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Mechanism of oleic acid-induced skin penetration enhancement in vivo in humans
journal of
ELSEVIER
controlled release Journal of Controlled Release 37 (1995) 299-306
Mechanism of oleic acid-induced skin penetration enhancement in vivo in humans Aarti Naik a,*, Louk A,R.M. Pechtold b,c, Russell O. Potts b, Richard H. Guy a "Departments of Pharmacy and Pharmaceutical Chemistry, University of California at San Francisco (UCSF), San Francisco, CA 94143, USA b Cygnus Therapeutic Systems, Redwood City, CA 94063, USA Leiden-Amsterdam Center for Drug Research, Department of Pharmaceutical Technology, University of Leiden, Leiden, The Netherlands
Received 13 September 1994; accepted 13 June 1995
Abstract The outermost layer of mammalian skin, the stratum corneum (SC), by virtue of its unique architecture, presents a significant barrier to the transdermal delivery of drugs. Penetration enhancers such as oleic acid (OA), which increase skin permeability, appear to act selectively on the extracellular lipids representing the principal regulatory channel for the penetration of small molecules. In vitro studies investigating the mode of action of OA, have generated two mechanistic scenarios, which may account for the action of this enhancer; (a) lipid fluidization, and (b) lipid phase separation. In the studies presented here, attenuated total reflectance infrared spectroscopy was used to determine the mode of action of OA in vivo, in man. The use of perdeuterated OA ( [2H] OA) enabled the behaviour of endogenous lipids to be observed independently to that of the exogenously applied enhancer as a result of their spectrally distinct methylene group vibrations. Human forearm was treated topically with 1 ml of either (a) a solution of 5% ( v / v ) [2H] OA in ethanol, or (b) ethanol alone, for a period of 16 h. After removal of the delivery system, the SC at the application site was progressively removed by adhesive tape-stripping, while sequential IR spectra were obtained at each newly exposed surface. In this way, we were able to monitor (a) the distribution profile of [2H] OA across the SC, (b) the conformational order of the SC lipids as a function of depth, and (c) the phase behaviour of the enhancer in the S C. Our results indicate that [ 2H ] OA induces lipid disordering only in the superficial layers of the SC, albeit of a smaller magnitude than that associated with a gel to liquid crystalline conformational change. Additionally, [2H] OA was found to exist in a liquid phase at all levels of the SC spectroscopically examined. These results suggest, therefore, that OA-induced skin penetration enhancement results from a mechanism involving both SC lipid fluidization and phase separation, with the latter probably predominating. Keywords: Stratum corneum lipid; Oleic acid; Percutaneous absorption; Skin penetration; Penetration enhancer; Attenuated total reflectance infrared spectroscopy
1. Introduction
* Corresponding author (present address): Department of Pharmaceutical and Biological Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK. Tel. 0121 359 3611; Fax 0121 359 0733. 0168-3659/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDIOI 6 8 - 3 6 5 9 ( 9 5 ) 00088-7
O f the m a n y diverse physiological functions that m a m m a l i a n skin is required to fulfil, its protective role is perhaps the m o s t demanding. R e c o g n i t i o n of the stratum c o r n e u m ( S C ) , the o u t e r m o s t epidermal layer, as the principal regulatory barrier to the transcutaneous traffic o f water and e x o g e n o u s substances, has b e e n
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succeeded by an array of structural and mechanistic insights. Central to these findings is the observation that the exceptionally thin, but remarkably impermeable, SC envelope is a complex composite of terminally differentiated ceils (corneocytes) embedded in a multilamellar lipid continuum - an assembly which is fundamental to the regulation of skin barrier function [ 1,21. The formidable SC barrier allows only a limited number of drugs to be delivered in therapeutic amounts through the skin. As a consequence, a number of chemical enhancers have been developed which, when coapplied with the drug, significantly and reversibly enhance skin flux. Oleic acid (OA) is a commonly used enhancer, the mechanism of action of which, has been widely studied in vitro [3-6]. Results of these studies indicate that OA primarily modulates the extracellular lipid domain of the SC. For example, the extent to which OA decreases the SC lipid phase transition temperature is directly (and highly) correlated with the amount of fatty acid taken up by the SC, as well as with its ability to enhance in vitro permeability of a coadministered permeant [4]. Permeation enhancers such as OA which reduce the diffusional resistance of the skin by interacting with the lipid matrix, have been postulated to act by increasing lipid fluidity [7,8], in a manner analogous to enhanced membrane permeability resulting from thermally induced SC lipid disordering [9-11 ]. However, recent infrared spectroscopic studies on porcine SC and extracted lipids treated in vitro have demonstrated, at physiologically relevant temperatures, the existence of OA in a liquid state, and therefore phase-separated from the endogenous solid lipids [ 12]. Since other phase-separated lipid systems have been reported to exhibit exceptionally high permeability at the interface formed at the boundary between the fluid and solid domains [ 13-16 ], the in vitro results to date suggest that OA enhances SC permeability through a similar mechanism. How does OA interact with SC lipids in vivo; is the observed scenario with excised SC and extracted lipids re-enacted in vivo? Attenuated total reflectance infrared (ATR-IR) spectroscopy is a non-invasive technique which has proven useful for the in vivo evaluation of topically applied enhancers in man. In a study designed to investigate the effect of OA on the transdermal delivery of a co-permeant, treatment with the enhancer was found to increase the rate of drug disappearance from
human SC [ 17]. These observations have been supported by further quantitative studies demonstrating the penetration enhancement properties of OA as a function of depth into the SC [ 18]. Here, we report the application of ATR-IR to ascertain the effect of OA on SC lipids, in vivo, in man. More specifically, we have utilized perdeuterated oleic acid ( [2H] OA), allowing the independent evaluation of endogenous SC lipids and exogenous OA, by virtue of their spectrally distinct CH2 and CD2 stretching absorbances, respectively.
