Introduction

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Opioids are used clinically to produce pharmacological effects such as antinociception. These drugs work via opioid receptors, which are found throughout the central and peripheral nervous systems. Endogenous ligands for these receptors (enkephalins, dynorphins, and -endorphin) can also produce such effects and play a role in controlling the perception of pain (Richardson and Akil, 1977; Bhargava, 1991). Three opioid receptors subtypes (µ, , ) have been identified and cloned (Kieffer et al., 1992; Chen et al., 1993; Yasuda et al., 1993), and each can be targeted by specific agonists and antagonists to study the role of each receptor type in pain perception. Morphine, a µ-opioid receptor agonist, has been seen to share several pharmacological effects (e.g., analgesia, euphoria) with -9 tetrahydrocannabinol (⁹-THC), the active constituent of marijuana.

Cannabinoids such as ⁹-THC produce a wide range of pharmacological effects such as antinociception. Previous studies have shown that the cannabinoids produce antinociception in mice and rats through spinal and supraspinal mechanisms (Smith and Martin, 1992; Welch and Stevens, 1992; Welch, 1993). The identification and cloning of specific cannabinoid receptors (CBs), CB1 in the brain (Matsuda et al., 1990) and CB2 in splenic macrophages (Munro et al., 1993), have led to the identification of specific antagonists for the CBs. Previous reports indicate that cannabinoids produce antinociception by indirect interaction with  opioids in the spinal cord (Smith et al., 1994; Pugh et al., 1996). Furthermore, the discovery of a bidirectional cross-tolerance of ⁹-THC and a synthetic cannabinoid, CP55,940, to  agonists in the tail-flick test (Smith et al., 1994) indicates that cannabinoids interact with  opioids. In addition, others have shown a functional relationship between  and µ opioid receptors in rat midbrain, suggesting a link between opioid receptors (Pan et al., 1997; Gutstein et al., 1998). ⁹-THC administered intrathecally (i.t.) has been shown to release endogenous opioids that stimulate both  and  opioid receptors (Welch, 1993; Smith et al., 1994; Pugh et al., 1996), which has been further substantiated by the findings that dynorphin antisera and nor-binaltorphimine (nor-BNI), a  receptor antagonist, block ⁹-THC-induced antinociception (Welch, 1993; Smith et al., 1994; Pugh et al., 1996; Reche et al., 1996a). However, other effects of ⁹-THC, such as hypothermia, catalepsy, and hypoactivity, remain unchanged after nor-BNI administration (Smith et al., 1994).

Many previous studies indicate that cannabinoids can enhance antinociceptive properties of opioids. The effects of morphine have been found to be enhanced by crude cannabis extract (Ghosh and Bhattacharya, 1979) and by p.o. ⁶-THC and ⁹-THC (Mechoulam et al., 1984). The administration of i.t. cannabinoids with i.t. morphine produces a greater-than-additive effect with respect to antinociception in mice (Smith and Martin, 1992; Welch and Stevens, 1992; Smith et al., 1994). Some cannabinoids have been found to enhance morphine in the brain, whereas others act predominantly in the spinal cord, as seen from a comparison of i.t. and i.c.v. administration (Welch et al., 1995). We recently reported that non-antinociceptive doses of ⁹-THC greatly enhance the antinociceptive effects of morphine. Furthermore, ⁹-THC and morphine administration by any two routes (i.t., i.c.v., s.c., p.o.) significantly enhances the potency of morphine (Smith et al., 1998). Other data suggest that ⁹-THC enhances the antinociception of morphine by triggering the release of endogenous dynorphin (Pugh et al., 1996). Corchero et al. (1997) report that 5-day treatment with ⁹-THC produces increases in both prodynorphin and proenkephalin gene expression in rat spinal cord.

Antagonist studies with CBs have implicated the CB1 receptor in the enhancement of morphine by ⁹-THC. We have shown that SR141716A blocks active doses of ⁹-THC but has no effect on morphine alone (Smith et al., 1998), demonstrating that SR141716A selectively antagonizes ⁹-THC. Furthermore, the enhanced antinociception due to a combination of a low p.o. dose of ⁹-THC and a low p.o. dose of morphine is blocked by SR141716A (Smith et al., 1998). Therefore, we propose that p.o. administration of ⁹-THC acts through the CB1 receptor to enhance the potency of morphine p.o.

