In vitro measurement of transepidermal water loss: a rapid alternative to tritiated water permeation for assessing skin barrier functions

Transepidermal water loss (TEWL), as measured with an evaporimeter, was used as a rapid assessment of the integrity of the barrier properties of skin as part of in vitro skin permeation studies. For a variety of physical and chemical treatments (i.e. solvent extraction, surfactants, mechanical abrasion and bases) TEWL correlated strongly with tritiated water permeation at short times. In contrast to the tedious process of measuring permeation of a finite dose of tritiated water, TEWL is a rapid, convenient measurement, and it provides a clear indication of the time dependence of barrier integrity.     

Comparison of tissue sources for the skin integrity function test (SIFT).

One of the in vitro models involved in an ECVAM-sponsored prevalidation study for acute skin irritation is the skin integrity function test (SIFT), which utilises full-thickness mouse skin. We have evaluated nine different skin types in order to identify the most useful model for assessing skin barrier function using transepidermal water loss (TEWL), electrical resistance (ER) and tritiated water flux (TWF) and sodium lauryl sulphate (SLS) as a standard skin irritant. Tissues were: human skin (epidermis and whole), reconstituted human epidermis (RHE), pig (dermatomed and whole), rabbit (whole), rat (epidermis and whole) and mouse (whole). Barrier function was measured following sodium lauryl sulphate (SLS) exposure and expressed as a damage ratio. Human epidermis gave good responses at high doses of SLS only. RHE had abnormally high permeability to water and therefore had little or no response to SLS. Pig skin gave low TEWL ratios and rabbit skin was a poor responder to SLS. Mouse whole skin performed best in this study, giving consistent high damage ratios to TEWL, ER and TWF following SLS treatment. Rat whole skin also performed well but was generally less responsive. We conclude that mouse skin is the best and most practical in vitro model for the SIFT approach for skin irritation prediction.     

Skin penetration enhancement by a microneedle device (Dermaroller) in vitro: Dependency on needle size and applied formulation

This study focused on the in vitro evaluation of skin perforation using a new microneedle device (Dermaroller^®) with different needle lengths (150, 500 and 1500 μm). The influence of the microneedle treatment on the morphology of the skin surface (studied by light and scanning electron microscopy), on the transepidermal water loss (TEWL) and on the penetration and permeation of hydrophilic model drugs was investigated using excised human full-thickness skin. Furthermore, invasomes – highly flexible phospholipid vesicles containing terpenes and ethanol as penetration enhancer – were compared with an aqueous solution.Elevated TEWL values were measured after Dermaroller^® treatment compared to untreated human skin with a gradual increase of the TEWL over the first hour whereas afterwards the TEWL values decreased probably caused by a reduction of the pore size with time. Skin perforation with the Dermarollers^® enhanced drug penetration and permeation for both formulations tested. Invasomes were more effective to deliver hydrophilic compounds into and through the skin compared to the aqueous drug solutions and the combination with skin perforation further enhanced drug penetration and permeation.In conclusion, Dermarollers^® being already commercially available for cosmetic purposes appear also promising for drug delivery purposes particularly those with medium (500 μm) and shorter (150 μm) needle lengths.     

Bacterial cellulose membranes as drug delivery systems: An in vivo skin compatibility study

Bacterial cellulose (BC) is a highly pure form of cellulose, produced in the form of a swollen membrane by several bacteria that demonstrated to be able to modulate the skin release of model drugs. In the present study, the skin irritation potential of BC was evaluated in human subjects. BC membranes with and without glycerin (acting as plasticizer) were tested. No significant differences were observed for transepidermal water loss (TEWL) measurements in comparison with negative control, 2 and 24 h after patch removal, which is an indicator of an absence of barrier disruption. Similar results were found for erythema. Clinical scores were zero at both times for all volunteers, with the exception of five volunteers that exhibited weak reactions. BC with glycerin provided a skin moisturizing effect statistically higher than the negative control (p = 0.044), which was not observed for BC alone. The good skin tolerance found after a single application under occlusion reinforces the putative interest of BC membranes as supports for drug topical delivery. Besides modifying the mechanical properties, the inclusion of glycerin results in a skin moisturizing effect which could be clinically relevant for the treatment for skin diseases characterized by dryness, such as psoriasis and atopic dermatitis.     

