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PHARMACOKINETICS AND PHARMACODYNAMICS |
From the Department of Pharmacy Practice and Science, College of Pharmacy, University of Arizona, Tucson, Arizona (Ms. Martin, Dr. Mayersohn); Scottsdale Police Department, Laboratory Division, Scottsdale, Arizona (Ms. Valdez); and Radiant Research, Inc., Tucson, Arizona (Dr. Boren).
Address for reprints: Michael Mayersohn, College of Pharmacy, University of Arizona, Tucson, AZ 85721.
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Key Words: Camphor • menthol • methyl salicylate • absorption • toxicity • dermal patches
A toxic dose of these compounds, especially camphor and methyl salicylate, can easily be ingested. Camphor has been associated with fatalities, especially in children. Many of these fatalities were associated with the ingestion of camphorated oil or CamphopheniqueTM. Sale of products containing more than 11% camphor was discontinued in 1983, but to this day, such products remain the cause of camphor poisonings.2 A nonlethal dose of camphor can result in hallucinations, tremors, fainting, cyanosis, arrhythmia, convulsions,3-7 and abortion.8 Chronic ingestion of camphor has led to hepatoxic effects.9 Menthol has been associated with allergic contact dermatitis10-12 and systemic allergic reactions.13 Local necrosis developed when menthol and methyl salicylate were applied to skin and a heating pad was allowed to cover the area.14 Methyl salicylate has an enticing wintergreen candy smell, and ingestion of as little as 4 mL has caused death in children.15 A pure form of methyl salicylate (oil of wintergreen) is readily available, and ingestion by children accounts for a large number of poisonings. In 1997, there were 7297 exposures to methyl salicylate in children younger than age 6 years.16
An adhesive patch impregnated with these compounds could eliminate the possibility of accidental oral ingestion, offer convenience in application, and still provide pain relief. Very little is known about the dermal absorption of these 3 compounds. One author has shown that the absorption of methyl salicylate could be increased in the presence of the other 2 compounds.17
The purpose of this study was to evaluate the systemic exposure to the 3 compounds following the application of a different number of adhesive patches to the skin of volunteer human subjects. Exposure was quantified by determining the plasma concentration-time profiles of the substances as a function of exposure to normal (2 patches) to very high doses (8 patches).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Subjects were randomly assigned to 1 of 3 dose groups, with each group made up of 4 men and 4 women. The 3 different dose groups were defined according to the number of dermal patches applied to the skin: 2, 4, or 8 patches. Each patch (SatogesicTM Medicated Adhesive patch; Sato Pharmaceutical Company, Ltd, Japan) contained the following active ingredients: 46.80 mg camphor, 37.44 mg menthol, and 74.88 mg methyl salicylate. The patches were supplied by the manufacturer.
On the morning of the study, an indwelling venous catheter (with a heparin lock) was placed into an ante-cubital vein. A blank or control blood sample was obtained for analytical purposes. At time 0, the appropriate number of patches (depending on the dose group) was applied to the skin of the arms and back. The entire margin along the outer circumference of the patches is made up of adhesive material, which forms a tight connection to the skin surface. The patches remained in place for the next 8 hours. All subjects were instructed to report any problems or adverse events immediately to the study investigator. Blood samples (10 mL) were obtained via the indwelling venous catheter (after removal of 1-mL fluid containing heparin and blood) at 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 12 hours. All times of sampling are relative to the placement of the patches. The catheter was removed at 12 hours, and the subjects were allowed to return home. The subjects returned to the clinic the following morning for a final (24-hour) blood sample to be taken by venipuncture.
All blood samples were collected into a 16 x 100-mm green-top Vacutainer® tube containing 143 U sodium heparin. Blood was immediately mixed by gentle inversion of the tube and centrifuged to obtain plasma. The plasma was stored in polypropylene tubes with snap-on caps at -12°C until analysis. All 3 analytes are stable for at least 8 weeks in frozen plasma.1
Analytical Methods
Plasma samples were analyzed for the 3 analytes using gas-chromatographic methods developed in this laboratory.1 Camphor and menthol concentrations were quantitated from a 2-mL plasma sample to which was added 25 µL of internal standard (3 µg/mL anethole in ethanol). Hexane (500 µL) was added and the mixture mechanically shaken for 10 minutes. Following centrifugation, the hexane layer was transferred into a tube containing 0.1 g anhydrous Na2SO4, and the tube was placed on ice. The extraction procedure was repeated 3 more times to achieve near-complete and consistent recovery of the compounds. The combined hexane extracts were evaporated under a gentle stream of nitrogen to a final volume of 30 to 100 µL. Samples were then analyzed by gas chromatography using a flame ionization detector. Standard curves were linear from 5 to 200 ng/mL. The lower limit of detection was 1 ng/mL, and the limit of quantitation was 5 ng/mL. The interday percent coefficient of variation (%CV) at 5 ng/mL was about 14% for camphor and 10% for menthol.
