Sébastien Grégoire

Quantitative 3D molecular cutaneous absorption in human skin using label free nonlinear microscopy.

Understanding the penetration mechanisms of drugs into human skin is a key issue in pharmaceutical and cosmetics research. To date, the techniques available for percutaneous penetration of compounds fail to provide a quantitative 3D map of molecular concentration distribution in complex tissues as the detected microscopy images are an intricate combination of concentration distribution and laser beam attenuation upon deep penetration. Here we introduce and validate a novel framework for imaging and reconstructing molecular concentration within the depth of artificial and human skin samples. Our approach combines the use of deuterated molecular compounds together with coherent anti-Stokes Raman scattering spectroscopy and microscopy that permits targeted molecules to be unambiguously discriminated within skin layers. We demonstrate both intercellular and transcellular pathways for different active compounds, together with in-depth concentration profiles reflecting the detailed skin barrier architecture. This method provides an enabling platform for establishing functional activity of topically applied products.

Improvement of the experimental setup for skin absorption screening studies with reconstructed skin EPISKIN.

Percutaneous penetration studies are usually performed in human skin samples set up in a Franz® cell device. The ability to perform these studies may depend on the availability of skin samples. Reconstructed skin models are an interesting alternative to overcome such limitations but are less easily mounted in diffusion cell devices. Previous data showed that EPISKIN® was a highly performing model to carry out such studies. However, the setup in a PermeGear® cell device is time consuming and therefore unsuitable for screening purposes. Another approach could be using EPISKIN in its cell culture insert. The aim of this study was to compare cutaneous penetration of chemicals applied to EPISKIN samples in a PermeGear cell versus in their own insert. Eight chemicals having widely different chemical structures and penetration potentials were studied. Six test chemicals showed a similar penetration level in both devices. Using the PermeGear cell device, the penetration level was overestimated for the other 2 tested chemicals. The results demonstrated that percutaneous studies with EPISKIN samples could be easily performed using the insert setup. The EPISKIN model has been greatly improved in the recent years and it is now possible to develop screening tests for the evaluation of skin penetration with a higher reliability.

Comparison of protocols for measuring cosmetic ingredient distribution in human and pig skin

The Cosmetics Europe Skin Bioavailability and Metabolism Task Force aims to improve the measurement and prediction of the bioavailability of topically-exposed compounds for risk assessment. Key parameters of the experimental design of the skin penetration studies were compared. Penetration studies with frozen human and pig skin were conducted in two laboratories, according to the SCCS and OECD 428 guidelines. The disposition in skin was measured 24h after finite topical doses of caffeine, resorcinol and 7-ethoxycoumarin. The bioavailability distribution in skin layers of cold and radiolabelled chemicals were comparable. Furthermore, the distribution of each chemical was comparable in human and pig skin. The protocol was reproducible across the two laboratories. There were small differences in the amount of chemical detected in the skin layers, which were attributed to differences in washing procedures and anatomical sites of the skin used. In conclusion, these studies support the use of pig skin as an alternative source of skin should the availability of human skin become a limiting factor. If radiolabelled chemicals are not available, cold chemicals can be used, provided that the influence of chemical stability, reactivity or metabolism on the experimental design and the relevance of the data obtained is considered.

Comparison of protocols measuring diffusion and partition coefficients in the stratum corneum

Partition (K) and diffusion (D) coefficients are important to measure for the modelling of skin penetration of chemicals through the stratum corneum (SC). We compared the feasibility of three protocols for the testing of 50 chemicals in our main studies, using three cosmetics‐relevant model chemicals with a wide range of logP values. Protocol 1: SC concentration‐depth profile using tape‐stripping (measures KSC/v and DSC/HSC2, where HSC is the SC thickness); Protocol 2A: incubation of isolated SC with chemical (direct measurement of KSC/v only) and Protocol 2B: diffusion through isolated SC mounted on a Franz cell (measures KSC/v and DSC/HSC2, and is based on Ficks laws). KSC/v values for caffeine and resorcinol using Protocol 1 and 2B were within 30% of each other, values using Protocol 2A were ~two‐fold higher, and all values were within 10‐fold of each other. Only indirect determination of KSC/v by Protocol 2B was different from the direct measurement of KSC/v by Protocol 2A and Protocol 1 for 7‐EC. The variability of KSC/v for all three chemicals using Protocol 2B was higher compared to Protocol 1 and 2A. DSC/HSC2 values for the three chemicals were of the same order of magnitude using all three protocols. Additionally, using Protocol 1, there was very little difference between parameters measured in pig and human SC. In conclusion, KSC/v, and DSC values were comparable using different methods. Pig skin might be a good surrogate for human skin for the three chemicals tested. Copyright

A Microfluidic Diffusion Cell for Fast and Easy Percutaneous Absorption Assays

ABSTRACTPurposePercutaneous absorption assays of molecules for pharmaceutical and cosmetology purposes are important to determine the bioavailability of new compounds, once topically applied. The current method of choice is to measure the rate of diffusion through excised human skin using a diffusion cell. This method however entails significant drawbacks such as scarce availability and poor reproducibility of the sample, low sampling rate, and tedious assay setup.MethodsThe objective of the present work is to propose an alternative method that overcomes these issues by integrating an experimental model of the skin (artificial stratum corneum) and online optical sensors into a microfluidic device.ResultsThe measurement of the diffusion profile followed by the calculation of the permeability coefficients and time lag were performed on seven different molecules and obtained data positively fit with those available from literature on human skin penetration. The coating of the lipid mixture to generate the artificial stratum corneum also proved robust and reproducible. The results show that the proposed device is able to give fast, real-time, accurate, and reproducible data in a user-friendly manner, and can be produced at a large scale.ConclusionThese assets should help both the cosmetics and pharmaceutics fields where the skin is the target or a pathway of a formulated compound, by allowing more candidate molecules or formulations to be assessed during the various stages of their development.