Our goal was to further study the administration of ⁹-THC p.o. and opioids such as morphine p.o., due to the clinical relevance of this route of administration for human patients. This study was designed to test other µ opioid compounds for their relative potencies in the presence of an inactive dose of p.o. ⁹-THC by testing for antinociception via the tail-flick latency test. In addition, we wanted to further identify which receptors are involved in the enhancement of opioids by ⁹-THC by using specific antagonists to each of the opioid receptor subtypes.

	Materials and Methods

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Animals. Male ICR mice (Harlan Laboratories, Indianapolis, IN) weighing 21 to 24 g were housed five 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. The mice were brought to the test room and allowed to acclimate for 24 h to recover from transportation and handling.

Drug Administration Protocol. For the generation of dose-response curves for the various opioids alone and in combination with ⁹-THC, all drugs were given p.o. Morphine and other opioids were prepared in distilled water. ⁹-THC was prepared in a solution of emulphor, ethanol, and saline at a 1:1:18 ratio. Drugs were administered by p.o. gavage with an inactive dose of ⁹-THC (20 mg/kg) or its 1:1:18 vehicle 15 to 30 min before the administration of the opioid, and the animals were tested 10 to 30 min later using the tail-flick test for antinociception. Time course studies performed previously determined the times between administrations for each opioid so as to obtain peak effects of the drugs. These times are consistent with previous studies, which have shown that ⁹-THC administered p.o. has limited activity for up to 2 h (Smith et al., 1998). The enhancement of morphine was found to be greatest when ⁹-THC was administered p.o. 15 to 30 min before morphine.

Tail-Flick Test. The tail-flick test for antinociception was designed by D'Amour and Smith (1941). Control reaction times were between 2 and 4 s, and the maximum or cutoff time was 10 s. Antinociception was quantified using the percentage of maximum possible effect (% MPE) calculated as developed by Harris and Pierson (1964) as follows: % MPE = [(test  control)/(10  control)] × 100.

Statistical Analysis. Dose-response curves were generated using at least four doses of test drug. ED₅₀ values and 95% confidence limits were determined using unweighted least-squares linear regression for the log dose-response curves as described by Tallarida and Murray (1986; procedures 8 and 9). Parallelism between the curves was calculated using procedure 6, and potency ratios were calculated using procedure 11 (Tallarida and Murray, 1986). Significance between treatment groups in the antagonist studies was determined using Student's t test.

Drugs. The following were obtained from the National Institute on Drug Abuse (Rockville, MD): ⁹-THC, morphine, codeine, oxymorphone, hydromorphone, methadone, l--acetylmethadol (LAAM), heroin, meperidine, fentanyl, and pentazocine. Naloxone, NTI, and nor-BNI were obtained from Sigma Chemical Co. (St. Louis, MO).

	Results

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Enhancement of Potency of Opioids by ⁹-THC. Several µ opioids were evaluated for their antinociceptive properties via p.o. administration alone and in conjunction with an inactive p.o. dose (20 mg/kg) of ⁹-THC. This dose of ⁹-THC was chosen because it was found to be the highest inactive dose after p.o. administration. Results for these dose-response analyses as determined by the tail-flick test are presented in Figs. 1 to 5, with lines determined by linear regression analysis. ED₅₀ values with 95% confidence limits are listed in Table 1. Figure 1A shows a dose-response curve generated for morphine, a prototypical µ opioid agonist. This is a confirmation of similar results found previously in our laboratory by Smith et al. (1998). ⁹-THC (20 mg/kg p.o.) alone did not elicit antinociception. The potency of morphine p.o. was enhanced 2.2-fold by ⁹-THC. The curve shift was not statistically different from parallel, and the respective ED₅₀ values are shown in Table 1. Codeine p.o. was also enhanced by ⁹-THC, as seen in Fig. 1B. The parallel curve shift yielded a potency ratio of 25.8, indicating a large increase in the potency of codeine. Both morphine and codeine were full agonists when administered p.o. However, codeine in doses above 500 mg/kg were toxic to the animals and could not be tested.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   The antinociceptive effects of morphine and codeine are enhanced by 9-THC. Vehicle (1:1:18) or 9-THC at an inactive dose of 20 mg/kg was administered p.o. 15 min before morphine p.o. (A) or 30 min before codeine p.o. (B), and the animals were tested 30 min later in the tail-flick test. The data are presented as % MPE with each data point representing data from five to seven mice. Both 1:1:18 vehicle and distilled water produced no antinociceptive effects. , vehicle pretreatment; , 9-THC pretreatment.

