0022-3565/03/3053-812817$7.

00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright 2003 by The American Society for Pharmacology and Experimental Therapeutics
JPET 305:812817, 2003

Vol. 305, No. 3

46870/1059085
Printed in U.S.A.

Modulation of Oral Morphine Antinociceptive Tolerance and

Naloxone-Precipitated Withdrawal Signs by Oral
9-Tetrahydrocannabinol
DIANA L. CICHEWICZ and SANDRA P. WELCH
Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia
Received November 21, 2002; accepted February 3, 2003

The clinical use of morphine for long periods of time is

limited by its propensity to cause tolerance and physical
dependence after repeated administration. To overcome tolerance to the analgesic effects of morphine, higher doses are
necessary for adequate pain relief but are often accompanied
by undesirable side effects such as constipation, nausea, and
respiratory depression (Ellison, 1993). Morphine tolerance
has been developed in rodent models using subcutaneously
implanted morphine pellets (Cicero and Meyer, 1973; Bhargava, 1978), but little is known about tolerance to oral doses
of morphine in mice. Mao et al. (1996) observed a decrease in
antinociception produced by oral morphine in rats as measured by the tail-flick test over a period of several days.
Our laboratory and others have shown that combinations
of cannabinoids with morphine profoundly enhance morphine-induced analgesia. 9-THC at a nonanalgetic dose administered p.o. to mice significantly enhances the potency of
This work was supported by National Institute on Drug Abuse Grants
DA-07027, DA-05274, and K02-DA-00186.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.046870.

naloxone-precipitated (1 mg/kg s.c.) platform jumping by 50%

but did not reduce diarrhea. In separate experiments, mice
treated chronically with high-dose morphine p.o. were not
cross-tolerant to 9-THC; in fact, these morphine-tolerant mice
were more sensitive to the acute antinociceptive effects of
9-THC. 9-THC (20 mg/kg p.o.) also reduced naloxone-precipitated jumping but not diarrhea when administered acutely
to morphine-tolerant mice. These results represent the first
evidence that oral morphine tolerance and dependence can be
circumvented by coadministration of a nonanalgetic dose of
9-THC p.o. In summary, cotreatment with a combination of
morphine and 9-THC may prove clinically beneficial in that
long-term morphine efficacy is maintained.

opioids such as morphine and codeine (Smith et al., 1998;

Cichewicz et al., 1999). This enhancement is theorized to be
due to a combination of morphines direct effects on the
-opioid receptor and the effect of endogenous opioids whose
release is stimulated by 9-THC (Smith et al., 1994; Pugh et
al., 1996). In addition, the CB1 cannabinoid receptor and
-opioid receptor have been found to be colocalized in areas
important for the expression of morphine abstinence: nucleus
accumbens, septum, striatum, periaqueductal gray area, and
amygdaloid nucleus (Navarro et al., 1998). Thus, we hypothesize that 9-THC might alter the expression of morphine
antinociceptive tolerance and/or dependence.
9-THC reduces naloxone-precipitated withdrawal in morphine-dependent rats and mice (Hine et al., 1975; Bhargava,
1976). The endogenous cannabinoid anandamide also decreases naloxone-precipitated morphine withdrawal (Vela et
al., 1995). Furthermore, several studies indicate that the
morphine withdrawal syndrome is markedly decreased in
CB1-receptor knockout mice (Ledent et al., 1999; Lichtman
et al., 2001). Blockade of the CB1 receptor by the antagonist
SR 141716A has been shown to precipitate morphine withdrawal signs such as weight loss, teeth chattering, and diar-

ABBREVIATIONS: 9-THC, 9-tetrahydrocannabinol; SR 141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1Hpyrazole-3-carboximide hydrochloride; % MPE, percentage of maximum possible effect; CL, confidence limits; ANOVA, analysis of variance;
DAMGO, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin; NMDA, N-methyl-D-aspartate; MK-801, dizocilpine maleate.
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ABSTRACT
Previous studies have demonstrated a functional interaction
between cannabinoid and opioid systems in the development
and expression of morphine tolerance and dependence. In
these experiments, we examined the effect of a low oral dose of
9-tetrahydrocannabinol (9-THC) on the development of oral
morphine tolerance and the expression of naloxone-precipitated morphine withdrawal signs of jumping and diarrhea in ICR
mice. Chronic treatment with high-dose oral morphine produced a 3.12-fold antinociceptive tolerance. Tolerance to morphine was prevented in groups receiving a daily cotreatment
with a nonanalgetic dose (20 mg/kg p.o.) of 9-THC, except
when challenged with a very high dose of morphine. The
chronic coadministration of low-dose 9-THC also reduced

