Topical niacinamide enhances hydrophobicity and resilience of corneocyte envelopes on different facial locations.

Age-related differences in maturation parameters of corneocyte envelopes (size, hydrophobicity and rigidity) were examined at several facial test sites in young and old female Caucasians. In addition, the effect of topically applied niacinamide on these parameters was evaluated in a 4-week placebo-controlled study.

3D-Printed Franz cells - update on optimization of manufacture and evaluation.

OBJECTIVES: Laboratory in vitro permeation processes require the use of modified Franz type diffusion cells which are conventionally fabricated from glass. Fragility and high cost are frequently associated with this type of laboratory apparatus. The purpose of our present research was to develop a simple, economical and versatile approach to manufacture Franz type cells using additive manufacturing (AM). METHODS: Graphical Franz diffusion cell designs were reproduced with a stereolithography (SLA) 3D printer and assessed over a minimum period of 24 h. The surface morphology of AM printouts was analysed before and after compatibility studies using scanning electron microscopy (SEM). Comparative permeation studies in both glass and AM Franz type diffusion cells were conducted using a caffeine solution (1.5 mg mL-1 ), applied to a model silicone membrane. RESULTS: Testing of the 3D printed scaffolds confirmed similar recovery of the permeant when compared to glass cells: 1.49 ± 0.01 and 1.50 ± 0.01 mg mL-1 , respectively, after 72 h. No significant differences were visible from the SEM micrographs demonstrating consistent, smooth and non-porous surfaces of the AM Franz cells' core structure. Permeation studies using transparent 3D printed constructs resulted in 12.85 ± 0.53 µg cm-2 caffeine recovery in the receptor solution after 180 min with comparable permeant recovery, 11.49 ± 1.04 µg cm-2 , for the glass homologues. CONCLUSION: AM constructs can be considered as viable alternatives to the use of conventional glass apparatus offering a simple, reproducible and cost-effective method of replicating specialised laboratory glassware. A wider range of permeants will be investigated in future studies with these novel 3D printed Franz diffusion cells.

OBJECTIF: les processus de perméation in vitro en laboratoire nécessitent l'utilisation de cellules de diffusion de type Franz modifiées, fabriquées traditionnellement en verre. La fragilité et un coût élevé sont fréquemment associés à ce type d'appareil de laboratoire. L'objectif de nos travaux de recherche actuels était de développer une approche simple, économique et polyvalente pour fabriquer des cellules de type Franz à l'aide de la fabrication additive (FA). MÉTHODES: les conceptions des cellules de diffusion Franz graphiques ont été reproduites avec une imprimante 3D stéréolithographie (SLA) et évaluées sur une période minimum de 24 h. La morphologie de surface des impressions FA a été analysée avant et après des études de compatibilité à l'aide de la microscopie électronique à balayage (MEB). Des études comparatives de perméation des cellules de diffusion de type Franz en verre et FA ont été réalisées à l'aide d'une solution de caféine (1,5 mg ml-1 ) appliquée à un modèle de membrane en silicone. RÉSULTATS: les tests des supports imprimés 3D ont confirmé une récupération similaire du perméant par rapport aux cellules de verre : 1,49 ± 0,01 et 1,50 ± 0,01 mg ml-1 , respectivement, après 72 h. Aucune différence significative n'a été observée sur les micrographiques MEB, montrant des surfaces cohérentes, lisses et non poreuses de la structure centrale des cellules Franz FA. Les études de perméation utilisant des constructions transparentes imprimées en 3D ont conduit à une récupération de la caféine de 12,85 ± 0,53 µg cm-2 dans la solution de récepteur après 180 min avec une récupération de perméant comparable, 11,49 ± 1,04 µg cm-2 , pour les homologues de verre. CONCLUSION: les constructions FA peuvent être considérées comme des alternatives viables à l'utilisation d'appareils de verre conventionnels offrant une méthode simple, reproductible et rentable de réplication de la verrerie de laboratoire spécialisée. Une gamme plus large de perméants sera étudiée dans de futures études avec ces nouvelles cellules de diffusion Franz imprimées en 3D.

The importance of 12R-lipoxygenase and transglutaminase activities in the hydration-dependent ex vivo maturation of corneocyte envelopes.

BACKGROUND: Terminally differentiated keratinocytes acquire corneocyte protein envelopes (CPE) complexed with corneocyte lipid envelopes (CLE). These two structural components of the corneocyte envelopes (CEs) undergo maturation by gaining in hydrophobicity, rigidity and surface area. Linoleoyl acylceramides are processed by 12R-lipoxygenase (12R-LOX) and other enzymes before transglutaminase (TG) attaches ω-hydroxyceramides to involucrin in the CPE. Concurrently, structural proteins are cross-linked by TG that has been activated by cathepsin D (CathD). OBJECTIVES: The primary aim of this work was to demonstrate the impact of relative humidity (RH) during ex vivo CE maturation. Low, optimal and high RH were selected to investigate the effect of protease inhibitors (PIs) on CE maturation and TG activity; in addition, 12R-LOX and CathD activity were measured at optimal RH. Finally, the effect of glycerol on ex vivo CE maturation was tested at low, optimal and high RH. METHODS: The first and ninth tape strip of photo-exposed (PE) cheek and photo-protected (PP) post-auricular sites of healthy volunteers were selected. Ex vivo CE maturation was assessed via the relative CE maturity (RCEM) approach based on CE rigidity and hydrophobicity. The second and eighth tapes were exposed to RH in the presence of inhibitors. RESULTS: Irrespective of tape stripping depth, CEs from PE samples attained CE rigidity to the same extent as mature CEs from the PP site, but such improvement was lacking for CE hydrophobicity. 70% RH was optimal for ex vivo CE maturation. The inhibition of 12R-LOX activity resulted in enhanced CE rigidity which was reduced by the TG inhibitor. CE hydrophobicity remained unchanged during ex vivo maturation in the presence of TG or 12R-LOX inhibition. CE hydrophobicity was enhanced in the presence of glycerol at 44% RH and 100% RH but not at 70% RH. Furthermore, TG activity was significantly diminished at 100% RH compared to the commercial inhibitor LDN-27219. However, a protease inhibitor mix reversed the negative effect of overhydration. CONCLUSION: The study adds to the understanding of the roles of 12R-LOX and TG activity in CE maturation and gives further insight into the effect of glycerol on the SC.