View this table: [in this window] [in a new window]   TABLE 1 ED50 values and potency ratios for various opioids in combination with vehicle or 9-THC (20 mg/kg) in the tail-flick test for antinociception Mice were injected with vehicle (1:1:18; emulphor/ethanol/saline) or 9-THC (20 mg/kg) p.o. before p.o. opioid treatment. Time courses for treatments were described in Figs. 1 to 5. Dose-response curves were generated for each opioid. ED50 values and 95% CL were determined along with potency ratios as described in Materials and Methods.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   The antinociceptive effects of oxymorphone and hydromorphone are enhanced by 9-THC. Vehicle (1:1:18) or 9-THC (20 mg/kg) was administered p.o. 30 min before oxymorphone p.o. (A) or 15 min before hydromorphone p.o. (B), and the animals were tested 30 min later in the tail-flick test. The data are presented as % MPE with each data point representing data from five to seven mice. Both vehicles produced no antinociceptive effects. , vehicle pretreatment; , 9-THC pretreatment.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   The antinociceptive effects of methadone and LAAM are enhanced by 9-THC. Vehicle (1:1:18) or 9-THC (20 mg/kg) was administered p.o. 30 min before methadone p.o. (A) or 30 min after LAAM p.o. (B) and the animals were tested 30 min later in the tail-flick test. The data are presented as % MPE with each data point representing data from five to seven mice. Both vehicles produced no antinociceptive effects. , vehicle pretreatment; , 9-THC pretreatment.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   The antinociceptive effects of heroin, meperidine, and fentanyl are enhanced by 9-THC. Vehicle (1:1:18) or 9-THC (20 mg/kg) was administered p.o. 30 min before heroin p.o. (A), 15 min before meperidine p.o. (B), or 60 min before fentanyl p.o. (C), and the animals were tested 30 min later in the tail-flick test. The data are presented as % MPE with each data point representing data from five to seven mice. Both vehicles produced no antinociceptive effects. , vehicle pretreatment; , 9-THC pretreatment.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   The antinociceptive effects of pentazocine are not significantly enhanced by 9-THC. Vehicle (1:1:18) or 9-THC (20 mg/kg) was administered p.o. 30 min before pentazocine p.o., and the animals were tested 30 min later in the tail-flick test. The data are presented as % MPE with each data point representing data from five to seven mice. Both vehicles produced no antinociceptive effects. , vehicle pretreatment; , 9-THC pretreatment.