2. Materials and m e t h o d s
Eight subjects (aged 25-50 years) in good health, with no history of dermatological disease, participated in these studies (approved by the UCSF Committee on Human Research). Experiments were performed on sites on the inner ventral forearm; one arm was treated while the other arm served as a control. Prior to treatment, the test sites were cleaned with a water-dampened swab, after which the subject remained in a room at constant temperature (21 _ 1°C) and relative humidity (30-40%) for 45 min, while pre-treatment spectra were recorded at 25, 35 and 45 min. An aliquot (1 ml) of a 5% (v/v) solution of perdeuterated oleic acid ( [ 2H ] OA) ( Cambridge Isotopes, Cambridge, MA) in ethanol was applied to a 12 cm 2 site for 16 h. The control site, on the contralateral arm, was treated with ethanol alone. Both formulations were applied on a surgical cotton pad ( 1.5 X 8 cm), occluded and covered with a Tegaderm ® (10X 12 cm) wrap. 16 h later, the occlusive patch was removed and both sites were cleaned with ethanol-soaked swabs. The sites were then exposed to air for 2 h to allow dehydration of the occluded skin. An IR spectrum of the dosed site was then recorded. After this initial spectral acquisition, a piece of Scotch® Book Tape no. 845 (3M, St. Paul, MN), was pressed against the application site and then removed, taking with it a layer of adherent SC. A further spectrum was recorded and the sequence repeated until a maximum of 20 incremental spectra were obtained. To quantify the mass of SC removed with each strip, the tape was weighed before and after the stripping procedure.
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3. Spectral analyses A T R spectroscopy is a non-invasive technique in which the IR beam is directed to an IR transparent crystal (internal reflection element; IRE) supporting a sample. The resulting spectrum describes a defined region of the surface of the sample in contact with the IRE; the application of this methodology to in vivo measurements of SC has been described in the literature [19,23]. In the study presented here, spectra were recorded with a Nicolet 730 Fourier Transform (FT) IR spectrophotometer (Nicolet, Madison, WI), equipped with a liquid N2 cooled mercury-cadmiumtelluride detector. The conventional transmission cell mount was replaced by a horizontal Out-of Compartment Contact Sampler (Spectra-Tech, Stamford, CT) supporting a zinc selenide (ZnSe) IRE (dimensions: 7 × 1 × 0.2 cm) having 45 ° entrance and exit faces and a refractive index of 2.4 at 1000 cm-1. To obtain a spectrum, 64 scans were collected at a resolution of 2 c m - 1 over a range of 4800-400 c m - 1, co-added, and transformed using a Happ-Genzel apodization function. While an internal laser referencing system in FT spectroscopy allows excellent wavenumber precision and accuracy, and thus direct comparison of peak positions, the intensity of A T R spectra is dependent on the degree of contact between the IRE and sample. Thus, in order to compare intensities, the absorbance of interest is normalized against a spectral parameter which also varies with contact. In this way, quantitative comparisons can be made. In the study described here, the absorbance was ratioed against the amide I absorbance near 1640 c m - i, due to the carbonyl stretching motions of the CO-NH group. The same concept has been used to quantitate the uptake of water [ 19] and drugs [ 17] by the skin. The frequencies of the CH2 and CD2 stretching absorbances were determined using a center-of-gravity algorithm [20] allowing positions to be determined with an uncertainty of less than 0.1 c m - 1. Since both the CH2 and CD2 absorbances overlap, to varying extents, with broad bands due to water and other IR absorbing materials, a baseline correction was required prior to further data analysis. For example, as shown in Fig. 1, the CH2 symmetric ( ~ 2850 cm ~) and asymmetric ( ~ 2920 c m - ~) peaks occur as shoulders on a broad absorbance with a maximum near 3200 cm-~.
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Fig. 1. Reflectance infrared spectrum of human stratum corneum treated with perdeuterated oleic acid. The spectral separation of CH2 and CD2 stretching bands is illustrated in the inset. A spectrum of untreated stratum comeum (not shown) appears identical to the above in every respect except for the presence of the CD2 stretching vibrations. The baseline here was established by extrapolating a straight line between 2993 and 2745 cm-1. The CD2 stretching absorbances near 2195 and 2095 cm -1, necessitated a 4th order polynomial baseline fit to the data due to the impact of a broad absorbance centering near 2100 c m - 1, directly beneath these regions.
4. Results and discussion An ATR-IR spectrum of human skin treated with perdeuterated oleic acid ( [ 2HI OA) is shown in Fig. 1, with the spectral region of particular interest ( 2 0 0 0 3000 c m - l ) magnified in the inset. Featured in this spectrum are peaks near 2850 and 2920 c m - ~ due to CH2 symmetric (us) and asymmetric (uas) stretching vibrations, respectively, originating primarily from the extracellular lipids of the SC [21]. The treated skin also contains [ 2H ] OA which, by virtue of the chemical substitution of deuterium for hydrogen, exhibits CD 2 symmetric and asymmetric stretching absorbances near 2095 and 2190 c m - l, respectively. Since spectral parameters associated with these absorbances, such as magnitude and frequency, report on the behaviour of the absorbing species, this spectrum clearly demonstrates the ability of the ATR-IR technique to distinguish, in the skin of human subjects, differences in the behaviour of endogenous SC lipids and the exogenously applied enhancer. In doing so, an in vivo assessment of [2H]OA uptake into human skin and its
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Fig. 2. Weight per unit area of SC removed by sequential tapestripping, followingtreatment with ethanol alone (O) or 5% (v/v) [2H]oleic acid in ethanol (O). Mean_+SE n=7 or 8. subsequent effect on SC lipids is facilitated. Furthermore, the combined use of ATR-IR spectroscopy and tape-stripping of the skin allows these measurements to be conducted as a function of depth within the SC. The distance into the membrane, probed by successive tape-stripping, was defined by the weight of SC removed [ 18]. Due to differing adhesive properties of the SC from site to site and as a function of depth at any one site, the mass removed by each tape strip was variable. Thus, the weight of SC removed, not the strip number, provided a more quantitative measure of the depth probed within the SC. Fig. 2 compares the mean mass removed with each strip for control and [ 2H ] OAtreated human skin. These results demonstrate that significantly greater amounts of SC were removed by the first three strips in the [ 2H ] OA-treated skin (ANOVA, p < 0.05), after which statistically equivalent amounts were removed per strip for treated and control groups. Primarily due to the greater mass removed in the outermost strips, the total amount of SC removed was significantly greater for the enhancer treated skin. Earlier studies of oleic acid-treated human skin have reported similar observations [ 18 ]. Since SC cohesion has been related to the nature of lipid constituents present within the membrane [22], these results suggest that the SC lipid network has been modified by [2H] OA. Finally, these data indicate that the amount removed per tape-strip was the same for treated and control sites at the deeper levels evaluated, implying
