In vitro to in vivo extrapolation to derive a metabolism factor for estimating the aggregate exposure to salicylic acid after dermal exposure of its esters.

As part of the safety assessment of salicylate esters in cosmetics, we developed a metabolism factor based on in vitro to in vivo extrapolation (IVIVE) to provide a better estimation of the aggregate internal exposure to the common metabolite, salicylic acid. Optimal incubation conditions using human liver S9 were identified before measuring salicylic acid formation from 31 substances. Four control substances, not defined as salicylic esters but which could be mistaken as such due to their nomenclature, did not form salicylic acid. For the remaining substances, higher in vitro intrinsic clearance (CLint, in vitro) values generally correlated with lower LogP values. A "High-Throughput Pharmacokinetic" (HTPK) model was used to extrapolate CLint, in vitro values to human in vivo clearance and half-lives. The latter were used to calculate the percentage of substance metabolised to salicylic acid in 24 h in vivo following human exposure to the ester, i.e. the "metabolism factor". The IVIVE model correctly reproduced the observed elimination rate of 3 substances using in silico or in vitro input parameters. For other substances, in silico only-based predictions generally resulted in lower metabolism factors than when in vitro values for plasma binding and liver S9 CLint, in vitro were used. Therefore, in vitro data input provides the more conservative metabolism factors compared to those derived using on in silico input. In conclusion, these results indicate that not all substances contribute equally (or at all) to the systemic exposure to salicylic acid. Therefore, we propose a realistic metabolism correction factor by which the potential contribution of salicylate esters to the aggregate consumer exposure to salicylic acid from cosmetic use can be estimated.

2-Mercaptonicotinoyl glycine, a new potent melanogenesis inhibitor, exhibits a unique mode of action while preserving melanocyte integrity.

Research on new ingredients that can prevent excessive melanin production in the skin while considering efficacy, safety but also environmental impact is of great importance to significantly improve the profile of existing actives on the market and avoid undesirable side effects. Here, the discovery of an innovative technology for the management of hyperpigmentation is described. High-throughput screening tests on a wide chemical diversity of molecules and in silico predictive methodologies were essential to design an original thiopyridinone backbone and select 2-mercaptonicotinoyl glycine (2-MNG) as exhibiting the most favorable balance between the impact on water footprint, skin penetration potential and performance. The effectiveness of 2-MNG was confirmed by topical application on pigmented reconstructed epidermis and human skin explants. In addition, experiments have shown that unlike most melanogenesis inhibitors on the market, this molecule is not a tyrosinase inhibitor. 2-MNG binds to certain melanin precursors, preventing their integration into growing melanin and leading to inhibition of eumelanin and pheomelanin synthesis, without compromising the integrity of melanocytes.

Use of in vitro ADME methods to identify suitable analogs of homosalate and octisalate for use in a read-across safety assessment.

Case studies are needed to demonstrate the use of human-relevant New Approach Methodologies in cosmetics ingredient safety assessments. For read-across assessments, it is crucial to compare the target chemical with the most appropriate analog; therefore, reliable analog selection should consider physicochemical properties, bioavailability, metabolism, as well as the bioactivity of potential analogs. To complement in vitro bioactivity assays, we evaluated the suitability of three potential analogs for the UV filters, homosalate and octisalate, according to their in vitro ADME properties. We describe how technical aspects of conducting assays for these highly lipophilic chemicals were addressed and interpreted. There were several properties that were common to all five chemicals: they all had similar stability in gastrointestinal fluids (in which no hydrolysis to salicylic occurred); were not substrates of the P-glycoprotein efflux transporter; were highly protein bound; and were hydrolyzed to salicylic acid (which was also a major metabolite). The main properties differentiating the chemicals were their permeability in Caco-2 cells, plasma stability, clearance in hepatic models, and the extent of hydrolysis to salicylic acid. Cyclohexyl salicylate, octisalate, and homosalate were identified suitable analogs for each other, whereas butyloctyl salicylate exhibited ADME properties that were markedly different, indicating it is unsuitable. Isoamyl salicylate can be a suitable analog with interpretation for octisalate. In conclusion, in vitro ADME properties of five chemicals were measured and used to pair target and potential analogs. This study demonstrates the importance of robust ADME data for the selection of analogs in a read-across safety assessment.