Receptor Identification via Antagonist Studies. It was found previously in our laboratory that the CB1-specific antagonist SR141716A successfully blocked the ⁹-THC-induced enhancement of morphine antinociception, implicating the CB1 receptor in the enhancement effect (Smith et al., 1998). These findings sparked our interest in using antagonists to the opioid receptors to determine which, if any, of those receptors are involved in the enhancement of opioids by ⁹-THC. Morphine and codeine were chosen as the representative opioid drugs because of their robust dose-response curves and significant shifts in ED₅₀. To confirm that the µ receptor was involved, we used naloxone at a dose of 1 mg/kg s.c. to antagonize the µ receptor. The animals received p.o. administrations of vehicle or ⁹-THC (20 mg/kg) and the opioid as per the time courses described in Materials and Methods. Naloxone was administered 5 min before the tail-flick test due to its short duration of action. As controls, the vehicle and naloxone were tested alone and did not show any antinociceptive effects; a high analgesic dose of ⁹-THC (150 mg/kg) was tested to show that the antagonists were specific for opioid receptors and not CBs. Figure 6 shows the effect of naloxone treatment on morphine (Fig. 6A) and codeine (Fig. 6B) alone and in combination with ⁹-THC. Both low and high doses of morphine and codeine were completely blocked by naloxone (P < .05). A high dose of 150 mg/kg ⁹-THC was not significantly attenuated by the µ antagonist. The enhancements of 20 mg/kg morphine and 25 mg/kg codeine by ⁹-THC were also significantly blocked by naloxone administration, indicating the involvement of the µ receptor in this enhancement.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   The effect of naloxone treatment on morphine (Mor; A) alone and in combination with 9-THC or codeine (Cod; B) alone and in combination with 9-THC. Naloxone (nalox; 1 mg/kg s.c.) or vehicle (veh) was administered 5 min before the tail-flick test, 25 min after opioid treatment p.o. or combination treatment p.o. series. The dotted bars represent vehicle (distilled water) treatment, and the striped bars represent naloxone treatment. The data are shown as % MPE ± S.E.M. with each bar representing data from 5 to 10 mice. Vehicle and naloxone alone are presented as controls. Bars denoted "20 THC/20 Mor" or "20 THC/25 Cod" represent the enhancement seen with low doses of opioid and an inactive 9-THC dose. 9-THC was administered 30 min before morphine or codeine. All doses are represented in mg/kg. *P < .05, **P < .01.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   The effect of NTI treatment on morphine (Mor; A) alone and in combination with 9-THC, or codeine (Cod; B) alone and in combination with 9-THC. NTI (2 mg/kg s.c.) or vehicle was administered 10 min before opioid treatment p.o. or 20 min after 9-THC administration in combination treatment p.o. series. The dotted bars represent vehicle (distilled water) treatment, and the solid bars represent NTI treatment. The data are shown as % MPE ± S.E.M. with each bar representing data from 5 to 10 mice. Vehicle and NTI alone are presented as controls. Bars denoted "20 THC/20 Mor" or "20 THC/25 Cod" represent the enhancement seen with low doses of opioid and an inactive 9-THC dose. 9-THC was administered 30 min before morphine or codeine. All doses are represented in mg/kg. *P < .05.

View larger version (28K): [in this window] [in a new window]   Fig. 8.   The effect of nor-BNI treatment on morphine (Mor; A) alone and in combination with 9-THC or codeine (Cod; B) alone and in combination with 9-THC. Nor-BNI (2 mg/kg s.c.) or vehicle was administered 90 min before opioid treatment p.o. or 60 min before 9-THC administration in combination treatment p.o. series. The dotted bars represent vehicle (distilled water) treatment, and the striped bars represent nor-BNI treatment. The data are shown as % MPE ± S.E.M. with each bar representing data from 5 to 10 mice. Vehicle and nor-BNI alone are presented as controls. Bars denoted "20 THC/20 Mor" or "20 THC/25 Cod" represent the enhancement seen with low doses of opioid and an inactive 9-THC dose. 9-THC was administered 30 min before morphine or codeine. All doses are represented in mg/kg. *P < .05.

View this table: [in this window] [in a new window]   TABLE 2 Comparison of antagonist effects on antinociception mediated by morphine, 9-THC, or a combination of 9-THC and morphine All antagonists were administered s.c. at appropriate time points so as to have peak action at the time of the tail-flick test. Naloxone was administered at a dose of 1 mg/kg, naltrindole at a dose of 2 mg/kg, and nor-BNI at a dose of 2 mg/kg. Morphine and 9-THC were administered p.o. Antinociception was measured using the tail-flick latency test as described in Materials and Methods. A designation of "Blocked" indicates that there was a significant decrease of antinociceptive effects by the antagonist (P < .05).

