Vol. 303, Issue 2, 557-562, November 2002

Synergy between µ Opioid Ligands: Evidence for Functional Interactions among µ Opioid Receptor Subtypes

Laboratory of Molecular Neuropharmacology, Memorial Sloan-Kettering Cancer Center and the Program in Neuroscience, the Weill Graduate School of Medical Sciences of Cornell University, New York, New York (E.A.B., G.W.P.), and Department of Pharmacology, Temple University Medical School, Philadelphia, Pennsylvania (R.J.T.)

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

Pharmacological differences among µ opioid drugs have been observed in in vitro and in vivo preclinical models, as well as clinically, implying that all µ opioids may not be working through the same mechanism of action. Here we demonstrate analgesic synergy between L-methadone and several µ opioid ligands. Of the compounds examined, L-methadone selectively synergizes with morphine, morphine-6-glucuronide, codeine, and the active metabolite of heroin, 6-acetylmorphine. Morphine synergizes only with L-methadone. In analgesic assays, D-methadone was inactive alone and did not enhance morphine analgesia when the two were given together, confirming that L-methadone was not acting through N-methyl-D-aspartate mechanisms. Both L-methadone and morphine displayed only additive effects when paired with oxymorphone, oxycodone, fentanyl, alfentanyl, or meperidine. Although it displays synergy in analgesic assays, the L-methadone/morphine combination does not exhibit synergy in the gastrointestinal transit assay. This analgesic synergy of L-methadone with selective µ opioid drugs and the differences in opioid-mediated actions suggest that these drugs may be acting via different mechanisms. These findings provide further evidence for the complexity of the pharmacology of µ opioids.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Opioids have long been the mainstay in the treatment of severe pain, and of these, morphine has long been the gold standard. Most of the opioids used clinically have been classified as µ opioid analgesics, including drugs such as morphine, codeine, methadone, heroin, morphine-6-glucuronide (M6G), and fentanyl. Originally defined by their pharmacological profile in vivo (Martin et al., 1976), opioids are now classified by their sensitivity and selectivity in receptor binding studies. The µ drugs have high affinity and selectivity for binding sites in the brain labeled by compounds such as [³H]morphine and the µ peptide ³H-[D-Ala²,Me(Phe)⁴,Gly(ol)⁵]enkephalin and for their high affinity and selectivity for the cloned µ opioid receptor (MOR-1). Yet, many of these µ opioid analgesics have interesting pharmacological differences clinically. When patients highly tolerant to one µ-selective opioid are switched to another opioid, a technique termed "opioid rotation", they can often be controlled by doses of the second drug that are far lower than predicted by their relative potencies in naive subjects (Mercadante, 1999; Cherny et al., 2001). Methadone is particularly interesting, in view of its persistent analgesic activity in patients highly tolerant to other µ opioids (Crews et al., 1993). Animal models also reveal incomplete cross-tolerance among many µ opioids (Sosnowski and Yaksh, 1990; Rossi et al., 1996; Nielsen et al., 2000; Neilan et al., 2001; Pasternak, 2001). These clinical observations suggesting differing responses to µ opioids among patients led us to examine the interactions between a series of µ opioid analgesics.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Male CD-1 mice (25-30 g) were purchased from Charles River Laboratories Inc. (Wilmington, MA). All drugs used were obtained from the Research Technology Branch of the National Institute on Drug Abuse (Rockville, MD). Drugs were administered systemically via subcutaneous injections. Gastrointestinal transit was assessed by measuring the distance traveled by a charcoal meal (Paul and Pasternak, 1988).

Analgesia was assessed 30 min postinjection (unless otherwise stated) using the radiant heat tail-flick assay. Baseline latencies ranged between 2.0 and 3.2 s. A maximal cutoff latency of 10 s was set to minimize tissue damage. Analgesia was assessed quantally as a doubling or greater of the baseline latency for each mouse. Quantal measures have long been used in this assay (D'Amour and Smith, 1941; Le Bars et al., 2001), as previously published by our group (Pasternak et al., 1980a,b; Rossi et al., 1995, 1996; Neilan et al., 2001). Groups of mice were compared using Fisher's exact test. ED₅₀ values and 95% confidence limits were calculated by probit analysis (Tallarida, 2000).

