FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

16,41-50

The Effects of Zwitterionic

(1991)

Surfactants

on Skin Barrier Function

GEOFFREY RIDOUT,**’ ROBERT S. HINZ,* JURIJ J. HOSTYNEK,~ A. KRISHNA REDDY,? RICHARD J. WIERSEMA,~ CONNIE D. HODSON,* CYNTHIA R. LORENCE,* AND RICHARD *Departments of Pharmacy and Pharmaceutical California 94143; and tClorox Technical

Received

December

H. GuY*,~

Chemistry, University of Cahyornia, San Francisco, Center, 7200 Johnson Drive. Pleasanton, California

28, 1989; accepted

August

31, 1990

The Effects of Zwitterionic Surfactants on Skin Barrier Function. HOSTYNEK,

J. J., REDDY,

A. K., WIERSEMA,

R. J., HODSON,

San Francisco, 94566

RIDOUT, C. D., LORENCE,

G., HINZ, C. R., AND

R. S., GUY,

R. H. (1991). Fundam. Appl. Toxicol. 16, 41-50. The action of five zwitterionic surfactants on the barrier function of hairless mouse skin has been studied in vitro. The surfactants considered were dodecylbetaine and hexadecylbctaine (C 12BET and C 16BET, respectively), hexadecylsulfobetaine (C 16SUB). N,Ndimethyl-N-dodecylamine oxide (C 12AO), and dodecyltrimethylammonium bromide (CI2TAB). Excised skin was pretreated with each surfactant, at various concentrations, for 16 hr, following which the permeation of a model compound, nicotinamide, was measured, The action of the surfactants was assessedby comparing nicotinamide flux through surfactant-pretreated skin with that across control membranes which were exposed to buffer alone for 16 hr. All surfactants decreased skin barrier function to some extent. The degree of nicotinamide penetration enhancement induced was correlated with the ratio of the surfactant pretreatment concentration to the surfactant critical micelle concentration, suggesting that solubilization of stratum comeum lipids may be an important mechanism in explaining the effectsobserved. More detailed studies with %radiolabeled C 12BET and Cl 6BET showed that the dodecyl analog was itself well absorbed, whereas the Cl6 compound partitioned into the skin favorably but then transferred only very slowly into the receptor phase. These observations were consistent with toxicity studies (albeit at much higher concentrations in a different animal model, the rat) which indicated that the dermal LD50 of C 12BET was significantly lessthan that of C 16BET (the value for which was so large that it could not be reliably determined). Overall, this study provides, we believe, useful information pertinent to the potential dermal toxicity of the surfactants considered following OCCupatiOnal or environmental exposure. 0 199 I Society of Toxicology.

A principal function of the skin is to act as a barrier to the ingress of toxic chemicals (Barry, 1983). This role is accomplished, in large part, by the skin’s outermost layer, the stratum corneum, which is a thin membrane (ca. 15 pm thick) consisting of fully differentiated keratinocytes embedded in a complex, apolar, lamellar lipid domain (Wertz and Downing, 1989). In general, the ability of the stratum

corneum to limit passive molecular transport restricts systemic toxicity following dermal exposure to chemicals which are very potent or to situations in which large surface areas of the body are contaminated. However, the scope of potentially hazardous occurrences is much greater when the skin barrier is damaged, for example, by abrasions or burns. There are, in addition, certain chemicals which can substantially compromise the integrity of the stratum comeum and, in consequence, lead to enhanced penetration of the chemicals and/or other contacting materials.

’ Present address: Syntex Research Center, Research Park, Heriot-Watt University, Riccarton, Edinburgh, Scotland, EH 14 4AP. ’ To whom correspondence should be addressed. 41

0272-059019 1 $3.00 Copyright 0 199 I by the Society of Toxicology. All rights of reproduction in any form reserved.