CONTEXTE: Les kératinocytes à différenciation terminale acquièrent les enveloppes protéiniques des cornéocytes (ECP) complexées aux enveloppes lipidiques des cornéocytes (ELC). Ces deux composants structurels des enveloppes cornéocytaires (EC) subissent un processus de maturation en gagnant en hydrophobicité, en rigidité et en surface. Les linoléoyl-acyle-céramides sont traités par 12R-lipoxygénase (12R-LOX) et d'autres enzymes avant que la transglutaminase (TG) ne fixe les x-hydroxy-céramides à l'involucrine dans les ECP. Les protéines structurelles sont simultanément réticulées par la TG qui a été activée par la cathepsine D (CathD). OBJECTIFS: L'objectif principal de ces travaux visait à démontrer l'impact de l'humidité relative (HR) pendant la maturation de l'EC ex vivo. Des humidités relatives faible, optimale et élevée ont été retenues pour étudier l'effet des inhibiteurs de la protéase (IP) sur la maturation de l'EC et l'activité de la TG ; l'activité de CathD et 12R-LOX a également été mesurée à une HR optimale. Finalement, l'effet du glycérol sur la maturation de l'EC ex vivo a été testé à des humidités relatives faible, optimale et élevée. MÉTHODES: La première et neuvième bandes adhésives sur un site à l'arrière de l'oreille protégé de la lumière (photo-protégé, PP) et sur une joue exposée à la lumière (photo-exposée, PE) de volontaires sains ont été sélectionnées. La maturation de l'EC ex vivo a été évaluée par l'approche de la maturité relative d'EC (RCEM) reposant sur l'hydrophobicité et la rigidité de l'EC. Les deuxième et huitième bandes ont été exposées à l'humidité relative en présence d'inhibiteurs. RÉSULTATS: Indépendamment de la profondeur de bande adhésive, les EC des échantillons EP ont atteint la rigidité d'EC de la même manière que les EC matures du site PP, mais ces améliorations faisaient défaut en ce qui concerne l'hydrophobicité des EC. Une HR à 70 % était optimale pour la maturation de l'EC ex vivo. L'inhibition de l'activité du 12R-LOX a entraîné une rigidité accrue de l'EC, laquelle était réduite par l'inhibiteur de la TG. L'hydrophobicité des EC est restée inchangée pendant la maturation ex vivo en présence de l'inhibition de la TG ou du 12R-LOX. L'hydrophobicité des EC a été améliorée en présence de glycérol à une HR de 44 % et à une HR de 100 %, mais non à une HR de 70 %. L'activité de la TG a par ailleurs significativement diminué à une HR de 100 % par rapport à l'inhibiteur commercial LDN-27219. Cependant, un mélange inhibiteur de la protéase a inversé l'effet négatif de la surhydratation. CONCLUSION: L'étude renforce la compréhension des rôles de l'activité de la TG et du 12R-LOX dans la maturation de l'EC et donne de plus amples détails sur l'effet du glycérol sur la couche cornée (stratum corneum, SC).

A proof-of-principle study comparing barrier function and cell morphology in face and body skin.

OBJECTIVE: The purpose of this pilot in vivo study was to investigate corneocyte size and transepidermal water loss (TEWL) in facial cheek and volar forearm skin as a function of consecutive tape stripping. Changes in corneocyte size and transepidermal water loss (TEWL) were measured as a function of stratum corneum (SC) depth at both anatomical sites. To our knowledge, this is the first published quantitative comparison based on these parameters. This work complements our previously published studies on face skin barrier recovery at 24 h and 4 weeks post-tape stripping [Gorcea et al., Skin Res. Technol., 19, 2013, e375-e382; Gorcea et al., Int. J. Cosmet. Sci. 35, 2013, 250]. METHODS: Transepidermal water loss in vivo measurements of forearm and facial skin sites were taken before tape stripping commenced (baseline) and after each tape was collected. Optical microscopy and image analysis techniques were employed to characterize corneocyte size as a function of skin depth (tape strip number) for both anatomical sites. RESULTS: Transepidermal water loss increased significantly from baseline with sequential tape stripping at both anatomical skin sites. Volar forearm skin required approximately three times as many tapes to 'damage' the SC barrier (arbitrarily defined as twice baseline TEWL) compared to facial cheek skin demonstrating significant differences in barrier properties between cheeks and forearms (P < 0.05). Corneocyte size decreased significantly with depth for both sites (P < 0.001). Corneocytes from face skin were significantly smaller than corneocytes from volar forearm skin. CONCLUSION: Statistically significant differences between facial and body skin stratum corneum cell morphology and transepidermal water loss were demonstrated and quantitatively measured as a function of tape stripping.

