In vivo transcutaneous penetration of nicotinates and sensitive skin

Proclivity to develop irritant reactions and transcutaneous penetration of nicotinates has been investigated in 20 subjects of both sexes, divided into reactors and nonreactors on the basis of the responses to irritant stimuli. 1% sodium lauryl sulphate (patch application for 24 h) and 5% lactic acid in aqueous solutions were used to detect chemical and sensory (subjective) irritation. The vasodilatation induced was measured using a chromameter for 1 h after topical application of the chemical. The area-under-the-curve response and the peak response was used to assess the in vivo penetration of methyl nicotinate (10 mM in aqueous solution). Significant differences were found between reactors and non-reactors. Non-reactors showed a significantly decreased area-under-the-curve response and peak response to methyl nicotinate compared to reactors. Nicotinate-induced vasodilatation has been used as a model to study transcutaneous penetration of chemicals; the correlation between increased penetration of nicotinates and skin hyperreactivity to irritant substances may suggest an increased transcutaneous penetration of water-soluble chemicals in individuals with sensitive skin.     

An in vivo model to evaluate the efficacy of barrier creams on the level of skin penetration of chemicals

The reservoir function and the barrier function are important properties of the skin. The reservoir function is dependent on the barrier function which, however, needs support by protective measures, in particular under working conditions. Barrier creams represent a possibility to protect the skin. In the present study, a method was developed to investigate the effectiveness of reservoir closure by different formulations. Patent Blue V in water was used as a model penetrant. Its penetration, with and without barrier cream treatment, was analyzed by tape stripping in combination with UV/VIS spectroscopic measurements. The investigations showed that the stratum corneum represents a reservoir for topically applied Patent Blue V in water. Furthermore, the barrier investigations showed that vaseline and bees wax form a 100% barrier on the skin surface. The third barrier cream, containing waxes and surfactant, only partially showed a protective effect against the penetration of Patent Blue V in water. Strong interindividual differences were observed for this barrier product. In conclusion, it was assumed that the application of barrier creams cannot replace other protective measures and should be maximally used to inhibit low-grade irritants or in combination with other protectants or in body areas where other protective measures are not applicable.     

Effect of barrier perturbation on cutaneous salicylic acid penetration in human skin: in vivo pharmacokinetics using microdialysis and non-invasive quantification of barrier function

We have used microdialysis in the dermis for assessing penetration kinetics of salicylic acid (SA) in healthy volunteers (n = 18), following application on the volar aspect of the left forearm. Penetration was monitored at four locations: in normal (unmodified) skin and in skin with perturbed barrier function from (i) repeated tape stripping (ii) irritant dermatitis from 1 or 2% sodium lauryl sulphate (SLS) for 24 h and (iii) delipidization by acetone. The order of the treatments was randomized according to a latin square design. Epidermal barrier function and skin irritation were assessed in each location using evaporimetry and colorimetry. Transepidermal water loss (TEWL) values confirmed that both mild (acetone), moderate (1% SLS) and severe barrier damage (tape stripping and 2% SLS) had occurred. Microdialysis sampling with two parallel probes in the dermis was performed in each of the four treatment areas for every subject. SA (5% in ethanol) was applied in a chamber glued to the skin overlying the microdialysis probes and sampling was continued for 4 h. SA was detectable in all samples and measurable in all samples from penetration through perturbed skin. Comparing the SA penetration in barrier-perturbed skin with the penetration in unmodified skin in the same subject, the mean SA penetration increase was 2.2-fold in acetone-treated skin (P = 0.012), 46-fold in mild dermatitis and 146- and 157-fold in severe dermatitis and tape stripped skin, respectively (P < 0.001). The penetration of SA significantly correlated with the measurements of barrier perturbation by TEWL (P = 0.01) and erythema (P = 0.02) for each individual. Microdialysis sampling of SA penetration was more sensitive than non-invasive measuring techniques in detecting significant barrier perturbation in acetone-treated skin. A positive dose-response relationship for the percutaneous penetration of SA in response to increasing SLS pretreatment concentrations and thus the degree of irritant dermatitis was found. When analysing data by location on the forearm, a tendency towards an intraregional variation in the reactivity to barrier damage was found, with the most proximal location displaying higher reactivity scores than the most distal location in response to the same barrier perturbation procedures. The penetration of SA was not significantly different between locations. In conclusion, using microdialysis in the dermis to obtain real-time dermal pharmacokinetics in the target organ, this study demonstrates highly increased and differentiated cutaneous penetration of SA in barrier-perturbed skin. The measured drug penetration was demonstrated to correlate with non-invasive quantification of barrier damage.     