42

RIDOUT

Skin permeation enhancers are diverse structural entities and include simple alkalis, organic solvents, unsaturated fatty acids, and surfactants (Walters, 1989). The latter category is particularly significant because of the presence of these amphiphiles in a variety of commonplace substances with which human skin is in frequent contact. For example, quaternary ammonium amphiphiles combine a number of cosmetic and dermatological attributes which lead to their wide application. Besides their traditional use in nonirritant shampoos, liquid soaps, and gels at 5- 10 wt% (Ernst and Miller, 1982; Linfield, 1970) the particular betaines selected for this study play a significant role in commercial formulation of hair care and wave setting products, are used in oral compositions for control of dental plaque or improved fluoride systems, and have potential uses in body deodorants and sanitary items, acne preparations, transdermal medications, suppositories, etc., as reflected in the patent literature. The amine oxide investigated has been reported to be a particularly effective keratolytic agent (Takahashi et al., 1987). The quarternary ammonium compound is representative of cationic amphiphiles commonly used as antimicrobials and laundry additives (l-2% in fabric softeners). While the damaging capabilities of certain surfactants have been demonstrated (Takahashi et al., 1987) quantitative relationships between structure and barrier derangement remain limited. The overall objective of the research described in this paper, therefore, was to probe the reduction of skin barrier function induced by representative amphoteric, cationic, and nonionic nitrogen-bearing amphiphiles. The effects of five surfactants on skin barrier function have been studied in a model system in vitro. The enhancement of nicotinamide permeation across excised hairless mouse skin following skin pretreatment with each of the surfactants has been assessed. Also, for two of the surfactants, dermal penetration levels have been determined and compared with their acute toxicities in vivo. The system used in this investigation to evaluate skin barrier.~

ET AL.

function and permeability was chosen for its simplicity and reproducibility. In vitro experiments employing excised skin tissue in simple diffusion cells are attractive alternatives to in vivo approaches @kelly et al., 1987): interpretation of permeation behavior is facile, the mechanics of the procedure are well established, and the results have demonstrated relevance to the in vivo situation (Poulsen and Flynn, 1985). Although hairless mouse skin is more permeable than its human counterpart, the murine tissue is readily available and shows relatively small inter-animal variation in barrier properties. The mouse provides a rather useful model, therefore, for structure-penetration studies relevant to dermatotoxicological situations. The per-meant selected to “report” on the barrier function changes induced by the surfactants was nicotinamide. This polar molecule penetrates only modestly across intact, unperturbed skin (Franz, 1975) and was considered an appropriate candidate to illustrate the induction of enhanced transport by the surfactants tested. The surfactants studied were N,NdimethylN-dodecylglycine (dodecylbetaine, C 12BET), N,N-dimethyl-N-hexadecylglycine (hexadecylbetaine, C 16BET), N,N-dimethyl-N-hexadecyl-3-ammonio- 1-propane sulfonate (C 16 propyl sulfobetaine, C 16SUB), N,N-dimethylN-dodecylamine oxide (C 12AO), and dodecyltrimethylammonium bromide (C 12TAB). The structures and selected properties of the surfactants are presented in Table 1. As well as their traditional use in shampoos, liquid soaps, and gels, the betaines and the amine oxide selected for this study are formulated with increasing frequency into hair conditioners, deodorants, and various topical medications. Dodecyltrimethylammonium bromide is used both for its biocidal activity and as a laundry additive for fabric softening. We believed, therefore, that the surfactants selected for this study were (a) relevant specific examples appropriate for risk assessment analysis, and (b) representative model compounds useful for the establishment of structure-pen-

EFFECTS OF ZWITTERIONIC

43

SURFACTANTS

TABLE 1 PROPERTIESAND STRUCIURES OF THE SURFACTANTS STUDIED Compound”

Molecular weight (da)

