Cationic membrane-active peptides - anticancer and antifungal activity as well as penetration into human skin.

Cationic antimicrobial peptides are ancient natural broad-spectrum antibiotics, and several compounds also exhibit anticancer activity. However, most applications pertain to bacterial infections, and treatment for skin cancer is less frequently considered. The cytotoxicity of melittin, cecropin A, protegrin-1 and histatin 5 against squamous skin cancer cell lines and normal human keratinocytes was evaluated and compared to established drugs. The results show that melittin clearly outperforms 5-fluorouracil regarding antitumor activity. Importantly, combined melittin and 5-fluorouracil enhanced cytotoxic effects on cancer cells and reduced toxicity on normal keratinocytes. Additionally, minimum inhibitory concentrations indicate that melittin also shows superior activity against clinical and laboratory strains of Candida albicans compared to amphotericin B. To evaluate its potential for topical applications, human skin penetration of melittin was investigated ex vivo and compared to two non-toxic cell-penetrating peptides (CPPs), low molecular weight protamine (LMWP) and penetratin. The stratum corneum prevents penetration into viable epidermis over 6 h; however, the peptides gain access to the viable skin after 24 h. Inhibition of digestive enzymes during skin penetration significantly enhances the availability of intact peptide. In conclusion, melittin may represent an innovative agent for non-melanoma skin cancer and infectious skin diseases. In order to develop a drug candidate, skin absorption and proteolytic digestion by skin enzymes need to be addressed.

Impact of age and body site on adult female skin surface pH.

BACKGROUND: pH is known as an important parameter in epidermal barrier function and homeostasis. AIM: The impact of age and body site on skin surface pH (pH(SS)) of women was evaluated in vivo. METHODS: Time domain dual lifetime referencing with luminescent sensor foils was used for pH(SS) measurements. pH(SS) was measured on the forehead, the temple, and the volar forearm of adult females (n = 97, 52.87 ± 18.58 years, 20-97 years). Every single measurement contained 2,500 pH values due to the luminescence imaging technique used. RESULTS: pH(SS) slightly increases with age on all three investigated body sites. There are no significant differences in pH(SS) between the three investigated body sites. CONCLUSION: Adult pH(SS) on the forehead, the temple and the volar forearm increases slightly with age. This knowledge is crucial for adapting medical skin care products.

The Munich outbreak of cutaneous cowpox infection: transmission by infected pet rats.

Cowpox virus infection of humans is an uncommon, potentially fatal, skin disease. It is largely confined to Europe, but is not found in Eire, or in the USA, Australasia, or the Middle or Far East. Patients having contact with infected cows, cats, or small rodents sporadically contract the disease from these animals. We report here clinical aspects of 8 patients from the Munich area who had purchased infected pet rats from a local supplier. Pet rats are a novel potential source of local outbreaks. The morphologically distinctive skin lesions are mostly restricted to the patients' necks, reflecting the infected animals' contact pattern. Individual lesions vaguely resemble orf or Milker's nodule, but show marked surrounding erythema, firm induration and local adenopathy. Older lesions develop eschar, leaving slow-healing, deep ulcerative defects after eschar separation. Severe flu-like illness may be present in the acute phase. Smallpox-vaccinated patients tend to develop less severe reactions and heal more quickly. The differential diagnosis may include other localized orthopoxvirus infections, herpes simplex, bacterial infection, anthrax, foreign body granuloma, and primary tuberculosis. Dermatologists should be aware of the diagnostic and therapeutic algorithms for handling this disease.

Medical devices in dermatology: topical semi-solid formulations for the treatment of skin diseases.

In recent years, topically applied semi-solid formulations certified as medicals devices and not as topical drugs are increasingly used for the treatment of skin diseases. Medical devices primarily unfold their therapeutic effect by physical means, not by pharmacological, immunological or metabolic means. Intensified placing of medical devices on the dermatological market may at least partly be explained by a less complex marketing authorization process compared to topical drugs. If the requirements are fulfilled to certify a product as a medical device the opportunity will be offered to quickly introduce innovations onto the market and propagate them. A variety of evidence-based medical devices for several dermatological indications are presented here.

