Vivien H. W. Mak

Percutaneous Penetration Enhancement in Vivo Measured by Attenuated Total Reflectance Infrared Spectroscopy

A novel application of attenuated total reflectance IR Spectroscopy (ATR-IR) was used to monitor the outer several microns of the stratum corneum (SC) and, thereby, demonstrate enhanced percutaneous absorption in vivo in man. 4-Cyanophenol (CP) as a model permeant yielded a unique IR signal, distinct from those of the stratum corneum and the vehicle components. CP was administered for 1, 2, or 3 hr as a 10% (w/v) solution either in propylene glycol or in propylene glycol containing 5% (v/v) oleic acid. The absorbance at 2230 cm−1, which corresponded to C≡N bond stretching, diminished significantly faster when CP was codelivered with oleic acid. An IR absorbance due primarily to propylene glycol at 1040 cm−1 (C–O stretching) also disappeared more quickly following application of the enhancer-containing solution. In addition, only the formulations with oleic acid induced a higher wavenumber shift in the frequency of the asymmetric C–H bond stretching absorbance. This change indicates increased lipid-chain disorder, the mechanism by which oleic acid is believed to cause enhanced drug transport across the stratum corneum. Therefore, ATR-IR permits one to examine noninvasively the kinetics, extent, and mechanism of percutaneous penetration enhancement in vivo in human subjects.

Oleic acid concentration and effect in human stratum corneum: non-invasive determination by attenuated total reflectance infrared spectroscopy in vivo

Attenuated total reflectance infrared (ATR-IR) spectroscopy was used to study, in vivo, the mechanism of action of a putative skin penetration enhancer, oleic acid. Specifically, the spectroscopic technique monitored structural changes and enhancer concentration in the stratum corneum (SC) (skins outermost and least permeable layer) following treatment with oleic acid in ethanol. As previously observed in vitro, oleic acid increased the hydrocarbon chain disorder of the inter-cellular lipid domains of the SC in vivo. The effects of the enhancer were prolonged but reversible. It also appeared that the treatment of the skin with oleic acid for 0.5 hour caused a gradient of effect (i.e. increased lipid disorder) that decreased from superficial to inner stratum corneum layers. However, this gradient was normalized by increasing the time of exposure of the skin to the enhancer. Although the oleic acid-induced lipid hydrocarbon chain disorder in vivo maximized at a concentration of 1% oleic acid in ethanol, the in vitro uptake of radiolabelied enhancer into excised SC increased linearly with concentration up to 10%. Further experiments showed that the saturation of response in vivo was not solubility limited. Precise analysis of the IR spectra enabled an absorption band to be assigned mainly to oleic acid and to be used, therefore, to quantity SC uptake of enhancer in vivo. A quite linear correlation between (radiochemical) in vitro and (spectroscopic) in vivo assessments of oleic acid in SC was found. Overall, then, the results reported support the conclusions: (a) that ATR-IR can monitor the effects of penetration enhancers on skin barrier function in vivo, and (b) that, in the case of oleic acid, the technique can estimate the level of enhancer within the SC as a function of time and, possibly, position.