CMC (mWb

Structure

Cl2BET Cl6BET C16SUB Cl2TAB Cl2AO

271 328 392 228 229

1.6 0.014 0.02 5.0 0.02

GJbsN+(CH,)FHKG C16HS,N+(CH,),CH2CO;C,bH33N+(CH3)*(CH*)3SO; C,bH33N+(CH3)2(CH2)3SO; CJ-W+(CHS)W C,zHzsN+(CH3hO-

’ C 12BET. dodecylbetaine; C 16BET. hexadecylbetaine; C 16SUB, C 16 propyl sulfobetaine; C 1ZTAB, dodecyltrimethylammonium bromide; Cl2A0, N,N-dimethyl-N-dodecylamine oxide. b CMC. critical micelle concentration.

etration enhancement taneous absorption.

relationships

in percu-

EXPERIMENTAL Materials

Female hairless mice (HRS/hr hr), aged 6-14 weeks, were obtained from Simonsen Laboratories (Gilroy, CA). Adult male rats (Sprague-Dawley) were supplied by Harlan Sprague-Dawley, Inc. (Indianapolis, IN). N,N-Dimethyl-N-dodecylglycine (CIZBET). This compound was prepared by treating methyl chloroacetate with N,N-dimethyldodecylamine. The resulting ammonium chloride compound was hydrolyzed using Bio-Rad AG l8x resin to give the final product. The structure was confirmed by “C NMR spectroscopy and the purity was determined to be 98.9% by dissolving the sample in acetic acid and titrating it with perchloric acid. The sample contained 1.O% water (Karl Fisher). N,N-Dimethyl-N-hexadecylglycine (C16BET). This compound was prepared by treating methyl chloroacetate with N,N-dimethylhexadecylamine. The resulting ammonium chloride compound was hydrolyzed using BioRad AG 1-8x resin to give the final product. The structure was confirmed by 13CNMR spectroscopy and the purity was determined to be 94.9% by dissolving the sample in acetic acid and titrating it with perchloric acid. The sample contained 3.8% water (Karl Fisher). N-Hexadecyl-N,N-dimethyl-S-ammonio-l-propanesulfonate (Cl6 sulfobetaine). This material

was purchased from Calbiochem (San Diego, CA). The structure was confirmed by “C NMR spectroscopy, and the purity was determined to be 95.5% and was assigned based on the average quantitative 13CNMR (acetonitrile standard) and quantitative ‘H NMR (phenol sulfonate standard).

N-Dodecyl-N,N,N-trimethylammonium bromide. This material was purchased from Eastman-Kodak Co. (Rochester, NY). The structure was confirmed by “C NMR spectroscopy and the purity was found to be 99.9% by two-phase (water-chloroform) titration using a standard sodium lauryl sulfate and the dimidium bromide-disulphine blue VN mixed indicator. N-Dodecyl-N,N-dimethylamine-N-oxide. This material was purchased from Fluka Chemical Corp. (Ronkonkoma, NY). This chemical was used as received; the label gave a purity of 91%. Radiolabeled chemicals. r4C-Labeled C 12BET and C 16BET were prepared by the above described procedures using methyl [carbonyl-‘4C]chloroacetate. Syntheses and purifications were performed by Wizard Laboratories (Davis, CA). The specific activities of the dodecyl and hexadecyl homologs were 52 and 51 mCi/mmol, respectively. 14C-Labeled nicotinamide (sp act, 56 mCi/mmol) was purchased from Amersham (Arlington Heights, IL) and purified before use by thin-layer chromatography. Other chemicals. Unlabeled nicotinamide (>98% pure) was obtained from J. T. Baker Co. (Phillipsburg, NJ). Surfactant solutions were prepared in pH 7.4 phosphatebuffered saline (PBS) using reagent grade chemicals and distilled water. Skin Permeation

Apparatus

and Procedures

In vitro experiments utilized previously described diffusion cells and equipment (Gummer et al., 1987; Knepp et al., 1988). Briefly, the experimental procedures were as follows. Immediately after euthanizing a hairless mouse by CO* asphyxiation, full-thickness dorsal skin was excised. Any subcutaneous fat adhering to the dermal surface was carefully removed. The skin was then mounted between the donor and the receptor compartments of glass permeation cells (Laboratory Glass Apparatus, Berkeley, CA).