Modulation of Morphine Tolerance and Dependence by 9-THC

Materials and Methods

Animals. Male ICR mice (Harlan, Indianapolis, IN) weighing 20
to 24 g were housed three per cage in an animal care facility maintained at 22 2C on a 12-h light/dark cycle. Food and water were
available ad libitum. Chronic drug administration was done in the
animal care facility, and then the mice were brought to the test room
12 h before testing to allow acclimation and recovery from transport
and handling. All experiments were conducted in accordance with
guidelines from the Institutional Animal Care and Use Committee at
Virginia Commonwealth University.
Chronic Drug Administration. Mice were administered distilled water vehicle or morphine by oral gavage in a ramped paradigm twice daily for 7 days (200 mg/kg, days 12; 300 mg/kg, days
37). This paradigm was used to prevent toxicity at the beginning of
the study from the high 300 mg/kg morphine dose that produces a
robust morphine tolerance. To confirm tolerance to morphine, groups
of six mice treated with the above paradigm were challenged 12 h
after the last chronic administration with varying doses of morphine
p.o. (one dose per group) and tested 30 min later in the tail-flick test
to obtain dose-response curves for morphine.
Prevention of Morphine Tolerance and Dependence. For
chronic combination studies, separate groups of six mice were administered a dose of 20 mg/kg 9-THC p.o., 15 min before each
morphine administration as described above (twice daily for 7 days).
This dose of 9-THC was previously determined to produce less than
20% antinociceptive effect in acute dose-response studies (Fig. 1). In
addition, previous work in the laboratory has determined that this
dose of 9-THC given orally twice daily for 7 days does not yield
antinociceptive tolerance (D. L. Cichewicz, unpublished observations). On the test day, the mice were challenged 12 h after the last
chronic administration with varying doses of morphine p.o. (one dose
per group) and tested 30 min later in the tail-flick test to obtain a
dose-response curve for morphine.
Reversal of Morphine Tolerance and Dependence. In the
studies of the acute effects of 9-THC on morphine tolerance and
withdrawal, separate groups of six mice were treated with the
chronic morphine paradigm described earlier (200 mg/kg, days 12;

Fig. 1. Dose-response analysis of 9-THC administered acutely to mice.

Note that a 20 mg/kg dose produces less than 20% MPE and therefore was
chosen as our low dose for subsequent experiments. Mice were administered various doses of 9-THC p.o. and tested 30 min later in the tail-flick
test for antinociception. Each data point represents an average % MPE
S.E.M. of six mice.

300 mg/kg, days 37) twice daily. The mice were then administered
various doses of 9-THC p.o. 12 h after their last chronic morphine
administration and tested 30 min later in the tail-flick test for
antinociception.
Evaluation of Physical Dependence to Morphine. Separate
groups of six mice were challenged with 1 mg/kg naloxone s.c. 2 h
after the last drug administration. Immediately after naloxone injection, each mouse was placed on an elevated pedestal, with a
platform 1 foot in height 4 inches in diameter, and observed for 10
min for the presence or absence of jumping and diarrhea, two signs
indicative of naloxone-precipitated morphine withdrawal (Way et al.,
1969). Data are presented as the percentage of mice (in a group of
six) that exhibited platform jumping or diarrhea.
Analgesic Assay. The tail-flick heat latency test for antinociception was designed by DAmour and Smith (1941). A radiant heat light
focused on the tail of the mouse automatically shuts off when the
mouse reflexively flicks its tail out of the light beam. Baseline
tail-flick latencies were determined prior to drug administration on
the test day and were between 2 and 4 s. During drug testing, a cutoff
time of 10 s was employed to prevent damage to the tail. Antinociception was quantified using the percentage of maximum possible
effect (% MPE) calculated as developed by Harris and Pierson (1964)
as follows: % MPE [(test baseline)/(10 baseline)] 100. A
mean % MPE value was determined for each group of six mice.
Statistical Analysis. ED50 values, potency ratios, and 95% confidence limits (CL) for morphine dose-response curves were determined using unweighted least-squares linear regression as modified
from procedures 5 and 8 described by Tallarida and Murray (1987).
Comparisons between groups in the dependence studies were performed using one-way analysis of variance (ANOVA) followed by the
Tukey-Kramer post hoc test for between-group comparisons. Statistical significance was set at p 0.05.
Drugs. 9-THC and morphine were obtained from the National
Institute on Drug Abuse (Bethesda, MD). 9-THC was prepared in
1:1:18 (ethanol/Emulphor/isotonic saline), and morphine was dissolved in distilled water.