View this table: [in this window] [in a new window]   TABLE 3 Comparison of antagonist effects on antinociception mediated by codeine, 9-THC, or a combination of 9-THC and codeine All antagonists were administered s.c. at appropriate time points so as to have peak action at the time of the tail-flick test. Naloxone was administered at a dose of 1 mg/kg, naltrindole at a dose of 2 mg/kg, and nor-BNI at a dose of 2 mg/kg. Codeine and 9-THC were both administered p.o. Antinociception was measured using the tail-flick test as described in Materials and Methods. A designation of "Blocked" indicates that there was a significant decrease of antinociceptive effects by the antagonist (P < .05).

	Discussion

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In the present study, we sought to continue the investigation of p.o. cannabinoid-opioid combinations in producing antinociception. The p.o. route was chosen as a focus due to its relevance to clinical situations. The two main goals of this research were to identify opioids other than morphine that show a similar enhancement with ⁹-THC and to determine which receptors are involved in the elicitation of the enhanced antinociceptive effect with typical µ opioids. We have shown that many other µ opioids are enhanced by an inactive p.o. dose (20 mg/kg) of ⁹-THC. This dose of ⁹-THC lacked antinociceptive and behavioral effects alone. The enhancement of these opioids by ⁹-THC indicates that there is some type of interaction leading to a greater-than-additive effect, most likely via release of endogenous ligands on administration of these drug combinations.

We hypothesized that both CBs and opioid receptors were activated when ⁹-THC was administered p.o. in combination with morphine, as previously seen with i.t. administration. As shown by Smith et al. (1998), SR141716A, the specific CB1 receptor antagonist, fully attenuated the enhancement of p.o. morphine by p.o. ⁹-THC. Thus, ⁹-THC is acting at the CB1 receptor to enhance the potency of morphine. Similarly, we have shown in the present study that the µ and possibly the  receptors are also involved in the enhancement of p.o. opioids. Naloxone (µ selective at a low dose) effectively attenuated low and high doses of morphine and codeine and also the enhancement of both morphine and codeine by ⁹-THC. This confirmed that the antinociceptive actions of morphine and codeine involve the µ opioid receptor. Naloxone did slightly block p.o. ⁹-THC at a high dose of 150 mg/kg, indicating that ⁹-THC may be acting to produce antinociception through a µ receptor mechanism.

Our antagonism studies with the -specific antagonist NTI indicate that NTI does not block morphine- or codeine-induced antinociception, which is in agreement with previous observations by Pugh et al. (1996). Yet, NTI did seem to attenuate the antinociceptive effects of ⁹-THC alone. This could be attributed to a  blockade of enkephalins released by ⁹-THC. However, we observed a differential block of ⁹-THC-induced enhancement of opioids; only the enhancement of codeine, not of morphine, was significantly blocked by NTI administration. Thus, some difference between morphine and codeine seems to be critical in the interaction with ⁹-THC. Codeine exhibits much less first-pass metabolism in the liver when given p.o. than morphine; in addition, 10% of the drug is demethylated to form morphine (Vree and Verwey-van-Wissen, 1992). If codeine is less potent than morphine, it may take more of the antagonist to block the morphine effects. In addition, codeine may exhibit different binding patterns in the brain than morphine. Neil (1984) showed that although both morphine and codeine are µ selective, codeine does not differentiate much between  and  sites, whereas morphine has a much higher affinity for the  than the  receptor. Therefore, it may be that codeine is exerting some of its analgesic effects via receptors other than morphine. Also, there is strong evidence that the µ/ interaction in the brain is specific at one dose ratio for the ₂ subtype (Porreca et al., 1992). We need to examine ₁- versus ₂-specific antagonists to further specify whether only one or both of these receptors could possibly play a role in this enhancement effect and whether the effects of different opioids with ⁹-THC are subtype specific.