Direct detection of five common dermatophyte species in clinical samples using a rapid and sensitive 24-h PCR-ELISA technique open to protocol transfer.

Identification of dermatophytes is usually based on morphological characteristics determined by time-consuming microscopic and cultural examinations. An effective PCR-ELISA method has been developed for rapid detection of dermatophyte species directly from clinical specimens within 24 h. Isolated genomic DNA of skin scrapings and nail samples from patients with suspected dermatophyte infections is amplified with species-specific digoxigenin-labelled primers targeting the topoisomerase II gene. The subsequent ELISA procedure with biotin-labelled probes allows a sensitive and specific identification of the five common dermatophytes -Trichophyton rubrum, T. interdigitale, T. violaceum, Microsporum canis and Epidermophyton floccosum. PCR-ELISA, based on the new polyphasic species concept, was assessed using 204 microscopy-positive samples in two university mycological laboratories in Munich and Tübingen, and 316 consecutive specimens - regardless of mycological findings - in a dermatological practice laboratory in Neu-Ulm. One of the five dermatophytes was confirmed by PCR-ELISA in 163 of 204 (79.9%) of the clinical samples from the university hospitals found positive using microscopy. Culture was positive for dermatophytes in 59.8% of the same cases. A significant difference between these two methods could be demonstrated using the McNemar test (P < 0.005). Analysis of specimens from Neu-Ulm confirmed the results in a dermatological practice laboratory as 25.0% of the specimens had positive PCR results, whereas only 7.3% were positive according to culture. Direct DNA isolation from clinical specimens and the PCR-ELISA method employed in this study provide a rapid, reproducible and sensitive tool for detection and discrimination of five major dermatophytes at species level, independent of morphological and biochemical characteristics.

Efficacy of cream-based novel formulations of hyaluronic acid of different molecular weights in anti-wrinkle treatment.

BACKGROUND: Due to its strong water-binding potential, hyaluronic acid (HA) is a well-known active ingredient for cosmetic applications. Native HA is proposed to help the skin to retain and maintain elasticity, turgor and moisture. OBJECTIVE: To observe the efficacy of topical application of 0.1% hyaluronan formulations of different molecular weights (MW) (50, 130, 300, 800 and 2000 kDa, respectively) in the periocular area as anti-wrinkle treatment. MATERIAL AND METHODS: Seventy-six female subjects between 30 and 60 years of age with clinical signs of periocular wrinkles applied one of the formulations twice-daily to the area of interest in a randomized fashion for 60 days. Around the other eye, a vehicle control cream was applied. Measurements of skin hydration and skin elasticity were performed before treatment, 30 and 60 days thereafter. At similar time points negative replicas were taken and evaluated by semi-automated morphometry. RESULTS: All HA-based creams utilized in this study demonstrated a significant improvement in skin hydration and overall elasticity values (R2) when compared to placebo. Measurements of wrinkle depth using mean roughness (Ra) and maximum roughness (Rz) values revealed significant improvement in the 130 and the 50 kDa HA group after 60 days of treatment compared to placebo-treated area. CONCLUSION: Topical application of all 0.1% HA formulations used in this study led to significant improvement in skin hydration and elasticity. Application of low-molecular-weight (LMW) HA was associated with significant reduction of wrinkle depth, which may be due to better penetration abilities of LMW HA.

Hallmarks of atopic skin mimicked in vitro by means of a skin disease model based on FLG knock-down.

