Confocal Raman investigation of diffusion processes in monolithic type transdermal drug delivery systems.

Release from a transdermal drug delivery system (TDDS) can either be controlled by diffusion in the adhesive, by diffusion processes in the stratum corneum of the skin or a combination of both. In this study, diffusion processes in monolithic type TDDS were investigated using confocal Raman microscopy. An acrylic adhesive (Duro-Tak 180-129a), a rubber adhesive (Duro-Tak H1540) and a silicone adhesive (BIO-PSA 7-4202) were used. Skin permeation of the model drug Paeonol from these adhesives was investigated. Release studies on porcine cadaver skin were carried out. Solubility of Paeonol in the different adhesives was measured. Diffusion coefficients of the drug in the TDDSs were calculated from confocal Raman depth scans, the diffusion coefficient in the stratum corneum was calculated using tape stripping. Solubility of Paeonol in the acrylic adhesive was the highest with 30 g/L among the tested systems. Paeonol had a solubility of 6 and 9 g/L in the silicone and rubber based system. Diffusion coefficient rank order was BIO-PSA 7-4204 > Duro-Tak 180-129a > Duro-Tak H1540. Release on porcine cadaver skin from the silicone was the highest followed by the rubber and the acrylic adhesive. During release studies on porcine skin with Duro-Tak H1540 no concentration gradient of Paeonol could be monitored in the Raman depth profiles, whereas in the stratum corneum an apparent diffusion gradient was detectable. Solubility of a drug in the adhesive dominated the release properties, high-diffusion coefficients of drugs in adhesives do not necessarily lead to high release rates from adhesives.

In vitro efficacy and release study with anti-inflammatory drugs incorporated in adhesive transdermal drug delivery systems.

The topical application of two different anti-inflammatory extracts incorporated in adhesive transdermal drug delivery systems (TDDSs) was investigated. Therefore, anti-inflammatory properties and percutaneous absorption behavior of adhesive TDDSs were characterized in vitro conducting experiments with a dermatologically relevant human skin model. Anti-inflammatory efficacy against UV irradiation of both TDDSs was determined in vitro with EpiDerm™. The reduction of the release of proinflammatory cytokines by topically applied TDDSs was compared with the reduction during the presence of the specific cyclooxygenase inhibitor diclofenac in the culture medium. A similar anti-inflammatory efficacy of the topically applied TDDSs in comparison with the use of diclofenac in the culture medium should be achieved. Furthermore, percutaneous absorption in efficacy tests was compared with percutaneous absorption in diffusion studies with porcine cadaver skin. Both the topically applied TDDSs showed a significant anti-inflammatory activity. Permeation coefficients through the stratum corneum and the epidermis gained from the release studies on porcine cadaver skin (Magnolia: 2.23·10(-5) cm/h, licorice: 4.68·10(-6) cm/h) were approximately five times lower than the permeation coefficients obtained with the EpiDerm™ skin model (Magnolia: 9.48·10(-5) cm/h, licorice: 24.0·10(-6) cm/h). Therefore, an adjustment of drug doses during experiments with the EpiDerm™ skin model because of weaker skin barrier properties should be considered.

Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase.

Mammalian tyrosinases are key enzymes of melanin formation. Their native forms undergo complex maturation and sorting processes before being integrated into the melanosomal membrane, which greatly complicates their heterologous expression in other cell types. In the present work, we constructed several differently truncated, soluble variants of human tyrosinase and studied their properties after expression in HEK 293 cells. In addition, we prepared two affinity-tagged forms of the enzyme for expression in the yeast Kluyveromyces lactis and HEK cells, respectively. A Strep-tagged variant was secreted by K. lactis in excellent yields but found to be inactive, whereas a His-tagged variant secreted by HEK 293 cells in an active state could be purified from cell supernatants to near homogeneity. The resulting preparation consisted of an inactive, probably unglycosylated species of about 57 kDa and several glycosylated forms with masses between 63 and 75 kDa, as confirmed by activity staining, Western blotting and mass spectrometry.

The deceptive nature of UVA tanning versus the modest protective effects of UVB tanning on human skin.

