John I. Ademola

Colorimetric method for quantifying human Stratum corneum removed by adhesive-tape stripping.

Tape-stripping of the skin is a useful method for removing the stratum corneum and obtaining more information about the function of this skill layer as the main barrier for skin penetration. The amount of stratum corneum removed is of relevance in establishing the concentration profile of chemicals within the stratum corneum after topical application. Weighing is the preferred method for measuring the amount stripped, but because it is often subject to artifacts, alternative methods are sought. We present a simple, colorimetric method for determining the amount stratum corneum removed by sequential adhesive-tape-stripping of human skin in vivo. The method is based on quantification of the sodium hydroxide soluble protein fraction using a commercially available protein assay similar to the Lowry assay. The method is shown to be an accurate and reproducible alternative to weighing, also demonstrating uniform removal of stratum corneum layers following the very initial strips.

Sodium lauryl sulfate (SLS) induced irritant contact dermatitis : a correlation study between ceramides and in vivo parameters of irritation

Sodium lauryl sulfate (SLS), a surfactant frequently used in the induction of experimental irritant contact dermatitis in animals and in humans, characterstically induces a dose‐related increase in TEWL (transepidermal water loss). Ceramides are considered to be important in the regulation of the skin barrier. We therefore examined the relationship between initial ceramide content of stratum corneum and induced changes in skin color (erythema) and barrier function, after SLS application under occlusion (1% and 3% in water) to the forearm of 14 volunteers. Stratum corneum sheets were removed, stratum corneum lipids extracted, and ceramide composition determined from chromatograms (TLC) using densitometry. After determining baseline skin color and TEWL at each area. 2 samples of stratum corneum were obtained from each volunteer. Clinical and instrumental controls of the SLS‐induced irritation were performed at 24, 48, 72 and 96 h. Erythema was evaluated by colorimetry; barrier impairment by changes in TEWL. We found inverse correlations between baseline ceramide 61 (weight) and the 24 h erythema score for SLS: between ceramide I and 24 h TEWL, and between ceramide 611 and 72 h TEWL for SLS 3%. Our findings suggest that low levels of these ceramides may determine a proclivity to SLS‐induced irritant contact dermatitis.

In vitro percutaneous absorption and metabolism in man of 2-chloro-4-ethylamino-6-isopropylamine-s-triazine (atrazine).

Atrazine is an extensively used herbicide in the USA. Our objective was to determine the absorption and metabolism (detoxification) of atrazine in human skin. Percutaneous absorption of atrazine in human skin from four sources was examined utilizing a flow-through in-vitro diffusion system. About 16.4% of the applied dose was absorbed by the skin. Radioactivity in the receptor fluid at 20 h was less than 5% of the administered dose. The highest concentration of the applied dose was found in the skin supernates, where 12.0% of the dose (68 nmol) was recovered. Some metabolites of atrazine were identified by thin layer and high pressure liquid chromatography after extraction of receptor fluid and the skin supernates. Two metabolites of atrazine [2-chloro-4-ethylamino-6-amino-striazine (desisopropylatrazine) and 2-chloro-4,6-diamino-s-triazine] were found in the receptor fluid and the skin supernates. An additional metabolite (2-chloro-4-amino-6-isopropylamino-s-triazine) was found in the skin supernates. Since desisopropylatrazine represented about 50% of the total metabolites formed during percutaneous absorption, cleavage of the N-isopropyl to the amino product was a key step in the metabolism of atrazine. Further metabolism may proceed by cleavage of the N-deethyl group to give totally dealkylated atrazine. The biotransformation of atrazine was studied in skin microsomal fraction supplemented with an NADPH-generating system. In analogy to metabolism during percutaneous absorption, atrazine was metabolized to its deisopropyl and deethylpropyl derivatives. In addition, 2-hydroxy derivatives of atrazine were formed by the skin microsomal fractions. The biotransformation of atrazine by skin microsomal enzymes indicates the metabolic capacity of the tissue. Cutaneous metabolism of atrazine may be an additional route by which human skin detoxifies the pesticide following topical exposure.

Effect of application time of betamethasone-17-valerate 0.1% cream on skin blanching and stratum corneum drug concentration