Evaluation of barrier creams - introduction and comparison of 3 in vivo methods

Often barrier creams (BC) do not fulfil their protecting behaviour, even when promised by the manufacturers. Therefore, it is of great importance to develop standardized in vivo techniques to prove the potency of BC in humans. In the present study, 3 promising techniques for the analysis of BC were evaluated: laser scanning microscopy, laser doppler flowmetry, and the tape-stripping procedure. Sodium fluorescein and glycerol trinitrate acted as hydrophilic model-penetrating substances. By means of these methods, 3 different BC were tested and compared for their potency. The investigations showed that primarily the tape-stripping procedure and also the laser scanning microscopy are promising tools for the evaluation of BC. In contrast, the laser doppler flowmetry represents a less feasible technique. In addition, the evaluation of BC showed that Vaseline enfolds a 100% barrier on the skin surface for the penetration of a hydrophilic dye, whereas BC1 only partially showed a protective effect and BC2 exhibited almost no potency. In conclusion, it can be referred that laser scanning microscopy and the tape-stripping procedure represent 2 non-invasive in vivo techniques, which enable a fast investigation concerning the potency of BC.     

Drug delivery through the skin barrier enhanced by treatment with tissue-tolerable plasma

Most treatments in dermatology and cosmetology are based on the penetration of topically applied drugs into the skin or through the skin barrier to the target structure in the living tissue. In the case of healthy skin, scarcely 1% of the applied drugs pass the skin barrier, depending on their chemical properties. Therefore, different physical and chemical methods have been developed to stimulate the penetration process. All these methods are based on the partial destruction of the barrier. In this study, an electrical tissue-tolerable plasma (TTP) was used to increase the penetration of a topically applied model drug (fluorescent dye) through the skin barrier. Using laser scanning microscopy, the distribution of the model drug in different depths of the skin was investigated. It was found that the plasma treatment of the skin is a very efficient process to deliver topically applied substances into the living tissue. In the case of the non-plasma-treated skin, it was found that the fluorescent dye could be detected exclusively on the skin surface. If the dye was applied to the TTP-treated skin, it could be observed in high concentration also in deeper parts of the skin extending down to the stratum basale and the papillary structure.     

Folic acid and creatine improve the firmness of human skin in vivo

Background The decrease in firmness is a hallmark of skin aging. Accelerated by chronic sun exposure, fundamental changes occur within the dermal extracellular matrix over the years, mainly impairing the collagenous network. Aims Based on the qualitative and quantitative assessment of skin firmness, in vitro and in vivo studies were carried out to elucidate the effects of topical folic acid and creatine to counteract this age‐dependent reduction in the amount of collagen. Patients/Methods Topical application of a commercially available formulation containing folic acid and creatine was performed to study effects on skin firmness in vivo using cutometric analysis. Imaging and quantification of collagen density were carried out using multiphoton laser scanning microscopy (MPLSM). To investigate the effects of these compounds on collagen gene expression, procollagen synthesis, and collagen fibril organization, complementary in vitro studies on cultured fibroblast‐populated collagen gels were carried out. Results The underlying structural changes in the collagen network of young and aged sun‐exposed facial skin in vivo were visualized by MPLSM. Topical application of a folic acid‐ and creatine‐containing formulation significantly improved firmness of mature skin in vivo. Treatment of fibroblast‐populated dermal equivalents with folic acid and creatine increased collagen gene expression and procollagen levels and improved collagen fiber density, suggesting that the in vivo effects are based on the overall improvement of the collagen metabolism. Conclusions Employing MPLSM, dermal changes occurring in photo‐aged human skin were visualized in an unprecedented manner and correlated to a loss of firmness. Treatment of aged skin with a topical formulation containing folic acid and creatine counteracted this age‐dependent decline by exerting sustained effects on collagen metabolism. Our results support previous findings on the efficacy of these actives.     

In vivo confocal laser scanning microscopy for non-invasive diagnosis of pemphigus foliaceus

Background/purpose: In vivo confocal laser scanning microscopy (CLSM) is a modern non-invasive method for investigation of the skin that allows real-time visualization of individual cells and sub-cellular structures at resolution similar to the one provided by routine histopathology. Our aim was to investigate the potential of CLSM for non-invasive diagnosis of pemphigus foliaceus (PF).
Methods: Pre-existing and mechanically induced lesions in two cases of PF were examined by means of CLSM, parallel to routine histology, direct immunofluorescence microscopy and enzyme-linked immunosorbent assay performed in the same patients.
Results: The morphological features characteristic for PF, namely an intraepidermal blister with acantholytic cells in the blister cavity, were readily detectable by means of CLSM. The findings were consistent in both patients and across the investigated lesions. The confocal images were consistent with the routine histology of the pre-existing lesions. No differences in the confocal images of pre-existing lesions compared with mechanically induced ones were observed.
Conclusions: Our findings suggest the potential of CLSM as a non-invasive tool for the diagnosis of pemphigus and differentiation of its subtype. Although at present the method cannot replace the current diagnostic standards for pemphigus, it may be successfully used as in vivo non-invasive screening tool to facilitate the diagnosis and point to the need for further investigation of the patient.     