The relationship between human skin pigmentation and protection from ultraviolet (UV) radiation is an important element underlying differences in skin carcinogenesis rates. The association between UV damage and the risk of skin cancer is clear, yet a strategic balance in exposure to UV needs to be met. Dark skin is protected from UV-induced DNA damage significantly more than light skin owing to the constitutively higher pigmentation, but an as yet unresolved and important question is what photoprotective benefit, if any, is afforded by facultative pigmentation (i.e. a tan induced by UV exposure). To address that and to compare the effects of various wavelengths of UV, we repetitively exposed human skin to suberythemal doses of UVA and/or UVB over 2 weeks after which a challenge dose of UVA and UVB was given. Although visual skin pigmentation (tanning) elicited by different UV exposure protocols was similar, the melanin content and UV-protective effects against DNA damage in UVB-tanned skin (but not in UVA-tanned skin) were significantly higher. UVA-induced tans seem to result from the photooxidation of existing melanin and its precursors with some redistribution of pigment granules, while UVB stimulates melanocytes to up-regulate melanin synthesis and increases pigmentation coverage, effects that are synergistically stimulated in UVA and UVB-exposed skin. Thus, UVA tanning contributes essentially no photoprotection, although all types of UV-induced tanning result in DNA and cellular damage, which can eventually lead to photocarcinogenesis.

The fibroblast-derived paracrine factor neuregulin-1 has a novel role in regulating the constitutive color and melanocyte function in human skin.

Interactions between melanocytes and neighboring cells in the skin are important in regulating skin color in humans. We recently demonstrated that the less pigmented and thicker skin on the palms and soles is regulated by underlying fibroblasts in those areas, specifically via a secreted factor (DKK1) that modulates Wnt signaling. In this study, we tested the hypothesis that dermal fibroblasts regulate the constitutive skin color of individuals ranging from very light to very dark. We used microarray analysis to compare gene expression patterns in fibroblasts derived from lighter skin types compared to darker skin types, with a focus on secreted proteins. We identified a number of genes that differ dramatically in expression and, among the expressed proteins, neuregulin-1, which is secreted by fibroblasts derived from dark skin, effectively increases the pigmentation of melanocytes in tissue culture and in an artificial skin model and regulates their growth, suggesting that it is one of the major factors determining human skin color.

Regulation of human skin pigmentation in situ by repetitive UV exposure: molecular characterization of responses to UVA and/or UVB.

UV radiation is a major environmental factor that affects pigmentation in human skin and can eventually result in various types of UV-induced skin cancers. The effects of various wavelengths of UV on melanocytes and other types of skin cells in culture have been studied, but little is known about gene expression patterns in situ following in situ exposure of human skin to different types of UV (UVA and/or UVB). Paracrine factors expressed by keratinocytes and/or fibroblasts that affect skin pigmentation might be regulated differently by UV, as might their corresponding receptors expressed on melanocytes. To test the hypothesis that different mechanisms are involved in the pigmentary responses of the skin to different types of UV, we used immunohistochemical and whole human genome microarray analyses to characterize human skin in situ to examine how melanocyte-specific proteins and paracrine melanogenic factors are regulated by repetitive exposure to different types of UV compared with unexposed skin as a control. The results show that gene expression patterns induced by UVA or UVB are distinct-UVB eliciting dramatic increases in a large number of genes involved in pigmentation as well as in other cellular functions, whereas UVA had little or no effect on these. The expression patterns characterize the distinct responses of the skin to UVA or UVB, and identify several potential previously unidentified factors involved in UV-induced responses of human skin.

Short- and long-term effects of UV radiation on the pigmentation of human skin.

The incidence of skin cancer, including cutaneous melanoma, has risen substantially in recent years, and epidemiological and laboratory studies show that UV radiation is a major causative factor of this increase. UV damage also underlies photoaging of the skin, and these deleterious effects of UV can be, in part, prevented in skin with higher levels of constitutive pigmentation. We review the clinical studies we have made in recent years regarding the rapid and the long-term responses of the pigmentary system in human skin to UV exposure.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 32-35; doi:10.1038/jidsymp.2009.10.

Microarray analysis sheds light on the dedifferentiating role of agouti signal protein in murine melanocytes via the Mc1r.