Methyl salicylate was analyzed from a 1.3-mL plasma sample to which was added 20 µL of the internal standard, ethyl salicylate (2250 ng/mL in ethanol). Acetonitrile (50 µL) and hexane (100 µL) were added to the plasma sample and mechanically shaken for 10 minutes. Following centrifugation, the hexane layer was transferred to an amber crimp-top vial containing 2 µL ethanol. The plasma was extracted one more time with 100 µL hexane. The hexane extracts were combined, and 25 µL BSTFA with 1% TMCS was added. The amber vials were sealed and heated in sand at 55°C for 30 minutes. Samples were analyzed by gas chromatography using a mass selective detector. The standard curve (area ratio vs methyl salicylate concentration) was linear over the range of 2.5 to 60 ng/mL. The lower limit of detection was 1 ng/mL, and the limit of quantitation was 5 ng/mL. The interday %CV at 5 ng/mL was about 6%.
All samples for an individual subject were prepared and analyzed on the same day. Generally, samples for 1 or 2 subjects were analyzed for camphor and menthol, and samples from up to 4 subjects were analyzed for methyl salicylate on 1 day. A minimum of 1 quality control sample was analyzed along with samples for 1 subject.
Any contamination peaks for menthol and/or camphor that were found in the subject's blank plasma were subtracted from the subject's samples. No corrections were needed for methyl salicylate concentrations.
Data Analysis
The plasmq concentration-time data were analyzed by noncompartmental methods. Individual concentration-time data were plotted on semi-logarithmic axes, and the data points best representing the terminal phase were selected. Those data points were then analyzed by linear regression to obtain the best estimate of the slope from which the terminal rate constant (k) and the corresponding half-life (t1/2) values were obtained. Those values were averaged to obtain the harmonic mean and pseudo-standard deviation.18 Values for the maximum plasma concentration (Cmax) and the time of its occurrence (tmax) were determined directly from the individual subject's data. The arithmetic mean and standard deviation were determined.
Average plasma concentration-time data across subjects for a given dose were analyzed by compartmental methods (WinNonlin®; Pharsight Corp, Mountain View, Calif), assuming linear kinetics and a 1-compartment model to provide a graphical illustration of the average data and the best-fitting relationship.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The average plasma concentration-time data for the 3 compounds as a function of dose are illustrated in Figure 1. The dermal application of 2 patches resulted in low plasma concentrations that are near the limits of quantitation for each compound at all times following application. The average Cmax values and standard deviation for camphor, menthol, and methyl salicylate were, respectively, 13.5 ± 4.8 ng/mL (range, 7.7-21.8 ng/mL), 7.6 ± 2.6 ng/mL (range, 5.1-10.8 ng/mL), and 8.6 ± 3.8 ng/mL (range, 4.0-12.7 ng/mL). The compounds could not be detected beyond 8 to 12 hours. Therefore, the application of 2 patches, containing the stated quantity of the 3 compounds, results in small plasma concentrations and low total systemic exposure.
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The 4-patch application resulted in plasma concentrations that were about twice those achieved from the 2-patch group. The average values for Cmax and standard deviation for camphor, menthol, and methyl salicylate were, respectively, 26.8 ± 7.2 ng/mL (range, 15.5-34.3 ng/mL), 19.0 ± 5.4 ng/mL (range, 9.0-24.8 ng/mL), and 16.8 ± 6.8 ng/mL (range, 8.9-25.7 ng/mL). The corresponding times of maximum plasma concentration were 3.0 ± 0.8 hours, 2.2 ± 1.2 hours, and 1.3 ± 0.6 hours.
The concentration-time profiles that were most reliable (from an analytical perspective) were those resulting from the 8-patch application. To provide a better appreciation for the between-subject variability in those profiles, Figure 2 illustrates the individual data for each compound. The clear impression that one obtains is that there is large intersubject variation in the concentration-time profiles for all 3 compounds. Plasma concentrations were, in general, measurable for 12 hours. An unusual exception was the rise in menthol concentrations from 12 to 24 hours in 3 subjects. Two other subjects who had immeasurable concentrations at 12 hours had quantifiable values at 24 hours (shown in Figure 2B). We have no explanation for this observation. The inset graphs illustrate the nonlinear regression fit of the average data.
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Table I summarizes several of the parameters that were determined for the 3 compounds following the 8-patch application. The average Cmax values evidenced about a 2-fold range for each compound, with the exception of camphor. In the latter instance, however, the values for 1 subject (#121) were not included in the calculation of the camphor averages. That same subject had the lowest Cmax values for the other compounds as well. The plasma concentrations of that subject (shown in Figure 2A) were beyond 2 standard deviations of the average values. The average Cmax values were somewhat less than twice those achieved with one-half the dose (ie, 4-patch application).
