mediated endocytosis
(see below for further examples of targeting moieties which
may be covalently or non-covalently bound to a target
molecule).
The term "subject" as used herein is intended to
include living organisms in which treatment with cannabinoid
receptor agonists can occur. Examples of subjects include
mammals such as humans, apes, monkeys, cows, sheep, goats,
dogs, cats, mice, rats, and transgenic species thereof. As
would be apparent to a person of skill in the art, the
animal subjects employed in the working examples set forth
below are reasonable models for human subjects with respect
to the tissues and biochemical pathways in question, and
consequently the methods, therapeutic compounds and
pharmaceutical compositions directed to same. As evidenced
by Mordenti (1986) and similar articles, dosage forms for
animals such as, for example, rats can be and are widely
used directly to establish dosage levels in therapeutic
applications in higher mammals, including humans. In
particular, the biochemical cascade initiated by many
physiological processes and conditions is generally accepted
to be identical in mammalian species (see, e.g., Mattson and
Scheff, 1994; Higashi et al., 1995). In light of this,
pharmacological agents that are efficacious in animal models
such as those described herein are believed to be predictive
of clinical efficacy in humans, after appropriate adjustment
of dosage.
CA 02517313 2005-08-26
22
Depending on the route of administration, the
therapeutic compound may be coated in a material to protect
the compound from the action of acids, enzymes and other
natural conditions which may inactivate the compound.
Insofar as the invention provides a combination therapy in
which two therapeutic compounds are administered, each of
the two compounds may be administered by the same route or
by a different route. Also, the compounds may be
administered either at the same time (i.e., simultaneously)
or each at different times. In some treatment regimes it
may be beneficial to administer one of the compounds more or
less frequently than the other.
The compounds of the invention can be formulated to
ensure proper distribution in vivo. For example, the blood-
brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds of the
invention cross the BBB, they can be formulated, for
example, in liposomes. For methods of manufacturing
liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and
5,399,331. The liposomes may comprise one or more moieties
which are selectively transported into specific cells or
organs ("targeting moieties"), thus providing targeted drug
delivery (see, e.g., Ranade et al., 1989). Exemplary
targeting moieties include folate and biotin (see, e.g.,
U.S. Patent 5,416,016 to Low et al.); mannosides (Umezawa et
al., 1988); antibodies (Bloeman et al., 1995; Owais et al.,
1995); and surfactant protein A receptor (Briscoe et al.,
1995). In a preferred embodiment, the therapeutic compounds
of the invention are formulated in liposomes; in a more
preferred embodiment, the liposomes include a targeting
moiety.
Delivery and in vivo distribution can also be affected
by alteration of an anionic group of compounds of the
CA 02517313 2005-08-26
23
invention. For example, anionic groups such as phosphonate
or carboxylate can be esterified to provide compounds with
desirable pharmocokinetic, pharmacodynamic, biodistributive,
or other properties.
To administer a therapeutic compound by other than
parenteral administration, it may be necessary to coat the
compound with, or co-administer the compound with, a
material to prevent its inactivation. For example, the
therapeutic compound may be administered to a subject in an
appropriate carrier, for example, liposomes, or a diluent.
Pharmaceutically acceptable diluents include saline and
aqueous buffer solutions. Liposomes include water-in-oil-
in-water CGF emulsions as well as conventional liposomes
(Strejan et al. , 1984) .
The therapeutic compound may also be administered
parenterally (e. g., intramuscularly, intravenously,
intraperitoneally, intraspinally, intrathecally, or
intracerebrally). Dispersions can be prepared in glycerol,
liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations may contain a preservative to prevent the
growth of microorganisms. Pharmaceutical compositions
suitable for injectable use include sterile aqueous
solutions (where water soluble) or dispersions and sterile
powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. In all cases, the
composition must be sterile and must be fluid to the extent
that easy syringability exists. It must be stable under the
conditions of manufacture and storage and must be preserved
against the contaminating action of microorganisms such as
bacteria and fungi. The vehicle can be a solvent or
dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, liquid
CA 02517313 2005-08-26
24
polyethylene glycol, and the like), suitable mixtures
thereof, and oils (e.g.,vegetable oil). The proper fluidity
can be maintained, for example, by the use of a coating such
as lecithin, by the maintenance of the required particle
size in the case of dispersion, and by the use of
surfactants.
Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. In some cases, it will be
preferable to include isotonic agents, for example, sugars,
sodium chloride, or polyalcohols such as mannitol and
sorbitol, in the composition. Prolonged absorption of the
injectable compositions can be brought about by including in
the composition an agent which delays absorption, for
example, aluminum monostearate or gelatin.