The melanocortin-1 receptor (MC1R) is a key regulator of pigmentation in mammals and is tightly linked to an increased risk of skin cancers, including melanoma, in humans. Physiologically activated by alpha-melanocyte stimulating hormone (alphaMSH), MC1R function can be antagonized by a secreted factor, agouti signal protein (ASP), which is responsible for the lighter phenotypes in mammals (including humans), and is also associated with increased risk of skin cancer. It is therefore of great interest to characterize the molecular effects elicited by those MC1R ligands. In this study, we determined the gene expression profiles of murine melan-a melanocytes treated with ASP or alphaMSH over a 4-day time course using genome-wide oligonucleotide microarrays. As expected, there were significant reductions in expression of numerous melanogenic proteins elicited by ASP, which correlates with its inhibition of pigmentation. ASP also unexpectedly modulated the expression of genes involved in various other cellular pathways, including glutathione synthesis and redox metabolism. Many genes up-regulated by ASP are involved in morphogenesis (especially in nervous system development), cell adhesion, and extracellular matrix-receptor interactions. Concomitantly, ASP enhanced the migratory potential and the invasiveness of melanocytic cells in vitro. These results demonstrate the role of ASP in the dedifferentiation of melanocytes, identify pigment-related genes targeted by ASP and by alphaMSH, and provide insights into the pleiotropic molecular effects of MC1R signaling that may function during development and may affect skin cancer risk.

Long-lasting molecular changes in human skin after repetitive in situ UV irradiation.

It is known that UV modulates the expression of paracrine factors that regulate melanocyte function in the skin. We investigated the consequences of repetitive UV exposure of human skin in biopsies of 10 subjects with phototypes 2-3.5 taken 1-4 years later. The expression of melanogenic factors (TYR, MART1, MITF), growth factors/receptors (SCF/KIT, bFGF/FGFR1, ET1/EDNRB, HGF, GM-CSF), adhesion molecules (beta-catenin, E-cadherin, N-cadherin), cell cycle proteins (PCNA, cyclins D1, E2) as well as Bcl-2, DKK1, and DKK3, were analyzed by immunohistochemistry. Most of those markers showed no detectable changes at > or = 1 year after the repetitive UV irradiation. Although increased expression of EDNRB protein was detected in 3 of 10 UV-irradiated subjects, there was no detectable change in the expression of ET1 protein or in EDNRB mRNA levels. In summary, only the expression of TYR, MART1, and/or EDNRB, and only in some subjects, was elevated at > or = 1 year after UV irradiation. Thus the long-term effects of repetitive UV irradiation on human skin did not lead to significant changes in skin morphology and there is considerable subject-to-subject variation in responses. The possibility that changes in the expression and function of EDNRB triggers downstream activation of abnormal melanocyte proliferation and differentiation deserves further investigation.

Pigmentation effects of solar-simulated radiation as compared with UVA and UVB radiation.

Different wavelengths of ultraviolet (UV) radiation elicit different responses in the skin. UVA induces immediate tanning and persistent pigment darkening through oxidation of pre-existing melanin or melanogenic precursors, while UVB induces delayed tanning which takes several days or longer to develop and requires activation of melanocytes. We compared the effects of a 2-week repetitive exposure of human skin to solar-simulated radiation (SSR), UVA or UVB at doses eliciting comparable levels of visible tanning and measured levels of melanins and melanin-related metabolites. Levels of eumelanin and pheomelanin were significantly higher in the order of SSR, UVB, UVA or unexposed control skin. Levels of free 5-S-cysteinyldopa (5SCD) were elevated about 4-fold in SSR- or UVB-exposed skin compared with UVA-exposed or control skin. Levels of protein-bound form of 5SCD tended to be higher in SSR- or UVB-exposed skin than in UVA-exposed or control skin. Total levels of 5-hydroxy-6-methoxyindole-2-carboxylic acid (5H6MI2C) and 6H5MI2C were higher in SSR- than in UVB-exposed or control skin. These results show that SSR is more effective in promoting delayed tanning than UVB radiation alone, suggesting a synergistic effect of UVA radiation. Furthermore, free 5SCD may serve as a good marker of the effect of SSR and UVB.

Regulation of eumelanin/pheomelanin synthesis and visible pigmentation in melanocytes by ligands of the melanocortin 1 receptor.

The production of melanin in the hair and skin is tightly regulated by the melanocortin 1 receptor (MC1R) whose activation is controlled by two secreted ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signal protein (ASP). As melanin is extremely stable, lasting years in biological tissues, the mechanism underlying the relatively rapid decrease in visible pigmentation elicited by ASP is of obvious interest. In this study, the effects of ASP and alphaMSH on the regulation of melanin synthesis and on visible pigmentation were assessed in normal murine melanocytes and were compared with the quick depigmenting effect of the tyrosinase inhibitor, phenylthiourea (PTU). alphaMSH increased pheomelanin levels prior to increasing eumelanin content over 4 days of treatment. Conversely, ASP switched off the pigment synthesis pathway, reducing eu- and pheo-melanin synthesis within 1 day of treatment that was proportional to the decrease in tyrosinase protein level and activity. These results demonstrate that the visible depigmentation of melanocytes induced by ASP does not require the degradation of existing melanin but rather is due to the dilution of existing melanin by melanocyte turnover, which emphasizes the importance of pigment distribution to visible color.

