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vehicle partition coefficient
Journal of Controlled Release 67 (2000) 211–221 www.elsevier.com / locate / jconrel
Reduction in skin permeation of N,N-diethyl-m-toluamide (DEET) by altering the skin / vehicle partition coefficient q a ,1 a, Julie S. Ross , Jaymin C. Shah * a
Department of Pharmaceutical Sciences, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA Received 2 November 1999; accepted 19 January 2000
Abstract Reported adverse side effects after using N,N-diethyl-m-toluamide (DEET)-containing mosquito repellent products appear to be the result of significant absorption of DEET through human skin. The overall objective was to develop formulations of DEET with significantly reduced permeation using the basic principles and model of skin permeation based on Fick’s laws of diffusion at steady state. Ternary phase diagrams of DEET with water and semipolar solvents, ethanol, PG and PEG 400, showed an increase in the aqueous solubility of DEET. This resulted in a linear decline in octanol / water PC with an increase in the concentration of the solvent. Permeation of DEET across human skin was studied from vehicles containing various amounts of PG and PEG 400 using an infinite dose technique and Franz diffusion cell. DEET’s flux reduced with increasing PG concentration and the flux from 90% PG was 9.962.1 mg / cm 2 h, 6-fold lower than flux of pure DEET control, 63.2624.5 mg / cm 2 h. Flux was reduced 6-fold from 60% PEG 400 solution, and permeation of DEET was totally prevented from 90% PEG 400 which was very viscous. However, a combination of 60% PEG 400 with 30% PG not only reduced permeation 9-fold but was suitable as a vehicle for formulation. The decrease in flux and permeability of DEET with increasing concentration of solvent appeared to be a direct result of decrease in skin / vehicle PC and octanol / water PC. This study clearly demonstrates that alternative formulations can be developed for DEET aimed at reduced permeation and toxicity unlike the current formulations some of which contain ethanol which has been shown to enhance permeation of DEET. A similar approach can be used for developing formulations of other industrial and occupational agents to prevent their skin permeation when a user may be exposed to them. 2000 Elsevier Science B.V. All rights reserved. Keywords: DEET; Mosquito repellent; Solvent; Skin permeation; Partition coefficient; Propylene glycol; Polyethylene glycol; Ethanol
1. Introduction q
This research was conducted as dissertation research by J. Stinecipher towards partial fulfillment of the requirements of doctor of philosophy degree at the Medical University of South Carolina. *Corresponding author. Present address: Pharmaceutical RandD, Pfizer Central Research, Ms 8156-39, Eastern Point Road, Groton, CT 06340, USA. Tel.: 11-860-715-2332; fax: 11-860441-0467. 1
Present address: IBAH Pharmaceutics Services, 525 Virginia Drive, Fort Washington, PA 19034, USA.
Use of N,N-diethyl-m-toluamide (DEET) containing mosquito repellent products have been associated with many adverse reactions [1–4]. Recently, DEET has also been implicated as a factor in neurotoxicity resulting from the Gulf War chemical exposure [5]. A review of the biodistribution and toxicity of DEET by Robbins and Cherniack suggested that there were several areas of toxicity that needed further evalua-
0168-3659 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 00 )00210-8
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tion [6]. DEET is absorbed through the skin very rapidly as shown by several investigators [7–10]. Our previous studies have shown not only significant absorption of DEET from all commercial products but enhanced permeation of DEET across human skin from 15 to 60% ethanol, a solvent for DEET in many products [11]. Therefore, it is very evident that dermal absorption of DEET may be responsible for the potential toxicity problems associated with DEET-containing products, and therefore, new or alternative formulations of DEET specifically aimed at decreased permeation and absorption will be safe and useful [12,13]. In order to develop these alternative formulations with decreased absorption of DEET, the basic principles of skin permeation should be applied to DEET. The simplest model to describe dermal absorption based on Fick’s laws of diffusion at steady state is as follows [14]: dc / dt ss 5 Jss 5 DAK /h (Cd 2 Cr )
(1)
where Jss 5steady state flux or the permeation rate; A5surface area of the skin exposed or of the application site; h5the thickness of the rate-limiting barrier, usually the stratum corneum; D5the diffusion coefficient of the diffusant, K5the skin / vehicle partition coefficient; Cd 5the thermodynamic activity of the diffusant in the vehicle and Cr 5the concentration of the diffusant in receptor, close to zero under sink conditions. Therefore, only D, K, and Cd may be altered to reduce the permeation of diffusant. By selecting an appropriate vehicle, the thermodynamic activity and the skin / vehicle partition coefficient can be reduced to minimize skin permeation, although, high thermodynamic activity of DEET in vehicle will be required to maintain its ability to repell mosquitos. The diffusion coefficient is relatively difficult to alter since it is dependent on a number of factors such as the skin characteristics, effect of vehicle on skin, skin hydration and interaction of diffusant with skin. Therefore, using an appropriate vehicle, either K, D, and / or the skin can be influenced to reduce permeation of DEET using this basic principle in order to develop improved formulations of DEET. The specific aim of the present study was to reduce and minimize skin permeation of DEET by reducing its skin / vehicle PC (K) using semipolar
solvents which may decrease DEET’s n-octanol / water PC. The permeation of DEET from each vehicle was evaluated by performing in vitro permeation studies across human skin with a Franz diffusion cell using an infinite dose technique. Permeation profiles were constructed in each case and the steady state flux, diffusion coefficient, lag time, permeability, and skin / vehicle partition coefficient were calculated to investigate the mechanism of reduction in flux. The permeation of DEET from each vehicle was compared to that of pure DEET in an effort to determine the reduction in flux and overall permeation.
2. Materials DEET (97%) was purchased from Aldrich Chemical Company. Propylene glycol, and methanol (HPLC grade) were purchased from Fisher (Fair Lawn, NJ, USA). Carbowax Sentry Polyethylene glycol 400 NF, FCC Grade was a gift from Union Carbide (Danbury, CT, USA). Phosphate buffer solution (0.1 M, pH 7.4) was prepared with sodium phosphate monobasic monohydrate (Mallinckrodt, St. Louis, MO, USA) and sodium phosphate dibasic anhydrous (Curtin Matheson Scientific,Houston, TX, USA).
3. Methods
3.1. Phase solubility diagram; effect of solvent on aqueous solubility of DEET Since DEET is a liquid at room temperature, a ternary phase diagram was constructed for DEET, water, and each solvent: ethanol, propylene glycol (PG) or polyethylene glycol 400 (PEG 400), to determine the miscibility of the three liquids. A pair of liquids such as DEET and water that are miscible with each other only to a slight extent would usually exist as two phases. The addition of a third component to the system that is miscible in all proportions with one of the solvents would be expected to produce a single phase in which all three components are miscible with each other. Solutions were prepared consisting of PG, PEG 400 or ethanol, and
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varying percentages of DEET from 1 to 99%. Water was added to these solutions in 100 ml increments until the solutions appeared turbid after vortexing for 30 s. A ternary plot was constructed in order to determine the binodal curve, or the boundary of the two-phase area as seen in Fig. 1. Regions above the binodal curve represent homogeneous solutions of the three liquids. Homogeneous or single phase solutions containing 10% DEET and varying percentages of PG and PEG 400 were selected for in vitro permeation experiments.
3.2. Effect of solvent on the n-octanol /water partition coefficient of DEET n-Octanol and water with various concentrations of each solvent were presaturated with each other in an amber colored bottle for 48 h. The two phases were separated using a separating funnel. DEET was added in small amounts to a mixture of equal volumes of the octanol and water phases in a scintillation vial, and allowed to agitate for 48 h to reach equilibrium. At the end of 48 h, the octanol and water phases were separated and individually analyzed for DEET concentration using UV spectrophotometric assay. The o / w partition coefficient was calculated from the ratio of concentrations in the two phases, octanol and water, and plotted as a function of solvent concentration (Fig. 2). However, the
Fig. 2. Effect of solvent on the apparent n-octanol / water partition coefficient of DEET.
experimentally determined PC is an apparent o / w PC since each solvent will itself partition between octanol and water and thus change composition of each phase and influence the PC of DEET directly.