Results
-THC Dose-Response Curve. To determine a nonanalgetic dose of 9-THC for use in combination studies, we
performed an acute dose-response analysis (Fig. 1). Based on
the data, we chose a 20 mg/kg dose, which produces less than
20% MPE, as our low inactive dose to combine with morphine
in further studies. This dose has been reported in the literature as the most effective for enhancement of morphine an9

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rhea in dependent rats (Navarro et al., 1998). Recently, acute

administration of 9-THC blocked some signs of morphine
withdrawal in mice (paw tremors and head shakes), but not
jumping or diarrhea (Lichtman et al., 2001). These findings
suggest a role of the endogenous cannabinoid system in opioid dependence. However, similar studies have never been
performed using orally administered cannabinoids. An oral
preparation of 9-THC (Marinol) is approved in the United
States for several indications including cachexia and emesis.
Our study of orally administered 9-THC for prevention of
tolerance or dependence to morphine was designed to provide
a potential new indication for Marinol, based on our previous
data with 9-THC/morphine combinations.
In the present study, we investigated the effect of an orally
administered nonanalgetic dose of 9-THC in altering the
behavioral expression of oral morphine tolerance and physical dependence by examining antinociception and naloxoneprecipitated withdrawal signs (platform jumping and diarrhea). In addition to examining the effects of acutely
administered 9-THC on the expression of morphine abstinence, we also evaluated the ability of the low-dose 9-THC
coadministered with morphine for 7 days to prevent development of tolerance to and/or dependence on morphine. A clinical extrapolation for such findings would be important, since
lower doses of drugs are desirable to maximize pain relief but
minimize side effects and drug tolerance.

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Cichewicz and Welch

Fig. 2. A dose of 20 mg/kg 9-THC p.o. attenuates the development of oral

morphine tolerance. Mice received distilled water vehicle, morphine (200
mg/kg p.o., days 12; 300 mg/kg p.o., days 37), or morphine plus 20
mg/kg 9-THC p.o. twice daily for 7 days. At 12 h after the last drug
administration, various doses of morphine were administered p.o. and
the mice were tested 30 min later in the tail-flick test. Each data point
represents an average % MPE S.E.M. of six mice.

TABLE 1
Morphine ED50 values after 7-day treatment with vehicle, morphine, or
a combination of 9-THC and morphine
Mice in groups of six were administered drugs twice daily for 7 days and tested for
morphine antinociception 12 h after the last drug administration. In the combination
group, 9-THC was administered 15 min prior to morphine. ED50 values were
calculated from at least three doses of morphine. Potency ratio indicates shift
between vehicle and drug-treated groups.
Treatment

Morphine ED50
(95% CL)

Vehicle (distilled water)

Morphinea
Morphinea 20 mg/kg 9-THC

40.3 (28.057.9)
126.7 (100.4160)*
48.5 (29.779.0)

Potency Ratio

mg/kg

3.12
1.28

a
Represents a 200 to 300 mg/kg p.o. regimen as described under Materials and
Methods.
* Significantly different from vehicle ED50 value.