Sterile injectable solutions can be prepared by
incorporating the therapeutic compound in the required
amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by
filter sterilization. Generally, dispersions are prepared
by incorporating the therapeutic compound into a sterile
vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In
the case of sterile powders for the preparation of sterile
injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yield a powder of
the active ingredient (i.e., the therapeutic compound)
optionally plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
Solid dosage forms for oral administration include
ingestible capsules, tablets, pills, lollipops, powders,
granules, elixirs, suspensions, syrups, wafers, buccal
CA 02517313 2005-08-26
tablets, troches, and the like. In such solid dosage forms
the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or diluent or
assimilable edible carrier such as sodium citrate or
5 dicalcium phosphate and/or a) fillers or extenders such as
starches, lactose, sucrose, glucose, mannitol, and silicic
acid, b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidone, sucrose, and acacia, c) humectants
10 such as glycerol, d) disintegrating agents such as agar-
agar, calcium carbonate, potato or tapioca starch, alginic
acid, certain silicates, and sodium carbonate, e) solution
retarding agents such as paraffin, f) absorption
accelerators such as quaternary ammonium compounds, g)
15 wetting agents such as, for example, cetyl alcohol and
glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium
stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate, and mixtures thereof, or incorporated
20 directly into the subject's diet. In the case, of capsules,
tablets and pills, the dosage form may also comprise
buffering agents. Solid compositions of a similar type may
also be employed as fillers in soft and hard-filled gelatin
capsules using such excipients as lactose or milk sugar as
25 well as high molecular weight polyethylene glycols and the
like. The percentage of the therapeutic compound in the
compositions and preparations may, of course, be varied.
The amount of the therapeutic compound in such
therapeutically useful compositions is such that a suitable
dosage will be obtained.
The solid dosage forms of tablets, dragees, capsules,
pills, and granules can be prepared with coatings and shells
such as enteric coatings and other coatings well-known in
CA 02517313 2005-08-26
26
the pharmaceutical formulating art. They may optionally
contain opacifying agents and can also be of a composition
that they release the active ingredients) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding
compositions which can be used include polymeric substances
and waxes. The active compounds can also be in micro-
encapsulated form, if appropriate, with one or more of the
above-mentioned excipients.
Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
compounds, the liquid dosage forms may contain inert
diluents commonly used in the art such as, for example,
water or other solvents, solubilizing agents and emulsifiers
such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,
ethyl acetate, benzyl alcohol, benzyl benzoate, propylene
glycol, 1,3-butylene glycol, dimethyl formamide, oils (in
particular, cottonseed, ground nut corn, germ olive, castor,
and sesame oils), glycerol, tetrahydrofurfuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof. Besides inert diluents, the oral
compositions can also include adjuvants such as wetting
agents, emulsifying and suspending agents, sweetening,
flavoring, and perfuming agents.
Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated
isostearyl alcohols, polyoxyethylene sorbitol and sorbitan
esters, microcrystalline cellulose, aluminum metahydroxide,
bentonite, agar-agar, and tragacanth, and mixtures thereof.
Therapeutic compounds can be administered in time-
release or depot form, to obtain sustained release of the
therapeutic compounds over time. The therapeutic compounds
CA 02517313 2005-08-26
27
of the invention can also be administered transdermally
(e.g., by providing the therapeutic compound, with a
suitable carrier, in patch form).
It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration
and uniformity of dosage. Dosage unit form as used herein
refers to physically discrete units suited as unitary
dosages for the subjects to be treated; each unit containing
a predetermined quantity of therapeutic compound calculated
to produce the desired therapeutic effect in association
with the required pharmaceutical vehicle. The specification
for the dosage unit forms of the invention are dictated by
and directly dependent on (a) the unique characteristics of
the therapeutic compound and the particular therapeutic
effect to be achieved, and (b) the limitations inherent in
the art of compounding such a therapeutic compound for the
treatment of neurological conditions in subjects.
Therapeutic compounds according to the invention are
administered at a therapeutically effective dosage
sufficient to achieve the desired therapeutic effect of the
cannabinoid receptor agonist, e.g. to mitigate pain and/or
to effect analgesia in a subject, to inhibit nausea and/or
vomiting in a subject, to control and/or inhibit spasticity
or a movement disorder or to alleviate a symptom of
glaucoma. For example, if the desired therapeutic effect is
analgesia, the "therapeutically effective dosage" mitigates
pain by about 40°s, preferably by about 60~, even more
preferably by about 80~, and still more preferably by about
100 relative to untreated subjects. Actual dosage levels
of active ingredients in the pharmaceutical compositions of
this invention may be varied so as to obtain an amount of
the active compounds) that is effective to achieve the
desired therapeutic response for a particular subject,
CA 02517313 2005-08-26
28
compositions, and mode of administration. The selected
dosage level will depend upon the activity of the particular
compound, the route of administration, the severity of the
condition being treated, the condition and prior medical
history of the subject being treated, the age, sex, and
weight of the subject, and the ability of the therapeutic
compound to produce the desired therapeutic effect in the
subject. Dosage regimens can be adjusted to provide the
optimum therapeutic response. For example, several divided
doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation.