Inter- and intra-individual variability in human skin barrier function: A large scale retrospective study

In vitro transepidermal tritiated water flux measurements are frequently used to evaluate skin barrier integrity for quality control purposes. However, research in this area to date has been largely based upon small-scale studies, each involving relatively few skin permeation measurements. In order to enhance our understanding in this area, we have conducted a much larger scale retrospective statistical analysis of tritiated water k_p values. These values reflected the permeability of 2400 skin samples that were derived from 112 female volunteers over a 4 year period. It was found that the population of tritiated water k_p values constituted a positively skewed, non-Normal distribution. Mean k_p was 2.04 × 10⁻³ cm/h while the 95th percentile was 4.50 × 10⁻³ cm/h. Both values are higher than those reported in previous smaller studies. Hence, our study indicates that previously suggested upper limits for tritiated water flux are too low and that they be revised upwards to a value of 4.5 × 10⁻³ cm/h. Analysis was also performed on smaller data subsets allowing inter-individual and intra-individual comparisons. For intra-individual k_p variability, site-related differences yielded a non-Normal, positively skewed pattern in most individuals. Inter-individual variability was Normally-distributed and showed scatter that was much smaller in magnitude.     

Effects of microneedle length,density, insertion time and multiple applications on human skin barrier function: Assessments by transepidermal water loss

Microneedle (MN) arrays have attracted considerable attention in recent years due to their ability to facilitate effective transdermal drug delivery. Despite appreciable research, there is still debate about how different MN dimensions or application modes influence permeabilization. This study aimed to investigate this issue by taking transepidermal water-loss measurements of dermatomed human skin samples following the insertion of solid polymeric MNs. Insertions caused an initial sharp drop in barrier function followed by a slower incomplete recovery – a paradigm consistent with MN-generation of microchannels that subsequently contract due to skin elasticity. While 600 μm-long MNs were more skin-perturbing than 400 μm MNs, insertion of 1000 μm-long MNs caused a smaller initial drop in integrity followed by a degree of long term permeabilization. This is explainable by the longest needles compacting the tissue, which then decompresses over subsequent hours. Multiple insertions had a similar effect as increasing MN length. There was some evidence that increasing MN density suppressed the partial barrier recovery caused by tissue contraction. Leaving MNs embedded in skin seemed to reduce the initial post-insertion drop in barrier function. Our results suggest that this in vitro TEWL approach can be used to rapidly screen MN-effects on skin.     

Characterization of Solid Maltose Microneedles and their Use for Transdermal Delivery

Purpose To characterize solid maltose microneedles and assess their ability to increase transdermal drug delivery. Materials and Methods Microneedles and microchannels were characterized using methylene blue staining and scanning electron microscopy. Diffusion pattern of calcein was observed using confocal scanning laser microscopy. Transepidermal water loss (TEWL) measurements were made to study the skin barrier recovery after treatment. Uniformity in calcein uptake by the pores was characterized and percutaneous penetration of nicardipine hydrochloride (NH) was studied in vitro and in vivo across hairless rat skin. Results Microneedles were measured to be 508.46 ± 9.32 μm long with a radius of curvature of 3 μm at the tip. They penetrated the skin while creating microchannels measuring about 55.42 ± 8.66 μm in diameter. Microchannels were visualized by methylene blue staining. Pretreatment with microneedles resulted in the migration of calcein into the microchannels. TEWL increased after pretreatment and uptake of calcein by the pores was uniform as measured by the pore permeability index values. NH in vitro transport across skin increased significantly after pretreatment (flux 7.05 μg/cm²/h) as compared to the untreated skin (flux 1.72 μg/cm²/h) and the enhanced delivery was also demonstrated in vivo in hairless rats. Conclusion Maltose microneedles were characterized and shown to create microchannels in the skin, which were also characterized and shown to improve the transdermal delivery of NH.     

TEWL measurements as a routine method for evaluating the integrity of epidermis sheets in static Franz type diffusion cells in vitro. Limitations shown by transport data testing.