Loss-of-function mutations in the filaggrin gene (FLG) are a strong predisposing factor for atopic dermatitis, although their relevance to the disease pathomechanism needs further elucidation. The generation of an in vitro model of atopic skin would not only permit further evaluation of the underlying pathogenetic mechanisms and the testing of new treatment options, but would also allow toxicological studies to be performed in a simple, rapid and inexpensive manner. In this study, we have knocked down FLG expression in human keratinocytes and created three-dimensional skin models, which we used to investigate the impact of FLG on epidermal maturation and on skin absorption and its response to irritation. Histopathological evaluation of the skin models showed impaired epidermal differentiation in the FLG knock-down model. In addition, skin irritation induced by an application of sodium dodecyl sulphate resulted in significantly higher lactate dehydrogenase leakage, and interleukin (IL)-6 and IL-8 levels, than in the control model. To assess the effect of filaggrin deficiency on skin absorption of topically applied agents, we quantified the percutaneous absorption of lipophilic and hydrophilic model drugs, finding clinical relevance only for lipophilic drugs. This study clearly demonstrates that important clinical characteristics of atopic skin can be mimicked by using in vitro skin models. The FLG knock-down construct is the first step toward an in vitro model that allows clinical and toxicological studies of atopic-like skin.

Human epithelial cells establish direct antifungal defense through TLR4-mediated signaling.

Mammalian TLRs are central mediators of the innate immune system that instruct cells of the innate and adaptive response to clear microbial infections. Here, we demonstrate that human epithelial TLR4 directly protected the oral mucosa from fungal infection via a process mediated by polymorphonuclear leukocytes (PMNs). In an in vitro epithelial model of oral candidiasis, the fungal pathogen Candida albicans induced a chemoattractive and proinflammatory cytokine response but failed to directly modulate the expression of genes encoding TLRs. However, the addition of PMNs to the C. albicans-infected model strongly upregulated cytoplasmic and cell-surface epithelial TLR4 expression, which correlated directly with protection against fungal invasion and cell injury. C. albicans invasion and cell injury was restored by the addition of TLR4-specific neutralizing antibodies and knockdown of TLR4 using RNA interference, even in the presence of PMNs, demonstrating the direct role of epithelial TLR4 in the protective process. Furthermore, treatment with neutralizing antibodies specific for TNF-alpha resulted in strongly reduced TLR4 expression accompanied by augmented epithelial cell damage and fungal invasion. To our knowledge, this is the first description of such a PMN-dependent, TLR4-mediated protective mechanism at epithelial surfaces, which may provide significant insights into how microbial infections are managed and controlled in the oral mucosa.

Carriers in the topical treatment of skin disease.

Topical drug application is less prone to severe systemic side-effects than systemic application. Starting with the liposomes, various types of nanosized and microsized drug carriers have been developed to increase the notoriously low penetration of active agents into the skin, which limits not only the topical therapy of skin disease but also transdermal therapy. Today, liposome- and microsponge-based preparations are approved for dermatomycosis, acne and actinic keratosis. Under investigation are drug carriers such as lipid nanoparticles, polymeric particles, dendrimers, and dendritic-core multi-shell nanotransporters. According to the rapidly increasing research in this field, both in academia and industry, a breakthrough appears likely, once stability problems (nanoparticles) and safety concerns (dendrimers) are overcome. Technical approaches and results of in vitro, ex vivo and in vivo testing are described, taking into account pharmacokinetic, efficacy and safety aspects.

Human polymerase alpha inhibitors for skin tumors. Part 2. Modeling, synthesis and influence on normal and transformed keratinocytes of new thymidine and purine derivatives.

Recently, the three-dimensional structure of the active site of human DNA polymerase alpha (pol alpha) was proposed based on the application of molecular modeling methods and molecular dynamic simulations. The modeled structure of the enzyme was used for docking selective inhibitors (nucleotide analogs and the non-nucleoside inhibitor aphidicolin) in its active site in order to design new drugs for actinic keratosis and squamous cell carcinoma (SCC). The resulting complexes explained the geometrical and physicochemical interactions of the inhibitors with the amino acid residues involved in binding to the catalytic site, and offered insight into the experimentally derived binding data. The proposed structures were synthesized and tested in vitro for their influence on human keratinocytes and relevant tumor cell lines. Effects were compared to aphidicolin which inhibits pol alpha in a non-competitive manner, as well as to diclofenac and 5-fluorouracil, both approved for therapy of actinic keratosis. Here we describe three new nucleoside analogs inhibiting keratinocyte proliferation by inhibiting DNA synthesis and inducing apoptosis and necrosis. Thus, the combination of modeling studies and in vitro tests should allow the derivation of new drug candidates for the therapy of skin tumors, given that the agents are not relevant substrates of nucleotide transporters expressed by skin cancer cells. Kinases for nucleoside activation were detected, too, corresponding with the observed effects of nucleoside analogs.