44

RIDOUT

The accessible skin area for transport was 0.95 cm2; one mouse, therefore, typically provided sufficient skin for four cells. The receptor compartment (volume = 3 ml) was perfused with pH 7.4 PBS at a rate of 3 ml&. The chamber temperature was regulated by a thermostat to maintain the skin surface temperature at 32 + 1“C. In the experiments, which measured the permeation of CIZBET and C16BET across the skin, 0.5 ml of surfactant solution (spiked with “‘C-labeled compound) in PBS was administered to the epidermal skin surface exposed in the donor compartment of the diffusion cell. The concentration of the applied C 12BET solution was 16 mM and that ofC16BET was 5.4 mM. These concentrations were chosen because they represented similar chemical doses (in Fmoles). For the next 24 hr perfusate samples, 3 ml at each hour, were collected on a fraction collector and then assayed for surfactant by liquid scintillation counting (Searle Model 6880). For each surfactant, seven or eight separate measurements of penetration (using skin from a minimum of four mice) were made. At the end of the transport experiment, the skin membrane was washed quickly in fresh buffer solution and was then dissolved in Soluene (Packard, Downers Grove, IL) before liquid scintillation counting. In this way, the amounts of C12BET and Cl6BET sequestered within the skin during the penetration process were determined. To assessthe effectsof surfactant pretreatment on skin barrier function, hairless mouse skin was excised and mounted in the diffusion cell as described above. The receptor chamber was filled with PBS and maintained at 32 f 1“C without perfusion. Surfactant solution (0.5 ml) was applied to the epidermal surface in the donor compartment for 16 hr (i.e., overnight). The concentrations of the different surfactants employed are given below. At the end of the pretreatment period, the surfactant solution was removed and the skin was gently washed three times with 0.5 ml of distilled water. The receptor compartment was drained, flushed with fresh PBS, and then perfused at 3 ml/hr. Nicotinamide solution (0.5 ml, concentration 100 pg/ml, spiked with %labeled compound) was administered to the epidermal surface and hourly 3-ml samples of the receptor phase were collected for the subsequent 12 hr. Nicotinamide permeation was determined by analyzing the fractions using liquid scintillation counting. Experiments were performed in triplicate or quadruplicate. For each surfactant solution tested, parallel control measurements were performed in which the pretreatment solution was PBS. Typically, skins from two mice were used such that half of the tissue from each mouse was pretreated with surfactant and the other half with PBS. In this way. a large body (n = 42) of control measurements was acquired. Toxicity

Studies

The following acute toxicology studies were performed on C 12BET and C 16BET:

ET AL. Acute oral toxicity. Adult male rats were acclimatized for 5 days before dosing. At the time of dosing (following an overnight fast) the animals weighed between 200 and 250 g. The test substances were administered in a single dose (as 25% w/v suspensions) by stomach intubation using a 16-gauge (3 in.) ball-end stainless steel needle attached to a disposable syringe. Following a preliminary range finding study, two dose levels for C 16BET and four dose levels for C 12BET were selected and groups of five rats were dosed at each dose level to determine the LD50. Acute dermal toxicity. Adult male rats were acclimatized for 5 days before dosing. Twenty-four hours before dosing the entire trunk area of each rat was clipped free of hair. At the time of dosing the animals weighed between 200 and 270 g. Appropriate amounts of test substances were weighed and applied on a body weight basis by moistening the sample with distilled water to form a paste. The sample was spread over as large an area of the trunk as possible and secured with double-layered gauze bandaging tape (3M Co., St. Paul, MN). After a 24-hr exposure period, all coverings were removed and any remaining test material was wiped off. Following a preliminary range finding study, two dose levels were selected and five animals per group were dosed to determine the LD50. Acute intraperitoneal toxicity. Adult male rats were acclimatized for 5 days before dosing. At the time of dosing, the animals weighed between 200 and 300 g. The test substances were administered as 25 or 5% (w/v) suspensions in distilled water, depending on the dose level required. The compounds were administered by a single intraperitoneal injection, using a 23-gauge needle. Following a preliminary range finding study, two dose levels were selected and five rats per group were dosed to determine the LD50. In all three studies, following the dosing, rats were observed for pharmacotoxic signs (including lethargy, abnormal exudates, or excretions, etc.) at hourly intervals on Day 1 and twice daily thereafter up to 30 days. Body weights were recorded on the day of dosing, once a week thereafter, and at euthanasia. All animals that died and all survivors were subjected to gross necroscopy, involving examination of all major organs (lungs, liver, pancreas, kidneys, and all regions of the GI tract). The LD50 values were calculated using the moving average method (Thompson, 1947). Statistics