Comparison of the Skin Penetration of 3 Metabolically Stable Chemicals Using Fresh and Frozen Human Skin

Background: The Cosmetics Europe ADME Task Force is developing in vitro and in silico tools for predicting skin and systemic concentrations after topical application of cosmetic ingredients. There are conflicting reports as to whether the freezing process affects the penetration of chemicals; therefore, we evaluated whether the storage of human skin used in our studies (8-12 weeks at -20°C) affected the penetration of model chemicals. Methods: Finite doses of trans-cinnamic acid (TCA), benzoic acid (BA), and 6-methylcoumarin (6MC) (non-volatile, non-protein reactive and metabolically stable in skin) were applied to fresh and thawed frozen skin from the same donors. The amounts of chemicals in different skin compartments were analysed after 24 h. Results: Although there were some statistical differences in some parameters for 1 or 2 donors, the penetration of TCA, BA, and 6MC was essentially the same in fresh and frozen skin, i.e., there were no biologically relevant differences in penetration values. Statistical differences that were evident indicated that penetration was marginally lower in frozen than in fresh skin, indicating that the barrier function of the skin was not lost. Conclusion: The penetration of the 3 chemicals was essentially unaffected by freezing the skin at -20°C for up to 12 weeks.

A strategy for systemic toxicity assessment based on non-animal approaches: The cosmetics Europe Long Range Science Strategy programme.

When performing safety assessment of chemicals, the evaluation of their systemic toxicity based only on non-animal approaches is a challenging objective. The Safety Evaluation Ultimately Replacing Animal Test programme (SEURAT-1) addressed this question from 2011 to 2015 and showed that further research and development of adequate tools in toxicokinetic and toxicodynamic are required for performing non-animal safety assessments. It also showed how to implement tools like thresholds of toxicological concern (TTCs) and read-across in this context. This paper shows a tiered scientific workflow and how each tier addresses the four steps of the risk assessment paradigm. Cosmetics Europe established its Long Range Science Strategy (LRSS) programme, running from 2016 to 2020, based on the outcomes of SEURAT-1 to implement this workflow. Dedicated specific projects address each step of this workflow, which is introduced here. It tackles the question of evaluating the internal dose when systemic exposure happens. The applicability of the workflow will be shown through a series of case studies, which will be published separately. Even if the LRSS puts the emphasis on safety assessment of cosmetic relevant chemicals, it remains applicable to any type of chemical.

Inter-laboratory skin distribution study of 4-n-butyl resorcinol: the importance of liquid chromatography/mass spectrometry (HPLC-MS/MS) bioanalytical validation

In the present study, three laboratories independently compared percutaneous absorption and distribution of 4-n-butylresorcinol, using human skin from five donors. Each laboratory used the same protocol for percutaneous absorption studies but different LC-MS/MS analytical methods to quantify the test compound. All laboratories respected the mass balance criteria (i.e. 100±15%; average 96.5-102% of applied dose). Regarding usual inter-lab variability, good agreement was observed for all compartments with the greatest difference in the epidermis: 3.3 fold increase. The data obtained demonstrate that robustness of skin absorption data rely on properly validated analytical methods including sample extraction and LC-MS/MS method. It also includes clearly defined cutaneous absorption protocol for dose skin preparation, application, washing and tape stripping.

Inter-laboratory study of the skin distribution of 4-n-butyl resorcinol in ex vivo pig and human skin

4-n-butyl resorcinol (4-nBR) is a highly effective tyrosinase inhibitor, and can be used in cosmetic product for depigmentation purpose. Its efficacy correlates with 4-nBR that absorbed by skin. In this study, skin distribution of 4-nBR within either human or pig skin ex vivo was studied and compared by three independent laboratories. Good agreement was observed in each compartment considering usual inter-lab variability. This study supports the use of pig skin as an alternative source of skin when the availability of human skin is a limiting factor.

Comparison of the metabolism of 10 chemicals in human and pig skin explants: Metabolism of chemicals in human and pig skin

Skin metabolism is important to consider when assessing local toxicity and/or penetration of chemicals and their metabolites. If human skin supply is limited, pig skin can be used as an alternative. To identify any species differences, we have investigated the metabolism of 10 chemicals in a pig and human skin explant model. Phase I metabolic pathways in skin from both species included those known to occur via cytochrome P450s, esterases, alcohol dehydrogenases and aldehyde dehydrogenases. Common Phase II pathways were glucuronidation and sulfation but other conjugation pathways were also identified. Chemicals not metabolized by pig skin (caffeine, IQ and 4‐chloroaniline) were also not metabolized by human skin. Six chemicals metabolized by pig skin were metabolized to a similar extent (percentage parent remaining) by human skin. Human skin metabolites were also detected in pig skin incubations, except for one unidentified minor vanillin metabolite. Three cinnamyl alcohol metabolites were unique to pig skin but represented minor metabolites. There were notable species differences in the relative amounts of common metabolites. The difference in the abundance of the sulfate conjugates of resorcinol and 4‐amino‐3‐nitrophenol was in accordance with the known lack of aryl sulfotransferase activity in pigs. In conclusion, while qualitative comparisons of metabolic profiles were consistent between pig and human skin, there were some quantitative differences in the percentage of metabolites formed. This preliminary assessment suggests that pig skin is metabolically competent and could be a useful tool for evaluating potential first‐pass metabolism before testing in human‐derived tissues.