However, it is well known within the medical art to
determine the proper dose for a particular subject by the
dose titration method. In this method, the patient is
started with a dose of the drug compound at a level lower
than that required to achieve the desired therapeutic
effect. The dose is then gradually increased until the
desired effect is achieved. Starting dosage levels for an
already commercially available therapeutic agent of the
classes discussed above can be derived from the information
already available on the dosages employed. Also, dosages
are routinely determined through preclinical ADME toxicology
studies and subsequent clinical trials as required by the
FDA or equivalent agency. The ability of a cannabinoid
receptor agonist to produce the desired therapeutic effect
may be demonstrated in various well known models for the
various conditions treated with these therapeutic compounds.
For example, mitigation of pain can be evaluated in model
systems that may be predictive of efficacy in mitigating
pain in human diseases and trauma, such as animal model
systems known in the art (including, e.g., the models
described herein).
CA 02517313 2005-08-26
29
Compounds of the invention may be formulated in such a
way as to reduce the potential for abuse of the compound.
For example, a compound may be combined with one or more
other agents that prevent or complicate separation of the
compound therefrom.
The following nonlimiting examples are provided to
further illustrate the present invention. The contents of
all references, pending patent applications, and published
patents cited throughout this application are hereby
expressly incorporated by reference.
EXAMPLES
Example 1: Animals
Male Long-Evans rats (N=175) from Charles River
(Montreal, QC, Canada) ranging from 230-380 grams, were
housed in polycarbonate cages in pairs and given free access
to food (Lab Diet, PMI Nutrition International, Inc.,
Brentwood, MO, USA) and water. Animal quarters were kept on
a reverse light-dark cycle (lights on from 7 pm to 7 am) and
maintained at 22 ~ 2 °C and 45 ~ 20 % relative humidity.
Animals were given a minimum of 3 days prior to the
experiment to acclimatize to the animal quarters.
Example 2: Drugs and Administration
All injections were administered in a volume of 1
ml/kg. All chemicals were dissolved in 5% dimethyl sulfoxide
(DMSO; Sigma, Oakville, ON, Canada), 0.3%
polyoxyethylenesorbitan monooleate (Tween° 80; Sigma,
Oakville, ON, Canada) and 94.7 saline vehicle, and
administered intravenously in the posterior 1/3 of the
lateral tail vein.
For single injection testing, vehicle alone was used as
a control injection (n=8). The non-specific CB receptor
CA 02517313 2005-08-26
agonist WIN 55 212-2 [(R)-(+)-[2,3-Dihydro-5-methyl-3-(4-
morpholinylmethyl)pyrrolo[1,2,3-de)-1,4-benzoxazin-6-yl]-1-
naphthalenylmethanone mesylate; Tocris Cookson, Ellisville,
MO, USA), was administered alone at doses of 0.0625 and
5 0.09375 mg/kg. These doses were chosen because they were
shown to produce sub-maximal antinociception in the tail-
flick test.
The CB1 receptor antagonist SR 141716 (SR) [N-
(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-
10 4-methyl-1H-pyrazole-3-carboxamide] was obtained from the
National Institute of Mental Health's Chemical Synthesis and
Drug Supply Program (Bethesda, MD, USA). Ultra-low doses
of SR (0.55 or 0.055 ng/kg) were combined with WIN (0.0625
mg/kg) and administered as a single injection. These
15 combinations produce WIN to SR molar ratios of 100,000:1 and
1,000,000:1, respectively. Also, WIN (0.09375 mg/kg) was
combined with ultra-low doses of SR (0.83 or 0.083 ng/kg)
producing WIN to SR molar ratios of 100,000:1 and
1,000,000:1, respectively. The control group received 0.55
20 and 0.83 ng/kg ultra-low dose of SR alone.
For repeated injection testing vehicle alone was used
as a control injection (n=8). WIN 55 212-2 was administered
alone at doses of 0.125 mg/kg. This dose was chosen because
it produces maximal antinociception in the tail-flick test.
25 Ultra-low doses of SR (1.1 or 0.11 ng/kg) were combined with
WIN (0.125 mg/kg) and administered as a single injection.
These combinations produce WIN to SR molar ratios of
100,000:1 and 1,000,000:1 respectively. The control group
received 1.1 ng/kg ultra-low dose of SR alone.
