Herbert Hönigsmann, Vienna, Austria Edward M. Jackson, Bonney Lake, Wash., USA Y.N. Kalia, Archamps, France Helena Kandárová, Ashland, Mass., USA S. Kezic, Amsterdam, The Netherlands Fanny Knorr, Bochum, Germany Hans-Christian Korting, Munich, Germany Marita Kosmadaki, Athens, Greece Axel Kramer, Greifswald-Eldena, Germany Jean Krutmann, Düsseldorf, Germany Christine Lang, Berlin, Germany David Leaper, Cardiff, UK J.J. Leyden, Philadelphia, Pa., USA Marie L. Lodén, Upplands Väsby, Sweden V. Luu-The, Sainte-Foy, Qué., Canada G. Mahrle, Köln, Germany Howard I. Maibach, San Francisco, Calif., USA Paul Matts, Egham, UK Hans Meffert, Berlin, Germany Martina Meinke, Berlin, Germany Hans F. Merk, Aachen, Germany Hasan Mukhtar, Madison, Wisc., USA Gerit D. Mulder, San Diego, Calif., USA Christel Müller-Goymann, Braunschweig, Germany Reinhard H.H. Neubert, Halle/Saale, Germany Gerhard J. Nohynek, Asnières, France Nina Otberg, Berlin, Germany M.C. Pardon, Paris, France José Luis Parra Juez, Barcelona, Spain Alexa Patzelt, Berlin, Germany I. Pechan, Bratislava, Czech Republic Giovanni Pellacani, Modena, Italy Andrea Peserico, Padova, Italy Neena Philips, Teaneck, N.J., USA M. Picardo, Rome, Italy Gerald E. Piérard, Liège, Belgium D. Pin, Lyon, France Nihal Ahmad, Madison, Wisc., USA Christina Antoniou, Kessariani, Greece Marco Ardigo, Rome, Italy Ojan Assadian, Vienna, Austria J.M. Baron, Aachen, Germany P. Bartak, Olomouc, Czech Republic Eva Benfeldt, Hellerup, Denmark Heather Benson, Perth, W.A., Australia Bruno A. Bernard, Clichy, France Hans Konrad Biesalski, Stuttgart, Germany T. Bito, Berkeley, Calif., USA Ulrike Blume-Peytavi, Berlin, Germany Zita Borbenyi, Szeged, Hungary Joke A. Bouwstra, Leiden, The Netherlands D. Bucks, Petaluma, Calif., USA C.N. Burkhart, Chapel Hill, N.C., USA Razvigor Darlenski, Sofia, Bulgaria Maxim E. Darvin, Berlin, Germany Lisa DeLouise, Rochester, N.Y., USA Kristien De Paepe, Brussels, Belgium M. De Reggi, Marseille, France Sandrine Dubrac, Innsbruck, Austria Anne E. Eady, Leeds, UK Peter Elsner, Jena, Germany Johan Engblom, Malmö, Sweden Alfred Fahr, Jena, Germany Arpad Farkas, Szeged, Hungary Manigé Fartasch, Bochum, Germany Tobias Fischer, Lübeck, Germany Joachim W. Fluhr, Berlin, Germany Wolfgang Gehring, Karlsruhe, Germany Curtis G. Gemmell, Glasgow, UK Heiner Gers-Barlag, Hamburg, Germany Malcolm W. Greaves, Singapore, Singapore Ramon Grimalt, Barcelona, Spain H. Gupta, New Delhi, India Richard H. Guy, Bath, UK Henning Hamm, Würzburg, Germany Steffi Hansen, Saarbrücken, Germany Ulrike Heinrich, Witten, Germany