Regulation of human skin pigmentation and responses to ultraviolet radiation.

Pigmentation of human skin is closely involved in protection against environmental stresses, in particular exposure to ultraviolet (UV) radiation. It is well known that darker skin is significantly more resistant to the damaging effects of UV, such as photocarcinogenesis and photoaging, than is lighter skin. Constitutive skin pigmentation depends on the amount of melanin and its distribution in that tissue. Melanin is significantly photoprotective and epidermal cells in darker skin incur less DNA damage than do those in lighter skin. This review summarizes current understanding of the regulation of constitutive human skin pigmentation and responses to UV radiation, with emphasis on physiological factors that influence those processes. Further research is needed to characterize the role of skin pigmentation to reduce photocarcinogenesis and to develop effective strategies to minimize such risks.

Anti-inflammatory efficacy of Licochalcone A: correlation of clinical potency and in vitro effects.

Licochalcone A (LicA), a major phenolic constituent of the licorice species Glycyrrhiza inflata, has recently been reported to have anti-inflammatory as well as anti-microbial effects. These anti-inflammatory properties might be exploited for topical applications of LicA. We conducted prospective randomized vehicle-controlled clinical trials to assess the anti-irritative efficacy of cosmetic formulations containing LicA in a post-shaving skin irritation model and on UV-induced erythema formation. The clinical trials were accompanied by a series of in vitro experiments to characterize anti-inflammatory properties of LicA on several dermatologically relevant cell types. Topical LicA causes a highly significant reduction in erythema relative to the vehicle control in both the shave- and UV-induced erythema tests, demonstrating the anti-irritative properties of LicA. Furthermore, LicA is a potent inhibitor of pro-inflammatory in vitro responses, including N-formyl-MET-LEU-PHE (fMLP)- or zymosan-induced oxidative burst of granulocytes, UVB-induced PGE(2) release by keratinocytes, lipopolysaccharide (LPS)-induced PGE(2) release by adult dermal fibroblasts, fMLP-induced LTB(4) release by granulocytes, and LPS-induced IL-6/TNF-alpha secretion by monocyte-derived dendritic cells. The reported data suggest therapeutic skin care benefits from LicA when applied to sensitive or irritated skin.

Solar-simulated radiation induces secretion of IL-6 and production of isoprostanes in human skin in vivo.

Cytokines represent an integral part of the large group of mediators involved in dermal inflammation. In this in vivo study, ultraviolet light which is one of the major environmental factors affecting cytokine release patterns in the skin was employed. The effects of repeated versus one-time irradiation with solar-simulated ultraviolet light was studied regarding the secretion of proinflammatory cytokines with a Bio-Plex cytokine assay using suction blisters as a model for localized inflammatory processes. The IL-6 concentration increased markedly after 24 h in skin areas irradiated with a twofold minimal erythemal dose (MED) compared to areas challenged repeatedly with 0.3 MED. In addition, we investigated the concentration of 8-isoprostane in the suction blister fluid as a marker of lipid peroxidation due to a UV-induced increase in free radical production. 8-Isoprostane was increased immediately after treatment but declined after 24 h with the exception of the skin area exposed to 2 MED. The differential expression and release of cytokines, and the extent of oxidative damage might therefore depend on the dose and regimen of exposure to solar-simulated radiation.

Mechanisms of skin tanning in different racial/ethnic groups in response to ultraviolet radiation.

Ultraviolet radiation stimulates pigmentation in human skin, but the mechanism(s) whereby this increase in melanin production (commonly known as tanning) occurs is not well understood. Few studies have examined the molecular consequences of UV on human skin of various racial backgrounds in situ. We investigated the effects of UV on human skin of various races before and at different times after a single 1 minimal erythemal dose UV exposure. We measured the distribution of DNA damage that results, as well as the melanin content/distribution and the expression of various melanocyte-specific genes. The density of melanocytes at the epidermal:dermal junction in different types of human skin are remarkably similar and do not change significantly within 1 wk after UV exposure. The expression of melanocyte-specific proteins (including TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), DCT (tyrosinase-related protein 2), MART1 (melanoma antigens recognized by T-cells) gp100 (Pmel17/silver), and MITF (micropthalmia transcription factor)) increased from 0 to 7 d after UV exposure, but the melanin content of the skin increased only slightly. The most significant change, however, was a change in the distribution of melanin from the lower layer upwards to the middle layer of the skin, which was more dramatic in the darker skin. These results provide a basis for understanding the origin of different skin colors and responses to UV within different races.