⁹-THC i.t. is known to release endogenous opioids, such as dynorphins, that act at opioid receptors to yield a greater-than-additive effect with morphine (Welch, 1993; Smith et al., 1994; Pugh et al., 1996). If this is so, then the metabolism of dynorphin to leu-enkephalin would indicate some activity at the  receptors that could be blocked by NTI. It was shown by Pugh et al. (1996) that enzymes that prevent dynorphin metabolism to leu-enkephalin blocked the enhancement of morphine by ⁹-THC when both drugs were given i.t. Our p.o. data show a slight but not significant block of ⁹-THC-induced antinociception with NTI, and therefore the presence of leu-enkephalin may be possible as a component of antinociceptive effects by ⁹-THC p.o. However, because nor-BNI fails to block the cannabinoid component and its enhancement of opioids, we could hypothesize that ⁹-THC p.o. does not release dynorphin as does ⁹-THC i.t. Thus, it is unlikely that any enhancement of opioids by ⁹-THC would occur via leu-enkephalin as a breakdown product of dynorphin. In fact, this points to the possibility that the NTI block of codeine in combination with ⁹-THC may actually occur due to leu-enkephalin release by codeine or, alternatively, the release of met-enkephalin by codeine or ⁹-THC. Met-enkephalin is also found to be blocked by NTI (Zachariou and Goldstein, 1996). A more detailed dose-response analysis with NTI in combination with ⁹-THC and the opioids may delineate further the involvement of the endogenous  receptor ligands in the enhancement effect.

It is likely that the enhancement via a p.o. route is mediated through the opioid receptors both spinally and supraspinally. The effect of ⁹-THC has been shown to differ in the enhancement of morphine i.t. versus i.c.v; thus, the  effect may be predominantly at a spinal site, whereas other mechanisms may come into play in the brain, as suggested by Reche et al. (1996b). Intracellular calcium concentration has been shown to be modulated by both morphine and ⁹-THC (Yamamoto et al., 1978; Harris and Stokes, 1982). Pugh et al. (1994) have also shown that a combination of inactive doses of ⁹-THC and morphine results in a greater-than-additive decrease in KCl-stimulated rises in intracellular calcium concentration and cAMP in the brain. The exact mechanism by which ⁹-THC p.o. enhances the potency of morphine p.o. is still largely unknown, and further studies may help to elucidate whether this enhancement is similar to that given i.t., i.c.v., or a combination of both.

In conclusion, we have shown that p.o. ⁹-THC at a low inactive dose enhanced the antinociceptive effects of several opioids; however, the receptors involved in this interaction have yet to be definitively identified. We see conflicting results from those seen in the spinal cord, indicating that perhaps ⁹-THC is not acting indirectly via both the  and  receptors to produce a greater-than-additive effect with morphine when the drugs are given p.o. There are several factors in regard to p.o. administration that have not been examined. For instance, the bioavailability of ⁹-THC may differ greatly from p.o. to i.t. administration, and thus we hypothesize that there may not be a large release of endogenous opioids because insufficient ⁹-THC is getting to its receptor fast enough. We also used a very low dose of ⁹-THC, so release of dynorphins may be negligible at this dose. Another factor is the diet and stomach contents of the animals; different results might be seen in selectively starved or water-deprived mice due to the faster uptake of the drug. Of course, there may be another distinct pathway that has not yet been identified by which p.o. ⁹-THC is enhancing p.o. morphine.

In this study, we have shown a potential use for low doses of ⁹-THC to enhance the potency of opioid drugs. Marketed for p.o. administration as dronabinol (Marinol), ⁹-THC is a schedule II drug currently used as an appetite stimulant in acquired immunodeficiency syndrome-wasting patients and as an antiemetic for cancer chemotherapy (Nelson et al., 1994; Beal et al., 1997; Timpone et al., 1997). Although high doses of ⁹-THC are analgesic, they can be accompanied by side effects such as anxiety, headache, euphoria, and tachycardia. Low doses of p.o. ⁹-THC have no analgesic effects; also, no behavioral changes such as ataxia, aggressiveness, or loss of righting reflex have been observed. Morphine is also commonly given in p.o. preparation, primarily to ease chronic pain. However, the continued administration of morphine can lead to tolerance and morphine-resistant pain, necessitating a steady increase in dosage that is potentially harmful to the patient. Also, high doses of morphine can have undesirable side effects such as respiratory depression, constipation, and nausea, but tolerance can develop to these effects of morphine (Ellison, 1993; de Stoutz et al., 1995). The administration of low doses of ⁹-THC in conjunction with low doses of morphine seems to be an alternative regimen for enhancing the pain-relieving effect of morphine without the side effects characteristic of either drug.