3.3. Skin preparation Human skin samples were obtained from elective plastic surgery, and the fat and other visceral debris were removed from the underside of the freshly excised skin. The skin was then washed with 0.1 M, pH 7.4 phosphate buffer solution before freezing at 2208C. The full thickness skin samples were cut into 2 cm 2 pieces and allowed to thaw overnight at room temperature in phosphate buffer solution prior to the in vitro percutaneous permeation experiments.
3.4. In vitro percutaneous permeation
Fig. 1. Ternary phase diagrams of solvent, water and DEET showing the homogeneous (single phase) and heterogeneous regions.
The permeation experiments were conducted with vertical Franz diffusion cells (Crown Glass Company, Somerville, NJ, USA) each having a receptor volume of 4.9 ml and a diameter of 0.9 cm. Pieces of full thickness human skin (2 cm 2 ), which had been previously thawed were mounted on the receptor compartment of the diffusion cells. The receptor compartment was filled with 0.1 M, pH 7.4 PBS which was stirred continuously to ensure uniform distribution and maintain sink conditions, and the temperature of the entire diffusion cell assembly was maintained at 378C using a recirculating water jacket.
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The permeation studies were conducted using the infinite dose technique in which a large excess of permeant (46.88 mg / cm 2 ) is applied to the skin in comparison to the amount permeating so that an approximately constant concentration of the permeant is maintained in the donor compartment during the entire course of the experiment. Homogeneous solutions consisting of 10% (100 mg / ml) DEET and 45, 60, 75, and 90% PG in water were evaluated for DEET permeation to study the effect of PG on the permeation of DEET. An aliquot of 300 ml (100 mg / ml DEET) of the above homogeneous solutions was applied to 0.64 cm 2 of skin in the donor compartment (46.88 mg / cm 2 ) and then covered with a glass slip to prevent evaporation. Aliquots of 300 ml of the receptor fluid were withdrawn and replaced periodically with fresh phosphate buffer for 36 h. All samples were refrigerated at 48C until analysis for DEET using HPLC. The permeation study for DEET from PEG 400 was conducted in a similar manner by applying homogeneous solutions consisting of 10% DEET and 60, 75, and 90% PEG 400 in water. In addition to vehicles containing PG or PEG, a homogeneous solution consisting of 10% DEET, 30% PG, and 60% PEG 400 was also studied for the permeation of DEET across human skin in a similar manner at the same exposure level (46.88 mg / cm 2 ). In all permeation studies, exposure of skin to DEET was constant at a level of 46.88 mg / cm 2 , and in no case did the total amount permeated exceed 10% of the applied DEET. The permeation profiles of DEET from PG, PEG 400 and the combination were constructed (Figs. 3–5). In an earlier study, the effect of ethanol on permeation of DEET from aqueous solutions was studied at the same exposure level [11]. The permeation parameters obtained from the previous ethanol study were compared to the PG and PEG 400 results to compare the effects of solvents on DEET’s skin permeation and understand the mechanism for reduction in flux (Figs. 6).
Fig. 3. Average permeation profiles of DEET from 45% PG (m), 60% PG (3), 75% PG (♦), and 90% PG (j) solutions, where n56, as compared to DEET control (*), where n58. All in vitro permeation studies were performed at 378C through full thickness human skin, and 46.88 mg / cm 2 of DEET was applied to the skin.
3.5. HPLC analysis DEET was analyzed by reverse phase HPLC on an Alltech C8 column (5 ml, 25 cm34.6 mm) and eluted with the mobile phase at a flow-rate of 0.7 ml / min. The mobile phase consisting of methanol–
Fig. 4. Average permeation profiles for DEET from DEET control (♦), 60% PEG 400 (j), 75% PEG 400 (m), and 90% PEG 400 (3), where (n53) except for the control where (n58). All in vitro permeation studies were performed at 378C through full thickness human skin, and 46.88 mg / cm 2 of DEET was applied to the skin.
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D 5 h 2 / 6t L
215
(3)
where h is the thickness of stratum corneum, the rate limiting barrier, and assumed to be 0.001 cm [15]. From the values of Jss , D, h, and Cd , permeability (P), and skin / vehicle partition coefficient (K) were calculated using the following equations:
Fig. 5. Average permeation profiles of DEET from PEG 400 / PG mixture (j) as compared to DEET control (♦), where n56 for PEG 400 / PG and n58 for the control. All in vitro permeation studies were performed at 378C through full thickness human skin, and 46.88 mg / cm 2 of DEET was applied to the skin.
P 5 Jss /Cd
(4)
K 5 Ph /D
(5)
The estimated parameters are presented as mean6standard deviation (S.D.) and were evaluated for differences using an ANOVA test at P,0.05. The flux, P and K were plotted as a function of solvent concentration to understand the mechanism for reduction in flux (Fig. 6a,b,c).
4. Results
water (80:20, v / v) was filtered through a Versapor 0.8 mm filter (Millipore) and degassed under vacuum. DEET was detected using UV absorbance at 240 nm at an average retention time of 8 min, and the minimum detectable level (MDL) for DEET was 24 ng / injection.
3.6. Data analysis The cumulative amount (A) in mg / cm 2 of DEET permeating into the receptor compartment was plotted against time (t) to obtain the permeation profile. The steady state flux (Jss ) was estimated from the slope of the linear portion of the profile. The J-shaped curve can be described by the following equation: A 5 Jss (t 2 t L ).
(2)
The lag time (t L ) was estimated from the xintercept of the linear portion of the profile and was used to calculate the apparent diffusion coefficient (D) as follows:
The ternary phase diagrams for DEET, water, and PG or PEG 400 are shown in Fig. 1. The binodal curve denoted by symbols separates the area where the three liquids are miscible with each other and the area where a heterogeneous mixture exists. The ternary phase diagrams for PG and PEG 400 are very similar and follow the same trend as that for ethanol obtained earlier (Fig. 1). As seen from the ternary diagrams, DEET and water are almost immiscible, however with addition of solvents, homogeneous solutions containing DEET and water can be prepared. Although all three solvents, ethanol, PG and PEG 400, behave similarly, from the relative size of the homogeneous area in the phase diagram, ethanol is a better solvent than PG, which is slightly better than PEG 400 for DEET. Thus, it is not surprising that many DEET products contain ethanol as the solvent [11]. The phase diagrams in Fig. 1 show that to obtain a homogeneous solution containing 10% DEET, it must contain at least 40% PG or at least 50% PEG 400. Therefore, several homogeneous solutions containing 10% DEET were chosen for the permeation studies to evaluate the effect of PG (.40%) and PEG 400 (.50%) on the permeation of DEET.
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Fig. 6. Effect of solvent on (a) steady state flux, (b) permeability, and (c) skin / vehicle partition coefficient (K 310 21 ) of DEET in permeation through whole thickness human skin at 378C from 10% DEET solutions in solvent–water mixtures.
4.1. Effect of solvent on n-octanol /water partition coefficient of DEET The octanol / water partition coefficients (o / w PC) of DEET are plotted as a function of solvent concentration in Fig. 2. Although, o / w PC were calculated from the ratio of concentrations in the two phases, the experimentally determined PC is an apparent o / w PC since each solvent partitioned between octanol and water and thus altered the composition of each phase and influence the PC of DEET directly. As expected, DEET is moderately lipophilic with an o / w PC of approximately 50, which decreases as the concentration of semipolar solvent increases for all three solvents; ethanol, PG
and PEG 400. At 60% level of solvent, o / w PC reduced to 6–10, and with further addition of solvent, no DEET was detected in the octanol phase suggesting solubilization of DEET in the water– solvent mixture. No significant differences were observed between the different cosolvents with respect to their effect on o / w PC of DEET.
