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PAIN MEDICINE

Cutaneous Analgesia After Transdermal Application of Amitriptyline Versus Lidocaine in Rats

Anna Haderer, MD^*,, Peter Gerner, MD, Grace Kao, BA, Venkatesh Srinivasa, MD, and Ging Kuo Wang, PhD

*Department of Anesthesiology, Ried General Hospital, Ried, Austria; and Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts

Address correspondence and reprint requests to Peter Gerner, MD, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Address e-mail to gerner{at}zeus.bwh.harvard.edu

	Abstract

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Amitriptyline, a tricyclic antidepressant, has potent local anesthetic properties. However, there is no report of cutaneous analgesic effects after transdermal application. We report here that transdermally applied amitriptyline is more potent than lidocaine in providing cutaneous analgesia in rats. Solutions of amitriptyline base in 50, 100, and 500 mM concentrations were applied as a patch to rats, and their effects were compared with those of lidocaine base at the same concentrations and of the vehicle alone (45% water, 45% isopropyl alcohol, and 10% glycerin). Rats in each test group developed a concentration-dependent cutaneous analgesic block in the areas to which the drugs were applied; however, amitriptyline produced a longer block than lidocaine at the same concentration. The development of amitriptyline as a longer-lasting topical analgesic may improve our ability to treat chronic pain, such as neuropathic pain and neuralgia, and to prevent pain in procedures such as venipuncture.

IMPLICATIONS: The tricyclic antidepressant amitriptyline, often used perorally for the management of chronic pain, is shown here to be more potent than lidocaine in providing cutaneous analgesia when applied transdermally with an occlusive dressing in rats.

	Introduction

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Amitriptyline, a tricyclic antidepressant, is frequently used perorally for the management of chronic pain such as neuropathic pain, diabetic neuropathy, postherpetic neuralgia, fibromyalgia, and peripheral neuropathy of other etiology (1). Proposed mechanisms for the action of amitriptyline include inhibition of the reuptake of serotonin and norepinephrine (2), block of voltage-gated ion channels such as Na⁺ channels (3), block of N-methyl-D-aspartate receptors (4), and interaction with adenosine receptors (5). Although the mechanism is still unclear, new research continues to elucidate novel indications and modes of application for amitriptyline. Overall, its site of action is probably both central and peripheral (6), with a smaller therapeutic plasma concentration necessary for treating chronic pain than the 0.3–0.8 µM used in treating depression (7).

Amitriptyline is used in pain clinics primarily perorally, but an IV form is also available for patients unable to take oral medication. A peripheral analgesia by amitriptyline has previously been demonstrated (5,8), and routes such as intrathecal (9) and peritoneal (8) administration are used for various pain models in animals. Transdermal application with the intent to obtain a systemic effect has been reported as an alternative mode of delivery in the case of a severely depressed patient to whom the drug could not be administered perorally or IV (10). There is, however, no report of local cutaneous analgesic effects after transdermal application of amitriptyline. Two formulations containing local anesthetics for topical analgesia of the intact skin are currently available: Lidoderm, a lidocaine patch produced by Endo Laboratories, and a eutectic mixture of local anesthetics cream, a lidocaine/prilocaine mixture available from AstraZeneca. Although useful for a variety of indications, including back pain and postherpetic neuralgia (11), these transdermal formulations are limited by the short duration of their analgesic effects; a longer-lasting cutaneous local anesthetic would thus be valuable. Amitriptyline has been shown to be more effective than lidocaine (12) and bupivacaine (13) in a rat sciatic nerve block model and more effective than bupivacaine in skin infiltration (14). It is also more potent than lidocaine, bupivacaine, and cocaine in blocking Na⁺ currents in µ1 Na⁺ channels during repetitive pulses under voltage-clamp conditions (15). On the basis of these in vivo and in vitro properties, we hypothesize that amitriptyline has more potent analgesic effects than lidocaine when applied transdermally.

	Methods

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Approval for these experiments was obtained from the Harvard Medical Area Standing Committee on Animals. Male Sprague-Dawley rats weighing 200–250 g were handled to familiarize them with the protocol and were maintained on a 12-h light/dark cycle with unlimited access to food and water. Amitriptyline hydrochloride and lidocaine base were obtained from Sigma Chemical Co. (St. Louis, MO). The base form of amitriptyline was obtained by basifying an aqueous solution of amitriptyline hydrochloride with sodium hydroxide, extracting with ethyl acetate, and concentrating the organic extracts in vacuo. Test solutions of amitriptyline base at 50, 100, and 500 mM concentrations were freshly prepared in a vehicle of 45% isopropyl alcohol, 45% water, and 10% glycerin, along with control solutions (lidocaine base at the same concentrations and vehicle alone). The vehicle chosen for this study was based on the identical solution used by Kissin et al. (16) in their trials of transdermal lidocaine in humans. Amitriptyline at a concentration of 500 mM was not fully soluble; therefore, a different vehicle composition (70% isopropyl alcohol/20% water/10% glycerin), in which the drug dissolved completely, was used. The pH of all solutions, including the vehicle-only group, was adjusted to 8.5, because acid-sensing ion channels might be activated at a low pH (17).

