Modulation Of Oral Morphine Antinociceptive Tolerance

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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 Δ9-tetrahydrocannabinol (Δ9-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 Δ9-THC, except when challenged with a very high dose of morphine. The chronic coadministration of low-dose Δ9-THC also reduced 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 Δ9-THC; in fact, these morphine-tolerant mice were more sensitive to the acute antinociceptive effects of Δ9-THC. Δ9-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 Δ9-THC p.o. In summary, cotreatment with a combination of morphine and Δ9-THC may prove clinically beneficial in that long-term morphine efficacy is maintained.

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. Δ9-THC at a nonanalgetic dose administered p.o. to mice significantly enhances the potency of 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 morphine's direct effects on the μ-opioid receptor and the effect of endogenous opioids whose release is stimulated by Δ9-THC (Smith et al., 1994; Pugh et al., 1996). In addition, the CB1 cannabinoid receptor and μ-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 Δ9-THC might alter the expression of morphine antinociceptive tolerance and/or dependence.

Δ9-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 diarrhea in dependent rats (Navarro et al., 1998). Recently, acute administration of Δ9-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 Δ9-THC (Marinol) is approved in the United States for several indications including cachexia and emesis. Our study of orally administered Δ9-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 Δ9-THC/morphine combinations.

In the present study, we investigated the effect of an orally administered nonanalgetic dose of Δ9-THC in altering the behavioral expression of oral morphine tolerance and physical dependence by examining antinociception and naloxone-precipitated withdrawal signs (platform jumping and diarrhea). In addition to examining the effects of acutely administered Δ9-THC on the expression of morphine abstinence, we also evaluated the ability of the low-dose Δ9-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.

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 ± 2°C 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 1—2; 300 mg/kg, days 3—7). 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 Δ9-THC p.o., 15 min before each morphine administration as described above (twice daily for 7 days). This dose of Δ9-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 Δ9-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 Δ9-THC on morphine tolerance and withdrawal, separate groups of six mice were treated with the chronic morphine paradigm described earlier (200 mg/kg, days 1—2; 300 mg/kg, days 3—7) twice daily. The mice were then administered various doses of Δ9-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 D'Amour 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. Δ9-THC and morphine were obtained from the National Institute on Drug Abuse (Bethesda, MD). Δ9-THC was prepared in 1:blushsmile:18 (ethanol/Emulphor/isotonic saline), and morphine was dissolved in distilled water.


Δ9-THC Dose-Response Curve. To determine a nonanalgetic dose of Δ9-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 analgesia without producing intrinsic antinociception (Smith et al., 1998; Cichewicz et al., 1999).

Effects of Chronic Δ9-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 1—2; 300 mg/kg on days 3—7). 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 Δ9-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 = 29.7—79.0) 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 Δ9-THC prevented the development of morphine tolerance in these animals.

Effect of Chronic Δ9-THC on the Prevention of Physical Dependence to Morphine. To evaluate whether Δ9-THC 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 Δ9-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 Δ9-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 Δ9-THC. Interestingly, naloxone-induced diarrhea was significantly increased in the morphine group treated daily with Δ9-THC (Fig. 4). Although locomotor activity was not directly measured in this study, we did not observe any distinct differences between any of the treatment groups during the 10-min period on the jumping platforms.

Effects of Acute Δ9-THC on the Reversal of Morphine Tolerance. The purpose of this experiment was to determine the antinociceptive efficacy of Δ9-THC or combinations of Δ9-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:blushsmile:18) or various doses of Δ9-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, Δ9-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 Δ9-THC than did their vehicle-treated counterparts, suggesting that morphine-tolerant mice were more sensitive to Δ9-THC-induced antinociception and were not cross-tolerant to Δ9-THC. A dose of 50 mg/kg Δ9-THC was equally effective in vehicle-treated and morphine-tolerant mice.

Effect of Acute Δ9-THC on the Expression of Physical Dependence on Morphine. To determine whether Δ9-THC could prevent the expression of naloxone-precipitated morphine withdrawal signs, we administered vehicle (1:blushsmile:18) or various doses of Δ9-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 Δ9-THC, and mice were observed for 10 min for jumping and diarrhea. All doses of Δ9-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.


There are several similarities in the pharmacology of Δ9-THC and morphine, and it has been shown that an interaction exists between cannabinoid and opioid pathways. Our aim was to investigate the effects of acute and repetitive oral treatment with the cannabinoid Δ9-THC on the expression of tolerance to and physical dependence on morphine. We show that cotreatment of mice with a low dose of Δ9-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 Δ9-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 Δ9-THC.

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 μ-opioid receptor and a reduction in μ-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 μ-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 Δ9-THC/morphine treatment would prevent morphine antinociceptive tolerance. Our data support that hypothesis, since after a 7-day treatment with both morphine and Δ9-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 Δ9-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 Δ9-THC and a potential for greater-than-normal stimulation of opioid receptors by Δ9-THC-induced release of endogenous opioids (Smith et al., 1994; Pugh et al., 1996). Since morphine tolerance is associated with desensitization of μ-opioid receptors (for review, see Williams et al., 2001), it is possible that Δ9-THC enhances the efficacy of G-protein coupling to μ-receptors and thus prevents desensitization. Corchero et al. (1999) report that administration of Δ9-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 Δ9-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 μ-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 Δ9-THC may enhance morphine's ability to endocytose and recycle its receptors, thus reducing the development of tolerance. The argument that an increase in Δ9-THC-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 Δ9-THC is contributing to provide analgesia. Further work with receptor antagonists may elucidate these mechanisms.

Other possibilities in defining the role of Δ9-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 Δ9-THC has been shown to reduce cAMP, it may be possible that Δ9-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 Δ9-THC may have actions similar to those of NMDA antagonists.

The potency of acute Δ9-THC is greatly increased in morphine-tolerant mice, indicating that these mice are more sensitive to the cannabinoid's antinociceptive effects and do not become tolerant to Δ9-THC, in contrast to previous reports in which morphine-pelleted mice demonstrated a significant decrease in the analgesic effects of Δ9-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 Δ9-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 Δ9-THC. These results suggest that effective antinociception can be produced in morphine-tolerant subjects utilizing 20 mg/kg Δ9-THC.

Combination therapy reduces not only morphine tolerance, but dependence as well. A 20 mg/kg p.o. dose of Δ9-THC administered for 7 days with morphine reduced naloxone-precipitated platform jumping by 50%. Previous studies show that repetitive pretreatment with Δ9-THC in mice treated chronically with morphine significantly reduces naloxone-precipitated 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). Δ9-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 μ-opioid receptors, thus reducing the development of dependence to morphine. Δ9-THC may serve the same function by usurping signaling pathways utilized by morphine to produce dependence at μ-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 Δ9-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 Δ9-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 Δ9-THC in the production of diarrhea in these mice.

Our results agree with previous reports that acute administration of Δ9-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 Δ9-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 Δ9-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 μ-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 Δ9-THC. This dose of Δ9-THC, although inactive on its own, is also sufficient to reduce the naloxone-precipitated morphine withdrawal sign of platform jumping in mice. The clinical implication of this work is the possibility that Δ9-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.

Source, Graphs and Figures: Modulation of Oral Morphine Antinociceptive Tolerance and Naloxone-Precipitated Withdrawal Signs by Oral
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