Herbert Hönigsmann, Vienna, Austria Edward M. Jackson, Bonney Lake, Wash., USA Y.N. Kalia, Archamps, France Helena Kandárová, Ashland, Mass., USA S. Kezic, Amsterdam, The Netherlands Fanny Knorr, Bochum, Germany Hans-Christian Korting, Munich, Germany Marita Kosmadaki, Athens, Greece Axel Kramer, Greifswald-Eldena, Germany Jean Krutmann, Düsseldorf, Germany Christine Lang, Berlin, Germany David Leaper, Cardiff, UK J.J. Leyden, Philadelphia, Pa., USA Marie L. Lodén, Upplands Väsby, Sweden V. Luu-The, Sainte-Foy, Qué., Canada G. Mahrle, Köln, Germany Howard I. Maibach, San Francisco, Calif., USA Paul Matts, Egham, UK Hans Meffert, Berlin, Germany Martina Meinke, Berlin, Germany Hans F. Merk, Aachen, Germany Hasan Mukhtar, Madison, Wisc., USA Gerit D. Mulder, San Diego, Calif., USA Christel Müller-Goymann, Braunschweig, Germany Reinhard H.H. Neubert, Halle/Saale, Germany Gerhard J. Nohynek, Asnières, France Nina Otberg, Berlin, Germany M.C. Pardon, Paris, France José Luis Parra Juez, Barcelona, Spain Alexa Patzelt, Berlin, Germany I. Pechan, Bratislava, Czech Republic Giovanni Pellacani, Modena, Italy Andrea Peserico, Padova, Italy Neena Philips, Teaneck, N.J., USA M. Picardo, Rome, Italy Gerald E. Piérard, Liège, Belgium D. Pin, Lyon, France Nihal Ahmad, Madison, Wisc., USA Christina Antoniou, Kessariani, Greece Marco Ardigo, Rome, Italy Ojan Assadian, Vienna, Austria J.M. Baron, Aachen, Germany P. Bartak, Olomouc, Czech Republic Eva Benfeldt, Hellerup, Denmark Heather Benson, Perth, W.A., Australia Bruno A. Bernard, Clichy, France Hans Konrad Biesalski, Stuttgart, Germany T. Bito, Berkeley, Calif., USA Ulrike Blume-Peytavi, Berlin, Germany Zita Borbenyi, Szeged, Hungary Joke A. Bouwstra, Leiden, The Netherlands D. Bucks, Petaluma, Calif., USA C.N. Burkhart, Chapel Hill, N.C., USA Razvigor Darlenski, Sofia, Bulgaria Maxim E. Darvin, Berlin, Germany Lisa DeLouise, Rochester, N.Y., USA Kristien De Paepe, Brussels, Belgium M. De Reggi, Marseille, France Sandrine Dubrac, Innsbruck, Austria Anne E. Eady, Leeds, UK Peter Elsner, Jena, Germany Johan Engblom, Malmö, Sweden Alfred Fahr, Jena, Germany Arpad Farkas, Szeged, Hungary Manigé Fartasch, Bochum, Germany Tobias Fischer, Lübeck, Germany Joachim W. Fluhr, Berlin, Germany Wolfgang Gehring, Karlsruhe, Germany Curtis G. Gemmell, Glasgow, UK Heiner Gers-Barlag, Hamburg, Germany Malcolm W. Greaves, Singapore, Singapore Ramon Grimalt, Barcelona, Spain H. Gupta, New Delhi, India Richard H. Guy, Bath, UK Henning Hamm, Würzburg, Germany Steffi Hansen, Saarbrücken, Germany Ulrike Heinrich, Witten, Germany

To learn about the interaction between drug agents and nanoparticular carrier systems, the physical analytical methods of parelectric, electron spin and fluorescence spectroscopy have proven helpful tools to yield descriptive models of such complex systems. For a deeper understanding of drug absorption from body surfaces and drug distribution into the tissues, however, the lack of knowledge about the interaction between such agents and membranes on different levels is a severe drawback. This gap can be closed by the application of atomic force microscopy at normal temperatures and under the admission of liquid surroundings. Moreover, this method allows the inspection of such system-membrane interactions in dependence on time. We studied membrane topography in liquid and gel-phase mixtures, structural changes of membranes during their destruction by aqueous peptide solutions as well as the stability of the membranes exposed to surfactants of increasing concentration and to lipid nanoparticles (solid lipid nanoparticles, nanostructured lipid carriers). For future modelling we can describe the geometry of lipid nanoparticles as well.