Reverse Iontophoresis: Noninvasive Assessment of Topical Drug Bioavailability.

Assessing drug disposition in the skin after the application of a topical formulation is difficult. It is hypothesized that reverse iontophoresis (RI), which can extract charged/polar molecules for monitoring purposes, may provide a noninvasive approach for the assessment of local drug bioavailability. The passive and RI extraction of salicylic acid (SA) and nicotine (NIC) from porcine skin in vitro was assessed after a simple solution of the former and a transdermal patch of the latter had been applied for 24 and 8 h, respectively. Immediately after this "passive skin loading", the amount of drug in the stratum corneum (SC) and "viable" tissue (VT) was measured either (a) after tape-stripping and subsequent solvent extraction of both skin layers or (b) following RI extraction over 4 h. Parallel experiments were then performed in vivo in healthy volunteers; in this case, the VT was not sampled and the skin loading period for NIC was only 4 h. RI extraction of both drugs was significantly higher (in vitro and in vivo) than that achieved passively, and the cumulative RI extraction profiles as a function of time were mathematically analyzed using a straightforward compartmental model. Best-fit estimates of drug amounts in the SC and VT (ASC,0 and AVT,0, respectively) at the end of "loading" and two first-order rate constants describing transfer between the model compartments were then determined. The in vitro predictions of ASC,0 and AVT,0 were in excellent agreement with the experimental results, as was the value of the former in vivo. The rate constants derived from the in vitro and in vivo results were also similar. In summary, the results provide proof-of-concept that the RI method has the potential to noninvasively assess relevant metrics of drug bioavailability in the skin.

Off to a good start? Review of the predictivity of reactivity methods modelling the molecular initiating event of skin sensitization.

The assessment of skin sensitizing properties of chemicals has moved away from animal methods to new approach methodologies (NAM), guided by qualitative mechanistic understanding operationalized in an adverse outcome pathway (AOP). As with any AOP, the molecular initiating event (MIE) of covalent binding of a chemical to skin proteins is particularly important. This MIE has been modelled by several test methods by measuring the reaction of a test chemical with model peptides in chemico. To better understand the similarities and differences, a data repository with publicly available data for the direct peptide reactivity assay (DPRA), amino acid derivative reactivity assay (ADRA) and kinetic DPRA (kDPRA), as well as the peroxidase peptide reactivity assay (PPRA) was assembled. The repository comprises 260 chemicals with animal and human reference data, data on four relevant physicochemical properties, and between 161 to 242 test chemical results per test method. First, an overview of the experimental conditions of the four test methods was compiled allowing to readily compare them. Second, data analyses demonstrated that the test methods' predictivity was consistently reduced for poorly watersoluble chemicals and that the DPRA and ADRA can be used interchangeably. It also revealed new categorization thresholds for the DPRA and ADRA that are potentially relevant for strategic uses. In summary, a detailed assessment of reactivity test methods is provided, highlighting their potential and limitations. The results presented are intended to stimulate scientific discussion around test methods modelling the MIE of the skin sensitization AOP.

Extraction of phytochemicals from the pomegranate (Punica granatum L., Punicaceae) by reverse iontophoresis.

