Anesth Analg 2004;98:1407-1412
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000107936.69436.5B
PAIN MEDICINE
Rapid Skin Anesthesia Using a New Topical Amethocaine Formulation: A Preclinical Study
M. I. Arévalo*,
E. Escribano
,
A. Calpena,
J. Domenech, and
J. Queralt*
*Departament de Fisiologia-Divisió IV and
Departament de Farmàcia, Unitat de Biofarmàcia i Farmacocinètica, Universitat de Barcelona, Spain
Address correspondence and reprint requests to Dr. Josep Queralt, Unitat de Fisiologia Divisió IV, Facultat de Farmàcia, Universitat de Barcelona, c/Joan XXIII s/n, 08028-Barcelona, Spain. Address e-mail to jregue{at}farmacia.far.ub.es
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Abstract
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We developed a fast-acting topical amethocaine emulsion and tested its analgesic activity against heat or mechanically induced pain in a rat paw model. The first experiment was performed in rats made hyperalgesic or allodynic after carrageenan-induced inflammation. Rats were distributed in five subgroups, each receiving topically one of the following: amethocaine microemulsion, amethocaine gel (Ametop®gel), EMLA (Eutectic Mixture of Local Anesthetics) cream, amethocaine infiltration, or nothing (controls). The second experiment was conducted on healthy, selected heat- or touch-hypersensitive rats, which were distributed as in the first experiment. Paw withdrawal time from a heat and a mechanical stimulus was used as a pain index. In the first experiment, antihyperalgesic activity appeared at 4.2, 13.8, and 14 min after amethocaine microemulsion, gel, or EMLA cream, respectively. Amethocaine microemulsion was the only topical formulation with an antiallodynic effects, although less than with amethocaine infiltration. In healthy rats (second experiment), all topical formulations produced similar analgesic effects in heat-induced pain of the ipsilateral paw. Activity in the contralateral paw appeared earlier with amethocaine microemulsion, which was also the only one that increased touch-induced withdrawal time in the ipsi- and contralateral paws. Therefore, the microemulsion could be valuable for improving amethocaine skin penetration and thus bringing rapid pain relief.
IMPLICATIONS: Topical anesthetics are used in several painful clinical procedures, but they tend to have a slow onset time. A new amethocaine microemulsion with a faster onset of analgesia than commercial formulations was developed and its activity tested in pain states induced by heat or mechanical stimulus in inflamed and healthy rat paws.
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Introduction
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The discomfort caused by infiltration of local anesthetics has stimulated research into developing topical anesthetics. These drugs relieve distress in children (1,2) and adults (3) during venepuncture and venous cannulation and for capillary heel prick blood sampling in neonates (4). They have also been used for minor superficial surgery (5,6).
However, the available formulations have a number of disadvantages, particularly a long delay between application and anesthetic effect and the need for an occlusive dressing. Thus, the instructions for use of EMLA cream (a eutectic mixture of 2.5% lidocaine and 2.5% prilocaine; Astra, Läkemedel, Sweden) or Ametop®gel (4% wt/wt amethocaine base preparation; Smith & Nephew, Dublin, Ireland) indicate that painful procedures should only be performed 60 min and 30–45 min, respectively, after application. With both preparations, the cream or gel must be covered with a plastic film to be effective.
Topical anesthetic preparation has also been effective for relieving neuropathic pain without the unacceptable side effects of conventional oral preparations (7).
We developed a 4% microemulsion of amethocaine in an attempt to achieve faster drug permeation and thus reduce the time for it to reach optimum anesthetic effect.
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Methods
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A microemulsion of amethocaine for topical application was prepared with oil (decane) (Fluka Chemie GmbH, Steinheim, Switzerland), water, and the low foam surfactant lauromacrogol 300 (synperonic A7, Uniqema, Wilmington, DE). The mean diameter of the disperse phase was 7.06 nm, as determined by light scattering (Photon correlation spectrometer Malvern 4700, Malvern Instruments, Malvern, United Kingdom) at the Department of Surfactants Technology of the Centre for Research and Development, CSIC (Barcelona, Spain). In addition, 0.5% amethocaine in saline was prepared and Ametop®Gel and EMLA were purchased.
Male Sprague-Dawley rats (220–250 g; Charles River, Santa Perpetua de la Mogoda, Barcelona, Spain) were housed 4 per cage with a 12-h light/dark cycle. Food and water were available ad libitum. The rats were acclimatized to temperature and humidity in controlled facilities for at least 8 days before the experiment. They were accustomed to handling (30 min of handling per day for 3 days) and to the testing equipment before the experiments.
