Visualisation of drug distribution in skin using correlative optical spectroscopy and mass spectrometry imaging.

A correlative methodology for label-free chemical imaging of soft tissue has been developed, combining non-linear optical spectroscopies and mass spectrometry to achieve sub-micron spatial resolution and critically improved drug detection sensitivity. The approach was applied to visualise the kinetics of drug reservoir formation within human skin following in vitro topical treatment with a commercial diclofenac gel. Non-destructive optical spectroscopic techniques, namely stimulated Raman scattering, second harmonic generation and two photon fluorescence microscopies, were used to provide chemical and structural contrast. The same tissue sections were subsequently analysed by secondary ion mass spectrometry, which offered higher sensitivity for diclofenac detection throughout the epidermis and dermis. A method was developed to combine the optical and mass spectrometric datasets using image registration techniques. The label-free, high-resolution visualisation of tissue structure coupled with sensitive chemical detection offers a powerful method for drug biodistribution studies in the skin that impact directly on topical pharmaceutical product development.

Optimization of topical formulations using a combination of in vitro methods to quantify the transdermal passive diffusion of drugs.

This paper describes a new approach to the early-stage optimization of topical products and selection of lead formulation candidates. It demonstrates the application of open flow microperfusion in vitro in conjunction with the Franz diffusion cell to compare time-resolved, 24-hour profiles of diclofenac passive diffusion through all skin layers (including the skin barrier, dermis, and subcutis) resulting from nine topical formulations of different composition. The technique was successfully validated for in vitro sampling of diclofenac in interstitial fluid. A multi-compartmental model integrating the two datasets was analyzed and revealed that the passive diffusion of diclofenac through the dermis and subcutis does not correlate with its diffusion through the skin barrier and cannot be predicted using Franz diffusion cell data alone. The combined application of the two techniques provides a new, convenient tool for product development and selection enabling the comparison of topical formulation candidates and their impact on drug delivery through all skin layers. This approach can also generate the experimental data required to improve the robustness of mechanistic PBPK models, and when combined with clinical sampling via open flow microperfusion - for the development of better in vivo-in vitro correlative models.

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 (RPARA) measured at different frequencies to the DC resistance (RskinA) and the steady-state skin permeability of tritiated water (kp). Using this analysis, kp and (RPARA) data from three laboratories were shown to be consistent with each other, and kp and (RskinA) estimated from (RPARA) were linearly correlated. Compared with urea and mannitol, which are known to permeate skin through a polar pathway, the value of kp 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 kp to (RPARA) and (RskinA) 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 (RskinA) 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.

Determining epidermal disposition kinetics for use in an integrated nonanimal approach to skin sensitization risk assessment.

Development of risk assessment methods for skin sensitization in the absence of toxicological data generated in animals represents a major scientific and technical challenge. The first step in human skin sensitization induction is the transport of sensitizer from the applied dose on the skin surface to the epidermis, where innate immune activation occurs. Building on the previous development of a time course in vitro human skin permeation assay, new kinetic data for 10 sensitizers and 2 nonsensitizers are reported. Multicompartmental modeling has been applied to analyze the data and determine candidate dose parameters for use in integrated risk assessment methods: the area under the curve (AUC) and maximum concentration (C(max)) in the epidermis. A model with two skin compartments, representing the stratum corneum and viable skin (epidermis and dermis), was chosen following a formal model selection process. Estimates of the uncertainty, as well as average values of the epidermal disposition kinetics parameters, were made by fitting to the time course skin permeation data from individual skin donors. A potential reduced time course method is proposed based on two time points at 4 and 24 h, which gives results close to those from the full time course for the current data sets. The time course data presented in this work have been provided as a resource for development of predictive in silico skin permeation models.

Development of a modified in vitro skin absorption method to study the epidermal/dermal disposition of a contact allergen in human skin.

In vitro skin absorption methods exist in Organisation for Economic Co-operation and Development (OECD) guideline form (No. 428) and are used to estimate the degree of systemic penetration of chemicals through skin. More detailed kinetics of permeation through skin compartments are not described well by existing methods. This study was designed to assess the practical feasibility of generating compartmental (stratum corneum/epidermal/dermal) disposition and kinetic data of topically applied chemicals. For chemically induced effects initiated in the skin (e.g., skin allergy), the delivery of tissue concentrations of chemical will impact the incidence and severity of biological effect. Explicit data on the kinetics of chemical disposition in skin have not traditionally been needed for skin allergy risk assessment: current in vivo assays embody delivery implicitly. Under the 7th Amendment to the European Cosmetics Directive, in vivo assays (such as the local lymph node assay for skin sensitization) will not be permitted to assess cosmetic ingredients. New in vitro and in silico alternative approaches and ways of predicting risk of adverse effects in humans need to be developed, and new methods such as that described here provide a way of estimating delivered concentrations and the effect of formulation changes on that delivery. As we continue to deconstruct the contributing factors of skin allergy in humans, it will be useful to have methods available that can measure skin tissue compartment exposure levels delivered from different exposure use scenarios. Here we provide such a method. The method could also be used to generate useful data for developing in silico kinetic models of compartmental skin delivery and for refining data for skin delivery in relation to the evaluation of systemic toxicity.

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 kp 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 kp values constituted a positively skewed, non-Normal distribution. Mean kp was 2.04 x 10(-3)cm/h while the 95th percentile was 4.50 x 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 x 10(-3)cm/h. Analysis was also performed on smaller data subsets allowing inter-individual and intra-individual comparisons. For intra-individual kp 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.

Absorption of [14C]-tetrabromodiphenyl ether (TeBDE) through human and rat skin in vitro.

The skin is the largest organ in the human body and has the potential to come into contact with a variety of xenobiotics both intentionally (e.g., drugs and cosmetics) or accidentally (e.g., agrochemicals and industrial chemicals). These chemicals may then cross the skin barrier (the stratum corneum) and enter into the systemic circulation where they may produce a desired or an undesired effect, or even no systemic effect at all. Tetrabromodiphenyl ether (TeBDE) is one congener in a mixture of polybrominated diphenyl ethers that makes up a flame-retardant commercial product called pentabromodiphenyl ether (PeBDE). TeBDE was used as a surrogate to assess the potential dermal absorption of this product. The physicochemical properties, including lipophilicity, of TeBDE and PeBDE are similar. Operator exposure of PeBDE product to human skin is possible during production and use. However, during these activities, operators wear protective clothing to protect from or minimize exposure. This study was designed to assess the rate and extent of absorption of [14C]-tetrabromodiphenyl ether ([14C]-TeBDE) through human and rat skin in vitro. [14C]-TeBDE was applied to human and rat split thickness skin membranes in vitro in a single test preparation: [14C]-TeBDE in acetone (ca. 20%, w/v). Dermal delivery and absorbed dose of TeBDE applied to human skin was 3.13% (313 microg equiv/cm(2)) and 1.94% (194 microg equiv/cm(2)) of the applied dose, respectively. Dermal delivery and absorbed dose of TeBDE applied to rat skin was 17.94% (1804 microg equiv/cm(2)) and 14.81% (1489 microg equiv/cm(2)) of the applied dose, respectively. These results confirm that the risk of systemic exposure due to external dermal exposure of the PeBDE product is low in the human. Consequently, based on the toxicological profile of these materials, the potential for undesirable effects is also quite low. The results also confirm that the rat is a conservative model overpredicting human absorption about eight fold.