Plant metabolic profiling can provide a wealth of information regarding the biochemical status of the organism, but sample acquisition typically requires an invasive and/or destructive extraction process. Reverse iontophoresis (RI) imposes a small electric field across a biological membrane to substantially enhance the transport of charged and polar compounds and has been employed, in particular, to extract biomarkers of interest across human skin. The objective of this work was to examine the capability of RI to sample phytochemicals in a minimally invasive fashion in fructo (i.e., from the intact fruit). RI was principally used to extract a model, bioactive compound - specifically, ellagic acid - from the fruit peel of Punica granatum L. The RI sampling protocol was refined using isolated peel, and a number of experimental factors were examined and optimised, including preparation of the peel samples, the current intensity applied and the pH of the medium into which samples were collected. The most favourable conditions (3 mA current for a period of 1 hour, into a buffer at pH 7.4) were then applied to the successful RI extraction of ellagic acid from intact pomegranates. Multiple additional phytochemicals were also extracted and identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS). A successful proof-of-concept has been achieved, demonstrating the capability to non-destructively extract phytochemicals of interest from intact fruit.

Non-destructive, reverse iontophoretic extraction of phytochemicals from Mangifera indica, Centella asiatica, Punica granatum, and Citrus sinensis.

For the commercial-scale isolation of phytochemicals, a suitable plant biomass source (including species, origin, growing season, etc.) must be identified, and frequent analytical verification is required to ensure that the phytochemicals are present at predefined minimum threshold concentrations. While the latter are typically assessed in the laboratory, a more efficient and less resource-intensive approach would involve non-destructive and environmentally friendly measurements in situ. Reverse iontophoretic (RI) sampling offers a potential solution to this challenge. OBJECTIVE: We aimed to demonstrate the non-destructive, RI sampling of phytochemicals of interest from biomass from four different sources. MATERIALS AND METHODS: RI experiments were performed in side-by-side diffusion cells using a current density of 0.5 mA/cm2 , for a predetermined time in a defined pH environment, using (1) fresh leaves from Mangifera indica and Centella asiatica and (2) isolated peel from Punica granatum and Citrus sinensis. RESULTS: Mangiferin, madecassoside, punicalagin, ellagic acid, and hesperidin were extracted from the different biomasses by RI. The amounts extracted ranged from 0.03 mg/100 mg of biomass for the cathodal extraction of madecassoside to 0.63 mg/100 mg of biomass for the anodal extraction of punicalagin. A linear relationship (r2  = 0.73) between the RI-extracted quantities of punicalagin and those determined using conventional methods was demonstrated. CONCLUSION: The non-destructive, in situ measurement of phytochemical levels by RI represents a feasible approach for timing the harvesting process.

An eco-friendly system for stabilization of retinol: A step towards attending performance with improved environmental respect.

OBJECTIVE: Retinol (Vitamin A) is one of the most effective molecules for the treatment of skin aging. However, it degrades rapidly under the influence of light, oxygen, metal ions, and oxidizing agents. To prevent this, stabilizing systems are used commonly. Notably, butylated hydroxytoluene (2,6-di-tert-butyl-p-cresol) (BHT) and ethylenediaminetetraacetic acid (EDTA) salts exhibit excellent antioxidant and metal-chelating properties but are not eco-friendly. In this study, our goal was to develop a new eco-friendly stabilization system for retinol-based formulations such that the system does not interfere with retinol skin absorption, nor its clinical efficacy. METHODS: An evaluation tool called the Sustainable Product Optimization Tool (SPOT) was used to assess the environmental performance of formulations containing retinol and the various stabilizers investigated. Accelerated stability tests were performed on formulations stored for 2 months at 4 and 45°C (ISO/TR Standard 18811 2018 directives). Long-term stability evaluation was done on formulations stored for 24-months at room temperature. Retinol skin absorption was assessed by the Franz cell method using human skin explants (OECD guideline 428). Finally, a clinical study was performed to evaluate the cosmetic performance of a 0.3% stabilized retinol formulation. RESULTS: N,N'-ethylenediamine disuccinic acid ([S,S]-EDDS isomer) and pentaerythritol tetrakis(3,5-di-tert-butyl-4-hydroxyhydrocinnamate) (PBHC) showed higher biodegradability and a reduced water footprint compared with those of BHT and EDTA. The SPOT simulation gave [S,S]-EDDS + PBHC a score of 10 versus 8.84 for EDTA + BHT. Moreover, [S,S]-EDDS + PBHC better controlled the chemical degradation of retinol compared with EDTA + BHT. Retinol skin absorption was also achieved in the case of a formulation containing [S,S]-EDDS + PBHC, and several skin attributes improved significantly after 12 weeks of product use, with over 75% of the panel perceiving benefits. CONCLUSION: Regarding retinol stabilization, the PBHC + [S,S]-EDDS combination is an eco-friendlier and more effective alternative to BHT + EDTA.