Age related changes of human skin investigated with histometric measurements by confocal laser scanning microscopy in vivo

Background/aims: The confocal laser scanning microscope Vivascope (Lucid, Henrietta) allows skin to be studied in real-time with a resolution of 0.5 µm horizontal and 1.3 µm vertical in vivo. In this study, we present the results of a comparison between the skin of an older and a younger group of volunteers by in vivo histometric measurements.
Methods: To investigate changes caused by age, 13 young (18–25 years) and 13 older (> 65 years) volunteers were examined. The following parameters were measured using the Vivascope at the volar forearm: minimal thickness of the epidermis (E_min), size of cells in the granular layer (A_gran), thickness of the horny layer (DSC), thickness of the basal layer (DSB) and number of dermal papillae per area (PapI). The image analysis program image tool was used to measure the size of the cells and the thickness of the basal layer.
Results: The older group of volunteers showed a significant increase in E_min, no significant change in DSC, a significant decrease in dermal papillae and in the thickness of the basal layer, and an increase in A_gran compared to the younger group.
Conclusions: Histometric measurements by in vivo confocal laser scanning microscopy are a sensitive and non-invasive tool for characterizing and quantifying histological changes of the epidermis and papillary dermis due to ageing.     

In vivo imaging of intradermal tattoos by confocal scanning laser microscopy

BACKGROUND/AIMS: In vivo confocal laser scanning microscopy (CLSM) is a new method for high-resolution imaging of intact skin in situ. Horizontal mapping of the outer skin is provided (magnification x 1000). OBJECTIVES: Tattooing is popular all over the world; however, tattooed skin has not been studied in using CLSM. RESULTS: Tattoos in two volunteers were studied using the Vivascope1500 of Lucid Inc. Subepidermal massive deposits of dense, clustered pigment granules up to about 3 mum in size corresponding to black tattoos, and more scarce and diffuse deposits, corresponding to red, blue and green tattoos, were observed. Diffuse pigment granules tended to accumulate in the outer dermis underneath the level of the basement membrane zone. CONCLUSIONS: Dermal pigments from tattoos can be imaged in vivo using CLSM. This application of CLSM has an important future potential for pre-evaluation of tattoos before laser removal, predicting good or poor outcome of laser removal.     

Correlation of image analysis features and visual morphology in melanocytic skin tumours using in vivo confocal laser scanning microscopy

Background/purpose: In vivo confocal laser scanning microscopy (CLSM) represents a novel imaging tool that allows the non-invasive examination of skin cancer morphology at a quasi histological resolution without biopsy. Previous studies dealt with the search for diagnostic, but subjective visual criteria. In this study we examined the correlation between objectively reproducible image-analysis features und visual morphology in melanocytic skin tumours using CLSM.
Methods: Eight hundred and fifty-seven CLSM tumour images including 408 benign nevi and 449 melanoma images were evaluated. Image analysis was based on features of the wavelet transform and classification tree analysis (CART) was used for classification purposes. In a second step, morphologic details of CLSM images, which have turned out to be of diagnostic significance by the classification algorithm were evaluated.
Results: CART analysis of the whole set of CLSM images correctly classified 97.55% of all melanoma images and 96.32% of all nevi images. Seven classification tree nodes seemed to indicate benign nevi, whereas six nodes were suggestive for melanoma morphology. The visual examination of selected nodes demonstrated that monomorphic melanocytic cells and melanocytic cell nests are characteristic for benign nevi whereas polymorphic melanocytic cells, disarray of melanocytic architecture and poorly defined or absent keratinocyte cell borders are characteristic for melanoma.
Conclusion: Well-known, but subjective CLSM criteria could be objectively reproduced by image analysis features and classification tree analysis. Moreover, features not accessible to the human eye seem to contribute to classification success.     

