A method for predicting steady-state rate of skin penetration in vivo

A simple in vivo method was proposed for predicting the steady-state rate of penetration of drugs across the stratum corneum. Both the diffusion coefficient and the partition coefficient in the stratum corneum can be determined by the amounts of drug in the stratum corneum at two time intervals under transient conditions after transdermal drug application. The amount of drug entering the stratum corneum is determined by 20 strippings with an adhesive tape. The steady-state rate of penetration was then calculated for the thickness of the stratum corneum and the concentration of the donor solution. The steady-state rates of penetration of ascorbic acid and estradiol across hairless mouse skin were evaluated from this in vivo approach and compared with those obtained from in vitro penetration experiment using excised hairless mouse skin. The data confirmed that the proposed in vivo method can predict the steady-state rate of penetration of these drugs across the stratum corneum in normal skin.     

Effect of physicochemical properties of cyclic terpenes on their ex vivo skin absorption and elimination kinetics

BACKGROUND: The terpenes disturb lipid arrangement in the intercellular region of the stratum corneum (SC) that leads to the increased permeability of the skin. This effect is used in technology of transdermal drug forms and depends on physicochemical properties of terpenes and their amounts penetrated to the stratum corneum; however terpenes do not need penetrate into viable skin tissue and this event is not even desired. OBJECTIVE: To correlate skin absorption and elimination kinetics of four cyclic terpenes, namely alpha-pinene, beta-pinene, eucalyptol and terpinen-4-ol, applied as neat substance with their physicochemical properties. METHODS: The terpenes were applied onto the human skin in vitro, and after 1-4 h their content in the separated by a tape-stripping method stratum corneum layers and in the epidermis/dermis was determined using GC. Similarly, the amounts of terpenes in the skin were analysed during 4 h following 1 h absorption. RESULTS: The fastest and progressive penetration into all skin layers was observed for terpinen-4-ol. All studied terpenes are absorbed in the viable epidermis/dermis, however penetration into this layers is time-dependent process, constantly increasing during 4 h. Like for stratum corneum, the largest cumulation in epidermis/dermis was observed for terpinen-4-ol. The elimination of terpenes from the stratum corneum was fast, especially in deeper layers, and much faster if the initial cumulation was small. CONCLUSION: Investigated cyclic terpenes represent different penetration and elimination characteristics and do not permeate across the skin to the acceptor medium due to large cumulation in the skin tissue. The penetration of terpenes into stratum corneum is greater if their log P-value is close to 3.     

By the grace of peeling: the brace function of the stratum corneum in the protection from photo-induced keratinocyte carcinogenesis

Chemical peeling with salicylic acid in polyethylene glycol vehicle (SA-PEG) is a safe and effective method for the rejuvenation of photo-damaged skin. The procedure removes photo-damaged stratum corneum, which consists of immature, fragile cornified envelopes (CEs) and stimulates the reconstruction of the stratum corneum with mature, rigid CEs. In UVB-irradiated hairless mice this procedure, which affects the stratum corneum only, suppresses skin tumor development. In addition, chemical peeling with SA-PAG suppresses p53 expression in mice and normalizes keratinocyte differentiation in both mice and humans. The stratum corneum functions as a barrier against physical and chemical insult and various infectious agents. Here, we hypothesize on a new function of the stratum corneum: a brace function that structurally protects keratinocytes from atypical differentiation or disordered proliferation. Although the precise mechanism remains to be elucidated, there is definite value to be gained from further investigation. This review discusses basic information about chemical peeling with SA-PEG, looks at its action on photo-induced tumor suppression, and proposes a new function for the stratum corneum in keratinocyte proliferation/differentiation.     

The content of free amino acids in the stratum corneum is increased in senile xerosis

Xerosis is one of the characteristics of aged skin. Xerosis may be caused by a decrease in the stratum corneum free amino acids which are natural moisturizing factors derived from filaggrin. In aged skin, filaggrin is immunohistochemically decreased compared with the levels in young skin. However, the differences in stratum corneum amino acids between aged and young skin have not been analyzed quantitatively. Therefore, in this study we determined the stratum corneum amino acids per 1000 stratum corneum cells in aged and young skin by high-performance liquid chromatography. The amount of filaggrin mRNA in the epidermis was also compared between aged and young skin using RT-PCR. The total amount of amino acids in the stratum corneum was larger in aged senile xerosis skin than in young skin. Only a few amino acids were found in the stratum corneum of ichthyosis vulgaris patients (control skin). The expression of filaggrin mRNA in aged skin was, however, similar to that in young skin. These findings suggest that the immunohistochemical decrease in filaggrin in aged skin may be caused by promotion of filaggrin proteolysis in the upper layers of the stratum spinulosum.     