4.2. Effect of propylene glycol ( PG) on the percutaneous permeation of DEET The permeation profiles of DEET from various PG solutions are shown in Fig. 3 as the cumulative amount of DEET permeated across the skin versus time. The profiles represent an average of six experi-
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ments for each solution and are compared to the permeation of pure DEET (n58). DEET was found to continuously permeate throughout the 36 h time period from each solution, however permeation profiles from all PG solutions were lower than that of DEET control. The permeation of DEET across human skin from the PG solutions decreased with an increase in the percentage of PG. At 36 h the cumulative amount of DEET permeated can be ranked as: pure DEET.45% PG.60% PG.75% PG.90% PG. The cumulative amounts permeated from the 75 and 90% PG solutions were significantly lower (P,0.05) than that from the 45, 60% PG solutions and DEET control. The permeation parameters of DEET were obtained from the typically J-shaped profiles using Eqs. (2)–(5), and listed in Table 1. DEET’s flux ranged from 59.5669.73 mg / cm 2 h at 60% PG to 9.9062.06 mg / cm 2 h at 90% PG, depending on the concentration of PG and was lower than the flux of DEET control, 63.20624.52 mg / cm 2 h. Flux of DEET from 75 and 90% PG were significantly lower than the flux from the 45, 60% PG solutions and the control (Table 1, Fig. 3). PG had no effect on the lag times and diffusion coefficients of DEET since they were similar for all of the solutions. The permeability of DEET through full thickness human skin decreased with increasing PG concentrations with highest permeability from 60% PG at 5.983 10 24 60.98310 24 cm / h and lowest permeability at 0.99310 24 60.21310 24 cm / h from 90% PG solution. Again, the permeability of DEET from the 75 and 90% PG solutions were significantly lower (P, 0.05) than that from the 45 and 60% PG solutions. The skin / vehicle partition coefficient (K) of DEET
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was significantly lower (Fig. 6c) at 75 and 90% PG compared to that from 45 and 60% PG (P,0.05). K of DEET was found to be 61.2612.8 from a 10% DEET solution containing 60% PG, however, K appeared to decrease as the percentage of PG in solution increased above 60%, and K for 90% PG solution was 8.362.7. Therefore, the relative affinity of DEET for skin as compared to that for vehicle decreases with an increase in PG concentration which results in parallel decreases in permeability and flux (Fig. 6a,b,c).
4.3. Effect of PEG 400 on the percutaneous permeation of DEET Fig. 4 shows the permeation profiles of DEET across human skin from various solutions of PEG 400. The average (n53) permeation profiles from PEG solutions when compared to pure DEET control (n58) show that the total amount of DEET permeating is significantly lower from 60 and 75% PEG 400. Furthermore, no permeation of DEET was detected from the 90% PEG 400 during the entire 36 h time period. Effectively pure PEG 400 (90%) prevents permeation of DEET. Thus, the total amount of DEET permeated at the end of 36 h decreased with an increase in PEG 400 concentration, and it can be ranked as: pure DEET . . .60% PEG 400.75% PEG 400.90% PEG 400¯0. The cumulative amounts permeated from the 60 and 75% PEG 400 solutions were significantly lower by 7-fold (P, 0.05) than that from DEET control. The permeation parameters, steady state flux (Jss ), lag time (t L ), diffusion coefficient (D), permeability (P), and skin / vehicle partition coefficient (K), ob-
Table 1 Permeation parameters of DEET from propylene glycol (PG) solutions through full thickness human skin (46.88 mg / cm 2 ) at 378C a % PG
Jss (mg / cm 2 h)
tL (h)
D 310 8 (cm 2 / h)
P310 4 (cm / h)
K 310 21
45 60 75 90 DEET (n58)
57.85626.85 59.5669.73 14.5963.33 b 9.9062.06 b 63.20624.52
12.6961.62 16.9861.21 14.9661.85 9.9062.06 12.6264.05
1.3360.16 0.9960.070 1.1360.13 1.2760.27 1.7360.95
5.8162.70 5.9860.98 1.4660.33 c 0.9960.21 c
4.3762.12 6.1261.28 1.2960.21 c 0.8360.2 7 c
a
Values are presented as mean6S.D. (n56); 100 mg / ml DEET was applied to 0.64 cm 2 of skin. Jss for DEET from 75 and 90% PG is significantly lower (P,0.05) than Jss for DEET from 45 and 60% PG and the DEET control. c P and K for DEET from 75 and 90% PG are significantly lower (P,0.05) than P and K for DEET from 45 and 60% PG. b
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Table 2 Permeation parameters of DEET from PEG 400 solutions through full thickness human skin (46.88 mg / cm 2 ) at 378C a % PEG 400
Jss (mg / cm 2 h)
TL (h)
D 310 8 (cm 2 / h)
P310 4 (cm / h)
K
60 75 90 DEET (n58)
11.9561.42 b 10.4566.12 b No permeation 63.20624.52
3.2061.66 b 3.9260.80 b
6.9164.94 4.3760.90
1.2060.14 1.0560.62
2.3961.38 2.2860.88
12.6264.05
1.7360.95
a b
Values are presented as mean6S.D. (n53); 100 mg / ml DEET was applied to 0.64 cm 2 of skin. Jss and t L for DEET from 60 and 75% PEG 400 are significantly lower (P,0.05) than Jss and t L from the DEET control.
tained from the permeation profiles are listed in Table 2. No significant difference (P.0.05) in the parameters exists between 60% PEG 400 and 75% PEG 400, however, the flux values for these solutions are significantly lower (6-fold) than the flux value for pure DEET. Since no permeation was observed from 90% PEG 400, the flux and skin / vehicle partition coefficient values for 90% PEG 400 must be significantly lower than those from the 60 and 75% PEG 400 (Fig. 4).
4.4. PEG 400 /PG combination (60:30, v /v) Although 90% PEG 400 prevented any permeation of DEET across human skin, the solution was too viscous. Therefore, a combination of 60% PEG 400 with 30% PG was studied to evaluate if the combination retained the synergistic effect of reducing DEET’s permeation since it will be practical for use due to its lower viscosity. The permeation profile for DEET across human skin from the PEG 400 / PG (60:30, v / v) solution, as compared to the pure DEET control, is shown in Fig. 5. Although some permeation of DEET was observed in contrast to 90% PEG 400, the permeation from the PEG 400 / PG combination was significantly lower (P,0.05) than that from pure DEET control. Table 3 lists the permeation parameters obtained from the profiles according to the methods presented in Section 3.6. The steady state flux (Jss ) from the PEG 400 / PG solution was significantly lower (P,0.05) at 7.2065.54 mg / cm 2 h than the flux of the DEET control (63.20624.52 mg / cm 2 h), and slightly lower than the flux from 90% PG solution also. The lag time and diffusion coefficients were similar for both, DEET control and PEG 400 / PG solution. The permeability of DEET from the PEG 400 / PG solution was lower at
7.2065.50310 25 cm / h than permeability for DEET from the 90% PG and 60% PEG 400 solutions previously evaluated suggesting a synergistic effect on reduction in permeability. Fig. 6a,b,c compares the average steady state flux, permeability and skin / vehicle partition coefficient of DEET, respectively, as a function of vehicle composition in terms of solvent levels for ethanol, PG and PEG 400. For ethanol and PG, the flux and permeability are at a higher plateau from 30 to 45% and 45–60% solvent levels, respectively, and then begin to decline significantly with further increase in solvent concentration. For PEG 400, the flux is lower to begin with, however with further increase in PEG level, flux decreases and no permeation was detected from the 90% PEG 400 for 36 h. A comparison of trends in Jss (Fig. 6a) and P (Fig. 6b) with K (Fig. 6c) clearly show that changes in flux and permeability are primarily due to changes in the skin / vehicle PC as expected. Also, this is consistent with the findings that no significant changes in D were observed from the three solvent systems studied. Table 3 Average permeation parameters for DEET across human skin (46.88 mg / cm 2 ) at 378C from PEG 400 / PG (60:30 v / v) solution a DEET control 2
Jss (mg / cm h) t L (h) D 310 8 (cm 2 / h) P310 5 (cm / h) K a
63.20624.52 12.6264.05 1.7360.95
b
PEG 400 / PG (60:30, v / v) 7.2065.54 16.4466.31 1.1260.34 7.2065.50 8.6861.79
The values were obtained from the permeation profile presented in Fig. 5. All values are presented as mean6S.D., where n56 for the PEG 400 / PG solutions and n58 for the DEET control; 100 mg / ml DEET was applied to 0.64 cm 2 of skin. b Jss for the DEET is significantly lower (P,0.05) from PEG 400 / PG solution than Jss for DEET control.
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The PEG 400 / PG combination exhibits the next lowest flux value (9-fold lower than the DEET control) and may be a better vehicle because of its lower viscosity. As previously observed, the reduction in permeation of DEET from the PEG 400 / PG combination was due to the lower skin / vehicle PC (K) of DEET.