OBJECTIF: L'objectif de cette étude pilote in vivo était d'étudier la taille des cornéocytes et la perte d'eau transépidermique (TEWL) dans la peau des joues et des avant-bras comme en fonction de l'épilation par bandelettes. Les modifications de la taille des cornéocytes et de la perte d'eau transépidermique (TEWL) ont été mesurées en fonction de la profondeur du stratum corneum (SC) au niveau des deux sites anatomiques. À notre connaissance, il s'agit de la première comparaison quantitative publiée sur la base de ces paramètres. Ce travail complète nos études publiées antérieurement sur la récupération de la barrière cutanée du visage 24 h et 4 semaines après l'application des bandelettes. [Gorcea et al., Skin Res. Technol., 19, 2013, e375-e382; Gorcea et al., Int. J. Cosmet. Sci. 35, 2013, 250]. MÉTHODES: Les mesures de la perte d'eau transépidermique in vivo au niveau des sites cutanés des avant-bras et du visage ont été effectuées avant le début du retrait des bandelettes (référence) et après le prélèvement de chaque bandelette. Des techniques de microscopie optique et d'analyse d'images ont été utilisées pour caractériser la taille des cornéocytes en fonction de la profondeur de la peau (numéro de bandelette) pour les deux sites anatomiques. RÉSULTATS: La perte d'eau transépidermique a augmenté de façon significative par rapport à la référence lors du retrait séquentiel des bandelettes au niveau des deux sites anatomiques de la peau. La peau de l'avant-bras a nécessité environ trois fois plus de bandelettes pour « endommager ¼ la barrière du SC (définie arbitrairement comme deux fois la TEWL de base) que la peau des joues du visage, ce qui démontre des différences significatives dans les propriétés barrières entre les joues et les avant-bras (P < 0,05). La taille des cornéocytes a diminué de façon significative avec la profondeur pour les deux sites (P < 0,001). Les cornéocytes de la peau du visage étaient significativement plus petits que les cornéocytes de la peau de l'avant-bras. CONCLUSION: Des différences statistiquement significatives de morphologie et de perte d'eau transépidermique entre les cellules du stratum corneum de la peau du visage et celles du corps ont été démontrées et mesurées quantitativement en fonction du retrait des bandelettes.

Characterization and topical delivery of phenylethyl resorcinol.

OBJECTIVE: Phenylethyl resorcinol (PR) has been used widely in the personal care industry as a novel skin lightening ingredient. Surprisingly, there is only limited information describing the physicochemical properties of this active. Therefore, the primary objective of this study was to perform a comprehensive characterization of PR. A secondary objective was to investigate the delivery of this molecule to mammalian skin. METHODS: Phenylethyl resorcinol was characterized using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and nuclear magnetic resonance (NMR). A new high-performance liquid chromatographic (HPLC) method for analysis of PR was developed and validated. The log P (octanol water partition coefficient), value, solubility and short-term stability of PR in a series of vehicles were also determined using HPLC. The evaporation of the selected vehicles was examined using dynamic vapour sorption (DVS). The permeation profiles of PR were investigated under finite dose conditions in porcine and human skin. RESULTS: The melting point of PR was determined to be 79.13 °C and the measured log P (octanol water partition coefficient) at 21 °C was 3.35 ± 0.03. The linearity of the HPLC analytical method was confirmed with an r2 value of 0.99. Accuracy of the method was evaluated by average recovery rates at three tested concentrations, and the values ranged from 99 to 106%. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.57 µg mL-1 , respectively. The solubility of PR in PG, DMI, glycerol was within the range of 367 to 877 mg mL-1 . The stability of PR in tested solvents was also confirmed by the 72 h stability studies. From the DVS studies, 70-125% of applied formulations were recovered at 24 h. The permeation through porcine skin at 24 h ranged from 4 to 13 µg cm-2 , while the corresponding amounts of PR delivered through human skin were 2 to 10 µg cm-2 . CONCLUSION: The physicochemical properties of PR confirm it is suitable for dermal delivery. In this study, propylene glycol was the most promising vehicle for PR delivery to human skin. Future work will expand the range of vehicles studied and explore the percutaneous absorption from more complex formulations.