The suitability of transepidermal water loss (TEWL) measurements in vitro as a barrier integrity test for human heat separated epidermis (HSE) was investigated. A model system consisting of a Teflon membrane mounted in Franz diffusion cells (FDC) filled with phosphate buffer saline (PBS) was set up. The membrane was used intact and punctured with a needle (up to five holes). After each puncturing the TEWL was measured. Only the TEWL of intact and punctured membrane differed significantly regardless of the number of holes. From three donors intact human HSE and punctured HSE were compared and no significant difference of the TEWL was found. Permeation experiments with flufenamic acid (FFA) showed a significantly higher diffusion rate through punctured HSE. TEWL and drug permeation were compared for skin stripped three, seven and 15 times prior to heat separation to an intact control group. Only the TEWL values of intact HSE and HSE stripped 15 times differed significantly. However, seven and 15 times stripping resulted in significantly higher diffusion rate. In conclusion, TEWL measurements can detect severe damage of the stratum corneum (SC) but not small changes, which nevertheless may already influence drug diffusion. Therefore, TEWL measurements appears to be of limited use as a barrier integrity test for human HSE in in vitro test systems.     

An investigation into the combination of low frequency ultrasound and liposomes on skin permeability

Antigen application onto skin that has been pre-treated with low frequency ultrasound leads to immunisation, and it was hypothesised that immunisation could be enhanced if antigens were entrapped within liposomes, the latter being known vaccine adjuvants. However, it has been suggested that liposomes can repair skin damage, which could limit antigen permeation and transcutaneous immunisation. The aim of the present work was therefore to investigate the influence of liposome application on subsequent: (i) in vitro antigen permeation through, and (ii) in vivo barrier properties of, ultrasound-treated skin. Sonication was conducted using either phosphate buffered saline (PBS) or an aqueous solution of sodium dodecyl sulphate (SDS) as the coupling medium, and rats were used as the animal models. Liposome application to sonicated skin reduced antigen penetration and transepidermal water loss (TEWL, used as an indication of skin integrity) when the skin had been sonicated using PBS coupling medium. The influence of liposome was evident within 5 min of its application, and smaller liposomes were more effective at repairing skin disruption caused by sonication. Such skin repair did not, however, take place when the skin had been sonicated in the presence of SDS (which caused greater skin disruption), and changes in in vitro antigen permeation and in vivo TEWL were negligible. Skin repair by liposomes seems to depend on the extent of the disruption caused by ultrasound application.     

Biophysical study of porcine ear skin in vitro and its comparison to human skin in vivo

The goal of this work was to establish, using biophysical characterization, that porcine ear skin in vitro is a valid model for its human counterpart. Specifically, stratum corneum (SC) barrier function was evaluated during its progressive removal by adhesive tape-stripping using the techniques of transepidermal water loss (TEWL) and impedance spectroscopy. TEWL increased slowly at first and then more rapidly with the degree of SC impairment. In contrast, low-frequency skin impedance declined exponentially as a function of progressive SC removal. The methods provide complementary and correlated information about SC barrier function. Biophysical parameters, including the diffusivity and permeability coefficient of water across the SC, and the thickness of the barrier were determined from the TEWL data using Fick's first law of diffusion. Furthermore, an ionic partition coefficient-mobility product was estimated from the skin impedance measurements. Comparison of the results with those previously reported for human skin in vivo strongly supports the validity of the porcine membrane as an in vitro model.     

Development of an in vitro model for premature neonatal skin: biophysical characterization using transepidermal water loss

The objective was to develop an in vitro model for the developing skin of the premature neonate. Barriers of different levels of efficiency were produced by differentially tape-stripping the stratum corneum (SC) from the skin of excised porcine ears, and were characterized by measurements of transepidermal water loss (TEWL). In this way, it was possible to express the recorded TEWL as a function of percentage SC thickness (F) generating the following relationship: TEWL = 2.7 + 41.exp [- 0.028.F]. These data were then compared to previously published in vivo measurements of TEWL obtained from a population of premature neonates at various post-conceptional ages (PCA). The latter conformed to a remarkably parallel relationship to that found in vitro with the porcine skin model, namely TEWL = 3.3 + 41.exp [-0.026.(PCA-160)]. It can be suggested, therefore, that the empirically adjusted PCA (i.e., PCA-160) correlates closely with the developing thickness of the neonate's SC. The corollary is that porcine skin, in vitro, tape-stripped to a particular level, can provide a barrier corresponding to a specific degree of neonate maturation and can serve, hence, as a useful tool with which to explore whether transdermal drug delivery in this unique patient population may be beneficial.     