Mechanism and enhancement of solute transport across the stratum corneum

Abstract This paper summarizes recent biophysical investigations of (a) stratum corneum (SC) barrier function, and (b) percutaneous penetration enhancement. Specifically, applications of differential scanning calorimetry (DSC) and infrared spectroscopy (IR) to probe the intercellular lipid domains of the SC are described. In vitro DSC experiments on isolated SC have determined the thermal melting behavior of the membrane and have indicated the presence of lipid phase transitions in the range 65–85 °C. Corresponding IR studies have confirmed this observation and, in conjunction with measurements of tritiated water permeability (Kp), have shown that solute flux increases with the number of gauche conformers along the lipid acyl hydrocarbon chains. There is an excellent correlation between Kp and the absorbance shift (to higher wavenumber) of the C-H antisymmetric stretching vibration associated with the SC lipids. Furthermore, it was found that certain putative penetration enhancers (e.g., cis-unsaturated fatty acids, such as oleic and vaccenic acids) induced similar shifts when applied to excised SC at ambient temperature. Concomitantly, the flux of another model solute (salicylic acid) was significantly enhanced by the treatment. The implied mechanism of penetration promotion (i.e., through the overall increased freedom of motion of the lipid acyl chains) was then examined in vivo, in humans, using attenuated total reflectance IR. Again, significant and sustained lipid disordering was induced by (in this case) oleic acid. In addition, through the assignment of unique absorbances, it was possible to obtain semi-quantitative measurements of (1) the enhancer in the upper layers of the SC, (2) the co-applied penetrant (4-cyanophenol, unequivocally identified by the intense C  N absorbance) and (3) the major vehicle component used (namely, propylene glycol, via the C-O stretching vibration). In this way, we have (i) documented the effect of the enhancer on the SC lipids, (ii) assessed the relative amount of the enhancer responsible for this action, and (iii) observed the kinetics of penetrant (solute) and solvent transport (in the presence and absence of enhancer) through the SC, from a single series of spectroscopic experiments in vivo in man. We suggest, therefore, that biophysical measurements of this type have considerable potential, often in clinically relevant situations, to reveal the crucial details of the mechanism and enhancement of solute transport across the SC.

Mechanism of percutaneous penetration enhancement: effect of n-alkanols on the permeability barrier of hairless mouse skin

To examine the action of alcohols on skin barrier function, we have: (a) determined, in vitro, how pretreatment of hairless mouse skin with a homologous series of n-alkanols (C2-C12) enhances the transport of a model penetrant (nicotinamide); (b) measured the skin uptake and permeation of the alkanols themselves during the pretreatment period; and (c) correlated the data from these two sets of experiments with biophysical evaluation of the pretreated murine stratum corneum using Fourier transform infrared spectroscopy (FT-IR). Following 3 or 6 h pretreatment, nicotinamide flux was increasingly enhanced with increasing alkanol carbon number up to C6. The effect of octanol was similar to that of hexanol, but decanol was less efficacious and dodecanol continued this diminishing trend. While skin uptake of the alkanols increased linearly with increasing lipophilicity (i.e., increasing chain length), the cumulative penetration (in 6 h) of the homologs was a parabolic function of Cn similar to the nicotinamide enhancement profile. FT-IR analysis of ethanol-pretreated hairless mouse skin revealed considerable extraction of stratum corneum intercellular lipids; the appearance of these lipids in the pretreatment liquid could also be measured spectroscopically. The same extraction phenomenon was also apparent when pretreatment was performed with butanol and octanol. For these homologues, removal of stratum corneum lipids was shown unequivocally by FT-IR through the use of perdeuterated alkanols, thereby separating the C-H(D) signals from intercellular lipid and penetration enhancer. Lipid extraction, therefore, is implicated as the mechanism by which alkanols promote transdermal penetration. Because of the efficiency of lipid removal in these experiments, it was not possible to determine if additional, and possibly more subtle, effects (e.g., “fluidization” of stratum corneum lipid domains) are also induced by alkanols.

Effect of Selective Lipid Extraction from Different Body Regions on Epidermal Barrier Function

AbstractPurpose. To assess the effects of selective lipid extraction and tape stripping on transepidermal water loss (TEWL) at three body regions in the pig. Methods. Lipids were extracted from the abdominal, inguinal, and back regions using three different solvent extraction procedures or cellophane tape stripping (15×) on Yorkshire pigs. Three solvent extraction stages were I, cyclohexane (5 ml for three, 1-min extractions); II, cyclohexane/ethanol (4:1) (5 ml for three, 1-min extractions); and III, cyclohexane/ethanol (1:4) (5 ml for three, 3-min extractions) extracted as follows: Site A, Stage I; Site B, Stage I and II; Site C, Stage I, II and III. Erythema, edema, and TEWL were assessed in control, tape-stripped, and extracted sites at 0, 6, and 24 h. The extracted lipids were analyzed by thin layer chromatography and quantified by densitometry for ceramide, cholesterol, cholesterol esters, fatty acids, and triglycerides. Results. The change in TEWL (Δ TEWL) in 14 of the 15 sites was the highest at 24 h and generally increased with each additional extraction. The greatest changes were present in the back. Each extraction stage removed specific lipids in reproducible quantities that caused the Δ TEWL to increase from 0 to 24 h. Lipid removal was verified by transmission electron microscopy. The mean total lipid concentration depended on extraction solvents and body region, and was reproducible across sites and regions at equivalent stages of lipid extraction. Relative proportions of individual lipids extracted were similar across all body regions. Higher concentrations of total lipids were extracted from the back. Conclusions. These studies demonstrate that extraction of lipids increased the Δ TEWL to a level similar to repeated tape stripping at all body sites in the pig. This study suggested that strategies that could biochemically alter epidermal lipid composition may increase absorption of simultaneously administered topical compounds and may be useful to enhance drug delivery.