By the grace of peeling: the brace function of the stratum corneum in the protection from photo-induced keratinocyte carcinogenesis

Chemical peeling with salicylic acid in polyethylene glycol vehicle (SA-PEG) is a safe and effective method for the rejuvenation of photo-damaged skin. The procedure removes photo-damaged stratum corneum, which consists of immature, fragile cornified envelopes (CEs) and stimulates the reconstruction of the stratum corneum with mature, rigid CEs. In UVB-irradiated hairless mice this procedure, which affects the stratum corneum only, suppresses skin tumor development. In addition, chemical peeling with SA-PAG suppresses p53 expression in mice and normalizes keratinocyte differentiation in both mice and humans. The stratum corneum functions as a barrier against physical and chemical insult and various infectious agents. Here, we hypothesize on a new function of the stratum corneum: a brace function that structurally protects keratinocytes from atypical differentiation or disordered proliferation. Although the precise mechanism remains to be elucidated, there is definite value to be gained from further investigation. This review discusses basic information about chemical peeling with SA-PEG, looks at its action on photo-induced tumor suppression, and proposes a new function for the stratum corneum in keratinocyte proliferation/differentiation.     

大鼠皮肤角质层对荧光素钠脂质体透皮吸收的影响

目的 探讨大鼠皮肤角质层对荧光素钠脂质体透皮吸收的影响。方法 采用Scotch胶带剥脱皮肤角质层,利用荧光光度计、Franz扩散池和荧光显微镜研究皮肤角质层和活性层中荧光素钠的含量;观察皮肤剥脱角质层后荧光素钠脂质体透皮吸收能力的变化和荧光素钠在皮肤中的分布。结果 应用脂质体后皮肤各层荧光素钠的分布量均较相应溶液和凝胶作用后增加(P<0.01);荧光素钠在皮肤各层的分布比例发生了变化;剥脱角质层后脂质体荧光素钠混悬液和荧光素钠溶液各时间点累积透皮浓度相比较,差异无显著性(P>0.05);脂质体荧光素钠凝胶和荧光素钠凝胶相比较,各时间段累积透皮浓度差异无显著性(P>0.05)。结论 脂质体能增加皮肤各层的荧光素钠含量,并能够改变皮肤角质层和去角质层皮肤之间的荧光素钠比例。剥脱角质层后脂质体制剂透皮量显著增加,与相应的普通制剂相比差异无显著性;剥脱角质层后脂质体制剂的毛囊扩散途径不发生改变。     

Hydration characteristics of pathologic stratum corneum--evaluation of bound water

We measured in vitro the hygroscopicity and bound (non-freezing) water of various samples of pathologic horny layer obtained from the lesions of senile xerotic skin and psoriasis vulgaris and the normal horny layer from glabrous skin and plantar horny layer. The amount of water taken up by pathologic stratum corneum was much smaller than that by normal horny layer in an environment at a high relative humidity (RH). Tightly bound primary water to stratum corneum measured by Karl Fischer's method was about 5 mg/100 mg of dry stratum corneum in all the samples studied, while less tightly bound secondary water was much smaller in amount in pathologic stratum corneum than in the controls, i.e., 31.7 mg/100 mg dry scale from senile xerosis and 27.2 mg/100 mg dry psoriatic scale as compared with 38.2 mg/100 mg dry normal stratum corneum from glabrous skin and 37.3 mg/100 mg dry normal plantar stratum corneum. We believe that the low hygroscopicity of the pathologic stratum corneum is due to this smaller capacity for secondary bound water, which is responsible for the development of a dry scaly appearance even at high RH.     

Composition of free long-chain (sphingoid) bases in stratum corneum of normal and pathologic human skin conditions.