OBJECTIF: Le rétinol est l'une des molécules les plus efficaces sur les signes du vieillissement. Cependant, il se dégrade rapidement, notamment sous l'influence de la lumière, de l'oxygène, des ions métalliques et des oxydants. Il existe des systèmes de stabilisation notamment à base d'hydroxytoluène butylé (2,6-di-tert-butyl-p-crésol) (BHT) et de sels d'acide éthylènediamine tétra acétique (EDTA) qui possèdent respectivement d'excellentes propriétés antioxydantes et chélatrices des métaux bien que certaines études aient montré que les deux présentaient un profil environnemental moins que parfait. Notre objectif était de développer un nouveau système de stabilisation ayant un meilleur respect environnemental. Ce nouveau système plus durable ne doit pas impacter l'absorption cutanée du rétinol, ni son efficacité clinique. MÉTHODES: Un outil d'évaluation appelé SPOT (Sustainable Product Optimization Tool) a été utilisé pour évaluer la performance environnementale des formules contenant du rétinol et des stabilisants. Des tests de stabilité accélérés ont été réalisés 2 mois après la formulation avec des produits gardés à 4°C et 45°C (norme ISO/TR 18811 directives 2018). Les stabilités de longue durée ont été évalués sur des produits gardés 24 mois à la température ambiante. L'absorption cutanée du rétinol a été réalisée par la méthode des cellules de Franz avec des explants de peau humaine (directive OCDE 428). Enfin, une étude clinique a prouvé la performance cosmétique d'une formule de rétinol stabilisé à 0,3%. RÉSULTATS: L'association de l'acide N,N'-éthylènediamine disuccinique (isomère [S,S]-EDDS) et du pentaérythritol tétrakis (3,5-di-tert-butyl-4-hydroxyhydrocinnamate) (PBHC) présente une biodégradabilité plus élevée et une empreinte eau réduite par rapport à BHT et EDTA. La simulation SPOT a donné au [S,S]-EDDS et au PBHC un score de 10 contre 8,84 pour l'EDTA et le BHT. De plus, le [S,S]-EDDS et le PBHC contrôlent mieux la dégradation chimique du rétinol par rapport à l'association BHT plus EDTA. L'absorption cutanée du rétinol a également été obtenue dans une formule contenant du [S,S]-EDDS et du PBHC et plusieurs attributs cutanés ont été significativement améliorés après 12 semaines d'utilisation du produit. Les bénéfices ont été perçus par plus de 75% du panel testé CONCLUSION: Concernant la stabilisation du rétinol, l'association PBHC et [S,S]-EDDS est une alternative plus écologique et plus performante que le BHT et l'EDTA.

Solubility of Foreign Molecules in Stratum Corneum Brick and Mortar Structure.

