Age-Related Changes to Human Stratum Corneum Lipids Detected Using Time-of-Flight Secondary Ion Mass Spectrometry Following in Vivo Sampling.

This work demonstrates the ability to detect changes in both quantity and spatial distribution of human stratum corneum (SC) lipids from samples collected in vivo. The SC functions as the predominant barrier to the body, protecting against the penetration of xenobiotic substances. Changes to the SC lipid composition have been associated with barrier impairment and consequent skin disorders, and it is therefore important to monitor and quantify changes to this structure. This work demonstrates the first reported use of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to assess physiological changes to human SC as a function of depth. This technique provides exceptional sensitivity and chemical specificity, allowing analysis of single tape stripped samples taken from volunteers. Using this methodology we were able to successfully identify chemical differences in human SC resulting from both intrinsic and extrinsic (photo) aging. Samples were collected from women of two age groups (under 27 and postmenopausal) and from two body sites with varying UV exposure (inner forearm and dorsal hand), and differences were identified using multivariate data analysis. The key finding was the significant aged-related increase and change in spatial distribution of the sterol cholesterol sulfate, a membrane stabilizing lipid. Significant changes in the prevalence of both lignoceric acid (C24:0) and hexacosanoic acid (C26:0) were also observed. This work describes previously unreported age-related chemical changes to human SC, providing an insight into aging mechanisms which may improve the design of both pharmaceutical and cosmetic topical products.

Melanin fate in the human epidermis: a reassessment of how best to detect and analyse histologically.

Melanin is the predominant pigment responsible for skin colour and is synthesized by the melanocyte in the basal layer of the epidermis and then transferred to surrounding keratinocytes. Despite its optical properties, melanin is barely detectable in unstained sections of human epidermis. However, identification and localization of melanin is of importance for the study of skin pigmentation in health and disease. Current methods for the histologic quantification of melanin are suboptimal and are associated with significant risk of misinterpretation. The aim of this study was to reassess the existing literature and to develop a more effective histological method of melanin quantification in human skin. Moreover, we confirm that Warthin-Starry (WS) stain provides a much more sensitive and more specific melanin detection method than the commonplace Fontana-Masson (FM) stain. For example, WS staining sensitivity allowed the visualization of melanin even in very pale Caucasian skin that was missed by FM or Von Kossa (VK) stains. From our reassessment of the histology-related literature, we conclude that so-called melanin dust is most likely an artifact of discoloration due to non-specific silver deposition in the stratum corneum. Unlike FM and VK, WS was not associated with this non-specific stratum corneum darkening, misinterpreted previously as 'degraded' melanin. Finally, WS melanin particle counts were largely similar to previously reported manual counts by transmission electron microscopy, in contrast to both FM and VK. Together these findings allow us to propose a new histology/Image J-informed method for the accurate and precise quantification of epidermal melanin in skin.

The effects of Sophora angustifolia and other natural plant extracts on melanogenesis and melanin transfer in human skin cells.

Skin pigmentation is a multistep process of melanin synthesis by melanocytes, its transfer to recipient keratinocytes and its degradation. As dyspigmentation is a prominent marker of skin ageing, novel effective agents that modulate pigmentation safely are being sought for both clinical and cosmetic use. Here, a number of plant extracts were examined for their effect on melanogenesis (by melanin assay and Western blotting) and melanin transfer (by confocal immunomicroscopy of gp100-positive melanin granules in cocultures and by SEM analysis of filopodia), in human melanocytes and in cocultures with phototype-matched normal adult epidermal keratinocytes. Mulberry, Kiwi and Sophora extracts were assessed against isobutylmethylxanthine, hydroquinone, vitamin C and niacinamide. Compared with unstimulated control, all extracts significantly reduced melanogenesis in human melanoma cells and normal adult epidermal melanocytes. These extracts also reduced melanin transfer and reduced filopodia expression on melanocytes, similar to hydroquinone and niacinamide, indicating their effectiveness as multimode pigmentation actives.

Enhanced vitamin C skin permeation from supramolecular hydrogels, illustrated using in situ ToF-SIMS 3D chemical profiling.

Vitamin C (ascorbic acid) is a naturally occurring, powerful anti-oxidant with the potential to deliver numerous benefits to the skin when applied topically. However, topical use of this compound is currently restricted by an instability in traditional formulations and the delivery and eventual fate of precursor compounds has been largely unexplored. Time of flight secondary ion mass spectrometry (ToF-SIMS) is an emerging technique in the field of skin research and offers detailed chemical analysis, with high mass and spatial resolution, as well as profiling capabilities that allow analysis as a function of sample depth. This work demonstrates the successful use of ToF-SIMS to obtain, in situ, accurate 3D permeation profiles of both ascorbic acid and a popular precursor, ascorbyl glucoside, from ex vivo porcine skin. The significant permeation enhancing effect of a supramolecular hydrogel formulation, produced from an amphiphilic gemini imidazolium-based surfactant, was also demonstrated for both compounds. Using ToF-SIMS, it was also possible to detect and track the breakdown of ascorbyl glucoside into ascorbic acid, elucidating the ability of the hydrogel formulation to preserve this important conversion until the targeted epidermal layer has been reached. This work demonstrates the potential of ToF-SIMS to provide 3D permeation profiles collected in situ from ex vivo tissue samples, offering detailed analysis on compound localisation and degradation. This type of analysis has significant advantages in the area of skin permeation, but can also be readily translated to other tissue types.