The composition of molecular species of free long-chain bases (FLCB) isolated from stratum corneum of various human skin conditions was analyzed by the high-performance liquid chromatographic and fast atom bombardment mass spectrometric methods. FLCB with carbon length ranging from 16 to 20 comprised about 0.3% of total lipids in stratum corneum of normal and pathologic skin conditions. Major FLCB included sphinganines and sphingenines with 18 to 20 carbons and some phytosphingosines such as t17:1, t18:1, t18:0, t20:1, and t20:0. Compared with stratum corneum of normal lower legs, molar percentages of FLCB having 18 carbons and those with 20 carbons were slightly higher and lower, respectively, in normal plantar epidermis, showing site-related differences in normal skin. Psoriatic scales and hyperkeratotic stratum corneum from clavus and plantar keratoderma contained increased levels of FLCB with 18 carbons and decreased levels of FLCB with 20 carbons. These findings may reflect abnormal keratinization in hyperkeratotic skin conditions.     

Sebaceous gland secretion is a major physiologic route of vitamin E delivery to skin

Skin plays an important part in the protection against oxidative stressors, such as ultraviolet radiation, ozone, and chemicals. This study was based on the observation that upper facial stratum corneum contained significantly higher levels of the antioxidant alpha-tocopherol than corresponding layers of arm stratum corneum. We hypothesized that the underlying mechanism involves sebaceous gland secretion of vitamin E. To test this, we examined in eight human volunteers: (i) stratum corneum levels and distribution profiles of vitamin E in sites with a different sebaceous gland density (arm versus cheek); (ii) whether vitamin E is a significant constituent of human sebum; and (iii) if there is a correlation between levels of vitamin E and squalene, a marker of sebum secretion, in skin surface lipids. Using standardized techniques for stratum corneum tape stripping and sebum collection, followed by high-performance liquid chromatography analysis of tocopherols and squalene, we found that: (i) the ratio of cheek versus upper arm alpha-tocopherol levels was 20 : 1 for the upper stratum corneum and decreased gradually with stratum corneum depth; (ii) vitamin E (alpha- and gamma-tocopherol forms) is a significant constituent of human sebum and is continuously secreted at cheek and forehead sites during a test period of 135 min; and (iii) vitamin E correlates well with levels of cosecreted squalene (r2 = 0.86, p < 0.001). In conclusion, sebaceous gland secretion is a relevant physiologic pathway for the delivery of vitamin E to upper layers of facial skin. This mechanism may serve to protect skin surface lipids and the upper stratum corneum from harmful oxidation.     

Antioxidant enzyme activity in human stratum corneum shows seasonal variation with an age-dependent recovery

The stratum corneum, as the body's principal barrier to the environment, is continuously exposed to environmental sources of reactive oxygen species like ultraviolet light, ozone, and pollution. Reactive oxygen species are believed to be involved in cancer, aging, and inflammatory skin disorders. We have developed a method to measure catalase and superoxide dismutase activity on tape strippings from the human stratum corneum and demonstrated a gradient of antioxidant enzyme activity across the stratum corneum with decreasing levels towards the skin surface. Sun exposure resulted in a seasonal variation of the catalase activity in stratum corneum, with low activities in summer and higher activities in winter for the same person, whereas superoxide dismutase activity in stratum corneum did not seem to vary in those conditions. Exposure of human skin to broadband ultraviolet-A resulted in a dose-dependent deactivation of the catalase activity in stratum corneum within 24 h, whereas exposure to ultraviolet-B had no effect. Superoxide dismutase activity in stratum corneum was not affected by ultraviolet-A or ultraviolet-B irradiation within 24 h. After exposure to a dose of 15 J per cm2 broadband ultraviolet-A, full recovery of the catalase activity occurred in 3-4 wk at an age-dependent rate. We conclude that sun exposure results in a disturbed catalase to superoxide dismutase ratio in the stratum corneum. This may lead to an increased vulnerability to oxidative damage in stratum corneum barrier components. These results therefore stress the importance of providing efficient protection for this internal defense mechanism in sun-exposed areas of the skin.     