that [2H] OA concentration decreased markedly with depth into the SC (see below). The uptake and distribution of [2H] OA in the SC was evaluated by measuring the area under the CD2 stretching absorbances; the magnitude of these bands being directly proportional to the amount of absorbing species. As shown by the results in Fig. 3, the CD 2 signal decreased with increasing mass of SC removed, reaching a limiting value near zero in the deepest layers of the SC probed. This concentration profile is consistent with sink conditions near the base of the SC, suggesting that the SC is the primary resistive barrier to the transdermal absorption of [ 2H ] OA. The amount of endogenous lipid in the SC was similarly determined from the area under the CH2 stretching bands. The data in Fig. 4, reporting the magnitude of the CH 2 stretching absorbances as a function of SC depth, were statistically indistinguishable for the treated versus control sites. These results indicate that the amount of lipid within the SC was (a) unaffected by [2H]OA treatment, and (b) decreased to a value of about 60% of the surface value following tape-stripping. Similar depthdependent results obtained by Bommannan et al. [23] have been interpreted to reflect decreased lipid content with increased distance into the SC. In addition to revealing lipid content and permeant distribution profiles, ATR-IR spectroscopy is also notably instructive with respect to the conformational and structural status of the membrane constituents. In par-
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Fig. 4. Relative lipid content within the SC (expressed as % of prestripping value for untreated SC) as a function of SC weight removed, followingtreatment with ethanol alone (O) or 5% (v/v) [ ZH] oleic acid in ethanol ( • ) . Mean _+SE; n = 7 or 8. ticular, we are concerned with discerning the influence, if any, which oleic acid exerts over the lipoidal domains within the SC. The key questions which we wish to address are: (a) does OA alter the conformational order of the endogenous SC lipids, and if so, to what extent, (b) is the fatty acid phase-separated from the endogenous lipids, and (c) are one or more of these effects dominant and/or consistently present across the entire membrane? The frequency positions of the CH2 stretching absorbances (/~CH2), observed near 2800-2950 c m in the IR spectrum, are sensitive indicators of the level of motional freedom and flexibility associated with the lipid alkyl chains and, as such, report on the average structural order of the SC lipid domains. For example, increased pCH2 is associated with decreased lipid order in a variety of lipid systems [ 2 4 - 2 6 ] , including SC [27,28]. More specifically, ~'CH2 is related to the ratio of t r a n s / g a u c h e conformational isomers along the hydrocarbon tail. Previous results obtained with porcine SC have shown a temperature-dependent increase in alkyl chain disorder manifested as an increase in vsCH 2 from a value near 2850.2 c m - ~ at 40°C where the chains were predominantly trans, to a value near 2853.3 c m - 1 at 90°C, above the phase transition temperature (50-80°C), reflecting a significant g a u c h e population [ 11 ]. The effect of [2H]OA on the endogenous lipid organization in these experiments was thus assessed by
determining the CH 2 symmetric and asymmetric stretching frequencies at each incremental spectral measurement across the SC. Fig. 5 illustrates the asymmetric stretching frequencies originating from the SC lipids plotted as a function of the average cumulative SC mass removed by tape-stripping. Note that since the amount of SC removed per strip differed for the treated and control groups, the abscissal data points for these two experimental groups do not precisely overlap. Nevertheless, the data in Fig. 5 show that the first two measurements (pre- and post- first tape-strip) for the [ 2H ] OA-treated group, were significantly (p = < 0.01 ) elevated with respect to the background (untreated skin) and control (ethanol treated) measurements. Thereafter, all vasCH 2 data conformed to a common mean value (_+SD) of 2915.60 ( + 0 . 3 3 ) cm-~, analogous to the corresponding control group mean frequency of 2915.60 (_+0.31) cm -1, characteristic of 'solid' alkyl chains in a predominantly trans configuration. The deduction from this profile, then, is that following topical administration of [ 2H ] OA, lipid disordering is sustained only by the surface and uppermost layers where the concentration of [2H]OA, and the intrinsic fluidity of the SC lipids is greatest [23]. The lipid viscosity in the remainder of the membrane is essentially unaffected by [2H]OA treatment. The results presented here are consistent with previous in vitro data derived from OA-treated porcine SC [ 12], 2920
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A. Naik et al./ Journal of Controlled Release 37 (1995) 299-306
304
Table 1 Asymmetric (v.~CH2) and symmetric (/.'syCH2) stretching frequencies for [ 2HI oleic acid treated human stratum corneum vasCH2
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demonstrating that the average conformational order of SC lipids was unaffected by [2H]OA. This is because the measured CH2 stretching absorbance is in fact an ensemble of overlapping vibrational bands, reflecting heterogeneity in the local conformational order. This is best illustrated by the case of a pure lipid undergoing a thermal phase transition; at the transition mid-point where the lipid is partially melted, the bandwidth of the-CH2 stretching absorbance increases due to the larger number of conformational states of the hydrocarbon chains [26]. The frequency reported by an IR spectrum, therefore, represents a weighted average for the population of methylene groups undergoing stretching oscillations, and will be dependent on the relative contribution (amount and conformational state) of each sub-population. Since transmission IR spectroscopy (used to scan the SC sample in vitro) examines and reports on the distribution of vibrational frequencies encountered across the entire membrane intersecting the path of the beam, the technique is insensitive to any localized or depth-dependent variations. Indeed, the mean ua~CH2 (of 21 successive measurements) for the SC examined in vivo, was calculated to be 2915.80 + 0,41 c m - 1, a value not significantly different from that of the control group. By contrast, our in vivo approach, using reflectance IR spectroscopy, facilitates the differentiation of any depth-dependent, though not lateral, disparities. A similar profile was noted for the CH2 symmetric stretching absorbances across the membrane (Table 1). The magnitude of the change in frequency has been related to the number of gauche conformers introduced,