Monophasic, cohesive-polydensified-matrix crosslinking-technology-based hyaluronic acid filler for the treatment of facial lipoatrophy in HIV-infected patients.

Human immunodeficiency virus (HIV)-associated facial lipoatrophy (FLA) represents a common and highly stigmatizing side effect of retroviral therapy. By causing loss of subcutaneous adipose tissue mainly in the cheek, temple and periocular area, FLA can significantly affect the patient's quality of life, both physically and psychologically. A limited quantity of data has been published on various filling substances for the management of FLA. Here, the authors present two patients with HIV-associated FLA successfully treated with a novel HA filler over a period of 24 months.

Safety and efficacy of sertaconazole nitrate cream 2% in the treatment of tinea pedis interdigitalis: a subgroup analysis.

Sertaconazole nitrate is a broad-spectrum antifungal agent indicated in the United States for the treatment of tinea pedis interdigitalis. The objective of this subgroup analysis was to evaluate the safety and efficacy of sertaconazole nitrate cream 2%, specifically in participants with tinea pedis interdigitalis (ie, fungal skin disease of the toe web) of dermatophyte origin. A total of 92 participants were included in this analysis. The primary end points were eradication of the pathogen (confirmed by fungal culture results) and reduction in total clinical score (TCS) of at least 2 points. Secondary end points included reducing signs and symptoms and reporting adverse events (AEs). After 4 weeks of treatment, 88.8% (79/89) of evaluable participants achieved success on the primary end points. Most participants also demonstrated substantial improvement in signs and symptoms after 4 weeks of treatment: 63.7% (58/91) were free of erythema, 33.0% (30/91) were free of desquamation, and 91.2% (83/91) were free of itch. The rate of reported AEs was low (8.7% [8/92]), and none were considered serious. These findings indicate that sertaconazole nitrate cream 2% is highly safe and effective in the treatment of tinea pedis interdigitalis.

Which plant for which skin disease? Part 2: Dermatophytes, chronic venous insufficiency, photoprotection, actinic keratoses, vitiligo, hair loss, cosmetic indications.

This paper continues our review of scientifically evaluated plant extracts in dermatology. After plants effective against dermatophytes, botanicals with anti-edema effects in chronic venous insufficiency are discussed. There is good evidence from randomized clinical studies that plant extracts from grape vine leaves (Vitis vinifera), horse chestnut (Aesculus hippocastanum), sea pine (Pinus maritima) and butcher's broom (Ruscus aculeatus) can reduce edema in chronic venous insufficiency. Plant extracts from witch hazel (Hamamelis virginiana), green tea (Camellia sinensis), the fern Polypodium leucotomos and others contain antioxidant polyphenolic compounds that may protect the skin from sunburn and photoaging when administered topically or systemically. Extracts from the garden spurge (Euphorbia peplus) and from birch bark (Betula alba) have been shown to be effective in the treatment of actinic keratoses in phase II studies. Some plant extracts have also been investigated in the treatment of vitiligo, various forms of hair loss and pigmentation disorders, and in aesthetic dermatology.

Bilateral areolar and periareolar pityriasis versicolor.

An adolescent boy presented with isolated, symmetrical, bilateral areolar and periareolar pityriasis versicolor. This extremely rare condition should be considered in the differential diagnosis of light brown patches on the areolae.

[Evidence-based cosmetics: concepts and applications in photoaging of the skin and xerosis]. / Evidenz-basierte Kosmetika: Konzept und Anwendung bei den Zielstellungen Licht-geschädigte Altershaut und Xerosis.