OBJECTIF: Le phényléthyl résorcinol (PR) est largement utilisé dans le secteur des soins personnels comme ingrédient éclaircissant pour la peau. Pour autant, on ne dispose que d'informations limitées concernant les propriétés physicochimiques de ce principe actif. C'est pourquoi cette étude avait pour objectif principal de réaliser une caractérisation exhaustive du PR. Son objectif secondaire était d'étudier l'administration de cette molécule à la peau de mammifères. MÉTHODES: Le phényléthyl résorcinol a été caractérisé par calorimétrie différentielle à balayage (CDB), analyse thermogravimétrique (ATG) et par résonance magnétique nucléaire (RMN). Pour analyser le PR, une nouvelle méthode de chromatographie liquide à haute performance (CLHP) a été développée et validée. On s'est servi de la CLHP pour déterminer les propriétés suivantes du PR : log P (coefficient de partage octanol/eau), valeur, solubilité et stabilité à court terme du PR dans plusieurs véhicules. L'évaporation des véhicules sélectionnés a été examinée par sorption de vapeur dynamique (DVS). Les profils de perméabilité du PR ont été étudiés dans des conditions de dose finie dans des peaux porcine et humaine. RÉSULTATS: On a pu déterminer que le point de fusion du PR était de 79,13 °C et le log P (coefficient de partage octanol/eau) à 21 °C était de 3,35 ± 0.03. La linéarité de la méthode analytique de la CLHP a été confirmée avec une valeur r2 de 0,99. L'exactitude de la méthode a été évaluée par les taux moyens de récupération à trois concentrations testées, avec des valeurs résultantes comprises entre 99 et 106 %. La limite de détection (LD) et la limite de quantification (LQ) ont été déterminées à 0,19 et 0,57 µg/ml_ 1, respectivement. La solubilité du PR dans le PG, le DMI et le glycérol reste dans une plage comprise entre 367 et 877 mg/ml _ 1. La stabilité du PR dans les solvants testés a également pu être confirmée par les études de stabilité à 72 h. Parmi les formulations appliquées lors des études de DVS, 70 à 125 % de celles-ci ont été récupérées à 24 h. La pénétration par la peau porcine à 24 h était comprise entre 4 et 13 µg/cm_ 2, tandis que les quantités de PR correspondantes délivrées à travers la peau humaine étaient de 2 à 10 µg/cm_ 2. CONCLUSION: Les propriétés physicochimiques du PR confirment qu'il est adapté à l'administration dermique. Dans le cadre de cette étude, le propylène glycol est apparu comme le véhicule le plus prometteur pour l'administration de PR dans la peau humaine. De futurs travaux étudieront davantage de véhicules et examineront l'absorption percutanée lors de l'emploi de formulations plus complexes.

12R-lipoxygenase activity is reduced in photodamaged facial stratum corneum. A novel activity assay indicates a key function in corneocyte maturation.

BACKGROUND: During the late stage of keratinocyte differentiation, corneocytes gain a strong protein-lipid structure: the corneocyte envelopes (CE), composed of the inner corneocyte protein envelope (CPE) and the outer corneocyte lipid envelope (CLE). The hydrophobicity of CEs depends on the covalent attachment of linoleoyl-acyl-ceramides by transglutaminases (TG). These ceramides are processed by a range of other enzymes, including 12R-lipoxygenase (12R-LOX), before the covalent attachment of the free ω-hydroxyceramides to the CPE surface to form the CLE. The mechanical strength of CE is obtained with the formation of isodipeptide bonds by TG. The increase in hydrophobicity and rigidity leads to CE maturation which supports the integrity and mechanical resistance of the stratum corneum (SC). OBJECTIVES: The aim of this work was to develop and validate a novel enzyme activity assay for 12R-LOX in tape strippings of photo-exposed (PE) cheek and photo-protected (PP) post-auricular SC of healthy Chinese volunteers (n = 12; age 25 ± 3 years). RESULTS: A fluorescence-based assay was developed with ethyl linoleic acid as the substrate and a polyclonal antibody against 12R-LOX as an inhibitor. The specificity was shown by the lack of effect by a LOX inhibitor (ML351) and an epidermal-type lipoxygenase 3 (eLOX3) antibody on the acquired 12R-LOX activity. Reduced 12R-LOX activity was observed in the outer compared to the inner SC layers. Moreover, dramatically lower activity was shown in the PE vs. PP samples. Furthermore, the enzyme activity has a positive correlation (r = 0.94 ± 0.03) with CE maturity, in particular hydrophobicity, and a negative correlation (r = -0.96 ± 0.01) with transepidermal water loss (TEWL). CONCLUSION: This novel enzyme assay revealed a lower 12R-LOX activity in tape strippings from PE cheek for the first time. This finding is in line with less mature CEs and higher TEWL compared to PP post-auricular samples. This study indicates a strong link between 12R-LOX activity and CE maturation and SC integrity.

CONTEXTE: Pendant le stade avancé de différenciation des kératinocytes, les cornéocytes acquièrent une solide structure protéines-lipides : l'enveloppe des cornéocytes (EC) composée de l'enveloppe protéinique des cornéocytes (EPC) interne et de l'enveloppe lipidique des cornéocytes (ELC) externe. L'hydrophobicité des EC dépend de la liaison covalente des linoléoyl-acyle-céramides par transglutaminases (TG). Ces céramides sont traités par un ensemble d'autres enzymes, y compris la 12R-lipoxygénase (12R-LOX), avant la liaison covalente des xhydroxycéramides à la surface de l'EPC pour former l'ELC. La force mécanique de l'EC est obtenue par la formation de liaisons isodipeptides par TG. L'augmentation de hydrophobicité et de la rigidité conduit à une maturation de l'EC qui soutient l'intégrité et la résistance mécanique de la couche cornée (stratum corneum, SC). OBJECTIFS: L'objectif de ce travail était de développer et de valider un test novateur de l'activité enzymatique pour le 12R-LOX par arrachage par bande sur une joue exposée à la lumière (photo-exposed, PE) et sur de la SC à l'arrière de l'oreille protégée de la lumière (photo-protected, PP) de volontaires sains chinois (n = 12; 25 ans ± 3 ans). RÉSULTATS: Un test de fluorescence a été développé avec de l'acide linoléique éthylique en tant que substrat et un anticorps polyclonal anti-12R-LOX en tant qu'inhibiteur. La spécificité a été démontrée par le manque d'effet d'un inhibiteur de LOX (ML351) et d'un anticorps anti-lipoxygénase 3 (eLOX3) de type épidermique sur l'activité du 12R-LOX acquise. Une réduction de l'activité du 12R-LOX a été observée dans les couches de SC externes par rapport aux couches internes. En outre, une activité considérablement plus faible a été démontrée dans les échantillons PE par rapport aux échantillons PP. De plus, l'activité enzymatique a une corrélation positive (r = 0,94 ± 0,03) avec la maturité de l'EC, en particulier l'hydrophobicité, et une corrélation négative (r = −0,96 ± 0,01) avec une perte d'eau transépidermique (transepidermal water los, TEWL). CONCLUSION: Ce dosage enzymatique novateur, à partir de tape stripping sur les joues exposée à la lumière, a révélé une activité 12RLOX plus faible pour la première fois. Cette découverte est cohérente avec des EC moins matures et une TEWL plus élevée par rapport aux échantillons PP de l'arrière de l'oreille. Cette étude indique un lien solide entre l'activité du 12R-LOX et la maturation de l'EC et l'intégrité de la SC.

