Anesth Analg 2003;97:1103-1107
© 2003 International Anesthesia Research Society
PAIN MEDICINE
Analgesic Synergy Between Topical Morphine and Butamben in Mice
Yuri A. Kolesnikov, MD PhD*,
Marcela Cristea, MD , and
Gavril W. Pasternak, MD PhD
*Department of Anesthesiology and
Laboratory of Molecular Neuropharmacology, Memorial-Sloan Kettering Cancer Center, New York, New York
Address correspondence and reprint requests to Yuri Kolesnikov, Department of Anesthesiology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021. Address email to kolesniy{at}mskcc.org
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Abstract
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Studies have revealed that lidocaine is an effective analgesic when applied topically to the tail of a mouse in the radiant heat tail-flick assay. In addition, the topical combination of lidocaine with morphine revealed synergistic interactions between the two drugs. In the current studies, we demonstrate that topical butamben, benzocaine, and bupivacaine are active in the radiant heat tail-flick assay. In this assay, topical lidocaine has a ceiling effect and displays a biphasic curve, with large doses markedly decreasing the responses almost to baseline levels. In contrast, butamben has an S-shape dose-dependent response in the assay and did not display a biphasic curve as seen with lidocaine, suggesting that topical butamben may have advantages over lidocaine. Both benzocaine and bupivacaine also showed dose-dependent analgesic activity in this model. Like lidocaine, butamben/morphine combinations displayed synergistic interactions. Indeed, the synergy appeared more prominent with a butamben/morphine combination. We also observed synergy between topical benzocaine and morphine. Although the bupivacaine/morphine combination was suggestive of synergy on isobolographic analysis, it did not achieve statistical significance. These studies indicate that a series of local anesthetics are all active topically in the radiant heat tail-flick assay in mice and that several interact synergistically with morphine. Of the local anesthetics tested, butamben seemed to have several pharmacological characteristics, alone and in combination with morphine, which suggest that it may be superior to the other local anesthetics. Together, these observations suggest that topical combinations of opioids and local anesthetics may prove clinically valuable.
IMPLICATIONS: Topical administration of the opioid µ-agonist morphine and the sodium channel inhibitors butamben and benzocaine results in a synergistic interaction for antinociception in radiant heat tail-flick assay in mice, suggesting that the combination of these drugs will enhance rather than detract from the analgesia of either alone.
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Introduction
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Local anesthetics are widely used for pain management (1–3). Local anesthetics produce a dose-related block of the voltage dependent sodium channels, and thereby reduce axonal conduction (4). Morphine is a mu opioid receptor agonist with central and peripheral actions (5–8). Opioid receptor activation activates an inwardly rectifying potassium channel and closes voltage sensitive calcium channels, resulting in membrane hyperpolarization, reduced calcium influx, and inhibition of neurotransmission (5). Numerous studies have shown the advantages of the combination of lidocaine and morphine (9–11). Although the mechanism of opioid and local anesthetic interaction are yet to be fully described, several groups have reported synergy between lidocaine and morphine given centrally. Intrathecal coadministration of lidocaine and morphine produces excellent antinociceptive actions, and addition of the local anesthetic can reduce the dose of opioid used and hence reduce adverse side effects (12,13). We also have observed a potent synergistic interaction between peripheral opioids and lidocaine in the radiant heat tail-flick assay (14)
There are a number of different local anesthetics. Bupivacaine is a widely used amide local anesthetic that has had the greatest influence on the practice of regional anesthesia since the introduction of lidocaine (4). Bupivacaine is a more potent sodium channel blocker and has a longer duration of action compared with lidocaine (4). Butamben (n-butyl-p-aminobenzoate) is an amino ester type of local anesthetic, with tetrodotoxin-like effects (15). When given epidurally to patients with chronic pain it has an unusual long-lasting effect (16). The preservation of motor functions during and after butamben treatment suggests that the epidural application of the local anesthetic has a selective effect on sensory nerve fibers (17). Benzocaine is a local anesthetic that is also from an ester group and it is widely used topically (4). As noted above, prior work from our laboratory has revealed that topical combinations of opioids and lidocaine synergize to induce a local analgesia. In the current study we have investigated whether topical butamben, benzocaine, and bupivacaine also have topical analgesic activity in the radiant heat tail-flick assay and whether they display similar synergy with morphine.
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Methods
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This study was approved by the Institutional Animal Care and Use Committee at Memorial Sloan-Kettering Cancer Center. Male CRL:CD-1(ICR)BR mice (25–30 g; Charles River Breeding Laboratory, Bloomington, MA) were maintained on 12-h light/dark cycle with food and water available ad libitum. Mice were housed in groups of five until tested. Morphine was generously provided by the Research Technology Branch of the National Institute on Drug Abuse (Rockville, MD). Lidocaine, butamben, bupivacaine, and benzocaine were purchased from Sigma Chemical (St. Louis, MO). Lidocaine base was used in all experiments unless indicated otherwise.
