Observations on the Tritiated Water and TEWL Skin Integrity Tests: Relevance to In Vitro Permeation Testing (IVPT).

BACKGROUND: The in vitro permeation test (IVPT) using ex vivo human skin is a sensitive and robust model system that has been vital in elucidating the fundamental parameters surrounding the absorption of both therapeutic agents and industrial chemicals through skin. FDA and OECD IVPT Guidances recommend that each skin section selected for study should be screened prior to use to ensure that the stratum corneum integrity is retained. Three methods are currently considered acceptable: 1) transepidermal water loss (TEWL), 2) electrical resistance, and 3) tritiated water (3H2O) absorption. METHODS: A retrospective analysis of data from the authors' laboratory has been performed with the objective of addressing a number of questions regarding the 3H2O and TEWL integrity tests, and the population attributes of a large database consisting of 17,330 individual skin sections obtained from 459 skin donors. The applicability and usefulness of these tests, when compared to companion permeation data obtained from 25 topical drug products, has also been examined. RESULTS: Both integrity tests found water permeability to be equal in White and Hispanic races but higher than in Blacks, 3H2O being more discriminating than TEWL. Male skin is more permeable than female and there is a slight decrease in permeability with advancing age in both groups. Correlation between 3H2O absorption and drug absorption revealed a minimal relationship between the two in most cases, the Pearson correlation coefficient ranging from -0.417 to 0.953. Additionally, drug outliers were not always identified with a failing integrity test. CONCLUSION: The results call for a critical reexamination of the value of the 3H2O integrity test, and by extension, TEWL, for use in IVPT studies.

Cutaneous Pharmacokinetic Approaches to Compare Bioavailability and/or Bioequivalence for Topical Drug Products.

The evaluation of bioequivalence (BE) involves comparing the test product to its reference product in a study whose fundamental scientific principles allow inferring of the clinical performance of the products. Several test methods have been discussed and developed to evaluate topical bioavailability (BA) and BE. Pharmacokinetics-based approaches characterize the rate and extent to which an active ingredient becomes available at or near its site of action in the skin. Such methodologies are considered to be among the most accurate, sensitive, and reproducible approaches for determining the BA or BE of a product.

The Tritiated Water Skin Barrier Integrity Test: Considerations for Acceptance Criteria with and Without 14C-Octanol.

PURPOSE: A study was designed to assess barrier integrity simultaneously using separate compounds (probes) for polar and non-polar pathways through the skin, 3H2O and 14C-octanol, respectively; and to determine whether the two probe approach could better define barrier integrity. METHODS: A 5-min dose of water containing 3H2O and 14C -octanol was applied to ex vivo human skin mounted in Franz diffusion cells. The receptor solution was sampled at 30 min, analyzed for 3H and 14C content, and the correlation between water and octanol absorption was determined by statistical tests suitable for non-normally distributed data. This study was conducted on skin from 37 donors with from 3 to 30 replicate skin sections per donor (a total of 426 sections). RESULTS: The correlation between 3H2O and 14C-octanol absorption was low (Pearson correlation coefficient = 0.3485). The 3H2O absorption cutoff used in this study to select for a normal skin barrier rejected some sections in which 14C-octanol absorption was within normal limits and accepted others in which 14C-octanol absorption was abnormally high. The converse was true for 3H2O absorption when the 14C-octanol-based cutoff was used. CONCLUSIONS: The results of the 3H2O test or of similar tests that primarily assess the permeability of polar pathways through the skin may not necessarily provide information relevant to the absorption of highly lipophilic compounds. Octanol, or another molecule that more closely matches the physicochemical attributes of the test compound, may characterize properties of the skin barrier that are more relevant to compounds of low water solubility.

Pharmacokinetics-Based Approaches for Bioequivalence Evaluation of Topical Dermatological Drug Products.

The pharmacokinetic approach has accelerated the development of high-quality generic medicines with extraordinary cost savings, transforming the pharmaceutical industry and healthcare system in the USA. While this is true for systemically absorbed drug products, the availability of generic versions of topical dermatological products remains constrained due to the limited methods accepted for bioequivalence evaluation of these products. The current review explores the possibility of developing appropriate bioequivalence approaches based on pharmacokinetic principles for topical dermatological products. This review focuses on the strengths and limitations of the three most promising pharmacokinetics-based methods to evaluate the performance and bioequivalence of topical dermatological products, which include in vivo skin stripping, in vivo microdialysis, and in vitro permeation testing (IVPT) with excised human skin. It is hoped that recent advances in pharmaceutical and regulatory science will facilitate the development of robust bioequivalence approaches for these dosage forms, enable more efficient methodologies to compare the performance of new drug products in certain pre-approval or post-approval change situations, and promote the availability of high-quality generic versions of topical dermatological products.

A simplified approach for estimating skin permeation parameters from in vitro finite dose absorption studies.

Historically, percutaneous absorption permeation parameters have been derived from in vitro infinite dose studies, yet there is uncertainty in their accuracy if the applied vehicle saturates or damages the stratum corneum, or when the permeation parameters are inappropriately derived from cumulative absorption data. An approach is provided for determining penetration parameters from in vitro finite dose data. Key variables, and equations for their derivation, are identified from the literature and provide permeation parameters that use only Tmax , AUC, and AUMC from finite dose data. The equations are tested with computer-generated model data and to actual study data. Derived permeation parameters obtained from the computer model data match those used in generating the simulated finite dose data. Parameters obtained from actual study data reasonably and acceptably model the penetration profile kinetics of the study data. From in vitro finite dose absorption data, three parameters can be obtained: the diffusion transit time (td ), which characterizes the diffusion coefficient, the partition volume (Vm P), which characterizes the partition coefficient, and the permeation coefficient (Kp ). These parameters can be obtained from finite dose data without having to know the length of the diffusion pathway through the membrane.