Safety of sodium fluorescein for in vivo study of skin

BACKGROUND/PURPOSE: Epicutaneous labeling or intradermal injection of the fluorescent sodium fluorescein is being used increasingly to investigate skin conditions in vivo when using non-invasive devices such as confocal scanning laser microscopy. Sodium fluorescein was used intravenously for decades for the examination of the vasculature of the ocular fundus (fluorescein angiography) and as eye drops for diagnosis of corneal erosions. The objective of this article is to systematically review the literature on fluorescein and conclude its safety in cutaneous research to support research planning and evaluations by ethics committees. METHODS: A number of databases and the literature about safety and toxicity of fluorescein in animal and human studies were searched and analyzed. RESULTS: Side effects or adverse events reported in the literature were related to intravenous bolus injection. Transient nausea and vomiting may occur. Other adverse events such as vasovagal reaction, cardiac or respiratory effects and anaphylaxes are extremely rare but may be fatal. Intradermal injection may cause mild itch or pain; systemic adverse event was reported. Epicutaneous labeling is associated with no reported problem. A typical local dose is several magnitudes of order smaller than a typical intravenous dose. CONCLUSION: Fluorescein has been used for many years in medicine for diagnostic purposes and is widely safe, albeit intravenous bolus injection may cause serious adverse reactions. In the literature, we could not trace reports of local or systemic side effects of topical sodium fluorescein except itch and pain on intradermal injection, however, dependent on the fluorescein preparation used. Local dermal application of fluorescein for in vivo study of skin may be considered widely safe.     

Orally administered ethanol: transepidermal pathways and effects on the human skin barrier

Ethanol intake is associated with a variety of skin diseases. The aim of the present study was (1) to identify the pathways of release of orally administered ethanol through the skin, and (2) to investigate the effects of a single oral dose of ethanol on the penetration of topically applied substances into the skin. Ethanol evaporation via the skin was measured using the new technique of ion mobility spectrometry (IMS). Transepidermal water loss (TEWL) and skin surface temperature were simultaneously measured before and after ethanol consumption. Measurements were performed on skin sites with different stratum corneum (SC) thickness, and density of follicles and sweat glands. These appendages were selectively sealed to investigate their participation in ethanol evaporation. The penetration of a topically applied UV filter substance was studied before and after ethanol consumption after removing the SC with adhesive tape. Ethanol evaporation was measured within 5 min of consumption, while the skin surface temperature remained nearly constant. The sealing of the appendages did not have a significant effect on ethanol evaporation. On the forehead, a higher TEWL value was measured than on the forearm. On both skin sites, an increase in TEWL was observed after ethanol ingestion. No influence of orally administered ethanol on the penetration of the topically applied UV filter substance was observed. The results indicate that ethanol evaporation occurs via the lipid layers without a significant effect on the penetration of the topically applied substance.     

Variation in barrier impairment and inflammation of human skin as determined by sodium lauryl sulphate penetration rate

BACKGROUND: Skin irritability after a brief exposure to the model skin irritant, sodium lauryl sulphate (SLS), is known to vary considerably between individuals. A difference in the skin barrier to SLS may contribute to this variation. To date, no human in vivo data have been available on SLS penetration into the skin. OBJECTIVES: We studied whether the SLS penetration rate into the stratum corneum (SC) is related to impairment of the water barrier function and inflammation of the skin. METHODS: The penetration of SLS into the SC was assessed using a noninvasive tape-stripping procedure in 20 volunteers after a 4-h exposure to 1% SLS. Additionally, the effect of a 24-h exposure to 1% SLS on the skin water barrier function was assessed by measuring the transepidermal water loss (TEWL). The accompanying inflammation was quantified by measuring erythema. RESULTS: The mean +/- SD diffusivity of SLS (D) and the SLS permeability coefficient (Kp) were 1.4 +/- 0.6 x 10(-8) cm2 h(-1) and 1.5 +/- 0.7 x 10(-3) cm h(-1), respectively. A multiple regression analysis showed that the baseline TEWL, SC thickness and SLS penetration parameters K (SC/water partition coefficient) and D clearly influenced the increase in TEWL after the 24-h irritation test (explained variance: r2 = 0.80). Change in erythema was mainly influenced by SC thickness. CONCLUSIONS: We found that variation in the barrier impairment and inflammation of human skin depends on the SLS penetration rate, which was mainly determined by SC thickness.     

Migration and penetration of a fluorescent textile dye into the skin –in vivo versus in vitro methods

Abstract:  The amount of textile dye migration from the textile and penetration into the skin is relevant when assessing the risk of textile dyes. In this paper, in vivo methods were developed using a harmless textile dye with a strong fluorescence and were then compared with in vitro methods. For the in vivo method, the textile was applied to the lower back of six volunteers wearing the textile 12 h and to the lower back of 12 volunteers during 30 min active sport. The maximum skin absorption of 55 ± 17 ng/cm² was obtained in the group engaged in sports. The in vitro methods, which involved the application of the textile to the pig ear skin, was shown to yield similar results to the 12 h in vivo group (31.2 ± 9.6 ng/cm² vs 27 ± 14 ng/cm²). The migration of the textiles into artificial sweat resulted in approximately 20  μ g/cm². The disadvantage of such textile extract applications on pig ear skin is discussed. It could be demonstrated that the absorption of the dye is strongly correlated to the amount of sweat, whereas the contact time was less important.