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The average tmax values were 3.5 hours, 3.4 hours, and 1.5 hours for camphor, menthol, and methyl salicylate, respectively. The relative rates of increase in concentrations of those compounds can be noted in the inset figures; methyl salicylate concentrations rose more rapidly than the other 2 compounds. The tmax values do not vary that markedly for camphor (2.0-4.0 hours), but there is a wider range of values for menthol (2.0-6.1 hours) and methyl salicylate (0.5-3.0 hours). These values are generally consistent with those following the 4-patch application, as noted above.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The total dermally applied dose varied over a 4-fold range (ie, 2-8 patches). The corresponding quantities of each compound contained in those patches, to which the skin was exposed, were as follows: 93.6 to 374.4 mg for camphor, 74.9 to 299.5 mg for menthol, and 149.8 to 599.0 mg for methyl salicylate. This dose range, although narrow for the examination of linearity in absorption and disposition, is unrealistically high for a typical pattern of use. The application of more than 2 patches (or its mass equivalent for the 3 compounds) at 
single time is unlikely, and the use of 8 patches at single time is improbable. Furthermore, leaving those patches attached to the skin for up to 8 hours is unlikely.
The application of 2 patches resulted in low plasma concentrations that were near the limits of quantitation. This was true during the entire 8-hour interval that the patches remained attached to the skin. In virtually all cases, the plasma concentrations of the 3 compounds were near or less than 10 ng/mL, and with rare exception, the compounds could not be detected beyond 8 to 12 hours postapplication.
The 4- and 8-patch applications resulted in plasma concentration-time profiles and parameter values (Cmax and AUC values) for all 3 compounds that suggested linearity among the doses tested. A definitive conclusion about linearity, however, cannot be reached because of the limited dose range examined, the relatively few subjects that participated, and the lack of a crossover design. Nonetheless, it is clear that camphor, menthol, and methyl salicylate undergo dermal absorption, resulting in some degree of systemic exposure.
Because the maximum plasma concentrations were achieved at times considerably shorter than the duration of the patch application (ie, tmax < 8 hours), it seems reasonable to conclude that whatever quantity is ultimately released from the patch has been released long before 8 hours. This appears to be particularly true for methyl salicylate, either because of its more rapid absorption and/or its loss from the patch via volatilization.
It is difficult to make definitive conclusions concerning the disposition kinetics of the compounds investigated. The terminal half-lives, for example, represent either absorption or disposition, whichever is the slower process. There is currently no basis for concluding one process over the other, nor are there published studies that would allow us to attribute unequivocal meaning to the terminal phase. With some individual exceptions, the terminal half-lives for a given compound tend to have a rather narrow range of values: 4 to 8 hours for camph or, 3 to 6 hours for menthol, and 2 to 4 hours for methyl salicylate. If dermal absorption contributes to or rate-limits the terminal phase, then the values for half-lives reported in Table I are overestimates of the correct, smaller values. Therefore, the half-lives reported are either overestimates or reasonable estimates of the correct terminal disposition half-lives. In any event, once-a-day or even twice-a-day application should produce little if any accumulation from continued use.
The only basis on which the meaning of the terminal phase may be discerned, at least for menthol, considers the results of a study by Gelal et al.19 Following the oral administration of menthol (100 mg) to human subjects, plasma concentrations of menthol were immeasurably low. In contrast, measurable plasma concentrations of a metabolite, menthol glucuronide, were found. These results suggest that menthol undergoes significant pre-systemic or first-pass (intestinal and/or hepatic) metabolism. The investigators reported that the terminal half-life for that metabolite averaged 1 hour. The terminal half-life for a metabolite must be equal to or longer than that for the parent compound, which acts as a precursor—in this case, menthol. The half-life of 1 hour for menthol glucuronide is considerably smaller than the average terminal half-life of menthol found here (
4.7 hours). Because the half-life for menthol must be equal to or shorter than 1 hour, the terminal half-life values noted here most likely reflect the absorption process. We are unable to make similar analyses for camphor and methyl salicylate.
In summary, the systemic absorption of camphor, menthol, and methyl salicylate from dermally applied patches has been examined. Plasma concentrations rise for several hours, and estimates of the terminal half-lives have been obtained. Concentration-time profiles are somewhat erratic and vary from subject to subject. Low-dose (2-patch) application results in plasma concentrations near the limits of quantitation. Extreme exposure to 8 patches for 8 hours (a duration of application unlikely to occur in practice) results in measurable concentrations of the 3 compounds, which average less than or about 40 ng/mL. Daily or even twice-daily application of these dermal patches (or other forms) is unlikely to result in substantial accumulation because the terminal half-lives are relatively short.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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Submitted for publication December 4, 2003; Revised version accepted June 18, 2004.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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2. Committee on Drugs, 1992-1993. Camphor revisited: focus on toxicity. Pediatrics. 1994;94: 127-128.
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), 4 (
), or 8 (
) patches. Each value is the mean obtained from all measurable concentrations within that group. The vertical bars represent the standard deviation of the mean.