The barrier function of the skin is mainly assured by its outermost layer, stratum corneum (SC). One key aspect in predicting dermal drug delivery and in safety assessment of skin exposure to chemicals is the need to determine the amount of chemical that is taken up into the SC. We here present a strategy that allows for direct measures of the amount of various solid chemicals that can be dissolved in the SC in any environmental relative humidity (RH). A main advantage of the presented method is that it distinguishes between molecules that are dissolved within the SC and molecules that are not dissolved but might be present at, for example, the skin surface. In addition, the method allows for studies of uptake of hydrophobic chemicals without the need to use organic solvents. The strategy relies on the differences in the molecular properties of the added molecules in the dissolved and the excess states, employing detection methods that act as a dynamic filter to spot only one of the fractions, either the dissolved molecules or the excess solid molecules. By measuring the solubility in SC and delipidized SC at the same RHs, the same method can be used to estimate the distribution of the added chemical between the extracellular lipids and corneocytes at different hydration conditions. The solubility in porcine SC is shown to vary with hydration, which has implications for the molecular uptake and transport across the skin. The findings highlight the importance of assessing the chemical uptake at hydration conditions relevant to the specific applications. The methodology presented in this study can also be generalized to study the solubility and partitioning of chemicals in other heterogeneous materials with complex composition and structure.

Hyaluronic acid skin penetration evaluated by tape stripping using ELISA kit assay.

Hyaluronic Acid (HA) is an endogenous skin matrix component with moisturizing and anti-inflammatory and healing properties. Cosmetic formulations containing HA aim to enhance skin structure, hydrate skin and reduce wrinkles. Therefore, the skin diffusion of HA into stratum corneum after application of a formulation containing two different size of HA, High Molecular weight (HMW-HA) and Low Molecular Weight (LMW-HA)) was evaluated. Ex vivo human skin samples were used to validate an ELISA assay measuring HA in the stratum corneum (SC), viable epidermis and dermis, and to identify optimal washing and extraction methods. These methods were used to measure HA levels in the SC of subjects before and after daily topical application of an HA-containing formulation for 7 days. Samples of SC (5 tape strips) were taken before and 2 h after the application on D0, D1 and D7. The ELISA assay was suitable for measuring HA in the SC but not epidermal or dermal layers. The upper and lower limits of quantitation were the same for both sizes of HA (200 and 3.1 ng/ml, respectively). In both ex vivo human skin and human volunteers, the "dry method" of removing the formulation led to much higher levels of HA in the SC samples, whereas the "wet method" involving one cotton swab soaked with an aqueous solution containing 10% soap and a second cotton swab for drying, was effective in removing the formulation and more relevant to simulate washing/showering. In the clinical study, the amount of HA in SC layers 3-5 were used to represent the HA level in the SC, whereas layers 1 and 2 were considered as surface "residual film". After each application, there was a significantly higher amount of HA compared to the amount before application, which was observed using both wash methods. The residual level 24 h after the first application was at least 8 times higher than before the first application and at least 31 times higher after 7 applications. In conclusion, these investigations validated the use of the ELISA method for the measurement of HA in SC samples. The ex vivo experiments provided recommendations for the clinical study, including the preferred cleansing and optimal sampling methods. The clinical study demonstrated the diffusion, accumulation and maintenance of HA levels in the SC after repeated application of the formulation containing HMW-HA and LMW-HA.

Membrane permeability based on mesh analysis.

The main function of a membrane is to control the exchange of matter between the surrounding regions. As such, accurate modeling of membranes is important to properly describe their properties. In many cases in both biological systems and technical applications, the membranes are composite structures where transport properties may vary between the different sub-regions of the membrane. In this work we develop a method based on Mesh analysis that is asymptotically exact and can describe diffusion in composite membrane structures. We do this by first reformulating a generalized Fick's law to include the effects from activity coefficient, diffusion coefficient, and solubility using a single condensed parameter. We then use the derived theory and Mesh analysis to, in essence, retrieve a finite element method approach. The calculated examples are based on a membrane structure that reassembles that of the brick and mortar structure of stratum corneum, the upper layer of our skin. Resulting concentration profiles from this procedure are then compared to experimental results for the distribution of different probes within intact stratum corneum, showing good agreement. Based on the derived approach we further investigate the impact from a gradient in the fluidity of the stratum corneum mortar lipids across the membrane, and find that it is substantial. We also show that anisotropic organisation of the lipid mortar can have large impact on the effective permeability compared to isotropic mortar lipids. Finally, we examine the effects of corneocyte swelling, and their lateral arrangement in the membrane on the overall membrane permeability.