Comparison between datasets, where appropriate, used Student’s t test.

RESULTS

Percutaneous C16BET

Penetration

of C12BET

and

The average permeation rates (*SD), as a function of time, of C 12BET and C 16BET are

EFFECTS OF ZWITTERIONIC

shown in Fig. 1 and 2, respectively. The cumulative amounts absorbed after 12 and 24 hr of exposure are given in Table 2. Penetration of C 12BET was relatively rapid, such that nearly 50% of the applied dose was absorbed in 24 hr. This substantial permeation is apparent in Fig. 1 which shows that the rate of penetration, following a pseudo-steady-state regime, reached a maximum at about 18 hr and then began to decline, indicative of significant depletion of surfactant in the donor phase. In contrast, only 1.3 f 0.4% of C 16BET was absorbed through the skin in 24 hr. It is not clear from Fig. 2 whether steady-state permeation of the surfactant had been reached by this time. The skin digestion experiments revealed that the average levels of C 12BET and ClBBET, associated with the skin at the end of the permeation runs, were approximately 25 and 15% of the applied dose, respectively. Eflects of Surfactant Pretreatment amide Permeation

on Nicotin-

The cumulative amounts of nicotinamide penetrating the surfactant-pretreated hairless mouse skin in 12 hr are summarized in Table 3. The data reveal that, with the exception of C 12A0, pretreatment by all surfactants at all concentrations significantly (p < 0.05) promotes nicotinamide permeation across hairless mouse skin when compared to the controls.

45

SURFACTANTS

0.1

0.0 Time

(hours)

FIG. 2. FIux of [‘%Z]C16BET (mean +_SD, n = 7) across hairless mow skin in vitro as a function of time.

For three of the surfactants (Cl6SUB, C16BET, and C12BET), the effects of pretreatment with three different concentrations were examined. The concentrations employed corresponded approximately to 10X the critical micelle concentrations (CMC), 100X CMC, and 500X CMC (see Table 3). On a molar basis, the C 16 surfactants are obviously greater perturbants than the C 12BET. However, when the concentrations are normalized with respect to the CMC, the penetration-enhancing potencies of the three surfactants appear to be remarkably equivalent. Parenthetically, the large number of control experiments performed allowed a relevant question to be examined; namely, does the age of the mouse, from which the skin is excised, have any influence on the unperturbed flux of nicotinamide? Figure 3 shows the cumulative amount of nicotinamide reaching the receptor phase in 12 hr as a function of the age of the mouse. It is clear that, under the experimental conditions of this study, no age-related dependence of nicotinamide penetration can be identified. Toxicity Studies

Time

(hours)

FIG. 1. FIux of [‘*C)C 12BET (mean -+ SD, n = 8) across hairless mouse skin in vitro as a function of time.

Oral administration of either C12BET or Cl 6BET caused sluggishness, diarrhea, and lacrimation in some of the animals. Weight gain of the surviving animals was within nor-

46

RIDOUT

ET AL.