Fig. 3. Effects of 9-THC on naloxone-precipitated morphine withdrawal

platform jumping. Mice were treated chronically with either distilled
water vehicle, morphine (200 mg/kg p.o., days 12; 300 mg/kg p.o., days
37), or morphine plus 20 mg/kg 9-THC p.o. twice daily for 7 days.
Naloxone (1 mg/kg s.c.) was injected 2 h after the last drug administration, and the mice were immediately placed on platforms and observed for
10 min. Results are presented as the percentage of mice in each group of
six that jumped from the platform. , significantly different from vehicletreated group (p 0.05). #, significantly different from morphine-treated
group (p 0.05), ANOVA and Tukey-Kramer post hoc test.

Fig. 4. Effects of 9-THC on naloxone-precipitated morphine withdrawal

diarrhea. Mice were treated chronically with either distilled water vehicle, morphine (200 mg/kg p.o., days 12; 300 mg/kg p.o., days 37), or
morphine plus 20 mg/kg 9-THC p.o. twice daily for 7 days. Naloxone (1
mg/kg s.c.) was injected 2 h after the last drug administration, and the
mice were immediately placed on platforms and observed for 10 min.
Results are presented as the percentage of mice in each group of six that
exhibited diarrhea. , significantly different from vehicle-treated group
(p 0.05), ANOVA and Tukey-Kramer post hoc test.

ment groups during the 10-min period on the jumping platforms.

Effects of Acute 9-THC on the Reversal of Morphine
Tolerance. The purpose of this experiment was to determine

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algesia without producing intrinsic antinociception (Smith et

al., 1998; Cichewicz et al., 1999).
Effects of Chronic 9-THC on the Prevention of Morphine Tolerance. Mice were treated twice daily for 7 days
with either distilled water vehicle p.o. or morphine p.o. (200
mg/kg on days 12; 300 mg/kg on days 37). Administration
of morphine p.o. on the test day produced a dose-response
relationship in chronic vehicle-treated mice (ED50 40.3
mg/kg; Fig. 2). In morphine-tolerant mice, the dose-response
curve of morphine was shifted to the right 3.12-fold, as determined by the ED50 values (Table 1). When 9-THC was
administered concurrently with morphine for 7 days, THC
prevented the development of morphine tolerance. Although
the curves showed a trend toward convergence at the highest
dose of morphine tested, the ED50 value of 48.5 mg/kg (95%
CL 29.779.0) for the combination was similar to that of
the vehicle-treated group, and the 95% CL overlapped, indicating that the vehicle curve and the combination curve were
not significantly different. Thus, the addition of 9-THC prevented the development of morphine tolerance in these animals.
Effect of Chronic 9-THC on the Prevention of Physical Dependence to Morphine. To evaluate whether 9THC would alter the expression of behavioral withdrawal
signs associated with morphine dependence, mice were
treated twice daily for 7 days with orally administered distilled water vehicle, morphine alone, or morphine in combination with a low 20 mg/kg dose of 9-THC as described
earlier. On the test day, the mice were challenged with 1
mg/kg naloxone s.c. 2 h after the last drug administration. As
shown in Fig. 3, vehicle-treated mice exhibited no platform
jumping behavior, whereas morphine-treated mice all exhibited platform jumping, indicating development of morphine
dependence. The concurrent administration of 9-THC with
morphine for 7 days significantly reduced the number of mice
showing platform jumping by 50%. However, the occurrence
of platform jumping was not completely prevented by the
daily administration of 9-THC. Interestingly, naloxone-induced diarrhea was significantly increased in the morphine
group treated daily with 9-THC (Fig. 4). Although locomotor
activity was not directly measured in this study, we did not
observe any distinct differences between any of the treat-

Modulation of Morphine Tolerance and Dependence by 9-THC

Discussion
There are several similarities in the pharmacology of 9THC and morphine, and it has been shown that an interaction exists between cannabinoid and opioid pathways. Our

Fig. 5. Effects of acutely administered 9-THC p.o. in vehicle-treated and

morphine-tolerant mice. Mice were administered distilled water vehicle
or morphine (200 mg/kg p.o., days 12; 300 mg/kg p.o., days 37) twice
daily for 7 days. At 12 h after the last drug treatment, 9-THC p.o. or
vehicle (1:1:18) p.o. was administered, and the mice were tested 30 min
later in the tail-flick test. Data are presented as mean S.E.M. with six
mice per group. , significantly different from corresponding vehicletreated group (p 0.05). $, significantly different from vehicle-treated/
vehicle-tested group (p 0.05). #, significantly different from morphinetreated/vehicle-tested group (p 0.05), ANOVA and Tukey-Kramer post
hoc test.