5. Discussion Suggestions of an incomplete toxicological assessment of N,N-diethyl-m-toluamide [6,16] and the several reported cases of toxicity and side effects in humans after using DEET-containing products [1–4], appear to warrant the development of alternative formulations of DEET designed to decrease permeation and absorption of DEET [12,13]. Although the actual mechanism for transport of DEET through human skin is not known, DEET being a moderately lipophilic small molecule will permeate through skin very rapidly. DEET’s facile permeation is confirmed by a relative large flux of 63.2 mg / cm 2 h which is in agreement with previous findings [7–11,17]. Therefore, an attempt was made to reduce its permeation by formulating DEET in a vehicle which results in reduced skin / vehicle partition coefficient with little or no direct effect of the vehicle on skin. Higuchi was the first one to develop equations relating thermodynamic activity and skin absorption from creams and ointments [18]. Barry has described in detail the drug–vehicle–skin interactions based on the theoretical model developed by Poulsen [19]. The flux of a permeant through skin should be constant at a maximum level from saturated binary solvent system (maximum thermodynamic activity) if it does not have a direct vehicle effect on the skin. However, decrease in flux should be observed with an increase in solubilizer concentration from vehicles of constant permeant concentration when the decrease in skin / vehicle PC is greater than the increase in solubility due to solubilizer in the vehicle. The above prediction assumes that the solvent acting as the solubilizer in the vehicle does not alter the D or solubility of permeant in skin. If the solvent increases the solubility of permeant in the skin with increasing concentration in vehicle, flux should increase initially as solubility increase is not offset
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by an equivalent decline in skin / vehicle PC. Increasing the solvent concentration beyond that point results in a decrease in flux due to the skin / vehicle PC declining more rapidly combined with below saturation concentration in the vehicle. Therefore, irrespective of the effect of solvent on the permeant’s solubility in skin, at high concentrations of solvent, flux should decline due to reduction in skin / vehicle PC. Ternary phase diagrams (Fig. 1) of DEET / water / solvent (ethanol, PG, PEG 400) do indeed show increasing solubility of DEET with addition of the solvent. This is also confirmed by an almost linear to exponential decline in octanol / water PC of DEET with an increasing concentration of solvent (Fig. 2). Thus, the decrease in flux (Fig. 6a) and permeability (Fig. 6b) of DEET with increasing concentrations of solvent appears to be a direct result of decrease in skin / vehicle PC (Fig. 6c). Although it is difficult to know the direct effect of solvents on DEET’s solubility in skin, from Fig. 6, it appears that solvent’s effect on DEET’s solubility in water is greater, particularly at higher concentrations. Ethanol is a known permeation enhancer and was shown to increase permeation of DEET compared to control except at high concentrations and is thus unsuitable as a vehicle solvent for DEET [11,17]. Propylene glycol (PG) is a semipolar cosolvent and although permeation of various drugs was enhanced by PG concentrations up to 50%, reduced permeation was seen at higher concentrations similar to the permeation changes observed for DEET [20–23]. Propylene glycol may be classified as a humectant, and the resultant dehydration effect on skin is especially noted at high concentrations of PG [19]. The dehydration effect of PG on the skin and the higher affinity of DEET for PG-water vehicle than for the skin may contribute to the reduced DEET permeation. The results of the in vitro percutaneous permeation studies with high PG concentrations suggest that PG may be a suitable substitution for ethanol as a solvent for DEET in commercial mosquito repellents aimed at reducing permeation. Polyethylene glycol 400 (PEG 400) was another potential vehicle that was evaluated for the permeation of DEET. PEG 400 is a fairly viscous, hygroscopic liquid that is often found in topical preparations. PEG 400 has been shown to reduce the permeation of drugs through human skin [24,25].
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The ternary phase diagram of DEET / water / PEG 400 (Fig. 1) although it showed increase in the aqueous solubility of DEET, it was not greater than that for the DEET / water / PG and DEET / water / ethanol systems, and therefore, DEET solutions containing only 60, 75, and 90% PEG 400 could be evaluated. The effect of concentration of PEG 400 on the steady state flux appears to follow a similar parabolic pattern to that of ethanol and PG [20–22,26–28]. Most strikingly, no permeation of DEET was detected from the 90% PEG 400 solution throughout the 36 h (Fig. 4). Similar results of negligible flux at the 90% PEG 400 level have been reported for oxaprozin and guanabenz solutions [25]. This decreased permeation was proposed to be due to the formation of PEG 400 association complex with oxaprozin and guanabenz, but this mechanism has not been proven. Since 90% PEG 400 eliminates the permeation of DEET through skin, it may be used as a solvent in new repellent formulations with reduced potential for toxicity. However, PEG 400 is a viscous liquid and may not have the customer appeal of the current ethanolic mosquito repellents. The high viscosity may also preclude the use of a spray pump, a common method of application. In order to reduce the viscosity, PG (30%) was added to a PEG 400 solution (60%) containing 10% DEET. Both vehicles, PG and PEG 400, effectively reduced the permeation of DEET, and similar results were observed with the combination (Fig. 5). The two solvents appear to exhibit a synergistic effect as the flux from the combination is lower than that from 60% PEG 400 alone or 30% PG alone (Tables 2 and 3). Thus, the 60:30 PEG 400 / PG solvent system appears to be a better alternative to ethanol than pure PEG 400, due to its lower viscosity and overall lower (9-fold reduction) permeation of DEET.
6. Summary Using a simple model of skin permeation based on Fick’s laws of diffusion at steady state, an approach of formulating DEET with significantly reduced permeation was successfully demonstrated. Semipolar solvents, ethanol, PG and PEG 400, significantly increased the aqueous solubility of DEET along with
a decreasing o / w PC with increase in concentration of the solvent. The permeation of DEET across human skin was reduced significantly from solutions containing 60% or greater amounts of PG and PEG 400. Permeation of DEET was totally prevented from 90% PEG 400 solution which was very viscous, however a combination of 60% PEG 400 with 30% PG not only reduced permeation 9-fold but was suitable as a solvent for vehicle. This study clearly demonstrates that alternative formulations can be developed for DEET aimed at reduced permeation and toxicity unlike the current formulations some of which contain ethanol which has been shown to enhance permeation of DEET. A similar approach can be used for developing formulations of other industrial and occupational agents to prevent their skin permeation and penetration when a user may be exposed.
Acknowledgements We would like to thank the American Foundation of Pharmaceutical Education for a predoctoral fellowship to J. Stinecipher, and Jayabharati Vaidyanathan and Kimberley Hill for technical help in the PC experiments.