After brief anesthetization by inhalation of sevoflurane, the rats (n = 6–10 per group for lidocaine; n = 5–7 per group for amitriptyline) were shaved, and a 2-cm² test area on the lumbar area of their backs was covered with gauze held in place by a transparent plastic adhesive dressing (Tegaderm). Great care was taken during these procedures not to injure the skin or break the skin barrier. Once the animals had fully recovered from the sevoflurane anesthesia, 0.3 mL of drug solution was injected through the dressing with a 30-gauge needle, directed obliquely to avoid the possibility of intradermal injection. The gauze was completely saturated by the solution, and no leakage was observed after withdrawal of the needle.

After 3 h, the dressings were removed, and the test area was delineated with a marker. The rats were evaluated by both nocifensive reaction and cutaneous trunci muscle reflex (CTMR) in response to stimulation with a blunt needle at the test area versus a contralateral control area. Nocifensive reaction consists of withdrawal and vocalization, and CTMR is characterized by reflexive movement of the skin over the back, produced by twitches of the lateral thoracispinal muscles in response to local dorsal cutaneous stimulation. After the animal’s normal reaction at the control area was observed, three sets of six pinpricks (at a frequency of 0.5–1 Hz) were applied to the test area, and the number of pinpricks to which the rat failed to respond was recorded. In the rare case of an unclear response to pinprick, the associated nocifensive reactions were taken into consideration to count or disregard a particular twitch response. Six pinpricks were found to be sufficient for consistent results without causing injury to the rat from excessive pinpricking. The analgesic effect was scored quantitatively by the number of times the pinprick failed to elicit a response and qualitatively by the resulting nocifensive behavior and CTMR, which was graded, averaged, and analyzed by an unpaired Student’s t-test at each time point by using methods similar to those previously described (18). The responses were graded in terms of maximum possible effect (MPE)—a complete absence of response to all six pinpricks (complete nociceptive block) was scored as 100% MPE, the absence of three responses out of six was scored as 50% MPE, and a response identical to the control was scored as 0% MPE. The pinprick testing was conducted 3, 5, 7, 10, 15, 20, and 25 h after application of the drug, ceasing when nocifensive behavior and CTMR indicated full recovery from the block. All testing was conducted by a single experimenter who was blinded to data recorded at previous time points.

	Results

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Two rats in the 100 mM amitriptyline group were excluded because of displacement of the occlusive dressing and leakage of the drug. Both of these rats displayed no block at all, probably because of an application time of <0.5 h with the drug-saturated gauze. All other rats in both study groups developed a concentration-dependent analgesic block in the areas of the skin to which the drugs were applied. Rats to which only vehicle was applied displayed no change over baseline at any time during the observation period. On removal of the patch, the skin that had been in contact with drug, vehicle, or both initially appeared slightly swollen and was more or less indistinguishable among the various groups. This irritation disappeared within the first hour and therefore most likely was caused by the patch itself. Amitriptyline and lidocaine elicited analgesic effects of different durations and degrees (% MPE). As seen in Figure 1, the dose-dependent analgesic effect of amitriptyline was greater than that of lidocaine at corresponding concentrations. For example, lidocaine at 100 mM had a maximal analgesic effect of 45.9 ± 4.2% (mean ± SEM) with full recovery at 15 h, and amitriptyline at the same concentration displayed a maximal analgesic effect of 70.8 ± 15.0% with complete recovery at 25 h.

View larger version (19K): [in this window] [in a new window]   Figure 1. Analgesic effects of transdermally applied amitriptyline and lidocaine. Patches containing solutions of 50, 100, and 500 mM lidocaine (A), n = 6–10, and amitriptyline (B), n = 5–7, were applied to rats for 3 h. The resulting nociceptive block was evaluated by nocifensive reaction and cutaneous trunci muscle reflex in response to stimulation with a blunt needle at the test area versus a contralateral control area. Stimulation of this control area, as well as the test area of rats in the vehicle-only group, consistently scored indistinguishable from 0% maximum possible effect (for clarity, data are omitted in the graph). The time course of the analgesic effect is presented in terms of maximum possible effect. Time points at which the analgesic effect of amitriptyline is significantly (P < 0.05) greater than that of lidocaine at the same concentration are denoted by * (Student’s t-test). Because of skin redness and skin induration at 25 h in the 500 mM amitriptyline group, testing was temporarily suspended; resumption of testing several days later indicated a complete return to baseline nociception.