3D-printed Franz type diffusion cells.

OBJECTIVE: Franz cells are routinely used to measure in vitro skin permeation of actives and must be inert to the permeant under study. The aim of the present work was to develop and manufacture transparent Franz-type diffusion cells using 3D printing. Printouts were then tested using a range of model active compounds. The study also aims to identify the critical 3D-printing parameters necessary for the process, including object design, choice of printing resin, printout curing and post-curing settings and introduction of model coatings. METHODS: Transparent Franz cells were constructed using an online computer aided design program and reproduced with different stereolithography 3D printers. The two acrylate-based resins used for the fabrication process were a commercially available product and a polymer synthesised in-house. Comparative studies between glass and 3D-printed Franz cells were conducted with selected model actives: terbinafine hydrochloride (TBF), niacinamide (NIA), diclofenac free acid (DFA) and n-methyl paraben (MPB). In preliminary studies, MPB showed the lowest recovery when exposed to the receptor compartment of 3D printed cells. Consequently, in vitro permeation studies were carried out using only MPB with polydimethylsiloxane (PDMS) membrane. RESULTS: A decrease in the amounts of selected compounds was observed for transparent 3D-printed Franz cells compared to glass cells. MPB showed the lowest recovery (53.8 ± 13.1%) when compared with NIA (74.9 ± 4.0%), TBF (81.5 ± 12.0%) and DFA (90.2 ± 12.9%) after 72 h. Permeation studies conducted using 3D-printed transparent cells with PDMS membrane also showed a decrease in MPB recovery of 51.4 ± 3.7% for the commercial resin and 94.4 ± 3.5% for the polymer synthesised in-house, when compared to glass cells. Although hydrophobic coatings were subsequently applied to the 3D-printed cells, the same reduction in MPB concentration was observed in the receptor solution. CONCLUSION: Transparent Franz cells were successfully prepared using 3D printing and were observed to be robust and leak-proof. There are few resins currently available for preparation of transparent materials and incompatibilities between the actives investigated and the 3D-printed cells were evident. Hydrophobic coatings applied as barriers to the printed materials did not prevent these interactions.

Use of LC-MS analysis to elucidate by-products of niacinamide transformation following in vitro skin permeation studies.

OBJECTIVE: To explore and elucidate the formation of niacinamide (NIA) by-products during in vitro skin permeation studies using liquid chromatography coupled to mass spectrometry (LC-MS) analysis. METHODS: Porcine skin permeation studies of various NIA formulations were conducted using Franz diffusion cells for a period of 24 hours. NIA by-products were identified by LC, extracted and further qualitatively analysed by LC-MS. RESULTS: Analysis and characterisation of NIA by-products using LC-MS resulted in the identification of different molecular entities with similar structures to NIA. The most prevalent molecular specie in this study was 1,4,5,6-tetrahydropyridine-3-carboxamide with the highest ion abundance. Other structural NIA analogues were also identified and reported, namely piperidine-3-carboxamide and 1,4-dihydropyridine-3-carboxamide. None of these NIA derivatives were detected in stability studies of NIA in the medium used as the receptor phase, phosphate buffered saline (PBS), that had not been in contact with skin. CONCLUSION: The comparatively low recovery of NIA following in vitro mass-balance and permeation studies for pseudo-finite and finite dosing of the active compared with infinite dosing is attributed to chemical derivatisation of the molecule during skin penetration. These findings reported here will allow the development of more sensitive methods to ensure full mass balance recovery of NIA following topical application of NIA preparations.

Early-life regional and temporal variation in filaggrin-derived natural moisturizing factor, filaggrin-processing enzyme activity, corneocyte phenotypes and plasmin activity: implications for atopic dermatitis.