and consequently to the level of disordering. Thermotropic phase transitions of human [28], porcine [ 10,11] and murine [27] SC are typically accompanied by a change in ua~CH2 and ~'syCH2 of approximately 6 and 3 cm -1, respectively. It is pertinent to point out that the shifts in maximum absorbance incurred following [2H] OA treatment are significantly smaller (Table 1; Fig. 5), and are thus indicative of a lower degree of perturbation. The small decrease in uasCH2 observed in both control and OA-treated (excluding pre- and post-firststripping) sites, relative to untreated skin, most likely reflects the combined outcome of two phenomena; (a) a heterogeneous gradient of lipid composition as revealed by repeated tape-stripping, and (b) the extraction of surface lipids (sebaceous in origin and liquid at physiological temperature) by prolonged contact with ethanol. The decrease in ua~CH2 is also accompanied by a reduction in bandwidth (data not shown), portraying increasing homogeneity among the lipid population below the surface of the SC. These inferences are supported by the findings of Bommannan et al. [24,30] reporting (a) a stratal gradient of lipid concentration and conformational order, with a predominance of less structured lipids near the surface, and (b) the concurrent percutaneous penetration of ethanol, extraction of SC lipids, and decrease in the intensity, bandwidth and frequency of the CH2 stretching vibrations, following in vivo dermal treatment with ethanol. The phase behaviour of the topically administered oleic acid within the SC can be similarly discerned from a survey of the CD2 stretching absorbance maxima. A plot of the measured CD2 asymmetric stretching frequencies (u,sCD2) as a function of depth into the SC (Fig. 6) illustrates a small, but gradual increase in vasCD2 with increasing membrane depth, being characterized by a slope (95% confidence interval; r 2) of 2 . 3 9 6 × 10 - 2 mg -1 ( +_9.309 X 10-3; 0.151). Within the membrane, the mean ( + SD) value of ua~CD2 is 2196.60 (+_0.3) cm ~. It becomes evident, on comparison with the b ' a s f D 2 value of 2197.17 cm-~ for pure [2H]OA at 25°C (above its melting point of 13.4°C), that this is consistent with [ 2HI OA in a liquid phase. The combined data from Fig. 5 and 6 demonstrate that [ 2H ] OA in human SC existed in a fluid state, while the endogenous lipids were both disordered (in the superficial layers) and solid (in the deeper regions). These in vivo results are therefore consistent
A. Naik et al./ Journal of Controlled Release 37 (1995) 299-306 2197.5
Acknowledgements
The authors w o u l d like to thank m e m b e r s o f the Skin B i o s c i e n c e Group, U C S F , and the Biophysical R e s e a r c h Group, C y g n u s Therapeutic S y s t e m s for their helpful c o m m e n t s and participatory role in these studies. This research was supported by a grant ( H D 23010) f r o m the U S National Institutes o f Health.
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Fig. 6. CD2 asymmetric stretching frequencies,/,'asCD2, (originating from the topically applied [ 2H] oleic acid) as a function of SC weight removed, following treatment with 5% (v/v) [2H]oleic acid in ethanol. Mean _+SD; n = 7 or 8. with lipid phase separation w h e r e solid and fluid d o m a i n s co-exist. As r e v i e w e d by Ongpipattanakul et al. [ 12], lipid phase separation can result in substantially e n h a n c e d permeability in m a m m a l i a n SC, as in other lamellar lipid barriers. Furthermore, it has b e e n s h o w n in s i m p l e r lipid systems that c i s - u n s a t u r a t e d fatty acids ( l i k e oleic acid) will f o r m a separate fluid d o m a i n w h e n introduced into a solid, saturated lipid mixture (like SC lipids) [ 2 9 ] . The possibility exists, therefore, that O A - i n d u c e d transdermal penetration e n h a n c e m e n t occurs as a result o f increased permeability at the interface b e t w e e n solid and fluid lipid d o m a i n s f o r m e d by the incorporation o f O A into the membrane. In s u m m a r y , the results presented here demonstrate that, w h e n applied to h u m a n skin in vivo, under conditions which e n h a n c e transdermal permeability, [2H] O A does not globally m o d i f y the c o n f o r m a t i o n a l order o f SC lipids; rather, it appears to decrease lipid viscosity only in the superficial layers. Additionally, while the intercellular lipids exist in a solid state, [ 2 H ] O A incorporated into the SC is found in fluid domains. T h e s e findings suggest, therefore, that O A m a y e n h a n c e transdermal p e r m e a b i l i t y through a dual m e c h a n i s m i n v o l v i n g lipid perturbation via both conformational permutations and phase separation, with the latter effect predominating.
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[ 13 ] M.C. Blok, E.C.M. Van der Neut-Ko, L.L.M. Van Deenen and J. DeGier, The effect of chain length and lipid phase transitions on the selective permeability properties of liposomes, Biochim. Biophys. Acta 406 (1975) 187-296. [ 14] D. Papahadjopoulos, K. Jacobson, S. Nir and T. Isac, Phase transitions in phospholipid vesicles. Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol, Biochim. Biophys. Acta 311 (1973) 330-348. [ 15] S.H.W. Wu and H.M. McConnell, Lateral phase separations and perpendicular transport in membranes, Biochem. Biophys. Res. Commun. 55 (1973) 484. [16] E.J. Shimshick, W. Kleeman, W.L. Hubbell and H.M. McConnell, Lateral phase separation in membranes, J. Supramol. Struct. 2 (1973) 285-295. [17] V.H.W. Mak, R.O. Potts and R.H. Guy, Percutaneous penetration enhancement in vivo measured by attenuated total reflectance infrared spectroscopy, Pharm. Res. 7 (1990) 835841. [ 18] N. Higo, A. Naik, D. Bommannan, R.O. Potts and R.H. Guy, Validation of reflectance infrared spectroscopy as a quantitative method to measure percutaneous absorption in vivo, Pharm. Res. 10 (1993) 1500-1506. [ 19] R.O. Potts, D.B. Guzek, R.R. Harris and J.E. McKie, A noninvasive, in vivo technique to quantitatively measure water concentration of the stratum corneum using attenuated totalreflectance infrared spectroscopy, Arch. Dermatol. Res. 277 (1985) 489-495. [20] D.G. Cameron, J.K. Kauppinen, D.J. Moffatt and H.H. Mantsch, Precision in condensed phase vibrational spectroscopy, Appl. Spectrosc. 36 (1982) 245-250.