The study protocol was approved by the Animal Experimentation Ethics Committee of the University of Barcelona, Spain. The animals were treated in accordance with the Ethical Guidelines for Investigation of Experimental Pain in Conscious Animals of the International Association for the Study of Pain (8).
Two separate experiments were performed to determine the analgesic activity of the formulations. The first experiment was performed in hyperalgesic or allodynic rat inflamed paws, and in the second, analgesic activity was tested in healthy rats. Paw withdrawal time from a heat and a mechanical stimulus was used as an index of nociception.
In the first experiment, 50 µL of a 0.5% saline solution of 
-carrageenan (Sigma-Aldrich, Química, Spain) was injected (9) with a 27-gauge needle coupled to a 50-µL Hamilton syringe into the palm of the left hindpaw. Control rats received 50 µL of saline into the left hindpaw. Three hours after carrageenan injection, rats were tested for the presence of hyperalgesia or allodynia. Rats with paw withdrawal latency of between 3 and 5 s for both heat and touch stimulus were selected for the study. Rats were randomly divided into 6 groups (5–9 rats each): Groups A, B, and C received topically (sole and palm surfaces of the left hindpaw) 200 µL of a 4% amethocaine microemulsion, 4% amethocaine gel (Ametop®gel), or EMLA cream, respectively. All these formulations were dispersed in the paw by continuous gentle friction for 30 s. No occlusive dressing was used in any case. Group D received 100 µL of 0.5% amethocaine in saline subcutaneously (sc) in the plantar surface of left hindpaw. Finally, Group E was the untreated control group.
In the second experiment, the topical anesthetic activity of the formulations was tested in healthy rats, distributed as outlined before for inflamed rat paws. Every group included rats spontaneously hypersensitive to heat or touch. The time elapsed until withdrawal of the paw from the stimulus was recorded automatically in seconds (see later for details).
We also measured the interdigital temperature of inflamed and contralateral healthy paws of untreated rats and that of inflamed paws before and at selected times after topical application of local anesthetic formulations. The effects of the vehicle administration either topically or sc were also determined. Temperature measurement was performed using a digital thermometer (Demestres® 305, Demestres SA, Barcelona, Spain) fitted with a probe.
Clinical measurements were performed as follows. Thermal stimulation was tested using a Plantar Test (Ugo Basile, Comerio, Italy) modeled on that described by Hargreaves et al. (10). Rats were placed individually in Plexiglas cubicles mounted on a glass surface maintained at 25°C, and a thermal stimulus, in the form of radiant heat emitted from a focused projection bulb, was applied to the midplantar surface of each hindpaw. Heat intensity was maintained at 4.5 amps, and the maximum time of exposure was set at 20 s to limit possible tissue damage.
Touch sensitivity was measured in both hindpaws using an automated apparatus for applying reproducible light touch (Dynamic plantar Aesthesiometer 37,400, Ugo Basile). Rats were placed in their compartments on a metal mesh surface. After a short period, during which they explored their surroundings, they remained still in a resting position until the test began. With the help of an adjustable angled mirror, the touch stimulator unit was placed beneath the selected hindpaw, with the filament below the plantar surface of the rat. When the unit was started, the electrodynamic actuator lifted the stainless-steel filament, which touched the plantar surface, and exerted a force less than the threshold of feeling. The force was then increased until the rat moved its paw or until a force of 50 g for 30 s was applied.
All the above-mentioned measurements were taken at 0, 2.5, 5, 12, 18, 24, and 30 min after anesthetic treatment. Data are expressed as mean ± SD. Because some variables increased to a peak and then returned to a baseline, results were analyzed by the method described by Matthews et al. (11). Pain threshold of each rat with or without treatment was measured as the area under the curve (AUC; time of paw withdrawal versus time from 0 to 30 min). The maximum effect (Emax) and the time required to reach the maximum effect (Tmax) were also determined. The results were compared using analysis of variance, with treatment and time as independent variables and response as the dependent variable. A value of P < 0.05 was considered statistically significant.