Practical application of the interim internal threshold of toxicological concern (iTTC): a case study based on clinical data.

We present a case study that provides a practical step-by-step example of how the internal Threshold of Toxicological Concern (iTTC) can be used as a tool to refine a TTC-based assessment for dermal exposures to consumer products. The case study uses a theoretical scenario where there are no systemic toxicity data for the case study chemicals (avobenzone, oxybenzone, octocrylene, homosalate, octisalate, octinoxate, and ecamsule). Human dermal pharmacokinetic data following single and repeat dermal exposure to products containing the case study chemicals were obtained from data published by the US FDA. The clinical studies utilized an application procedure that followed maximal use conditions (product applied as 2 mg/cm2 to 75% of the body surface area, 4 times a day). The case study chemicals were first reviewed to determine if they were in the applicability domain of the iTTC, and then, the human plasma concentrations were compared to an iTTC limit of 1 µM. When assessed under maximum usage, the external exposure of all chemicals exceeded the external dose TTC limits. By contrast, the internal exposure to all chemicals, except oxybenzone, was an order of magnitude lower than the 1 µM interim iTTC threshold. This work highlights the importance of understanding internal exposure relative to external dose and how the iTTC can be a valuable tool for assessing low-level internal exposures; additionally, the work demonstrates how to use an iTTC, and highlights considerations and refinement opportunities for the approach.

3D Molecular Imaging of Stratum Corneum by Mass Spectrometry Suggests Distinct Distribution of Cholesteryl Esters Compared to Other Skin Lipids.

The crucial barrier properties of the stratum corneum (SC) depend critically on the design and integrity of its layered molecular structure. However, analysis methods capable of spatially resolved molecular characterization of the SC are scarce and fraught with severe limitations, e.g., regarding molecular specificity or spatial resolution. Here, we used 3D time-of-flight secondary ion mass spectrometry to characterize the spatial distribution of skin lipids in corneocyte multilayer squams obtained by tape stripping. Depth profiles of specific skin lipids display an oscillatory behavior that is consistent with successive monitoring of individual lipid and corneocyte layers of the SC structure. Whereas the most common skin lipids, i.e., ceramides, C24:0 and C26:0 fatty acids and cholesteryl sulfate, are similarly organized, a distinct 3D distribution was observed for cholesteryl oleate, suggesting a different localization of cholesteryl esters compared to the lipid matrix separating the corneocyte layers. The possibility to monitor the composition and spatial distribution of endogenous lipids as well as active drug and cosmetic substances in individual lipid and corneocyte layers has the potential to provide important contributions to the basic understanding of barrier function and penetration in the SC.

In-Vivo Tape Stripping Study with Caffeine for Comparisons on Body Sites, Age and Washing.

PURPOSE: Assessing the percutaneous absorption of cosmetic ingredients using in-vitro human skin reveals certain limitations, such as restricted anatomical sites and repeated exposure, and to overcome these issues, in-vivo studies are required. The aim of the study is to develop a robust non-invasive in-vivo protocol that should be applicable to a wide range of application. METHODS: A robust tape stripping protocol was therefore designed according to recent recommendations, and the impact of two different washing procedures on caffeine distribution in tape strips was investigated to optimise the protocol. The optimised protocol was then used to study the effect of age and anatomical area on the percutaneous absorption of caffeine, including facial areas which are not readily available for in-vitro studies. RESULTS: With tape stripping, a difference between the percutaneous absorption on the face (forehead, cheek) and the volar forearm was observed. No obvious difference was observed between percutaneous absorption in young and post-menopausal women, but this could be due to the limited number of subjects. CONCLUSION: This tape stripping protocol is now to be deployed to address many other factors, such as percutaneous absorption in other anatomical areas (e.g. abdomen, axilla, etc.), impact of repeated applications and effect of formulation.