On the correlation between single-frequency impedance measurements and human skin permeability to water

The objective of this study was to quantitatively compare measurements of tritiated water permeability with impedance determined at either 100 or 1000 Hz using an LCR databridge on the same pieces of skin. A previously published expression based on a simple circuit of a parallel resistor and constant phase element (CPE) was used to relate (R_PARA) measured at different frequencies to the DC resistance (R_skinA) and the steady-state skin permeability of tritiated water (k_p). Using this analysis, k_p and (R_PARA) data from three laboratories were shown to be consistent with each other, and k_p and (R_skinA) estimated from (R_PARA) were linearly correlated. Compared with urea and mannitol, which are known to permeate skin through a polar pathway, the value of k_p for water was found to be about two times larger than expected for transport through only the polar pathway, suggesting an approximately equal contribution from the lipophilic pathway. Equations relating k_p to (R_PARA) and (R_skinA) were used to compare on a consistent basis proposed tests for identifying and excluding damaged skin from chemical absorption studies. The criterion of 20 kΩ cm² for (R_skinA) corresponds to a tritiated water permeability of 3.2 × 10⁻³ cm/h, which should exclude damaged skin without screening undamaged but higher permeability skin samples from study.     

Porcine Ear Skin as a Model for the Assessment of Transdermal Drug Delivery to Premature Neonates

PURPOSE: The purpose of this study was (i) to validate differentially tape-stripped, porcine skin as an in vitro model for the evaluation of transdermal drug delivery (TDD) to premature neonates, (ii) to determine whether the model could estimate neonatal skin permeability as a function of postconceptional age (PCA), and (iii) to demonstrate that iontophoretic delivery permits precise control of drug input independent of skin barrier function. METHODS: Passive permeation of caffeine, phenobarbital, and lidocaine across tape-stripped porcine skin barriers was measured. Iontophoretic delivery of lidocaine across skins with different barrier competencies was also evaluated. RESULTS: For all drugs, passive permeation correlated with skin barrier function; that is, with transepidermal water loss (TEWL): Jss = A x exp[B x TEWL]. Combining this result with a previously derived dependence of TEWL upon the PCA of premature neonates in vivo allowed a relative value of Jss to be predicted for a given PCA. Comparison of these predictions showed excellent agreement with experimental data reported for diamorphine. Iontophoretic lidocaine delivery was precisely controllable independent of barrier competency. CONCLUSIONS: Porcine skin, in vitro, differentially tape-stripped to specific barrier competencies, is a useful model to explore TDD in premature neonates. The potential for iontophoresis to provide improved dose control and adjustment, irrespective of skin barrier maturity, is established.     

Effect of ethanol/propylene glycol on the in vitro percutaneous absorption of aspirin, biophysical changes and macroscopic barrier properties of the skin.

The effect of the solvent systems ethanol (EtOH), propylene glycol (PG) and combinations thereof was examined on the in vitro percutaneous absorption of the antithrombotic, aspirin, through porcine epidermis. Biophysical changes in the stratum corneum lipids were studied through the use of Fourier transform infrared (FTIR) spectroscopy. Macroscopic barrier properties of the epidermis were examined through the use of in vitro transepidermal water loss (TEWL). The flux of aspirin increased with increasing concentrations of EtOH in the solvent systems. The maximum flux of aspirin was achieved by 80% EtOH in combination with 20% PG beyond which (i.e. 100% EtOH) there was no increase in the flux. FTIR spectroscopic study was enacted in order to determine the biophysical properties of the stratum corneum when the solvents were applied. The FTIR spectra of the stratum corneum treated with 80% EtOH/20% PG showed a maximum decrease in absorbance for the asymmetric and symmetric C&z. sbnd;H peaks, which suggests a greater loss of the lipids in the stratum corneum layers. In vitro TEWL studies allowed an investigation into the macroscopic barrier integrity properties of the stratum corneum. The TEWL results indicated that each of the solvent systems significantly enhanced (P<0.05) in vitro TEWL in comparison to the control. In conclusion, 80% EtOH/20% PG enhanced the percutaneous absorption of aspirin by perturbing the macroscopic barrier integrity of the stratum corneum and through a loss of stratum corneum lipids. Copyright     