Changes in environmental humidity affect the water-holding property of the stratum corneum and its free amino acid content,and the expression of filaggrin in the epidermis of hairless mice

BACKGROUND: Seasonal changes affect the condition of skin and may trigger various cutaneous disorders. OBJECTIVE: To clarify the effects of the environmental humidity on the skin pathology, we studied the effects of the humidity on a water-holding function of the stratum corneum. METHODS: We evaluated the skin surface conductance, amino acid in the stratum corneum, and immunoreactivity of filaggrin of the epidermis of hairless mice kept in different environmental humidity. RESULTS: Skin surface conductance in the stratum corneum of hairless mice 3-7 days after transfer from a humid environment (>80% relative humidity) to a dry (<10% relative humidity) environment, was significantly lower than that of the mice transferred from a normal environment (relative humidity=40-70%) to a dry environment. The free amino acid content in the stratum corneum significantly decreased 24 h after we transferred the mice from a normal to a dry condition, then it recovered to the original level within 3 days, while the mice transferred from a humid to a dry condition showed a significantly lower amino acid content even 7 days after the transfer. No obvious change was observed in the relative composition of the major components of the free amino acids during the experiments. Immunoreactivity of filaggrin, which is the main precursor of free amino acids in the stratum corneum, also became faint in the epidermis of the mice transferred from a humid or normal to a dry environment. CONCLUSION: These results suggested that a drastic decrease in the environmental humidity reduced the total free amino acid generation and consequently induced skin surface dryness in the stratum corneum.     

Controlled removal of human stratum corneum by pulsed laser

A new method is presented for controlled removal of the stratum corneum of human skin. An excimer laser (193 nm wavelength, 14 ns pulsewidth) was used to remove stratum corneum from in vitro human skin samples by an ablative process. The tritiated water (3H2O) permeability constant and electrical resistance of skin samples were measured in a diffusion chamber apparatus to quantify the enhancement of skin permeability. Each laser pulse ablates about a micrometer of stratum corneum, which allows controlled removal of tissue. The maximum specific enhancement of the 3H2O permeability constant obtained after complete stratum corneum removal depends on the laser pulse energy used. The most gentle laser ablation, achieved with a radiant exposure of 70 mJ/cm2 per pulse, produced a 124-fold enhancement, which is comparable to that achieved after stratum corneum removal by tape-stripping or removal of epidermis by mild heat treatment. Rapid tissue ablation occurred at higher radiant exposures of 170-480 mJ/cm2 per pulse, but only a 45-fold enhancement of permeability was achieved. The precision with which stratum corneum can be ablated using excimer laser pulses may allow further basic research on the internal structure of stratum corneum and on the re-epithelization in controlled wounds. The technique may prove useful clinically to enhance percutaneous transport in applications such as topical delivery of drugs, patch testing, and percutaneous blood gas monitoring.     

Correlations between epithelial cells and bacterial populations in bacteriological skin samples

The purposes of this study were to determine whether the variability in populations of skin bacteria observed between individuals is related to the number of epithelial cells removed during the skin scrubbing procedure and also whether any particular group of microorganisms can be directly associated with epithelial cells. Foreheads of 31 subjects were sampled using the cup scrubbing method, and bacterial populations were quantified as: total anaerobes, aerobes, Propionibacterium spp., aerobic coryneforms, and Micrococcaceae. Epithelial cells were counted in a haemocytometer. Correlation coefficients were positive between total bacterial populations and epithelial cell counts, with the largest values of 0.64 for Propionibacterium spp., 0.58 for Micrococcaceae, and 0.15 for aerobic coryneforms. High epithelial cell counts were always associated with high bacterial populations, but high bacterial counts occurred, in some instances, with low epithehal cell counts; epithelial cells are not, therefore, responsible for variations in bacterial populations in all cases. Stained smears of epithelial cells from subjects from whom aerobic coryneforms were not cultured showed microcolonies of presumptive anaerobic diphtheroids, closely associated with epithelial cells. These findings and the high correlation between Propionibacterium spp. and epithelial cells suggests a re-examination of the location of anaerobes in the stratum corneum.     