Use of an In Vitro Skin Parallel Artificial Membrane Assay (Skin-PAMPA) as a Screening Tool to Compare Transdermal Permeability of Model Compound 4-Phenylethyl-Resorcinol Dissolved in Different Solvents.

Absorption through the skin of topically applied chemicals is relevant for both formulation development and safety assessment, especially in the early stages of development. However, the supply of human skin is limited, and the traditional in vitro methods are of low throughput. As an alternative, an artificial membrane-based Skin Parallel Artificial Membrane Permeability Assay (Skin-PAMPA) has been developed to mimic the permeability through the stratum corneum. In this study, this assay was used to measure the permeability of a model compound, 4-phenylethyl-resorcinol (PER), dissolved in 13 different solvents that are commonly used in cosmetic formulation development. The study was performed at concentrations close to the saturated solution of PER in each solvent to investigate the maximum thermodynamic potential of the solvents. The permeability of PER in selected solvents was also measured on ex vivo pig skin for comparison. Pig ear skin is an accepted alternative model of human skin. The permeability coefficient, which is independent of the concentration of the applied solution, showed a good correlation (R2 = 0.844) between the Skin-PAMPA and the pig skin permeation data. Our results support the use of the Skin-PAMPA to screen the suitability of different solvents for non-polar compounds at an early stage of formulation development.

Elongated microparticles tuned for targeting hyaluronic acid delivery to specific skin strata.

OBJECTIVE: Microneedle or fractional laser applications are the most common topical delivery enhancement platforms. However, these methods of drug delivery are not skin strata specific. Drug delivery approaches which could target specific stratum of the skin remains a challenge. Elongated microparticles (EMPs) have been used in enhancing drug delivery into the skin. The aim of this study was to evaluate, for the first time, elongated silica microparticles with two different length profiles to enhance delivery of hyaluronic acid into different strata of human skin. METHODS: Two types of EMPs-long (milled EMPs) or short (etched EMPs) length ranges were characterized. A prototypical liquid formulation (Fluorescent hyaluronic acid) with and without EMP enhancement were evaluated for hyaluronic acid delivery in ex-vivo human skin. High performance liquid chromatography, Typhoon fluorescence scanning system, laser scanning confocal microscopy and reflectance confocal microscopy (RCM) were used to validate F-HA stability, visualize fluorescein in the skin, image the depth of F-HA delivery in the skin and define EMP penetration in skin strata, respectively. Statistical analysis was conducted using GraphPad Prism 6 software (GraphPad Software Inc, USA). RESULTS: Fluorescein-hyaluronic acid was stable and EMP enhanced skin penetration. RCM revealed that 'etched EMP' penetrated the skin to the stratum spinosum level. The vast majority (97.8%; p < 0.001) of the etched EMP did not penetrate completely through the viable epidermis and no obvious penetration into the dermis. In contrast, milled EMP showed 41-fold increase in penetration compared to the etched EMP but penetrated beyond the dermoepidermal junction. CONCLUSION: EMPs can enhance delivery of hyaluronic acid. Using EMPs with defined length distributions, which can be tuned for a specific stratum of the skin, can achieve targeted hyaluronic acid delivery.