References [1] J.C. Veltri, T.G. Osmitiz, D.C. Bradford, B.C. Page, Retrospective analysis of calls to poison control centers resulting from exposure to the insect repellent N,N-diethyl-mtoluamide (DEET) from 1985 to 1989, Clin. Toxicol. 32 (1994) 1–16. [2] D.L. Edwards, C.E. Johnson, Insect-repellent-induced toxic encephalopathy in a child, Clin. Pharmacol. 6 (1987) 496– 498. [3] J.W. Lipscomb, J.E. Kramer, J.B. Leiken, Seizure following brief exposure to the insect repellent N,N-diethyl-mtoluamide, Ann. Emerg. Med. 21 (1992) 315–317. [4] J.P. deGarbino, A. Laborde, Toxicity of an insect repellent: N,N-diethyltoluamide, Vet. Hum. Toxicol. 25 (1983) 422– 423. [5] M.B. Abou-Donia, K.R. Wilmarth, K.F. Jensen, F.W. Oehme, T.L. Kurt, Neurotoxicity resulting from coexposure to pyridostigmine bromide, DEET, and permethrin: Implications of gulf war chemical exposures, J. Toxicol. Environ. Health 48 (1996) 35–56. [6] P.J. Robbins, M.G. Cherniack, Review of the biodistribution
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ortho, and para isomers of N,N-diethyltoluamide (DEET), Int. J. Pharmaceut. 160 (1) (1998) 31–41. T. Higuchi, Thermodynamic analysis of percutaneous absorption process from creams and ointments, J. Cos. Chem. 11 (1960) 85–87. B.W. Barry, in: Dermatological Formulations – Percutaneous Absorption, Marcel Dekker, Inc, New York, 1983, pp. 49– 233. J.W. Hadgraft, Recent progress in the formulation of vehicles for topical applications, Br. J. Derm. 87 (1972) 386–389. J. Ostrenga, C. Steinmitz, B. Poulsen, Significance of vehicle composition. I: Relationship between topical vehicle composition, skin penetrability, and clinical efficacy, J. Pharm. Sci. 60 (1971) 1175–1179. J. Ostrenga, C. Steinmitz, B. Poulsen, S. Yett, Significance of vehicle composition. II: Prediction of optimal vehicle composition, J. Pharm. Sci. 60 (1971) 1180–1183. G.L. Flynn, R.W. Smith, Membrane diffusion. III: Influence of solvent composition and permeant solubility on membrane transport, J. Pharm. Sci. 61 (1972) 61–66. S. Kumar, A.W. Malick, N.M. Meltzer, J.D. Mouskountakis, C.R. Behl, In vitro skin permeation and retention of a leukotriene antagonist from topical vehicles using hairless guinea pig model, J. Pharm. Sci. 69 (1992) 631–638. P.P. Sarpotdar, J.L. Gaskill, R.P. Giannini, Effect of polyethylene glycol 400 on the penetration of drugs through human cadaver skin in vitro, J. Pharm. Sci. 75 (1986) 26–28. B. Berner, J.H. Otte, G.C. Mazzenga, R.J. Steffens, C.D. Ebert, Ethanol:water mutually enhanced transdermal therapeutic system II: Skin permeation of ethanol and nitroglycerin, J. Pharm. Sci. 78 (1989) 402–407. T. Kurihara-Bergstrom, K. Knutson, L.J. DeNoble, C.Y. Goates, Percutaneous absorption enhancement of an ionic molecule by ethanol–water systems in human skin, Pharm. Res. 7 (1990) 762–766. Y. Wada, K. Nakajima, J. Yamazaki, T. Seki, K. Sugibayashi, Y. Morimota, Influence of composition of l-menthol–ethanol–water ternary solvent system on the transdermal delivery of morphine hydrochloride, Biol. Pharm. Bull. 16 (1993) 600–603.
Reduction in skin permeation of N,N-diethyl-m-toluamide (DEET) by altering the skin / vehicle partition coefficient q a ,1 a, Julie S. Ross , Jaymin C. Shah * a
Department of Pharmaceutical Sciences, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA Received 2 November 1999; accepted 19 January 2000
Abstract Reported adverse side effects after using N,N-diethyl-m-toluamide (DEET)-containing mosquito repellent products appear to be the result of significant absorption of DEET through human skin. The overall objective was to develop formulations of DEET with significantly reduced permeation using the basic principles and model of skin permeation based on Fick’s laws of diffusion at steady state. Ternary phase diagrams of DEET with water and semipolar solvents, ethanol, PG and PEG 400, showed an increase in the aqueous solubility of DEET. This resulted in a linear decline in octanol / water PC with an increase in the concentration of the solvent. Permeation of DEET across human skin was studied from vehicles containing various amounts of PG and PEG 400 using an infinite dose technique and Franz diffusion cell. DEET’s flux reduced with increasing PG concentration and the flux from 90% PG was 9.962.1 mg / cm 2 h, 6-fold lower than flux of pure DEET control, 63.2624.5 mg / cm 2 h. Flux was reduced 6-fold from 60% PEG 400 solution, and permeation of DEET was totally prevented from 90% PEG 400 which was very viscous. However, a combination of 60% PEG 400 with 30% PG not only reduced permeation 9-fold but was suitable as a vehicle for formulation. The decrease in flux and permeability of DEET with increasing concentration of solvent appeared to be a direct result of decrease in skin / vehicle PC and octanol / water PC. This study clearly demonstrates that alternative formulations can be developed for DEET aimed at reduced permeation and toxicity unlike the current formulations some of which contain ethanol which has been shown to enhance permeation of DEET. A similar approach can be used for developing formulations of other industrial and occupational agents to prevent their skin permeation when a user may be exposed to them. 2000 Elsevier Science B.V. All rights reserved. Keywords: DEET; Mosquito repellent; Solvent; Skin permeation; Partition coefficient; Propylene glycol; Polyethylene glycol; Ethanol
1. Introduction q
This research was conducted as dissertation research by J. Stinecipher towards partial fulfillment of the requirements of doctor of philosophy degree at the Medical University of South Carolina. *Corresponding author. Present address: Pharmaceutical RandD, Pfizer Central Research, Ms 8156-39, Eastern Point Road, Groton, CT 06340, USA. Tel.: 11-860-715-2332; fax: 11-860441-0467. 1
Present address: IBAH Pharmaceutics Services, 525 Virginia Drive, Fort Washington, PA 19034, USA.
Use of N,N-diethyl-m-toluamide (DEET) containing mosquito repellent products have been associated with many adverse reactions [1–4]. Recently, DEET has also been implicated as a factor in neurotoxicity resulting from the Gulf War chemical exposure [5]. A review of the biodistribution and toxicity of DEET by Robbins and Cherniack suggested that there were several areas of toxicity that needed further evalua-
0168-3659 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 00 )00210-8
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tion [6]. DEET is absorbed through the skin very rapidly as shown by several investigators [7–10]. Our previous studies have shown not only significant absorption of DEET from all commercial products but enhanced permeation of DEET across human skin from 15 to 60% ethanol, a solvent for DEET in many products [11]. Therefore, it is very evident that dermal absorption of DEET may be responsible for the potential toxicity problems associated with DEET-containing products, and therefore, new or alternative formulations of DEET specifically aimed at decreased permeation and absorption will be safe and useful [12,13]. In order to develop these alternative formulations with decreased absorption of DEET, the basic principles of skin permeation should be applied to DEET. The simplest model to describe dermal absorption based on Fick’s laws of diffusion at steady state is as follows [14]: dc / dt ss 5 Jss 5 DAK /h (Cd 2 Cr )
(1)
where Jss 5steady state flux or the permeation rate; A5surface area of the skin exposed or of the application site; h5the thickness of the rate-limiting barrier, usually the stratum corneum; D5the diffusion coefficient of the diffusant, K5the skin / vehicle partition coefficient; Cd 5the thermodynamic activity of the diffusant in the vehicle and Cr 5the concentration of the diffusant in receptor, close to zero under sink conditions. Therefore, only D, K, and Cd may be altered to reduce the permeation of diffusant. By selecting an appropriate vehicle, the thermodynamic activity and the skin / vehicle partition coefficient can be reduced to minimize skin permeation, although, high thermodynamic activity of DEET in vehicle will be required to maintain its ability to repell mosquitos. The diffusion coefficient is relatively difficult to alter since it is dependent on a number of factors such as the skin characteristics, effect of vehicle on skin, skin hydration and interaction of diffusant with skin. Therefore, using an appropriate vehicle, either K, D, and / or the skin can be influenced to reduce permeation of DEET using this basic principle in order to develop improved formulations of DEET. The specific aim of the present study was to reduce and minimize skin permeation of DEET by reducing its skin / vehicle PC (K) using semipolar
solvents which may decrease DEET’s n-octanol / water PC. The permeation of DEET from each vehicle was evaluated by performing in vitro permeation studies across human skin with a Franz diffusion cell using an infinite dose technique. Permeation profiles were constructed in each case and the steady state flux, diffusion coefficient, lag time, permeability, and skin / vehicle partition coefficient were calculated to investigate the mechanism of reduction in flux. The permeation of DEET from each vehicle was compared to that of pure DEET in an effort to determine the reduction in flux and overall permeation.
2. Materials DEET (97%) was purchased from Aldrich Chemical Company. Propylene glycol, and methanol (HPLC grade) were purchased from Fisher (Fair Lawn, NJ, USA). Carbowax Sentry Polyethylene glycol 400 NF, FCC Grade was a gift from Union Carbide (Danbury, CT, USA). Phosphate buffer solution (0.1 M, pH 7.4) was prepared with sodium phosphate monobasic monohydrate (Mallinckrodt, St. Louis, MO, USA) and sodium phosphate dibasic anhydrous (Curtin Matheson Scientific,Houston, TX, USA).