The effect of application time on skin blanching response and stratum corneum concentration after topical application of 0.1% betamethasone-17-valerate cream on healthy volunteers was assessed. Total corticosteroid content in the stratum corneum (SC) was determined at different application times (0.5, 8, 10 and 24 h) from five subjects in whom blanching was also evaluated at the same application times by visual score, colorimetry (L-, a- and b-values) and Laser Doppler flowmetry. No significant differences between corticosteroid concentration in the SC at 8, 10 and 24 h was observed when compared using ANOVA and t-test (P > 0.05) while drug content at 0.5 h was significantly lower (P < 0.05). Significant differences between the blanching response at 0.5 h and the other time points (8, 10 and 24 h) were observed by visual assessment and a-value readings from a chromameter. However, at 24 h visual score and †a-values were lower than those measured at 8 and 10 h. but this difference was significant only for †a-values. This findings suggest that skin blanching may not be related to drug concentration in the stratum corneum and that †a-readings may be more sensitive and accurate than visual score in evaluating skin blanching. L-values were not significantly different at all the time points while b-values at 0.5 h were significantly different only from those at 8 and 10 h. Skin blanching was not observed when laser Doppler flow was measured while the concentration parameter was capable of detecting blanching; however, the concentration values were not significantly different at all the application times. The results of this study suggest that the duration of application should be carefully chosen to assess betamethasone-17- valerate topical bioavailability since after long application time skin blanching response may not be related to drug concentration in the stratum corneum.

Metabolism and degradation of betamethasone 17-valerate in homogenized living skin equivalent.

The metabolism of betamethasone 17-valerate was estimated using an artificial living skin equivalent (LSE). Betamethasone 17-valerate, betamethasone 21-valerate and betamethasone were measured by a normal-phase high-performance liquid chromatographic (HPLC) method. Betamethasone 17-valerate was added to the culture medium with or without LSE homogenate. Degradation profiles (%) of betamethasone 17-valerate remaining in the culture medium with skin homogenate did not differ from those without homogenate. However, the conversion of betamethasone 21-valerate to betamethasone was accelerated by skin homogenate, indicating that LSE has a sufficient level of esterase.

Effects of freezing and azide treatment of in vitro human skin on the flux and metabolism of 8-methoxypsoralen

Clinical and in vitro evidence suggests that the physicochemical properties of the skin influence the process by which drugs are transported through skin. The effects of skin storage, preparation and pretreatment on the permeation and metabolism of (8-methoxypsoralen (8-MOP), as a model penetrant, were studied using the flow-through in vitro cell diffusion system. The metabolites and unchanged drug were estimated by thin-layer chromatography. While the permeability of 8-MOP was similar in fresh (445 cm.h-1) and azide-treated (449 cm.h-1) skin (p < 0.01), decreased permeability was observed in frozen skin (406 cm.h-1, p < 0.01). A 2.8-fold increase in the cumulative flux of 8-MOP at 24 h through azide-pretreated (2.5 x 10(-3) mumol.h-1.cm-1) versus fresh skin (9.1 x 10(-4) mumol.h-1.cm-1) was observed (p < 0.01). There was a slight increase in the flux of 8-MOP at 24 h when skin was frozen, compared with untreated skin. Increase in the flux of 8-MOP in frozen skin might result from the alteration of the molecular arrangement of the skin components during freezing. In addition to the obvious differences between frozen and fresh skin, these observations discourage the use of frozen skin. There is a moderate relationship between the permeability and flux of 8-MOP through frozen skin. A similar but nonrelated correlation was observed between the permeability and flux of 8-MOP through azide-treated skin samples (r = 0.6). These findings suggest that azide and freezing treatments lower the skin barrier properties to the transport of 8-MOP. Apparently, factors that may affect the inherent permeability of human skin, particularly those related to the handling, storage and pretreatment of skin with solvents and chemicals, can also influence topical drug delivery. The metabolic capacity of frozen skin and fresh skin remained constant during the period of study. These data may be of value in the development of topical methoxypsoralen systems. Further in vitro and in vivo studies are required to ascertain the generalization of this process.

Skin Penetration and Metabolism: Comparative Evaluation of Skin Equivalent, Cell Culture, and Human Skin

AbstractUsing an organotypic model of human skin, living skin equivalent (LSE) and its homogenate, monolayer cell culture, and human skin, we have studied the simultaneous transport and metabolic fate of two compounds. The LSE was maintained in an assay culture medium. When the model compounds were applied to LSE at dosages of 9.0 ± 1.2 µg/cm2, the transport of salicylic acid through human skin was 0.12 ±0.1µg/cm2/hr. Salicylic acid flux was 5.6-fold greater in LSE than in human skin. Shorter lag time of absorption was observed in LSE (∼ 1.5 hr) than in human skin (7–9 hr). Percutaneous transport across LSE was accompanied by metabolism of the compounds and there were quantitative and qualitative similarities between the metabolites produced by the LSE and human skin. When compounds were added to homogenates (LSE or skin) and to human cell cultures, the activities of the LSE and human fractions were similar. Data from the present study demonstrate that although LSE is more permeable than human skin, the a...

Pharmacodynamic Measurements of 8-Methoxypsoralen in Human Skin

The efficiency of the epicutaneous microcirculation is important to the elicitation of pharmacological effects and percutaneous absorption of drugs. Laser Doppler flowmeter, chroma-meter and measureme