Fig. 6. Effects of acutely administered 9-THC on naloxone-precipitated

morphine withdrawal platform jumping. Mice were treated chronically
with either distilled water vehicle or morphine (200 mg/kg p.o., days 12;
300 mg/kg p.o., days 37) p.o. twice daily for 7 days. At 12 h after the last
drug administration, vehicle (1:1:18) or 9-THC p.o. was administered.
Naloxone (1 mg/kg s.c.) was injected 2 h later, and the mice were immediately placed on platforms and observed for 10 min. Results are presented as the percentage of mice in each group of six that jumped from the
platform. , significantly different from corresponding vehicle-treated
group (p 0.05). #, significantly different from morphine-treated/vehicletested group (p 0.05), ANOVA and Tukey-Kramer post hoc test.

aim was to investigate the effects of acute and repetitive oral

treatment with the cannabinoid 9-THC on the expression of
tolerance to and physical dependence on morphine. We show
that cotreatment of mice with a low dose of 9-THC prevents
the development of morphine tolerance and attenuates the
level of physical dependence as measured by withdrawal
jumping and diarrhea. These results support an interaction
between endogenous cannabinoid and opioid systems. It was
important for us to explore the oral route of administration,
since both 9-THC and morphine are administered orally in
clinics. The data presented here are the first report of the
prevention of morphine tolerance by a low oral dose of 9THC.
Morphine tolerance has been demonstrated in many different models (Way et al., 1969; Huidobro et al., 1976). Cellular
events that occur in morphine tolerance include desensitization of the -opioid receptor and a reduction in -opioid
agonist potency (Yoburn et al., 1993; Bernstein and Welch,
1998). The development of tolerance to morphine is also
consistent with down-regulation of the -opioid receptor at
spinal and supraspinal sites (Bernstein and Welch, 1998;
Cichewicz et al., 2001). However, observation of receptor
down-regulation is highly variable with in vivo studies.
We hypothesized that replacing morphine treatment with
combination 9-THC/morphine treatment would prevent
morphine antinociceptive tolerance. Our data support that
hypothesis, since after a 7-day treatment with both morphine
and 9-THC, no antinociceptive tolerance to morphine occurred. At the highest challenge dose of morphine, there was
a trend for the combination-treated curve to resemble the
morphine-tolerant curve. Yet, statistically, the ED50 values
of these two curves were different. The mechanism by which
9-THC prevents morphine tolerance at low doses but not
high doses has yet to be defined. However, it seems logical
that the two drugs together might yield an increase, not a
decrease, in the likelihood of tolerance development, due to
enhancement of morphine-induced antinociception by
9-THC and a potential for greater-than-normal stimulation
of opioid receptors by 9-THC-induced release of endogenous

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the antinociceptive efficacy of 9-THC or combinations of

9-THC and morphine after the development of morphine
tolerance. Mice received distilled water vehicle or morphine
twice daily for 7 days according to the above paradigms. On
the test day, mice received vehicle (1:1:18) or various doses of
9-THC p.o. 12 h after the last morphine administration and
were tested for antinociception 30 min later in the tail-flick
test. In vehicle-treated mice, 9-THC produced a dose-response relationship, with the 1 mg/kg dose producing a low
degree of antinociception similar to vehicle, whereas the 50
mg/kg dose produced significant antinociception (Fig. 5).
Morphine-tolerant mice showed a much greater antinociceptive response to lower doses of 9-THC than did their vehicletreated counterparts, suggesting that morphine-tolerant
mice were more sensitive to 9-THC-induced antinociception
and were not cross-tolerant to 9-THC. A dose of 50 mg/kg
9-THC was equally effective in vehicle-treated and morphine-tolerant mice.
Effect of Acute 9-THC on the Expression of Physical
Dependence on Morphine. To determine whether 9-THC
could prevent the expression of naloxone-precipitated morphine withdrawal signs, we administered vehicle (1:1:18) or
various doses of 9-THC to chronic vehicle-treated and morphine-treated mice 12 h after their last vehicle or morphine
administration. A 1 mg/kg s.c. dose of naloxone was injected
2 h after 9-THC, and mice were observed for 10 min for
jumping and diarrhea. All doses of 9-THC tested reduced
naloxone-precipitated platform jumping behavior in morphine-dependent mice by 50% (Fig. 6). However, there were
still a significant number of morphine-treated animals exhibiting jumping as compared with vehicle-treated animals.
There were no differences in naloxone-precipitated diarrhea
among these groups (data not shown). Again, no differences
in locomotor activity were observed.