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Rilopirox and piroctone belong to the class of hydroxypyridone antimycotics. This class is not related to other antimycotics. In contrast to azole antimycotics and polyene antimycotics the mode of action of hydroxypyridone antimycotics is not fully understood. Inhibition of cellular uptake of essential compounds as well as loss of other compounds seems to be only a secondary effect of a primary not known action of these drugs. The antifungal effect in vitro depends on the medium used. The hyphal induction of Candida albicans is inhibited by hydroxypyridone antimycotics, but this effect is compensated by iron ions. A damage of the cell membrane and a direct influence on adenosine triphosphate synthesis, respectively, do not seem to be part of the mode of action. But there are clear hints that reactive oxygen species (ROS) and available metabolic activity are important parts of the mode of action of the hydroxypyridone antimycotics rilopirox and piroctone.

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.

For testing cutaneous absorption of drugs, ingredients of cosmetics and also for risk assessment of industrial compounds predictable in vitro test protocols are under investigation using excised skin or reconstructed human epidermis. Since the metabolizing enzymes expressed by viable skin can influence the absorption behaviour of substances by changing their structure and thereby their physicochemical characteristics, the metabolic capacity should be considered in the design of the test protocols of compounds susceptible to metabolism. Then data, generated using viable reconstructed epidermis may reflect the in vivo situation. Interestingly, bovine serum albumin (BSA) commonly used in receptor media in permeation studies to facilitate solubility of highly lipophilic substances strongly inhibited the metabolism of topically applied prednicarbate in reconstructed epidermis. Here, we show that 5% BSA is toxic to reconstructed epidermis and keratinocytes which was consistent with the earlier findings. While media toxicity (deficiency media) was at least partly the cause of both apoptotic and necrotic processes in keratinocytes, BSA only slightly increased the rate of necrotic cells. Moreover, caspase inhibitors did not reduce BSA toxicity. Yet, the results show that BSA toxicity on keratinocytes has to be carefully considered if this protein is used in permeation studies with reconstructed epidermis.

Aim and Methods: Drug development in dermatotherapy and also development of transdermal therapeutic systems (TTS) demand high-predictive in vitro models to estimate drug levels in skin and systemic uptake. Here we compare three ready-to-use models, reconstructed human epidermis, split porcine skin and the perfused porcine forelimb. 17β-Estradiol (E2), which is highly metabolized by skin cells, serves as model drug since E2 application is of high relevance in hormone replacement therapy while topical E2 may promote wound healing. E2 TTS, gel and an ethanolic solution were investigated for cutaneous penetration, permeation and metabolism. Results: E2 TTS enabled an E2 uptake of 42.9% of the applied dose accompanied by a high percentage of E2 metabolism (30% of the penetrated dose) in the perfused porcine forelimb. In Franz cell experiments with reconstructed human epidermis and split porcine skin, the gel allowed an E2 uptake of 41.7 and 22.9% of the applied dose accompanied by a high E2 metabolism (42.6 and 28.6% of the penetrated dose). Due to toxic effects of the vehicle, this was not true with an ethanolic solution, then E2 permeation and metabolism were clearly diminished. Most importantly, the in vitro models proved to be predictive with respect to the E2/estrone ratio in female plasma under transdermal hormone replacement therapy. Conclusion: In vitro tests should reduce the need for both animal and human studies for cutaneous uptake and metabolism in the future.

Acne and androgenetic alopecia are linked to androgen effects and therefore should improve following topical application of antiandrogens. We present a new antiandrogen prodrug, RU 58841-myristate (RUM) for topical therapy. Almost devoid of affinity to the androgen receptor, as derived from investigations in the mouse fibroblast cell line 29+/GR+, RUM is rapidly metabolised to the potent antiandrogen RU 58841 by cultured human foreskin fibroblasts and keratinocytes, male occipital scalp skin dermal papilla cells, and by cells of the sebaceous gland cell line SZ95. In order to improve a specific targeting of the hair follicle, RUM was loaded on solid lipid nanoparticles (SLN), which are already known to support dermal targeting effects. Physically stable RUM loaded SLN were produced by hot homogenization. Penetration/permeation studies carried out using the Franz diffusion cell proved only negligible permeation of reconstructed epidermis and excised porcine skin within 6 h, implying a more topical action of the drug. Targeting to the hair follicle using SLN was visualised by fluorescence microscopy, following the application of Nile Red labelled SLN to human scalp skin. Transmission electron microscopy (TEM) allowed to detect intact silver labelled SLN in porcine hair follicles of preparations applied to the skin for 24 h. RUM loaded SLN should be considered for topical antiandrogen therapy of acne and androgenetic alopecia.