[21 ] R.O. Potts, G.M. Golden, M.L. Francoeur, V.H.W. Mak and R.H. Guy, Mechanism and enhancement of solute transport across the stratum corneum. J. Control. Release 15 (1991) 249-260. [22] N.Y. Schurer and P.M. Elias, The biochemistry and function of stratum corneum lipids, in: P.M. Elias (Ed.), Advances in Lipid Research, Vo124, Academic Press, San Diego, 1991, pp. 27-56. [23] D. Bommannan, R.O. Potts and R.H. Guy, Examination of stratum corneum barrier function in vivo by infrared spectroscopy, J. Invest. Dermatol. 95 (1990) 403--408. [24] D.C. Lee and D. Chapman, Infrared spectroscopic studies of biomembranes and model membranes, B iosci. Rep. 6 (1986) 235-256. [25] H.L. Casal and H.H. Mantsch, Polymorphic phase behaviour of phospholipid membranes studied by infrared spectroscopy, Biochim. Biophys. Acta 779 (1984) 381-401. [26] H.H. Mantsch and R.N. McElhaney, Phospholipid phase transitions in model and biological membranes as studied by infrared spectroscopy, Chem. Phys. Lipids 57 (1991) 213226. [27] S.L. Krill, K. Knutson and W.l. Higuchi, The stratum corneum lipid thermotropic phase behavior, Biochim. Biophys. Acta 1112 (1992) 281-286. [28] G.M. Golden, D.B. Guzek, R.R. Harris, J.E. McKie and R.O. Potts, Lipid thermotropic transitions in human stratum corneum, J. Invest. Dermatol. 86 (1986) 255-259. [29] A. Ortiz and J.C. Gomez-Fernandez, A differential scanning calorimetry study of the interaction of free fatty acids with phospholipid membranes, Chem. Phys. Lipids 45 (1987) 7591. [30] D. Bommannan, R.O. Potts and R,H. Guy, Examination of the effect of ethanol on human stratum corneum in vivo using infrared spectroscopy, J. Control. Release 16 ( 1991 ) 299-304.
ELSEVIER
controlled release Journal of Controlled Release 37 (1995) 299-306
Mechanism of oleic acid-induced skin penetration enhancement in vivo in humans Aarti Naik a,*, Louk A,R.M. Pechtold b,c, Russell O. Potts b, Richard H. Guy a "Departments of Pharmacy and Pharmaceutical Chemistry, University of California at San Francisco (UCSF), San Francisco, CA 94143, USA b Cygnus Therapeutic Systems, Redwood City, CA 94063, USA Leiden-Amsterdam Center for Drug Research, Department of Pharmaceutical Technology, University of Leiden, Leiden, The Netherlands
Received 13 September 1994; accepted 13 June 1995
Abstract The outermost layer of mammalian skin, the stratum corneum (SC), by virtue of its unique architecture, presents a significant barrier to the transdermal delivery of drugs. Penetration enhancers such as oleic acid (OA), which increase skin permeability, appear to act selectively on the extracellular lipids representing the principal regulatory channel for the penetration of small molecules. In vitro studies investigating the mode of action of OA, have generated two mechanistic scenarios, which may account for the action of this enhancer; (a) lipid fluidization, and (b) lipid phase separation. In the studies presented here, attenuated total reflectance infrared spectroscopy was used to determine the mode of action of OA in vivo, in man. The use of perdeuterated OA ( [2H] OA) enabled the behaviour of endogenous lipids to be observed independently to that of the exogenously applied enhancer as a result of their spectrally distinct methylene group vibrations. Human forearm was treated topically with 1 ml of either (a) a solution of 5% ( v / v ) [2H] OA in ethanol, or (b) ethanol alone, for a period of 16 h. After removal of the delivery system, the SC at the application site was progressively removed by adhesive tape-stripping, while sequential IR spectra were obtained at each newly exposed surface. In this way, we were able to monitor (a) the distribution profile of [2H] OA across the SC, (b) the conformational order of the SC lipids as a function of depth, and (c) the phase behaviour of the enhancer in the S C. Our results indicate that [ 2H ] OA induces lipid disordering only in the superficial layers of the SC, albeit of a smaller magnitude than that associated with a gel to liquid crystalline conformational change. Additionally, [2H] OA was found to exist in a liquid phase at all levels of the SC spectroscopically examined. These results suggest, therefore, that OA-induced skin penetration enhancement results from a mechanism involving both SC lipid fluidization and phase separation, with the latter probably predominating. Keywords: Stratum corneum lipid; Oleic acid; Percutaneous absorption; Skin penetration; Penetration enhancer; Attenuated total reflectance infrared spectroscopy
1. Introduction
* Corresponding author (present address): Department of Pharmaceutical and Biological Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK. Tel. 0121 359 3611; Fax 0121 359 0733. 0168-3659/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDIOI 6 8 - 3 6 5 9 ( 9 5 ) 00088-7
O f the m a n y diverse physiological functions that m a m m a l i a n skin is required to fulfil, its protective role is perhaps the m o s t demanding. R e c o g n i t i o n of the stratum c o r n e u m ( S C ) , the o u t e r m o s t epidermal layer, as the principal regulatory barrier to the transcutaneous traffic o f water and e x o g e n o u s substances, has b e e n
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succeeded by an array of structural and mechanistic insights. Central to these findings is the observation that the exceptionally thin, but remarkably impermeable, SC envelope is a complex composite of terminally differentiated ceils (corneocytes) embedded in a multilamellar lipid continuum - an assembly which is fundamental to the regulation of skin barrier function [ 1,21. The formidable SC barrier allows only a limited number of drugs to be delivered in therapeutic amounts through the skin. As a consequence, a number of chemical enhancers have been developed which, when coapplied with the drug, significantly and reversibly enhance skin flux. Oleic acid (OA) is a commonly used enhancer, the mechanism of action of which, has been widely studied in vitro [3-6]. Results of these studies indicate that OA primarily modulates the extracellular lipid domain of the SC. For example, the extent to which OA decreases the SC lipid phase transition temperature is directly (and highly) correlated with the amount of fatty acid taken up by the SC, as well as with its ability to enhance in vitro permeability of a coadministered permeant [4]. Permeation enhancers such as OA which reduce the diffusional resistance of the skin by interacting with the lipid matrix, have been postulated to act by increasing lipid fluidity [7,8], in a manner analogous to enhanced membrane permeability resulting from thermally induced SC lipid disordering [9-11 ]. However, recent infrared spectroscopic studies on porcine SC and extracted lipids treated in vitro have demonstrated, at physiologically relevant temperatures, the existence of OA in a liquid state, and therefore phase-separated from the endogenous solid lipids [ 12]. Since other phase-separated lipid systems have been reported to exhibit exceptionally high permeability at the interface formed at the boundary between the fluid and solid domains [ 13-16 ], the in vitro results to date suggest that OA enhances SC permeability through a similar mechanism. How does OA interact with SC lipids in vivo; is the observed scenario with excised SC and extracted lipids re-enacted in vivo? Attenuated total reflectance infrared (ATR-IR) spectroscopy is a non-invasive technique which has proven useful for the in vivo evaluation of topically applied enhancers in man. In a study designed to investigate the effect of OA on the transdermal delivery of a co-permeant, treatment with the enhancer was found to increase the rate of drug disappearance from
human SC [ 17]. These observations have been supported by further quantitative studies demonstrating the penetration enhancement properties of OA as a function of depth into the SC [ 18]. Here, we report the application of ATR-IR to ascertain the effect of OA on SC lipids, in vivo, in man. More specifically, we have utilized perdeuterated oleic acid ( [2H] OA), allowing the independent evaluation of endogenous SC lipids and exogenous OA, by virtue of their spectrally distinct CH2 and CD2 stretching absorbances, respectively.