Assessing topical bioavailability and bioequivalence: a comparison of the in vitro permeation test and the vasoconstrictor assay.

PURPOSE: To compare the sensitivity of a pharmacokinetic assay, the in vitro permeation test (IVPT), with that of a pharmacodynamic assay, the human skin blanching or vasoconstrictor (VC) assay, in assessing the relative bioavailability of topical clobetasol propionate products. METHODS: The percutaneous absorption of clobetasol propionate from five commercial products was measured in vitro using cryopreserved human skin. The pharmacodynamic potency of the same five products was also assessed in vivo using the VC assay, the surrogate method by which regulatory authorities in the United States establish the bioequivalence of generic topical glucocorticoid products. RESULTS: IVPT found total clobetasol absorption varying ten-fold from highest to lowest product, whereas the VC assay found this same difference was less than two-fold. The coefficient of variation ranged from 78 to 126% in the VC assay, but only 30-43% for IVPT. Statistically, IVPT could separate the 5 products into three groups: 1) ointment, 2) cream and gel, 3) emollient cream and solution). Due to its greater variability as well as saturation of the pharmacodynamic response at higher flux levels, the VC assay found all products except the solution to be equipotent. CONCLUSIONS: IVPT was found to be substantially more sensitive and less variable than the VC assay for assessing clobetasol bioavailability.

Effect of induced acute diabetes and insulin therapy on stratum corneum barrier function in rat skin.

BACKGROUND: A wide variety of cutaneous manifestations are associated with diabetes. However, there is a paucity of information on stratum corneum barrier function in diabetics, with and without insulin therapy. METHODS: To assess for alteration of the stratum corneum, its barrier function was tested by evaluating the percutaneous absorption of water, ethanol, lidocaine and hydrocortisone, in vitro, on normal control, 4-week diabetic and 8-day insulin-treated diabetic Sprague-Dawley CD rats. RESULTS: Total water penetration was not different between the 3 groups though flux profiles were different. Both total penetration and peak flux of lidocaine and hydrocortisone increased slightly in the diabetic rats over the control group. However, total penetration and peak flux (including ethanol) were significantly increased in the insulin-treated rats. CONCLUSION: The data indicate that diabetes modestly alters stratum corneum physiology but less so than that seen following insulin therapy.

In vitro percutaneous absorption of ketoprofen and testosterone: comparison of pluronic lecithin organogel vs. pentravan cream.

An in vitro human percutaneous absorption study was conducted to assess the delivery of ketoprofen and testosterone from two base formulations, a Pluronic lecithin organogel and Pentravan Cream. Each formulation was applied to ex vivo human trunk skin (from three skin donors) on triplicate sections mounted onto Franz Diffusion Cells. Following a 5-mcL/cm2 applied dose, serial dermal receptor solutions were collected over 48 hours. For both compounds, a greater rate and extent of absorption was found from the Pentravan formulation than from the Pluronic lecithin organogel formulation: 3.8-fold greater for ketoprofen, 1.7-fold greater for testosterone, for amount absorbed.

Impact of order of application of moisturizers on percutaneous absorption kinetics: evaluation of sequential application of moisturizer lotions and azelaic acid gel 15% using a human skin model.

The medical management of rosacea increasingly has involved not only the appropriate selection of topical medication but also patient education and specific recommendations regarding appropriate skin care. The recognition that epidermal barrier dysfunction and transepidermal water loss (TEWL) play a pathophysiologic role in rosacea and that skin moisturization may help to mitigate signs and symptoms of the disease has led to a deeper appreciation of the importance of proper skin care in the treatment of rosacea. Data from a percutaneous penetration study performed using human skin suggest that any of the tested moisturizer lotions may be applied either before or after azelaic acid gel 15% without a major change in the percutaneous absorption profile of azelaic acid.

Evaluation of the percutaneous absorption of promethazine hydrochloride, in vitro, using the human ex vivo skin model.

For several years, pharmacists have been using vehicles such as Pluronic lecithin organogel and Lipoderm for the delivery of medication, including promethazine, through the skin. This study evaluated the percutaneous absorption pharmacokinetics of promethazine hydrochloride 25 mg/g in Pluronic lecithin organogel and the commercial base Lipoderm,in vitro, in human ex vivo skin, using the finite dose technique and Franz Diffusion Cells. Due to the polar nature of promethazine hydrochloride, it was theorized that this drug would present a transdermal challenge for these two commonly utilized compounding bases. An amount of 5 mcL/cm squared of each formulation was applied to triplicate sections of skin from three different ex vivo skin donors. At 4, 8, 12, 24, 32, and 48 hours post-application, the reservoir solution in the Franz Cell was removed and analyzed. Lipoderm performed better than Pluronic lecithin organogel based on total absorption into the reservoir solution (2.86% +/- 0.79 vs. 1.73% +/-0.85) (P less than 0.2). Total permeation through and beyond the stratum corneum (epidermis, dermis, and reservoir solution) after 48 hours was significantly greater for Lipoderm than for Pluronic lecithin organogel (23.06% +/- 3.33 vs. 10.31% +/- 5.44), with the Student's t-test showing statistical significance (P less than 0.001). These data indicate that promethazine hydrochloride does penetrate into and through human ex vivo skin in vitro using Lipoderm and Pluronic lecithin organogel, with Lipoderm showing better results.