To assess the statistical significance of the combinations, complete dose-response data were determined for each compound and were examined with probit regression analysis with the aid of PharmTools Pro (The McCary Group, Elkins Park, PA). Each compound was paired in a fixed-ratio combination with methadone to assess whether the combination displayed enhanced potency indicative of synergism. That assessment was made by determining the composite line of additivity for the combination and comparing that line to the dose-response regression line of the experimentally determined combination using ANOVA (Tallarida, 2000). A graphical assessment of synergy was also presented using isobolographic analysis (Roerig et al., 1984; Kolesnikov et al., 1996, 2000; Tallarida and Raffa, 1996; Tallarida et al., 1997).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

First, we established the relative potencies of a series of µ opioids by determining their ED₅₀ values from dose-response curves (Table 1). We then examined the interactions between a fixed dose of a number of µ opioids and a fixed dose of either L-methadone (Fig. 1A) or morphine (Fig. 1B). We chose the dose of each of the drugs that would give an analgesic response of approximately 10% alone, which corresponded to approximately a quarter of the ED₅₀ value. We administered each drug alone and compared each to the response seen with the coadministered pair.

View this table: [in this window] [in a new window]   TABLE 1 ED50 values of drugs alone and in combination with L-methadone Groups of mice (n  20) were injected subcutaneously with a fixed ratio of the stated drug and L-methadone and tested 30 min later for analgesia, with the exception of fentanyl, which was given 20 min after L-methadone injection (10 min before testing) in order for the peak effects to coincide with those of L-methadone. The following methadone/drug ratios, on a weight basis, were used: morphine, 0.5:1; M6G, 0.5:1; codeine, 0.5:1; 6-acetylcodeine, 9.5:1; fentanyl, 95:1. Values for the ED50 ± S.E.M. were determined using regression probit analysis, as described under Materials and Methods. The D-isomer of methadone showed no analgesia at a dose equivalent to the ED80 of L-methadone. In the combinations, both the observed and the predicted additive total doses of both drugs together are presented. ED50 values for the combination and the predicted additive values represent the total dose of the two drugs together. The amount of each component represents its fraction of the total dose, based upon the ratios of the drugs used to generate the dose-response curve. The enhanced potency, indicative of synergism, is indicated by a significant difference between the additive and combination ED50 values using ANOVA. All shown combinations, with the exception of fentanyl, indicated a significant potency enhancement. Isobolograms, with insets and confirming ANOVA tests, are shown in Fig. 3.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Interactions of L-methadone and morphine with other µ agonists. L-Methadone (A; 0.5 mg/kg s.c.) or morphine (B; 1 mg/kg s.c.) was administered alone or in combination with a variety of selective µ drugs. Dotted bars represent L-methadone or morphine alone, open bars represent the indicated drug alone, and stacked bars represent the added effect of the two drugs given alone. The filled bar represents the coadministration of the two drugs. Groups of mice (n  20) were injected subcutaneously with drug or drug plus L-methadone or morphine. Mice were tested 30 min after injection of L-methadone, morphine, M6G (1 mg/kg), codeine (1 mg/kg), 6-acetylmorphine (0.05 mg/kg), oxymorphone (0.01 mg/kg), oxycodone (0.5 mg/kg), or meperidine (2 mg/kg). Fentanyl (0.006 mg/kg) and alfentanyl (0.01 mg/kg) were given 20 min after morphine or L-methadone injection (10 min before testing) in order for the peak effects to coincide. Statistical significance was determined with Fisher's exact test.

Most of the single-dose combinations with L-methadone revealed interactions that appeared to be additive, including oxymorphone, oxycodone, fentanyl, meperidine, and alfentanyl (Fig. 1A). However, L-methadone gave responses with several other drugs that were far greater than anticipated. The combination of morphine and methadone clearly was greater than the sum of their independent actions (P < 0.001), as was the combination of M6G with L-methadone (P < 0.001). The combinations of L-methadone with either codeine (P < 0.001) or 6-acetylmorphine, the active component of heroin, also generated greater than additive responses (P < 0.001). Morphine, on the other hand, showed greater than additive interactions only with L-methadone (Fig. 1B). The interactions of morphine with all the other opioids tested yielded only additive effects. The interaction between morphine and methadone was seen only with L-methadone. D-Methadone, which has poor affinity for opioid receptors but which does interact with NMDA receptors (Davis and Inturrisi, 1999), did not show any effects alone at doses up to 4 mg/kg s.c., a dose corresponding to the ED₈₀ dose of L-methadone, and did not influence morphine responses when both were given together (Fig. 2). This was particularly impressive, since the dose of D-methadone was 8-fold higher than the dose of L-methadone that did reveal synergy.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Effect of the D- and L-isomers of methadone on analgesia in conjunction with morphine. Dotted bars represent morphine (1 mg/kg) alone, and open bars represent the D- or L-isomer of methadone (4 mg/kg and 0.5 mg/kg, respectively), together representing the expected additive effect of the two drugs. The filled bar represents the coadministration of morphine with either D- or L-methadone. Groups of mice (n  20) were injected subcutaneously and tested 30 min later for analgesia. Statistical significance was determined with Fisher's exact test.