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Results
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Hyperalgesia appeared in carrageenan-inflamed paws (Table 1). The mean reaction time was 5.04 ± 0.2 s and 15.85 ± 0.3 s for inflamed ipsilateral versus contralateral healthy paw, respectively. The microemulsion applied to ipsilateral paw increased reaction time to heat. The AUC of paw withdrawal time versus time and the Emax increased by 89% (P < 0.0130) and 97% (P < 0.0004), respectively, with respect to inflamed untreated paws (control untreated). After application of Ametop® gel, AUC increased by 73% (P = 0.0099) and Emax by 58% (nonsignificant) compared with inflamed, untreated paws. In rats treated with EMLA cream, AUC increased by 58% and Emax by 43%, both increases being nonsignificant. The greatest analgesic effect was obtained after infiltration of amethocaine, with an increase in AUC and Emax of 239% (P < 0.00001) and 163% (P < 0.00001), respectively.
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Table 1. Antihyperalgesic and Antiallodynic Effect of Anesthetic Formulations on Carrageenan- Induced Paw Inflammation (ipsilateral paw)
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The peak effect (Tmax) occurred significantly faster after application of the amethocaine microemulsion than after amethocaine gel, EMLA cream, or amethocaine infiltration. In none of the rats tested was there an increased pain tolerance to heat in the contralateral healthy paws.
Allodynia was clearly seen in carrageenan-treated edematous paws. The mean reaction time was 3.98 ± 0.3 s and 22.8 ± 0.3 s for ipsilateral inflamed and contralateral-healthy paws, respectively. Only topical microemulsion and amethocaine infiltration increased the paw withdrawal time after a light touch stimulus. Contralateral paws of inflamed rats were not affected by any treatment (data not shown).
As shown in Figure 1, from 3 h to 3.5 h after carrageenan injection (0 to 30 min of treatment), the skin temperature of the ipsilateral paw was higher than that of the contralateral healthy paw (P < 0.001). After ipsilateral topical treatment with amethocaine microemulsion or vehicle, paw skin temperature decreased to a mean value that did not differ from that of contralateral healthy paws. Vehicle infiltration in the inflamed paw did not change its temperature. None of the amethocaine gel, EMLA cream, or the vehicle alone affected the temperature of the inflamed paw (data not shown).
Healthy rats selected for high sensitivity to heat-induced pain had paw withdrawal latency of 12.2 ± 0.5 s and 11.6 ± 0.4 s in the right and left paw, respectively (Tables 2 and 3 ). As seen in Table 2, all formulations tested produced analgesia in both ipsilateral and contralateral paws. All formulations showed activity in the ipsilateral paw, as evidenced by similar and significant increases in AUC and Emax as compared with healthy untreated paws. However, the effect of the microemulsion and amethocaine infiltration appeared before that of the gel. In the contralateral paw, all topical formulations produced similar increases in the AUC and Emax; however, the microemulsion acted faster than the gel or EMLA. A greater effect was seen with amethocaine infiltration, which always peaked at 5 min.
Healthy rats selected for high sensitivity to light touch-induced paw withdrawal had a reaction time of 5.8 ± 0.3 s and 6.7 ± 0.6 s for the right and left paw, respectively. In the ipsilateral paw, the amethocaine microemulsion increased the time of paw flick withdrawal after a light-touch stimulus. After amethocaine gel and EMLA cream, a nonsignificant increase in both AUC and Emax was seen. Tmax was similar for all topical formulations. Amethocaine infiltration produced greater effects and peaked before that of topical formulations. In the contralateral paw, only the microemulsion and amethocaine infiltration produced significant increases in the touch-paw flick. In both cases, the effect appeared early at 6.8 ± 3.2 min and 7.0 ± 3.4 min, respectively.
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Discussion
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The prolonged onset of maximum analgesia is a significant handicap of current commercial formulations of topical anesthetics. In an attempt to overcome this, we developed a new amethocaine microemulsion, and our results using animal models confirmed that it has a much faster onset than the two commercial formulations tested.
There are few preclinical reports regarding the efficacy of topical anesthetic formulations in animal models. Flecknell et al. (12) reported that the application of EMLA to the tail in rats before cannulation of the lateral tail vein did not significantly reduce behavioral responses to venepuncture. Because of the paucity of literature on this topic, our first task, and second goal, was to define an experimental model to test topical anesthetic activity. Hyperalgesia, and particularly allodynia, have been considered manifestations of neuropathic pain. However, hypersensitivity to heat or light touch stimulus has been reported in acute inflammatory models, such as carrageenan-induced edema (13–15), and in chronic experimental arthritis (16). Thus, in our first experiment, analgesic activity was tested in rats made hyperalgesic and allodynic by carrageenan. However, we used a smaller carrageenan dose than usual (50 µL of a 0.5% solution) (10,17,18) because at larger doses, rats showed spontaneous pain behavior. Moreover, hyperalgesia or allodynia induced by large doses was not suppressed by infiltration with 100 µL of amethocaine 1%. However, hypersensitive rats used in the second experiment were selected independently for their sensitivity to heat or touch because animals hypersensitive to one stimulus are not necessarily hypersensitive to another. They were usually younger than nonhypersensitive healthy ones (19).