2. Materials and m e t h o d s
Eight subjects (aged 25-50 years) in good health, with no history of dermatological disease, participated in these studies (approved by the UCSF Committee on Human Research). Experiments were performed on sites on the inner ventral forearm; one arm was treated while the other arm served as a control. Prior to treatment, the test sites were cleaned with a water-dampened swab, after which the subject remained in a room at constant temperature (21 _ 1°C) and relative humidity (30-40%) for 45 min, while pre-treatment spectra were recorded at 25, 35 and 45 min. An aliquot (1 ml) of a 5% (v/v) solution of perdeuterated oleic acid ( [ 2H ] OA) ( Cambridge Isotopes, Cambridge, MA) in ethanol was applied to a 12 cm 2 site for 16 h. The control site, on the contralateral arm, was treated with ethanol alone. Both formulations were applied on a surgical cotton pad ( 1.5 X 8 cm), occluded and covered with a Tegaderm ® (10X 12 cm) wrap. 16 h later, the occlusive patch was removed and both sites were cleaned with ethanol-soaked swabs. The sites were then exposed to air for 2 h to allow dehydration of the occluded skin. An IR spectrum of the dosed site was then recorded. After this initial spectral acquisition, a piece of Scotch® Book Tape no. 845 (3M, St. Paul, MN), was pressed against the application site and then removed, taking with it a layer of adherent SC. A further spectrum was recorded and the sequence repeated until a maximum of 20 incremental spectra were obtained. To quantify the mass of SC removed with each strip, the tape was weighed before and after the stripping procedure.
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3. Spectral analyses A T R spectroscopy is a non-invasive technique in which the IR beam is directed to an IR transparent crystal (internal reflection element; IRE) supporting a sample. The resulting spectrum describes a defined region of the surface of the sample in contact with the IRE; the application of this methodology to in vivo measurements of SC has been described in the literature [19,23]. In the study presented here, spectra were recorded with a Nicolet 730 Fourier Transform (FT) IR spectrophotometer (Nicolet, Madison, WI), equipped with a liquid N2 cooled mercury-cadmiumtelluride detector. The conventional transmission cell mount was replaced by a horizontal Out-of Compartment Contact Sampler (Spectra-Tech, Stamford, CT) supporting a zinc selenide (ZnSe) IRE (dimensions: 7 × 1 × 0.2 cm) having 45 ° entrance and exit faces and a refractive index of 2.4 at 1000 cm-1. To obtain a spectrum, 64 scans were collected at a resolution of 2 c m - 1 over a range of 4800-400 c m - 1, co-added, and transformed using a Happ-Genzel apodization function. While an internal laser referencing system in FT spectroscopy allows excellent wavenumber precision and accuracy, and thus direct comparison of peak positions, the intensity of A T R spectra is dependent on the degree of contact between the IRE and sample. Thus, in order to compare intensities, the absorbance of interest is normalized against a spectral parameter which also varies with contact. In this way, quantitative comparisons can be made. In the study described here, the absorbance was ratioed against the amide I absorbance near 1640 c m - i, due to the carbonyl stretching motions of the CO-NH group. The same concept has been used to quantitate the uptake of water [ 19] and drugs [ 17] by the skin. The frequencies of the CH2 and CD2 stretching absorbances were determined using a center-of-gravity algorithm [20] allowing positions to be determined with an uncertainty of less than 0.1 c m - 1. Since both the CH2 and CD2 absorbances overlap, to varying extents, with broad bands due to water and other IR absorbing materials, a baseline correction was required prior to further data analysis. For example, as shown in Fig. 1, the CH2 symmetric ( ~ 2850 cm ~) and asymmetric ( ~ 2920 c m - ~) peaks occur as shoulders on a broad absorbance with a maximum near 3200 cm-~.
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Fig. 1. Reflectance infrared spectrum of human stratum corneum treated with perdeuterated oleic acid. The spectral separation of CH2 and CD2 stretching bands is illustrated in the inset. A spectrum of untreated stratum comeum (not shown) appears identical to the above in every respect except for the presence of the CD2 stretching vibrations. The baseline here was established by extrapolating a straight line between 2993 and 2745 cm-1. The CD2 stretching absorbances near 2195 and 2095 cm -1, necessitated a 4th order polynomial baseline fit to the data due to the impact of a broad absorbance centering near 2100 c m - 1, directly beneath these regions.