We next determined the ED₅₀ values from dose-response curves using fixed ratios of L-methadone with a series of opioids (Table 1; Fig. 3). To determine whether the observed ED₅₀ values were synergistic, we compared them to predicted additive results. Isobolographic analysis provides a graphic approach toward assessing the possibility of synergy (Tallarida and Raffa, 1996). Graphical analysis of the data suggested synergistic L-methadone isobolograms with morphine, M6G, codeine, and 6-acetylmorphine (Fig. 3). We then assessed the synergy statistically by comparing a composite additive line to the experimental data, as described under Materials and Methods. The insets in Fig. 3 show the experimental lines and the calculated composite additive line. There was marked, statistically significant synergy between methadone and all the opioids tested, with the exception of fentanyl (Fig. 3).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Isobolographic analysis of combinations of L-methadone and several other µ-selective ligands. Dose-response curves were generated using fixed ratios of L-methadone with either morphine (A), M6G (B), codeine (C), 6-acetylmorphine (D), or fentanyl (E). ED50 values ± S.E.M. were determined, as reported in Table 1, and plotted. Isobolograms provide a visual approach to assessing the possibility of synergy. The points on the axes represent the ED50 values for either L-methadone or the second drug when each was administered alone. Combination drug points falling on or around the line connecting the ED50 values alone represent additivity, whereas points falling below the line suggest synergy. Although the isobolographic presentation is helpful in assessing the data, statistical significance was determined by comparing the dose-response curve for the combination to the composite additive curve determined from each agent alone, as described in Materials and Methods and shown in the insets. The experimental dose-response, plotted as a log-probit graph, was then compared with the composite additive line using ANOVA. Total doses of the two drugs combined were used in the plots. The combination of morphine and methadone was significantly greater than the composite additive line (F2,12 = 15.00; p < 0.002). The combination of M6G and methadone was significantly greater than the composite additive line (F2,11 = 17.03; p < 0.001). The combination of codeine and methadone was significantly greater than the composite additive line (F2,14 = 17.03; p < 0.001). The combination of 6-acetylmorphine and methadone was significantly greater than the composite additive line (F2,13 = 41.6; p < 0.001). The combination of fentanyl and methadone was not significantly greater than the composite additive line (F2,12 = 3.36).

Finally, we examined whether the interactions between L-methadone and morphine were restricted to analgesia or whether they extended to other mu opioid receptor actions. Both morphine and L-methadone inhibit gastrointestinal transit in the mouse. In contrast to the analgesic synergy between the two drugs, which was confirmed, the combination failed to show anything suggesting more than simple additive interactions for the combination with regard to the inhibition of gastrointestinal transit (Fig. 4). This observation is important, since it suggests that the therapeutic index for analgesia compared with at least one potential side effect should be significantly increased by using the combination of the two drugs.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   L-Methadone/morphine interactions on analgesia and the inhibition of gastrointestinal transit. Groups of mice (n = 10) received either L-methadone (0.25 mg/kg s.c.) or morphine (0.5 mg/kg s.c.) alone or in combination and were tested for analgesia and then for inhibition of gastrointestinal transit 30 min later. The anticipated values for additivity are indicated by the dashed lines. The analgesic activity of the combination was significantly greater than additive (P < 0.05), but inhibition of transit by the combination in the gastrointestinal transit assay was not.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Multiple µ receptor subtypes were initially defined pharmacologically with the use of selective antagonists (Wolozin and Pasternak, 1981) and then through incomplete tolerance among them (Pasternak et al., 1980a,b; Heyman et al., 1988; Paul and Pasternak, 1988; Paul et al., 1989; Rossi et al., 1996; Nielsen et al., 2000). The cloning of the µ opioid receptor MOR-1 quickly led to antisense mapping approaches that also revealed different profiles among µ opioids (Pasternak and Standifer, 1995; Rossi et al., 1995). Furthermore, M6G and heroin analgesia was retained in a knockout mouse lacking exon 1 of MOR-1 (Schuller et al., 1999).