Occlusive Properties of Monolayer Patches: In Vitro and in Vivo Evaluation

Purpose. Patches can cause a different grade of skin occlusion, depending on matrix composition and thickness, backing layer material. The aim of this work was to verify if in vitro water vapour permeability (WVP) values are predictive of transepidermal water loss (TEWL) and Fourier-transform infrared (FTIR) spectroscopy values measured in vivo after 24 h of methacrylic or acrylic monolayer patches application. The correlation between both in vivo methods has been evaluated. Methods. The WVP, TEWL and FTIR measurements were performed by using four patches made of a methacrylic or an acrylic polymeric system (250 and 500 m thickness on a polyurethane backing layer). A fifth patch was made of the methacrylic matrix on a polyvinyl chloride backing layer. Results. A good correlation was found between TEWL values and IR water/lipid absorbance ratios. The in vitro WVP values are in a good correlation with the results of both in vivo methods: TEWL = –0.01WVP + 21.31 (R² = 0.9312); FTIR water/lipid ratio = –0.01WVP + 27.15 (R² = 0.9447). Conclusions. The in vitro method proposed for measuring the WVP is predictive of the degree of occlusion resulting from the in vivo application of monolayer patches.     

Assessment of Skin Barrier Function Using Transepidermal Water Loss: Effect of Age

To probe age-related changes in skin barrier function, transepidermal water loss (TEWL) rates have been measured in young (19–42 years) and old (69–85 years) subjects. TEWL was determined at ventral forearm skin sites, which had been occluded for 24 hr with polypropylene chambers. Baseline TEWL rates (J , which showed no dependence on age, were measured for each subject before and after the experiment. Following removal of the occlusive chamber, TEWL was monitored continuously from t = 0.5 min until its return to the baseline (preocclusion) level, which was typically in the range of 2–7 g/m²/hr. Initial TEWL rates (mean ± SD) were found to differ significantly between young (28.6 ± 7.5 g/m²/hr; n = 26) and old (36.9 ± 10.5 g/m²/hr; n = 18) subjects (P < 0.01). Relaxation of TEWL to J was significantly slower in the aged cohort, such that the characteristic time for diffusion of water in the stratum corneum was estimated to be (mean ± SD) 176 ± 59 min for the young subjects, compared to 360 ± 76 min for the old (P < 0.001.). Thus, the initial TEWL value following removal of occlusion is significantly greater, and the excessive stratum corneum hydration produced by occlusion is dissipated more slowly, in old skin than in young. A hypothesis to explain the slower relaxation of perturbed TEWL in old skin is proposed.     

Alteration of skin hydration and its barrier function by vehicle and permeation enhancers: a study using TGA, FTIR, TEWL and drug permeation as markers

Vehicles and permeation enhancers (PEs) used in transdermal drug delivery (TDD) of a drug can affect skin hydration, integrity and permeation of the solute administered. This investigation was designed to study the effect of the most commonly used vehicles and PEs on rat skin hydration, barrier function and permeation of an amphiphilic drug, imipramine hydrochloride (IMH). An array of well-established techniques were used to confirm the findings of the study. Thermogravimetric analysis (TGA) and Fourier transform infrared (FTIR) spectroscopy were used to determine changes in skin hydration. Alteration of the stratum corneum (SC) structure was investigated using FTIR studies. To monitor the barrier function alteration, transepidermal water loss (TEWL) measurement and permeation studies were performed. Our findings indicate that with hydration, there was an increase in the bound water content of the skin, and pseudoequilibrium of hydration (a drastic decrease in hydration rate) was achieved at around 12 h. Hydration increased the ratio between amide-I and amide-II peaks in FTIR and reduced the C-H stretching peak area. Both propylene glycol (PG) and ethanol (EtOH) dehydrated skin, with the latter showing a predominant effect. Furthermore, it was confirmed that PG and EtOH decreased the bound water content due to alteration in the protein domains and extraction of SC lipids, respectively. The effect of hydration on the SC was found to be similar to that reported for temperature. Permeation studies revealed that the dehydration caused by vehicles decreased IMH flux, whereas the flux was enhanced by PEs. The role of partition was predominant for the permeation of IMH through dehydrated skin. A synergistic effect was observed for PG and menthol in the enhancement of IMH. Further findings provided strong evidence that PG affects protein domains and EtOH extracts lipids from the bilayer. Both PG and EtOH, with or without PEs, increased TEWL. Initial TEWL was well correlated with the flux of IMH through the same skin. It was found that both PG and EtOH affect the permeation of solute and TEWL by dehydration. The experiments also proved that the initial TEWL value has a strong potential as a predictive tool for the permeation of the solute.     