Example 3: Apparatus
The tail-flick apparatus consists of a projection lamp
that creates radiant heat located just below the animal-
CA 02517313 2005-08-26
31
testing surface (D'Amour, E.E. and Smith, D.L. J. Pharmacol.
Exp. Ther. 1941 72:74-79). The light from the lamp
projected through a small hole in the testing surface and
was aimed at a photocell located 25 cm above the testing
surface. A digital timer, connected to the apparatus,
started when the heat source was activated. When the animal
flicked its tail away from the heat source, the light from
the projection lamp activated the photocell, simultaneously
stopping the timer and turning off the lamp. The heat
intensity was calibrated to result in baseline tail flick
latencies of 2-3 seconds and a l0 second cutoff was used to
minimize tissue damage.
Example 4: Nociceptive testing
Nociceptive reflexes to a thermal stimulus were tested
using the tail-flick analgesia meter. This apparatus
focuses a hot beam on the animal's tail. The time it takes
for the rat to flick its tail away from the heat source is a
measure of pain; the longer the animal leaves its tail on
the hotspot, the greater the degree of pain relief. On the
day prior to tail flick testing, animals were handled on the
tail flick apparatus for 5-10 minutes to reduce stress-
induced analgesia (Kelly, S.J. and Franklin, K.B.,
Neuropharmacology 1985 24:1019-1025; Terman et al., Science
1984 226: 1270-1277). For single injection tested groups,
animals were restrained in a small towel and a baseline tail
flick latency was measured. Following the baseline measure,
animals were given a drug injection and tail-flick latencies
were assessed every 10 minutes for 90 minutes. For the
repeated injection groups, animals were given one drug
injection every day for seven days. Prior to the first
injection, a baseline tail-flick latency was measured.
Following this measure, animals were given a drug injection
CA 02517313 2005-08-26
32
and tail flick latencies were assessed 10 minutes post-
injection. This time point was selected because it is the
point at which maximal WIN-induced antinociception is
detected in this protocol. Post injection tail flick
latencies were assessed on days 1, 3, 5, and 7. For all
animals, tail-flick latencies were converted into a percent
of maximal possible effect (MPE) using the equation:
MPE = ((post-injection latency - baseline latency) / (10 s
cutoff - baseline latency)] - 100
Example 5: Tissue sampling and analysis
On the day following the last injection, animals were
sedated with CO2, and then decapitated. The brain and lumbar
spinal cord were quickly extracted on ice, and a sample of
the striatum, periaqueductal gray and dorsal horn were
extracted, immersed in liquid nitrogen, and stored at -80°C
until the immunoassays could be performed.
Co-immunoprecipitation experiments were performed on
this tissue to determine the G-protein sub-type coupling
profile of activated CB1 receptors. Tissue was bathed in
vitro with either vehicle or cannabinoid agonist and then
solubilized. Samples were then divided and exposed to
immobilized antibodies for Gas, Gai, or Gao to isolate
cannabinoid receptors bound to these G-protein sub-types.
These samples were later subjected to western blotting using
a specific CB1 antibody. Comparisons were made between
tissue from animals previously treated chronically vehicle,
WIN 55,212-2, ultra-low dose SR 141716 or combination
treatment of WIN and ultra-low dose SR 141716.
CA 02517313 2005-08-26
33
Example 6: Reversal of the pre-existing cannabinoid receptor
agonist analgesic tolerance by ultra-low dose of cannabinoid
receptor antagonist
Chronic tolerance is induced in rats by intravenous
injection of WIN 55 212-2 (0.125 mg/kg) once daily for 5-
days. Animals are divided into two groups and nociceptive
testing is performed 10 minutes after the daily drug
injection using the tail-flick test. On day 6, one group
will continue on the cannabinoid receptor agonist dose for
an additional 5 days whereas the other group will receive
the cannabinoid receptor agonist in combination with a low
dose of the cannabinoid receptor antagonist (0.11 ng/kg) for
the same period. Nociception will be assessed on a daily
basis as described above.
Example 7: Statistics
Separate two-way repeated measure ANOVAs were performed
on the single injection groups, and repeated injection
groups. Post-injection time (10-90 minutes for single
injection groups) or day of injection (1-7 days for the
repeated injection groups) were the within-subjects factors
and drug group was the between-subjects factor. Whenever
there were violations of sphericity, Huynh-Feldt corrections
to the within-group degrees of freedom were reported.
Because many drug group comparisons were irrelevant, a
priori multiple comparisons were used to analyze the main
drug effect using the Dunn's critical t-ratio. A corrected
a of 0.005 was used for the single injection groups because
there were 8 comparisons, and a corrected a of 0.01 was used
for the repeated injection groups because there were 4
comparisons.