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Cichewicz and Welch

but dependence as well. A 20 mg/kg p.o. dose of 9-THC

administered for 7 days with morphine reduced naloxoneprecipitated platform jumping by 50%. Previous studies show
that repetitive pretreatment with 9-THC in mice treated
chronically with morphine significantly reduces naloxoneprecipitated withdrawal signs (Valverde et al., 2001). Morphine withdrawal is associated with decreased dopamine
levels in various brain regions associated with reward such
as the ventral tegmental area and nucleus accumbens (Tokuyama et al., 2000; Walters et al., 2000). 9-THC may be
reducing the severity of morphine withdrawal by increasing
dopamine levels in these areas (Melis et al., 2000). However,
reports have shown that chronic administration of SR
141716A, the CB1 antagonist, also lessens several morphine
withdrawal symptoms (Mas-Nieto et al., 2001). These authors hypothesize that stabilization of CB1 receptors upon
antagonist binding may sequester a pool of Gi/o-proteins
away from -opioid receptors, thus reducing the development
of dependence to morphine. 9-THC may serve the same
function by usurping signaling pathways utilized by morphine to produce dependence at -receptors. These data, in
which both an agonist and an antagonist at the CB1 receptor
reduce the signs of morphine withdrawal, support an interaction between endogenous opioid and cannabinoid systems.
However, we can conclude that there is a clear separation of
morphine tolerance and physical dependence, since 9-THC
was able to completely prevent morphine tolerance but only
partially reduced signs of morphine withdrawal.
Only 20% of morphine-tolerant mice exhibited diarrhea
upon injection of naloxone. Furthermore, mice that received
9-THC daily with morphine for 7 days exhibited an increased occurrence of diarrhea upon naloxone injection. However, Lichtman et al. (2001) report that a 1 mg/kg s.c. dose of
naloxone precipitates diarrhea in all morphine-tolerant mice
tested. The reason for the increase in naloxone-precipitated
diarrhea in combination-treated mice is unclear; however, it
may be that diarrhea is not as reliable as platform jumping to
measure morphine withdrawal. Studies using the cannabinoid antagonist SR 141716A would further help to elucidate
the role of 9-THC in the production of diarrhea in these
mice.
Our results agree with previous reports that acute administration of 9-THC and other cannabinoids also decreases
naloxone-precipitated jumping in morphine-dependent rodents (Hine et al., 1975; Bhargava, 1976). Yamaguchi et al.
(2001) suggest that the appearance of withdrawal signs in
morphine-dependent mice results from an inactivation of
CB1 receptors and/or the inhibition of release or synthesis of
endocannabinoids. Thus, during morphine dependence, the
endocannabinoid system may be activated, whereas withdrawal may inactivate the system. The acute administration
of 9-THC prior to precipitation of morphine withdrawal by
naloxone may prevent inactivation of the endocannabinoid
system by providing an alternate way of stimulating CB1
receptors. However, others have failed to ameliorate jumping
with acute 9-THC (Lichtman et al., 2001). Differences in
naloxone dose may account for the disparity. The dose of
naloxone used in the present studies (1 mg/kg s.c.) has consistently been shown to precipitate morphine withdrawal
symptoms (Campbell et al., 2000; Mas-Nieto et al., 2001).
Other researchers have used heroic doses of naloxone up to
50 mg/kg s.c., far beyond the selectivity of antagonizing