3. Methods
3.1. Phase solubility diagram; effect of solvent on aqueous solubility of DEET Since DEET is a liquid at room temperature, a ternary phase diagram was constructed for DEET, water, and each solvent: ethanol, propylene glycol (PG) or polyethylene glycol 400 (PEG 400), to determine the miscibility of the three liquids. A pair of liquids such as DEET and water that are miscible with each other only to a slight extent would usually exist as two phases. The addition of a third component to the system that is miscible in all proportions with one of the solvents would be expected to produce a single phase in which all three components are miscible with each other. Solutions were prepared consisting of PG, PEG 400 or ethanol, and
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213
varying percentages of DEET from 1 to 99%. Water was added to these solutions in 100 ml increments until the solutions appeared turbid after vortexing for 30 s. A ternary plot was constructed in order to determine the binodal curve, or the boundary of the two-phase area as seen in Fig. 1. Regions above the binodal curve represent homogeneous solutions of the three liquids. Homogeneous or single phase solutions containing 10% DEET and varying percentages of PG and PEG 400 were selected for in vitro permeation experiments.
3.2. Effect of solvent on the n-octanol /water partition coefficient of DEET n-Octanol and water with various concentrations of each solvent were presaturated with each other in an amber colored bottle for 48 h. The two phases were separated using a separating funnel. DEET was added in small amounts to a mixture of equal volumes of the octanol and water phases in a scintillation vial, and allowed to agitate for 48 h to reach equilibrium. At the end of 48 h, the octanol and water phases were separated and individually analyzed for DEET concentration using UV spectrophotometric assay. The o / w partition coefficient was calculated from the ratio of concentrations in the two phases, octanol and water, and plotted as a function of solvent concentration (Fig. 2). However, the
Fig. 2. Effect of solvent on the apparent n-octanol / water partition coefficient of DEET.
experimentally determined PC is an apparent o / w PC since each solvent will itself partition between octanol and water and thus change composition of each phase and influence the PC of DEET directly.
3.3. Skin preparation Human skin samples were obtained from elective plastic surgery, and the fat and other visceral debris were removed from the underside of the freshly excised skin. The skin was then washed with 0.1 M, pH 7.4 phosphate buffer solution before freezing at 2208C. The full thickness skin samples were cut into 2 cm 2 pieces and allowed to thaw overnight at room temperature in phosphate buffer solution prior to the in vitro percutaneous permeation experiments.
3.4. In vitro percutaneous permeation
Fig. 1. Ternary phase diagrams of solvent, water and DEET showing the homogeneous (single phase) and heterogeneous regions.
The permeation experiments were conducted with vertical Franz diffusion cells (Crown Glass Company, Somerville, NJ, USA) each having a receptor volume of 4.9 ml and a diameter of 0.9 cm. Pieces of full thickness human skin (2 cm 2 ), which had been previously thawed were mounted on the receptor compartment of the diffusion cells. The receptor compartment was filled with 0.1 M, pH 7.4 PBS which was stirred continuously to ensure uniform distribution and maintain sink conditions, and the temperature of the entire diffusion cell assembly was maintained at 378C using a recirculating water jacket.
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The permeation studies were conducted using the infinite dose technique in which a large excess of permeant (46.88 mg / cm 2 ) is applied to the skin in comparison to the amount permeating so that an approximately constant concentration of the permeant is maintained in the donor compartment during the entire course of the experiment. Homogeneous solutions consisting of 10% (100 mg / ml) DEET and 45, 60, 75, and 90% PG in water were evaluated for DEET permeation to study the effect of PG on the permeation of DEET. An aliquot of 300 ml (100 mg / ml DEET) of the above homogeneous solutions was applied to 0.64 cm 2 of skin in the donor compartment (46.88 mg / cm 2 ) and then covered with a glass slip to prevent evaporation. Aliquots of 300 ml of the receptor fluid were withdrawn and replaced periodically with fresh phosphate buffer for 36 h. All samples were refrigerated at 48C until analysis for DEET using HPLC. The permeation study for DEET from PEG 400 was conducted in a similar manner by applying homogeneous solutions consisting of 10% DEET and 60, 75, and 90% PEG 400 in water. In addition to vehicles containing PG or PEG, a homogeneous solution consisting of 10% DEET, 30% PG, and 60% PEG 400 was also studied for the permeation of DEET across human skin in a similar manner at the same exposure level (46.88 mg / cm 2 ). In all permeation studies, exposure of skin to DEET was constant at a level of 46.88 mg / cm 2 , and in no case did the total amount permeated exceed 10% of the applied DEET. The permeation profiles of DEET from PG, PEG 400 and the combination were constructed (Figs. 3–5). In an earlier study, the effect of ethanol on permeation of DEET from aqueous solutions was studied at the same exposure level [11]. The permeation parameters obtained from the previous ethanol study were compared to the PG and PEG 400 results to compare the effects of solvents on DEET’s skin permeation and understand the mechanism for reduction in flux (Figs. 6).
Fig. 3. Average permeation profiles of DEET from 45% PG (m), 60% PG (3), 75% PG (♦), and 90% PG (j) solutions, where n56, as compared to DEET control (*), where n58. All in vitro permeation studies were performed at 378C through full thickness human skin, and 46.88 mg / cm 2 of DEET was applied to the skin.
3.5. HPLC analysis DEET was analyzed by reverse phase HPLC on an Alltech C8 column (5 ml, 25 cm34.6 mm) and eluted with the mobile phase at a flow-rate of 0.7 ml / min. The mobile phase consisting of methanol–
Fig. 4. Average permeation profiles for DEET from DEET control (♦), 60% PEG 400 (j), 75% PEG 400 (m), and 90% PEG 400 (3), where (n53) except for the control where (n58). All in vitro permeation studies were performed at 378C through full thickness human skin, and 46.88 mg / cm 2 of DEET was applied to the skin.
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D 5 h 2 / 6t L
215
(3)
where h is the thickness of stratum corneum, the rate limiting barrier, and assumed to be 0.001 cm [15]. From the values of Jss , D, h, and Cd , permeability (P), and skin / vehicle partition coefficient (K) were calculated using the following equations:
Fig. 5. Average permeation profiles of DEET from PEG 400 / PG mixture (j) as compared to DEET control (♦), where n56 for PEG 400 / PG and n58 for the control. All in vitro permeation studies were performed at 378C through full thickness human skin, and 46.88 mg / cm 2 of DEET was applied to the skin.
P 5 Jss /Cd
(4)
K 5 Ph /D
(5)
The estimated parameters are presented as mean6standard deviation (S.D.) and were evaluated for differences using an ANOVA test at P,0.05. The flux, P and K were plotted as a function of solvent concentration to understand the mechanism for reduction in flux (Fig. 6a,b,c).
4. Results
water (80:20, v / v) was filtered through a Versapor 0.8 mm filter (Millipore) and degassed under vacuum. DEET was detected using UV absorbance at 240 nm at an average retention time of 8 min, and the minimum detectable level (MDL) for DEET was 24 ng / injection.
3.6. Data analysis The cumulative amount (A) in mg / cm 2 of DEET permeating into the receptor compartment was plotted against time (t) to obtain the permeation profile. The steady state flux (Jss ) was estimated from the slope of the linear portion of the profile. The J-shaped curve can be described by the following equation: A 5 Jss (t 2 t L ).
(2)
The lag time (t L ) was estimated from the xintercept of the linear portion of the profile and was used to calculate the apparent diffusion coefficient (D) as follows:
The ternary phase diagrams for DEET, water, and PG or PEG 400 are shown in Fig. 1. The binodal curve denoted by symbols separates the area where the three liquids are miscible with each other and the area where a heterogeneous mixture exists. The ternary phase diagrams for PG and PEG 400 are very similar and follow the same trend as that for ethanol obtained earlier (Fig. 1). As seen from the ternary diagrams, DEET and water are almost immiscible, however with addition of solvents, homogeneous solutions containing DEET and water can be prepared. Although all three solvents, ethanol, PG and PEG 400, behave similarly, from the relative size of the homogeneous area in the phase diagram, ethanol is a better solvent than PG, which is slightly better than PEG 400 for DEET. Thus, it is not surprising that many DEET products contain ethanol as the solvent [11]. The phase diagrams in Fig. 1 show that to obtain a homogeneous solution containing 10% DEET, it must contain at least 40% PG or at least 50% PEG 400. Therefore, several homogeneous solutions containing 10% DEET were chosen for the permeation studies to evaluate the effect of PG (.40%) and PEG 400 (.50%) on the permeation of DEET.