With topical treatment of skin diseases, the requirement of a high and reproducible drug uptake often still is not met. Moreover, drug targeting to specific skin strata may improve the use of agents which are prone to cause local unwanted effects. Recent investigations have indicated that improved uptake and skin targeting may become feasible by means of nanoparticular systems such as solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and nanoemulsions (NE). Here we describe techniques to characterize drug loading to carrier systems and skin penetration profiles by using the lipophilic dye nile red as a model agent. Since the mode of drug association with the particle matrix may strongly influence the efficiency of skin targeting, parelectric spectroscopy (PS) was used to differentiate between matrix incorporation and attachment to the particle surface and fluorescence spectroscopy (FS) to solve dye distribution within NLC particles. Nile red was incorporated into the lipid matrix or the covering tensed shell, respectively, of SLN and NLC with all the lipids studied (Compritol, Precirol, oleic acid, Miglyol). In NLC, the dye was enriched in the liquid phase. Next, nile red concentrations were followed by image analysis of vertical sections of pigskin treated with dye-loaded nanoparticular dispersions and an oil-in-water cream for 4 and 8 h in vitro. Following the SLN dispersions, dye penetration increased about fourfold over the uptake obtained following the cream. NLC turned out less potent (< threefold increase) and penetration appeared even reduced when applying a NE. In contrast to previous studies with glucocorticoids attached to the surface of SLN, a targeting effect was not detected here. Therefore, drug targeting appears to be more strictly related to the mode of interaction of drug and particle than penetration enhancement.

OBJECTIVES:Ciclopirox is a topical antifungal agent of the hydroxypyridone class whose mode of action is poorly understood. In order to elucidate the mechanism of action of ciclopirox, we analysed the growth, cellular integrity, biochemical properties, viability and transcriptional profile of the polymorphic yeast Candida albicans following exposure to this antifungal agent.METHODS:Multiple biochemical assays served to identify factors that were critical for antifungal activity and to identify proteins whose activities changed in drug-exposed cells. Genome-wide transcriptional profiling was used to identify genes that were up-regulated in response to the cellular effects of the drug.RESULTS:Ciclopirox inhibited growth of C. albicans yeast and hyphal cells in a dose-dependent manner. This effect was reduced (i) by the addition of iron ions or the metabolic inhibitor 2-deoxy-D-glucose to growth media, (ii) in media that lacked glucose, and (iii) for cells that were pre-incubated with hydrogen peroxide or menadione [which caused induction of proteins involved in detoxification of reactive oxygen species (ROS)]. In contrast, cells pre-cultured under poor oxygen conditions (which had decreased activity of proteins involved in ROS detoxification) were more susceptible to ciclopirox. Treatment with ciclopirox did not directly cause cell membrane damage and did not change intracellular levels of ATP. Finally, the transcriptional profiling pattern of drug-treated cells strongly resembled iron-limited conditions.CONCLUSIONS:These data indicate that metabolic activity, oxygen accessibility and iron levels are critical parameters in the mode of action of ciclopirox olamine.