TABLE CUMULATIVE

Time (hr)

Surfactant ClZBET’

OF C 12BET AND C 16BET THROUGH AFTER 12- AND 24-hr EXWSURES Cumulative

12 24 12 24

C16BETb

‘Applied b Applied

PENETRATION

2

amount (fig) absorbed (mean f SD) 220 1010 1.9 11.8

SKIN

10.3 46.5 0.21 1.33

f f + +

3.5 8.2 0.10 0.38

dose = 8 pmol = 2168 pg = 0.5 ml of 16 mM solution; n = 8. dose = 2.7 ymol = 886 rg = 0.5 ml of 5.4 mM solution; n = 7.

included sluggishness and reddish nasal and ocular discharges. Body weight gains of animals subjected to C 12BET and C 16BET were within normal limits. Gross necropsy did not reveal any significant changes related to the test compounds. Intraperitoneal administration of both C12BET and C16BET resulted in general symptoms of sluggishness, diarrhea, lacrimation, and distended abdomen. Weight gains of animals were within normal limits for both

TABLE CUMULATIVE

PENETRATION

3

OF NICOTINAMIDE (MEAN f SD) IN 12 hr FOLLOWING OF HAIRLESS MOUSESKIN WITH~URFACTANTS

Applied concentration Surfactant

b-4

ControlC C12BET”

0 16 100 800 0.14 1.0 5.4 0.2 2.0 10 10 10

C12TAB C12AO

MOUSE

Cumulative % dose absorbed (mean f SD)

f 80 + 180 + 0.9 + 3.4

ma1 limits. Gross necropsy of those that died revealed that the gastrointestinal tract was distended with red fluid and that the lungs appeared mottled and red. There were no remarkable differences in the pharmacotoxic signs or gross necropsy findings between the two compounds. Topical administration of either of the two betaines caused localized effects, including erythema, edema, desquamation, necrosis, and scab formation; generalized toxic symptoms

C16BET C16SUB

HAIRLESS

Normalized concentration cc*)’

L?C* = Applied concentration divided by the CMC. b N = number of replicates. ’ Pretreatment with pH 7.4 PBS containing no surfactant.

0 10 62.5 500 10 71.4 457 10 100 500 50:

PRETREATMENT

Cumulative % dose absorbed 0.32 5.1 8.0 33 1.2 11 33 4.7 6.7 26 4.3 0.71

+ zk f k + f + f f * f +

0.26 1.7 3.1 7.4 I.0 2.4 6.1 0.9 1.7 6.6 3.7 0.10

Nb 42 4 4 4 4 3 4 3 4 4 4 4

EFFECTS OF ZWITTERIONIC 1.5 1

s

4

4

6

8 Age

10 of

12 mice

14 (weeks)

16

18

FIG. 3. Cumulative penetration of [‘4C]nicotinamide in 12 hr following pretreatment of hairless mouse skin with pH 7.4 PBS. The results of 42 separate experiments are plotted as a function of mouse age. The line of linear regression through the data is Y = 0.76-0.041 X, with rz = 0.20.

test compounds. Necropsy revealed no test material-related findings. The LDSO values for the two compounds were calculated for the three routes of exposure and are presented in Table 4. DISCUSSION The experiments performed have demonstrated that surfactant solutions are capable of significantly promoting the percutaneous transport of a model penetrant. In addition, the surfactants themselves also permeate the skin, but do so to different extents. We have found that C12BET crosses hairless mouse skin more easily than the more lipophilic homologue, C16BET. The C12BET compound