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Fig. 6. Effect of solvent on (a) steady state flux, (b) permeability, and (c) skin / vehicle partition coefficient (K 310 21 ) of DEET in permeation through whole thickness human skin at 378C from 10% DEET solutions in solvent–water mixtures.
4.1. Effect of solvent on n-octanol /water partition coefficient of DEET The octanol / water partition coefficients (o / w PC) of DEET are plotted as a function of solvent concentration in Fig. 2. Although, o / w PC were calculated from the ratio of concentrations in the two phases, the experimentally determined PC is an apparent o / w PC since each solvent partitioned between octanol and water and thus altered the composition of each phase and influence the PC of DEET directly. As expected, DEET is moderately lipophilic with an o / w PC of approximately 50, which decreases as the concentration of semipolar solvent increases for all three solvents; ethanol, PG
and PEG 400. At 60% level of solvent, o / w PC reduced to 6–10, and with further addition of solvent, no DEET was detected in the octanol phase suggesting solubilization of DEET in the water– solvent mixture. No significant differences were observed between the different cosolvents with respect to their effect on o / w PC of DEET.
4.2. Effect of propylene glycol ( PG) on the percutaneous permeation of DEET The permeation profiles of DEET from various PG solutions are shown in Fig. 3 as the cumulative amount of DEET permeated across the skin versus time. The profiles represent an average of six experi-
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ments for each solution and are compared to the permeation of pure DEET (n58). DEET was found to continuously permeate throughout the 36 h time period from each solution, however permeation profiles from all PG solutions were lower than that of DEET control. The permeation of DEET across human skin from the PG solutions decreased with an increase in the percentage of PG. At 36 h the cumulative amount of DEET permeated can be ranked as: pure DEET.45% PG.60% PG.75% PG.90% PG. The cumulative amounts permeated from the 75 and 90% PG solutions were significantly lower (P,0.05) than that from the 45, 60% PG solutions and DEET control. The permeation parameters of DEET were obtained from the typically J-shaped profiles using Eqs. (2)–(5), and listed in Table 1. DEET’s flux ranged from 59.5669.73 mg / cm 2 h at 60% PG to 9.9062.06 mg / cm 2 h at 90% PG, depending on the concentration of PG and was lower than the flux of DEET control, 63.20624.52 mg / cm 2 h. Flux of DEET from 75 and 90% PG were significantly lower than the flux from the 45, 60% PG solutions and the control (Table 1, Fig. 3). PG had no effect on the lag times and diffusion coefficients of DEET since they were similar for all of the solutions. The permeability of DEET through full thickness human skin decreased with increasing PG concentrations with highest permeability from 60% PG at 5.983 10 24 60.98310 24 cm / h and lowest permeability at 0.99310 24 60.21310 24 cm / h from 90% PG solution. Again, the permeability of DEET from the 75 and 90% PG solutions were significantly lower (P, 0.05) than that from the 45 and 60% PG solutions. The skin / vehicle partition coefficient (K) of DEET
217
was significantly lower (Fig. 6c) at 75 and 90% PG compared to that from 45 and 60% PG (P,0.05). K of DEET was found to be 61.2612.8 from a 10% DEET solution containing 60% PG, however, K appeared to decrease as the percentage of PG in solution increased above 60%, and K for 90% PG solution was 8.362.7. Therefore, the relative affinity of DEET for skin as compared to that for vehicle decreases with an increase in PG concentration which results in parallel decreases in permeability and flux (Fig. 6a,b,c).
4.3. Effect of PEG 400 on the percutaneous permeation of DEET Fig. 4 shows the permeation profiles of DEET across human skin from various solutions of PEG 400. The average (n53) permeation profiles from PEG solutions when compared to pure DEET control (n58) show that the total amount of DEET permeating is significantly lower from 60 and 75% PEG 400. Furthermore, no permeation of DEET was detected from the 90% PEG 400 during the entire 36 h time period. Effectively pure PEG 400 (90%) prevents permeation of DEET. Thus, the total amount of DEET permeated at the end of 36 h decreased with an increase in PEG 400 concentration, and it can be ranked as: pure DEET . . .60% PEG 400.75% PEG 400.90% PEG 400¯0. The cumulative amounts permeated from the 60 and 75% PEG 400 solutions were significantly lower by 7-fold (P, 0.05) than that from DEET control. The permeation parameters, steady state flux (Jss ), lag time (t L ), diffusion coefficient (D), permeability (P), and skin / vehicle partition coefficient (K), ob-
Table 1 Permeation parameters of DEET from propylene glycol (PG) solutions through full thickness human skin (46.88 mg / cm 2 ) at 378C a % PG
Jss (mg / cm 2 h)
tL (h)
D 310 8 (cm 2 / h)
P310 4 (cm / h)
K 310 21
45 60 75 90 DEET (n58)
57.85626.85 59.5669.73 14.5963.33 b 9.9062.06 b 63.20624.52
12.6961.62 16.9861.21 14.9661.85 9.9062.06 12.6264.05
1.3360.16 0.9960.070 1.1360.13 1.2760.27 1.7360.95
5.8162.70 5.9860.98 1.4660.33 c 0.9960.21 c
4.3762.12 6.1261.28 1.2960.21 c 0.8360.2 7 c
a
Values are presented as mean6S.D. (n56); 100 mg / ml DEET was applied to 0.64 cm 2 of skin. Jss for DEET from 75 and 90% PG is significantly lower (P,0.05) than Jss for DEET from 45 and 60% PG and the DEET control. c P and K for DEET from 75 and 90% PG are significantly lower (P,0.05) than P and K for DEET from 45 and 60% PG. b
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Table 2 Permeation parameters of DEET from PEG 400 solutions through full thickness human skin (46.88 mg / cm 2 ) at 378C a % PEG 400
Jss (mg / cm 2 h)
TL (h)
D 310 8 (cm 2 / h)
P310 4 (cm / h)
K
60 75 90 DEET (n58)
11.9561.42 b 10.4566.12 b No permeation 63.20624.52
3.2061.66 b 3.9260.80 b
6.9164.94 4.3760.90
1.2060.14 1.0560.62
2.3961.38 2.2860.88
12.6264.05
1.7360.95
a b
Values are presented as mean6S.D. (n53); 100 mg / ml DEET was applied to 0.64 cm 2 of skin. Jss and t L for DEET from 60 and 75% PEG 400 are significantly lower (P,0.05) than Jss and t L from the DEET control.
tained from the permeation profiles are listed in Table 2. No significant difference (P.0.05) in the parameters exists between 60% PEG 400 and 75% PEG 400, however, the flux values for these solutions are significantly lower (6-fold) than the flux value for pure DEET. Since no permeation was observed from 90% PEG 400, the flux and skin / vehicle partition coefficient values for 90% PEG 400 must be significantly lower than those from the 60 and 75% PEG 400 (Fig. 4).
4.4. PEG 400 /PG combination (60:30, v /v) Although 90% PEG 400 prevented any permeation of DEET across human skin, the solution was too viscous. Therefore, a combination of 60% PEG 400 with 30% PG was studied to evaluate if the combination retained the synergistic effect of reducing DEET’s permeation since it will be practical for use due to its lower viscosity. The permeation profile for DEET across human skin from the PEG 400 / PG (60:30, v / v) solution, as compared to the pure DEET control, is shown in Fig. 5. Although some permeation of DEET was observed in contrast to 90% PEG 400, the permeation from the PEG 400 / PG combination was significantly lower (P,0.05) than that from pure DEET control. Table 3 lists the permeation parameters obtained from the profiles according to the methods presented in Section 3.6. The steady state flux (Jss ) from the PEG 400 / PG solution was significantly lower (P,0.05) at 7.2065.54 mg / cm 2 h than the flux of the DEET control (63.20624.52 mg / cm 2 h), and slightly lower than the flux from 90% PG solution also. The lag time and diffusion coefficients were similar for both, DEET control and PEG 400 / PG solution. The permeability of DEET from the PEG 400 / PG solution was lower at
7.2065.50310 25 cm / h than permeability for DEET from the 90% PG and 60% PEG 400 solutions previously evaluated suggesting a synergistic effect on reduction in permeability. Fig. 6a,b,c compares the average steady state flux, permeability and skin / vehicle partition coefficient of DEET, respectively, as a function of vehicle composition in terms of solvent levels for ethanol, PG and PEG 400. For ethanol and PG, the flux and permeability are at a higher plateau from 30 to 45% and 45–60% solvent levels, respectively, and then begin to decline significantly with further increase in solvent concentration. For PEG 400, the flux is lower to begin with, however with further increase in PEG level, flux decreases and no permeation was detected from the 90% PEG 400 for 36 h. A comparison of trends in Jss (Fig. 6a) and P (Fig. 6b) with K (Fig. 6c) clearly show that changes in flux and permeability are primarily due to changes in the skin / vehicle PC as expected. Also, this is consistent with the findings that no significant changes in D were observed from the three solvent systems studied. Table 3 Average permeation parameters for DEET across human skin (46.88 mg / cm 2 ) at 378C from PEG 400 / PG (60:30 v / v) solution a DEET control 2
Jss (mg / cm h) t L (h) D 310 8 (cm 2 / h) P310 5 (cm / h) K a
63.20624.52 12.6264.05 1.7360.95
b
PEG 400 / PG (60:30, v / v) 7.2065.54 16.4466.31 1.1260.34 7.2065.50 8.6861.79
The values were obtained from the permeation profile presented in Fig. 5. All values are presented as mean6S.D., where n56 for the PEG 400 / PG solutions and n58 for the DEET control; 100 mg / ml DEET was applied to 0.64 cm 2 of skin. b Jss for the DEET is significantly lower (P,0.05) from PEG 400 / PG solution than Jss for DEET control.