Strategies to Enhance Permeability via Stratum Corneum Lipid Pathways

Publisher Summary Stratum corneum (SC) lipids, like those of other biomembranes, can be studied by a variety of biophysical techniques. Methods that have been used to investigate lipid membrane biophysics include X-ray diffraction, differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), electron spin resonance (ESR), and infrared (IR) spectroscopy. This chapter discusses the techniques of IR spectroscopy and DSC. These techniques have been particularly useful in establishing the phase behavior of a number of lipid membrane systems. The SC lipid pathway is important to the permeability of water and numerous drugs. Despite differences in lipid composition and distribution, the mechanisms of molecular permeation through the SC and other biomembranes are similar. The technique of Fourier transform IR (FTIR) spectroscopy has been extensively used to study the phase behavior of lipid membranes. The lipids of the SC can also be studied by FTIR techniques.

Stratum Corneum Lipids of Human Epidermal Keratinocyte Air-Liquid Cultures: Implications for Barrier Function

AbstractPurpose. The purpose of this study is to investigate the permeability barrier, i.e., the stratum corneum (SC) lipids, of human epidermal keratinocyte air-liquid cultures and compare them with those of human SC. Methods. The SC lipids composition was analyzed by TLC technique, the organization by electron microscopic procedure, and the phase transition temperature by Infrared spectroscopic method. Results. Electron microscopy demonstrated that The SC lipids of cultures were largely retained inside the corneocytes, and that the intercellular lipids lack both the basic unit repetition (i.e., broad : narrow : broad : broad : narrow : broad of electron lucent bands) and the covalently-bound lipid envelope normally found in human SC. These characteristics are similar to those found in SC from patients with atopic dermatitis or psoriasis, or from animals with essential fatty acid deficiency, suggesting that the cultures may be hyperproliferative. In addition, the high free sterol content and the altered fatty acid/ceramide composition of these cultures argue that the compromised barrier function is linked to hyperproliferation and lipid synthesis, or vice versa. Infrared spectroscopic analyses confirm that there are major conformational differences between the lipids of human and cultured SC. Conclusions. The profound differences in SC lipid composition, organization and conformational properties attest that permeability alone is not a sufficiently sensitive marker to define barrier equivalence between cultures and human skin.

ARE WATER PERMEABILITY MEASUREMENTS SUFFICIENT TO CHARACTERIZE IN VITRO CULTURED HUMAN SKIN SURROGATES

AbstractWe have found previously that human neonatal foreskin keratinocyte air/liquid (A/L) cultures developed normal-appearing epidermis, and that water flux was only two to three times higher than that found with intact human skin. To understand further the barrier properties of these A/L cultures, we have analyzed the composition and the gross conformational structures of the cultured stratum corneum (SC) lipids, and compared them with those of human SC. Electron microscopic examination demonstrated that cultured SC has high intracellular lipid content, but that it lacks both the basic unit repetition (e.g., broad/narrow/broad/broad/narrow/ broad pattern of electronlucent bands) normally found in human SC intercellular lipids and the covalently bound lipid envelope. These results indicate that the cultures are hyperproliferative, with characteristics similar to those found in SC obtained from patients with atopic dermatitis or psoriasis, or from animals whose diets lack essential fatty acids. In additi...