47

SURFACTANTS

also shows much greater dermal toxicity than the Cl6 homologue. The skin penetration of C 16BET in 24 hr amounts to approximately 1.5% of the applied dose. However, when the skin is digested at the end of the transport experiment, about 15% of the administered radioactivity is associated with the tissue. For Cl 2BET, approximately 50% of the administered surfactant penetrates in 24 hr, with somewhat less than half this amount remaining associated with the skin at this time. The flux of surfactant across the skin is a function of a number of variables. Assuming that monomers transport in preference to micelles, the concentration of monomeric surfactant is one parameter that will affect the flux. Given the relative CMC values of C 12BET and C 16BET there is clearly a much higher concentration of free C 12BET available compared to C 16BET and this must partially, at least, account for the relative flux values observed. The greater lipophilicity of C 16BET is also likely to result in a smaller flux for this homologue. While the chemical will partition favorably into the stratum corneum, its subsequent transfer from this lipid layer into the underlying, and much more aqueous in nature, viable epidermis will be slow. This hypothesis is supported by the 24-hr disposition of C16BET in the transport experiments: There were 10X more molecules in the skin than had accumulated in the hourly fractions, The percutaneous transport of surfactants and the effects of these amphiphiles on skin barrier function have been reviewed in the lit-

TABLE 4 ACUTE ORAL, DERMAL, AND INTRAPERITONEAL (IP) TOXICITIES OF C 12BET AND C 16BET IN ADULT MALE RATS LD50 (mg/kg)= Betaine

Oral

Cl2BET C16BET

7 1 (52-96)b 1620 (1010-2620)

Dermal 1300 (960-1750)

> 16000

0 Calculated by the moving average method of Thompson (1947). b Values in parentheses are 95% confidence limits.

IP 53 (35-79) 150 (84-260)

48

RIDOUT

erature (see, for example, Ashton et al., 1986; Walters, 1989). There is evidence that an alkyl chain length of 12 carbons confers optimal skin permeation behavior. For example, maximum naloxone flux through human skin was observed with Cl2 adjuvants in a comparison of eight saturated fatty acids (C,-C,s) and six fatty alcohols (C&,s) (Aungst et al., 1986). Of the surfactants considered in this study, the percutaneous absorption of C12TAB (Bartnik and Wingen, 1979) and C12AO (Rice, 1977) have been evaluated. Percutaneous absorption in rabbits and mice has been observed also for a topical antimicrobial agent which contains a mixture of alkyl dimethyl Nbetaines, alkyl dimethyl amine oxides, and alkyl dimethyl amines (Michaels et al., 1983). C12TAB was very poorly absorbed in the rat when administered either in a hair-care formulation (0.01% dose in 48 hr) or as an aqueous solution (0.6% dose in 24 hr). Cl2AO was relatively well absorbed in the rat (18% dose in 24 hr), mouse (18% dose in 24 hr), and rabbit (24% dose in 24 hr; 46% dose in 72 hr). However, penetration of Cl 2A0 in humans following an 8-hr exposure was at the limit of detection. With respect to skin barrier compromisation, the CIZ chain length seems to be close to optimal. For example, a much publicized compound, l-dodecylazacycloheptan-2-one (Azone) has been shown to act as an effective penetration enhancer for a number of topically applied drugs (Morimoto et al., 1986; Stoughton, 1982; Sugibayashi et al., 1985; Wotton et al., 1985). Although variations of the structure of Azone have been prepared and tested (Mirejovsky and Takruri, 1986; Sugibayashi, et al., 1985). the Cl2 hydrocarbon “tail” remains the lipophilic portion of choice. Other indirect evidence comes from recent work examining the penetrationpromoting ability of various cis-octadecenoic acid isomers (Golden, 1987b). It has been shown that the acid with the c&unsaturated C=C bond at the C- 1I position enhances salicylic acid flux across the porcine stratum corneum to the greatest extent.

ET

AL.