Drugs were applied topically and analgesia assessed as previously described (18). Briefly, the distal portion of the tail (2–2.5) was immersed in a 90% dimethyl sulfoxide (DMSO) solution containing the indicated drugs for the stated time, usually 2 min (18). In our initial studies we have demonstrated that DMSO alone has no effect when tested in this manner in the radiant heat tail-flick assay (18). Furthermore, DMSO provides an effective way of solubilizing a wide range of drugs and facilitating their transport into the skin. The onset of analgesia is rapid, with peak effects seen immediately after the removal of the tail from the treatment solution. Therefore, we tested animals immediately after termination of topical administration.
Testing was performed on the portion of the tail immersed in the treatment solution because the analgesic actions of drugs administered in this manner are restricted to the exposed part of the tail; proximal regions are not affected (14,18). Analgesia was defined quantally as a tail flick latency for an individual animal that was twice its baseline latency or more. Baseline latencies typically ranged from 2.5 to 3.5 s, with a maximum cutoff latency of 10 s to minimize tissue damage in analgesic animals. In some groups of mice, to ensure a local effect, testing was also done with a more proximal segment of the tail that was not exposed to the drug solution.
Because analgesia was assessed quantally, group comparisons were performed with Fisher’s exact test (8,18). The 50% effective dose (ED50) values were determined with the Bliss program (19,20).
Isobolographic analyses were used to determine drug interactions (21,22). ED50 values were determined for each drug alone. They were then tested together at various doses at a constant ratio based on their respective ED50 values. In Figure 1, values on the axes represent the ED50 values for the indicated drug alone and the lines connecting these values correspond to simple additive interactions. Points lying below the line of additivity indicate synergism. Significance was assumed when there was no overlap between the confidence limits of the combination value and the confidence limits of the line of additivity.
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Figure 1. Topical morphine and local anesthetics in the mouse. Groups of mice (20 mice per dose) were exposed for 2 min to the various doses of drugs. A, Lidocaine (free base), morphine, and butamben. B, Other groups of animals received benzocaine or bupivacaine (20 mice per dose). Analgesia was tested immediately after termination of the treatment (see Methods). Lidocaine produced a biphasic analgesic effect, with larger doses showing less response. Butamben produced a full dose-dependent analgesic effect. Both bupivacaine and benzocaine were effective peripheral analgesics in this paradigm.
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Results
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Topical Morphine and Local Anesthetics
Tail flick latencies from the unexposed (proximal) portion of the tail were similar to baseline latencies of the exposed (distal) portion (2.9 ± 0.3 and 2.7 ± 0.2 s, respectively). Furthermore, no analgesic response was seen with large doses of these drugs in the proximal portions of the tail, which was not exposed to the local anesthetic solutions, confirming the local effects (Table 1). Lidocaine is effective topically in the radiant heat tail-flick assay (Fig. 1A), achieving a maximal response of nearly 75% with a potency more than morphine (Table 2). However, the dose-response curve was biphasic, with larger doses showing less response. Indeed, the analgesic actions were almost completely lost at the largest lidocaine dose tested (43 mM or 10 mg/mL). We also examined several other local anesthetics. Butamben also was effective, with potency similar to that of lidocaine (Table 2, Fig. 1A). However, butamben differed in several respects from lidocaine. It was able to produce a classical S-shape dose-dependent response. Although there was a slight decline in its activities at the largest doses, it did not show the profound loss of activity at large concentrations seen with lidocaine.
We next explored whether the responses seen with butamben and lidocaine extended to the local anesthetics benzocaine and bupivacaine. Both bupivacaine and benzocaine administered topically elicited a dose-dependent analgesic effect (Fig. 1B). It is interesting that the curves for these compounds were more shallow than those seen with either lidocaine or butamben and there was a suggestion of a plateau for both as well.
Previous studies have shown that the peak effects after topical administration are seen immediately after removal of the tail from the drug solution (14). We saw the same peak effect immediately after removal of the tail from butamben, benzocaine, and bupivacaine. Their responses gradually declined to baseline levels over the next 60 min (Fig. 2). This response was longer in duration than equipotent topical morphine and lidocaine doses (14).
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Figure 2. Time-course action of topical drugs. Groups of mice received a 2-min topical exposure to butamben (5.2 mM; n = 20), bupivacaine (3.0 mM; n = 10), or benzocaine (12 mM; n = 10) and were tested in the tail-flick assay at the indicated time over 60 min (see Methods). Their analgesic effects gradually declined to baseline levels over the next 60 min.
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Morphine/Local Anesthetic Combinations
We next examined combinations of the local anesthetics and morphine. Previous studies have documented synergy between lidocaine and morphine (14). Initially, we performed dose-response curves of the local anesthetics and morphine alone. We next determined the ED50 values from dose-response curves using fixed ratios of morphine with a series of local anesthetics. 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 (21). Coadministration of morphine with butamben enhanced their combined relative potencies approximately fivefold, a greater enhancement than that seen with the lidocaine/morphine combination (Table 2). Isobolographic analysis was consistent with synergy, as shown by the lack of overlap of the 95% confidence limits (Fig. 3A). The morphine and benzocaine combination also revealed synergy, with an approximate sixfold shift of their individual ED50 values (Fig. 3B, Table 2). Combining morphine and bupivacaine produced approximately a threefold shift for the 2 drugs (Fig. 3C, Table 2). Although suggestive, the enhanced activity of the combination did not achieve statistical significance.