4. Results and discussion An ATR-IR spectrum of human skin treated with perdeuterated oleic acid ( [ 2HI OA) is shown in Fig. 1, with the spectral region of particular interest ( 2 0 0 0 3000 c m - l ) magnified in the inset. Featured in this spectrum are peaks near 2850 and 2920 c m - ~ due to CH2 symmetric (us) and asymmetric (uas) stretching vibrations, respectively, originating primarily from the extracellular lipids of the SC [21]. The treated skin also contains [ 2H ] OA which, by virtue of the chemical substitution of deuterium for hydrogen, exhibits CD 2 symmetric and asymmetric stretching absorbances near 2095 and 2190 c m - l, respectively. Since spectral parameters associated with these absorbances, such as magnitude and frequency, report on the behaviour of the absorbing species, this spectrum clearly demonstrates the ability of the ATR-IR technique to distinguish, in the skin of human subjects, differences in the behaviour of endogenous SC lipids and the exogenously applied enhancer. In doing so, an in vivo assessment of [2H]OA uptake into human skin and its
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Fig. 2. Weight per unit area of SC removed by sequential tapestripping, followingtreatment with ethanol alone (O) or 5% (v/v) [2H]oleic acid in ethanol (O). Mean_+SE n=7 or 8. subsequent effect on SC lipids is facilitated. Furthermore, the combined use of ATR-IR spectroscopy and tape-stripping of the skin allows these measurements to be conducted as a function of depth within the SC. The distance into the membrane, probed by successive tape-stripping, was defined by the weight of SC removed [ 18]. Due to differing adhesive properties of the SC from site to site and as a function of depth at any one site, the mass removed by each tape strip was variable. Thus, the weight of SC removed, not the strip number, provided a more quantitative measure of the depth probed within the SC. Fig. 2 compares the mean mass removed with each strip for control and [ 2H ] OAtreated human skin. These results demonstrate that significantly greater amounts of SC were removed by the first three strips in the [ 2H ] OA-treated skin (ANOVA, p < 0.05), after which statistically equivalent amounts were removed per strip for treated and control groups. Primarily due to the greater mass removed in the outermost strips, the total amount of SC removed was significantly greater for the enhancer treated skin. Earlier studies of oleic acid-treated human skin have reported similar observations [ 18 ]. Since SC cohesion has been related to the nature of lipid constituents present within the membrane [22], these results suggest that the SC lipid network has been modified by [2H] OA. Finally, these data indicate that the amount removed per tape-strip was the same for treated and control sites at the deeper levels evaluated, implying
that [2H] OA concentration decreased markedly with depth into the SC (see below). The uptake and distribution of [2H] OA in the SC was evaluated by measuring the area under the CD2 stretching absorbances; the magnitude of these bands being directly proportional to the amount of absorbing species. As shown by the results in Fig. 3, the CD 2 signal decreased with increasing mass of SC removed, reaching a limiting value near zero in the deepest layers of the SC probed. This concentration profile is consistent with sink conditions near the base of the SC, suggesting that the SC is the primary resistive barrier to the transdermal absorption of [ 2H ] OA. The amount of endogenous lipid in the SC was similarly determined from the area under the CH2 stretching bands. The data in Fig. 4, reporting the magnitude of the CH 2 stretching absorbances as a function of SC depth, were statistically indistinguishable for the treated versus control sites. These results indicate that the amount of lipid within the SC was (a) unaffected by [2H]OA treatment, and (b) decreased to a value of about 60% of the surface value following tape-stripping. Similar depthdependent results obtained by Bommannan et al. [23] have been interpreted to reflect decreased lipid content with increased distance into the SC. In addition to revealing lipid content and permeant distribution profiles, ATR-IR spectroscopy is also notably instructive with respect to the conformational and structural status of the membrane constituents. In par-
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Fig. 4. Relative lipid content within the SC (expressed as % of prestripping value for untreated SC) as a function of SC weight removed, followingtreatment with ethanol alone (O) or 5% (v/v) [ ZH] oleic acid in ethanol ( • ) . Mean _+SE; n = 7 or 8. ticular, we are concerned with discerning the influence, if any, which oleic acid exerts over the lipoidal domains within the SC. The key questions which we wish to address are: (a) does OA alter the conformational order of the endogenous SC lipids, and if so, to what extent, (b) is the fatty acid phase-separated from the endogenous lipids, and (c) are one or more of these effects dominant and/or consistently present across the entire membrane? The frequency positions of the CH2 stretching absorbances (/~CH2), observed near 2800-2950 c m in the IR spectrum, are sensitive indicators of the level of motional freedom and flexibility associated with the lipid alkyl chains and, as such, report on the average structural order of the SC lipid domains. For example, increased pCH2 is associated with decreased lipid order in a variety of lipid systems [ 2 4 - 2 6 ] , including SC [27,28]. More specifically, ~'CH2 is related to the ratio of t r a n s / g a u c h e conformational isomers along the hydrocarbon tail. Previous results obtained with porcine SC have shown a temperature-dependent increase in alkyl chain disorder manifested as an increase in vsCH 2 from a value near 2850.2 c m - ~ at 40°C where the chains were predominantly trans, to a value near 2853.3 c m - 1 at 90°C, above the phase transition temperature (50-80°C), reflecting a significant g a u c h e population [ 11 ]. The effect of [2H]OA on the endogenous lipid organization in these experiments was thus assessed by
determining the CH 2 symmetric and asymmetric stretching frequencies at each incremental spectral measurement across the SC. Fig. 5 illustrates the asymmetric stretching frequencies originating from the SC lipids plotted as a function of the average cumulative SC mass removed by tape-stripping. Note that since the amount of SC removed per strip differed for the treated and control groups, the abscissal data points for these two experimental groups do not precisely overlap. Nevertheless, the data in Fig. 5 show that the first two measurements (pre- and post- first tape-strip) for the [ 2H ] OA-treated group, were significantly (p = < 0.01 ) elevated with respect to the background (untreated skin) and control (ethanol treated) measurements. Thereafter, all vasCH 2 data conformed to a common mean value (_+SD) of 2915.60 ( + 0 . 3 3 ) cm-~, analogous to the corresponding control group mean frequency of 2915.60 (_+0.31) cm -1, characteristic of 'solid' alkyl chains in a predominantly trans configuration. The deduction from this profile, then, is that following topical administration of [ 2H ] OA, lipid disordering is sustained only by the surface and uppermost layers where the concentration of [2H]OA, and the intrinsic fluidity of the SC lipids is greatest [23]. The lipid viscosity in the remainder of the membrane is essentially unaffected by [2H]OA treatment. The results presented here are consistent with previous in vitro data derived from OA-treated porcine SC [ 12], 2920