It is worth noting that amethocaine microemulsion always produced an immediate antihyperalgesic effect. In contrast, the peak effect of amethocaine gel and EMLA cream appeared later. One possible explanation for the faster onset of analgesia is that local anesthetics such as amethocaine are more easily released from a microemulsion than from a gel or cream. This, and the smaller particle size of amethocaine, may facilitate its percutaneous absorption.
Only topical amethocaine microemulsion released enough amethocaine to increase the reaction time after a light touch stimulus in the ipsilateral paw. On the whole, reduced touch threshold after inflammation seems to be more difficult to reverse with anesthetics than heat threshold. This follows the general rule that the small nonmyelinated nerve fibers (C and A
), which transmit pain sensations, are more susceptible to the action of local anesthetics than the large myelinated Aß fibers that transmit touch stimulus. As a result, the sensation of pain disappears before that of touch (20).
Topical, but not sc, application of the microemulsion or its vehicle produced an immediate decrease in the previously increased temperature of the inflamed paw by 4°C. Thus, heat loss, which could be attributed to vehicle-induced structural changes in the stratum corneum, was clearly seen. The vehicle may momentarily open channels to allow the passage of the drug and then close immediately. The effect disappeared rapidly, and the decrease in skin temperatures only lasted 5 min. This permeation was also favored by small particle size in the disperse phase because vehicles passed through the narrow (
30 nm) intercellular passages (virtual pores) in the outer skin layers (21).
In healthy rats, all the anesthetic formulations were effective in reducing heat-induced pain in both the ipsilateral-treated paw and in the nontreated contralateral paw. Because of the relatively high stimulus threshold in healthy rat paws, and because a cutoff of 20 s in heat stimulus was fixed to avoid tissue damage, only a narrow range of pain had to be overcome in these rats. It was, therefore, not surprising that all the formulations tested showed an effect. It was more surprising that the basal reaction time of contralateral paws (similar to that of the ipsilateral paw) also increased after both topical and infiltration anesthesia. A similar finding has been reported for lidocaine in a rat mononeuropathic model (22). This antinociceptive effect was reduced when the contralateral sciatic nerve was ligated (23–25). Thus, ipsilateral anesthesia of sciatic nerve afferents from one paw may affect contralateral touch input, probably when communication of the inputs coming from both paws occurs in the spinal cord. In our rats, the more rapid release of amethocaine from the microemulsion may have contributed to this effect.
Touch sensitivity in healthy rats of both ipsilateral and contralateral paws increased only after amethocaine microemulsion and amethocaine infiltration. To understand these results, we have to remember that rats used in this study were selected because their threshold to touch stimulus was very low in both paws. Thus, both healthy paws reacted to touch in approximately 5 s, whereas the majority of contralateral paws made allodynic reacted only to touch after 23 s. Therefore, it is possible that only small concentrations of amethocaine are required to increase the lower threshold of contralateral paws. Even so, these data reveal the rapid permeation of amethocaine from microemulsions as opposed to the other topical formulations.
Our results highlight two disadvantages of using healthy animals to test local anesthetic activity. First, the low pain range that can be tested and, second, the fact that both ipsi- and contralateral paws increase their heat sensitivity after both topical and sc anesthetic application. Thus, the increase in the threshold of both paws needs to be considered when assessing the effect of an anesthetic formulation. An additional consideration is that the contralateral paw should not be used as a control. In conclusion, in our rat models, the amethocaine microemulsion proved to be a fast-acting promising analgesic in experimental preclinical studies.
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Acknowledgments
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Supported by a grant from the Spanish Ministry of Science and Technology (SAF2001–3402).
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Accepted for publication October 31, 2003.
Top
Abstract
Introduction
). Changes in paw temperature of carrageenan-inflamed paws after its topical treatment with amethocaine 4% microemulsion (
) or vehicle (
) and after subcutaneous treatment with the vehicle (
). *
P < 0.05 are differences with respect to inflamed paw or contralateral healthy paw, respectively.