J.S. Ross, J.C. Shah / Journal of Controlled Release 67 (2000) 211 – 221
The PEG 400 / PG combination exhibits the next lowest flux value (9-fold lower than the DEET control) and may be a better vehicle because of its lower viscosity. As previously observed, the reduction in permeation of DEET from the PEG 400 / PG combination was due to the lower skin / vehicle PC (K) of DEET.
5. Discussion Suggestions of an incomplete toxicological assessment of N,N-diethyl-m-toluamide [6,16] and the several reported cases of toxicity and side effects in humans after using DEET-containing products [1–4], appear to warrant the development of alternative formulations of DEET designed to decrease permeation and absorption of DEET [12,13]. Although the actual mechanism for transport of DEET through human skin is not known, DEET being a moderately lipophilic small molecule will permeate through skin very rapidly. DEET’s facile permeation is confirmed by a relative large flux of 63.2 mg / cm 2 h which is in agreement with previous findings [7–11,17]. Therefore, an attempt was made to reduce its permeation by formulating DEET in a vehicle which results in reduced skin / vehicle partition coefficient with little or no direct effect of the vehicle on skin. Higuchi was the first one to develop equations relating thermodynamic activity and skin absorption from creams and ointments [18]. Barry has described in detail the drug–vehicle–skin interactions based on the theoretical model developed by Poulsen [19]. The flux of a permeant through skin should be constant at a maximum level from saturated binary solvent system (maximum thermodynamic activity) if it does not have a direct vehicle effect on the skin. However, decrease in flux should be observed with an increase in solubilizer concentration from vehicles of constant permeant concentration when the decrease in skin / vehicle PC is greater than the increase in solubility due to solubilizer in the vehicle. The above prediction assumes that the solvent acting as the solubilizer in the vehicle does not alter the D or solubility of permeant in skin. If the solvent increases the solubility of permeant in the skin with increasing concentration in vehicle, flux should increase initially as solubility increase is not offset
219
by an equivalent decline in skin / vehicle PC. Increasing the solvent concentration beyond that point results in a decrease in flux due to the skin / vehicle PC declining more rapidly combined with below saturation concentration in the vehicle. Therefore, irrespective of the effect of solvent on the permeant’s solubility in skin, at high concentrations of solvent, flux should decline due to reduction in skin / vehicle PC. Ternary phase diagrams (Fig. 1) of DEET / water / solvent (ethanol, PG, PEG 400) do indeed show increasing solubility of DEET with addition of the solvent. This is also confirmed by an almost linear to exponential decline in octanol / water PC of DEET with an increasing concentration of solvent (Fig. 2). Thus, the decrease in flux (Fig. 6a) and permeability (Fig. 6b) of DEET with increasing concentrations of solvent appears to be a direct result of decrease in skin / vehicle PC (Fig. 6c). Although it is difficult to know the direct effect of solvents on DEET’s solubility in skin, from Fig. 6, it appears that solvent’s effect on DEET’s solubility in water is greater, particularly at higher concentrations. Ethanol is a known permeation enhancer and was shown to increase permeation of DEET compared to control except at high concentrations and is thus unsuitable as a vehicle solvent for DEET [11,17]. Propylene glycol (PG) is a semipolar cosolvent and although permeation of various drugs was enhanced by PG concentrations up to 50%, reduced permeation was seen at higher concentrations similar to the permeation changes observed for DEET [20–23]. Propylene glycol may be classified as a humectant, and the resultant dehydration effect on skin is especially noted at high concentrations of PG [19]. The dehydration effect of PG on the skin and the higher affinity of DEET for PG-water vehicle than for the skin may contribute to the reduced DEET permeation. The results of the in vitro percutaneous permeation studies with high PG concentrations suggest that PG may be a suitable substitution for ethanol as a solvent for DEET in commercial mosquito repellents aimed at reducing permeation. Polyethylene glycol 400 (PEG 400) was another potential vehicle that was evaluated for the permeation of DEET. PEG 400 is a fairly viscous, hygroscopic liquid that is often found in topical preparations. PEG 400 has been shown to reduce the permeation of drugs through human skin [24,25].
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The ternary phase diagram of DEET / water / PEG 400 (Fig. 1) although it showed increase in the aqueous solubility of DEET, it was not greater than that for the DEET / water / PG and DEET / water / ethanol systems, and therefore, DEET solutions containing only 60, 75, and 90% PEG 400 could be evaluated. The effect of concentration of PEG 400 on the steady state flux appears to follow a similar parabolic pattern to that of ethanol and PG [20–22,26–28]. Most strikingly, no permeation of DEET was detected from the 90% PEG 400 solution throughout the 36 h (Fig. 4). Similar results of negligible flux at the 90% PEG 400 level have been reported for oxaprozin and guanabenz solutions [25]. This decreased permeation was proposed to be due to the formation of PEG 400 association complex with oxaprozin and guanabenz, but this mechanism has not been proven. Since 90% PEG 400 eliminates the permeation of DEET through skin, it may be used as a solvent in new repellent formulations with reduced potential for toxicity. However, PEG 400 is a viscous liquid and may not have the customer appeal of the current ethanolic mosquito repellents. The high viscosity may also preclude the use of a spray pump, a common method of application. In order to reduce the viscosity, PG (30%) was added to a PEG 400 solution (60%) containing 10% DEET. Both vehicles, PG and PEG 400, effectively reduced the permeation of DEET, and similar results were observed with the combination (Fig. 5). The two solvents appear to exhibit a synergistic effect as the flux from the combination is lower than that from 60% PEG 400 alone or 30% PG alone (Tables 2 and 3). Thus, the 60:30 PEG 400 / PG solvent system appears to be a better alternative to ethanol than pure PEG 400, due to its lower viscosity and overall lower (9-fold reduction) permeation of DEET.
6. Summary Using a simple model of skin permeation based on Fick’s laws of diffusion at steady state, an approach of formulating DEET with significantly reduced permeation was successfully demonstrated. Semipolar solvents, ethanol, PG and PEG 400, significantly increased the aqueous solubility of DEET along with
a decreasing o / w PC with increase in concentration of the solvent. The permeation of DEET across human skin was reduced significantly from solutions containing 60% or greater amounts of PG and PEG 400. Permeation of DEET was totally prevented from 90% PEG 400 solution which was very viscous, however a combination of 60% PEG 400 with 30% PG not only reduced permeation 9-fold but was suitable as a solvent for vehicle. This study clearly demonstrates that alternative formulations can be developed for DEET aimed at reduced permeation and toxicity unlike the current formulations some of which contain ethanol which has been shown to enhance permeation of DEET. A similar approach can be used for developing formulations of other industrial and occupational agents to prevent their skin permeation and penetration when a user may be exposed.
Acknowledgements We would like to thank the American Foundation of Pharmaceutical Education for a predoctoral fellowship to J. Stinecipher, and Jayabharati Vaidyanathan and Kimberley Hill for technical help in the PC experiments.
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