BACKGROUND: Filaggrin is central to the pathogenesis of atopic dermatitis (AD). The cheeks are a common initiation site of infantile AD. Regional and temporal expression of levels of filaggrin degradation products [natural moisturizing factors (NMFs)], activities of filaggrin-processing enzymes [bleomycin hydrolase (BH) and calpain-1 (C-1)] and plasmin, and corneocyte envelope (CE) maturity in early life are largely unknown. OBJECTIVES: We conducted a cross-sectional, observational study investigating regional and age-dependent variations in NMF levels, activity of proteases and CE maturity in stratum corneum (SC) from infants to determine whether these factors could explain the observed predilection sites for AD in early life. METHODS: We measured NMF using a tape-stripping method at seven sites in the SC of 129 children (aged < 12 months to 72 months) and in three sites in 56 neonates and infants (< 48 h to 3 months). In 37 of these neonates and infants, corneocyte size, maturity, BH, C-1 and plasmin activities were determined. RESULTS: NMF levels are low at birth and increase with age. Cheek SC, compared with elbow flexure and nasal tip, has the lowest NMF in the first year of life and is the slowest to reach stable levels. Cheek corneocytes remain immature. Plasmin, BH and C-1 activities are all elevated by 1 month of age in exposed cheek skin, but not in elbow skin. CONCLUSIONS: Regional and temporal differences in NMF levels, CE maturity and protease activities may explain the predilection for AD to affect the cheeks initially and are supportive of this site as key for allergen priming in early childhood. These observations will help design early intervention and treatment strategies for AD.

A new approach to assess the effect of photodamage on corneocyte envelope maturity using combined hydrophobicity and mechanical fragility assays.

BACKGROUND: The maturity of the corneocyte envelope (CE) provides information about the barrier functionality of the stratum corneum (SC). Corneocytes are enclosed by the CE, a protein-lipid matrix, contributing to mechanical resistance and hydrophobicity of the SC. OBJECTIVES: The aim of the work was to develop a novel and robust approach to characterize CE maturity based on rigidity, hydrophobicity and surface area. This offers an alternative approach to the Nile red staining and antigenicity of involucrin to characterize the CE. The photoexposed (PE) cheek and photoprotected (PP) post-auricular sites were selected for investigation. METHODS: Nine tape strips were obtained from the cheek and post-auricular sites of healthy Caucasians. CEs on the first and last tape strip were subjected to sonication to assess rigidity, and Nile red staining to determine hydrophobicity per unit surface area. In addition, the presence of involucrin and lipids was assessed to determine CE maturity by examination of the red/green pixel ratio, percentage of involucrin expressing CEs and alternatively the ratio of fluorescence density. RESULTS: The CE rigidity was lower in the deeper SC layers of the cheek, whereas post-auricular CEs were mechanically more resistant. Post-auricular CEs from the superficial SC had a larger surface area with a stronger fluorescence signal than those from the cheek. Interestingly, those CEs from the deeper SC layers had similar surface areas in both anatomical sites but were significantly different in hydrophobicity. These three parameters can be summarized as a relative CE maturity index that expresses CE maturity more precisely with a higher sensitivity than the conventional involucrin and Nile red staining approach. CEs of the cheek surface are more mature than CEs in the deeper SC layer, whereas CEs obtained from the post-auricular surface are more mature than those from the cheek surface. CONCLUSION: The combined method developed allows characterization of CE maturity based on hydrophobicity per unit surface area and rigidity rather than a simple ratio of lipid to involucrin. A more robust and sensitive measurement has therefore been developed addressing the limitations of earlier protocols.

Hexamidine salts - applications in skin health and personal care products.

Hexamidine (HEX) has been used as a preservative in topical preparations since the 1950s. A number of studies also indicate that the molecule plays a beneficial role in skin homoeostasis. In this review, we describe the physicochemical properties of hexamidine diisethionate (HEX D) and the corresponding hydrochloride salt (HEX H). The biocidal and protease inhibition properties of HEX are outlined as well as the effects of HEX on lipid processing enzymes, corneocyte maturity, stratum corneum thickness and transepidermal water loss (TEWL). Skin permeation properties of HEX D and HEX H are summarized, and formulation approaches for effective dermal targeting of HEX are discussed.

Effect of different alcohols on stratum corneum kallikrein 5 and phospholipase A2 together with epidermal keratinocytes and skin irritation.

OBJECTIVES: The aim of this exploratory study was to investigate the effect of ethanol, isopropanol and n-propanol on stratum corneum (SC) enzymes and keratinocytes in vitro together with their effects on skin condition and function. METHODS: Activities of kallikrein 5 (KLK5) and phospholipase A2 (PLA2) as well as keratinocyte metabolic activity, interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α) were measured in vitro in the presence and absence of the different alcohols. We also measured transepidermal water loss (TEWL), skin capacitance, visual dryness and visual redness on the volar forearms of 25 Caucasian women following application of the alcohols 20 and 100 times per day over a period of 14 days in a clinical study. RESULTS: Reduced activities of KLK5 and PLA2 were observed in the presence of the alcohols. The greatest denaturing effect was always observed for n-propanol (P < 0.001), and in the case of PLA2, the effect of isopropanol was greater than ethanol (P < 0.001). Equally, ethanol had the mildest effects on keratinocyte metabolic activity and cytokine secretion (P < 0.001) and n-propanol always produced the most severe changes in normal and differentiated keratinocytes. These in vitro findings supported the clinical results where the major effects were on the induction of skin irritation (increased dropout rates) and ranked the intolerance of the different alcohols as follows: n-propanol > isopropanol > ethanol. At the high application frequencies, the effect of the different alcohols on transepidermal water loss (TEWL) and skin capacitance was similar, but at the low application frequencies, n-propanol had a significant effect on TEWL and capacitance values (P < 0.05). Equally, n-propanol and isopropanol produced significantly more skin redness at the low application frequencies. CONCLUSIONS: Clearly, isopropanol and n-propanol caused significant SC and keratinocyte perturbation in vitro together with damage to skin condition and function in vivo whereas ethanol did not. As a result, we show that ethanol-based sanitizers are better tolerated by skin, particularly in high-use settings, than other alcohols and should be the active ingredient of choice.