麻风患者愈后表皮某些生理功能障碍研究

目的：了解麻风愈后者其原受累及部位是否存在角质层生理功能障碍。方法：利用多功能皮肤生理仪对麻风患者痊愈后前臂原皮损处角质层含水量、酸碱度、透过皮肤水分丢失率及屏障功能的恢复速度进行测量。结果：与正常对照组相比，麻风愈后者原皮损处角质层含水量明显降低（P〈0．002），以瘤型麻风患者降低幅度最大；皮肤表面的pH明显增高（P〈0．0001），界线类偏结核型患者pH改变不甚明显；麻风愈后者原皮损处的基础表皮通透屏障功能好于正常人，以界线类偏瘤型患者水分丢失量降低最明显：麻风愈后者原皮损处表皮通透屏障功能的恢复速度无明显改变。结论：麻风患者痊愈后仍然有角质层生理功能障碍。     

pH directly regulates epidermal permeability barrier homeostasis,and stratum corneum integrity/cohesion

Both exposure of stratum corneum to neutral pH buffers and blockade of acidification mechanisms disturb cutaneous permeability barrier homeostasis and stratum corneum integrity/cohesion, but these approaches all introduce potentially confounding variables. To study the consequences of stratum corneum neutralization, independent of hydration, we applied two chemically unrelated superbases, 1,1,3,3-tetramethylguanidine or 1,8-diazabicyclo [5,4,0] undec-7-ene, in propylene glycol:ethanol (7:3) to hairless mouse skin and assessed whether discrete pH changes alone regulate cutaneous permeability barrier function and stratum corneum integrity/cohesion, as well as the responsible mechanisms. Both 1,1,3,3-tetramethylguanidine and 1,8-diazabicyclo [5,4,0] undec-7-ene applications increased skin surface pH in parallel with abnormalities in both barrier homeostasis and stratum corneum integrity/cohesion. The latter was attributable to rapid activation (<20 min) of serine proteases, assessed by in situ zymography, followed by serine-protease-mediated degradation of corneodesmosomes. Western blotting revealed degradation of desmoglein 1, a key corneodesmosome structural protein, in parallel with loss of corneodesmosomes. Coapplication of serine protease inhibitors with the superbase normalized stratum corneum integrity/cohesion. The superbases also delayed permeability barrier recovery, attributable to decreased beta-glucocerebrosidase activity, assessed zymographically, resulting in a lipid-processing defect on electron microscopy. These studies demonstrate unequivocally that stratum corneum neutralization alone provokes stratum corneum functional abnormalities, including aberrant permeability barrier homeostasis and decreased stratum corneum integrity/cohesion, as well as the mechanisms responsible for these abnormalities.     

Electron paramagnetic resonance: a new techniquein skin research

Electron paramagnetic resonance (EPR) spectra of nitroxide spin probes have been used for studying biological membranes and chemical‐membrane interaction. We have investigated the influence of surfactants on the intercellular lipid structure of cadaver stratum corneum and the possibility of EPR spectral measurements on the stripped stratum corneum utilizing cyanoacrylate resin, which might reflect the actual skin lipid conditions. Conclusion: EPR spectra are useful in evaluating the fluidity measurement of stratum corneum of cadaver skin and stripped stratum corneum.     

Measuring depth depends on frequency in electrical skin impedance measurements

Background/aims: Multi-frequency electrical measurements in vivo on stratum corneum are frequently reported in the literature. It is well known that the measured volume of skin is dependent on measuring frequency, and we wanted to investigate to what extent viable skin influences the measured impedance as a function of frequency.
Methods: Finite element modelling (FEM) was used to investigate the current and potential distribution in the skin when measuring impedance with two concentric surface electrodes.
Results: The study showed that the impedance measured reflects mainly the stratum corneum at low frequencies, in this case below 1 kHz, and that the viable skin dominates at higher frequencies.
Conclusion: It is concluded that a multi-frequency measurement on isolated stratum corneum is not possible in vivo with conventional techniques, except within a very narrow and low frequency range.     