In contrast to the absolute permeabilities of the Cl 2 and Cl6 betaines, the skin perturbation effect of C16BET, as measured on a molar basis, is significantly greater. However, when the surfactant concentrations employed are normalized with respect to the corresponding CMC, the enhancing effects on nicotinamide penetration are quite similar. The sulfobetaine (Cl6SUB) and, to a certain extent, the quaternary ammonium salt (C 12TAB) conform to the same pattern of behavior. These observations suggest the hypothesis that a common mechanism of surfactant-enhanced skin penetration is operative in this experimental system: viz, that surfactant micelles are solubilizing the intercellular lipids of the hairless mouse stratum comeum and thereby reducing the diffusional and partitioning resistance of the barrier to the passage of nicotinamide. Parenthetically, it should be pointed out that the sensitivity of murine skin to lipid extraction by alkanols has been demonstrated in similar experiments conducted in our laboratory (Kai et al., 1990). It should be pointed out that the amount of stratum comeum lipid that can be solubilized is proportional to the concentration of micellar surfactant in the pretreatment solution and also to the capacity of the micelles to solubilize the lipid. For example, pretreatment with solutions of C 12BET and C 16BET at concentrations 500X their respective CMCs results in similar nicotinamide absorption, even though the actual concentration of C16BET is much less (160X) than that of C 12BET. This indicates that C 16BET micelles are larger than C 12BET micelles and have a much greater solubilization capacity. The fact that the concentration/capacity effects seem to balance one another out may only be coincidental. C 12AO did not induce significant enhancement of nicotinamide absorption. Despite pretreatment of the skin with a concentration of 500X CMC, nicotinamide flux did not increase above control levels. It would appear that C 12AO does not show a particularly high affinity for mouse stratum comeum and is in-

EFFECTS OF ZWITTERIONIC

capable of efficiently solubilizing the intercellular lipids. An alternative hypothesis for the mechanism of action is that the surfactant monomers partition into the SC and disorder the intercellular lipid domains, as has been demonstrated for cis-unsaturated fatty acids (Golden et al., 1987b, Mak et al., 1990). While the concentration-dependence of the results presented in this paper are inconsistent with the pattern of behavior associated with the lipid disordering phenomenon (i.e., a saturable process at relatively low levels of fatty acid) (Mak et al., 1990), it should be possible to evaluate the contribution, if any, of this mechanism to the enhanced permeation of nicotinamide by further biophysical measurements using, for example, infrared spectroscopy (Golden et al., 1987a,b; Knutson et al., 1985). Finally, it is appropriate to comment upon the importance of this paper’s findings with respect to dermatotoxicity and, in passing, to transdermally delivered drug therapy. In the former case, the research described here has in many ways been designed to examine the “worst-case” scenario. The surfactant pretreatments have been prolonged and have involved high concentrations of the agents in intimate and continuous contact with the skin. The model transport system has been the skin of the hairless mouse, a membrane known to be more permeable than its human counterpart and to be particularly sensitive to hydration and chemical damage (Bond and Barry, 1988a,b; Hinz et al., 1989). Nevertheless, the controls have revealed that the tissue holds up well for the duration of the measurement period and, for this reason, the data may be helpful in the risk assessment process. In particular, the procedures employed will certainly not underestimate dermal exposure in man. From a therapeutic standpoint, the results discussed here may be significant. It is clear that the surfactants investigated are capable of inducing a range of skin-penetration enhancing effects. Given that transdermal drug delivery is presently limited to only very potent

SURFACTANTS

49

pharmacological agents (because the skin is such a substantial barrier), there is considerable interest in the development of adjuvants which can reversibly modify stratum comeum resistance, yet cause negligible toxicity (Guy and Hadgraft, 1988). In this regard, the surfactants studied, in particular the betaines, are worthy of further examination. ACKNOWLEDGMENTS This research was supported in part by NIH Grant HD23010 and by a Cooperative Agreement (CR-812474) with the U.S. Environmental Protection Agency. We thank Dr. Larry L. Hall for his constructive comments and suggestions.

REFERENCES ASHTON, P., HADGRAFT, J., AND WALTERS, K. A. (1986). Effects of surfactants in percutaneous absorption. Pharm.

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