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opioids (Smith et al., 1994; Pugh et al., 1996). Since morphine

tolerance is associated with desensitization of -opioid receptors (for review, see Williams et al., 2001), it is possible that
9-THC enhances the efficacy of G-protein coupling to -receptors and thus prevents desensitization. Corchero et al.
(1999) report that administration of 9-THC for 1, 3, 7, or 14
days causes an increase in DAMGO-stimulated guanosine
5-O-(3-[35S]thio)triphosphate binding in the caudate-putamen, supporting the theory that 9-THC increases opioid
receptor/transduction coupling. The RAVE theory (relative
activity versus endocytosis) by Whistler et al. (1999) suggests
that morphine, although a high-efficacy agonist, has little
ability to induce endocytosis of -opioid receptors that would
attenuate prolonged signaling by chronic administration of
morphine. However, in combination with DAMGO, an enkephalin derivative, cells containing morphine-bound receptors showed profound endocytosis (He et al., 2002). Therefore, in our combination treatment, endogenous opioids
released by 9-THC may enhance morphines ability to endocytose and recycle its receptors, thus reducing the development of tolerance. The argument that an increase in 9THC-induced analgesia over 7 days may contribute to the
reduction in morphine tolerance can be countered by the fact
that there is no analgesic response at the lowest challenge
dose of morphine, indicating that neither morphine nor 9THC is contributing to provide analgesia. Further work with
receptor antagonists may elucidate these mechanisms.
Other possibilities in defining the role of 9-THC in prevention of morphine tolerance involve second-messenger systems. Acute administration of morphine decreases adenylyl
cyclase activity, thus reducing intracellular levels of cAMP,
whereas chronic morphine causes an up-regulation of adenylyl cyclase activity (Sharma et al., 1975). In morphine withdrawal, an increase in neurotransmitter release is initiated
through a cAMP-dependent mechanism (Valverde et al.,
1996; Williams et al., 2001), and so cAMP levels remain
elevated. Since acute 9-THC has been shown to reduce
cAMP, it may be possible that 9-THC acts in opposition to
morphine to maintain basal cAMP levels, thus preventing
tolerance to morphine. NMDA receptor activation has also
been implicated in the development of morphine tolerance
and dependence (for review, see Mao, 1999). NMDA receptor
antagonists such as MK-801 and dextromethorphan have
been effective in preventing tolerance to and dependence on
morphine. It is possible that 9-THC may have actions similar to those of NMDA antagonists.
The potency of acute 9-THC is greatly increased in morphine-tolerant mice, indicating that these mice are more
sensitive to the cannabinoids antinociceptive effects and do
not become tolerant to 9-THC, in contrast to previous reports in which morphine-pelleted mice demonstrated a significant decrease in the analgesic effects of 9-THC i.p. (Thorat and Bhargava, 1994). These two studies differ in the
route of administration of drugs and mouse strain, as well as
length of drug administration. However, others report that
antinociceptive effects of 9-THC are potentiated in morphine-dependent rats (Rubino et al., 1997). It is possible that
the induction of oral morphine tolerance causes an alteration
in the CB1 system, increasing the potency of 9-THC. These
results suggest that effective antinociception can be produced
in morphine-tolerant subjects utilizing 20 mg/kg 9-THC.
Combination therapy reduces not only morphine tolerance,

Modulation of Morphine Tolerance and Dependence by 9-THC

-opioid receptors, and could clearly result in discrepancies

from our results.
In summary, we have presented the first evidence that oral
morphine tolerance can be prevented by coadministration of
a low dose of oral 9-THC. This dose of 9-THC, although
inactive on its own, is also sufficient to reduce the naloxoneprecipitated morphine withdrawal sign of platform jumping
in mice. The clinical implication of this work is the possibility
that 9-THC and morphine in combination therapy may be
more effective analgesics for a longer period of time than
morphine alone, without tolerance to subsequent opioid
treatments. In addition, the potential cross-talk between cannabinoid and opioid systems in the development of morphine
dependence may indicate new strategies for treatment of
opioid addiction.
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Address correspondence to: Dr. Sandra P. Welch, P.O. Box 980613, MCV
Station, Richmond, VA 23298-0613. E-mail: Swelch@hsc.vcu.edu

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