Image analysis of the distribution of turnover rate in the stratum corneum

Background/aims: A common method to evaluate turnover rate in the stratum corneum is to measure the change in fluorescence intensity with time after dyeing the stratum corneum with fluorescent pigments. If these changes in fluorescence over time are carefully observed, the rate of decline in fluorescence intensity differs among different small areas on the skin surface. A possible relationship between these differences and dry skin has been reported. The purpose of this research was to develop a method for analyzing turnover rate in the stratum corneum in each small area on the surface of the skin as well as to investigate the variations in the inconsistencies of turnover rate. Methods: The stratum corneum at six body regions (forehead, cheek, forearm, opisthenar, back and lower leg) was dyed with dansyl chloride (DC), and the change in fluorescence intensity over time was imaged with a highly sensitive television camera through special filters. Then, the fluorescent distribution in the images was analyzed to measure the change in fluorescence intensity with time among the small areas. Also, the decline in fluorescence intensity observed was categorized using specific characteristics into six different types. Results: By attaching a filter to an ultraviolet (UV) light source in order to transmit light at the excitation wavelength and a filter to the camera lens to transmit light at the wavelength of DC fluorescence, we could image the low intensity fluorescent light from the DC without interference from the UV light exciting the DC. The characteristics of the variation in the decline in fluorescence intensity were categorized into six patterns. Type I: pattern showing a uniform decline in fluorescence intensity. Type II: pattern showing sporadic areas where fluorescence intensity declines quickly. Type III: pattern showing relatively large areas where fluorescence intensity declines slowly. Type IV: pattern showing sporadic areas of fluorescence intensity, matched with locations of keratotic plugs. Type V: pattern showing sporadic fluorescent areas, not matched with locations of keratotic plugs. Type VI: pattern showing a partial, drastic decline in fluorescence intensity occurring on inflamed skin after sunburn. Conclusions: By analyzing the image generated from a highly sensitive television camera equipped with special filters, we could measure turnover rate of the stratum corneum at any small area. The variations in Types IV and V were believed to be derived from keratotic plugs and closed comedo. Except for Type VI, observed on significant skin inflammation, Type II and Type III were believed to be the patterns that reflected variations in turnover rate in stratum corneum itself.     

Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin?

Stratum corneum lipids are an important determinant for both water-retention function and permeability-barrier function in the stratum corneum. However, their major constituent, ceramides, have not been analyzed in detail in skin diseases such as atopic dermatitis that show defective water-retention and permeability-barrier function. In an attempt to assess the quantity of ceramides per unit mass of the stratum corneum in atopic dermatitis, stratum corneum sheet was removed from the forearm skin by stripping with cyanoacrylate resin and placed in hexane/ethanol extraction to yield stratum corneum lipids. The stratum corneum was dispersed by solubilization of cyanoacrylate resin with dimethylformamide, and after membrane filtration, the weight of the stratum corneum mass was measured. The ceramides were quantified by thin-layer chromatography and evaluated as microgram/mg stratum corneum. In the forearm skin of healthy individuals (n = 65), the total ceramide content significantly declined with increasing age. In atopic dermatitis (n = 32-35), there was a marked reduction in the amount of ceramides in the lesional forearm skin compared with those of healthy individuals of the same age. Interestingly, the non-lesional skin also exhibited a similar and significant decrease of ceramides. Among six ceramide fractions, ceramide 1 was most significantly reduced in both lesional and non-lesional skin. These findings suggest that an insufficiency of ceramides in the stratum corneum is an etiologic factor in atopic dry skin.     

Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature.

The outermost layer of mammalian skin, the stratum corneum, provides the body with a barrier against transepidermal water loss and penetration of agents from outside. The lipid-rich extracellular matrix surrounding the corneocytes in the stratum corneum is mainly responsible for this barrier function. In this study (cryo-) electron diffraction was applied to obtain information about the local lateral lipid organization in the extracellular matrix in relation to depth in human stratum corneum. For this purpose, stratum corneum grid-strips were prepared from native skin in vivo and ex vivo. It was found that the lipid packing in samples prepared at room temperature is predominantly orthorhombic. In samples prepared at 32 degrees C the presence of a hexagonal packing is more pronounced in the outer layers of the stratum corneum. Gradually increasing the specimen temperature from 30 to 40 degrees C induced a further transition from an orthorhombic to a hexagonal sublattice. At 90 degrees C all lipids were present in a fluid phase. These results are in good agreement with previously reported wide angle X-ray diffraction and Fourier transformed infrared spectroscopy studies. We conclude that the lipids in human stratum corneum are highly ordered throughout the stratum corneum and that electron diffraction allows monitoring of the local lipid organization, which contributes to the understanding of stratum corneum barrier function.