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Figure 3. Topical morphine and butamben and benzocaine interaction. Using fixed ratios of morphine/butamben (2.5) and morphine/benzocaine (1) or morphine/bupivacaine (4), the ED50 value of combinations were determined with the 95% confidence limits and plotted. Isobologram provide a visual approach to assessing the possibility of synergy. The points on the axes represent the ED50 values of either morphine or the second drug when each was administered alone. Combination drug points occurring on or around the line connecting the ED50 values alone represent additivity, whereas points below the line suggest synergy. Statistical significance was confirmed by the lack of the overlap between the 95% confidence limits. Both the morphine/butamben (A) and the morphine/benzocaine (B) combinations showed statistical significance. The ED50 values of the morphine/bupivacaine (C) combination were below the theoretical line of additivity, but did not achieve statistical significance because there was overlap of the confidence limits.
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Discussion
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The paradigm was selective for local mechanisms. The analgesic actions seen with topical local anesthetic and morphine were limited to the region of the tail exposed to the drug and were not seen in more proximal areas not exposed to the drug. All the local anesthetics tested were potent and effective topical analgesics in the radiant heat tail-flick assay. Both butamben and benzocaine are amino ester local anesthetics and are insoluble in water (4). However, current studies indicate that they can be used topically with other solvents or delivery approaches. In in vitro studies, it has been reported that butamben selectively affects inactivation of fast sodium currents in cultured dorsal root ganglion neurons, thus substantially reducing membrane excitability resulting from a "hyperpolarization" type of block (15). This mechanism is similar to that produced by the other ester local anesthetic, benzocaine, and the typical fast sodium channel blocker tetrodotoxin (15). In our paradigm, butamben was a potent analgesic with several advantages over lidocaine. Unlike lidocaine, butamben did not have a ceiling effect, and large doses did not have the dramatic decline in analgesic activity seen with lidocaine. The lidocaine dose-response curve was biphasic, with concentrations larger than 30 mM giving a progressive decrease in response. The reasons for this are not clear, but it is interesting that lidocaine concentrations larger than 10 mM can act directly on rat sensory neurons to cause membrane depolarization, an increase in [Ca2+] I, and neurotoxicity (23). However, the incidence of nerve injury resulting from the clinical use of lidocaine intrathecally is relatively small, possibly because of dilution and diffusion of the local anesthetic in the relatively large volume of subarachnoid space (3). These are major factors contributing to the minimization of lidocaine concentration at the neuronal membrane after spinal administration and potential toxicity. Theoretically, in our paradigm, using large doses of topical lidocaine (>10 mg/mL) may result in depolarization of nerve endings and loss of analgesic activity of local anesthetic. More studies are needed to elucidate the mechanism(s) of topical lidocaine-induced biphasic effect.
Both lidocaine and bupivacaine are amino amide local anesthetics that block sodium channels. Bupivacaine is approximately four times more potent than lidocaine (4) and is better suited for phasic blocks because it dissociates more slowly from the closed channels than does lidocaine (24). In our study, topical bupivacaine was approximately twice as potent as lidocaine as well. The combination of bupivacaine and morphine was more effective than the drugs used alone, and the ED50 of the combination occurred below the line of additivity; however, overlap between the 95% confidence limits for the drugs alone and the combination implies the synergy is not significant. Additional studies will be required to determine if bupivacaine is mechanistically different from the other local anesthetics or whether our ability to detect synergy was impaired by the type of study.
Many drugs modulate the actions of central opioids, raising the question of whether similar interactions can be observed in the periphery. We found that topical lidocaine has synergistic interactions with morphine, levorphanol, and buprenorphine (14). This was not unexpected. Synergistic interactions might be more likely when drugs act on different mechanisms, as shown here with morphine and butamben as well with morphine and benzocaine. Isobolographic analysis confirmed synergy between morphine and the local anesthetics tested. The most impressive interaction was between morphine and butamben. However, it is not clear whether this resulted from its receptor selectivity or other factors, such as its greater lipophilicity, which would enhance its ability to become diffused through the skin. Combined with its advantages over lidocaine alone, the butamben/opioid combination may prove clinically superior to lidocaine/opioid formulations.
The demonstration of synergy between morphine and local anesthetics deserves further study. However, even without a full understanding of how these drugs interact, the demonstration of topical synergy between morphine and butamben opens many clinical possibilities in pain management.
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Acknowledgments
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Supported, in part, by a research grant (DA07241) and a Senior Scientist Award (DA00220) (to G.W.P.) and a Mentored Scientist Award (DA00405) (to Y.A.K) from the National Institute on Drug Abuse, as well as a grant from EpiCept Corporation (Y.A.K).
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Accepted for publication May 21, 2003.
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Top
Abstract
Introduction