As well as for topically used dermatological agents, studies performed according to the rules of evidence-based medicine (EBM) are also needed for cosmetics. Although the concept of evidence-based cosmetics has been only partly developed so far, there are some agents and preparations available that can be considered as evidence-based. In this paper we present data from several studies that claim to have examined and demonstrated the efficacy of cosmetic preparations for the management of solar damage and aging skin as well as lentigo and melanosis according to EBM criteria. Certainly, further controlled studies are needed to cover the main application areas of dermocosmetics. Retinol and antioxidant agents such as vitamin C and coenzymes that positively act via several mechanisms on collagen biosynthesis can be considered evidence-based substances for the management of aging skin. According to the same criteria, the preventive effect of regularly applied dermocosmetic sun screens on the development of actinic keratosis could also be shown. Dermocosmetic sun screens should offer adequate protection against UV-B and UV-A light by combining compatible organic and/or non-organic UV-filters and at the same time be well tolerated. Furthermore, they may contain some additional agents such as antioxidants, DNA repair enzymes, dexpanthenol, glycerin or hamamelis distillate. In the treatment of melanosis, a substantial bleaching effect corresponding to that of 0.1% topical tretinoin can be achieved with 10% all-trans-retinol gel. Preparations containing urea, ammonium lactate or glycerol in different concentrations are considered the best characterized and most effective substances for the care of dry skin. However, the lack of controlled studies confirming the efficacy of dermocosmetic products as well as the superiority of the preparation incorporating the active agent over the corresponding base is a problem yet to be solved. Undoubtedly, the efficacy and the sustainability of the achieved effects have to be examined and proven accordingly to EBM criteria in further active cosmetic agents. Moreover, generally accepted guidelines for the examination of efficacy and tolerability of dermocosmetics have to be developed.

The significance of itraconazole for treatment of fungal infections of skin, nails and mucous membranes.

Itraconazole is an antifungal drug from the triazole group with distinct in vitro activity against dermatophytes, yeasts and some molds. Itraconazole has a primarily fungistatic activity. Itraconazole accumulates in the stratum corneum and in nail material due to its high affinity to keratin, as well as in sebum and vaginal mucosa. Together with terbinafine and fluconazole, itraconazole belongs to the modern highly effective systemic antifungal drugs with a favorable risk-benefit ratio and for this reason is a preferred therapy option for fungal infections of skin, nails and mucous membranes. Compared to terbinafine in the treatment of fingernail and toenail fungal infections, itraconazole offers the advantage of a broad antifungal spectrum and better effectiveness against onychomycosis caused by yeasts yet appears inferior with regard to the more common dermatophyte infections. Itraconazole constitutes an important therapy option, along with fluconazole, terbinafine, ketoconazole and griseofulvin, for the treatment of dermatophyte infections of glabrous skin (tinea pedis, tinea manuum, tinea corporis and tinea cruris) in adults following unsuccessful topical therapy. In the oral therapy of tinea capitis, itraconazole plays an especially important role, in particular for disease caused by Microsporum canis (for children, however, only off-label use is feasible currently). In the treatment of oropharyngeal candidiasis, candidiasis of the skin and vulvovaginal candidiasis, itraconazole and fluconazole are the preferred treatment options in cases in which topical therapy has proven unsuccessful.

X-ray structures of Sap1 and Sap5: structural comparison of the secreted aspartic proteinases from Candida albicans.