Contribution of UVB and UVA to UV-dependent stimulation of cyclooxygenase-2 expression in artificial epidermis.

Both UVB (280-320 nm) and UVA (320-400 nm) radiation lead to an enhanced expression of cyclooxygenase-2 (COX-2) in epidermal cells in various in-vitro and in-vivo models. It is demonstrated here that the expression of COX-2 is induced in artificial human epidermis exposed to simulated solar light (>290 nm). Employing filters eliminating specified regions from the simulated solar spectrum, the UVB and UVA-2 (320-350 nm) regions are shown to fully account for induction of COX-2 mRNA and protein as well as the enhanced production of prostaglandin E(2) after irradiation. At the protein level, approximately 70% of the total induction by solar light is due to light in the UVA-2 region. UVA-1 (350-400 nm), visible light and IR radiation are practically ineffective. COX-2 induction by simulated solar light is attenuated in the presence of inhibitors of p38(MAPK) or of c-Jun-N-terminal kinases (JNK), whereas COX-2 induction by UVA is blocked only by inhibition of JNK. UV-induced COX-2 expression is not affected by inhibition of the MEK 1,2/ERK 1,2 pathways.

[Alpha-glucosylrutin, a highly effective flavonoid for protection against oxidative stress]. / Alpha-Glucosylrutin: ein hochwirksames Flavonoid zum Schutz vor oxidativem Stress.

The flavonoid alpha-glucosylrutin (AGR) is a potent antioxidant with a high epidermal bioavailability. This makes this substance particularly suitable for various dermato-cosmetic applications. Flavonoids are phytamines with a common chemical structure and a broad range of activities, the most prominent being their radical scavenging ability. Reactive oxygen species (ROS) damage cells by different mechanisms. Direct cytotoxic effects include destruction of the cell membrane by causing radical chain reactions or induction of mutagenic changes in the nuclear and mitochondrial DNA. Indirect changes involve modification of intracellular signal transduction pathways that regulate inflammatory or proliferative activities. The excellent antioxidant efficacy of AGR has been shown in various experimental studies, both in vitro and in vivo. Subsequent clinical studies have demonstrated that AGR is also effective in the prevention of dermatologic diseases in which oxidative stress is of pathogenetic relevance, e.g. in polymorphous light eruption (PLE). Other promising dermato-cosmetic areas for AGR application are aging of the skin, especially photoaging. All in vivo evaluations indicate that AGR in the applied concentrations is a very well-tolerated ingredient for medical skin care preparations.

Reconstituted 3-dimensional human skin of various ethnic origins as an in vitro model for studies of pigmentation.

Reconstituted 3-dimensional human skin equivalents containing melanocytes and keratinocytes on an artificial dermal substitute are gaining popularity for studies of skin metabolism because they exhibit morphological and growth characteristics similar to human epidermis. In this study, we show that such a pigmented epidermis model can be used to assess the regulation of pigmentation by known melanogenic compounds. In monolayers or in melanocyte-keratinocyte co-cultures, melanocyte-keratinocyte interactions are missing or are spatially limited. The commercial skin equivalents used in this study were derived from epidermal cells obtained from donors of three different ethnic origins (African- American, Asian, and Caucasian), and they reflect those distinct skin phenotypes. We used these pigmented human epidermis models to test compounds for potential effects on pigmentation in a more physiologically relevant context, which allows further characterization and validation of interesting melanogenic factors. We used known melanogenic stimulators (alpha-melanocyte-stimulating hormone and 3,4-dihydroxyphenylalanine) and inhibitors (hydroquinone, arbutin, kojic acid, and niacinamide) and examined their effects on the production of melanin and its distribution in upper layers of the skin. Our studies indicate that commercial skin equivalents provide a convenient and cost-effective alternative to animal testing for evaluating the regulation of mammalian pigmentation by melanogenic factors and for elucidating their mechanisms of action.