Transepidermal water loss (TEWL) and corneometry with hydrogel vehicle in the treatment of atopic dermatitis: a randomized, investigator-blind pilot study

Disruption of the epidermal barrier, as indicated by a reduction in skin hydration and an increase in transepidermal water loss (TEWL) is a feature of atopic dermatitis (AD). Novel formulations of dermatologic therapies may enhance patient satisfaction and adherence and may possibly preserve and enhance epidermal barrier function. A single-center, investigator-blinded, randomized, split-body exploratory study was undertaken to assess the hydrating and barrier preserving effects of a water-based hydrogel vehicle. Subjects (n=20) with mild to moderate disease at baseline applied hydrogel vehicle or a moisturizing lotion (Eucerin Lotion?, Beiersdorf, Inc.) in a split-body fashion for two weeks. Corneometry and TEWL measurements were taken at baseline and week 2. Hydrogel vehicle produced a statistically significant improvement in skin hydration from baseline, as compared to a moisturizing lotion control. Hydrogel produced no statistically significant change in TEWL, while comparator lotion increased TEWL. Data from this pilot study indicate that the water-based hydrogel vehicle improves skin hydration and does not further impair epidermal barrier function, suggesting that it is an appropriate vehicle choice for patients with mild-to-moderate atopic dermatitis.     

Development of an in vitro skin permeation model simulating atopic dermatitis skin for the evaluation of dermatological products

Atopic dermatitis (AD) skin has a defective barrier function as indicated by increased transepidermal water loss (TEWL). In order to test potential new formulations for AD, it was our aim to develop a skin permeation model simulating AD skin by inducing barrier impairment to otherwise healthy skin simulating the barrier properties of AD skin as evaluated by TEWL measurements. Pig ear skin was mounted to Franz-type diffusion cells. Skin barrier impairment was induced by tape strippings. As the number of strips increased, higher TEWL values were obtained. By performing 25 tape strippings, the TEWL value within the range reported for involved skin of AD patients was reached. The in vitro skin permeation of fusidic acid and betamethasone-17-valerate was found to correlate with the number of tape strippings used to remove stratum corneum cell layers. A comparison of the permeability of fusidic acid and betamethasone-17-valerate from Fucicort cream to a new Fucicort Lipid formulation was studied with intact (0 strippings) and barrier-impaired skin simulating involved AD skin (25 strippings). As opposed to intact skin, no statistically significant difference through barrier-impaired skin was found for fusidic acid and betamethasone-17-valerate for the two formulations. This is in accordance with the clinical results of an international multicentre study and thus confirms the predictability of the model.     

Novel transdermal delivery of Timolol maleate using sugar esters: Preclinical and clinical studies

The feasibility of matrix controlled transdermal patch based on sugar fatty acid ester (SE) as penetration and absorption enhancer containing Timolol maleate (TM) was investigated. The influence of fatty acid type, chain length and hydrophile-lipophile balance (HLB) on the in vitro drug release as well as its permeation across hairless rat skin were studied and compared aiming to select a patch formula for clinical performance. Skin irritation induced by SE patch was evaluated by visual scoring, color reflectance measurements and non-invasive transepidermal water loss (TEWL) technique. The results indicated that among different SEs tried, laurate SE with shorter fatty acid chain length and higher HLB value significantly increased the amount of TM liberated from the patch (99 ± 2.1%) and its permeation across rat skin (86 ± 4.3%). The total drug permeation and flux values were approximately 5-fold greater compared to SE free patch. The extent of absorption of TM-SE patch expressed by AUC was 64% larger as compared to the oral solution with steady plasma concentration over 18 h and relative bioavailability (F_rel) of 163%. The developed patch was well tolerated by all the subjects with only moderate skin irritation, which was recovered in 24 h after patch removal. The results are very encouraging and offer an alternative approach to maintain higher, prolonged and controlled blood level profile of the drug over 18-24 h.