Proteolytic activity is an important virulence factor for Candida albicans (C. albicans). It is attributed to the family of the secreted aspartic proteinases (Saps) from C. albicans with a minimum of 10 members. Saps show controlled expression and regulation for the individual stages of the infection process. Distinct isoenzymes can be responsible for adherence and tissue damage of local infections, while others cause systemic diseases. Earlier, only the structures of Sap2 and Sap3 were known. In our research, we have now succeeded in solving the X-ray crystal structures of the apoenzyme of Sap1 and Sap5 in complex with pepstatin A at 2.05 and 2.5 A resolution, respectively. With the structure of Sap1, we have completed the set of structures of isoenzyme subgroup Sap1-3. Of subgroup Sap4-6, the structure of the enzyme Sap5 is the first structure that has been described up to now. This facilitates comparison of structural details as well as inhibitor binding modes among the different subgroup members. Structural analysis reveals a highly conserved overall secondary structure of Sap1-3 and Sap5. However, Sap5 clearly differs from Sap1-3 by its electrostatic overall charge as well as through structural conformation of its entrance to the active site cleft. Design of inhibitors specific for Sap5 should concentrate on the S4 and S3 pockets, which significantly differ from Sap1-3 in size and electrostatic charge. Both Sap1 and Sap5 seem to play a major part in superficial Candida infections. Determination of the isoenzymes' structures can contribute to the development of new Sap-specific inhibitors for the treatment of superficial infections with a structure-based drug design program.

The use of reconstructed human epidermis for skin absorption testing: Results of the validation study.

A formal validation study was performed, in order to investigate whether the commercially-available reconstructed human epidermis (RHE) models, EPISKIN, EpiDerm and SkinEthic, are suitable for in vitro skin absorption testing. The skin types currently recommended in the OECD Test Guideline 428, namely, ex vivo human epidermis and pig skin, were used as references. Based on the promising outcome of the prevalidation study, the panel of test substances was enlarged to nine substances, covering a wider spectrum of physicochemical properties. The substances were tested under both infinite-dose and finite-dose conditions, in ten laboratories, under strictly controlled conditions. The data were subjected to independent statistical analyses. Intra-laboratory and inter-laboratory variability contributed almost equally to the total variability, which was in the same range as that in preceding studies. In general, permeation of the RHE models exceeded that of human epidermis and pig skin (the SkinEthic RHE was found to be the most permeable), yet the ranking of substance permeation through the three tested RHE models and the pig skin reflected the permeation through human epidermis. In addition, both infinite-dose and finite-dose experiments are feasible with RHE models. The RHE models did not show the expected significantly better reproducibility, as compared to excised skin, despite a tendency toward lower variability of the data. Importantly, however, the permeation data showed a sufficient correlation between all the preparations examined. Thus, the RHE models, EPISKIN, EpiDerm and SkinEthic, are appropriate alternatives to human and pig skin, for the in vitro assessment of the permeation and penetration of substances when applied as aqueous solutions.

Lipid nanoparticles for improved topical application of drugs for skin diseases.

Due to the lower risk of systemic side effects topical treatment of skin disease appears favourable, yet the stratum corneum counteracts the penetration of xenobiotics into viable skin. Particulate carrier systems may mean an option to improve dermal penetration. Since epidermal lipids are found in high amounts within the penetration barrier, lipid carriers attaching themselves to the skin surface and allowing lipid exchange between the outermost layers of the stratum corneum and the carrier appear promising. Besides liposomes, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have been studied intensively. Here we describe the potential of these carrier systems and compare the dermal uptake from SLN and NLC to the one of alternative vehicle systems. A special focus is upon the interactions of active ingredients and the lipid matrix as well as the quantification of dermal penetration.

The crystal structure of the secreted aspartic proteinase 3 from Candida albicans and its complex with pepstatin A.

The family of secreted aspartic proteinases (Sap) encoded by 10 SAP genes is an important virulence factor during Candida albicans (C. albicans) infections. Antagonists to Saps could be envisioned to help prevent or treat candidosis in immunocompromised patients. The knowledge of several Sap structures is crucial for inhibitor design; only the structure of Sap2 is known. We report the 1.9 and 2.2 A resolution X-ray crystal structures of Sap3 in a stable complex with pepstatin A and in the absence of an inhibitor, shedding further light on the enzyme inhibitor binding. Inhibitor binding causes active site closure by the movement of a flap segment. Comparison of the structures of Sap3 and Sap2 identifies elements responsible for the specificity of each isoenzyme.