Molecular biological approaches have now identified 15 splice variants of the µ opioid receptor gene Oprm, with at least 10 showing high affinity and selectivity for µ opioids in receptor binding assays (Bare et al., 1994; Zimprich et al., 1995; Pan et al., 1999, 2000, 2001). These MOR-1 splice variants also have been implicated in µ opioid analgesia through antisense mapping approaches and show unique regional distributions (Abbadie et al., 2000a,b,c, 2001; Abbadie and Pasternak, 2001).

Synergy was first described for morphine given both supraspinally and spinally (Yeung and Rudy, 1980), clearly showing the importance of regional interactions in morphine action. Regional morphine synergy is present among brainstem nuclei (Rossi et al., 1993) and even between the periphery and the central nervous system (Kolesnikov et al., 1996). Synergy also exists between µ and  opioids (Sutters et al., 1990; Adams et al., 1993; Rossi et al., 1994; Negri et al., 1995; He and Lee, 1998; Gomes et al., 2000). Thus, synergy is commonplace in opioid pharmacology.

Our current results show synergy between L-methadone and a number of other µ opioids. The results with single-dose combinations suggested the possibility of synergy, which was then confirmed using fixed-ratio dose-response studies of the combinations. L-Methadone significantly potentiated the analgesic activity of morphine, M6G, codeine, and 6-acetylmorphine. Although these drugs share synergistic interactions with L-methadone, distinguishing them from the other µ analgesics tested, they also differ pharmacologically among themselves. For example, morphine and M6G actions can be distinguished in antisense mapping studies that show different exon sensitivities for the two drugs (Rossi et al., 1995, 1996, 1997) and in MOR-1 knockout mice that are insensitive to morphine and remain sensitive to M6G (Schuller et al., 1999). Despite the insensitivity of CXBK mice to systemic morphine (Pick et al., 1993), these same mice respond normally to M6G (Rossi et al., 1996; Chang et al., 1998). Thus, the pharmacology of these two drugs differs, both from each other and from L-methadone.

Not all µ analgesics with differing pharmacological profiles revealed synergy when given in combination. Despite their pharmacological differences, as noted above, the combination of morphine and M6G showed only additive interactions. We also were surprised to see only additive interactions between L-methadone and fentanyl, two drugs that both retain analgesic activity in the CXBK mice.

NMDA receptors have been intimately associated with opioid analgesia and tolerance. Since methadone blocks NMDA receptors (Davis and Inturrisi, 1999), it was important to determine whether NMDA interactions might explain the synergy between morphine and methadone. Although D-methadone has poor affinity for opioid receptors, it still interacts well with NMDA receptors (Davis and Inturrisi, 1999). The inability of D-methadone to enhance the analgesic actions of morphine implies that NMDA receptors are not involved. Thus, L-methadone is modulating morphine actions through µ opioid receptors.

The combination of methadone with morphine offers a number of potential advantages, particularly since these interactions seem to be restricted to analgesia. The lack of synergy in the gastrointestinal transit studies is important, since the combination of the two drugs should provide a greater therapeutic index. Although we have not yet examined respiratory depression, earlier studies suggest a common mechanism as for the inhibition of gastrointestinal transit (Ling et al., 1985; Heyman et al., 1988; Paul and Pasternak, 1988).

In conclusion, our findings suggest the presence of functional interactions among µ opioid analgesics. These observations seem most consistent with the involvement of multiple subpopulations of µ opioid receptors, as originally proposed over 20 years ago (Pasternak and Snyder, 1975; Pasternak et al., 1980a,b; Wolozin and Pasternak, 1981). These findings also raise the possibility of potential clinical advantages of combining several different opioids in pain management. However, it remains to be seen whether morphine/methadone synergy can also be demonstrated in patients and to define the specific drug combinations that may be effective. Further work is needed to define at the molecular level the receptors responsible for these actions. Understanding the receptors involved in these actions and their drug selectivities will provide a major step forward in the development of rational pain management.

	Footnotes

Accepted for publication July 26, 2002.

Received for publication March 26, 2002.

This work was supported in part by a grant (DA07242) and a Senior Scientist Award (DA00220) to G.W.P., a grant (DA09793) to R.J.T. from the National Institute on Drug Abuse, and a core grant from the National Cancer Institute (CA08748) to Memorial Sloan-Kettering Cancer Center. E.A.B. was supported through a training grant (DA007274) from the National Institute on Drug Abuse.

DOI: 10.1124/jpet.102.035881

Address correspondence to: Dr. Gavril W. Pasternak, Department of Neurology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. E-mail: pasterng{at}mskcc.org

	Abbreviations

MOR-1, µ opioid receptor 1; ANOVA, analysis of variance; M6G, morphine-6-glucuronide; NMDA, N-methyl-D-aspartate.

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