A fundamental investigation into aspects of the physiology and biochemistry of the stratum corneum in subjects with sensitive skin.

BACKGROUND: Sensitive skin is a poorly understood skin condition. Defects in stratum corneum (SC) barrier function and/or extrasensory neuronal networks in the epidermis are believed to be involved in the problem. OBJECTIVES: This study aimed to unravel the relationships between bleomycin hydrolase (BH) and calpain-1 (C-1), pyrrolidone carboxylic acid (PCA) levels, corneocyte maturation, transglutaminase (TG) and plasmin activities on the cheeks of subjects with sensitive skin. METHODS: Forty-eight female Caucasian subjects, Fitzpatrick skin phototypes II-III, with self-perceived sensitive facial skin, were assessed and underwent a capsaicin reactivity test. Expert grading of skin condition was conducted as well as the measurement of transepidermal water loss (TEWL), skin capacitance, SC cohesion and SC integrity. BH, C-1 and plasmin activities were measured as well as PCA levels, plasmin and TG activity. Differential Nile red and involucrin immunostaining was performed to assess corneocyte maturation and size. RESULTS: About 52% of the subjects reacted to capsaicin. There were no significant differences between the capsaicin-sensitive and non-capsaicin-sensitive subjects with reference to skin grading, TEWL, skin capacitance and SC cohesion. PCA levels and BH activity were lowest in the capsaicin-sensitive panel (P < 0.05) and were correlated in non-capsaicin-sensitive subjects (r = 0.72). The activity of TG was significantly lower (48%) in the capsaicin-sensitive subjects (P < 0.001) and their corneocytes were less mature and smaller (P ≤ 0.05). SC was estimated to be thinner (6.87 ± 0.28 vs. 8.68 ± 0.26 µm; P = 0.001) in the capsaicin-sensitive subjects with a corresponding shorter SC path length (83.2 ± 4.4 µm and 113.1 ± 4.5 µm; P = 0.001). CONCLUSIONS: Despite the physiological similarities between the two groups of sensitive skin subjects, differences in their biochemistry were clearly evident. Lower levels of PCA, BH and TG activities together with a greater number of smaller and immature corneocytes indicate inferior SC maturation in the capsaicin-sensitive subjects. The reduced maturation of corneocytes and thinner SC likely contributes to a greater penetration of capsaicin and the associated increased skin sensitivity.

Chemical ultraviolet absorbers topically applied in a skin barrier mimetic formulation remain in the outer stratum corneum of porcine skin.

The objective of the present study was to evaluate the fate of three chemical sunscreens, isoamyl p-methoxycinnamate (IPMC), diethylamino hydroxybenzoyl hexyl benzoate (DHHB), and bis-ethylhexylphenol methoxyphenyl triazine (BEMT), topically applied to mammalian skin from a skin barrier mimetic oil-in-water formulation. High Performance Liquid Chromatography (HPLC) methods were developed for the analysis of each molecule and validated. Franz cell permeation studies were conducted following application of finite doses of the formulations to excised porcine skin. A vehicle formulation containing no sunscreens was evaluated as a control. Permeation studies were conducted for 12h after which full mass balance studies were carried out. Analysis of individual UV sunscreens was achieved with HPLC following application of the formulation to the skin with no interference from the vehicle components. No skin permeation of any of the chemical sunscreens was evident after 12h. While sunscreens were detected in up to 12 tape strips taken from the SC, 87% or more of the applied doses recovered in the first 5 tape strips. When corrected for the amount of protein removed per tape strip this corresponded to a penetration depth in porcine stratum corneum of ∼1.7µm. Mass balance studies indicated total recovery values were within accepted guidelines for cosmetic formulations. Overall, only superficial penetration into the SC was observed for each compound. These findings are consistent with the physicochemical properties of the selected UV absorbing molecules and their formulation into an ordered biomimetic barrier formulation thus support their intended use in topical consumer formulations designed to protect from UV exposure. To our knowledge this is the first report of depth profiling of chemical sunscreens in the SC that combines tape stripping and protein determination following in vitro Franz cell studies.

Variation in the activities of late stage filaggrin processing enzymes, calpain-1 and bleomycin hydrolase, together with pyrrolidone carboxylic acid levels, corneocyte phenotypes and plasmin activities in non-sun-exposed and sun-exposed facial stratum corneum of different ethnicities.