Glucocorticoids are potent anti-inflammatory and immunomodulatory drugs which also induce growth inhibition in a variety of cell types. For this reason long-term treatment of inflammatory skin diseases may result in irreversible skin atrophy. To elucidate whether the antiproliferative action of glucocorticoids in fibroblasts is accompanied by induction of apoptosis we investigated the influence of dexamethasone (DEX) on both parameters. Interestingly, we revealed that growth inhibitory concentrations of this glucocorticoid did not induce fibroblast apoptosis. Moreover, DEX protected these cells from apoptosis induced by tumor necrosis factor alpha (TNFalpha)/actinomycin, UV-irradiation, and cell permeable ceramides. These findings are in contrast to the lack of anti-apoptotic effects detected in keratinocytes. Although DEX inhibited TNFalpha mediated nuclear factor-kappa (NF-kappaB) activity in fibroblasts, this mechanism was not involved in its cytoprotection as it was verified by specific NF-kappaB inhibitors. Therefore, we looked for alternative intracellular mediators. Coincubation of fibroblasts with the sphingosine kinase inhibitor N,N-dimethylsphingosine, which blocks formation of the sphingolipid degradation product sphingosine-1-phosphate (S1P), abrogated the protective glucocorticoid effect almost completely. As preincubation with S1P reduced the number of apoptotic cells after stimulation with TNFalpha/actinomycin and moreover DEX increased the intracellular S1P content a role of this sphingolipid in the cytoprotection by DEX is suggested.

Tazarotene is a member of the new generation of receptor-selective, synthetic retinoids for the topical treatment of mild to moderate plaque psoriasis, acne vulgaris and photoaging. Though they are effective in monotherapy, clinical studies with a focus on novel combination treatments and a comparison of different agents for these skin disorders are accumulating. The concomitant use of tazarotene with a mid-potency or high-potency corticosteroid enhances the efficacy in psoriatic plaques and reduces the risk of steroid-induced skin atrophy. Combining phototherapy with adjunctive tazarotene accelerates the clinical response and reduces the cumulative UVB or PUVA exposure load. Tazarotene applied once daily is superior to adapalene monotherapy in acne vulgaris and is efficacious in the treatment of photodamage. Novel therapeutic regimens such as short-contact therapy have been developed for both acne and psoriasis in order to diminish the major adverse events like pruritus, burning, local skin irritation and erythema.

Lysophosphatidic acid (LPA, 1-acyl-glycerol-3-phosphate) plays an important role in diverse biological responses including cell proliferation, differentiation, survival, migration, and tumor cell invasion. The most prominent source of LPA is platelets from which it is released after thrombin activation and is assumed to be an essential function of this lysophospholipid in cutaneous wound closure. Therefore, we examined the role of LPA on biological responses of keratinocytes. Although LPA potently enhances keratinocyte migration, it strongly induces growth arrest of proliferating epidermal cells. Thus, LPA possesses analogous actions to transforming growth factor-beta (TGF-beta), which is also released from degranulating platelets at wounded sites. In contrast to LPA, the intracellular signaling events of TGF-beta have been clearly identified and indicate that Smad3 is involved in chemotaxis and cell growth arrest of keratinocytes induced by this cytokine. Here we show that LPA, although it does not alter TGF-beta release is capable to activate Smad3 and results in a heteromerization with Smad4 and binding of the complex to its specific DNA-promoter elements. LPA completely fails to induce chemotaxis in Smad3-deficient cells, whereas growth inhibition is at least in part reduced. These findings indicate an essential role of Smad3 in diverse biological properties of LPA-stimulated keratinocytes.

The glycosaminoglycan hyaluronic acid (HA), or hyaluronan, is a major component of the extracellular matrix of skin, joints, eye and many other tissues and organs. In spite of its simple structure, HA demonstrates remarkable rheological, viscoelastic and hygroscopic properties which are relevant for dermal tissue function. Biological activities in skin, however, are also due to its interaction with various binding proteins (hyaladherins). Due to an influence on signaling pathways, HA is involved in the wound-healing process and scarless fetal healing. Increased HA concentrations have been associated with inflammatory skin diseases. In clinical trials, topical application of HA improved wound healing; in particular, acute radioepithelitis, venous leg ulcers or diabetic foot lesions responded to HA treatment. Moreover, as a topical drug delivery system for diclofenac, an HA gel has recently been approved for the treatment of actinic keratoses. Finally, chemical modifications led to new HA derivates and biomaterials, which may be introduced into therapy in the future. Therefore, ongoing research offers new horizons for the therapeutic use of this glycosaminoglycan which has been regarded as an inert structural component until recently.