OBJECTIF: Les microaiguilles ou le laser fractionné sont couramment utilisés pour augmenter l'absorption d'actif appliqué par voie topique. Toutefois, ces approches ne permettent de cibler une strate spécifique de la peau. Ainsi les méthodes permettant de cibler spécifiquement l'épiderme reste un défi. Les microparticules allongées (EMP) ont déjà été utilisé pour augmenter l'absorption cutanée. L'objectif de l'étude est d'évaluer pour la première fois, la capacité de microparticules allongées de silice (de deux longueurs différentes) à délivrer l'acide hyaluronique dans les différentes couches de la peau. MÉTHODES: Deux types d'EMP, longues (EMP broyé) ou courtes (EMP gravé), ont été caractérisées. Une formulation liquide contenant de l'acide hyaluronique marquée avec une sonde fluorescente (F-HA) a été évaluée avec et sans EMP sur peau humaine ex vivo. La chromatographie liquide haute performance, le scanner à fluorescence Typhoon, la microscopie laser confocal à balayage et la microscopie confocale par réflectance (RCM) ont été utilisées respectivement pour contrôler la stabilité de la F-HA, visualiser le signal de la fluorescéine dans la peau, imager l'absorption du F-HA dans la peau en fonction de la profondeur et caractériser la pénétration des EMP. Les analyses statistiques ont été réalisées avec le logiciel GraphPad Prims 6 (GraphPad Software Inc, USA). RÉSULTATS: L'acide hyaluronique marquée avec la fluorescéine est stable et les EMP permettent d'augmenter son absorption cutanée. Le RCM a montré que les EMP gravées pénètrent dans la peau jusqu'au niveau du stratum spinosum. La grande majorité des EMP gravés (97.8% ; p < 0,001) ne pénètre pas complétement dans l'épiderme viable et aucune pénétration mesurable dans le derme. Au contraire, les EMP broyées ont montrées une pénétration 41 fois plus importantes que les EMP gravées et peuvent aller au-delà de la jonction derme-épiderme. CONCLUSION: Les EMP peuvent augmenter l'absorption cutanée de l'acide hyaluronique. En utilisant des EMP de longueur définie et en ajustant celle-ci, il est même possible de cibler spécifiquement une strate cutanée.

Skin hydration as a tool to control the distribution and molecular effects of intermediate polarity compounds in intact stratum corneum.

The barrier function of the skin is mainly assured by its outermost layer, stratum corneum (SC), which consists of dead keratin-filled cells embedded in a lipid matrix. The skin is daily exposed to an environment with changing conditions in terms of hydration and different chemicals. Here we investigate how a molecule that has reasonable solubility in both hydrophobic and hydrophilic environments can be directed to certain regions in SC by changing the skin hydration. We use 1,2,3-trimethoxy propane (TMP) as a model substance and solid-state NMR on natural abundance 13C to obtain atomically resolved information on the molecular dynamics of TMP as well as SC lipid and protein components at varying hydration conditions. Upon dehydration, TMP redistributes from the hydrophilic corneocytes to the hydrophobic SC lipid regions. In this way, TMP can act to prevent the fluid-solid lipid transition in drying conditions and be present in the corneocytes in more humid conditions. Hydration can thereby be used as a switch to control the location and action of TMP or similar compounds in complex materials like SC. The general principles described here can also have impact on other applications including lipid-based formulations in food, drug delivery and cosmetics.

Effective exposure of chemicals in in vitro cell systems: A review of chemical distribution models.

Nominal effect concentrations from in vitro toxicity assays may lead to inaccurate estimations of in vivo toxic doses because the nominal concentration poorly reflects the concentration at the molecular target in cells in vitro, which is responsible for initiating effects and can be referred to as the biologically effective dose. Chemicals can differentially distribute between in vitro assay compartments, including serum constituents in exposure medium, microtitre plate plastic, headspace and extracellular matrices. The partitioning of test chemicals to these extracellular compartments reduces the concentration at the molecular target. Free concentrations in medium and cell-associated concentrations are considered better proxies of the biologically effective dose. This paper reviews the mechanisms by which test chemicals distribute between in vitro assay compartments, and also lists the physicochemical properties driving the extent of this distribution. The mechanisms and physicochemical properties driving the distribution of test chemical in vitro help explain the makeup of mass balance models that estimate free concentrations and cell-associated concentrations in in vitro toxicity assays. A thorough understanding of the distribution processes and assumptions underlying these mass balance models helps define chemical and biological applicability domains of individual models, as well as provide a perspective on how to improve model predictivity and quantitative in vitro-in vivo extrapolations.