BACKGROUND: Knowledge of the ethnic differences and effects of photodamage on the relative amounts of natural moisturizing factor (NMF) together with filaggrin processing enzymes in facial stratum corneum is limited. Our aim was to characterize the activities of calpain-1 (C-1), bleomycin hydrolase (BH) and the levels of pyrrolidone carboxylic acid (PCA) as a marker for total NMF levels and to relate them to plasmin activities and corneocyte maturation. METHODS: Enzyme activities, PCA levels and corneocyte maturation were determined from facial tape strippings of photoexposed cheek and photoprotected post-auricular areas (PA) of healthy Caucasian (C), Black African (BA) and albino African (AA) female subjects living in South Africa. RESULTS: PCA concentration levels were of the order AA > BA > C subjects, and the highest activities of BH were present in the AA subjects. BH activities were greater on the photoexposed sites for the BA and C subjects, but they were only numerically elevated in the AA subjects. Photoprotected sites had an increase in C-1 activity in pigmented groups (C and BA), whereas in the AA subjects, the opposite was measured. Plasmin activities were greater on the cheek compared with the PA site for the AA and C subjects, but the activity was low in the BA subjects. In both test sites, the AA, but not the BA and C subjects, had smaller, parakeratotic and less mature corneocytes. CONCLUSION: Variation in PCA levels has been found for different ethnic groups in this study (AA > BA > C subjects). The values in the AA subjects are surprising as one might expect that the lack of pigmentation, and thereby increased photodamage, might lead to lower levels. Increased BH, but not C-1 activity, was observed in the AA subjects indicating that BH is associated with PCA production to a greater extent. Surprisingly, corneocyte maturation is still impaired with elevated PCA levels in AA subjects. The higher levels of plasmin and BH activities on the cheeks, especially for AA and C subjects, suggest that they can be used as markers for epidermal photodamage.

Variation in stratum corneum protein content as a function of anatomical site and ethnic group.

OBJECTIVES: Quantification of stratum corneum (SC) protein levels from tape strippings is frequently used to investigate skin conditions, to correct for amounts of SC protein removed in SC biomarker studies and to determine distribution of topically applied ingredients. In recent years, a rapid and convenient method for SC protein quantification from tape strippings has become available using infrared densitometry (IRD). However, standard curves have only been generated for Caucasian forearm and shoulder SC and have been assumed to be correct not only for facial SC but also for SC samples of other ethnic groups. The aim of this study was to investigate whether the use of IRD for SC protein measurement is valid for other body sites such as the cheek and for measuring SC protein content of darkly pigmented skin types. METHODS: Ten Caucasian and ten Black African female subjects with self-assessed normal skin participated in the study. Tape strippings were collected from two different body sites (forearm and cheek). First from the tape strippings, the SC optical absorption was determined densitometrically. This obtained absorption (%) was compared with absolute SC protein extracted from the same tapes using a colorimetric microbicinchoninic acid (µBCA) assay. RESULTS: Higher amounts of SC protein were removed from the forearm compared with the cheek (P < 0.01). The absolute SC protein concentration quantified by µBCA assay and the absorption of SC proteins by IRD followed a similar profile. There was no significant difference found between the two ethnicities in SC protein (P > 0.05). The overall coefficient of determination (R(2) ) shows a good fit to the regression line between the two methods in both sites (forearm = 0.82, cheek = 0.77). Also, both ethnicities showed good correlation (R(2) ≥ 0.69, P = 0.01). CONCLUSIONS: Facial SC is morphologically distinct from the forearm, as demonstrated by the differences in amounts of SC removed. Although the data distribution in different subject groups varied, the regression was always quite similar between the two body sites and both ethnic groups. Also, the correlations were similar to previously published data on other body sites. The resultant calibration curves can be used as a rapid indirect protein assessment of tape strippings from the cheek.

In vitro-in vivo correlation in skin permeation.

PURPOSE: In vitro skin permeation studies have been used extensively in the development and optimisation of delivery of actives in vivo. However, there are few reported correlations of such in vitro studies with in vivo data. The aim of this study was to investigate the skin permeation of a model active, niacinamide, both in vitro and in vivo. METHODS: Conventional diffusion cell studies were conducted in human skin to determine niacinamide permeation from a range of vehicles which included dimethyl isosorbide (DMI), propylene glycol (PG), propylene glycol monolaurate (PGML), N-methyl 2-pyrrolidone (NMP), Miglyol 812N® (MG), and mineral oil (MO). Single, binary or ternary systems were examined. The same vehicles were subsequently examined to investigate niacinamide delivery in vivo. For this proof-of-concept study one donor was used for the in vitro studies and one volunteer for the in vivo investigations to minimise biovariability. Analysis of in vitro samples was conducted using HPLC and in vivo uptake of niacinamide was evaluated using Confocal Raman spectroscopy (CRS). RESULTS: The amount of niacinamide permeated through skin in vitro was linearly proportional to the intensity of the niacinamide signal determined in the stratum corneum in vivo. A good correlation was observed between the signal intensities of selected vehicles and niacinamide signal intensity. CONCLUSIONS: The findings provide further support for the use of CRS to monitor drug delivery into and across the skin. In addition, the results highlight the critical role of the vehicle and its disposition in skin for effective dermal delivery.

Percutaneous penetration paradigms: the contribution of Jonathan Hadgraft.

This paper addresses the application of physical chemistry towards understanding the processes of percutaneous penetration. Over the years, this approach has been used by the subject of this review to advance our knowledge of the skin and to interpret, model and predict dermal delivery. The basic laws of diffusion provide a framework for the comprehension of skin permeability and, as a corollary, how the barrier function can be modulated. The importance of understanding the physicochemical properties of potential skin permeants is highlighted. Rational formulation strategies for passive skin delivery are outlined. However, the overriding emphasis is that multidisciplinary approaches must be used to advance the field. This is exemplified in the work and references cited.