A comparative study of the in vitro permeation of ibuprofen in mammalian skin, the PAMPA model and silicone membrane.

Human skin remains the membrane of choice when conducting in vitro studies to determine dermal penetration of active pharmaceutical ingredients or xenobiotics. However there are ethical and safety issues associated with obtaining human tissue. For these reasons synthetic membranes, cell culture models or in silico predictive algorithms have been researched intensively as alternative approaches to predict dermal exposure in man. Porcine skin has also been recommended as an acceptable surrogate for topical or transdermal delivery research. Here we examine the in vitro permeation of a model active, ibuprofen, using human or porcine skin, as well as the Parallel Artificial Membrane Permeation Assay (PAMPA) model and silicone membrane. Finite dose studies were conducted in all models using commercial ibuprofen formulations and simple volatile ibuprofen solutions. The dose applied in the PAMPA model was also varied in order to determine the amount of applied formulation which best simulates typical amounts of topical products applied by patients or consumers. Permeation studies were conducted up to 6h for PAMPA and silicone and up to 48h for human and porcine skin. Cumulative amounts permeated at 6h were comparable for PAMPA and silicone, ranging from 91 to 136µg/cm(2) across the range of formulations studied. At 48h, maximum ibuprofen permeation in human skin ranged from 11 to 38µg/cm(2) and corresponding values in porcine skin were 59-81µg/cm(2). A dose of 1µL was confirmed as appropriate for finite dose studies in the PAMPA model. The formulation which delivered the greatest amount of ibuprofen in human skin was also significantly more efficient than other formulations when evaluated in the PAMPA model. The PAMPA model also discriminated between different formulation types (i.e. gel versus solution) compared with other models. Overall, the results confirm the more permeable nature of the PAMPA, silicone membrane and porcine tissue models to ibuprofen compared with human skin. Further finite dose studies to elucidate the effects of individual excipients on the barrier properties of the PAMPA model are needed to expand the applications of this model. The range of actives that are suitable for study using the model also needs to be delineated.

Delivery of ibuprofen to the skin.

Ibuprofen (IBU) has been available as a topical skin preparation for more than two decades. Its primary indication is for the relief of pain and inflammation in rheumatic disease and other musculoskeletal conditions. This article reviews the various formulation strategies which have been investigated for percutaneous IBU delivery to date. The focus is on studies which have been conducted with IBU in the free base form on human or porcine skin as data from other species are known to overestimate likely permeation in man. Emerging technologies for enhanced dermal delivery of IBU are considered including methods which require physical disruption of the membrane. The role of biophysical techniques such as Confocal Raman Spectroscopy in the rational development of IBU formulations is also discussed.

Discovery of 2-(6-{[(6-fluoroquinolin-2-yl)methyl]amino}bicyclo[3.1.0]hex-3-yl)-N-hydroxypyrimidine-5-carboxamide (CHR-3996), a class I selective orally active histone deacetylase inhibitor.

A novel series of HDAC inhibitors demonstrating class I subtype selectivity and good oral bioavailability is described. The compounds are potent enzyme inhibitors (IC50 values less than 100 nM), and improved activity in cell proliferation assays was achieved by modulation of polar surface area (PSA) through the introduction of novel linking groups. Employing oral pharmacokinetic studies in mice, comparing drug levels in spleen to plasma, we selected compounds that were tested for efficacy in human tumor xenograft studies based on their potential to distribute into tumor. One compound, 21r (CHR-3996), showed good oral activity in these models, including dose-related activity in a LoVo xenograft. In addition 21r showed good activity in combination with other anticancer agents in in vitro studies. On the basis of these results, 21r was nominated for clinical development.

Gastric juice, gastric tissue and blood antibiotic concentrations following omeprazole, amoxicillin and clarithromycin triple therapy.

BACKGROUND: Amoxicillin and clarithromycin are key antibiotics in proton pump inhibitor-based Helicobacter pylori eradication therapies. AIMS: To study gastric mucus and tissue concentrations and collect basic data about optimal antibacterial doses. METHODS: Plasma, gastric mucosa and gastric juice antibiotic concentrations were measured following either low- or high-dose amoxicillin (750 or 1000 mg b.i.d.) and clarithromycin (400 or 500 mg b.i.d.) given in combination with omeprazole 20 mg bid to 12 male volunteers in an open crossover design. Gastric juice and mucosal biopsy collection was performed either 2 (n=6) or 6 hours (n=6) after dosing. RESULTS: Amoxicillin concentrations 2 hours after high dosage were gastric juice > gastric body > antral mucosa > plasma. At 6 hours, plasma and gastric juice concentrations were still above the MIC for amoxicillin-susceptible bacteria but no antibiotic was detectable in mucosa samples. Clarithromycin concentrations after high dosage were gastric juice > mucosa > serum; all above the MIC for clarithromycin-susceptible bacteria at both 2 and 6 hours. CONCLUSIONS: Both dosage regimens provided effective antibiotic concentrations in gastric juice at 2 hours. After dosing, both antibiotics demonstrated high gastric tissue concentrations via local diffusion while clarithromycin also provided sustained delivery (6 hours) via gastric mucosa penetration.