	Discussion

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This is the first report of amitriptyline base providing analgesia of the skin when applied transdermally; however, amitriptyline HCl, the salt form, has been shown to provide local analgesia when injected peripherally (5,8). Although lidocaine products with similar modes of application are currently available, they require long application times and provide analgesic effects of relatively short duration. Our experiments in rats show that three hours of application under an occlusive dressing is necessary for adequate efficacy; however, a shorter application time may be sufficient in humans because of differences in the rate of permeation between rat and human skin (19). The discovery of a more effective vehicle may also decrease the application time required. In any case, the identification of a drug with longer cutaneous analgesic effects than lidocaine represents a potentially important step in the search for longer-lasting local anesthetics.

The mechanism of amitriptyline causing analgesia of the skin is unclear and beyond the scope of this study. We speculate that, in addition to Na⁺ channel blockade, other known mechanisms of amitriptyline that were primarily confirmed in the central nervous system may also, in part, contribute to peripheral analgesia, including blockade of Ca⁺ channels and block of histamine, cholinergic, and -adrenergic receptors (20).

For these experiments, the base form of amitriptyline, rather than amitriptyline hydrochloride, was chosen. In a pilot experiment, solutions of amitriptyline hydrochloride, unadjusted for pH, were applied in the same manner as above, but with little or no resulting analgesic effect (Haderer et al., unpublished observations, 2002). Most likely, the charged nature of amitriptyline hydrochloride prevents it from penetrating the skin, whereas the lipid solubility of amitriptyline base, as evidenced by its high log P value of 4.92, allows it to more easily penetrate the relatively lipophilic stratum corneum.

Because skin toxicity becomes evident at a concentration of 500 mM, we attempted to determine whether this toxicity is a consequence of the amitriptyline itself or of other components in the test solution. The formulation used in this set of experiments contains the base form of amitriptyline, the preparation of which involves ethyl acetate, an organic solvent known to cause skin irritation. We speculate that residual ethyl acetate may have contributed to the skin toxicity observed. To address this possibility, we prepared the drug solution via an alternative route, dissolving amitriptyline hydrochloride directly in the water/isopropyl alcohol/glycerin vehicle. Because the ratio of molecules in the neutral base form to those in the charged hydrochloride form is dependent on the pH of the solution, it was necessary to titrate the amitriptyline hydrochloride solution to a pH of 8.5 with sodium hydroxide to match the measured pH of the 100 mM solution prepared from amitriptyline base. The analgesic effects from this new preparation were comparable to those obtained previously; however, both skin induration and redness were reduced, and redness disappeared completely by 25 hours in the 500 mM group. It is therefore likely that residual ethyl acetate contributed to the skin irritation.

The largest dosage of amitriptyline administered in these experiments was 0.3 mL of a 500 mM solution, containing 47 mg of the drug. Previous studies have shown, though, that IV injection of 7.5 mg/kg of amitriptyline in rats, or approximately 1.875 mg per 250-g rat, causes severe electrocardiogram changes leading to death within 10 minutes (21). Because no overt cardiac impairment or sedation was observed in any of the rats, it is clear that only a minute fraction of the transdermally applied amitriptyline was absorbed into the bloodstream. Cardiac toxicity, commencing at plasma levels >3 µM in humans (7), is the most serious adverse effect of amitriptyline. However, because even a dose as large as 47 mg was tolerated by rats, it is unlikely that the transdermal application of amitriptyline in humans will present any safety concerns regarding systemic adverse effects.

In summary, the antidepressant amitriptyline, often used perorally for the management of pain, is more potent than lidocaine in providing cutaneous analgesia when applied transdermally with an occlusive dressing in rats. Future investigations with different vehicle compositions, application times, or both may improve skin tolerance to larger concentrations of amitriptyline. On the basis of these initial studies, the development of new therapeutic uses of transdermal amitriptyline appears to be feasible. A longer-lasting topical analgesic would be useful in the treatment of chronic pain, such as neuropathic pain and neuralgia, and in the prevention of pain in procedures such as venipuncture, IV cannulation, vaccination, circumcision, dermatological procedures, and skin grafting. Further testing will be necessary to determine the potential of this drug in humans.

	Acknowledgments

 
Supported by Grants GM48090 (GKW) and GM07592 (PG) from the National Institutes of Health, Bethesda, MD.

	References

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Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999