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A. Naik et al./ Journal of Controlled Release 37 (1995) 299-306
304
Table 1 Asymmetric (v.~CH2) and symmetric (/.'syCH2) stretching frequencies for [ 2HI oleic acid treated human stratum corneum vasCH2
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Values represent m e a n _ SD, for n = 8 or n = 16a; bmeasurement of SC surface, prior to tape-stripping; Cmeasurement o f SC surface, following two tape-strippings,
demonstrating that the average conformational order of SC lipids was unaffected by [2H]OA. This is because the measured CH2 stretching absorbance is in fact an ensemble of overlapping vibrational bands, reflecting heterogeneity in the local conformational order. This is best illustrated by the case of a pure lipid undergoing a thermal phase transition; at the transition mid-point where the lipid is partially melted, the bandwidth of the-CH2 stretching absorbance increases due to the larger number of conformational states of the hydrocarbon chains [26]. The frequency reported by an IR spectrum, therefore, represents a weighted average for the population of methylene groups undergoing stretching oscillations, and will be dependent on the relative contribution (amount and conformational state) of each sub-population. Since transmission IR spectroscopy (used to scan the SC sample in vitro) examines and reports on the distribution of vibrational frequencies encountered across the entire membrane intersecting the path of the beam, the technique is insensitive to any localized or depth-dependent variations. Indeed, the mean ua~CH2 (of 21 successive measurements) for the SC examined in vivo, was calculated to be 2915.80 + 0,41 c m - 1, a value not significantly different from that of the control group. By contrast, our in vivo approach, using reflectance IR spectroscopy, facilitates the differentiation of any depth-dependent, though not lateral, disparities. A similar profile was noted for the CH2 symmetric stretching absorbances across the membrane (Table 1). The magnitude of the change in frequency has been related to the number of gauche conformers introduced,
and consequently to the level of disordering. Thermotropic phase transitions of human [28], porcine [ 10,11] and murine [27] SC are typically accompanied by a change in ua~CH2 and ~'syCH2 of approximately 6 and 3 cm -1, respectively. It is pertinent to point out that the shifts in maximum absorbance incurred following [2H] OA treatment are significantly smaller (Table 1; Fig. 5), and are thus indicative of a lower degree of perturbation. The small decrease in uasCH2 observed in both control and OA-treated (excluding pre- and post-firststripping) sites, relative to untreated skin, most likely reflects the combined outcome of two phenomena; (a) a heterogeneous gradient of lipid composition as revealed by repeated tape-stripping, and (b) the extraction of surface lipids (sebaceous in origin and liquid at physiological temperature) by prolonged contact with ethanol. The decrease in ua~CH2 is also accompanied by a reduction in bandwidth (data not shown), portraying increasing homogeneity among the lipid population below the surface of the SC. These inferences are supported by the findings of Bommannan et al. [24,30] reporting (a) a stratal gradient of lipid concentration and conformational order, with a predominance of less structured lipids near the surface, and (b) the concurrent percutaneous penetration of ethanol, extraction of SC lipids, and decrease in the intensity, bandwidth and frequency of the CH2 stretching vibrations, following in vivo dermal treatment with ethanol. The phase behaviour of the topically administered oleic acid within the SC can be similarly discerned from a survey of the CD2 stretching absorbance maxima. A plot of the measured CD2 asymmetric stretching frequencies (u,sCD2) as a function of depth into the SC (Fig. 6) illustrates a small, but gradual increase in vasCD2 with increasing membrane depth, being characterized by a slope (95% confidence interval; r 2) of 2 . 3 9 6 × 10 - 2 mg -1 ( +_9.309 X 10-3; 0.151). Within the membrane, the mean ( + SD) value of ua~CD2 is 2196.60 (+_0.3) cm ~. It becomes evident, on comparison with the b ' a s f D 2 value of 2197.17 cm-~ for pure [2H]OA at 25°C (above its melting point of 13.4°C), that this is consistent with [ 2HI OA in a liquid phase. The combined data from Fig. 5 and 6 demonstrate that [ 2H ] OA in human SC existed in a fluid state, while the endogenous lipids were both disordered (in the superficial layers) and solid (in the deeper regions). These in vivo results are therefore consistent
A. Naik et al./ Journal of Controlled Release 37 (1995) 299-306 2197.5
Acknowledgements
The authors w o u l d like to thank m e m b e r s o f the Skin B i o s c i e n c e Group, U C S F , and the Biophysical R e s e a r c h Group, C y g n u s Therapeutic S y s t e m s for their helpful c o m m e n t s and participatory role in these studies. This research was supported by a grant ( H D 23010) f r o m the U S National Institutes o f Health.
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Cumulative SC w e i g h t (mg)
Fig. 6. CD2 asymmetric stretching frequencies,/,'asCD2, (originating from the topically applied [ 2H] oleic acid) as a function of SC weight removed, following treatment with 5% (v/v) [2H]oleic acid in ethanol. Mean _+SD; n = 7 or 8. with lipid phase separation w h e r e solid and fluid d o m a i n s co-exist. As r e v i e w e d by Ongpipattanakul et al. [ 12], lipid phase separation can result in substantially e n h a n c e d permeability in m a m m a l i a n SC, as in other lamellar lipid barriers. Furthermore, it has b e e n s h o w n in s i m p l e r lipid systems that c i s - u n s a t u r a t e d fatty acids ( l i k e oleic acid) will f o r m a separate fluid d o m a i n w h e n introduced into a solid, saturated lipid mixture (like SC lipids) [ 2 9 ] . The possibility exists, therefore, that O A - i n d u c e d transdermal penetration e n h a n c e m e n t occurs as a result o f increased permeability at the interface b e t w e e n solid and fluid lipid d o m a i n s f o r m e d by the incorporation o f O A into the membrane. In s u m m a r y , the results presented here demonstrate that, w h e n applied to h u m a n skin in vivo, under conditions which e n h a n c e transdermal permeability, [2H] O A does not globally m o d i f y the c o n f o r m a t i o n a l order o f SC lipids; rather, it appears to decrease lipid viscosity only in the superficial layers. Additionally, while the intercellular lipids exist in a solid state, [ 2 H ] O A incorporated into the SC is found in fluid domains. T h e s e findings suggest, therefore, that O A m a y e n h a n c e transdermal p e r m e a b i l i t y through a dual m e c h a n i s m i n v o l v i n g lipid perturbation via both conformational permutations and phase separation, with the latter effect predominating.
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